Sample records for affects mitochondrial respiration

  1. Oxidative stress negatively affects human sperm mitochondrial respiration.

    PubMed

    Ferramosca, Alessandra; Pinto Provenzano, Sara; Montagna, Daniela Domenica; Coppola, Lamberto; Zara, Vincenzo

    2013-07-01

    To correlate the level of oxidative stress in serum and seminal fluid and the level of sperm deoxyribonucleic acid (DNA) fragmentation with sperm mitochondrial respiratory efficiency. Sperm mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. A possible relationship between sperm mitochondrial respiratory efficiency, the level of oxidative stress, and the level of sperm DNA fragmentation was investigated. Sperm motility was positively correlated with mitochondrial respiration but negatively correlated with oxidative stress and DNA fragmentation. Interestingly, sperm mitochondrial respiratory activity was negatively affected by oxidative stress and DNA fragmentation. Our data indicate that sperm mitochondrial respiration is decreased in patients with high levels of reactive oxygen species by an uncoupling between electron transport and adenosine triphosphate synthesis. This reduction in mitochondrial functionality might be 1 of the reasons responsible for the decrease in spermatozoa motility. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    PubMed

    Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M

    2016-11-07

    An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.

  3. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

    PubMed

    Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W

    2015-01-15

    Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.

  4. Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap Thickness.

    PubMed

    Yu, Emma P K; Reinhold, Johannes; Yu, Haixiang; Starks, Lakshi; Uryga, Anna K; Foote, Kirsty; Finigan, Alison; Figg, Nichola; Pung, Yuh-Fen; Logan, Angela; Murphy, Michael P; Bennett, Martin

    2017-12-01

    Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis. Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E-deficient (ApoE -/- ) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE -/- mice overexpressing the mitochondrial helicase Twinkle (Tw + /ApoE -/- ). Tw + /ApoE -/- mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw + /ApoE -/- mice had decreased necrotic core and increased fibrous cap areas, and Tw + /ApoE -/- bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress-induced apoptosis. Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in

  5. Mitochondrial flash as a novel biomarker of mitochondrial respiration in the heart.

    PubMed

    Gong, Guohua; Liu, Xiaoyun; Zhang, Huiliang; Sheu, Shey-Shing; Wang, Wang

    2015-10-01

    Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions. Copyright © 2015 the American Physiological Society.

  6. Metabolic depression during warm torpor in the Golden spiny mouse (Acomys russatus) does not affect mitochondrial respiration and hydrogen peroxide release.

    PubMed

    Grimpo, Kirsten; Kutschke, Maria; Kastl, Anja; Meyer, Carola W; Heldmaier, Gerhard; Exner, Cornelia; Jastroch, Martin

    2014-01-01

    Small mammals actively decrease metabolism during daily torpor and hibernation to save energy. Recently, depression of mitochondrial substrate oxidation in isolated liver mitochondria was observed and associated to hypothermic/hypometabolic states in Djungarian hamsters, mice and hibernators. We aimed to clarify whether hypothermia or hypometabolism causes mitochondrial depression during torpor by studying the Golden spiny mouse (Acomys russatus), a desert rodent which performs daily torpor at high ambient temperatures of 32°C. Notably, metabolic rate but not body temperature is significantly decreased under these conditions. In isolated liver, heart, skeletal muscle or kidney mitochondria we found no depression of respiration. Moderate cold exposure lowered torpor body temperature but had minor effects on minimal metabolic rate in torpor. Neither decreased body temperature nor metabolic rate impacted mitochondrial respiration. Measurements of mitochondrial proton leak kinetics and determination of P/O ratio revealed no differences in mitochondrial efficiency. Hydrogen peroxide release from mitochondria was not affected. We conclude that interspecies differences of mitochondrial depression during torpor do not support a general relationship between mitochondrial respiration, body temperature and metabolic rate. In Golden spiny mice, reduction of metabolic rate at mild temperatures is not triggered by depression of substrate oxidation as found in liver mitochondria from other cold-exposed rodents. © 2013.

  7. SMG-1 kinase attenuates mitochondrial ROS production but not cell respiration deficits during hyperoxia.

    PubMed

    Resseguie, Emily A; Brookes, Paul S; O'Reilly, Michael A

    Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia. Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes. Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53. Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.

  8. Targeting mitochondrial respiration as a therapeutic strategy for cervical cancer.

    PubMed

    Tian, Shenglan; Chen, Heng; Tan, Wei

    2018-05-23

    Targeting mitochondrial respiration has been documented as an effective therapeutic strategy in cancer. However, the impact of mitochondrial respiration inhibition on cervical cancer cells are not well elucidated. Using a panel of cervical cancer cell lines, we show that an existing drug atovaquone is active against the cervical cancer cells with high profiling of mitochondrial biogenesis. Atovaquone inhibited proliferation and induced apoptosis with varying efficacy among cervical cancer cell lines regardless of HPV infection, cellular origin and their sensitivity to paclitaxel. We further demonstrated that atovaquone acts on cervical cancer cells via inhibiting mitochondrial respiration. In particular, atovaquone specifically inhibited mitochondrial complex III but not I, II or IV activity, leading to respiration inhibition and energy crisis. Importantly, we found that the different sensitivity of cervical cancer cell lines to atovaquone were due to their differential level of mitochondrial biogenesis and dependency to mitochondrial respiration. In addition, we demonstrated that the in vitro observations were translatable to in vivo cervical cancer xenograft mouse model. Our findings suggest that the mitochondrial biogenesis varies among patients with cervical cancer. Our work also suggests that atovaquone is a useful addition to cervical cancer treatment, particularly to those with high dependency on mitochondrial respiration. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Effect of Simvastatin, Coenzyme Q10, Resveratrol, Acetylcysteine and Acetylcarnitine on Mitochondrial Respiration.

    PubMed

    Fišar, Z; Hroudová, J; Singh, N; Kopřivová, A; Macečková, D

    2016-01-01

    Some therapeutic and/or adverse effects of drugs may be related to their effects on mitochondrial function. The effects of simvastatin, resveratrol, coenzyme Q10, acetylcysteine, and acetylcarnitine on Complex I-, Complex II-, or Complex IV-linked respiratory rate were determined in isolated brain mitochondria. The protective effects of these biologically active compounds on the calcium-induced decrease of the respiratory rate were also studied. We observed a significant inhibitory effect of simvastatin on mitochondrial respiration (IC50 = 24.0 μM for Complex I-linked respiration, IC50 = 31.3 μM for Complex II-linked respiration, and IC50 = 42.9 μM for Complex IV-linked respiration); the inhibitory effect of resveratrol was found at very high concentrations (IC50 = 162 μM for Complex I-linked respiration, IC50 = 564 μM for Complex II-linked respiration, and IC50 = 1454 μM for Complex IV-linked respiration). Concentrations required for effective simvastatin- or resveratrol-induced inhibition of mitochondrial respiration were found much higher than concentrations achieved under standard dosing of these drugs. Acetylcysteine and acetylcarnitine did not affect the oxygen consumption rate of mitochondria. Coenzyme Q10 induced an increase of Complex I-linked respiration. The increase of free calcium ions induced partial inhibition of the Complex I+II-linked mitochondrial respiration, and all tested drugs counteracted this inhibition. None of the tested drugs showed mitochondrial toxicity (characterized by respiratory rate inhibition) at drug concentrations achieved at therapeutic drug intake. Resveratrol, simvastatin, and acetylcarnitine had the greatest neuroprotective potential (characterized by protective effects against calcium-induced reduction of the respiratory rate).

  10. SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake.

    PubMed

    Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

    2017-05-01

    Mitochondrial calcium ([Ca 2+ ] m ) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca 2+ ] m uptake upon SK channel activation as detected by time lapse mitochondrial Ca 2+ measurements with the Ca 2+ -binding mitochondria-targeted aequorin and FRET-based [Ca 2+ ] m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca 2+ ] m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death.

  11. SK2 channels regulate mitochondrial respiration and mitochondrial Ca2+ uptake

    PubMed Central

    Honrath, Birgit; Matschke, Lina; Meyer, Tammo; Magerhans, Lena; Perocchi, Fabiana; Ganjam, Goutham K; Zischka, Hans; Krasel, Cornelius; Gerding, Albert; Bakker, Barbara M; Bünemann, Moritz; Strack, Stefan; Decher, Niels; Culmsee, Carsten; Dolga, Amalia M

    2017-01-01

    Mitochondrial calcium ([Ca2+]m) overload and changes in mitochondrial metabolism are key players in neuronal death. Small conductance calcium-activated potassium (SK) channels provide protection in different paradigms of neuronal cell death. Recently, SK channels were identified at the inner mitochondrial membrane, however, their particular role in the observed neuroprotection remains unclear. Here, we show a potential neuroprotective mechanism that involves attenuation of [Ca2+]m uptake upon SK channel activation as detected by time lapse mitochondrial Ca2+ measurements with the Ca2+-binding mitochondria-targeted aequorin and FRET-based [Ca2+]m probes. High-resolution respirometry revealed a reduction in mitochondrial respiration and complex I activity upon pharmacological activation and overexpression of mitochondrial SK2 channels resulting in reduced mitochondrial ROS formation. Overexpression of mitochondria-targeted SK2 channels enhanced mitochondrial resilience against neuronal death, and this effect was inhibited by overexpression of a mitochondria-targeted dominant-negative SK2 channel. These findings suggest that SK channels provide neuroprotection by reducing [Ca2+]m uptake and mitochondrial respiration in conditions, where sustained mitochondrial damage determines progressive neuronal death. PMID:28282037

  12. Changes in mitochondrial respiration in the human placenta over gestation.

    PubMed

    Holland, Olivia J; Hickey, Anthony J R; Alvsaker, Anna; Moran, Stephanie; Hedges, Christopher; Chamley, Lawrence W; Perkins, Anthony V

    2017-09-01

    Placental mitochondria are subjected to micro-environmental changes throughout gestation, in particular large variations in oxygen. How placental mitochondrial respiration adapts to changing oxygen concentrations remains unexplored. Additionally, placental tissue is often studied in culture; however, the effect of culture on placental mitochondria is unclear. Placental tissue was obtained from first trimester and term (laboured and non-laboured) pregnancies, and selectively permeabilized to access mitochondria. Respirometry was used to compare respiration states and substrate use in mitochondria. Additionally, explants of placental tissue were cultured for four, 12, 24, 48, or 96 h and respiration measured. Mitochondrial respiration decreased at 11 weeks compared to earlier gestations (p = 0.05-0.001), and mitochondrial content increased at 12-13 weeks compared to 7-10 weeks (p = 0.042). In term placentae, oxidative phosphorylation (OXPHOS) through mitochondrial complex IV (p < 0.001), the relative proportion of OXPHOS CI (p < 0.001), the total capacity of the respiratory system (p = 0.003), and mitochondrial content (p < 0.001) were higher compared to first trimester. Respiration was increased (p ≤ 0.006-0.001) in laboured compared to non-laboured placenta. After four hours of culture, respiration was depressed compared to fresh tissue from the same placenta and continued to decline with time in culture. Markers of apoptosis were increased, while markers of autophagy, mitochondrial biogenesis, and mitochondrial membrane potential were decreased after four hours of culture. Respiration and mitochondrial content alter over gestation/with labour. Decreased respiration at 11 weeks and increased mitochondrial content at 12-13 weeks may relate to onset of maternal blood flow, and increased respiration as a result of labour may be an adaptation to ischaemia-reperfusion. At term, mitochondria were more susceptible to changes in respiratory function

  13. A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration

    PubMed Central

    Zhang, Huiliang; Wang, Pei; Bisetto, Sara; Yoon, Yisang; Chen, Quan; Sheu, Shey-Shing; Wang, Wang

    2017-01-01

    Aims Mitochondria in adult cardiomyocytes exhibit static morphology and infrequent dynamic changes, despite the high abundance of fission and fusion regulatory proteins in the heart. Previous reports have indicated that fusion proteins may bear functions beyond morphology regulation. Here, we investigated the role of fission protein, dynamin-related protein 1 (DRP1), on mitochondrial respiration regulation in adult cardiomyocytes. Methods and results By using genetic or pharmacological approaches, we manipulated the activity or protein level of fission and fusion proteins and found they mildly influenced mitochondrial morphology in adult rodent cardiomyocytes, which is in contrast to their significant effect in H9C2 cardiac myoblasts. Intriguingly, inhibiting endogenous DRP1 by dominant-negative DRP1 mutation (K38A), shRNA, or Mdivi-1 suppressed maximal respiration and respiratory control ratio in isolated mitochondria from adult mouse heart or in adult cardiomyocytes from rat. Meanwhile, basal respiration was increased due to increased proton leak. Facilitating mitofusin-mediated fusion by S3 compound, however, failed to inhibit mitochondrial respiration in adult cardiomyocytes. Mechanistically, DRP1 inhibition did not affect the maximal activity of individual respiratory chain complexes or the assembly of supercomplexes. Knocking out cyclophilin D, a regulator of mitochondrial permeability transition pore (mPTP), abolished the effect of DRP1 inhibition on respiration. Finally, DRP1 inhibition decreased transient mPTP-mediated mitochondrial flashes, delayed laser-induced mPTP opening and suppressed mitochondrial reactive oxygen species (ROS). Conclusion These results uncover a novel non-canonical function of the fission protein, DRP1 in maintaining or positively stimulating mitochondrial respiration, bioenergetics and ROS signalling in adult cardiomyocyte, which is likely independent of morphological changes. PMID:27794519

  14. Betaine is a positive regulator of mitochondrial respiration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Icksoo, E-mail: icksoolee@dankook.ac.kr

    2015-01-09

    Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro.more » Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.« less

  15. A novel fission-independent role of dynamin-related protein 1 in cardiac mitochondrial respiration.

    PubMed

    Zhang, Huiliang; Wang, Pei; Bisetto, Sara; Yoon, Yisang; Chen, Quan; Sheu, Shey-Shing; Wang, Wang

    2017-02-01

    Mitochondria in adult cardiomyocytes exhibit static morphology and infrequent dynamic changes, despite the high abundance of fission and fusion regulatory proteins in the heart. Previous reports have indicated that fusion proteins may bear functions beyond morphology regulation. Here, we investigated the role of fission protein, dynamin-related protein 1 (DRP1), on mitochondrial respiration regulation in adult cardiomyocytes. By using genetic or pharmacological approaches, we manipulated the activity or protein level of fission and fusion proteins and found they mildly influenced mitochondrial morphology in adult rodent cardiomyocytes, which is in contrast to their significant effect in H9C2 cardiac myoblasts. Intriguingly, inhibiting endogenous DRP1 by dominant-negative DRP1 mutation (K38A), shRNA, or Mdivi-1 suppressed maximal respiration and respiratory control ratio in isolated mitochondria from adult mouse heart or in adult cardiomyocytes from rat. Meanwhile, basal respiration was increased due to increased proton leak. Facilitating mitofusin-mediated fusion by S3 compound, however, failed to inhibit mitochondrial respiration in adult cardiomyocytes. Mechanistically, DRP1 inhibition did not affect the maximal activity of individual respiratory chain complexes or the assembly of supercomplexes. Knocking out cyclophilin D, a regulator of mitochondrial permeability transition pore (mPTP), abolished the effect of DRP1 inhibition on respiration. Finally, DRP1 inhibition decreased transient mPTP-mediated mitochondrial flashes, delayed laser-induced mPTP opening and suppressed mitochondrial reactive oxygen species (ROS). These results uncover a novel non-canonical function of the fission protein, DRP1 in maintaining or positively stimulating mitochondrial respiration, bioenergetics and ROS signalling in adult cardiomyocyte, which is likely independent of morphological changes. Published on behalf of the European Society of Cardiology. All rights reserved. © The

  16. Insulin resistance in HIV-infected youth is associated with decreased mitochondrial respiration.

    PubMed

    Takemoto, Jody K; Miller, Tracie L; Wang, Jiajia; Jacobson, Denise L; Geffner, Mitchell E; Van Dyke, Russell B; Gerschenson, Mariana

    2017-01-02

    To identify relationships between insulin resistance (IR) and mitochondrial respiration in perinatally HIV-infected youth. Case-control study. Mitochondrial respiration was assessed in perinatally HIV-infected youth in Tanner stages 2-5, 25 youth with IR (IR+) and 50 without IR (IR-) who were enrolled in the Pediatric HIV/AIDS Cohort Study. IR was defined as a homeostatic model of assessment for IR value at least 4.0. A novel, high-throughput oximetry method was used to evaluate cellular respiration in peripheral blood mononuclear cells. Unadjusted and adjusted differences in mitochondrial respiration markers between IR+ and IR- were evaluated, as were correlations between mitochondrial respiration markers and biochemical measurements. IR+ and IR- youth were similar on age, sex, and race/ethnicity. Mean age was 16.5 and 15.6 years in IR+ and IR-, respectively. The IR+ group had significantly higher mean BMI and metabolic analytes (fasting glucose, insulin, cholesterol, triglycerides, and venous lactate and pyruvate) compared with the IR-. Mitochondrial respiration markers were, on average, lower in the IR+ compared with IR-, including basal respiration (417.5 vs. 597.5 pmol, P = 0.074), ATP production (11 513 vs. 15 202 pmol, P = 0.078), proton leak (584.6 vs. 790.0 pmol, P = 0.033), maximal respiration (1815 vs. 2399 pmol, P = 0.025), and spare respiration capacity (1162 vs. 2017 pmol, P = 0.032). Nonmitochondrial respiration did not differ by IR status. The results did not change when adjusted for age. HIV-infected youth with IR have lower mitochondrial respiration markers when compared to youth without IR. Disordered mitochondrial respiration may be a potential mechanism for IR in this population.

  17. Dynamics of enhanced mitochondrial respiration in female compared with male rat cerebral arteries.

    PubMed

    Rutkai, Ibolya; Dutta, Somhrita; Katakam, Prasad V; Busija, David W

    2015-11-01

    Mitochondrial respiration has never been directly examined in intact cerebral arteries. We tested the hypothesis that mitochondrial energetics of large cerebral arteries ex vivo are sex dependent. The Seahorse XFe24 analyzer was used to examine mitochondrial respiration in isolated cerebral arteries from adult male and female Sprague-Dawley rats. We examined the role of nitric oxide (NO) on mitochondrial respiration under basal conditions, using N(ω)-nitro-l-arginine methyl ester, and following pharmacological challenge using diazoxide (DZ), and also determined levels of mitochondrial and nonmitochondrial proteins using Western blot, and vascular diameter responses to DZ. The components of mitochondrial respiration including basal respiration, ATP production, proton leak, maximal respiration, and spare respiratory capacity were elevated in females compared with males, but increased in both male and female arteries in the presence of the NOS inhibitor. Although acute DZ treatment had little effect on mitochondrial respiration of male arteries, it decreased the respiration in female arteries. Levels of mitochondrial proteins in Complexes I-V and the voltage-dependent anion channel protein were elevated in female compared with male cerebral arteries. The DZ-induced vasodilation was greater in females than in males. Our findings show that substantial sex differences in mitochondrial respiratory dynamics exist in large cerebral arteries and may provide the mechanistic basis for observations that the female cerebral vasculature is more adaptable after injury. Copyright © 2015 the American Physiological Society.

  18. Abnormal mitochondrial respiration in failed human myocardium.

    PubMed

    Sharov, V G; Todor, A V; Silverman, N; Goldstein, S; Sabbah, H N

    2000-12-01

    Chronic heart failure (HF) is associated with morphologic abnormalities of cardiac mitochondria including hyperplasia, reduced organelle size and compromised structural integrity. In this study, we examined whether functional abnormalities of mitochondrial respiration are also present in myocardium of patients with advanced HF. Mitochondrial respiration was examined using a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles obtained from myocardium of failed explanted human hearts due to ischemic (ICM, n=9) or idiopathic dilated (IDC, n=9) cardiomyopathy. Myocardial specimens from five normal donor hearts served as controls (CON). Basal respiratory rate, respiratory rate after addition of the substrates glutamate and malate (V(SUB)), state 3 respiration (after addition of ADP, V(ADP)) and respiration after the addition of atractyloside (V(AT)) were measured in scar-free muscle bundles obtained from the subendocardial (ENDO) and subepicardial (EPI) thirds of the left ventricular (LV) free wall, interventricular septum and right ventricular (RV) free wall. There were no differences in basal and substrate-supported respiration between CON and HF regardless of etiology. V(ADP)was significantly depressed both in ICM and IDC compared to CON in all the regions studied. The respiratory control ratio, V(ADP)/V(AT), was also significantly decreased in HF compared to CON. In both ICM and IDC, V(ADP)was significantly lower in ENDO compared to EPI. The results indicate that mitochondrial respiration is abnormal in the failing human heart. The findings support the concept of low myocardial energy production in HF via oxidative phosphorylation, an abnormality with a potentially impact on global cardiac performance. Copyright 2000 Academic Press.

  19. Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

    PubMed

    Smolina, Natalia; Bruton, Joseph; Kostareva, Anna; Sejersen, Thomas

    2017-01-01

    Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.

  20. Uncoupling Mitochondrial Respiration for Diabesity.

    PubMed

    Larrick, James W; Larrick, Jasmine W; Mendelsohn, Andrew R

    2016-08-01

    Until recently, the mechanism of adaptive thermogenesis was ascribed to the expression of uncoupling protein 1 (UCP1) in brown and beige adipocytes. UCP1 is known to catalyze a proton leak of the inner mitochondrial membrane, resulting in uncoupled oxidative metabolism with no production of adenosine triphosphate and increased energy expenditure. Thus increasing brown and beige adipose tissue with augmented UCP1 expression is a viable target for obesity-related disorders. Recent work demonstrates an UCP1-independent pathway to uncouple mitochondrial respiration. A secreted enzyme, PM20D1, enriched in UCP1+ adipocytes, exhibits catalytic and hydrolytic activity to reversibly form N-acyl amino acids. N-acyl amino acids act as endogenous uncouplers of mitochondrial respiration at physiological concentrations. Administration of PM20D1 or its products, N-acyl amino acids, to diet-induced obese mice improves glucose tolerance by increasing energy expenditure. In short-term studies, treated animals exhibit no toxicity while experiencing 10% weight loss primarily of adipose tissue. Further study of this metabolic pathway may identify novel therapies for diabesity, the disease state associated with diabetes and obesity.

  1. Gastrocnemius mitochondrial respiration: are there any differences between men and women?

    PubMed

    Thompson, Jonathan R; Swanson, Stanley A; Casale, George P; Johanning, Jason M; Papoutsi, Evlampia; Koutakis, Panagiotis; Miserlis, Dimitrios; Zhu, Zhen; Pipinos, Iraklis I

    2013-11-01

    Work on human and mouse skeletal muscle by our group and others has demonstrated that aging and age-related degenerative diseases are associated with mitochondrial dysfunction, which may be more prevalent in males. There have been, however, no studies that specifically examine the influence of male or female sex on human skeletal muscle mitochondrial respiration. The purpose of this study was to compare mitochondrial respiration in the gastrocnemius of adult men and women. Gastrocnemius muscle was obtained from male (n = 19) and female (n = 11) human subjects with healthy lower-extremity musculoskeletal and arterial systems and normal ambulatory function. All patients were undergoing operations for the treatment of varicose veins in their legs. Mitochondrial respiration was determined with a Clark electrode in an oxygraph cell containing saponin-skinned muscle bundles. Complex I-, II-, III-, and IV-dependent respiration was measured individually and normalized to muscle weight, total protein content, and citrate synthase (CS, index of mitochondrial content). Male and female patients had no evidence of musculoskeletal or arterial disease and did not differ with regard to age, race, body mass index, or other clinical characteristics. Complex I-, II-, III-, and IV-dependent respiration normalized to muscle weight, total protein content, and CS did not statistically differ for males compared with females. Our study evaluates, for the first time, gastrocnemius mitochondrial respiration of adult men and women who have healthy musculoskeletal and arterial systems and normal ambulatory function. Our data demonstrate there are no differences in the respiration of gastrocnemius mitochondria between men and women. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production.

    PubMed

    Krumschnabel, Gerhard; Fontana-Ayoub, Mona; Sumbalova, Zuzana; Heidler, Juliana; Gauper, Kathrin; Fasching, Mario; Gnaiger, Erich

    2015-01-01

    Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2 (•-)) and particularly hydrogen peroxide (H2O2). Since H2O2 plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H2O2 production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. Fundamental instrumental and methodological parameters were optimized for analysis of the effects of various substrate, uncoupler, and inhibitor titrations (SUIT) on respiration versus H2O2 production. The sensitivity of the H2O2 assay was strongly influenced by compounds contained in different mitochondrial respiration media, which also exerted significant effects on chemical background fluorescence changes. Near linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H2O2 production in SUIT protocols.

  3. Pig Brain Mitochondria as a Biological Model for Study of Mitochondrial Respiration.

    PubMed

    Fišar, Z; Hroudová, J

    2016-01-01

    Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

  4. Microchambers with Solid-State Phosphorescent Sensor for Measuring Single Mitochondrial Respiration.

    PubMed

    Pham, Ted D; Wallace, Douglas C; Burke, Peter J

    2016-07-09

    It is now well established that, even within a single cell, multiple copies of the mitochondrial genome may be present (genetic heteroplasmy). It would be interesting to develop techniques to determine if and to what extent this genetic variation results in functional variation from one mitochondrion to the next (functional heteroplasmy). Measuring mitochondrial respiration can reveal the organelles' functional capacity for Adenosine triphosphate (ATP) production and determine mitochondrial damage that may arise from genetic or age related defects. However, available technologies require significant quantities of mitochondria. Here, we develop a technology to assay the respiration of a single mitochondrion. Our "micro-respirometer" consists of micron sized chambers etched out of borofloat glass substrates and coated with an oxygen sensitive phosphorescent dye Pt(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP) mixed with polystyrene. The chambers are sealed with a polydimethylsiloxane layer coated with oxygen impermeable Viton rubber to prevent diffusion of oxygen from the environment. As the mitochondria consume oxygen in the chamber, the phosphorescence signal increases, allowing direct determination of the respiration rate. Experiments with coupled vs. uncoupled mitochondria showed a substantial difference in respiration, confirming the validity of the microchambers as single mitochondrial respirometers. This demonstration could enable future high-throughput assays of mitochondrial respiration and benefit the study of mitochondrial functional heterogeneity, and its role in health and disease.

  5. Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

    PubMed Central

    Park, Song-Young; Gifford, Jayson R.; Andtbacka, Robert H. I.; Trinity, Joel D.; Hyngstrom, John R.; Garten, Ryan S.; Diakos, Nikolaos A.; Ives, Stephen J.; Dela, Flemming; Larsen, Steen; Drakos, Stavros

    2014-01-01

    Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s−1·mg−1, P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g−1·min−1, P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s−1·mg−1, P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production. PMID:24906913

  6. Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

    PubMed

    Park, Song-Young; Gifford, Jayson R; Andtbacka, Robert H I; Trinity, Joel D; Hyngstrom, John R; Garten, Ryan S; Diakos, Nikolaos A; Ives, Stephen J; Dela, Flemming; Larsen, Steen; Drakos, Stavros; Richardson, Russell S

    2014-08-01

    Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s(-1)·mg(-1), P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g(-1)·min(-1), P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s(-1)·mg(-1), P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.

  7. The role of p38 in mitochondrial respiration in male and female mice.

    PubMed

    Ju, Xiaohua; Wen, Yi; Metzger, Daniel; Jung, Marianna

    2013-06-07

    p38 is a mitogen-activated protein kinase and mediates cell growth, cell differentiation, and synaptic plasticity. The aim of this study is to determine the extent to which p38 plays a role in maintaining mitochondrial respiration in male and female mice under a normal condition. To achieve this aim, we have generated transgenic mice that lack p38 in cerebellar Purkinje neurons by crossing Pcp2 (Purkinje cell protein 2)-Cre mice with p38(loxP/loxP) mice. Mitochondria from cerebellum were then isolated from the transgenic and wild-type mice to measure mitochondrial respiration using XF24 respirometer. The mRNA and protein expression of cytochrome c oxidase (COX) in cerebellum were also measured using RT-PCR and immunoblot methods. Separately, HT22 cells were used to determine the involvement of 17β-estradiol (E2) and COX in mitochondrial respiration. The genetic knockout of p38 in Purkinje neurons suppressed the mitochondrial respiration only in male mice and increased COX expression only in female mice. The inhibition of COX by sodium azide (SA) sharply suppressed mitochondrial respiration of HT22 cells in a manner that was protected by E2. These data suggest that p38 is required for the mitochondrial respiration of male mice. When p38 is below a normal level, females may maintain mitochondrial respiration through COX up-regulation. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  8. Fluoride decreased the sperm ATP of mice through inhabiting mitochondrial respiration.

    PubMed

    Sun, Zilong; Zhang, Wen; Xue, Xingchen; Zhang, Yuliang; Niu, Ruiyan; Li, Xuying; Li, Baojun; Wang, Xiaowen; Wang, Jundong

    2016-02-01

    Fluoride-induced low sperm motility was observed in accumulated investigations. However, the effect of fluoride exposure on ATP generation which is essential to sperm motility remains to be elucidated. In this study, 120 healthy male mice were orally administrated with 0, 25, 50, and 100 mg L(-1) NaF for 90 d. Results showed that compared with controls, fluoride ingestion significantly reduced sperm count, survival, as well as mobility and total ATP level in sperm untreated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) or pyruvate, which was used to establish glycolysis or mitochondrial respiration model, respectively. Data further revealed that sperm mobility and ATP level under mitochondrial respiration condition were significantly suppressed, while no statistical difference occurred in the model of glycolysis, indicating ATP derived from mitochondria was affected. Moreover, mRNA expressions of mitochondrial cytochrome b (mt-Cytb) and cytochrome c oxidase subunit 2 (mt-COX2), two important molecules in mitochondrial electron transport chain (ETC), were down-regulated in all fluoride treatment groups. Mitochondria in sperm of mice exposed to 100 mg L(-1) NaF appeared to be irregular and vacuolated. These findings suggested that decreased sperm motility induced by fluoride may result from low ATP generation due to the disturbed ETC in sperm mitochondrial. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Influence of menhaden oil on mitochondrial respiration in BHE rats.

    PubMed

    Kim, M J; Berdanier, C D

    1989-11-01

    The effects of corn or menhaden oil and thyroxine treatment on hepatic mitochondrial respiration was studied. BHE rats were fed a 64% sucrose, 6% corn, or menhaden oil diet until they were 60-70 days of age. Succinate-supported mitochondrial respiration was studied at 3 degrees C intervals from 4 to 40 degrees C. Upper and lower activation energies and transition temperatures were determined through the calculation of Arrhenius plot. Menhaden oil plus daily thyroxine injection resulted in higher and lower activation energies than the other treatments. This combined treatment also resulted in lower state 3 and higher state 4 respiration rates and tighter coupling of respiration to ATP synthesis. These effects were thought to be due to the effect this treatment combination had on membrane fluidity.

  10. Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite.

    PubMed

    Jones, Charles I; Han, Zhaosheng; Presley, Tennille; Varadharaj, Saradhadevi; Zweier, Jay L; Ilangovan, Govindasamy; Alevriadou, B Rita

    2008-07-01

    Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

  11. Microbiopsies versus Bergström needle for skeletal muscle sampling: impact on maximal mitochondrial respiration rate.

    PubMed

    Isner-Horobeti, M E; Charton, A; Daussin, F; Geny, B; Dufour, S P; Richard, R

    2014-05-01

    Microbiopsies are increasingly used as an alternative to the standard Bergström technique for skeletal muscle sampling. The potential impact of these two different procedures on mitochondrial respiration rate is unknown. The objective of this work was to compare microbiopsies versus Bergström procedure on mitochondrial respiration in skeletal muscle. 52 vastus lateralis muscle samples were obtained from 13 anesthetized pigs, either with a Bergström [6 gauges (G)] needle or with microbiopsy needles (12, 14, 18G). Maximal mitochondrial respiration (V GM-ADP) was assessed using an oxygraphic method on permeabilized fibers. The weight of the muscle samples and V GM-ADP decreased with the increasing gauge of the needles. A positive nonlinear relationship was observed between the weight of the muscle sample and the level of maximal mitochondrial respiration (r = 0.99, p < 0.05) and between needle size and maximal mitochondrial respiration (r = 0.99, p < 0.05). Microbiopsies give lower muscle sample weight and maximal rate of mitochondrial respiration compared to the standard Bergström needle.Therefore, the higher the gauge (i.e. the smaller the size) of the microbiopsy needle, the lower is the maximal rate of respiration. Microbiopsies of skeletal muscle underestimate the maximal mitochondrial respiration rate, and this finding needs to be highlighted for adequate interpretation and comparison with literature data.

  12. Low Concentrations of Cationic PAMAM Dendrimers Affect Lymphocyte Respiration in In vitro Studies.

    PubMed

    Labieniec-Watala, Magdalena; Szwed, Marzena; Hertel, Joanna; Wisnik, Ewelina

    2017-01-01

    In this study, the effect of low concentrations of poly(amido)amine dendrimers (G2-G4) on human lymphocytes was studied. Some works revealed that PAMAMs can adversely affect the morphology of blood components and mitochondria functions. In this context, the present report aimed to investigate the in vitro cationic dendrimers' effect on mitochondrial respiration and cell morphology in lymphocytes isolated from human blood. To monitor the mitochondrial changes, the high-resolution respirometer was used, whereas the cell morphology was analyzed using a flow cytometer and fluorescence microscopy. The concentration-dependent dendrimers' influence on lymphocytes morphology was shown. Changes in mitochondrial respiration revealed the concentration- and generation-dependent differences between dendrimer activity. There were no alterations in the routine respiration and in the state of the inner mitochondrial membrane (L/E), but decreased ADP- and FCCP-stimulated respirations were detected after treatment with G3 and G4 dendrimers. The markers of mitochondrial membrane integrity (RCR) and OXPHOS efficiency (P/E) significantly decreased regardless of the dendrimer generation used. Based on these in vitro evaluations, we state that cationic PAMAM dendrimers can impair both the morphology and the bioenergetics of human lymphocytes, even when used at low concentrations and in a short time (up to 1 h). However, these results do not imply that similar findings could be possible for in vivo observations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Thermal sensitivity of mitochondrial respiration efficiency and protein phosphorylation in the clam Mercenaria mercenaria.

    PubMed

    Ulrich, P N; Marsh, A G

    2009-01-01

    The mitochondria of intertidal invertebrates continue to function when organisms are exposed to rapid substantial shifts in temperature. To test if mitochondrial physiology of the clam Mercenaria mercenaria is compromised under elevated temperatures, we measured mitochondrial respiration efficiency at 15 degrees C, 18 degrees C, and 21 degrees C using a novel, high-throughput, microplate respirometry methodology developed for this study. Though phosphorylating (state 3) and resting (state 4) respiration rates were unaffected over this temperature range, respiratory control ratios (RCRs: ratio of state 3 to state 4 respiration rates) decreased significantly above 18 degrees C (p < 0.05). The drop in RCR was not associated with reduction of phosphorylation efficiency, suggesting that, while aerobic scope of mitochondrial respiration is limited at elevated temperatures, mitochondria continue to efficiently produce adenosine triphosphate. We further investigated the response of clam mitochondria to elevated temperatures by monitoring phosphorylation of mitochondrial protein. Three proteins clearly demonstrated significant time- and temperature-specific phosphorylation patterns. The protein-specific patterns of phosphorylation may suggest that a suite of protein kinases and phosphatases regulate mitochondrial physiology in response to temperature. Thus, while aerobic scope of clam mitochondrial respiration is reduced at moderate temperatures, specific protein phosphorylation responses reflect large shifts in function that are initiated within the organelle at higher temperatures.

  14. The axon-protective WLD(S) protein partially rescues mitochondrial respiration and glycolysis after axonal injury.

    PubMed

    Godzik, Katharina; Coleman, Michael P

    2015-04-01

    The axon-protective Wallerian degeneration slow (WLD(S)) protein can ameliorate the decline in axonal ATP levels after neurite transection. Here, we tested the hypothesis that this effect is associated with maintenance of mitochondrial respiration and/or glycolysis. We used isolated neurites of superior cervical ganglion (SCG) cultures in the Seahorse XF-24 Metabolic Flux Analyser to determine mitochondrial respiration and glycolysis under different conditions. We observed that both mitochondrial respiration and glycolysis declined significantly during the latent phase of Wallerian degeneration. WLD(S) partially reduced the decline both in glycolysis and in mitochondrial respiration. In addition, we found that depleting NAD levels in uncut cultures led to changes in mitochondrial respiration and glycolysis similar to those rescued by WLD(S) after cut, suggesting that the maintenance of NAD levels in Wld(S) neurites after axonal injury at least partially underlies the maintenance of ATP levels. However, by using another axon-protective mutation (Sarm1(-/-)), we could demonstrate that rescue of basal ECAR (and hence probably glycolysis) rather than basal OCR (mitochondrial respiration) may be part of the protective phenotype to delay Wallerian degeneration. These findings open new routes to study glycolysis and the connection between NAD and ATP levels in axon degeneration, which may help to eventually develop therapeutic strategies to treat neurodegenerative diseases.

  15. Dose Response of Endotoxin on Hepatocyte and Muscle Mitochondrial Respiration In Vitro

    PubMed Central

    Brandt, Sebastian; Porta, Francesca; Jakob, Stephan M.; Takala, Jukka; Djafarzadeh, Siamak

    2015-01-01

    Introduction. Results on mitochondrial dysfunction in sepsis are controversial. We aimed to assess effects of LPS at wide dose and time ranges on hepatocytes and isolated skeletal muscle mitochondria. Methods. Human hepatocellular carcinoma cells (HepG2) were exposed to placebo or LPS (0.1, 1, and 10 μg/mL) for 4, 8, 16, and 24 hours and primary human hepatocytes to 1 μg/mL LPS or placebo (4, 8, and 16 hours). Mitochondria from porcine skeletal muscle samples were exposed to increasing doses of LPS (0.1–100 μg/mg) for 2 and 4 hours. Respiration rates of intact and permeabilized cells and isolated mitochondria were measured by high-resolution respirometry. Results. In HepG2 cells, LPS reduced mitochondrial membrane potential and cellular ATP content but did not modify basal respiration. Stimulated complex II respiration was reduced time-dependently using 1 μg/mL LPS. In primary human hepatocytes, stimulated mitochondrial complex II respiration was reduced time-dependently using 1 μg/mL LPS. In isolated porcine skeletal muscle mitochondria, stimulated respiration decreased at high doses (50 and 100 μg/mL LPS). Conclusion. LPS reduced cellular ATP content of HepG2 cells, most likely as a result of the induced decrease in membrane potential. LPS decreased cellular and isolated mitochondrial respiration in a time-dependent, dose-dependent and complex-dependent manner. PMID:25649304

  16. Quadriceps exercise intolerance in patients with chronic obstructive pulmonary disease: the potential role of altered skeletal muscle mitochondrial respiration.

    PubMed

    Gifford, Jayson R; Trinity, Joel D; Layec, Gwenael; Garten, Ryan S; Park, Song-Young; Rossman, Matthew J; Larsen, Steen; Dela, Flemming; Richardson, Russell S

    2015-10-15

    This study sought to determine if qualitative alterations in skeletal muscle mitochondrial respiration, associated with decreased mitochondrial efficiency, contribute to exercise intolerance in patients with chronic obstructive pulmonary disease (COPD). Using permeabilized muscle fibers from the vastus lateralis of 13 patients with COPD and 12 healthy controls, complex I (CI) and complex II (CII)-driven State 3 mitochondrial respiration were measured separately (State 3:CI and State 3:CII) and in combination (State 3:CI+CII). State 2 respiration was also measured. Exercise tolerance was assessed by knee extensor exercise (KE) time to fatigue. Per milligram of muscle, State 3:CI+CII and State 3:CI were reduced in COPD (P < 0.05), while State 3:CII and State 2 were not different between groups. To determine if this altered pattern of respiration represented qualitative changes in mitochondrial function, respiration states were examined as percentages of peak respiration (State 3:CI+CII), which revealed altered contributions from State 3:CI (Con 83.7 ± 3.4, COPD 72.1 ± 2.4%Peak, P < 0.05) and State 3:CII (Con 64.9 ± 3.2, COPD 79.5 ± 3.0%Peak, P < 0.05) respiration, but not State 2 respiration in COPD. Importantly, a diminished contribution of CI-driven respiration relative to the metabolically less-efficient CII-driven respiration (CI/CII) was also observed in COPD (Con 1.28 ± 0.09, COPD 0.81 ± 0.05, P < 0.05), which was related to exercise tolerance of the patients (r = 0.64, P < 0.05). Overall, this study indicates that COPD is associated with qualitative alterations in skeletal muscle mitochondria that affect the contribution of CI and CII-driven respiration, which potentially contributes to the exercise intolerance associated with this disease.

  17. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

    PubMed

    Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

    2016-02-01

    Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Is cell aging caused by respiration-dependent injury to the mitochondrial genome

    NASA Technical Reports Server (NTRS)

    Fleming, J. E.; Yengoyan, L. S.; Miquel, J.; Cottrell, S. F.; Economos, A. C.

    1982-01-01

    Though intrinsic mitochondrial aging has been considered before as a possible cause of cellular senescence, the mechanisms of such mitochondrial aging have remained obscure. In this article, the hypothesis of free-radical-induced inhibition of mitochondrial replenishment in fixed postmitotic cells is expanded. It is maintained that the respiration-dependent production of superoxide and hydroxyl radicals may not be fully counteracted, leading to a continuous production of lipoperoxides and malonaldehyde in actively respiring mitochondria. These compounds, in turn, can easily react with the mitochondrial DNA which is in close spatial relationship with the inner mitochondrial membrane, producing an injury that the mitochondria may be unable to counteract because of their apparent lack of adequate repair mechanisms. Mitochondrial division may thus be inhibited leading to age-related reduction of mitochondrial numbers, a deficit in energy production with a concomitant decrease in protein synthesis, deterioration of physiological performance, and, therefore, of organismic performance.

  19. Varicocele Negatively Affects Sperm Mitochondrial Respiration.

    PubMed

    Ferramosca, Alessandra; Albani, Denise; Coppola, Lamberto; Zara, Vincenzo

    2015-10-01

    To evaluate the effect of varicocele on oxidative stress, sperm mitochondrial respiratory efficiency, sperm morphology, and semen parameters. A total of 20 patients with varicocele and 20 normozoospermic subjects without varicocele (control group) were recruited from a medical center for reproductive biology. The levels of serum reactive oxygen metabolites and seminal lipid peroxides were assessed for both control and varicocele subjects. Sperm deoxyribonucleic acid fragmentation was measured by sperm chromatin dispersion test. Mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. In this study, varicocele patients were compared with men without varicoceles. Oxidative stress was observed in the serum and seminal fluid of varicocele patients. These patients showed an increase of 59% (P <.05) in serum reactive oxygen metabolites and a 3-fold increase in the level of sperm lipid peroxides. A parallel and significant increase (a 2-fold increase; P <.05) in the degree of sperm deoxyribonucleic acid fragmentation was also observed. Varicocele patients showed a 27% decrease (P <.05) in mitochondrial respiratory activity in comparison to the control group. A 32% increase (P <.05) in sperm midpiece defects and a 41% decrease (P <.05) in sperm concentration and motility were also observed. Men with varicocele have increased markers of oxidative stress and decreased mitochondrial respiratory activity. These results correlated with abnormalities in semen parameters. For morphology, these correlated with midpiece defects. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia

    PubMed Central

    Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar

    2015-01-01

    Objective To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Methods and Design Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20–22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Results Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Conclusions Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of

  1. Mitochondrial Respiration in Insulin-Producing β-Cells: General Characteristics and Adaptive Effects of Hypoxia.

    PubMed

    Hals, Ingrid K; Bruerberg, Simon Gustafson; Ma, Zuheng; Scholz, Hanne; Björklund, Anneli; Grill, Valdemar

    2015-01-01

    To provide novel insights on mitochondrial respiration in β-cells and the adaptive effects of hypoxia. Insulin-producing INS-1 832/13 cells were exposed to 18 hours of hypoxia followed by 20-22 hours re-oxygenation. Mitochondrial respiration was measured by high-resolution respirometry in both intact and permeabilized cells, in the latter after establishing three functional substrate-uncoupler-inhibitor titration (SUIT) protocols. Concomitant measurements included proteins of mitochondrial complexes (Western blotting), ATP and insulin secretion. Intact cells exhibited a high degree of intrinsic uncoupling, comprising about 50% of oxygen consumption in the basal respiratory state. Hypoxia followed by re-oxygenation increased maximal overall respiration. Exploratory experiments in peremabilized cells could not show induction of respiration by malate or pyruvate as reducing substrates, thus glutamate and succinate were used as mitochondrial substrates in SUIT protocols. Permeabilized cells displayed a high capacity for oxidative phosphorylation for both complex I- and II-linked substrates in relation to maximum capacity of electron transfer. Previous hypoxia decreased phosphorylation control of complex I-linked respiration, but not in complex II-linked respiration. Coupling control ratios showed increased coupling efficiency for both complex I- and II-linked substrates in hypoxia-exposed cells. Respiratory rates overall were increased. Also previous hypoxia increased proteins of mitochondrial complexes I and II (Western blotting) in INS-1 cells as well as in rat and human islets. Mitochondrial effects were accompanied by unchanged levels of ATP, increased basal and preserved glucose-induced insulin secretion. Exposure of INS-1 832/13 cells to hypoxia, followed by a re-oxygenation period increases substrate-stimulated respiratory capacity and coupling efficiency. Such effects are accompanied by up-regulation of mitochondrial complexes also in pancreatic islets

  2. Protective effect of creatine against inhibition by methylglyoxal of mitochondrial respiration of cardiac cells.

    PubMed Central

    Roy, Soumya Sinha; Biswas, Swati; Ray, Manju; Ray, Subhankar

    2003-01-01

    Previous publications from our laboratory have shown that methylglyoxal inhibits mitochondrial respiration of malignant and cardiac cells, but it has no effect on mitochondrial respiration of other normal cells [Biswas, Ray, Misra, Dutta and Ray (1997) Biochem. J. 323, 343-348; Ray, Biswas and Ray (1997) Mol. Cell. Biochem. 171, 95-103]. However, this inhibitory effect of methylglyoxal is not significant in cardiac tissue slices. Moreover, post-mitochondrial supernatant (PMS) of cardiac cells could almost completely protect the mitochondrial respiration against the inhibitory effect of methylglyoxal. A systematic search indicated that creatine present in cardiac cells is responsible for this protective effect. Glutathione has also some protective effect. However, creatine phosphate, creatinine, urea, glutathione disulphide and beta-mercaptoethanol have no protective effect. The inhibitory and protective effects of methylglyoxal and creatine respectively on cardiac mitochondrial respiration were studied with various concentrations of both methylglyoxal and creatine. Interestingly, neither creatine nor glutathione have any protective effect on the inhibition by methylglyoxal on the mitochondrial respiration of Ehrlich ascites carcinoma cells. The creatine and glutathione contents of several PMS, which were tested for the possible protective effect, were measured. The activities of two important enzymes, namely glyoxalase I and creatine kinase, which act upon glutathione plus methylglyoxal and creatine respectively, were also measured in different PMS. Whether mitochondrial creatine kinase had any role in the protective effect of creatine had also been investigated using 1-fluoro-2,4-dinitrobenzene, an inhibitor of creatine kinase. The differential effect of creatine on mitochondria of cardiac and malignant cells has been discussed with reference to the therapeutic potential of methylglyoxal. PMID:12605598

  3. Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics.

    PubMed

    Scandurra, Francesca M; Gnaiger, Erich

    2010-01-01

    When oxygen supply to tissues is limiting, mitochondrial respiration and ATP production are compromised. To assess the bioenergetic consequences under normoxia and hypoxia, quantitative evaluation of mitochondrial oxygen kinetics is required. Using high-resolution respirometry, the "apparent K (m)" for oxygen or p (50) of respiration in 32D cells was determined at 0.05 +/- 0.01 kPa (0.4 mmHg, 0.5 microM, 0.25% air saturation). Close agreement with p (50) of isolated mitochondria indicates that intracellular gradients are small in small cells at routine activity. At intracellular p (O2) <2 kPa (15 mmHg, 10% air saturation) in various tissues under normoxia, respiration is limited by >2% with a p (50) of 0.05 kPa. Over-estimation of p (50) at 0.4 kPa (3 mmHg) would imply significant (>17%) oxygen limitation of respiration under intracellular normoxia. Based on a critical review, we conclude that p (50) ranges from 0.01 to 0.10 kPa in mitochondria and small cells in the absence of inhibitors of cytochrome c oxidase, whereas experimental artefacts explain the controversial >200-fold range of p (50) in the literature on mitochondrial oxygen kinetics.

  4. Ammonium Assimilation Requires Mitochondrial Respiration in the Light 1

    PubMed Central

    Weger, Harold G.; Birch, Douglas G.; Elrifi, Ivor R.; Turpin, David H.

    1988-01-01

    Mass spectrometric analysis of O2 and CO2 exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH4Cl resulted in nearly a 250% increase in the rate of TCA cycle CO2 efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O2 consumption in both light and dark. Anaerobiosis inhibited the CO2 release caused by NH4Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O2 consumption in the light. This implied that the Mehler reaction did not play a significant role in O2 consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions. PMID:16665971

  5. Increased platelet mitochondrial respiration after cardiac arrest and resuscitation as a potential peripheral biosignature of cerebral bioenergetic dysfunction.

    PubMed

    Ferguson, Michael A; Sutton, Robert M; Karlsson, Michael; Sjövall, Fredrik; Becker, Lance B; Berg, Robert A; Margulies, Susan S; Kilbaugh, Todd J

    2016-06-01

    Cardiac arrest (CA) results in a sepsis-like syndrome with activation of the innate immune system and increased mitochondrial bioenergetics. To determine if platelet mitochondrial respiration increases following CA in a porcine pediatric model of asphyxia-associated ventricular fibrillation (VF) CA, and if this readily obtained biomarker is associated with decreased brain mitochondrial respiration. CA protocol: 7 min of asphyxia, followed by VF, protocolized titration of compression depth to systolic blood pressure of 90 mmHg and vasopressor administration to a coronary perfusion pressure greater than 20 mmHg. platelet integrated mitochondrial electron transport system (ETS) function evaluated pre- and post-CA/ROSC four hours after return of spontaneous circulation (ROSC). Secondary outcome: correlation of platelet mitochondrial bioenergetics to cerebral bioenergetic function. Platelet maximal oxidative phosphorylation (OXPHOSCI+CII), P < 0.02, and maximal respiratory capacity (ETSCI+CII), P < 0.04, were both significantly increased compared to pre-arrest values. This was primarily due to a significant increase in succinate-supported respiration through Complex II (OXPHOSCII, P < 0.02 and ETSCII, P < 0.03). Higher respiration was not due to uncoupling, as the LEAKCI + CII respiration (mitochondrial respiration independent of ATP-production) was unchanged after CA/ROSC. Larger increases in platelet mitochondrial respiratory control ratio (RCR) compared to pre-CA RCR were significantly correlated with lower RCRs in the cortex (P < 0.03) and hippocampus (P < 0.04) compared to sham respiration. Platelet mitochondrial respiration is significantly increased four hours after ROSC. Future studies will identify mechanistic relationships between this serum biomarker and altered cerebral bioenergetics function following cardiac arrest.

  6. Mitochondrial dynamics and respiration within cells with increased open pore cytoskeletal meshes

    PubMed Central

    Jang, David H.; Seeger, Sarah C.; Grady, Martha E.; Shofer, Frances S.

    2017-01-01

    ABSTRACT The cytoskeletal architecture directly affects the morphology, motility, and tensional homeostasis of the cell. In addition, the cytoskeleton is important for mitosis, intracellular traffic, organelle motility, and even cellular respiration. The organelle responsible for a majority of the energy conversion for the cell, the mitochondrion, has a dependence on the cytoskeleton for mobility and function. In previous studies, we established that cytoskeletal inhibitors altered the movement of the mitochondria, their morphology, and their respiration in human dermal fibroblasts. Here, we use this protocol to investigate applicability of power law diffusion to describe mitochondrial locomotion, assessment of rates of fission and fusion in healthy and diseased cells, and differences in mitochondria locomotion in more open networks either in response to cytoskeletal destabilizers or by cell line. We found that mitochondria within fibrosarcoma cells and within fibroblast cells treated with an actin-destabilizing toxin resulted in increased net travel, increased average velocity, and increased diffusion of mitochondria when compared to control fibroblasts. Although the mitochondria within the fibrosarcoma travel further than mitochondria within their healthy counterparts, fibroblasts, the dependence on mitochondria for respiration is much lower with higher rates ofhydrogen peroxide production and was confirmed using the OROBOROS O2K. We also found that rates of fission and fusion of the mitochondria equilibrate despite significant alteration of the cytoskeleton. Rates ranged from 15% to 25%, where the highest rates were observed within the fibrosarcoma cell line. This result is interesting because the fibrosarcoma cell line does not have increased respiration metrics including when compared to fibroblast. Mitochondria travel further, faster, and have an increase in percent mitochondria splitting or joining while not dependent on the mitochondria for a majority of

  7. Improved brain and muscle mitochondrial respiration with CoQ. An in vivo study by 31P-MR spectroscopy in patients with mitochondrial cytopathies.

    PubMed

    Barbiroli, B; Iotti, S; Lodi, R

    1999-01-01

    We used in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) to study the effect of CoQ10 on the efficiency of brain and skeletal muscle mitochondrial respiration in ten patients with mitochondrial cytopathies. Before CoQ, brain [PCr] was remarkably lower in patients than in controls, while [Pi] and [ADP] were higher. Brain cytosolic free [Mg2+] and delta G of ATP hydrolysis were also abnormal in all patients. MRS also revealed abnormal mitochondrial function in the skeletal muscles of all patients, as shown by a decreased rate of PCr recovery from exercise. After six-months of treatment with CoQ (150 mg/day), all brain MRS-measurable variables as well as the rate of muscle mitochondrial respiration were remarkably improved in all patients. These in vivo findings show that treatment with CoQ in patients with mitochondrial cytopathies improves mitochondrial respiration in both brain and skeletal muscles, and are consistent with Lenaz's view that increased CoQ concentration in the mitochondrial membrane increases the efficiency of oxidative phosphorylation independently of enzyme deficit.

  8. Is Reliance on Mitochondrial Respiration a “Chink in the Armor” of Therapy-Resistant Cancer?

    PubMed Central

    Wolf, Dieter A.

    2016-01-01

    Summary A series of recent reports has suggested PGC1α-driven upregulation of mitochondrial oxidative phosphorylation as a selective vulnerability of drug-resistant cancers. Accordingly, chemical inhibitors of respiration led to selective eradication of such cancer cells due to their preferential sensitivity to mitochondrial production of reactive oxygen species. These novel insights create a timely opportunity for a biomarker guided application of already existing and newly emerging mitochondrial inhibitors in recurrent drug resistant cancer, including lymphomas, melanomas, and other malignant diseases marked by increased mitochondrial respiration. PMID:25490445

  9. The proline metabolism intermediate Δ1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast.

    PubMed

    Nishimura, Akira; Nasuno, Ryo; Takagi, Hiroshi

    2012-07-30

    The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  10. Short Communication: HIV Patient Systemic Mitochondrial Respiration Improves with Exercise.

    PubMed

    Kocher, Morgan; McDermott, Mindy; Lindsey, Rachel; Shikuma, Cecilia M; Gerschenson, Mariana; Chow, Dominic C; Kohorn, Lindsay B; Hetzler, Ronald K; Kimura, Iris F

    2017-10-01

    In HIV-infected individuals, impaired mitochondrial function may contribute to cardiometabolic disease as well as to fatigue and frailty. Aerobic exercise improves total body energy reserves; however, its impact at the cellular level is unknown. We assessed alterations in cellular bioenergetics in peripheral blood mononuclear cells (PBMC) before and after a 12-week aerobic exercise study in sedentary HIV-infected subjects on stable antiretroviral therapy who successfully completed a 12-week aerobic exercise program. In this prospective study, participants underwent supervised 20-40 min of light aerobic exercise (walking or jogging) performed three times per week for 12 weeks, gradually increasing to maintain an intensity of 50%-80% of heart rate reserve. Maximal aerobic capacity (VO 2MAX ) was assessed by a graded exercise test on a cycle ergometer before and after completion of the study. PBMC from compliant subjects (attended at least 70% of exercise sessions) were assessed for mitochondrial respiration using the Seahorse XF24 Bio-Analyzer. Seven of 24 enrolled subjects were compliant with the exercise regimen. In these individuals, a significant increase (p = .04) in VO 2MAX over 12 weeks was found with a median increase of 14%. During the same interval, a 2.45-fold increase in PBMC mitochondrial respiratory capacity (p = .04), a 5.65-fold increase in spare respiratory capacity (p = .01), and a 3.15-fold (p = .04) increase in nonmitochondrial respiration was observed. Aerobic exercise improves respiration at the cellular level. The diagnostic and prognostic value of such improved cellular respiration in the setting of chronic HIV warrants further investigation.

  11. Secreted Human Adipose Leptin Decreases Mitochondrial Respiration in HCT116 Colon Cancer Cells

    PubMed Central

    Yehuda-Shnaidman, Einav; Nimri, Lili; Tarnovscki, Tanya; Kirshtein, Boris; Rudich, Assaf; Schwartz, Betty

    2013-01-01

    Obesity is a key risk factor for the development of colon cancer; however, the endocrine/paracrine/metabolic networks mediating this connection are poorly understood. Here we hypothesize that obesity results in secreted products from adipose tissue that induce malignancy-related metabolic alterations in colon cancer cells. Human HCT116 colon cancer cells, were exposed to conditioned media from cultured human adipose tissue fragments of obese vs. non-obese subjects. Oxygen consumption rate (OCR, mostly mitochondrial respiration) and extracellular acidification rate (ECAR, mostly lactate production via glycolysis) were examined vis-à-vis cell viability and expression of related genes and proteins. Our results show that conditioned media from obese (vs. non-obese) subjects decreased basal (40%, p<0.05) and maximal (50%, p<0.05) OCR and gene expression of mitochondrial proteins and Bax without affecting cell viability or expression of glycolytic enzymes. Similar changes could be recapitulated by incubating cells with leptin, whereas, leptin-receptor specific antagonist inhibited the reduced OCR induced by conditioned media from obese subjects. We conclude that secreted products from the adipose tissue of obese subjects inhibit mitochondrial respiration and function in HCT116 colon cancer cells, an effect that is at least partly mediated by leptin. These results highlight a putative novel mechanism for obesity-associated risk of gastrointestinal malignancies, and suggest potential new therapeutic avenues. PMID:24073224

  12. Creatine kinase and mitochondrial respiration in hearts of trout, cod and freshwater turtle.

    PubMed

    Birkedal, R; Gesser, H

    2003-08-01

    The importance of the creatine kinase system in the cardiac muscle of ectothermic vertebrates is unclear. Mammalian cardiac muscle seems to be structurally organized in a manner that compartmentalizes the intracellular environment as evidenced by the substantially higher mitochondrial apparent Km for ADP in skinned fibres compared to isolated mitochondria. A mitochondrial fraction of creatine kinase is functionally coupled to the mitochondrial respiration, and the transport of phosphocreatine and creatine as energy equivalents of ATP and ADP, respectively, increases the mitochondrial apparent ADP affinity, i.e. lowers the Km. This function of creatine kinase seems to be absent in hearts of frog species. To find out whether this applies to hearts of ectothermic vertebrate species in general, we investigated the effect of creatine on the mitochondrial respiration of saponin-skinned fibres from the ventricle of rainbow trout, Atlantic cod and freshwater turtle. For all three species, the apparent Km for ADP appeared to be substantially higher than for isolated mitochondria. Creatine lowered this Km in trout and turtle, thus indicating a functional coupling between mitochondrial creatine kinase and respiration. However, creatine had no effect on Km in cod ventricle. In conclusion, the creatine kinase-system in trout and turtle hearts seems to fulfil the same functions as in the mammalian heart, i.e. facilitating energy transport and communication between cellular compartments. In cod heart, however, this does not seem to be the case.

  13. Investigation of Mitochondrial Dysfunction by Sequential Microplate-Based Respiration Measurements from Intact and Permeabilized Neurons

    PubMed Central

    Clerc, Pascaline; Polster, Brian M.

    2012-01-01

    Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria. PMID:22496810

  14. Metabolic Characterization of Intact Cells Reveals Intracellular Amyloid Beta but Not Its Precursor Protein to Reduce Mitochondrial Respiration

    PubMed Central

    Schaefer, Patrick M.; von Einem, Bjoern; Walther, Paul; Calzia, Enrico; von Arnim, Christine A. F.

    2016-01-01

    One hallmark of Alzheimer´s disease are senile plaques consisting of amyloid beta (Aβ), which derives from the processing of the amyloid precursor protein (APP). Mitochondrial dysfunction has been linked to the pathogenesis of Alzheimer´s disease and both Aβ and APP have been reported to affect mitochondrial function in isolated systems. However, in intact cells, considering a physiological localization of APP and Aβ, it is pending what triggers the mitochondrial defect. Thus, the aim of this study was to dissect the impact of APP versus Aβ in inducing mitochondrial alterations with respect to their subcellular localization. We performed an overexpression of APP or beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), increasing APP and Aβ levels or Aβ alone, respectively. Conducting a comprehensive metabolic characterization we demonstrate that only APP overexpression reduced mitochondrial respiration, despite lower extracellular Aβ levels compared to BACE overexpression. Surprisingly, this could be rescued by a gamma secretase inhibitor, oppositionally indicating an Aβ-mediated mitochondrial toxicity. Analyzing Aβ localization revealed that intracellular levels of Aβ and an increased spatial association of APP/Aβ with mitochondria are associated with reduced mitochondrial respiration. Thus, our data provide marked evidence for a prominent role of intracellular Aβ accumulation in Alzheimer´s disease associated mitochondrial dysfunction. Thereby it highlights the importance of the localization of APP processing and intracellular transport as a decisive factor for mitochondrial function, linking two prominent hallmarks of neurodegenerative diseases. PMID:28005987

  15. The effect of respiration buffer composition on mitochondrial metabolism and function.

    PubMed

    Wollenman, Lucas C; Vander Ploeg, Matthew R; Miller, Mackinzie L; Zhang, Yizhu; Bazil, Jason N

    2017-01-01

    Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffers and substrate combinations to determine the optimal conditions to support mitochondrial function through ADP-stimulated respiration and uncoupled respiration using FCCP. The buffers consisted of a standard KCl based buffer (B1) and three modified buffers with chloride replaced by the K-lactobionate, sucrose, and the antioxidant taurine (B2) or K-gluconate (B3). The fourth buffer (B4) was identical to B2 except that K-lactobionate was replaced with K-gluconate. The substrate combinations consisted of metabolites that utilize different pathways of mitochondrial metabolism. To test mitochondrial function, we used isolated cardiac guinea pig mitochondria and measured oxygen consumption for three respiratory states using an Oroboros Oxygraph-2k. These states were the leak state (energized mitochondria in the absence of adenylates), ADP-stimulated state (energized mitochondria in the presence of saturating ADP concentrations), and uncoupled state (energized mitochondria in the presence of FCCP). On average across all substrate combinations, buffers B2, B3, and B4 had an increase of 16%, 26%, and 35% for the leak state, ADP-simulated state, and uncoupled state, respectively, relative to rates using B1. The common feature distinguishing these buffers from B1 is the notable lack of high chloride concentrations. Based on the respiratory rate metrics obtained with the substrate combinations, we conclude that the adenine nucleotide translocase, the dicarboxylate carrier, and the alpha

  16. The effect of respiration buffer composition on mitochondrial metabolism and function

    PubMed Central

    Wollenman, Lucas C.; Vander Ploeg, Matthew R.; Miller, Mackinzie L.; Zhang, Yizhu

    2017-01-01

    Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffers and substrate combinations to determine the optimal conditions to support mitochondrial function through ADP-stimulated respiration and uncoupled respiration using FCCP. The buffers consisted of a standard KCl based buffer (B1) and three modified buffers with chloride replaced by the K-lactobionate, sucrose, and the antioxidant taurine (B2) or K-gluconate (B3). The fourth buffer (B4) was identical to B2 except that K-lactobionate was replaced with K-gluconate. The substrate combinations consisted of metabolites that utilize different pathways of mitochondrial metabolism. To test mitochondrial function, we used isolated cardiac guinea pig mitochondria and measured oxygen consumption for three respiratory states using an Oroboros Oxygraph-2k. These states were the leak state (energized mitochondria in the absence of adenylates), ADP-stimulated state (energized mitochondria in the presence of saturating ADP concentrations), and uncoupled state (energized mitochondria in the presence of FCCP). On average across all substrate combinations, buffers B2, B3, and B4 had an increase of 16%, 26%, and 35% for the leak state, ADP-simulated state, and uncoupled state, respectively, relative to rates using B1. The common feature distinguishing these buffers from B1 is the notable lack of high chloride concentrations. Based on the respiratory rate metrics obtained with the substrate combinations, we conclude that the adenine nucleotide translocase, the dicarboxylate carrier, and the alpha

  17. Effect of intermittent phrenic nerve stimulation during cardiothoracic surgery on mitochondrial respiration in the human diaphragm

    PubMed Central

    Martin, A. Daniel; Joseph, Anna M.; Beaver, Thomas M.; Smith, Barbra K.; Martin, Tomas D.; Berg, Kent; Hess, Philip J.; Deoghare, Harsha V.; Leeuwenburgh, Christiaan

    2013-01-01

    Objectives: Recent studies have shown that brief periods of mechanical ventilation (MV) in animals and humans can lead to ventilator induced diaphragmatic dysfunction (VIDD), which includes muscle atrophy, reduced force development and impaired mitochondrial function. Animal work has shown that short periods of increased diaphragm activity during MV support can attenuate VIDD, but corresponding human data are lacking. The purpose of this study was to examine the effect of intermittent diaphragm contractions during cardiothoracic surgery, including controlled MV, on mitochondrial respiration in the human diaphragm. Method: In five patients (age 65.6 ± 6.3 yrs) undergoing cardiothoracic surgery, one phrenic nerve was stimulated hourly (30 pulses per minute, 1.5 msec duration, 17.0 ± 4.4 mA) during the surgery. Subjects received 3.4 ± 0.6 stimulation bouts during surgery. Thirty minutes following the last stimulation bout, samples of diaphragm muscle were obtained from the antero-lateral costal regions of the stimulated and inactive hemidiaphragms. Mitochondrial respiration was measured in permeabilized muscle fibers with high-resolution respirometry. Results: State III mitochondrial respiration rates (pmol O2/sec/mg wet weight) were 15.05 ± 3.92 and 11.42 ± 2.66 for the stimulated and unstimulated samples respectively, p < 0.05. State IV mitochondrial respiration rates were 3.59 ± 1.25 and 2.11 ± 0.97 in the stimulated samples and controls samples, respectively, p < 0.05. Conclusion: These are the first data examining the effect of intermittent contractions on mitochondrial respiration rates in the human diaphragm following surgery/MV. Our results indicate that very brief periods (duty cycle ~1.7%) of activity can improve mitochondrial function in the human diaphragm following surgery/MV. PMID:24126442

  18. Mitochondrial respiration controls lysosomal function during inflammatory T cell responses

    PubMed Central

    Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Dolores Ledesma, Maria; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

    2016-01-01

    Summary The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4+ T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration-deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward pro-inflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD+ levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify novel strategies for intervention in mitochondrial-related diseases. PMID:26299452

  19. Mitochondrial ultrastructure and tissue respiration of pea leaves under clinorotation

    NASA Astrophysics Data System (ADS)

    Brykov, Vasyl

    2016-07-01

    Respiration is essential for growth, maintenance, and carbon balance of all plant cells. Mitochondrial respiration in plants provides energy for biosynthesis, and its balance with photosynthesis determines the rate of plant biomass accumulation (production). Mitochondria are not only the energetic organelles in a cell but they play an essential regulatory role in many basic cellular processes. As plants adapt to real and simulated microgravity, it is very important to understand the state of mitochondria in these conditions. Disturbance of respiratory metabolism can significantly affect the productivity of plants in long-term space flights. We have established earlier that the rate of respiration in root apices of pea etiolated seedlings rose after 7 days of clinorotation. These data indicate the oxygen increased requirement by root apices under clinorotation, that confirms the necessity of sufficient substrate aeration in space greenhouses to provide normal respiratory metabolism and supply of energy for root growth. In etiolated seedlings, substrate supply of mitochondria occurs at the expense of the mobilization of cotyledon nutrients. A goal of our work was to study the ultrastructure and respiration of mitochondria in pea leaves after 12 days of clinorotation during (2 rpm/min). Plants grew at a light level of 180 μµmol m ^{-2} s ^{-1} PAR and a photoperiod of 16 h light/4 h dark. It was showed an essential increase in the mitochondrion area on 53% in palisade parenchyma cells at the sections. Such phenomenon can not be described as swelling of mitochondria, since enlarged mitochondria contained a more quantity of crista 1.76 times. In addition, the cristae total area per organelle also increased in comparison with that in control. An increase in a size of mitochondria in the experimental conditions is supposed to occur by a partial alteration of the chondriom. Thus, a size of 49% mitochondria in control was 0.1 - 0.3 μµm ^{2}, whereas only 26

  20. High dietary fat intake increases fat oxidation and reduces skeletal muscle mitochondrial respiration in trained humans.

    PubMed

    Leckey, Jill J; Hoffman, Nolan J; Parr, Evelyn B; Devlin, Brooke L; Trewin, Adam J; Stepto, Nigel K; Morton, James P; Burke, Louise M; Hawley, John A

    2018-06-01

    High-fat, low-carbohydrate (CHO) diets increase whole-body rates of fat oxidation and down-regulate CHO metabolism. We measured substrate utilization and skeletal muscle mitochondrial respiration to determine whether these adaptations are driven by high fat or low CHO availability. In a randomized crossover design, 8 male cyclists consumed 5 d of a high-CHO diet [>70% energy intake (EI)], followed by 5 d of either an isoenergetic high-fat (HFAT; >65% EI) or high-protein diet (HPRO; >65% EI) with CHO intake clamped at <20% EI. During the intervention, participants undertook daily exercise training. On d 6, participants consumed a high-CHO diet before performing 100 min of submaximal steady-state cycling plus an ∼30-min time trial. After 5 d of HFAT, skeletal muscle mitochondrial respiration supported by octanoylcarnitine and pyruvate, as well as uncoupled respiration, was decreased at rest, and rates of whole-body fat oxidation were higher during exercise compared with HPRO. After 1 d of high-CHO diet intake, mitochondrial respiration returned to baseline values in HFAT, whereas rates of substrate oxidation returned toward baseline in both conditions. These findings demonstrate that high dietary fat intake, rather than low-CHO intake, contributes to reductions in mitochondrial respiration and increases in whole-body rates of fat oxidation after a consuming a high-fat, low-CHO diet.-Leckey, J. J., Hoffman, N. J., Parr, E. B., Devlin, B. L., Trewin, A. J., Stepto, N. K., Morton, J. P., Burke, L. M., Hawley, J. A. High dietary fat intake increases fat oxidation and reduces skeletal muscle mitochondrial respiration in trained humans.

  1. A mitochondrial-targeted ubiquinone modulates muscle lipid profile and improves mitochondrial respiration in obesogenic diet-fed rats.

    PubMed

    Coudray, Charles; Fouret, Gilles; Lambert, Karen; Ferreri, Carla; Rieusset, Jennifer; Blachnio-Zabielska, Agnieszka; Lecomte, Jérôme; Ebabe Elle, Raymond; Badia, Eric; Murphy, Michael P; Feillet-Coudray, Christine

    2016-04-14

    The prevalence of the metabolic syndrome components including abdominal obesity, dyslipidaemia and insulin resistance is increasing in both developed and developing countries. It is generally accepted that the development of these features is preceded by, or accompanied with, impaired mitochondrial function. The present study was designed to analyse the effects of a mitochondrial-targeted lipophilic ubiquinone (MitoQ) on muscle lipid profile modulation and mitochondrial function in obesogenic diet-fed rats. For this purpose, twenty-four young male Sprague-Dawley rats were divided into three groups and fed one of the following diets: (1) control, (2) high fat (HF) and (3) HF+MitoQ. After 8 weeks, mitochondrial function markers and lipid metabolism/profile modifications in skeletal muscle were measured. The HF diet was effective at inducing the major features of the metabolic syndrome--namely, obesity, hepatic enlargement and glucose intolerance. MitoQ intake prevented the increase in rat body weight, attenuated the increase in adipose tissue and liver weights and partially reversed glucose intolerance. At the muscle level, the HF diet induced moderate TAG accumulation associated with important modifications in the muscle phospholipid classes and in the fatty acid composition of total muscle lipid. These lipid modifications were accompanied with decrease in mitochondrial respiration. MitoQ intake corrected the lipid alterations and restored mitochondrial respiration. These results indicate that MitoQ protected obesogenic diet-fed rats from some features of the metabolic syndrome through its effects on muscle lipid metabolism and mitochondrial activity. These findings suggest that MitoQ is a promising candidate for future human trials in the metabolic syndrome prevention.

  2. Homocysteine activates T cells by enhancing endoplasmic reticulum-mitochondria coupling and increasing mitochondrial respiration.

    PubMed

    Feng, Juan; Lü, Silin; Ding, Yanhong; Zheng, Ming; Wang, Xian

    2016-06-01

    Hyperhomocysteinemia (HHcy) accelerates atherosclerosis by increasing proliferation and stimulating cytokine secretion in T cells. However, whether homocysteine (Hcy)-mediated T cell activation is associated with metabolic reprogramming is unclear. Here, our in vivo and in vitro studies showed that Hcy-stimulated splenic T-cell activation in mice was accompanied by increased levels of mitochondrial reactive oxygen species (ROS) and calcium, mitochondrial mass and respiration. Inhibiting mitochondrial ROS production and calcium signals or blocking mitochondrial respiration largely blunted Hcy-induced T-cell interferon γ (IFN-γ) secretion and proliferation. Hcy also enhanced endoplasmic reticulum (ER) stress in T cells, and inhibition of ER stress with 4-phenylbutyric acid blocked Hcy-induced T-cell activation. Mechanistically, Hcy increased ER-mitochondria coupling, and uncoupling ER-mitochondria by the microtubule inhibitor nocodazole attenuated Hcy-stimulated mitochondrial reprogramming, IFN-γ secretion and proliferation in T cells, suggesting that juxtaposition of ER and mitochondria is required for Hcy-promoted mitochondrial function and T-cell activation. In conclusion, Hcy promotes T-cell activation by increasing ER-mitochondria coupling and regulating metabolic reprogramming.

  3. Gallic acid targets acute myeloid leukemia via Akt/mTOR-dependent mitochondrial respiration inhibition.

    PubMed

    Gu, Ruixin; Zhang, Minqin; Meng, Hu; Xu, Dandan; Xie, Yonghua

    2018-06-05

    Gallic acid is one of the many phenolic acids that can be found in dietary substances and traditional medicine herbs. The anti-cancer activities of gallic acid have been shown in various cancers but its underlying molecular mechanisms are not well understood. In this study, we show Akt/mammalian target of rapamycin (mTOR)-dependent inhibition of mitochondrial respiration as a mechanism of gallic acid's action in acute myeloid leukemia (AML). Gallic acid significantly induces apoptosis of AML cell lines, primary mononuclear cells (MNC) and CD34 stem/progenitors isolated form AML patients via caspase-dependent pathway. It also significantly enhances two standard AML chemotherapeutic agents' efficacy in vitro cell culture system and in vivo xenograft model. Gallic acid inhibits dose- and time-dependent mitochondrial respiration, leading to decreased ATP production and oxidative stress. Overexpression of constitutively active Akt restores gallic acid-mediated inhibition of mTOR signaling, mitochondrial dysfunction, energy crisis and apoptosis. Our results demonstrate that mitochondrial respiration inhibition by gallic acid is a consequence of Akt/mTOR signaling suppression. Our findings suggest that combination therapy with gallic acid may enhance antileukemic efficacy of standard chemotherapeutic agents in AML. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  4. Newly identified protein Imi1 affects mitochondrial integrity and glutathione homeostasis in Saccharomyces cerevisiae.

    PubMed

    Kowalec, Piotr; Grynberg, Marcin; Pająk, Beata; Socha, Anna; Winiarska, Katarzyna; Fronk, Jan; Kurlandzka, Anna

    2015-09-01

    Glutathione homeostasis is crucial for cell functioning. We describe a novel Imi1 protein of Saccharomyces cerevisiae affecting mitochondrial integrity and involved in controlling glutathione level. Imi1 is cytoplasmic and, except for its N-terminal Flo11 domain, has a distinct solenoid structure. A lack of Imi1 leads to mitochondrial lesions comprising aberrant morphology of cristae and multifarious mtDNA rearrangements and impaired respiration. The mitochondrial malfunctioning is coupled to significantly decrease the level of intracellular reduced glutathione without affecting oxidized glutathione, which decreases the reduced/oxidized glutathione ratio. These defects are accompanied by decreased cadmium sensitivity and increased phytochelatin-2 level. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated withmore » reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.« less

  6. Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

    PubMed

    Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

    2015-05-04

    Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  7. Transferrin receptor regulates pancreatic cancer growth by modulating mitochondrial respiration and ROS generation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jeong, Seung Min, E-mail: smjeong@catholic.ac.kr; Institute for Aging and Metabolic Diseases, College of Medicine, The Catholic University of Korea, Seoul 137-701; Hwang, Sunsook

    2016-03-11

    The transferrin receptor (TfR1) is upregulated in malignant cells and its expression is associated with cancer progression. Because of its pre-eminent role in cell proliferation, TfR1 has been an important target for the development of cancer therapy. Although TfR1 is highly expressed in pancreatic cancers, what it carries out in these refractory cancers remains poorly understood. Here we report that TfR1 supports mitochondrial respiration and ROS production in human pancreatic ductal adenocarcinoma (PDAC) cells, which is required for their tumorigenic growth. Elevated TfR1 expression in PDAC cells contributes to oxidative phosphorylation, which allows for the generation of ROS. Importantly, mitochondrial-derivedmore » ROS are essential for PDAC growth. However, exogenous iron supplement cannot rescue the defects caused by TfR1 knockdown. Moreover, we found that TfR1 expression determines PDAC cells sensitivity to oxidative stress. Together, our findings reveal that TfR1 can contribute to the mitochondrial respiration and ROS production, which have essential roles in growth and survival of pancreatic cancer. - Highlights: • Pancreatic ductal adenocarcinoma (PDAC) exhibits an elevated transferrin receptor (TfR1) expression in comparison with non-transformed pancreatic cells. • TfR1 is required for PDAC growth by regulating mitochondrial respiration and ROS production. • TfR1 functions as a determinant of cell viability to oxidative stress in PDAC cells.« less

  8. Age-Dependent Reductions in Mitochondrial Respiration are Exacerbated by Calcium in the Female Rat Heart

    PubMed Central

    Hunter, J. Craig; Machikas, Alexandra M.; Korzick, Donna H.

