Science.gov

Sample records for brain liver mitochondria

  1. Distinct characteristics of Ca(2+)-induced depolarization of isolated brain and liver mitochondria.

    PubMed

    Vergun, Olga; Reynolds, Ian J

    2005-09-05

    Ca(2+)-induced mitochondrial depolarization was studied in single isolated rat brain and liver mitochondria. Digital imaging techniques and rhodamine 123 were used for mitochondrial membrane potential measurements. Low Ca(2+) concentrations (about 30--100 nM) initiated oscillations of the membrane potential followed by complete depolarization in brain mitochondria. In contrast, liver mitochondria were less sensitive to Ca(2+); 20 microm Ca(2+) was required to depolarize liver mitochondria. Ca(2+) did not initiate oscillatory depolarizations in liver mitochondria, where each individual mitochondrion depolarized abruptly and irreversibly. Adenine nucleotides dramatically reduced the oscillatory depolarization in brain mitochondria and delayed the onset of the depolarization in liver mitochondria. In both type of mitochondria, the stabilizing effect of adenine nucleotides completely abolished by an inhibition of adenine nucleotide translocator function with carboxyatractyloside, but was not sensitive to bongkrekic acid. Inhibitors of mitochondrial permeability transition cyclosporine A and bongkrekic acid also delayed Ca(2+)-depolarization. We hypothesize that the oscillatory depolarization in brain mitochondria is associated with the transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore, whereas the non-oscillatory depolarization in liver mitochondria is caused by the irreversible opening of the pore.

  2. Mitochondrial complex I dysfunction induced by cocaine and cocaine plus morphine in brain and liver mitochondria.

    PubMed

    Cunha-Oliveira, Teresa; Silva, Lisbeth; Silva, Ana Maria; Moreno, António J; Oliveira, Catarina R; Santos, Maria S

    2013-06-07

    Mitochondrial function and energy metabolism are affected in brains of human cocaine abusers. Cocaine is known to induce mitochondrial dysfunction in cardiac and hepatic tissues, but its effects on brain bioenergetics are less documented. Furthermore, the combination of cocaine and opioids (speedball) was also shown to induce mitochondrial dysfunction. In this work, we compared the effects of cocaine and/or morphine on the bioenergetics of isolated brain and liver mitochondria, to understand their specific effects in each tissue. Upon energization with complex I substrates, cocaine decreased state-3 respiration in brain (but not in liver) mitochondria and decreased uncoupled respiration and mitochondrial potential in both tissues, through a direct effect on complex I. Morphine presented only slight effects on brain and liver mitochondria, and the combination cocaine+morphine had similar effects to cocaine alone, except for a greater decrease in state-3 respiration. Brain and liver mitochondrial respirations were differentially affected, and liver mitochondria were more prone to proton leak caused by the drugs or their combination. This was possibly related with a different dependence on complex I in mitochondrial populations from these tissues. In summary, cocaine and cocaine+morphine induce mitochondrial complex I dysfunction in isolated brain and liver mitochondria, with specific effects in each tissue.

  3. Effects of Various Kynurenine Metabolites on Respiratory Parameters of Rat Brain, Liver and Heart Mitochondria

    PubMed Central

    Baran, Halina; Staniek, Katrin; Bertignol-Spörr, Melanie; Attam, Martin; Kronsteiner, Carina; Kepplinger, Berthold

    2016-01-01

    Previously, we demonstrated that the endogenous glutamate receptor antagonist kynurenic acid dose-dependently and significantly affected rat heart mitochondria. Now we have investigated the effects of L-tryptophan, L-kynurenine, 3-hydroxykynurenine and kynurenic, anthranilic, 3-hydroxyanthranilic, xanthurenic and quinolinic acids on respiratory parameters (ie, state 2, state 3), respiratory control index (RC) and ADP/oxygen ratio in brain, liver and heart mitochondria of adult rats. Mitochondria were incubated with glutamate/malate (5 mM) or succinate (10 mM) and in the presence of L-tryptophan metabolites (1 mM) or in the absence, as control. Kynurenic and anthranilic acids significantly reduced RC values of heart mitochondria in the presence of glutamate/malate. Xanthurenic acid significantly reduced RC values of brain mitochondria in the presence of glutamate/malate. Furthermore, 3-hydroxykynurenine and 3-hydroxyanthranilic acid decreased RC values of brain, liver and heart mitochondria using glutamate/malate. In the presence of succinate, 3-hydroxykynurenine and 3-hydroxyanthranilic acid affected RC values of brain mitochondria, whereas in liver and heart mitochondria only 3-hydroxykynurenine lowered RC values significantly. Furthermore, lowered ADP/oxygen ratios were observed in brain mitochondria in the presence of succinate with 3-hydroxykynurenine and 3-hydroxyanthranilic acid, and to a lesser extent with glutamate/malate. In addition, 3-hydroxyanthranilic acid significantly lowered the ADP/oxygen ratio in heart mitochondria exposed to glutamate/malate, while in the liver mitochondria only a mild reduction was found. Tests of the influence of L-tryptophan and its metabolites on complex I in liver mitochondria showed that only 3-hydroxykynurenine, 3-hydroxyanthranilic acid and L-kynurenine led to a significant acceleration of NADH-driven complex I activities. The data indicate that L-tryptophan metabolites had different effects on brain, liver and heart

  4. Cyclophilin D-Sensitive Mitochondrial Permeability Transition in Adult Human Brain and Liver Mitochondria

    PubMed Central

    Morota, Saori; Chen, Li; Matsuyama, Nagahisa; Suzuki, Yoshiaki; Nakajima, Satoshi; Tanoue, Tadashi; Omi, Akibumi; Shibasaki, Futoshi; Shimazu, Motohide; Ikeda, Yukio; Uchino, Hiroyuki; Elmér, Eskil

    2011-01-01

    Abstract The mitochondrial permeability transition (mPT) is considered to be a major cause of cell death under a variety of pathophysiological conditions of the central nervous system (CNS) and other organs. Pharmacological inhibition or genetic knockout of the matrix protein cyclophilin D (CypD) prevents mPT and cell degeneration in several models of brain injury. If these findings in animal models are translatable to human disease, pharmacological inhibition of mPT offers a promising therapeutic target. The objective of this study was to validate the presence of a CypD-sensitive mPT in adult human brain and liver mitochondria. In order to perform functional characterization of human mitochondria, fresh tissue samples were obtained during hemorrhage or tumor surgery and mitochondria were rapidly isolated. Mitochondrial calcium retention capacity, a quantitative assay for mPT, was significantly increased by the CypD inhibitor cyclosporin A in both human brain and liver mitochondria, whereas thiol-reactive compounds and oxidants sensitized mitochondria to calcium-induced mPT. Brain mitochondria underwent swelling upon calcium overload, which was reversible upon calcium removal. To further explore mPT of human mitochondria, liver mitochondria were demonstrated to exhibit several classical features of the mPT phenomenon, such as calcium-induced loss of membrane potential and respiratory coupling, as well as release of the pro-apoptotic protein cytochrome c. We concluded that adult viable human brain and liver mitochondria possess an active CypD-sensitive mPT. Our findings support the rationale of CypD and mPT inhibition as pharmacological targets in acute and chronic neurodegeneration. PMID:21121808

  5. Mitochondrial oxidative stress and dysfunction induced by isoniazid: study on isolated rat liver and brain mitochondria.

    PubMed

    Ahadpour, Morteza; Eskandari, Mohammad Reza; Mashayekhi, Vida; Haj Mohammad Ebrahim Tehrani, Kamaleddin; Jafarian, Iman; Naserzadeh, Parvaneh; Hosseini, Mir-Jamal

    2016-01-01

    Isoniazid (INH or isonicotinic hydrazide) is used for the treatment and prophylaxis of tuberculosis. Liver and brain are two important target organs in INH toxicity. However, the exact mechanisms behind the INH hepatotoxicity or neurotoxicity have not yet been completely understood. Considering the mitochondria as one of the possible molecular targets for INH toxicity, the aim of this study was to evaluate the mechanisms of INH mitochondrial toxicity on isolated mitochondria. Mitochondria were isolated by differential ultracentrifugation from male Sprague-Dawley rats and incubated with different concentrations of INH (25-2000 μM) for the investigation of mitochondrial parameters. The results indicated that INH could interact with mitochondrial respiratory chain and inhibit its activity. Our results showed an elevation in mitochondrial reactive oxygen species (ROS) formation, lipid peroxidation and mitochondrial membrane potential collapse after exposure of isolated liver mitochondria in INH. However, different results were obtained in brain mitochondria. Noteworthy, significant glutathione oxidation, adenosine triphosphate (ATP) depletion and lipid peroxidation were observed in higher concentration of INH, as compared to liver mitochondria. In conclusion, our results suggest that INH may initiate its toxicity in liver mitochondria through interaction with electron transfer chain, lipid peroxidation, mitochondrial membrane potential decline and cytochrome c expulsion which ultimately lead to cell death signaling.

  6. 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.

  7. Mechanistic approach for the toxic effects of perfluorooctanoic acid on isolated rat liver and brain mitochondria.

    PubMed

    Mashayekhi, V; Tehrani, K Haj Mohammad Ebrahim; Hashemzaei, M; Tabrizian, K; Shahraki, J; Hosseini, M-J

    2015-10-01

    Perfluorooctanoic acid (PFOA) is one of the most widely used perfluoroalkanes as surfactants, lubricants and processing aids in the production of polymers, which has also been detected in the environment, wildlife and human body. Animal studies indicated that PFOA caused a wide array of toxic effects including liver and brain dysfunction, carcinogenicity and reproductive and developmental toxicity. Based on the established role of mitochondria-mediated pathways in the observed toxic effects of many drugs and chemicals, in this study, the potential toxic effects of PFOA on mitochondria isolated from rat liver and brain have been investigated. Mitochondria were isolated by differential centrifugation method and incubated with different concentrations of PFOA (0.5-1.5 mM). The effects of PFOA were assessed on a series of mitochondrial parameters including reactive oxygen species (ROS) formation, activities of mitochondrial complexes I/II/III, reduced glutathione (GSH) content, adenosine triphosphate (ATP) level, membrane potential, lipid peroxidation (LPO), mitochondrial swelling and cytochrome c release. The data on liver mitochondria indicated that PFOA-induced ROS elevation in both mitochondrial complexes I and III, mitochondrial membrane potential collapse, swelling, cytochrome c release and decreased ATP level which induces apoptosis or necrosis. On brain mitochondria, PFOA showed fairly similar effects on the above-mentioned parameters. However, different results were obtained when the effect of PFOA was assessed on LPO and complex II activity. Due to the fact that PFOA had toxic effects on the mitochondria isolated, it could be suggested that mitochondrial toxicity could be a plausible mechanism for the toxic effects of this fluorochemical on liver and brain function. © The Author(s) 2015.

  8. Energetic, oxidative and ionic exchange in rat brain and liver mitochondria at experimental audiogenic epilepsy (Krushinsky-Molodkina model).

    PubMed

    Venediktova, Natalya I; Gorbacheva, Olga S; Belosludtseva, Natalia V; Fedotova, Irina B; Surina, Natalia M; Poletaeva, Inga I; Kolomytkin, Oleg V; Mironova, Galina D

    2017-01-09

    The role of brain and liver mitochondria at epileptic seizure was studied on Krushinsky-Molodkina (KM) rats which respond to sound with an intensive epileptic seizure (audiogenic epilepsy). We didn't find significant changes in respiration rats of brain and liver mitochondria of KM and control rats; however the efficiency of АТР synthesis in the KM rat mitochondria was 10% lower. In rats with audiogenic epilepsy the concentration of oxidative stress marker malondialdehyde in mitochondria of the brain (but not liver) was 2-fold higher than that in the control rats. The rate of H2O2 generation in brain mitochondria of КМ rats was twofold higher than in the control animals when using NAD-dependent substrates. This difference was less pronounced in liver mitochondria. In KM rats, the activity of mitochondrial ATP-dependent potassium channel was lower than in liver mitochondria of control rats. The comparative study of the mitochondria ability to retain calcium ions revealed that in the case of using the complex I and complex II substrates, permeability transition pore is easier to trigger in brain and liver mitochondria of KM and КМs rats than in the control ones. The role of the changes in the energetic, oxidative, and ionic exchange in the mechanism of audiogenic epilepsy generation in rats and the possible correction of the epilepsy seizures are discussed.

  9. Effect of peroxides on spermine transport in rat brain and liver mitochondria.

    PubMed

    Battaglia, Valentina; Tibaldi, Elena; Grancara, Silvia; Zonta, Francesca; Brunati, Anna Maria; Martinis, Pamela; Bragadin, Marcantonio; Grillo, Maria Angelica; Tempera, Giampiero; Agostinelli, Enzo; Toninello, Antonio

    2012-02-01

    The polyamine spermine is transported into the matrix of various types of mitochondria by a specific uniporter system identified as a protein channel. This mechanism is regulated by the membrane potential; other regulatory effectors are unknown. This study analyzes the transport of spermine in the presence of peroxides in both isolated rat liver and brain mitochondria, in order to evaluate the involvement of the redox state in this mechanism, and to compare its effect in both types of mitochondria. In liver mitochondria peroxides are able to inhibit spermine transport. This effect is indicative of redox regulation by the transporter, probably due to the presence of critical thiol groups along the transport pathway, or in close association with it, with different accessibility for the peroxides and performing different functions. In brain mitochondria, peroxides have several effects, supporting the hypothesis of a different regulation of spermine transport. The fact that peroxovanadate can inhibit tyrosine phosphatases in brain mitochondria suggests that mitochondrial spermine transport is regulated by tyrosine phosphorylation in this organ. In this regard, the evaluation of spermine transport in the presence of Src inhibitors suggests the involvement of Src family kinases in this process. It is possible that phosphorylation sites for Src kinases are present in the channel pathway and have an inhibitory effect on spermine transport under regulation by Src kinases. The results of this study suggest that the activity of the spermine transporter probably depends on the redox and/or tyrosine phosphorylation state of mitochondria, and that its regulation may be different in distinct organs.

  10. Effect of estrogens on base excision repair in brain and liver mitochondria of aged female rats.

    PubMed

    Leclère, R; Torregrosa-Muñumer, R; Kireev, R; García, C; Vara, E; Tresguerres, J A F; Gredilla, R

    2013-08-01

    Changes in the endocrine system have been suggested to act as signaling factors in the regulation of age-related events. Among the different hormones that have been linked to the aging process, estrogens have been widely investigated. They have been associated with inflammatory and oxidative processes and several investigations have established a relationship between the protective effects of estrogens and the mitochondrial function. Mitochondrial DNA is subjected to continuous oxidative attack by free radicals, and the base excision repair (BER) pathway is the main DNA repair route present in mitochondria. We have investigated the effect of estrogen levels on some of the key enzymes of BER in brain and liver mitochondria. In both tissues, depletion of estrogens led to an increased mitochondrial AP endonuclease (mtAPE1) activity, while restoration of estrogen levels by exogenous supplementation resulted in restitution of control APE1 activity only in liver. Moreover, in hepatic mitochondria, changes in estrogen levels affected the processing of oxidative lesions but not deaminations. Our results suggest that changes in mtAPE1 activity are related to specific translocation of the enzyme from the cytosol into the mitochondria probably due to oxidative stress changes as a consequence of changes in estrogen levels.

  11. An analysis of the effects of Mn{sup 2+} on oxidative phosphorylation in liver, brain, and heart mitochondria using state 3 oxidation rate assays

    SciTech Connect

    Gunter, Thomas E.; Gerstner, Brent; Lester, Tobias; Wojtovich, Andrew P.; Malecki, Jon; Swarts, Steven G.; Brookes, Paul S.; Gavin, Claire E. Gunter, Karlene K.

    2010-11-15

    Manganese (Mn) toxicity is partially mediated by reduced ATP production. We have used oxidation rate assays-a measure of ATP production-under rapid phosphorylation conditions to explore sites of Mn{sup 2+} inhibition of ATP production in isolated liver, brain, and heart mitochondria. This approach has several advantages. First, the target tissue for Mn toxicity in the basal ganglia is energetically active and should be studied under rapid phosphorylation conditions. Second, Mn may inhibit metabolic steps which do not affect ATP production rate. This approach allows identification of inhibitions that decrease this rate. Third, mitochondria from different tissues contain different amounts of the components of the metabolic pathways potentially resulting in different patterns of ATP inhibition. Our results indicate that Mn{sup 2+} inhibits ATP production with very different patterns in liver, brain, and heart mitochondria. The primary Mn{sup 2+} inhibition site in liver and heart mitochondria, but not in brain mitochondria, is the F{sub 1}F{sub 0} ATP synthase. In mitochondria fueled by either succinate or glutamate + malate, ATP production is much more strongly inhibited in brain than in liver or heart mitochondria; moreover, Mn{sup 2+} inhibits two independent sites in brain mitochondria. The primary site of Mn-induced inhibition of ATP production in brain mitochondria when succinate is substrate is either fumarase or complex II, while the likely site of the primary inhibition when glutamate plus malate are the substrates is either the glutamate/aspartate exchanger or aspartate aminotransferase.

  12. Behavioral tests and oxidative stress evaluation in mitochondria isolated from the brain and liver of mice treated with riparin A.

    PubMed

    Nunes, Geandra Batista Lima; Costa, Luciana Muratori; Gutierrez, Stanley Juan Chávez; Satyal, Prabodh; de Freitas, Rivelilson Mendes

    2015-01-15

    This study aimed at evaluating the oxidative stress in mitochondria isolated from the brain and liver of mice treated with riparin A, as well as the locomotor activity and myorelaxant effect of this compound. Behavioral models of rota rod and open field tests were used for locomotor activity and myorelaxant effect evaluation. The animals were divided into five groups (n=8), which were treated with: diazepam (1mg/kg, i.p), riparin A (5, 10, and 20mg/kg, o.r.) or vehicle (0.9% saline, o.r.). The oxidative stress evaluation was carried out in mitochondria isolated from the brain and liver of mice from five experimental groups (n=8), which were treated with: ascorbic acid (250 mg/kg; positive control), vehicle (0.9% saline; negative control) and riparin A (5, 10 and 20mg/kg). In an open field and rota rod test a significant difference in the number of crossings, in time of permanence on the swivel bar and in the number of falls in riparin A treated animals (5, 10 and 20mg/kg) was not observed, when compared with negative control (vehicle) (p>0.05). In comparison to the negative control, there was a reduction of lipid peroxidation levels and nitrite content in mice treated with riparin A (p<0.05). Reduced glutathione (GSH) levels (p<0.05), superoxide dismutase (SOD) and catalase activities increased in the brain (rip A 5mg/kg; p<0.05), while in the liver SOD remained unchanged (p>0.05) and catalase activity (p<0.05) was reduced. Riparin A was presented as a bioactive molecule devoid of adverse effects of alteration of motor activity. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Effect of the CRAC Peptide, VLNYYVW, on mPTP Opening in Rat Brain and Liver Mitochondria

    PubMed Central

    Azarashvili, Tamara; Krestinina, Olga; Baburina, Yulia; Odinokova, Irina; Akatov, Vladimir; Beletsky, Igor; Lemasters, John; Papadopoulos, Vassilios

    2016-01-01

    The translocator protein (TSPO; 18 kDa) is a high-affinity cholesterol-binding protein located in the outer membrane of mitochondria. A domain in the C-terminus of TSPO was characterized as the cholesterol recognition/interaction amino acid consensus (CRAC). The ability of the CRAC domain to bind to cholesterol led us to hypothesize that this peptide may participate in the regulation of mitochondrial membrane permeability. Herein, we report the effect of the synthetic CRAC peptide, VLNYYVW, on mitochondrial permeability transition pore (mPTP) opening. It was found that the CRAC peptide alone prevents the mPTP from opening, as well as the release of apoptotic factors (cytochrome c, AIF, and EndoG) in rat brain mitochondria (RBM). Co-incubation of CRAC, together with the TSPO drug ligand, PK 11195, resulted in the acceleration of mPTP opening and in the increase of apoptotic factor release. VLNYYVW did not induce swelling in rat liver mitochondria (RLM). 3,17,19-androsten-5-triol (19-Atriol; an inhibitor of the cholesterol-binding activity of the CRAC peptide) alone and in combination with the peptide was able to stimulate RLM swelling, which was Ca2+- and CsA-sensitive. Additionally, a combination of 19-Atriol with 100 nM PK 11195 or with 100 µM PK 11195 displayed the opposite effect: namely, the addition of 19-Atriol with 100 µM PK 11195 in a suspension of RLM suppressed the Ca2+-induced swelling of RLM by 40%, while the presence of 100 nM PK 11195 with 19-Atriol enhanced the swelling of RLM by 60%. Taken together, these data suggest the participation of the TSPO’s CRAC domain in the regulation of permeability transition. PMID:27983605

  14. Ameliorating reactive oxygen species-induced in vitro lipid peroxidation in brain, liver, mitochondria and DNA damage by Zingiber officinale Roscoe.

    PubMed

    Ajith, T A

    2010-01-01

    Iron is an essential nutrient for a number of cellular activities. However, excess cellular iron can be toxic by producing reactive oxygen species (ROS) such as superoxide anion (O(2) (-)) and hydroxyl radical (HO(·)) that damage proteins, lipids and DNA. Mutagenic and genotoxic end products of lipid peroxidation can induce the decline of mitochondrial respiration and are associated with various human ailments including aging, neurodegenerative disorders, cancer etc. Zingiber officinale Roscoe (ginger) is a widely used spice around the world. The protective effect of aqueous ethanol extract of Z. officinale against ROS-induced in vitro lipid peroxidation and DNA damage was evaluated in this study. The lipid peroxidation was induced by hydroxyl radical generated from Fenton's reaction in rat liver and brain homogenates and mitochondrial fraction (isolated from rat liver). The DNA protection was evaluated using H(2)O(2)-induced changes in pBR-322 plasmid and Fenton reaction-induced DNA fragmentation in rat liver. The results indicated that Z. officinale significantly (P<0.001) protected the lipid peroxidation in all the tissue homogenate/mitochondria. The extract at 2 and 0.5 mg/ml could protect 92 % of the lipid peroxidation in brain homogenate and liver mitochondria respectively. The percent inhibition of lipid peroxidation at 1mg/ml of Z. officinale in the liver homogenate was 94 %. However, the extract could partially alleviate the DNA damage. The protective mechanism can be correlated to the radical scavenging property of Z. officinale. The results of the study suggest the possible nutraceutical role of Z. officinale against the oxidative stress induced human ailments.

  15. OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA FROM LIVERS SHOWING CLOUDY SWELLING

    PubMed Central

    Fonnesu, Alberto; Severi, Clara

    1956-01-01

    Using succinate and α-ketoglutarate as substrates, oxidative phosphorylation has been measured in mitochondria isolated from livers showing cloudy swelling. This cellular change was obtained by injecting rats with S. typhi murium toxin and guinea pigs with diphtheria toxin. It has been found that phosphorylation associated with the oxidation of either of these substrates was partially inhibited in mitochondria from livers showing cloudy swelling, while the oxygen consumption was unchanged. Thus, the P:O ratios for both succinate and α-ketoglutarate were lower in mitochondria from treated animals than they were in normal mitochondria. Dephosphorylation of ATP was not significantly modified in mitochondria from livers showing cloudy swelling as compared with normal controls. No dephosphorylation of AMP and G-6-P was observed either in normal mitochondria or in mitochondria from treated animals. PMID:13331961

  16. Resveratrol and Brain Mitochondria: a Review.

    PubMed

    Jardim, Fernanda Rafaela; de Rossi, Fernando Tonon; Nascimento, Marielle Xavier; da Silva Barros, Renata Gabriele; Borges, Paula Agrizzi; Prescilio, Isabella Cristina; de Oliveira, Marcos Roberto

    2017-03-10

    Resveratrol (3,4',5-trihydroxystilbene; C14H12O3) is a polyphenolic phytoalexin found in grapes, berries, peanuts, and wines. Resveratrol has been viewed as an antioxidant, anti-inflammatory, anti-apoptotic, and anticancer agent. Moreover, it has been reported that resveratrol modulates mitochondrial function, redox biology, and dynamics in both in vitro and in vivo experimental models. Resveratrol also attenuates mitochondrial impairment induced by certain stressors. Resveratrol upregulates, for example, mitochondria-located antioxidant enzymes, decreasing the production of reactive species by these organelles. Resveratrol also triggers mitochondrial biogenesis, ameliorating the mitochondria-related bioenergetics status in mammalian cells. In the present work, we discuss about the effects of resveratrol on brain mitochondria. Brain cells (both neuronal and glial) are susceptible to mitochondrial dysfunction due to their high demand for adenosine triphosphate (ATP). Additionally, brain cells consume oxygen (O2) at very high rates, leading to a proportionally high mitochondrial production of reactive species. Therefore, strategies focusing on the maintenance of mitochondrial function in these cell types are of pharmacological interest in the case of neurodegenerative diseases, which involve mitochondrial impairment and increased generation of reactive species, leading to neuroinflammation and cell death. The mechanism by which resveratrol protects mitochondrial function and dynamics is not completely understood, and further research would be necessary in order to investigate exactly how resveratrol affects mitochondria-related parameters. Furthermore, it is particularly important because resveratrol is able to induce cytotoxicity depending on its dosage.

  17. Metabolism of deoxypyrimidines and deoxypyrimidine antiviral analogs in isolated brain mitochondria

    PubMed Central

    McCann, Kathleen A.; Williams, David W.; McKee, Edward E.

    2012-01-01

    The goal of this project was to characterize deoxypyrimidine salvage pathways used to maintain deoxynucleoside triphosphate pools in isolated brain mitochondria and to determine the extent that antiviral pyrimidine analogs utilize or affect these pathways. Mitochondria from rat brains were incubated in media with labeled and unlabeled deoxynucleosides and deoxynucleoside analogs. Products were analyzed by HPLC coupled to an inline UV monitor and liquid scintillation counter. Isolated mitochondria transported thymidine and deoxycytidine into the matrix, and readily phosphorylated both of these to mono-, di, and tri-phosphate nucleotides. Rates of phosphorylation were much higher than rates observed in mitochondria from heart and liver. Deoxyuridine was phosphorylated much more slowly than thymidine and only to dUMP. AZT, an antiviral thymidine analog, was phosphorylated to AZT-MP as readily as thymidine was phosphorylated to TMP, but little if any AZT-DP or AZT-TP was observed. AZT at 5.5 ± 1.7 μM was shown to inhibit thymidine phosphorylation by 50%, but was not observed to inhibit deoxycytidine phosphorylation except at levels > 100 μM. Stavudine and lamivudine were inert when incubated with isolated brain mitochondria. The kinetics of phosphorylation of thymidine, dC and AZT were significantly different in brain mitochondria compared to mitochondria from liver and heart. PMID:22530558

  18. Transferring Xenogenic Mitochondria Provides Neural Protection Against Ischemic Stress in Ischemic Rat Brains.

    PubMed

    Huang, Po-Jui; Kuo, Chi-Chung; Lee, Hsiu-Chin; Shen, Ching-I; Cheng, Fu-Chou; Wu, Shih-Fang; Chang, Jui-Chih; Pan, Hung-Chuan; Lin, Shinn-Zong; Liu, Chin-San; Su, Hong-Lin

    2016-01-01

    Transferring exogenous mitochondria has therapeutic effects on damaged heart, liver, and lung tissues. Whether this protective effect requires the symbiosis of exogenous mitochondria in host cells remains unknown. Here xenogenic mitochondria derived from a hamster cell line were applied to ischemic rat brains and rat primary cortical neurons. Isolated hamster mitochondria, either through local intracerebral or systemic intra-arterial injection, significantly restored the motor performance of brain-ischemic rats. The brain infarct area and neuronal cell death were both attenuated by the exogenous mitochondria. Although internalized mitochondria could be observed in neurons and astrocytes, the low efficacy of mitochondrial internalization could not completely account for the high rate of rescue of the treated neural cells. We further illustrated that disrupting electron transport or ATPase synthase in mitochondria significantly attenuated the protective effect, suggesting that intact respiratory activity is essential for the mitochondrial potency on neural protection. These results emphasize that nonsymbiotic extracellular mitochondria can provide an effective cell defense against acute injurious ischemic stress in the central nervous system.

  19. Oxidation of hydrogen sulfide by human liver mitochondria.

    PubMed

    Helmy, Nada; Prip-Buus, Carina; Vons, Corinne; Lenoir, Véronique; Abou-Hamdan, Abbas; Guedouari-Bounihi, Hala; Lombès, Anne; Bouillaud, Frédéric

    2014-09-15

    Hydrogen sulfide (H2S) is the third gasotransmitter discovered. Sulfide shares with the two others (NO and CO) the same inhibiting properties towards mitochondrial respiration. However, in contrast with NO or CO, sulfide at concentrations lower than the toxic (μM) level is an hydrogen donor and a substrate for mitochondrial respiration. This is due to the activity of a sulfide quinone reductase found in a large majority of mitochondria. An ongoing study of the metabolic state of liver in obese patients allowed us to evaluate the sulfide oxidation capacity with twelve preparations of human liver mitochondria. The results indicate relatively high rates of sulfide oxidation with a large variability between individuals. These observations made with isolated mitochondria appear in agreement with the main characteristics of sulfide oxidation as established before with the help of cellular models.

  20. Isolation and characterization of intact mitochondria from neonatal rat brain.

    PubMed

    Rajapakse, N; Shimizu, K; Payne, M; Busija, D

    2001-12-01

    Poor outcome after neonatal brain injury may be associated with alterations in mitochondrial function. Thus, isolated mitochondria have been a useful tool in understanding the underlying mechanisms of mitochondrial dysfunction. However, isolation and characterization of mitochondria from neonatal rat brain are not fully described. Thus, the aim of this study was to develop a rapid method for the isolation and characterization of functional mitochondria from neonatal rat brain. Mitochondria were isolated from 7-day-old rat brain weighing approximately 500 mg using a discontinuous Percoll density gradient. Brains were homogenized in 12% Percoll/sucrose buffer and layered onto a 26% Percoll/40% Percoll gradient followed by centrifugation. Four methods were used for assessing mitochondrial integrity and function: (1) electron microscopy to assess the morphology of the mitochondria and to determine the relative purity of the preparation; (2) fluorescence of chloromethyl-X-rosamine (Mito Tracker Red) in mitochondria as an indicator of mitochondrial membrane potential (Delta psi(m)); (3) state 3 and 4 respiration; and (4) protein import into mitochondria using an in vitro-synthesized mitochondrial malate dehydrogenase (mMDH). These studies demonstrated that the morphology of mitochondria is maintained with intact outer membranes and well-developed cristae, and Delta psi(m) is preserved. Respiration measurements revealed tightly coupled mitochondria with a respiration control ratio (RCR) of 4.1+/-0.18 (n=6). Import of precursor mMDH into mitochondria increased in a time-dependent manner maximizing at 15 min. The results indicate that neonatal brain mitochondria isolated using this method are well coupled, morphologically intact and are capable of protein import across the outer and inner mitochondrial membranes.