    2012-01-01

    Cardiovascular disease mortality increases rapidly following menopause by poorly defined mechanisms. Since mitochondrial function and Ca2+ sensitivity are important regulators of cell death following myocardial ischemia, we sought to determine if aging and/or estrogen deficiency (ovx) increased mitochondrial Ca2+ sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6mo; n=26) and aged (24mo; n=25), intact or ovariectomized female rats using the substrates: α-ketoglutarate/malate (Complex I); succinate/rotenone (Complex II); ascorbate/TMPD/Antimycin (Complex IV). State 2 and State 3 respiration was initiated by sequential addition of mitochondria and ADP. Ca2+ sensitivity was assessed by Ca2+-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered i.p. 45 min prior to euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for Complexes I and II by 12% and 8%, respectively, independent of ovary status (p<0.05). Of interest, Ca2+ induced a greater decrease (18–30%; p<0.05) in Complex I state 3 respiration in aged and ovx animals, and mitochondrial swelling occurred twice as quickly in aged (vs. adult) female rats (p<0.05). Pretreatment with PPT increased RCI by 8% and 7% at Complexes I and II, respectively (p<0.05) but surprisingly increased Ca2+ sensitivity. Age-dependent decreases in RCI and sensitization to Ca2+ may explain in part the age-associated reductions in female ischemic tolerance; however protection afforded by ER agonism involves more complex mechanisms. PMID:22555015

  9. Mitochondrial Respiration after One Session of Calf Raise Exercise in Patients with Peripheral Vascular Disease and Healthy Older Adults.

    PubMed

    van Schaardenburgh, Michel; Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J R

    2016-01-01

    Mitochondria are essential for energy production in the muscle cell and for this they are dependent upon a sufficient supply of oxygen by the circulation. Exercise training has shown to be a potent stimulus for physiological adaptations and mitochondria play a central role. Whether changes in mitochondrial respiration are seen after exercise in patients with a reduced circulation is unknown. The aim of the study was to evaluate the time course and whether one session of calf raise exercise stimulates mitochondrial respiration in the calf muscle of patients with peripheral vascular disease. One group of patients with peripheral vascular disease (n = 11) and one group of healthy older adults (n = 11) were included. Patients performed one session of continuous calf raises followed by 5 extra repetitions after initiation of pain. Healthy older adults performed 100 continuous calf raises. Gastrocnemius muscle biopsies were collected at baseline and 15 minutes, one hour, three hours and 24 hours after one session of calf raise exercise. A multi substrate (octanoylcarnitine, malate, adp, glutamate, succinate, FCCP, rotenone) approach was used to analyze mitochondrial respiration in permeabilized fibers. Mixed-linear model for repeated measures was used for statistical analyses. Patients with peripheral vascular disease have a lower baseline respiration supported by complex I and they increase respiration supported by complex II at one hour post-exercise. Healthy older adults increase respiration supported by electron transfer flavoprotein and complex I at one hour and 24 hours post-exercise. Our results indicate a shift towards mitochondrial respiration supported by complex II as being a pathophysiological component of peripheral vascular disease. Furthermore exercise stimulates mitochondrial respiration already after one session of calf raise exercise in patients with peripheral vascular disease and healthy older adults. ClinicalTrials.gov NCT01842412.

  10. Mitochondrial Respiration after One Session of Calf Raise Exercise in Patients with Peripheral Vascular Disease and Healthy Older Adults

    PubMed Central

    Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J. R.

    2016-01-01

    Purpose Mitochondria are essential for energy production in the muscle cell and for this they are dependent upon a sufficient supply of oxygen by the circulation. Exercise training has shown to be a potent stimulus for physiological adaptations and mitochondria play a central role. Whether changes in mitochondrial respiration are seen after exercise in patients with a reduced circulation is unknown. The aim of the study was to evaluate the time course and whether one session of calf raise exercise stimulates mitochondrial respiration in the calf muscle of patients with peripheral vascular disease. Methods One group of patients with peripheral vascular disease (n = 11) and one group of healthy older adults (n = 11) were included. Patients performed one session of continuous calf raises followed by 5 extra repetitions after initiation of pain. Healthy older adults performed 100 continuous calf raises. Gastrocnemius muscle biopsies were collected at baseline and 15 minutes, one hour, three hours and 24 hours after one session of calf raise exercise. A multi substrate (octanoylcarnitine, malate, adp, glutamate, succinate, FCCP, rotenone) approach was used to analyze mitochondrial respiration in permeabilized fibers. Mixed-linear model for repeated measures was used for statistical analyses. Results Patients with peripheral vascular disease have a lower baseline respiration supported by complex I and they increase respiration supported by complex II at one hour post-exercise. Healthy older adults increase respiration supported by electron transfer flavoprotein and complex I at one hour and 24 hours post-exercise. Conclusion Our results indicate a shift towards mitochondrial respiration supported by complex II as being a pathophysiological component of peripheral vascular disease. Furthermore exercise stimulates mitochondrial respiration already after one session of calf raise exercise in patients with peripheral vascular disease and healthy older adults. Trial

  11. Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) Signaling Regulates Mitochondrial Biogenesis and Respiration via Estrogen-related Receptor α (ERRα)*

    PubMed Central

    Li, Yang; He, Lina; Zeng, Ni; Sahu, Divya; Cadenas, Enrique; Shearn, Colin; Li, Wei; Stiles, Bangyan L.

    2013-01-01

    Mitochondrial abnormalities are associated with cancer development, yet how oncogenic signals affect mitochondrial functions has not been fully understood. In this study, we investigate the relationship between mitochondrial alterations and PI3K/protein kinase B (AKT) signaling activation using hepatocytes and liver tissues as our experimental models. We show here that liver-specific deletion of Pten, which leads to activation of PI3K/AKT, is associated with elevated oxidative stress, increased mitochondrial mass, and augmented respiration accompanied by enhanced glycolysis. Consistent with these observations, estrogen-related receptor α (ERRα), an orphan nuclear receptor known for its role in mitochondrial biogenesis, is up-regulated in the absence of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Our pharmacological and genetic studies show that PI3K/AKT activity regulates the expression of ERRα and mitochondrial biogenesis/respiration. Furthermore, cAMP-response element-binding protein, as a downstream target of AKT, plays a role in the regulation of ERRα, independent of PKA signaling. ERRα regulates reactive oxygen species production, and ERRα knockdown attenuates proliferation and colony-forming potential in Pten-null hepatocytes. Finally, analysis of clinical datasets from liver tissues showed a negative correlation between expressions of ERRα and PTEN in patients with liver cancer. Therefore, this study has established a previously unrecognized link between a growth signal and mitochondrial metabolism. PMID:23836899

  12. Age-dependent reductions in mitochondrial respiration are exacerbated by calcium in the female rat heart.

    PubMed

    Hunter, J Craig; Machikas, Alexandra M; Korzick, Donna H

    2012-06-01

    Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms. Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.

  13. In Vitro Effects of Cognitives and Nootropics on Mitochondrial Respiration and Monoamine Oxidase Activity.

    PubMed

    Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

    2017-10-01

    Impairment of mitochondrial metabolism, particularly the electron transport chain (ETC), as well as increased oxidative stress might play a significant role in pathogenesis of Alzheimer's disease (AD). Some effects of drugs used for symptomatic AD treatment may be related to their direct action on mitochondrial function. In vitro effects of pharmacologically different cognitives (galantamine, donepezil, rivastigmine, 7-MEOTA, memantine) and nootropic drugs (latrepirdine, piracetam) were investigated on selected mitochondrial parameters: activities of ETC complexes I, II + III, and IV, citrate synthase, monoamine oxidase (MAO), oxygen consumption rate, and hydrogen peroxide production of pig brain mitochondria. Complex I activity was decreased by galantamine, donepezil, and memantine; complex II + III activity was increased by galantamine. None of the tested drugs caused significant changes in the rate of mitochondrial oxygen consumption, even at high concentrations. Except galantamine, all tested drugs were selective MAO-A inhibitors. Latrepirdine, donepezil, and 7-MEOTA were found to be the most potent MAO-A inhibitors. Succinate-induced mitochondrial hydrogen peroxide production was not significantly affected by the drugs tested. The direct effect of cognitives and nootropics used in the treatment of AD on mitochondrial respiration is relatively small. The safest drugs in terms of disturbing mitochondrial function appear to be piracetam and rivastigmine. The MAO-A inhibition by cognitives and nootropics may also participate in mitochondrial neuroprotection. The results support the future research aimed at measuring the effects of currently used drugs or newly synthesized drugs on mitochondrial functioning in order to understand their mechanism of action.

  14. Effect of the antitumoral alkylating agent 3-bromopyruvate on mitochondrial respiration: role of mitochondrially bound hexokinase.

    PubMed

    Rodrigues-Ferreira, Clara; da Silva, Ana Paula Pereira; Galina, Antonio

    2012-02-01

    The alkylating agent 3-Bromopyruvate (3-BrPA) has been used as an anti-tumoral drug due to its anti-proliferative property in hepatomas cells. This propriety is believed to disturb glycolysis and respiration, which leads to a decreased rate of ATP synthesis. In this study, we evaluated the effects of the alkylating agent 3-BrPA on the respiratory states and the metabolic steps of the mitochondria of mice liver, brain and in human hepatocarcinoma cell line HepG2. The mitochondrial membrane potential (ΔΨ(m)), O(2) consumption and dehydrogenase activities were rapidly dissipated/or inhibited by 3-BrPA in respiration medium containing ADP and succinate as respiratory substrate. 3-BrPA inhibition was reverted by reduced glutathione (GSH). Respiration induced by yeast soluble hexokinase (HK) was rapidly inhibited by 3-BrPA. Similar results were observed using mice brain mitochondria that present HK naturally bound to the outer mitochondrial membrane. When the adenine nucleotide transporter (ANT) was blocked by the carboxyatractiloside, the 3-BrPA effect was significantly delayed. In permeabilized human hepatoma HepG2 cells that present HK type II bound to mitochondria (mt-HK II), the inhibiting effect occurred faster when the endogenous HK activity was activated by 2-deoxyglucose (2-DOG). Inhibition of mt-HK II by glucose-6-phosphate retards the mitochondria to react with 3-BrPA. The HK activities recovered in HepG2 cells treated or not with 3-BrPA were practically the same. These results suggest that mitochondrially bound HK supporting the ADP/ATP exchange activity levels facilitates the 3-BrPA inhibition reaction in tumors mitochondria by a proton motive force-dependent dynamic equilibrium between sensitive and less sensitive SDH in the electron transport system.

  15. MacroH2A1.1 regulates mitochondrial respiration by limiting nuclear NAD+ consumption.

    PubMed

    Posavec Marjanović, Melanija; Hurtado-Bagès, Sarah; Lassi, Maximilian; Valero, Vanesa; Malinverni, Roberto; Delage, Hélène; Navarro, Miriam; Corujo, David; Guberovic, Iva; Douet, Julien; Gama-Perez, Pau; Garcia-Roves, Pablo M; Ahel, Ivan; Ladurner, Andreas G; Yanes, Oscar; Bouvet, Philippe; Suelves, Mònica; Teperino, Raffaele; Pospisilik, J Andrew; Buschbeck, Marcus

    2017-11-01

    Histone variants are structural components of eukaryotic chromatin that can replace replication-coupled histones in the nucleosome. The histone variant macroH2A1.1 contains a macrodomain capable of binding NAD + -derived metabolites. Here we report that macroH2A1.1 is rapidly induced during myogenic differentiation through a switch in alternative splicing, and that myotubes that lack macroH2A1.1 have a defect in mitochondrial respiratory capacity. We found that the metabolite-binding macrodomain was essential for sustained optimal mitochondrial function but dispensable for gene regulation. Through direct binding, macroH2A1.1 inhibits basal poly-ADP ribose polymerase 1 (PARP-1) activity and thus reduces nuclear NAD + consumption. The resultant accumulation of the NAD + precursor NMN allows for maintenance of mitochondrial NAD + pools that are critical for respiration. Our data indicate that macroH2A1.1-containing chromatin regulates mitochondrial respiration by limiting nuclear NAD + consumption and establishing a buffer of NAD + precursors in differentiated cells.

  16. Parkin loss leads to PARIS-dependent declines in mitochondrial mass and respiration

    PubMed Central

    Stevens, Daniel A.; Lee, Yunjong; Kang, Ho Chul; Lee, Byoung Dae; Lee, Yun-Il; Bower, Aaron; Jiang, Haisong; Kang, Sung-Ung; Andrabi, Shaida A.; Dawson, Valina L.; Shin, Joo-Ho; Dawson, Ted M.

    2015-01-01

    Mutations in parkin lead to early-onset autosomal recessive Parkinson’s disease (PD) and inactivation of parkin is thought to contribute to sporadic PD. Adult knockout of parkin in the ventral midbrain of mice leads to an age-dependent loss of dopamine neurons that is dependent on the accumulation of parkin interacting substrate (PARIS), zinc finger protein 746 (ZNF746), and its transcriptional repression of PGC-1α. Here we show that adult knockout of parkin in mouse ventral midbrain leads to decreases in mitochondrial size, number, and protein markers consistent with a defect in mitochondrial biogenesis. This decrease in mitochondrial mass is prevented by short hairpin RNA knockdown of PARIS. PARIS overexpression in mouse ventral midbrain leads to decreases in mitochondrial number and protein markers and PGC-1α–dependent deficits in mitochondrial respiration. Taken together, these results suggest that parkin loss impairs mitochondrial biogenesis, leading to declining function of the mitochondrial pool and cell death. PMID:26324925

  17. Cardiomyocyte mitochondrial respiration is reduced by receptor for advanced glycation end-product signaling in a ceramide-dependent manner.

    PubMed

    Nelson, Michael B; Swensen, Adam C; Winden, Duane R; Bodine, Jared S; Bikman, Benjamin T; Reynolds, Paul R

    2015-07-01

    Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function. Copyright © 2015 the American Physiological Society.

  18. β-Cell deletion of Nr4a1 and Nr4a3 nuclear receptors impedes mitochondrial respiration and insulin secretion.

    PubMed

    Reynolds, Merrick S; Hancock, Chad R; Ray, Jason D; Kener, Kyle B; Draney, Carrie; Garland, Kevin; Hardman, Jeremy; Bikman, Benjamin T; Tessem, Jeffery S

    2016-07-01

    β-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic β-cells. In this study, we examined whether Nr4a expression impacts pancreatic β-cell mitochondrial function. Here, we show that β-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in β-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for β-cell mitochondrial function and insulin secretion. Copyright © 2016 the American Physiological Society.

  19. Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy.

    PubMed

    Czajka, Anna; Malik, Afshan N

    2016-12-01

    Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

    PubMed

    Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

    2017-11-18

    Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Mitochondrial gene therapy improves respiration, biogenesis, and transcription in G11778A Leber's hereditary optic neuropathy and T8993G Leigh's syndrome cells.

    PubMed

    Iyer, Shilpa; Bergquist, Kristen; Young, Kisha; Gnaiger, Erich; Rao, Raj R; Bennett, James P

    2012-06-01

    Many incurable mitochondrial disorders result from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh's syndrome (LS) is a fatal neurodegenerative disorder of infants, and Leber's hereditary optic neuropathy (LHON) causes blindness in young adults. Treatment of LHON and LS cells harboring G11778A and T8993G mutant mtDNA, respectively, by >90%, with healthy donor mtDNA complexed with recombinant human mitochondrial transcription factor A (rhTFAM), improved mitochondrial respiration by ∼1.2-fold in LHON cells and restored >50% ATP synthase function in LS cells. Mitochondrial replication, transcription, and translation of key respiratory genes and proteins were increased in the short term. Increased NRF1, TFAMB1, and TFAMA expression alluded to the activation of mitochondrial biogenesis as a mechanism for improving mitochondrial respiration. These results represent the development of a therapeutic approach for LHON and LS patients in the near future.

  2. High-throughput assessment of oxidative respiration in fish embryos: Advancing adverse outcome pathways for mitochondrial dysfunction.

    PubMed

    Souders, Christopher L; Liang, Xuefang; Wang, Xiaohong; Ector, Naomi; Zhao, Yuan H; Martyniuk, Christopher J

    2018-06-01

    Mitochondrial dysfunction is a prevalent molecular event that can result in multiple adverse outcomes. Recently, a novel high throughput method to assess metabolic capacity in fish embryos following exposure to chemicals has been adapted for environmental toxicology. Assessments of oxygen consumption rates using the Seahorse XF(e) 24/96 Extracellular Flux Analyzer (Agilent Technologies) can be used to garner insight into toxicant effects at early stages of development. Here we synthesize the current state of the science using high throughput metabolic profiling in zebrafish embryos, and present considerations for those wishing to adopt high throughput methods for mitochondrial bioenergetics into their research. Chemicals that have been investigated in zebrafish using this metabolic platform include herbicides (e.g. paraquat, diquat), industrial compounds (e.g. benzo-[a]-pyrene, tributyltin), natural products (e.g. quercetin), and anti-bacterial chemicals (i.e. triclosan). Some of these chemicals inhibit mitochondrial endpoints in the μM-mM range, and reduce basal respiration, maximum respiration, and spare capacity. We present a theoretical framework for how one can use mitochondrial performance data in zebrafish to categorize chemicals of concern and prioritize mitochondrial toxicants. Noteworthy is that our studies demonstrate that there can be considerable variation in basal respiration of untreated zebrafish embryos due to clutch-specific effects as well as individual variability, and basal oxygen consumption rates (OCR) can vary on average between 100 and 300 pmol/min/embryo. We also compare OCR between chorionated and dechorionated embryos, as both models are employed to test chemicals. After 24 h, dechorionated embryos remain responsive to mitochondrial toxicants, although they show a blunted response to the uncoupling agent carbonylcyanide-4-trifluoromethoxyphenylhydrazone (FCCP); dechorionated embryos are therefore a viable option for investigations

  3. Tributyltin (TBT) and mitochondrial respiration in mussel digestive gland.

    PubMed

    Nesci, Salvatore; Ventrella, Vittoria; Trombetti, Fabiana; Pirini, Maurizio; Pagliarani, Alessandra

    2011-06-01

    The toxicity of organotins and especially tri-n-butyltin (TBT) on mitochondria is well known. However as far as we are aware, effects on mitochondrial respiration are unexplored in mollusks. In this work mitochondria isolated from the digestive gland of Mytilus galloprovincialis and susceptive to the classical respiratory chain inhibitors, were assayed in the presence of micromolar TBT concentrations to investigate mitochondrial respiratory activities. Intact and freeze-thawed mitochondria were used. TBT significantly inhibited oxygen consumption in the presence of glutamate/malate or succinate as substrates. Conversely cytochrome c oxidase activity (complex IV), assayed both polarographically and spectrophotometrically, was unaffected. The addition of 1,4-dithioerythritol (DTE) decreased the TBT-driven inhibition of complexes I and III. The TBT capability of covalent binding to thiol groups of mitochondrial proteins in a dose-dependent manner was confirmed by the aid of Ellman's reagent. Data strongly suggests that TBT may prevent the electron transfer from complexes I and III to downhill respiratory chain complexes by binding to critical SH residues. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Sulfide-inhibition of mitochondrial respiration at very low oxygen concentrations.

    PubMed

    Matallo, J; Vogt, J; McCook, O; Wachter, U; Tillmans, F; Groeger, M; Szabo, C; Georgieff, M; Radermacher, P; Calzia, E

    2014-09-15

    Our aim was to study the ability of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the rate of elimination of sulfide through the pathway linked to the mitochondrial respiratory chain and therefore operating under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide's inhibition of cellular respiration would occur faster under continuous sulfide exposure when the oxygen concentration is in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5-1×10(6) cells in 2 ml of continuously stirred respiration medium at 37 °C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73±0.05 μM, 3.1±0.2 μM, and 6.2±0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3;3.5] and 11.7 [6.2;21.2]min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6 [15.5;28.1]min (coupled) and 35.9 [27.4;59.2]min (uncoupled), as well as 42.4 [27.5;42.4]min (coupled) and 51.5 [46.4;51.7]min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results confirm that the onset of inhibition of cell respiration by sulfide occurs earlier under a continuous exposure when approaching

  5. Altered sterol metabolism in budding yeast affects mitochondrial iron-sulfur (Fe-S) cluster synthesis.

    PubMed

    Ward, Diane M; Chen, Opal S; Li, Liangtao; Kaplan, Jerry; Bhuiyan, Shah Alam; Natarajan, Selvamuthu K; Bard, Martin; Cox, James E

    2018-05-17

    Ergosterol synthesis is essential for cellular growth and viability of the budding yeast Saccharomyces cerevisiae, and intracellular sterol distribution and homeostasis are therefore highly regulated in this species. Erg25 is an iron-containing C4-methyl sterol oxidase that contributes to the conversion of 4,4-dimethylzymosterol to zymosterol, a precursor of ergosterol. The ERG29 gene encodes an endoplasmic reticulum (ER)-associated protein, and here we identified a role for Erg29 in the methyl sterol oxidase step of ergosterol synthesis. ERG29 deletion resulted in lethality in respiring cells, but respiration-incompetent (Rho- or Rho0) cells survived, suggesting that Erg29 loss leads to accumulation of oxidized sterol metabolites that affect cell viability. Down-regulation of ERG29 expression in Δerg29 cells indeed led to accumulation of methyl sterol metabolites, resulting in increased mitochondrial oxidants and a decreased ability of mitochondria to synthesize iron-sulfur (Fe-S) clusters due to reduced levels of Yfh1, the mammalian frataxin homolog, which is involved in mitochondrial Fe metabolism. Using a high-copy genomic library, we identified suppressor genes that permitted growth of Δerg29 cells on respiratory substrates, and these included genes encoding the mitochondrial proteins Yfh1, Mmt1, Mmt2, and Pet20, which reversed all phenotypes associated with loss of ERG29. Of note, loss of Erg25 also resulted in accumulation of methyl sterol metabolites and also increased mitochondrial oxidants and degradation of Yfh1. We propose that accumulation of toxic intermediates of the methyl sterol oxidase reaction increase mitochondrial oxidants, which affect Yfh1 protein stability. These results indicate an interaction between sterols generated by ER proteins and mitochondrial iron metabolism. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Formation of a Snf1-Mec1-Atg1 Module on Mitochondria Governs Energy Deprivation-Induced Autophagy by Regulating Mitochondrial Respiration.

    PubMed

    Yi, Cong; Tong, Jingjing; Lu, Puzhong; Wang, Yizheng; Zhang, Jinxie; Sun, Chen; Yuan, Kangning; Xue, Renyu; Zou, Bing; Li, Nianzhong; Xiao, Shuhua; Dai, Chong; Huang, Yuwei; Xu, Liling; Li, Lin; Chen, She; Miao, Di; Deng, Haiteng; Li, Hongliang; Yu, Li

    2017-04-10

    Autophagy is essential for maintaining glucose homeostasis, but the mechanism by which energy deprivation activates autophagy is not fully understood. We show that Mec1/ATR, a member of the DNA damage response pathway, is essential for glucose starvation-induced autophagy. Mec1, Atg13, Atg1, and the energy-sensing kinase Snf1 are recruited to mitochondria shortly after glucose starvation. Mec1 is recruited through the adaptor protein Ggc1. Snf1 phosphorylates Mec1 on the mitochondrial surface, leading to recruitment of Atg1 to mitochondria. Furthermore, the Snf1-mediated Mec1 phosphorylation and mitochondrial recruitment of Atg1 are essential for maintaining mitochondrial respiration during glucose starvation, and active mitochondrial respiration is required for energy deprivation-activated autophagy. Thus, formation of a Snf1-Mec1-Atg1 module on mitochondria governs energy deprivation-induced autophagy by regulating mitochondrial respiration. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Sulfide-Inhibition of Mitochondrial Respiration at Very Low Oxygen Concentrations

    PubMed Central

    Matallo, J; Vogt, J; McCook, O; Wachter, U; Tillmans, F; Groeger, M; Szabo, C; Georgieff, M; Radermacher, P; Calzia, E

    2014-01-01

    Our aim was to study the capacity of an immortalized cell line (AMJ2-C11) to sustain aerobic cell respiration at decreasing oxygen concentrations under continuous sulfide exposure. We assumed that the capacity of the pathway metabolizing and eliminating sulfide, which is linked to the mitochondrial respiratory chain and therefore operates under aerobic conditions, should decrease with limiting oxygen concentrations. Thus, sulfide’s inhibition of cellular respiration would be dependent of the oxygen concentration in the very low range. The experiments were performed with an O2K-oxygraph (Oroboros Instruments) by suspending 0.5 – 1 × 106 cells in 2 ml of continuously stirred respiration medium at 37°C and calculating the oxygen flux (JO2) as the negative derivative of the oxygen concentration in the medium. The cells were studied in two different metabolic states, namely under normal physiologic respiration (1) and after uncoupling of mitochondrial respiration (2). Oxygen concentration was controlled by means of a titration-injection pump, resulting in average concentration values of 0.73 ± 0.05 μM, 3.1 ± 0.2 μM, and 6.2 ± 0.2 μM. Simultaneously we injected a 2 mM Na2S solution at a continuous rate of 10 μl/s in order to quantify the titration-time required to reduce the JO2 to 50% of the initial respiratory activity. Under the lowest oxygen concentration this effect was achieved after 3.5 [0.3; 3.5] and 11.7 [6.2;21.2] min in the uncoupled and coupled state, respectively. This time was statistically significantly shorter when compared to the intermediate and the highest O2 concentrations tested, which yielded values of 24.6[15.5;28.1] min (coupled) and 35.9[27.4;59.2] min (uncoupled), as well as 42.4 [27.5;42.4] min (coupled) and 51.5 [46.4;51.7] min (uncoupled). All data are medians [25%, and 75% percentiles]. Our results suggest that elimination of sulfide in these cells is limited by oxygen availability when approaching the anoxic condition. This

  8. Zinc and calcium alter the relationship between mitochondrial respiration, ROS and membrane potential in rainbow trout (Oncorhynchus mykiss) liver mitochondria.

    PubMed

    Sharaf, Mahmoud S; Stevens, Don; Kamunde, Collins

    2017-08-01

    At excess levels, zinc (Zn) disrupts mitochondrial functional integrity and induces oxidative stress in aquatic organisms. Although much is known about the modulation of Zn toxicity by calcium (Ca) in fish, their interactions at the mitochondrial level have scarcely been investigated. Here we assessed the individual and combined effects of Zn and Ca on the relationship between mitochondrial respiration, ROS and membrane potential (ΔΨ mt ) in rainbow trout liver mitochondria. We tested if cation uptake through the mitochondrial calcium uniporter (MCU) is a prerequisite for Zn- and/or Ca-induced alteration of mitochondrial function. Furthermore, using our recently developed real-time multi-parametric method, we investigated the changes in respiration, ΔΨ mt , and reactive oxygen species (ROS, as hydrogen peroxide (H 2 O 2 )) release associated with Ca-induced mitochondrial depolarization imposed by transient and permanent openings of the mitochondrial permeability transition pore (mPTP). We found that independent of the MCU, Zn precipitated an immediate depolarization of the ΔΨ mt that was associated with relatively slow enhancement of H 2 O 2 release, inhibition of respiration and reversal of the positive correlation between ROS and ΔΨ mt . In contrast, an equitoxic dose of Ca caused transient depolarization, and stimulation of both respiration and H 2 O 2 release, effects that were completely abolished when the MCU was blocked. Contrary to our expectation that mitochondrial transition ROS Spike (mTRS) would be sensitive to both Zn and Ca, only Ca suppressed it. Moreover, Zn and Ca in combination immediately depolarized the ΔΨ mt , and caused transient and sustained stimulation of respiration and H 2 O 2 release, respectively. Lastly, we uncovered and characterized an mPTP-independent Ca-induced depolarization spike that was associated with exposure to moderately elevated levels of Ca. Importantly, we showed the stimulation of ROS release associated with

  9. Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

    NASA Astrophysics Data System (ADS)

    Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

    2001-06-01

    An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (<20 μM). In this study, we present evidence for a consumption of NO in mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

  10. Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential

    PubMed Central

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B.; Khan, Ikhlas A.; Nagle, Dale G.; Zhou, Yu-Dong

    2014-01-01

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “Black Box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3) exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage. PMID:24328138

  11. Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration.

    PubMed

    Rowley, Thomas J; Bitner, Benjamin F; Ray, Jason D; Lathen, Daniel R; Smithson, Andrew T; Dallon, Blake W; Plowman, Chase J; Bikman, Benjamin T; Hansen, Jason M; Dorenkott, Melanie R; Goodrich, Katheryn M; Ye, Liyun; O'Keefe, Sean F; Neilson, Andrew P; Tessem, Jeffery S

    2017-11-01

    A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13-derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients. Copyright

  12. Intracellular diffusion of oxygen and hypoxic sensing: role of mitochondrial respiration.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2010-01-01

    In vivo, diffusional O(2) gradients from the capillary blood to the intracellular space determine O(2) availability at the O(2) sensing molecules in the cell. With a novel technique for imaging intracellular O(2) levels using green fluorescent protein (GFP), we examined the possibility that diffusional O(2) concentration gradients might be involved in the cellular hypoxic sensing in cultured Hep3B cells. In the present study, we failed to demonstrate significant gradients of intracellular O(2) when mitochondrial respiration was maximally elevated by an uncoupler of oxidative phosphorylation. Thus, we conclude that intracellular O(2) gradients may be negligible at normal mitochondrial O(2) demand in these cells.

  13. Characterization of the respiration-induced yeast mitochondrial permeability transition pore.

    PubMed

    Bradshaw, Patrick C; Pfeiffer, Douglas R

    2013-12-01

    When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.

  14. [Endoplasmic-mitochondrial Ca(2+)-functional unit: dependence of respiration of secretory cells on activity of ryanodine- and IP3 - sensitive Ca(2+)-channels].

    PubMed

    Velykopols'ka, O Iu; Man'ko, B O; Man'ko, V V

    2012-01-01

    Using Clark oxygen electrode, dependence of mitochondrial functions on Ca(2+)-release channels activity of Chironomus plumosus L. larvae salivary glands suspension was investigated. Cells were ATP-permeabilized in order to enable penetration of exogenous oxidative substrates. Activation of plasmalemmal P2X-receptors (as well as P2Y-receptors) per se does not modify the endogenous respiration of salivary gland suspension. That is, Ca(2+)-influx from extracellular medium does not influence functional activity of mitochondria, although they are located along the basal part of the plasma membrane. Activation of RyRs intensifies endogenous respiration and pyruvate-malate-stimulated respiration, but not succinate-stimulated respiration. Neither activation of IP3Rs (via P2Y-receptors activation), nor their inhibition alters endogenous respiration. Nevertheless, IP3Rs inhibition by 2-APB intensifies succinate-stimulated respiration. All abovementioned facts testify that Ca2+, released from stores via channels, alters functional activity of mitochondria, and undoubtedly confirm the existence of endoplasmic-mitochondrial Ca(2+)-functional unit in Ch. plumosus larvae salivary glands secretory cells. In steady state of endoplasmic-mitochondrial Ca(2+)-functional unit the spontaneous activity of IP3Rs is observed; released through IP3Rs, Ca2+ is accumulated in mitochondria via uniporter and modulates oxidative processes. Activation of RyRs induces the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to the active state, which is required to intensify cell respiration and oxidative phosphorylation. As expected, the transition of endoplasmic-mitochondrial Ca(2+)-functional unit to inactivated state (i. e. inhibition of Ca(2+)-release channels at excessive [Ca2+]i) limits the duration of signal transduction, has protective nature and prevents apoptosis.

  15. Mitochondrial Respiration in Human Colorectal and Breast Cancer Clinical Material Is Regulated Differently

    PubMed Central

    Koit, Andre; Ounpuu, Lyudmila; Klepinin, Aleksandr; Chekulayev, Vladimir; Timohhina, Natalja; Tepp, Kersti; Puurand, Marju; Truu, Laura; Heck, Karoliina; Valvere, Vahur; Guzun, Rita

    2017-01-01

    We conducted quantitative cellular respiration analysis on samples taken from human breast cancer (HBC) and human colorectal cancer (HCC) patients. Respiratory capacity is not lost as a result of tumor formation and even though, functionally, complex I in HCC was found to be suppressed, it was not evident on the protein level. Additionally, metabolic control analysis was used to quantify the role of components of mitochondrial interactosome. The main rate-controlling steps in HBC are complex IV and adenine nucleotide transporter, but in HCC, complexes I and III. Our kinetic measurements confirmed previous studies that respiratory chain complexes I and III in HBC and HCC can be assembled into supercomplexes with a possible partial addition from the complex IV pool. Therefore, the kinetic method can be a useful addition in studying supercomplexes in cell lines or human samples. In addition, when results from culture cells were compared to those from clinical samples, clear differences were present, but we also detected two different types of mitochondria within clinical HBC samples, possibly linked to two-compartment metabolism. Taken together, our data show that mitochondrial respiration and regulation of mitochondrial membrane permeability have substantial differences between these two cancer types when compared to each other to their adjacent healthy tissue or to respective cell cultures. PMID:28781720

  16. Metabolic compartmentation in rainbow trout cardiomyocytes: coupling of hexokinase but not creatine kinase to mitochondrial respiration.

    PubMed

    Karro, Niina; Sepp, Mervi; Jugai, Svetlana; Laasmaa, Martin; Vendelin, Marko; Birkedal, Rikke

    2017-01-01

    Rainbow trout (Oncorhynchus mykiss) cardiomyocytes have a simple morphology with fewer membrane structures such as sarcoplasmic reticulum and t-tubules penetrating the cytosol. Despite this, intracellular ADP diffusion is restricted. Intriguingly, although diffusion is restricted, trout cardiomyocytes seem to lack the coupling between mitochondrial creatine kinase (CK) and respiration. Our aim was to study the distribution of diffusion restrictions in permeabilized trout cardiomyocytes and verify the role of CK. We found a high activity of hexokinase (HK), which led us to reassess the situation in trout cardiomyocytes. We show that diffusion restrictions are more prominent than previously thought. In the presence of a competitive ADP-trapping system, ADP produced by HK, but not CK, was channeled to the mitochondria. In agreement with this, we found no positively charged mitochondrial CK in trout heart homogenate. The results were best fit by a simple mathematical model suggesting that trout cardiomyocytes lack a functional coupling between ATPases and pyruvate kinase. The model simulations show that diffusion is restricted to almost the same extent in the cytosol and by the outer mitochondrial membrane. Furthermore, they confirm that HK, but not CK, is functionally coupled to respiration. In perspective, our results suggest that across a range of species, cardiomyocyte morphology and metabolism go hand in hand with cardiac performance, which is adapted to the circumstances. Mitochondrial CK is coupled to respiration in adult mammalian hearts, which are specialized to high, sustained performance. HK associates with mitochondria in hearts of trout and neonatal mammals, which are more hypoxia-tolerant.

  17. CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through upregulating L-type calcium channel activity.

    PubMed

    Sun, Meiqun; Liu, Hongli; Xu, Huanbai; Wang, Hongtao; Wang, Xiaojing

    2016-09-01

    A specialized culture medium termed ciliary neurotrophic factor-treated astrocyte-conditioned medium (CNTF-ACM) allows investigators to assess the peripheral effects of CNTF-induced activated astrocytes upon cultured neurons. CNTF-ACM has been shown to upregulate neuronal L-type calcium channel current activity, which has been previously linked to changes in mitochondrial respiration and oxidative stress. Therefore, the aim of this study was to evaluate CNTF-ACM's effects upon mitochondrial respiration and oxidative stress in rat cortical neurons. Cortical neurons, CNTF-ACM, and untreated control astrocyte-conditioned medium (UC-ACM) were prepared from neonatal Sprague-Dawley rat cortical tissue. Neurons were cultured in either CNTF-ACM or UC-ACM for a 48-h period. Changes in the following parameters before and after treatment with the L-type calcium channel blocker isradipine were assessed: (i) intracellular calcium levels, (ii) mitochondrial membrane potential (ΔΨm), (iii) oxygen consumption rate (OCR) and adenosine triphosphate (ATP) formation, (iv) intracellular nitric oxide (NO) levels, (v) mitochondrial reactive oxygen species (ROS) production, and (vi) susceptibility to the mitochondrial complex I toxin rotenone. CNTF-ACM neurons displayed the following significant changes relative to UC-ACM neurons: (i) increased intracellular calcium levels (p < 0.05), (ii) elevation in ΔΨm (p < 0.05), (iii) increased OCR and ATP formation (p < 0.05), (iv) increased intracellular NO levels (p < 0.05), (v) increased mitochondrial ROS production (p < 0.05), and (vi) increased susceptibility to rotenone (p < 0.05). Treatment with isradipine was able to partially rescue these negative effects of CNTF-ACM (p < 0.05). CNTF-ACM promotes mitochondrial respiration and oxidative stress in cortical neurons through elevating L-type calcium channel activity.

  18. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    PubMed

    de Moura, Michelle Barbi; Uppala, Radha; Zhang, Yuxun; Van Houten, Bennett; Goetzman, Eric S

    2014-01-01

    SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  19. Augmentation of aerobic respiration and mitochondrial biogenesis in skeletal muscle by hypoxia preconditioning with cobalt chloride

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Saxena, Saurabh; Shukla, Dhananjay; Bansal, Anju, E-mail: anjubansaldipas@gmail.com

    High altitude/hypoxia training is known to improve physical performance in athletes. Hypoxia induces hypoxia inducible factor-1 (HIF-1) and its downstream genes that facilitate hypoxia adaptation in muscle to increase physical performance. Cobalt chloride (CoCl{sub 2}), a hypoxia mimetic, stabilizes HIF-1, which otherwise is degraded in normoxic conditions. We studied the effects of hypoxia preconditioning by CoCl{sub 2} supplementation on physical performance, glucose metabolism, and mitochondrial biogenesis using rodent model. The results showed significant increase in physical performance in cobalt supplemented rats without (two times) or with training (3.3 times) as compared to control animals. CoCl{sub 2} supplementation in rats augmentedmore » the biological activities of enzymes of TCA cycle, glycolysis and cytochrome c oxidase (COX); and increased the expression of glucose transporter-1 (Glut-1) in muscle showing increased glucose metabolism by aerobic respiration. There was also an increase in mitochondrial biogenesis in skeletal muscle observed by increased mRNA expressions of mitochondrial biogenesis markers which was further confirmed by electron microscopy. Moreover, nitric oxide production increased in skeletal muscle in cobalt supplemented rats, which seems to be the major reason for peroxisome proliferator activated receptor-gamma coactivator-1α (PGC-1α) induction and mitochondrial biogenesis. Thus, in conclusion, we state that hypoxia preconditioning by CoCl{sub 2} supplementation in rats increases mitochondrial biogenesis, glucose uptake and metabolism by aerobic respiration in skeletal muscle, which leads to increased physical performance. The significance of this study lies in understanding the molecular mechanism of hypoxia adaptation and improvement of work performance in normal as well as extreme conditions like hypoxia via hypoxia preconditioning. -- Highlights: ► We supplemented rats with CoCl{sub 2} for 15 days along with training.

  20. Melanogenesis inhibits respiration in B16-F10 melanoma cells whereas enhances mitochondrial cell content

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meira, Willian Vanderlei; Heinrich, Tassiele Andréa; Cadena, Silvia Maria Suter Correia

    Melanoma is a rare and aggressive skin tumor; the survival of patients diagnosed late is fairly low. This high mortality rate is due to the characteristics of the cells that allow them to be resistant to radiotherapy and conventional chemotherapy, besides of being able to evade the immune system. Melanin, the pigment responsible for skin, hair and eye color, seems to be involved in this resistance. The main function of melanin is to protect the cells against ultraviolet (UV) light by absorbing this radiation and reactive oxygen species (ROS) scavenging. But this pigment may have also a role as photosensitizer,more » because when it is irradiated with UVA light (320-400 nm), the generation of ROS was detected. Besides, the melanogenesis stimulation on B16-F10 cells resulted in cell cycle arrest, induction of a quiescent state, change in the expression of several proteins and alterations on ADP/ATP ratio. The present study aimed to investigate the influence of melanogenesis stimulation in mitochondrial function of B16-F10 melanoma cells. Therefore, we analyzed cells respiration, mitochondrial membrane potential (Δψ{sub m}) and mitochondria mass in B16-F10 melanoma cells stimulated with 0.4 mM L-tyrosine and 10 mM NH{sub 4}Cl. Our results showed that the induction of melanin synthesis was able to reduce significantly the oxygen consumption after 48 h of stimulation, without changes of mitochondrial membrane potential when compared to non-stimulated cells. Despite of respiration inhibition, the mitochondria mass was higher in cells with melanogenesis stimulation. We suggest that the stimulation in the melanin synthesis might be promoting the inhibition of electrons transport chain by some intermediate compound from the synthesis of the pigment and this effect could contribute to explain the entry in the quiescent state. - Highlights: • Melanoma pigmentation alters mitochondrial respiration. • Induction of melanin synthesis by 48 h do not change mitochondrial

  1. Glycolysis-respiration relationships in a neuroblastoma cell line.

    PubMed

    Swerdlow, Russell H; E, Lezi; Aires, Daniel; Lu, Jianghua

    2013-04-01

    Although some reciprocal glycolysis-respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized. We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions. Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased. These data document a novel intracellular glycolysis-respiration effect in which restricting glycolysis flux increases mitochondrial respiration. Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis-respiration effect can practically inform the development of new mitochondrial medicine approaches. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-Mediated Inhibition of Mitochondrial Respiration

    PubMed Central

    Banks, Courtney J.; Rodriguez, Nathan W.; Gashler, Kyle R.; Pandya, Rushika R.; Mortenson, Jeffrey B.; Whited, Matthew D.; Soderblom, Erik J.; Thompson, J. Will; Moseley, M. Arthur; Reddi, Amit R.; Tessem, Jeffery S.; Torres, Matthew P.; Bikman, Benjamin T.

    2017-01-01

    ABSTRACT In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival. PMID:28739857

  3. Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum.

    PubMed

    Weger, H G; Birch, D G; Elrifi, I R; Turpin, D H

    1988-03-01

    Mass spectrometric analysis of O(2) and CO(2) exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH(4)Cl resulted in nearly a 250% increase in the rate of TCA cycle CO(2) efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O(2) consumption in both light and dark. Anaerobiosis inhibited the CO(2) release caused by NH(4)Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O(2) consumption in the light. This implied that the Mehler reaction did not play a significant role in O(2) consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions.

  4. Septic Shock Alters Mitochondrial Respiration of Lymphoid Cell-Lines and Human Peripheral Blood Mononuclear Cells: The Role of Plasma.

    PubMed

    Clere-Jehl, Raphael; Helms, Julie; Kassem, Mohamad; Le Borgne, Pierrick; Delabranche, Xavier; Charles, Anne-Laure; Geny, Bernard; Meziani, Ferhat; Bilbault, Pascal

    2018-02-14

    In septic shock patients, post-septic immunosuppression state following the systemic inflammatory response syndrome is responsible for nosocomial infections, with subsequent increased mortality. The aim of the present study was to assess the underlying cellular mechanisms of the post-septic immunosuppression state, by investigating mitochondrial functions of peripheral blood mononuclear cells (PBMCs) from septic shock patients over 7 days. Eighteen patients admitted to a French intensive care unit for septic shock were included. At days 1 and 7, PBMCs were isolated by Ficoll density gradient centrifugation. Mitochondrial respiration of intact septic PBMCs was assessed versus control group PBMCs, by measuring O2 consumption in plasma, using high-resolution respirometry. Mitochondrial respiration was then compared between septic plasmas and control plasmas for control PBMCs, septic PBMCs and lymphoid cell-line (CEM). To investigate the role of plasma, we measured several plasma cytokines, among them HMGB1, by ELISA. Basal O2 consumption of septic shock PBMCs was of 8.27 ± 3.39 and 10.48 ± 3.99 pmol/s/10 cells at days 1 and 7 respectively, significantly higher than in control PBMCs (5.37 ± 1.46 pmol/s/10 cells, p < 0.05). Septic patient PBMCs showed a lower response to oligomycin, suggesting a reduced ATP-synthase activity, as well as an increased response to FCCP suggesting an increased mitochondrial respiratory capacity. At 6 hours, septic plasmas showed a decreased O2 consumption of CEM (4.73 ± 1.46 vs. 6.58 ± 1.53, p < 0.05) as well as in control group PBMCs (1.76 ± 0.36 vs. 2.70 ± 0.42, p < 0.05), and triggered a decreased ATP-synthase activity but an increased response to FCCP. These differences are not explained by different cell survival. High HMGB1 levels were significantly associated with reduced PBMCs mitochondrial respiration. Septic plasma impairs mitochondrial respiration in immune cells, with a

  5. Initial brain aging: heterogeneity of mitochondrial size is associated with decline in complex I-linked respiration in cortex and hippocampus.

    PubMed

    Thomsen, Kirsten; Yokota, Takashi; Hasan-Olive, Md Mahdi; Sherazi, Niloofar; Fakouri, Nima Borhan; Desler, Claus; Regnell, Christine Elisabeth; Larsen, Steen; Rasmussen, Lene Juel; Dela, Flemming; Bergersen, Linda Hildegard; Lauritzen, Martin

    2018-01-01

    Brain aging is accompanied by declining mitochondrial respiration. We hypothesized that mitochondrial morphology and dynamics would reflect this decline. Using hippocampus and frontal cortex of a segmental progeroid mouse model lacking Cockayne syndrome protein B (CSB m/m ) and C57Bl/6 (WT) controls and comparing young (2-5 months) to middle-aged mice (13-14 months), we found that complex I-linked state 3 respiration (CI) was reduced at middle age in CSB m/m hippocampus, but not in CSB m/m cortex or WT brain. In hippocampus of both genotypes, mitochondrial size heterogeneity increased with age. Notably, an inverse correlation between heterogeneity and CI was found in both genotypes, indicating that heterogeneity reflects mitochondrial dysfunction. The ratio between fission and fusion gene expression reflected age-related alterations in mitochondrial morphology but not heterogeneity. Mitochondrial DNA content was lower, and hypoxia-induced factor 1α mRNA was greater at both ages in CSB m/m compared to WT brain. Our findings show that decreased CI and increased mitochondrial size heterogeneity are highly associated and point to declining mitochondrial quality control as an initial event in brain aging. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Calcium-regulation of mitochondrial respiration maintains ATP homeostasis and requires ARALAR/AGC1-malate aspartate shuttle in intact cortical neurons.

    PubMed

    Llorente-Folch, Irene; Rueda, Carlos B; Amigo, Ignacio; del Arco, Araceli; Saheki, Takeyori; Pardo, Beatriz; Satrústegui, Jorgina

    2013-08-28

    Neuronal respiration is controlled by ATP demand and Ca2+ but the roles played by each are unknown, as any Ca2+ signal also impacts on ATP demand. Ca2+ can control mitochondrial function through Ca2+-regulated mitochondrial carriers, the aspartate-glutamate and ATP-Mg/Pi carriers, ARALAR/AGC1 and SCaMC-3, respectively, or in the matrix after Ca2+ transport through the Ca2+ uniporter. We have studied the role of Ca2+ signaling in the regulation of mitochondrial respiration in intact mouse cortical neurons in basal conditions and in response to increased workload caused by increases in [Na+]cyt (veratridine, high-K+ depolarization) and/or [Ca2+]cyt (carbachol). Respiration in nonstimulated neurons on 2.5-5 mm glucose depends on ARALAR-malate aspartate shuttle (MAS), with a 46% drop in aralar KO neurons. All stimulation conditions induced increased OCR (oxygen consumption rate) in the presence of Ca2+, which was prevented by BAPTA-AM loading (to preserve the workload), or in Ca2+-free medium (which also lowers cell workload). SCaMC-3 limits respiration only in response to high workloads and robust Ca2+ signals. In every condition tested Ca2+ activation of ARALAR-MAS was required to fully stimulate coupled respiration by promoting pyruvate entry into mitochondria. In aralar KO neurons, respiration was stimulated by veratridine, but not by KCl or carbachol, indicating that the Ca2+ uniporter pathway played a role in the first, but not in the second condition, even though KCl caused an increase in [Ca2+]mit. The results suggest a requirement for ARALAR-MAS in priming pyruvate entry in mitochondria as a step needed to activate respiration by Ca2+ in response to moderate workloads.

  7. Measurement of Mitochondrial Respiration and Motility in Acute Care: Sepsis, Trauma, and Poisoning.

    PubMed

    Jang, David H; Greenwood, John C; Spyres, Meghan B; Eckmann, David M

    2017-01-01

    Metabolic biomarkers have potentially wider use in disease diagnosis and prognosis as well as in monitoring disease response to treatment. While biomarkers such as interleukins, microRNA, and lactate have been proposed for disease surveillance, there are still conflicting results regarding their clinical utility. Treatment of commonly encountered disease of acute care such as sepsis, trauma, and poisoning often relies on clinical diagnosis and therapy guided by use of surrogate markers of illness severity. The measurement of mitochondrial function, including respiration and motility, may offer superior alternatives to such markers. Assessing mitochondrial function in a clinical context has the potential to impact the area of acute care in terms of diagnosis, prognosis, and treatment. The study of mitochondrial bioenergetics has become critical in understanding the pathophysiology and treatment of complex diseases such as diabetes and cardiovascular disorders. © The Author(s) 2016.

  8. Glycolysis and Mitochondrial Respiration in Mouse LDHC-Null Sperm1

    PubMed Central

    Odet, Fanny; Gabel, Scott; London, Robert E.; Goldberg, Erwin; Eddy, Edward M.

    2013-01-01

    ABSTRACT We demonstrated previously that a knockout (KO) of the lactate dehydrogenase type C (Ldhc) gene disrupted male fertility and caused a considerable reduction in sperm glucose consumption, ATP production, and motility. While that study used mice with a mixed genetic background, the present study used C57BL/6 (B6) and 129S6 (129) Ldhc KO mice. We found that B6 KO males were subfertile and 129 KO males were infertile. Sperm from 129 wild-type (WT) mice have a lower glycolytic rate than sperm from B6 WT mice, resulting in a greater reduction in ATP production in 129 KO sperm than in B6 KO sperm. The lower glycolytic rate in 129 sperm offered a novel opportunity to examine the role of mitochondrial respiration in sperm ATP production and motility. We observed that in media containing a mitochondrial substrate (pyruvate or lactate) as the sole energy source, ATP levels and progressive motility in 129 KO sperm were similar to those in 129 WT sperm. However, when glucose was added, lactate was unable to maintain ATP levels or progressive motility in 129 KO sperm. The rate of respiration (ZO2) was high when 129 KO or WT sperm were incubated with lactate alone, but addition of glucose caused a reduction in ZO2. These results indicate that in the absence of glucose, 129 sperm can produce ATP via oxidative phosphorylation, but in the presence of glucose, oxidative phosphorylation is suppressed and the sperm utilize aerobic glycolysis, a phenomenon known as the Crabtree effect. PMID:23486916

  9. Redox-optimized ROS balance and the relationship between mitochondrial respiration and ROS.

    PubMed

    Cortassa, Sonia; O'Rourke, Brian; Aon, Miguel A

    2014-02-01

    The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO2) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~400 to ~900mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~500 to ~300mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO2, the ROS dependence on RE was strong between ~550 and ~350mV·mM, when VO2 is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO2 is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO2 and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Growth and mitochondrial respiration of mungbeans (Phaseolus aureus Roxb.) germinated at low pressure

    NASA Technical Reports Server (NTRS)

    Musgrave, M. E.; Gerth, W. A.; Scheld, H. W.; Strain, B. R.

    1988-01-01

    Mungbean (Phaseolus aureus Roxb.) seedlings were grown hypobarically to assess the effects of low pressure (21-24 kilopascals) on growth and mitochondrial respiration. Control seedlings grown at ambient pressure (101 kilopascals) were provided amounts of O2 equivalent to those provided experimental seedlings at reduced pressure to factor out responses to O2 concentration and to total pressure. Respiration was assayed using washed mitochondria, and was found to respond only to O2 concentration. Regardless of total pressure, seedlings grown at 2 millimoles O2 per liter had higher state 3 respiration rates and decreased percentages of alternative respiration compared to ambient (8.4 millimoles O2 per liter) controls. In contrast, seedling growth responded to total pressure but not to O2 concentration. Seedlings were significantly larger when grown under low pressure. While low O2 (2 millimoles O2 per liter) diminished growth at ambient pressure, growth at low pressure in the same oxygen concentration was enhanced. Respiratory development and growth of mungbean seedlings under low pressure is unimpaired whether oxygen or air is used as the chamber gas, and further, low pressure can improve growth under conditions of poor aeration.

  11. Herba Cistanches stimulates cellular glutathione redox cycling by reactive oxygen species generated from mitochondrial respiration in H9c2 cardiomyocytes.

    PubMed

    Wong, Hoi Shan; Ko, Kam Ming

    2013-01-01

    Earlier findings demonstrated that pretreatment of Herba Cistanches [the dried whole plant of Cistanche deserticola Y.C. Ma (Orobanchaceae)], a "Yang-invigorating" Chinese tonic herb, stimulated the ATP-generation capacity (ATP-GC) in mitochondria isolated from rat heart ex vivo. The enhancement of mitochondrial ATP-GC by Herba Cistanches was associated with induction of glutathione antioxidant status and protection against ischemia/reperfusion (I/R) injury in rat hearts. This study investigated the relationship between enhancements in mitochondrial ATP-GC and glutathione antioxidant status in H9c2 cardiomyocytes using a semipurified fraction of Herba Cistanches (HCF1). HCF1 (10-300 ng/mL) was tested for its effects on mitochondrial ATP generation, glutathione antioxidant status and protection against oxidant injury in H9c2 cardiomyocytes and rat hearts. HCF1 at 30 ng/mL increased mitochondrial ATP-GC and ADP-stimulated state 3 respiration (by 50 and 100%, respectively) in H9c2 cardiomyocytes. The stimulation of mitochondrial respiration was associated with the induction of mitochondrial uncoupling (27%) and enhancement of cellular glutathione redox cycling as well as protection against hypoxia/reoxygenation (hypox/reoxy)-induced apoptosis (by 60%). While HCF1 treatment increased reactive oxygen species generation from mitochondrial respiration in H9c2 cardiomyocytes, pretreatment with antioxidants (DMTU) abrogated the HCF1-induced cellular responses and the associated cytoprotective effect. HCF1 pretreatment (1.14 and 3.41 mg/kg × 14) also protected against myocardial I/R injury in rats (by 13 and 32%), presumably mediated by the induction of glutathione antioxidant response. The long-term intake of HCF1 may offer a prospect for the prevention of ischemic heart disease.

  12. A somatic T15091C mutation in the Cytb gene of mouse mitochondrial DNA dominantly induces respiration defects.

    PubMed

    Hayashi, Chisato; Takibuchi, Gaku; Shimizu, Akinori; Mito, Takayuki; Ishikawa, Kaori; Nakada, Kazuto; Hayashi, Jun-Ichi

    2015-08-07

    Our previous studies provided evidence that mammalian mitochondrial DNA (mtDNA) mutations that cause mitochondrial respiration defects behave in a recessive manner, because the induction of respiration defects could be prevented with the help of a small proportion (10%-20%) of mtDNA without the mutations. However, subsequent studies found the induction of respiration defects by the accelerated accumulation of a small proportion of mtDNA with various somatic mutations, indicating the presence of mtDNA mutations that behave in a dominant manner. Here, to provide the evidence for the presence of dominant mutations in mtDNA, we used mouse lung carcinoma P29 cells and examined whether some mtDNA molecules possess somatic mutations that dominantly induce respiration defects. Cloning and sequence analysis of 40-48 mtDNA molecules from P29 cells was carried out to screen for somatic mutations in protein-coding genes, because mutations in these genes could dominantly regulate respiration defects by formation of abnormal polypeptides. We found 108 missense mutations existing in one or more of 40-48 mtDNA molecules. Of these missense mutations, a T15091C mutation in the Cytb gene was expected to be pathogenic due to the presence of its orthologous mutation in mtDNA from a patient with cardiomyopathy. After isolation of many subclones from parental P29 cells, we obtained subclones with various proportions of T15091C mtDNA, and showed that the respiration defects were induced in a subclone with only 49% T15091C mtDNA. Because the induction of respiration defects could not be prevented with the help of the remaining 51% mtDNA without the T15091C mutation, the results indicate that the T15091C mutation in mtDNA dominantly induced the respiration defects. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. TGF-β1-Mediated Differentiation of Fibroblasts Is Associated with Increased Mitochondrial Content and Cellular Respiration

    PubMed Central

    Negmadjanov, Ulugbek; Godic, Zarko; Rizvi, Farhan; Emelyanova, Larisa; Ross, Gracious; Richards, John; Holmuhamedov, Ekhson L.; Jahangir, Arshad

    2015-01-01

    Objectivs Cytokine-dependent activation of fibroblasts to myofibroblasts, a key event in fibrosis, is accompanied by phenotypic changes with increased secretory and contractile properties dependent on increased energy utilization, yet changes in the energetic profile of these cells are not fully described. We hypothesize that the TGF-β1-mediated transformation of myofibroblasts is associated with an increase in mitochondrial content and function when compared to naive fibroblasts. Methods Cultured NIH/3T3 mouse fibroblasts treated with TGF-β1, a profibrotic cytokine, or vehicle were assessed for transformation to myofibroblasts (appearance of α-smooth muscle actin [α-SMA] stress fibers) and associated changes in mitochondrial content and functions using laser confocal microscopy, Seahorse respirometry, multi-well plate reader and biochemical protocols. Expression of mitochondrial-specific proteins was determined using western blotting, and the mitochondrial DNA quantified using Mitochondrial DNA isolation kit. Results Treatment with TGF-β1 (5 ng/mL) induced transformation of naive fibroblasts into myofibroblasts with a threefold increase in the expression of α-SMA (6.85 ± 0.27 RU) compared to cells not treated with TGF-β1 (2.52 ± 0.11 RU). TGF-β1 exposure increased the number of mitochondria in the cells, as monitored by membrane potential sensitive dye tetramethylrhodamine, and expression of mitochondria-specific proteins; voltage-dependent anion channels (0.54 ± 0.05 vs. 0.23 ± 0.05 RU) and adenine nucleotide transporter (0.61 ± 0.11 vs. 0.22 ± 0.05 RU), as well as mitochondrial DNA content (530 ± 12 μg DNA/106 cells vs. 307 ± 9 μg DNA/106 cells in control). TGF-β1 treatment was associated with an increase in mitochondrial function with a twofold increase in baseline oxygen consumption rate (2.25 ± 0.03 vs. 1.13 ± 0.1 nmol O2/min/106 cells) and FCCP-induced mitochondrial respiration (2.87 ± 0.03 vs. 1.46 ± 0.15 nmol O2/min/106 cells

  14. Lower Intrinsic ADP-Stimulated Mitochondrial Respiration Underlies In Vivo Mitochondrial Dysfunction in Muscle of Male Type 2 Diabetic Patients

    PubMed Central

    Phielix, Esther; Schrauwen-Hinderling, Vera B.; Mensink, Marco; Lenaers, Ellen; Meex, Ruth; Hoeks, Joris; Kooi, Marianne Eline; Moonen-Kornips, Esther; Sels, Jean-Pierre; Hesselink, Matthijs K.C.; Schrauwen, Patrick

    2008-01-01

    OBJECTIVE—A lower in vivo mitochondrial function has been reported in both type 2 diabetic patients and first-degree relatives of type 2 diabetic patients. The nature of this reduction is unknown. Here, we tested the hypothesis that a lower intrinsic mitochondrial respiratory capacity may underlie lower in vivo mitochondrial function observed in diabetic patients. RESEARCH DESIGN AND METHODS—Ten overweight diabetic patients, 12 first-degree relatives, and 16 control subjects, all men, matched for age and BMI, participated in this study. Insulin sensitivity was measured with a hyperinsulinemic-euglycemic clamp. Ex vivo intrinsic mitochondrial respiratory capacity was determined in permeabilized skinned muscle fibers using high-resolution respirometry and normalized for mitochondrial content. In vivo mitochondrial function was determined by measuring phosphocreatine recovery half-time after exercise using 31P-magnetic resonance spectroscopy. RESULTS—Insulin-stimulated glucose disposal was lower in diabetic patients compared with control subjects (11.2 ± 2.8 vs. 28.9 ± 3.7 μmol · kg−1 fat-free mass · min−1, respectively; P = 0.003), with intermediate values for first-degree relatives (22.1 ± 3.4 μmol · kg−1 fat-free mass · min−1). In vivo mitochondrial function was 25% lower in diabetic patients (P = 0.034) and 23% lower in first-degree relatives, but the latter did not reach statistical significance (P = 0.08). Interestingly, ADP-stimulated basal respiration was 35% lower in diabetic patients (P = 0.031), and fluoro-carbonyl cyanide phenylhydrazone–driven maximal mitochondrial respiratory capacity was 31% lower in diabetic patients (P = 0.05) compared with control subjects with intermediate values for first-degree relatives. CONCLUSIONS—A reduced basal ADP-stimulated and maximal mitochondrial respiratory capacity underlies the reduction in in vivo mitochondrial function, independent of mitochondrial content. A reduced capacity at both the

  15. Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration.

    PubMed

    Zampol, Mariana A; Busso, Cleverson; Gomes, Fernando; Ferreira-Junior, Jose Ribamar; Tzagoloff, Alexander; Barros, Mario H

    2010-11-05

    COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q(2). Rescue of respiration by Q(2) is a characteristic of mutants blocked in coenzyme Q(6) synthesis. Unlike Q(6) deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q(6). The physiological significance of earlier observations that purified Coq10p contains bound Q(6) was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q(2). This suggests that in vivo binding of Q(6) by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. [State of mitochondrial respiration and calcium capacity in livers of rats with different resistance to hypoxia after injections of L-arginine].

    PubMed

    Kurhaliuk, N M

    2001-01-01

    In experiments on rats with different resistance to hypoxia are investigated processes of mitochondrial respiration, oxidative phosphorylation and calcium capacity in liver under precursor nitric oxide L-arginine (600 mg/kg) and blockator nitric oxide synthase L-NNA (35 mg/kg) injections. We are used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate, 1 mM alpha-ketoglutarate and 2 mM malonic acid. Increasing of ADP-stimulation respiration states under exogenous L-arginine injection, decreasing efficacy of respiration processes (respiration control on Chance and ADP/O) under such substrates oxidation, testify to oxide energy support decreasing and reversing nitric oxide inhibit in such conditions. This will be used as mechanism cell regulation succinate dehydrogenase activity. It has shown that L-arginine injection increase calcium mitochondrial capacity low resistance to hypoxia rats using substrates of oxidation succinate and alpha-ketoglutarate to control meanings of high resistance rats. Effects of nitric oxide precursor influence on this processes limit NO-synthase inhibitor L-NNA.

  17. Secondary metabolites of the grapevine pathogen Eutypa lata inhibit mitochondrial respiration, based on a model bioassay using the yeast Saccharomyces cerevisiae.

    PubMed

    Kim, Jong H; Mahoney, Noreen; Chan, Kathleen L; Molyneux, Russell J; Campbell, Bruce C

    2004-10-01

    Acetylenic phenols and a chromene isolated from the grapevine fungal pathogen Eutypa lata were examined for mode of toxicity. The compounds included eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl aldehyde), eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), eulatachromene, 2- isoprenyl-5-formyl-benzofuran, siccayne, and eulatinol. A bioassay using the yeast Saccharomyces cerevisiae showed that all compounds were either lethal or inhibited growth. A respiratory assay using 2,3,5-triphenyltetrazolium (TTC) indicated that eutypinol and eulatachromene inhibited mitochondrial respiration in wild-type yeast. Bioassays also showed that 2- isoprenyl-5-formyl-benzofuran and siccayne inhibited mitochondrial respiration in the S. cerevisiae deletion mutant vph2Delta, lacking a vacuolar type H (+) ATPase (V-ATPase) assembly protein. Cell growth of tsa1Delta, a deletion mutant of S. cerevisiae lacking a thioredoxin peroxidase (cTPx I), was greatly reduced when grown on media containing eutypinol or eulatachromene and exposed to hydrogen peroxide (H(2)O(2)) as an oxidative stress. This reduction in growth establishes the toxic mode of action of these compounds through inhibition of mitochondrial respiration.

  18. Decreased hydrogen peroxide production and mitochondrial respiration in skeletal muscle but not cardiac muscle of the green-striped burrowing frog, a natural model of muscle disuse.

    PubMed

    Reilly, Beau D; Hickey, Anthony J R; Cramp, Rebecca L; Franklin, Craig E

    2014-04-01

    Suppression of disuse-induced muscle atrophy has been associated with altered mitochondrial reactive oxygen species (ROS) production in mammals. However, despite extended hindlimb immobility, aestivating animals exhibit little skeletal muscle atrophy compared with artificially immobilised mammalian models. Therefore, we studied mitochondrial respiration and ROS (H2O2) production in permeabilised muscle fibres of the green-striped burrowing frog, Cyclorana alboguttata. Mitochondrial respiration within saponin-permeabilised skeletal and cardiac muscle fibres was measured concurrently with ROS production using high-resolution respirometry coupled to custom-made fluorometers. After 4 months of aestivation, C. alboguttata had significantly depressed whole-body metabolism by ~70% relative to control (active) frogs, and mitochondrial respiration in saponin-permeabilised skeletal muscle fibres decreased by almost 50% both in the absence of ADP and during oxidative phosphorylation. Mitochondrial ROS production showed up to an 88% depression in aestivating skeletal muscle when malate, succinate and pyruvate were present at concentrations likely to reflect those in vivo. The percentage ROS released per O2 molecule consumed was also ~94% less at these concentrations, indicating an intrinsic difference in ROS production capacities during aestivation. We also examined mitochondrial respiration and ROS production in permeabilised cardiac muscle fibres and found that aestivating frogs maintained respiratory flux and ROS production at control levels. These results show that aestivating C. alboguttata has the capacity to independently regulate mitochondrial function in skeletal and cardiac muscles. Furthermore, this work indicates that ROS production can be suppressed in the disused skeletal muscle of aestivating frogs, which may in turn protect against potential oxidative damage and preserve skeletal muscle structure during aestivation and following arousal.

  19. Adaptive aneuploidy protects against thiol peroxidase deficiency by increasing respiration via key mitochondrial proteins.

    PubMed

    Kaya, Alaattin; Gerashchenko, Maxim V; Seim, Inge; Labarre, Jean; Toledano, Michel B; Gladyshev, Vadim N

    2015-08-25

    Aerobic respiration is a fundamental energy-generating process; however, there is cost associated with living in an oxygen-rich environment, because partially reduced oxygen species can damage cellular components. Organisms evolved enzymes that alleviate this damage and protect the intracellular milieu, most notably thiol peroxidases, which are abundant and conserved enzymes that mediate hydrogen peroxide signaling and act as the first line of defense against oxidants in nearly all living organisms. Deletion of all eight thiol peroxidase genes in yeast (∆8 strain) is not lethal, but results in slow growth and a high mutation rate. Here we characterized mechanisms that allow yeast cells to survive under conditions of thiol peroxidase deficiency. Two independent ∆8 strains increased mitochondrial content, altered mitochondrial distribution, and became dependent on respiration for growth but they were not hypersensitive to H2O2. In addition, both strains independently acquired a second copy of chromosome XI and increased expression of genes encoded by it. Survival of ∆8 cells was dependent on mitochondrial cytochrome-c peroxidase (CCP1) and UTH1, present on chromosome XI. Coexpression of these genes in ∆8 cells led to the elimination of the extra copy of chromosome XI and improved cell growth, whereas deletion of either gene was lethal. Thus, thiol peroxidase deficiency requires dosage compensation of CCP1 and UTH1 via chromosome XI aneuploidy, wherein these proteins support hydroperoxide removal with the reducing equivalents generated by the electron transport chain. To our knowledge, this is the first evidence of adaptive aneuploidy counteracting oxidative stress.

  20. Mitochondrial peptides modulate mitochondrial function during cellular senescence.

    PubMed

    Kim, Su-Jeong; Mehta, Hemal H; Wan, Junxiang; Kuehnemann, Chisaka; Chen, Jingcheng; Hu, Ji-Fan; Hoffman, Andrew R; Cohen, Pinchas

    2018-06-10

    Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

  1. Allotopic expression of a mitochondrial alternative oxidase confers cyanide resistance to human cell respiration

    PubMed Central

    Hakkaart, Gerrit A J; Dassa, Emmanuel P; Jacobs, Howard T; Rustin, Pierre

    2006-01-01

    Human mitochondrial respiration is distinct from that of most plants, microorganisms and even some metazoans in that it reduces molecular oxygen only through the highly cyanide-sensitive enzyme cytochrome c oxidase. Here we show that expression of the cyanide-insensitive alternative oxidase (AOX), recently identified in the ascidian Ciona intestinalis, is well tolerated by cultured human cells and confers spectacular cyanide resistance to mitochondrial substrate oxidation. The expressed AOX seems to be confined to mitochondria. AOX involvement in electron flow is triggered by a highly reduced redox status of the respiratory chain (RC) and enhanced by pyruvate; otherwise, the enzyme remains essentially inactive. AOX expression promises to be a valuable tool to limit the deleterious consequences of RC deficiency in human cells and whole animals. PMID:16322757

  2. Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome.

    PubMed

    Kumar, Maneesh G; Rowley, Shane; Fulton, Ruth; Dinday, Matthew T; Baraban, Scott C; Patel, Manisha

    2016-01-01

    Altered metabolism is an important feature of many epileptic syndromes but has not been reported in Dravet syndrome (DS), a catastrophic childhood epilepsy associated with mutations in a voltage-activated sodium channel, Nav1.1 (SCN1A). To address this, we developed novel methodology to assess real-time changes in bioenergetics in zebrafish larvae between 4 and 6 d postfertilization (dpf). Baseline and 4-aminopyridine (4-AP) stimulated glycolytic flux and mitochondrial respiration were simultaneously assessed using a Seahorse Biosciences extracellular flux analyzer. Scn1Lab mutant zebrafish showed a decrease in baseline glycolytic rate and oxygen consumption rate (OCR) compared to controls. A ketogenic diet formulation rescued mutant zebrafish metabolism to control levels. Increasing neuronal excitability with 4-AP resulted in an immediate increase in glycolytic rates in wild-type zebrafish, whereas mitochondrial OCR increased slightly and quickly recovered to baseline values. In contrast, scn1Lab mutant zebrafish showed a significantly slower and exaggerated increase of both glycolytic rates and OCR after 4-AP. The underlying mechanism of decreased baseline OCR in scn1Lab mutants was not because of altered mitochondrial DNA content or dysfunction of enzymes in the electron transport chain or tricarboxylic acid cycle. Examination of glucose metabolism using a PCR array identified five glycolytic genes that were downregulated in scn1Lab mutant zebrafish. Our findings in scn1Lab mutant zebrafish suggest that glucose and mitochondrial hypometabolism contribute to the pathophysiology of DS.

  3. Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F₀-ATP synthase

    PubMed Central

    Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

    2013-01-01

    We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53+/+ cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria. PMID:23966169

  4. Temperature and cell-type dependency of sulfide effects on mitochondrial respiration.

    PubMed

    Groeger, Michael; Matallo, Jose; McCook, Oscar; Wagner, Florian; Wachter, Ulrich; Bastian, Olga; Gierer, Saskia; Reich, Vera; Stahl, Bettina; Huber-Lang, Markus; Szabó, Csaba; Georgieff, Michael; Radermacher, Peter; Calzia, Enrico; Wagner, Katja

    2012-10-01

    Previous studies suggest that sulfide-induced inhibition of cytochrome c oxidase (cCox) and, consequently, the metabolic and toxic effects of sulfide are less pronounced at low body temperature. Because the temperature-dependent effects of sulfide on the inflammatory response are still a matter of debate, we investigated the impact of varying temperature on the cCox excess capacity and the mitochondrial sulfide oxidation by the sulfide-ubiquinone oxidoreductase in macrophage-derived cell lines (AMJ2-C11 and RAW 264.7). Using an oxygraph chamber, the inhibition of mitochondrial respiration was measured by stepwise titrations with sulfide and the nonmetabolizable cCox inhibitor sodium azide at 25°C and 37°C. Using the latter of the two inhibitors, the excess capacity of the cCox was obtained. Furthermore, we quantified the capacity of these cells to withstand sulfide inhibition by measuring the amount required to inhibit respiration by 50% and 90% and the viability of the cells after 24-h exposure to 100 ppm of hydrogen sulfide. At low titration rates, the AMJ2-C11 cells, but not the RAW 264.7 cells, increased their capacity to withstand exogenously added sulfide. This effect was even greater at 25°C than at 37°C. Furthermore, only the AMJ2-C11 cells remained viable after sulfide exposure for 24 h. In contrast, only in the RAW 264.7 cells that an increase in cCox excess capacity was found at low temperatures. In macrophage-derived cell lines, both the excess capacity of cCox and the efficiency of sulfide elimination may increase at low temperatures. These properties may modify the effects of sulfide in immune cells and, potentially, the inflammatory response during sulfide exposure at different body temperatures.

  5. Does Short-term Litter Input Manipulation Affect Soil Respiration and the Carbon-isotopic Signature of Soil Respired CO2

    NASA Astrophysics Data System (ADS)

    Cheng, X.; Wu, J.

    2016-12-01

    Global change greatly alters the quality and quantity of plant litter inputs to soils, and further impacts soil organic matter (SOM) dynamics and soil respiration. However, the process-based understanding of how soil respiration may change with future shift in litter input is not fully understood. The Detritus Input and Removal Treatment (DIRT) experiment was conducted in coniferous forest (Platycladus orientalis (Linn.) Franco) ecosystem of central China to investigate the impact of above- and belowground litter input on soil respiration and the carbon-isotopic signature of soil respired CO2. Short-term (1-2 years) litter input manipulation significantly affected soil respiration, based on annual flux values, soil respiration was 31.9%, 20.5% and 37.2% lower in no litter (NL), no root (NR) and no input (NRNL), respectively, compared to control (CK). Whereas double litter (DL) treatment increased soil respiration by 9.1% compared to CK. The recalcitrance index of carbon (RIC) and the relative abundance of fungi increased under litter removal or root exclusion treatment (NL, NR and NRNL) compared to CK. Basal soil respiration was positively related to liable C and microbial biomass and negatively related to RIC and fungi to bacteria (F: B) ratio. The carbon-isotopic signature of soil respired CO2 enriched under litter removal and no input treatment, and slightly depleted under litter addition treatment compared to CK. Our results suggest that short-term litter input manipulation can affect the soil respiration by altering substrate availability and microbial community structure, and also impact the carbon-isotopic signature of soil respired CO2 possibly duo to change in the component of soil respiration and soil microclimate.

  6. Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

    PubMed

    Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

    2017-03-06

    Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used

  7. Mitochondrial Energy and Redox Signaling in Plants

    PubMed Central

    Schwarzländer, Markus

    2013-01-01

    Abstract Significance: For a plant to grow and develop, energy and appropriate building blocks are a fundamental requirement. Mitochondrial respiration is a vital source for both. The delicate redox processes that make up respiration are affected by the plant's changing environment. Therefore, mitochondrial regulation is critically important to maintain cellular homeostasis. This involves sensing signals from changes in mitochondrial physiology, transducing this information, and mounting tailored responses, by either adjusting mitochondrial and cellular functions directly or reprogramming gene expression. Recent Advances: Retrograde (RTG) signaling, by which mitochondrial signals control nuclear gene expression, has been a field of very active research in recent years. Nevertheless, no mitochondrial RTG-signaling pathway is yet understood in plants. This review summarizes recent advances toward elucidating redox processes and other bioenergetic factors as a part of RTG signaling of plant mitochondria. Critical Issues: Novel insights into mitochondrial physiology and redox-regulation provide a framework of upstream signaling. On the other end, downstream responses to modified mitochondrial function have become available, including transcriptomic data and mitochondrial phenotypes, revealing processes in the plant that are under mitochondrial control. Future Directions: Drawing parallels to chloroplast signaling and mitochondrial signaling in animal systems allows to bridge gaps in the current understanding and to deduce promising directions for future research. It is proposed that targeted usage of new technical approaches, such as quantitative in vivo imaging, will provide novel leverage to the dissection of plant mitochondrial signaling. Antioxid. Redox Signal. 18, 2122–2144. PMID:23234467

  8. Mitochondrial p53 mediates a transcription-independent regulation of cell respiration and interacts with the mitochondrial F₁F0-ATP synthase.

    PubMed

    Bergeaud, Marie; Mathieu, Lise; Guillaume, Arnaud; Moll, Ute M; Mignotte, Bernard; Le Floch, Nathalie; Vayssière, Jean-Luc; Rincheval, Vincent

    2013-09-01

    We and others previously reported that endogenous p53 can be located at mitochondria in the absence of stress, suggesting that p53 has a role in the normal physiology of this organelle. The aim of this study was to characterize in unstressed cells the intramitochondrial localization of p53 and identify new partners and functions of p53 in mitochondria. We find that the intramitochondrial pool of p53 is located in the intermembrane space and the matrix. Of note, unstressed HCT116 p53(+/+) cells simultaneously show increased O₂ consumption and decreased mitochondrial superoxide production compared with their p53-null counterpart. This data was confirmed by stable H1299 cell lines expressing low levels of p53 specifically targeted to the matrix. Using immunoprecipitation and mass spectrometry, we identified the oligomycin sensitivity-conferring protein (OSCP), a subunit of the F₁F₀-ATP synthase complex, as a new partner of endogenous p53, specifically interacting with p53 localized in the matrix. Interestingly, this interaction seems implicated in mitochondrial p53 localization. Moreover, p53 localized in the matrix promotes the assembly of F₁F₀-ATP synthase. Taking into account that deregulations of mitochondrial respiration and reactive oxygen species production are tightly linked to cancer development, we suggest that mitochondrial p53 may be an important regulator of normal mitochondrial and cellular physiology, potentially exerting tumor suppression activity inside mitochondria.

  9. High Fat Diet-Induced Changes in Mouse Muscle Mitochondrial Phospholipids Do Not Impair Mitochondrial Respiration Despite Insulin Resistance

    PubMed Central

    Hulshof, Martijn F. M.; van den Berg, Sjoerd A. A.; Schaart, Gert; van Dijk, Ko Willems; Smit, Egbert; Mariman, Edwin C. M.

    2011-01-01

    Background Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance. Methodology C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR. Principal Findings At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks. Conclusions/Interpretation Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in

  10. DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

    PubMed Central

    LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

    2012-01-01

    Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

  11. Mitochondrial threshold effects.

    PubMed Central

    Rossignol, Rodrigue; Faustin, Benjamin; Rocher, Christophe; Malgat, Monique; Mazat, Jean-Pierre; Letellier, Thierry

    2003-01-01

    The study of mitochondrial diseases has revealed dramatic variability in the phenotypic presentation of mitochondrial genetic defects. To attempt to understand this variability, different authors have studied energy metabolism in transmitochondrial cell lines carrying different proportions of various pathogenic mutations in their mitochondrial DNA. The same kinds of experiments have been performed on isolated mitochondria and on tissue biopsies taken from patients with mitochondrial diseases. The results have shown that, in most cases, phenotypic manifestation of the genetic defect occurs only when a threshold level is exceeded, and this phenomenon has been named the 'phenotypic threshold effect'. Subsequently, several authors showed that it was possible to inhibit considerably the activity of a respiratory chain complex, up to a critical value, without affecting the rate of mitochondrial respiration or ATP synthesis. This phenomenon was called the 'biochemical threshold effect'. More recently, quantitative analysis of the effects of various mutations in mitochondrial DNA on the rate of mitochondrial protein synthesis has revealed the existence of a 'translational threshold effect'. In this review these different mitochondrial threshold effects are discussed, along with their molecular bases and the roles that they play in the presentation of mitochondrial diseases. PMID:12467494

  12. Structures and mechanisms of antitumor agents: xestoquinones uncouple cellular respiration and disrupt HIF signaling in human breast tumor cells.