  1. Role of JNK Translocation to Mitochondria Leading to Inhibition of Mitochondria Bioenergetics in Acetaminophen-induced Liver Injury*S⃞

    PubMed Central

    Hanawa, Naoko; Shinohara, Mie; Saberi, Behnam; Gaarde, William A.; Han, Derick; Kaplowitz, Neil

    2008-01-01

    Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165–178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria

  2. Metabolism of propionate by sheep-liver mitochondria

    PubMed Central

    Smith, R. M.; Russell, G. R.

    1967-01-01

    1. A study has been made of the effects of ATP and α-oxoglutarate on the rate of metabolism of propionate by whole mitochondria from sheep liver, and by mitochondria disrupted with ultrasonic energy or by freezing and thawing. Whole mitochondria metabolized propionate aerobically; the rate was increased and stabilized by 0·5mm-ATP, and increased at least a further 50% by 1·67mm-α-oxoglutarate. 2. Anaerobically, externally added ATP at high concentrations permitted slow consumption of propionate. 3. In the presence of 1·3mm-ATP, but in the absence of α-oxoglutarate, there was no significant lag phase in the removal of propionate by whole mitochondria, and the rate declined at concentrations below 2mm. In the additional presence of 1·67mm-α-oxoglutarate or -glutamate, propionate was removed at linear rates until the residual propionate concentration was about 0·1mm. 4. Maximum rates of metabolism of propionate by whole mitochondria with 1·3mm-ATP occurred with alkali-metal chloride concentrations of 65–95mm and with K+/Na+ ratios 5–10, both in the presence and absence of α-oxoglutarate. 5. With disrupted mitochondria stimulatory effects of α-oxoglutarate were obtained only aerobically, only with propionate and not propionyl-CoA as substrate, and only when sufficient mitochondrial structure remained to permit unsupplemented metabolism of propionate to occur. 6. In the presence of ATP and CoA, disrupted mitochondria fixed [2-14C]propionate at a rate adequate to explain the rate with whole mitochondria stimulated with ATP and α-oxoglutarate. 7. With both whole and partially disrupted mitochondria in the absence of ATP, the rate of metabolism of propionate was inhibited by about 80% by 3·3mm-AMP. The inhibition was partly overcome by α-oxoglutarate plus CoA. 8. It is concluded that the ultimate effect of α-oxoglutarate was to increase the rate of supply of ATP within the mitochondria. Reasons are given why it is premature to conclude that the extra

  3. Citrate synthesis in intact rat-liver mitochondria is irreversible.

    PubMed

    Greksák, M; Lopes-Cardozo, M; van den Bergh, S G

    1982-02-01

    Rat-liver mitochondria were incubated with [1,5-14C]citrate in the presence of fluorocitrate to block its oxidation in the Krebs cycle. The reaction products were analysed enzymatically and by anion-exchange chromatography. Incorporation of 14C into acetyl-L-carnitine or ketone bodies via a backward action of citrate synthase was not observed. The optimal rate of citrate synthesis from pyruvate and malate in the presence of fluorocitrate was 15 nmol . mg-1 min-1. In the absence of fluorocitrate, but in the presence of malonate, citrate was oxidized to succinate at a rate of 4 nmol . mg-1 . min-1. We conclude that the synthesis of citrate by intact rat liver mitochondria is an irreversible process. The possible mechanism underlying this phenomenon and the consequence for metabolic regulation are discussed.

  4. Fractionation of human liver mitochondria: enzymic and morphological characterization of the inner and outer membranes as compared to rat liver mitochondria.

    PubMed

    Benga, G; Hodarnau, A; Tilinca, R; Porutiu, D; Dancea, S; Pop, V; Wrigglesworth, J

    1979-02-01

    The fractionation of human liver mitochondria into inner membrane, outer membrane and matrix material is reported. Compared with rat, human liver mitochondria are more fragile. Fractionation can be achieved in only 2 steps, a digitonin treatment for removal of the outer membrane and centrifugation of the inner membrane plus matrix particles through a linear sucrose gradient resulting in purified inner membranes and matrix.

  5. Effects of fasting on oxidative stress in rat liver mitochondria.

    PubMed

    Sorensen, M; Sanz, A; Gómez, J; Pamplona, R; Portero-Otín, M; Gredilla, R; Barja, G

    2006-04-01

    While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O(2) consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N(epsilon)-malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.

  6. The reversible Ca2+-induced permeabilization of rat liver mitochondria.

    PubMed Central

    Al-Nasser, I; Crompton, M

    1986-01-01

    Rat liver mitochondria became permeabilized to sucrose according to an apparent first-order process after accumulating 35 nmol of Ca2+/mg of protein in the presence of 2.5 mM-Pi, but not in its absence. A fraction (24-32%) of the internal space remains sucrose-inaccessible. The rate constant for permeabilization to sucrose decreases slightly when the pH is decreased from 7.5 to 6.5, whereas the rate of inner-membrane potential (delta psi) dissipation is markedly increased, which indicates that H+ permeation precedes sucrose permeation. Permeabilization does not release mitochondrial proteins. [14C]Sucrose appears to enter permeabilized mitochondria instantaneously. Chelation of Ca2+ with EGTA restores delta psi and entraps sucrose in the matrix space. With 20 mM-sucrose at the instant of resealing, about 21 nmol of sucrose/mg of protein becomes entrapped. The amount of sucrose entrapped is proportional to the degree of permeabilization. Entrapped sucrose is not removed by dilution of the mitochondrial suspension. Resealed mitochondria washed three times retain about 74% of the entrapped sucrose. In the presence of Ruthenium Red and Ca2+ buffers permeabilized mitochondria reseal only partially with free [Ca2+] greater than 3 microM. [14C]Sucrose enters partially resealed mitochondria continuously with time, despite maintenance of delta psi, in accordance with continued interconversion of permeable and impermeable forms. Kinetic analyses of [14C]sucrose entry indicate two Ca2+-sensitive reactions in permeabilization. This conclusion is supported by the biphasic time courses of resealing and repolarization of permeabilized mitochondria and the acute dependence of the rapid repolarization on the free [Ca2+]. A hypothetical model of permeabilization and resealing is suggested and the potential of the procedure for matrix entrapment of substances is discussed. PMID:3099778

  7. The transport of sulphate and sulphite in rat liver mitochondria.

    PubMed

    Crompton, M; Palmieri, F; Capano, M; Quagliariello, E

    1974-07-01

    1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO(2) or an exchange of the sulphite anion for hydroxyl ion(s).

  8. The transport of sulphate and sulphite in rat liver mitochondria

    PubMed Central

    Crompton, M.; Palmieri, F.; Capano, Michela; Quagliariello, E.

    1974-01-01

    1. The mechanism of sulphite and sulphate permeation into rat liver mitochondria was investigated. 2. Extramitochondrial sulphite and sulphate elicit efflux of intramitochondrial phosphate, malate, succinate and malonate. The sulphate-dependent effluxes and the sulphite-dependent efflux of dicarboxylate anions are inhibited by butylmalonate, phenylsuccinate and mersalyl. Inhibition of the phosphate efflux produced by sulphite is caused by mersalyl alone and by N-ethylmaleimide and butylmalonate when present together. 3. External sulphite and sulphate cause efflux of intramitochondrial sulphate, and this is inhibited by butylmalonate, phenylsuccinate and mersalyl. 4. External sulphite and sulphate do not cause efflux of oxoglutarate or citrate. 5. Mitochondria swell when suspended in an iso-osmotic solution of ammonium sulphite; this is not inhibited by N-ethylmaleimide or mersalyl. 6. Low concentrations of sulphite, but not sulphate, produce mitochondrial swelling in iso-osmotic solutions of ammonium malate, succinate, malonate, sulphate, or phosphate in the presence of N-ethylmaleimide. 7. It is concluded that both sulphite and sulphate may be transported by the dicarboxylate carrier of rat liver mitochondria and also that sulphite may permeate by an additional mechanism; the latter may involve the permeation of sulphurous acid or SO2 or an exchange of the sulphite anion for hydroxyl ion(s). PMID:4441366

  9. Proteomic analysis of liver mitochondria from rats with nonalcoholic steatohepatitis

    PubMed Central

    Li, Lin; Lu, De-Zhao; Li, You-Ming; Zhang, Xue-Qun; Zhou, Xin-Xin; Jin, Xi

    2014-01-01

    AIM: To explore mitochondrial dysfunction in nonalcoholic steatohepatitis (NASH) by analyzing the proteome of liver mitochondria from a NASH model. METHODS: The NASH rat model was established by feeding rats a fat-rich diet for 24 wk and was confirmed using hematoxylin and eosin staining of liver tissue and by changes in the levels of serum alanine transaminase, aspartate aminotransferase, triglyceride, total cholesterol and other markers. Liver mitochondria from each group were isolated using differential centrifugation. The mitochondrial samples were lyzed, purified and further analyzed using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Bioinformatic analyses of assigned gene ontology and biological pathway was used to study functional enrichments in the abundant proteomic data. RESULTS: Eight up-regulated and sixteen down-regulated proteins were identified that showed greater than 1.5-fold differences between the controls and the NASH group. These dysregulated proteins were predicted to be involved in different metabolic processes including fatty acid β-oxidation processes, lipid metabolic processes, cell-cycle arrest, cell polarity maintenance, and adenosine triphosphate/sex hormone metabolic processes. Novel proteins that may be involved in NASH pathogenesis including the trifunctional enzyme Hadha, thyroxine, prohibitin, aldehyde dehydrogenase ALDH1L2, UDP-glucuronosyltransferase 2B31, and carbamoyl-phosphate synthase were identified using bioinformatics tools. The decreased expression of Hadha in NASH liver was verified by Western blotting, which was used as a complementary technique to confirm the proteomic results. CONCLUSION: This novel report on the liver mitochondrial proteome of a NASH model may provide a reservoir of information on the pathogenesis and treatment of NASH. PMID:24782632

  10. Interaction of the antibiotic minocycline with liver mitochondria - role of membrane permeabilization in the impairment of respiration.

    PubMed

    Schönfeld, Peter; Siemen, Detlef; Kreutzmann, Peter; Franz, Claudia; Wojtczak, Lech

    2013-12-01

    Several studies have proposed that the antibiotic minocycline (MC) has cytoprotective activities. Nevertheless, when cells have been exposed to MC at micromolar concentrations, detrimental effects have been also observed. Despite the known inhibitory activity of MC on ATP synthesis and the Ca(2+) retention capacity of isolated rat liver and brain mitochondria, the underlying mechanism is still debated. Here, we present further arguments supporting our concept that MC acting on rat liver mitochondria suspended in KCl medium permeabilizes the inner membrane. Supplementation of the medium with cytochrome c and NAD(+) strongly enhanced the respiration of MC-treated mitochondria, thus partly preventing or reversing the inhibitory effect of MC on state 3 or uncoupled respiration. These results indicate that MC produced depletion of mitochondrial cytochrome c and NAD(+) , thus impairing mitochondrial respiration. In addition, NADH oxidation by alamethicin-permeabilized mitochondria supplemented with cytochrome c was insensitive to 200 μm MC, arguing against direct impairment of respiratory chain complexes by MC. Finally, a surprising feature of MC was its accumulation or binding by intact rat liver mitochondria, but not by mitochondria permeabilized with alamethicin or disrupted by freezing and thawing. © 2013 FEBS.

  11. Toxicity of valproic acid in isolated rat liver mitochondria.

    PubMed

    Jafarian, Iman; Eskandari, Mohammad Reza; Mashayekhi, Vida; Ahadpour, Morteza; Hosseini, Mir-Jamal

    2013-10-01

    Valproic acid (VPA), an anticonvulsant and mood-stabilizing drug, is widely used for the treatment of different types of seizures and myoclonic epilepsy. Several mechanisms have been suggested for VPA hepatotoxicity, and most of them are associated with oxidative stress. It seems that oxidative stress by VPA treatment has been associated with mitochondrial dysfunction. Therefore, this study investigated the mitochondrial toxicity mechanisms of VPA on freshly isolated rat mitochondria for better understanding pathogenesis of VPA in mitochondrial toxicity. Rat liver mitochondria were obtained by differential ultracentrifugation and were then incubated with different concentrations of VPA (25-200 µM). Our results showed that VPA could induce oxidative stress via rising in mitochondrial reactive oxygen species formation, lipid peroxidation, mitochondrial membrane potential collapse, mitochondrial swelling and finally release of cytochrome c. These effects were well inhibited by pretreatment of isolated mitochondria with cyclosporin A and butylated hydroxytoluene. Based on these results, it is clear that VPA exerts mitochondrial toxicity by impairing mitochondrial functions leading to oxidative stress and cytochrome c expulsion, which start cell death signaling.

  12. Transport and oxidation of choline by liver mitochondria

    PubMed Central

    Tyler, D. D.

    1977-01-01

    1. Rapid choline oxidation and the onset of Pi-induced swelling by liver mitochondria, incubated in a sucrose medium at or above pH7.0, required the addition of both Pi and an uncoupling agent. Below pH7.0, Pi alone was required for rapid choline oxidation and swelling. 2. Choline oxidation was inhibited by each of several reagents that also inhibited Pi-induced swelling under similar conditions of incubation, including EGTA, mersalyl, Mg2+, the Ca2+-ionophore A23187, rotenone and nupercaine. None of these reagents had any significant effect on the rate of choline oxidation by sonicated mitochondria. There was therefore a close correlation between the conditions required for rapid choline oxidation and for Pi-induced swelling to occur, suggesting that in the absence of mitochondrial swelling the rate of choline oxidation is regulated by the rate of choline transport across the mitochondrial membrane. 3. Respiratory-chain inhibitors, uncoupling agents (at pH6.5) and ionophore A23187 caused a loss of endogenous Ca2+ from mitochondria, whereas nupercaine and Mg2+ had no significant effect on the Ca2+ content. Inhibition of choline oxidation and mitochondrial swelling by ionophore A23187 was reversed by adding Ca2+, but not by Mg2+. It is concluded that added Pi promotes the Ca2+-dependent activation of mitochondrial membrane phospholipase activity in respiring mitochondria, causing an increase in the permeability of the mitochondrial inner membrane to choline and therefore enabling rapid choline oxidation to occur. Nupercaine and Mg2+ appear to block choline oxidation and swelling by inhibiting phospholipase activity. 4. Choline was oxidized slowly by tightly coupled mitochondria largely depleted of their endogenous adenine nucleotides, suggesting that these compounds are not directly concerned in the regulation of choline oxidation. 5. The results are discussed in relation to the possible mechanism of choline transport across the mitochondrial membrane in vivo and

  13. Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria.

    PubMed

    Jesina, P; Kholová, D; Bolehovská, R; Cervinková, Z; Drahota, Z; Houstek, J

    2004-01-01

    We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.

  14. Catalase takes part in rat liver mitochondria oxidative stress defense.

    PubMed

    Salvi, Mauro; Battaglia, Valentina; Brunati, Anna Maria; La Rocca, Nicoletta; Tibaldi, Elena; Pietrangeli, Paola; Marcocci, Lucia; Mondovì, Bruno; Rossi, Carlo A; Toninello, Antonio

    2007-08-17

    Highly purified rat liver mitochondria (RLM) when exposed to tert-butylhydroperoxide undergo matrix swelling, membrane potential collapse, and oxidation of glutathione and pyridine nucleotides, all events attributable to the induction of mitochondrial permeability transition. Instead, RLM, if treated with the same or higher amounts of H2O2 or tyramine, are insensitive or only partially sensitive, respectively, to mitochondrial permeability transition. In addition, the block of respiration by antimycin A added to RLM respiring in state 4 conditions, or the addition of H2O2, results in O2 generation, which is blocked by the catalase inhibitors aminotriazole or KCN. In this regard, H2O2 decomposition yields molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism with a rate constant of 0.0346 s(-1). The rate of H2O2 consumption is not influenced by respiratory substrates, succinate or glutamate-malate, nor by N-ethylmaleimide, suggesting that cytochrome c oxidase and the glutathione-glutathione peroxidase system are not significantly involved in this process. Instead, H2O2 consumption is considerably inhibited by KCN or aminotriazole, indicating activity by a hemoprotein. All these observations are compatible with the presence of endogenous heme-containing catalase with an activity of 825 +/- 15 units, which contributes to mitochondrial protection against endogenous or exogenous H2O2. Mitochondrial catalase in liver most probably represents regulatory control of bioenergetic metabolism, but it may also be proposed for new therapeutic strategies against liver diseases. The constitutive presence of catalase inside mitochondria is demonstrated by several methodological approaches as follows: biochemical fractionating, proteinase K sensitivity, and immunogold electron microscopy on isolated RLM and whole rat liver tissue.

  15. Enhanced oxidative capacity of ground squirrel brain mitochondria during hibernation.

    PubMed

    Ballinger, Mallory A; Schwartz, Christine; Andrews, Matthew T

    2017-03-01

    During hibernation, thirteen-lined ground squirrels (Ictidomys tridecemlineatus) regularly cycle between bouts of torpor and interbout arousal (IBA). Most of the brain is electrically quiescent during torpor but regains activity quickly upon arousal to IBA, resulting in extreme oscillations in energy demand during hibernation. We predicted increased functional capacity of brain mitochondria during hibernation compared with spring to accommodate the variable energy demands of hibernation. To address this hypothesis, we examined mitochondrial bioenergetics in the ground squirrel brain across three time points: spring (SP), torpor (TOR), and IBA. Respiration rates of isolated brain mitochondria through complex I of the electron transport chain were more than twofold higher in TOR and IBA than in SP (P < 0.05). We also found a 10% increase in membrane potential between hibernation and spring (P < 0.05), and that proton leak was lower in TOR and IBA than in SP. Finally, there was a 30% increase in calcium loading in SP brain mitochondria compared with TOR and IBA (P < 0.01). To analyze brain mitochondrial abundance between spring and hibernation, we measured the ratio of copy number in a mitochondrial gene (ND1) vs. a nuclear gene (B2M) in frozen cerebral cortex samples. No significant differences were observed in DNA copies between SP and IBA. These data show that brain mitochondrial bioenergetics are not static across the year and suggest that brain mitochondria function more effectively during the hibernation season, allowing for rapid production of energy to meet demand when extreme physiological changes are occurring.

  16. Role of Mitochondria in Nonalcoholic Fatty Liver Disease

    PubMed Central

    Nassir, Fatiha; Ibdah, Jamal A.

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) affects about 30% of the general population in the United States and includes a spectrum of disease that includes simple steatosis, non-alcoholic steatohepatitis (NASH), fibrosis and cirrhosis. Significant insight has been gained into our understanding of the pathogenesis of NALFD; however the key metabolic aberrations underlying lipid accumulation in hepatocytes and the progression of NAFLD remain to be elucidated. Accumulating and emerging evidence indicate that hepatic mitochondria play a critical role in the development and pathogenesis of steatosis and NAFLD. Here, we review studies that document a link between the pathogenesis of NAFLD and hepatic mitochondrial dysfunction with particular focus on new insights into the role of impaired fatty acid oxidation, the transcription factor peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and sirtuins in development and progression of NAFLD. PMID:24837835

  17. Scavenging of H2O2 by mouse brain mitochondria

    PubMed Central

    Starkov, Anatoly A.; Andreyev, Alexander Yu; Zhang, Steven F.; Starkova, Natalia N.; Korneeva, Maria; Syromyatnikov, Mikhail; Popov, Vasily N.

    2015-01-01

    Mitochondrial reactive oxygen species (ROS) metabolism is unique in that mitochondria both generate and scavenge ROS. Recent estimates of ROS scavenging capacity of brain mitochondria are surprisingly high, ca. 9-12 nmol H2O2/min/mg, which is ~100 times higher than the rate of ROS generation. This raises a question whether brain mitochondria are a source or a sink of ROS. We studied the interaction between ROS generation and scavenging in mouse brain mitochondria by measuring the rate of removal of H2O2 added at a concentration of 0.4 μM, which is close to the reported physiological H2O2 concentrations in tissues, under conditions of low and high levels of mitochondrial H2O2 generation. With NAD-linked substrates, the rate of H2O2 generation by mitochondria was ~50–70 pmol/min/mg. The H2O2 scavenging dynamics was best approximated by the first order reaction equation. H2O2 scavenging was not affected by the uncoupling of mitochondria, phosphorylation of added ADP, or the genetic ablation of glutathione peroxidase 1, but decreased in the absence of respiratory substrates, in the presence of thioredoxin reductase inhibitor auranofin, or in partially disrupted mitochondria. With succinate, the rate of H2O2 generation was ~2,200–2,900 pmol/min/mg; the scavenging of added H2O2 was masked by a significant accumulation of generated H2O2 in the assay medium. The obtained data were fitted into a simple model that reasonably well described the interaction between H2O2 scavenging and production. It showed that mitochondria are neither a sink nor a source of H2O2, but can function as both at the same time, efficiently stabilizing exogenous H2O2 concentration at a level directly proportional to the ratio of the H2O2 generation rate to the rate constant of the first order scavenging reaction. PMID:25248416

  18. Scavenging of H2O2 by mouse brain mitochondria.

    PubMed

    Starkov, Anatoly A; Andreyev, Alexander Yu; Zhang, Steven F; Starkova, Natalia N; Korneeva, Maria; Syromyatnikov, Mikhail; Popov, Vasily N

    2014-12-01

    Mitochondrial reactive oxygen species (ROS) metabolism is unique in that mitochondria both generate and scavenge ROS. Recent estimates of ROS scavenging capacity of brain mitochondria are surprisingly high, ca. 9-12 nmol H2O2/min/mg, which is ~100 times higher than the rate of ROS generation. This raises a question whether brain mitochondria are a source or a sink of ROS. We studied the interaction between ROS generation and scavenging in mouse brain mitochondria by measuring the rate of removal of H2O2 added at a concentration of 0.4 μM, which is close to the reported physiological H2O2 concentrations in tissues, under conditions of low and high levels of mitochondrial H2O2 generation. With NAD-linked substrates, the rate of H2O2 generation by mitochondria was ~50-70 pmol/min/mg. The H2O2 scavenging dynamics was best approximated by the first order reaction equation. H2O2 scavenging was not affected by the uncoupling of mitochondria, phosphorylation of added ADP, or the genetic ablation of glutathione peroxidase 1, but decreased in the absence of respiratory substrates, in the presence of thioredoxin reductase inhibitor auranofin, or in partially disrupted mitochondria. With succinate, the rate of H2O2 generation was ~2,200-2,900 pmol/min/mg; the scavenging of added H2O2 was masked by a significant accumulation of generated H2O2 in the assay medium. The obtained data were fitted into a simple model that reasonably well described the interaction between H2O2 scavenging and production. It showed that mitochondria are neither a sink nor a source of H2O2, but can function as both at the same time, efficiently stabilizing exogenous H2O2 concentration at a level directly proportional to the ratio of the H2O2 generation rate to the rate constant of the first order scavenging reaction.

  19. Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention

    PubMed Central

    Kristian, Tibor; Pivovarova, Natalia B.; Fiskum, Gary; Andrews, S. Brian

    2008-01-01

    Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca2+-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca2+ uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP) increased mitochondrial Ca2+ loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca2+ capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca2+ is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods. PMID:17663756

  20. Calcium-induced precipitate formation in brain mitochondria: composition, calcium capacity, and retention.

    PubMed

    Kristian, Tibor; Pivovarova, Natalia B; Fiskum, Gary; Andrews, S Brian

    2007-08-01

    Both isolated brain mitochondria and mitochondria in intact neurons are capable of accumulating large amounts of calcium, which leads to formation in the matrix of calcium- and phosphorus-rich precipitates, the chemical composition of which is largely unknown. Here, we have used inhibitors of the mitochondrial permeability transition (MPT) to determine how the amount and rate of mitochondrial calcium uptake relate to mitochondrial morphology, precipitate composition, and precipitate retention. Using isolated rat brain (RBM) or liver mitochondria (RLM) Ca(2+)-loaded by continuous cation infusion, precipitate composition was measured in situ in parallel with Ca(2+) uptake and mitochondrial swelling. In RBM, the endogenous MPT inhibitors adenosine 5'-diphosphate (ADP) and adenosine 5'-triphosphate (ATP) increased mitochondrial Ca(2+) loading capacity and facilitated formation of precipitates. In the presence of ADP, the Ca/P ratio approached 1.5, while ATP or reduced infusion rates decreased this ratio towards 1.0, indicating that precipitate chemical form varies with the conditions of loading. In both RBM and RLM, the presence of cyclosporine A in addition to ADP increased the Ca(2+) capacity and precipitate Ca/P ratio. Following MPT and/or depolarization, the release of accumulated Ca(2+) is rapid but incomplete; significant residual calcium in the form of precipitates is retained in damaged mitochondria for prolonged periods.

  1. Injurious effect on rat liver mitochondria by lymphocytes from patients with primary biliary cirrhosis.

    PubMed Central

    Bootello, A; Fernandez-Cruz, E; Escartin, P; Blanco, M F; Gosalvez, M; Segovia De Arana, J M

    1976-01-01

    Lymphocytes from primary biliary cirrhosis (PBC) patients were shown to have an injurious effect on rat liver mitochondria, as was demonstrated by the inhibition of mitochondrial respiratory control by these cells. The incubation of the PBC patients' lymphocytes with isolated rat liver mitochondria produced a significant inhibition of mitochondrial respiration in the presence of ADP. However, no significant effect on respiration was seen with control lymphocytes of normal persons or with lymphocytes from patients with alcoholic cirrhosis and miscellaneous liver diseases. The results suggest that this injurious effect of PBC lymphocytes on mitochondria might be a consequence of sensitization in vivo of the PBC patients' lymphocytes by the mitochondrial antigens. PMID:1277585

  2. Mitotherapy for Fatty Liver by Intravenous Administration of Exogenous Mitochondria in Male Mice.

    PubMed

    Fu, Ailing; Shi, Xianxun; Zhang, Huajing; Fu, Bin

    2017-01-01

    Mitochondrial dysfunction is a major and common mechanism in developing non-alcoholic fatty liver disease (NAFLD). Replacement of dysfunctional mitochondria by functional exogenous mitochondria may attenuate intrahepatic excessive lipid and recover hepatocyte function. However, no data shows that mitochondria can be systemically administrated to animals to date. Here we suggest that mitochondria isolated from hepatoma cells are used as a mitotherapy agent to treat mouse fatty liver induced by high-fat diets. When the mitochondria were intravenously injected into the mice, serum aminotransferase activity and cholesterol level decreased in a dose-dependent manner. Also, the mitotherapy reduced lipid accumulation and oxidation injury of the fatty liver mice, improved energy production, and consequently restored hepatocyte function. The mitotherapy strategy offers a new potential therapeutic approach for treating NAFLD.

  3. Dicarboxylate carrier-mediated glutathione transport is essential for reactive oxygen species homeostasis and normal respiration in rat brain mitochondria

    PubMed Central

    Kamga, Christelle K.; Zhang, Shelley X.

    2010-01-01

    Glutathione transport into mitochondria is mediated by oxoglutarate (OGC) and dicarboxylate carrier (DIC) in the kidney and liver. However, transport mechanisms in brain mitochondria are unknown. We found that both carriers were expressed in the brain. Using cortical mitochondria incubated with physiological levels of glutathione, we found that butylmalonate, a DIC inhibitor, reduced mitochondrial glutathione to levels similar to those seen in mitochondria incubated without extramitochondrial glutathione (59% of control). In contrast, phenylsuccinate, an OGC inhibitor, had no effect (97% of control). Additional experiments with DIC and OGC short hairpin RNA in neuronal-like PC12 cells resulted in similar findings. Significantly, DIC inhibition resulted in increased reactive oxygen species (ROS) content in and H2O2 release from mitochondria. It also led to decreased membrane potential, increased basal respiration rates, and decreased phosphorus-to-oxygen (P/O) ratios, especially when electron transport was initiated from complex I. Accordingly, we found that DIC inhibition impaired complex I activity, but not those for complexes II and III. This impairment was not associated with dislodgment of complex subunits. These results suggest that DIC is the main glutathione transporter in cortical mitochondria and that DIC-mediated glutathione transport is essential for these mitochondria to maintain ROS homeostasis and normal respiratory functions. PMID:20538765

  4. Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds.

    PubMed Central

    Brand, Martin D; Turner, Nigel; Ocloo, Augustine; Else, Paul L; Hulbert, A J

    2003-01-01

    The proton conductance of isolated liver mitochondria correlates significantly with body mass in mammals, but not in ectotherms. To establish whether the correlation in mammals is general for endotherms or mammal-specific, we measured proton conductance in mitochondria from birds, the other main group of endotherms, using birds varying in mass over a wide range (nearly 3000-fold), from 13 g zebra finches to 35 kg emus. Respiratory control ratios were higher in mitochondria from larger birds. Mitochondrial proton conductance in liver mitochondria from birds correlated strongly with body mass [respiration rate per mg of protein driving proton leak at 170 mV being 44.7 times (body mass in g)(-0.19)], thus suggesting a general relationship between body mass and proton conductance in endotherms. Mitochondria from larger birds had the same or perhaps greater surface area per mg of protein than mitochondria from smaller birds. Hence, the lower proton conductance was caused not by surface area changes but by some change in the properties of the inner membrane. Liver mitochondria from larger birds had phospholipid fatty acyl chains that were less polyunsaturated and more monounsaturated when compared with those from smaller birds. Phospholipid fatty acyl polyunsaturation correlated positively and monounsaturation correlated negatively with proton conductance. These correlations echo those seen in mammalian liver mitochondria, suggesting that they too are general for endotherms. PMID:12943530

  5. Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds.

    PubMed

    Brand, Martin D; Turner, Nigel; Ocloo, Augustine; Else, Paul L; Hulbert, A J

    2003-12-15

    The proton conductance of isolated liver mitochondria correlates significantly with body mass in mammals, but not in ectotherms. To establish whether the correlation in mammals is general for endotherms or mammal-specific, we measured proton conductance in mitochondria from birds, the other main group of endotherms, using birds varying in mass over a wide range (nearly 3000-fold), from 13 g zebra finches to 35 kg emus. Respiratory control ratios were higher in mitochondria from larger birds. Mitochondrial proton conductance in liver mitochondria from birds correlated strongly with body mass [respiration rate per mg of protein driving proton leak at 170 mV being 44.7 times (body mass in g)(-0.19)], thus suggesting a general relationship between body mass and proton conductance in endotherms. Mitochondria from larger birds had the same or perhaps greater surface area per mg of protein than mitochondria from smaller birds. Hence, the lower proton conductance was caused not by surface area changes but by some change in the properties of the inner membrane. Liver mitochondria from larger birds had phospholipid fatty acyl chains that were less polyunsaturated and more monounsaturated when compared with those from smaller birds. Phospholipid fatty acyl polyunsaturation correlated positively and monounsaturation correlated negatively with proton conductance. These correlations echo those seen in mammalian liver mitochondria, suggesting that they too are general for endotherms.