    PubMed

    Du, Lin; Mahdi, Fakhri; Datta, Sandipan; Jekabsons, Mika B; Zhou, Yu-Dong; Nagle, Dale G

    2012-09-28

    The organic extract of a marine sponge, Petrosia alfiani, selectively inhibited iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in a human breast tumor T47D cell-based reporter assay. Bioassay-guided fractionation yielded seven xestoquinones (1-7) including three new compounds: 14-hydroxymethylxestoquinone (1), 15-hydroxymethylxestoquinone (2), and 14,15-dihydroxestoquinone (3). Compounds 1-7 were evaluated for their effects on HIF-1 signaling, mitochondrial respiration, and tumor cell proliferation/viability. The known metabolites adociaquinones A (5) and B (6), which possess a 3,4-dihydro-2H-1,4-thiazine-1,1-dioxide moiety, potently and selectively inhibited iron chelator-induced HIF-1 activation in T47D cells, each with an IC(50) value of 0.2 μM. Mechanistic studies revealed that adociaquinones promote oxygen consumption without affecting mitochondrial membrane potential. Compound 1 both enhances respiration and decreases mitochondrial membrane potential, suggesting that it acts as a protonophore that uncouples mitochondrial respiration.

  13. Acute exercise alters skeletal muscle mitochondrial respiration and H2O2 emission in response to hyperinsulinemic-euglycemic clamp in middle-aged obese men

    PubMed Central

    Trewin, Adam J.; Levinger, Itamar; Parker, Lewan; Shaw, Christopher S.; Serpiello, Fabio R.; Anderson, Mitchell J.; McConell, Glenn K.; Hare, David L.

    2017-01-01

    Obesity, sedentary lifestyle and aging are associated with mitochondrial dysfunction and impaired insulin sensitivity. Acute exercise increases insulin sensitivity in skeletal muscle; however, whether mitochondria are involved in these processes remains unclear. The aim of this study was to investigate the effects of insulin stimulation at rest and after acute exercise on skeletal muscle mitochondrial respiratory function (JO2) and hydrogen peroxide emission (JH2O2), and the associations with insulin sensitivity in obese, sedentary men. Nine men (means ± SD: 57 ± 6 years; BMI 33 ± 5 kg.m2) underwent hyperinsulinemic-euglycemic clamps in two separate trials 1–3 weeks apart: one under resting conditions, and another 1 hour after high-intensity exercise (4x4 min cycling at 95% HRpeak). Muscle biopsies were obtained at baseline, and pre/post clamp to measure JO2 with high-resolution respirometry and JH2O2 via Amplex UltraRed from permeabilized fibers. Post-exercise, both JO2 and JH2O2 during ADP stimulated state-3/OXPHOS respiration were lower compared to baseline (P<0.05), but not after subsequent insulin stimulation. JH2O2 was lower post-exercise and after subsequent insulin stimulation compared to insulin stimulation in the rest trial during succinate supported state-4/leak respiration (P<0.05). In contrast, JH2O2 increased during complex-I supported leak respiration with insulin after exercise compared with resting conditions (P<0.05). Resting insulin sensitivity and JH2O2 during complex-I leak respiration were positively correlated (r = 0.77, P<0.05). We conclude that in obese, older and sedentary men, acute exercise modifies skeletal muscle mitochondrial respiration and H2O2 emission responses to hyperinsulinemia in a respiratory state-specific manner, which may have implications for metabolic diseases involving insulin resistance. PMID:29161316

  14. The MEF2 gene is essential for yeast longevity, with a dual role in cell respiration and maintenance of mitochondrial membrane potential.

    PubMed

    Callegari, Sylvie; McKinnon, Ross A; Andrews, Stuart; de Barros Lopes, Miguel A

    2011-04-20

    The Saccharomyces cerevisiae MEF2 gene is a mitochondrial protein translation factor. Formerly believed to catalyze peptide elongation, evidence now suggests its involvement in ribosome recycling. This study confirms the role of the MEF2 gene for cell respiration and further uncovers a slow growth phenotype and reduced chronological lifespan. Furthermore, in comparison with cytoplasmic ρ(0) strains, mef2Δ strains have a marked reduction of the inner mitochondrial membrane potential and mitochondria show a tendency to aggregate, suggesting an additional role for the MEF2 gene in maintenance of mitochondrial health, a role that may also be shared by other mitochondrial protein synthesis factors. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  15. Nutrient-gene interactions determine mitochondrial function: effect of dietary fat.

    PubMed

    Kim, M J; Berdanier, C D

    1998-02-01

    The effect on mitochondrial respiration of feeding hydrogenated coconut oil, corn oil, or menhaden oil (MO) to diabetes-prone BHE/cdb rats and normal Sprague Dawley (SD) rats was studied. Both fat source and strain affected the temperature dependence of succinate-supported respiration. The transition temperature was greater in BHE/cdb rats than in the SD rats. The efficiency of ATP synthesis as reflected by the ADP:O ratio was decreased in the BHE/cdb rats compared to SD rats, with the exception of the comparison made at 37 degrees C with the MO-fed rats; at this temperature, the ADP:O ratios were similar. The diet and strain differences suggest a dietary lipid-gene interaction with respect to the mobility of subunit 6 of the F1F0ATPase. This subunit has two errors in its gene: one that affects the proton channel and another that likely affects its mobility within the inner mitochondrial membrane.

  16. Enhanced Heme Function and Mitochondrial Respiration Promote the Progression of Lung Cancer Cells

    PubMed Central

    Alam, Md Maksudul; Shah, Ajit; Cao, Thai M.; Sullivan, Laura A.; Brekken, Rolf; Zhang, Li

    2013-01-01

    Lung cancer is the leading cause of cancer-related mortality, and about 85% of the cases are non-small-cell lung cancer (NSCLC). Importantly, recent advance in cancer research suggests that altering cancer cell bioenergetics can provide an effective way to target such advanced cancer cells that have acquired mutations in multiple cellular regulators. This study aims to identify bioenergetic alterations in lung cancer cells by directly measuring and comparing key metabolic activities in a pair of cell lines representing normal and NSCLC cells developed from the same patient. We found that the rates of oxygen consumption and heme biosynthesis were intensified in NSCLC cells. Additionally, the NSCLC cells exhibited substantially increased levels in an array of proteins promoting heme synthesis, uptake and function. These proteins include the rate-limiting heme biosynthetic enzyme ALAS, transporter proteins HRG1 and HCP1 that are involved in heme uptake, and various types of oxygen-utilizing hemoproteins such as cytoglobin and cytochromes. Several types of human tumor xenografts also displayed increased levels of such proteins. Furthermore, we found that lowering heme biosynthesis and uptake, like lowering mitochondrial respiration, effectively reduced oxygen consumption, cancer cell proliferation, migration and colony formation. In contrast, lowering heme degradation does not have an effect on lung cancer cells. These results show that increased heme flux and function are a key feature of NSCLC cells. Further, increased generation and supply of heme and oxygen-utilizing hemoproteins in cancer cells will lead to intensified oxygen consumption and cellular energy production by mitochondrial respiration, which would fuel cancer cell proliferation and progression. The results show that inhibiting heme and respiratory function can effectively arrest the progression of lung cancer cells. Hence, understanding heme function can positively impact on research in lung cancer

  17. Effect of endogenous nitric oxide on mitochondrial respiration of rat hepatocytes in vitro and in vivo

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stadler, J.; Curran, R.D.; Ochoa, J.B.

    1991-02-01

    Nitric oxide, a highly reactive radical, was recently identified as an intermediate of L-arginine metabolism in mammalian cells. We have shown that nitric oxide synthesis is induced in vitro in cultured hepatocytes by supernatants from activated Kupffer cells or in vivo by injecting rats with nonviable Corynebacterium parvum. In both cases, nitric oxide biosynthesis in hepatocytes was associated with suppression of total protein synthesis. This study attempts to determine the effect of nitric oxide biosynthesis on the activity of specific hepatocytic mitochondrial enzymes and to determine whether inhibition of protein synthesis is caused by suppression of energy metabolism. Exposure ofmore » hepatocytes to supernatants from activated Kupffer cells led to a 30% decrease of aconitase (Krebs cycle) and complex I (mitochondrial electron transport chain) activity. Using NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis, we demonstrated that the inhibition of mitochondrial aconitase activity was due, in part, to the action of nitric oxide. In contrast, in vivo nitric oxide synthesis of hepatocytes from Corynebacterium parvum-treated animals had no effect on mitochondrial respiration. This suggests that inhibition of protein synthesis by nitric oxide is not likely to be mediated by inhibition of energy metabolism.« less

  18. Targeting mitochondria by Zn(II)N-alkylpyridylporphyrins: the impact of compound sub-mitochondrial partition on cell respiration and overall photodynamic efficacy.

    PubMed

    Odeh, Ahmad M; Craik, James D; Ezzeddine, Rima; Tovmasyan, Artak; Batinic-Haberle, Ines; Benov, Ludmil T

    2014-01-01

    Mitochondria play a key role in aerobic ATP production and redox control. They harness crucial metabolic pathways and control cell death mechanisms, properties that make these organelles essential for survival of most eukaryotic cells. Cancer cells have altered cell death pathways and typically show a shift towards anaerobic glycolysis for energy production, factors which point to mitochondria as potential culprits in cancer development. Targeting mitochondria is an attractive approach to tumor control, but design of pharmaceutical agents based on rational approaches is still not well established. The aim of this study was to investigate which structural features of specially designed Zn(II)N-alkylpyridylporphyrins would direct them to mitochondria and to particular mitochondrial targets. Since Zn(II)N-alkylpyridylporphyrins can act as highly efficient photosensitizers, their localization can be confirmed by photodamage to particular mitochondrial components. Using cultured LS174T adenocarcinoma cells, we found that subcellular distribution of Zn-porphyrins is directed by the nature of the substituents attached to the meso pyridyl nitrogens at the porphyrin ring. Increasing the length of the aliphatic chain from one carbon (methyl) to six carbons (hexyl) increased mitochondrial uptake of the compounds. Such modifications also affected sub-mitochondrial distribution of the Zn-porphyrins. The amphiphilic hexyl derivative (ZnTnHex-2-PyP) localized in the vicinity of cytochrome c oxidase complex, causing its inactivation during illumination. Photoinactivation of critical cellular targets explains the superior efficiency of the hexyl derivative in causing mitochondrial photodamage, and suppressing cellular respiration and survival. Design of potent photosensitizers and redox-active scavengers of free radicals should take into consideration not only selective organelle uptake and localization, but also selective targeting of critical macromolecular structures.

  19. Hybrid respiration in the denitrifying mitochondria of Fusarium oxysporum.

    PubMed

    Takaya, Naoki; Kuwazaki, Seigo; Adachi, Yoshiaki; Suzuki, Sawako; Kikuchi, Tomoko; Nakamura, Hiro; Shiro, Yoshitsugu; Shoun, Hirofumi

    2003-04-01

    Induction of the mitochondrial nitrate-respiration (denitrification) system of the fungus Fusarium oxysporum requires the supply of low levels of oxygen (O(2)). Here we show that O(2) and nitrate (NO(3)(-)) respiration function simultaneously in the mitochondria of fungal cells incubated under hypoxic, denitrifying conditions in which both O(2) and NO(3)(-) act as the terminal electron acceptors. The NO(3)(-) and nitrite (NO(2)(-)) reductases involved in fungal denitrification share the mitochondrial respiratory chain with cytochrome oxidase. F. oxysporum cytochrome c(549) can serve as an electron donor for both NO(2)(-) reductase and cytochrome oxidase. We are the first to demonstrate hybrid respiration in respiring eukaryotic mitochondria.

  20. Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase.

    PubMed

    Monge, Claire; Beraud, Nathalie; Kuznetsov, Andrey V; Rostovtseva, Tatiana; Sackett, Dan; Schlattner, Uwe; Vendelin, Marko; Saks, Valdur A

    2008-11-01

    The role of ubiquitous mitochondrial creatine kinase (uMtCK) reaction in regulation of mitochondrial respiration was studied in purified preparations of rat brain synaptosomes and mitochondria. In permeabilized synaptosomes, apparent Km for exogenous ADP, Km (ADP), in regulation of respiration in situ was rather high (110 +/- 11 microM) in comparison with isolated brain mitochondria (9 +/- 1 microM). This apparent Km for ADP observed in isolated mitochondria in vitro dramatically increased to 169 +/- 52 microM after their incubation with 1 muM of dimeric tubulin showing that in rat brain, particularly in synaptosomes, mitochondrial outer membrane permeability for ADP, and ATP may be restricted by tubulin binding to voltage dependent anion channel (VDAC). On the other hand, in synaptosomes apparent Km (ADP) decreased to 25 +/- 1 microM in the presence of 20 mM creatine. To fully understand this effect of creatine on kinetics of respiration regulation, complete kinetic analysis of uMtCK reaction in isolated brain mitochondria was carried out. This showed that oxidative phosphorylation specifically altered only the dissociation constants for MgATP, by decreasing that from ternary complex MtCK.Cr.MgATP (K (a)) from 0.13 +/- 0.02 to 0.018 +/- 0.007 mM and that from binary complex MtCK.MgATP (K (ia)) from 1.1 +/- 0.29 mM to 0.17 +/- 0.07 mM. Apparent decrease of dissociation constants for MgATP reflects effective cycling of ATP and ADP between uMtCK and adenine nucleotide translocase (ANT). These results emphasize important role and various pathophysiological implications of the phosphocreatine-creatine kinase system in energy transfer in brain cells, including synaptosomes.

  1. Mitochondrial remodeling in the liver following chronic alcohol feeding to rats.

    PubMed

    Han, Derick; Johnson, Heather S; Rao, Madhuri P; Martin, Gary; Sancheti, Harsh; Silkwood, Kai H; Decker, Carl W; Nguyen, Kim Tho; Casian, Joseph G; Cadenas, Enrique; Kaplowitz, Neil

    2017-01-01

    The feeding of alcohol orally (Lieber-DeCarli diet) to rats has been shown to cause declines in mitochondrial respiration (state III), decreased expression of respiratory complexes, and decreased respiratory control ratios (RCR) in liver mitochondria. These declines and other mitochondrial alterations have led to the hypothesis that alcohol feeding causes "mitochondrial dysfunction" in the liver. If oral alcohol feeding leads to mitochondrial dysfunction, one would predict that increasing alcohol delivery by intragastric (IG) alcohol feeding to rats would cause greater declines in mitochondrial bioenergetics in the liver. In this study, we examined the mitochondrial alterations that occur in rats fed alcohol both orally and intragastrically. Oral alcohol feeding decreased glutamate/malate-, acetaldehyde- and succinate-driven state III respiration, RCR, and expression of respiratory complexes (I, III, IV, V) in liver mitochondria, in agreement with previous results. IG alcohol feeding, on the other hand, caused a slight increase in glutamate/malate-driven respiration, and significantly increased acetaldehyde-driven respiration in liver mitochondria. IG feeding also caused liver mitochondria to experience a decline in succinate-driven respiration, but these decreases were smaller than those observed with oral alcohol feeding. Surprisingly, oral and IG alcohol feeding to rats increased mitochondrial respiration using other substrates, including glycerol-3-phosphate (which delivers electrons from cytoplasmic NADH to mitochondria) and octanoate (a substrate for beta-oxidation). The enhancement of glycerol-3-phosphate- and octanoate-driven respiration suggests that liver mitochondria remodeled in response to alcohol feeding. In support of this notion, we observed that IG alcohol feeding also increased expression of mitochondrial glycerol phosphate dehydrogenase-2 (GPD2), transcription factor A (TFAM), and increased mitochondrial NAD + -NADH and NADP + -NADPH levels in

  2. Evolutionary implications of mitochondrial genetic variation: mitochondrial genetic effects on OXPHOS respiration and mitochondrial quantity change with age and sex in fruit flies.

    PubMed

    Wolff, J N; Pichaud, N; Camus, M F; Côté, G; Blier, P U; Dowling, D K

    2016-04-01

    The ancient acquisition of the mitochondrion into the ancestor of modern-day eukaryotes is thought to have been pivotal in facilitating the evolution of complex life. Mitochondria retain their own diminutive genome, with mitochondrial genes encoding core subunits involved in oxidative phosphorylation. Traditionally, it was assumed that there was little scope for genetic variation to accumulate and be maintained within the mitochondrial genome. However, in the past decade, mitochondrial genetic variation has been routinely tied to the expression of life-history traits such as fertility, development and longevity. To examine whether these broad-scale effects on life-history trait expression might ultimately find their root in mitochondrially mediated effects on core bioenergetic function, we measured the effects of genetic variation across twelve different mitochondrial haplotypes on respiratory capacity and mitochondrial quantity in the fruit fly, Drosophila melanogaster. We used strains of flies that differed only in their mitochondrial haplotype, and tested each sex separately at two different adult ages. Mitochondrial haplotypes affected both respiratory capacity and mitochondrial quantity. However, these effects were highly context-dependent, with the genetic effects contingent on both the sex and the age of the flies. These sex- and age-specific genetic effects are likely to resonate across the entire organismal life-history, providing insights into how mitochondrial genetic variation may contribute to sex-specific trajectories of life-history evolution. © 2016 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2016 European Society For Evolutionary Biology.

  3. In yeast redistribution of Sod1 to the mitochondrial intermembrane space provides protection against respiration derived oxidative stress.

    PubMed

    Klöppel, Christine; Michels, Christine; Zimmer, Julia; Herrmann, Johannes M; Riemer, Jan

    2010-12-03

    The antioxidative enzyme copper-zinc superoxide dismutase (Sod1) is an important cellular defence system against reactive oxygen species (ROS). While the majority of this enzyme is localized to the cytosol, about 1% of the cellular Sod1 is present in the intermembrane space (IMS) of mitochondria. These amounts of mitochondrial Sod1 are increased for certain Sod1 mutants that are linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). To date, only little is known about the physiological function of mitochondrial Sod1. Here, we use the model system Saccharomyces cerevisiae to generate cells in which Sod1 is exclusively localized to the IMS. We find that IMS-localized Sod1 can functionally substitute wild type Sod1 and that it even exceeds the protective capacity of wild type Sod1 under conditions of mitochondrial ROS stress. Moreover, we demonstrate that upon expression in yeast cells the common ALS-linked mutant Sod1(G93A) becomes enriched in the mitochondrial fraction and provides an increased protection of cells from mitochondrial oxidative stress. Such an effect cannot be observed for the catalytically inactive mutant Sod1(G85R). Our observations suggest that the targeting of Sod1 to the mitochondrial IMS provides an increased protection against respiration-derived ROS. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Molecular chaperone TRAP1 regulates a metabolic switch between mitochondrial respiration and aerobic glycolysis

    PubMed Central

    Yoshida, Soichiro; Tsutsumi, Shinji; Muhlebach, Guillaume; Sourbier, Carole; Lee, Min-Jung; Lee, Sunmin; Vartholomaiou, Evangelia; Tatokoro, Manabu; Beebe, Kristin; Miyajima, Naoto; Mohney, Robert P.; Chen, Yang; Hasumi, Hisashi; Xu, Wanping; Fukushima, Hiroshi; Nakamura, Ken; Koga, Fumitaka; Kihara, Kazunori; Trepel, Jane; Picard, Didier; Neckers, Leonard

    2013-01-01

    TRAP1 (TNF receptor-associated protein), a member of the HSP90 chaperone family, is found predominantly in mitochondria. TRAP1 is broadly considered to be an anticancer molecular target. However, current inhibitors cannot distinguish between HSP90 and TRAP1, making their utility as probes of TRAP1-specific function questionable. Some cancers express less TRAP1 than do their normal tissue counterparts, suggesting that TRAP1 function in mitochondria of normal and transformed cells is more complex than previously appreciated. We have used TRAP1-null cells and transient TRAP1 silencing/overexpression to show that TRAP1 regulates a metabolic switch between oxidative phosphorylation and aerobic glycolysis in immortalized mouse fibroblasts and in human tumor cells. TRAP1-deficiency promotes an increase in mitochondrial respiration and fatty acid oxidation, and in cellular accumulation of tricarboxylic acid cycle intermediates, ATP and reactive oxygen species. At the same time, glucose metabolism is suppressed. TRAP1-deficient cells also display strikingly enhanced invasiveness. TRAP1 interaction with and regulation of mitochondrial c-Src provide a mechanistic basis for these phenotypes. Taken together with the observation that TRAP1 expression is inversely correlated with tumor grade in several cancers, these data suggest that, in some settings, this mitochondrial molecular chaperone may act as a tumor suppressor. PMID:23564345

  5. From the Cover: Arsenite Uncouples Mitochondrial Respiration and Induces a Warburg-like Effect in Caenorhabditis elegans

    PubMed Central

    Godebo, Tewodros R.; Bhatt, Dhaval P.; Ilkayeva, Olga R.; Maurer, Laura L.; Hirschey, Matthew D.; Meyer, Joel N.

    2016-01-01

    Millions of people worldwide are chronically exposed to arsenic through contaminated drinking water. Despite decades of research studying the carcinogenic potential of arsenic, the mechanisms by which arsenic causes cancer and other diseases remain poorly understood. Mitochondria appear to be an important target of arsenic toxicity. The trivalent arsenical, arsenite, can induce mitochondrial reactive oxygen species production, inhibit enzymes involved in energy metabolism, and induce aerobic glycolysis in vitro, suggesting that metabolic dysfunction may be important in arsenic-induced disease. Here, using the model organism Caenorhabditis elegans and a novel metabolic inhibition assay, we report an in vivo induction of aerobic glycolysis following arsenite exposure. Furthermore, arsenite exposure induced severe mitochondrial dysfunction, including altered pyruvate metabolism; reduced steady-state ATP levels, ATP-linked respiration and spare respiratory capacity; and increased proton leak. We also found evidence that induction of autophagy is an important protective response to arsenite exposure. Because these results demonstrate that mitochondria are an important in vivo target of arsenite toxicity, we hypothesized that deficiencies in mitochondrial electron transport chain genes, which cause mitochondrial disease in humans, would sensitize nematodes to arsenite. In agreement with this, nematodes deficient in electron transport chain complexes I, II, and III, but not ATP synthase, were sensitive to arsenite exposure, thus identifying a novel class of gene-environment interactions that warrant further investigation in the human populace. PMID:27208080

  6. Cardiac mitochondrial oxidative capacity is partly preserved after cryopreservation with dimethyl sulfoxide.

    PubMed

    Meyer, A; Charles, A L; Singh, F; Zoll, J; Talha, S; Enache, I; Chaarloux, A; Inser-Horobeti, M E; Geny, B

    2016-01-01

    Cardiac muscle cryopreservation is a challenge for both diagnostic procedure requiring viable tissues and therapeutic advance in regenerative medicine. Mitochondria are targets of both direct and indirect damages, secondary to congelation per se and/or to cryoprotectant's toxic effects, which participate to diminution of viability and/or functioning of cells after freezing. At the cardiac muscle level, only one study had investigated mitochondrial respiration after cryopreservation. To determine the effect of cryopreservation on mitochondrial respiration of cardiac muscle. We recorded mitochondrial respiration through complexes I, II, III and IV along with mitochondrial coupling in fresh and cryopreserved rat left ventricles samples and assessed difference of the means, correlation and agreement between the measures in all samples. Mitochondrial respiration was partly maintained up to 70% in cryopreserved samples whatever the substrate. A significant correlation was observed between fresh and cryopreserved samples (r = 0.71, p < 0.0001). However, mitochondrial coupling significantly decreased after cryopreservation (- 1.44 ± 0.15; p < 0.005) suggesting that mitochondrial intactness was not totally preserved by cryopreservation. Further, the fluctuations around the mean difference were wide (-14.06, +5.08 µmol/min/g), increasing with respiration rates (p < 0.0001). Thus, fresh samples extemporaneous analysis should be preferred when available despite the fact that cryopreservation using DMSO partly protect cardiac mitochondrial respiration and coupling. These data support the interest to further refine cryopreservation methods.

  7. Calcium Co-regulates Oxidative Metabolism and ATP Synthase-dependent Respiration in Pancreatic Beta Cells

    PubMed Central

    De Marchi, Umberto; Thevenet, Jonathan; Hermant, Aurelie; Dioum, Elhadji; Wiederkehr, Andreas

    2014-01-01

    Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)+ ratio. PMID:24554722

  8. Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction.

    PubMed

    Varela, Ana T; Gomes, Ana P; Simões, Anabela M; Teodoro, João S; Duarte, Filipe V; Rolo, Anabela P; Palmeira, Carlos M

    2008-12-01

    Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10microM, significantly depresses the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25microM stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.

  9. ARALAR/AGC1 deficiency, a neurodevelopmental disorder with severe impairment of neuronal mitochondrial respiration, does not produce a primary increase in brain lactate.

    PubMed

    Juaristi, Inés; García-Martín, María L; Rodrigues, Tiago B; Satrústegui, Jorgina; Llorente-Folch, Irene; Pardo, Beatriz

    2017-07-01

    ARALAR/AGC1 (aspartate-glutamate mitochondrial carrier 1) is an important component of the NADH malate-aspartate shuttle (MAS). AGC1-deficiency is a rare disease causing global cerebral hypomyelination, developmental arrest, hypotonia, and epilepsy (OMIM ID #612949); the aralar-KO mouse recapitulates the major findings in humans. This study was aimed at understanding the impact of ARALAR-deficiency in brain lactate levels as a biomarker. We report that lactate was equally abundant in wild-type and aralar-KO mouse brain in vivo at postnatal day 17. We find that lactate production upon mitochondrial blockade depends on up-regulation of lactate formation in astrocytes rather than in neurons. However, ARALAR-deficiency decreased cell respiration in neurons, not astrocytes, which maintained unchanged respiration and lactate production. As the primary site of ARALAR-deficiency is neuronal, this explains the lack of accumulation of brain lactate in ARALAR-deficiency in humans and mice. On the other hand, we find that the cytosolic and mitochondrial components of the glycerol phosphate shuttle are present in astrocytes with similar activities. This suggests that glycerol phosphate shuttle is the main NADH shuttle in astrocytes and explains the absence of effects of ARALAR-deficiency in these cells. © 2017 International Society for Neurochemistry.

  10. Methoxychlor causes mitochondrial dysfunction and oxidative damage in the mouse ovary

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gupta, R.K.; Schuh, R.A.; Department of Anesthesiology, University of Maryland, Baltimore, MD

    2006-11-01

    Methoxychlor (MXC) is an organochlorine pesticide that reduces fertility in female rodents by causing ovarian atrophy, persistent estrous cyclicity, and antral follicle atresia (apoptotic cell death). Oxidative damage resulting from reactive oxygen species (ROS) generation has been demonstrated to lead to toxicant-induced cell death. Thus, this work tested the hypothesis that MXC causes oxidative damage to the mouse ovary and affects mitochondrial respiration in a manner that stimulates ROS production. For the in vitro experiments, mitochondria were collected from adult cycling mouse ovaries, treated with vehicle (dimethyl sulfoxide; DMSO) or MXC, and subjected to polarographic measurements of respiration. For themore » in vivo experiments, adult cycling CD-1 mice were dosed with either vehicle (sesame oil) or MXC for 20 days. After treatment, ovarian mitochondria were isolated and subjected to measurements of respiration and fluorimetric measurements of H{sub 2}O{sub 2} production. Some ovaries were also fixed and processed for immunohistochemistry using antibodies for ROS production markers: nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHG). Ovaries from in vivo experiments were also used to measure the mRNA expression and activity of antioxidants such as Cu/Zn superoxide dismutase (SOD1), glutathione peroxidase (GPX), and catalase (CAT). The results indicate that MXC significantly impairs mitochondrial respiration, increases production of H{sub 2}O{sub 2}, causes more staining for nitrotyrosine and 8-OHG in antral follicles, and decreases the expression and activity of SOD1, GPX, and CAT as compared to controls. Collectively, these data indicate that MXC inhibits mitochondrial respiration, causes ROS production, and decreases antioxidant expression and activity in the ovary, specifically in the antral follicles. Therefore, it is possible that MXC causes atresia of ovarian antral follicles by inducing oxidative stress through mitochondrial production of ROS.« less

  11. A single nucleotide polymorphism in COQ9 affects mitochondrial and ovarian function and fertility in Holstein cows.

    PubMed

    Ortega, M Sofia; Wohlgemuth, Stephanie; Tribulo, Paula; Siqueira, Luiz G B; Cole, John B; Hansen, Peter J

    2017-03-01

    A single missense mutation at position 159 of coenzyme Q9 (COQ9) (G→A; rs109301586) has been associated with genetic variation in fertility in Holstein cattle, with the A allele associated with higher fertility. COQ9 is involved in the synthesis of coenzyme COQ10, a component of the electron transport system of the mitochondria. Here we tested whether reproductive phenotype is associated with the mutation and evaluated functional consequences for cellular oxygen metabolism, body weight changes, and ovarian function. The mutation in COQ9 modifies predicted tertiary protein structure and affected mitochondrial respiration of peripheral blood mononuclear cells. The A allele was associated with low resting oxygen consumption and high electron transport system capacity. Phenotypic measurements for fertility were evaluated for up to five lactations in a population of 2273 Holstein cows. There were additive effects of the mutation (P < 0.05) in favor of the A allele for pregnancy rate, interval from calving to conception, and services per conception. There was no association of genotype with milk production or body weight changes postpartum. The mutation in COQ9 affected ovarian function; the A allele was associated with increased mitochondrial DNA copy number in oocytes, and there were overdominance effects for COQ9 expression in oocytes, follicle number, and antimullerian hormone concentrations. Overall, results show how a gene involved in mitochondrial function is associated with overall fertility, possibly in part by affecting oocyte quality. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. The Ability of PAS, Acetylsalicylic Acid and Calcium Disodium EDTA to Protect Against the Toxic Effects of Manganese on Mitochondrial Respiration in Gill of Crassostrea virginica.

    PubMed

    Crawford, Sherine; Davis, Kiyya; Saddler, Claudette; Joseph, Jevaun; Catapane, Edward J; Carroll, Margaret A

    2011-01-01

    Manganese (Mn) is an essential metal that at excessive levels in brain causes Manganism, a condition similar to Parkinson's disease. Previously we showed that Mn had a neurotoxic effect on the dopaminergic, but not serotonergic, innervation of the lateral ciliated cells in the gill of the Eastern Oyster, Crassostrea virginica. While the mechanism of action of Mn toxicity is not completely understood, studies suggest that Mn toxicity may involve mitochondrial damage and resulting neural dysfunction in the brain's dopaminergic system. In this study we utilized micro-batch chambers and oxygen probes to measure oyster gill mitochondrial respiration in the presence of Mn and potential Mn blockers. The addition of Mn to respiring mitochondria caused a dose dependent decrease in mitochondrial O(2) consumption. Pretreating mitochondria with calcium disodium EDTA (caEDTA), p aminosalicylic acid (PAS) or acetylsalicylic acid (ASA) before Mn additions, provided full protection against the toxic effects of Mn. While mitochondrial pretreatment with any of the 3 drugs effectively blocked Mn toxicity, none of the drugs tested was able to reverse the decrease in mitochondrial O(2) consumption seen in Mn treated mitochondria. The study found that high levels of Mn had a toxic effect on gill mitochondrial O(2) consumption and that this effect could be blocked by the drugs caEDTA, PAS and ASA. C. virginica continues to be a good model with which to investigate the mechanism that underlies manganese neurotoxcity and in the pharmacological study of drugs to treat or prevent Manganism.

  13. The Ability of PAS, Acetylsalicylic Acid and Calcium Disodium EDTA to Protect Against the Toxic Effects of Manganese on Mitochondrial Respiration in Gill of Crassostrea virginica

    PubMed Central

    Crawford, Sherine; Davis, Kiyya; Saddler, Claudette; Joseph, Jevaun; Catapane, Edward J.; Carroll, Margaret A.

    2011-01-01

    Manganese (Mn) is an essential metal that at excessive levels in brain causes Manganism, a condition similar to Parkinson's disease. Previously we showed that Mn had a neurotoxic effect on the dopaminergic, but not serotonergic, innervation of the lateral ciliated cells in the gill of the Eastern Oyster, Crassostrea virginica. While the mechanism of action of Mn toxicity is not completely understood, studies suggest that Mn toxicity may involve mitochondrial damage and resulting neural dysfunction in the brain’s dopaminergic system. In this study we utilized micro-batch chambers and oxygen probes to measure oyster gill mitochondrial respiration in the presence of Mn and potential Mn blockers. The addition of Mn to respiring mitochondria caused a dose dependent decrease in mitochondrial O2 consumption. Pretreating mitochondria with calcium disodium EDTA (caEDTA), p aminosalicylic acid (PAS) or acetylsalicylic acid (ASA) before Mn additions, provided full protection against the toxic effects of Mn. While mitochondrial pretreatment with any of the 3 drugs effectively blocked Mn toxicity, none of the drugs tested was able to reverse the decrease in mitochondrial O2 consumption seen in Mn treated mitochondria. The study found that high levels of Mn had a toxic effect on gill mitochondrial O2 consumption and that this effect could be blocked by the drugs caEDTA, PAS and ASA. C. virginica continues to be a good model with which to investigate the mechanism that underlies manganese neurotoxcity and in the pharmacological study of drugs to treat or prevent Manganism. PMID:21977482

  14. Mitochondrial Metabolism in Aging Heart

    PubMed Central

    Lesnefsky, Edward J.; Chen, Qun; Hoppel, Charles L.

    2016-01-01

    Altered mitochondrial metabolism is the underlying basis for the increased sensitivity in the aged heart to stress. The aged heart exhibits impaired metabolic flexibility, with a decreased capacity to oxidize fatty acids and enhanced dependence on glucose metabolism. Aging impairs mitochondrial oxidative phosphorylation, with a greater role played by the mitochondria located between the myofibrils, the interfibrillar mitochondria. With aging, there is a decrease in activity of complexes III and IV, which account for the decrease in respiration. Furthermore, aging decreases mitochondrial content among the myofibrils. The end result is that in the interfibrillar area there is an approximate 50% decrease in mitochondrial function, affecting all substrates. The defective mitochondria persist in the aged heart, leading to enhanced oxidant production and oxidative injury and the activation of oxidant signaling for cell death. Aging defects in mitochondria represent new therapeutic targets, whether by manipulation of the mitochondrial proteome, modulation of electron transport, activation of biogenesis or mitophagy, or the regulation of mitochondrial fission and fusion. These mechanisms provide new ways to attenuate cardiac disease in elders by preemptive treatment of age-related defects, in contrast to the treatment of disease-induced dysfunction. PMID:27174952

  15. Human Cytomegalovirus Infection Upregulates the Mitochondrial Transcription and Translation Machineries

    PubMed Central

    Weekes, M. P.; Antrobus, R.; Rorbach, J.; van Haute, L.; Umrania, Y.; Smith, D. L.; Minczuk, M.; Lehner, P. J.; Sinclair, J. H.

    2016-01-01

    ABSTRACT Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication. PMID:27025248

  16. Indirubin-3'-oxime impairs mitochondrial oxidative phosphorylation and prevents mitochondrial permeability transition induction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Varela, Ana T.; Gomes, Ana P.; Simoes, Anabela M.

    2008-12-01

    Indirubin, a red colored 3,2'-bisindole isomer, is a component of Indigo naturalis and is an active ingredient used in traditional Chinese medicine for the treatment of chronic diseases. The family of indirubin derivatives, such as indirubin-3'-oxime, has been suggested for various therapeutic indications. However, potential toxic interactions such as indirubin effects on mitochondrial bioenergetics are still unknown. This study evaluated the action of indirubin-3'-oxime on the function of isolated rat liver mitochondria contributing to a better understanding of the biochemical mechanisms underlying the multiple effects of indirubin. Indirubin-3'-oxime incubated with isolated rat liver mitochondria, at concentrations above 10{mu}M, significantly depressesmore » the phosphorylation efficiency of mitochondria as inferred from the decrease in the respiratory control and ADP/O ratios, the perturbations in mitochondrial membrane potential and in the phosphorylative cycle induced by ADP. Furthermore, indirubin-3'-oxime at up to 25{mu}M stimulates the rate of state 4 respiration and inhibits state 3 respiration. The increased lag phase of repolarization was associated with a direct inhibition of the mitochondrial ATPase. Indirubin-3'-oxime significantly inhibited the activity of complex II and IV thus explaining the decreased FCCP-stimulated mitochondrial respiration. Mitochondria pre-incubated with indirubin-3'-oxime exhibits decreased susceptibility to calcium-induced mitochondrial permeability transition. This work shows for the first time multiple effects of indirubin-3'-oxime on mitochondrial bioenergetics thus indicating a potential mechanism for indirubin-3'-oxime effects on cell function.« less

  17. Mitochondrial respiration and ROS emission during β-oxidation in the heart: An experimental-computational study

    PubMed Central

    Sollott, Steven J.

    2017-01-01

    Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze β-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO2) and hydrogen peroxide (H2O2) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H2O2 emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of β-oxidation of long-chain fatty acids and main energy-redox processes is able to simulate the relationship between VO2 and H2O2 emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H2O2 emission as a function of VO2. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply. PMID:28598967

  18. Mitochondrial respiration and ROS emission during β-oxidation in the heart: An experimental-computational study.

    PubMed

    Cortassa, Sonia; Sollott, Steven J; Aon, Miguel A

    2017-06-01

    Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze β-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO2) and hydrogen peroxide (H2O2) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H2O2 emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of β-oxidation of long-chain fatty acids and main energy-redox processes is able to simulate the relationship between VO2 and H2O2 emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H2O2 emission as a function of VO2. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply.

  19. Acute Ethanol Causes Hepatic Mitochondrial Depolarization in Mice: Role of Ethanol Metabolism

    PubMed Central

    Zhong, Zhi; Ramshesh, Venkat K.; Rehman, Hasibur; Liu, Qinlong; Theruvath, Tom P.; Krishnasamy, Yasodha; Lemasters, John J.

    2014-01-01

    Background/Aims An increase of ethanol metabolism and hepatic mitochondrial respiration occurs in vivo after a single binge of alcohol. Here, our aim was to determine how ethanol intake affects hepatic mitochondrial polarization status in vivo in relation to ethanol metabolism and steatosis. Methods Hepatic mitochondrial polarization, permeability transition (MPT), and reduce pyridine nucleotides, and steatosis in mice were monitored by intravital confocal/multiphoton microscopy of the fluorescence of rhodamine 123 (Rh123), calcein, NAD(P)H, and BODIPY493/503, respectively, after gavage with ethanol (1–6 g/kg). Results Mitochondria depolarized in an all-or-nothing fashion in individual hepatocytes as early as 1 h after alcohol. Depolarization was dose- and time-dependent, peaked after 6 to 12 h and maximally affected 94% of hepatocytes. This mitochondrial depolarization was not due to onset of the MPT. After 24 h, mitochondria of most hepatocytes recovered normal polarization and were indistinguishable from untreated after 7 days. Cell death monitored by propidium iodide staining, histology and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was low throughout. After alcohol, mitochondrial NAD(P)H autofluorescence increased and decreased, respectively, in hepatocytes with polarized and depolarized mitochondria. Ethanol also caused steatosis mainly in hepatocytes with depolarized mitochondria. Depolarization was linked to ethanol metabolism, since deficiency of alcohol dehydrogenase and cytochrome-P450 2E1 (CYP2E1), the major ethanol-metabolizing enzymes, decreased mitochondrial depolarization by ∼70% and ∼20%, respectively. Activation of aldehyde dehydrogenase decreased depolarization, whereas inhibition of aldehyde dehydrogenase enhanced depolarization. Activation of aldehyde dehydrogenase also markedly decreased steatosis. Conclusions Acute ethanol causes reversible hepatic mitochondrial depolarization in vivo that may contribute to

  20. Mitochondrial respiration inhibition after exposure to UWB pulses as a possible mechanism of antitumor action

    NASA Astrophysics Data System (ADS)

    Zharkova, L.; Romanchenko, I.; Bolshakov, M.; Rostov, V.

    2017-05-01

    The respiration of isolated mice liver mitochondria after exposure to nanosecond UWB pulses (0.15 - 36 kV/cm, 0.6 - 1.0 GHz centre frequency, 3 - 20 ns pulse duration) has been investigated. The respiratory control (RC, the ratio of oxygen consumption) was estimated. The possibility of mitochondrial membrane electroporation was detected as the decrease in the electrical resistance, according to the β-dispersion of the electric current. The monotonous decrease of RC after 1000 UWB pulses from 0.15 kV/cm was observed, the ohmic resistance of mitochondria suspension was reduced. The obtained data indicate the inhibitory effect of UWB pulses on a state of irradiated mitochondria and its membrane.

  1. Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging.

    PubMed

    Lores-Arnaiz, Silvia; Lombardi, Paulina; Karadayian, Analía G; Orgambide, Federico; Cicerchia, Daniela; Bustamante, Juanita

    2016-02-01

    Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decreased by 26 and 33 % in synaptosomes from 17-months old mice, but spare respiratory capacity was not modified by aging. Succinate supported state 3 respiratory rate was decreased by 45 % in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animals, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non-synaptic mitochondria would not be affected by calcium overload. UCP-2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.

  2. The Responses of Tissues from the Brain, Heart, Kidney, and Liver to Resuscitation following Prolonged Cardiac Arrest by Examining Mitochondrial Respiration in Rats

    PubMed Central

    Kim, Junhwan; Perales Villarroel, José Paul; Zhang, Wei; Yin, Tai; Shinozaki, Koichiro; Hong, Angela; Lampe, Joshua W.; Becker, Lance B.

    2016-01-01

    Cardiac arrest induces whole-body ischemia, which causes damage to multiple organs. Understanding how each organ responds to ischemia/reperfusion is important to develop better resuscitation strategies. Because direct measurement of organ function is not practicable in most animal models, we attempt to use mitochondrial respiration to test efficacy of resuscitation on the brain, heart, kidney, and liver following prolonged cardiac arrest. Male Sprague-Dawley rats are subjected to asphyxia-induced cardiac arrest for 30 min or 45 min, or 30 min cardiac arrest followed by 60 min cardiopulmonary bypass resuscitation. Mitochondria are isolated from brain, heart, kidney, and liver tissues and examined for respiration activity. Following cardiac arrest, a time-dependent decrease in state-3 respiration is observed in mitochondria from all four tissues. Following 60 min resuscitation, the respiration activity of brain mitochondria varies greatly in different animals. The activity after resuscitation remains the same in heart mitochondria and significantly increases in kidney and liver mitochondria. The result shows that inhibition of state-3 respiration is a good marker to evaluate the efficacy of resuscitation for each organ. The resulting state-3 respiration of brain and heart mitochondria following resuscitation reenforces the need for developing better strategies to resuscitate these critical organs following prolonged cardiac arrest. PMID:26770657

  3. The Responses of Tissues from the Brain, Heart, Kidney, and Liver to Resuscitation following Prolonged Cardiac Arrest by Examining Mitochondrial Respiration in Rats.

    PubMed

    Kim, Junhwan; Villarroel, José Paul Perales; Zhang, Wei; Yin, Tai; Shinozaki, Koichiro; Hong, Angela; Lampe, Joshua W; Becker, Lance B

    2016-01-01

    Cardiac arrest induces whole-body ischemia, which causes damage to multiple organs. Understanding how each organ responds to ischemia/reperfusion is important to develop better resuscitation strategies. Because direct measurement of organ function is not practicable in most animal models, we attempt to use mitochondrial respiration to test efficacy of resuscitation on the brain, heart, kidney, and liver following prolonged cardiac arrest. Male Sprague-Dawley rats are subjected to asphyxia-induced cardiac arrest for 30 min or 45 min, or 30 min cardiac arrest followed by 60 min cardiopulmonary bypass resuscitation. Mitochondria are isolated from brain, heart, kidney, and liver tissues and examined for respiration activity. Following cardiac arrest, a time-dependent decrease in state-3 respiration is observed in mitochondria from all four tissues. Following 60 min resuscitation, the respiration activity of brain mitochondria varies greatly in different animals. The activity after resuscitation remains the same in heart mitochondria and significantly increases in kidney and liver mitochondria. The result shows that inhibition of state-3 respiration is a good marker to evaluate the efficacy of resuscitation for each organ. The resulting state-3 respiration of brain and heart mitochondria following resuscitation reenforces the need for developing better strategies to resuscitate these critical organs following prolonged cardiac arrest.

  4. Comparative Analysis of the Mitochondrial Physiology of Pancreatic β Cells

    PubMed Central

    Kim, Chul; Patel, Pinal; Gouvin, Lindsey M.; Brown, Melissa L.; Khalil, Ahmed; Henchey, Elizabeth M; Heuck, Alejandro P.; Yadava, Nagendra

    2014-01-01

    The mitochondrial metabolism of β cells is thought to be highly specialized. Its direct comparison with other cells using isolated mitochondria is limited by the availability of islets/β cells in sufficient quantity. In this study, we have compared mitochondrial metabolism of INS1E/β cells with other cells in intact and permeabilized states. To selectively permeabilize the plasma membrane, we have evaluated the use of perfringolysin-O (PFO) in conjunction with microplate-based respirometry. PFO is a protein that binds membranes based on a threshold level of active cholesterol. Therefore, unless active cholesterol reaches a threshold level in mitochondria, they are expected to remain untouched by PFO. Cytochrome c sensitivity tests showed that in PFO-permeabilized cells, the mitochondrial integrity was completely preserved. Our data show that a time-dependent decline of the oligomycin-insensitive respiration observed in INS1E cells was due to a limitation in substrate supply to the respiratory chain. We predict that it is linked with the β cell-specific metabolism involving metabolites shuttling between the cytoplasm and mitochondria. In permeabilized β cells, the Complex l-dependent respiration was either transient or absent because of the inefficient TCA cycle. The TCA cycle insufficiency was confirmed by analysis of the CO2 evolution. This may be linked with lower levels of NAD+, which is required as a co-factor for CO2 producing reactions of the TCA cycle. β cells showed comparable OxPhos and respiratory capacities that were not affected by the inorganic phosphate (Pi) levels in the respiration medium. They showed lower ADP-stimulation of the respiration on different substrates. We believe that this study will significantly enhance our understanding of the β cell mitochondrial metabolism. PMID:25309834

  5. Exercise training improves vascular mitochondrial function

    PubMed Central

    Park, Song-Young; Rossman, Matthew J.; Gifford, Jayson R.; Bharath, Leena P.; Bauersachs, Johann; Richardson, Russell S.; Abel, E. Dale; Symons, J. David

    2016-01-01

    Exercise training is recognized to improve cardiac and skeletal muscle mitochondrial respiratory capacity; however, the impact of chronic exercise on vascular mitochondrial respiratory function is unknown. We hypothesized that exercise training concomitantly increases both vascular mitochondrial respiratory capacity and vascular function. Arteries from both sedentary (SED) and swim-trained (EX, 5 wk) mice were compared in terms of mitochondrial respiratory function, mitochondrial content, markers of mitochondrial biogenesis, redox balance, nitric oxide (NO) signaling, and vessel function. Mitochondrial complex I and complex I + II state 3 respiration and the respiratory control ratio (complex I + II state 3 respiration/complex I state 2 respiration) were greater in vessels from EX relative to SED mice, despite similar levels of arterial citrate synthase activity and mitochondrial DNA content. Furthermore, compared with the SED mice, arteries from EX mice displayed elevated transcript levels of peroxisome proliferative activated receptor-γ coactivator-1α and the downstream targets cytochrome c oxidase subunit IV isoform 1, isocitrate dehydrogenase (Idh) 2, and Idh3a, increased manganese superoxide dismutase protein expression, increased endothelial NO synthase phosphorylation (Ser1177), and suppressed reactive oxygen species generation (all P < 0.05). Although there were no differences in EX and SED mice concerning endothelium-dependent and endothelium-independent vasorelaxation, phenylephrine-induced vasocontraction was blunted in vessels from EX compared with SED mice, and this effect was normalized by NOS inhibition. These training-induced increases in vascular mitochondrial respiratory capacity and evidence of improved redox balance, which may, at least in part, be attributable to elevated NO bioavailability, have the potential to protect against age- and disease-related challenges to arterial function. PMID:26825520

  6. Motor neuron mitochondrial dysfunction in spinal muscular atrophy

    PubMed Central

    Miller, Nimrod; Shi, Han; Zelikovich, Aaron S.; Ma, Yong-Chao

    2016-01-01

    Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, predominantly affects high metabolic tissues including motor neurons, skeletal muscles and the heart. Although the genetic cause of SMA has been identified, mechanisms underlying tissue-specific vulnerability are not well understood. To study these mechanisms, we carried out a deep sequencing analysis of the transcriptome of spinal motor neurons in an SMA mouse model, in which we unexpectedly found changes in many genes associated with mitochondrial bioenergetics. Importantly, functional measurement of mitochondrial activities showed decreased basal and maximal mitochondrial respiration in motor neurons from SMA mice. Using a reduction-oxidation sensitive GFP and fluorescence sensors specifically targeted to mitochondria, we found increased oxidative stress level and impaired mitochondrial membrane potential in motor neurons affected by SMA. In addition, mitochondrial mobility was impaired in SMA disease conditions, with decreased retrograde transport but no effect on anterograde transport. We also found significantly increased fragmentation of the mitochondrial network in primary motor neurons from SMA mice, with no change in mitochondria density. Electron microscopy study of SMA mouse spinal cord revealed mitochondria fragmentation, edema and concentric lamellar inclusions in motor neurons affected by the disease. Intriguingly, these functional and structural deficiencies in the SMA mouse model occur during the presymptomatic stage of disease, suggesting a role in initiating SMA. Altogether, our findings reveal a critical role for mitochondrial defects in SMA pathogenesis and suggest a novel target for improving tissue health in the disease. PMID:27488123

  7. Dengue virus induces mitochondrial elongation through impairment of Drp1-triggered mitochondrial fission

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Barbier, Vincent; Lang, Diane; Valois, Sierra

    Mitochondria are highly dynamic organelles that undergo continuous cycles of fission and fusion to maintain essential cellular functions. An imbalance between these two processes can result in many pathophysiological outcomes. Dengue virus (DENV) interacts with cellular organelles, including mitochondria, to successfully replicate in cells. This study used live-cell imaging and found an increase in mitochondrial length and respiration during DENV infection. The level of mitochondrial fission protein, Dynamin-related protein 1 (Drp1), was decreased on mitochondria during DENV infection, as well as Drp1 phosphorylated on serine 616, which is important for mitochondrial fission. DENV proteins NS4b and NS3 were also associatedmore » with subcellular fractions of mitochondria. Induction of fission through uncoupling of mitochondria or overexpression of Drp1 wild-type and Drp1 with a phosphomimetic mutation (S616D) significantly reduced viral replication. These results demonstrate that DENV infection causes an imbalance in mitochondrial dynamics by inhibiting Drp1-triggered mitochondrial fission, which promotes viral replication. - Highlights: •Mitochondrial length and respiration are increased during DENV infection. •DENV inhibits Drp1-triggered mitochondrial fission. •DENV titers are reduced by mitochondrial fragmentation, Drp1 WT and S616D expression. •Viral proteins NS4b and NS3 are associated with subcellular fractions of mitochondria.« less

  8. coq7/clk-1 regulates mitochondrial respiration and the generation of reactive oxygen species via coenzyme Q.

    PubMed

    Nakai, Daisuke; Shimizu, Takahiko; Nojiri, Hidetoshi; Uchiyama, Satoshi; Koike, Hideo; Takahashi, Mayumi; Hirokawa, Katsuiku; Shirasawa, Takuji

    2004-10-01

    coq7/clk-1 was isolated from a long-lived mutant of Caenorhabditis elegans, and shows sluggish behaviours and an extended lifespan. In C. elegans and Saccharomyces cerevisiae, coq7/clk-1 is required for the biosynthesis of coenzyme Q (CoQ), an essential co-factor in mitochondrial respiration. The clk-1 mutant contains dietary CoQ(8) from Escherichia coli and demethoxyubiquinone 9 (DMQ9) instead of CoQ(9). In a previous study, we generated COQ7-deficient mice by targeted disruption of the coq7 gene and reported that mouse coq7/clk-1 is also essential for CoQ synthesis, maintenance of mitochondrial integrity and neurogenesis. In the present study, we rescued COQ7-deficient mice from embryonic lethality and established a mouse model with decreased CoQ level by transgene expression of COQ7/CLK-1. A biochemical analysis showed a concomitant decrease in CoQ(9), mitochondrial respiratory enzyme activity and the generation of reactive oxygen species (ROS) in the mitochondria of CoQ-insufficient mice. This implied that the depressed activity of respiratory enzymes and the depressed production of ROS may play a physiological role in the control of lifespan in mammalian species and of C. elegans.

  9. Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

    PubMed

    Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

    2015-06-01

    Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

  10. Developmental plasticity of mitochondrial function in American alligators, Alligator mississippiensis

    PubMed Central

    Crossley, Janna; Elsey, Ruth M.; Dzialowski, Edward M.; Shiels, Holly A.; Crossley, Dane A.

    2016-01-01

    The effect of hypoxia on cellular metabolism is well documented in adult vertebrates, but information is entirely lacking for embryonic organisms. The effect of hypoxia on embryonic physiology is particularly interesting, as metabolic responses during development may have life-long consequences, due to developmental plasticity. To this end, we investigated the effects of chronic developmental hypoxia on cardiac mitochondrial function in embryonic and juvenile American alligators (Alligator mississippiensis). Alligator eggs were incubated in 21% or 10% oxygen from 20 to 90% of embryonic development. Embryos were either harvested at 90% development or allowed to hatch and then reared in 21% oxygen for 3 yr. Ventricular mitochondria were isolated from embryonic/juvenile alligator hearts. Mitochondrial respiration and enzymatic activities of electron transport chain complexes were measured with a microrespirometer and spectrophotometer, respectively. Developmental hypoxia induced growth restriction and increased relative heart mass, and this phenotype persisted into juvenile life. Embryonic mitochondrial function was not affected by developmental hypoxia, but at the juvenile life stage, animals from hypoxic incubations had lower levels of Leak respiration and higher respiratory control ratios, which is indicative of enhanced mitochondrial efficiency. Our results suggest developmental hypoxia can have life-long consequences for alligator morphology and metabolic function. Further investigations are necessary to reveal the adaptive significance of the enhanced mitochondrial efficiency in the hypoxic phenotype. PMID:27707718

  11. Mitochondrial Redox Dysfunction and Environmental Exposures

    PubMed Central

    Caito, Samuel W.

    2015-01-01

    Abstract Significance: Mitochondria are structurally and biochemically diverse, even within a single type of cell. Protein complexes localized to the inner mitochondrial membrane synthesize ATP by coupling electron transport and oxidative phosphorylation. The organelles produce reactive oxygen species (ROS) from mitochondrial oxygen and ROS can, in turn, alter the function and expression of proteins used for aerobic respiration by post-translational and transcriptional regulation. Recent Advances: New interest is emerging not only into the roles of mitochondria in disease development and progression but also as a target for environmental toxicants. Critical Issues: Dysregulation of respiration has been linked to cell death and is a major contributor to acute neuronal trauma, peripheral diseases, as well as chronic neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Future Directions: Here, we discuss the mechanisms underlying the sensitivity of the mitochondrial respiratory complexes to redox modulation, as well as examine the effects of environmental contaminants that have well-characterized mitochondrial toxicity. The contaminants discussed in this review are some of the most prevalent and potent environmental contaminants that have been linked to neurological dysfunction, altered cellular respiration, and oxidation. Antioxid. Redox Signal. 23, 578–595. PMID:25826672

  12. Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

    PubMed Central

    Le, Anne; Stine, Zachary E.; Nguyen, Christopher; Afzal, Junaid; Sun, Peng; Hamaker, Max; Siegel, Nicholas M.; Gouw, Arvin M.; Kang, Byung-hak; Yu, Shu-Han; Cochran, Rory L.; Sailor, Kurt A.; Song, Hongjun; Dang, Chi V.

    2014-01-01

    Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity. PMID:25114222

  13. Loss of Pink1 modulates synaptic mitochondrial bioenergetics in the rat striatum prior to motor symptoms: concomitant complex I respiratory defects and increased complex II-mediated respiration.

    PubMed

    Stauch, Kelly L; Villeneuve, Lance M; Purnell, Phillip R; Ottemann, Brendan M; Emanuel, Katy; Fox, Howard S

    2016-12-01

    Mutations in PTEN-induced putative kinase 1 (Pink1), a mitochondrial serine/threonine kinase, cause a recessive inherited form of Parkinson's disease (PD). Pink1 deletion in rats results in a progressive PD-like phenotype, characterized by significant motor deficits starting at 4 months of age. Despite the evidence of mitochondrial dysfunction, the pathogenic mechanism underlying disease due to Pink1-deficiency remains obscure. Striatal synaptic mitochondria from 3-month-old Pink1-deficient rats were characterized using bioenergetic and mass spectroscopy (MS)-based proteomic analyses. Striatal synaptic mitochondria from Pink1-deficient rats exhibit decreased complex I-driven respiration and increased complex II-mediated respiration compared with wild-type rats. MS-based proteomics revealed 69 of the 811 quantified mitochondrial proteins were differentially expressed between Pink1-deficient rats and controls. Down-regulation of several electron carrier proteins, which shuttle electrons to reduce ubiquinone at complex III, in the Pink1-knockouts suggests disruption of the linkage between fatty acid, amino acid, and choline metabolism and the mitochondrial respiratory system. These results suggest that complex II activity is increased to compensate for loss of electron transfer mechanisms due to reduced complex I activity and loss of electron carriers within striatal nerve terminals early during disease progression. This may contribute to the pathogenesis of PD. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Myoglobin and the regulation of mitochondrial respiratory chain complex IV.

    PubMed

    Yamada, Tatsuya; Takakura, Hisashi; Jue, Thomas; Hashimoto, Takeshi; Ishizawa, Rie; Furuichi, Yasuro; Kato, Yukio; Iwanaka, Nobumasa; Masuda, Kazumi

    2016-01-15

    Mitochondrial respiration is regulated by multiple elaborate mechanisms. It has been shown that muscle specific O2 binding protein, Myoglobin (Mb), is localized in mitochondria and interacts with respiratory chain complex IV, suggesting that Mb could be a factor that regulates mitochondrial respiration. Here, we demonstrate that muscle mitochondrial respiration is improved by Mb overexpression via up-regulation of complex IV activity in cultured myoblasts; in contrast, suppression of Mb expression induces a decrease in complex IV activity and mitochondrial respiration compared with the overexpression model. The present data are the first to show the biological significance of mitochondrial Mb as a potential modulator of mitochondrial respiratory capacity. Mitochondria are important organelles for metabolism, and their respiratory capacity is a primary factor in the regulation of energy expenditure. Deficiencies of cytochrome c oxidase complex IV, which reduces O2 in mitochondria, are linked to several diseases, such as mitochondrial myopathy. Moreover, mitochondrial respiration in skeletal muscle tissue tends to be susceptible to complex IV activity. Recently, we showed that the muscle-specific protein myoglobin (Mb) interacts with complex IV. The precise roles of mitochondrial Mb remain unclear. Here, we demonstrate that Mb facilitates mitochondrial respiratory capacity in skeletal muscles. Although mitochondrial DNA copy numbers were not altered in Mb-overexpressing myotubes, O2 consumption was greater in these myotubes than that in mock cells (Mock vs. Mb-Flag::GFP: state 4, 1.00 ± 0.09 vs. 1.77 ± 0.34; state 3, 1.00 ± 0.29; Mock: 1.60 ± 0.53; complex 2-3-4: 1.00 ± 0.30 vs. 1.50 ± 0.44; complex IV: 1.00 ± 0.14 vs. 1.87 ± 0.27). This improvement in respiratory capacity could be because of the activation of enzymatic activity of respiratory complexes. Moreover, mitochondrial respiration was up-regulated in myoblasts transiently

  15. Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways.

    PubMed

    Shen, Yao; Yang, Jianbo; Li, Juan; Shi, Xiaojie; Ouyang, Li; Tian, Yueyang; Lu, Jianxin

    2014-01-01

    Carnosine, a naturally occurring dipeptide, has been recently demonstrated to possess anti-tumor activity. However, its underlying mechanism is unclear. In this study, we investigated the effect and mechanism of carnosine on the cell viability and proliferation of the cultured human gastric cancer SGC-7901 cells. Carnosine treatment did not induce cell apoptosis or necrosis, but reduced the proliferative capacity of SGC-7901 cells. Seahorse analysis showed SGC-7901 cells cultured with pyruvate have active mitochondria, and depend on mitochondrial oxidative phosphorylation more than glycolysis pathway for generation of ATP. Carnosine markedly decreased the absolute value of mitochondrial ATP-linked respiration, and reduced the maximal oxygen consumption and spare respiratory capacity, which may reduce mitochondrial function correlated with proliferative potential. Simultaneously, carnosine also reduced the extracellular acidification rate and glycolysis of SGC-7901 cells. Our results suggested that carnosine is a potential regulator of energy metabolism of SGC-7901 cells both in the anaerobic and aerobic pathways, and provided a clue for preclinical and clinical evaluation of carnosine for gastric cancer therapy.

  16. The impact of the thermal sensitivity of cytochrome c oxidase on the respiration rate of Arctic charr red muscle mitochondria. pierre_blier@uqar.qc.ca.

    PubMed

    Blier, P U; Lemieux, H

    2001-04-01

    To assess if cytochrome c oxidase could determine the response of mitochondrial respiration to changes in environmental temperature in ectotherms, we performed KCN titration of the respiration rate and cytochrome c oxidase activity in mitochondria from Arctic charr (Salvelinusfontinalis) muscle at four different temperatures (1 degrees C, 6 degrees C, 12 degrees C, and 18 degrees C). Our data showed an excess of cytochrome c oxidase activity over the mitochondrial state 3 respiration rate. Mitochondrial oxygen consumption rates reached approximately 12% of the cytochrome c oxidase maximal capacity at every temperature. Also, following titration, the mitochondrial respiration rate significantly decreased when KCN reached concentrations that inhibit almost 90% of the cytochrome c oxidase activity. This strongly supports the idea that the thermal sensitivity of the maximal mitochondrial respiration rate cannot be dictated by the effect of temperature on cytochrome c oxidase catalytic capacity. Furthermore, the strong similarity of the Q10s of mitochondrial respiration and cytochrome c oxidase activity suggests a functional or structural link between the two. The functional link could be coevolution of parts of the mitochondrial system to maintain optimal functions in most of the temperature range encountered by organisms.

  17. Lactate is oxidized outside of the mitochondrial matrix in rodent brain.

    PubMed

    Herbst, Eric A F; George, Mitchell A J; Brebner, Karen; Holloway, Graham P; Kane, Daniel A

    2018-05-01

    The nature and existence of mitochondrial lactate oxidation is debated in the literature. Obscuring the issue are disparate findings in isolated mitochondria, as well as relatively low rates of lactate oxidation observed in permeabilized muscle fibres. However, respiration with lactate has yet to be directly assessed in brain tissue with the mitochondrial reticulum intact. To determine if lactate is oxidized in the matrix of brain mitochondria, oxygen consumption was measured in saponin-permeabilized mouse brain cortex samples, and rat prefrontal cortex and hippocampus (dorsal) subregions. While respiration in the presence of ADP and malate increased with the addition of lactate, respiration was maximized following the addition of exogenous NAD + , suggesting maximal lactate metabolism involves extra-matrix lactate dehydrogenase. This was further supported when NAD + -dependent lactate oxidation was significantly decreased with the addition of either low-concentration α-cyano-4-hydroxycinnamate or UK-5099, inhibitors of mitochondrial pyruvate transport. Mitochondrial respiration was comparable between glutamate, pyruvate, and NAD + -dependent lactate oxidation. Results from the current study demonstrate that permeabilized brain is a feasible model for assessing lactate oxidation, and support the interpretation that lactate oxidation occurs outside the mitochondrial matrix in rodent brain.

  18. Lopimune-induced mitochondrial toxicity is attenuated by increased uncoupling protein-2 level in treated mouse hepatocytes.

    PubMed

    El Hoss, Sara; Bahr, Georges M; Echtay, Karim S

    2015-06-15

    Although the protease inhibitor (PI) Lopimune has proven to be effective, no studies have examined the side effects of Lopimune on mitochondrial bioenergetics in hepatocytes. The objective of the present study is to evaluate mitochondrial respiration, production of reactive oxygen species (ROS) and expression of uncoupling protein-2 (UCP2) in mouse hepatocytes following Lopimune administration. Mitochondria were extracted from mouse liver using differential centrifugation and hepatocytes were isolated by the collagenase perfusion procedure. Mitochondrial respiration was measured using a Rank Brothers oxygen electrode. ROS production in hepatocytes was monitored by flow cytometry using a 2',7'-dichlorofluorescin diacetate probe and UCP2 protein expression was detected by Western blotting. We found that Lopimune induced a significant decrease of approximately 30% in the respiratory control ratio (RCR) starting from day 4 until day 9 of treatment. This decrease was due to an increase in state 4 respiration, reflecting an increase in mitochondrial proton leak. State 2 and state 3 respirations were not affected. Moreover, ROS production significantly increased by about 2-fold after day 1 of treatment and decreased after day 3, returning to the resting level on day 5. Interestingly, UCP2 which is absent from control hepatocytes, was expressed starting from day 4 of treatment. Our findings indicate that Lopimune-induced proton leak, mediated by UCP2, may represent a response to inhibit the production of ROS as a negative feedback regulatory mechanism. These results imply a potential involvement of UCP2 in the regulation of oxidative stress and add new insights into the understanding of mitochondrial toxicity induced by PIs. © The Authors Journal compilation © 2015 Biochemical Society.

  19. Plasma Amino Acids Stimulate Uncoupled Respiration of Muscle Subsarcolemmal Mitochondria in Lean but Not Obese Humans.

    PubMed

    Kras, Katon A; Hoffman, Nyssa; Roust, Lori R; Patel, Shivam H; Carroll, Chad C; Katsanos, Christos S

    2017-12-01

    Obesity is associated with mitochondrial dysfunction in skeletal muscle. Increasing the plasma amino acid (AA) concentrations stimulates mitochondrial adenosine triphosphate (ATP) production in lean individuals. To determine whether acute elevation in plasma AAs enhances muscle mitochondrial respiration and ATP production in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in obese adults. Assessment of SS and IMF mitochondrial function during saline (i.e., control) and AA infusions. Eligible participants were healthy lean (body mass index, <25 kg/m2; age, 37 ± 3 years; n = 10) and obese (body mass index >30 kg/m2; age 35 ± 3 years; n = 11) subjects. Single trial of saline infusion followed by AA infusion. SS and IMF mitochondria were isolated from muscle biopsies collected at the end of the saline and AA infusions. Mitochondrial respiration and ATP production. AA infusion increased adenosine 5'-diphosphate (ADP)-stimulated respiration and ATP production rates of SS mitochondria in the lean (P < 0.05), but not obese, subjects. Furthermore, AA infusion increased the uncoupled (i.e., non-ADP-stimulated) respiration of SS mitochondria in the lean subjects only (P < 0.05). AA infusion had no effect on any of these parameters in IMF mitochondria in either lean or obese subjects (P > 0.05). Increasing the plasma AA concentrations enhances the capacity for respiration and ATP production of muscle SS, but not IMF, mitochondria in lean individuals, in parallel with increases in uncoupled respiration. However, neither of these parameters increases in muscle SS or IMF mitochondria in obese individuals. Copyright © 2017 Endocrine Society

  20. Mic60/Mitofilin Overexpression Alters Mitochondrial Dynamics and Attenuates Vulnerability of Dopaminergic Cells to Dopamine and Rotenone

    PubMed Central

    Van Laar, Victor S.; Berman, Sarah B.; Hastings, Teresa G.

    2017-01-01

    Mitochondrial dysfunction has been implicated in Parkinson’s disease (PD) neuropathology. Mic60, also known as mitofilin, is a protein of the inner mitochondrial membrane and a key component of the mitochondrial contact site and cristae junction organizing system (MICOS). Mic60 is critical for maintaining mitochondrial membrane structure and function. We previously demonstrated that mitochondrial Mic60 protein is susceptible to both covalent modification and loss in abundance following exposure to dopamine quinone. In this study, we utilized neuronally-differentiated SH-SY5Y and PC12 dopaminergic cell lines to examine the effects of altered Mic60 levels on mitochondrial function and cellular vulnerability in response to PD-relevant stressors. Short hairpin RNA (shRNA)-mediated knockdown of endogenous Mic60 protein in neuronal SH-SY5Y cells significantly potentiated dopamine-induced cell death, which was rescued by co-expressing shRNA-insensitive Mic60. Conversely, in PC12 and SH-SY5Y cells, Mic60 overexpression significantly attenuated both dopamine- and rotenone-induced cell death as compared to controls. Mic60 overexpression in SH-SY5Y cells was also associated with increased mitochondrial respiration, and, following rotenone exposure, increased spare respiratory capacity. Mic60 knockdown cells exhibited suppressed respiration and, following rotenone treatment, decreased spare respiratory capacity. Mic60 overexpression also affected mitochondrial fission/fusion dynamics. PC12 cells overexpressing Mic60 exhibited increased mitochondrial interconnectivity. Further, both PC12 cells and primary rat cortical neurons overexpressing Mic60 displayed suppressed mitochondrial fission and increased mitochondrial length in neurites. These results suggest that altering levels of Mic60 in dopaminergic neuronal cells significantly affects both mitochondrial homeostasis and cellular vulnerability to the PD-relevant stressors dopamine and rotenone, carrying implications for PD

  1. Disruption of the GABA shunt affects mitochondrial respiration and virulence in the cereal pathogen Fusarium graminearum.

    PubMed

    Bönnighausen, Jakob; Gebhard, Daniel; Kröger, Cathrin; Hadeler, Birgit; Tumforde, Thomas; Lieberei, Reinhard; Bergemann, Jörg; Schäfer, Wilhelm; Bormann, Jörg

    2015-12-01

    The cereal pathogen Fusarium graminearum threatens food and feed production worldwide. It reduces the yield and poisons the remaining kernels with mycotoxins, notably deoxynivalenol (DON). We analyzed the importance of gamma-aminobutanoic acid (GABA) metabolism for the life cycle of this fungal pathogen. GABA metabolism in F. graminearum is partially regulated by the global nitrogen regulator AreA. Genetic disruption of the GABA shunt by deletion of two GABA transaminases renders the pathogen unable to utilize the plant stress metabolites GABA and putrescine. The mutants showed increased sensitivity against oxidative stress, GABA accumulation in the mycelium, downregulation of two key enzymes of the TCA cycle, disturbed potential gradient in the mitochondrial membrane and lower mitochondrial oxygen consumption. In contrast, addition of GABA to the wild type resulted in its rapid turnover and increased mitochondrial steady state oxygen consumption. GABA concentrations are highly upregulated in infected wheat tissues. We conclude that GABA is metabolized by the pathogen during infection increasing its energy production, whereas the mutants accumulate GABA intracellularly resulting in decreased energy production. Consequently, the GABA mutants are strongly reduced in virulence but, because of their DON production, are able to cross the rachis node. © 2015 John Wiley & Sons Ltd.

  2. Short-term sleep deprivation with exposure to nocturnal light alters mitochondrial bioenergetics in Drosophila.

    PubMed

    Rodrigues, Nathane Rosa; Macedo, Giulianna Echeverria; Martins, Illana Kemmerich; Gomes, Karen Kich; de Carvalho, Nélson Rodrigues; Posser, Thaís; Franco, Jeferson Luis

    2018-05-20

    Many studies have shown the effects of sleep deprivation in several aspects of health and disease. However, little is known about how mitochondrial bioenergetics function is affected under this condition. To clarify this, we developed a simple model of short-term sleep deprivation, in which fruit-flies were submitted to a nocturnal light condition and then mitochondrial parameters were assessed by high resolution respirometry (HRR). Exposure of flies to constant light was able to alter sleep patterns, causing locomotor deficits, increasing ROS production and lipid peroxidation, affecting mitochondrial activity, antioxidant defense enzymes and caspase activity. HRR analysis showed that sleep deprivation affected mitochondrial bioenergetics capacity, decreasing respiration at oxidative phosphorylation (OXPHOS) and electron transport system (ETS). In addition, the expression of genes involved in the response to oxidative stress and apoptosis were increased. Thus, our results suggest a connection between sleep deprivation and oxidative stress, pointing to mitochondria as a possible target of this relationship. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Mechanisms of respiration intensification of rat pancreatic acini upon carbachol-induced Ca(2+) release.

    PubMed

    Manko, B O; Manko, V V

    2013-08-01

    Acetylcholine as one of the main secretagogues modulates mitochondrial functions in acinar pancreacytes, presumably due to increase in ATP hydrolysis or Ca(2+) transport into mitochondria. The aim of this work was to investigate the mechanisms of carbachol (CCh) action on respiration and oxidative phosphorylation of isolated pancreatic acini. Respiration of intact or permeabilized rat pancreatic acini was studied at 37 °C using a Clark oxygen electrode. Respiration rate of isolated acini in rest was 0.27 ± 0.01 nmol O2 s(-1) 10(-6) cells. Addition of 10 μM CCh into respiration chamber evoked biphasic stimulation of respiration. Rapid increase of respiration by 20.1% lasted for approx. 1 min, followed by decrease to level by 11.5% higher than control. Addition of 1 μm CCh caused monophasic increase by 11.5%. Preincubation (5 min) with 1 or 10 μm CCh elevated respiration rate by 12.5 or 11.2% respectively. FCCP prevented the effect of CCh. Preincubation with 1 (but not 10) μm CCh increased FCCP-uncoupled respiration rate. Thapsigargin slightly elevated respiration, but ryanodine did not. Application of 2-aminoethoxydiphenyl borate or ruthenium red prevented the effects of CCh on respiration, while oligomycin abolished them. Preincubation with 1 μm CCh prior to cell permeabilization increased respiration rate at pyruvate+malate oxidation, but not at succinate oxidation. In contrast, preincubation with 10 μm CCh decreased pyruvate+malate oxidation. Medium CCh dose (1 μm) intensifies respiration and oxidative phosphorylation of acinar pancreacytes by feedforward mechanism via Ca(2+) transport into mitochondria and activation of Ca(2+) /ADP-sensitive mitochondrial dehydrogenases. Prolonged action of high CCh dose (10 μm) might impair mitochondrial functions. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

  4. Mitochondrial Targeted Coenzyme Q, Superoxide, and Fuel Selectivity in Endothelial Cells

    PubMed Central

    Fink, Brian D.; O'Malley, Yunxia; Dake, Brian L.; Ross, Nicolette C.; Prisinzano, Thomas E.; Sivitz, William I.

    2009-01-01

    Background Previously, we reported that the “antioxidant” compound “mitoQ” (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. Methods and Results To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. Conclusions In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells

  5. Mitochondrial targeted coenzyme Q, superoxide, and fuel selectivity in endothelial cells.

    PubMed

    Fink, Brian D; O'Malley, Yunxia; Dake, Brian L; Ross, Nicolette C; Prisinzano, Thomas E; Sivitz, William I

    2009-01-01

    Previously, we reported that the "antioxidant" compound "mitoQ" (mitochondrial-targeted ubiquinol/ubiquinone) actually increased superoxide production by bovine aortic endothelial (BAE) cell mitochondria incubated with complex I but not complex II substrates. To further define the site of action of the targeted coenzyme Q compound, we extended these studies to include different substrate and inhibitor conditions. In addition, we assessed the effects of mitoquinone on mitochondrial respiration, measured respiration and mitochondrial membrane potential in intact cells, and tested the intriguing hypothesis that mitoquinone might impart fuel selectivity in intact BAE cells. In mitochondria respiring on differing concentrations of complex I substrates, mitoquinone and rotenone had interactive effects on ROS consistent with redox cycling at multiple sites within complex I. Mitoquinone increased respiration in isolated mitochondria respiring on complex I but not complex II substrates. Mitoquinone also increased oxygen consumption by intact BAE cells. Moreover, when added to intact cells at 50 to 1000 nM, mitoquinone increased glucose oxidation and reduced fat oxidation, at doses that did not alter membrane potential or induce cell toxicity. Although high dose mitoquinone reduced mitochondrial membrane potential, the positively charged mitochondrial-targeted cation, decyltriphenylphosphonium (mitoquinone without the coenzyme Q moiety), decreased membrane potential more than mitoquinone, but did not alter fuel selectivity. Therefore, non-specific effects of the positive charge were not responsible and the quinone moiety is required for altered nutrient selectivity. In summary, the interactive effects of mitoquinone and rotenone are consistent with redox cycling at more than one site within complex I. In addition, mitoquinone has substrate dependent effects on mitochondrial respiration, increases repiration by intact cells, and alters fuel selectivity favoring glucose over

  6. Desmin Cytoskeleton Linked to Muscle Mitochondrial Distribution and Respiratory Function

    PubMed Central

    Milner, Derek J.; Mavroidis, Manolis; Weisleder, Noah; Capetanaki, Yassemi

    2000-01-01

    Ultrastructural studies have previously suggested potential association of intermediate filaments (IFs) with mitochondria. Thus, we have investigated mitochondrial distribution and function in muscle lacking the IF protein desmin. Immunostaining of skeletal muscle tissue sections, as well as histochemical staining for the mitochondrial marker enzymes cytochrome C oxidase and succinate dehydrogenase, demonstrate abnormal accumulation of subsarcolemmal clumps of mitochondria in predominantly slow twitch skeletal muscle of desmin-null mice. Ultrastructural observation of desmin-null cardiac muscle demonstrates in addition to clumping, extensive mitochondrial proliferation in a significant fraction of the myocytes, particularly after work overload. These alterations are frequently associated with swelling and degeneration of the mitochondrial matrix. Mitochondrial abnormalities can be detected very early, before other structural defects become obvious. To investigate related changes in mitochondrial function, we have analyzed ADP-stimulated respiration of isolated muscle mitochondria, and ADP-stimulated mitochondrial respiration in situ using saponin skinned muscle fibers. The in vitro maximal rates of respiration in isolated cardiac mitochondria from desmin-null and wild-type mice were similar. However, mitochondrial respiration in situ is significantly altered in desmin-null muscle. Both the maximal rate of ADP-stimulated oxygen consumption and the dissociation constant (K m) for ADP are significantly reduced in desmin-null cardiac and soleus muscle compared with controls. Respiratory parameters for desmin-null fast twitch gastrocnemius muscle were unaffected. Additionally, respiratory measurements in the presence of creatine indicate that coupling of creatine kinase and the adenine translocator is lost in desmin-null soleus muscle. This coupling is unaffected in cardiac muscle from desmin-null animals. All of these studies indicate that desmin IFs play a significant

  7. Niemann-Pick Type C2 Protein Regulates Free Cholesterol Accumulation and Influences Hepatic Stellate Cell Proliferation and Mitochondrial Respiration Function.