  6. Characterization of cationic acid phosphatase isozyme from rat liver mitochondria.

    PubMed

    Fujimoto, S; Murakami, K; Hosoda, T; Yamamoto, Y; Watanabe, K; Morinaka, Y; Ohara, A

    1992-05-01

    Acid phosphatase isozyme was highly purified from rat liver mitochondrial fraction. The enzyme showed an isoelectric point value of above 9.5 on isoelectric focusing, and the apparent molecular weight was estimated to be 32000 by Sephadex G-100 gel filtration or 16000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed the hydrolysis of adenosine 5'-triphosphate, adenosine 5'-diphosphate, thiamine pyrophosphate, inorganic pyrophosphate, and phosphoprotein such as casein and phosvitin, but not of several phosphomonoesters, except for p-nitrophenyl phosphate and o-phosphotyrosine. The enzyme was not inhibited by L-(+)-tartrate, and was significantly activated by Fe2+ and reducing agents such as ascorbic acid, L-cysteine,and dithiothreitol. The enzyme was found to be distributed in various rat tissues including liver, spleen, kidney, small intestine, lung, stomach, brain and heart, but not in skeletal muscle.

  7. Proton leak in hepatocytes and liver mitochondria from archosaurs (crocodiles) and allometric relationships for ectotherms.

    PubMed

    Hulbert, A J; Else, P L; Manolis, S C; Brand, M D

    2002-07-01

    It has previously been shown that mitochondrial proton conductance decreases with increasing body mass in mammals and is lower in a 250-g lizard than the laboratory rat. To examine whether mitochondrial proton conductance is extremely low in very large reptiles, hepatocytes and mitochondria were prepared from saltwater crocodiles ( Crocodylus porosus) and freshwater crocodiles ( Crocodylus johnstoni). Respiration rates of hepatocytes and liver mitochondria were measured at 37 degrees C and compared with values obtained for rat or previously measured for other species. Respiration rates of hepatocytes from either species of crocodile were similar to those reported for lizards and approximately one fifth of the rates measured using cells from mammals (rat and sheep). Ten-to-thirty percent of crocodile hepatocyte respiration was used to drive mitochondrial proton leak, similar to the proportion in other species. Respiration rates of crocodile liver mitochondria were similar to those of mammalian species. Proton leak rate in isolated liver mitochondria was measured as a function of membrane potential. Contrary to our prediction, the mitochondrial proton conductance of liver mitochondria from crocodiles was greater than that of liver mitochondria from lizards and was similar to that of rats. The acyl composition of liver mitochondrial phospholipids from the crocodiles was more similar to that in mitochondria from rats than in mitochondria from lizards. The relatively high mitochondrial proton conductance was associated with a relatively small liver, which seems to be characteristic of crocodilians. Comparison of data from a number of diverse ectothermic species suggested that hepatocyte respiration rate may decrease with body mass, with an allometric exponent of about -0.2, similar to the exponent in mammalian hepatocytes. However, unlike mammals, liver mitochondrial proton conductance in ectotherms showed no allometric relationship with body size.

  8. The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria.

    PubMed

    Macchioni, Lara; Davidescu, Magdalena; Roberti, Rita; Corazzi, Lanfranco

    2014-10-01

    Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H(2)O(2) release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.

  9. Thiamine triphosphate synthesis in rat brain occurs in mitochondria and is coupled to the respiratory chain.

    PubMed

    Gangolf, Marjorie; Wins, Pierre; Thiry, Marc; El Moualij, Benaïssa; Bettendorff, Lucien

    2010-01-01

    In animals, thiamine deficiency leads to specific brain lesions, generally attributed to decreased levels of thiamine diphosphate, an essential cofactor in brain energy metabolism. However, another far less abundant derivative, thiamine triphosphate (ThTP), may also have a neuronal function. Here, we show that in the rat brain, ThTP is essentially present and synthesized in mitochondria. In mitochondrial preparations from brain (but not liver), ThTP can be produced from thiamine diphosphate and P(i). This endergonic process is coupled to the oxidation of succinate or NADH through the respiratory chain but cannot be energized by ATP hydrolysis. ThTP synthesis is strongly inhibited by respiratory chain inhibitors, such as myxothiazol and inhibitors of the H(+) channel of F(0)F(1)-ATPase. It is also impaired by disruption of the mitochondria or by depolarization of the inner membrane (by protonophores or valinomycin), indicating that a proton-motive force (Deltap) is required. Collapsing Deltap after ThTP synthesis causes its rapid disappearance, suggesting that both synthesis and hydrolysis are catalyzed by a reversible H(+)-translocating ThTP synthase. The synthesized ThTP can be released from mitochondria in the presence of external P(i). However, ThTP probably does not accumulate in the cytoplasm in vivo, because it is not detected in the cytosolic fraction obtained from a brain homogenate. Our results show for the first time that a high energy triphosphate compound other than ATP can be produced by a chemiosmotic type of mechanism. This might shed a new light on our understanding of the mechanisms of thiamine deficiency-induced brain lesions.

  10. Role of Mitochondria in Neonatal Hypoxic-Ischemic Brain Injury

    PubMed Central

    Lu, Yujiao; Tucker, Donovan; Dong, Yan; Zhao, Ningjun; Zhuo, Xiaoying; Zhang, Quanguang

    2016-01-01

    Hypoxic-ischemia (HI) causes severe brain injury in neonates. It’s one of the leading causes to neonatal death and pediatric disability, resulting in devastating consequences, emotionally and economically, to their families. A series of events happens in this process, e.g. excitatory transmitter release, extracelluar Ca2+ influxing, mitochondrial dysfunction, energy failure, and neuron death. There are two forms of neuron death after HI insult: necrosis and apoptosis, apoptosis being the more prevalent form. Mitochondria handle a series of oxidative reactions, and yield energy for various cellular activities including the maintainance of membrane potential and preservation of intracellular ionic homeostasis. Therefore mitochondria play a critical role in neonatal neurodegeneration following HI, and mitochondrial dysfunction is the key point in neurodegenerative evolution. Because of this, exploring effective mitochondria-based clinical strategies is crucial. Today the only efficacious clinic treatment is hypothermia. However, due to its complex management, clinical complication and autoimmune decrease, its clinical application is limited. So far, many mitochondria-based strategies have been reported neuroprotective in animal models, which offers promise on neonatal therapy. However, since their clinical effectiveness are still unclear, plenty of studies need to be continued in the future. According to recent reports, two novel strategies have been proposed: methylene blue (MB) and melatonin. Although they are still in primary stage, the underlying mechanisms indicate promising clinical applications. Every neurological therapeutic strategy has its intrinsic deficit and limited efficacy, therefore in the long run, the perfect clinical therapy for hypoxic-ischemic neonatal brain injury will be based on the combination of multiple strategies. PMID:27441209

  11. Mouse Liver Mitochondria Isolation, Size Fractionation, and Real-time MOMP Measurement

    PubMed Central

    Renault, Thibaud T.; Luna-Vargas, Mark P.A.; Chipuk, Jerry E.

    2016-01-01

    The mitochondrial pathway of apoptosis involves a complex interplay between dozens of proteins and lipids, and is also dependent on the shape and size of mitochondria. The use of cellular models in past studies has not been ideal for investigating how the complex multi-factor interplay regulates the molecular mechanisms of mitochondrial outer membrane permeabilization (MOMP). Isolated systems have proven to be a paradigm to deconstruct MOMP into individual steps and to study the behavior of each subset of MOMP regulators. In particular, isolated mitochondria are key to in vitro studies of the BCL-2 family proteins, a complex family of pro-survival and pro-apoptotic proteins that directly control the mitochondrial pathway of apoptosis (Renault et al., 2013). In this protocol, we describe three complementary procedures for investigating in real-time the effects of MOMP regulators using isolated mitochondria. The first procedure is “Liver mitochondria isolation” in which the liver is dissected from mice to obtain mitochondria. “Mitochondria labeling with JC-1 and size fractionation” is the second procedure that describes a method to label, fractionate by size and standardize subpopulations of mitochondria. Finally, the “Real-time MOMP measurements” protocol allows to follow MOMP in real-time on isolated mitochondria. The aforementioned procedures were used to determine in vitro the role of mitochondrial membrane shape at the level of isolated cells and isolated mitochondria (Renault et al., 2015). PMID:28093578

  12. The effects of ammonium chloride and bicarbonate on the activity of glutaminase in isolated liver mitochondria.

    PubMed Central

    Joseph, S K; McGivan, J D

    1978-01-01

    1. Glutamine hydrolysis in liver mitochondria was studied by measuring the production of glutamate under conditions where this compound could not be further metabolized. 2. Glutaminase activity in intact mitochondria was very low in the absence of activators. 3. Glutamine hydrolysis was markedly stimulated by NH4Cl and also by HCO3- ions. 4. The stimulation by each of these compounds was much decreased if the mitochondria were uncoupled. 5. Maximum rates of glutamine hydrolysis required the addition of phosphate. A correlation was observed between the activity of glutaminase in the presence of NH4Cl plus HCO3- and the intramitochondrial content of ATP. 6. In disrupted mitochondria, NH4Cl stimulated glutaminase to a much smaller extent than in intact mitochondria. The NH4Cl stimulation in disrupted mitochondria was much increased by the addition of ATP. KHCO3 also stimulated glutaminase activity in disrupted mitochondria, and ATP increased the magnitude of this stimulation. 7. It was concluded that maximum rates of glutaminase activity in liver mitochondria require the presence of phosphate, ATP and either HCO3- or NH4+. A comparison of the results obtained on intact and broken mitochondria indicates that these effectors have a direct effect on the glutaminase enzyme system rather than an indirect effect mediated by changes in transmembrane ion gradients or in the concentrations of intramitochondrial metabolites. PMID:747656

  13. Paradoxical effect of methimazole on liver mitochondria: In vitro and in vivo.

    PubMed

    Niknahad, Hossein; Jamshidzadeh, Akram; Heidari, Reza; Hosseini, Zeynab; Mobini, Keivan; Khodaei, Forouzan; Ommati, Mohammad Mehdi; Abdoli, Narges; Keshavarz, Nahid; Bazyari, Mandana; Najibi, Asma

    2016-09-30

    Methimazole is the most frequently prescribed antithyroid agent. On the other hand, several cases of liver injury are attributed to this drug. The mechanism of methimazole-induced liver injury is obscure. Hepatocytes mitochondria seem to be a target for methimazole cytotoxicity. Current investigation aimed to evaluate the effects of methimazole on the hepatocytes mitochondria in different experimental models. In the in vivo model, methimazole (100, 200 and 400mg/kg, i.p) was administered to mice and liver mitochondria were isolated and assessed. In the in vitro experiments, intact isolated liver mitochondria were incubated with increasing methimazole concentrations (10μM-100mM). It was found that methimazole decreased liver mitochondrial ATP and glutathione, increased mitochondrial swelling, lipid peroxidation and reactive oxygen species (ROS), and collapsed mitochondrial membrane potential when administered to mice. Paradoxically, methimazole not only caused no significant injury toward isolated liver mitochondria in vitro but improved mitochondrial function and protected this organelle. The differences between two investigated models in the current study might be associated with drug bioactivation and reactive metabolites formation. These findings suggest mitochondrial dysfunction as a mechanism for methimazole-induced liver injury. Moreover, methimazole seems to be a novel mitochondrial protecting agent in vitro. Copyright © 2016. Published by Elsevier Ireland Ltd.

  14. 3-nitropropionic acid-induced mitochondrial permeability transition: comparative study of mitochondria from different tissues and brain regions.

    PubMed

    Mirandola, Sandra R; Melo, Daniela R; Saito, Angela; Castilho, Roger F

    2010-02-15

    The adult rat striatum is particularly vulnerable to systemic administration of the succinate dehydrogenase inhibitor 3-nitropropionic acid (3NP), which is known to induce degeneration of the caudate-putamen, as occurs in Huntington's disease. The aim of the present study was to compare the susceptibility of isolated mitochondria from different rat brain regions (striatum, cortex, and cerebellum) as well as from the liver, kidney, and heart to mitochondrial permeability transition (MPT) induced by 3NP and Ca(2+). In the presence of micromolar Ca(2+) concentrations, 3NP induces MPT in a dose-dependent manner, as estimated by mitochondrial swelling and a decrease in the transmembrane electrical potential. A 3NP concentration capable of promoting a 10% inhibition of ADP-stimulated, succinate-supported respiration was sufficient to stimulate Ca(2+)-induced MPT. Brain and heart mitochondria were generally more sensitive to 3NP and Ca(2+)-induced MPT than mitochondria from liver and kidney. In addition, a partial inhibition of mitochondrial respiration by 3NP resulted in more pronounced MPT in striatal mitochondria than in cortical or cerebellar organelles. A similar inhibition of succinate dehydrogenase activity was observed in rat tissue homogenates obtained from various brain regions as well as from liver, kidney, and heart 24 hr after a single i.p. 3NP dose. Mitochondria isolated from forebrains of 3NP-treated rats were also more susceptible to Ca(2+)-induced MPT than those of control rats. We propose that the increased susceptibility of the striatum to 3NP-induced neurodegeneration may be partially explained by its susceptibility to MPT, together with the greater vulnerability of this brain region to glutamate receptor-mediated Ca(2+) influx.

  15. Dynamic Adaptation of Liver Mitochondria to Chronic Alcohol Feeding in Mice

    PubMed Central

    Han, Derick; Ybanez, Maria D.; Johnson, Heather S.; McDonald, Jeniece N.; Mesropyan, Lusine; Sancheti, Harsh; Martin, Gary; Martin, Alanna; Lim, Atalie M; Dara, Lily; Cadenas, Enrique; Tsukamoto, Hidekazu; Kaplowitz, Neil

    2012-01-01

    Liver mitochondria undergo dynamic alterations following chronic alcohol feeding to mice. Intragastric alcohol feeding to mice resulted in 1) increased state III respiration (109% compared with control) in isolated liver mitochondria, probably due to increased levels of complexes I, IV, and V being incorporated into the respiratory chain; 2) increased mitochondrial NAD+ and NADH levels (∼2-fold), with no change in the redox status; 3) alteration in mitochondrial morphology, with increased numbers of elongated mitochondria; and 4) enhanced mitochondrial biogenesis in the liver, which corresponded with an up-regulation of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α). Oral alcohol feeding to mice, which is associated with less liver injury and steatosis, slightly enhanced respiration in isolated liver mitochondria (30.8% compared with control), lower than the striking increase caused by intragastric alcohol feeding. Mitochondrial respiration increased with both oral and intragastric alcohol feeding despite extensive N-acetylation of mitochondrial proteins. The alcohol-induced mitochondrial alterations are probably an adaptive response to enhance alcohol metabolism in the liver. Isolated liver mitochondria from alcohol-treated mice had a greater rate of acetaldehyde metabolism and respiration when treated with acetaldehyde than control. Aldehyde dehydrogenase-2 levels were unaltered in response to alcohol, suggesting that the greater acetaldehyde metabolism by isolated mitochondria from alcohol-treated mice was due to increased mitochondrial respiration that regenerated NAD+, the rate-limiting substrate in alcohol/acetaldehyde metabolism. Overall, our work suggests that mitochondrial plasticity in the liver may be an important adaptive response to the metabolic stress caused by alcohol intake and could potentially play a role in many other vital functions performed by the liver. PMID:23086958

  16. 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.

  17. Effect of hexavalent chromium on electron leakage of respiratory chain in mitochondria isolated from rat liver.

    PubMed

    Xie, Ying; Zhong, Caigao; Zeng, Ming; Guan, Lan; Luo, Lei

    2013-01-01

    In the present study, we explored reactive axygen species (ROS) production in mitochondria, the mechanism of hexavalent chromium (Cr(VI)) hepatotoxicity, and the role of protection by GSH. Intact mitochondria were isolated from rat liver tissues and mitochondrial basal respiratory rates of NADH and FADH2 respiratory chains were determined. Mitochondria were treated with Cr(VI), GSH and several complex inhibitors. Mitochondria energized by glutamate/malate were separately or jointly treated with Rotenone (Rot), diphenyleneiodonium (DPI) and antimycinA (Ant), while mitochondria energized by succinate were separately or jointly treated with Rot, DPI ' thenoyltrifluoroacetone (TTFA) and Ant. Cr(VI) concentration-dependently induced ROS production in the NADH and FADH2 respiratory chain in liver mitochondria. Basal respiratory rate of the mitochondrial FADH2 respiratory chain was significantly higher than that of NADH respiratory chain. Hepatic mitochondrial electron leakage induced by Cr(VI) from NADH respiratory chain were mainly from ubiquinone binding sites of complex I and complex III. Treatment with 50µM Cr(VI) enhances forward movement of electrons through FADH2 respiratory chain and leaking through the ubiquinone binding site of complex III. Moreover, the protective effect of GSH on liver mitochondria electron leakage is through removing excess H2O2 and reducing total ROS. Copyright © 2013 S. Karger AG, Basel.

  18. Influence of aging on membrane permeability transition in brain mitochondria.

    PubMed

    Toman, Julia; Fiskum, Gary

    2011-02-01

    The mitochondrial inner membrane permeability transition (MPT) plays an important role in the pathophysiology of acute disorders of the central nervous systems, including ischemic and traumatic brain injury, and possibly in neurodegenerative diseases. Opening of the permeability transition pore (PTP) by a combination of abnormally elevated intramitochondrial Ca2+ and oxidative stress induces the collapse of transmembrane ion gradients, resulting in membrane depolarization and uncoupling of oxidative phosphorylation. This loss of ATP synthesis eventually results in cellular metabolic failure and necrotic cell death. Drugs, e.g., cyclosporin A, can inhibit the permeability transition through their interaction with the mitochondria-specific protein, cyclophilin D, and demonstrate neuroprotection in several animal models. These characteristics of the MPT were developed almost exclusively from experiments performed with young, mature rodents whereas the neuropathologies associated with the MPT are most prevalent in the elderly population. Some evidence indicates that the sensitivity of mitochondria to Ca2+-induced PTP opening is greater in the aged compared to the young mature brain; however, the basis for this difference is unknown. Based on knowledge of factors that regulate the MPT and on other comparisons between cells and mitochondria from young and old animals, several features may be important. These aging-related features include impaired neuronal Ca2+ homeostasis, increased oxidative stress, increased cyclophilin D protein levels, oxidative modification of the adenine nucleotide translocase and of cardiolipin, and changes in the levels of anti-death mitochondrial proteins, e.g., Bcl-2. The influence of aging on both the contribution of the MPT to neuropathology and the neuroprotective efficacy of MPT inhibitors is a substantial knowledge gap that requires extensive research at the subcellular, cellular, and animal model levels.

  19. Effect of chronic ethanol consumption on the maximal respiratory capacity of rat liver mitochondria

    SciTech Connect

    Spach, P.I.; Cunningham, C.C.

    1986-05-01

    Previous observations suggest strongly that lowered ATP synthetase activity is responsible for the decrease in succinate-driven state 3 (+ ADP) respiration in liver mitochondria from rats fed ethanol chronically (ethanol mitochondria). In the present study uncoupler-stimulated respiration was measured to determine if ethanol-induced decreases in the concentrations of electron transport components were rate limiting for respiration in coupled ethanol mitochondria. Mitochondria were isolated from pair fed rats. Lowered state 3 respiration was observed in ethanol mitochondria with succinate, glutamate-malate, ..cap alpha..-ketoglutarate, and ascorbate-TMPD; cytochrome oxidase activity was decreased 25%. In contrast, uncoupler-stimulated rates were unchanged. These results demonstrate that maximal respiratory capacity is maintained in ethanol mitochondria, even when cytochrome oxidase is lowered. State 3 respiration at site 3, while significantly lowered in ethanol mitochondria, is still significantly higher than succinate-driven state 3 respiration in control mitochondria. These observations are, therefore, consistent with the suggestion that respiratory activity in coupled ethanol mitochondria (state 3 respiration) is limited by activities of other components of the oxidative-phosphorylation system, possibly the ATP synthetase complex.

  20. Synaptic Mitochondria Sustain More Damage than Non-Synaptic Mitochondria after Traumatic Brain Injury and Are Protected by Cyclosporine A.

    PubMed

    Kulbe, Jacqueline R; Hill, Rachel L; Singh, Indrapal N; Wang, Juan A; Hall, Edward D

    2016-10-13

    Currently, there are no Food and Drug Administration (FDA)-approved pharmacotherapies for the treatment of those with traumatic brain injury (TBI). As central mediators of the secondary injury cascade, mitochondria are promising therapeutic targets for prevention of cellular death and dysfunction after TBI. One of the most promising and extensively studied mitochondrial targeted TBI therapies is inhibition of the mitochondrial permeability transition pore (mPTP) by the FDA-approved drug, cyclosporine A (CsA). A number of studies have evaluated the effects of CsA on total brain mitochondria after TBI; however, no study has investigated the effects of CsA on isolated synaptic and non-synaptic mitochondria. Synaptic mitochondria are considered essential for proper neurotransmission and synaptic plasticity, and their dysfunction has been implicated in neurodegeneration. Synaptic and non-synaptic mitochondria have heterogeneous characteristics, but their heterogeneity can be masked in total mitochondrial (synaptic and non-synaptic) preparations. Therefore, it is essential that mitochondria targeted pharmacotherapies, such as CsA, be evaluated in both populations. This is the first study to examine the effects of CsA on isolated synaptic and non-synaptic mitochondria after experimental TBI. We conclude that synaptic mitochondria sustain more damage than non-synaptic mitochondria 24 h after severe controlled cortical impact injury (CCI), and that intraperitoneal administration of CsA (20 mg/kg) 15 min after injury improves synaptic and non-synaptic respiration, with a significant improvement being seen in the more severely impaired synaptic population. As such, CsA remains a promising neuroprotective candidate for the treatment of those with TBI.

  1. Xanthommatin formation in rat liver mitochondria and its inhibition by respiratory chain substrates.

    PubMed

    Ogawa, H; Nagamura, Y; Ishiguro, I

    1983-11-01

    3-Hydroxykynurenine is condensed to xanthommatin by cytochrome c and cytochrome oxidase in rat liver mitochondria. In intact mitochondria the reaction is inhibited by respiratory chain substrates. However, this was not the case with preincubated mitochondria or with isolated cytochrome c and cytochrome oxidase. The inhibition of xanthommatin formation in native mitochondria by succinate was abolished by addition of antimycin A or malonate, whereas the inhibition by citrate, glutamate or fumarate was not impaired by antimycin A or amobarbital. However, after preincubation of mitochondria at 37 degrees C for 30 min the inhibition disappeared in these cases too. It is suggested that the inhibition by succinate is due to the supply of reduced cytochrome b which competes with 3-hydroxykynurenine for ferricytochrome c, while the other respiratory chain substrates inhibit xanthommatin formation only in the case of intact mitochondria by a yet unknown mechanism. These inhibition mechanisms prevent xanthommatin formation in rat liver mitochondria, even though 3-hydroxykynurenine is synthesized in the outer mitochondrial membrane.

  2. Bufothionine induced the mitochondria-mediated apoptosis in H22 liver tumor and acute liver injury.

    PubMed

    Xie, Rui-Fang; Li, Zhi-Cheng; Chen, Pei-Pei; Zhou, Xin

    2015-01-01

    Bufothionine is an alkaloid in Cinobufacini (Huachansu). This study aims to investigate the effects of bufothionine on liver tumors and acute liver injury. In the hepatoprotective experiment, fifty rats were randomly divided into five groups (n = 10): normal saline group, model group, compound glycyrrhizin injection (9.14 mL/kg); cinobufacini injection (3.42 mL/kg) (InjA) and bufothionine (9.77 mL/kg) (BufoA) group. Liver weight indices were recorded to judge the degree of liver swelling, hematoxylin and eosin (H&E) staining of liver tissues was carried out to observe liver histological morphology injury and biochemical indicators including aspartate aminotransferase (AST); alanine aminotransferase (ALT); alkaline phosphatase (ALP); and total bilirubin (TBIL) were determined by modular auto-analyzer. In anti-tumor experiment, H22-tumor-bearing mice were randomly divided into five groups (n = 10): normal saline group, model group, cinobufacini injection (InjB) (5.14 mL/kg), bufothionine (8.02 mL/kg) (BufoB) and 5-fluorouracil (5-Fu) (3.42 mL/kg). Tumors were picked out and determined with vernier calipers. Histological morphology of tumors was observed by H&E staining. In SMMC-7721 cells, expressions of proteins related to mitochondria-mediated apoptosis pathway including Bcl-2, Bax, caspase-3, caspase-9, cyto-c, Bid, and p53 were analyzed by western blotting at low, medium, high concentrations of bufothione (3.62 μg/mL, 18.12 μg/mL,90.62 μg/mL). Butothionine relieved CCl4-induced liver morphology, decreased the level of ALT (P =2.46 × 10(-2)) and expressed tendency to decrease other biochemical markers including AST, ALP and TBIL. Butothionine could also promote necrosis of tumor tissue in H22-tumor-bearing mice and restrained tumor growth with 65.16% inhibition rate. Its mechanism might relate to up-regulation of p53 (at low, mediate and high concentration, corresponding P values were 0.142, 0.0257, 0.0162), caspase-3 (P = 0.246, 0

  3. Mitochondria-controlled signaling mechanisms of brain protection in hypoxia

    PubMed Central

    Lukyanova, Ludmila D.; Kirova, Yulia I.

    2015-01-01

    The article is focused on the role of the cell bioenergetic apparatus, mitochondria, involved in development of immediate and delayed molecular mechanisms for adaptation to hypoxic stress in brain cortex. Hypoxia induces reprogramming of respiratory chain function and switching from oxidation of NAD-related substrates (complex I) to succinate oxidation (complex II). Transient, reversible, compensatory activation of respiratory chain complex II is a major mechanism of immediate adaptation to hypoxia necessary for (1) succinate-related energy synthesis in the conditions of oxygen deficiency and formation of urgent resistance in the body; (2) succinate-related stabilization of HIF-1α and initiation of its transcriptional activity related with formation of long-term adaptation; (3) succinate-related activation of the succinate-specific receptor, GPR91. This mechanism participates in at least four critical regulatory functions: (1) sensor function related with changes in kinetic properties of complex I and complex II in response to a gradual decrease in ambient oxygen concentration; this function is designed for selection of the most efficient pathway for energy substrate oxidation in hypoxia; (2) compensatory function focused on formation of immediate adaptive responses to hypoxia and hypoxic resistance of the body; (3) transcriptional function focused on activated synthesis of HIF-1 and the genes providing long-term adaptation to low pO2; (4) receptor function, which reflects participation of mitochondria in the intercellular signaling system via the succinate-dependent receptor, GPR91. In all cases, the desired result is achieved by activation of the succinate-dependent oxidation pathway, which allows considering succinate as a signaling molecule. Patterns of mitochondria-controlled activation of GPR-91- and HIF-1-dependent reaction were considered, and a possibility of their participation in cellular-intercellular-systemic interactions in hypoxia and adaptation was

  4. Liver and heart mitochondria obtained from Adelie penguin (Pygoscelis adeliae) offers high resistance to lipid peroxidation.

    PubMed

    Gavazza, Mariana; Marmunti, Mónica; Montalti, D; Gutiérrez, Ana María

    2008-06-01

    Lipid peroxidation is generally thought to be a major mechanism of cell injury in aerobic organisms subjected to oxidative stress. All cellular membranes are especially vulnerable to oxidation due to their high concentration of polyunsaturated fatty acids. However, birds have special adaptations for preventing membrane damage caused by reactive oxygen species. This study examines fatty acid profiles and susceptibility to lipid peroxidation in liver and heart mitochondria obtained from Adelie penguin (Pygoscelis adeliae). The saturated fatty acids in these organelles represent approximately 40-50% of total fatty acids whereas the polyunsaturated fatty acid composition was highly distinctive, characterized by almost equal amounts of 18:2 n-6; 20:4 n-6 and 22:6 n-3 in liver mitochondria, and a higher proportion of 18:2 n-6 compared to 20:4 n-6 and 22:6 n-3 in heart mitochondria. The concentration of total unsaturated fatty acids of liver and heart mitochondria was approximately 50% and 60%, respectively, with a prevalence of oleic acid C18:1 n9. The rate C20:4 n6/C18:2 n6 and the unsaturation index was similar in liver and heart mitochondria; 104.33 +/- 6.73 and 100.09 +/- 3.07, respectively. Light emission originating from these organelles showed no statistically significant differences and the polyunsaturated fatty acid profiles did not change during the lipid peroxidation process.

  5. Cannabinoid-induced changes in respiration of brain mitochondria.

    PubMed

    Fišar, Zdeněk; Singh, Namrata; Hroudová, Jana

    2014-11-18

    Cannabinoids exert various biological effects that are either receptor-mediated or independent of receptor signaling. Mitochondrial effects of cannabinoids were interpreted either as non-receptor-mediated alteration of mitochondrial membranes, or as indirect consequences of activation of plasma membrane type 1 cannabinoid receptors (CB1). Recently, CB1 receptors were confirmed to be localized to the membranes of neuronal mitochondria, where their activation directly regulates respiration and energy production. Here, we performed in-depth analysis of cannabinoid-induced changes of mitochondrial respiration using both an antagonist/inverse agonist of CB1 receptors, AM251 and the cannabinoid receptor agonists, Δ(9)-tetrahydrocannabinol (THC), cannabidiol, anandamide, and WIN 55,212-2. Relationships were determined between cannabinoid concentration and respiratory rate driven by substrates of complex I, II or IV in pig brain mitochondria. Either full or partial inhibition of respiratory rate was found for the tested drugs, with an IC50 in the micromolar range, which verified the significant role of non-receptor-mediated mechanism in inhibiting mitochondrial respiration. Effect of stepwise application of THC and AM251 evidenced protective role of AM251 and corroborated the participation of CB1 receptor activation in the inhibition of mitochondrial respiration. We proposed a model, which includes both receptor- and non-receptor-mediated mechanisms of cannabinoid action on mitochondrial respiration. This model explains both the inhibitory effect of cannabinoids and the protective effect of the CB1 receptor inverse agonist. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

    PubMed

    Benga, G; Poruţiu, D; Hodârnău, A; Ferdinand, W

    1992-05-01

    1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.

  7. Isolation and Functional Assessment of Mitochondria from Small Amounts of Mouse Brain Tissue

    PubMed Central

    Chinopoulos, Christos; Zhang, Steven F.; Thomas, Bobby; Ten, Vadim; Starkov, Anatoly A.

    2013-01-01

    Recent discoveries have brought mitochondria functions in focus of the neuroscience research community and greatly stimulated the demand for approaches to study mitochondria dysfunction in neurodegenerative diseases. Many mouse disease models have been generated, but studying mitochondria isolated from individual mouse brain regions is a challenge because of small amount of the available brain tissue. Conventional techniques for isolation and purification of mitochondria from mouse brain subregions, such as ventral midbrain, hippocampus, or striatum, require pooling brain tissue from six to nine animals for a single mitochondrial preparation. Working with pooled tissue significantly decreases the quality of data because of the time required to dissect several brains. It also greatly increases the labor intensity and the cost of experiments as several animals are required per single data point. We describe a method for isolation of brain mitochondria from mouse striata or other 7–12 mg brain samples. The method utilizes a refrigerated table-top microtube centrifuge, and produces research grade quality mitochondria in amounts sufficient for performing multiple enzymatic and functional assays, thereby eliminating the necessity for pooling mouse brain tissue. We also include a method of measuring ADP-ATP exchange rate as a function of mitochondrial membrane potential (ΔΨm) in small amounts of isolated mitochondria, adapted to a plate reader format. PMID:21913109

  8. Isolation and functional assessment of mitochondria from small amounts of mouse brain tissue.

    PubMed

    Chinopoulos, Christos; Zhang, Steven F; Thomas, Bobby; Ten, Vadim; Starkov, Anatoly A

    2011-01-01

    Recent discoveries have brought mitochondria functions in focus of the neuroscience research community and greatly stimulated the demand for approaches to study mitochondria dysfunction in neurodegenerative diseases. Many mouse disease models have been generated, but studying mitochondria isolated from individual mouse brain regions is a challenge because of small amount of the available brain tissue. Conventional techniques for isolation and purification of mitochondria from mouse brain subregions, such as ventral midbrain, hippocampus, or striatum, require pooling brain tissue from six to nine animals for a single mitochondrial preparation. Working with pooled tissue significantly decreases the quality of data because of the time required to dissect several brains. It also greatly increases the labor intensity and the cost of experiments as several animals are required per single data point. We describe a method for isolation of brain mitochondria from mouse striata or other 7-12 mg brain samples. The method utilizes a refrigerated table-top microtube centrifuge, and produces research grade quality mitochondria in amounts sufficient for performing multiple enzymatic and functional assays, thereby eliminating the necessity for pooling mouse brain tissue. We also include a method of measuring ADP-ATP exchange rate as a function of mitochondrial membrane potential (ΔΨm) in small amounts of isolated mitochondria, adapted to a plate reader format.

  9. Inducible cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Raza, H.; Shayiq, F.M.; Avadhani, N.G.

    1987-05-01

    In the present study they have purified US -naphthoflavone (BNF, which induces isotypes similar to 3-MC) and PB induced mitochondrial isoforms. They have been able to purify two isoforms with molecular weights of 54 Kd and 52 Kd from BNF induced mitochondria. Only the 54 KD form, but not the 52 KD species reacts with the polyclonal antibody to microsomal P-450c, though, both show arylhydrocarbon hydroxylase activity in an in vitro system reconstituted with adrenodoxin and adrenodoxin-reductase. Fingerprint analyses, N-terminal sequencing and use of monoclonal antibody probes show that the two mitochondrial isoforms are different from the microsomal P-450c. Further, the 54 Kd mitochondrial isoform is not detected in control mitochondria indicating that it is truly an induced form. Similarly, a PB induced mitochondrial form which exhibits physical, immunochemical and enzymatic properties different from the microsomal P-450b has also been purified.

  10. Hypothyroidism renders liver mitochondria resistant to the opening of membrane permeability transition pore.

    PubMed

    Chávez, E; Franco, M; Reyes-Vivas, H; Zazueta, C; Ramírez, J; Carrillo, R

    1998-09-30

    Membrane permeability was examined in liver mitochondria isolated from hypothyroid rats. It was found that such a thyroid status provides substantial protection from membrane leakiness as induced by Ca2+ loading. Thus, these mitochondria are less prone to undergoing permeability transition than mitochondria from euthyroid rats. The above conclusion was reached on the basis of the following two facts: (1) hypothyroid mitochondria are not strictly dependent on the addition of ADP to retain high matrix Ca2+ concentrations, and (2) carboxyatractyloside, antimycin A or carbonyl cyanide-m-chlorophenyl hydrazone failed to promote Ca2+ efflux. We discuss the possible relevance of the low content of membrane cardiolipin as well as the low expression of the adenine nucleotide translocase as responsible for the resistance to membrane damage.

  11. [Oxidative phosphorylation in liver mitochondria in toxic form of experimental form of influenza].

    PubMed

    Bykova, N O; Gorbunov, N V; Bolgarev, A A; Savina, M V; Ivanova, T I; Egoiants, M A; Vanin, V F; Prozorovskaia, M P

    1991-12-01

    The effect of free radical processes on the oxidation-phosphorylation activity in the liver of CBA-mice has been studied, using a model of toxic form viral infection. The EPR-spectroscopic and electrochemical methods applied in the study of animals infected with pathogenic form of the influenza virus made it possible to reveal a decrease in the activity of the respiratory chain in mitochondria. This seems likely to be due to accumulation of endogenic nitric oxide in the liver tissue.

  12. Some features of mitochondria and fluffy layer in regenerating rat liver

    PubMed Central

    Gear, A. R. L.

    1965-01-01

    1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD–isocitrate dehydrogenase and NADP–isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18·4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4·7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD– and NADP–isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP–isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1·198, light mitochondria 1·193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed. PMID:14333547

  13. SOME FEATURES OF MITOCHONDRIA AND FLUFFY LAYER IN REGENERATING RAT LIVER.

    PubMed

    GEAR, A R

    1965-04-01

    1. Mitochondria and fluffy layer were prepared from control and regenerating rat liver. Differential and density-gradient centrifugation were used to fractionate the preparations, which were examined for protein content, density and the activity of cytochrome c oxidase, succinate dehydrogenase, NAD-isocitrate dehydrogenase and NADP-isocitrate dehydrogenase. 2. During regeneration the mitochondrial protein content of the liver fell by 18% from the control value of 18.4mg. of protein/g. of liver (wet wt.) and by 3 weeks had risen to 130% of the control value. It then declined slowly. 3. The fluffy-layer protein content (4.7mg./g. of liver) varied inversely as the mitochondrial content and increased by 70% in the early stages (10 days) of liver regeneration. The results suggest that fluffy layer may partially represent both partly formed and broken-down mitochondria. 4. NAD- and NADP-isocitrate dehydrogenases differed in their behaviour during liver regeneration. 5. The succinate-dehydrogenase and NADP-isocitrate-dehydrogenase activity of fluffy layer was high and rose during the early stages of liver regeneration (1 week). Succinate dehydrogenase and cytochrome c oxidase were concentrated in the lighter fluffy-layer particles 10 days to 3 weeks after partial hepatectomy. The significance of this with respect to mitochondrial formation is discussed. 6. Mitochondrial fractions possessed a certain degree of heterogeneity in enzymic activity when separated according to size and density. The mean density of heavy mitochondria was 1.198, light mitochondria 1.193. Fluffy layer was nearly homogeneous in control liver, but during regeneration considerable heterogeneity became evident. The significance of the heterogeneity is discussed.

  14. Calcium-induced Cytochrome c release from rat brain mitochondria is altered by digitonin.

    PubMed

    Brustovetsky, Nickolay; Jemmerson, Ronald; Dubinsky, Janet M

    2002-10-31

    To determine if calcium could release Cytochrome c (Cyt c) from brain mitochondria without activating the permeability transition (mPT), brain mitochondria were prepared in two different ways. Digitonin was used to lyse synaptosomes and release synaptosomal mitochondria or a Percoll gradient was used to separate non-synaptosomal mitochondria from the synaptosomes. In gradient-purified mitochondria, low levels of added digitonin produced swelling and Cyt c release. Digitonin augmented Ca(2+)-induced Cyt c release that was insensitive to the mPT inhibitors, cyclosporin A CsA and ADP. Similarly, in mitochondria prepared with digitonin, these inhibitors also failed to prevent Ca(2+)-induced Cyt c release. Thus the mPT-independent, Ca(2+)-induced Cyt c release pathway was attributable to alteration of the permeability properties of the outer mitochondrial membrane by digitonin. Copyright 2002 Elsevier Science Ireland Ltd.

  15. Ameliorative efficacy of quercetin against cisplatin‑induced mitochondrial dysfunction: Study on isolated rat liver mitochondria.

    PubMed

    Waseem, Mohammad; Tabassum, Heena; Bhardwaj, Monica; Parvez, Suhel

    2017-09-01

    The present study aimed to investigate the hepatoprotective effects of the bioflavonoid quercetin (QR) on cisplatin (CP)‑induced mitochondrial oxidative stress in the livers of rats, to elucidate the role of mitochondria in CP‑induced hepatotoxicity, and its underlying mechanism. Isolated liver mitochondria were incubated with 100 µg/ml CP and/or 50 µM QR in vitro. CP treatment triggered a significant increase in membrane lipid peroxidation (LPO) levels, protein carbonyl (PC) contents, and a decrease in reduced glutathione (GSH) and non‑protein thiol (NP‑SH) levels. In addition, CP caused a marked decline in the activities of enzymatic antioxidants and mitochondrial complexes (I, II, III and V) in liver mitochondria. QR pre‑treatment significantly modulated the activities of enzymatic antioxidants and mitochondrial complex enzymes. Furthermore, QR reversed the alterations in LPO and PC levels, and GSH and NP‑SH contents in liver mitochondria. The results of the present study suggested that QR supplementation may suppress CP‑induced mitochondrial toxicity during chemotherapy, and provides a potential prophylactic and defensive candidate for anticancer agent‑induced oxidative stress.

  16. EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA

    PubMed Central

    Greenawalt, John W.; Rossi, Carlo S.; Lehninger, Albert L.

    1964-01-01

    Rat liver mitochondria allowed to accumulate maximal amounts of Ca++ and HPO4= ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca++ and HPO4= from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca++-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca++-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca++-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca++ and phosphate from the mitochondria into the medium. PMID:14228516

  17. EFFECT OF ACTIVE ACCUMULATION OF CALCIUM AND PHOSPHATE IONS ON THE STRUCTURE OF RAT LIVER MITOCHONDRIA.

    PubMed

    GREENAWALT, J W; ROSSI, C S; LEHNINGER, A L

    1964-10-01

    Rat liver mitochondria allowed to accumulate maximal amounts of Ca(++) and HPO(4) (=) ions from the suspending medium in vitro during respiration have a considerably higher specific gravity than normal mitochondria and may be easily separated from the latter by isopycnic centrifugation in density gradients of sucrose or cesium chloride. When the mitochondria are allowed to accumulate less than maximal amounts of Ca(++) and HPO(4) (=) from the medium, they have intermediate specific gravities which are roughly proportional to their content of calcium phosphate. Maximally "loaded" mitochondria are relatively homogeneous with respect to specific gravity. Correlated biochemical and electron microscopic studies show that Ca(++)-loaded mitochondria contain numerous dense granules, of which some 85 per cent are over 500 A in diameter. These granules are electron-opaque not only following fixation and staining with heavy metal reagents, but also following fixation with formaldehyde, demonstrating that the characteristic granules in Ca(++)-loaded mitochondria have intrinsic electron-opacity. The dense granules are almost always located within the inner compartment of the mitochondria and not in the space between the inner and outer membranes. They are frequently located at or near the cristae and they often show electron-transparent "cores." Such granules appear to be made up of clusters of smaller dense particles, but preliminary x-ray diffraction analysis and electron diffraction studies have revealed no evidence of crystallinity in the deposits. The electron-opaque granules decrease in number when the Ca(++)-loaded mitochondria are incubated with 2,4-dinitrophenol; simultaneously there is discharge of Ca(++) and phosphate from the mitochondria into the medium.

  18. Arsenic induces apoptosis in mouse liver is mitochondria dependent and is abrogated by N-acetylcysteine

    SciTech Connect

    Santra, Amal . E-mail: asantra2000@yahoo.co.in; Chowdhury, Abhijit; Ghatak, Subhadip; Biswas, Ayan; Dhali, Gopal Krishna

    2007-04-15

    Arsenicosis, caused by arsenic contamination of drinking water supplies, is a major public health problem in India and Bangladesh. Chronic liver disease, often with portal hypertension occurs in chronic arsenicosis, contributes to the morbidity and mortality. The early cellular events that initiate liver cell injury due to arsenicosis have not been studied. Our aim was to identify the possible mechanisms related to arsenic-induced liver injury in mice. Liver injury was induced in mice by arsenic treatment. The liver was used for mitochondrial oxidative stress, mitochondrial permeability transition (MPT). Evidence of apoptosis was sought by TUNEL test, caspase assay and histology. Pretreatment with N-acetyl-L-cysteine (NAC) was done to modulate hepatic GSH level. Arsenic treatment in mice caused liver injury associated with increased oxidative stress in liver mitochondria and alteration of MPT. Altered MPT facilitated cytochrome c release in the cytosol, activation of caspase 9 and caspase 3 activities and apoptotic cell death. Pretreatment of NAC to arsenic-treated mice abrogated all these alteration suggesting a glutathione (GSH)-dependent mechanism. Oxidative stress in mitochondria and inappropriate MPT are important in the pathogenesis of arsenic induced apoptotic liver cell injury. The phenomenon is GSH dependent and supplementation of NAC might have beneficial effects.

  19. Study on Biological Effects of La(3+) on Rat Liver Mitochondria by Microcalorimetric and Spectroscopic Methods.

    PubMed

    Wu, Man; Gao, Jia-Ling; Feng, Zhi-Jiang; Liu, Wen; Zhang, Ye-Zhong; Liu, Yi; Dai, Jie

    2015-09-01

    The effects of lanthanum on heat production of mitochondria isolated from Wistar rat liver were investigated with microcalorimetry; simultaneously, the effects on mitochondrial swelling and membrane potential (Δψ) were determined by spectroscopic methods. La(3+) showed only inhibitory action on mitochondrial energy turnover with IC50 being 55.8 μmol L(-1). In the spectroscopic experiments, La(3+), like Ca(2+), induced rat liver mitochondrial swelling and decreased membrane potential (Δψ), which was inhibited by the specific permeability transition inhibitor, cyclosporine A (CsA). The induction ability of La(3+) was stronger than that of Ca(2+). These results demonstrated that La(3+) had some biotoxicity effect on mitochondria; the effects of La(3+) and Ca(2+) on rat liver mitochondrial membrane permeability transition (MPT) are different, and La represents toxic action rather than Ca analogy.

  20. Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition.

    PubMed

    Hansson, Magnus J; Månsson, Roland; Morota, Saori; Uchino, Hiroyuki; Kallur, Thérese; Sumi, Tetsuo; Ishii, Nagao; Shimazu, Motohide; Keep, Marcus F; Jegorov, Alexandr; Elmér, Eskil

    2008-08-01

    Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

  1. OSMOTICALLY-INDUCED ALTERATIONS IN VOLUME AND ULTRASTRUCTURE OF MITOCHONDRIA ISOLATED FROM RAT LIVER AND BOVINE HEART

    PubMed Central

    Stoner, Clinton D.; Sirak, Howard D.

    1969-01-01

    Detailed studies correlating changes in mitochondrial optical density, packed volume, and ultrastructure associated with osmotically-induced swelling were performed. Various swelling states were established by incubating mitochondria (isolated in 0.25 M sucrose) at 0°C for 5 min in series of KCl and sucrose solutions ranging in tonicity from 250 to 3 milliosmols. Reversibility of swelling was determined by examining mitochondria exposed to 250 milliosmols media after they had been induced to swell. Swelling induced by lowering the ambient tonicity to approximately 130 (liver mitochondria) and 90 (heart mitochondria) milliosmols involves primarily swelling of the inner compartment within the intact outer membrane. Decreasing the ambient tonicity beyond this level results in rupture of the outer membrane and expansion of the inner compartment through the break. The maximum extent of swelling, corresponding with complete unfolding of the cristae and an increase in over-all mitochondrial volume of approximately 6-fold (liver mitochondria) and 11-fold (heart mitochondria), is reached at approximately 15 (liver mitochondria) and 3 (heart mitochondria) milliosmols. Exposure of liver mitochondria to media of lower tonicity results in irreversibility of inner compartment swelling and escape of matrix material. These changes appear to result from increased inner membrane permeability, possibly due to stretching. PMID:5351404

  2. [The effect of fenibut on the ultrastructure of the brain mitochondria in traumatic edema and swelling].

    PubMed

    Novikov, V E; Naperstnikov, V V

    1994-01-01

    Rat experiments using electron microscopy have established that profound destructive changes occur in the mitochondria in the intra- and perifocal traumatic area in dynamics of traumatic edema-swelling. With phenibut, 50 mg/kg, there is an increase in the number of mitochondria in the brain tissue of the perifocal area, their destructive changes are less pronounced. It is assumed that the positive effect of phenibut on brain bioenergetic processes in the posttraumatic period is associated with the changes.

  3. Membrane effects of Vitamin E deficiency: bioenergetic and surface-charge-density studies of skeletal muscle and liver mitochondria

    SciTech Connect

    Quintanilha, A.T.; Packer, L.; Szyszlo Davies, J.M.; Racanelli, T.L.; Davies, K.J.A.

    1981-12-01

    Vitamin E (dl-..cap alpha..-tocopherol) deficiency in rats increased the sensitivity of liver and muscle mitochondria to damage during incubation at various temperatures, irradiation with visible light, or steady state respiration with substrates. In all cases, vitamin E deficient mitochondria exhibited increased lipid peroxidation, reduced transmembrane potential, decreased respiratory coupling, and lower rates of electron transport, compared to control mitochondria. Muscle mitochondria always showed greater negative inner membrane surface charge density, and were also more sensitive to damage than were liver mitochondria. Vitamin E deficient mitochondria also showed slightly more negative inner membrane surface charge density compared to controls. The relationship between greater negative surface potential and increased sensitivity to damage observed, provides for a new and sensitive method to further probe the role of surface charge in membrane structure and function. Implications of these new findings for the well known human muscle myopathies and those experimentally induced by Vitamin E deficiency in animals, are discussed.

  4. CONTROL OF GLUTAMATE OXIDATION IN BRAIN AND LIVER MITOCHONDRIAL SYSTEMS.

    PubMed

    BALAZS, R

    1965-05-01

    1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the alpha-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-(14)C]- and [5-(14)C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of alpha-oxoglutarate or after decarboxylation to form gamma-aminobutyrate. There is no indication of gamma-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD(+), the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the ;free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields alpha-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is

  5. Effects of butyric acid and arsenic on isolated pancreatic islets and liver mitochondria of male mouse

    PubMed Central

    Ahangarpour, Akram; Oroojan, Ali Akbar; Rezae, Mohsen; Khodayar, Mohammad Javad; Alboghobeish, Soheila; Zeinvand, Marzieh

    2017-01-01

    Aim: The aim of the present study was to evaluate the different doses of Butyric acid (BA) and Arsenic (As) in liver mitochondria oxidative stress and pancreatic islet insulin secretion of male mouse. Background: BA is found in many foods and As as a toxic metal is present in drinking water. They can induce oxidative stress in tissues. Methods: In this experimental study, Liver mitochondria were isolated by administration of the different centrifugation method and pancreatic islets were isolated by collagenase method. Mitochondria were incubated by BA (35, 75, 150, 300 μM) and As (20, 50, 100, 200 μM) as the islets were incubated by BA (250, 500, 1000, 1500 μM) and As (50, 100, 200 μM) for 1 hour. At the end of the experiment, mitochondrial viability and membrane potential, ROS, MDA, GSH and islets insulin secretion were measured by their specific methods. Results: BA and As administration increased mitochondrial levels of ROS, MDA and decreased GSH and pancreatic islet insulin secretion in a dose dependent manner (p<0.05). The doses of BA 75μM and As 100μM have been revealed the most mitochondria toxic concentrations. Also, the doses of 1000μM for BA and 100μM for As were considered as reducing concentrations for islets insulin secretion. Additionally, co administration of them intensified more these effects Conclusion: Alone or in combination administration of BA and As induced oxidative stress in liver mitochondria and decreased insulin secretion of pancreatic islets. PMID:28331564

  6. Toxicity of Arsenic (III) on Isolated Liver Mitochondria: A New Mechanistic Approach

    PubMed Central

    Hosseini, Mir-Jamal; Shaki, Fatemeh; Ghazi-Khansari, Mahmoud; Pourahmad, Jalal

    2013-01-01

    Arsenic exposure mainly through food and water has been shown to be associated with increased incidence of numerous cancers and non-cancer harmful health. It is also used in cancer chemotherapy and treatment of several cancer types due to its apoptogenic effects in the various cancer and normal cell lines. We have already reported that liver is the storage site and important target organ in As (III) toxicity and recently, it has been suggested that hepatic toxicity of arsenic could be resulted from impairment of the liver mitochondria. In this study, interaction of As (III) with freshly isolated rat mitochondria was investigated. We determined different mitochondrial toxicity factors as well as mitochondrial sources of ROS formation using specific substrates and inhibitors following addition of As (III) to the mitochondria. Our results showed that arsenic (III) increased mitochondrial ROS formation, lipid peroxidation and mitochondrial membrane potential collapse, cytochrome c release and mitochondrial swelling in a concentration dependent manner. Addition of As (III) in to the isolated mitochondria, inhibited complexes I and II leading to disruption of mitochondrial electron transfer chain, decreased mitochondrial ATP content and ROS formation. PMID:24250680

  7. Oxidative phosphorylation of liver mitochondria from mice acclimatized to hypobaric hypoxia

    NASA Astrophysics Data System (ADS)

    Leon-Velarde, F.; Whittembury, J.; Monge, C.

    1986-09-01

    Mice exposed to intermittent hypobaric hypoxia for 20 hours a day, 6 days a week, develop extracellular adaptive responses similar to those found in humans exposed to oxygen tension equivalent to that found at an altitude of 4500 m. Isolated liver mitochondria from these animals show no significant differences in rates of substrate-stimulated respiration, ADP-stimulated respiration and the respiratory control ratio (RCR), when compared with sea level controls. Undetectable or negligible differences in these parameters are also noted when sea level animals are exposed for one hour to severe hypoxia (7% O2). We therefore conclude that the oxidative phosphorylation capacity of the isolated mouse liver mitochondria remains unaltered in both acute and chronic hypoxia. However the in vivo oxygen consumption by mice at this degree of hypoxia was markedly reduced. Lack of observable changes in oxidative phosphorylation could be accounted for by extracellular adaptations in mitochondria isolated from acclimatized animals. This explanation, however, is not consistent with the lack of changes on oxidative phosphorylation in mitochondria isolated from mice undergoing acute hypoxia at sea level. It is then suggested that isolated mitochondrial preparations are of limited value for investigating biochemical mechanisms underlying the variation of cellular respiration occurring in vivo.

  8. Nonalcoholic Fatty Liver Disease: Pathogenesis and Therapeutics from a Mitochondria-Centric Perspective

    PubMed Central

    Gusdon, Aaron M.; Song, Ke-xiu; Qu, Shen

    2014-01-01

    Nonalcoholic fatty liver disease (NAFLD) describes a spectrum of disorders characterized by the accumulation of triglycerides within the liver. The global prevalence of NAFLD has been increasing as the obesity epidemic shows no sign of relenting. Mitochondria play a central role in hepatic lipid metabolism and also are affected by upstream signaling pathways involved in hepatic metabolism. This review will focus on the role of mitochondria in the pathophysiology of NAFLD and touch on some of the therapeutic approaches targeting mitochondria as well as metabolically important signaling pathways. Mitochondria are able to adapt to lipid accumulation in hepatocytes by increasing rates of beta-oxidation; however increased substrate delivery to the mitochondrial electron transport chain (ETC) leads to increased reactive oxygen species (ROS) production and eventually ETC dysfunction. Decreased ETC function combined with increased rates of fatty acid beta-oxidation leads to the accumulation of incomplete products of beta-oxidation, which combined with increased levels of ROS contribute to insulin resistance. Several related signaling pathways, nuclear receptors, and transcription factors also regulate hepatic lipid metabolism, many of which are redox sensitive and regulated by ROS. PMID:25371775

  9. Reversible depression of oxygen consumption in isolated liver mitochondria during hibernation.

    PubMed

    Martin, S L; Maniero, G D; Carey, C; Hand, S C

    1999-01-01

    The biochemical mechanisms by which hibernators cool as they enter torpor are not fully understood. In order to examine whether rates of substrate oxidation vary as a function of hibernation, liver mitochondria were isolated from telemetered ground squirrels (Spermophilus lateralis) in five phases of their annual hibernation cycle: summer active, and torpid, interbout aroused, entrance, and arousing hibernators. Rates of state 3 and state 4 respiration were measured in vitro at 25 degrees C. Relative to mitochondria from summer-active animals, rates of state 3 respiration were significantly depressed in mitochondria from torpid animals yet fully restored during interbout arousals. These findings indicate that a depression of ADP-dependent respiration in liver mitochondria occurs during torpor and is reversed during the interbout arousals to euthermia. Because this inhibition was determined to be temporally independent of entrance and arousal, it is unlikely that active suppression of state 3 respiration causes entrance into torpor by facilitating metabolic depression. In contrast to the observed depression of state 3 respiration in torpid animals, state 4 respiration did not differ significantly among any of the five groups, suggesting that alterations in proton leak are not contributing appreciably to downregulation of respiration in hibernation.

  10. [The antioxidant enzyme activity in mouse liver mitochondria after nanosecond pulsed periodic X-ray exposure].

    PubMed

    Kniazeva, I R; Ivanov, V V; Bol'shakov, M A; Zharkova, L P; Kereia, A V; Kutenkov, O P; Rostov, V V

    2013-01-01

    The effect of repetitive pulsed X-ray (4 ns pulse duration, 300 kV accelerating voltage; 2.5 kA electron beam current) on the antioxidant enzyme activity in mouse liver mitochondria has been investigated. The mitochondrial suspension was exposed to single 4000 pulse X-ray radiation with repetition rates ranging between 10 and 22 pps (pulsed dose was 0.3-1.8 x 10(-6) Gy/pulse, the total absorbed dose following a single exposure was 7.2 x 10(-3) Gy). It was shown that a short-time exposure to X-ray radiation changes the antioxidant enzyme activity in mouse liver mitochondria. The greatest effect was observed in the changes of the activity of the metal-containing enzymes: superoxide dismutase and glutathione peroxidase. The effect depends on the pulse repetition frequency and radiation dose.

  11. Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas.

    PubMed

    Valenti, Daniela; de Bari, Lidia; De Filippis, Bianca; Ricceri, Laura; Vacca, Rosa Anna

    2014-01-01

    Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses.

  12. Studies on essential fatty acid deficiency. Effect of the deficiency on the lipids in liver mitochondria and oxidative phosphorylation

    PubMed Central

    Biran, L. A.; Bartley, W.; Carter, C. W.; Renshaw, A.

    1965-01-01

    1. Dietary deficiency of essential fatty acids results in a twofold increase in the neutral lipid content of liver mitochondria as compared with the corresponding value for stock-fed rats. 2. Deficiency produces changes in the pattern of the constituent fatty acids of the main phospholipid fractions of liver mitochondria which are similar to those previously reported for the lipids of whole liver. There is a fall in the content of C18:2 acid and to a smaller extent of C20:4 acid associated with a rise of C16:1, C18:1 and C20:3 acids. 3. Deficiency results in small decreases in the phosphorylation quotients of liver mitochondria during oxidation of succinate and pyruvate, but the values lie within the range reported for normal mitochondria. Mitochondrial respiration with succinate is decreased as a result of deficiency but no change was observed with pyruvate as substrate. PMID:14342237

  13. The Cooperative Effect of Local Angiotensin-II in Liver with Adriamycin Hepatotoxicity on Mitochondria.

    PubMed

    Taskin, Eylem; Guven, Celal; Sahin, Leyla; Dursun, Nurcan

    2016-03-28

    Adriamycin (ADR) is a drug used clinically for anticancer treatment; however, it causes adverse effects in the liver. The mechanism by which these adverse effects occur remains unclear, impeding efforts to enhance the therapeutic effects of ADR. Its hepatotoxicity might be related to increasing reactive oxygen species (ROS) and mitochondrial dysfunction. The interaction between ADR and the local renin-angiotensin system (RAS) in the liver is unclear. ADR might activate the RAS. Angiotensin-II (Ang-II) leads to ROS production and mitochondrial dysfunction. In the present study we investigated whether ADR's hepatotoxicity interacts with local RAS in causing oxidative stress resulting from mitochondrial dysfunction in the rat liver. Rats were divided into 5 groups: control, ADR, co-treated ADR with captopril, co-treated ADR with Aliskiren, and co-treated ADR with both captopril and Aliskiren. Mitochondria and cytosol were separated from the liver, then biochemical measurements were made from them. Mitochondrial membrane potential (MMP) and ATP levels were evaluated. ADR remarkably decreased MMP and ATP in liver mitochondria (p<0.05). Co-administration with ADR and Aliskiren and captopril improved the dissipation of MMP (p<0.05). The decreased ATP level was restored by treatment with inhibitors of ACE and renin. Angiotensin-II may contribute to hepatotoxicity of in the ADR via mitochondrial oxidative production, resulting in the attenuation of MMP and ATP production.

  14. The Cooperative Effect of Local Angiotensin-II in Liver with Adriamycin Hepatotoxicity on Mitochondria

    PubMed Central

    Taskin, Eylem; Guven, Celal; Sahin, Leyla; Dursun, Nurcan

    2016-01-01

    Background Adriamycin (ADR) is a drug used clinically for anticancer treatment; however, it causes adverse effects in the liver. The mechanism by which these adverse effects occur remains unclear, impeding efforts to enhance the therapeutic effects of ADR. Its hepatotoxicity might be related to increasing reactive oxygen species (ROS) and mitochondrial dysfunction. The interaction between ADR and the local renin-angiotensin system (RAS) in the liver is unclear. ADR might activate the RAS. Angiotensin-II (Ang-II) leads to ROS production and mitochondrial dysfunction. In the present study we investigated whether ADR’s hepatotoxicity interacts with local RAS in causing oxidative stress resulting from mitochondrial dysfunction in the rat liver. Material/Methods Rats were divided into 5 groups: control, ADR, co-treated ADR with captopril, co-treated ADR with Aliskiren, and co-treated ADR with both captopril and Aliskiren. Mitochondria and cytosol were separated from the liver, then biochemical measurements were made from them. Mitochondrial membrane potential (MMP) and ATP levels were evaluated. Results ADR remarkably decreased MMP and ATP in liver mitochondria (p<0.05). Co-administration with ADR and Aliskiren and captopril improved the dissipation of MMP (p<0.05). The decreased ATP level was restored by treatment with inhibitors of ACE and renin. Conclusions Angiotensin-II may contribute to hepatotoxicity of in the ADR via mitochondrial oxidative production, resulting in the attenuation of MMP and ATP production. PMID:27019222

  15. One-year high fat diet affects muscle-but not brain mitochondria.

    PubMed

    Jørgensen, Tenna; Grunnet, Niels; Quistorff, Bjørn

    2015-06-01

    It is well known that few weeks of high fat (HF) diet may induce metabolic disturbances and mitochondrial dysfunction in skeletal muscle. However, little is known about the effects of long-term HF exposure and effects on brain mitochondria are unknown. Wistar rats were fed either chow (13E% fat) or HF diet (60E% fat) for 1 year. The HF animals developed obesity, dyslipidemia, insulin resistance, and dysfunction of isolated skeletal muscle mitochondria: state 3 and state 4 were 30% to 50% increased (P<0.058) with palmitoyl carnitine (PC), while there was no effect with pyruvate as substrate. Adding also succinate in state 3 resulted in a higher substrate control ratio (SCR) with PC, but a lower SCR with pyruvate (P<0.05). The P/O2 ratio was lower with PC (P<0.004). However, similar tests on isolated brain mitochondria from the same animal showed no changes with the substrates relevant for brain (pyruvate and 3-hydroxybutyrate). Thus, long-term HF diet was associated with obesity, dyslipidemia, insulin resistance, and significantly altered mitochondrial function in skeletal muscle. Yet, brain mitochondria were unaffected. We suggest that the relative isolation of the brain due to the blood-brain barrier may play a role in this strikingly different phenotype of mitochondria from the two tissues of the same animal.