    PubMed

    Wang, Yuan-Hsi; Twu, Yuh-Ching; Wang, Chung-Kwe; Lin, Fu-Zhen; Lee, Chun-Ya; Liao, Yi-Jen

    2018-06-05

    Liver fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. A high-cholesterol diet is associated with liver fibrosis via the accumulation of free cholesterol in hepatic stellate cells (HSCs). Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular free cholesterol homeostasis via direct binding with free cholesterol. Previously, we reported that NPC2 was downregulated in liver cirrhosis tissues. Loss of NPC2 enhanced the accumulation of free cholesterol in HSCs and made them more susceptible to transforming growth factor (TGF)-β1. In this study, we showed that knockdown of NPC2 resulted in marked increases in platelet-derived growth factor BB (PDGF-BB)-induced HSC proliferation through enhanced extracellular signal-regulated kinases (ERK), p38, c-Jun N-terminal kinases (JNK), and protein kinase B (AKT) phosphorylation. In contrast, NPC2 overexpression decreased PDGF-BB-induced cell proliferation by inhibiting p38, JNK, and AKT phosphorylation. Although NPC2 expression did not affect caspase-related apoptosis, the autophagy marker light chain 3β (LC3B) was decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs. The mitochondrial respiration functions (such as oxygen consumption rate, ATP production, and maximal respiratory capacity) were decreased in NPC2 knockdown, and free cholesterol accumulated in the HSCs, while NPC2-overexpressed cells remained normal. In addition, NPC2 expression did not affect the susceptibility of HSCs to lipopolysaccharides (LPS), and U18666A treatment induced free cholesterol accumulation, which enhanced LPS-induced Toll-like receptor 4 (TLR4), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 phosphorylation, interleukin (IL)-1 and IL-6 expression. Our study demonstrated that NPC2-mediated free cholesterol homeostasis controls HSC proliferation and mitochondrial function.

  8. The Role of Mitochondrial Dysfunction in Psychiatric Disease

    ERIC Educational Resources Information Center

    Scaglia, Fernando

    2010-01-01

    Mitochondrial respiratory chain disorders are a group of genetically and clinically heterogeneous disorders caused by the biochemical complexity of mitochondrial respiration and the fact that two genomes, one mitochondrial and one nuclear, encode the components of the respiratory chain. These disorders can manifest at birth or present later in…

  9. A high throughput respirometric assay for mitochondrial biogenesis and toxicity

    PubMed Central

    Beeson, Craig C.; Beeson, Gyda C.; Schnellmann, Rick G.

    2010-01-01

    Mitochondria are a common target of toxicity for drugs and other chemicals, and results in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput, real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTC) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Biosciences analyzer to measure mitochondrial function in real time in multi-well plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl2 and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTC. The merger of the RPTC model and multi-well respirometry results in a single high throughput assay to measure mitochondrial biogenesis and toxicity, and nephrotoxic potential. PMID:20465991

  10. Decreased carbon limitation of litter respiration in a mortality-affected pinon-juniper woodland

    Treesearch

    Erin Berryman; John D. Marshall; Thom Rahn; Marcie Litvak; John Butnor

    2013-01-01

    Microbial respiration depends on microclimatic variables and carbon (C) substrate availability, all of which are altered when ecosystems experience major disturbance. Widespread tree mortality, currently affecting pinon-juniper ecosystems in southwestern North America, may affect C substrate availability in several ways, for example, via litterfall pulses and loss of...

  11. Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals.

    PubMed

    Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K; Waagepetersen, Helle S

    2017-01-01

    Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.

  12. MELAS syndrome with mitochondrial tRNA(Leu(UUR)) gene mutation in a Chinese family.

    PubMed Central

    Huang, C C; Chen, R S; Chen, C M; Wang, H S; Lee, C C; Pang, C Y; Hsu, H S; Lee, H C; Wei, Y H

    1994-01-01

    The clinical features of a patient in a Chinese family with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS syndrome) are reported. The study revealed that hearing and visual impairments and miscarriages may be early clinical presentations in MELAS. A heteroplasmic A to G transition in the tRNA(Leu(UUR)) gene was noted at the nucleotide pair 3243 in the mitochondrial DNA of muscle, blood, and hair follicles of the proband and his maternal relatives. Quantitative analysis of the mutated mitochondrial DNA revealed variable proportions in different tissues and subjects of maternal lineage in the family. Muscle tissue contained a higher proportion of the mutant mitochondria than other tissues examined. The function of the reproductive system of the proband seems to be impaired. In one clinically healthy sibling, the 3243rd point mutation was found in sperm mitochondrial DNA, although sperm motility was not affected. It seems that biochemical defects in mitochondrial respiration and oxidative phosphorylation are tissue specific expressions of the 3243rd point mutation in the mitochondrial DNA of the affected target tissues. Images PMID:8201329

  13. Paraquat affects mitochondrial bioenergetics, dopamine system expression, and locomotor activity in zebrafish (Danio rerio).

    PubMed

    Wang, Xiao H; Souders, Christopher L; Zhao, Yuan H; Martyniuk, Christopher J

    2018-01-01

    The dipyridyl herbicide paraquat induces oxidative stress in cells and is implicated in adult neurodegenerative diseases. However, less is known about paraquat toxicity in early stages of vertebrate development. To address this gap, zebrafish (Danio rerio) embryos were exposed to 1, 10 and 100 μM paraquat for 96 h. Paraquat did not induce significant mortality nor deformity in embryos and larvae, but it did accelerate time to hatch. To evaluate whether mitochondrial respiration was related to earlier hatch times, oxygen consumption rate was measured in whole embryos. Maximal respiration of embryos exposed to 100 μM paraquat for 24 h was reduced by more than 70%, suggesting that paraquat negatively impacts mitochondrial bioenergetics in early development. Based upon this evidence for mitochondrial dysfunction, transcriptional responses of oxidative stress- and apoptosis-related genes were measured. Fish exposed to 1 μM paraquat showed higher expression levels of superoxide dismutase 2, heat shock protein 70, Bcl-2-associated X protein, and B-cell CLL/lymphoma 2a compared to control fish. No differences among groups were detected in larvae exposed to 10 and 100 μM paraquat, suggesting a non-monotonic response. We also measured endpoints related to larval behavior and dopaminergic signaling as paraquat is associated with degeneration of dopamine neurons. Locomotor activity was stimulated with 100 μM paraquat and dopamine transporter and dopamine receptor 3 mRNA levels were increased in larvae exposed to 1 μM paraquat, interpreted to be a compensatory response at lower concentrations. This study improves mechanistic understanding into the toxic actions of paraquat on early developmental stages. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. The Natural Product Osthole Attenuates Yeast Growth by Extensively Suppressing the Gene Expressions of Mitochondrial Respiration Chain.

    PubMed

    Wang, Zhe; Shen, Yan

    2017-03-01

    The fast growing evidences have indicated that the natural product osthole is a promising drug candidate for fighting several serious human diseases, for example, cancer and inflammation. However, the mode-of-action (MoA) of osthole remains largely incomplete. In this study, we investigated the growth inhibition activity of osthole using fission yeast as a model, with the goal of understanding the osthole's mechanism of action, especially from the molecular level. Microarray analysis indicated that osthole has significant impacts on gene transcription levels (In total, 214 genes are up-regulated, and 97 genes are down-regulated). Gene set enrichment analysis (GSEA) indicated that 11 genes belong to the "Respiration module" category, especially including the components of complex III and V of mitochondrial respiration chain. Based on GSEA and network analysis, we also found that 54 up-regulated genes belong to the "Core Environmental Stress Responses" category, particularly including many transporter genes, which suggests that the rapidly activated nutrient exchange between cell and environment is part of the MoA of osthole. In summary, osthole can greatly impact on fission yeast transcriptome, and it primarily represses the expression levels of the genes in respiration chain, which next causes the inefficiency of ATP production and thus largely explains osthole's growth inhibition activity in Schizosaccharomyces pombe (S. pombe). The complexity of the osthole's MoA shown in previous studies and our current research demonstrates that the omics approach and bioinformatics tools should be applied together to acquire the complete landscape of osthole's growth inhibition activity.

  15. Methylene blue improves mitochondrial respiration and decreases oxidative stress in a substrate-dependent manner in diabetic rat hearts.

    PubMed

    Duicu, Oana M; Privistirescu, Andreea; Wolf, Adrian; Petruş, Alexandra; Dănilă, Maria D; Raţiu, Corina D; Muntean, Danina M; Sturza, Adrian

    2017-11-01

    Diabetic cardiomyopathy has been systematically associated with compromised mitochondrial energetics and increased generation of reactive oxygen species (ROS) that underlie its progression to heart failure. Methylene blue is a redox drug with reported protective effects mainly on brain mitochondria. The purpose of the present study was to characterize the effects of acute administration of methylene blue on mitochondrial respiration, H 2 O 2 production, and calcium sensitivity in rat heart mitochondria isolated from healthy and 2 months (streptozotocin-induced) diabetic rats. Mitochondrial respiratory function was assessed by high-resolution respirometry. H 2 O 2 production and calcium retention capacity were measured spectrofluorimetrically. The addition of methylene blue (0.1 μmol·L -1 ) elicited an increase in oxygen consumption of mitochondria energized with complex I and II substrates in both normal and diseased mitochondria. Interestingly, methylene blue elicited a significant increase in H 2 O 2 release in the presence of complex I substrates (glutamate and malate), but had an opposite effect in mitochondria energized with complex II substrate (succinate). No changes in the calcium retention capacity of healthy or diabetic mitochondria were found in the presence of methylene blue. In conclusion, in cardiac mitochondria isolated from diabetic and nondiabetic rat hearts, methylene blue improved respiratory function and elicited a dichotomic, substrate-dependent effect on ROS production.

  16. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

    PubMed Central

    Kathiresan, Meena; Martins, Dorival; English, Ann M.

    2014-01-01

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification. PMID:25422453

  17. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

    PubMed

    Kathiresan, Meena; Martins, Dorival; English, Ann M

    2014-12-09

    In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

  18. Evidence that the antiproliferative effects of auranofin in Saccharomyces cerevisiae arise from inhibition of mitochondrial respiration.

    PubMed

    Gamberi, Tania; Fiaschi, Tania; Modesti, Alessandra; Massai, Lara; Messori, Luigi; Balzi, Manuela; Magherini, Francesca

    2015-08-01

    Auranofin is a gold based drug in clinical use since 1985 for the treatment of rheumatoid arthritis. Beyond its antinflammatory properties, auranofin exhibits other attractive biological and pharmacological actions such as a potent in vitro cytotoxicity and relevant antimicrobial and antiparasitic effects that make it amenable for new therapeutic indications. For instance, auranofin is currently tested as an anticancer agent in four independent clinical trials; yet, its mode of action is highly controversial. With the present study, we explore the effects of auranofin in Saccharomyces cerevisiae and its likely mechanism. Notably, auranofin is reported to induce remarkable yeast growth inhibition. Solid evidence is provided that growth inhibition is the consequence of a direct cytotoxic insult occurring at the mitochondrial level; a profound depression of cell respiration is indeed clearly documented as the main cause of cell death while induction of ROS plays only a secondary role. More in detail, the mitochondrial NADH kinase Pos5 is identified as a primary target for auranofin. The implications of these results are discussed in the frame of current mechanistic knowledge on the cellular effects of auranofin and of its role as a prospective anticancer drug. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Impaired Mitochondrial Transcription Termination Disrupts the Stromal Redox Poise in Chlamydomonas1[OPEN

    PubMed Central

    Uhmeyer, Andreas

    2017-01-01

    In photosynthetic eukaryotes, the metabolite exchange between chloroplast and mitochondria ensures efficient photosynthesis under saturating light conditions. The Chlamydomonas reinhardtii mutant stm6 is devoid of the mitochondrial transcription termination factor MOC1 and aberrantly expresses the mitochondrial genome, resulting in enhanced photosynthetic hydrogen production and diminished light tolerance. We analyzed the modulation of mitochondrial and chlororespiration during the acclimation of stm6 and the MOC1-complemented strain to excess light. Although light stress stimulated mitochondrial respiration via the energy-conserving cytochrome c pathway in both strains, the mutant was unable to fine-tune the expression and activity of oxidative phosphorylation complex I in excess light, which was accompanied by an increased mitochondrial respiration via the alternative oxidase pathway. Furthermore, stm6 failed to fully activate chlororespiration and cyclic electron flow due to a more oxidized state of the chloroplast stroma, which is caused by an increased mitochondrial electron sink capacity. Increased susceptibility to photoinhibition of PSII in stm6 demonstrates that the MOC1-dependent modulation of mitochondrial respiration helps control the stromal redox poise as a crucial part of high-light acclimation in C. reinhardtii. PMID:28500267

  20. Effect of biosolid waste compost on soil respiration in salt-affected soils

    NASA Astrophysics Data System (ADS)

    Raya, Silvia; Gómez, Ignacio; García, Fuensanta; Navarro, José; Jordán, Manuel Miguel; Belén Almendro, María; Martín Soriano, José

    2013-04-01

    A great part of mediterranean soils are affected by salinization. This is an important problem in semiarid areas increased by the use of low quality waters, the induced salinization due to high phreatic levels and adverse climatology. Salinization affects 25% of irrigated agriculture, producing important losses on the crops. In this situation, the application of organic matter to the soil is one of the possible solutions to improve their quality. The main objective of this research was to asses the relation between the salinity level (electrical conductivity, EC) in the soil and the response of microbial activity (soil respiration rate) after compost addition. The study was conducted for a year. Soil samples were collected near to an agricultural area in Crevillente and Elche, "El Hondo" Natural Park (Comunidad de Regantes from San Felipe Neri). The experiment was developed to determine and quantify the soil respiration rate in 8 different soils differing in salinity. The assay was done in close pots -in greenhouse conditions- containing soil mixed with different doses of sewage sludge compost (2, 4 and 6%) besides the control. They were maintained at 60% of water holding capacity (WHC). Soil samples were analyzed every four months for a year. The equipment used to estimate the soil respiration was a Bac-Trac and CO2 emitted by the soil biota was measured and quantified by electrical impedance changes. It was observed that the respiration rate increases as the proportion of compost added to each sample increases as well. The EC was incremented in each sampling period from the beginning of the experiment, probably due to the fact that soils were in pots and lixiviation was prevented, so the salts couldńt be lost from soil. Over time the compost has been degraded and, it was more susceptible to be mineralized. Salts were accumulated in the soil. Also it was observed a decrease of microbial activity with the increase of salinity in the soil. Keywords: soil

  1. Aspirin increases mitochondrial fatty acid oxidation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Uppala, Radha; Dudiak, Brianne; Beck, Megan E.

    The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse themore » mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 h incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. - Highlights: • Aspirin increases mitochondrial—but inhibits peroxisomal—fatty acid oxidation. • Aspirin acetylates mitochondrial proteins including fatty acid oxidation enzymes. • SIRT3 does not influence the effect of aspirin on fatty acid oxidation. • Increased fatty acid oxidation is likely due to altered mitochondrial morphology and respiration.« less

  2. Response of mitochondrial function to hypothyroidism in normal and regenerated rat skeletal muscle.

    PubMed

    Zoll, J; Ventura-Clapier, R; Serrurier, B; Bigard, A X

    2001-01-01

    Although thyroid hormones induce a well known decrease in muscle oxidative capacity, nothing is known concerning their effects on mitochondrial function and regulation in situ. Similarly, the influence of regeneration process is not completely understood. We investigated the effects of hypothyroidism on mitochondrial function in fast gastrocnemius (GS) and slow soleus (SOL) muscles either intact or having undergone a cycle of degeneration/regeneration (Rg SOL) following a local injection of myotoxin. Thyroid hormone deficiency was induced by thyroidectomy and propylthiouracyl via drinking water. Respiration was measured in muscle fibres permeabilised by saponin in order to assess the oxidative capacity of the muscles and the regulation of mitochondria in situ. Oxidative capacities were 8.9 in SOL, 8.5 in Rg SOL and 5.9 micromol O2/min/g dry weight in GS and decreased by 52, 42 and 39% respectively (P < 0.001) in hypothyroid rats. Moreover, the Km of mitochondrial respiration for the phosphate acceptor ADP exhibited a two-fold decrease in Rg SOL and intact SOL by hypothyroidism (P < 0.01), while mitochondrial creatine kinase activity and sensitivity of mitochondrial respiration to creatine were not altered. The results of this study demonstrate that hypothyroidism markedly altered the sensitivity of mitochondrial respiration to ADP but not to creatine in SOL muscles, suggesting that mitochondrial regulation could be partially controlled by thyroid hormones. On the other hand, mitochondrial function completely recovered following regeneration/degeneration, suggesting that thyroid hormones are not involved in the regeneration process per se.

  3. Suppression of mitochondrial respiration with auraptene inhibits the progression of renal cell carcinoma: involvement of HIF-1α degradation.

    PubMed

    Jang, Yunseon; Han, Jeongsu; Kim, Soo Jeong; Kim, Jungim; Lee, Min Joung; Jeong, Soyeon; Ryu, Min Jeong; Seo, Kang-Sik; Choi, Song-Yi; Shong, Minho; Lim, Kyu; Heo, Jun Young; Kweon, Gi Ryang

    2015-11-10

    Renal cell carcinoma (RCC) progression resulting from the uncontrolled migration and enhanced angiogenesis is an obstacle to effective therapeutic intervention. Tumor metabolism has distinctive feature called Warburg effect, which enhances the aerobic glycolysis rapidly supplying the energy for migration of tumor. To manipulate this metabolic change characteristic of aggressive tumors, we utilized the citrus extract, auraptene, known as a mitochondrial inhibitor, testing its anticancer effects against the RCC4 cell line. We found that auraptene impaired RCC4 cell motility through reduction of mitochondrial respiration and glycolytic pathway-related genes. It also strongly disrupted VEGF-induced angiogenesis in vitro and in vivo. Hypoxia-inducible factor 1a (HIF-1a), a key regulator of cancer metabolism, migration and angiogenesis that is stably expressed in RCCs by virtue of a genetic mutation in the von Hippel-Lindau (VHL) tumor-suppressor protein, was impeded by auraptene, which blocked HIF-1a translation initiation without causing cytotoxicity. We suggest that blockade HIF-1a and reforming energy metabolism with auraptene is an effective approach for suspension RCC progression.

  4. Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle.

    PubMed

    Lefort, Natalie; Glancy, Brian; Bowen, Benjamin; Willis, Wayne T; Bailowitz, Zachary; De Filippis, Elena A; Brophy, Colleen; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J

    2010-10-01

    The contribution of mitochondrial dysfunction to skeletal muscle insulin resistance remains elusive. Comparative proteomics are being applied to generate new hypotheses in human biology and were applied here to isolated mitochondria to identify novel changes in mitochondrial protein abundance present in insulin-resistant muscle. Mitochondria were isolated from vastus lateralis muscle from lean and insulin-sensitive individuals and from obese and insulin-resistant individuals who were otherwise healthy. Respiration and reactive oxygen species (ROS) production rates were measured in vitro. Relative abundances of proteins detected by mass spectrometry were determined using a normalized spectral abundance factor method. NADH- and FADH(2)-linked maximal respiration rates were similar between lean and obese individuals. Rates of pyruvate and palmitoyl-DL-carnitine (both including malate) ROS production were significantly higher in obesity. Mitochondria from obese individuals maintained higher (more negative) extramitochondrial ATP free energy at low metabolic flux, suggesting that stronger mitochondrial thermodynamic driving forces may underlie the higher ROS production. Tandem mass spectrometry identified protein abundance differences per mitochondrial mass in insulin resistance, including lower abundance of complex I subunits and enzymes involved in the oxidation of branched-chain amino acids (BCAA) and fatty acids (e.g., carnitine palmitoyltransferase 1B). We provide data suggesting normal oxidative capacity of mitochondria in insulin-resistant skeletal muscle in parallel with high rates of ROS production. Furthermore, we show specific abundance differences in proteins involved in fat and BCAA oxidation that might contribute to the accumulation of lipid and BCAA frequently associated with the pathogenesis of insulin resistance.

  5. An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes.

    PubMed

    Traba, Javier; Miozzo, Pietro; Akkaya, Billur; Pierce, Susan K; Akkaya, Munir

    2016-11-21

    Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

  6. Mitochondrial Respiration Inhibitors Suppress Protein Translation and Hypoxic Signaling via the Hyperphosphorylation and Inactivation of Translation Initiation Factor eIF2α and Elongation Factor eEF2

    PubMed Central

    Li, Jun; Mahdi, Fakhri; Du, Lin; Datta, Sandipan; Nagle, Dale G.; Zhou, Yu-Dong

    2011-01-01

    Over 20000 lipid extracts of plants and marine organisms were evaluated in a human breast tumor T47D cell-based reporter assay for hypoxia-inducible factor-1 (HIF-1) inhibitory activity. Bioassay-guided isolation and dereplication-based structure elucidation of an active extract from the Bael tree (Aegle marmelos) afforded two protolimonoids, skimmiarepin A (1) and skimmiarepin C (2). In T47D cells, 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC50 values of 0.063 µM and 0.068 µM, respectively. Compounds 1 and 2 also suppressed hypoxic induction of the HIF-1 target genes GLUT-1 and VEGF. Mechanistic studies revealed that 1 and 2 inhibited HIF-1 activation by blocking the hypoxia-induced accumulation of HIF-1α protein. At the range of concentrations that inhibited HIF-1 activation, 1 and 2 suppressed cellular respiration by selectively inhibiting the mitochondrial electron transport chain at complex I (NADH dehydrogenase). Further investigation indicated that mitochondrial respiration inhibitors such as 1 and rotenone induced the rapid hyperphosphorylation and inhibition of translation initiation factor eIF2α and elongation factor eEF2. The inhibition of protein translation may account for the short-term exposure effects exerted by mitochondrial inhibitors on cellular signaling, while the suppression of cellular ATP production may contribute to the inhibitory effects following extended treatment periods. PMID:21875114

  7. Maintenance of Mitochondrial Oxygen Homeostasis by Cosubstrate Compensation

    PubMed Central

    Kueh, Hao Yuan; Niethammer, Philipp; Mitchison, Timothy J.

    2013-01-01

    Mitochondria maintain a constant rate of aerobic respiration over a wide range of oxygen levels. However, the control strategies underlying oxygen homeostasis are still unclear. Using mathematical modeling, we found that the mitochondrial electron transport chain (ETC) responds to oxygen level changes by undergoing compensatory changes in reduced electron carrier levels. This emergent behavior, which we named cosubstrate compensation (CSC), enables the ETC to maintain homeostasis over a wide of oxygen levels. When performing CSC, our ETC models recapitulated a classic scaling relationship discovered by Chance [Chance B (1965) J. Gen. Physiol. 49:163-165] relating the extent of oxygen homeostasis to the kinetics of mitochondrial electron transport. Analysis of an in silico mitochondrial respiratory system further showed evidence that CSC constitutes the dominant control strategy for mitochondrial oxygen homeostasis during active respiration. Our findings indicate that CSC constitutes a robust control strategy for homeostasis and adaptation in cellular biochemical networks. PMID:23528093

  8. Drosophila mitochondrial topoisomerase III alpha affects the aging process via maintenance of mitochondrial function and genome integrity.

    PubMed

    Tsai, Han-Zen; Lin, Ren-Kuo; Hsieh, Tao-Shih

    2016-04-12

    Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III alpha (Top3α) contributes to the maintenance of the mitochondrial genome and male germ-line stem cells. However, the involvement of mitochondrial Top3α in the mitochondrion-mediated aging process remains unclear. In this study, the M1L flies, in which Top3α protein lacks the mitochondrial import sequence and is thus present in cell nuclei but not in mitochondria, is used as a model system to examine the role of mitochondrial Top3α in the aging of fruit flies. Here, we reported that M1L flies exhibit mitochondrial defects which affect the aging process. First, we observed that M1L flies have a shorter life span, which was correlated with a significant reduction in the mitochondrial DNA copy number, the mitochondrial membrane potential, and ATP content compared with those of both wildtype and transgene-rescued flies of the same age. Second, we performed a mobility assay and electron microscopic analysis to demonstrate that the locomotion defect and mitophagy of M1L flies were enhanced with age, as compared with the controls. Finally, we showed that the correlation between the mtDNA deletion level and aging in M1L flies resembles what was reported in mammalian systems. The results reported here demonstrate that mitochondrial Top3α ablation results in mitochondrial genome instability and its dysfunction, thereby accelerating the aging process.

  9. Caloric Restriction-Induced Extension of Chronological Lifespan Requires Intact Respiration in Budding Yeast.

    PubMed

    Kwon, Young-Yon; Lee, Sung-Keun; Lee, Cheol-Koo

    2017-04-01

    Caloric restriction (CR) has been shown to extend lifespan and prevent cellular senescence in various species ranging from yeast to humans. Many effects of CR may contribute to extend lifespan. Specifically, CR prevents oxidative damage from reactive oxygen species (ROS) by enhancing mitochondrial function. In this study, we characterized 33 single electron transport chain (ETC) gene-deletion strains to identify CR-induced chronological lifespan (CLS) extension mechanisms. Interestingly, defects in 17 of these 33 ETC gene-deleted strains showed loss of both respiratory function and CR-induced CLS extension. On the contrary, the other 16 respiration-capable mutants showed increased CLS upon CR along with increased mitochondrial membrane potential (MMP) and intracellular adenosine triphosphate (ATP) levels, with decreased mitochondrial superoxide generation. We measured the same parameters in the 17 non-respiratory mutants upon CR. CR simultaneously increased MMP and mitochondrial superoxide generation without altering intracellular ATP levels. In conclusion, respiration is essential for CLS extension by CR and is important for balancing MMP, ROS, and ATP levels.

  10. Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

    PubMed

    Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

    2015-08-01

    High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  11. A novel mitochondrial carrier protein Mme1 acts as a yeast mitochondrial magnesium exporter.

    PubMed

    Cui, Yixian; Zhao, Shanke; Wang, Juan; Wang, Xudong; Gao, Bingquan; Fan, Qiangwang; Sun, Fei; Zhou, Bing

    2015-03-01

    The homeostasis of magnesium (Mg2+), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously, two mitochondrial Mg2+ importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg2+ uptake. We now show that mitochondrial Mg2+ homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased steady-state mitochondrial Mg2+ concentration, while overexpression decreased it. Measurements of Mg2+ exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg2+ exporter. Our studies identified, for the first time, a mitochondrial Mg2+ exporter that works together with mitochondrial importers to ensure the precise control of mitochondrial Mg2+ homeostasis. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. The mTOR inhibitor sirolimus suppresses renal, hepatic, and cardiac tissue cellular respiration.

    PubMed

    Albawardi, Alia; Almarzooqi, Saeeda; Saraswathiamma, Dhanya; Abdul-Kader, Hidaya Mohammed; Souid, Abdul-Kader; Alfazari, Ali S

    2015-01-01

    The purpose of this in vitro study was to develop a useful biomarker (e.g., cellular respiration, or mitochondrial O2 consumption) for measuring activities of mTOR inhibitors. It measured the effects of commonly used immunosuppressants (sirolimus-rapamycin, tacrolimus, and cyclosporine) on cellular respiration in target tissues (kidney, liver, and heart) from C57BL/6 mice. The mammalian target of rapamycin (mTOR), a serine/ threonine kinase that supports nutrient-dependent cell growth and survival, is known to control energy conversion processes within the mitochondria. Consistently, inhibitors of mTOR (e.g., rapamycin, also known as sirolimus or Rapamune®) have been shown to impair mitochondrial function. Inhibitors of the calcium-dependent serine/threonine phosphatase calcineurin (e.g., tacrolimus and cyclosporine), on the other hand, strictly prevent lymphokine production leading to a reduced T-cell function. Sirolimus (10 μM) inhibited renal (22%, P=0.002), hepatic (39%, P<0.001), and cardiac (42%, P=0.005) cellular respiration. Tacrolimus and cyclosporine had no or minimum effects on cellular respiration in these tissues. Thus, these results clearly demonstrate that impaired cellular respiration (bioenergetics) is a sensitive biomarker of the immunosuppressants that target mTOR.

  13. The antidiabetic drug metformin decreases mitochondrial respiration and tricarboxylic acid cycle activity in cultured primary rat astrocytes.

    PubMed

    Hohnholt, Michaela C; Blumrich, Eva-Maria; Waagepetersen, Helle S; Dringen, Ralf

    2017-11-01

    Metformin is an antidiabetic drug that is used daily by millions of patients worldwide. Metformin is able to cross the blood-brain barrier and has recently been shown to increase glucose consumption and lactate release in cultured astrocytes. However, potential effects of metformin on mitochondrial tricarboxylic acid (TCA) cycle metabolism in astrocytes are unknown. We investigated this by mapping 13 C labeling in TCA cycle intermediates and corresponding amino acids after incubation of primary rat astrocytes with [U- 13 C]glucose. The presence of metformin did not compromise the viability of cultured astrocytes during 4 hr of incubation, but almost doubled cellular glucose consumption and lactate release. Compared with control cells, the presence of metformin dramatically lowered the molecular 13 C carbon labeling (MCL) of the cellular TCA cycle intermediates citrate, α-ketoglutarate, succinate, fumarate, and malate, as well as the MCL of the TCA cycle intermediate-derived amino acids glutamate, glutamine, and aspartate. In addition to the total molecular 13 C labeling, analysis of the individual isotopomers of TCA cycle intermediates confirmed a severe decline in labeling and a significant lowering in TCA cycling ratio in metformin-treated astrocytes. Finally, the oxygen consumption of mitochondria isolated from metformin-treated astrocytes was drastically reduced in the presence of complex I substrates, but not of complex II substrates. These data demonstrate that exposure to metformin strongly impairs complex I-mediated mitochondrial respiration in astrocytes, which is likely to cause the observed decrease in labeling of mitochondrial TCA cycle intermediates and the stimulation of glycolytic lactate production. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  14. Reversible temperature-dependent differences in brown adipose tissue respiration during torpor in a mammalian hibernator.

    PubMed

    McFarlane, Sarah V; Mathers, Katherine E; Staples, James F

    2017-03-01

    Although seasonal modifications of brown adipose tissue (BAT) in hibernators are well documented, we know little about functional regulation of BAT in different phases of hibernation. In the 13-lined ground squirrel, liver mitochondrial respiration is suppressed by up to 70% during torpor. This suppression is reversed during arousal and interbout euthermia (IBE), and corresponds with patterns of maximal activities of electron transport system (ETS) enzymes. Uncoupling of BAT mitochondria is controlled by free fatty acid release stimulated by sympathetic activation of adipocytes, so we hypothesized that further regulation at the level of the ETS would be of little advantage. As predicted, maximal ETS enzyme activities of isolated BAT mitochondria did not differ between torpor and IBE. In contrast to this pattern, respiration rates of mitochondria isolated from torpid individuals were suppressed by ~60% compared with rates from IBE individuals when measured at 37°C. At 10°C, however, mitochondrial respiration rates tended to be greater in torpor than IBE. As a result, the temperature sensitivity (Q 10 ) of mitochondrial respiration was significantly lower in torpor (~1.4) than IBE (~2.4), perhaps facilitating energy savings during entrance into torpor and thermogenesis at low body temperatures. Despite the observed differences in isolated mitochondria, norepinephrine-stimulated respiration rates of isolated BAT adipocytes did not differ between torpor and IBE, perhaps because the adipocyte isolation requires lengthy incubation at 37°C, potentially reversing any changes that occur in torpor. Such changes may include remodeling of BAT mitochondrial membrane phospholipids, which could change in situ enzyme activities and temperature sensitivities. Copyright © 2017 the American Physiological Society.

  15. [Characteristics of soil respiration in Phyllostachys edulis forest in Wanmulin Natural Reserve and related affecting factors].

    PubMed

    Wang, Chao; Yang, Zhi-Jie; Chen, Guang-Shui; Fan, Yue-Xin; Liu, Qiang; Tian, Hao

    2011-05-01

    By using Li-Cor 8100 open soil carbon flux system, the dynamic changes of soil respiration rate in Phyllostachys edulis forest in Wanmulin Natural Reserve in Fujian Province of China were measured from January 2009 to December 2009, with the relationships between the dynamic changes and related affecting factors analyzed. The monthly variation of soil respiration rate in the forest presented a double peak curve, with the peaks appeared in June 2009 (6. 83 micromol x m(-2) x s(-1)) and September 2009 (5.59 micromol x m(-2) x s(-1)), and the seasonal variation of the soil respiration rate was significant, with the maximum in summer and the minimum in winter. The soil respiration rate had significant correlation with the soil temperature at depth 5 cm (P < 0.05), but no significant correlation with soil moisture (P > 0.05). The monthly variation of litter fall mass in the forest was in single peak shape, and there was a significantly positive correlation between the monthly litter fall mass and soil respiration rate (P < 0.05). Two-factor model of soil temperature and litter fall mass could explain 93.2% variation of the soil respiration rate.

  16. p63 supports aerobic respiration through hexokinase II

    PubMed Central

    Viticchiè, Guiditta; Agostini, Massimiliano; Lena, Anna Maria; Mancini, Mara; Zhou, Huiqing; Zolla, Lello; Dinsdale, David; Saintigny, Gaelle; Melino, Gerry; Candi, Eleonora

    2015-01-01

    Short p63 isoform, ΔNp63, is crucial for epidermis formation, and it plays a pivotal role in controlling the turnover of basal keratinocytes by regulating the expression of a subset of genes involved in cell cycle and cell adhesion programs. The glycolytic enzyme hexokinase 2 (HK2) represents the first step of glucose utilization in cells. The family of HKs has four isoforms that differ mainly in their tissue and subcellular distribution. The preferential mitochondrial localization of HK2 at voltage-dependent anion channels provides access to ATP generated by oxidative phosphorylation and generates an ADP/ATP recycling mechanism to maintain high respiration rates and low electron leak. Here, we report that ΔNp63 depletion in human keratinocytes impairs mitochondrial basal respiration and increases mitochondrial membrane polarization and intracellular reactive oxygen species. We show ΔNp63-dependent regulation of HK2 expression, and we use ChIP, validated by p63-Chip sequencing genomewide profiling analysis, and luciferase assays to demonstrate the presence of one p63-specific responsive element within the 15th intronic region of the HK2 gene, providing evidence of a direct interaction. Our data support the notion of ΔNp63 as a master regulator in epithelial cells of a combined subset of molecular mechanisms, including cellular energy metabolism and respiration. The ΔNp63–HK2 axis is also present in epithelial cancer cells, suggesting that ΔNp63 could participate in cancer metabolic reprogramming. PMID:26324887

  17. Measurement of Mitochondrial Respiration in Isolated Protoplasts: Cytochrome and Alternative Pathways.

    PubMed

    Sunil, Bobba; Raghavendra, Agepati S

    2017-01-01

    The electron partitioning between COX and AOX pathways of mitochondria and their coordination is necessary to meet the energy demands as well as to maintain optimized redox status in plants under varying environmental conditions. The relative contribution of these two pathways to total respiration is an important measure during a given stress condition. We describe in detail the procedure that allows the measurement of the parameters of COX and AOX pathway of respiration in mesophyll protoplasts using Clark-type O 2 electrode. This chapter also lists the steps for rapid isolation procedure for mesophyll protoplasts from pea leaves. The advantages and limitations of the use of metabolic inhibitors and the protoplasts for measuring the respiration are also briefly discussed.

  18. Mitochondrial respiratory control is lost during growth factor deprivation.

    PubMed

    Gottlieb, Eyal; Armour, Sean M; Thompson, Craig B

    2002-10-01

    The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized in vitro. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome c and cytochrome c-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome c. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-x(L), restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control.

  19. Mitochondrial respiratory control is lost during growth factor deprivation

    PubMed Central

    Gottlieb, Eyal; Armour, Sean M.; Thompson, Craig B.

    2002-01-01

    The ability of cells to maintain a bioenergetically favorable ATP/ADP ratio confers a tight balance between cellular events that consume ATP and the rate of ATP production. However, after growth factor withdrawal, the cellular ATP/ADP ratio declines. To investigate these changes, mitochondria from growth factor-deprived cells isolated before the onset of apoptosis were characterized in vitro. Mitochondria from growth factor-deprived cells have lost their ability to undergo matrix condensation in response to ADP, which is accompanied by a failure to perform ADP-coupled respiration. At the time of analysis, mitochondria from growth factor-deprived cells were not depleted of cytochrome c and cytochrome c-dependent respiration was unaffected, demonstrating that the inhibition of the respiratory rate is not due to loss of cytochrome c. Agents that disrupt the mitochondrial outer membrane, such as digitonin, or maintain outer membrane exchange of adenine nucleotide, such as Bcl-xL, restored ADP-dependent control of mitochondrial respiration. Together, these data suggest that the regulation of mitochondrial outer membrane permeability contributes to respiratory control. PMID:12228733

  20. Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP-linked respiration in cultured cortical astrocytes.

    PubMed

    Shen, Yao; Tian, Yueyang; Shi, Xiaojie; Yang, Jianbo; Ouyang, Li; Gao, Jieqiong; Lu, Jianxin

    2014-08-01

    Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate-induced gliotoxicity. Exposure to 10-mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non-mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP-linked respiration of astrocytes was reduced. The glutamate-induced astrocyte damage can be mimicked by the non-metabolized substrate d-aspartate but reversed by the non-selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate-induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.

  1. Mitochondrial myopathy, lactic acidosis, and sideroblastic anemia (MLASA) plus associated with a novel de novo mutation (m.8969G>A) in the mitochondrial encoded ATP6 gene.

    PubMed

    Burrage, Lindsay C; Tang, Sha; Wang, Jing; Donti, Taraka R; Walkiewicz, Magdalena; Luchak, J Michael; Chen, Li-Chieh; Schmitt, Eric S; Niu, Zhiyv; Erana, Rodrigo; Hunter, Jill V; Graham, Brett H; Wong, Lee-Jun; Scaglia, Fernando

    2014-11-01

    Mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA) is a rare mitochondrial disorder that has previously been associated with mutations in PUS1 and YARS2. In the present report, we describe a 6-year old male with an MLASA plus phenotype. This patient had features of MLASA in the setting of developmental delay, sensorineural hearing loss, epilepsy, agenesis of the corpus callosum, failure to thrive, and stroke-like episodes. Sequencing of the mitochondrial genome identified a novel de novo, heteroplasmic mutation in the mitochondrial DNA (mtDNA) encoded ATP6 gene (m.8969G>A, p.S148N). Whole exome sequencing did not identify mutations or variants in PUS1 or YARS2 or any known nuclear genes that could affect mitochondrial function and explain this phenotype. Studies of fibroblasts derived from the patient revealed a decrease in oligomycin-sensitive respiration, a finding which is consistent with a complex V defect. Thus, this mutation in MT-ATP6 may represent the first mtDNA point mutation associated with the MLASA phenotype. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells.

    PubMed

    Masci, Alessandra; Mastronicola, Daniela; Arese, Marzia; Piane, Maria; De Amicis, Andrea; Blanck, Thomas J J; Chessa, Luciana; Sarti, Paolo

    2008-01-01

    Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P< or =0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.

  3. Mitochondrial tRNA cleavage by tRNA-targeting ribonuclease causes mitochondrial dysfunction observed in mitochondrial disease

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ogawa, Tetsuhiro, E-mail: atetsu@mail.ecc.u-tokyo.ac.jp; Shimizu, Ayano; Takahashi, Kazutoshi

    2014-08-15

    Highlights: • MTS-tagged ribonuclease was translocated successfully to the mitochondrial matrix. • MTS-tagged ribonuclease cleaved mt tRNA and reduced COX activity. • Easy and reproducible method of inducing mt tRNA dysfunction. - Abstract: Mitochondrial DNA (mtDNA) is a genome possessed by mitochondria. Since reactive oxygen species (ROS) are generated during aerobic respiration in mitochondria, mtDNA is commonly exposed to the risk of DNA damage. Mitochondrial disease is caused by mitochondrial dysfunction, and mutations or deletions on mitochondrial tRNA (mt tRNA) genes are often observed in mtDNA of patients with the disease. Hence, the correlation between mt tRNA activity and mitochondrialmore » dysfunction has been assessed. Then, cybrid cells, which are constructed by the fusion of an enucleated cell harboring altered mtDNA with a ρ{sup 0} cell, have long been used for the analysis due to difficulty in mtDNA manipulation. Here, we propose a new method that involves mt tRNA cleavage by a bacterial tRNA-specific ribonuclease. The ribonuclease tagged with a mitochondrial-targeting sequence (MTS) was successfully translocated to the mitochondrial matrix. Additionally, mt tRNA cleavage, which resulted in the decrease of cytochrome c oxidase (COX) activity, was observed.« less

  4. Relationships between Respiration and Susceptibility to Azole Antifungals in Candida glabrata

    PubMed Central

    Brun, Sophie; Aubry, Christophe; Lima, Osana; Filmon, Robert; Bergès, Thierry; Chabasse, Dominique; Bouchara, Jean-Philippe

    2003-01-01

    Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain. PMID:12604511

  5. Chicken or the egg: Warburg effect and mitochondrial dysfunction

    PubMed Central

    Senyilmaz, Deniz

    2015-01-01

    Compared with normal cells, cancer cells show alterations in many cellular processes, including energy metabolism. Studies on cancer metabolism started with Otto Warburg's observation at the beginning of the last century. According to Warburg, cancer cells rely on glycolysis more than mitochondrial respiration for energy production. Considering that glycolysis yields much less energy compared with mitochondrial respiration, Warburg hypothesized that mitochondria must be dysfunctional and this is the initiating factor for cancer formation. However, this hypothesis did not convince every scientist in the field. Some believed the opposite: the reduction in mitochondrial activity is a result of increased glycolysis. This discrepancy of opinions is ongoing. In this review, we will discuss the alterations in glycolysis, pyruvate metabolism, and the Krebs cycle in cancer cells and focus on cause and consequence. PMID:26097714

  6. Immunometabolic profiling of T cells from patients with relapsing-remitting multiple sclerosis reveals an impairment in glycolysis and mitochondrial respiration.

    PubMed

    La Rocca, Claudia; Carbone, Fortunata; De Rosa, Veronica; Colamatteo, Alessandra; Galgani, Mario; Perna, Francesco; Lanzillo, Roberta; Brescia Morra, Vincenzo; Orefice, Giuseppe; Cerillo, Ilaria; Florio, Ciro; Maniscalco, Giorgia Teresa; Salvetti, Marco; Centonze, Diego; Uccelli, Antonio; Longobardi, Salvatore; Visconti, Andrea; Matarese, Giuseppe

    2017-12-01

    Metabolic reprogramming is shaped to support specific cell functions since cellular metabolism controls the final outcome of immune response. Multiple sclerosis (MS) is an autoimmune disease resulting from loss of immune tolerance against central nervous system (CNS) myelin. Metabolic alterations of T cells occurring during MS are not yet well understood and their studies could have relevance in the comprehension of the pathogenetic events leading to loss of immune tolerance to self and to develop novel therapeutic strategies aimed at limiting MS progression. In this report, we observed that extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), indicators of glycolysis and oxidative phosphorylation, respectively, were impaired during T cell activation in naïve-to-treatment relapsing remitting (RR)MS patients when compared with healthy controls. These results were also corroborated at biochemical level by a reduced expression of the glycolitic enzymes aldolase, enolase 1, hexokinase I, and by reduction of Krebs cycle enzymes dihydrolipoamide-S-acetyl transferase (DLAT) and dihydrolipoamide-S-succinyl transferase (DLST). Treatment of RRMS patients with interferon beta-1a (IFN beta-1a) was able to restore T cell glycolysis and mitochondrial respiration as well as the amount of the metabolic enzymes to a level comparable to that of healthy controls. These changes associated with an up-regulation of the glucose transporter-1 (GLUT-1), a key element in intracellular transport of glucose. Our data suggest that T cells from RRMS patients display a reduced engagement of glycolysis and mitochondrial respiration, reversible upon IFN beta-1a treatment, thus suggesting an involvement of an altered metabolism in the pathogenesis of MS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells.

    PubMed

    Santo-Domingo, Jaime; Chareyron, Isabelle; Broenimann, Charlotte; Lassueur, Steve; Wiederkehr, Andreas

    2017-08-15

    Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed

  8. Decreased mitochondrial respiration in aneurysmal aortas of Fibulin-4 mutant mice is linked to PGC1A regulation.

    PubMed

    van der Pluijm, I; Burger, J; van Heijningen, P M; IJpma, A; van Vliet, N; Milanese, C; Schoonderwoerd, K; Sluiter, W; Ringuette, L J; Dekkers, D H W; Que, I; Kaijzel, E L; Te Riet, L; MacFarlane, E; Das, D; van der Linden, R; Vermeij, M; Demmers, J A; Mastroberardino, P G; Davis, E C; Yanagisawa, H; Dietz, H; Kanaar, R; Essers, J

    2018-06-21

    Thoracic aortic aneurysms are a life-threatening condition often diagnosed too late. To discover novel robust biomarkers, we aimed to better understand the molecular mechanisms underlying aneurysm formation. In Fibulin-4R/R mice, the extracellular matrix protein Fibulin-4 is 4-fold reduced, resulting in progressive ascending aneurysm formation and early death around 3 months of age. We performed proteomics and genomics studies on Fibulin-4R/R mouse aortas. Intriguingly, we observed alterations in mitochondrial protein composition in Fibulin-4R/R aortas. Consistently, functional studies in Fibulin-4R/R vascular smooth muscle cells (VSMCs) revealed lower oxygen consumption rates, but increased acidification rates. Yet, mitochondria in Fibulin-4R/R VSMCs showed no aberrant cytoplasmic localization. We found similar reduced mitochondrial respiration in Tgfbr-1M318R/+ VSMCs, a mouse model for Loeys-Dietz syndrome. Interestingly, also human fibroblasts from Marfan (FBN1) and Loeys-Dietz syndrome (TGFBR2 and SMAD3) patients showed lower oxygen consumption. While individual mitochondrial complex I-V activities were unaltered in Fibulin-4R/R heart and muscle, these tissues showed similar decreased oxygen consumption. Furthermore, aortas of aneurysmal Fibulin-4R/R mice displayed increased ROS levels. Consistent with these findings, gene expression analyses revealed dysregulation of metabolic pathways. Accordingly, blood ketone levels of Fibulin-4R/R mice were reduced and liver fatty acids were decreased, while liver glycogen was increased, indicating dysregulated metabolism at the organismal level. As predicted by gene expression analysis, the activity of PGC1α, a key regulator between mitochondrial function and organismal metabolism, was downregulated in Fibulin-4R/R VSMCs. Increased TGFβ reduced PGC1α levels, indicating involvement of TGFβ signalling in PGC1α regulation. Activation of PGC1α restored the decreased oxygen consumption in Fibulin-4R/R VSMCs and improved

  9. MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload.

    PubMed

    Ribeiro Junior, Rogério Faustino; Dabkowski, Erinne Rose; Shekar, Kadambari Chandra; O Connell, Kelly A; Hecker, Peter A; Murphy, Michael P

    2018-03-01

    Heart failure remains a major public-health problem with an increase in the number of patients worsening from this disease. Despite current medical therapy, the condition still has a poor prognosis. Heart failure is complex but mitochondrial dysfunction seems to be an important target to improve cardiac function directly. Our goal was to analyze the effects of MitoQ (100 µM in drinking water) on the development and progression of heart failure induced by pressure overload after 14 weeks. The main findings are that pressure overload-induced heart failure in rats decreased cardiac function in vivo that was not altered by MitoQ. However, we observed a reduction in right ventricular hypertrophy and lung congestion in heart failure animals treated with MitoQ. Heart failure also decreased total mitochondrial protein content, mitochondrial membrane potential in the intermyofibrillar mitochondria. MitoQ restored membrane potential in IFM but did not restore mitochondrial protein content. These alterations are associated with the impairment of basal and stimulated mitochondrial respiration in IFM and SSM induced by heart failure. Moreover, MitoQ restored mitochondrial respiration in heart failure induced by pressure overload. We also detected higher levels of hydrogen peroxide production in heart failure and MitoQ restored the increase in ROS production. MitoQ was also able to improve mitochondrial calcium retention capacity, mainly in the SSM whereas in the IFM we observed a small alteration. In summary, MitoQ improves mitochondrial dysfunction in heart failure induced by pressure overload, by decreasing hydrogen peroxide formation, improving mitochondrial respiration and improving mPTP opening. Published by Elsevier Inc.

  10. Cytochrome and Alternative Pathway Respiration in Green Algae 1

    PubMed Central

    Weger, Harold G.; Guy, Robert D.; Turpin, David H.

    1990-01-01

    Inhibitor titration curves and discrimination against 18O2 by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities. PMID:16667462

  11. γ-Tocotrienol Protects against Mitochondrial Dysfunction and Renal Cell Death

    PubMed Central

    Bakajsova, Diana; Hayes, Corey; Hauer-Jensen, Martin; Compadre, Cesar M.

    2012-01-01

    Oxidative stress is a major mechanism of a variety of renal diseases. Tocopherols and tocotrienols are well known antioxidants. This study aimed to determine whether γ-tocotrienol (GT3) protects against mitochondrial dysfunction and renal proximal tubular cell (RPTC) injury caused by oxidants. Primary cultures of RPTCs were injured by using tert-butyl hydroperoxide (TBHP) in the absence and presence of GT3 or α-tocopherol (AT). Reactive oxygen species (ROS) production increased 300% in TBHP-injured RPTCs. State 3 respiration, oligomycin-sensitive respiration, and respiratory control ratio (RCR) decreased 50, 63, and 47%, respectively. The number of RPTCs with polarized mitochondria decreased 54%. F0F1-ATPase activity and ATP content decreased 31 and 65%, respectively. Cell lysis increased from 3% in controls to 26 and 52% at 4 and 24 h, respectively, after TBHP exposure. GT3 blocked ROS production, ameliorated decreases in state 3 and oligomycin-sensitive respirations and F0F1-ATPase activity, and maintained RCR and mitochondrial membrane potential (ΔΨm) in injured RPTCs. GT3 maintained ATP content, blocked RPTC lysis at 4 h, and reduced it to 13% at 24 h after injury. Treatment with equivalent concentrations of AT did not block ROS production and cell lysis and moderately improved mitochondrial respiration and coupling. This is the first report demonstrating the protective effects of GT3 against RPTC injury by: 1) decreasing production of ROS, 2) improving mitochondrial respiration, coupling, ΔΨm, and F0F1-ATPase function, 3) maintaining ATP levels, and 4) preventing RPTC lysis. Our data suggest that GT3 is superior to AT in protecting RPTCs against oxidant injury and may prove therapeutically valuable for preventing renal injury associated with oxidative stress. PMID:22040679

  12. A nuclear mutation defective in mitochondrial recombination in yeast.

    PubMed

    Ling, F; Makishima, F; Morishima, N; Shibata, T

    1995-08-15

    Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

  13. Do Mitochondrial Replacement Techniques Affect Qualitative or Numerical Identity?

    PubMed

    Liao, S Matthew

    2017-01-01

    Mitochondrial replacement techniques (MRTs), known in the popular media as 'three-parent' or 'three-person' IVFs, have the potential to enable women with mitochondrial diseases to have children who are genetically related to them but without such diseases. In the debate regarding whether MRTs should be made available, an issue that has garnered considerable attention is whether MRTs affect the characteristics of an existing individual or whether they result in the creation of a new individual, given that MRTs involve the genetic manipulation of the germline. In other words, do MRTs affect the qualitative identity or the numerical identity of the resulting child? For instance, a group of panelists on behalf of the UK Human Fertilisation and Embryology Authority (HFEA) has claimed that MRTs affect only the qualitative identity of the resulting child, while the Working Group of the Nuffield Council on Bioethics (NCOB) has argued that MRTs would create a numerically distinct individual. In this article, I shall argue that MRTs do create a new and numerically distinct individual. Since my explanation is different from the NCOB's explanation, I shall also offer reasons why my explanation is preferable to the NCOB's explanation. © 2016 John Wiley & Sons Ltd.

  14. Direct effects of Vaccinium myrtillus L. fruit extracts on rat heart mitochondrial functions.

    PubMed

    Trumbeckaitė, S; Burdulis, D; Raudonė, L; Liobikas, J; Toleikis, A; Janulis, V

    2013-04-01

    In this study, the direct influence of bilberry (Vaccinium myrtillus) fruit extracts (aqueous and ethanolic) rich in anthocyanins on the oxidative phosphorylation of isolated rat heart mitochondria was investigated in vitro. Higher concentrations of bilberry extracts concentration-dependently inhibited mitochondrial state 3 respiration (by 23%-61%) with pyruvate plus malate, mildly (by 1.2- to 1.3-fold) uncoupled the oxidative phosphorylation, and increased (by 30%-87%) the state 4 respiration rate in the presence of exogenous cytochrome c. Succinate oxidation was less affected. Pure anthocyanins, the main components of used extracts, malvidin-3-glucoside, malvidin-3-galactoside, and cyanidin-3-galactoside, had no effect on oxidation of pyruvate plus malate. A statistically significant decrease in H2 O2 production by mitochondria was found in the presence of bilberry fruit extracts. Our findings show that bilberry fruit anthocyanin-rich extracts possess direct effects on rat heart mitochondrial function in vitro. These findings give the first insights into the mechanism(s) of their action on cellular energy metabolism. Copyright © 2012 John Wiley & Sons, Ltd.

  15. Does the increased air humidity affect soil respiration and carbon stocks?

    NASA Astrophysics Data System (ADS)

    Kukumägi, Mai; Celi, Luisella; Said-Pullicino, Daniel; Kupper, Priit; Sõber, Jaak; Lõhmus, Krista; Kutti, Sander; Ostonen, Ivika

    2013-04-01

    contents of the more stable MOM. These results strongly suggest that, apart from the predicted increase in temperature and atmospheric carbon and nitrogen concentrations, an increase in free air humidity as a result of climate change may significantly influence the complex belowground carbon cycling by affecting biomass production, soil respiration and organic matter turnover.

  16. Long-term rates of mitochondrial protein synthesis are increased in mouse skeletal muscle with high-fat feeding regardless of insulin-sensitizing treatment.

    PubMed

    Newsom, Sean A; Miller, Benjamin F; Hamilton, Karyn L; Ehrlicher, Sarah E; Stierwalt, Harrison D; Robinson, Matthew M

    2017-11-01

    Skeletal muscle mitochondrial protein synthesis is regulated in part by insulin. The development of insulin resistance with diet-induced obesity may therefore contribute to impairments to protein synthesis and decreased mitochondrial respiration. Yet the impact of diet-induced obesity and insulin resistance on mitochondrial energetics is controversial, with reports varying from decreases to increases in mitochondrial respiration. We investigated the impact of changes in insulin sensitivity on long-term rates of mitochondrial protein synthesis as a mechanism for changes to mitochondrial respiration in skeletal muscle. Insulin resistance was induced in C57BL/6J mice using 4 wk of a high-fat compared with a low-fat diet. For 8 additional weeks, diets were enriched with pioglitazone to restore insulin sensitivity compared with nonenriched control low-fat or high-fat diets. Skeletal muscle mitochondrial protein synthesis was measured using deuterium oxide labeling during weeks 10-12 High-resolution respirometry was performed using palmitoyl-l-carnitine, glutamate+malate, and glutamate+malate+succinate as substrates for mitochondria isolated from quadriceps. Mitochondrial protein synthesis and palmitoyl- l-carnitine oxidation were increased in mice consuming a high-fat diet, regardless of differences in insulin sensitivity with pioglitazone treatment. There was no effect of diet or pioglitazone treatment on ADP-stimulated respiration or H 2 O 2 emission using glutamate+malate or glutamate+malate+succinate. The results demonstrate no impairments to mitochondrial protein synthesis or respiration following induction of insulin resistance. Instead, mitochondrial protein synthesis was increased with a high-fat diet and may contribute to remodeling of the mitochondria to increase lipid oxidation capacity. Mitochondrial adaptations with a high-fat diet appear driven by nutrient availability, not intrinsic defects that contribute to insulin resistance. Copyright © 2017 the

  17. Time-controlled fasting prevents aging-like mitochondrial changes induced by persistent dietary fat overload in skeletal muscle

    PubMed Central

    Lettieri-Barbato, Daniele; Cannata, Stefano Maria; Casagrande, Viviana; Ciriolo, Maria Rosa

    2018-01-01

    A large body of evidence suggests that persistent dietary fat overload causes mitochondrial dysfunction and systemic metabolic gridlock. Mitochondrial and lipid metabolism in skeletal muscle (SkM) are severely affected upon persistent high fat diet (HFD) leading to premature tissue aging. Here, we designed weekly cycles of fasting (called as time-controlled fasting, TCF) and showed that they were effective in limiting mitochondrial damage and metabolic disturbances induced by HFD. Specifically, TCF was able to prevent the decline of adipose triglyceride lipase (Atgl), maintain efficient mitochondrial respiration in SkM as well as improve blood glucose and lipid profile. Atgl was found to be the mediator of such preventive effects as its downregulation or up-regulation in C2C12 myotubes triggers mitochondrial alteration or protects against the deleterious effects of high fat levels respectively. In conclusion, TCF could represent an effective strategy to limit mitochondrial impairment and metabolic inflexibility that are typically induced by modern western diets or during aging. PMID:29742122

  18. Time-controlled fasting prevents aging-like mitochondrial changes induced by persistent dietary fat overload in skeletal muscle.

    PubMed

    Lettieri-Barbato, Daniele; Cannata, Stefano Maria; Casagrande, Viviana; Ciriolo, Maria Rosa; Aquilano, Katia

    2018-01-01

    A large body of evidence suggests that persistent dietary fat overload causes mitochondrial dysfunction and systemic metabolic gridlock. Mitochondrial and lipid metabolism in skeletal muscle (SkM) are severely affected upon persistent high fat diet (HFD) leading to premature tissue aging. Here, we designed weekly cycles of fasting (called as time-controlled fasting, TCF) and showed that they were effective in limiting mitochondrial damage and metabolic disturbances induced by HFD. Specifically, TCF was able to prevent the decline of adipose triglyceride lipase (Atgl), maintain efficient mitochondrial respiration in SkM as well as improve blood glucose and lipid profile. Atgl was found to be the mediator of such preventive effects as its downregulation or up-regulation in C2C12 myotubes triggers mitochondrial alteration or protects against the deleterious effects of high fat levels respectively. In conclusion, TCF could represent an effective strategy to limit mitochondrial impairment and metabolic inflexibility that are typically induced by modern western diets or during aging.

  19. Resveratrol stimulates mitochondrial fusion by a mechanism requiring mitofusin-2.

    PubMed

    Robb, Ellen L; Moradi, Fereshteh; Maddalena, Lucas A; Valente, Andrew J F; Fonseca, Joao; Stuart, Jeffrey A

    2017-04-01

    Resveratrol (RES) is a plant-derived stilbene associated with a wide range of health benefits. Mitochondria are a key downstream target of RES, and in some cell types RES promotes mitochondrial biogenesis, altered cellular redox status, and a shift toward oxidative metabolism. Mitochondria exist as a dynamic network that continually remodels via fusion and fission processes, and the extent of fusion is related to cellular redox status and metabolism. We investigated RES's effects on mitochondrial network morphology in several cell lines using a quantitative approach to measure the extent of network fusion. 48 h continuous treatment with 10-20 μM RES stimulated mitochondrial fusion in C2C12 myoblasts, PC3 cancer cells, and mouse embryonic fibroblasts stimulated significant increases in fusion in all instances, resulting in larger and more highly branched mitochondrial networks. Mitofusin-2 (Mfn2) is a key protein facilitating mitochondrial fusion, and its expression was also stimulated by RES. Using Mfn2-null cells we demonstrated that RES's effects on mitochondrial fusion, cellular respiration rates, and cell growth are all dependent upon the presence of Mfn2. Taken together, these results demonstrate that Mfn2 and mitochondrial fusion are affected by RES in ways that appear to relate to RES's known effects on cellular metabolism and growth. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. The methyl donor S-adenosylmethionine prevents liver hypoxia and dysregulation of mitochondrial bioenergetic function in a rat model of alcohol-induced fatty liver disease.

    PubMed

    King, Adrienne L; Mantena, Sudheer K; Andringa, Kelly K; Millender-Swain, Telisha; Dunham-Snary, Kimberly J; Oliva, Claudia R; Griguer, Corinne E; Bailey, Shannon M

    2016-10-01

    Mitochondrial dysfunction and bioenergetic stress play an important role in the etiology of alcoholic liver disease. Previous studies from our laboratory show that the primary methyl donor S-Adenosylmethionine (SAM) minimizes alcohol-induced disruptions in several mitochondrial functions in the liver. Herein, we expand on these earlier observations to determine whether the beneficial actions of SAM against alcohol toxicity extend to changes in the responsiveness of mitochondrial respiration to inhibition by nitric oxide (NO), induction of the mitochondrial permeability transition (MPT) pore, and the hypoxic state of the liver. For this, male Sprague-Dawley rats were pair-fed control and alcohol-containing liquid diets with and without SAM for 5 weeks and liver hypoxia, mitochondrial respiration, MPT pore induction, and NO-dependent control of respiration were examined. Chronic alcohol feeding significantly enhanced liver hypoxia, whereas SAM supplementation attenuated hypoxia in livers of alcohol-fed rats. SAM supplementation prevented alcohol-mediated decreases in mitochondrial state 3 respiration and cytochrome c oxidase activity. Mitochondria isolated from livers of alcohol-fed rats were more sensitive to calcium-mediated MPT pore induction (i.e., mitochondrial swelling) than mitochondria from pair-fed controls, whereas SAM treatment normalized sensitivity for calcium-induced swelling in mitochondria from alcohol-fed rats. Liver mitochondria from alcohol-fed rats showed increased sensitivity to NO-dependent inhibition of respiration compared with pair-fed controls. In contrast, mitochondria isolated from the livers of SAM treated alcohol-fed rats showed no change in the sensitivity to NO-mediated inhibition of respiration. Collectively, these findings indicate that the hepato-protective effects of SAM against alcohol toxicity are mediated, in part, through a mitochondrial mechanism involving preservation of key mitochondrial bioenergetic parameters and the

  1. Effects of different acute high ambient temperatures on function of hepatic mitochondrial respiration, antioxidative enzymes, and oxidative injury in broiler chickens.

    PubMed

    Tan, G-Y; Yang, L; Fu, Y-Q; Feng, J-H; Zhang, M-H

    2010-01-01

    This study investigated the effects of different acute high ambient temperatures on dysfunction of hepatic mitochondrial respiration, the antioxidative enzyme system, and oxidative injury in broiler chickens. One hundred twenty-eight 6-wk-old broiler chickens were assigned randomly to 4 groups and subsequently exposed to 25 (control), 32, 35, and 38 degrees C (RH, 70 +/- 5%) for 3 h, respectively. The rectal temperatures, activity of antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase), content of malondialdehyde and protein carbonyl, and the activity of mitochondrial respiratory enzymes were determined. The results showed that exposure to high ambient temperature induced a significant elevation of rectal temperature, antioxidative enzyme activity, and formation of malondialdehyde and protein carbonyl, as well as dysfunction of the mitochondrial respiratory chain in comparison with control (P < 0.05). Almost all of the indicators changed in a temperature-dependent manner with the gradual increase of ambient temperature from 32 to 38 degrees C; differences in each parameter (except catalase) among the groups exposed to different high ambient temperatures were also statistically significant (P < 0.05). The results of the present study suggest that, in the broiler chicken model used here, acute exposure to high temperatures may depress the activity of the mitochondrial respiratory chain. This inactivation results subsequently in overproduction of reactive oxygen species, which ultimately results in oxidative injury. However, this hypothesis needs to be evaluated more rigorously in future studies. It has also been shown that, with the gradual increase in temperature, the oxidative injury induced by heat stress in broiler chickens becomes increasingly severe, and this stress response presents in a temperature-dependent manner in the temperature range of 32 to 38 degrees C.

  2. Early mitochondrial dysfunction in glycolytic muscle, but not oxidative muscle, of the fructose-fed insulin-resistant rat

    PubMed Central

    Warren, Blair E.; Lou, Phing-How; Lucchinetti, Eliana; Zhang, Liyan; Clanachan, Alexander S.; Affolter, Andreas; Hersberger, Martin; Zaugg, Michael

    2014-01-01

    Although evidence that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle has been accumulating, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I and II respiration in the presence of pyruvate but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional β-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity, and increased fatty acid oxidation, as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was reduced exclusively in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. Here, we show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle. PMID:24425766

  3. Supporting aspartate biosynthesis is an essential function of respiration in proliferating cells

    PubMed Central

    Sullivan, Lucas B.; Gui, Dan Y.; Hosios, Aaron M.; Bush, Lauren N.; Freinkman, Elizaveta; Vander Heiden, Matthew G.

    2015-01-01

    Summary Mitochondrial respiration is important for cell proliferation, however the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. PMID:26232225

  4. Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

    PubMed

    Sullivan, Lucas B; Gui, Dan Y; Hosios, Aaron M; Bush, Lauren N; Freinkman, Elizaveta; Vander Heiden, Matthew G

    2015-07-30

    Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Pollen tube energetics: respiration, fermentation and the race to the ovule

    PubMed Central

    Rounds, Caleb M.; Winship, Lawrence J.; Hepler, Peter K.

    2011-01-01

    Background Pollen tubes grow by transferring chemical energy from stored cellular starch and newly assimilated sugars into ATP. This drives myriad processes essential for cell elongation, directly or through the creation of ion gradients. Respiration plays a central role in generating and regulating this energy flow and thus in the success of plant reproduction. Pollen tubes are easily grown in vitro and have become an excellent model for investigating the contributions of respiration to plant cellular growth and morphogenesis at the molecular, biochemical and physiological levels. Scope In recent decades, pollen tube research has become increasingly focused on the molecular mechanisms involved in cellular processes. Yet, effective growth and development requires an intact, integrated set of cellular processes, all supplied with a constant flow of energy. Here we bring together information from the current and historical literature concerning respiration, fermentation and mitochondrial physiology in pollen tubes, and assess the significance of more recent molecular and genetic investigations in a physiological context. Conclusions The rapid growth of the pollen tube down the style has led to the evolution of high rates of pollen tube respiration. Respiration rates in lily predict a total energy turnover of 40–50 fmol ATP s−1 per pollen grain. Within this context we examine the energetic requirements of cell wall synthesis, osmoregulation, actin dynamics and cyclosis. At present, we can only estimate the amount of energy required, because data from growing pollen tubes are not available. In addition to respiration, we discuss fermentation and mitochondrial localization. We argue that the molecular pathways need to be examined within the physiological context to understand better the mechanisms that control tip growth in pollen tubes. PMID:22476489

  6. Long-term fasting decreases mitochondrial avian UCP-mediated oxygen consumption in hypometabolic king penguins

    PubMed Central

    Rey, Benjamin; Halsey, Lewis G.; Dolmazon, Virginie; Rouanet, Jean-Louis; Roussel, Damien; Handrich, Yves; Butler, Patrick J.; Duchamp, Claude

    2008-01-01

    In endotherms, regulation of the degree of mitochondrial coupling affects cell metabolic efficiency. Thus it may be a key contributor to minimizing metabolic rate during long periods of fasting. The aim of the present study was to investigate whether variation in mitochondrial avian uncoupling proteins (avUCP), as putative regulators of mitochondrial oxidative phosphorylation, may contribute to the ability of king penguins (Aptenodytes patagonicus) to withstand fasting for several weeks. After 20 days of fasting, king penguins showed a reduced rate of whole animal oxygen consumption (V̇o2; −33%) at rest, together with a reduced abundance of avUCP and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1-α) mRNA in pectoralis muscle (−54%, −36%, respectively). These parameters were restored after the birds had been refed for 3 days. Furthermore, in recently fed, but not in fasted penguins, isolated muscle mitochondria showed a guanosine diphosphate-inhibited, fatty acid plus superoxide-activated respiration, indicating the presence of a functional UCP. It was calculated that variation in mitochondrial UCP-dependent respiration in vitro may contribute to nearly 20% of the difference in resting V̇o2 between fed or refed penguins and fasted penguins measured in vivo. These results suggest that the lowering of avUCP activity during periods of long-term energetic restriction may contribute to the reduction in metabolic rate and hence the ability of king penguins to face prolonged periods of fasting. PMID:18495832

  7. Long-term fasting decreases mitochondrial avian UCP-mediated oxygen consumption in hypometabolic king penguins.

    PubMed

    Rey, Benjamin; Halsey, Lewis G; Dolmazon, Virginie; Rouanet, Jean-Louis; Roussel, Damien; Handrich, Yves; Butler, Patrick J; Duchamp, Claude

    2008-07-01

    In endotherms, regulation of the degree of mitochondrial coupling affects cell metabolic efficiency. Thus it may be a key contributor to minimizing metabolic rate during long periods of fasting. The aim of the present study was to investigate whether variation in mitochondrial avian uncoupling proteins (avUCP), as putative regulators of mitochondrial oxidative phosphorylation, may contribute to the ability of king penguins (Aptenodytes patagonicus) to withstand fasting for several weeks. After 20 days of fasting, king penguins showed a reduced rate of whole animal oxygen consumption (Vo2; -33%) at rest, together with a reduced abundance of avUCP and peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC1-alpha) mRNA in pectoralis muscle (-54%, -36%, respectively). These parameters were restored after the birds had been refed for 3 days. Furthermore, in recently fed, but not in fasted penguins, isolated muscle mitochondria showed a guanosine diphosphate-inhibited, fatty acid plus superoxide-activated respiration, indicating the presence of a functional UCP. It was calculated that variation in mitochondrial UCP-dependent respiration in vitro may contribute to nearly 20% of the difference in resting Vo2 between fed or refed penguins and fasted penguins measured in vivo. These results suggest that the lowering of avUCP activity during periods of long-term energetic restriction may contribute to the reduction in metabolic rate and hence the ability of king penguins to face prolonged periods of fasting.

  8. Disruption of mitochondrial electron transport chain function potentiates the pro-apoptotic effects of MAPK inhibition.

    PubMed

    Trotta, Andrew P; Gelles, Jesse D; Serasinghe, Madhavika N; Loi, Patrick; Arbiser, Jack L; Chipuk, Jerry E

    2017-07-14

    The mitochondrial network is a major site of ATP production through the coupled integration of the electron transport chain (ETC) with oxidative phosphorylation. In melanoma arising from the V600E mutation in the kinase v-RAF murine sarcoma viral oncogene homolog B (BRAF V600E ), oncogenic signaling enhances glucose-dependent metabolism while reducing mitochondrial ATP production. Likewise, when BRAF V600E is pharmacologically inhibited by targeted therapies ( e.g. PLX-4032/vemurafenib), glucose metabolism is reduced, and cells increase mitochondrial ATP production to sustain survival. Therefore, collateral inhibition of oncogenic signaling and mitochondrial respiration may help enhance the therapeutic benefit of targeted therapies. Honokiol (HKL) is a well tolerated small molecule that disrupts mitochondrial function; however, its underlying mechanisms and potential utility with targeted anticancer therapies remain unknown. Using wild-type BRAF and BRAF V600E melanoma model systems, we demonstrate here that HKL administration rapidly reduces mitochondrial respiration by broadly inhibiting ETC complexes I, II, and V, resulting in decreased ATP levels. The subsequent energetic crisis induced two cellular responses involving cyclin-dependent kinases (CDKs). First, loss of CDK1-mediated phosphorylation of the mitochondrial division GTPase dynamin-related protein 1 promoted mitochondrial fusion, thus coupling mitochondrial energetic status and morphology. Second, HKL decreased CDK2 activity, leading to G 1 cell cycle arrest. Importantly, although pharmacological inhibition of oncogenic MAPK signaling increased ETC activity, co-treatment with HKL ablated this response and vastly enhanced the rate of apoptosis. Collectively, these findings integrate HKL action with mitochondrial respiration and shape and substantiate a pro-survival role of mitochondrial function in melanoma cells after oncogenic MAPK inhibition.

  9. Mitochondrial divergence between slow- and fast-aging garter snakes.

    PubMed

    Schwartz, Tonia S; Arendsee, Zebulun W; Bronikowski, Anne M

    2015-11-01

    Mitochondrial function has long been hypothesized to be intimately involved in aging processes--either directly through declining efficiency of mitochondrial respiration and ATP production with advancing age, or indirectly, e.g., through increased mitochondrial production of damaging free radicals with age. Yet we lack a comprehensive understanding of the evolution of mitochondrial genotypes and phenotypes across diverse animal models, particularly in species that have extremely labile physiology. Here, we measure mitochondrial genome-types and transcription in ecotypes of garter snakes (Thamnophis elegans) that are adapted to disparate habitats and have diverged in aging rates and lifespans despite residing in close proximity. Using two RNA-seq datasets, we (1) reconstruct the garter snake mitochondrial genome sequence and bioinformatically identify regulatory elements, (2) test for divergence of mitochondrial gene expression between the ecotypes and in response to heat stress, and (3) test for sequence divergence in mitochondrial protein-coding regions in these slow-aging (SA) and fast-aging (FA) naturally occurring ecotypes. At the nucleotide sequence level, we confirmed two (duplicated) mitochondrial control regions one of which contains a glucocorticoid response element (GRE). Gene expression of protein-coding genes was higher in FA snakes relative to SA snakes for most genes, but was neither affected by heat stress nor an interaction between heat stress and ecotype. SA and FA ecotypes had unique mitochondrial haplotypes with amino acid substitutions in both CYTB and ND5. The CYTB amino acid change (Isoleucine → Threonine) was highly segregated between ecotypes. This divergence of mitochondrial haplotypes between SA and FA snakes contrasts with nuclear gene-flow estimates, but correlates with previously reported divergence in mitochondrial function (mitochondrial oxygen consumption, ATP production, and reactive oxygen species consequences). Copyright © 2015

  10. GPA protects the nigrostriatal dopamine system by enhancing mitochondrial function.

    PubMed

    Horvath, Tamas L; Erion, Derek M; Elsworth, John D; Roth, Robert H; Shulman, Gerald I; Andrews, Zane B

    2011-07-01

    Guanidinopropionic acid (GPA) increases AMPK activity, mitochondrial function and biogenesis in muscle and improves physiological function, for example during aging. Mitochondrial dysfunction is a major contributor to the pathogenesis of Parkinson's disease. Here we tested whether GPA prevents neurodegeneration of the nigrostriatal dopamine system in MPTP-treated mice. Mice were fed a diet of 1% GPA or normal chow for 4 weeks and then treated with either MPTP or saline. Indices of nigrostriatal function were examined by HPLC, immunohistochemistry, stereology, electron microscopy and mitochondrial respiration. MPTP intoxication decreased TH neurons in the SNpc of normal chow-fed mice; however GPA-fed mice remarkably exhibited no loss of TH neurons in the SNpc. MPTP caused a decrease in striatal dopamine of both normal chow- and GPA-fed mice, although this effect was significantly attenuated in GPA-fed mice. GPA-fed mice showed increased AMPK activity, mitochondrial respiration and mitochondrial number in nigrostriatal TH neurons, suggesting that the neuroprotective effects of GPA involved AMPK-dependent increases in mitochondrial function and biogenesis. MPTP treatment produced a decrease in mitochondrial number and volume in normal chow-fed mice but not GPA-fed mice. Our results show the neuroprotective properties of GPA in a mouse model of Parkinson's disease are partially mediated by AMPK and mitochondrial function. Mitochondrial dysfunction is a common problem in neurodegeneration and thus GPA may slow disease progression in other models of neurodegeneration. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. Rat liver mitochondrial dysfunction by addition of copper(II) or iron(III) ions.

    PubMed

    Saporito-Magriñá, Christian; Musacco-Sebio, Rosario; Acosta, Juan M; Bajicoff, Sofía; Paredes-Fleitas, Paola; Boveris, Alberto; Repetto, Marisa G

    2017-01-01

    Increased copper (Cu) and iron (Fe) levels in liver and brain are associated to oxidative stress and damage with increased phospholipid oxidation process. The aim of this work was to assess the toxic effects of Cu 2+ and Fe 3+ addition to rat liver mitochondria by determining mitochondrial respiration in states 3 (active respiration) and 4 (resting respiration), and phospholipid peroxidation. Both, Cu 2+ and Fe 3+ produced decreases in O 2 consumption in a concentration-dependent manner in active state 3: both ions by 42% with malate-glutamate as complex I substrate (concentration for half maximal response (C 50 ) 60μM Cu 2+ and 1.25mM Fe 3+ ), and with succinate as complex II substrate: 64-69% with C 50 of 50μM Cu 2+ and with C 50 of 1.25mM of Fe 3+ . Respiratory control decreased with Cu 2+ (C 50 50μM) and Fe 3+ (C 50 1.25-1-75mM) with both substrates. Cu 2+ produced a 2-fold increase and Fe 3+ a 5-fold increase of thiobarbituric acid-reactive substances (TBARS) content from 25μM Cu 2+ (C 50 40μM) and from 100μM Fe 3+ (C 50 1.75mM). Supplementations with Cu 2+ and Fe 3+ ions induce mitochondrial dysfunction with phospholipid peroxidation in rat liver mitochondria. Although is proved that a Fenton/Haber Weiss mechanism of oxidative damage occurs in metal-ion induced mitochondrial toxicity, slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. The decreased rates of mitochondrial respiration and the alteration of mitochondrial function by phospholipid and protein oxidations lead to mitochondrial dysfunction, cellular dyshomeostasis and cell death. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration

    PubMed Central

    Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

    2018-01-01

    The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders. PMID:29451229

  13. m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

    PubMed

    Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

    2018-03-01

    The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

  14. Mitochondrial fatty acid synthesis, fatty acids and mitochondrial physiology.

    PubMed

    Kastaniotis, Alexander J; Autio, Kaija J; Kerätär, Juha M; Monteuuis, Geoffray; Mäkelä, Anne M; Nair, Remya R; Pietikäinen, Laura P; Shvetsova, Antonina; Chen, Zhijun; Hiltunen, J Kalervo

    2017-01-01

    Mitochondria and fatty acids are tightly connected to a multiplicity of cellular processes that go far beyond mitochondrial fatty acid metabolism. In line with this view, there is hardly any common metabolic disorder that is not associated with disturbed mitochondrial lipid handling. Among other aspects of mitochondrial lipid metabolism, apparently all eukaryotes are capable of carrying out de novo fatty acid synthesis (FAS) in this cellular compartment in an acyl carrier protein (ACP)-dependent manner. The dual localization of FAS in eukaryotic cells raises the questions why eukaryotes have maintained the FAS in mitochondria in addition to the "classic" cytoplasmic FAS and what the products are that cannot be substituted by delivery of fatty acids of extramitochondrial origin. The current evidence indicates that mitochondrial FAS is essential for cellular respiration and mitochondrial biogenesis. Although both β-oxidation and FAS utilize thioester chemistry, CoA acts as acyl-group carrier in the breakdown pathway whereas ACP assumes this role in the synthetic direction. This arrangement metabolically separates these two pathways running towards opposite directions and prevents futile cycling. A role of this pathway in mitochondrial metabolic sensing has recently been proposed. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Immortalized Parkinson's disease lymphocytes have enhanced mitochondrial respiratory activity

    PubMed Central

    Annesley, Sarah J.; Lay, Sui T.; De Piazza, Shawn W.; Sanislav, Oana; Hammersley, Eleanor; Allan, Claire Y.; Francione, Lisa M.; Bui, Minh Q.; Chen, Zhi-Ping; Ngoei, Kevin R. W.; Tassone, Flora; Kemp, Bruce E.; Storey, Elsdon; Evans, Andrew; Loesch, Danuta Z.

    2016-01-01

    ABSTRACT In combination with studies of post-mortem Parkinson's disease (PD) brains, pharmacological and genetic models of PD have suggested that two fundamental interacting cellular processes are impaired – proteostasis and mitochondrial respiration. We have re-examined the role of mitochondrial dysfunction in lymphoblasts isolated from individuals with idiopathic PD and an age-matched control group. As previously reported for various PD cell types, the production of reactive oxygen species (ROS) by PD lymphoblasts was significantly elevated. However, this was not due to an impairment of mitochondrial respiration, as is often assumed. Instead, basal mitochondrial respiration and ATP synthesis are dramatically elevated in PD lymphoblasts. The mitochondrial mass, genome copy number and membrane potential were unaltered, but the expression of indicative respiratory complex proteins was also elevated. This explains the increased oxygen consumption rates by each of the respiratory complexes in experimentally uncoupled mitochondria of iPD cells. However, it was not attributable to increased activity of the stress- and energy-sensing protein kinase AMPK, a regulator of mitochondrial biogenesis and activity. The respiratory differences between iPD and control cells were sufficiently dramatic as to provide a potentially sensitive and reliable biomarker of the disease state, unaffected by disease duration (time since diagnosis) or clinical severity. Lymphoblasts from control and PD individuals thus occupy two distinct, quasi-stable steady states; a ‘normal’ and a ‘hyperactive’ state characterized by two different metabolic rates. The apparent stability of the ‘hyperactive’ state in patient-derived lymphoblasts in the face of patient ageing, ongoing disease and mounting disease severity suggests an early, permanent switch to an alternative metabolic steady state. With its associated, elevated ROS production, the ‘hyperactive’ state might not cause pathology

  16. Regulation by magnesium of potato tuber mitochondrial respiratory activities.

    PubMed

    Vicente, Joaquim A F; Madeira, Vítor M C; Vercesi, Anibal E

    2004-12-01

    Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg2+. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg2+ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg2+. However, respiration of malate, citrate, and alpha-ketoglutarate requires at least 4-mM Mg2+ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg2+ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or alpha-ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg2+. Respiration of succinate is active without external and matrix Mg2+, although stimulated by the cation. Respiration of alpha-ketoglutarate was strictly dependent on external Mg2+ required for substrate transport into mitochondria, and internal Mg2+ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg2+ but, unlike alpha-ketoglutarate, some activity still remains without external Mg2+. All the substrates revealed insensitive to external and internal mitochondrial Ca2+, except the exogenous NADH dehydrogenase, which requires either external Ca2+ or Mg2+ for detectable activity. Calcium is more efficient than Mg2+, both having cumulative stimulation. Unlike Ca2+, Mn2+ could substitute for Mg2+, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg2+.

  17. Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death.

    PubMed

    Lu, Yapeng; Liu, Siyuan; Wang, Ying; Wang, Dang; Gao, Jing; Zhu, Li

    2016-09-05

    Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca(2+) release from Ca(2+)-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca(2+)-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca(2+) release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Understanding D-Ribose and Mitochondrial Function.

    PubMed

    Mahoney, Diane E; Hiebert, John B; Thimmesch, Amanda; Pierce, John T; Vacek, James L; Clancy, Richard L; Sauer, Andrew J; Pierce, Janet D

    2018-01-01

    Mitochondria are important organelles referred to as cellular powerhouses for their unique properties of cellular energy production. With many pathologic conditions and aging, mitochondrial function declines, and there is a reduction in the production of adenosine triphosphate. The energy carrying molecule generated by cellular respiration and by pentose phosphate pathway, an alternative pathway of glucose metabolism. D-ribose is a naturally occurring monosaccharide found in the cells and particularly in the mitochondria is essential in energy production. Without sufficient energy, cells cannot maintain integrity and function. Supplemental D-ribose has been shown to improve cellular processes when there is mitochondrial dysfunction. When individuals take supplemental D-ribose, it can bypass part of the pentose pathway to produce D-ribose-5-phosphate for the production of energy. In this article, we review how energy is produced by cellular respiration, the pentose pathway, and the use of supplemental D-ribose.

  19. Human mitochondrial DNA replication machinery and disease

    PubMed Central

    Young, Matthew J.; Copeland, William C.

    2016-01-01

    The human mitochondrial genome is replicated by DNA polymerase γ in concert with key components of the mitochondrial DNA (mtDNA) replication machinery. Defects in mtDNA replication or nucleotide metabolism cause deletions, point mutations, or depletion of mtDNA. The resulting loss of cellular respiration ultimately induces mitochondrial genetic diseases, including mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. Here we review the current literature regarding human mtDNA replication and heritable disorders caused by genetic changes of the POLG, POLG2, Twinkle, RNASEH1, DNA2 and MGME1 genes. PMID:27065468

  20. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: Unravelling the role of Mg2+ in cell respiration

    PubMed Central

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-01-01

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg2+ concentrations must be considered as well. Here we developed in vivo/in vitro techniques using 31P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg2+ concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg2+ in the mitochondrial matrix, where [Mg2+] is tenfold higher. In contrast, owing to a much higher affinity for Mg2+, ATP is mostly complexed by Mg2+ in both compartments. Mg2+ starvation used to alter cytosolic and mitochondrial [Mg2+] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg2+ concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis. PMID:25313036

  1. Interplay of Mg2+, ADP, and ATP in the cytosol and mitochondria: unravelling the role of Mg2+ in cell respiration.

    PubMed

    Gout, Elisabeth; Rébeillé, Fabrice; Douce, Roland; Bligny, Richard

    2014-10-28

    In animal and plant cells, the ATP/ADP ratio and/or energy charge are generally considered key parameters regulating metabolism and respiration. The major alternative issue of whether the cytosolic and mitochondrial concentrations of ADP and ATP directly mediate cell respiration remains unclear, however. In addition, because only free nucleotides are exchanged by the mitochondrial ADP/ATP carrier, whereas MgADP is the substrate of ATP synthase (EC 3.6.3.14), the cytosolic and mitochondrial Mg(2+) concentrations must be considered as well. Here we developed in vivo/in vitro techniques using (31)P-NMR spectroscopy to simultaneously measure these key components in subcellular compartments. We show that heterotrophic sycamore (Acer pseudoplatanus L.) cells incubated in various nutrient media contain low, stable cytosolic ADP and Mg(2+) concentrations, unlike ATP. ADP is mainly free in the cytosol, but complexed by Mg(2+) in the mitochondrial matrix, where [Mg(2+)] is tenfold higher. In contrast, owing to a much higher affinity for Mg(2+), ATP is mostly complexed by Mg(2+) in both compartments. Mg(2+) starvation used to alter cytosolic and mitochondrial [Mg(2+)] reversibly increases free nucleotide concentration in the cytosol and matrix, enhances ADP at the expense of ATP, decreases coupled respiration, and stops cell growth. We conclude that the cytosolic ADP concentration, and not ATP, ATP/ADP ratio, or energy charge, controls the respiration of plant cells. The Mg(2+) concentration, remarkably constant and low in the cytosol and tenfold higher in the matrix, mediates ADP/ATP exchange between the cytosol and matrix, [MgADP]-dependent mitochondrial ATP synthase activity, and cytosolic free ADP homeostasis.

  2. Can plant phloem properties affect the link between ecosystem assimilation and respiration?

    NASA Astrophysics Data System (ADS)

    Mencuccini, M.; Hölttä, T.; Sevanto, S.; Nikinmaa, E.

    2012-04-01

    Phloem transport of carbohydrates in plants under field conditions is currently not well understood. This is largely the result of the lack of techniques suitable for measuring phloem physiological properties continuously under field conditions. This lack of knowledge is currently hampering our efforts to link ecosystem-level processes of carbon fixation, allocation and use, especially belowground. On theoretical grounds, the properties of the transport pathway from canopy to roots must be important in affecting the link between carbon assimilation and respiration, but it is unclear whether their effect is partially or entirely masked by processes occurring in other parts of the ecosystem. One can also predict the characteristic time scales over which these effects should occur and, as consequence, predict whether the transfer of turgor and osmotic signals from the site of carbon assimilation to the sites of carbon use are likely to control respiration. We will present two sources of evidence suggesting that the properties of the phloem transport system may affect processes that are dependent on the supply of carbon substrate, such as root or soil respiration. Firstly, we will summarize the results of a literature survey on soil and ecosystem respiration where the speed of transfer of photosynthetic sugars from the plant canopy to the soil surface was determined. Estimates of the transfer speed could be grouped according to whether the study employed isotopic or canopy soil flux-based techniques. These two groups provided very different estimates of transfer times likely because transport of sucrose molecules, and pressure-concentration waves, in phloem differed. Secondly, we will argue that simultaneous measurements of bark and xylem diameters provide a novel tool to determine the continuous variations of phloem turgor in vivo in the field. We will present a model that interprets these changes in xylem and live bark diameters and present data testing the model

  3. Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases

    PubMed Central

    DeBalsi, Karen L.; Hoff, Kirsten E.; Copeland, William C.

    2016-01-01

    As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined. PMID:27143693

  4. Atmospheric CO2 mole fraction affects stand-scale carbon use efficiency of sunflower by stimulating respiration in light.

    PubMed

    Gong, Xiao Ying; Schäufele, Rudi; Lehmeier, Christoph Andreas; Tcherkez, Guillaume; Schnyder, Hans

    2017-03-01

    Plant carbon-use-efficiency (CUE), a key parameter in carbon cycle and plant growth models, quantifies the fraction of fixed carbon that is converted into net primary production rather than respired. CUE has not been directly measured, partly because of the difficulty of measuring respiration in light. Here, we explore if CUE is affected by atmospheric CO 2 . Sunflower stands were grown at low (200 μmol mol -1 ) or high CO 2 (1000 μmol mol -1 ) in controlled environment mesocosms. CUE of stands was measured by dynamic stand-scale 13 C labelling and partitioning of photosynthesis and respiration. At the same plant age, growth at high CO 2 (compared with low CO 2 ) led to 91% higher rates of apparent photosynthesis, 97% higher respiration in the dark, yet 143% higher respiration in light. Thus, CUE was significantly lower at high (0.65) than at low CO 2 (0.71). Compartmental analysis of isotopic tracer kinetics demonstrated a greater commitment of carbon reserves in stand-scale respiratory metabolism at high CO 2 . Two main processes contributed to the reduction of CUE at high CO 2 : a reduced inhibition of leaf respiration by light and a diminished leaf mass ratio. This work highlights the relevance of measuring respiration in light and assessment of the CUE response to environment conditions. © 2016 John Wiley & Sons Ltd.

  5. Associations between fatty acid oxidation, hepatic mitochondrial function, and plasma acylcarnitine levels in mice.

    PubMed

    Bjørndal, Bodil; Alterås, Eva Katrine; Lindquist, Carine; Svardal, Asbjørn; Skorve, Jon; Berge, Rolf K

    2018-01-01

    The 4-thia fatty acid tetradecylthiopropionic acid (TTP) is known to inhibit mitochondrial β-oxidation, and can be used as chemically induced hepatic steatosis-model in rodents, while 3-thia fatty acid tetradecylthioacetic acid (TTA) stimulates fatty acid oxidation through activation of peroxisome proliferator activated receptor alpha (PPARα). We wished to determine how these two compounds affected in vivo respiration and mitochondrial efficiency, with an additional goal to elucidate whether mitochondrial function is reflected in plasma acylcarnitine levels. C57BL/6 mice were divided in 4 groups of 10 mice and fed a control low-fat diet, low-fat diets with 0.4% ( w /w) TTP, 0.4% TTA or a combination of these two fatty acids for three weeks ( n  = 10). At sacrifice, β-oxidation and oxidative phosphorylation (OXPHOS) capacity was analysed in fresh liver samples. Hepatic mitochondria were studied using transmission electron microscopy. Lipid classes were measured in plasma, heart and liver, acylcarnitines were measured in plasma, and gene expression was measured in liver. The TTP diet resulted in hepatic lipid accumulation, plasma L-carnitine and acetylcarnitine depletion and elevated palmitoylcarnitine and non-esterified fatty acid levels. No significant lipid accumulation was observed in heart. The TTA supplement resulted in enhanced hepatic β-oxidation, accompanied by an increased level of acetylcarnitine and palmitoylcarnitine in plasma. Analysis of mitochondrial respiration showed that TTP reduced oxidative phosphorylation, while TTA increased the maximum respiratory capacity of the electron transport system. Combined treatment with TTP and TTA resulted in a profound stimulation of genes involved in the PPAR-response and L-carnitine metabolism, and partly prevented triacylglycerol accumulation in the liver concomitant with increased peroxisomal β-oxidation and depletion of plasma acetylcarnitines. Despite an increased number of mitochondria in the liver

  6. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

    PubMed

    Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C; Fernyhough, Paul

    2015-12-08

    Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances. © 2016 Authors.

  7. Overexpression of Mitochondrial Phosphate Transporter 3 Severely Hampers Plant Development through Regulating Mitochondrial Function in Arabidopsis.

    PubMed

    Jia, Fengjuan; Wan, Xiaomin; Zhu, Wei; Sun, Dan; Zheng, Chengchao; Liu, Pei; Huang, Jinguang

    2015-01-01

    Mitochondria are abundant and important organelles present in nearly all eukaryotic cells, which maintain metabolic communication with the cytosol through mitochondrial carriers. The mitochondrial membrane localized phosphate transporter (MPT) plays vital roles in diverse development and signaling processes, especially the ATP biosynthesis. Among the three MPT genes in Arabidopsis genome, AtMPT3 was proven to be a major member, and its overexpression gave rise to multiple developmental defects including curly leaves with deep color, dwarfed stature, and reduced fertility. Transcript profiles revealed that genes involved in plant metabolism, cellular redox homeostasis, alternative respiration pathway, and leaf and flower development were obviously altered in AtMPT3 overexpression (OEMPT3) plants. Moreover, OEMPT3 plants also accumulated higher ATP content, faster respiration rate and more reactive oxygen species (ROS) than wild type plants. Overall, our studies showed that AtMPT3 was indispensable for Arabidopsis normal growth and development, and provided new sights to investigate its possible regulation mechanisms.

  8. Overexpression of Mitochondrial Phosphate Transporter 3 Severely Hampers Plant Development through Regulating Mitochondrial Function in Arabidopsis

    PubMed Central

    Jia, Fengjuan; Wan, Xiaomin; Zhu, Wei; Sun, Dan; Zheng, Chengchao; Liu, Pei; Huang, Jinguang

    2015-01-01

    Mitochondria are abundant and important organelles present in nearly all eukaryotic cells, which maintain metabolic communication with the cytosol through mitochondrial carriers. The mitochondrial membrane localized phosphate transporter (MPT) plays vital roles in diverse development and signaling processes, especially the ATP biosynthesis. Among the three MPT genes in Arabidopsis genome, AtMPT3 was proven to be a major member, and its overexpression gave rise to multiple developmental defects including curly leaves with deep color, dwarfed stature, and reduced fertility. Transcript profiles revealed that genes involved in plant metabolism, cellular redox homeostasis, alternative respiration pathway, and leaf and flower development were obviously altered in AtMPT3 overexpression (OEMPT3) plants. Moreover, OEMPT3 plants also accumulated higher ATP content, faster respiration rate and more reactive oxygen species (ROS) than wild type plants. Overall, our studies showed that AtMPT3 was indispensable for Arabidopsis normal growth and development, and provided new sights to investigate its possible regulation mechanisms. PMID:26076137

  9. Nutritional Ketosis and Mitohormesis: Potential Implications for Mitochondrial Function and Human Health

    PubMed Central

    Villamena, Frederick A.

    2018-01-01

    Impaired mitochondrial function often results in excessive production of reactive oxygen species (ROS) and is involved in the etiology of many chronic diseases, including cardiovascular disease, diabetes, neurodegenerative disorders, and cancer. Moderate levels of mitochondrial ROS, however, can protect against chronic disease by inducing upregulation of mitochondrial capacity and endogenous antioxidant defense. This phenomenon, referred to as mitohormesis, is induced through increased reliance on mitochondrial respiration, which can occur through diet or exercise. Nutritional ketosis is a safe and physiological metabolic state induced through a ketogenic diet low in carbohydrate and moderate in protein. Such a diet increases reliance on mitochondrial respiration and may, therefore, induce mitohormesis. Furthermore, the ketone β-hydroxybutyrate (BHB), which is elevated during nutritional ketosis to levels no greater than those resulting from fasting, acts as a signaling molecule in addition to its traditionally known role as an energy substrate. BHB signaling induces adaptations similar to mitohormesis, thereby expanding the potential benefit of nutritional ketosis beyond carbohydrate restriction. This review describes the evidence supporting enhancement of mitochondrial function and endogenous antioxidant defense in response to nutritional ketosis, as well as the potential mechanisms leading to these adaptations. PMID:29607218

  10. Impaired mitochondrial oxidative phosphorylation and supercomplex assembly in rectus abdominis muscle of diabetic obese individuals.

    PubMed

    Antoun, Ghadi; McMurray, Fiona; Thrush, A Brianne; Patten, David A; Peixoto, Alyssa C; Slack, Ruth S; McPherson, Ruth; Dent, Robert; Harper, Mary-Ellen

    2015-12-01

    Skeletal muscle mitochondrial dysfunction has been documented in patients with type 2 diabetes mellitus; however, specific respiratory defects and their mechanisms are poorly understood. The aim of the current study was to examine oxidative phosphorylation and electron transport chain (ETC) supercomplex assembly in rectus abdominis muscles of 10 obese diabetic and 10 obese non-diabetic individuals. Twenty obese women undergoing Roux-en-Y gastric bypass surgery were recruited for this study. Muscle samples were obtained intraoperatively and subdivided for multiple analyses, including high-resolution respirometry and assessment of supercomplex assembly. Clinical data obtained from referring physicians were correlated with laboratory findings. Participants in both groups were of a similar age, weight and BMI. Mitochondrial respiration rates were markedly reduced in diabetic vs non-diabetic patients. This defect was observed during maximal ADP-stimulated respiration in the presence of complex I-linked substrates and complex I- and II-linked substrates, and during maximal uncoupled respiration. There were no differences in fatty acid (octanoyl carnitine) supported respiration, leak respiration or isolated activity of cytochrome c oxidase. Intriguingly, significant correlations were found between glycated haemoglobin (HbA1c) levels and maximal respiration or respiration supported by complex I, complex I and II or fatty acid. In the muscle of diabetic patients, blue native gel electrophoresis revealed a striking decrease in complex I, III and IV containing ETC supercomplexes. These findings support the hypothesis that ETC supercomplex assembly may be an important underlying mechanism of muscle mitochondrial dysfunction in type 2 diabetes mellitus.

  11. Resveratrol Co-Treatment Attenuates the Effects of HIV Protease Inhibitors on Rat Body Weight and Enhances Cardiac Mitochondrial Respiration

    PubMed Central

    Symington, Burger; Mapanga, Rudo F.; Norton, Gavin R.

    2017-01-01

    Since the early 1990s human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) emerged as a global health pandemic, with sub-Saharan Africa the hardest hit. While the successful roll-out of antiretroviral (ARV) therapy provided significant relief to HIV-positive individuals, such treatment can also elicit damaging side-effects. Here especially HIV protease inhibitors (PIs) are implicated in the onset of cardio-metabolic complications such as type-2 diabetes and coronary heart disease. As there is a paucity of data regarding suitable co-treatments within this context, this preclinical study investigated whether resveratrol (RSV), aspirin (ASP) or vitamin C (VitC) co-treatment is able to blunt side-effects in a rat model of chronic PI exposure (Lopinavir/Ritonavir treatment for 4 months). Body weights and weight gain, blood metabolite levels (total cholesterol, HDL, LDL, triglycerides), echocardiography and cardiac mitochondrial respiration were assessed in PI-treated rats ± various co-treatments. Our data reveal that PI treatment significantly lowered body weight and cardiac respiratory function while no significant changes were found for heart function and blood metabolite levels. Moreover, all co-treatments ameliorated the PI-induced decrease in body weight after 4 months of PI treatment, while RSV co-treatment enhanced cardiac mitochondrial respiratory capacity in PI-treated rats. This pilot study therefore provides novel hypotheses regarding RSV co-treatment that should be further assessed in greater detail. PMID:28107484

  12. Focal adhesion kinase-promoted tumor glucose metabolism is associated with a shift of mitochondrial respiration to glycolysis.

    PubMed

    Zhang, J; Gao, Q; Zhou, Y; Dier, U; Hempel, N; Hochwald, S N

    2016-04-14

    Cancer cells often gains a growth advantage by taking up glucose at a high rate and undergoing aerobic glycolysis through intrinsic cellular factors that reprogram glucose metabolism. Focal adhesion kinase (FAK), a key transmitter of growth factor and anchorage stimulation, is aberrantly overexpressed or activated in most solid tumors, including pancreatic ductal adenocarcinomas (PDACs). We determined whether FAK can act as an intrinsic driver to promote aerobic glycolysis and tumorigenesis. FAK inhibition decreases and overexpression increases intracellular glucose levels during unfavorable conditions, including growth factor deficiency and cell detachment. Amplex glucose assay, fluorescence and carbon-13 tracing studies demonstrate that FAK promotes glucose consumption and glucose-to-lactate conversion. Extracellular flux analysis indicates that FAK enhances glycolysis and decreases mitochondrial respiration. FAK increases key glycolytic proteins, including enolase, pyruvate kinase M2 (PKM2), lactate dehydrogenase and monocarboxylate transporter. Furthermore, active/tyrosine-phosphorylated FAK directly binds to PKM2 and promotes PKM2-mediated glycolysis. On the other hand, FAK-decreased levels of mitochondrial complex I can result in reduced oxidative phosphorylation (OXPHOS). Attenuation of FAK-enhanced glycolysis re-sensitizes cancer cells to growth factor withdrawal, decreases cell viability and reduces growth of tumor xenografts. These observations, for the first time, establish a vital role of FAK in cancer glucose metabolism through alterations in the OXPHOS-to-glycolysis balance. Broadly targeting the common phenotype of aerobic glycolysis and more specifically FAK-reprogrammed glucose metabolism will disrupt the bioenergetic and biosynthetic supply for uncontrolled growth of tumors, particularly glycolytic PDAC.

  13. Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1

    PubMed Central

    Chouchani, Edward T.; Kazak, Lawrence; Jedrychowski, Mark P.; Lu, Gina Z.; Erickson, Brian K.; Szpyt, John; Pierce, Kerry A.; Laznik-Bogoslavski, Dina; Vetrivelan, Ramalingam; Clish, Clary B.; Robinson, Alan J.; Gygi, Steve P.; Spiegelman, Bruce M.

    2017-01-01

    Brown adipose tissue (BAT) can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1)1,2. Thermogenesis from BAT and beige adipose can combat obesity and diabetes3, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Herein we show that acutely activated BAT thermogenesis is defined by a substantial increase in mitochondrial reactive oxygen species (ROS) levels. Remarkably, this process supports in vivo BAT thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole body energy expenditure. We further establish that thermogenic ROS alter BAT cysteine thiol redox status to drive increased respiration, and Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine nucleotide inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify BAT mitochondrial ROS induction as a mechanism that drives UCP1-dependent thermogenesis and whole body energy expenditure, which opens the way to develop improved therapeutic strategies for combating metabolic disorders. PMID:27027295

  14. A mutation in MT-TW causes a tRNA processing defect and reduced mitochondrial function in a family with Leigh syndrome.

    PubMed

    Duff, Rachael M; Shearwood, Anne-Marie J; Ermer, Judith; Rossetti, Giulia; Gooding, Rebecca; Richman, Tara R; Balasubramaniam, Shanti; Thorburn, David R; Rackham, Oliver; Lamont, Phillipa J; Filipovska, Aleksandra

    2015-11-01

    Leigh syndrome (LS) is a progressive mitochondrial neurodegenerative disorder, whose symptoms most commonly include psychomotor delay with regression, lactic acidosis and a failure to thrive. Here we describe three siblings with LS, but with additional manifestations including hypertrophic cardiomyopathy, hepatosplenomegaly, cholestatic hepatitis, and seizures. All three affected siblings were found to be homoplasmic for an m. 5559A>G mutation in the T stem of the mitochondrial DNA-encoded MT-TW by next generation sequencing. The m.5559A>G mutation causes a reduction in the steady state levels of tRNA(Trp) and this decrease likely affects the stability of other mitochondrial RNAs in the patient fibroblasts. We observe accumulation of an unprocessed transcript containing tRNA(Trp), decreased de novo protein synthesis and consequently lowered steady state levels of mitochondrial DNA-encoded proteins that compromise mitochondrial respiration. Our results show that the m.5559A>G mutation at homoplasmic levels causes LS in association with severe multi-organ disease (LS-plus) as a consequence of dysfunctional mitochondrial RNA metabolism. Copyright © 2015 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  15. Differential regulation of mitochondrial pyruvate carrier genes modulates respiratory capacity and stress tolerance in yeast.

    PubMed

    Timón-Gómez, Alba; Proft, Markus; Pascual-Ahuir, Amparo

    2013-01-01

    Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.

  16. Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling

    PubMed Central

    Dhingra, Rimpy; Margulets, Victoria; Chowdhury, Subir Roy; Thliveris, James; Jassal, Davinder; Fernyhough, Paul; Dorn, Gerald W.; Kirshenbaum, Lorrie A.

    2014-01-01

    Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3−/− mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy. PMID:25489073

  17. An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

    PubMed

    Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

    2016-04-19

    Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Common effects of lithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrial damage.

    PubMed

    Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K

    2009-07-01

    Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

  19. In Vivo Mitochondrial Oxygen Tension Measured by a Delayed Fluorescence Lifetime Technique

    PubMed Central

    Mik, Egbert G.; Johannes, Tanja; Zuurbier, Coert J.; Heinen, Andre; Houben-Weerts, Judith H. P. M.; Balestra, Gianmarco M.; Stap, Jan; Beek, Johan F.; Ince, Can

    2008-01-01

    Mitochondrial oxygen tension (mitoPO2) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO2 in vivo exists. Here we report in vivo measurement of mitoPO2 and the recovery of mitoPO2 histograms in rat liver by a novel optical technique under normal and pathological circumstances. The technique is based on oxygen-dependent quenching of the delayed fluorescence lifetime of protoporphyrin IX. Application of 5-aminolevulinic acid enhanced mitochondrial protoporphyrin IX levels and induced oxygen-dependent delayed fluorescence in various tissues, without affecting mitochondrial respiration. Using fluorescence microscopy, we demonstrate in isolated hepatocytes that the signal is of mitochondrial origin. The delayed fluorescence lifetime was calibrated in isolated hepatocytes and isolated perfused livers. Ultimately, the technique was applied to measure mitoPO2 in rat liver in vivo. The results demonstrate mitoPO2 values of ∼30–40 mmHg. mitoPO2 was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO2 distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications. PMID:18641065

  20. β-Sitosterol enhances cellular glutathione redox cycling by reactive oxygen species generated from mitochondrial respiration: protection against oxidant injury in H9c2 cells and rat hearts.

    PubMed

    Wong, Hoi Shan; Chen, Na; Leong, Pou Kuan; Ko, Kam Ming

    2014-07-01

    Herba Cistanches (Cistanche deserticola Y. C. Ma) is a 'Yang-invigorating' tonic herb in Chinese medicine. Preliminary chemical analysis indicated that β-sitosterol (BS) is one of the chemical constituents in an active fraction of Herba Cistanches. To investigate whether BS is an active ingredient of Herba Cistanches, the effects of BS on H9c2 cells and rat hearts were examined. The results indicated that BS stimulated the mitochondrial ATP generation capacity in H9c2 cells, which was associated with the increased production of mitochondrial reactive oxygen species. BS also stimulated mitochondrial state 3 and state 4 respiration, with the resultant decrease in coupling efficiency. BS produced an up-regulation of cellular glutathione redox cycling and protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. However, the protective effect of BS against myocardial ischemia/reperfusion injury was seen in female but not male rats ex vivo. The cardioprotection afforded by BS was likely mediated by an up-regulation of mitochondrial glutathione redox cycling in female rat hearts. In conclusion, the ensemble of results suggests that BS is an active ingredient of Herba Cistanches. The gender-dependent effect of BS on myocardial protection will further be investigated. Copyright © 2013 John Wiley & Sons, Ltd.

  1. Combined effects of cadmium, temperature and hypoxia-reoxygenation on mitochondrial function in rainbow trout (Oncorhynchus mykiss).

    PubMed

    Onukwufor, John O; Stevens, Don; Kamunde, Collins

    2017-01-01

    Although aquatic organisms face multiple environmental stressors that may interact to alter adverse outcomes, our knowledge of stressor-stressor interaction on cellular function is limited. We investigated the combined effects of cadmium (Cd), hypoxia-reoxygenation (H-R) and temperature on mitochondrial function. Liver mitochondria from juvenile rainbow trout were exposed to Cd (0-20μM) and H-R (0 and 5min) at 5, 13 and 25°C followed by measurements of mitochondrial Cd load, volume, complex І active (A)↔deactive (D) transition, membrane potential, ROS release and ultrastructural changes. At high temperature Cd exacerbated H-R-imposed reduction of maximal complex I (CI) respiration whereas at low temperature 5 and 10μM stimulated maximal CI respiration post H-R. The basal respiration showed a biphasic response at high temperatures with low Cd concentrations reducing the stimulatory effect of H-R and high concentrations enhancing this effect. At low temperature Cd monotonically enhanced H-R-induced stimulation of basal respiration. Cd and H-R reduced both the P/O ratio and the RCR at all 3 temperatures. Temperature rise alone increased mitochondrial Cd load and toxicity, but combined H-R and temperature exposure reduced mitochondrial Cd load but surprisingly exacerbated the mitochondrial dysfunction. Mitochondrial dysfunction induced by H-R was associated with swelling of the organelle and blocking of conversion of CІ D to A form. However, low amounts of Cd protected against H-R induced swelling and prevented the inhibition of H-R-induced CI D to A transition. Both H-R and Cd dissipated mitochondrial membrane potential Δψ m and damaged mitochondrial structure. We observed increased reactive oxygen species (H 2 O 2 ) release that together with the protection afforded by EGTA, vitamin E and N-acetylcysteine against the Δψ m dissipation suggested direct involvement of Cd and oxidative stress. Overall, our findings indicate that mitochondrial sensitivity to Cd

  2. Changes in Respiratory Mitochondrial Machinery and Cytochrome and Alternative Pathway Activities in Response to Energy Demand Underlie the Acclimation of Respiration to Elevated CO2 in the Invasive Opuntia ficus-indica1[OA

    PubMed Central

    Gomez-Casanovas, Nuria; Blanc-Betes, Elena; Gonzalez-Meler, Miquel A.; Azcon-Bieto, Joaquim

    2007-01-01

    Studies on long-term effects of plants grown at elevated CO2 are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO2, the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO2 concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO2 during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO2 also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO2, the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO2. Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO2, the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO2 suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO2. However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO2, total mitochondrial ATP production was decreased by plant growth at elevated CO2 when compared to ambient-grown plants. Because plant growth at elevated CO2 increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O2 consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO2 results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested. PMID:17660349

  3. An mtDNA mutation accelerates liver aging by interfering with the ROS response and mitochondrial life cycle.

    PubMed

    Niemann, Jan; Johne, Cindy; Schröder, Susanne; Koch, Franziska; Ibrahim, Saleh M; Schultz, Julia; Tiedge, Markus; Baltrusch, Simone

    2017-01-01

    Mitochondrial dysfunction affects liver metabolism, but it remains unclear whether this interferes with normal liver aging. We investigated several mitochondrial pathways in hepatocytes and liver tissue from a conplastic mouse strain compared with the control C57BL/6NTac strain over 18 months of life. The C57BL/6NTac-mtNODLtJ mice differed from C57BL/6NTac mice by a point mutation in mitochondrial-encoded subunit 3 of cytochrome c oxidase. Young C57BL/6NTac-mtNODLtJ mice showed reduced mitochondrial metabolism but similar reactive oxygen species (ROS) production to C57BL/6NTac mice. Whereas ROS increased almost equally up to 9 months in both strains, different mitochondrial adaptation strategies resulted in decreasing ROS in advanced age in C57BL/6NTac mice, but persistent ROS production in C57BL/6NTac-mtNODLtJ mice. Only the conplastic strain developed elongated mitochondrial networks with artificial loop structures, depressed autophagy, high mitochondrial respiration and up-regulated antioxidative response. Our results indicate that mtDNA mutations accelerate liver ballooning degeneration and carry a serious risk of premature organ aging. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

    PubMed Central

    Mitsopoulos, Panagiotis; Chang, Yu-Han; Wai, Timothy; König, Tim; Dunn, Stanley D.; Langer, Thomas

    2015-01-01

    Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III2 and I-III2-IV1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function. PMID:25776552

  5. MOF Acetyl Transferase Regulates Transcription and Respiration in Mitochondria.

    PubMed

    Chatterjee, Aindrila; Seyfferth, Janine; Lucci, Jacopo; Gilsbach, Ralf; Preissl, Sebastian; Böttinger, Lena; Mårtensson, Christoph U; Panhale, Amol; Stehle, Thomas; Kretz, Oliver; Sahyoun, Abdullah H; Avilov, Sergiy; Eimer, Stefan; Hein, Lutz; Pfanner, Nikolaus; Becker, Thomas; Akhtar, Asifa

    2016-10-20

    A functional crosstalk between epigenetic regulators and metabolic control could provide a mechanism to adapt cellular responses to environmental cues. We report that the well-known nuclear MYST family acetyl transferase MOF and a subset of its non-specific lethal complex partners reside in mitochondria. MOF regulates oxidative phosphorylation by controlling expression of respiratory genes from both nuclear and mtDNA in aerobically respiring cells. MOF binds mtDNA, and this binding is dependent on KANSL3. The mitochondrial pool of MOF, but not a catalytically deficient mutant, rescues respiratory and mtDNA transcriptional defects triggered by the absence of MOF. Mof conditional knockout has catastrophic consequences for tissues with high-energy consumption, triggering hypertrophic cardiomyopathy and cardiac failure in murine hearts; cardiomyocytes show severe mitochondrial degeneration and deregulation of mitochondrial nutrient metabolism and oxidative phosphorylation pathways. Thus, MOF is a dual-transcriptional regulator of nuclear and mitochondrial genomes connecting epigenetics and metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves.

    PubMed

    Martí, María C; Florez-Sarasa, Igor; Camejo, Daymi; Pallol, Beatriz; Ortiz, Ana; Ribas-Carbó, Miquel; Jiménez, Ana; Sevilla, Francisca

    2013-02-01

    Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA-NONOate, a pure NO slow generator, and of SIN-1 (3-morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non-enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA-NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn-SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN-1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn-SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO-resistant AP and mitochondrial APX may be important components of the H(2) O(2) -signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn-SOD, against NO and ONOO(-) stress in plant mitochondria. Copyright © Physiologia Plantarum 2012.

  7. Top-down control analysis of the cadmium effects on molluscan mitochondria and the mechanisms of cadmium-induced mitochondrial dysfunction.

    PubMed

    Kurochkin, Ilya O; Etzkorn, Markus; Buchwalter, David; Leamy, Larry; Sokolova, Inna M

    2011-01-01

    Cadmium (Cd) is a toxic metal and an important environmental pollutant that can strongly affect mitochondrial function and bioenergetics in animals. We investigated the mechanisms of Cd action on mitochondrial function of a marine mollusk (the eastern oyster Crassostrea virginica) by performing a top-down control analysis of the three major mitochondrial subsystems (substrate oxidation, proton leak, and phosphorylation). Our results showed that the substrate oxidation and proton leak subsystems are the main targets for Cd toxicity in oyster mitochondria. Exposure to 12.5 μM Cd strongly inhibited the substrate oxidation subsystem and stimulated the proton conductance across the inner mitochondrial membrane. Proton conductance was also elevated and substrate oxidation inhibited by Cd in the presence of a mitochondrially targeted antioxidant, MitoVitE, indicating that Cd effects on these subsystems were to a large extent ROS independent. Cd did not affect the kinetics of the phosphorylation system, indicating that it has negligible effects on F₁, F(O) ATP synthase and/or the adenine nucleotide transporter in oyster mitochondria. Cd exposure altered the patterns of control over mitochondrial respiration, increasing the degree of control conferred by the substrate oxidation subsystem, especially in resting (state 4) mitochondria. Taken together, these data suggest that Cd-induced decrease of mitochondrial efficiency and ATP production are predominantly driven by the high sensitivity of substrate oxidation and proton leak subsystems to this metal.

  8. Inhibition of free radical scavenging enzymes affects mitochondrial membrane permeability transition during growth and aging of yeast cells.

    PubMed

    Deryabina, Yulia; Isakova, Elena; Sekova, Varvara; Antipov, Alexey; Saris, Nils-Erik L

    2014-12-01

    In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.

  9. Sorafenib and FH535 in combination act synergistically on hepatocellular carcinoma by targeting cell bioenergetics and mitochondrial function.

    PubMed

    Turcios, Lilia; Vilchez, Valery; Acosta, Luis F; Poyil, Pratheeshkumar; Butterfield, David Allan; Mitov, Mihail; Marti, Francesc; Gedaly, Roberto

    2017-06-01

    Treatment of advanced hepatocellular carcinoma (HCC) remains a challenge due to the high tumor heterogeneity. In the present study, we aim to evaluate the impact of the β-catenin inhibitor, FH535, alone or in combination with the Ras/Raf/MAPK inhibitor Sorafenib, on the bioenergetics profiles of the HCC cell lines Huh7 and PLC/PRF/5. Single low-dose treatments with FH535 or Sorafenib promoted different effects on mitochondrial respiration and glycolysis in a cell type specific manner. However, the combination of these drugs significantly reduced both mitochondrial respiration and glycolytic rates regardless of the HCC cells. The significant changes in mitochondrial respiration observed in cells treated with the Sorafenib-FH535 combination may correspond to differential targeting of ETC complexes and changes in substrate utilization mediated by each drug. Moreover, the bioenergetics changes and the loss of mitochondrial membrane potential that were evidenced by treatment of HCC cells with the combination of FH535 and Sorafenib, preceded the induction of cell apoptosis. Overall, our results demonstrated that Sorafenib-FH535 drug combination induce the disruption of the bioenergetics of HCC by the simultaneous targeting of mitochondrial respiration and glycolytic flux that leads the synergistic effect on inhibition of cell proliferation. These findings support the therapeutic potential of combinatory FH535-Sorafenib treatment of the HCC heterogeneity by the simultaneous targeting of different molecular pathways. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  10. Depressed mitochondrial function and electron transport Complex II-mediated H2O2 production in the cortex of type 1 diabetic rodents.

    PubMed

    Chowdhury, Subir Roy; Djordjevic, Jelena; Thomson, Ella; Smith, Darrell R; Albensi, Benedict C; Fernyhough, Paul

    2018-05-23

    Abnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess simultaneously mitochondrial respiration rates and membrane potential or H 2 O 2 generation and proteins involved in mitochondrial dynamics, antioxidants and AMPK/SIRT/PGC-1α pathway activity in cortex under diabetic conditions. Cortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-match controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H 2 O 2 using OROBOROS oxygraph and measurements of enzymatic activities by a spectrophotometer. Protein levels in cortical mitochondria and homogenates were determined by Western blotting. Mitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of STZ-diabetic cortical rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H 2 O 2 were significantly diminished in mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1α were significantly diminished in diabetic cortex. Deficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1α signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions. Copyright © 2017. Published by Elsevier Inc.

  11. Muscle mitochondrial metabolism and calcium signaling impairment in patients treated with statins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sirvent, P., E-mail: pascal.sirvent@univ-bpclermont.fr; CHRU Montpellier, 34295 Montpellier; Clermont Université, Université Blaise Pascal, EA 3533, Laboratoire des Adaptations Métaboliques à l'Exercice en conditions Physiologiques et Pathologiques

    2012-03-01

    The most common and problematic side effect of statins is myopathy. To date, the patho-physiological mechanisms of statin myotoxicity are still not clearly understood. In previous studies, we showed that acute application in vitro of simvastatin caused impairment of mitochondrial function and dysfunction of calcium homeostasis in human and rat healthy muscle samples. We thus evaluated in the present study, mitochondrial function and calcium signaling in muscles of patients treated with statins, who present or not muscle symptoms, by oxygraphy and recording of calcium sparks, respectively. Patients treated with statins showed impairment of mitochondrial respiration that involved mainly the complexmore » I of the respiratory chain and altered frequency and amplitude of calcium sparks. The muscle problems observed in statin-treated patients appear thus to be related to impairment of mitochondrial function and muscle calcium homeostasis, confirming the results we previously reported in vitro. -- Highlights: ► The most common and problematic side effect of statins is myopathy. ► Patients treated with statins showed impairment of mitochondrial respiration. ► Statins-treated patients showed altered frequency and amplitude of calcium sparks.« less

  12. Alteration of respiration capacity and transcript accumulation level of alternative oxidase genes in necrosis lines of common wheat.

    PubMed

    Sugie, Atsushi; Murai, Koji; Takumi, Shigeo

    2007-06-01

    Mitochondrial alternative oxidase (AOX) is the terminal oxidase responsible for cyanide-insensitive and salicylhydroxamic acid-sensitive respiration in plants. AOX is a key enzyme of the alternative respiration pathway. To study the effects of necrotic cell death on the mitochondrial function, production of reactive oxygen species (ROS), respiration capacities and accumulation patterns of mitochondria-targeted protein-encoding gene transcripts were compared between wild-type, lesion-mimic mutant and hybrid necrosis wheat plants. Around cells with the necrosis symptom, ROS accumulated abundantly in the intercellular spaces. The ratio of the alternative pathway to the cytochrome pathway was markedly enhanced in the necrotic leaves. Transcripts of a wheat AOX gene, Waox1a, were more abundant in a novel lesion-mimic mutant of common wheat than in the wild-type plants. An increased level of the Waox1a transcripts was also observed in hybrid plants containing Ne1 and Ne2 genes. These results indicated that an increase of the wheat AOX transcript level resulted in enhancement of respiration capacity of the alternative pathway in the necrotic cells.

  13. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Cong; Sekine, Shuichi, E-mail: ssekine@facult

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and inmore » hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.« less

  14. Role of the putative structural protein Sed1p in mitochondrial genome maintenance.

    PubMed

    Phadnis, Naina; Ayres Sia, Elaine

    2004-09-24

    The nuclear gene MIP1 encodes the mitochondrial DNA polymerase responsible for replicating the mitochondrial genome in Saccharomyces cerevisiae. A number of other factors involved in replicating and segregating the mitochondrial genome are yet to be identified. Here, we report that a bacterial two-hybrid screen using the mitochondrial polymerase, Mip1p, as bait identified the yeast protein Sed1p. Sed1p is a cell surface protein highly expressed in the stationary phase. We find that several modified forms of Sed1p are expressed and the largest of these forms interacts with the mitochondrial polymerase in vitro. Deletion of SED1 causes a 3.5-fold increase in the rate of mitochondrial DNA point mutations as well as a 4.3-fold increase in the rate of loss of respiration. In contrast, we see no change in the rate of nuclear point mutations indicating the specific role of Sed1p function in mitochondrial genome stability. Indirect immunofluorescence analysis of Sed1p localization shows that Sed1p is targeted to the mitochondria. Moreover, Sed1p is detected in purified mitochondrial fractions and the localization to the mitochondria of the largest modified form is insensitive to the action of proteinase K. Deletion of the sed1 gene results in a reduction in the quantity of Mip1p and also affects the levels of a mitochondrially-expressed protein, Cox3p. Our results point towards a role for Sed1p in mitochondrial genome maintenance.

  15. Phosphatidylserine transport by Ups2-Mdm35 in respiration-active mitochondria.

    PubMed

    Miyata, Non; Watanabe, Yasunori; Tamura, Yasushi; Endo, Toshiya; Kuge, Osamu

    2016-07-04

    Phosphatidylethanolamine (PE) is an essential phospholipid for mitochondrial functions and is synthesized mainly by phosphatidylserine (PS) decarboxylase at the mitochondrial inner membrane. In Saccharomyces cerevisiae, PS is synthesized in the endoplasmic reticulum (ER), such that mitochondrial PE synthesis requires PS transport from the ER to the mitochondrial inner membrane. Here, we provide evidence that Ups2-Mdm35, a protein complex localized at the mitochondrial intermembrane space, mediates PS transport for PE synthesis in respiration-active mitochondria. UPS2- and MDM35-null mutations greatly attenuated conversion of PS to PE in yeast cells growing logarithmically under nonfermentable conditions, but not fermentable conditions. A recombinant Ups2-Mdm35 fusion protein exhibited phospholipid-transfer activity between liposomes in vitro. Furthermore, UPS2 expression was elevated under nonfermentable conditions and at the diauxic shift, the metabolic transition from glycolysis to oxidative phosphorylation. These results demonstrate that Ups2-Mdm35 functions as a PS transfer protein and enhances mitochondrial PE synthesis in response to the cellular metabolic state. © 2016 Miyata et al.

  16. The Mitochondrial Genome Impacts Respiration but Not Fermentation in Interspecific Saccharomyces Hybrids

    PubMed Central

    Rigoulet, Michel; Salin, Benedicte; Masneuf-Pomarede, Isabelle; de Vienne, Dominique; Sicard, Delphine; Bely, Marina; Marullo, Philippe

    2013-01-01

    In eukaryotes, mitochondrial DNA (mtDNA) has high rate of nucleotide substitution leading to different mitochondrial haplotypes called mitotypes. However, the impact of mitochondrial genetic variant on phenotypic variation has been poorly considered in microorganisms because mtDNA encodes very few genes compared to nuclear DNA, and also because mitochondrial inheritance is not uniparental. Here we propose original material to unravel mitotype impact on phenotype: we produced interspecific hybrids between S. cerevisiae and S. uvarum species, using fully homozygous diploid parental strains. For two different interspecific crosses involving different parental strains, we recovered 10 independent hybrids per cross, and allowed mtDNA fixation after around 80 generations. We developed PCR-based markers for the rapid discrimination of S. cerevisiae and S. uvarum mitochondrial DNA. For both crosses, we were able to isolate fully isogenic hybrids at the nuclear level, yet possessing either S. cerevisiae mtDNA (Sc-mtDNA) or S. uvarum mtDNA (Su-mtDNA). Under fermentative conditions, the mitotype has no phenotypic impact on fermentation kinetics and products, which was expected since mtDNA are not necessary for fermentative metabolism. Alternatively, under respiratory conditions, hybrids with Sc-mtDNA have higher population growth performance, associated with higher respiratory rate. Indeed, far from the hypothesis that mtDNA variation is neutral, our work shows that mitochondrial polymorphism can have a strong impact on fitness components and hence on the evolutionary fate of the yeast populations. We hypothesize that under fermentative conditions, hybrids may fix stochastically one or the other mt-DNA, while respiratory environments may increase the probability to fix Sc-mtDNA. PMID:24086452

  17. Mitochondrial uncoupling proteins in unicellular eukaryotes.

    PubMed

    Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

    2010-01-01

    Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

  18. Ntg1p, the base excision repair protein, generates mutagenic intermediates in yeast mitochondrial DNA.

    PubMed

    Phadnis, Naina; Mehta, Reema; Meednu, Nida; Sia, Elaine A

    2006-07-13

    Mitochondrial DNA is predicted to be highly prone to oxidative damage due to its proximity to free radicals generated by oxidative phosphorylation. Base excision repair (BER) is the primary repair pathway responsible for repairing oxidative damage in nuclear and mitochondrial genomes. In yeast mitochondria, three N-glycosylases have been identified so far, Ntg1p, Ogg1p and Ung1p. Ntg1p, a broad specificity N-glycosylase, takes part in catalyzing the first step of BER that involves the removal of the damaged base. In this study, we examined the role of Ntg1p in maintaining yeast mitochondrial genome integrity. Using genetic reporters and assays to assess mitochondrial mutations, we found that loss of Ntg1p suppresses mitochondrial point mutation rates, frameshifts and recombination rates. We also observed a suppression of respiration loss in the ntg1-Delta cells in response to ultraviolet light exposure implying an overlap between BER and UV-induced damage in the yeast mitochondrial compartment. Over-expression of the BER AP endonuclease, Apn1p, did not significantly affect the mitochondrial mutation rate in the presence of Ntg1p, whereas Apn1p over-expression in an ntg1-Delta background increased the frequency of mitochondrial mutations. In addition, loss of Apn1p also suppressed mitochondrial point mutations. Our work suggests that both Ntg1p and Apn1p generate mutagenic intermediates in the yeast mitochondrial genome.

  19. Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT−/− mouse hearts

    PubMed Central

    Branovets, Jelena; Sepp, Mervi; Kotlyarova, Svetlana; Jepihhina, Natalja; Sokolova, Niina; Aksentijevic, Dunja; Lygate, Craig A.; Neubauer, Stefan; Birkedal, Rikke

    2013-01-01

    Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes. PMID:23792673

  20. Low levels of lipopolysaccharide modulate mitochondrial oxygen consumption in skeletal muscle

    PubMed Central

    Frisard, Madlyn I.; Wu, Yaru; McMillan, Ryan P.; Voelker, Kevin A.; Wahlberg, Kristin A.; Anderson, Angela S.; Boutagy, Nabil; Resendes, Kyle; Ravussin, Eric; Hulver, Matthew W.

    2014-01-01

    Objective We have previously demonstrated that activation of toll-like receptor 4 (TLR4) in skeletal muscle results in an increased reliance on glucose as an energy source and a concomitant decrease in fatty acid oxidation under basal conditions. Herein, we examined the effects of lipopolysaccharide (LPS), the primary ligand for TLR4, on mitochondrial oxygen consumption in skeletal muscle cell culture and isolated mitochondria. Materials/ methods Skeletal muscle cell cultures were exposed to LPS and oxygen consumption was assessed using a Seahorse Bioscience extracellular flux analyzer. Mice were also exposed to LPS and oxygen consumption was assessed in mitochondria isolated from skeletal muscle. Results Acute LPS exposure resulted in significant reductions in cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP)-stimulated maximal respiration (state 3u) and increased oligomycin induced state 4 (state 4O) respiration in C2C12 and human primary myotubes. These findings were observed in conjunction with increased mRNA of uncoupling protein 3 (UCP3), superoxide dismutase 2 (SOD2), and pyruvate dehydrogenase activity. The LPS-mediated changes in substrate oxidation and maximal mitochondrial respiration were prevented in the presence of the antioxidants N-acetylcysteine and catalase, suggesting a potential role of reactive oxygen species in mediating these effects. Mitochondria isolated from red gastrocnemius and quadriceps femoris muscle from mice injected with LPS also demonstrated reduced respiratory control ratio (RCR), and ADP- and FCCP-stimulated respiration. Conclusion LPS exposure in skeletal muscle alters mitochondrial oxygen consumption and substrate preference, which is absent when antioxidants are present. PMID:25528444

  1. Stimulated Leaf Dark Respiration in Tomato in an Elevated Carbon Dioxide Atmosphere

    PubMed Central

    Li, Xin; Zhang, Guanqun; Sun, Bo; Zhang, Shuai; Zhang, Yiqing; Liao, Yangwenke; Zhou, Yanhong; Xia, Xiaojian; Shi, Kai; Yu, Jingquan

    2013-01-01

    It is widely accepted that leaf dark respiration is a determining factor for the growth and maintenance of plant tissues and the carbon cycle. However, the underlying effect and mechanism of elevated CO2 concentrations ([CO2]) on dark respiration remain unclear. In this study, tomato plants grown at elevated [CO2] showed consistently higher leaf dark respiratory rate, as compared with ambient control plants. The increased respiratory capacity was driven by a greater abundance of proteins, carbohydrates, and transcripts involved in pathways of glycolysis carbohydrate metabolism, the tricarboxylic acid cycle, and mitochondrial electron transport energy metabolism. This study provides substantial evidence in support of the concept that leaf dark respiration is increased by elevated [CO2] in tomato plants and suggests that the increased availability of carbohydrates and the increased energy status are involved in the increased rate of dark respiration in response to elevated [CO2]. PMID:24305603

  2. Cardioprotection by modulation of mitochondrial respiration during ischemia–reperfusion: Role of apoptosis-inducing factor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu, Aijun; Department of Anesthesiology, Tongji Hospital, Huazhong University of Science and Technology, Wuhan 430030; Szczepanek, Karol

    Highlights: •Blockade of electron transport prevents the loss of AIF from mitochondria during IR. •Blockade of electron transport decreases caspase-independent cell death during IR. •Mitochondrial AIF content is down-regulated in Harlequin mice. •Blockade of electron transport protects Harlequin mouse hearts during IR. •Amobarbital protection is partially dependent on mitochondrial AIF content. -- Abstract: The transient, reversible blockade of electron transport (BET) during ischemia or at the onset of reperfusion protects mitochondria and decreases cardiac injury. Apoptosis inducing factor (AIF) is located within the mitochondrial intermembrane space. A release of AIF from mitochondria into cytosol and nucleus triggers caspase-independent cell death.more » We asked if BET prevents the loss of AIF from mitochondria as a mechanism of protection in the buffer perfused heart. BET during ischemia with amobarbital, a rapidly reversible inhibitor of mitochondrial complex I, attenuated a release of AIF from mitochondria into cytosol, in turn decreasing the formation of cleaved and activated PARP-1. These results suggest that BET-mediated protection may occur through prevention of the loss of AIF from mitochondria during ischemia–reperfusion. In order to further clarify the role of mitochondrial AIF in BET-mediated protection, Harlequin (Hq) mice, a genetic model with mitochondrial AIF deficiency, were used to test whether BET could still decrease cell injury in Hq mouse hearts during reperfusion. BET during ischemia protected Hq mouse hearts against ischemia–reperfusion injury and improved mitochondrial function in these hearts during reperfusion. Thus, cardiac injury can still be decreased in the presence of down-regulated mitochondrial AIF content. Taken together, BET during ischemia protects both hearts with normal mitochondrial AIF content and hearts with mitochondrial AIF deficiency. Although preservation of mitochondrial AIF content plays a key role

  3. Mitochondrial vasculopathy

    PubMed Central

    Finsterer, Josef; Zarrouk-Mahjoub, Sinda

    2016-01-01

    Mitochondrial disorders (MIDs) are usually multisystem disorders (mitochondrial multiorgan disorder syndrome) either on from onset or starting at a point during the disease course. Most frequently affected tissues are those with a high oxygen demand such as the central nervous system, the muscle, endocrine glands, or the myocardium. Recently, it has been shown that rarely also the arteries may be affected (mitochondrial arteriopathy). This review focuses on the type, diagnosis, and treatment of mitochondrial vasculopathy in MID patients. A literature search using appropriate search terms was carried out. Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy. Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy, migraine-like headache, stroke-like episodes, or peripheral retinopathy. Mitochondrial macroangiopathy manifests as atherosclerosis, ectasia of arteries, aneurysm formation, dissection, or spontaneous rupture of arteries. The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes. Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes. Mitochondrial vasculopathy exists and manifests as micro- or macroangiopathy. Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications. PMID:27231520

  4. Disruption of hepatic mitochondrial bioenergetics is not a primary mechanism for the toxicity of methoprene - relevance for toxicological assessment.

    PubMed

    Monteiro, J P; Oliveira, P J; Moreno, A J M; Jurado, A S

    2008-07-01

    Methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate) is an insect growth regulator generally used to control insect populations by preventing insect maturation. So far, the effects of the insecticide on mitochondrial bioenergetics were not investigated. In the present work, liver mitochondria from Wistar rats were isolated and features of mitochondrial physiology were characterized in the presence of methoprene. High concentrations of methoprene, in the range of 40-100 nmol/mg of protein could decrease the transmembrane electric potential (Delta Psi) developed by mitochondria and, at the highest concentration, methoprene prevented complete Delta Psi repolarization after ADP addition. The effect was more evident using succinate than with ascorbate+TMPD as substrate. State 3 respiration was approximately 60% inhibited by 80 nmol of methoprene/mg of protein, while state 4 respiration, within the same range of methoprene concentrations, showed a slight increase, when both glutamate-malate and succinate were used as substrates. Additionally, FCCP-stimulated respiration was inhibited to an extent comparable to the effect on state 3, which suggests an interaction of methoprene with the respiratory chain, more evident with glutamate/malate as substrate. The activity of complex I (NADH-ubiquinone oxidorreductase) and that of the segment comprehending complexes II and III (succinate-cytochrome c reductase) were decreased in the presence of methoprene (approximately 60% and 85% of inhibition, respectively, with 300 nmol of methoprene/mg of protein), while the activities of cytochrome c oxidase and ATPase do not seem to be affected. Furthermore, the action of methoprene on the mitochondrial permeability transition was also studied, showing that the insecticide (in the range of 30-80 nmol mg(-1) of protein) decreases the susceptibility of liver mitochondria to the opening of the transition pore, even in non-energized mitochondria. These results lead to

  5. Effect of selected dimethylaminochalcones on some mitochondrial activities.

    PubMed

    Vašková, Janka; Reisch, Renáta; Vaško, Ladislav; Poškrobová, Martina; Kron, Ivan; Guzy, Juraj; Perjési, Pál

    2013-05-01

    Chalcones and their synthetic cyclic analogues have been shown to possess a full scale of biological activities in a variety of experimental systems. They were assessed to be mostly effective in defense against free radicals in the organism, but several compounds exhibited cytotoxic pro-oxidant activities. The respiratory response and antioxidant status in mitochondria were investigated upon addition of 4'-dimethylaminochalcone (1a) and its cyclic analogues, (E)-2-(4'-((CH3)2 N)-benzylidene)-1-indanone (1b), -1-tetralone (1c), and -1-benzosuberone (1d). Selected structures were able to change the respiratory response of mitochondria and showed an ability to modify mitochondrial metabolic and redox efficiency, though they did not indicate redox reactivity towards glutathione in adduct-free incubations. The results of the study indicate that -chalcone and -tetralone derivatives cause suppression of reactive oxygen species affecting mitochondrial respiration by mild uncoupling. In addition, (E)-2-(4'-((CH3)2 N)-indanone (1b), and to a greater extent, -benzosuberone (1d), showed pro-oxidant effects, which partially explain their cytotoxicity.

  6. Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

    PubMed Central

    Zabielski, Piotr; Lanza, Ian R.; Gopala, Srinivas; Holtz Heppelmann, Carrie J.; Bergen, H. Robert; Dasari, Surendra

    2016-01-01

    Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress. PMID:26718503

  7. Contributions of photosynthetic and non-photosynthetic cell types to leaf respiration in Vicia faba L. and their responses to growth temperature.

    PubMed

    Long, Benedict M; Bahar, Nur H A; Atkin, Owen K

    2015-11-01

    In intact leaves, mitochondrial populations are highly heterogeneous among contrasting cell types; how such contrasting populations respond to sustained changes in the environment remains, however, unclear. Here, we examined respiratory rates, mitochondrial protein composition and response to growth temperature in photosynthetic (mesophyll) and non-photosynthetic (epidermal) cells from fully expanded leaves of warm-developed (WD) and cold-developed (CD) broad bean (Vicia faba L.). Rates of respiration were significantly higher in mesophyll cell protoplasts (MCPs) than epidermal cell protoplasts (ECPs), with both protoplast types exhibiting capacity for cytochrome and alternative oxidase activity. Compared with ECPs, MCPs contained greater relative quantities of porin, suggesting higher mitochondrial surface area in mesophyll cells. Nevertheless, the relative quantities of respiratory proteins (normalized to porin) were similar in MCPs and ECPs, suggesting that ECPs have lower numbers of mitochondria yet similar protein complement to MCP mitochondria (albeit with lower abundance serine hydroxymethyltransferase). Several mitochondrial proteins (both non-photorespiratory and photorespiratory) exhibited an increased abundance in response to cold in both protoplast types. Based on estimates of individual protoplast respiration rates, combined with leaf cell abundance data, epidermal cells make a small but significant (2%) contribution to overall leaf respiration which increases twofold in the cold. Taken together, our data highlight the heterogeneous nature of mitochondrial populations in leaves, both among contrasting cell types and in how those populations respond to growth temperature. © 2015 John Wiley & Sons Ltd.

  8. Gem1 and ERMES Do Not Directly Affect Phosphatidylserine Transport from ER to Mitochondria or Mitochondrial Inheritance

    PubMed Central

    Nguyen, Tammy T; Lewandowska, Agnieszka; Choi, Jae-Yeon; Markgraf, Daniel F; Junker, Mirco; Bilgin, Mesut; Ejsing, Christer S; Voelker, Dennis R; Rapoport, Tom A; Shaw, Janet M

    2012-01-01

    In yeast, a protein complex termed the ER-Mitochondria Encounter Structure (ERMES) tethers mitochondria to the endoplasmic reticulum. ERMES proteins are implicated in a variety of cellular functions including phospholipid synthesis, mitochondrial protein import, mitochondrial attachment to actin, polarized mitochondrial movement into daughter cells during division, and maintenance of mitochondrial DNA (mtDNA). The mitochondrial-anchored Gem1 GTPase has been proposed to regulate ERMES functions. Here, we show that ERMES and Gem1 have no direct role in the transport of phosphatidylserine (PS) from the ER to mitochondria during the synthesis of phosphatidylethanolamine (PE), as PS to PE conversion is not affected in ERMES or gem1 mutants. In addition, we report that mitochondrial inheritance defects in ERMES mutants are a secondary consequence of mitochondrial morphology defects, arguing against a primary role for ERMES in mitochondrial association with actin and mitochondrial movement. Finally, we show that ERMES complexes are long-lived, and do not depend on the presence of Gem1. Our findings suggest that the ERMES complex may have primarily a structural role in maintaining mitochondrial morphology. PMID:22409400

  9. How does warming affect carbon allocation, respiration and residence time in trees? An isotope tracer approach in a eucalypt

    NASA Astrophysics Data System (ADS)

    Pendall, E.; Drake, J. E.; Furze, M.; Barton, C. V.; Carillo, Y.; Richter, A.; Tjoelker, M. G.

    2017-12-01

    Climate warming has the potential to alter the balance between photosynthetic carbon assimilation and respiratory losses in forest trees, leading to uncertainty in predicting their future physiological functioning. In a previous experiment, warming decreased canopy CO2 assimilation (A) rates of Eucalyptus tereticornis trees, but respiration (R) rates were usually not significantly affected, due to physiological acclimation to temperature. This led to a slight increase in (R/A) and thus decrease in plant carbon use efficiency with climate warming. In contrast to carbon fluxes, the effect of warming on carbon allocation and residence time in trees has received less attention. We conducted a study to test the hypothesis that warming would decrease the allocation of C belowground owing to reduced cost of nutrient uptake. E. parramattensis trees were grown in the field in unique whole-tree chambers operated at ambient and ambient +3 °C temperature treatments (n=3 per treatment). We applied a 13CO2 pulse and followed the label in CO2 respired from leaves, roots, canopy and soil, in plant sugars, and in rhizosphere microbes over a 3-week period in conjunction with measurements of tree growth. The 9-m tall, 57 m3 whole-tree chambers were monitored for CO2 concentrations in independent canopy and below ground (root and soil) compartments; periodic monitoring of δ13C values in air in the compartments allowed us to quantify the amount of 13CO2 assimilated and respired by each tree. Warmed trees grew faster and assimilated more of the label than control trees, but the 13C allocation to canopy, root and soil respiration was not altered. However, warming appeared to reduce the residence time of carbon respired from leaves, and especially from roots and soil, indicating that autotrophic respiration has the potential to feedback to climate change. This experiment provides insights into how warming may affect the fate of assimilated carbon from the leaf to the ecosystem scale.

  10. Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance

    PubMed Central

    Hoeks, Joris; van Herpen, Noud A.; Mensink, Marco; Moonen-Kornips, Esther; van Beurden, Denis; Hesselink, Matthijs K.C.; Schrauwen, Patrick

    2010-01-01

    OBJECTIVE Type 2 diabetes and insulin resistance have been associated with mitochondrial dysfunction, but it is debated whether this is a primary factor in the pathogenesis of the disease. To test the concept that mitochondrial dysfunction is secondary to the development of insulin resistance, we employed the unique model of prolonged fasting in humans. Prolonged fasting is a physiologic condition in which muscular insulin resistance develops in the presence of increased free fatty acid (FFA) levels, increased fat oxidation and low glucose and insulin levels. It is therefore anticipated that skeletal muscle mitochondrial function is maintained to accommodate increased fat oxidation unless factors secondary to insulin resistance exert negative effects on mitochondrial function. RESEARCH DESIGN AND METHODS While in a respiration chamber, twelve healthy males were subjected to a 60 h fast and a 60 h normal fed condition in a randomized crossover design. Afterward, insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp, and mitochondrial function was quantified ex vivo in permeabilized muscle fibers using high-resolution respirometry. RESULTS Indeed, FFA levels were increased approximately ninefold after 60 h of fasting in healthy male subjects, leading to elevated intramuscular lipid levels and decreased muscular insulin sensitivity. Despite an increase in whole-body fat oxidation, we observed an overall reduction in both coupled state 3 respiration and maximally uncoupled respiration in permeabilized skeletal muscle fibers, which could not be explained by changes in mitochondrial density. CONCLUSIONS These findings confirm that the insulin-resistant state has secondary negative effects on mitochondrial function. Given the low insulin and glucose levels after prolonged fasting, hyperglycemia and insulin action per se can be excluded as underlying mechanisms, pointing toward elevated plasma FFA and/or intramuscular fat accumulation as possible

  11. Alterations in mitochondrial dynamics induced by tebufenpyrad and pyridaben in a dopaminergic neuronal cell culture model

    PubMed Central

    Charli, Adhithiya; Jin, Huajun; Anantharam, Vellareddy; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

    2015-01-01

    Tebufenpyrad and pyridaben are two agro-chemically important acaricides that function like the known mitochondrial toxicant rotenone. Although these two compounds have been commonly used to kill populations of mites and ticks in commercial greenhouses, their neurotoxic profiles remain largely unknown. Therefore, we investigated the effects of these two pesticides on mitochondrial structure and function in an in vitro cell culture model using the Seahorse bioanalyzer and confocal fluorescence imaging. The effects were compared with rotenone. Exposing rat dopaminergic neuronal cells (N27 cells) to tebufenpyrad and pyridaben for 3 h induced dose-dependent cell death with an EC50 of 3.98 μM and 3.77 μM, respectively. Also, tebufenpyrad and pyridaben (3 μM) exposure induced reactive oxygen species (ROS) generation and m-aconitase damage, suggesting that the pesticide toxicity is associated with oxidative damage. Morphometric image analysis with the MitoTracker red fluorescent probe indicated that tebufenpyrad and pyridaben, as well as rotenone, caused abnormalities in mitochondrial morphology, including reduced mitochondrial length and circularity. Functional bioenergetic experiments using the Seahorse XF96 analyzer revealed that tebufenpyrad and pyridaben very rapidly suppressed the basal mitochondrial oxygen consumption rate similar to that of rotenone. Further analysis of bioenergetic curves also revealed dose-dependent decreases in ATP-linked respiration and respiratory capacity. The luminescence-based ATP measurement further confirmed that pesticide-induced mitochondrial inhibition of respiration is accompanied by the loss of cellular ATP. Collectively, our results suggest that exposure to the pesticides tebufenpyrad and pyridaben induces neurotoxicity by rapidly initiating mitochondrial dysfunction and oxidative damage in dopaminergic neuronal cells. Our findings also reveal that monitoring the kinetics of mitochondrial respiration with Seahorse could be used

  12. Glucose Modulates Respiratory Complex I Activity in Response to Acute Mitochondrial Dysfunction

    PubMed Central

    Cannino, Giuseppe; El-Khoury, Riyad; Pirinen, Marja; Hutz, Bettina; Rustin, Pierre; Jacobs, Howard T.; Dufour, Eric

    2012-01-01

    Proper coordination between glycolysis and respiration is essential, yet the regulatory mechanisms involved in sensing respiratory chain defects and modifying mitochondrial functions accordingly are unclear. To investigate the nature of this regulation, we introduced respiratory bypass enzymes into cultured human (HEK293T) cells and studied mitochondrial responses to respiratory chain inhibition. In the absence of respiratory chain inhibitors, the expression of alternative respiratory enzymes did not detectably alter cell physiology or mitochondrial function. However, in permeabilized cells NDI1 (alternative NADH dehydrogenase) bypassed complex I inhibition, whereas alternative oxidase (AOX) bypassed complex III or IV inhibition. In contrast, in intact cells the effects of the AOX bypass were suppressed by growth on glucose, whereas those produced by NDI1 were unaffected. Moreover, NDI1 abolished the glucose suppression of AOX-driven respiration, implicating complex I as the target of this regulation. Rapid Complex I down-regulation was partly released upon prolonged respiratory inhibition, suggesting that it provides an “emergency shutdown” system to regulate metabolism in response to dysfunctions of the oxidative phosphorylation. This system was independent of HIF1, mitochondrial superoxide, or ATP synthase regulation. Our findings reveal a novel pathway for adaptation to mitochondrial dysfunction and could provide new opportunities for combatting diseases. PMID:23007390

  13. Mitochondrial-nuclear genome interactions in nonalcoholic fatty liver disease in mice

    PubMed Central

    Betancourt, Angela M.; King, Adrienne L.; Fetterman, Jessica L.; Millender-Swain, Telisha; Finley, Rachel D.; Oliva, Claudia R.; Crowe, David Ralph; Ballinger, Scott W.; Bailey, Shannon M.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation, and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. Herein, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, Mitochondrial-Nuclear eXchange (MNX) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared to wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation, and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD. PMID:24758559

  14. Soil respiration as affected by long-term broiler litter application to a udult in the ozark highlands.

    PubMed

    McMullen, Richard L; Brye, Kristofor R; Gbur, Edward E

    2015-01-01

    The United States produced 8.4 billion broiler chickens () and an estimated 10.1 to 14.3 million Mg of broiler litter (BL) in 2012. Arkansas' production of 1 billion broilers in 2012 produced an estimated 1.2 to 1.7 million Mg of BL, most of which was concentrated in the Ozark Highlands region of northwest Arkansas. Increased CO release from soils associated with agricultural practices has generated concerns regarding the contribution of certain agricultural management practices to global warming. The objectives of this study were to evaluate the effects of long-term (>6 yr) BL application to a Udult on soil respiration and annual C emissions and to determine the predictability of soil respiration based on soil temperature and moisture in the Ozark Highlands region of northwest Arkansas. Soil respiration was measured routinely between May 2009 and May 2012 in response to annual BL application rates of 0, 5.6, and 11.2 Mg dry litter ha that began in 2003. Soil respiration varied ( < 0.01) with BL rate, measurement date, and year. Additions of BL stimulated respiration after application, and rainfall events after dry-soil conditions stimulated respiration in all years. Soil temperature at the 10-cm depth, 0- to 6-cm soil volumetric water content (VWC), and annual CO-C emissions were unaffected ( > 0.05) by BL application rate but differed ( < 0.01) among study years. Multiple regression indicated that soil respiration could be reasonably predicted using 2-cm-depth soil temperature (T) and the product of T and VWC as predictors ( = 0.52; < 0.01). Results indicate that organic amendments, such as BL, can stimulate release of CO from the soil to the atmosphere, potentially negatively affecting atmospheric greenhouse gas concentrations; thus, there may be application rates above which the benefits of organic amendments may be diminished by adverse environmental effects. Improved BL management strategies are needed to lessen the loss of CO from BL-amended soils. Copyright

  15. Mitochondrial genomic variation associated with higher mitochondrial copy number: the Cache County Study on Memory Health and Aging.

    PubMed

    Ridge, Perry G; Maxwell, Taylor J; Foutz, Spencer J; Bailey, Matthew H; Corcoran, Christopher D; Tschanz, JoAnn T; Norton, Maria C; Munger, Ronald G; O'Brien, Elizabeth; Kerber, Richard A; Cawthon, Richard M; Kauwe, John S K

    2014-01-01

    The mitochondria are essential organelles and are the location of cellular respiration, which is responsible for the majority of ATP production. Each cell contains multiple mitochondria, and each mitochondrion contains multiple copies of its own circular genome. The ratio of mitochondrial genomes to nuclear genomes is referred to as mitochondrial copy number. Decreases in mitochondrial copy number are known to occur in many tissues as people age, and in certain diseases. The regulation of mitochondrial copy number by nuclear genes has been studied extensively. While mitochondrial variation has been associated with longevity and some of the diseases known to have reduced mitochondrial copy number, the role that the mitochondrial genome itself has in regulating mitochondrial copy number remains poorly understood. We analyzed the complete mitochondrial genomes from 1007 individuals randomly selected from the Cache County Study on Memory Health and Aging utilizing the inferred evolutionary history of the mitochondrial haplotypes present in our dataset to identify sequence variation and mitochondrial haplotypes associated with changes in mitochondrial copy number. Three variants belonging to mitochondrial haplogroups U5A1 and T2 were significantly associated with higher mitochondrial copy number in our dataset. We identified three variants associated with higher mitochondrial copy number and suggest several hypotheses for how these variants influence mitochondrial copy number by interacting with known regulators of mitochondrial copy number. Our results are the first to report sequence variation in the mitochondrial genome that causes changes in mitochondrial copy number. The identification of these variants that increase mtDNA copy number has important implications in understanding the pathological processes that underlie these phenotypes.

  16. Mitochondrial myopathies.

    PubMed

    DiMauro, Salvatore

    2006-11-01

    Our understanding of mitochondrial diseases (defined restrictively as defects of the mitochondrial respiratory chain) is expanding rapidly. In this review, I will give the latest information on disorders affecting predominantly or exclusively skeletal muscle. The most recently described mitochondrial myopathies are due to defects in nuclear DNA, including coenzyme Q10 deficiency and mutations in genes controlling mitochondrial DNA abundance and structure, such as POLG, TK2, and MPV17. Barth syndrome, an X-linked recessive mitochondrial myopathy/cardiopathy, is associated with decreased amount and altered structure of cardiolipin, the main phospholipid of the inner mitochondrial membrane, but a secondary impairment of respiratory chain function is plausible. The role of mutations in protein-coding genes of mitochondrial DNA in causing isolated myopathies has been confirmed. Mutations in tRNA genes of mitochondrial DNA can also cause predominantly myopathic syndromes and--contrary to conventional wisdom--these mutations can be homoplasmic. Defects in the mitochondrial respiratory chain impair energy production and almost invariably involve skeletal muscle, causing exercise intolerance, cramps, recurrent myoglobinuria, or fixed weakness, which often affects extraocular muscles and results in droopy eyelids (ptosis) and progressive external ophthalmoplegia.

  17. The mitochondrial-targeted antioxidant, MitoQ, increases liver mitochondrial cardiolipin content in obesogenic diet-fed rats.

    PubMed

    Fouret, Gilles; Tolika, Evanthia; Lecomte, Jérôme; Bonafos, Béatrice; Aoun, Manar; Murphy, Michael P; Ferreri, Carla; Chatgilialoglu, Chryssostomos; Dubreucq, Eric; Coudray, Charles; Feillet-Coudray, Christine

    2015-10-01

    Cardiolipin (CL), a unique mitochondrial phospholipid, plays a key role in several processes of mitochondrial bioenergetics as well as in mitochondrial membrane stability and dynamics. The present study was designed to determine the effect of MitoQ, a mitochondrial-targeted antioxidant, on the content of liver mitochondrial membrane phospholipids, in particular CL, and its fatty acid composition in obesogenic diet-fed rats. To do this, twenty-four 6week old male Sprague Dawley rats were randomized into three groups of 8 animals and fed for 8weeks with either a control diet, a high fat diet (HF), or a HF diet with MitoQ (HF+MitoQ). Phospholipid classes and fatty acid composition were assayed by chromatographic methods in liver and liver mitochondria. Mitochondrial bioenergetic function was also evaluated. While MitoQ had no or slight effects on total liver fatty acid composition and phospholipid classes and their fatty acid composition, it had major effects on liver mitochondrial phospholipids and mitochondrial function. Indeed, MitoQ both increased CL synthase gene expression and CL content of liver mitochondria and increased 18:2n-6 (linoleic acid) content of mitochondrial phospholipids by comparison to the HF diet. Moreover, mitochondrial CL content was positively correlated to mitochondrial membrane fluidity, membrane potential and respiration, as well as to ATP synthase activity, while it was negatively correlated to mitochondrial ROS production. These findings suggest that MitoQ may decrease pathogenic alterations to CL content and profiles, thereby preserving mitochondrial function and attenuating the development of some of the features of metabolic syndrome in obesogenic diet-fed rats. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Apolipoprotein E4 (1-272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells.

    PubMed

    Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

    2009-08-20

    Apolipoprotein E allele epsilon4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1-272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1-272) more strongly than intact apoE4(1-299). Further analysis showed that in Neuro-2a cells expressing apoE4(1-272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1-299). ApoE4(1-272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1-272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration.

  19. Mitochondrial role in cell aging

    NASA Technical Reports Server (NTRS)

    Miquel, J.; Fleming, J.; Economos, A. C.; Johnson, J. E., Jr.

    1980-01-01

    The experimental studies on the mitochondria of insect and mammalian cells are examined with a view to an analysis of intrinsic mitochondrial senescence, and its relation to the age-related changes in other cell organelles. The fine structural and biochemical data support the concept that the mitochondria of fixed postmitotic cells may be the site of intrinsic aging because of the attack by free radicals and lipid peroxides originating in the organelles as a by-product of oxygen reduction during respiration. Although the cells have numerous mechanisms for counteracting lipid peroxidation injury, there is a slippage in the antioxidant protection. Intrinsic mitochondrial aging could thus be considered as a specific manifestation of oxygen toxicity. It is proposed that free radical injury renders an increasing number of the mitochondria unable to divide, probably because of damage to the lipids of the inner membrane and to mitochondrial DNA.

  20. Investigating mitochondrial dysfunction in human lung cells exposed to redox-active PM components.

    PubMed

    Lavrich, Katelyn S; Corteselli, Elizabeth M; Wages, Phillip A; Bromberg, Philip A; Simmons, Steven O; Gibbs-Flournoy, Eugene A; Samet, James M

    2018-03-01

    Exposure to ambient particulate matter (PM) causes cardiopulmonary morbidity and mortality through mechanisms that involve oxidative stress. 1,2-naphthoquinone (1,2-NQ) is a ubiquitous component of PM and a potent redox-active electrophile. We previously reported that 1,2-NQ increases mitochondrial H 2 O 2 production through an unidentified mechanism. We sought to characterize the effects of 1,2-NQ exposure on mitochondrial respiration as a source of H 2 O 2 in human airway epithelial cells. We measured the effects of acute exposure to 1,2-NQ on oxygen consumption rate (OCR) in the human bronchial epithelial cell line BEAS-2B and mitochondrial preparations using extracellular flux analysis. Complex-specific assays and NADPH depletion by glucose deprivation distinguished between mitochondrial and non-mitochondrial oxygen utilization. 1,2-NQ exposure of BEAS cells caused a rapid, marked dose-dependent increase in OCR that was independent of mitochondrial respiration, exceeded the OCR observed after mitochondrial uncoupling, and remained sensitive to NADPH depletion, implicating extra-mitochondrial redox cycling processes. Similar effects were observed with the environmentally relevant redox-cycling quinones 1,4-naphthoquinone and 9,10-phenanthrenequinone, but not with quinones that do not redox cycle, such as 1,4-benzoquinone. In mitochondrial preparations, 1,2-NQ caused a decrease in Complex I-linked substrate oxidation, suggesting impairment of pyruvate utilization or transport, a novel mechanism of mitochondrial inhibition by an environmental exposure. This study also highlights the methodological utility and challenges in the use of extracellular flux analysis to elucidate the mechanisms of action of redox-active electrophiles present in ambient air. Published by Elsevier Inc.