  16. One-year high fat diet affects muscle-but not brain mitochondria

    PubMed Central

    Jørgensen, Tenna; Grunnet, Niels; Quistorff, Bjørn

    2015-01-01

    It is well known that few weeks of high fat (HF) diet may induce metabolic disturbances and mitochondrial dysfunction in skeletal muscle. However, little is known about the effects of long-term HF exposure and effects on brain mitochondria are unknown. Wistar rats were fed either chow (13E% fat) or HF diet (60E% fat) for 1 year. The HF animals developed obesity, dyslipidemia, insulin resistance, and dysfunction of isolated skeletal muscle mitochondria: state 3 and state 4 were 30% to 50% increased (P<0.058) with palmitoyl carnitine (PC), while there was no effect with pyruvate as substrate. Adding also succinate in state 3 resulted in a higher substrate control ratio (SCR) with PC, but a lower SCR with pyruvate (P<0.05). The P/O2 ratio was lower with PC (P<0.004). However, similar tests on isolated brain mitochondria from the same animal showed no changes with the substrates relevant for brain (pyruvate and 3-hydroxybutyrate). Thus, long-term HF diet was associated with obesity, dyslipidemia, insulin resistance, and significantly altered mitochondrial function in skeletal muscle. Yet, brain mitochondria were unaffected. We suggest that the relative isolation of the brain due to the blood-brain barrier may play a role in this strikingly different phenotype of mitochondria from the two tissues of the same animal. PMID:25757754

  17. Catalase, a target of glycation damage in rat liver mitochondria with aging.

    PubMed

    Bakala, Hilaire; Hamelin, Maud; Mary, Jean; Borot-Laloi, Caroline; Friguet, Bertrand

    2012-10-01

    Aging is characterized by progressive decline of major cell functions, associated with accumulation of altered macromolecules, particularly proteins. This deterioration parallels age-related dysfunction of mitochondria, thought to be a major determinant of this decline in cell function, since these organelles are both the main sources of reactive oxygen species and targets for their damaging effects. To investigate the link between glycation damages that accumulate with aging and the status of mitochondrial antioxidant enzymes, we identified, by mass spectrometry after two dimensional-gel electrophoresis and western blotting, advanced glycation end product-modified matrix proteins in rat liver mitochondria. Catalase appeared to be the only antioxidant enzyme markedly glycated in old rats. Immunogold labeling performed on isolated mitochondria confirmed the mitochondrial matrix location of this enzyme. The content of catalase protein in mitochondrial extract increased with aging whereas the catalase activity was not significantly modified, in spite of a significant increase rate of glycation. Treatment of catalase with the glycating agent fructose led to significant time-dependent inactivation of the enzyme, while methylglyoxal had no noticeable effect. Catalase was co-identified with unglycated glutathione peroxidase-1 in the mitochondrial extracts. Taken together, these results indicate that both anti-oxidant enzymes catalase and glutathione peroxidase-1 housed in liver mitochondria, exhibited a differential sensitivity to glycation; moreover, they lend support to the hypothesis that glycation damages targeting catalase with aging may severely affect its activity, suggesting a link between glycation stress and the age-related decline in antioxidant defense in the mitochondria. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Some properties of purified hepatoredoxin from bovine liver mitochondria

    SciTech Connect

    Gilevich, S.N.; Gurev, O.L.; Shkumatov, V.M.; Chashchin, V.L.; Akhrem, A.A.

    1986-02-20

    Some of the most important physicochemical properties of hepatoredoxin from bovine liver, purified to a homogeneous state, were determined for the first time. The protein contains a (2Fe-2S) cluster in its active site and in an oxidized state has absorption maxima at 280, 320, 415, and 455 nm. The spectrophotometric index of purity (A/sub 415//A/sub 280/) of the homogeneous native preparation is 0.84; the extinction coefficient (epsilon/sub 415/) is equal to 9800 M/sup -1/ cm/sup -1/. According to the data of gel electrophoresis in the presence of SDS, hepatoredoxin has an M/sub r/ of 12,500; its isoelectric point (pI) is equal to 4.2. Hepatoredoxin is necessary for the reconstitution of the C/sub 27/-steroid hydroxylase activity and can be replaced by the related protein, adrenodoxin. All the parameters listed above, as well as the CD spectra, the immunochemical properties, and sequence of the first five N-terminal amino acids of hepatoredoxin and adrenodoxin are very similar of identical. At the same time, the amino acid composition of the two ferredoxins, along with common properties, has some differences.

  19. Mitochondria in traumatic brain injury and mitochondrial-targeted multipotential therapeutic strategies

    PubMed Central

    Cheng, Gang; Kong, Rong-hua; Zhang, Lei-ming; Zhang, Jian-ning

    2012-01-01

    Traumatic brain injury (TBI) is a major health and socioeconomic problem throughout the world. It is a complicated pathological process that consists of primary insults and a secondary insult characterized by a set of biochemical cascades. The imbalance between a higher energy demand for repair of cell damage and decreased energy production led by mitochondrial dysfunction aggravates cell damage. At the cellular level, the main cause of the secondary deleterious cascades is cell damage that is centred in the mitochondria. Excitotoxicity, Ca2+ overload, reactive oxygen species (ROS), Bcl-2 family, caspases and apoptosis inducing factor (AIF) are the main participants in mitochondria-centred cell damage following TBI. Some preclinical and clinical results of mitochondria-targeted therapy show promise. Mitochondria- targeted multipotential therapeutic strategies offer new hope for the successful treatment of TBI and other acute brain injuries. PMID:23003569

  20. Melatonin modulates permeability transition pore and 5-hydroxydecanoate induced KATP channel inhibition in isolated brain mitochondria.

    PubMed

    Waseem, Mohammad; Tabassum, Heena; Parvez, Suhel

    2016-11-01

    There is increasing recognition of the magnitude of mitochondria in neurodegenerative disorders. Mitochondria play a key role in apoptotic and necrotic cell death. Melatonin (Mel), an indoleamine produced in several organs including the pineal gland has been known for its neuroprotective actions. In our study, we have investigated whether the mitochondrial ATP sensitive potassium (mtKATP) channel blocker 5-hydroxydecanoate (5-HD) and calcium (Ca(2+)) affects permeability transition pore (PTP) alterations in isolated brain mitochondria treated with melatonin (Mel) and cyclosporin A (CsA). Mitochondrial swelling, mitochondrial membrane potential (Δψm), ROS measurement and mitochondrial respiration were evaluated in isolated brain mitochondria. In our results, mitochondrial swelling stimulated by exposing Ca(2+) ions and 5-HD associated by mPTP opening as depicted by modulation of CsA and Mel. In addition, Ca(2+) and 5-HD decreased Δψm, depleted intracellular ROS, and inhibition of mitochondrial respiration (state 3 and state 4) in isolated brain mitochondria. Addition of Mel and CsA has shown significant restoration in mitochondrial swelling, Δψm, intracellular ROS measurement and mitochondrial respiration in isolated brain mitochondria. Therefore, we speculate the modulatory effect of Mel and CsA in mitochondria treated with 5-HD and Ca(2+) hinders the mPTP-mediated mitochondrial dysfunction and cellular oxidative stress. We conclude that inhibition of mPT is one likely mechanism of CsA's and its neuroprotective actions. Development of neuroprotective agents including Mel targeting the mPTP therefore bears hope for future treatment of severe neurodegenerative diseases. Copyright © 2016 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  1. Why to compare absolute numbers of mitochondria.

    PubMed

    Schmitt, Sabine; Schulz, Sabine; Schropp, Eva-Maria; Eberhagen, Carola; Simmons, Alisha; Beisker, Wolfgang; Aichler, Michaela; Zischka, Hans

    2014-11-01

    Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility toward inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility toward inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations. Copyright © 2014 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

  2. Thyroid hormones regulate the onset of osmotic activity of rat liver mitochondria after birth.

    PubMed

    Almeida, A; Lopez-Mediavilla, C; Medina, J M

    1997-02-01

    The effect of thyroid hormone deprivation on the osmotic activity of liver mitochondria from early newborn rats was studied. Experimentally induced hypothyroidism prevented the increase in the osmotic activity of mitochondria observed immediately after birth. Osmotic activity was restored by T4 and T3 treatment to hypothyroid newborns but not when this treatment was supplemented with cycloheximide. Under the same circumstances, streptomycin had no effect. Hypothyroidism abolished the change in the slope of the osmotic curve (plot of inverse absorbance of mitochondrial suspensions incubated in sucrose solutions vs. inverse sucrose concentration) observed in mitochondria from euthyroid newborns at 110-120 mOsm sucrose, suggesting that hypothyroidism prevents the formation of tight physical connections between mitochondrial outer and inner membranes. Thyroid hormone deprivation increased the passive permeability of the mitochondrial inner membrane to protons, resulting in a decreased respiratory control ratio. Hypothyroidism prevented the sharp decrease in the affinity of mitochondria for ATP observed in euthyroid newborns immediately after birth. These results corroborate our previous suggestion (Endocrinology, 1995, 136:4448) that, during the early neonatal period, thyroid hormones control the synthesis of some nucleus-coded protein(s) involved in the assembly of F0,F1-ATPase.

  3. Calcium uptake in rat liver mitochondria accompanied by activation of ATP-dependent potassium channel.

    PubMed

    Akopova, O V; Nosar, V I; Mankovskaya, I N; Sagach, V F

    2008-10-01

    The influence of potassium ions on calcium uptake in rat liver mitochondria is studied. It is shown that an increase in K+ and Ca2+ concentrations in the incubation medium leads to a decrease in calcium uptake in mitochondria together with a simultaneous increase in potassium uptake due to the potential-dependent transport of K+ in the mitochondrial matrix. Both effects are more pronounced in the presence of an ATP-dependent K+-channel (K+(ATP)-channel) opener, diazoxide (Dz). Activation of the K+(ATP)-channel by Dz alters the functional state of mitochondria and leads to an increase in the respiration rate in state 2 and a decrease in the oxygen uptake and the rate of ATP synthesis in state 3. The effect of Dz on oxygen consumption in state 3 is mimicked by valinomycin, but it is opposite to that of the classical protonophore uncoupler CCCP. It is concluded that the potential-dependent uptake of potassium is closely coupled to calcium transport and is an important parameter of energy coupling responsible for complex changes in oxygen consumption and Ca2+-transport properties of mitochondria.

  4. Lipidomic analysis and electron transport chain activities in C57BL/6J mouse brain mitochondria.

    PubMed

    Kiebish, Michael A; Han, Xianlin; Cheng, Hua; Lunceford, Adam; Clarke, Catherine F; Moon, Hwi; Chuang, Jeffrey H; Seyfried, Thomas N

    2008-07-01

    The objective of this study was to characterize the lipidome and electron transport chain activities in purified non-synaptic (NS) and synaptic (Syn) mitochondria from C57BL/6J mouse cerebral cortex. Contamination from subcellular membranes, especially myelin, has hindered past attempts to accurately characterize the lipid composition of brain mitochondria. An improved Ficoll and sucrose discontinuous gradient method was employed that yielded highly enriched mitochondrial populations free of myelin contamination. The activities of Complexes I, II, III, and II/III were lower in Syn than in NS mitochondria, while Complexes I/III and IV activities were similar in both populations. Shotgun lipidomics showed that levels of cardiolipin (Ptd(2)Gro) were lower, whereas levels of ceramide and phosphatidylserine were higher in Syn than in NS mitochondria. Coenzyme Q(9) and Q(10) was also lower in Syn than in NS mitochondria. Gangliosides, phosphatidic acid, sulfatides, and cerebrosides were undetectable in brain mitochondria. The distribution of Ptd(2)Gro molecular species was similar in both populations and formed a unique pattern, consisting of seven major molecular species groups, when arranged according to mass to charge ratios. Remodeling involving choline and ethanolamine phosphoglycerides could explain Ptd(2)Gro heterogeneity. NS and Syn mitochondrial lipidomic heterogeneity could influence energy metabolism, which may contribute to metabolic compartmentation of the brain.

  5. Expression of mitochondria-related genes is elevated in overfeeding-induced goose fatty liver.

    PubMed

    Osman, Rashid H; Shao, Dan; Liu, Long; Xia, Lili; Sun, Xiaoxian; Zheng, Yun; Wang, Laidi; Zhang, Rui; Zhang, Yihui; Zhang, Jun; Gong, Daoqing; Geng, Tuoyu

    2016-02-01

    Mitochondrion, the power house of the cell, is an important organelle involving in energy homeostasis. Change in mitochondrial mass and function may lead to metabolic disorders. Previous studies indicate that mitochondrial mass loss and dysfunction are associated with non-alcoholic fatty liver disease (NAFLD) in human and mouse. However, it is unclear whether mitochondrial genes are involved in the development of goose fatty liver. To address this, we determined the response of goose mitochondrial genes to overfeeding and other fatty liver-related factors (e.g., hyperinsulinemia, hyperglycemia, and hyperlipidemia). We first employed RNA-seq technology to determine the differentially expressed genes in the livers from normally-fed vs. overfed geese, followed by bioinformatics analysis and quantitative PCR validation. Data indicated that a majority of mitochondrial genes in the liver were induced by overfeeding. To understand how these genes are regulated in the context of fatty liver, we treated goose primary hepatocytes with high levels of glucose, fatty acids and insulin. The results indicated that these factors had an influence on the expression of some mitochondria related genes. Together, these findings suggest that the induction of mitochondrial gene expression by overfeeding is required for the development of goose fatty liver, and this induction is partially attributable to hyperglycemia, hyperlipidemia and hyperinsulinemia. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

    PubMed

    Brand, M D; Lehninger, A L

    1975-10-10

    The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.

  7. Oxidative phosphorylation accompanying oxidation of short-chain fatty acids by rat-liver mitochondria

    PubMed Central

    Hird, F. J. R.; Weidemann, M. J.

    1966-01-01

    1. The factors concerned in the estimation of P/O ratios when fatty acids are oxidized by rat-liver mitochondria have been assessed. 2. The oxidation of butyrate, hexanoate and octanoate is accompanied by ATP synthesis. At low concentrations of the fatty acids, P/O ratios approximately 2·5 are obtained. 3. Oxidative phosphorylation is uncoupled, respiratory control ratios are lowered and respiration is inhibited when the concentration of the fatty acid in the incubating medium is raised (to 5–10mm); octanoate is a more potent uncoupler than either hexanoate or butyrate. 4. Serum albumin and carnitine, either singly or in combination, protect the mitochondria from the effect exerted by the fatty acids. 5. The rate of oxidation of short-chain fatty acids in the presence of ADP is increased in the presence of carnitine. PMID:4223170

  8. Mitochondria-associated microRNAs in rat hippocampus following traumatic brain injury.

    PubMed

    Wang, Wang-Xia; Visavadiya, Nishant P; Pandya, Jignesh D; Nelson, Peter T; Sullivan, Patrick G; Springer, Joe E

    2015-03-01

    Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI; however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI; however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12h following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI.

  9. Mitochondria-associated microRNAs in rat hippocampus following traumatic brain injury

    PubMed Central

    Wang, Wang-Xia; Visavadiya, Nishant P.; Pandya, Jignesh D.; Nelson, Peter T.; Sullivan, Patrick G.; Springer, Joe E.

    2015-01-01

    Traumatic brain injury (TBI) is a major cause of death and disability. However, the molecular events contributing to the pathogenesis are not well understood. Mitochondria serve as the powerhouse of cells, respond to cellular demands and stressors, and play an essential role in cell signaling, differentiation, and survival. There is clear evidence of compromised mitochondrial function following TBI, however, the underlying mechanisms and consequences are not clear. MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression post-transcriptionally, and function as important mediators of neuronal development, synaptic plasticity, and neurodegeneration. Several miRNAs show altered expression following TBI, however, the relevance of mitochondria in these pathways is unknown. Here, we present evidence supporting the association of miRNA with hippocampal mitochondria, as well as changes in mitochondria-associated miRNA expression following a controlled cortical impact (CCI) injury in rats. Specifically, we found that the miRNA processing proteins Argonaute (AGO) and Dicer are present in mitochondria fractions from uninjured rat hippocampus, and immunoprecipitation of AGO associated miRNA from mitochondria suggests the presence of functional RNA-induced silencing complexes. Interestingly, RT-qPCR miRNA array studies revealed that a subset of miRNA is enriched in mitochondria relative to cytoplasm. At 12 hour following CCI, several miRNAs are significantly altered in hippocampal mitochondria and cytoplasm. In addition, levels of miR-155 and miR-223, both of which play a role in inflammatory processes, are significantly elevated in both cytoplasm and mitochondria. We propose that mitochondria-associated miRNAs may play an important role in regulating the response to TBI. PMID:25562527

  10. 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.

  11. Taurine treatment preserves brain and liver mitochondrial function in a rat model of fulminant hepatic failure and hyperammonemia.

    PubMed

    Jamshidzadeh, Akram; Heidari, Reza; Abasvali, Mozhgan; Zarei, Mehdi; Ommati, Mohammad Mehdi; Abdoli, Narges; Khodaei, Forouzan; Yeganeh, Yasaman; Jafari, Faezeh; Zarei, Azita; Latifpour, Zahra; Mardani, Elnaz; Azarpira, Negar; Asadi, Behnam; Najibi, Asma

    2017-02-01

    Ammonia-induced mitochondrial dysfunction and energy crisis is known as a critical consequence of hepatic encephalopathy (HE). Hence, mitochondria are potential targets of therapy in HE. The current investigation was designed to evaluate the role of taurine treatment on the brain and liver mitochondrial function in a rat model of hepatic encephalopathy and hyperammonemia. The animals received thioacetamide (400mg/kg, i.p, for three consecutive days at 24-h intervals) as a model of acute liver failure and hyperammonemia. Several biochemical parameters were investigated in the serum, while the animals' cognitive function and locomotor activity were monitored. Mitochondria was isolated from the rats' brain and liver and several indices were assessed in isolated mitochondria. Liver failure led to cognitive dysfunction and impairment in locomotor activity in the rats. Plasma and brain ammonia was high and serum markers of liver injury were drastically elevated in the thioacetamide-treated group. An assessment of brain and liver mitochondrial function in the thioacetamide-treated animals revealed an inhibition of succinate dehydrogenase activity (SDA), collapsed mitochondrial membrane potential, mitochondrial swelling, and increased reactive oxygen species (ROS). Furthermore, a significant decrease in mitochondrial ATP was detected in the brain and liver mitochondria isolated from thioacetamide-treated animals. Taurine treatment (250, 500, and 1000mg/kg) decreased mitochondrial swelling, ROS, and LPO. Moreover, the administration of this amino acid restored brain and liver mitochondrial ATP. These data suggest taurine to be a potential protective agent with therapeutic capability against hepatic encephalopathy and hyperammonemia-induced mitochondrial dysfunction and energy crisis.

  12. Deleterious effects of disulfiram on the respiratory electron transport system of liver mitochondria.

    PubMed

    Kuroda, M A; Cuéllar, A

    1993-01-01

    1. The mechanism of action of disulfiram on the respiratory electron transport system of the liver mitochondria was studied in vitro. 2. Disulfiram inhibited the respiration supported by malate-glutamate as well as succinate. 3. Mitochondrial respiration inhibition was dependent upon alteration of -SH groups. 4. The inhibitory action of disulfiram might be related to the crosslinking of several proteins of the inner mitochondrial membrane. 5. The effects described above could be attributed to disulfiram per se and not to the main metabolite diethyldithiocarbamate.

  13. [Respiration and oxidative phosphorylation in liver mitochondria of bass (Morone labrax) and their dependence on temperature].

    PubMed

    Ventrella, V; Pagliarani, A; Trigari, G; Borgatti, A R

    1982-12-15

    In the present paper the respiratory function in bass liver mitochondria is studied over the temperature range 6-34 degrees C. The respiratory rate in state 3 and state 4 at the temperatures examined agrees with data from poikiloterms reported elsewhere by other investigators ICR and ADP/O values with glutamate and succinate as substrates are considered and their variations as a function of the experimental temperature are discussed. ICR shows a maximum at 20 degrees C which approximatively corresponds with the temperature of the seawater where these fishes were bred. On the contrary ADP/O ratio does not show any meaningful variation.

  14. [Arrhenius diagrams of respiratory enzymes of liver mitochondria from bass (Morone labrax)].

    PubMed

    Borgatti, A R; Trigari, G; Pagliarani, A; Ventrella, V

    1982-12-15

    Arrhenius plot of glutamate, succinate and ascorbate+TMPD oxidation in bass liver mitochondria show a break at different temperatures. Above the break activation energies (Ea) of the three enzymes. Above the break activation energies (Ea) of the three enzymes examined are similar and comparable with literature data in poikilotermic and homeothermic animals. Below the break the Ea are again comparable with poikiloterm and homeotherm ones except for succinate-oxidase whose Ea is surprisingly higher. The data are suggested to be due to the features of the enzymes or to the microenvironmental physical state.

  15. Brain mitochondria from rats treated with sulforaphane are resistant to redox-regulated permeability transition.

    PubMed

    Greco, Tiffany; Fiskum, Gary

    2010-12-01

    Oxidative stress promotes Ca2+-dependent opening of the mitochondrial inner membrane permeability transition pore (PTP), causing bioenergetic failure and subsequent cell death in many paradigms, including those related to acute brain injury. One approach to pre-conditioning against oxidative stress is pharmacologic activation of the Nrf2/ARE pathway of antioxidant gene expression by agents such as sulforaphane (SFP). This study tested the hypothesis that administration of SFP to normal rats increases resistance of isolated brain mitochondria to redox-sensitive PTP opening. SFP or DMSO vehicle was administered intraperitoneally to adult male rats at 10 mg/kg 40 h prior to isolation of non-synaptic brain mitochondria. Mitochondria were suspended in medium containing a respiratory substrate and were exposed to an addition of Ca2+ below the threshold for PTP opening. Subsequent addition of tert-butyl hydroperoxide (tBOOH) resulted in a cyclosporin A-inhibitable release of accumulated Ca2+ into the medium, as monitored by an increase in fluorescence of Calcium Green 5N within the medium, and was preceded by a decrease in the autofluorescence of mitochondrial NAD(P)H. SFP treatment significantly reduced the rate of tBOOH-induced Ca2+ release but did not affect NAD(P)H oxidation or inhibit PTP opening induced by the addition of phenylarsine oxide, a direct sulfhydryl oxidizing agent. SFP treatment had no effect on respiration by brain mitochondria and had no effect on PTP opening or respiration when added directly to isolated mitochondria. We conclude that SFP confers resistance of brain mitochondria to redox-regulated PTP opening, which could contribute to neuroprotection observed with SFP.

  16. 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.

  17. Butylated hydroxytoluene prevents cumene hydroperoxide-induced Ca2+ release from liver mitochondria by inhibiting pyridine nucleotide hydrolysis.

    PubMed

    Gogvadze, V; Kass, G E; Boyer, C S; Zhukova, A; Kim, Y; Orrenius, S

    1992-06-15

    The mechanism by which the free radical scavenger butylated hydroxytoluene (BHT) prevents cumene hydroperoxide-induced Ca2+ release from rat liver mitochondria was studied. In Ca(2+)-loaded mitochondria cumene hydroperoxide induced a rapid oxidation and subsequent hydrolysis of the pyridine nucleotides. In the presence of BHT, pyridine nucleotide oxidation by cumene hydroperoxide occurred but was reversible as hydrolysis was prevented by BHT. However, the addition of BHT directly to rat liver submitochondrial particles did not inhibit NAD+ hydrolysis or the formation of ADP-ribose from NAD+. Thus, whilst BHT prevented NAD+ hydrolysis in isolated mitochondria, this appeared not to be due to a direct effect of BHT on the NADase. It is concluded that the mechanism of action of BHT on cumene hydroperoxide-induced Ca2+ release from mitochondria involves the inhibition of pyridine nucleotide hydrolysis by an indirect mechanism rather than the radical scavenging properties of BHT.

  18. Nicotinamide nucleotide transhydrogenase (Nnt) links the substrate requirement in brain mitochondria for hydrogen peroxide removal to the thioredoxin/peroxiredoxin (Trx/Prx) system.

    PubMed

    Lopert, Pamela; Patel, Manisha

    2014-05-30

    Mitochondrial reactive oxygen species are implicated in the etiology of multiple neurodegenerative diseases, including Parkinson disease. Mitochondria are known to be net producers of ROS, but recently we have shown that brain mitochondria can consume mitochondrial hydrogen peroxide (H2O2) in a respiration-dependent manner predominantly by the thioredoxin/peroxiredoxin system. Here, we sought to determine the mechanism linking mitochondrial respiration with H2O2 catabolism in brain mitochondria and dopaminergic cells. We hypothesized that nicotinamide nucleotide transhydrogenase (Nnt), which utilizes the proton gradient to generate NADPH from NADH and NADP(+), provides the link between mitochondrial respiration and H2O2 detoxification through the thioredoxin/peroxiredoxin system. Pharmacological inhibition of Nnt in isolated brain mitochondria significantly decreased their ability to consume H2O2 in the presence, but not absence, of respiration substrates. Nnt inhibition in liver mitochondria, which do not require substrates to detoxify H2O2, had no effect. Pharmacological inhibition or lentiviral knockdown of Nnt in N27 dopaminergic cells (a) decreased H2O2 catabolism, (b) decreased NADPH and increased NADP(+) levels, and (c) decreased basal, spare, and maximal mitochondrial oxygen consumption rates. Nnt-deficient cells possessed higher levels of oxidized mitochondrial Prx, which rendered them more susceptible to steady-state increases in H2O2 and cell death following exposure to subtoxic levels of paraquat. These data implicate Nnt as the critical link between the metabolic and H2O2 antioxidant function in brain mitochondria and suggests Nnt as a potential therapeutic target to improve the redox balance in conditions of oxidative stress associated with neurodegenerative diseases.

  19. Protective effect of Cichorium glandulosum seeds from ultraviolet B-induced damage in rat liver mitochondria.

    PubMed

    Huang, Bo; Chen, Yuxin; Ma, Bingxin; Zhou, Gao; Tong, Jing; He, Jingsheng; Wang, Youwei

    2014-05-01

    Cichorium glandulosum Boiss. et Huet, a common herb for treating hepatitis, is indigenous to Europe, Western Asia, and the Xinjiang Uygur Autonomous Region of China. This study aims at evaluating the protective activity of different extracts from C. glandulosum seeds against experimental oxidation- and ultraviolet B (UVB)-induced damage in rat liver mitochondria. The antioxidant property of different extracts from C. glandulosum seeds was investigated by employing various established in vitro systems, such as α,α-diphenyl-β-picrylhydrazyl, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid), and reducing power assay. The protective effects of different C. glandulosum seed extracts against UVB-induced phototoxicity in a mitochondria model were also evaluated by measuring thiobarbituric acid reactive substances, glutathione, lipid hydroperoxide, conjugated diene, and 4-hydroxynonenal. The main compounds in C. glandulosum seeds were identified by HPLC-PDA-ESI-MS/MS. The results showed that C. glandulosum seed extracts have strong antioxidant activity, in which the ethyl acetate extract (EE) and n-butanol extract (BE) showed better activity than other extracts. In a UVB-induced mitochondria model, both EE and BE have better antioxidant activity and protective effects against phototoxicity than the petroleum ether extract, chloroform extract, and water extract. The differences in antioxidant activity and photoprotective capacity among these five extracts are associated with their phenolic compound content. Therefore, research on this function of C. glandulosum seeds may broaden their applications in the food and medical industry.

  20. Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.

    PubMed

    Mukherjee, Konark; Clark, Helen R; Chavan, Vrushali; Benson, Emily K; Kidd, Grahame J; Srivastava, Sarika

    2016-07-09

    Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer's and Parkinson's diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on

  1. Glucagon treatment of rats activates the respiratory chain of liver mitochondria at more than one site.

    PubMed

    Halestrap, A P

    1987-02-18

    The rate of reduction of ferricyanide in the presence and absence of antimycin and ubiquinone-1 was measured using liver mitochondria from control and glucagon treated rats. Glucagon treatment was shown to increase electron flow from both NADH and succinate to ubiquinone, and from ubiquinone to cytochrome c. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was shown to inhibit the oxidation of glutamate + malate to a much greater extent than that of succinate or duroquinol. Spectral and kinetic studies confirmed that electron flow between NADH and ubiquinone was the primary site of action but that the interaction of the ubiquinone pool with complex 3 was also affected. The effects of various respiratory chain inhibitors on the rate of uncoupled oxidation of succinate and glutamate + malate by control and glucagon treated mitochondria were studied. The stimulation of respiration seen in the mitochondria from glucagon treated rats was maintained or increased as respiration was progressively inhibited with DCMU, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO) and colletotrichin, but greatly reduced when inhibition was produced with malonate or antimycin. These data were also shown to support the conclusion that glucagon treatment may cause some stimulation of electron flow through NADH dehydrogenase, succinate dehydrogenase and through the bc1 complex, probably at the point of interaction of the complexes with the ubiquinone pool. The effects of glucagon treatment on duroquinol oxidation and the inhibitor titrations could not be mimicked by increasing the matrix volume, nor totally reversed by aging of mitochondria. These are both processes that have been suggested as the means by which glucagon exerts its effects on the respiratory chain (Armston, A.E., Halestrap, A.P. and Scott, R.D., 1982, Biochim. Biophys. Acta 681, 429-439). It is concluded that an additional mechanism for regulating electron flow must exist and a

  2. Glucagon effects on the membrane potential and calcium uptake rate of rat liver mitochondria

    SciTech Connect

    Wingrove, D.E.; Amatruda, J.M.; Gunter, T.E.

    1984-08-10

    It has been widely reported that the in vivo administration of glucagon to rats results in the stimulation of calcium influx in subsequently isolated liver mitochondria. The mechanism of this effect is investigated through simultaneous measurements of calcium uptake rate and mitochondrial membrane potential. This allows the measurement of the calcium uniporter conductance independent of hormonal effects on electron transport or respiration. Two experimental approaches are used. The first involves measuring the uptake of 40-50 nmol of Ca/sup 2 +//mg of mitochondrial protein with the calcium dye antipyrylazo III; the second uses /sup 45/Ca/sup 2 +/ to follow uptake in the presence of 0.5 to 1.5 ..mu..M free calcium, buffered with HEDTA. In both cases a tetraphenyl phosphonium electrode is used to follow membrane potential, and membrane potential is varied using either malonate or butylmalonate in the presence of rotenone. The relative merits of these two approaches are discussed. The conductance of the calcium uniporter is found not to be stimulated by glucagon pretreatment. Also, the relative glucagon stimulation of both calcium influx and membrane potential is found to increase with increasing malonate concentration. These results imply that there is no direct stimulation of calcium uptake into liver mitochondria following glucagon treatment. The results are consistent with a glucagon stimulation of substrate transport, substrate oxidation, or a stimulation of electron transport resulting in an increased membrane potential and secondary stimulation of calcium uptake.

  3. [Study of DNA-binding proteins in mitochondria of rat liver gamma-irradiation].

    PubMed

    Kutsyĭ, M P; Guliaeva, N A; Kuznetsova, E A; Gaziev, A I

    2005-01-01

    Acid-soluble proteins were isolated from the liver mitochondria of control and irradiated (8 Gy) rats. By means of electrophoresis in 15% polyacrylamide gel, these proteins were separated into more than 20 polypeptides of molecular masses between 10 and 120 kDa. The irradiation of rats with a dose of 8 Gy led to changes in the polypeptide content of mitochondrial acid-soluble proteins in the postradiation period. It was found that the liver acid-soluble proteins of control and irradiated rats were able to form nucleoproteid complexes with DNA at the physiological NaCl concentration. It was shown that along with mitochondrial acid-soluble proteins, proteases were also released, their activity increased in the presence of DNA. Twenty four hours after irradiation of rats with 8 Gy, the activity of proteases cleaving mitochondrial acid-soluble proteins decreased. Probably, the acid-soluble proteins and DNA-activated proteases of mitochondria are involved in the regulation of the structural organization and functional activity of mitochondrial DNA.

  4. Catabolism of 5-aminolevulinic acid to CO2 by rat liver mitochondria.

    PubMed

    Medeiros, M H; Di Mascio, P; Gründel, S; Soboll, S; Sies, H; Bechara, E J

    1994-04-01

    5-Aminolevulinic acid (ALA), the heme precursor accumulated in plasma and several organs of carriers of acute intermittent porphyria, hereditary tyrosinemia, and saturnism, was previously shown to yield reactive oxygen species upon metal-catalyzed aerobic oxidation and to cause the in vivo and in vitro impairment of rat liver mitochondrial functions. We have studied the uptake and catabolism of [5-14C]ALA to CO2 by isolated rat liver mitochondria (RLM) with the aim of determining whether possible ALA-driven oxidative injury to mitochondria can also occur into the matrix. Using silicone oil centrifugation of [5-14C]ALA-treated RLM, ALA was found to partition evenly into the intra- and extramatrix space of the mitochondrial preparations. The yield of evolved 14CO2 is very low (0.2%), responds to the concentration of added ADP, and is inhibited by malonate (75% at 2 mM), iproniazid (45% at 2 mM), beta-chloroalanine (36% at 1 mM), and aminooxyacetate (55% at 0.1 mM). With both iproniazid and aminooxyacetate, the percentage of inhibition is the same as that observed with the latter inhibitor alone. These data indicate that ALA decarboxylation by the Krebs cycle is a minor process and that it is initiated enzymically (transaminase) and not by metal-catalyzed ALA autoxidation.

  5. Regulation of free Ca2+ by liver mitochondria and endoplasmic reticulum.

    PubMed

    Becker, G L; Fiskum, G; Lehninger, A L

    1980-10-10

    Electrode measurements were made of the free Ca2+ concentration maintained by suspensions of isolated rat liver mitochondria and microsomes, as well as by hepatocytes whose plasma membranes had been made permeable by treatment with digitonin. When the KCl, ATP, Mg2+, and phosphate concentrations were made similar to that of cytosol, the steady state free Ca2+ concentration in the presence of respiring mitochondria alone was about 0.5 microM. The additional presence of rat liver microsomes resulted in a steady state level of close to 0.2 microM, which was maintaied for greater than 1 h at 25 degrees C. This concentration of Ca2+ was also maintained by suspensions of hepatocytes permeabilized by digitonin and thus may approximate the actual cytosolic free Ca2+ concentration in vivo. The "set point" for free Ca2+ homeostasis in these systems is determined by mitochondrial Ca2+ influx-efflux cycling, which is dependent on the level of intramitochondrial Ca2+ and can be adjusted by sequestration of Ca2+ in microsomes.

  6. Purified acetaminophen-glutathione conjugate is able to induce oxidative stress in rat liver mitochondria.

    PubMed

    Roušar, T; Nýdlová, E; Česla, P; Staňková, P; Kučera, O; Pařík, P; Červinková, Z

    2012-01-01

    Acetaminophen overdose is the most often cause of acute liver injury. The toxic mechanism is linked to formation of an active metabolite that reacts with glutathione generating acetaminophen-glutathione conjugate (APAP-SG). This compound has been recognized to be non-toxic generally. Our preliminary results showed, however, that APAP-SG could possess a toxic effect too. Therefore, the aim of our study was to prepare, purify and to test possible toxicity of APAP-SG. We prepared APAP-SG using organic synthesis. The conjugate was purified by preparative HPLC and its structure was confirmed using mass spectrometry. Final purity of APAP-SG was >98 %. We estimated a toxic effect of APAP-SG in isolated rat liver mitochondria using a fluorescent ROS probe. We assessed ROS production in presence of complex I or complex II substrates. The increase of ROS-dependent fluorescence in presence of glutamate/malate was 104 ± 13 % and 130 ± 10 % in 1 mM and 5 mM APAP-SG, respectively, in comparison with controls. ROS production related to presence of complex II substrate was enhanced 4-times in APAP-SG (5 mM) treated mitochondria (compared to controls). We conclude, we proved our hypothesis that APAP-SG conjugate is able to induce a mitochondrial impairment leading to enhanced ROS production.

  7. Inhibition of ATP synthesis by fenbufen and its conjugated metabolites in rat liver mitochondria.

    PubMed

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-03-01

    Fenbufen is an arylpropionic acid derivative belonging to the group of non-steroidal anti-inflammatory drugs (NSAIDs). Even though fenbufen is considered a safe drug, some adverse reactions including hepatic events have been reported. To investigate whether mitochondrial damage could be involved in the drug induced liver injury (DILI) by fenbufen, the inhibitory effect of fenbufen and its conjugated metabolites on oxidative phosphorylation (ATP synthesis) in rat liver mitochondria was investigated. Fenbufen glucuronide (F-GlcA), fenbufen-N-acetyl cysteine-thioester (F-NAC) and fenbufen-S-glutathione thioester (F-SG) were found to be more potent inhibitors compared to parent fenbufen (F), whereas fenbufen-O-carnitine (F-carn), fenbufen-glycine (F-gly) and fenbufen-N-acetyl lysine amide (F-NAL) were less potent compared to fenbufen. Fenbufen-CoA thioester (F-CoA) was equally potent as fenbufen in inhibiting ATP synthesis. Fenbufen showed time and concentration dependent inhibition of ATP synthesis with Kinact of 4.4 min(-1) and KI of 0.88 μM and Kinact/KI ratio of 5.01 min(-1) μM(-1). Data show that fenbufen did not act through opening MPT pore, nor did incubation of mitochondria with reduced GSH and fenbufen show any protective effect on fenbufen mediated inhibition of oxidative phosphorylation. Inclusion of NADPH in mitochondrial preparations with fenbufen did not modulate the inhibitory effects, suggesting no role of CYP mediated oxidative metabolites on the ATP synthesis in isolated mitochondria. The results from the present experiments provide evidence that fenbufen and its metabolites could be involved in mitochondrial toxicity through inhibition of ATP synthesis.

  8. Comparison of three methods for mitochondria isolation from the human liver cell line (HepG2)

    PubMed Central

    Azimzadeh, Pedram; Asadzadeh Aghdaei, Hamid; Tarban, Peyman; Akhondi, Mohammad Mahdi; Shirazi, Abolfazl; Khorram Khorshid, Hamid Reza

    2016-01-01

    Aim: The aim of this study was to evaluate and compare three available methods for mitochondrial isolation from a human cell line to predict the best method for each probable application. Background: Organelle isolation is gaining importance in experimental laboratory settings. Mitochondrial dysfunction may affect tumorgenesis process. There are some evidences that transplantation of healthy, intact and active mitochondria into cells containing defective mitochondria may reduce the proliferation. Therefore, isolated mitochondria could be considered as an effective tool for assessment and management of mitochondrial related disorders. Patients and methods: Mitochondrial isolation from the human liver cell line (HepG2) was performed using two commercially available kits, including Qproteome (Qiagen) and MITOISO2 (Sigma-Aldrich), as well as a manual method. Integrity of inner membrane of mitochondria was assessed by JC-1 staining. Activity of isolated mitochondria was evaluated by DCFH-DA staining, and total yield of isolated mitochondria determined by micro-Lowry method. Finally, relative quantification using Real-time PCR was conducted to compare the mtDNA copy number of mitochondria isolated by three different methods. Results: Compared to other methods, manual kit resulted in higher yields of total amount of mitochondrial protein and mtDNA copy numbers. Isolated mitochondria by Qproteome kit, showed a higher activity. Finally, the integrity of inner-membrane of isolated mitochondria was significantly higher in Qproteome when compared with the other two methods. Conclusion: Due to differences in quality, quantity and activity of isolated mitochondria using three techniques discussed here, the method in which best-suited to each research project should be selected according to the distinct features of isolated mitochondria. PMID:27099670

  9. Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria.

    PubMed

    Tretter, Laszlo; Takacs, Katalin; Hegedus, Vera; Adam-Vizi, Vera

    2007-02-01

    Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.

  10. An Isolation Method for Assessment of Brain Mitochondria Function in Neonatal Mice with Hypoxic-Ischemic Brain Injury

    PubMed Central

    Caspersen, Casper S.; Sosunov, Alexander; Utkina-Sosunova, Irina; Ratner, Veniamin I.; Starkov, Anatoly A.; Ten, Vadim S.

    2010-01-01

    This work was undertaken to develop a method for the isolation of mitochondria from a single cerebral hemisphere in neonatal mice. Mitochondria from the normal mouse brain hemisphere isolated by the proposed method exhibited a good respiratory control ratio of 6.39 ± 0.53 during glutamate-malate-induced phosphorylating respiration. Electron microscopy showed intact mitochondria. The applicability of this method was tested on mitochondria isolated from naïve mice and their littermates subjected to hypoxic-ischemic insult. Hypoxic-ischemic insult prior to reperfusion resulted in a significant (p < 0.01) inhibition of phosphorylating respiration compared to naïve littermates. This was associated with a profound depletion of the ATP content in the ischemic hemisphere. The expression for Mn superoxide dismutase and cytochrome C (markers for the integrity of the mitochondrial matrix and outer membrane) was determined by Western blot to control for mitochondrial integrity and quantity in the compared samples. Thus, we have developed a method for the isolation of the cerebral mitochondria from a single hemisphere adapted to neonatal mice. This method may serve as a valuable tool to study mitochondrial function in a mouse model of immature brain injury. In addition, the suggested method enables us to examine the mitochondrial functional phenotype in immature mice with a targeted genetic alteration. PMID:18349523

  11. Proton leak and hydrogen peroxide production in liver mitochondria from energy-restricted rats.

    PubMed

    Ramsey, Jon J; Hagopian, Kevork; Kenny, Teresa M; Koomson, Edward K; Bevilacqua, Lisa; Weindruch, Richard; Harper, Mary-Ellen

    2004-01-01

    Energy restriction (ER), without malnutrition, is the only environmental intervention that consistently increases maximum life span in laboratory rodents. One theory proposes that a reduction in energy expenditure and reactive oxygen species production is the mechanism responsible for this action of ER. To further test this theory, proton leak, H2O2 production, lipid peroxidation, and protein carbonyls were measured in mitochondria from FBNF1 rats fed either a control or 40% ER diet (onset at 6 mo of age). Liver mitochondria were isolated at 7 and 12 mo of age. Liver weight decreased 25 and 36% at 1 and 6 mo of ER, respectively (P < 0.05). ER resulted in an increase (P < 0.05) in percent total polyunsaturates, n-6 polyunsaturates, and total unsaturates (6 mo only) in mitochondrial lipids. These changes, however, were not associated with significant alterations in mitochondrial function. State 4 respiration and membrane potential were not different (P > 0.05) between groups at either assessment period. Similarly, proton leak kinetics were not different between control and ER animals. Top-down metabolic control analysis and its extension, elasticity analysis, were used at the 6-mo assessment and revealed no difference in control of the oxidative phosphorylation system between control and ER rats. H2O2 production with either succinate or pyruvate/malate substrates was also not different (P > 0.05) between groups at either time point. In conclusion, ER did not alter proton leak or H2O2 production at this age or stage of restriction in liver.

  12. Inner- and outer-membrane enzymes of mitochondria during liver regeneration

    PubMed Central

    Gear, A. R. L.

    1970-01-01

    1. Marker enzymes for the mitochondrial matrix, inner membrane, inter-membrane space and outer membrane were measured in mitochondria isolated from control and regenerating rat liver. The specific activity of these enzymes was then followed for up to 30 days after operation. 2. The specific activity of marker enzymes for the matrix, inner membrane and inter-membrane space remained constant during liver regeneration. 3. However, the specific activities of monoamine oxidase and kynurenine hydroxylase, both outer-membrane markers, fell by 67% and 49% respectively from their control values at 4 days after operation, and returned to normal by about 3 weeks. 4. The repression of kynurenine hydroxylase activity was shown to be unrelated to any independent variation in tryptophan catabolism, based on tryptophan pyrrolase assays. 5. These results are considered to indicate that enzymes of the inner and outer mitochondrial membranes are synthesized asynchronously during morphogenesis. 6. The enzyme complement of purified outer membrane at 4 days after operation was about 50% of that of the appropriate control. Thus the composition of the outer membrane itself may vary dramatically, and supports the concept that constitutive enzymes may turn over independently of a membrane's existence. 7. The behaviour of the rotenone-insensitive, NADH cytochrome c reductase did not parallel the other outer-membrane enzymes for intact mitochondria, but did so when assayed in highly purified fractions of outer membrane. This suggests a labile binding to the outer membrane during the early stages of morphogenesis. 8. Electrophoresis of inner- and outer-membrane proteins revealed little difference between control and experimental mitochondria at 4 days, except for an increase in several, high-molecular-weight components of the outer membrane. These bands closely correspond to similar bands derived from smooth endoplasmic reticulum. 9. The results are discussed in relation to the biogenesis and

  13. The Swelling of Rat Liver Mitochondria by Thyroxine and its Reversal

    PubMed Central

    Lehninger, Albert L.; Ray, Betty Lou; Schneider, Marion

    1959-01-01

    The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20°. Aging of mitochondria at 0° causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn++, and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the contractility and

  14. The swelling of rat liver mitochondria by thyroxine and its reversal.

    PubMed

    LEHNINGER, A L; RAY, B L; SCHNEIDER, M

    1959-01-25

    The in vitro swelling action of L-thyroxine on rat liver mitochondria as examined photometrically represents an acceleration of a process which the mitochondria are already inherently capable of undergoing spontaneously, as indicated by the identical kinetic characteristics and the extent of thyroxine-induced and spontaneous swelling, the nearly identical pH dependence, and the fact that sucrose has a specific inhibitory action on both types of swelling. However, thyroxine does not appear to be a "catalyst" or coenzyme since it does not decrease the temperature coefficient of spontaneous swelling. The temperature coefficient is very high, approximately 6.0 near 20 degrees . Aging of mitochondria at 0 degrees causes loss of thyroxine sensitivity which correlates closely with the loss of bound DPN from the mitochondria, but not with loss of activity of the respiratory chain or with the efficiency of oxidative phosphorylation. Tests with various respiratory chain inhibitors showed that the oxidation state of bound DPN may be a major determinant of thyroxine sensitivity; the oxidation state of the other respiratory carriers does not appear to influence sensitivity to thyroxine. These facts and other considerations suggest that a bound form of mitochondrial DPN is the "target" of the action of thyroxine. The thyroxine-induced swelling is not reversed by increasing the osmolar concentration of external sucrose, but can be "passively" or osmotically reversed by adding the high-particle weight solute polyvinylpyrrolidone. The mitochondrial membrane becomes more permeable to sucrose during the swelling reaction. On the other hand, thyroxine-induced swelling can be "actively" reversed by ATP in a medium of 0.15 M KCl or NaCl but not in a 0.30 M sucrose medium. The action of ATP is specific; ADP, Mn(++), and ethylenediaminetetraacetate are not active. It is concluded that sucrose is an inhibitor of the enzymatic relationship between oxidative phosphorylation and the

  15. Demethyleneberberine, a natural mitochondria-targeted antioxidant, inhibits mitochondrial dysfunction, oxidative stress, and steatosis in alcoholic liver disease mouse model.

    PubMed

    Zhang, Pengcheng; Qiang, Xiaoyan; Zhang, Miao; Ma, Dongshen; Zhao, Zheng; Zhou, Cuisong; Liu, Xie; Li, Ruiyan; Chen, Huan; Zhang, Yubin

    2015-01-01

    Excessive alcohol consumption induces oxidative stress and lipid accumulation in the liver. Mitochondria have long been recognized as the key target for alcoholic liver disease (ALD). Recently, the artificial mitochondria-targeted antioxidant MitoQ has been used to treat ALD effectively in mice. Here, we introduce the natural mitochondria-targeted antioxidant demethyleneberberine (DMB), which has been found in Chinese herb Cortex Phellodendri chinensis. The protective effect of DMB on ALD was evaluated with HepG2 cells and acutely/chronically ethanol-fed mice, mimicking two common patterns of drinking in human. The results showed that DMB, which is composed of a potential antioxidant structure, could penetrate the membrane of mitochondria and accumulate in mitochondria either in vitro or in vivo. Consequently, the acute drinking-caused oxidative stress and mitochondrial dysfunction were significantly ameliorated by DMB. Moreover, we also found that DMB suppressed CYP2E1, hypoxia inducible factor α, and inducible nitric oxide synthase, which contributed to oxidative stress and restored sirtuin 1/AMP-activated protein kinase/peroxisome proliferator-activated receptor-γ coactivator-1α pathway-associated fatty acid oxidation in chronic ethanol-fed mice, which in turn ameliorated lipid peroxidation and macrosteatosis in the liver. Taking these findings together, DMB could serve as a novel and potential therapy for ALD in human beings.

  16. Protein targets for carbonylation by 4-hydroxy-2-nonenal in rat liver mitochondria

    PubMed Central

    Guo, Jia; Prokai-Tatrai, Katalin; Ngyuen, Vien; Rauniyar, Navin; Ughy, Bettina; Prokai, Laszlo

    2011-01-01

    Protein carbonylation has been associated with various pathophysiological processes. A representative reactive carbonyl species (RCS), 4-hydroxy-2-nonenal (HNE), has been implicated specifically as a causative factor for the initiation and/or progression of various diseases. To date, however, little is known about the proteins and their modification sites susceptible to “carbonyl stress” by this RCS, especially in the liver. Using chemoprecipitation based on a solid phase hydrazine chemistry coupled with LC-MS/MS bottom-up approach and database searching, we identified several protein-HNE adducts in isolated rat liver mitochondria upon HNE exposure. The identification of selected major protein targets, such as the ATP synthase β-subunit, was further confirmed by immunoblotting and a gel-based approach in combination with LC–MS/MS. A network was also created based on the identified protein targets that showed that the main protein interactions were associated with cell death, tumor morphology and drug metabolism, implicating the toxic nature of HNE in the liver mitoproteome. The functional consequence of carbonylation was illustrated by its detrimental impact on the activity of ATP synthase, a representative major mitochondrial protein target for HNE modifications. PMID:21801862

  17. Kinetics and control of oxidative phosphorylation in rat liver mitochondria after chronic ethanol feeding.

    PubMed Central

    Marcinkeviciute, A; Mildaziene, V; Crumm, S; Demin, O; Hoek, J B; Kholodenko, B

    2000-01-01

    Changes in the kinetics and regulation of oxidative phosphorylation were characterized in isolated rat liver mitochondria after 2 months of ethanol consumption. Mitochondrial energy metabolism was conceptually divided into three groups of reactions, either producing protonmotive force (Deltap) (the respiratory subsystem) or consuming it (the phosphorylation subsystem and the proton leak). Manifestation of ethanol-induced mitochondrial malfunctioning of the respiratory subsystem was observed with various substrates; the respiration rate in State 3 was inhibited by 27+/-4% with succinate plus amytal, by 20+/-4% with glutamate plus malate, and by 17+/-2% with N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate. The inhibition of the respiratory activity correlated with the lower activities of cytochrome c oxidase, the bc(1) complex, and the ATP synthase in mitochondria of ethanol-fed rats. The block of reactions consuming the Deltap to produce ATP (the phosphorylating subsystem) was suppressed after 2 months of ethanol feeding, whereas the mitochondrial proton leak was not affected. The contributions of Deltap supply (the respiratory subsystem) and Deltap demand (the phosphorylation and the proton leak) to the control of the respiratory flux were quantified as the control coefficients of these subsystems. In State 3, the distribution of control exerted by different reaction blocks over respiratory flux was not significantly affected by ethanol diet, despite the marked changes in the kinetics of individual functional units of mitochondrial oxidative phosphorylation. This suggests the operation of compensatory mechanisms, when control redistributes among the different components within the same subsystem. PMID:10880351

  18. Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase.

    PubMed

    Masola, B; Devlin, T M

    1995-12-01

    The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.

  19. [Effect of cold and cool herbs on liver mitochondria proteome of rats with heat symptom].

    PubMed

    Li, Yi; Lu, De-Zhao; Tang, Li-Hua; Wo, Xing-De; Yang, Zhen

    2013-12-01

    In the 1960s, modern science began involving the essence of heat syndrome, but there have still no in-depth systematic studies on pathological mechanisms of heat syndrome and action mechanisms of cold and cool herbs. In this study, the animal model with heat syndrome was set up by feeding herbs with hot property, and then cold and cool herbs was applied in the experimental therapy. The two-dimensional electrophoresis and mass spectrometry technologies were adopted to compare the liver mitochondria proteome of the rats of the heat syndrome model and the ones treated with cold and cool herbs, so as to discover specificity-related proteins after heat syndrome and treatment with cold and cool herbs.

  20. Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology

    PubMed Central

    Kubalik, Nataliya; Zinchenko, Natalia; Ridings, Daisy M.; Radoff, David A.; Hemendinger, Richelle; Brooks, Benjamin R.; Bonkovsky, Herbert L.

    2011-01-01

    Mitochondrial dysfunctions contribute to neurodegeneration, the locations of which vary among neurodegenerative diseases. To begin to understand what mechanisms may underlie higher vulnerability of the spinal cord motor neurons in amyotrophic lateral sclerosis, compared with brain mitochondria, we studied three major functions of rat brain mitochondria (BM) and spinal cord mitochondria (SCM) mitochondria: oxidative phosphorylation, Ca2+ sequestration, and production of reactive oxygen species (ROS), using a new metabolic paradigm (Panov et al., J. Biol. Chem. 284: 14448–14456, 2009). We present data that SCM share some unique metabolic properties of the BM. However, SCM also have several distinctions from the BM: 1) With the exception of succinate, SCM show significantly lower rates of respiration with all substrates studied; 2) immunoblotting analysis showed that this may be due to 30–40% lower contents of respiratory enzymes and porin; 3) compared with BM, SCM sequestered 40–50% less Ca2+, and the total tissue calcium content was 8 times higher in the spinal cord; 4) normalization for mitochondria from 1 g of tissue showed that BM can sequester several times more Ca2+ than was available in the brain tissue, whereas SCM had the capacity to sequester only 10–20% of the total tissue Ca2+; and 5) with succinate and succinate-containing substrate mixtures, SCM showed significantly higher state 4 respiration than BM and generated more ROS associated with the reverse electron transport. We conclude that SCM have an intrinsically higher risk of oxidative damage and overload with calcium than BM, and thus spinal cord may be more vulnerable under some pathologic conditions. (250) PMID:21248309

  1. Glutamine transport in rat brain synaptic and non-synaptic mitochondria.

    PubMed

    Roberg, B; Torgner, I A; Kvamme, E

    1999-03-01

    Glutamine transport into rat brain mitochondria (synaptic and non-synaptic) was monitored by the uptake of [3H]glutamine as well as by mitochondrial swelling. The uptake is inversely correlated to medium osmolarity, temperature-dependent, saturable and inhibited by mersalyl, and glutamine is upconcentrated in the mitochondria. These results indicate that glutamine is transported into an osmotically active space by a protein catalyzed mechanism. The uptake is slightly higher in synaptic mitochondria than in non-synaptic ones. It is inhibited both by rotenone and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the latter at pH 6.5, showing that the transport is activated by an electrochemical proton gradient. The K+/H+ ionophore nigericin also inhibits the uptake at pH 6.5 in the presence of external K+, which indicates that glutamine, at least in part, is taken up by a proton symport transporter. In addition, glutamine uptake as measured by the swelling technique revealed an additional glutamine transport activity with at least 10 times higher Km value. This uptake is inhibited by valinomycin in the presence of K+ and is thus also activated by the membrane potential. Otherwise, the two methods show similar results. These data indicate that glutamine transport in brain mitochondria cannot be described by merely a simple electroneutral uniport mechanism, but are consistent with the uptake of both the anionic and the zwitterionic glutamine.

  2. Effect of chronic alcohol exposure on folate uptake by liver mitochondria.

    PubMed

    Biswas, Arundhati; Senthilkumar, Sundar Rajan; Said, Hamid M

    2012-01-01

    Mammalian cells obtain folate, a water-soluble vitamin, from their surroundings via transport across cell membrane. Intracellular folate is compartmentalized between the cytoplasm and the mitochondria. Transport of folate from the cytoplasm into the mitochondria is via a specific carrier-mediated process involving the mitochondrial folate transporter (MFT). Chronic alcohol use negatively impacts folate homeostasis, but its effect on mitochondrial folate uptake is not clear. We addressed this issue using mitochondrial preparations isolated from the liver of rats chronically fed an alcohol liquid diet and from human liver HepG2 cells chronically exposed to alcohol. The results showed that chronic alcohol feeding of rats leads to a significant inhibition in mitochondrial carrier-mediated folate uptake. This inhibition was associated with a significant reduction in the level of expression of the MFT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Similarly, chronic alcohol exposure (96 h) of HepG2 cells led to significant inhibition in mitochondrial carrier-mediated folate uptake, which was associated with a marked reduction in the level of expression of the human MFT (hMFT). To determine whether the latter effect is, in part, being exerted at the transcriptional level, we cloned the 5'-regulatory region of the human SLC25A32 gene (which encodes the hMFT) and showed that chronic alcohol exposure of HepG2 cells leads to a significant inhibition in its promoter activity. These studies show for the first time that chronic alcohol feeding/exposure leads to a significant inhibition in mitochondrial carrier-mediated folate uptake and that the inhibition is, in part, being exerted at the level of transcription of the SLC25A32 gene.

  3. Ca(2+) handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease.

    PubMed

    Pellman, Jessica J; Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay

    2015-08-01

    We investigated Ca(2+) handling in isolated brain synaptic and non-synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non-synaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca(2+) uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca(2+) capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca(2+) uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen because of mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca(2+) followed by recovery to near resting levels. Following recovery of cytosolic Ca(2+), mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca(2+), suggesting similar Ca(2+) release and, consequently, Ca(2+) loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca(2+) handling in brain mitochondria of YAC128 mice. We demonstrate that mutant huntingtin (mHtt) binds to brain synaptic and nonsynaptic mitochondria and the amount of mitochondria-bound mHtt correlates with increased mitochondrial Ca(2+) uptake capacity. We propose that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage. © 2015 International Society for Neurochemistry.

  4. Pro-Apoptotic Changes in Brain Mitochondria After Toxin Exposure

    DTIC Science & Technology

    2004-10-01

    with failure of functional recovery in brain slices (Rosenthal, 1995; Perez- Pinzon , 1998). The loss of intramitochondrial was calcium-dependent...Perez- Pinzon , 1998) and was inhibited with antioxidants (Perez- Pinzon , 1997). In the following experiments, we monitored intramitochondrial NADH by...approach Reportable Outcomes S. Hagioka, M.D. Ginsberg, M.A. Perez- Pinzon , T.J. Sick. Poly(adp-ribose) polymerase inhibitors prevent loss of mitochondrial

  5. 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.

  6. Brain cholinergic impairment in liver failure

    PubMed Central

    García-Ayllón, María-Salud; Cauli, Omar; Silveyra, María-Ximena; Rodrigo, Regina; Candela, Asunción; Compañ, Antonio; Jover, Rodrigo; Pérez-Mateo, Miguel; Martínez, Salvador; Felipo, Vicente

    2008-01-01

    The cholinergic system is involved in specific behavioural responses and cognitive processes. Here, we examined potential alterations in the brain levels of key cholinergic enzymes in cirrhotic patients and animal models with liver failure. An increase (∼30%) in the activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE) is observed in the brain cortex from patients deceased from hepatic coma, while the activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase, remains unaffected. In agreement with the human data, AChE activity in brain cortical extracts of bile duct ligated (BDL) rats was increased (∼20%) compared to controls. A hyperammonemic diet did not result in any further increase of AChE levels in the BDL model, and no change was observed in hyperammonemic diet rats without liver disease. Portacaval shunted rats which display increased levels of cerebral ammonia did not show any brain cholinergic abnormalities, confirming that high ammonia levels do not play a role in brain AChE changes. A selective increase of tetrameric AChE, the major AChE species involved in hydrolysis of acetylcholine in the brain, was detected in both cirrhotic humans and BDL rats. Histological examination of BDL and non-ligated rat brains shows that the subcellular localization of both AChE and choline acetyltransferase, and thus the accessibility to their substrates, appears unaltered by the pathological condition. The BDL-induced increase in AChE activity was not parallelled by an increase in mRNA levels. Increased AChE in BDL cirrhotic rats leads to a pronounced decrease (∼50–60%) in the levels of acetylcholine. Finally, we demonstrate that the AChE inhibitor rivastigmine is able to improve memory deficits in BDL rats. One week treatment with rivastigmine (0.6 mg/kg; once a day, orally, for a week) resulted in a 25% of inhibition in the enzymatic activity of AChE with no change in protein composition, as assessed by sucrose density

  7. Depleted uranium induces disruption of energy homeostasis and oxidative stress in isolated rat brain mitochondria.

    PubMed

    Shaki, Fatemeh; Hosseini, Mir-Jamal; Ghazi-Khansari, Mahmoud; Pourahmad, Jalal

    2013-06-01

    Depleted uranium (DU) is emerging as an environmental pollutant primarily due to its military applications. Gulf War veterans with embedded DU showed cognitive disorders that suggest that the central nervous system is a target of DU. Recent evidence has suggested that DU could induce oxidative stress and mitochondrial dysfunction in brain tissue. However, the underlying mechanisms of DU toxicity in brain mitochondria are not yet well understood. Brain mitochondria were obtained using differential centrifugation and were incubated with different concentrations (50, 100 and 200 μM) of uranyl acetate (UA) as a soluble salt of U(238) for 1 h. In this research, mitochondrial ROS production, collapse of mitochondrial membrane potential and mitochondrial swelling were examined by flow cytometry following the addition of UA. Meanwhile, mitochondrial sources of ROS formation were determined using specific substrates and inhibitors. Complex II and IV activity and also the extent of lipid peroxidation and glutathione (GSH) oxidation were detected via spectroscopy. Furthermore, we investigated the concentration of ATP and ATP/ADP ratio using luciferase enzyme and cytochrome c release from mitochondria which was detected by ELISA kit. UA caused concentration-dependent elevation of succinate-linked mitochondrial ROS production, lipid peroxidation, GSH oxidation and inhibition of mitochondrial complex II. UA also induced mitochondrial permeability transition, ATP production decrease and increase in cytochrome c release. Pre-treatment with antioxidants significantly inhibited all the above mentioned toxic effects of UA. This study suggests that mitochondrial oxidative stress and impairment of oxidative phosphorylation in brain mitochondria may play a key role in DU neurotoxicity as reported in Gulf War Syndrome.