  1. Unraveling Biochemical Pathways Affected by Mitochondrial Dysfunctions Using Metabolomic Approaches

    PubMed Central

    Demine, Stéphane; Reddy, Nagabushana; Renard, Patricia; Raes, Martine; Arnould, Thierry

    2014-01-01

    Mitochondrial dysfunction(s) (MDs) can be defined as alterations in the mitochondria, including mitochondrial uncoupling, mitochondrial depolarization, inhibition of the mitochondrial respiratory chain, mitochondrial network fragmentation, mitochondrial or nuclear DNA mutations and the mitochondrial accumulation of protein aggregates. All these MDs are known to alter the capacity of ATP production and are observed in several pathological states/diseases, including cancer, obesity, muscle and neurological disorders. The induction of MDs can also alter the secretion of several metabolites, reactive oxygen species production and modify several cell-signalling pathways to resolve the mitochondrial dysfunction or ultimately trigger cell death. Many metabolites, such as fatty acids and derived compounds, could be secreted into the blood stream by cells suffering from mitochondrial alterations. In this review, we summarize how a mitochondrial uncoupling can modify metabolites, the signalling pathways and transcription factors involved in this process. We describe how to identify the causes or consequences of mitochondrial dysfunction using metabolomics (liquid and gas chromatography associated with mass spectrometry analysis, NMR spectroscopy) in the obesity and insulin resistance thematic. PMID:25257998

  2. The effect of crataegus fruit extract and some of its flavonoids on mitochondrial oxidative phosphorylation in the heart.

    PubMed

    Bernatoniene, J; Trumbeckaite, S; Majiene, D; Baniene, R; Baliutyte, G; Savickas, A; Toleikis, A

    2009-12-01

    Crataegus (Hawthorn) fruit extracts (CE) are widely used for the treatment of various cardiovascular diseases (arrhythmias, heart failure, myocardial weakness, etc). Despite the fact that many of these diseases are associated with disturbances of the mitochondria, no data have been found on the effect of CE on their function. The aim of this study was to perform an oxygraphic investigation of the effect of CE (in concentration range from 70 ng/mL to 13.9 microg/mL of Crataegus phenolic compounds (PC)) and its several pure flavonoids on isolated rat heart mitochondria respiring on pyruvate+malate, succinate and palmitoyl-L-carnitine+malate. CE at doses under 278 ng/mL of PC had no effect on mitochondrial functions. At concentrations from 278 ng/mL to 13.9 microg/mL of PC, CE stimulated State 2 respiration by 11%-34% with all used substrates, and decreased the mitochondrial membrane potential by 1.2-4.4 mV measured with a tetraphenylphosphonium-selective electrode and H2O2 production measured fluorimetrically. Similar uncoupling effects on mitochondrial respiration were observed with several pure CE flavonoids. The highest CE concentration also slightly reduced the maximal ADP-stimulated and uncoupled respiration, which might be due to inhibition of the mitochondrial respiratory chain between flavoprotein and cytochrome c. Whether or not the uncoupling and other effects of CE on mitochondria may be realized in vivo remains to be determined. Copyright (c) 2009 John Wiley & Sons, Ltd.

  3. Long-Term Skeletal Muscle Mitochondrial Dysfunction is Associated with Hypermetabolism in Severely Burned Children.

    PubMed

    Porter, Craig; Herndon, David N; Børsheim, Elisabet; Bhattarai, Nisha; Chao, Tony; Reidy, Paul T; Rasmussen, Blake B; Andersen, Clark R; Suman, Oscar E; Sidossis, Labros S

    2016-01-01

    The long-term impact of burn trauma on skeletal muscle bioenergetics remains unknown. Here, the authors determined respiratory capacity and function of skeletal muscle mitochondria in healthy individuals and in burn victims for up to 2 years postinjury. Biopsies were collected from the m. vastus lateralis of 16 healthy men (26 ± 4 years) and 69 children (8 ± 5 years) with burns encompassing ≥30% of their total BSA. Seventy-nine biopsies were collected from cohorts of burn victims at 2 weeks (n = 18), 6 months (n = 18), 12 months (n = 25), and 24 months (n = 18) postburn. Hypermetabolism was determined by the difference in predicted and measured metabolic rate. Mitochondrial respiration was determined in saponin-permeabilized myofiber bundles. Outcomes were modeled by analysis of variance, with differences in groups assessed by Tukey-adjusted contrasts. Burn patients were hypermetabolic for up to 2 years postinjury. Coupled mitochondrial respiration was lower at 2 weeks (17 [8] pmol/sec/mg; P < .001), 6 months (41 [30] pmol/sec/mg; P = .03), and 12 months (35 [14] pmol/sec/mg; P < .001) postburn compared with healthy controls (58 [13] pmol/sec/mg). Coupled respiration was greater at 6, 12, and 24 months postburn vs 2 weeks postburn (P < .001). Mitochondrial adenosine diphosphate and oligomycin sensitivity (measures of coupling control) were lower at all time-points postburn vs control (P < .05), but greater at 6, 12, and 24 months postburn vs 2 weeks postburn (P < .05). Muscle mitochondrial respiratory capacity remains significantly lower in burn victims for 1-year postinjury. Mitochondrial coupling control is diminished for up to 2 years postinjury in burn victims, resulting in greater mitochondrial thermogenesis. These quantitative and qualitative derangements in skeletal muscle bioenergetics likely contribute to the long-term pathophysiological stress response to burn trauma.

  4. Synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression.

    PubMed

    Bellanti, Francesco; Villani, Rosanna; Tamborra, Rosanna; Blonda, Maria; Iannelli, Giuseppina; di Bello, Giorgia; Facciorusso, Antonio; Poli, Giuseppe; Iuliano, Luigi; Avolio, Carlo; Vendemiale, Gianluigi; Serviddio, Gaetano

    2018-05-01

    The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs) and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol) combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial biogenesis. Finally, a lower protein content in the mitochondrial respiratory chain complexes was observed in human non-alcoholic steatohepatitis. In conclusion, hepatic accumulation of FFAs and non-enzymatic oxysterols synergistically facilitates development and progression of NAFLD by impairing mitochondrial function, energy balance and biogenesis adaptation to chronic injury. Copyright © 2017. Published by Elsevier B.V.

  5. Mitochondrial functions of THP-1 monocytes following the exposure to selected natural compounds.

    PubMed

    Schultze, Nadin; Wanka, Heike; Zwicker, Paula; Lindequist, Ulrike; Haertel, Beate

    2017-02-15

    The immune system is an important target of various xenobiotics, which may lead to severe adverse effects including immunosuppression or inappropriate immunostimulation. Mitochondrial toxicity is one possibility by which xenobiotics exert their toxic effects in cells or organs. In this study, we investigated the impact of three natural compounds, cyclosporine A (CsA), deoxynivalenol (DON) and cannabidiol (CBD) on mitochondrial functions in the THP-1 monocytic cell line. The cells were exposed for 24h to two different concentrations (IC 10 and IC 50 determined by MTT) of each compound. The cells showed concentration-dependent elevated intracellular reactive oxygen species (iROS) and induction of apoptosis (except DON) in response to the three test compounds. Mitochondrial functions were characterized by using bioenergetics profiling experiments. In THP-1 monocytes, the IC 50 of CsA decreased basal and maximal respiration as well as ATP production with an impact on spare capacity indicating a mitochondrial dysfunction. Similar reaction patterns were observed following CBD exposure. The basal respiration level and ATP-production decreased in the THP-1 cells exposed to the IC 50 of DON with no major impact on mitochondrial function. In conclusion, impaired mitochondrial function was accompanied by elevated iROS and apoptosis level in a monocytic cell line exposed to CsA and CBD. Mitochondrial dysfunction may be one explanation for the cytotoxicity of CBD and CsA also in other in immune cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Permeabilization of brain tissue in situ enables multiregion analysis of mitochondrial function in a single mouse brain.

    PubMed

    Herbst, Eric A F; Holloway, Graham P

    2015-02-15

    Mitochondrial function in the brain is traditionally assessed through analysing respiration in isolated mitochondria, a technique that possesses significant tissue and time requirements while also disrupting the cooperative mitochondrial reticulum. We permeabilized brain tissue in situ to permit analysis of mitochondrial respiration with the native mitochondrial morphology intact, removing the need for isolation time and minimizing tissue requirements to ∼2 mg wet weight. The permeabilized brain technique was validated against the traditional method of isolated mitochondria and was then further applied to assess regional variation in the mouse brain with ischaemia-reperfusion injuries. A transgenic mouse model overexpressing catalase within mitochondria was applied to show the contribution of mitochondrial reactive oxygen species to ischaemia-reperfusion injuries in different brain regions. This technique enhances the accessibility of addressing physiological questions in small brain regions and in applying transgenic mouse models to assess mechanisms regulating mitochondrial function in health and disease. Mitochondria function as the core energy providers in the brain and symptoms of neurodegenerative diseases are often attributed to their dysregulation. Assessing mitochondrial function is classically performed in isolated mitochondria; however, this process requires significant isolation time, demand for abundant tissue and disruption of the cooperative mitochondrial reticulum, all of which reduce reliability when attempting to assess in vivo mitochondrial bioenergetics. Here we introduce a method that advances the assessment of mitochondrial respiration in the brain by permeabilizing existing brain tissue to grant direct access to the mitochondrial reticulum in situ. The permeabilized brain preparation allows for instant analysis of mitochondrial function with unaltered mitochondrial morphology using significantly small sample sizes (∼2 mg), which permits

  7. Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

    PubMed

    Zhdanov, Alexander V; Waters, Alicia H C; Golubeva, Anna V; Dmitriev, Ruslan I; Papkovsky, Dmitri B

    2014-01-01

    Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment. © 2013.

  8. Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

    PubMed

    Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

    2013-01-01

    Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and

  9. Frost Induces Respiration and Accelerates Carbon Depletion in Trees.

    PubMed

    Sperling, Or; Earles, J Mason; Secchi, Francesca; Godfrey, Jessie; Zwieniecki, Maciej A

    2015-01-01

    Cellular respiration depletes stored carbohydrates during extended periods of limited photosynthesis, e.g. winter dormancy or drought. As respiration rate is largely a function of temperature, the thermal conditions during such periods may affect non-structural carbohydrate (NSC) availability and, ultimately, recovery. Here, we surveyed stem responses to temperature changes in 15 woody species. For two species with divergent respirational response to frost, P. integerrima and P. trichocarpa, we also examined corresponding changes in NSC levels. Finally, we simulated respiration-induced NSC depletion using historical temperature data for the western US. We report a novel finding that tree stems significantly increase respiration in response to near freezing temperatures. We observed this excess respiration in 13 of 15 species, deviating 10% to 170% over values predicted by the Arrhenius equation. Excess respiration persisted at temperatures above 0 °C during warming and reoccurred over multiple frost-warming cycles. A large adjustment of NSCs accompanied excess respiration in P. integerrima, whereas P. trichocarpa neither excessively respired nor adjusted NSCs. Over the course of the years included in our model, frost-induced respiration accelerated stem NSC consumption by 8.4 mg (glucose eq.) cm(-3) yr(-1) on average in the western US, a level of depletion that may continue to significantly affect spring NSC availability. This novel finding revises the current paradigm of low temperature respiration kinetics.

  10. Frost Induces Respiration and Accelerates Carbon Depletion in Trees

    PubMed Central

    Sperling, Or; Earles, J. Mason; Secchi, Francesca; Godfrey, Jessie; Zwieniecki, Maciej A.

    2015-01-01

    Cellular respiration depletes stored carbohydrates during extended periods of limited photosynthesis, e.g. winter dormancy or drought. As respiration rate is largely a function of temperature, the thermal conditions during such periods may affect non-structural carbohydrate (NSC) availability and, ultimately, recovery. Here, we surveyed stem responses to temperature changes in 15 woody species. For two species with divergent respirational response to frost, P. integerrima and P. trichocarpa, we also examined corresponding changes in NSC levels. Finally, we simulated respiration-induced NSC depletion using historical temperature data for the western US. We report a novel finding that tree stems significantly increase respiration in response to near freezing temperatures. We observed this excess respiration in 13 of 15 species, deviating 10% to 170% over values predicted by the Arrhenius equation. Excess respiration persisted at temperatures above 0°C during warming and reoccurred over multiple frost-warming cycles. A large adjustment of NSCs accompanied excess respiration in P. integerrima, whereas P. trichocarpa neither excessively respired nor adjusted NSCs. Over the course of the years included in our model, frost-induced respiration accelerated stem NSC consumption by 8.4 mg (glucose eq.) cm-3 yr-1 on average in the western US, a level of depletion that may continue to significantly affect spring NSC availability. This novel finding revises the current paradigm of low temperature respiration kinetics. PMID:26629819

  11. The inter-relationship of ascorbate transport, metabolism and mitochondrial, plastidic respiration.

    PubMed

    Szarka, András; Bánhegyi, Gábor; Asard, Han

    2013-09-20

    Ascorbate, this multifaceted small molecular weight carbohydrate derivative, plays important roles in a range of cellular processes in plant cells, from the regulation of cell cycle, through cell expansion and senescence. Beyond these physiological functions, ascorbate has a critical role in responses to abiotic stresses, such as high light, high salinity, or drought. The biosynthesis, recycling, and intracellular transport are important elements of the balancing of ascorbate level to the always-changing conditions and demands. A bidirectional tight relationship was described between ascorbate biosynthesis and the mitochondrial electron transfer chain (mETC), since L-galactono-1,4-lactone dehydrogenase (GLDH), the enzyme catalyzing the ultimate step of ascorbate biosynthesis, uses oxidized cytochrome c as the only electron acceptor and has a role in the assembly of Complex I. A similar bidirectional relationship was revealed between the photosynthetic apparatus and ascorbate biosynthesis since the electron flux through the photosynthetic ETC affects the biosynthesis of ascorbate and the level of ascorbate could affect photosynthesis. The details of this regulatory network of photosynthetic electron transfer, respiratory electron transfer, and ascorbate biosynthesis are still not clear, as are the potential regulatory role and the regulation of intracellular ascorbate transport and fluxes. The elucidation of the role of ascorbate as an important element of the network of photosynthetic, respiratory ETC and tricarboxylic acid cycle will contribute to understanding plant cell responses to different stress conditions.

  12. Optical tweezers and non-ratiometric fluorescent-dye-based studies of respiration in sperm mitochondria

    NASA Astrophysics Data System (ADS)

    Chen, Timothy; Shi, Linda Z.; Zhu, Qingyuan; Chandsawangbhuwana, Charlie; Berns, Michael W.

    2011-04-01

    The purpose of this study is to investigate how the mitochondrial membrane potential affects sperm motility using laser tweezers and a non-ratiometric fluorescent probe, DiOC6(3). A 1064 nm Nd:YVO4 continuous wave laser was used to trap motile sperm at a power of 450 mW in the trap spot. Using customized tracking software, the curvilinear velocity (VCL) and the escape force from the laser tweezers were measured. Human (Homo sapiens), dog (Canis lupis familiaris) and drill (Mandrillus leucophaeus) sperm were treated with DiOC6(3) to measure the membrane potential in the mitochondria-rich sperm midpieces. Sperm from all three species exhibited an increase in fluorescence when treated with the DiOC6(3). When a cyanide inhibitor (CCCP) of aerobic respiration was applied, sperm of all three species exhibited a reduction in fluorescence to pre-dye levels. With respect to VCL and escape force, the CCCP had no effect on dog or human sperm, suggesting a major reliance upon anaerobic respiration (glycolysis) for ATP in these two species. Based on the preliminary study on drill sperm, CCCP caused a drop in the VCL, suggesting potential reliance on both glycolysis and aerobic respiration for motility. The results demonstrate that optical trapping in combination with DiOC6(3) is an effective way to study sperm motility and energetics.

  13. Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

    PubMed

    Vanlerberghe, G C; McIntosh, L

    1992-12-01

    In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

  14. Mitochondrial-nuclear genome interactions in non-alcoholic fatty liver disease in mice.

    PubMed

    Betancourt, Angela M; King, Adrienne L; Fetterman, Jessica L; Millender-Swain, Telisha; Finley, Rachel D; Oliva, Claudia R; Crowe, David R; Ballinger, Scott W; Bailey, Shannon M

    2014-07-15

    NAFLD (non-alcoholic fatty liver disease) involves significant changes in liver metabolism characterized by oxidative stress, lipid accumulation and fibrogenesis. Mitochondrial dysfunction and bioenergetic defects also contribute to NAFLD. In the present study, we examined whether differences in mtDNA influence NAFLD. To determine the role of mitochondrial and nuclear genomes in NAFLD, MNX (mitochondrial-nuclear exchange) mice were fed an atherogenic diet. MNX mice have mtDNA from C57BL/6J mice on a C3H/HeN nuclear background and vice versa. Results from MNX mice were compared with wild-type C57BL/6J and C3H/HeN mice fed a control or atherogenic diet. Mice with the C57BL/6J nuclear genome developed more macrosteatosis, inflammation and fibrosis compared with mice containing the C3H/HeN nuclear genome when fed the atherogenic diet. These changes were associated with parallel alterations in inflammation and fibrosis gene expression in wild-type mice, with intermediate responses in MNX mice. Mice with the C57BL/6J nuclear genome had increased State 4 respiration, whereas MNX mice had decreased State 3 respiration and RCR (respiratory control ratio) when fed the atherogenic diet. Complex IV activity and most mitochondrial biogenesis genes were increased in mice with the C57BL/6J nuclear or mitochondrial genome, or both fed the atherogenic diet. These results reveal new interactions between mitochondrial and nuclear genomes and support the concept that mtDNA influences mitochondrial function and metabolic pathways implicated in NAFLD.

  15. Negative regulation of STAT3 protein-mediated cellular respiration by SIRT1 protein.

    PubMed

    Bernier, Michel; Paul, Rajib K; Martin-Montalvo, Alejandro; Scheibye-Knudsen, Morten; Song, Shaoming; He, Hua-Jun; Armour, Sean M; Hubbard, Basil P; Bohr, Vilhelm A; Wang, Lili; Zong, Yaping; Sinclair, David A; de Cabo, Rafael

    2011-06-03

    In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions.

  16. Rejuvenating cellular respiration for optimizing respiratory function: targeting mitochondria.

    PubMed

    Agrawal, Anurag; Mabalirajan, Ulaganathan

    2016-01-15

    Altered bioenergetics with increased mitochondrial reactive oxygen species production and degradation of epithelial function are key aspects of pathogenesis in asthma and chronic obstructive pulmonary disease (COPD). This motif is not unique to obstructive airway disease, reported in related airway diseases such as bronchopulmonary dysplasia and parenchymal diseases such as pulmonary fibrosis. Similarly, mitochondrial dysfunction in vascular endothelium or skeletal muscles contributes to the development of pulmonary hypertension and systemic manifestations of lung disease. In experimental models of COPD or asthma, the use of mitochondria-targeted antioxidants, such as MitoQ, has substantially improved mitochondrial health and restored respiratory function. Modulation of noncoding RNA or protein regulators of mitochondrial biogenesis, dynamics, or degradation has been found to be effective in models of fibrosis, emphysema, asthma, and pulmonary hypertension. Transfer of healthy mitochondria to epithelial cells has been associated with remarkable therapeutic efficacy in models of acute lung injury and asthma. Together, these form a 3R model--repair, reprogramming, and replacement--for mitochondria-targeted therapies in lung disease. This review highlights the key role of mitochondrial function in lung health and disease, with a focus on asthma and COPD, and provides an overview of mitochondria-targeted strategies for rejuvenating cellular respiration and optimizing respiratory function in lung diseases. Copyright © 2016 the American Physiological Society.

  17. Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Montaigne, David; Marechal, Xavier; Baccouch, Riadh

    2010-05-01

    The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solutionmore » containing 1 muM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt{sub max} of 105 +- 8 mN/s in control hearts vs. 49 +- 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 +- 0.2 in control hearts vs. 2.2 +- 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 +- 1 muM cytochrome c/min/mg in control hearts vs. 14 +- 3 muM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.« less

  18. Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase.

    PubMed

    Bridges, Hannah R; Sirviö, Ville A; Agip, Ahmed-Noor A; Hirst, Judy

    2016-08-09

    The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known. Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments. Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.

  19. Glutamate Impairs Mitochondria Aerobic Respiration Capacity and Enhances Glycolysis in Cultured Rat Astrocytes.

    PubMed

    Yan, Xu; Shi, Zhong Fang; Xu, Li Xin; Li, Jia Xin; Wu, Min; Wang, Xiao Xuan; Jia, Mei; Dong, Li Ping; Yang, Shao Hua; Yuan, Fang

    2017-01-01

    To study the effect of glutamate on metabolism, shifts in glycolysis and lactate release in rat astrocytes. After 10 days, secondary cultured astrocytes were treated with 1 mmol/L glutamate for 1 h, and the oxygen consumption rates (OCR) and extra cellular acidification rate (ECAR) was analyzed using a Seahorse XF 24 Extracellular Flux Analyzer. Cell viability was then evaluated by MTT assay. Moreover, changes in extracellular lactate concentration induced by glutamate were tested with a lactate detection kit. Compared with the control group, treatment with 1 mmol/L glutamate decreased the astrocytes' maximal respiration and spare respiratory capacity but increased their glycolytic capacity and glycolytic reserve. Further analysis found that 1-h treatment with different concentrations of glutamate (0.1-1 mmol/L) increased lactate release from astrocytes, however the cell viability was not affected by the glutamate treatment. The current study provided direct evidence that exogenous glutamate treatment impaired the mitochondrial respiration capacity of astrocytes and enhanced aerobic glycolysis, which could be involved in glutamate injury or protection mechanisms in response to neurological disorders. Copyright © 2017 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  20. Mitochondrial O-GlcNAc Transferase (mOGT) Regulates Mitochondrial Structure, Function, and Survival in HeLa Cells*

    PubMed Central

    Sacoman, Juliana L.; Dagda, Raul Y.; Burnham-Marusich, Amanda R.; Dagda, Ruben K.; Berninsone, Patricia M.

    2017-01-01

    O-Linked N-acetylglucosamine transferase (OGT) catalyzes O-GlcNAcylation of target proteins and regulates numerous biological processes. OGT is encoded by a single gene that yields nucleocytosolic and mitochondrial isoforms. To date, the role of the mitochondrial isoform of OGT (mOGT) remains largely unknown. Using high throughput proteomics, we identified 84 candidate mitochondrial glycoproteins, of which 44 are novel. Notably, two of the candidate glycoproteins identified (cytochrome oxidase 2 (COX2) and NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4)) are encoded by mitochondrial DNA. Using siRNA in HeLa cells, we found that reducing endogenous mOGT expression leads to alterations in mitochondrial structure and function, including Drp1-dependent mitochondrial fragmentation, reduction in mitochondrial membrane potential, and a significant loss of mitochondrial content in the absence of mitochondrial ROS. These defects are associated with a compensatory increase in oxidative phosphorylation per mitochondrion. mOGT is also critical for cell survival; siRNA-mediated knockdown of endogenous mOGT protected cells against toxicity mediated by rotenone, a complex I inhibitor. Conversely, reduced expression of both nucleocytoplasmic (ncOGT) and mitochondrial (mOGT) OGT isoforms is associated with increased mitochondrial respiration and elevated glycolysis, suggesting that ncOGT is a negative regulator of cellular bioenergetics. Last, we determined that mOGT is probably involved in the glycosylation of a restricted set of mitochondrial targets. We identified four proteins implicated in mitochondrial biogenesis and metabolism regulation as candidate substrates of mOGT, including leucine-rich PPR-containing protein and mitochondrial aconitate hydratase. Our findings suggest that mOGT is catalytically active in vivo and supports mitochondrial structure, health, and survival, whereas ncOGT predominantly regulates cellular bioenergetics. PMID:28100784

  1. Cytochrome and alternative pathway respiration in green algae : measurements using inhibitors and o(2) discrimination.

    PubMed

    Weger, H G; Guy, R D; Turpin, D H

    1990-05-01

    Inhibitor titration curves and discrimination against (18)O(2) by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for alternative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and alternative pathway capacities.

  2. The life of plant mitochondrial complex I.

    PubMed

    Braun, Hans-Peter; Binder, Stefan; Brennicke, Axel; Eubel, Holger; Fernie, Alisdair R; Finkemeier, Iris; Klodmann, Jennifer; König, Ann-Christine; Kühn, Kristina; Meyer, Etienne; Obata, Toshihiro; Schwarzländer, Markus; Takenaka, Mizuki; Zehrmann, Anja

    2014-11-01

    The mitochondrial NADH dehydrogenase complex (complex I) of the respiratory chain has several remarkable features in plants: (i) particularly many of its subunits are encoded by the mitochondrial genome, (ii) its mitochondrial transcripts undergo extensive maturation processes (e.g. RNA editing, trans-splicing), (iii) its assembly follows unique routes, (iv) it includes an additional functional domain which contains carbonic anhydrases and (v) it is, indirectly, involved in photosynthesis. Comprising about 50 distinct protein subunits, complex I of plants is very large. However, an even larger number of proteins are required to synthesize these subunits and assemble the enzyme complex. This review aims to follow the complete "life cycle" of plant complex I from various molecular perspectives. We provide arguments that complex I represents an ideal model system for studying the interplay of respiration and photosynthesis, the cooperation of mitochondria and the nucleus during organelle biogenesis and the evolution of the mitochondrial oxidative phosphorylation system. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  3. Mitochondrial protein hyperacetylation in the failing heart.

    PubMed

    Horton, Julie L; Martin, Ola J; Lai, Ling; Riley, Nicholas M; Richards, Alicia L; Vega, Rick B; Leone, Teresa C; Pagliarini, David J; Muoio, Deborah M; Bedi, Kenneth C; Margulies, Kenneth B; Coon, Joshua J; Kelly, Daniel P

    2016-02-01

    Myocardial fuel and energy metabolic derangements contribute to the pathogenesis of heart failure. Recent evidence implicates posttranslational mechanisms in the energy metabolic disturbances that contribute to the pathogenesis of heart failure. We hypothesized that accumulation of metabolite intermediates of fuel oxidation pathways drives posttranslational modifications of mitochondrial proteins during the development of heart failure. Myocardial acetylproteomics demonstrated extensive mitochondrial protein lysine hyperacetylation in the early stages of heart failure in well-defined mouse models and the in end-stage failing human heart. To determine the functional impact of increased mitochondrial protein acetylation, we focused on succinate dehydrogenase A (SDHA), a critical component of both the tricarboxylic acid (TCA) cycle and respiratory complex II. An acetyl-mimetic mutation targeting an SDHA lysine residue shown to be hyperacetylated in the failing human heart reduced catalytic function and reduced complex II-driven respiration. These results identify alterations in mitochondrial acetyl-CoA homeostasis as a potential driver of the development of energy metabolic derangements that contribute to heart failure.

  4. Acute maneb exposure significantly alters both glycolysis and mitochondrial function in neuroblastoma cells.

    PubMed

    Anderson, Colin C; Aivazidis, Stefanos; Kuzyk, Crystal L; Jain, Abhilasha; Roede, James R

    2018-05-14

    The pesticides paraquat (PQ) and maneb (MB) have been described as environmental risk factors for Parkinson's disease (PD), with mechanisms associated with mitochondrial dysfunction and reactive oxygen species (ROS) generation. A combined exposure of PQ and MB in murine models and neuroblastoma cells has been utilized to further advance understanding of the PD phenotype. MB acts as a redox modulator through alkylation of protein thiols and has been previously characterized to inhibit complex III of the electron transport chain (ETC) and uncouple the mitochondrial proton gradient. The purpose of this study was to analyze ATP-linked respiration and glycolysis in human neuroblastoma cells utilizing the Seahorse extracellular flux (XFp) platform. Employing an acute, subtoxic exposure of MB, this investigation revealed a MB-mediated decrease in mitochondrial oxygen consumption at baseline and maximal respiration, with inhibition of ATP synthesis and coupling efficiency. Additionally, MB treated cells showed an increase in non-mitochondrial respiration and proton leak. Further investigation into mitochondrial fuel flex revealed an elimination of fuel flexibility across all three major substrates, with a decrease in pyruvate capacity as well as glutamine dependency. Analyses of glycolytic function showed a substantial decrease in glycolytic acidification caused by lactic acid export. This inhibition of glycolytic parameters was also observed after titrating the MB dose as low as 6 μM, and appears to be dependent on the dithiocarbamate functional group, with manganese possibly potentiating the effect. Further studies into cellular ATP and NAD levels revealed a drastic decrease in cells treated with MB. In summary, MB significantly impacted both aerobic and anaerobic energy production; therefore, further characterization of MB's effect on cellular energetics may provide insight into the specificity of PD to dopaminergic neurons.

  5. Regulation of thermogenesis in plants: the interaction of alternative oxidase and plant uncoupling mitochondrial protein.

    PubMed

    Zhu, Yan; Lu, Jianfei; Wang, Jing; Chen, Fu; Leng, Feifan; Li, Hongyu

    2011-01-01

    Thermogenesis is a process of heat production in living organisms. It is rare in plants, but it does occur in some species of angiosperm. The heat is generated via plant mitochondrial respiration. As possible involvement in thermogenesis of mitochondrial factors, alternative oxidases (AOXs) and plant uncoupling mitochondrial proteins (PUMPs) have been well studied. AOXs and PUMPs are ubiquitously present in the inner membrane of plant mitochondria. They serve as two major energy dissipation systems that balance mitochondrial respiration and uncoupled phosphorylation by dissipating the H+ redox energy and proton electrochemical gradient (ΔμH+) as heat, respectively. AOXs and PUMPs exert similar physiological functions during homeothermic heat production in thermogenic plants. AOXs have five isoforms, while PUMPs have six. Both AOXs and PUMPs are encoded by small nuclear multigene families. Multiple isoforms are expressed in different tissues or organs. Extensive studies have been done in the area of thermogenesis in higher plants. In this review, we focus on the involvement and regulation of AOXs and PUMPs in thermogenesis.

  6. Mitochondrial energy deficiency leads to hyperproliferation of skeletal muscle mitochondria and enhanced insulin sensitivity.

    PubMed

    Morrow, Ryan M; Picard, Martin; Derbeneva, Olga; Leipzig, Jeremy; McManus, Meagan J; Gouspillou, Gilles; Barbat-Artigas, Sébastien; Dos Santos, Carlos; Hepple, Russell T; Murdock, Deborah G; Wallace, Douglas C

    2017-03-07

    Diabetes is associated with impaired glucose metabolism in the presence of excess insulin. Glucose and fatty acids provide reducing equivalents to mitochondria to generate energy, and studies have reported mitochondrial dysfunction in type II diabetes patients. If mitochondrial dysfunction can cause diabetes, then we hypothesized that increased mitochondrial metabolism should render animals resistant to diabetes. This was confirmed in mice in which the heart-muscle-brain adenine nucleotide translocator isoform 1 (ANT1) was inactivated. ANT1-deficient animals are insulin-hypersensitive, glucose-tolerant, and resistant to high fat diet (HFD)-induced toxicity. In ANT1-deficient skeletal muscle, mitochondrial gene expression is induced in association with the hyperproliferation of mitochondria. The ANT1-deficient muscle mitochondria produce excess reactive oxygen species (ROS) and are partially uncoupled. Hence, the muscle respiration under nonphosphorylating conditions is increased. Muscle transcriptome analysis revealed the induction of mitochondrial biogenesis, down-regulation of diabetes-related genes, and increased expression of the genes encoding the myokines FGF21 and GDF15. However, FGF21 was not elevated in serum, and FGF21 and UCP1 mRNAs were not induced in liver or brown adipose tissue (BAT). Hence, increased oxidation of dietary-reducing equivalents by elevated muscle mitochondrial respiration appears to be the mechanism by which ANT1-deficient mice prevent diabetes, demonstrating that the rate of mitochondrial oxidation of calories is important in the etiology of metabolic disease.

  7. Apolipoprotein E4 (1–272) fragment is associated with mitochondrial proteins and affects mitochondrial function in neuronal cells

    PubMed Central

    Nakamura, Toshiyuki; Watanabe, Atsushi; Fujino, Takahiro; Hosono, Takashi; Michikawa, Makoto

    2009-01-01

    Background Apolipoprotein E allele ε4 (apoE4) is a strong risk factor for developing Alzheimer's disease (AD). Secreted apoE has a critical function in redistributing lipids among central nervous system cells to maintain normal lipid homeostasis. In addition, previous reports have shown that apoE4 is cleaved by a protease in neurons to generate apoE4(1–272) fragment, which is associated with neurofibrillary tanglelike structures and mitochondria, causing mitochondrial dysfunction. However, it still remains unclear how the apoE fragment associates with mitochondria and induces mitochondrial dysfunction. Results To clarify the molecular mechanism, we carried out experiments to identify intracellular apoE-binding molecules and their functions in modulating mitochondria function. Here, we found that apoE4 binds to ubiquinol cytochrome c reductase core protein 2 (UQCRC2) and cytochrome C1, both of which are components of mitochondrial respiratory complex III, and cytochrome c oxidase subunit 4 isoform 1 (COX IV 1), which is a component of complex IV, in Neuro-2a cells. Interestingly, these proteins associated with apoE4(1–272) more strongly than intact apoE4(1–299). Further analysis showed that in Neuro-2a cells expressing apoE4(1–272), the enzymatic activities of mitochondrial respiratory complexes III and IV were significantly lower than those in Neuro-2a cells expressing apoE4(1–299). Conclusion ApoE4(1–272) fragment expressed in Neuro2a cells is associated with mitochondrial proteins, UQCRC2 and cytochrome C1, which are component of respiratory complex III, and with COX IV 1, which is a member of complex IV. Overexpression of apoE4(1–272) fragment impairs activities of complex III and IV. These results suggest that the C-terminal-truncated fragment of apoE4 binds to mitochondrial complexes and affects their activities, and thereby leading to neurodegeneration. PMID:19695092

  8. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

    PubMed

    Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

    2012-07-15

    Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that

  9. Down-regulation of respiration in pear fruit depends on temperature.

    PubMed

    Ho, Quang Tri; Hertog, Maarten L A T M; Verboven, Pieter; Ambaw, Alemayehu; Rogge, Seppe; Verlinden, Bert E; Nicolaï, Bart M

    2018-04-09

    The respiration rate of plant tissues decreases when the amount of available O2 is reduced. There is, however, a debate on whether the respiration rate is controlled either by diffusion limitation of oxygen or through regulatory processes at the level of the transcriptome. We used experimental and modelling approaches to demonstrate that both diffusion limitation and metabolic regulation affect the response of respiration of bulky plant organs such as fruit to reduced O2 levels in the surrounding atmosphere. Diffusion limitation greatly affects fruit respiration at high temperature, but at low temperature respiration is reduced through a regulatory process, presumably a response to a signal generated by a plant oxygen sensor. The response of respiration to O2 is time dependent and is highly sensitive, particularly at low O2 levels in the surrounding atmosphere. Down-regulation of the respiration at low temperatures may save internal O2 and relieve hypoxic conditions in the fruit.

  10. Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

    PubMed

    Romero-Moya, Damià; Santos-Ocaña, Carlos; Castaño, Julio; Garrabou, Gloria; Rodríguez-Gómez, José A; Ruiz-Bonilla, Vanesa; Bueno, Clara; González-Rodríguez, Patricia; Giorgetti, Alessandra; Perdiguero, Eusebio; Prieto, Cristina; Moren-Nuñez, Constanza; Fernández-Ayala, Daniel J; Victoria Cascajo, Maria; Velasco, Iván; Canals, Josep Maria; Montero, Raquel; Yubero, Delia; Jou, Cristina; López-Barneo, José; Cardellach, Francesc; Muñoz-Cánoves, Pura; Artuch, Rafael; Navas, Plácido; Menendez, Pablo

    2017-07-01

    Coenzyme Q 10 (CoQ 10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ 10 biosynthesis (COQ genes) cause primary CoQ 10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ 10 ], CoQ 10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4 ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4 ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ 10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ 10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703. © 2017 AlphaMed Press.

  11. Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration.

    PubMed

    Goswami, Ishan; Perry, Justin B; Allen, Mitchell E; Brown, David A; von Spakovsky, Michael R; Verbridge, Scott S

    2018-06-19

    Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used

  12. Mitochondrial uncoupling, ROS generation and cardioprotection.

    PubMed

    Cadenas, Susana

    2018-05-31

    Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase -a process known as proton leak- generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis. Copyright © 2018. Published by Elsevier B.V.

  13. Protection of rat skeletal muscle fibers by either L-carnitine or coenzyme Q10 against statins toxicity mediated by mitochondrial reactive oxygen generation

    PubMed Central

    La Guardia, P. G.; Alberici, L. C.; Ravagnani, F. G.; Catharino, R. R.; Vercesi, A. E.

    2013-01-01

    Mitochondrial redox imbalance has been implicated in mechanisms of aging, various degenerative diseases and drug-induced toxicity. Statins are safe and well-tolerated therapeutic drugs that occasionally induce myotoxicity such as myopathy and rhabdomyolysis. Previous studies indicate that myotoxicity caused by statins may be linked to impairment of mitochondrial functions. Here, we report that 1-h incubation of permeabilized rat soleus muscle fiber biopsies with increasing concentrations of simvastatin (1–40 μM) slowed the rates of ADP-or FCCP-stimulated respiration supported by glutamate/malate in a dose-dependent manner, but caused no changes in resting respiration rates. Simvastatin (1 μM) also inhibited the ADP-stimulated mitochondrial respiration supported by succinate by 24% but not by TMPD/ascorbate. Compatible with inhibition of respiration, 1 μM simvastatin stimulated lactate release from soleus muscle samples by 26%. Co-incubation of muscle samples with 1 mM L-carnitine, 100 μM mevalonate or 10 μM coenzyme Q10 (Co-Q10) abolished simvastatin effects on both mitochondrial glutamate/malate-supported respiration and lactate release. Simvastatin (1 μM) also caused a 2-fold increase in the rate of hydrogen peroxide generation and a decrease in Co-Q10 content by 44%. Mevalonate, Co-Q10 or L-carnitine protected against stimulation of hydrogen peroxide generation but only mevalonate prevented the decrease in Co-Q10 content. Thus, independently of Co-Q10 levels, L-carnitine prevented the toxic effects of simvastatin. This suggests that mitochondrial respiratory dysfunction induced by simvastatin, is associated with increased generation of superoxide, at the levels of complexes-I and II of the respiratory chain. In all cases the damage to these complexes, presumably at the level of 4Fe-4S clusters, is prevented by L-carnitine. PMID:23720630

  14. Negative Regulation of STAT3 Protein-mediated Cellular Respiration by SIRT1 Protein*

    PubMed Central

    Bernier, Michel; Paul, Rajib K.; Martin-Montalvo, Alejandro; Scheibye-Knudsen, Morten; Song, Shaoming; He, Hua-Jun; Armour, Sean M.; Hubbard, Basil P.; Bohr, Vilhelm A.; Wang, Lili; Zong, Yaping; Sinclair, David A.; de Cabo, Rafael

    2011-01-01

    In mammals, the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) is regulated by the deacetylase SIRT1. However, whether the newly described nongenomic actions of STAT3 toward mitochondrial oxidative phosphorylation are dependent on SIRT1 is unclear. In this study, Sirt1 gene knock-out murine embryonic fibroblast (MEF) cells were used to delineate the role of SIRT1 in the regulation of STAT3 mitochondrial function. Here, we show that STAT3 mRNA and protein levels and the accumulation of serine-phosphorylated STAT3 in mitochondria were increased significantly in Sirt1-KO cells as compared with wild-type MEFs. Various mitochondrial bioenergetic parameters, such as the oxygen consumption rate in cell cultures, enzyme activities of the electron transport chain complexes in isolated mitochondria, and production of ATP and lactate, indicated that Sirt1-KO cells exhibited higher mitochondrial respiration as compared with wild-type MEFs. Two independent approaches, including ectopic expression of SIRT1 and siRNA-mediated knockdown of STAT3, led to reduction in intracellular ATP levels and increased lactate production in Sirt1-KO cells that were approaching those of wild-type controls. Comparison of profiles of phospho-antibody array data indicated that the deletion of SirT1 was accompanied by constitutive activation of the pro-inflammatory NF-κB pathway, which is key for STAT3 induction and increased cellular respiration in Sirt1-KO cells. Thus, SIRT1 appears to be a functional regulator of NF-κB-dependent STAT3 expression that induces mitochondrial biogenesis. These results have implications for understanding the interplay between STAT3 and SIRT1 in pro-inflammatory conditions. PMID:21467030

  15. Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation.

    PubMed

    Teplova, Vera V; Belosludtsev, Konstantin N; Kruglov, Alexey G

    2017-06-05

    Triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Q d -binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Seahorse Xfe24 Extracellular Flux Analyzer-based analysis of cellular respiration in Caenorhabditis elegans

    PubMed Central

    Luz, Anthony L.; Smith, Latasha L.; Rooney, John P.

    2015-01-01

    Mitochondria are critical for their role in ATP production as well as multiple nonenergetic functions, and mitochondrial dysfunction is causal in myriad human diseases. Less well appreciated is the fact that mitochondria integrate environmental and inter- as well as intracellular signals to modulate function. Because mitochondria function in an organismal milieu, there is need for assays capable of rapidly assessing mitochondrial health in vivo. Here, using the Seahorse XFe24 Extracellular Flux Analyzer and the pharmacological inhibitors dicyclohexylcarbodiimide (DCCD, ATP synthase inhibitor), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupler) and sodium azide (cytochrome c oxidase inhibitor), we describe how to obtain in vivo measurements of the fundamental parameters (basal oxygen consumption rate (OCR), ATP-linked respiration, maximal OCR, spare respiratory capacity and proton leak) of the mitochondrial respiratory chain in the model organism Caenorhabditis elegans. PMID:26523474

  17. Sulforaphane is anticonvulsant and improves mitochondrial function.

    PubMed

    Carrasco-Pozo, Catalina; Tan, Kah Ni; Borges, Karin

    2015-12-01

    The nuclear factor erythroid 2-related factor 2 pathway (Nrf2) has been previously identified to protect the brain against various impacts. Here, we investigated the effect of the Nrf2 activator sulforaphane in various seizure models and hippocampal mitochondrial bioenergetics. We found that daily injections of sulforaphane for 5 days elevated the seizure thresholds to 6 Hz stimulation and fluorothyl-, but not pentylenetetrazole-induced tonic seizures and protected mice against pilocarpine-induced status epilepticus (SE). Also, sulforaphane increased the antioxidant defences within hippocampal formations and blood plasma. In addition, sulforaphane treatment reduced the extent of hippocampal lipid peroxidation 24 h post-SE and protected hippocampal mitochondria against SE-induced reduction in state 2 and uncoupler-stimulated state 3 respiration. SE-mediated partial loss of rotenone-sensitive and complex II-driven respiration was reduced, consistent with the enhanced activities of complexes I and II in sulforaphane-treated SE mice. In mitochondria isolated from both no SE and SE mice, sulforaphane increased state 3 respiration and respiration linked to ATP synthesis, which may contribute to its anticonvulsant and antioxidant effects by providing more ATP for cellular vital and protective functions. However, sulforaphane did not prevent SE-induced hippocampal cell death. In conclusion, sulforaphane and/or Nrf2 activation are viable anticonvulsant strategies, which are antioxidant and enhance mitochondrial function, especially the ability to produce ATP. Sulforaphane was anticonvulsant in two acute mouse models of epilepsy and protected mice against pilocarpine-induced status epilepticus (SE). We also found antioxidant effects of sulforaphane in mouse plasma and hippocampal formations, exhibited by increased catalase and superoxide dismutase (SOD) activity, as well as increased abilities of hippocampal mitochondria to produce ATP. These effects likely underlie

  18. Skeletal Muscle Mitochondrial Energetics Are Associated With Maximal Aerobic Capacity and Walking Speed in Older Adults

    PubMed Central

    2013-01-01

    Background. Lower ambulatory performance with aging may be related to a reduced oxidative capacity within skeletal muscle. This study examined the associations between skeletal muscle mitochondrial capacity and efficiency with walking performance in a group of older adults. Methods. Thirty-seven older adults (mean age 78 years; 21 men and 16 women) completed an aerobic capacity (VO2 peak) test and measurement of preferred walking speed over 400 m. Maximal coupled (State 3; St3) mitochondrial respiration was determined by high-resolution respirometry in saponin-permeabilized myofibers obtained from percutanous biopsies of vastus lateralis (n = 22). Maximal phosphorylation capacity (ATPmax) of vastus lateralis was determined in vivo by 31P magnetic resonance spectroscopy (n = 30). Quadriceps contractile volume was determined by magnetic resonance imaging. Mitochondrial efficiency (max ATP production/max O2 consumption) was characterized using ATPmax per St3 respiration (ATPmax/St3). Results. In vitro St3 respiration was significantly correlated with in vivo ATPmax (r 2 = .47, p = .004). Total oxidative capacity of the quadriceps (St3*quadriceps contractile volume) was a determinant of VO2 peak (r 2 = .33, p = .006). ATPmax (r 2 = .158, p = .03) and VO2 peak (r 2 = .475, p < .0001) were correlated with preferred walking speed. Inclusion of both ATPmax/St3 and VO2 peak in a multiple linear regression model improved the prediction of preferred walking speed (r 2 = .647, p < .0001), suggesting that mitochondrial efficiency is an important determinant for preferred walking speed. Conclusions. Lower mitochondrial capacity and efficiency were both associated with slower walking speed within a group of older participants with a wide range of function. In addition to aerobic capacity, lower mitochondrial capacity and efficiency likely play roles in slowing gait speed with age. PMID:23051977

  19. Skeletal muscle mitochondrial energetics are associated with maximal aerobic capacity and walking speed in older adults.

    PubMed

    Coen, Paul M; Jubrias, Sharon A; Distefano, Giovanna; Amati, Francesca; Mackey, Dawn C; Glynn, Nancy W; Manini, Todd M; Wohlgemuth, Stephanie E; Leeuwenburgh, Christiaan; Cummings, Steven R; Newman, Anne B; Ferrucci, Luigi; Toledo, Frederico G S; Shankland, Eric; Conley, Kevin E; Goodpaster, Bret H

    2013-04-01

    Lower ambulatory performance with aging may be related to a reduced oxidative capacity within skeletal muscle. This study examined the associations between skeletal muscle mitochondrial capacity and efficiency with walking performance in a group of older adults. Thirty-seven older adults (mean age 78 years; 21 men and 16 women) completed an aerobic capacity (VO2 peak) test and measurement of preferred walking speed over 400 m. Maximal coupled (State 3; St3) mitochondrial respiration was determined by high-resolution respirometry in saponin-permeabilized myofibers obtained from percutanous biopsies of vastus lateralis (n = 22). Maximal phosphorylation capacity (ATPmax) of vastus lateralis was determined in vivo by (31)P magnetic resonance spectroscopy (n = 30). Quadriceps contractile volume was determined by magnetic resonance imaging. Mitochondrial efficiency (max ATP production/max O2 consumption) was characterized using ATPmax per St3 respiration (ATPmax/St3). In vitro St3 respiration was significantly correlated with in vivo ATPmax (r (2) = .47, p = .004). Total oxidative capacity of the quadriceps (St3*quadriceps contractile volume) was a determinant of VO2 peak (r (2) = .33, p = .006). ATPmax (r (2) = .158, p = .03) and VO2 peak (r (2) = .475, p < .0001) were correlated with preferred walking speed. Inclusion of both ATPmax/St3 and VO2 peak in a multiple linear regression model improved the prediction of preferred walking speed (r (2) = .647, p < .0001), suggesting that mitochondrial efficiency is an important determinant for preferred walking speed. Lower mitochondrial capacity and efficiency were both associated with slower walking speed within a group of older participants with a wide range of function. In addition to aerobic capacity, lower mitochondrial capacity and efficiency likely play roles in slowing gait speed with age.

  20. Fiber-type differences in muscle mitochondrial profiles.

    PubMed

    Leary, S C; Lyons, C N; Rosenberger, A G; Ballantyne, J S; Stillman, J; Moyes, C D

    2003-10-01

    Although striated muscles differ in mitochondrial content, the extent of fiber-type specific mitochondrial specializations is not well known. To address this issue, we compared mitochondrial structural and functional properties in red muscle (RM), white muscle (WM), and cardiac muscle of rainbow trout. Overall preservation of the basic relationships between oxidative phosphorylation complexes among fiber types was confirmed by kinetic analyses, immunoblotting of native holoproteins, and spectroscopic measurements of cytochrome content. Fiber-type differences in mitochondrial properties were apparent when parameters were expressed per milligram mitochondrial protein. However, the differences diminished when expressed relative to cytochrome oxidase (COX), possibly a more meaningful denominator than mitochondrial protein. Expressed relative to COX, there were no differences in oxidative phosphorylation enzyme activities, pyruvate-based respiratory rates, H2O2 production, or state 4 proton leak respiration. These data suggest most mitochondrial qualitative properties are conserved across fiber types. However, there remained modest differences ( approximately 50%) in stoichiometries of selected enzymes of the Krebs cycle, beta-oxidation, and antioxidant enzymes. There were clear differences in membrane fluidity (RM > cardiac, WM) and proton conductance (H+/min/mV/U COX: WM > RM > cardiac). The pronounced differences in mitochondrial content between fiber types could be attributed to a combination of differences in myonuclear domain and modest effects on the expression of nuclear- and mitochondrially encoded respiratory genes. Collectively, these studies suggest constitutive pathways that transcend fiber types are primarily responsible for determining most quantitative and qualitative properties of mitochondria.

  1. Thyroid hormone action on intermediary metabolism. Part I: respiration, thermogenesis and carbohydrate metabolism.

    PubMed

    Müller, M J; Seitz, H J

    1984-01-02

    The effect of thyroid hormones on mitochondrial respiration are summarized: T3 directly stimulates mitochondrial respiration and the synthesis of adenosine 5'-triphosphate (ATP). Cytosolic ATP availability is increased by a thyroid hormone-induced increase in adenine nucleotide translocation across the mitochondrial membrane; the steady state ATP concentration and the cytosolic ATP/adenosine 5'-diphosphate (ADP) ratio is even decreased in hyperthyroid tissues because of the simultaneous stimulation of the synthesis and consumption of ATP. With regard to the thyroid hormone-induced energy wasting processes, heart work, intra- and interorgan futile cycling and Na+/K+-ATPase are involved to varying degrees. As a consequence of the thyroid hormone-induced hydrolysis of ATP, thermogenesis is increased in hyper- and decreased in hypothyroidism. Despite an increased rate of glucose utilization, clinical and experimental hyperthyroidism is often characterized by an abnormal oral glucose tolerance test. This finding is due to the thyroid hormone-induced increase in intestinal glucose absorption as well as the still enhanced endogenous glucose production in the liver. Hypothyroid patients show a reduced glucose tolerance test because of a decrease in intestinal glucose absorption and a sometimes reduced glucose turnover. The thyroid hormone-induced alterations in glucose metabolism are most probably not due to alterations in serum insulin levels and/or to a peripheral insulin resistance at the receptor level.

  2. Defects in Mitochondrial Fatty Acid Synthesis Result in Failure of Multiple Aspects of Mitochondrial Biogenesis in Saccharomyces cerevisiae

    PubMed Central

    Kursu, V. A. Samuli; Pietikäinen, Laura P.; Fontanesi, Flavia; Aaltonen, Mari J.; Suomi, Fumi; Nair, Remya Raghavan; Schonauer, Melissa S.; Dieckmann, Carol L.; Barrientos, Antoni; Hiltunen, J. Kalervo; Kastaniotis, Alexander J.

    2014-01-01

    Summary Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild heme deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a coordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell. PMID:24102902

  3. Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae.

    PubMed

    Kursu, V A Samuli; Pietikäinen, Laura P; Fontanesi, Flavia; Aaltonen, Mari J; Suomi, Fumi; Raghavan Nair, Remya; Schonauer, Melissa S; Dieckmann, Carol L; Barrientos, Antoni; Hiltunen, J Kalervo; Kastaniotis, Alexander J

    2013-11-01

    Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis. In our ensuing analysis, we show that mtFAS, but not lipoylation per se, is required for respiratory competence. We demonstrate that mtFAS is required for mRNA splicing, mitochondrial translation and respiratory complex assembly, and provide evidence that not LA per se, but fatty acids longer than C8 play a role in these processes. We also show that mtFAS- and LA-deficient strains suffer from a mild haem deficiency that may contribute to the respiratory complex assembly defect. Based on our data and previously published information, we propose a model implicating mtFAS as a sensor for mitochondrial acetyl-CoA availability and a co-ordinator of nuclear and mitochondrial gene expression by adapting the mitochondrial compartment to changes in the metabolic status of the cell. © 2013 John Wiley & Sons Ltd.

  4. Beating oxygen: chronic anoxia exposure reduces mitochondrial F1FO-ATPase activity in turtle (Trachemys scripta) heart

    PubMed Central

    Galli, Gina L. J.; Lau, Gigi Y.; Richards, Jeffrey G.

    2013-01-01

    SUMMARY The freshwater turtle Trachemys scripta can survive in the complete absence of O2 (anoxia) for periods lasting several months. In mammals, anoxia leads to mitochondrial dysfunction, which culminates in cellular necrosis and apoptosis. Despite the obvious clinical benefits of understanding anoxia tolerance, little is known about the effects of chronic oxygen deprivation on the function of turtle mitochondria. In this study, we compared mitochondrial function in hearts of T. scripta exposed to either normoxia or 2 weeks of complete anoxia at 5°C and during simulated acute anoxia/reoxygenation. Mitochondrial respiration, electron transport chain activities, enzyme activities, proton conductance and membrane potential were measured in permeabilised cardiac fibres and isolated mitochondria. Two weeks of anoxia exposure at 5°C resulted in an increase in lactate, and decreases in ATP, glycogen, pH and phosphocreatine in the heart. Mitochondrial proton conductance and membrane potential were similar between experimental groups, while aerobic capacity was dramatically reduced. The reduced aerobic capacity was the result of a severe downregulation of the F1FO-ATPase (Complex V), which we assessed as a decrease in enzyme activity. Furthermore, in stark contrast to mammalian paradigms, isolated turtle heart mitochondria endured 20 min of anoxia followed by reoxygenation without any impact on subsequent ADP-stimulated O2 consumption (State III respiration) or State IV respiration. Results from this study demonstrate that turtle mitochondria remodel in response to chronic anoxia exposure and a reduction in Complex V activity is a fundamental component of mitochondrial and cellular anoxia survival. PMID:23926310

  5. Mitochondrial Physiology in the Major Arbovirus Vector Aedes aegypti: Substrate Preferences and Sexual Differences Define Respiratory Capacity and Superoxide Production

    PubMed Central

    Soares, Juliana B. R. Correa; Gaviraghi, Alessandro; Oliveira, Marcus F.

    2015-01-01

    Adult females of Aedes aegypti are facultative blood sucking insects and vectors of Dengue and yellow fever viruses. Insect dispersal plays a central role in disease transmission and the extremely high energy demand posed by flight is accomplished by a very efficient oxidative phosphorylation process, which take place within flight muscle mitochondria. These organelles play a central role in energy metabolism, interconnecting nutrient oxidation to ATP synthesis, but also represent an important site of cellular superoxide production. Given the importance of mitochondria to cell physiology, and the potential contributions of this organelle for A. aegypti biology and vectorial capacity, here, we conducted a systematic assessment of mitochondrial physiology in flight muscle of young adult A. aegypti fed exclusively with sugar. This was carried out by determining the activities of mitochondrial enzymes, the substrate preferences to sustain respiration, the mitochondrial bioenergetic efficiency and capacity, in both mitochondria-enriched preparations and mechanically permeabilized flight muscle in both sexes. We also determined the substrates preferences to promote mitochondrial superoxide generation and the main sites where it is produced within this organelle. We observed that respiration in A. aegypti mitochondria was essentially driven by complex I and glycerol 3 phosphate dehydrogenase substrates, which promoted distinct mitochondrial bioenergetic capacities, but with preserved efficiencies. Respiration mediated by proline oxidation in female mitochondria was strikingly higher than in males. Mitochondrial superoxide production was essentially mediated through proline and glycerol 3 phosphate oxidation, which took place at sites other than complex I. Finally, differences in mitochondrial superoxide production among sexes were only observed in male oxidizing glycerol 3 phosphate, exhibiting higher rates than in female. Together, these data represent a significant step

  6. Rearing Tenebrio molitor in BLSS: Dietary fiber affects larval growth, development, and respiration characteristics

    NASA Astrophysics Data System (ADS)

    Li, Leyuan; Stasiak, Michael; Li, Liang; Xie, Beizhen; Fu, Yuming; Gidzinski, Danuta; Dixon, Mike; Liu, Hong

    2016-01-01

    Rearing of yellow mealworm (Tenebrio molitor L.) will provide good animal nutrition for astronauts in a bioregenerative life support system. In this study, growth and biomass conversion data of T. molitor larvae were tested for calculating the stoichiometric equation of its growth. Result of a respiratory quotient test proved the validity of the equation. Fiber had the most reduction in mass during T. molitor‧s consumption, and thus it is speculated that fiber is an important factor affecting larval growth of T. molitor. In order to further confirm this hypothesis and find out a proper feed fiber content, T. molitor larvae were fed on diets with 4 levels of fiber. Larval growth, development and respiration in each group were compared and analyzed. Results showed that crude-fiber content of 5% had a significant promoting effect on larvae in early instars, and is beneficial for pupa eclosion. When fed on feed of 5-10% crude-fiber, larvae in later instars reached optimal levels in growth, development and respiration. Therefore, we suggest that crude fiber content in feed can be controlled within 5-10%, and with the consideration of food palatability, a crude fiber of 5% is advisable.

  7. Sulforaphane Inhibits Mitochondrial Permeability Transition and Oxidative Stress

    PubMed Central

    Greco, Tiffany; Shafer, Jonathan; Fiskum, Gary

    2012-01-01

    Exposure of mitochondria to oxidative stress and elevated Ca2+ promotes opening of the mitochondrial permeability transition pore (PTP), resulting in membrane depolarization, uncoupling of oxidative phosphorylation, and potentially cell death. This study tested the hypothesis that treatment of rats with sulforaphane (SFP), an activator of the Nrf2 pathway of antioxidant gene expression, increases the resistance of liver mitochondria to redox-regulated PTP opening and elevates mitochondrial levels of antioxidants. Rats were injected with SFP or drug vehicle and liver mitochondria were isolated 40 hr later. Respiring mitochondria actively accumulated added Ca2+, which was then released through PTP opening induced by agents that either cause an oxidized shift in the mitochondrial redox state or that directly oxidize protein thiol groups. SFP treatment of rats inhibited the rate of pro-oxidant-induced mitochondrial Ca2+ release and increased expression of the glutathione peroxidase/reductase system, thioredoxin, and malic enzyme. These results are the first to demonstrate that SFP treatment of animals increases liver mitochondrial antioxidant defenses and inhibits redox-sensitive PTP opening. This novel form of preconditioning could protect against a variety of pathologies that include oxidative stress and mitochondrial dysfunction in their etiologies. PMID:21986339

  8. Exercise in claudicants increase or decrease walking ability and the response relates to mitochondrial function.

    PubMed

    van Schaardenburgh, Michel; Wohlwend, Martin; Rognmo, Øivind; Mattsson, Erney J R

    2017-06-07

    Exercise of patients with intermittent claudication improves walking performance. Exercise does not usually increase blood flow, but seems to increase muscle mitochondrial enzyme activities. Although exercise is beneficial in most patients, it might be harmful in some. The mitochondrial response to exercise might therefore differ between patients. Our hypothesis was that changes in walking performance relate to changes in mitochondrial function after 8 weeks of exercise. At a subgroup level, negative responders decrease and positive responders increase mitochondrial capacity. Two types of exercise were studied, calf raising and walking (n = 28). We wanted to see whether there were negative and positive responders, independent of type of exercise. Measurements of walking performance, peripheral hemodynamics, mitochondrial respiration and content (citrate synthase activity) were obtained on each patient before and after the intervention period. Multiple linear regression was used to test whether changes in peak walking time relate to mitochondrial function. Subgroups of negative (n = 8) and positive responders (n = 8) were defined as those that either decreased or increased peak walking time following exercise. Paired t test and analysis of covariance was used to test changes within and between subgroups. Changes in peak walking time were related to changes in mitochondrial respiration supported by electron transferring flavoprotein (ETF + CI) P (p = 0.004), complex I (CI + ETF) P (p = 0.003), complex I + complex II (CI + CII + ETF) P (p = 0.037) and OXPHOS coupling efficiency (p = 0.046) in the whole group. Negative responders had more advanced peripheral arterial disease. Mitochondrial respiration supported by electron transferring flavoprotein (ETF + CI) P (p = 0.0013), complex I (CI + ETF) P (p = 0.0005), complex I + complex II (CI + CII + ETF) P (p = 0.011) and electron transfer system capacity (CI + CII + ETF) E (p

  9. Mitochondrial Proteome Studies in Seeds during Germination

    PubMed Central

    Czarna, Malgorzata; Kolodziejczak, Marta; Janska, Hanna

    2016-01-01

    Seed germination is considered to be one of the most critical phases in the plant life cycle, establishing the next generation of a plant species. It is an energy-demanding process that requires functioning mitochondria. One of the earliest events of seed germination is progressive development of structurally simple and metabolically quiescent promitochondria into fully active and cristae-containing mitochondria, known as mitochondrial biogenesis. This is a complex and tightly regulated process, which is accompanied by sequential and dynamic gene expression, protein synthesis, and post-translational modifications. The aim of this review is to give a comprehensive summary of seed mitochondrial proteome studies during germination of various plant model organisms. We describe different gel-based and gel-free proteomic approaches used to characterize mitochondrial proteomes of germinating seeds as well as challenges and limitations of these proteomic studies. Furthermore, the dynamic changes in the abundance of the mitochondrial proteomes of germinating seeds are illustrated, highlighting numerous mitochondrial proteins involved in respiration, tricarboxycylic acid (TCA) cycle, metabolism, import, and stress response as potentially important for seed germination. We then review seed mitochondrial protein carbonylation, phosphorylation, and S-nitrosylation as well as discuss the possible link between these post-translational modifications (PTMs) and the regulation of seed germination. PMID:28248229

  10. Mitochondrial rhodanese: membrane-bound and complexed activity.

    PubMed

    Ogata, K; Volini, M

    1990-05-15

    We have proposed that phosphorylated and dephosphorylated forms of the mitochondrial sulfurtransferase, rhodanese, function as converter enzymes that interact with membrane-bound iron-sulfur centers of the electron transport chain to modulate the rate of mitochondrial respiration (Ogata, K., Dai, X., and Volini, M. (1989) J. Biol. Chem. 204, 2718-2725). In the present studies, we have explored some structural aspects of the mitochondrial rhodanese system. By sequential extraction of lysed mitochondria with phosphate buffer and phosphate buffer containing 20 mM cholate, we have shown that 30% of the rhodanese activity of bovine liver is membrane-bound. Resolution of cholate extracts on Sephadex G-100 indicates that part of the bound rhodanese is complexed with other mitochondrial proteins. Tests with the complex show that it forms iron-sulfur centers when incubated with the rhodanese sulfur-donor substrate thiosulfate, iron ions, and a reducing agent. Experiments on the rhodanese activity of rat liver mitochondria give similar results. Taken together, the findings indicate that liver rhodanese is in part bound to the mitochondrial membrane as a component of a multiprotein complex that forms iron-sulfur centers. The findings are consistent with the role we propose for rhodanese in the modulation of mitochondrial respiratory activity.

  11. Treating SCA1 Mice with Water-Soluble Compounds to Non-Specifically Boost Mitochondrial Function.

    PubMed

    Ferro, Austin; Carbone, Emily; Marzouk, Evan; Siegel, Asher; Nguyen, Donna; Polley, Kailen; Hartman, Jessilyn; Frederick, Kimberley; Ives, Stephen; Lagalwar, Sarita

    2017-01-22

    Mitochondrial dysfunction plays a significant role in the aging process and in neurodegenerative diseases including several hereditary spinocerebellar ataxias and other movement disorders marked by progressive degeneration of the cerebellum. The goal of this protocol is to assess mitochondrial dysfunction in Spinocerebellar ataxia type 1 (SCA1) and assess the efficacy of pharmacological targeting of metabolic respiration via the water-soluble compound succinic acid to slow disease progression. This approach is applicable to other cerebellar diseases and can be adapted to a host of water-soluble therapies. Ex vivo analysis of mitochondrial respiration is used to detect and quantify disease-related changes in mitochondrial function. With genetic evidence (unpublished data) and proteomic evidence of mitochondrial dysfunction in the SCA1 mouse model, we evaluate the efficacy of treatment with the water-soluble metabolic booster succinic acid by dissolving this compound directly into the home cage drinking water. The ability of the drug to pass the blood brain barrier can be deduced using high performance liquid chromatography (HPLC). The efficacy of these compounds can then be tested using multiple behavioral paradigms including the accelerating rotarod, balance beam test and footprint analysis. Cytoarchitectural integrity of the cerebellum can be assessed using immunofluorescence assays that detect Purkinje cell nuclei and Purkinje cell dendrites and soma. These methods are robust techniques for determining mitochondrial dysfunction and the efficacy of treatment with water-soluble compounds in cerebellar neurodegenerative disease.

  12. Scaling with body mass of mitochondrial respiration from the white muscle of three phylogenetically, morphologically and behaviorally disparate teleost fishes.

    PubMed

    Burpee, Jessica L; Bardsley, Elise L; Dillaman, Richard M; Watanabe, Wade O; Kinsey, Stephen T

    2010-10-01

    White muscle (WM) fibers in many fishes often increase in size from <50 μm in juveniles to >250 μm in adults. This leads to increases in intracellular diffusion distances that may impact the scaling with body mass of muscle metabolism. We have previously found similar negative scaling of aerobic capacity (mitochondrial volume density, V(mt)) and the rate of an aerobic process (post-contractile phosphocreatine recovery) in fish WM. In the present study, we examined the scaling with body mass of oxygen consumption rates of isolated mitochondria (VO(2mt)) from WM in three species from different families that vary in morphology and behavior: an active, pelagic species (bluefish, Pomatomus saltatrix), a relatively inactive demersal species (black sea bass, Centropristis striata), and a sedentary, benthic species (southern flounder, Paralichthys lethostigma). In contrast to our prior studies, the measurement of respiration in isolated mitochondria is not influenced by the diffusion of oxygen or metabolites. V(mt) was measured in WM and in high-density isolates used for VO(2mt) measurements. WM V(mt) was significantly higher in the bluefish than in the other two species and VO(2mt) was independent of body mass when expressed per milligram protein or per milliliter mitochondria. The size-independence of VO(2mt) indicates that differences in WM aerobic function result from variation in V(mt) and not to changes in VO(2mt). This is consistent with our prior work that indicated that while diffusion constraints influence mitochondrial distribution, the negative scaling of aerobic processes like post-contractile PCr recovery can largely be attributed to the body size dependence of V(mt).

  13. Alterations in mitochondrial electron transport system activity in response to warm acclimation, hypoxia-reoxygenation and copper in rainbow trout, Oncorhynchus mykiss.

    PubMed

    Sappal, Ravinder; MacDougald, Michelle; Fast, Mark; Stevens, Don; Kibenge, Fred; Siah, Ahmed; Kamunde, Collins

    2015-08-01

    Fish expend significant amounts of energy to handle the numerous potentially stressful biotic and abiotic factors that they commonly encounter in aquatic environments. This universal requirement for energy singularizes mitochondria, the primary cellular energy transformers, as fundamental drivers of responses to environmental change. Our study probed the interacting effects of thermal stress, hypoxia-reoxygenation (HRO) and copper (Cu) exposure in rainbow trout to test the prediction that they act jointly to impair mitochondrial function. Rainbow trout were acclimated to 11 (controls) or 20°C for 2 months. Liver mitochondria were then isolated and their responses in vitro to Cu (0-20μM) without and with HRO were assessed. Sequential inhibition and activation of mitochondrial electron transport system (ETS) enzyme complexes permitted the measurement of respiratory activities supported by complex I-IV (CI-IV) in one run. The results showed that warm acclimation reduced fish and liver weights but increased mitochondrial protein indicating impairment of energy metabolism, increased synthesis of defense proteins and/or reduced liver water content. Whereas acute rise (11→20°C) in temperature increased mitochondrial oxidation rates supported by CI-IV, warm acclimation reduced the maximal (state 3) and increased the basal (state 4) respiration leading to global uncoupling of oxidative phosphorylation (OXPHOS). HRO profoundly inhibited both maximal and basal respiration rates supported by CI-IV, reduced RCR for all except CII and lowered CI:CII respiration ratio, an indication of decreased OXPHOS efficiency. The effects of Cu were less pronounced but more variable and included inhibition of CII-IV maximal respiration rates and stimulation of both CI and CIII basal respiration rates. Surprisingly, only CII and CIII indices exhibited significant 3-way interactions whereas 2-way interactions of acclimation either with Cu or HRO were portrayed mostly by CIV, and those of

  14. Respiration in heterotrophic unicellular eukaryotic organisms.

    PubMed

    Fenchel, Tom

    2014-08-01

    Surface:volume quotient, mitochondrial volume fraction, and their distribution within cells were investigated and oxygen gradients within and outside cells were modelled. Cell surface increases allometrically with cell size. Mitochondrial volume fraction is invariant with cell size and constitutes about 10% and mitochondria are predominantly found close to the outer membrane. The results predict that for small and medium sized protozoa maximum respiration rates should be proportional to cell volume (scaling exponent ≈1) and access to intracellular O2 is not limiting except at very low ambient O2-tensions. Available data do not contradict this and some evidence supports this interpretation. Cell size is ultimately limited because an increasing fraction of the mitochondria becomes exposed to near anoxic conditions with increasing cell size. The fact that mitochondria cluster close to the cell surface and the allometric change in cell shape with increasing cell size alleviates the limitation of aerobic life at low ambient O2-tension and for large cell size. Copyright © 2014 Elsevier GmbH. All rights reserved.

  15. The Effect of Glucose Concentration and Sodium Phenylbutyrate Treatment on Mitochondrial Bioenergetics and ER Stress in 3T3-L1 Adipocytes

    PubMed Central

    Tanis, Ross M.; Piroli, Gerardo G.; Day, Stani D.; Frizzell, Norma

    2016-01-01

    While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5 mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ∼1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose. PMID:25448036

  16. Brain mitochondrial bioenergetics change with rapid and prolonged shifts in aggression in the honey bee, Apis mellifera.

    PubMed

    Rittschof, Clare C; Vekaria, Hemendra J; Palmer, Joseph H; Sullivan, Patrick G

    2018-04-25

    Neuronal function demands high-level energy production, and as such, a decline in mitochondrial respiration characterizes brain injury and disease. A growing number of studies, however, link brain mitochondrial function to behavioral modulation in non-diseased contexts. In the honey bee, we show for the first time that an acute social interaction, which invokes an aggressive response, may also cause a rapid decline in brain mitochondrial bioenergetics. The degree and speed of this decline has only been previously observed in the context of brain injury. Furthermore, in the honey bee, age-related increases in aggressive tendency are associated with increased baseline brain mitochondrial respiration, as well as increased plasticity in response to metabolic fuel type in vitro Similarly, diet restriction and ketone body feeding, which commonly enhance mammalian brain mitochondrial function in vivo , cause increased aggression. Thus, even in normal behavioral contexts, brain mitochondria show a surprising degree of variation in function over both rapid and prolonged time scales, with age predicting both baseline function and plasticity in function. These results suggest that mitochondrial function is integral to modulating aggression-related neuronal signaling. We hypothesize that variation in function reflects mitochondrial calcium buffering activity, and that shifts in mitochondrial function signal to the neuronal soma to regulate gene expression and neural energetic state. Modulating brain energetic state is emerging as a critical component of the regulation of behavior in non-diseased contexts. © 2018. Published by The Company of Biologists Ltd.

  17. Mitochondrial respiratory chain Complex I defects in Fanconi anemia complementation group A.

    PubMed

    Ravera, Silvia; Vaccaro, Daniele; Cuccarolo, Paola; Columbaro, Marta; Capanni, Cristina; Bartolucci, Martina; Panfoli, Isabella; Morelli, Alessandro; Dufour, Carlo; Cappelli, Enrico; Degan, Paolo

    2013-10-01

    Fanconi anemia (FA) is a rare and complex inherited blood disorder of the child. At least 15 genes are associated with the disease. The highest frequency of mutations belongs to groups A, C and G. Genetic instability and cytokine hypersensitivity support the selection of leukemic over non-leukemic stem cells. FA cellular phenotype is characterized by alterations in red-ox state, mitochondrial functionality and energy metabolism as reported in the past however a clear picture of the altered biochemical phenotype in FA is still elusive and the final biochemical defect(s) still unknown. Here we report an analysis of the respiratory fluxes in FANCA primary fibroblasts, lymphocytes and lymphoblasts. FANCA mutants show defective respiration through Complex I, diminished ATP production and metabolic sufferance with an increased AMP/ATP ratio. Respiration in FANCC mutants is normal. Treatment with N-acetyl-cysteine (NAC) restores oxygen consumption to normal level. Defective respiration in FANCA mutants appear correlated with the FA pro-oxidative phenotype which is consistent with the altered morphology of FANCA mitochondria. Electron microscopy measures indeed show profound alterations in mitochondrial ultrastructure and shape. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  18. Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

    PubMed

    Cavalheiro, R A; Fortes, F; Borecký, J; Faustinoni, V C; Schreiber, A Z; Vercesi, A E

    2004-10-01

    The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

  19. Mitochondrial metabolism and stress response of yeast: Applications in fermentation technologies.

    PubMed

    Kitagaki, Hiroshi; Takagi, Hiroshi

    2014-04-01

    Mitochondria are sites of oxidative respiration. During sake brewing, sake yeasts are exposed to long periods of hypoxia; the structure, role, and metabolism of mitochondria of sake yeasts have not been studied in detail. It was first elucidated that the mitochondrial structure of sake yeast transforms from filamentous to dotted structure during sake brewing, which affects malate metabolism. Based on the information of yeast mitochondria during sake brewing, practical technologies have been developed; (i) breeding pyruvate-underproducing sake yeast by the isolation of a mutant resistant to an inhibitor of mitochondrial pyruvate transport; and (ii) modifying malate and succinate production by manipulating mitochondrial activity. During the bread-making process, baker's yeast cells are exposed to a variety of baking-associated stresses, such as freeze-thaw, air-drying, and high sucrose concentrations. These treatments induce oxidative stress generating reactive oxygen species due to mitochondrial damage. A novel metabolism of proline and arginine catalyzed by N-acetyltransferase Mpr1 in the mitochondria eventually leads to synthesis of nitric oxide, which confers oxidative stress tolerance on yeast cells. The enhancement of proline and arginine metabolism could be promising for breeding novel baker's yeast strains that are tolerant to multiple baking-associated stresses. These new and practical methods provide approaches to improve the processes in the field of industrial fermentation technologies. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Mitochondrial oxidative stress is the achille's heel of melanoma cells resistant to Braf-mutant inhibitor

    PubMed Central

    André, Fanny; Jonneaux, Aurélie; Scalbert, Camille; Garçon, Guillaume; Malet-Martino, Myriam; Balayssac, Stéphane; Rocchi, Stephane; Savina, Ariel; Formstecher, Pierre; Mortier, Laurent; Kluza, Jérome; Marchetti, Philippe

    2013-01-01

    Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition. PMID:24161908

  1. Oxidative stress induces mitochondrial dysfunction in a subset of autistic lymphoblastoid cell lines

    PubMed Central

    Rose, S; Frye, R E; Slattery, J; Wynne, R; Tippett, M; Melnyk, S; James, S J

    2014-01-01

    There is an increasing recognition that mitochondrial dysfunction is associated with autism spectrum disorders. However, little attention has been given to the etiology of mitochondrial dysfunction and how mitochondrial abnormalities might interact with other physiological disturbances such as oxidative stress. Reserve capacity is a measure of the ability of the mitochondria to respond to physiological stress. In this study, we demonstrate, for the first time, that lymphoblastoid cell lines (LCLs) derived from children with autistic disorder (AD) have an abnormal mitochondrial reserve capacity before and after exposure to reactive oxygen species (ROS). Ten (44%) of 22 AD LCLs exhibited abnormally high reserve capacity at baseline and a sharp depletion of reserve capacity when challenged with ROS. This depletion of reserve capacity was found to be directly related to an atypical simultaneous increase in both proton-leak respiration and adenosine triphosphate-linked respiration in response to increased ROS in this AD LCL subgroup. In this AD LCL subgroup, 48-hour pretreatment with N-acetylcysteine, a glutathione precursor, prevented these abnormalities and improved glutathione metabolism, suggesting a role for altered glutathione metabolism associated with this type of mitochondrial dysfunction. The results of this study suggest that a significant subgroup of AD children may have alterations in mitochondrial function, which could render them more vulnerable to a pro-oxidant microenvironment as well as intrinsic and extrinsic sources of ROS such as immune activation and pro-oxidant environmental toxins. These findings are consistent with the notion that AD is caused by a combination of genetic and environmental factors. PMID:24690598

  2. Actin dynamics affect mitochondrial quality control and aging in budding yeast.

    PubMed

    Higuchi, Ryo; Vevea, Jason D; Swayne, Theresa C; Chojnowski, Robert; Hill, Vanessa; Boldogh, Istvan R; Pon, Liza A

    2013-12-02

    Actin cables of budding yeast are bundles of F-actin that extend from the bud tip or neck to the mother cell tip, serve as tracks for bidirectional cargo transport, and undergo continuous movement from buds toward mother cells [1]. This movement, retrograde actin cable flow (RACF), is similar to retrograde actin flow in lamellipodia, growth cones, immunological synapses, dendritic spines, and filopodia [2-5]. In all cases, actin flow is driven by the push of actin polymerization and assembly at the cell cortex, and myosin-driven pulling forces deeper within the cell [6-10]. Therefore, for movement and inheritance from mothers to buds, mitochondria must "swim upstream" against the opposing force of RACF [11]. We find that increasing RACF rates results in increased fitness of mitochondria inherited by buds and that the increase in mitochondrial fitness leads to extended replicative lifespan and increased cellular healthspan. The sirtuin SIR2 is required for normal RACF and mitochondrial fitness, and increasing RACF rates in sir2Δ cells increases mitochondrial fitness and cellular healthspan but does not affect replicative lifespan. These studies support the model that RACF serves as a filter for segregation of fit from less-fit mitochondria during inheritance, which controls cellular lifespan and healthspan. They also support a role for Sir2p in these processes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Mitochondrial protein hyperacetylation in the failing heart

    PubMed Central

    Horton, Julie L.; Martin, Ola J.; Lai, Ling; Richards, Alicia L.; Vega, Rick B.; Leone, Teresa C.; Pagliarini, David J.; Muoio, Deborah M.; Bedi, Kenneth C.; Coon, Joshua J.