  8. 2-Methylcitric acid impairs glutamate metabolism and induces permeability transition in brain mitochondria.

    PubMed

    Amaral, Alexandre Umpierrez; Cecatto, Cristiane; Castilho, Roger Frigério; Wajner, Moacir

    2016-04-01

    Accumulation of 2-methylcitric acid (2MCA) is observed in methylmalonic and propionic acidemias, which are clinically characterized by severe neurological symptoms. The exact pathogenetic mechanisms of brain abnormalities in these diseases are poorly established and very little has been reported on the role of 2MCA. In the present work we found that 2MCA markedly inhibited ADP-stimulated and uncoupled respiration in mitochondria supported by glutamate, with a less significant inhibition in pyruvate plus malate respiring mitochondria. However, no alterations occurred when α-ketoglutarate or succinate was used as respiratory substrates, suggesting a defect on glutamate oxidative metabolism. It was also observed that 2MCA decreased ATP formation in glutamate plus malate or pyruvate plus malate-supported mitochondria. Furthermore, 2MCA inhibited glutamate dehydrogenase activity at concentrations as low as 0.5 mM. Kinetic studies revealed that this inhibitory effect was competitive in relation to glutamate. In contrast, assays of osmotic swelling in non-respiring mitochondria suggested that 2MCA did not significantly impair mitochondrial glutamate transport. Finally, 2MCA provoked a significant decrease in mitochondrial membrane potential and induced swelling in Ca(2+)-loaded mitochondria supported by different substrates. These effects were totally prevented by cyclosporine A plus ADP or ruthenium red, indicating induction of mitochondrial permeability transition. Taken together, our data strongly indicate that 2MCA behaves as a potent inhibitor of glutamate oxidation by inhibiting glutamate dehydrogenase activity and as a permeability transition inducer, disturbing mitochondrial energy homeostasis. We presume that 2MCA-induced mitochondrial deleterious effects may contribute to the pathogenesis of brain damage in patients affected by methylmalonic and propionic acidemias. We propose that brain glutamate oxidation is disturbed by 2-methylcitric acid (2MCA), which

  9. Energy metabolism in rat brain: inhibition of pyruvate decarboxylation by 3-hydroxybutyrate in neonatal mitochondria.

    PubMed

    Booth, R F; Clark, J B

    1981-07-01

    The effect of 3-hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1-14C]pyruvate decarboxylation (1 mM final concentration) by brain synaptosomes derived from 8-day-old rats was inhibited by 10% in the presence of 2 mM-D,L-3-hydroxybutyrate and by more than 20% in the presence of 20 mM D,L-3-hydroxybutyrate. The presence of 2 mM-D,L-3-hydroxybutyrate did not affect the rate of [1-14C]pyruvate decarboxylation (1 mM final concentration) by brain mitochondria; however, at a concentration of 20 mM-D,L-3-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-day-old (35% inhibition) and 21-day-old (24% inhibition) but not in those from adult rats. Although the presence of 100 mM-K+ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in presence of 1 mM-K+, the presence of 20 mM-D,L-3-hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mM-K+). The presence of 20 mM-D,L-3-hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8-day-old rats. The concentrations of ATP, ADP, NAD+, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8-day-old rats incubated in the presence of 1 mM-pyruvate alone or 1 mM-pyruvate plus 20 mM-D,L-3-hydroxybutyrate. Neither the APT/ADP nor the NADH/NAD+ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3-hydroxybutyrate. The possible mechanisms and physiological significance of 3-hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain rat mitochondria are discussed.

  10. Tl+ induces the permeability transition pore in Ca2+-loaded rat liver mitochondria energized by glutamate and malate.

    PubMed

    Korotkov, Sergey M; Emelyanova, Larisa V; Konovalova, Svetlana A; Brailovskaya, Irina V

    2015-08-01

    It is known that Ca2+ and heavy metals more actively induce MPTP opening in mitochondria, energized by the I complex substrates. Thus, a rise in a Tl+-induced MPTP was proposed in experiments on isolated rat liver mitochondria energized by the complex I substrate (glutamate and malate). Expose of the mitochondria to Ca2+ into a medium containing TlNO3, glutamate, and malate as well as sucrose or KNO3 resulted in a decrease in state 3, state 4, or DNP-stimulated respiration as well as an increase of both mitochondrial swelling and ΔΨmito dissipation. The MPTP inhibitors, CsA and ADP, almost completely eliminated the effect of Ca2+, which was more pronounced in the presence of the complex I substrates than the complex II substrate (succinate) and rotenone (Korotkov and Saris, 2011). The present study concludes that Tl+-induced MPTP opening is more appreciable in mitochondria energized by glutamate and malate but not succinate in the presence of rotenone. We assume that the Tl+-induced MPTP opening along with followed swelling and possible structural deformations of the complex I in Ca2+-loaded mitochondria may be a part of the thallium toxicity mechanism on mitochondria in living organisms. At the same time, oxidation of Tl+ to Tl3+ by mitochondrial oxygen reactive species is proposed for the mechanism. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Proteomic analysis of mitochondria from embryonic and postnatal rat brains reveals response to developmental changes in energy demands

    PubMed Central

    Villeneuve, Lance M.; Stauch, Kelly L.; Fox, Howard S.

    2014-01-01

    Many biological processes converge on the mitochondria. In such systems, where many pathways converge, manipulation of the components can produce varied and far-reaching effects. Due to the centrality of the mitochondria in many cellular pathways, we decided to investigate the brain mitochondrial proteome during early development. Using a SWATH mass spectrometry-based technique, we were able to identify vast proteomic alterations between whole brain mitochondria from rats at embryonic day 18 compared to postnatal day 7. These findings include statistically significant alterations in proteins involved in glycolysis and mitochondrial trafficking/dynamics. Additionally, bioinformatic analysis enabled the identification of HIF1A and XBP1 as upstream transcriptional regulators of many of the differentially expressed proteins. These data suggest that the cell is rearranging mitochondria to accommodate special energy demands and that cytosolic proteins exert mitochondrial effects through dynamic interactions with mitochondria. PMID:25046836

  12. Acute treatment of constant darkness increases the efficiency of ATP synthase in rat liver mitochondria.

    PubMed

    Ramírez-Iñiguez, Ana Laura; Ortiz, Genaro Gabriel; El Hafidi, Mohammed; Rincón-Sánchez, Ana Rosa; Macías-Rodríguez, Ernesto; Pacheco-Moisés, Fermín Paul

    2009-01-01

    The circadian oscillations of many physiological processes provide an endogenous temporal program for the adaptive synchronization of mammals to the fluctuating external world. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system. The aim of the present work was investigate the effect of acute treatment of constant darkness on mitochondrial ATP synthase activities and membrane fluidity in liver from male rat. We found that ATP synthase activity was not changed by the treatment. However ATPase activity and membrane fluidity were significantly diminished and pH gradient driven by ATP hydrolysis was incremented, in comparison from samples from rats kept on normal light/dark cycles. Additionally, the treatment of constant darkness diminishes the passive proton permeability of the inner mitochondrial membrane. In conclusion constant darkness induces a more efficient coupling between proton transport and catalysis, and increment the efficiency of the enzyme because the ratio of ATP synthase/ATPase activity was higher. These results exhibited the physiological adaptation of liver mitochondria to acute treatment of constant darkness in order to satisfy the cellular energy demand.

  13. Effects of Epigallocatechin Gallate on Tert-Butyl Hydroperoxide-Induced Mitochondrial Dysfunction in Rat Liver Mitochondria and Hepatocytes

    PubMed Central

    Endlicher, Rene; Sobotka, Ondrej; Drahota, Zdenek

    2016-01-01

    Epigallocatechin gallate (EGCG) is a green tea antioxidant with adverse effects on rat liver mitochondria and hepatocytes at high doses. Here, we assessed whether low doses of EGCG would protect these systems from damage induced by tert-butyl hydroperoxide (tBHP). Rat liver mitochondria or permeabilized rat hepatocytes were pretreated with EGCG and then exposed to tBHP. Oxygen consumption, mitochondrial membrane potential (MMP), and mitochondrial retention capacity for calcium were measured. First, 50 μM EGCG or 0.25 mM tBHP alone increased State 4 Complex I-driven respiration, thus demonstrating uncoupling effects; tBHP also inhibited State 3 ADP-stimulated respiration. Then, the coexposure to 0.25 mM tBHP and 50 μM EGCG induced a trend of further decline in the respiratory control ratio beyond that observed upon tBHP exposure alone. EGCG had no effect on MMP and no effect, in concentrations up to 50 μM, on mitochondrial calcium retention capacity. tBHP led to a decline in both MMP and mitochondrial retention capacity for calcium; these effects were not changed by pretreatment with EGCG. In addition, EGCG dose-dependently enhanced hydrogen peroxide formation in a cell- and mitochondria-free medium. Conclusion. Moderate nontoxic doses of EGCG were not able to protect rat liver mitochondria and hepatocytes from tBHP-induced mitochondrial dysfunction. PMID:28074116

  14. Oxidative phosphorylation efficiency, proton conductance and reactive oxygen species production of liver mitochondria correlates with body mass in frogs.

    PubMed

    Roussel, Damien; Salin, Karine; Dumet, Adeline; Romestaing, Caroline; Rey, Benjamin; Voituron, Yann

    2015-10-01

    Body size is a central biological parameter affecting most biological processes (especially energetics) and the mitochondrion is a key organelle controlling metabolism and is also the cell's main source of chemical energy. However, the link between body size and mitochondrial function is still unclear, especially in ectotherms. In this study, we investigated several parameters of mitochondrial bioenergetics in the liver of three closely related species of frog (the common frog Rana temporaria, the marsh frog Pelophylax ridibundus and the bull frog Lithobates catesbeiana). These particular species were chosen because of their differences in adult body mass. We found that mitochondrial coupling efficiency was markedly increased with animal size, which led to a higher ATP production (+70%) in the larger frogs (L. catesbeiana) compared with the smaller frogs (R. temporaria). This was essentially driven by a strong negative dependence of mitochondrial proton conductance on body mass. Liver mitochondria from the larger frogs (L. catesbeiana) displayed 50% of the proton conductance of mitochondria from the smaller frogs (R. temporaria). Contrary to our prediction, the low mitochondrial proton conductance measured in L. catesbeiana was not associated with higher reactive oxygen species production. Instead, liver mitochondria from the larger individuals produced significantly lower levels of radical oxygen species than those from the smaller frogs. Collectively, the data show that key bioenergetics parameters of mitochondria (proton leak, ATP production efficiency and radical oxygen species production) are correlated with body mass in frogs. This research expands our understanding of the relationship between mitochondrial function and the evolution of allometric scaling in ectotherms.

  15. ATP synthase-mediated proton fluxes and phosphorylation in rat liver mitochondria: dependence on delta mu H.

    PubMed

    Zoratti, M; Petronilli, V; Azzone, G F

    1986-08-13

    The dependence of the proton flux through the ATP synthases of rat liver mitochondria on a driving force composed mainly of a potassium diffusion potential was determined and compared with the relationship between rate of phosphorylation and delta mu H given by titrations with the respiratory inhibitor malonate. The two functions are in good agreement in the lower part of the delta mu H range covered. However, the maximal proton fluxes through the ATP synthases are much lower than needed to account for the rate of State 3 phosphorylation sustained by the same mitochondria oxidizing succinate. Possible reasons for this behavior are discussed.

  16. Mitochondrial nitric oxide synthase, mitochondrial brain dysfunction in aging, and mitochondria-targeted antioxidants.

    PubMed

    Navarro, Ana; Boveris, Alberto

    2008-01-01

    This paper reviews the current ideas on nitric oxide (NO) physiology in brain and other mammalian organs and on the subcellular distribution of nitric oxide synthases (NOS) emphasizing on the evidence of a mitochondrial NOS isoform (mtNOS) that exhibits a mean activity of 0.86+/-0.09 nmol NO/min x mg protein in 13 mouse and rat organs. Mammalian brain aging is associated with mitochondrial dysfunction, determined as decreased electron transfer and enzymatic activities and as an increased content of phospholipid oxidation products and of protein oxidation/nitration products. Brain mtNOS is the most decreased enzymatic activity upon aging; decreased levels of NO are interpreted as the cause of decreased mitochondrial biogenesis in aged brain. The beneficial effect of high doses of vitamin E on mice survival and neurological function are related to its effect as antioxidant in brain mitochondria and to the preservation of mtNOS activity. Mitochondria-targeted antioxidants, phosphonium cation derivatives and antioxidant tetrapeptides, are reviewed in terms of structures and biological effects.

  17. Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax.

    PubMed

    Starkov, Anatoly A; Polster, Brian M; Fiskum, Gary

    2002-10-01

    Abnormal accumulation of Ca2+ and exposure to pro-apoptotic proteins, such as Bax, is believed to stimulate mitochondrial generation of reactive oxygen species (ROS) and contribute to neural cell death during acute ischemic and traumatic brain injury, and in neurodegenerative diseases, e.g. Parkinson's disease. However, the mechanism by which Ca2+ or apoptotic proteins stimulate mitochondrial ROS production is unclear. We used a sensitive fluorescent probe to compare the effects of Ca2+ on H2O2 emission by isolated rat brain mitochondria in the presence of physiological concentrations of ATP and Mg2+ and different respiratory substrates. In the absence of respiratory chain inhibitors, Ca2+ suppressed H2O2 generation and reduced the membrane potential of mitochondria oxidizing succinate, or glutamate plus malate. In the presence of the respiratory chain Complex I inhibitor rotenone, accumulation of Ca2+ stimulated H2O2 production by mitochondria oxidizing succinate, and this stimulation was associated with release of mitochondrial cytochrome c. In the presence of glutamate plus malate, or succinate, cytochrome c release and H2O2 formation were stimulated by human recombinant full-length Bax in the presence of a BH3 cell death domain peptide. These results indicate that in the presence of ATP and Mg2+, Ca2+ accumulation either inhibits or stimulates mitochondrial H2O2 production, depending on the respiratory substrate and the effect of Ca2+ on the mitochondrial membrane potential. Bax plus a BH3 domain peptide stimulate H2O2 production by brain mitochondria due to release of cytochrome c and this stimulation is insensitive to changes in membrane potential.

  18. In aging, the vulnerability of rat brain mitochondria is enhanced due to reduced level of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNP) and subsequently increased permeability transition in brain mitochondria in old animals.

    PubMed

    Krestinina, Olga; Azarashvili, Tamara; Baburina, Yulia; Galvita, Anastasia; Grachev, Dmitry; Stricker, Rolf; Reiser, Georg

    2015-01-01

    Aging is accompanied by progressive dysfunction of mitochondria associated with a continuous decrease of their capacity to produce ATP. Mitochondria isolated from brain of aged animals show an increased mitochondrial permeability transition pore (mPTP) opening. We recently detected new regulators of mPTP function in brain mitochondria, the enzyme 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and its substrates 2', 3'-cAMP and 2', 3'-cNADP, and the neuronal protein p42(IP4). Here, we compared parameters of mPTP opening in non-synaptic brain mitochondria isolated from young and old rats. In mitochondria from old rats (>18 months), mPTP opening occurred at a lower threshold of Ca(2+) concentration than in mitochondria from young rats (<3 months). mPTP opening in mitochondria from old rats was accelerated by 2', 3'-cAMP, which further lowered the threshold Ca(2+) concentration. In non-synaptic mitochondria from old rats, the CNP level was decreased by 34%. Lowering of the CNP level in non-synaptic mitochondria with aging was accompanied by decreased levels of voltage-dependent anion channel (VDAC; by 69%) and of p42(IP4) (by 59%). Thus, reduced levels of CNP in mitochondria could lead to a rise in the concentration of the mPTP promoter 2', 3'-cAMP. The level of CNP and p42(IP4) and, probably VDAC, might be essential for myelination and electrical activity of axons. We propose that in aging the reduction in the level of these proteins leads to mitochondrial dysfunction, in particular, to a decreased threshold Ca(2+) concentration to induce mPTP opening. This might represent initial steps of age-related mitochondrial dysfunction, resulting in myelin and axonal pathology.

  19. Mechanism of the acceleration of CO2 production from pyruvate in liver mitochondria by HCO3-.

    PubMed

    Taguchi, Y; Ono, Y; Lin, L; Storey, B T; Dodgson, S J; Forster, R E

    1997-07-01

    To investigate the mechanism by which HCO3- accelerates pyruvate metabolism in guinea pig liver mitochondria, we measured continuously, at pH 7.4 and 37 degrees C, 13C16O2 production from [1-13C]pyruvate by mass spectrometry and NADH concentration by fluorescence and analyzed total malate, citrate, and beta-hydroxybutyrate produced by standard biochemical methods. When [1-13C]pyruvate is added to the mitochondrial suspension, 13C16O2 concentration rises steeply in the first seconds and then slows to a steady lower rate. Carbonic anhydrase (CA) eliminates this initial phase, which shows that decarboxylation of pyruvate produces CO2, not HCO3-, and it does this more rapidly than it can equilibrate without CA. HCO3- (25 mM) increased 13C16O2 production, O2 consumption and total malate and citrate production and decreased NADH concentration and total beta-hydroxybutyrate production. After obtaining the total amount of 13C16O2, malate, citrate, and beta-hydroxybutyrate produced, we calculated that the addition of 25 mM HCO3- to the suspension medium increased the amount of pyruvate decarboxylated by pyruvate dehydrogenase (PDH) 16% and increased the amount carboxylated by pyruvate carboxylase 300%. This supports our initial proposal that HCO3- accelerates the pyruvate carboxylation, which in turn consumes ATP directly and NADH and acetyl CoA secondarily, all of which increase PDH activity. However, we found no acceleration of pyruvate decarboxylation by 0.5 and 1 microM free Ca2+ concentration, unless the mitochondria were uncoupled and ATP was added.

  20. Melatonin attenuates brain mitochondria DNA damage induced by potassium cyanide in vivo and in vitro.

    PubMed

    Yamamoto, Hiro-aki; Mohanan, Parayanthala V

    2002-09-30

    The effect of potassium cyanide on mitochondria DNA (mtDNA) in mouse brain was investigated in vivo and in vitro. When potassium cyanide (0, 0.1, 1.0 or 2.0 mM) was incubated with a crude mitochondria fraction prepared from mouse brain at 37 degrees C for 60 min, the damage of mtDNA was observed in a concentration-dependent manner. However, the mtDNA damage was prevented by a co-treatment with melatonin (1.5 mM), a scavenger of hydroxyl radicals (*OH). Furthermore, a subcutaneous injection of potassium cyanide (7mg/kg) caused both brain mtDNA damage and severe seizures in mouse. The damage of mtDNA and seizures induced by potassium cyanide were abolished by the pre-injection of melatonin (20 mg/kg). Hydrogen peroxide (1.5 mM) inflicted damage to brain mtDNA in the presence of Fe(2+) (3.0 microM). The damage was abolished by the co-treatment with melatonin. Furthermore, when cyanide (0, 0.1 or 1.0 mM) was incubated with the crude mitochondria fraction prepared from mouse brain, the lipid peroxidation was significantly increased in a concentration-dependent manner. The increased lipid peroxidation was completely inhibited by the co-treatment with melatonin (1.0 mM). These results suggest that reactive oxygen species including the *OH may play a cardinal role for mtDNA damage induced by potassium cyanide. Hence, the present study concluded that melatonin protects against DNA damage induced by the *OH produced by cyanide or hydrogen peroxide.

  1. Dithiothreitol abrogates the effect of arsenic trioxide on normal rat liver mitochondria and human hepatocellular carcinoma cells

    SciTech Connect

    Paul, Manash K. Kumar, Rajinder; Mukhopadhyay, Anup K.

    2008-01-15

    Arsenic trioxide (ATO) is a known environmental toxicant and a potent chemotherapeutic agent. Significant correlation has been reported between consumption of arsenic-contaminated water and occurrence of liver cancer; moreover, ATO-treated leukemia patients also suffers from liver toxicity. Hence, modulation of ATO action may help to prevent populations suffering from arsenic toxicity as well as help reduce the drug-related side effects. Dithiothreitol (DTT) is a well-known dithiol agent reported to modulate the action of ATO. Controversial reports exist regarding the effect of DTT on ATO-induced apoptosis in leukemia cells. To the best of our knowledge, no report illustrates the modulatory effect of DTT on ATO-induced liver toxicity, the prime target for arsenic. Mitochondria serve as the doorway to apoptosis and have been implicated in ATO-induced cell death. Hence, we attempted to study the modulatory effect of DTT on ATO-induced dysfunction of mammalian liver mitochondria and human hepatocellular carcinoma cell line (Hep3B). We, for the first time, report that ATO produces complex I-mediated electron transfer inhibition, reactive oxygen species (ROS) generation, respiration inhibition, and ATO-induced ROS-mediated mitochondrial permeability transition (MPT) opening. DTT at low concentration (100 {mu}M and less) prevents the effect of ATO-induced complex I-malfunctions. DTT protects mitochondria from ATO-mediated opening of MPT and membrane potential depolarization. DTT also prevented ATO-induced Hep3B cell death. Thus, at low concentrations DTT abrogates the effect of ATO on rat liver mitochondria and Hep3B cell line. Therefore, the present result suggests, that use of low concentration of dithiols as food supplement may prevent arsenic toxicity in affected population.

  2. Mitochondria: A crossroads for lipid metabolism defect in neurodegeneration with brain iron accumulation diseases.

    PubMed

    Aoun, Manar; Tiranti, Valeria

    2015-06-01

    Neurodegeneration with brain iron accumulation (NBIA) comprises a group of brain iron deposition syndromes that lead to mixed extrapyramidal features and progressive dementia. Exact pathologic mechanism of iron deposition in NBIA remains unknown. However, it is becoming increasingly evident that many neurodegenerative diseases are hallmarked by metabolic dysfunction that often involves altered lipid profile. Among the identified disease genes, four encode for proteins localized in mitochondria, which are directly or indirectly implicated in lipid metabolism: PANK2, CoASY, PLA2G6 and C19orf12. Mutations in PANK2 and CoASY, both implicated in CoA biosynthesis that acts as a fatty acyl carrier, lead, respectively, to PKAN and CoPAN forms of NBIA. Mutations in PLA2G6, which plays a key role in the biosynthesis and remodeling of membrane phospholipids including cardiolipin, lead to PLAN. Mutations in C19orf12 lead to MPAN, a syndrome similar to that caused by mutations in PANK2 and PLA2G6. Although the function of C19orf12 is largely unknown, experimental data suggest its implication in mitochondrial homeostasis and lipid metabolism. Altogether, the identified mutated proteins localized in mitochondria and associated with different NBIA forms support the concept that dysfunctions in mitochondria and lipid metabolism play a crucial role in the pathogenesis of NBIA. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  3. Mitochondria-targeted antioxidants as promising drugs for treatment of age-related brain diseases.

    PubMed

    Skulachev, Vladimir P

    2012-01-01

    Much experimental evidence suggests that age-related brain pathologies are most often mediated by reactive oxygen species primarily originating from mitochondria (mROS). Two papers with such evidence have been recently published in the Journal of Alzheimer's Disease (Stefanova et al., J Alzheimers Dis 21, 476-491, 2010; Lloret et al., J Alzheimers Dis, doi: 10.3233/JAD-2011-110890). In the first paper, it was shown that appearance of a typical behavioral trait of aging in rats (that old animals do not enter an open arm in a maze) was completely reversed by ten weeks treatment of the old rats with the mitochondria-targeted antioxidant SkQ1. In the second article, the authors identified molecular mechanisms by which amyloid-β-induced mROS can mediate hyperphosphorylation of the tau protein, a key event in Alzheimer's disease. Conventional antioxidants prevented such hyperphosphorylation. In this article, I will summarize the present state of the art in this field. I conclude that mitochondria-targeted rechargeable antioxidants are promising as tools to treat brain pathologies developing in elderly humans.

  4. Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

    PubMed

    Wilkins, Heather M; Koppel, Scott J; Weidling, Ian W; Roy, Nairita; Ryan, Lauren N; Stanford, John A; Swerdlow, Russell H

    2016-12-01

    Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aβ1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

  5. Effects of salvianolic acid B on liver mitochondria of rats with nonalcoholic steatohepatitis

    PubMed Central

    Wang, Ying-Chun; Kong, Wei-Zong; Jin, Qing-Mei; Chen, Juan; Dong, Lei

    2015-01-01

    AIM: To investigate the effects of salvianolic acid B (Sal B) on the morphological characteristics and functions of liver mitochondria of rats with nonalcoholic steatohepatitis (NASH). METHODS: A total of 60 male Sprague-Dawley rats were randomly divided into three groups: (1) a normal group fed a normal diet; (2) an NASH model group; and (3) a Sal B-treated group fed a high-fat diet. Two rats from each group were executed at the end of the 12th week to detect pathological changes. The rats in the Sal B-treated group were gavaged with 20 mL/kg Sal B (1 mg/mL) daily. The model group received an equal volume of distilled water as a control. At the end of the 24th weekend, the remaining rats were executed. Serum biochemical parameters and liver histological characteristics were observed. Malondialdehyde (MDA) and superoxide dismutase (SOD) in the liver were determined. Protein expression of CytC and caspase-3 was determined by immunohistochemistry. The mRNA transcripts of mitofusin-2 (Mfn2) and NF-κB in the liver tissue were detected by real-time PCR. Mitochondrial membrane potential was detected using a fluorescence spectrophotometer. Mitochondrial respiratory function was detected using a Clark oxygen electrode. RESULTS: The model group showed significantly higher ALT, AST, TG, TC and MDA but significantly lower SOD than the normal group. In the model group, the histological characteristics of inflammation and steatosis were also evident; mitochondrial swelling and crest were shortened or even disappeared. CytC (18.46 ± 1.21 vs 60.01 ± 3.43, P < 0.01) and caspase-3 protein expression (30.26 ± 2.56 vs 83.31 ± 5.12, P < 0.01) increased significantly. The mRNA expression of NF-κB increased (0.81 ± 0.02 vs 0.91 ± 0.03, P < 0.05), whereas the mRNA expression of Mfn2 decreased (1.65 ± 0.31 vs 0.83 ± 0.16, P < 0.05). Mitochondrial membrane potential also decreased and breathing of rats was weakened. Steatosis and inflammation degrees in the treatment group were

  6. COMPARATIVE STUDIES ON MITOCHONDRIA ISOLATED FROM NEURON-ENRICHED AND GLIA-ENRICHED FRACTIONS OF RABBIT AND BEEF BRAIN

    PubMed Central

    Hamberger, Anders; Blomstrand, Christian; Lehninger, Albert L.

    1970-01-01

    Fractions enriched in neuronal and glial cells were obtained from dispersions of whole beef brain and rabbit cerebral cortex by large-scale density gradient centrifugation procedures. The fractions were characterized by appropriate microscopic observation. Mitochondria were then isolated from these fractions by differential centrifugation of their homogenates. The two different types of mitochondria were characterized with respect to certain enzyme activities, respiratory rate, rate of protein synthesis, and their buoyant density in sucrose gradients. The mitochondria from the neuron-enriched fraction were distinguished by a higher rate of incorporation of amino acids into protein, higher cytochrome oxidase activity, and a higher buoyant density in sucrose density gradients. Mitochondria from the glia-enriched fraction showed relatively high monoamine oxidase and Na+- and K+-stimulated ATPase activities. The rates of oxidation of various substrates and the acceptor control ratios did not differ appreciably between the two types of mitochondria. The difference in the buoyant density of mitochondria isolated from the neuron-enriched and glia-enriched cell fractions was utilized in attempts to separate neuronal and glial mitochondria from the mixed mitochondria obtained from whole brain homogenates in shallow sucrose gradients. The appearance of two peaks of cytochrome oxidase, monoamine oxidase, and protein concentration in such gradients shows the potential feasibility of such an approach. PMID:5513605

  7. Comparative studies on mitochondria isolated from neuron-enriched and glia-enriched fractions of rabbit and beef brain.

    PubMed

    Hamberger, A; Blomstrand, C; Lehninger, A L

    1970-05-01

    Fractions enriched in neuronal and glial cells were obtained from dispersions of whole beef brain and rabbit cerebral cortex by large-scale density gradient centrifugation procedures. The fractions were characterized by appropriate microscopic observation. Mitochondria were then isolated from these fractions by differential centrifugation of their homogenates. The two different types of mitochondria were characterized with respect to certain enzyme activities, respiratory rate, rate of protein synthesis, and their buoyant density in sucrose gradients. The mitochondria from the neuron-enriched fraction were distinguished by a higher rate of incorporation of amino acids into protein, higher cytochrome oxidase activity, and a higher buoyant density in sucrose density gradients. Mitochondria from the glia-enriched fraction showed relatively high monoamine oxidase and Na(+)- and K(+)-stimulated ATPase activities. The rates of oxidation of various substrates and the acceptor control ratios did not differ appreciably between the two types of mitochondria. The difference in the buoyant density of mitochondria isolated from the neuron-enriched and glia-enriched cell fractions was utilized in attempts to separate neuronal and glial mitochondria from the mixed mitochondria obtained from whole brain homogenates in shallow sucrose gradients. The appearance of two peaks of cytochrome oxidase, monoamine oxidase, and protein concentration in such gradients shows the potential feasibility of such an approach.