    2016-01-01

    Myocardial fuel and energy metabolic derangements contribute to the pathogenesis of heart failure. Recent evidence implicates posttranslational mechanisms in the energy metabolic disturbances that contribute to the pathogenesis of heart failure. We hypothesized that accumulation of metabolite intermediates of fuel oxidation pathways drives posttranslational modifications of mitochondrial proteins during the development of heart failure. Myocardial acetylproteomics demonstrated extensive mitochondrial protein lysine hyperacetylation in the early stages of heart failure in well-defined mouse models and the in end-stage failing human heart. To determine the functional impact of increased mitochondrial protein acetylation, we focused on succinate dehydrogenase A (SDHA), a critical component of both the tricarboxylic acid (TCA) cycle and respiratory complex II. An acetyl-mimetic mutation targeting an SDHA lysine residue shown to be hyperacetylated in the failing human heart reduced catalytic function and reduced complex II–driven respiration. These results identify alterations in mitochondrial acetyl-CoA homeostasis as a potential driver of the development of energy metabolic derangements that contribute to heart failure. PMID:26998524

  4. Mitochondrial flashes: From indicator characterization to in vivo imaging.

    PubMed

    Wang, Wang; Zhang, Huiliang; Cheng, Heping

    2016-10-15

    Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP targeted to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Effects of a human recombinant alkaline phosphatase during impaired mitochondrial function in human renal proximal tubule epithelial cells.

    PubMed

    Peters, Esther; Schirris, Tom; van Asbeck, Alexander H; Gerretsen, Jelle; Eymael, Jennifer; Ashikov, Angel; Adjobo-Hermans, Merel J W; Russel, Frans; Pickkers, Peter; Masereeuw, Rosalinde

    2017-02-05

    Sepsis-associated acute kidney injury is a multifactorial syndrome in which inflammation and renal microcirculatory dysfunction play a profound role. Subsequently, renal tubule mitochondria reprioritize cellular functions to prevent further damage. Here, we investigated the putative protective effects of human recombinant alkaline phosphatase (recAP) during inhibition of mitochondrial respiration in conditionally immortalized human proximal tubule epithelial cells (ciPTEC). Full inhibition of mitochondrial oxygen consumption was obtained after 24h antimycin A treatment, which did not affect cell viability. While recAP did not affect the antimycin A-induced decreased oxygen consumption and increased hypoxia-inducible factor-1α or adrenomedullin gene expression levels, the antimycin A-induced increase of pro-inflammatory cytokines IL-6 and IL-8 was attenuated. Antimycin A tended to induce the release of detrimental purines ATP and ADP, which reached statistical significance when antimycin A was co-incubated with lipopolysaccharide, and were completely converted into cytoprotective adenosine by recAP. As the adenosine A 2A receptor was up-regulated after antimycin A exposure, an adenosine A 2A receptor knockout ciPTEC cell line was generated in which recAP still provided protection. Together, recAP did not affect oxygen consumption but attenuated the inflammatory response during impaired mitochondrial function, an effect suggested to be mediated by dephosphorylating ATP and ADP into adenosine. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. VDAC electronics: 3. VDAC-Creatine kinase-dependent generation of the outer membrane potential in respiring mitochondria.

    PubMed

    Lemeshko, Victor V

    2016-07-01

    Mitochondrial energy in cardiac cells has been reported to be channeled into the cytosol through the intermembrane contact sites formed by the adenine nucleotide translocator, creatine kinase and VDAC. Computational analysis performed in this study showed a high probability of the outer membrane potential (OMP) generation coupled to such a mechanism of energy channeling in respiring mitochondria. OMPs, positive inside, calculated at elevated concentrations of creatine are high enough to restrict ATP release from mitochondria, to significantly decrease the apparent K(m,ADP) for state 3 respiration and to maintain low concentrations of Ca(2+) in the mitochondrial intermembrane space. An inhibition by creatine of Ca(2+)-induced swelling of isolated mitochondria and other protective effects of creatine reported in the literature might be explained by generated positive OMP. We suggest that VDAC-creatine kinase-dependent generation of OMP represents a novel physiological factor controlling metabolic state of mitochondria, cell energy channeling and resistance to death. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Energy metabolism determines the sensitivity of human hepatocellular carcinoma cells to mitochondrial inhibitors and biguanide drugs.

    PubMed

    Hsu, Chia-Chi; Wu, Ling-Chia; Hsia, Cheng-Yuan; Yin, Pen-Hui; Chi, Chin-Wen; Yeh, Tien-Shun; Lee, Hsin-Chen

    2015-09-01

    Human hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide particularly in Asia. Deregulation of cellular energetics was recently included as one of the cancer hallmarks. Compounds that target the mitochondria in cancer cells were proposed to have therapeutic potential. Biguanide drugs which inhibit mitochondrial complex I and repress mTOR signaling are clinically used to treat type 2 diabetes mellitus patients (T2DM) and were recently found to reduce the risk of HCC in T2DM patients. However, whether alteration of energy metabolism is involved in regulating the sensitivity of HCC to biguanide drugs is still unclear. In the present study, we treated four HCC cell lines with mitochondrial inhibitors (rotenone and oligomycin) and biguanide drugs (metformin and phenformin), and found that the HCC cells which had a higher mitochondrial respiration rate were more sensitive to these treatments; whereas the HCC cells which exhibited higher glycolysis were more resistant. When glucose was replaced by galactose in the medium, the altered energy metabolism from glycolysis to mitochondrial respiration in the HCC cells enhanced the cellular sensitivity to mitochondrial inhibitors and biguanides. The energy metabolism change enhanced AMP-activated protein kinase (AMPK) activation, mTOR repression and downregulation of cyclin D1 and Mcl-1 in response to the mitochondrial inhibitors and biguanides. In conclusion, our results suggest that increased mitochondrial oxidative metabolism upregulates the sensitivity of HCC to biguanide drugs. Enhancing the mitochondrial oxidative metabolism in combination with biguanide drugs may be a therapeutic strategy for HCC.

  8. Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression.

    PubMed

    Zhang, Tiantian; Bu, Pengli; Zeng, Joey; Vancura, Ales

    2017-10-13

    Regulation of mitochondrial biogenesis and respiration is a complex process that involves several signaling pathways and transcription factors as well as communication between the nuclear and mitochondrial genomes. Under aerobic conditions, the budding yeast Saccharomyces cerevisiae metabolizes glucose predominantly by glycolysis and fermentation. We have recently shown that altered chromatin structure in yeast induces respiration by a mechanism that requires transport and metabolism of pyruvate in mitochondria. However, how pyruvate controls the transcriptional responses underlying the metabolic switch from fermentation to respiration is unknown. Here, we report that this pyruvate effect involves heme. We found that heme induces transcription of HAP4 , the transcriptional activation subunit of the Hap2/3/4/5p complex, required for growth on nonfermentable carbon sources, in a Hap1p- and Hap2/3/4/5p-dependent manner. Increasing cellular heme levels by inactivating ROX1 , which encodes a repressor of many hypoxic genes, or by overexpressing HEM3 or HEM12 induced respiration and elevated ATP levels. Increased heme synthesis, even under conditions of glucose repression, activated Hap1p and the Hap2/3/4/5p complex and induced transcription of HAP4 and genes required for the tricarboxylic acid (TCA) cycle, electron transport chain, and oxidative phosphorylation, leading to a switch from fermentation to respiration. Conversely, inhibiting metabolic flux into the TCA cycle reduced cellular heme levels and HAP4 transcription. Together, our results indicate that the glucose-mediated repression of respiration in budding yeast is at least partly due to the low cellular heme level. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Proton MR spectroscopy in the diagnostic evaluation of suspected mitochondrial disease.

    PubMed

    Lin, Doris D M; Crawford, Thomas O; Barker, Peter B

    2003-01-01

    Mitochondrial diseases are a group of inherited disorders caused by a derangement of mitochondrial respiration. The clinical manifestations are heterogeneous, and the diagnosis is often based on information acquired from multiple levels of inquiry. MR spectroscopy has previously been shown to help detect an abnormal accumulation of lactate in brain parenchyma and CSF in association with mitochondrial disorders, but the frequency of detection is largely unknown. We sought to examine the frequency of detectable elevations of CNS lactate by proton MR spectroscopy in a population of children and young adults with suspected mitochondrial disease. MR spectroscopy data evaluated for the presence or absence of abnormal brain or CSF lactate were compared with other clinical indicators of mitochondrial dysfunction for 29 patients with suspected mitochondrial disease during the years 1990 to 2000. Based on an independent review of the final diagnoses, the patients were divided into groups based on the probability of mitochondrial disorder. A total of 32 scans from 29 patients were reviewed. Of eight patients thought to have a definitive mitochondrial disorder on the basis of genetic, biochemical, or pathologic features, five were found to have abnormal brain or CSF lactate levels revealed by MR spectroscopy (for one patient in whom two images were acquired, one was negative and the other positive). Among the studies conducted using a multisection spectroscopic imaging technique, five of six showed elevated lactate in the brain parenchyma, six of six showed elevated lactate in the CSF, and five of six showed elevated lactate in both brain and CSF. Of 16 patients who were highly suspected of having mitochondrial disorders on the basis of clinical grounds alone but who were lacking genetic, biochemical, or pathologic confirmation, four had abnormal lactate levels shown by MR spectroscopy. Mitochondrial disorder was excluded for five patients, none of whom had CNS lactate shown

  10. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase.

    PubMed

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan; Trnka, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70-4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes.

  11. Mitochondrial Probe Methyltriphenylphosphonium (TPMP) Inhibits the Krebs Cycle Enzyme 2-Oxoglutarate Dehydrogenase

    PubMed Central

    Elkalaf, Moustafa; Tůma, Petr; Weiszenstein, Martin; Polák, Jan

    2016-01-01

    Methyltriphenylphosphonium (TPMP) salts have been widely used to measure the mitochondrial membrane potential and the triphenylphosphonium (TPP+) moiety has been attached to many bioactive compounds including antioxidants to target them into mitochondria thanks to their high affinity to accumulate in the mitochondrial matrix. The adverse effects of these compounds on cellular metabolism have been insufficiently studied and are still poorly understood. Micromolar concentrations of TPMP cause a progressive inhibition of cellular respiration in adherent cells without a marked effect on mitochondrial coupling. In permeabilized cells the inhibition was limited to NADH-linked respiration. We found a mixed inhibition of the Krebs cycle enzyme 2-oxoglutarate dehydrogenase complex (OGDHC) with an estimated IC50 3.93 [3.70–4.17] mM, which is pharmacologically plausible since it corresponds to micromolar extracellular concentrations. Increasing the lipophilic character of the used TPP+ compound further potentiates the inhibition of OGDHC activity. This effect of TPMP on the Krebs cycle ought to be taken into account when interpreting observations on cells and mitochondria in the presence of TPP+ derivatives. Compounds based on or similar to TPP+ derivatives may also be used to alter OGDHC activity for experimental or therapeutic purposes. PMID:27537184

  12. Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal.

    PubMed

    SinhaRoy, Soumya; Banerjee, Sambhunath; Ray, Manju; Ray, Subhankar

    2005-03-01

    We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661-669,2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3'-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine.

  13. Regulation of mitochondrial bioenergetics by the non-canonical roles of mitochondrial dynamics proteins in the heart.

    PubMed

    Wang, Wang; Fernandez-Sanz, Celia; Sheu, Shey-Shing

    2018-05-01

    Recent advancement in mitochondrial research has significantly extended our knowledge on the role and regulation of mitochondria in health and disease. One important breakthrough is the delineation of how mitochondrial morphological changes, termed mitochondrial dynamics, are coupled to the bioenergetics and signaling functions of mitochondria. In general, it is believed that fusion leads to an increased mitochondrial respiration efficiency and resistance to stress-induced dysfunction while fission does the contrary. This concept seems not applicable to adult cardiomyocytes. The mitochondria in adult cardiomyocytes exhibit fragmented morphology (tilted towards fission) and show less networking and movement as compared to other cell types. However, being the most energy-demanding cells, cardiomyocytes in the adult heart possess vast number of mitochondria, high level of energy flow, and abundant mitochondrial dynamics proteins. This apparent discrepancy could be explained by recently identified new functions of the mitochondrial dynamics proteins. These "non-canonical" roles of mitochondrial dynamics proteins range from controlling inter-organelle communication to regulating cell viability and survival under metabolic stresses. Here, we summarize the newly identified non-canonical roles of mitochondrial dynamics proteins. We focus on how these fission and fusion independent roles of dynamics proteins regulate mitochondrial bioenergetics. We also discuss potential molecular mechanisms, unique intracellular location, and the cardiovascular disease relevance of these non-canonical roles of the dynamics proteins. We propose that future studies are warranted to differentiate the canonical and non-canonical roles of dynamics proteins and to identify new approaches for the treatment of heart diseases. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and

  14. Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: effect of catalase overexpression.

    PubMed

    Barbosa, Marina R; Sampaio, Igor H; Teodoro, Bruno G; Sousa, Thais A; Zoppi, Claudio C; Queiroz, André L; Passos, Madla A; Alberici, Luciane C; Teixeira, Felipe R; Manfiolli, Adriana O; Batista, Thiago M; Cappelli, Ana Paula Gameiro; Reis, Rosana I; Frasson, Danúbia; Kettelhut, Isis C; Parreiras-e-Silva, Lucas T; Costa-Neto, Claudio M; Carneiro, Everardo M; Curi, Rui; Silveira, Leonardo R

    2013-10-01

    The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits.

    PubMed

    Masand, Ruchi; Paulo, Esther; Wu, Dongmei; Wang, Yangmeng; Swaney, Danielle L; Jimenez-Morales, David; Krogan, Nevan J; Wang, Biao

    2018-03-06

    Brown adipose tissue (BAT) thermogenesis is critical for thermoregulation and contributes to total energy expenditure. However, whether BAT has non-thermogenic functions is largely unknown. Here, we describe that BAT-specific liver kinase b1 knockout (Lkb1 BKO ) mice exhibited impaired BAT mitochondrial respiration and thermogenesis but reduced adiposity and liver triglyceride accumulation under high-fat-diet feeding at room temperature. Importantly, these metabolic benefits were also present in Lkb1 BKO mice at thermoneutrality, where BAT thermogenesis was not required. Mechanistically, decreased mRNA levels of mtDNA-encoded electron transport chain (ETC) subunits and ETC proteome imbalance led to defective BAT mitochondrial respiration in Lkb1 BKO mice. Furthermore, reducing mtDNA gene expression directly in BAT by removing mitochondrial transcription factor A (Tfam) in BAT also showed ETC proteome imbalance and the trade-off between BAT thermogenesis and systemic metabolism at room temperature and thermoneutrality. Collectively, our data demonstrate that ETC proteome imbalance in BAT regulates systemic metabolism independently of thermogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Expanding the functional human mitochondrial DNA database by the establishment of primate xenomitochondrial cybrids

    PubMed Central

    Kenyon, Lesley; Moraes, Carlos T.

    1997-01-01

    The nuclear and mitochondrial genomes coevolve to optimize approximately 100 different interactions necessary for an efficient ATP-generating system. This coevolution led to a species-specific compatibility between these genomes. We introduced mitochondrial DNA (mtDNA) from different primates into mtDNA-less human cells and selected for growth of cells with a functional oxidative phosphorylation system. mtDNA from common chimpanzee, pigmy chimpanzee, and gorilla were able to restore oxidative phosphorylation in the context of a human nuclear background, whereas mtDNA from orangutan, and species representative of Old-World monkeys, New-World monkeys, and lemurs were not. Oxygen consumption, a sensitive index of respiratory function, showed that mtDNA from chimpanzee, pigmy chimpanzee, and gorilla replaced the human mtDNA and restored respiration to essentially normal levels. Mitochondrial protein synthesis was also unaltered in successful “xenomitochondrial cybrids.” The abrupt failure of mtDNA from primate species that diverged from humans as recently as 8–18 million years ago to functionally replace human mtDNA suggests the presence of one or a few mutations affecting critical nuclear–mitochondrial genome interactions between these species. These cellular systems provide a demonstration of intergenus mtDNA transfer, expand more than 20-fold the number of mtDNA polymorphisms that can be analyzed in a human nuclear background, and provide a novel model for the study of nuclear–mitochondrial interactions. PMID:9256447

  17. Curcumin Rescues a PINK1 Knock Down SH-SY5Y Cellular Model of Parkinson's Disease from Mitochondrial Dysfunction and Cell Death.

    PubMed

    van der Merwe, Celia; van Dyk, Hayley Christy; Engelbrecht, Lize; van der Westhuizen, Francois Hendrikus; Kinnear, Craig; Loos, Ben; Bardien, Soraya

    2017-05-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of dopaminergic neurons in the substantia nigra. Mutations in the PINK1 gene result in an autosomal recessive form of early-onset PD. PINK1 plays a vital role in mitochondrial quality control via the removal of dysfunctional mitochondria. The aim of the present study was to create a cellular model of PD using siRNA-mediated knock down of PINK1 in SH-SY5Y neuroblastoma cells The possible protective effects of curcumin, known for its many beneficial properties including antioxidant and anti-inflammatory effects, was tested on this model in the presence and absence of paraquat, an additional stressor. PINK1 siRNA and control cells were separated into four treatment groups: (i) untreated, (ii) treated with paraquat, (iii) pre-treated with curcumin then treated with paraquat, or (iv) treated with curcumin. Various parameters of cellular and mitochondrial function were then measured. The PINK1 siRNA cells exhibited significantly decreased cell viability, mitochondrial membrane potential (MMP), mitochondrial respiration and ATP production, and increased apoptosis. Paraquat-treated cells exhibited decreased cell viability, increased apoptosis, a more fragmented mitochondrial network and decreased MMP. Curcumin pre-treatment followed by paraquat exposure rescued cell viability and increased MMP and mitochondrial respiration in control cells, and significantly decreased apoptosis and increased MMP and maximal respiration in PINK1 siRNA cells. These results highlight a protective effect of curcumin against mitochondrial dysfunction and apoptosis in PINK1-deficient and paraquat-exposed cells. More studies are warranted to further elucidate the potential neuroprotective properties of curcumin.

  18. Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

    PubMed Central

    Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Houlden, Henry

    2017-01-01

    Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. PMID:28360103

  19. Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

    PubMed

    Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

    2017-05-26

    Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Using Yeast to Easily Determine Mitochondrial Functionality with 1-(4,5-Dimethylthiazol-2-yl)-3,5-Diphenyltetrazolium Bromide (MTT) Assay

    ERIC Educational Resources Information Center

    Sanchez, Norma Silvia; Konigsberg, Mina

    2006-01-01

    Mitochondrial respiration is an important topic in biochemical courses, but at the same time it is a very arduous and challenging subject for the students to comprehend. In teaching laboratories, experiments are also difficult to perform because mitochondrial isolation as well as oxygen consumption determinations require expensive equipment,…

  1. Sleep disorders associated with primary mitochondrial diseases.

    PubMed

    Ramezani, Ryan J; Stacpoole, Peter W

    2014-11-15

    Primary mitochondrial diseases are caused by heritable or spontaneous mutations in nuclear DNA or mitochondrial DNA. Such pathological mutations are relatively common in humans and may lead to neurological and neuromuscular complication that could compromise normal sleep behavior. To gain insight into the potential impact of primary mitochondrial disease and sleep pathology, we reviewed the relevant English language literature in which abnormal sleep was reported in association with a mitochondrial disease. We examined publication reported in Web of Science and PubMed from February 1976 through January 2014, and identified 54 patients with a proven or suspected primary mitochondrial disorder who were evaluated for sleep disturbances. Both nuclear DNA and mitochondrial DNA mutations were associated with abnormal sleep patterns. Most subjects who underwent polysomnography had central sleep apnea, and only 5 patients had obstructive sleep apnea. Twenty-four patients showed decreased ventilatory drive in response to hypoxia and/ or hyperapnea that was not considered due to weakness of the intrinsic muscles of respiration. Sleep pathology may be an underreported complication of primary mitochondrial diseases. The probable underlying mechanism is cellular energy failure causing both central neurological and peripheral neuromuscular degenerative changes that commonly present as central sleep apnea and poor ventilatory response to hyperapnea. Increased recognition of the genetics and clinical manifestations of mitochondrial diseases by sleep researchers and clinicians is important in the evaluation and treatment of all patients with sleep disturbances. Prospective population-based studies are required to determine the true prevalence of mitochondrial energy failure in subjects with sleep disorders, and conversely, of individuals with primary mitochondrial diseases and sleep pathology. © 2014 American Academy of Sleep Medicine.

  2. Synergism of antifungal activity between mitochondrial respiration inhibitors and kojic acid

    USDA-ARS?s Scientific Manuscript database

    Co-application of certain types of compounds with conventional antimicrobial drugs results in the enhancement of efficacy of drugs through a mechanism termed chemosensitization. We show that kojic acid (KA), a natural product, is a potent chemosensitizer to complex III inhibitors of mitochondrial re...

  3. Mitochondrial angiotensin receptors in dopaminergic neurons. Role in cell protection and aging-related vulnerability to neurodegeneration

    PubMed Central

    Valenzuela, Rita; Costa-Besada, Maria A; Iglesias-Gonzalez, Javier; Perez-Costas, Emma; Villar-Cheda, Begoña; Garrido-Gil, Pablo; Melendez-Ferro, Miguel; Soto-Otero, Ramon; Lanciego, Jose L; Henrion, Daniel; Franco, Rafael; Labandeira-Garcia, Jose L

    2016-01-01

    The renin–angiotensin system (RAS) was initially considered as a circulating humoral system controlling blood pressure, being kidney the key control organ. In addition to the ‘classical' humoral RAS, a second level in RAS, local or tissular RAS, has been identified in a variety of tissues, in which local RAS play a key role in degenerative and aging-related diseases. The local brain RAS plays a major role in brain function and neurodegeneration. It is normally assumed that the effects are mediated by the cell-surface-specific G-protein-coupled angiotensin type 1 and 2 receptors (AT1 and AT2). A combination of in vivo (rats, wild-type mice and knockout mice) and in vitro (primary mesencephalic cultures, dopaminergic neuron cell line cultures) experimental approaches (confocal microscopy, electron microscopy, laser capture microdissection, transfection of fluorescent-tagged receptors, treatments with fluorescent angiotensin, western blot, polymerase chain reaction, HPLC, mitochondrial respirometry and other functional assays) were used in the present study. We report the discovery of AT1 and AT2 receptors in brain mitochondria, particularly mitochondria of dopaminergic neurons. Activation of AT1 receptors in mitochondria regulates superoxide production, via Nox4, and increases respiration. Mitochondrial AT2 receptors are much more abundant and increase after treatment of cells with oxidative stress inducers, and produce, via nitric oxide, a decrease in mitochondrial respiration. Mitochondria from the nigral region of aged rats displayed altered expression of AT1 and AT2 receptors. AT2-mediated regulation of mitochondrial respiration represents an unrecognized primary line of defence against oxidative stress, which may be particularly important in neurons with increased levels of oxidative stress such as dopaminergic neurons. Altered expression of AT1 and AT2 receptors with aging may induce mitochondrial dysfunction, the main risk factor for neurodegeneration

  4. Shrubs stimulate heterotrophic respiration in arctic soils

    NASA Astrophysics Data System (ADS)

    Phillips, C. A.; Wurzburger, N.

    2016-12-01

    The response of arctic ecosystems to global change will have critical effects on future climate. Climate warming has already triggered the expansion of shrubs across tundra, raising questions about how shrubs will affect ecosystem carbon balance. Shrub litter quality and mycorrhizal symbionts may accelerate the activity of soil microorganisms that facilitate the release of large stores of soil carbon. We investigated how shrubs affect the activity of soil microorganisms by creating soil mesocosms from areas with and without shrub species as dominants of the plant community in arctic Alaska. We hypothesized that relative to their non-shrub counterparts, heterotrophic respiration of shrub soils would: (1) be greater, (2) demonstrate greater response to additions of shrub litter, and (3) be less nutrient limited. We created mesocosms with root-free soils at constant moisture and temperature, and quantified basal heterotrophic soil respiration rates, and the response of respiration to litter and nutrient inputs in a series of laboratory experiments inputs. (1) We found that the presence of shrubs generally produced higher rates of basal soil respiration in both horizons, suggesting that shrubs stimulate microbial activity. (2) Litter addition increased respiration across both horizons with no differences in response between shrub and non-shrub soils. (3) N additions did not increase heterotrophic respiration, but P and N+P additions induced a short respiratory pulse in all soils, suggesting mild P limitation. Collectively, these findings provide evidence that shrubs stimulate heterotrophic microbial activity to enhance carbon loss, but generate new questions about the mechanisms driving these patterns.

  5. [Respiration of wheat root cells under simultaneous inhibition of parts I and III of the electron transport chain of mitochondria by rotenone and antimycine A].

    PubMed

    Rakhmatullina, D F; Gordon, L Kh; Ogorodnikova, T I

    2005-01-01

    Respiration of excised roots of 5 day old wheat seedlings with blocked mitochondrial oxidation under simultaneous action of rotenone and antimycine A was studied. A reduced rate of oxygen uptake was observed within the first hour of root treatment inhibitors. However, after a 5 h exposure there was an increase in oxygen uptake, which was prevented by KCN but amplified by malate and ascorbate. The application of inhibitors caused a considerable increase in the respiratory coefficient (RC) up to 2.1, that suggests a significant CO2 release, when the initial sites of mitochondrial electron transport chain were inhibited. RC did not raise, when ascorbate was added in the presence of inhibitors. We assume that inhibition of mitochondrial oxidation at I and III sites of electron transport chain facilitates switching on the alternative paths of reductant translocation to oxygen. Participation of ATPases and redox system of plasma membrane in the response reactions of respiration directed to the restoration of ion, particularly, proton homeostasis in conditions of inhibited mitochondrial oxidation is discussed.

  6. Carnosine decreased neuronal cell death through targeting glutamate system and astrocyte mitochondrial bioenergetics in cultured neuron/astrocyte exposed to OGD/recovery.

    PubMed

    Ouyang, Li; Tian, Yueyang; Bao, Yun; Xu, Huijuan; Cheng, Jiaoyan; Wang, Bingyu; Shen, Yao; Chen, Zhong; Lyu, Jianxin

    2016-06-01

    Previously, we showed that carnosine upregulated the expression level of glutamate transporter 1 (GLT-1), which has been recognized as an important participant in the astrocyte-neuron lactate shuttle (ANLS), with ischemic model in vitro and in vivo. This study was designed to investigate the protective effect of carnosine on neuron/astrocyte co-cultures exposed to OGD/recovery, and to explore whether the ANLS or any other mechanism contributes to carnosine-induced neuroprotection on neuron/astrocyte. Co-cultures were treated with carnosine and exposed to OGD/recovery. Cell death and the extracellular levels of glutamate and GABA were measured. The mitochondrial respiration and glycolysis were detected by Seahorse Bioscience XF96 Extracellular Flux Analyzer. Results showed that carnosine decreased neuronal cell death, increased extracellular GABA level, and abolished the increase in extracellular glutamate and reversed the mitochondrial energy metabolism disorder induced by OGD/recovery. Carnosine also upregulated the mRNA level of neuronal glutamate transporter EAAC1 at 2h after OGD. Dihydrokainate, a specific inhibitor of GLT-1, decreased glycolysis but it did not affect mitochondrial respiration of the cells, and it could not reverse the increase in mitochondrial OXPHOS induced by carnosine in the co-cultures. The levels of mRNAs for monocarboxylate transporter1, 4 (MCT1, 4), which were expressed in astrocytes, and MCT2, the main neuronal MCT, were significantly increased at the early stage of recovery. Carnosine only partly reversed the increased expression of astrocytic MCT1 and MCT4. These results suggest that regulating astrocytic energy metabolism and extracellular glutamate and GABA levels but not the ANLS are involved in the carnosine-induced neuroprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Single nucleotide polymorphisms linked to mitochondrial uncoupling protein genes UCP2 and UCP3 affect mitochondrial metabolism and healthy aging in female nonagenarians

    PubMed Central

    Kim, Sangkyu; Myers, Leann; Ravussin, Eric; Cherry, Katie E.; Jazwinski, S. Michal

    2016-01-01

    Energy expenditure decreases with age, but in the oldest-old, energy demand for maintenance of body functions increases with declining health. Uncoupling proteins have profound impact on mitochondrial metabolic processes; therefore, we focused attention on mitochondrial uncoupling protein genes. Alongside resting metabolic rate (RMR), two SNPs in the promoter region of UCP2 were associated with healthy aging. These SNPs mark potential binding sites for several transcription factors; thus, they may affect expression of the gene. A third SNP in the 3′-UTR of UCP3 interacted with RMR. This UCP3 SNP is known to impact UCP3 expression in tissue culture cells, and it has been associated with body weight and mitochondrial energy metabolism. The significant main effects of the UCP2 SNPs and the interaction effect of the UCP3 SNP were also observed after controlling for fat-free mass (FFM) and physical-activity related energy consumption. The association of UCP2/3 with healthy aging was not found in males. Thus, our study provides evidence that the genetic risk factors for healthy aging differ in males and females, as expected from the differences in the phenotypes associated with healthy aging between the two sexes. It also has implications for how mitochondrial function changes during aging. PMID:26965008

  8. Single nucleotide polymorphisms linked to mitochondrial uncoupling protein genes UCP2 and UCP3 affect mitochondrial metabolism and healthy aging in female nonagenarians.

    PubMed

    Kim, Sangkyu; Myers, Leann; Ravussin, Eric; Cherry, Katie E; Jazwinski, S Michal

    2016-08-01

    Energy expenditure decreases with age, but in the oldest-old, energy demand for maintenance of body functions increases with declining health. Uncoupling proteins have profound impact on mitochondrial metabolic processes; therefore, we focused attention on mitochondrial uncoupling protein genes. Alongside resting metabolic rate (RMR), two SNPs in the promoter region of UCP2 were associated with healthy aging. These SNPs mark potential binding sites for several transcription factors; thus, they may affect expression of the gene. A third SNP in the 3'-UTR of UCP3 interacted with RMR. This UCP3 SNP is known to impact UCP3 expression in tissue culture cells, and it has been associated with body weight and mitochondrial energy metabolism. The significant main effects of the UCP2 SNPs and the interaction effect of the UCP3 SNP were also observed after controlling for fat-free mass (FFM) and physical-activity related energy consumption. The association of UCP2/3 with healthy aging was not found in males. Thus, our study provides evidence that the genetic risk factors for healthy aging differ in males and females, as expected from the differences in the phenotypes associated with healthy aging between the two sexes. It also has implications for how mitochondrial function changes during aging.

  9. The effect of substrate, ADP and uncoupler on the respiration of tomato pollen during incubation in vitro at moderately high temperature.

    PubMed

    Karapanos, I C; Akoumianakis, K A; Olympios, C M; Passam, H C

    2009-09-01

    Pollen of tomato cv. Supermarmande was collected from greenhouse-grown plants at various intervals throughout the year and arbitrarily classified as of high, medium or low respiratory activity on the basis of CO(2) production during 8 h incubation in vitro at 30 degrees C, a temperature that is considered to be moderately high for tomato fruit set. After an initial burst of respiration during the first stage of hydration at 30 degrees C (>1 h), the respiration rate of pollen of all three categories declined, the decrease being greater in the lots with a low or medium respiratory activity than in the high category. During hydration (10 min after the start of incubation), the addition of succinate or reduced beta-nicotinamide adenine dinucleotide (NADH) to the substrate increased the respiratory rate of slowly-respiring pollen more than that of fast-respiring pollen, but carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and adenosine 5'-diphosphate (ADP) had less effect. After 1-4 h incubation, the respiration rate of the slow- or medium-respiring pollen lots had decreased, but was stimulated by succinate or NADH, and to a lesser degree by ADP. By 7 h, the respiration rate of all pollen lots had declined and was stimulated less by substrate, ADP or CCCP. The oxidation of NADH by tomato pollen contrasts with the failure of other pollen species to utilize this substrate; moreover, a synergistic effect of NADH and succinate was consistently observed. We conclude that the decline in respiration during incubation for up to 4 h at 30 degrees C may reflect a lack of respiratory substrate. After 7 h, however, the decreased response to substrate indicates a loss of mitochondrial integrity or an accumulation of metabolic inhibitors. It is concluded that at 30 degrees C (a moderately high temperature for tomato pollen), the initially high rate of respiration leads to exhaustion of the endogenous respiratory substrates (particularly in pollen with low to medium respiratory

  10. Mitochondrial activity in the regulation of stem cell self-renewal and differentiation.

    PubMed

    Khacho, Mireille; Slack, Ruth S

    2017-12-01

    Mitochondria are classically known as the essential energy producers in cells. As such, the activation of mitochondrial metabolism upon cellular differentiation was deemed a necessity to fuel the high metabolic needs of differentiated cells. However, recent studies have revealed a direct role for mitochondrial activity in the regulation of stem cell fate and differentiation. Several components of mitochondrial metabolism and respiration have now been shown to regulate different aspects of stem cell differentiation through signaling, transcriptional, proteomic and epigenetic modulations. In light of these findings mitochondrial metabolism is no longer considered a consequence of cellular differentiation, but rather a key regulatory mechanism of this process. This review will focus on recent progress that defines mitochondria as the epicenters for the regulation of stem cell fate decisions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes.

    PubMed

    Tanis, Ross M; Piroli, Gerardo G; Day, Stani D; Frizzell, Norma

    2015-01-01

    While the 3T3-L1 adipocyte model is routinely used for the study of obesity and diabetes, the mitochondrial respiratory profile in normal versus high glucose has not been examined in detail. We matured adipocytes in normal (5mM) or high (30 mM) glucose and insulin and examined the mitochondrial bioenergetics. We also assessed the requirement for the Unfolded Protein Response (UPR) and ER stress under these conditions. Basal respiration was ~1.7-fold greater in adipocytes that had matured in 30 mM glucose; however, their ability to increase oxygen consumption in response to stress was impaired. Adipogenesis proceeded in both normal and high glucose with concomitant activation of the UPR, but only high glucose was associated with increased levels of ER stress and mitochondrial stress as observed by parallel increases in CHOP and protein succination. Treatment of adipocytes with sodium phenylbutyrate relieved mitochondrial stress through a reduction in mitochondrial respiration. Our data suggests that mitochondrial stress, protein succination and ER stress are uniquely linked in adipocytes matured in high glucose. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Bicarbonate alters cellular responses in respiration assays.

    PubMed

    Krycer, James R; Fisher-Wellman, Kelsey H; Fazakerley, Daniel J; Muoio, Deborah M; James, David E

    2017-08-05

    Metabolic assay buffers often omit bicarbonate, which is susceptible to alkalinisation in an open environment. Here, we assessed the effect of including bicarbonate in respirometry experiments. By supplementing HEPES-buffered media with low concentrations of bicarbonate, we found increased respiration in adipocytes and hepatocytes, but not myotubes. This was observed across multiple respirometry platforms and was independent of effects on enhanced insulin sensitivity, pH drift, or mitochondrial function. Permeabilised cell experiments suggest that bicarbonate increases substrate availability, likely by acting as a cofactor for carboxylase enzymes. This emphasises the importance of buffer choice in experimental biology. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast.

    PubMed

    Aris, John P; Alvers, Ashley L; Ferraiuolo, Roy A; Fishwick, Laura K; Hanvivatpong, Amanda; Hu, Doreen; Kirlew, Christine; Leonard, Michael T; Losin, Kyle J; Marraffini, Michelle; Seo, Arnold Y; Swanberg, Veronica; Westcott, Jennifer L; Wood, Michael S; Leeuwenburgh, Christiaan; Dunn, William A

    2013-10-01

    We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Autophagy and leucine promote chronological longevity and respiration proficiency during calorie restriction in yeast

    PubMed Central

    Aris, John P.; Alvers, Ashley L.; Ferraiuolo, Roy A.; Fishwick, Laura K.; Hanvivatpong, Amanda; Hu, Doreen; Kirlew, Christine; Leonard, Michael T.; Losin, Kyle J.; Marraffini, Michelle; Seo, Arnold Y.; Swanberg, Veronica; Westcott, Jennifer L.; Wood, Michael S.; Leeuwenburgh, Christiaan; Dunn, William A.

    2013-01-01

    We have previously shown that autophagy is required for chronological longevity in the budding yeast Saccharomyces cerevisiae. Here we examine the requirements for autophagy during extension of chronological life span (CLS) by calorie restriction (CR). We find that autophagy is upregulated by two CR interventions that extend CLS: water wash CR and low glucose CR. Autophagy is required for full extension of CLS during water wash CR under all growth conditions tested. In contrast, autophagy was not uniformly required for full extension of CLS during low glucose CR, depending on the atg allele and strain genetic background. Leucine status influenced CLS during CR. Eliminating the leucine requirement in yeast strains or adding supplemental leucine to growth media extended CLS during CR. In addition, we observed that both water wash and low glucose CR promote mitochondrial respiration proficiency during aging of autophagy-deficient yeast. In general, the extension of CLS by water wash or low glucose CR was inversely related to respiration deficiency in autophagy-deficient cells. Also, autophagy is required for full extension of CLS under non-CR conditions in buffered media, suggesting that extension of CLS during CR is not solely due to reduced medium acidity. Thus, our findings show that autophagy is: (1) induced by CR, (2) required for full extension of CLS by CR in most cases (depending on atg allele, strain, and leucine availability) and, (3) promotes mitochondrial respiration proficiency during aging under CR conditions. PMID:23337777

  15. Improving respiration measurements with gas exchange analyzers.

    PubMed

    Montero, R; Ribas-Carbó, M; Del Saz, N F; El Aou-Ouad, H; Berry, J A; Flexas, J; Bota, J

    2016-12-01

    Dark respiration measurements with open-flow gas exchange analyzers are often questioned for their low accuracy as their low values often reach the precision limit of the instrument. Respiration was measured in five species, two hypostomatous (Vitis Vinifera L. and Acanthus mollis) and three amphistomatous, one with similar amount of stomata in both sides (Eucalyptus citriodora) and two with different stomata density (Brassica oleracea and Vicia faba). CO 2 differential (ΔCO 2 ) increased two-fold with no change in apparent R d , when the two leaves with higher stomatal density faced outside. These results showed a clear effect of the position of stomata on ΔCO 2 . Therefore, it can be concluded that leaf position is important to guarantee the improvement of respiration measurements increasing ΔCO 2 without affecting the respiration results by leaf or mass units. This method will help to increase the accuracy of leaf respiration measurements using gas exchange analyzers. Copyright © 2016 Elsevier GmbH. All rights reserved.

  16. High-resolution respirometry of fine-needle muscle biopsies in pre-manifest Huntington's disease expansion mutation carriers shows normal mitochondrial respiratory function.

    PubMed

    Buck, Eva; Zügel, Martina; Schumann, Uwe; Merz, Tamara; Gumpp, Anja M; Witting, Anke; Steinacker, Jürgen M; Landwehrmeyer, G Bernhard; Weydt, Patrick; Calzia, Enrico; Lindenberg, Katrin S

    2017-01-01

    Alterations in mitochondrial respiration are an important hallmark of Huntington's disease (HD), one of the most common monogenetic causes of neurodegeneration. The ubiquitous expression of the disease causing mutant huntingtin gene raises the prospect that mitochondrial respiratory deficits can be detected in skeletal muscle. While this tissue is readily accessible in humans, transgenic animal models offer the opportunity to cross-validate findings and allow for comparisons across organs, including the brain. The integrated respiratory chain function of the human vastus lateralis muscle was measured by high-resolution respirometry (HRR) in freshly taken fine-needle biopsies from seven pre-manifest HD expansion mutation carriers and nine controls. The respiratory parameters were unaffected. For comparison skeletal muscle isolated from HD knock-in mice (HdhQ111) as well as a broader spectrum of tissues including cortex, liver and heart muscle were examined by HRR. Significant changes of mitochondrial respiration in the HdhQ knock-in mouse model were restricted to the liver and the cortex. Mitochondrial mass as quantified by mitochondrial DNA copy number and citrate synthase activity was stable in murine HD-model tissue compared to control. mRNA levels of key enzymes were determined to characterize mitochondrial metabolic pathways in HdhQ mice. We demonstrated the feasibility to perform high-resolution respirometry measurements from small human HD muscle biopsies. Furthermore, we conclude that alterations in respiratory parameters of pre-manifest human muscle biopsies are rather limited and mirrored by a similar absence of marked alterations in HdhQ skeletal muscle. In contrast, the HdhQ111 murine cortex and liver did show respiratory alterations highlighting the tissue specific nature of mutant huntingtin effects on respiration.

  17. Mitochondrial function is altered in horse atypical myopathy.

    PubMed

    Lemieux, Hélène; Boemer, François; van Galen, Gaby; Serteyn, Didier; Amory, Hélène; Baise, Etienne; Cassart, Dominique; van Loon, Gunther; Marcillaud-Pitel, Christel; Votion, Dominique-M

    2016-09-01

    Equine atypical myopathy in Europe is a fatal rhabdomyolysis syndrome that results from the ingestion of hypoglycin A contained in seeds and seedlings of Acer pseudoplatanus (sycamore maple). Acylcarnitine concentrations in serum and muscle OXPHOS capacity were determined in 15 atypical myopathy cases. All but one acylcarnitine were out of reference range and mitochondrial respiratory capacity was severely decreased up to 49% as compared to 10 healthy controls. The hallmark of atypical myopathy thus consists of a severe alteration in the energy metabolism including a severe impairment in muscle mitochondrial respiration that could contribute to its high death rate. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  18. Mitochondrial Respiratory Chain Dysfunction in Dorsal Root Ganglia of Streptozotocin-Induced Diabetic Rats and Its Correction by Insulin Treatment

    PubMed Central

    Chowdhury, Subir K. Roy; Zherebitskaya, Elena; Smith, Darrell R.; Akude, Eli; Chattopadhyay, Sharmila; Jolivalt, Corinne G.; Calcutt, Nigel A.; Fernyhough, Paul

    2010-01-01

    OBJECTIVE Impairments in mitochondrial physiology may play a role in diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in sensory neurons is due to abnormal mitochondrial respiratory function. RESEARCH DESIGN AND METHODS Rates of oxygen consumption were measured in mitochondria from dorsal root ganglia (DRG) of 12- to- 22-week streptozotocin (STZ)-induced diabetic rats, diabetic rats treated with insulin, and age-matched controls. Activities and expression of components of mitochondrial complexes and reactive oxygen species (ROS) were analyzed. RESULTS Rates of coupled respiration with pyruvate + malate (P + M) and with ascorbate + TMPD (Asc + TMPD) in DRG were unchanged after 12 weeks of diabetes. By 22 weeks of diabetes, respiration with P + M was significantly decreased by 31–44% and with Asc + TMPD by 29–39% compared with control. Attenuated mitochondrial respiratory activity of STZ-diabetic rats was significantly improved by insulin that did not correct other indices of diabetes. Activities of mitochondrial complexes I and IV and the Krebs cycle enzyme, citrate synthase, were decreased in mitochondria from DRG of 22-week STZ-diabetic rats compared with control. ROS levels in perikarya of DRG neurons were not altered by diabetes, but ROS generation from mitochondria treated with antimycin A was diminished compared with control. Reduced mitochondrial respiratory function was associated with downregulation of expression of mitochondrial proteins. CONCLUSIONS Mitochondrial dysfunction in sensory neurons from type 1 diabetic rats is associated with impaired rates of respiratory activity and occurs without a significant rise in perikaryal ROS. PMID:20103706

  19. Effects of acute lipid overload on skeletal muscle insulin resistance, metabolic flexibility, and mitochondrial performance

    PubMed Central

    Coen, Paul M.; DiStefano, Giovanna; Chacon, Alexander C.; Helbling, Nicole L.; Desimone, Marisa E.; Stafanovic-Racic, Maja; Hames, Kazanna C.; Despines, Alex A.; Toledo, Frederico G. S.; Goodpaster, Bret H.

    2014-01-01

    We hypothesized that acute lipid-induced insulin resistance would be attenuated in high-oxidative muscle of lean trained (LT) endurance athletes due to their enhanced metabolic flexibility and mitochondrial capacity. Lean sedentary (LS), obese sedentary (OS), and LT participants completed two hyperinsulinemic euglycemic clamp studies with and without (glycerol control) the coinfusion of Intralipid. Metabolic flexibility was measured by indirect calorimetry as the oxidation of fatty acids and glucose during fasted and insulin-stimulated conditions, the latter with and without lipid oversupply. Muscle biopsies were obtained for mitochondrial and insulin-signaling studies. During hyperinsulinemia without lipid, glucose infusion rate (GIR) was lowest in OS due to lower rates of nonoxidative glucose disposal (NOGD), whereas state 4 respiration was increased in all groups. Lipid infusion reduced GIR similarly in all subjects and reduced state 4 respiration. However, in LT subjects, fat oxidation was higher with lipid oversupply, and although glucose oxidation was reduced, NOGD was better preserved compared with LS and OS subjects. Mitochondrial performance was positively associated with better NOGD and insulin sensitivity in both conditions. We conclude that enhanced mitochondrial performance with exercise is related to better metabolic flexibility and insulin sensitivity in response to lipid overload. PMID:25352435

  20. Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart: ROLE OF MITOCHONDRIAL PYRUVATE CARRIER 2 (MPC2) ACETYLATION.

    PubMed

    Vadvalkar, Shraddha S; Matsuzaki, Satoshi; Eyster, Craig A; Giorgione, Jennifer R; Bockus, Lee B; Kinter, Caroline S; Kinter, Michael; Humphries, Kenneth M

    2017-03-17

    Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Clinical pulmonary function and industrial respirator wear

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raven, P.B.; Moss, R.F.; Page, K.

    1981-12-01

    This investigation was the initial step in determining a clinical pulmonary test which could be used to evaluate workers as to their suitability to industrial respirator wear. Sixty subjects, 12 superior, 37 normal, and 11 moderately impaired with respect to lung function tests were evaluated with a battery of clinical pulmonary tests while wearing an industrial respirator. The respirator was a full-face mask (MSA-Ultravue) demand breathing type equipped with an inspiratory resistance of 85mm H/sub 2/O at 85 L/min air flow and an expiratory resistance of 25mm H/sub 2/O at 85 L/min air flow. Comparisons of these tests were mademore » between the three groups of subjects both with and without a respirator. It appears that those lung tests which measure the flow characteristics of the lung especially those that are effort dependant are more susceptible to change as a result of respirator wear. Hence, the respirator affects the person with superior lung function to a greater degree than the moderately impaired person. It was suggested that the clinical test of 15 second maximum voluntary ventilations (MVV./sub 25/) may be the test of choice for determining worker capability in wearing an industrial respirator.« less

  2. Mitochondrial Toxicity of Cadmium Telluride Quantum Dot Nanoparticles in Mammalian Hepatocytes

    PubMed Central

    Nguyen, Kathy C.; Rippstein, Peter; Tayabali, Azam F.; Willmore, William G.

    2015-01-01

    There are an increasing number of studies indicating that mitochondria are relevant targets in nanomaterial-induced toxicity. However, the underlying mechanisms by which nanoparticles (NPs) interact with these organelles and affect their functions are unknown. The aim of this study was to investigate the effects of cadmium telluride quantum dot (CdTe-QD) NPs on mitochondria in human hepatocellular carcinoma HepG2 cells. CdTe-QD treatment resulted in the enlargement of mitochondria as examined with transmission electron microscopy and confocal microscopy. CdTe-QDs appeared to associate with the isolated mitochondria as detected by their inherent fluorescence. Further analyses revealed that CdTe-QD caused disruption of mitochondrial membrane potential, increased intracellular calcium levels, impaired cellular respiration, and decreased adenosine triphosphate synthesis. The effects of CdTe-QDs on mitochondrial oxidative phosphorylation were evidenced by changes in levels and activities of the enzymes of the electron transport chain. Elevation of peroxisome proliferator-activated receptor-γ coactivator levels after CdTe-QD treatment suggested the effects of CdTe-QDs on mitochondrial biogenesis. Our results also showed that the effects of CdTe-QDs were similar or greater to those of cadmium chloride at equivalent concentrations of cadmium, suggesting that the toxic effects of CdTe-QDs were not solely due to cadmium released from the NPs. Overall, the study demonstrated that CdTe-QDs induced multifarious toxicity by causing changes in mitochondrial morphology and structure, as well as impairing their function and stimulating their biogenesis. PMID:25809595

  3. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats.

    PubMed

    Ortiz-Avila, Omar; Esquivel-Martínez, Mauricio; Olmos-Orizaba, Berenice Eridani; Saavedra-Molina, Alfredo; Rodriguez-Orozco, Alain R; Cortés-Rojo, Christian

    2015-01-01

    Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨ m ), besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

  4. Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

    PubMed

    Sakai, Chika; Tomitsuka, Eriko; Esumi, Hiroyasu; Harada, Shigeharu; Kita, Kiyoshi

    2012-05-01

    Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Mitochondrial ribosome assembly in health and disease

    PubMed Central

    De Silva, Dasmanthie; Tu, Ya-Ting; Amunts, Alexey; Fontanesi, Flavia; Barrientos, Antoni

    2015-01-01

    The ribosome is a structurally and functionally conserved macromolecular machine universally responsible for catalyzing protein synthesis. Within eukaryotic cells, mitochondria contain their own ribosomes (mitoribosomes), which synthesize a handful of proteins, all essential for the biogenesis of the oxidative phosphorylation system. High-resolution cryo-EM structures of the yeast, porcine and human mitoribosomal subunits and of the entire human mitoribosome have uncovered a wealth of new information to illustrate their evolutionary divergence from their bacterial ancestors and their adaptation to synthesis of highly hydrophobic membrane proteins. With such structural data becoming available, one of the most important remaining questions is that of the mitoribosome assembly pathway and factors involved. The regulation of mitoribosome biogenesis is paramount to mitochondrial respiration, and thus to cell viability, growth and differentiation. Moreover, mutations affecting the rRNA and protein components produce severe human mitochondrial disorders. Despite its biological and biomedical significance, knowledge on mitoribosome biogenesis and its deviations from the much-studied bacterial ribosome assembly processes is scarce, especially the order of rRNA processing and assembly events and the regulatory factors required to achieve fully functional particles. This article focuses on summarizing the current available information on mitoribosome assembly pathway, factors that form the mitoribosome assembly machinery, and the effect of defective mitoribosome assembly on human health. PMID:26030272

  6. Beetroot juice supplementation reduces whole body oxygen consumption but does not improve indices of mitochondrial efficiency in human skeletal muscle.

    PubMed

    Whitfield, J; Ludzki, A; Heigenhauser, G J F; Senden, J M G; Verdijk, L B; van Loon, L J C; Spriet, L L; Holloway, G P

    2016-01-15

    Oral consumption of nitrate (NO3(-)) in beetroot juice has been shown to decrease the oxygen cost of submaximal exercise; however, the mechanism of action remains unresolved. We supplemented recreationally active males with beetroot juice to determine if this altered mitochondrial bioenergetics. Despite reduced submaximal exercise oxygen consumption, measures of mitochondrial coupling and respiratory efficiency were not altered in muscle. In contrast, rates of mitochondrial hydrogen peroxide (H2O2) emission were increased in the absence of markers of lipid or protein oxidative damage. These results suggest that improvements in mitochondrial oxidative metabolism are not the cause of beetroot juice-mediated improvements in whole body oxygen consumption. Ingestion of sodium nitrate (NO3(-)) simultaneously reduces whole body oxygen consumption (V̇O2) during submaximal exercise while improving mitochondrial efficiency, suggesting a causal link. Consumption of beetroot juice (BRJ) elicits similar decreases in V̇O2 but potential effects on the mitochondria remain unknown. Therefore we examined the effects of 7-day supplementation with BRJ (280 ml day(-1), ∼26 mmol NO3(-)) in young active males (n = 10) who had muscle biopsies taken before and after supplementation for assessments of mitochondrial bioenergetics. Subjects performed 20 min of cycling (10 min at 50% and 70% V̇O2 peak) 48 h before 'Pre' (baseline) and 'Post' (day 5 of supplementation) biopsies. Whole body V̇O2 decreased (P < 0.05) by ∼3% at 70% V̇O2 peak following supplementation. Mitochondrial respiration in permeabilized muscle fibres showed no change in leak respiration, the content of proteins associated with uncoupling (UCP3, ANT1, ANT2), maximal substrate-supported respiration, or ADP sensitivity (apparent Km). In addition, isolated subsarcolemmal and intermyofibrillar mitochondria showed unaltered assessments of mitochondrial efficiency, including ADP consumed/oxygen consumed (P/O ratio

  7. Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion.

    PubMed

    Whittington, Hannah J; Ostrowski, Philip J; McAndrew, Debra J; Cao, Fang; Shaw, Andrew; Eykyn, Thomas R; Lake, Hannah; Tyler, Jack; Schneider, Jurgen E; Neubauer, Stefan; Zervou, Sevasti; Lygate, Craig A

    2018-03-02

    Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesised that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury. Mice were created overexpressing the sarcomeric MtCK gene with αMHC promoter at the Rosa26 locus (MtCK-OE) and compared with wild-type (WT) littermates. MtCK activity was 27% higher than WT, with no change in other CK isoenzymes or creatine levels. Electron microscopy confirmed normal mitochondrial cell density and mitochondrial localisation of transgenic protein. Respiration in isolated mitochondria was unaltered and metabolomic analysis by 1H-NMR suggests that cellular metabolism was not grossly affected by transgene expression. There were no significant differences in cardiac structure or function under baseline conditions by cine-MRI or LV haemodynamics. In Langendorff-perfused hearts subjected to 20min ischaemia and 30 min reperfusion, MtCK-OE exhibited less ischaemic contracture and improved functional recovery (Rate pressure product 58% above WT; P < 0.001). These hearts had reduced myocardial infarct size, which was confirmed in vivo: 55±4% in WT vs 29±4% in MtCK-OE; P < 0.0001). Isolated cardiomyocytes from MtCK-OE hearts exhibited delayed opening of the mitochondrial permeability transition pore (mPTP) compared to WT, which was confirmed by reduced mitochondrial swelling in response to calcium. There was no detectable change in the structural integrity of the mitochondrial membrane. Modest elevation of MtCK activity in the heart does not adversely affect cellular metabolism, mitochondrial or in vivo cardiac function, but modifies mPTP opening to protect against I/R injury and improve functional recovery. Our findings support MtCK as a prime therapeutic target in myocardial ischaemia.

  8. Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome

    PubMed Central

    Garone, Caterina; D’Souza, Aaron R; Dallabona, Cristina; Lodi, Tiziana; Rebelo-Guiomar, Pedro; Rorbach, Joanna; Donati, Maria Alice; Procopio, Elena; Montomoli, Martino; Guerrini, Renzo; Zeviani, Massimo; Calvo, Sarah E; Mootha, Vamsi K; DiMauro, Salvatore; Ferrero, Ileana; Minczuk, Michal

    2017-01-01

    Abstract Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encephalomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a human disease and encodes an enzyme responsible for 2’-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2’-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead completely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation. PMID:28973171

  9. Defective mitochondrial rRNA methyltransferase MRM2 causes MELAS-like clinical syndrome.

    PubMed

    Garone, Caterina; D'Souza, Aaron R; Dallabona, Cristina; Lodi, Tiziana; Rebelo-Guiomar, Pedro; Rorbach, Joanna; Donati, Maria Alice; Procopio, Elena; Montomoli, Martino; Guerrini, Renzo; Zeviani, Massimo; Calvo, Sarah E; Mootha, Vamsi K; DiMauro, Salvatore; Ferrero, Ileana; Minczuk, Michal

    2017-11-01

    Defects in nuclear-encoded proteins of the mitochondrial translation machinery cause early-onset and tissue-specific deficiency of one or more OXPHOS complexes. Here, we report a 7-year-old Italian boy with childhood-onset rapidly progressive encephalomyopathy and stroke-like episodes. Multiple OXPHOS defects and decreased mtDNA copy number (40%) were detected in muscle homogenate. Clinical features combined with low level of plasma citrulline were highly suggestive of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, however, the common m.3243 A > G mutation was excluded. Targeted exome sequencing of genes encoding the mitochondrial proteome identified a damaging mutation, c.567 G > A, affecting a highly conserved amino acid residue (p.Gly189Arg) of the MRM2 protein. MRM2 has never before been linked to a human disease and encodes an enzyme responsible for 2'-O-methyl modification at position U1369 in the human mitochondrial 16S rRNA. We generated a knockout yeast model for the orthologous gene that showed a defect in respiration and the reduction of the 2'-O-methyl modification at the equivalent position (U2791) in the yeast mitochondrial 21S rRNA. Complementation with the mrm2 allele carrying the equivalent yeast mutation failed to rescue the respiratory phenotype, which was instead completely rescued by expressing the wild-type allele. Our findings establish that defective MRM2 causes a MELAS-like phenotype, and suggests the genetic screening of the MRM2 gene in patients with a m.3243 A > G negative MELAS-like presentation. © The Author 2017. Published by Oxford University Press.

  10. Respiromics - An integrative analysis linking mitochondrial bioenergetics to molecular signatures.

    PubMed

    Walheim, Ellen; Wiśniewski, Jacek R; Jastroch, Martin

    2018-03-01

    Energy metabolism is challenged upon nutrient stress, eventually leading to a variety of metabolic diseases that represent a major global health burden. Here, we combine quantitative mitochondrial respirometry (Seahorse technology) and proteomics (LC-MS/MS-based total protein approach) to understand how molecular changes translate to changes in mitochondrial energy transduction during diet-induced obesity (DIO) in the liver. The integrative analysis reveals that significantly increased palmitoyl-carnitine respiration is supported by an array of proteins enriching lipid metabolism pathways. Upstream of the respiratory chain, the increased capacity for ATP synthesis during DIO associates strongest to mitochondrial uptake of pyruvate, which is routed towards carboxylation. At the respiratory chain, robust increases of complex I are uncovered by cumulative analysis of single subunit concentrations. Specifically, nuclear-encoded accessory subunits, but not mitochondrial-encoded or core units, appear to be permissive for enhanced lipid oxidation. Our integrative analysis, that we dubbed "respiromics", represents an effective tool to link molecular changes to functional mechanisms in liver energy metabolism, and, more generally, can be applied for mitochondrial analysis in a variety of metabolic and mitochondrial disease models. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

  11. Mitochondrial NUDIX hydrolases: A metabolic link between NAD catabolism, GTP and mitochondrial dynamics.

    PubMed

    Long, Aaron; Klimova, Nina; Kristian, Tibor

    2017-10-01

    NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.

  12. Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

    PubMed

    Woodman, Andrew G; Mah, Richard; Keddie, Danae; Noble, Ronan M N; Panahi, Sareh; Gragasin, Ferrante S; Lemieux, Hélène; Bourque, Stephane L

    2018-06-01

    Prenatal iron deficiency alters fetal developmental trajectories, which results in persistent changes in organ function. Here, we studied the effects of prenatal iron deficiency on fetal kidney and liver mitochondrial function. Pregnant Sprague-Dawley rats were fed partially or fully iron-restricted diets to induce a state of moderate or severe iron deficiency alongside iron-replete control rats. We assessed mitochondrial function via high-resolution respirometry and reactive oxygen species generation via fluorescence microscopy on gestational d 21. Hemoglobin levels were reduced in dams in the moderate (-31%) and severe groups (-54%) compared with controls, which was accompanied by 55% reductions in fetal hemoglobin levels in both moderate and severe groups versus controls. Male iron-deficient kidneys exhibited globally reduced mitochondrial content and respiration, as well as increased cytosolic superoxide and decreased NO. Female iron-deficient kidneys exhibited complex II down-regulation and increased mitochondrial oxidative stress. Male iron-deficient livers exhibited reduced complex IV respiration and increased cytosolic superoxide, whereas female liver tissues exhibited no alteration in oxidant levels or mitochondrial function. These findings indicate that prenatal iron deficiency causes changes in mitochondrial content and function as well as oxidant status in a sex- and organ-dependent manner, which may be an important mechanism that underlies the programming of cardiovascular disease.-Woodman, A. G., Mah, R., Keddie, D., Noble, R. M. N., Panahi, S., Gragasin, F. S., Lemieux, H., Bourque, S. L. Prenatal iron deficiency causes sex-dependent mitochondrial dysfunction and oxidative stress in fetal rat kidneys and liver.

  13. Vulnerable Parkin Loss-of-Function Drosophila Dopaminergic Neurons Have Advanced Mitochondrial Aging, Mitochondrial Network Loss and Transiently Reduced Autophagosome Recruitment.

    PubMed

    Cackovic, Juliana; Gutierrez-Luke, Susana; Call, Gerald B; Juba, Amber; O'Brien, Stephanie; Jun, Charles H; Buhlman, Lori M

    2018-01-01

    Selective degeneration of substantia nigra dopaminergic (DA) neurons is a hallmark pathology of familial Parkinson's disease (PD). While the mechanism of degeneration is elusive, abnormalities in mitochondrial function and turnover are strongly implicated. An Autosomal Recessive-Juvenile Parkinsonism (AR-JP) Drosophila melanogaster model exhibits DA neurodegeneration as well as aberrant mitochondrial dynamics and function. Disruptions in mitophagy have been observed in parkin loss-of-function models, and changes in mitochondrial respiration have been reported in patient fibroblasts. Whether loss of parkin causes selective DA neurodegeneration in vivo as a result of lost or decreased mitophagy is unknown. This study employs the use of fluorescent constructs expressed in Drosophila DA neurons that are functionally homologous to those of the mammalian substantia nigra. We provide evidence that degenerating DA neurons in parkin loss-of-function mutant flies have advanced mitochondrial aging, and that mitochondrial networks are fragmented and contain swollen organelles. We also found that mitophagy initiation is decreased in park ( Drosophila parkin/PARK2 ortholog) homozygous mutants, but autophagosome formation is unaffected, and mitochondrial network volumes are decreased. As the fly ages, autophagosome recruitment becomes similar to control, while mitochondria continue to show signs of damage, and climbing deficits persist. Interestingly, aberrant mitochondrial morphology, aging and mitophagy initiation were not observed in DA neurons that do not degenerate. Our results suggest that parkin is important for mitochondrial homeostasis in vulnerable Drosophila DA neurons, and that loss of parkin-mediated mitophagy may play a role in degeneration of relevant DA neurons or motor deficits in this model.

  14. Antibiotic tigecycline enhances cisplatin activity against human hepatocellular carcinoma through inducing mitochondrial dysfunction and oxidative damage

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tan, Jun; Song, Meijun; Zhou, Mi

    Targeting mitochondrial metabolism has been recently demonstrated to be a promising therapeutic strategy for the treatment of various cancer. In this work, we demonstrate that antibiotic tigecycline is selectively against hepatocellular carcinoma (HCC) through inducing mitochondrial dysfunction and oxidative damage. Tigecycline is more effective in inhibiting proliferation and inducing apoptosis of HCC than normal liver cells. Importantly, tigecycline significantly enhances the inhibitory effects of chemotherapeutic drug cisplatin in HCC in vitro and in vivo. Mechanistically, tigecycline specifically inhibits mitochondrial translation as shown by the decreased protein levels of Cox-1 and -2 but not Cox-4 or Grp78, and increased mRNA levels of Cox-1more » and -2 but not Cox-4 in HCC cells exposed to tigecycline. In addition, tigecycline significantly induces mitochondrial dysfunction in HCC cells via decreasing mitochondrial membrane potential, complex I and IV activities, mitochondrial respiration and ATP levels. Tigecycline also increases levels of mitochondrial superoxide, hydrogen peroxide and ROS levels. Consistent with oxidative stress, oxidative damage on DNA, protein and lipid are also observed in tigecycline-treated cells. Importantly, antioxidant N-acetyl-L-cysteine (NAC) reverses the effects of tigecycline, suggesting that oxidative stress is required for the action of tigecycline in HCC cells. We further show that HCC cells have higher level of mitochondrial biogenesis than normal liver cells which might explain the different sensitivity to tigecycline between HCC and normal liver cells. Our work is the first to demonstrate that tigecycline is a promising candidate for HCC treatment and highlight the therapeutic value of targeting mitochondrial metabolism in HCC. - Highlights: • Tigecycline selectively targets HCC in vitro and in vivo. • Tigecycline enhances HCC cell response to chemotherapeutic drug. • Tigecycline inhibits mitochondrial

  15. Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

    PubMed

    Gusdon, Aaron M; Fernandez-Bueno, Gabriel A; Wohlgemuth, Stephanie; Fernandez, Jenelle; Chen, Jing; Mathews, Clayton E

    2015-09-10

    Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice. We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II-III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria. We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.

  16. [Mitochondrial and microcirculatory distress syndrome in the critical patient. Therapeutic implications].

    PubMed

    Navarrete, M L; Cerdeño, M C; Serra, M C; Conejero, R

    2013-10-01

    Mitochondrial and microcirculatory distress syndrome (MMDS) can occur during systemic inflammatory response syndrome (SIRS), and is characterized by cytopathic tissue hypoxia uncorrected by oxygen transport optimization, and associated with an acquired defect in the use of oxygen and energy production in mitochondria, leading to multiple organ dysfunction (MOD). We examine the pathogenesis of MMDS, new diagnostic methods, and recent therapeutic approaches adapted to each of the three phases in the evolution of the syndrome. In the initial phase, the aim is prevention and early reversal of mitochondrial dysfunction. Once the latter is established, the aim is to restore flow of the electron chain, mitochondrial respiration, and to avoid cellular energy collapse. Finally, in the third (resolution) stage, treatment should focus on stimulating mitochondrial biogenesis and the repair or replacement of damaged mitochondria. Copyright © 2012 Elsevier España, S.L. and SEMICYUC. All rights reserved.

  17. VDAC electronics: 4. Novel electrical mechanism and thermodynamic estimations of glucose repression of yeast respiration.

    PubMed

    Lemeshko, Victor V

    2017-11-01

    Inhibition of cell respiration by high concentrations of glucose (glucose repression), known as "Crabtree effect", has been demonstrated for various cancerous strains, highly proliferating cells and yeast lines. Although significant progress in understanding metabolic events associated with the glucose repression of cell respiration has been achieved, it is not yet clear whether the Crabtree effect is the result of a limited activity of the respiratory chain, or of some glucose-mediated regulation of mitochondrial metabolic state. In this work we propose an electrical mechanism of glucose repression of the yeast S. cerevisiae, resulting from generation of the mitochondrial outer membrane potential (OMP) coupled to the direct oxidation of cytosolic NADH in mitochondria. This yeast-type mechanism of OMP generation is different from the earlier proposed VDAC-hexokinase-mediated voltage generation of cancer-type, associated with the mitochondrial outer membrane. The model was developed assuming that VDAC is more permeable to NADH than to NAD + . Thermodynamic estimations of OMP, generated as a result of NADH(2-)/NAD + (1-) turnover through the outer membrane, demonstrated that the values of calculated negative OMP match the known range of VDAC voltage sensitivity, thus suggesting a possibility of OMP-dependent VDAC-mediated regulation of cell energy metabolism. According to the proposed mechanism, we suggest that the yeast-type Crabtree effect is the result of a fast VDAC-mediated electrical repression of mitochondria due to a decrease in the outer membrane permeability to charged metabolites and owing their redistribution between the mitochondrial intermembrane space and the cytosol, both controlled by metabolically-derived OMP. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. 4-Hydroxytamoxifen induces slight uncoupling of mitochondrial oxidative phosphorylation system in relation to the deleterious effects of tamoxifen.

    PubMed

    Cardoso, Carla M P; Moreno, António J M; Almeida, Leonor M; Custódio, José B A

    2002-10-15

    The use of tamoxifen (TAM) has been questioned on the chemotherapy and chemoprevention of breast cancer due to several estrogen receptor-independent cytotoxic effects. As an alternative, its more active metabolite 4-hydroxytamoxifen (OHTAM) has been proposed with presumed lower side effects. In this work, the potential OHTAM toxicity on rat liver mitochondrial bioenergetics in relation to the multiple deleterious effects of TAM was evaluated. OHTAM, at concentrations lower than those putatively reached in tissues following the administration of TAM, does not induce significant perturbations on the respiratory control ratio (RCR), ADP/O, transmembrane potential (DeltaPsi), phosphorylative capacity and membrane integrity of mitochondria. However, at high concentrations, OHTAM depresses the DeltaPsi, RCR and ADP/O, affecting the phosphorylation efficiency, as also inferred from the DeltaPsi fluctuations and pH changes associated with ADP phosphorylation. Moreover, OHTAM, at concentrations that stimulate the rate of state 4 respiration in parallel to the decrease in the DeltaPsi and phosphorylation rate, causes mitochondrial swelling and stimulates both ATPase and citrate synthase activities. However, the OHTAM-observed effects, at high concentrations, are not significant relatively to the damaging effects promoted by TAM and suggest alterations to mitochondrial functions due to proton leak across the mitochondrial inner membrane.

  19. Effects of exercise on obesity-induced mitochondrial dysfunction in skeletal muscle

    PubMed Central

    Heo, Jun-Won; No, Mi-Hyun; Park, Dong-Ho; Kang, Ju-Hee; Seo, Dae Yun; Han, Jin; Neufer, P. Darrell

    2017-01-01

    Obesity is known to induce inhibition of glucose uptake, reduction of lipid metabolism, and progressive loss of skeletal muscle function, which are all associated with mitochondrial dysfunction in skeletal muscle. Mitochondria are dynamic organelles that regulate cellular metabolism and bioenergetics, including ATP production via oxidative phosphorylation. Due to these critical roles of mitochondria, mitochondrial dysfunction results in various diseases such as obesity and type 2 diabetes. Obesity is associated with impairment of mitochondrial function (e.g., decrease in O2 respiration and increase in oxidative stress) in skeletal muscle. The balance between mitochondrial fusion and fission is critical to maintain mitochondrial homeostasis in skeletal muscle. Obesity impairs mitochondrial dynamics, leading to an unbalance between fusion and fission by favorably shifting fission or reducing fusion proteins. Mitophagy is the catabolic process of damaged or unnecessary mitochondria. Obesity reduces mitochondrial biogenesis in skeletal muscle and increases accumulation of dysfunctional cellular organelles, suggesting that mitophagy does not work properly in obesity. Mitochondrial dysfunction and oxidative stress are reported to trigger apoptosis, and mitochondrial apoptosis is induced by obesity in skeletal muscle. It is well known that exercise is the most effective intervention to protect against obesity. Although the cellular and molecular mechanisms by which exercise protects against obesity-induced mitochondrial dysfunction in skeletal muscle are not clearly elucidated, exercise training attenuates mitochondrial dysfunction, allows mitochondria to maintain the balance between mitochondrial dynamics and mitophagy, and reduces apoptotic signaling in obese skeletal muscle. PMID:29200899

  20. Mitochondrial Recombination and Introgression during Speciation by Hybridization.

    PubMed

    Leducq, Jean-Baptiste; Henault, Mathieu; Charron, Guillaume; Nielly-Thibault, Lou; Terrat, Yves; Fiumera, Heather L; Shapiro, B Jesse; Landry, Christian R

    2017-08-01

    Genome recombination is a major source of genotypic diversity and contributes to adaptation and speciation following interspecies hybridization. The contribution of recombination in these processes has been thought to be largely limited to the nuclear genome because organelles are mostly uniparentally inherited in animals and plants, which prevents recombination. Unicellular eukaryotes such as budding yeasts do, however, transmit mitochondria biparentally, suggesting that during hybridization, both parents could provide alleles that contribute to mitochondrial functions such as respiration and metabolism in hybrid populations or hybrid species. We examined the dynamics of mitochondrial genome transmission and evolution during speciation by hybridization in the natural budding yeast Saccharomyces paradoxus. Using population-scale mitochondrial genome sequencing in two endemic North American incipient species SpB and SpC and their hybrid species SpC*, we found that both parental species contributed to the hybrid mitochondrial genome through recombination. We support our findings by showing that mitochondrial recombination between parental types is frequent in experimental crosses that recreate the early step of this speciation event. In these artificial hybrids, we observed that mitochondrial genome recombination enhances phenotypic variation among diploid hybrids, suggesting that it could play a role in the phenotypic differentiation of hybrid species. Like the nuclear genome, the mitochondrial genome can, therefore, also play a role in hybrid speciation. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Laccase-13 Regulates Seed Setting Rate by Affecting Hydrogen Peroxide Dynamics and Mitochondrial Integrity in Rice.

    PubMed

    Yu, Yang; Li, Quan-Feng; Zhang, Jin-Ping; Zhang, Fan; Zhou, Yan-Fei; Feng, Yan-Zhao; Chen, Yue-Qin; Zhang, Yu-Chan

    2017-01-01

    Seed setting rate is one of the most important components of rice grain yield. To date, only several genes regulating setting rate have been identified in plant. In this study, we showed that laccase-13 ( OsLAC13 ), a member of laccase family genes which are known for their roles in modulating phenylpropanoid pathway and secondary lignification in cell wall, exerts a regulatory function in rice seed setting rate. OsLAC13 expressed in anthers and promotes hydrogen peroxide production both in vitro and in the filaments and anther connectives. Knock-out of OsLAC13 showed significantly increased seed setting rate, while overexpression of this gene exhibited induced mitochondrial damage and suppressed sugar transportation in anthers, which in turn affected seed setting rate. OsLAC13 also induced H 2 O 2 production and mitochondrial damage in the root tip cells which caused the lethal phenotype. We also showed that high abundant of OsmiR397, the suppressor of OsLAC13 mRNA, increased the seed setting rate of rice plants, and restrains H 2 O 2 accumulation in roots during oxidative stress. Our results suggested a novel regulatory role of OsLAC13 gene in regulating seed setting rate by affecting H 2 O 2 dynamics and mitochondrial integrity in rice.

  2. Linear programming model can explain respiration of fermentation products.

    PubMed

    Möller, Philip; Liu, Xiaochen; Schuster, Stefan; Boley, Daniel

    2018-01-01

    Many differentiated cells rely primarily on mitochondrial oxidative phosphorylation for generating energy in the form of ATP needed for cellular metabolism. In contrast most tumor cells instead rely on aerobic glycolysis leading to lactate to about the same extent as on respiration. Warburg found that cancer cells to support oxidative phosphorylation, tend to ferment glucose or other energy source into lactate even in the presence of sufficient oxygen, which is an inefficient way to generate ATP. This effect also occurs in striated muscle cells, activated lymphocytes and microglia, endothelial cells and several mammalian cell types, a phenomenon termed the "Warburg effect". The effect is paradoxical at first glance because the ATP production rate of aerobic glycolysis is much slower than that of respiration and the energy demands are better to be met by pure oxidative phosphorylation. We tackle this question by building a minimal model including three combined reactions. The new aspect in extension to earlier models is that we take into account the possible uptake and oxidation of the fermentation products. We examine the case where the cell can allocate protein on several enzymes in a varying distribution and model this by a linear programming problem in which the objective is to maximize the ATP production rate under different combinations of constraints on enzymes. Depending on the cost of reactions and limitation of the substrates, this leads to pure respiration, pure fermentation, and a mixture of respiration and fermentation. The model predicts that fermentation products are only oxidized when glucose is scarce or its uptake is severely limited.

  3. Linear programming model can explain respiration of fermentation products

    PubMed Central

    Möller, Philip; Liu, Xiaochen; Schuster, Stefan

    2018-01-01

    Many differentiated cells rely primarily on mitochondrial oxidative phosphorylation for generating energy in the form of ATP needed for cellular metabolism. In contrast most tumor cells instead rely on aerobic glycolysis leading to lactate to about the same extent as on respiration. Warburg found that cancer cells to support oxidative phosphorylation, tend to ferment glucose or other energy source into lactate even in the presence of sufficient oxygen, which is an inefficient way to generate ATP. This effect also occurs in striated muscle cells, activated lymphocytes and microglia, endothelial cells and several mammalian cell types, a phenomenon termed the “Warburg effect”. The effect is paradoxical at first glance because the ATP production rate of aerobic glycolysis is much slower than that of respiration and the energy demands are better to be met by pure oxidative phosphorylation. We tackle this question by building a minimal model including three combined reactions. The new aspect in extension to earlier models is that we take into account the possible uptake and oxidation of the fermentation products. We examine the case where the cell can allocate protein on several enzymes in a varying distribution and model this by a linear programming problem in which the objective is to maximize the ATP production rate under different combinations of constraints on enzymes. Depending on the cost of reactions and limitation of the substrates, this leads to pure respiration, pure fermentation, and a mixture of respiration and fermentation. The model predicts that fermentation products are only oxidized when glucose is scarce or its uptake is severely limited. PMID:29415045

  4. Mössbauer Spectra of Mouse Hearts Reveal Age-dependent Changes in Mitochondrial and Ferritin Iron Levels.

    PubMed

    Wofford, Joshua D; Chakrabarti, Mrinmoy; Lindahl, Paul A

    2017-03-31

    Cardiac function requires continuous high levels of energy, and so iron, a critical player in mitochondrial respiration, is an important component of the heart. Hearts from 57 Fe-enriched mice were evaluated by Mössbauer spectroscopy. Spectra consisted of a sextet and two quadrupole doublets. One doublet was due to residual blood, whereas the other was due to [Fe 4 S 4 ] 2+ clusters and low-spin Fe II hemes, most of which were associated with mitochondrial respiration. The sextet was due to ferritin; there was no evidence of hemosiderin, a ferritin decomposition product. Iron from ferritin was nearly absent in young hearts, but increased steadily with age. EPR spectra exhibited signals similar to those of brain, liver, and human cells. No age-dependent EPR trends were apparent. Hearts from HFE -/- mice with hemochromatosis contained slightly more iron overall than controls, including more ferritin and less mitochondrial iron; these differences typify slightly older hearts, perhaps reflecting the burden due to this disease. HFE -/- livers were overloaded with ferritin but had low mitochondrial iron levels. IRP2 -/- hearts contained less ferritin than controls but normal levels of mitochondrial iron. Hearts of young mice born to an iron-deficient mother contained normal levels of mitochondrial iron and no ferritin; the heart from the mother contained low ferritin and normal levels of mitochondrial iron. High-spin Fe II ions were nearly undetectable in heart samples; these were evident in brains, livers, and human cells. Previous Mössbauer spectra of unenriched diseased human hearts lacked mitochondrial and blood doublets and included hemosiderin features. This suggests degradation of iron-containing species during sample preparation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Steps Toward Understanding Mitochondrial Fe/S Cluster Biogenesis.

    PubMed

    Melber, Andrew; Winge, Dennis R

    2018-01-01

    Iron-sulfur clusters (Fe/S clusters) are essential cofactors required throughout the clades of biology for performing a myriad of unique functions including nitrogen fixation, ribosome assembly, DNA repair, mitochondrial respiration, and metabolite catabolism. Although Fe/S clusters can be synthesized in vitro and transferred to a client protein without enzymatic assistance, biology has evolved intricate mechanisms to assemble and transfer Fe/S clusters within the cellular environment. In eukaryotes, the foundation of all cellular clusters starts within the mitochondria. The focus of this review is to detail the mitochondrial Fe/S biogenesis (ISC) pathway along with the Fe/S cluster transfer steps necessary to mature Fe/S proteins. New advances in our understanding of the mitochondrial Fe/S biogenesis machinery will be highlighted. Additionally, we will address various experimental approaches that have been successful in the identification and characterization of components of the ISC pathway. © 2018 Elsevier Inc. All rights reserved.

  6. Tridecanoin is anticonvulsant, antioxidant, and improves mitochondrial function

    PubMed Central

    Tan, Kah Ni; Carrasco-Pozo, Catalina; McDonald, Tanya S; Puchowicz, Michelle

    2016-01-01

    The hypothesis that chronic feeding of the triglycerides of octanoate (trioctanoin) and decanoate (tridecanoin) in “a regular non-ketogenic diet” is anticonvulsant was tested and possible mechanisms of actions were subsequently investigated. Chronic feeding of 35E% of calories from tridecanoin, but not trioctanoin, was reproducibly anticonvulsant in two acute CD1 mouse seizure models. The levels of beta-hydroxybutyrate in plasma and brain were not significantly increased by either treatment relative to control diet. The respective decanoate and octanoate levels are 76 µM and 33 µM in plasma and 1.17 and 2.88 nmol/g in brain. Tridecanoin treatment did not alter the maximal activities of several glycolytic enzymes, suggesting that there is no reduction in glycolysis contributing to anticonvulsant effects. In cultured astrocytes, 200 µM of octanoic and decanoic acids increased basal respiration and ATP turnover, suggesting that both medium chain fatty acids are used as fuel. Only decanoic acid increased mitochondrial proton leak which may reduce oxidative stress. In mitochondria isolated from hippocampal formations, tridecanoin increased respiration linked to ATP synthesis, indicating that mitochondrial metabolic functions are improved. In addition, tridecanoin increased the plasma antioxidant capacity and hippocampal mRNA levels of heme oxygenase 1, and FoxO1. PMID:27418037

  7. Fungal mitochondrial oxygen consumption induces the growth of strict anaerobic bacteria.

    PubMed

    Lambooij, Joost M; Hoogenkamp, Michel A; Brandt, Bernd W; Janus, Marleen M; Krom, Bastiaan P

    2017-12-01

    Fungi are commonly encountered as part of a healthy oral ecosystem. Candida albicans is the most often observed and investigated fungal species in the oral cavity. The role of fungi in the oral ecosystem has remained enigmatic for decades. Recently, it was shown that C. albicans, in vitro, influences the bacterial composition of young oral biofilms, indicating it possibly plays a role in increasing diversity in the oral ecosystem. C. albicans favored growth of strictly anaerobic species under aerobic culture conditions. In the present study, the role of mitochondrial respiration, as mechanism by which C. albicans modifies its environment, was investigated. Using oxygen sensors, a rapid depletion of dissolved oxygen (dO 2 ) was observed. This decrease was not C. albicans specific as several non-albicans Candida species showed similar oxygen consumption. Heat inactivation as well as addition of the specific mitochondrial respiration inhibitor Antimycin A inhibited depletion of dO 2 . Using 16S rDNA sequencing, it is shown that mitochondrial activity, more than physical presence of C. albicans is responsible for inducing growth of strictly anaerobic oral bacteria in aerobic growth conditions. The described mechanism of dO 2 depletion may be a general mechanism by which fungi modulate their direct environment. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Rewiring carbohydrate catabolism differentially affects survival of pancreatic cancer cell lines with diverse metabolic profiles

    PubMed Central

    Tataranni, Tiziana; Agriesti, Francesca; Ruggieri, Vitalba; Mazzoccoli, Carmela; Simeon, Vittorio; Laurenzana, Ilaria; Scrima, Rosella; Pazienza, Valerio; Capitanio, Nazzareno; Piccoli, Claudia

    2017-01-01

    An increasing body of evidence suggests that targeting cellular metabolism represents a promising effective approach to treat pancreatic cancer, overcome chemoresistance and ameliorate patient's prognosis and survival. In this study, following whole-genome expression analysis, we selected two pancreatic cancer cell lines, PANC-1 and BXPC-3, hallmarked by distinct metabolic profiles with specific concern to carbohydrate metabolism. Functional comparative analysis showed that BXPC-3 displayed a marked deficit of the mitochondrial respiratory and oxidative phosphorylation activity and a higher production of reactive oxygen species and a reduced NAD+/NADH ratio, indicating their bioenergetic reliance on glycolysis and a different redox homeostasis as compared to PANC-1. Both cell lines were challenged to rewire their metabolism by substituting glucose with galactose as carbon source, a condition inhibiting the glycolytic flux and fostering full oxidation of the sugar carbons. The obtained data strikingly show that the mitochondrial respiration-impaired-BXPC-3 cell line was unable to sustain the metabolic adaptation required by glucose deprivation/substitution, thereby resulting in a G2\\M cell cycle shift, unbalance of the redox homeostasis, apoptosis induction. Conversely, the mitochondrial respiration-competent-PANC-1 cell line did not show clear evidence of cell sufferance. Our findings provide a strong rationale to candidate metabolism as a promising target for cancer therapy. Defining the metabolic features at time of pancreatic cancer diagnosis and likely of other tumors, appears to be crucial to predict the responsiveness to therapeutic approaches or coadjuvant interventions affecting metabolism. PMID:28476035

  9. Mitochondrial gene polymorphisms alter hepatic cellular energy metabolism and aggravate diet-induced non-alcoholic steatohepatitis.

    PubMed

    Schröder, Torsten; Kucharczyk, David; Bär, Florian; Pagel, René; Derer, Stefanie; Jendrek, Sebastian Torben; Sünderhauf, Annika; Brethack, Ann-Kathrin; Hirose, Misa; Möller, Steffen; Künstner, Axel; Bischof, Julia; Weyers, Imke; Heeren, Jörg; Koczan, Dirk; Schmid, Sebastian Michael; Divanovic, Senad; Giles, Daniel Aaron; Adamski, Jerzy; Fellermann, Klaus; Lehnert, Hendrik; Köhl, Jörg; Ibrahim, Saleh; Sina, Christian

    2016-04-01

    Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with an enhanced risk for liver and cardiovascular diseases and mortality. NAFLD can progress from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). However, the mechanisms predisposing to this progression remain undefined. Notably, hepatic mitochondrial dysfunction is a common finding in patients with NASH. Due to a lack of appropriate experimental animal models, it has not been evaluated whether this mitochondrial dysfunction plays a causative role for the development of NASH. To determine the effect of a well-defined mitochondrial dysfunction on liver physiology at baseline and during dietary challenge, C57BL/6J-mt(FVB/N) mice were employed. This conplastic inbred strain has been previously reported to exhibit decreased mitochondrial respiration likely linked to a non-synonymous gene variation (nt7778 G/T) of the mitochondrial ATP synthase protein 8 (mt-ATP8). At baseline conditions, C57BL/6J-mt(FVB/N) mice displayed hepatic mitochondrial dysfunction characterized by decreased ATP production and increased formation of reactive oxygen species (ROS). Moreover, genes affecting lipid metabolism were differentially expressed, hepatic triglyceride and cholesterol levels were changed in these animals, and various acyl-carnitines were altered, pointing towards an impaired mitochondrial carnitine shuttle. However, over a period of twelve months, no spontaneous hepatic steatosis or inflammation was observed. On the other hand, upon dietary challenge with either a methionine and choline deficient diet or a western-style diet, C57BL/6J-mt(FVB/N) mice developed aggravated steatohepatitis as characterized by lipid accumulation, ballooning of hepatocytes and infiltration of immune cells. We observed distinct metabolic alterations in mice with a mitochondrial polymorphism associated hepatic mitochondrial dysfunction. However, a second hit, such as dietary stress

  10. Mitochondrial gene polymorphisms alter hepatic cellular energy metabolism and aggravate diet-induced non-alcoholic steatohepatitis

    PubMed Central

    Schröder, Torsten; Kucharczyk, David; Bär, Florian; Pagel, René; Derer, Stefanie; Jendrek, Sebastian Torben; Sünderhauf, Annika; Brethack, Ann-Kathrin; Hirose, Misa; Möller, Steffen; Künstner, Axel; Bischof, Julia; Weyers, Imke; Heeren, Jörg; Koczan, Dirk; Schmid, Sebastian Michael; Divanovic, Senad; Giles, Daniel Aaron; Adamski, Jerzy; Fellermann, Klaus; Lehnert, Hendrik; Köhl, Jörg; Ibrahim, Saleh; Sina, Christian

    2016-01-01

    Objective Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is associated with an enhanced risk for liver and cardiovascular diseases and mortality. NAFLD can progress from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). However, the mechanisms predisposing to this progression remain undefined. Notably, hepatic mitochondrial dysfunction is a common finding in patients with NASH. Due to a lack of appropriate experimental animal models, it has not been evaluated whether this mitochondrial dysfunction plays a causative role for the development of NASH. Methods To determine the effect of a well-defined mitochondrial dysfunction on liver physiology at baseline and during dietary challenge, C57BL/6J-mtFVB/N mice were employed. This conplastic inbred strain has been previously reported to exhibit decreased mitochondrial respiration likely linked to a non-synonymous gene variation (nt7778 G/T) of the mitochondrial ATP synthase protein 8 (mt-ATP8). Results At baseline conditions, C57BL/6J-mtFVB/N mice displayed hepatic mitochondrial dysfunction characterized by decreased ATP production and increased formation of reactive oxygen species (ROS). Moreover, genes affecting lipid metabolism were differentially expressed, hepatic triglyceride and cholesterol levels were changed in these animals, and various acyl-carnitines were altered, pointing towards an impaired mitochondrial carnitine shuttle. However, over a period of twelve months, no spontaneous hepatic steatosis or inflammation was observed. On the other hand, upon dietary challenge with either a methionine and choline deficient diet or a western-style diet, C57BL/6J-mtFVB/N mice developed aggravated steatohepatitis as characterized by lipid accumulation, ballooning of hepatocytes and infiltration of immune cells. Conclusions We observed distinct metabolic alterations in mice with a mitochondrial polymorphism associated hepatic mitochondrial dysfunction. However, a

  11. Cycling efficiency in humans is related to low UCP3 content and to type I fibres but not to mitochondrial efficiency.

    PubMed

    Mogensen, M; Bagger, M; Pedersen, P K; Fernström, M; Sahlin, K

    2006-03-15

    The purpose of this study was to investigate the hypothesis that cycling efficiency in vivo is related to mitochondrial efficiency measured in vitro and to investigate the effect of training status on these parameters. Nine endurance trained and nine untrained male subjects (V(O2peak) = 60.4 +/- 1.4 and 37.0 +/- 2.0 ml kg(-1) min(-1), respectively) completed an incremental submaximal efficiency test for determination of cycling efficiency (gross efficiency, work efficiency (WE) and delta efficiency). Muscle biopsies were taken from m. vastus lateralis and analysed for mitochondrial respiration, mitochondrial efficiency (MEff; i.e. P/O ratio), UCP3 protein content and fibre type composition (% MHC I). MEff was determined in isolated mitochondria during maximal (state 3) and submaximal (constant rate of ADP infusion) rates of respiration with pyruvate. The rates of mitochondrial respiration and oxidative phosphorylation per muscle mass were about 40% higher in trained subjects but were not different when expressed per unit citrate synthase (CS) activity (a marker of mitochondrial density). Training status had no influence on WE (trained 28.0 +/- 0.5, untrained 27.7 +/- 0.8%, N.S.). Muscle UCP3 was 52% higher in untrained subjects, when expressed per muscle mass (P < 0.05 versus trained). WE was inversely correlated to UCP3 (r = -0.57, P < 0.05) and positively correlated to percentage MHC I (r = 0.58, P < 0.05). MEff was lower (P < 0.05) at submaximal respiration rates (2.39 +/- 0.01 at 50% V(O2max)) than at state 3 (2.48 +/- 0.01) but was neither influenced by training status nor correlated to cycling efficiency. In conclusion cycling efficiency was not influenced by training status and not correlated to MEff, but was related to type I fibres and inversely related to UCP3. The inverse correlation between WE and UCP3 indicates that extrinsic factors may influence UCP3 activity and thus MEff in vivo.

  12. Mitochondrial and Morphologic Alterations in Native Human Corneal Endothelial Cells Associated With Diabetes Mellitus.

    PubMed

    Aldrich, Benjamin T; Schlötzer-Schrehardt, Ursula; Skeie, Jessica M; Burckart, Kimberlee A; Schmidt, Gregory A; Reed, Cynthia R; Zimmerman, M Bridget; Kruse, Friedrich E; Greiner, Mark A

    2017-04-01

    To characterize changes in the energy-producing metabolic activity and morphologic ultrastructure of corneal endothelial cells associated with diabetes mellitus. Transplant suitable corneoscleral tissue was obtained from donors aged 50 to 75 years. We assayed 3-mm punches of endothelium-Descemet membrane for mitochondrial respiration and glycolysis activity using extracellular flux analysis of oxygen and pH, respectively. Transmission electron microscopy was used to assess qualitative and quantitative ultrastructural changes in corneal endothelial cells and associated Descemet membrane. For purposes of analysis, samples were divided into four groups based on a medical history of diabetes regardless of type: (1) nondiabetic, (2) noninsulin-dependent diabetic, (3) insulin-dependent diabetic, and (4) insulin-dependent diabetic with specified complications due to diabetes (advanced diabetic). In total, 229 corneas from 159 donors were analyzed. Insulin-dependent diabetic samples with complications due to diabetes displayed the lowest spare respiratory values compared to all other groups (P ≤ 0.002). The remaining mitochondrial respiration and glycolysis metrics did not differ significantly among groups. Compared to nondiabetic controls, the endothelium from advanced diabetic samples had alterations in mitochondrial morphology, pronounced Golgi bodies associated with abundant vesicles, accumulation of lysosomal bodies/autophagosomes, and focal production of abnormal long-spacing collagen. Extracellular flux analysis suggests that corneal endothelial cells of donors with advanced diabetes have impaired mitochondrial function. Metabolic findings are supported by observed differences in mitochondrial morphology of advanced diabetic samples but not controls. Additional studies are needed to determine the precise mechanism(s) by which mitochondria become impaired in diabetic corneal endothelial cells.

  13. Hypothalamic mitochondrial abnormalities occur downstream of inflammation in diet-induced obesity.

    PubMed

    Carraro, Rodrigo S; Souza, Gabriela F; Solon, Carina; Razolli, Daniela S; Chausse, Bruno; Barbizan, Roberta; Victorio, Sheila C; Velloso, Licio A

    2018-01-15

    Hypothalamic dysfunction is a common feature of experimental obesity. Studies have identified at least three mechanisms involved in the development of hypothalamic neuronal defects in diet-induced obesity: i, inflammation; ii, endoplasmic reticulum stress; and iii, mitochondrial abnormalities. However, which of these mechanisms is activated earliest in response to the consumption of large portions of dietary fats is currently unknown. Here, we used immunoblot, real-time PCR, mitochondrial respiration assays and transmission electron microscopy to evaluate markers of inflammation, endoplasmic reticulum stress and mitochondrial abnormalities in the hypothalamus of Swiss mice fed a high-fat diet for up to seven days. In the present study we show that the expression of the inflammatory chemokine fractalkine was the earliest event detected. Its hypothalamic expression increased as early as 3 h after the introduction of a high-fat diet and was followed by the increase of cytokines. GPR78, an endoplasmic reticulum chaperone, was increased 6 h after the introduction of a high-fat diet, however the actual triggering of endoplasmic reticulum stress was only detected three days later, when IRE-1α was increased. Mitofusin-2, a protein involved in mitochondrial fusion and tethering of mitochondria to the endoplasmic reticulum, underwent a transient reduction 24 h after the introduction of a high-fat diet and then increased after seven days. There were no changes in hypothalamic mitochondrial respiration during the experimental period, however there were reductions in mitochondria/endoplasmic reticulum contact sites, beginning three days after the introduction of a high-fat diet. The inhibition of TNF-α with infliximab resulted in the normalization of mitofusin-2 levels 24 h after the introduction of the diet. Thus, inflammation is the earliest mechanism activated in the hypothalamus after the introduction of a high-fat diet and may play a mechanistic role in the

  14. Role of organic cation/carnitine transporter 1 in uptake of phenformin and inhibitory effect on complex I respiration in mitochondria.

    PubMed

    Shitara, Yoshihisa; Nakamichi, Noritaka; Norioka, Misaki; Shima, Hiroyo; Kato, Yukio; Horie, Toshiharu

    2013-03-01

    Phenformin causes lactic acidosis in clinical situations due to inhibition of mitochondrial respiratory chain complex I. It is reportedly taken up by hepatocytes and exhibits mitochondrial toxicity in the liver. In this study, uptake of phenformin and [(14)C]tetraethylammonium (TEA) and complex I inhibition by phenformin were examined in isolated liver and heart mitochondria. Uptake of phenformin into isolated rat liver mitochondria was higher than that into heart mitochondria. It was inhibited by several cat ionic compounds, which suggests the involvement of multispecific transport system(s). Similar characteristics were also observed for uptake of TEA; however, uptake of phenformin into mitochondria of organic cation/carnitine transporter 1 (OCTN1) knockout mice was lower than that in wild-type mice, whereas uptake of TEA was comparable between the two strains, suggesting the involvement of distinct transport mechanisms for these two cations in mitochondria. Inhibition by phenformin of oxygen consumption via complex I respiration in isolated rat liver mitochondria was greater than that in heart mitochondria, whereas inhibitory effect of phenformin on complex I respiration was similar in inside-out structured submitochondrial particles prepared from rat livers and hearts. Lactic acidosis provoked by iv infusion of phenformin was weaker in octn1(-/-) mice than that in wild-type mice. These observations suggest that uptake of phenformin into liver mitochondria is at least partly mediated by OCTN1 and functionally relevant to its inhibition potential of complex I respiration. This study was, thus, the first to demonstrate OCTN1-mediated mitochondrial transport and toxicity of biguanide in vivo in rodents.

  15. Mitochondrial functions mediate cellulase gene expression in Trichoderma reesei.

    PubMed

    Abrahão-Neto, J; Rossini, C H; el-Gogary, S; Henrique-Silva, F; Crivellaro, O; el-Dorry, H

    1995-08-22

    We examined the effects of inhibition of mitochondrial functions on the expression of two nuclear genes encoding the extracellular cellobiohydrolase I (cbh1) and endoglucanase I (egl1) of the cellulase system of the filamentous fungus Trichoderma reesei. The cbh1 and egl1 transcripts are repressed at a low oxygen tension, and by glucose at a concentration known to repress mitochondrial respiration. The transcripts are also down-regulated by chemical agents known to dissipate the proton electrochemical gradient of the inner mitochondrial membrane and blocking of the electron-transport chain, such as DNP and KCN, respectively. These results suggest that expression of those transcripts is influenced by the physiological state of the mitochondria. In addition, heterologous gene fusion shows that the sensitivity of the expression of those transcripts to the functional state of the mitochondria is transcriptionally controlled through the 5'-flanking DNA sequence of those genes.

  16. Crystal structure of mitochondrial respiratory membrane protein complex II.

    PubMed

    Sun, Fei; Huo, Xia; Zhai, Yujia; Wang, Aojin; Xu, Jianxing; Su, Dan; Bartlam, Mark; Rao, Zihe

    2005-07-01

    The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.

  17. Soil respiration patterns and rates at three Taiwanese forest plantations: dependence on elevation, temperature, precipitation, and litterfall.

    PubMed

    Huang, Yu-Hsuan; Hung, Chih-Yu; Lin, I-Rhy; Kume, Tomonori; Menyailo, Oleg V; Cheng, Chih-Hsin

    2017-11-15

    Soil respiration contributes to a large quantity of carbon emissions in the forest ecosystem. In this study, the soil respiration rates at three Taiwanese forest plantations (two lowland and one mid-elevation) were investigated. We aimed to determine how soil respiration varies between lowland and mid-elevation forest plantations and identify the relative importance of biotic and abiotic factors affecting soil respiration. The results showed that the temporal patterns of soil respiration rates were mainly influenced by soil temperature and soil water content, and a combined soil temperature and soil water content model explained 54-80% of the variation. However, these two factors affected soil respiration differently. Soil temperature positively contributed to soil respiration, but a bidirectional relationship between soil respiration and soil water content was revealed. Higher soil moisture content resulted in higher soil respiration rates at the lowland plantations but led to adverse effects at the mid-elevation plantation. The annual soil respiration rates were estimated as 14.3-20.0 Mg C ha -1  year -1 at the lowland plantations and 7.0-12.2 Mg C ha -1  year -1 at the mid-elevation plantation. When assembled with the findings of previous studies, the annual soil respiration rates increased with the mean annual temperature and litterfall but decreased with elevation and the mean annual precipitation. A conceptual model of the biotic and abiotic factors affecting the spatial and temporal patterns of the soil respiration rate was developed. Three determinant factors were proposed: (i) elevation, (ii) stand characteristics, and (iii) soil temperature and soil moisture. The results indicated that changes in temperature and precipitation significantly affect soil respiration. Because of the high variability of soil respiration, more studies and data syntheses are required to accurately predict soil respiration in Taiwanese forests.

  18. Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

    PubMed

    Barel, Ortal; Malicdan, May Christine V; Ben-Zeev, Bruria; Kandel, Judith; Pri-Chen, Hadass; Stephen, Joshi; Castro, Inês G; Metz, Jeremy; Atawa, Osama; Moshkovitz, Sharon; Ganelin, Esther; Barshack, Iris; Polak-Charcon, Sylvie; Nass, Dvora; Marek-Yagel, Dina; Amariglio, Ninette; Shalva, Nechama; Vilboux, Thierry; Ferreira, Carlos; Pode-Shakked, Ben; Heimer, Gali; Hoffmann, Chen; Yardeni, Tal; Nissenkorn, Andreea; Avivi, Camila; Eyal, Eran; Kol, Nitzan; Glick Saar, Efrat; Wallace, Douglas C; Gahl, William A; Rechavi, Gideon; Schrader, Michael; Eckmann, David M; Anikster, Yair

    2017-03-01

    Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder. © Published by Oxford University Press on behalf of the Guarantors of Brain 2017. This work is written by US Government employees and is in the public domain in the US.

  19. Supply and demand: How does variation in atmospheric oxygen during development affect insect tracheal and mitochondrial networks?

    PubMed

    VandenBrooks, John M; Gstrein, Gregory; Harmon, Jason; Friedman, Jessica; Olsen, Matthew; Ward, Anna; Parker, Gregory

    2018-04-01

    Atmospheric oxygen is one of the most important atmospheric component for all terrestrial organisms. Variation in atmospheric oxygen has wide ranging effects on animal physiology, development, and evolution. This variation in oxygen has the potential to affect both respiratory systems (the supply side) and mitochondrial networks (the demand side) in animals. Insect respiratory systems supplying oxygen to tissues in the gas phase through blind ended tracheal systems are particularly susceptible to this variation. While the large conducting tracheae have previously been shown to respond developmentally to changes in rearing oxygen, the effect of oxygen on the tracheolar network has been relatively unexplored, especially in adult insects. Similarly, mitochondrial networks that meet energy demand in insects and other animals are dynamic and their enzyme activities have been shown to vary in the presence of oxygen. These two systems together should be under selective pressure to meet the aerobic metabolic requirements of insects. To test this hypothesis, we reared Mito-YFP Drosophila under three different oxygen concentrations hypoxia (12%), normoxia (21%), and hyperoxia (31%) and imaged their tracheolar and mitochondrial networks within their flight muscle using confocal microscopy. In terms of oxygen supply, hypoxia increased mean (mid-length) tracheolar diameters, tracheolar tip diameters, the number of tracheoles per main branch and affected tracheal branching patterns, while the opposite was observed in hyperoxia. In terms of oxygen demand, hypoxia increased mitochondrial investment and mitochondrial to tracheolar volume ratios; while the opposite was observed in hyperoxia. Generally, hypoxia had a stronger effect on both systems than hyperoxia. These results show that insects are capable of developmentally changing investment in both their supply and demand networks to increase overall fitness. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. [Parameters of fibers cell respiration and desmin content in rat soleus muscle at early stages of gravitational unloading].

    PubMed

    Mirzoev, T M; Biriukov, N S; Veselova, O M; Larina, I M; Shenkman, B S; Ogneva, I V

    2012-01-01

    The aim of the work was to study the parameters of fibers cell respiration and desmin content in Wistar rat soleus muscle after 1, 3, 7 and 14 days of gravitational unloading. Gravitational unloading was simulated by antiorthostatic hindlimb suspension. The parameters of cell respiration were determined using the polarography, and desmin content was assessed by means of Western blotting. The results showed that the intensity of cell respiration is reduced after three days of gravitational unloading, reaches a minimum level after seven days and slightly increases by the fourteenth day of hindlimb unloading, as well as the content of desmin, which, however, to the fourteenth day returns to the control level. Taking into account that mitochondrial function depends on the state of cytoskeleton the data allow us to assume that early reduction of the intensity of cell respiration under unloading could be caused by degradation of the protein desmin that determines intracellular localization of mitochondria.

  1. Assessment of Mitochondrial Dysfunction Arising from Treatment with Hepatotoxicants

    PubMed Central

    King, Adrienne L.; Bailey, Shannon M.

    2010-01-01

    Studies demonstrate that mitochondrial dysfunction is a key causative factor in liver disease. Indeed, defects in mitochondrial energy metabolism, disrupted calcium handling, and increased reactive oxygen/nitrogen species production are observed in many metabolic disorders and diseases induced by toxicants. Mitochondria have emerged as a main research focus through work defining new functions of this key organelle in normal cellular physiology and pathophysiology. Specifically, studies show a critical role of mitochondrial reactive oxygen/nitrogen species production in regulating cellular signaling pathways involved in cell survival and death. Given this, along with advances made in proteomics technologies, mitochondria are recognized as top candidates for proteomics analysis. However, assessment of mitochondrial function and it’s proteome following toxicant exposure are not trivial undertakings. In this chapter a technique used to isolate mitochondria from liver tissue is presented along with methods needed to assess mitochondria functionality. The methods described include measurement of mitochondrial respiration, calcium accumulation, and reactive oxygen species production. A presentation of proteomics approaches is also included to allow researchers the basic tools needed to identify alterations in the mitochondrial proteome that contribute to toxicant-mediated diseases. Specifically, methods are presented that demonstrate how thiol labeling reagents in combination with electrophoresis and western blotting can be used to detect oxidant-mediated alterations in mitochondrial protein thiols. A few select pieces data are presented highlighting the power of proteomics to identify mitochondrial targets that contribute to mitochondrial dysfunction and hepatotoxicity in response to specific toxicant exposures and metabolic stressors such as alcohol and environmental tobacco smoke. PMID:23045017

  2. Autophagy Deficiency Compromises Alternative Pathways of Respiration following Energy Deprivation in Arabidopsis thaliana.

    PubMed

    Barros, Jessica A S; Cavalcanti, João Henrique F; Medeiros, David B; Nunes-Nesi, Adriano; Avin-Wittenberg, Tamar; Fernie, Alisdair R; Araújo, Wagner L

    2017-09-01

    Under heterotrophic conditions, carbohydrate oxidation inside the mitochondrion is the primary energy source for cellular metabolism. However, during energy-limited conditions, alternative substrates are required to support respiration. Amino acid oxidation in plant cells plays a key role in this by generating electrons that can be transferred to the mitochondrial electron transport chain via the electron transfer flavoprotein/ubiquinone oxidoreductase system. Autophagy, a catabolic mechanism for macromolecule and protein recycling, allows the maintenance of amino acid pools and nutrient remobilization. Although the association between autophagy and alternative respiratory substrates has been suggested, the extent to which autophagy and primary metabolism interact to support plant respiration remains unclear. To investigate the metabolic importance of autophagy during development and under extended darkness, Arabidopsis ( Arabidopsis thaliana ) mutants with disruption of autophagy ( atg mutants) were used. Under normal growth conditions, atg mutants showed lower growth and seed production with no impact on photosynthesis. Following extended darkness, atg mutants were characterized by signatures of early senescence, including decreased chlorophyll content and maximum photochemical efficiency of photosystem II coupled with increases in dark respiration. Transcript levels of genes involved in alternative pathways of respiration and amino acid catabolism were up-regulated in atg mutants. The metabolite profiles of dark-treated leaves revealed an extensive metabolic reprogramming in which increases in amino acid levels were partially compromised in atg mutants. Although an enhanced respiration in atg mutants was observed during extended darkness, autophagy deficiency compromises protein degradation and the generation of amino acids used as alternative substrates to the respiration. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. Exosomes derived from B16F0 melanoma cells alter the transcriptome of cytotoxic T cells that impacts mitochondrial respiration.

    PubMed

    Bland, Cassidy L; Byrne-Hoffman, Christina N; Fernandez, Audry; Rellick, Stephanie L; Deng, Wentao; Klinke, David J

    2018-03-01

    While recent clinical studies demonstrate the promise of cancer immunotherapy, a barrier for broadening the clinical benefit is identifying how tumors locally suppress cytotoxic immunity. As an emerging mode of intercellular communication, exosomes secreted by malignant cells can deliver a complex payload of coding and noncoding RNA to cells within the tumor microenvironment. Here, we quantified the RNA payload within tumor-derived exosomes and the resulting dynamic transcriptomic response to cytotoxic T cells upon exosome delivery to better understand how tumor-derived exosomes can alter immune cell function. Exosomes derived from B16F0 melanoma cells were enriched for a subset of coding and noncoding RNAs that did not reflect the abundance in the parental cell. Upon exosome delivery, RNAseq revealed the dynamic changes in the transcriptome of CTLL2 cytotoxic T cells. In analyzing transiently coexpressed gene clusters, pathway enrichment suggested that the B16F0 exosomal payload altered mitochondrial respiration, which was confirmed independently, and upregulated genes associated with the Notch signaling pathway. Interestingly, exosomal miRNA appeared to have no systematic effect on downregulating target mRNA levels. Gene expression data are available in the GEO database under the accession SuperSeries number GSE102951. © 2018 Federation of European Biochemical Societies.

  4. Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

    PubMed

    Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

    2016-04-01

    Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  5. The control of iron-induced oxidative damage in isolated rat-liver mitochondria by respiration state and ascorbate.

    PubMed

    Burkitt, M J; Gilbert, B C

    1989-01-01

    The reaction of iron (II) with H2O2 is believed to generate highly reactive species (e.g. .OH) capable of initiating biological damage. This study investigates the possibility that the severity of oxidative damage induced by iron in hepatic mitochondria is determined by the level of mitochondrial-H2O2 generation, which is believed to be particularly prominent in state-4 respiration. Iron-induced damage is found to be greater in state-4 than in state-3 respiration. Experiments using uncoupling agents and Ca++ to mimic state-3 conditions indicate that this effect reflects differences in the steady-state oxidation-level of the electron carriers of the respiratory chain (and hence the level of H2O2-generation), rather than changes in redox potential or transportation of the metal-ion. Evidence is also presented for a mechanism in which Fe(II) and H2O2 react inside the mitochondrial matrix. Ascorbate (vitamin C) is shown to be pro-oxidant in this system, except when present at very high concentration when it becomes antioxidant in nature.

  6. miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor.

    PubMed

    Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

    2014-11-28

    Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3'-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF.

  7. Measuring mitochondrial uncoupling protein-2 level and activity in insulinoma cells.

    PubMed

    Barlow, Jonathan; Hirschberg, Verena; Brand, Martin D; Affourtit, Charles

    2013-01-01

    Mitochondrial uncoupling protein-2 (UCP2) regulates glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells-the physiological role of the beta cell UCP2 remains a subject of debate. Experimental studies informing this debate benefit from reliable measurements of UCP2 protein level and activity. In this chapter, we describe how UCP2 protein can be detected in INS-1 insulinoma cells and how it can be knocked down by RNA interference. We demonstrate briefly that UCP2 knockdown lowers glucose-induced rises in mitochondrial respiratory activity, coupling efficiency of oxidative phosphorylation, levels of mitochondrial reactive oxygen species, and insulin secretion. We provide protocols for the detection of the respective UCP2 phenotypes, which are indirect, but invaluable measures of UCP2 activity. We also introduce a convenient method to normalize cellular respiration to cell density allowing measurement of UCP2 effects on specific mitochondrial oxygen consumption. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Skeletal Muscle-Specific Overexpression of PGC-1α Induces Fiber-Type Conversion through Enhanced Mitochondrial Respiration and Fatty Acid Oxidation in Mice and Pigs.

    PubMed

    Zhang, Lin; Zhou, Ying; Wu, Wangjun; Hou, Liming; Chen, Hongxing; Zuo, Bo; Xiong, Yuanzhu; Yang, Jinzeng

    2017-01-01

    Individual skeletal muscles in the animal body are heterogeneous, as each is comprised of different fiber types. Type I muscle fibers are rich with mitochondria, and have high oxidative metabolisms while type IIB fibers have few mitochondria and high glycolytic metabolic capacity. Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), a transcriptional co-activator that regulates mitochondrial biogenesis and respiratory function, is implicated in muscle fiber-type switching. Over-expression of PGC-1α in transgenic mice increased the proportion of red/oxidative type I fiber. During pig muscle growth, an increased number of type I fibers can give meat more red color. To explore the roles of PGC-1α in regulation of muscle fiber type conversion, we generated skeletal muscle-specific PGC-1α transgenic mice and pig. Ectopic over-expression of PGC-1α was detected in both fast and slow muscle fibers. The transgenic animals displayed a remarkable amount of red/oxidative muscle fibers in major skeletal muscle tissues. Skeletal muscles from transgenic mice and pigs have increased expression levels of oxidative fiber markers such as MHC1, MHC2x, myoglobin and Tnni1, and decreased expressions of glycolytic fiber genes (MHC2a, MHC2b, CASQ-1 and Tnni2). The genes responsible for the TCA cycle and oxidative phosphorylation, cytochrome coxidase 2 and 4, and citrate synthase were also increased in the transgenic mice and pigs. These results suggested that transgenic over-expressed PGC-1α significantly increased muscle mitochondrial biogenesis, resulting in qualitative changes from glycolytic to oxidative energy generation. The transgenic animals also had elevated levels of PDK4 and PPARγ proteins in muscle tissue, which can lead to increased glycogen deposition and fatty acid oxidation. Therefore, the results support a significant role of PGC-1α in conversion of fast glycolytic fibers to slow and oxidative fiber through enhanced mitochondrial respiration

  9. The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore.

    PubMed

    Zago, E B; Castilho, R F; Vercesi, A E

    2000-07-28

    Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca(2+) efflux without significant release of state-4 respiration, disruption of membrane potential (Deltapsi) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca(2+) efflux associated with a release of state-4 respiration, a disruption of Deltapsi and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.

  10. The mitochondrial gene orfH79 plays a critical role in impairing both male gametophyte development and root growth in CMS-Honglian rice.

    PubMed

    Peng, Xiaojue; Wang, Kun; Hu, Chaofeng; Zhu, Youlin; Wang, Ting; Yang, Jing; Tong, Jiping; Li, Shaoqing; Zhu, Yingguo

    2010-06-24

    Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames. The mitochondrial gene orfH79 is a candidate gene for causing the CMS trait in CMS-Honglian (CMS-HL) rice. However, whether the orfH79 expression can actually induce CMS in rice remains unclear. Western blot analysis revealed that the ORFH79 protein is mainly present in mitochondria of CMS-HL rice and is absent in the fertile line. To investigate the function of ORFH79 protein in mitochondria, this gene was fused to a mitochondrial transit peptide sequence and used to transform wild type rice, where its expression induced the gametophytic male sterile phenotype. In addition, excessive accumulation of reactive oxygen species (ROS) in the microspore, a reduced ATP/ADP ratio, decreased mitochondrial membrane potential and a lower respiration rate in the transgenic plants were found to be similar to those in CMS-HL rice. Moreover, retarded growth of primary and lateral roots accompanied by abnormal accumulation of ROS in the root tip was observed in both transgenic rice and CMS-HL rice (YTA). These results suggest that the expression of orfH79 in mitochondria impairs mitochondrial function, which affects the development of both male gametophytes and the roots of CMS-HL rice.

  11. Decreased Mitochondrial Pyruvate Transport Activity in the Diabetic Heart

    PubMed Central

    Vadvalkar, Shraddha S.; Matsuzaki, Satoshi; Eyster, Craig A.; Giorgione, Jennifer R.; Bockus, Lee B.; Kinter, Caroline S.; Kinter, Michael

    2017-01-01

    Alterations in mitochondrial function contribute to diabetic cardiomyopathy. We have previously shown that heart mitochondrial proteins are hyperacetylated in OVE26 mice, a transgenic model of type 1 diabetes. However, the universality of this modification and its functional consequences are not well established. In this study, we demonstrate that Akita type 1 diabetic mice exhibit hyperacetylation. Functionally, isolated Akita heart mitochondria have significantly impaired maximal (state 3) respiration with physiological pyruvate (0.1 mm) but not with 1.0 mm pyruvate. In contrast, pyruvate dehydrogenase activity is significantly decreased regardless of the pyruvate concentration. We found that there is a 70% decrease in the rate of pyruvate transport in Akita heart mitochondria but no decrease in the mitochondrial pyruvate carriers 1 and 2 (MPC1 and MPC2). The potential role of hyperacetylation in mediating this impaired pyruvate uptake was examined. The treatment of control mitochondria with the acetylating agent acetic anhydride inhibits pyruvate uptake and pyruvate-supported respiration in a similar manner to the pyruvate transport inhibitor α-cyano-4-hydroxycinnamate. A mass spectrometry selective reactive monitoring assay was developed and used to determine that acetylation of lysines 19 and 26 of MPC2 is enhanced in Akita heart mitochondria. Expression of a double acetylation mimic of MPC2 (K19Q/K26Q) in H9c2 cells was sufficient to decrease the maximal cellular oxygen consumption rate. This study supports the conclusion that deficient pyruvate transport activity, mediated in part by acetylation of MPC2, is a contributor to metabolic inflexibility in the diabetic heart. PMID:28154187

  12. Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: prevention by thiol group protecting agents.

    PubMed

    Custódio, José B A; Cardoso, Carla M P; Santos, Maria S; Almeida, Leonor M; Vicente, Joaquim A F; Fernandes, Maria A S

    2009-05-02

    Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca(2+)-induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20nmol/mg protein) induced Ca(2+)-dependent mitochondrial swelling, depolarization of membrane potential (DeltaPsi), Ca(2+) release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the DeltaPsi, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H(2)O(2) generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca(2+)-induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane

  13. Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase.

    PubMed

    Reyes-Prieto, Adrián; El-Hafidi, Mohammed; Moreno-Sánchez, Rafael; González-Halphen, Diego

    2002-07-01

    The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.

  14. miR-27 regulates mitochondrial networks by directly targeting the mitochondrial fission factor

    PubMed Central

    Tak, Hyosun; Kim, Jihye; Jayabalan, Aravinth Kumar; Lee, Heejin; Kang, Hoin; Cho, Dong-Hyung; Ohn, Takbum; Nam, Suk Woo; Kim, Wook; Lee, Eun Kyung

    2014-01-01

    Mitochondrial morphology is dynamically regulated by forming small, fragmented units or interconnected networks, and this is a pivotal process that is used to maintain mitochondrial homeostasis. Although dysregulation of mitochondrial dynamics is related to the pathogenesis of several human diseases, its molecular mechanism is not fully elucidated. In this study, we demonstrate the potential role of miR-27 in the regulation of mitochondrial dynamics. Mitochondrial fission factor (MFF) mRNA is a direct target of miR-27, whose ectopic expression decreases MFF expression through binding to its 3′-untranslated region. Expression of miR-27 results in the elongation of mitochondria as well as an increased mitochondrial membrane potential and mitochondrial ATP level. Our results suggest that miR-27 is a novel regulator affecting morphological mitochondrial changes by targeting MFF. PMID:25431021

  15. Physical exercise in aging human skeletal muscle increases mitochondrial calcium uniporter expression levels and affects mitochondria dynamics.

    PubMed

    Zampieri, Sandra; Mammucari, Cristina; Romanello, Vanina; Barberi, Laura; Pietrangelo, Laura; Fusella, Aurora; Mosole, Simone; Gherardi, Gaia; Höfer, Christian; Löfler, Stefan; Sarabon, Nejc; Cvecka, Jan; Krenn, Matthias; Carraro, Ugo; Kern, Helmut; Protasi, Feliciano; Musarò, Antonio; Sandri, Marco; Rizzuto, Rosario

    2016-12-01

    Age-related sarcopenia is characterized by a progressive loss of muscle mass with decline in specific force, having dramatic consequences on mobility and quality of life in seniors. The etiology of sarcopenia is multifactorial and underlying mechanisms are currently not fully elucidated. Physical exercise is known to have beneficial effects on muscle trophism and force production. Alterations of mitochondrial Ca 2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) have been recently shown to affect muscle trophism in vivo in mice. To understand the relevance of MCU-dependent mitochondrial Ca 2+ uptake in aging and to investigate the effect of physical exercise on MCU expression and mitochondria dynamics, we analyzed skeletal muscle biopsies from 70-year-old subjects 9 weeks trained with either neuromuscular electrical stimulation (ES) or leg press. Here, we demonstrate that improved muscle function and structure induced by both trainings are linked to increased protein levels of MCU Ultrastructural analyses by electron microscopy showed remodeling of mitochondrial apparatus in ES-trained muscles that is consistent with an adaptation to physical exercise, a response likely mediated by an increased expression of mitochondrial fusion protein OPA1. Altogether these results indicate that the ES-dependent physiological effects on skeletal muscle size and force are associated with changes in mitochondrial-related proteins involved in Ca 2+ homeostasis and mitochondrial shape. These original findings in aging human skeletal muscle confirm the data obtained in mice and propose MCU and mitochondria-related proteins as potential pharmacological targets to counteract age-related muscle loss. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  16. Do mitochondrial properties explain intraspecific variation in thermal tolerance?

    PubMed

    Fangue, Nann A; Richards, Jeffrey G; Schulte, Patricia M

    2009-02-01

    As global temperatures rise, there is a growing need to understand the physiological mechanisms that determine an organism's thermal niche. Here, we test the hypothesis that increases in mitochondrial capacity with cold acclimation and adaptation are associated with decreases in thermal tolerance using two subspecies of killifish (Fundulus heteroclitus) that differ in thermal niche. We assessed whole-organism metabolic rate, mitochondrial amount and mitochondrial function in killifish acclimated to several temperatures. Mitochondrial enzyme activities and mRNA levels were greater in fish from the northern subspecies, particularly in cold-acclimated fish, suggesting that the putatively cold-adapted northern subspecies has a greater capacity for increases in mitochondrial amount in response to cold acclimation. When tested at the fish's acclimation temperature, maximum ADP-stimulated (State III) rates of mitochondrial oxygen consumption in vitro were greater in cold-acclimated northern fish than in southern fish but did not differ between subspecies at higher acclimation temperatures. Whole-organism metabolic rate was greater in fish of the northern subspecies at all acclimation temperatures. Cold acclimation also changed the response of mitochondrial respiration to acute temperature challenge. Mitochondrial oxygen consumption was greater in cold-acclimated northern fish than in southern fish at low test temperatures, but the opposite was true at high test temperatures. These differences were reflected in whole-organism oxygen consumption. Our data indicate that the plasticity of mitochondrial function and amount differs between killifish subspecies, with the less high-temperature tolerant, and putatively cold adapted, northern subspecies having greater ability to increase mitochondrial capacity in the cold. However, there were few differences in mitochondrial properties between subspecies at warm acclimation temperatures, despite differences in both whole

  17. Comparing organic versus conventional soil management on soil respiration.

    PubMed

    Mátyás, Bence; Chiluisa Andrade, Maritza Elizabeth; Yandun Chida, Nora Carmen; Taipe Velasco, Carina Maribel; Gavilanes Morales, Denisse Estefania; Miño Montero, Gisella Nicole; Ramirez Cando, Lenin Javier; Lizano Acevedo, Ronnie Xavier

    2018-01-01

    Soil management has great potential to affect soil respiration. In this study, we investigated the effects of organic versus conventional soil management on soil respiration.  We measured the main soil physical-chemical properties from conventional and organic managed soil in Ecuador. Soil respiration was determined using alkaline absorption according to Witkamp.  Soil properties such as organic matter, nitrogen, and humidity, were comparable between conventional and organic soils in the present study, and in a further analysis there was no statically significant correlation with soil respiration. Therefore, even though organic farmers tend to apply more organic material to their fields, but this did not result in a significantly higher CO2 production in their soils in the present study.

  18. Comparing organic versus conventional soil management on soil respiration

    PubMed Central

    Mátyás, Bence; Chiluisa Andrade, Maritza Elizabeth; Yandun Chida, Nora Carmen; Taipe Velasco, Carina Maribel; Gavilanes Morales, Denisse Estefania; Miño Montero, Gisella Nicole; Ramirez Cando, Lenin Javier; Lizano Acevedo, Ronnie Xavier

    2018-01-01

    Soil management has great potential to affect soil respiration. In this study, we investigated the effects of organic versus conventional soil management on soil respiration.  We measured the main soil physical-chemical properties from conventional and organic managed soil in Ecuador. Soil respiration was determined using alkaline absorption according to Witkamp.  Soil properties such as organic matter, nitrogen, and humidity, were comparable between conventional and organic soils in the present study, and in a further analysis there was no statically significant correlation with soil respiration. Therefore, even though organic farmers tend to apply more organic material to their fields, but this did not result in a significantly higher CO2 production in their soils in the present study. PMID:29623193

  19. Respirable dust and respirable silica exposure in Ontario gold mines.

    PubMed

    Verma, Dave K; Rajhans, Gyan S; Malik, Om P; des Tombe, Karen

    2014-01-01

    A comprehensive survey of respirable dust and respirable silica in Ontario gold mines was conducted by the Ontario Ministry of Labor during 1978-1979. The aim was to assess the feasibility of introducing gravimetric sampling to replace the assessment method which used konimeters, a device which gave results in terms of number of particles per cubic centimeter (ppcc) of air. The study involved both laboratory and field assessments. The field assessment involved measurement of airborne respirable dust and respirable silica at all eight operating gold mines of the time. This article describes the details of the field assessment. A total of 288 long-term (7-8 hr) personal respirable dust air samples were collected from seven occupational categories in eight gold mines. The respirable silica (α-quartz) was determined by x-ray diffraction method. The results show that during 1978-1979, the industry wide mean respirable dust was about 1 mg/m(3), and the mean respirable silica was 0.08 mg/m(3.)The mean% silica in respirable dust was 7.5%. The data set would be useful in future epidemiological and health studies, as well as in assessment of workers' compensation claims for occupational diseases such as silicosis, chronic obstructive pulmonary disease (COPD), and autoimmune diseases such as renal disease and rheumatoid arthritis.

  20. Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart

    PubMed Central

    Ashmore, Tom; Fernandez, Bernadette O; Branco-Price, Cristina; West, James A; Cowburn, Andrew S; Heather, Lisa C; Griffin, Julian L; Johnson, Randall S; Feelisch, Martin; Murray, Andrew J

    2014-01-01

    Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l−1 NaCl (as control) or 0.7 mmol l−1 NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics. PMID:25172947

  1. Evidence for involvement of uncoupling proteins in cerebral mitochondrial oxidative phosphorylation deficiency of rats exposed to 5,000 m high altitude.

    PubMed

    Xu, Yu; Liu, Yuliang; Xia, Chen; Gao, Pan; Liu, Jun-Ze

    2013-02-01

    The present study aimed to investigate the change of proton leak and discuss the role of cerebral uncoupling proteins (UCPs) and its regulatory molecules non-esterified fatty acid (NEFA) in high altitude mitochondrial oxidative phosphorylation deficiency. The model group animals were exposed to acute high altitude hypoxia, and the mitochondrial respiration, protein leak, UCPs abundance/activity and cerebral NEFA concentration were measured. We found that in the model group, cerebral mitochondrial oxidative phosphorylation was severely impaired with decreased ST3 respiration rate and ATP pool. Proton leak kinetics curves demonstrated an increase in proton leak; GTP binding assay pointed out that total cerebral UCPs activity significantly increased; Q-PCR and western blot showed upregulated expression of UCP4 and UCP5. Moreover, cerebral NEFA concentration increased. In conclusion, UCPs mediated proton leak is closely related to cerebral mitochondria oxidative phosphorylation deficiency during acute high altitude hypoxia and NEFA is involved in this signaling pathway.

  2. Lack of Parkin Anticipates the Phenotype and Affects Mitochondrial Morphology and mtDNA Levels in a Mouse Model of Parkinson's Disease.

    PubMed

    Pinto, Milena; Nissanka, Nadee; Moraes, Carlos T

    2018-01-24

    PARK2 is the most common gene mutated in monogenic recessive familial cases of Parkinson's disease (PD). Pathogenic mutations cause a loss of function of the encoded protein Parkin. ParkinKO mice, however, poorly represent human PD symptoms as they only exhibit mild motor phenotypes, minor dopamine metabolism abnormalities, and no signs of dopaminergic neurodegeneration. Parkin has been shown to participate in mitochondrial turnover, by targeting damaged mitochondria with low membrane potential to mitophagy. We studied the role of Parkin on mitochondrial quality control in vivo by knocking out Parkin in the PD-mito- Pst I mouse (males), where the mitochondrial DNA (mtDNA) undergoes double-strand breaks only in dopaminergic neurons. The lack of Parkin promoted earlier onset of dopaminergic neurodegeneration and motor defects in the PD-mito- Pst I mice, but it did not worsen the pathology. The lack of Parkin affected mitochondrial morphology in dopaminergic axons and was associated with an increase in mtDNA levels (mutant and wild type). Unexpectedly, it did not cause a parallel increase in mitochondrial mass or mitophagy. Our results suggest that Parkin affects mtDNA levels in a mitophagy-independent manner. SIGNIFICANCE STATEMENT Parkinson's disease is characterized by progressive motor symptoms due to the selective loss of dopaminergic neurons in the substantia nigra. Loss-of-function mutations of Parkin cause some monogenic forms of Parkinson's disease, possibly through its role in mitochondrial turnover and quality control. To study whether Parkin has a role in vivo in the context of mitochondrial damage, we knocked out Parkin in a mouse model in which the mitochondrial DNA is damaged in dopaminergic neurons. We found that the loss of Parkin did not exacerbate the parkinsonian pathology already present in the mice, but it was associated with an increase in mtDNA levels (mutant and wild-type) without altering mitochondrial mass. These results shed new light on

  3. Fas cell surface death receptor controls hepatic lipid metabolism by regulating mitochondrial function.

    PubMed

    Item, Flurin; Wueest, Stephan; Lemos, Vera; Stein, Sokrates; Lucchini, Fabrizio C; Denzler, Rémy; Fisser, Muriel C; Challa, Tenagne D; Pirinen, Eija; Kim, Youngsoo; Hemmi, Silvio; Gulbins, Erich; Gross, Atan; O'Reilly, Lorraine A; Stoffel, Markus; Auwerx, Johan; Konrad, Daniel

    2017-09-07

    Nonalcoholic fatty liver disease is one of the most prevalent metabolic disorders and it tightly associates with obesity, type 2 diabetes, and cardiovascular disease. Reduced mitochondrial lipid oxidation contributes to hepatic fatty acid accumulation. Here, we show that the Fas cell surface death receptor (Fas/CD95/Apo-1) regulates hepatic mitochondrial metabolism. Hepatic Fas overexpression in chow-fed mice compromises fatty acid oxidation, mitochondrial respiration, and the abundance of mitochondrial respiratory complexes promoting hepatic lipid accumulation and insulin resistance. In line, hepatocyte-specific ablation of Fas improves mitochondrial function and ameliorates high-fat-diet-induced hepatic steatosis, glucose tolerance, and insulin resistance. Mechanistically, Fas impairs fatty acid oxidation via the BH3 interacting-domain death agonist (BID). Mice with genetic or pharmacological inhibition of BID are protected from Fas-mediated impairment of mitochondrial oxidation and hepatic steatosis. We suggest Fas as a potential novel therapeutic target to treat obesity-associated fatty liver and insulin resistance.Hepatic steatosis is a common disease closely associated with metabolic syndrome and insulin resistance. Here Item et al. show that Fas, a member of the TNF receptor superfamily, contributes to mitochondrial dysfunction, steatosis development, and insulin resistance under high fat diet.

  4. Kidney-targeted inhibition of protein kinase C-α ameliorates nephrotoxic nephritis with restoration of mitochondrial dysfunction.

    PubMed

    Kvirkvelia, Nino; McMenamin, Malgorzata; Warren, Marie; Jadeja, Ravirajsinh N; Kodeboyina, Sai Karthik; Sharma, Ashok; Zhi, Wenbo; O'Connor, Paul M; Raju, Raghavan; Lucas, Rudolf; Madaio, Michael P

    2018-05-04

    To investigate the role of protein kinase C-α (PKC-α) in glomerulonephritis, the capacity of PKC-α inhibition to reverse the course of established nephrotoxic nephritis (NTN) was evaluated. Nephritis was induced by a single injection of nephrotoxic serum and after its onset, a PKC-α inhibitor was administered either systemically or by targeted glomerular delivery. By day seven, all mice with NTN had severe nephritis, whereas mice that received PKC-α inhibitors in either form had minimal evidence of disease. To further understand the underlying mechanism, label-free shotgun proteomic analysis of the kidney cortexes were performed, using quantitative mass spectrometry. Ingenuity pathway analysis revealed 157 differentially expressed proteins and mitochondrial dysfunction as the most modulated pathway. Functional protein groups most affected by NTN were mitochondrial proteins associated with respiratory processes. These proteins were down-regulated in the mice with NTN, while their expression was restored with PKC-α inhibition. This suggests a role for proteins that regulate oxidative phosphorylation in recovery. In cultured glomerular endothelial cells, nephrotoxic serum caused a decrease in mitochondrial respiration and membrane potential, mitochondrial morphologic changes and an increase in glycolytic lactic acid production; all normalized by PKC-α inhibition. Thus, PKC-α has a critical role in NTN progression, and the results implicate mitochondrial processes through restoring oxidative phosphorylation, as an essential mechanism underlying recovery. Importantly, our study provides additional support for targeted therapy to glomeruli to reverse the course of progressive disease. Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  5. Commonalities and differences in plants deficient in autophagy and alternative pathways of respiration on response to extended darkness.

    PubMed

    Barros, Jessica A S; Cavalcanti, João Henrique F; Medeiros, David B; Nunes-Nesi, Adriano; Avin-Wittenberg, Tamar; Fernie, Alisdair R; Araújo, Wagner L

    2017-11-02

    Autophagy is a highly conserved cellular mechanism in eukaryotes allowing the degradation of cell constituents. It is of crucial significance in both cellular homeostasis and nutrient recycling. During energy limited conditions plant cells can metabolize alternative respiratory substrates, such as amino acids, providing electrons to the mitochondrial metabolism via the tricarboxylic acid (TCA) cycle or electron transfer flavoprotein/ electron transfer flavoprotein ubiquinone oxidoreductase (ETF/ETFQO) system. Our recent study reveals the importance of autophagy in the supply of amino acids to provide energy through alternative pathways of respiration during carbon starvation. This fact apart, autophagy seems to have more generalized effects related not only to amino acid catabolism but also to metabolism in general. By further comparing the metabolic data obtained with atg mutants with those of mutants involved in the alternative pathways of respiration, we observed clear differences between these mutants, pointing out additional effects of the autophagy deficiency on metabolism of Arabidopsis leaves. Collectively, our data point to an interdependence between mitochondrial metabolism and autophagy and suggest an exquisite regulation of primary metabolism under low energetic conditions.

  6. Triclosan is a Mitochondrial Uncoupler in Live Zebrafish

    PubMed Central

    Shim, Juyoung; Weatherly, Lisa M.; Luc, Richard H.; Dorman, Maxwell T.; Neilson, Andy; Ng, Ryan; Kim, Carol H.; Millard, Paul J.; Gosse, Julie A.

    2016-01-01

    Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24 hour post fertilization zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, following acute exposure to TCS, basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96 well assay format. The method developed in this study provides a high-throughput tool to identify previously-unknown mitochondrial uncouplers in a living organism. PMID:27111768

  7. Age-and Brain Region-Specific Differences in Mitochondrial ...

    EPA Pesticide Factsheets

    Mitochondria are central regulators of energy homeostasis and play a pivotal role in mechanisms of cellular senescence. The objective of the present study was to evaluate mitochondrial bio­-energetic parameters in five brain regions [brainstem (BS), frontal cortex (FC), cerebellum (CER), striatum (STR), hippocampus (HIP)] of four diverse age groups [1 Month (young), 4 Month (adult), 12 Month (middle-aged), 24 Month (old age)] to understand age-related differences in selected brain regions and their contribution to age-related chemical sensitivity. Mitochondrial bioenergetics parameters and enzyme activity were measured under identical conditions across multiple age groups and brain regions in Brown Norway rats (n = 5). The results indicate age- and brain region-specific patterns in mitochondrial functional endpoints. For example, an age-specific decline in ATP synthesis (State 111 respiration) was observed in BS and HIP. Similarly, the maximal respiratory capacities (State V1 and V2) showed age-specific declines in all brain regions examined (young > adult > middle-aged > old age). Amongst all regions, HIP had the greatest change in mitochondrial bioenergetics, showing declines in the 4, 12 and 24 Month age groups. Activities of mitochondrial pyruvate dehydrogenase complex (PDHC) and electron transport chain (ETC) complexes I, II, and IV enzymes were also age- and brain-region specific. In general changes associated with age were more pronounced, with

  8. Quercetin, a natural product supplement, impairs mitochondrial bioenergetics and locomotor behavior in larval zebrafish (Danio rerio).

    PubMed

    Zhang, Ji-Liang; Laurence Souders, Christopher; Denslow, Nancy D; Martyniuk, Christopher J

    2017-07-15

    Quercetin is a natural product that is sold as a supplement in health food stores. While there are reported benefits for this flavonoid as a dietary supplement due to antioxidant properties, the full scope of its biological interactions has not been fully addressed. To learn more about the mechanisms of action related to quercetin, we exposed zebrafish (Danio rerio) embryos to 1 and 10μg/L quercetin for 96h starting at 3h post fertilization. Quercetin up to 10μg/L did not induce significant mortality in developing fish, but did increase prevalence of an upward-curved dorsal plane in hatched larvae. To determine whether this developmental defect was potentially related to mitochondrial bioenergetics during development, we measured oxygen consumption rate in whole embryos following a 24-hour exposure to quercetin. Basal mitochondrial and ATP-linked respiration were decreased at 1 and 10μg/L quercetin, and maximal respiration was decreased at 10μg/L quercetin, suggesting that quercetin impairs mitochondrial bioenergetics. This is proposed to be related to the deformities observed during development. Due to the fact that ATP production was affected by quercetin, larval behaviors related to locomotion were investigated, as well as transcriptional responses of six myogenesis transcripts. Quercetin at 10μg/L significantly reduced the swimming velocity of zebrafish larvae. The expression levels of both myostatin A (mstna) and myogenic differentiation (myoD) were also altered by quercetin. Mstna, an inhibitory factor for myogenesis, was significantly increased at 1μg/L quercetin exposure, while myoD, a stimulatory factor for myogenesis, was significantly increased at 10μg/L quercetin exposure. There were no changes in transcripts related to apoptosis (bcl2, bax, casp3, casp7), but we did observe a decrease in mRNA levels for catalase (cat) in fish exposed to each dose, supporting an oxidative stress response. Our data support the hypothesis that quercetin may affect

  9. Selective inhibition of endogenous antioxidants with Auranofin causes mitochondrial oxidative stress which can be countered by selenium supplementation.

    PubMed

    Radenkovic, Filip; Holland, Olivia; Vanderlelie, Jessica J; Perkins, Anthony V

    2017-12-15

    Auranofin is a thiol-reactive gold (I)-containing compound with potential asa chemotherapeutic. Auranofin has the capacity to selectively inhibit endogenous antioxidant enzymes thioredoxin reductase (TrxR) and glutathione peroxidase (GPx), resulting in oxidative stress and the initiation of a pro-apoptotic cascade. The effect of Auranofin exposure on TrxR and GPx, and the potential for cellular protection through selenium supplementation was examined in the non-cancerous human cell line Swan-71. Auranofin exposure resulted in a concentration dependent differential inhibition of selenoprotein antioxidants. Significant inhibition of TrxR was observed at 20nM Auranofin with inhibition of GPx from 10µM. Significant increases in reactive oxygen species (ROS) were associated with antioxidant inhibition at Auranofin concentrations of 100nM (TrxR inhibition) and 10µM (TrxR and GPx inhibition), respectively. Evaluation of mitochondrial respiration demonstrated significant reductions in routine and maximal respiration at both 100nM and 10μM Auranofin. Auranofin treatment at concentrations of 10μM and higher concentrations resulted in a ∼68% decrease in cellular viability and was associated with elevations in pro-apoptotic markers cytochrome c flux control factor (FCFc) at concentration of 100nM and mitochondrial Bax at 10μM. The supplementation of selenium (100nM) prior to treatment had a generalized protective affect through the restoration of antioxidant activity with a significant increase in TrxR and GPx activity, a significant reduction in ROS and associated improvement in mitochondrial respiration and cellular viability (10µM ∼48% increase). Selenium supplementation reduced the FCFc at low doses of Auranofin (<10μM) however no effect was noted on either FCFc or Bax at concentrations above 10μM. The inhibition of antioxidant systems in non-cancerous cells by Auranofin is strongly dose dependent, and this inhibition can be altered by selenium exposure

  10. Mitochondrial transcription factor A (Tfam) gene sequencing and mitochondrial evaluation in inherited retinal dysplasia in miniature schnauzer dogs.

    PubMed

    Bauer, Bianca S; Forsyth, George W; Sandmeyer, Lynne S; Grahn, Bruce H

    2011-04-01

    Mitochondrial transcription factor A (Tfam) has been implicated in the pathogenesis of retinal dysplasia in miniature schnauzer dogs and it has been proposed that affected dogs have altered mitochondrial numbers, size, and morphology. To test these hypotheses the Tfam gene of affected and normal miniature schnauzer dogs with retinal dysplasia was sequenced and lymphocyte mitochondria were quantified, measured, and the morphology was compared in normal and affected dogs using transmission electron microscopy. For Tfam sequencing, retina, retinal pigment epithelium (RPE), and whole blood samples were collected. Total RNA was isolated from the retina and RPE and reverse transcribed to make cDNA. Genomic DNA was extracted from white blood cell pellets obtained from the whole blood samples. The Tfam coding sequence, 5' promoter region, intron1 and the 3' non-coding sequence of normal and affected dogs were amplified using polymerase chain reaction (PCR), cloned and sequenced. For electron microscopy, lymphocytes from affected and normal dogs were photographed and the mitochondria within each cross-section were identified, quantified, and the mitochondrial area (μm²) per lymphocyte cross-section was calculated. Lastly, using a masked technique, mitochondrial morphology was compared between the 2 groups. Sequencing of the miniature schnauzer Tfam gene revealed no functional sequence variation between affected and normal dogs. Lymphocyte and mitochondrial area, mitochondrial quantification, and morphology assessment also revealed no significant difference between the 2 groups. Further investigation into other candidate genes or factors causing retinal dysplasia in the miniature schnauzer is warranted.

  11. Effect of a high dose of simvastatin on muscle mitochondrial metabolism and calcium signaling in healthy volunteers

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Galtier, F., E-mail: f-galtier@chu-montpellier.fr; INSERM, CIC 1001, 80 Avenue Augustin Fliche, 34295 Montpellier Cedex 5; CPID, Faculté de Pharmacie, 15 Av. Charles Flahault, BP 14491, 34093 Montpellier Cedex 5, Montpellier

    Statin use may be limited by muscle side effects. Although incompletely understood to date, their pathophysiology may involve oxidative stress and impairments of mitochondrial function and of muscle Ca{sup 2+} homeostasis. In order to simultaneously assess these mechanisms, 24 male healthy volunteers were randomized to receive either simvastatin for 80 mg daily or placebo for 8 weeks. Blood and urine samples and a stress test were performed at baseline and at follow-up, and mitochondrial respiration and Ca{sup 2+} spark properties were evaluated on a muscle biopsy 4 days before the second stress test. Simvastatin-treated subjects were separated according to theirmore » median creatine kinase (CK) increase. Simvastatin treatment induced a significant elevation of aspartate amino transferase (3.38 ± 5.68 vs − 1.15 ± 4.32 UI/L, P < 0.001) and CK (− 24.3 ± 99.1 ± 189.3vs 48.3 UI/L, P = 0.01) and a trend to an elevation of isoprostanes (193 ± 408 vs12 ± 53 pmol/mmol creatinine, P = 0.09) with no global change in mitochondrial respiration, lactate/pyruvate ratio or Ca{sup 2+} sparks. However, among statin-treated subjects, those with the highest CK increase displayed a significantly lower Vmax rotenone succinate and an increase in Ca{sup 2+} spark amplitude vs both subjects with the lowest CK increase and placebo-treated subjects. Moreover, Ca{sup 2+} spark amplitude was positively correlated with treatment-induced CK increase in the whole group (r = 0.71, P = 0.0045). In conclusion, this study further supports that statin induced muscular toxicity may be related to alterations in mitochondrial respiration and muscle calcium homeostasis independently of underlying disease or concomitant medication. -- Highlights: ► Statin use may be limited by side effects, particularly myopathy. ► Statins might impair mitochondrial function and muscle Ca2+ signaling in muscle. ► This was tested among healthy volunteers receiving simvastatin 80 mg daily for 8 weeks.

  12. Maintained peak leg and pulmonary VO2 despite substantial reduction in muscle mitochondrial capacity.

    PubMed

    Boushel, R; Gnaiger, E; Larsen, F J; Helge, J W; González-Alonso, J; Ara, I; Munch-Andersen, T; van Hall, G; Søndergaard, H; Saltin, B; Calbet, J A L

    2015-12-01

    We recently reported the circulatory and muscle oxidative capacities of the arm after prolonged low-intensity skiing in the arctic (Boushel et al., 2014). In the present study, leg VO2 was measured by the Fick method during leg cycling while muscle mitochondrial capacity was examined on a biopsy of the vastus lateralis in healthy volunteers (7 male, 2 female) before and after 42 days of skiing at 60% HR max. Peak pulmonary VO2 (3.52 ± 0.18 L.min(-1) pre vs 3.52 ± 0.19 post) and VO2 across the leg (2.8 ± 0.4L.min(-1) pre vs 3.0 ± 0.2 post) were unchanged after the ski journey. Peak leg O2 delivery (3.6 ± 0.2 L.min(-1) pre vs 3.8 ± 0.4 post), O2 extraction (82 ± 1% pre vs 83 ± 1 post), and muscle capillaries per mm(2) (576 ± 17 pre vs 612 ± 28 post) were also unchanged; however, leg muscle mitochondrial OXPHOS capacity was reduced (90 ± 3 pmol.sec(-1) .mg(-1) pre vs 70 ± 2 post, P < 0.05) as was citrate synthase activity (40 ± 3 μmol.min(-1) .g(-1) pre vs 34 ± 3 vs P < 0.05). These findings indicate that peak muscle VO2 can be sustained with a substantial reduction in mitochondrial OXPHOS capacity. This is achieved at a similar O2 delivery and a higher relative ADP-stimulated mitochondrial respiration at a higher mitochondrial p50. These findings support the concept that muscle mitochondrial respiration is submaximal at VO2max , and that mitochondrial volume can be downregulated by chronic energy demand. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  13. Aluminum Toxicity Is Associated with Mitochondrial Dysfunction and the Production of Reactive Oxygen Species in Plant Cells1

    PubMed Central

    Yamamoto, Yoko; Kobayashi, Yukiko; Devi, S. Rama; Rikiishi, Sanae; Matsumoto, Hideaki

    2002-01-01

    Potential mechanisms of Al toxicity measured as Al-induced inhibition of growth in cultured tobacco cells (Nicotiana tabacum, nonchlorophyllic cell line SL) and pea (Pisum sativum) roots were investigated. Compared with the control treatment without Al, the accumulation of Al in tobacco cells caused instantaneously the repression of mitochondrial activities [monitored by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and the uptake of Rhodamine 123] and, after a lag of about 12 h, triggered reactive oxygen species (ROS) production, respiration inhibition, ATP depletion, and the loss of growth capability almost simultaneously. The presence of an antioxidant, butylated hydroxyanisol, during Al treatment of SL cells prevented not only ROS production but also ATP depletion and the loss of growth capability, suggesting that the Al-triggered ROS production seems to be a cause of ATP depletion and the loss of growth capability. Furthermore, these three late events were similarly repressed in an Al-tolerant cell line (ALT301) isolated from SL cells, suggesting that the acquisition of antioxidant functions mimicking butylated hydroxyanisol can be a mechanism of Al tolerance. In the pea root, Al also triggered ROS production, respiration inhibition, and ATP depletion, which were all correlated with inhibition of root elongation. Taken together, we conclude that Al affects mitochondrial functions, which leads to ROS production, probably the key critical event in Al inhibition of cell growth. PMID:11788753

  14. Mitochondria and FOXO3: breath or die.

    PubMed

    Hagenbuchner, Judith; Ausserlechner, Michael J

    2013-01-01

    Forkhead box O (FOXO) transcription factors are regulators of cell-type specific apoptosis and cell cycle arrest but also control longevity and reactive oxygen species (ROS). ROS-control by FOXO is mediated by transcriptional activation of detoxifying enzymes such as Superoxide dismutase 2 (SOD2), Catalase or Sestrins or by the repression of mitochondrial respiratory chain proteins resulting in reduced mitochondrial activity. FOXO3 also regulates the adaptation to hypoxia by reducing mitochondrial mass and oxygen consumption during HIF-1α activation. In neuronal tumor cells, FOXO3 triggers ROS-accumulation as a consequence of transient mitochondrial outer membrane permeabilization, which is essential for FOXO3-induced apoptosis in these cells. Cellular ROS levels are affected by the FOXO-targets Bim, BclxL, and Survivin. All three proteins localize to mitochondria and affect mitochondrial membrane potential, respiration and cellular ROS levels. Bim-activation by FOXO3 causes mitochondrial depolarization resulting in a transitory decrease of respiration and ROS production. Survivin, on the other hand, actively changes mitochondrial architecture, respiration-efficacy and energy metabolism. This ability distinguishes Survivin from other anti-apoptotic proteins such as BclxL, which inhibits ROS by inactivating Bim but does not alter mitochondrial function. Importantly, FOXO3 simultaneously also activates ROS-detoxification via induction of SESN3. In this paper we discuss the hypothesis that the delicate balance between ROS-accumulation by Bim-triggered mitochondrial damage, mitochondrial architecture and ROS-detoxifying proteins determines cell fate. We provide evidence for a FOXO self-reactivating loop and for novel functions of FOXO3 in controlling mitochondrial respiration of neuronal cells, which further supports the current view that FOXO transcription factors are information-integrating sentinels of cellular stress and critical modulators of cell homeostasis.

  15. Tai Chi training reduced coupling between respiration and postural control.

    PubMed

    Holmes, Matthew L; Manor, Brad; Hsieh, Wan-hsin; Hu, Kun; Lipsitz, Lewis A; Li, Li

    2016-01-01

    In order to maintain stable upright stance, the postural control system must account for the continuous perturbations to the body's center-of-mass including those caused by spontaneous respiration. Both aging and disease increase "posturo-respiratory synchronization;" which reflects the degree to which respiration affects postural sway fluctuations over time. Tai Chi training emphasizes the coordination of respiration and bodily movements and may therefore optimize the functional interaction between these two systems. The purpose of the project was to examine the effect of Tai Chi training on the interaction between respiration and postural control in older adults. We hypothesized that Tai Chi training would improve the ability of the postural control system to compensate for respiratory perturbations and thus, reduce posturo-respiratory synchronization. Participants were recruited from supportive housing facilities and randomized to a 12-week Tai Chi intervention (n=28; 86 ± 5 yrs) or educational-control program (n=34, 85 ± 6 yrs). Standing postural sway and respiration were simultaneously recorded with a force plate and respiratory belt under eyes-open and eyes-closed conditions. Posturo-respiratory synchronization was determined by quantifying the variation of the phase relationship between the dominant oscillatory mode of respiration and corresponding oscillations within postural sway. Groups were similar in age, gender distribution, height, body mass, and intervention compliance. Neither intervention altered average sway speed, sway magnitude or respiratory rate. As compared to the education-control group, however, Tai Chi training reduced posturo-respiratory synchronization when standing with eyes open or closed (p<0.001). Tai Chi training did not affect traditional parameters of standing postural control or respiration, yet reduced the coupling between respiration and postural control. The beneficial effects of Tai Chi training may therefore stem in part

  16. Tai Chi training reduced coupling between respiration and postural control

    PubMed Central

    Holmes, Matthew L; Manor, Brad; Hsieh, Wan-hsin; Hu, Kun; Lipsitz, Lewis A; Li, Li

    2015-01-01

    In order to maintain stable upright stance, the postural control system must account for the continuous perturbations to the body’s center-of-mass including those caused by spontaneous respiration. Both aging and disease increase “posturo-respiratory synchronization;” which reflects the degree to which respiration affects postural sway fluctuations over time. Tai Chi training emphasizes the coordination of respiration and bodily movements and may therefore optimize the functional interaction between these two systems. The purpose of the project was to examine the effect of Tai Chi training on the interaction between respiration and postural control in older adults. We hypothesized that Tai Chi training would improve the ability of the postural control system to compensate for respiratory perturbations and thus, reduce posturo-respiratory synchronization. Participants were recruited from supportive housing facilities and randomized to a 12-week Tai Chi intervention (n=28; 86±5yrs) or educational-control program (n=34, 85±6yrs). Standing postural sway and respiration were simultaneously recorded with a force plate and respiratory belt under eyes-open and eyes-closed conditions. Posturo-respiratory synchronization was determined by quantifying the variation of the phase relationship between the dominant oscillatory mode of respiration and corresponding oscillations within postural sway. Groups were similar in age, gender distribution, height, body mass, and intervention compliance. Neither intervention altered average sway speed, sway magnitude or respiratory rate. As compared to the education-control group, however, Tai Chi training reduced posturo-respiratory synchronization when standing with eyes open or closed (p<0.001). Tai Chi training did not affect traditional parameters of standing postural control or respiration, yet reduced the coupling between respiration and postural control. The beneficial effects of Tai Chi training may therefore stem in part

  17. Aspergillus fumigatus mitochondrial electron transport chain mediates oxidative stress homeostasis, hypoxia responses and fungal pathogenesis.

    PubMed

    Grahl, Nora; Dinamarco, Taisa Magnani; Willger, Sven D; Goldman, Gustavo H; Cramer, Robert A

    2012-04-01

    We previously observed that hypoxia is an important component of host microenvironments during pulmonary fungal infections. However, mechanisms of fungal growth in these in vivo hypoxic conditions are poorly understood. Here, we report that mitochondrial respiration is active in hypoxia (1% oxygen) and critical for fungal pathogenesis. We generated Aspergillus fumigatus alternative oxidase (aoxA) and cytochrome C (cycA) null mutants and assessed their ability to tolerate hypoxia, macrophage killing and virulence. In contrast to ΔaoxA, ΔcycA was found to be significantly impaired in conidia germination, growth in normoxia and hypoxia, and displayed attenuated virulence. Intriguingly, loss of cycA results in increased levels of AoxA activity, which results in increased resistance to oxidative stress, macrophage killing and long-term persistence in murine lungs. Thus, our results demonstrate a previously unidentified role for fungal mitochondrial respiration in the pathogenesis of aspergillosis, and lay the foundation for future research into its role in hypoxia signalling and adaptation. © 2012 Blackwell Publishing Ltd.

  18. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro.

    PubMed

    Dykens, James A; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A; Will, Yvonne

    2008-12-01

    As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.

  19. On the role of plant mitochondrial metabolism and its impact on photosynthesis in both optimal and sub-optimal growth conditions.

    PubMed

    Araújo, Wagner L; Nunes-Nesi, Adriano; Fernie, Alisdair R

    2014-02-01

    Given that the pathways of photosynthesis and respiration catalyze partially opposing processes, it follows that their relative activities must be carefully regulated within plant cells. Recent evidence has shown that the components of the mitochondrial electron transport chain are essential for the proper maintenance of intracellular redox gradients, to allow considerable rates of photorespiration and in turn efficient photosynthesis. Thus considerable advances have been made in understanding the interaction between respiration and photosynthesis during the last decades and the potential mechanisms linking mitochondrial function and photosynthetic efficiency will be reviewed. Despite the fact that manipulation of various steps of mitochondrial metabolism has been demonstrated to alter photosynthesis under optimal growth conditions, it is likely that these changes will, by and large, not be maintained under sub-optimal situations. Therefore producing plants to meet this aim remains a critical challenge. It is clear, however, that although there have been a range of studies analysing changes in respiratory and photosynthetic rates in response to light, temperature and CO2, our knowledge of the environmental impact on these processes and its linkage still remains fragmented. We will also discuss the metabolic changes associated to plant respiration and photosynthesis as important components of the survival strategy as they considerably extend the period that a plant can withstand to a stress situation.

  20. [Assessment of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H].

    PubMed

    Cheng, Ying; Ren, Mingming; Niu, Yanyan; Qiao, Jianhua; Aneba, S; Chorvat, D; Chorvatova, A

    2009-12-01

    The primary function of cardiac mitochondria is the production of ATP to support heart contraction. Examination of the mitochondrial redox state is therefore crucially important to sensitively detect early signs of mitochondrial function in pathophysiological conditions, such as ischemia, diabetes and heart failure. We study fingerprinting of mitochondrial metabolic oxidative state in living cardiomyocytes with spectrally-resolved fluorescence lifetime spectroscopy of NAD(P)H, the principal electron donor in mitochondrial respiration responsible for vital ATP supply. Here NAD(P)H is studied as a marker for non-invasive fluorescent probing of the mitochondrial function. NAD(P) H fluorescence is recorded in cardiac cells following excitation with 375nm UV-light and detection by spectrally-resolved time-correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and fluorescence lifetimes. Modulation of NADH production and/or mitochondrial respiration is tested to study dynamic characteristics of NAD(P) H fluorescence decay. Our results show that at least a 3-exponential decay model, with 0.4-0.7ns, 1.2-1.9ns and 8.0-13. Ons lifetime pools is necessary to describe cardiomyocyte autofluorescence (AF) within 420-560nm spectral range. Increased mitochondrial NADH production by ketone bodies enhanced the fluorescence intensity, without significant change in fluorescent lifetimes. Rotenone, the inhibitor of Complex I of the mitochondrial respiratory chain, increased AF intensity and shortened the average fluorescence lifetime. Dinitrophenol (DNP), an uncoupling agent of the mitochondrial oxidative phosphorylation, lowered AF intensity, broadened the spectral shoulder at 520 nm and increased the average fluorescence lifetime. These effects are comparable to the study of NADH fluorescence decay in vitro. In the present contribution we demonstrated that spectrally-resolved fluorescence lifetime technique provides promising new