  8. Prediction of Liver Injury Induced by Chemicals in Human With a Multiparametric Assay on Isolated Mouse Liver Mitochondria

    PubMed Central

    Porceddu, Mathieu; Buron, Nelly; Borgne-Sanchez, Annie

    2012-01-01

    Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In this study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release), and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cutoff) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (p < 0.001). Moreover, this multiparametric assay allowed identifying several compounds for which mitochondrial toxicity had never been described before and even helped to clarify mechanisms with some drugs already known to be mitochondriotoxic. Investigation of drug-induced loss of mitochondrial integrity and function with this multiparametric assay should be considered for integration into basic screening processes at early stage to select drug candidates with lower risk of DILI in human. This assay is also a valuable tool for assessing the mitochondrial toxicity profile and investigating the mechanism of action of new

  9. Mitochondria-targeted plastoquinone derivative SkQ(1) decreases ischemia-reperfusion injury during liver hypothermic storage for transplantation.

    PubMed

    Cherkashina, D V; Sosimchik, I A; Semenchenko, O A; Volina, V V; Petrenko, A Yu

    2011-09-01

    The ability of the mitochondria-targeted plastoquinone derivative 10-(6'-plastoquinonyl)decyl triphenylphosphonium (SkQ(1)) to decrease ischemia-reperfusion injury in isolated liver during hypothermic storage (HS) was studied. Rat liver was stored for 24 h at 4°C without or in the presence of 1 μM SkQ(1) with following reperfusion for 60 min at 37°C. The presence in the storage medium of SkQ(1) significantly decreased spontaneous production of reactive oxygen species and intensity of lipid peroxidation in the liver during HS and reperfusion. The GSH level after HS in solution with SkQ(1) was reliably higher, but reperfusion leveled this effect. At all stages of experiment the presence of SkQ(1) did not prevent the decrease of antioxidant enzyme activities such as catalase, GSH peroxidase, GSH reductase, and glucose-6-phosphate dehydrogenase. The addition of SkQ(1) to the storage medium improved energetic function of the liver, as was revealed in increased respiratory control index of mitochondria and ATP level. SkQ(1) exhibited positive effect on the liver secretory function and morphology after HS as revealed in enhanced bile flow rate during reperfusion and partial recovery of organ architectonics and state of liver sinusoids and hepatocytes. The data point to promising application of mitochondria-targeted antioxidants for correction of the ischemia-reperfusion injury of isolated liver during long-term cold storage before transplantation.

  10. Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria

    PubMed Central

    Garland, P. B.; Yates, D. W.; Haddock, B. A.

    1970-01-01

    1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.–) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with εmM 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg2+ and corresponded to a previously described `external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg2+. Of these two `internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.–), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do

  11. The influence of dietary iodine and enviromental temperature on the activity of mitochondria in liver and kidney.

    PubMed

    Chaiyabutr, N; Jakobsen, P E

    1978-08-01

    It was found that both effect of temperatures and diets influence metabolic changes in rabbits. In animals fed basal and PTU diets (propyl-thiouracil diets) at 34 degrees C for 4 weeks the metabolic response showed a marked reduction in feed intake and body weight, compared with animals fed at normal temperatures. In the animals fed the iodine diet, there was an increase in daily food consumption and weekly body weight gain at 34 degrees C. This indicates a rise in metabolic activity in this case. Studying the activity of kidney mitochondria of the three groups of animals using succinate as a substrate revealed that the P/O ratio tends to decrease in animals kept at 6 degrees C while the RCR value was not altered by changing conditions or produced by the different diets. At the temperature of 6 degrees C both the P/O ratios and the RCR values of liver mitochondria using succinate as a substrate decreased in the group of rabbits fed the basal and iodine diets, but were not significantly different in the group fed the PTU diet. In the experiment on kidney mitochondrial activity using alpha-ketoglutarate as a substrate it was found that both the P/O ratios and the RCR values from animals fed basal and PTU diets at 6 degrees C decreased slightly as compared with animals fed at 20 degrees C and 34 degrees C. In liver mitochondria, using alpha-ketoglutarate as a substrate a significant decrease in the P/O ratio and the RCR value was found for both rabbits fed the basal and the iodine diets at 6 degrees C. In the group of rabbits fed the PTU diet, the P/O ratio also decreased but the fall was not significant. These results suggested that the activity of succinate dehydrogenase in liver mitochondria increases in animals fed basal and iodine diets at 6 degrees C. The enzyme dehydrogenase involved in oxidation of alpha-ketoglutarate which is localized in the outer membrane of mitochondria seems to be affected by different temperatures and diets as compared with succinate

  12. Acetaldehyde metabolism by brain mitochondria from UChA and UChB rats.

    PubMed

    Quintanilla, M E; Tampier, L

    1995-01-01

    The acetaldehyde (AcH) oxidizing capacity of total brain homogenates from the genetically high-ethanol consumer (UChB) appeared to be greater than that of the low-ethanol consumer (UChA) rats. To gain further information about this strain difference, the activity of aldehyde dehydrogenase (AIDH) in different subcellular fractions of whole brain homogenates from naive UChA and UChB rat strains of both sexes has been studied by measuring the rate of AcH disappearance and by following the reduction of NAD to NADH. The results demonstrated that the higher capacity of brain homogenates from UChB rats to oxidize AcH when compared to UChA ones was because the UChB mitochondrial low Km AIDH exhibits a much greater affinity for NAD than that of the UChA rats, as evidenced by four-to fivefold differences in the Km values for NAD. But the dehydrogenases from both strains exhibited a similar maximum rate at saturating NAD concentrations. Because intact brain mitochondria isolated from UChB rats oxidized AcH at a higher rate than did mitochondria from UChA rats only in state 4, but not in state 3, this strain difference in AIDH activity might be restricted in vivo to NAD disposition.

  13. On the mechanism(s) of membrane permeability transition in liver mitochondria of lamprey, Lampetra fluviatilis L.: insights from cadmium.

    PubMed

    Belyaeva, Elena A; Emelyanova, Larisa V; Korotkov, Sergey M; Brailovskaya, Irina V; Savina, Margarita V

    2014-01-01

    Previously we have shown that opening of the mitochondrial permeability transition pore in its low conductance state is the case in hepatocytes of the Baltic lamprey (Lampetra fluviatilis L.) during reversible metabolic depression taking place in the period of its prespawning migration when the exogenous feeding is switched off. The depression is observed in the last year of the lamprey life cycle and is conditioned by reversible mitochondrial dysfunction (mitochondrial uncoupling in winter and coupling in spring). To further elucidate the mechanism(s) of induction of the mitochondrial permeability transition pore in the lamprey liver, we used Cd(2+) and Ca(2+) plus Pi as the pore inducers. We found that Ca(2+) plus Pi induced the high-amplitude swelling of the isolated "winter" mitochondria both in isotonic sucrose and ammonium nitrate medium while both low and high Cd(2+) did not produce the mitochondrial swelling in these media. Low Cd(2+) enhanced the inhibition of basal respiration rate of the "winter" mitochondria energized by NAD-dependent substrates whereas the same concentrations of the heavy metal evoked its partial stimulation on FAD-dependent substrates. The above changes produced by Cd(2+) or Ca(2+) plus Pi in the "winter" mitochondria were only weakly (if so) sensitive to cyclosporine A (a potent pharmacological desensitizer of the nonselective pore) added alone and they were not sensitive to dithiothreitol (a dithiol reducing agent). Under monitoring of the transmembrane potential of the "spring" lamprey liver mitochondria, we revealed that Cd(2+) produced its decrease on both types of the respiratory substrates used that was strongly hampered by cyclosporine A, and the membrane potential was partially restored by dithiothreitol. The effects of different membrane permeability modulators on the lamprey liver mitochondria function and the seasonal changes in their action are discussed.

  14. On the Mechanism(s) of Membrane Permeability Transition in Liver Mitochondria of Lamprey, Lampetra fluviatilis L.: Insights from Cadmium

    PubMed Central

    Belyaeva, Elena A.; Emelyanova, Larisa V.; Korotkov, Sergey M.; Brailovskaya, Irina V.; Savina, Margarita V.

    2014-01-01

    Previously we have shown that opening of the mitochondrial permeability transition pore in its low conductance state is the case in hepatocytes of the Baltic lamprey (Lampetra fluviatilis L.) during reversible metabolic depression taking place in the period of its prespawning migration when the exogenous feeding is switched off. The depression is observed in the last year of the lamprey life cycle and is conditioned by reversible mitochondrial dysfunction (mitochondrial uncoupling in winter and coupling in spring). To further elucidate the mechanism(s) of induction of the mitochondrial permeability transition pore in the lamprey liver, we used Cd2+ and Ca2+ plus Pi as the pore inducers. We found that Ca2+ plus Pi induced the high-amplitude swelling of the isolated “winter” mitochondria both in isotonic sucrose and ammonium nitrate medium while both low and high Cd2+ did not produce the mitochondrial swelling in these media. Low Cd2+ enhanced the inhibition of basal respiration rate of the “winter” mitochondria energized by NAD-dependent substrates whereas the same concentrations of the heavy metal evoked its partial stimulation on FAD-dependent substrates. The above changes produced by Cd2+ or Ca2+ plus Pi in the “winter” mitochondria were only weakly (if so) sensitive to cyclosporine A (a potent pharmacological desensitizer of the nonselective pore) added alone and they were not sensitive to dithiothreitol (a dithiol reducing agent). Under monitoring of the transmembrane potential of the “spring” lamprey liver mitochondria, we revealed that Cd2+ produced its decrease on both types of the respiratory substrates used that was strongly hampered by cyclosporine A, and the membrane potential was partially restored by dithiothreitol. The effects of different membrane permeability modulators on the lamprey liver mitochondria function and the seasonal changes in their action are discussed. PMID:24995321

  15. 4,6-Dinitro-o-cresol uncouples oxidative phosphorylation and induces membrane permeability transition in rat liver mitochondria.

    PubMed

    Castilho, R F; Vicente, J A; Kowaltowski, A J; Vercesi, A E

    1997-07-01

    The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.

  16. Hemin inhibits the large conductance potassium channel in brain mitochondria: a putative novel mechanism of neurodegeneration.

    PubMed

    Augustynek, Bartłomiej; Kudin, Alexei P; Bednarczyk, Piotr; Szewczyk, Adam; Kunz, Wolfram S

    2014-07-01

    Intracerebral hemorrhage (ICH) is a pathological condition that accompanies certain neurological diseases like hemorrhagic stroke or brain trauma. Its effects are severely destructive to the brain and can be fatal. There is an entire spectrum of harmful factors which are associated with the pathogenesis of ICH. One of them is a massive release of hemin from the decomposed erythrocytes. It has been previously shown, that hemin can inhibit the large-conductance Ca(2+)-regulated potassium channel in the plasma membrane. However, it remained unclear whether this phenomenon applies also to the mitochondrial large-conductance Ca(2+)-regulated potassium channel. The aim of the present study was to determine the impact of hemin on the activity of the large conductance Ca(2+)-regulated potassium channel in the brain mitochondria (mitoBKCa). In order to do so, we have used a patch-clamp technique and shown that hemin inhibits mitoBKCa in human astrocytoma U-87 MG cell line mitochondria. Since opening of the mitochondrial potassium channels is known to be cytoprotective, we have elucidated whether hemin can attenuate some of the beneficiary effects of potassium channel opening. We have studied the effect of hemin on reactive oxygen species synthesis, and mild mitochondrial uncoupling in isolated rat brain mitochondria. Taken together, our data show that hemin inhibits mitoBKCa and partially abolishes some of the cytoprotective properties of potassium channel opening. Considering the role of the mitoBKCa in cytoprotection, it can be presumed that its inhibition by hemin may be a novel mechanism contributing to the severity of the ICH symptoms. However, the validity of the presented results shall be further verified in an experimental model of ICH. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. THE LARGE-SCALE SEPARATION OF PEROXISOMES, MITOCHONDRIA, AND LYSOSOMES FROM THE LIVERS OF RATS INJECTED WITH TRITON WR-1339

    PubMed Central

    Leighton, Federico; Poole, Brian; Beaufay, Henri; Baudhuin, Pierre; Coffey, John W.; Fowler, Stanley; De Duve, Christian

    1968-01-01

    Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-α-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage. PMID:4297786

  18. Entry and exit pathways of CO2 in rat liver mitochondria respiring in a bicarbonate buffer system.

    PubMed

    Balboni, E; Lehninger, A L

    1986-03-15

    The dynamics and pathways of CO2 movements across the membranes of mitochondria respiring in vitro in a CO2/HCO-3 buffer at concentrations close to that in intact rat tissues were continuously monitored with a gas-permeable CO2-sensitive electrode. O2 uptake and pH changes were monitored simultaneously. Factors affecting CO2 entry were examined under conditions in which CO2 uptake was coupled to electrophoretic influx of K+ (in the presence of valinomycin) or Ca2+. The role of mitochondrial carbonic anhydrase (EC 4.2.1.1) in CO2 entry was evaluated by comparison of CO2 uptake by rat liver mitochondria, which possess carbonic anhydrase, versus rat heart mitochondria, which lack carbonic anhydrase. Such studies showed that matrix carbonic anhydrase activity is essential for rapid net uptake of CO2 with K+ or Ca2+. Studies with acetazolamide (Diamox), a potent inhibitor of carbonic anhydrase, confirmed the requirement of matrix carbonic anhydrase for net CO2 uptake. It was shown that at pH 7.2 the major species leaving respiring mitochondria is dissolved CO2, rather than HCO-3 or H2CO3 suggested by earlier reports. Efflux of endogenous CO2/HCO-3 is significantly inhibited by inhibitors of the dicarboxylate and tricarboxylate transport systems of the rat liver inner membrane. The possibility that these anion carriers mediate outward transport of HCO-3 is discussed.

  19. Studies on the mechanism of pyrophosphate-mediated uptake of iron from transferrin by isolated rat-liver mitochondria.

    PubMed

    Konopka, K; Romslo, I

    1981-07-01

    1. Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1--1.5 nmol mg protein-1 h-1, or approximately 60% of the amount of iron mobilized from transferrin. 2. The uptake declines if respiration is inhibited, substrate is deleted, or the experiments are run under anaerobic conditions. Substrate depletion and respiratory inhibitors are less inhibitory under anaerobic conditions. 3. More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bathophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration-inhibited mitochondria under aerobic conditions is not available for heme synthesis. 4. With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate. 5. The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous iron thus formed is available for heme synthesis.

  20. Reduction in energy efficiency induced by expression of the uncoupling protein, UCP1, in mouse liver mitochondria.

    PubMed

    González-Muniesa, Pedro; Milagro, Fermín I; Campión, Javier; Martínez, J Alfredo

    2006-04-01

    Uncoupling Protein 1 (UCP1) is an inner mitochondrial membrane protein, uniquely expressed in brown adipocytes, which uncouples the mitochondrial respiration impairing ATP production and energy efficiency. The aim of the present study was to express UCP1 in liver mitochondria using a non-viral system in order to affect energy utilization. The effect of ectopic protein expression on liver energy metabolism, which was evaluated 42 h after DNA transfer, showed that mitochondria expressing UCP1 presented decreased ATP production, lasted more time in membrane potential state 3, and consumed more molecular oxygen to produce the same amount of ATP than the control group. In summary, the successful functionality of the mitochondrial protein, UCP1, after hydrodynamic delivery is a novel and significant finding. This approach could be useful to ectopically express mitochondrial proteins and, in this particular case, to manage metabolic disorders related to energy efficiency and expenditure, such as obesity.

  1. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine inhibits proton motive force in energized liver mitochondria

    SciTech Connect

    Singh, Y.; Bhatnagar, R.; Sidhu, G.S.; Batra, J.K.; Krishna, G. )

    1989-05-15

    It is known that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which induces Parkinson's-like disease in primates and humans, depletes hepatocytes of ATP and subsequently causes cell death. Incubation of rat liver mitochondria with MPTP and 1-methyl-4-phenyl pyridinium ion (MPP+) significantly inhibited incorporation of {sup 32}Pi into ATP. MPTP and MPP+ inhibited the development of membrane potential and pH gradient in energized rat liver mitochondria, suggesting that reduction of the proton motive force may have reduced ATP synthesis. Since deprenyl, an inhibitor of monoamine oxidase, prevented the formation of MPP+ and inhibited the decrease in membrane potential caused by MPTP, but not that caused by MPP+, these effects of MPTP, as well as cell death, probably were mediated by MPP+. This mechanism may play a role in the specific loss of dopaminergic neurons resulting in MPTP-induced Parkinson's disease.

  2. The effect of diet on the fatty acid compositions of serum, brain, brain mitochondria and myelin in the rat

    PubMed Central

    Rathbone, L.

    1965-01-01

    1. Three groups of female rats (8–12 weeks old) were maintained respectively on a linoleic acid-rich diet, a linoleic acid-poor predominantly saturated-fatty acid diet and a normal diet. Changes in the fatty acid compositions of serum, brain, brain mitochondria-rich fraction and myelin were observed. 2. Of the serum fatty acids, linoleic acid showed the greatest change in the percentage of the total acids in response to diet; the change in the proportion of oleic acid was considerable. The percentages of arachidonic acid in serum fatty acids in the groups on the linoleic acid-rich and linoleic acid-poor diets were similar, but higher than those in the normal group. 3. Changes in the proportions of linoleic acid, arachidonic acid and docosahexaenoic acid occurred in brain fatty acids that to some extent paralleled those occurring in the serum. Changes in the proportions of most other acids in the serum fatty acids were not accompanied by corresponding changes in the brain fatty acids. 4. The percentage fatty acid compositions of a mitochondria-rich fraction and myelin are given, and changes in the relative proportions of linoleic acid, arachidonic acid and possibly some docosapolyenoic acids were demonstrated to occur as a result of diet. 5. The results are discussed in relation to the possible aetiology of multiple sclerosis. PMID:5881652

  3. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water.

    PubMed

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury.

  4. Death receptor and mitochondria-mediated hepatocyte apoptosis underlies liver dysfunction in rats exposed to organic pollutants from drinking water

    PubMed Central

    Yang, Guanghong; Zhou, Zhiwei; Cen, Yanli; Gui, Xiaolin; Zeng, Qibing; Ao, Yunxia; Li, Qian; Wang, Shiran; Li, Jun; Zhang, Aihua

    2015-01-01

    Persistent organic pollutants in drinking water impose a substantial risk to the health of human beings, but the evidence for liver toxic effect and the underlying mechanism is scarce. This study aimed to examine the liver toxicity and elucidate the molecular mechanism of organic pollutants in drinking water in normal human liver cell line L02 cells and rats. The data showed that organic extraction from drinking water remarkably impaired rat liver function, evident from the increase in the serum level of alanine aminotransferase, aspartate aminotransferase, and cholinesterase, and decrease in the serum level of total protein and albumin. Organic extraction dose-dependently induced apoptotic cell death in rat liver and L02 cells. Administration of rats with organic extraction promoted death receptor signaling pathway through the increase in gene and protein expression level of Fas and FasL. Treatment of rats with organic extraction also induced mitochondria-mediated apoptosis via increasing the expression level of proapoptotic protein, Bax, but decreasing the expression level of antiapoptotic protein, Bcl-2, resulting in an upregulation of cytochrome c and activation of caspase cascade at both transcriptional and post-transcriptional levels. Moreover, organic extraction enhanced rat liver glutathione S-transferases activity and reactive oxygen species generation, and upregulated aryl hydrocarbon receptor and glutathione S-transferase A1 at both transcriptional and translational levels. Collectively, the results indicate that organic extraction from drinking water impairs liver function, with the involvement of death receptor and mitochondria-mediated apoptosis in rats. The results provide evidence and molecular mechanisms for organic pollutants in drinking water-induced liver dysfunction, which may help prevent and treat organic extraction-induced liver injury. PMID:26316710

  5. Purification and characterization of a benzene hydroxylase: A cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Karaszkiewicz, J.W.

    1989-01-01

    This laboratory previously demonstrated that incubation of ({sup 14}C)benzene with isolated mitochondria resulted in the formation of mtDNA adducts. Since benzene is incapable of spontaneously covalently binding to nuclei acids, it was hypothesized that enzyme(s) present in the organelle metabolized benzene to reactive derivatives. We have purified, to electrophoretic homogeneity, a 52 kDa cytochrome P-450 from liver mitoplasts which metabolizes benzene to phenol. The enzyme has a K{sub M} for benzene of 0.012 mM, and a V{sub MAX} of 22.6 nmol phenol/nmol P-450/10 min, and requires NADPH, adrenodoxin, and adrenodoxin reductase for activity. Activity also can be reconstituted with microsomal cytochrome P-450 reductase. Benzene hydroxylase activity could be inhibited by carbon monoxide and SKF-525A, and by specific inhibitors of microsomal benzene metabolism. The purified enzyme oxidized phenol, forming catechol; aminopyrine N-demethylase activity was also demonstrated. These data confirm that a cytochrome P-450 of mitochondrial origin is involved in benzene metabolism, and indicate a role for the mitochondrion in xenobiotic activation.

  6. Liposomes from mammalian liver mitochondria are more polyunsaturated and leakier to protons than those from reptiles.

    PubMed

    Brand, M D; Couture, P; Hulbert, A J

    1994-06-01

    Liposomes were prepared from phospholipids extracted from liver mitochondria of the rat (Rattus norvegicus) and an agamid lizard, the bearded dragon (Amphibolurus vitticeps) and liposome proton conductance was measured at an imposed membrane potential of 160 mV as well as the fatty acid composition of the liposomes. Despite presumed changes in fatty acid composition during liposome preparation, the mammalian liposomes had a significantly lower content of the monounsaturated oleic acid and a significantly greater content of the omega-3 polyunsaturated docosahexaenoic acid. There were significant direct correlations between the liposome arachidonic and docosahexanoic acid content and bilayer proton flux and a significant inverse correlation between liposome oleic acid content and bilayer proton flux. "Apparent valinomycin-catalysed proton flux" was significantly directly correlated with liposome docosahexaenoic acid content and inversely correlated with oleic acid content. It is suggested that the high content of long-chain polyunsaturates in the mammalian mitochondrial membrane is responsible for an increased proton leak across the mitochondrial inner membrane and thus partly responsible for the high metabolic rate in endothermic mammals compared to their ectothermic reptilian predecessors.

  7. Malaria parasite liver stages render host hepatocytes susceptible to mitochondria-initiated apoptosis

    PubMed Central

    Kaushansky, A; Metzger, P G; Douglass, A N; Mikolajczak, S A; Lakshmanan, V; Kain, H S; Kappe, S HI

    2013-01-01

    Intracellular eukaryotic parasites and their host cells constitute complex, coevolved cellular interaction systems that frequently cause disease. Among them, Plasmodium parasites cause a significant health burden in humans, killing up to one million people annually. To succeed in the mammalian host after transmission by mosquitoes, Plasmodium parasites must complete intracellular replication within hepatocytes and then release new infectious forms into the blood. Using Plasmodium yoelii rodent malaria parasites, we show that some liver stage (LS)-infected hepatocytes undergo apoptosis without external triggers, but the majority of infected cells do not, and can also resist Fas-mediated apoptosis. In contrast, apoptosis is dramatically increased in hepatocytes infected with attenuated parasites. Furthermore, we find that blocking total or mitochondria-initiated host cell apoptosis increases LS parasite burden in mice, suggesting that an anti-apoptotic host environment fosters parasite survival. Strikingly, although LS infection confers strong resistance to extrinsic host hepatocyte apoptosis, infected hepatocytes lose their ability to resist apoptosis when anti-apoptotic mitochondrial proteins are inhibited. This is demonstrated by our finding that B-cell lymphoma 2 family inhibitors preferentially induce apoptosis in LS-infected hepatocytes and significantly reduce LS parasite burden in mice. Thus, targeting critical points of susceptibility in the LS-infected host cell might provide new avenues for malaria prophylaxis. PMID:23928701

  8. N-arachidonylglycine causes ROS production and cytochrome c release in liver mitochondria.

    PubMed

    Zaccagnino, Patrizia; Saltarella, Maddalena; D'Oria, Susanna; Corcelli, Angela; Saponetti, Matilde Sublimi; Lorusso, Michele

    2009-09-01

    N-arachidonylglycine (NA-Gly) is an amino acid derivative of arachidonic acid. This compound is structurally related to anandamide (arachidonylethanolamine), which is considered an endogenous ligand of the cannabinoid receptor. NA-Gly is present at relatively high levels in the spinal cord, small intestine, and kidneys and at lower, but remarkable, levels in testes, lungs, and liver. The presence of varying levels in different organs suggests multiple functions in addition to the reported anti-inflammatory and pain suppression actions. Here a study on the interaction of NA-Gly with isolated mitochondria is reported. The results show that micromolar concentrations of NA-Gly cause: (i) an increase in the resting state respiration with both glutamate plus malate and succinate as substrates and (ii) a decrease in either ADP- or uncoupler-activated respiration. Whereas the stimulated resting state respiration was substantially reduced by cyclosporin A (CsA), the NA-Gly-inhibited State 3 respiration was almost unaffected. Measurements by blot analysis showed that NA-Gly caused a CsA-sensitive cytochrome c release. Under these conditions no matrix swelling could be detected. Experiments are also presented showing that NA-Gly caused a respiration-dependent large ROS production, which seems in turn to be responsible for the inhibition of electron transport activity and cytochrome c release.

  9. Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria.

    PubMed

    Brand, M D; Chen, C H; Lehninger, A L

    1976-02-25

    We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxtbutyrate were accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis.

  10. Mevalonolactone disrupts mitochondrial functions and induces permeability transition pore opening in rat brain mitochondria: Implications for the pathogenesis of mevalonic aciduria.

    PubMed

    Cecatto, Cristiane; Amaral, Alexandre Umpierrez; da Silva, Janaína Camacho; Wajner, Alessandro; Godoy, Kálita Dos Santos; Ribeiro, Rafael Teixeira; Gonçalves, Aline de Mello; Vargas, Carmen Regla; Wajner, Moacir

    2017-03-09

    Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and the capacity to retain Ca(2+) in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca(2+)-loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca(2+) in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML-induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA.

  11. Fructose-Drinking Water Induced Nonalcoholic Fatty Liver Disease and Ultrastructural Alteration of Hepatocyte Mitochondria in Male Wistar Rat.

    PubMed

    Mamikutty, Norshalizah; Thent, Zar Chi; Haji Suhaimi, Farihah

    2015-01-01

    Nonalcoholic fatty liver disease (NAFLD) is one of the complications of the metabolic syndrome. It encompasses a wide range of disease spectrum from simple steatosis to liver cirrhosis. Structural alteration of hepatic mitochondria might be involved in the pathogenesis of NAFLD. In the present study, we used a newly established model of fructose-induced metabolic syndrome in male Wistar rats in order to investigate the ultrastructural changes in hepatic mitochondria that occur with fructose consumption and their association with NAFLD pathogenesis. The concentration of fructose-drinking water (FDW) used in this study was 20%. Six male Wistar rats were supplemented with FDW 20% for eight weeks. Body composition and metabolic parameters were measured before and after 8 weeks of FDW 20%. Histomorphology of the liver was evaluated and ultrastructural changes of mitochondria were assessed with transmission electron micrograph. After 8 weeks of fructose consumption, the animals developed several features of the metabolic syndrome. Moreover, fructose consumption led to the development of macrovesicular hepatic steatosis and mitochondrial ultrastructural changes, such as increase in mitochondrial size, disruption of the cristae, and reduction of matrix density. We conclude that in male Wistar rat 8-week consumption of FDW 20% leads to NAFLD likely via mitochondrial structural alteration.

  12. Fructose-Drinking Water Induced Nonalcoholic Fatty Liver Disease and Ultrastructural Alteration of Hepatocyte Mitochondria in Male Wistar Rat

    PubMed Central

    Thent, Zar Chi; Haji Suhaimi, Farihah

    2015-01-01

    Background. Nonalcoholic fatty liver disease (NAFLD) is one of the complications of the metabolic syndrome. It encompasses a wide range of disease spectrum from simple steatosis to liver cirrhosis. Structural alteration of hepatic mitochondria might be involved in the pathogenesis of NAFLD. Aims. In the present study, we used a newly established model of fructose-induced metabolic syndrome in male Wistar rats in order to investigate the ultrastructural changes in hepatic mitochondria that occur with fructose consumption and their association with NAFLD pathogenesis. Methods. The concentration of fructose-drinking water (FDW) used in this study was 20%. Six male Wistar rats were supplemented with FDW 20% for eight weeks. Body composition and metabolic parameters were measured before and after 8 weeks of FDW 20%. Histomorphology of the liver was evaluated and ultrastructural changes of mitochondria were assessed with transmission electron micrograph. Results. After 8 weeks of fructose consumption, the animals developed several features of the metabolic syndrome. Moreover, fructose consumption led to the development of macrovesicular hepatic steatosis and mitochondrial ultrastructural changes, such as increase in mitochondrial size, disruption of the cristae, and reduction of matrix density. Conclusion. We conclude that in male Wistar rat 8-week consumption of FDW 20% leads to NAFLD likely via mitochondrial structural alteration. PMID:26273656

  13. Ultrastructural and biochemical aspects of liver mitochondria during recovery from ethanol-induced alterations. Experimental evidence of mitochondrial division.

    PubMed Central

    Koch, O. R.; Roatta de Conti, L. L.; Bolaños, L. P.; Stoppani, A. O.

    1978-01-01

    To study the morphologic and biochemical changes occuring in liver mitochondria during recovery from ethanol-induced injury, rats fed a 6-month high-alcohol regimen plus a nutritionally adequate diet which did not induce fatty liver were compared with isocalorically fed controls. After this period the alcohol-fed animals displayed striking ultrastructural changes of liver mitochondria and a decreased respiratory activity with succinate or malate-glutamate as substrate. On the contrary, the respiratory rate with I-glycerophosphate was 50% increased. Regression changes were studied after alcohol was withdrawn from the diet. Enlarged mitochondria rapidly disappeared (in 24 hours), although a few megamitochondria were still present after 8 days of abstinence. A similar recovery was observed for the functional alterations. At the end of the experimental period, only a slight decrease of the maximal respiratory rate using malate-glutamate as a substrate was noted. The ultrastructural findings and the morphometric data suggest that the way in which mitochondrial normalization takes place is based on partition of these organelles. Images Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 1 Figure 2 Figure 13 PMID:623205

  14. Mitochondrial dysfunction in brain cortex mitochondria of STZ-diabetic rats: effect of l-Arginine.

    PubMed

    Ortiz, M Del Carmen; Lores-Arnaiz, Silvia; Albertoni Borghese, M Florencia; Balonga, Sabrina; Lavagna, Agustina; Filipuzzi, Ana Laura; Cicerchia, Daniela; Majowicz, Monica; Bustamante, Juanita

    2013-12-01

    Mitochondrial dysfunction has been implicated in many diseases, including diabetes. It is well known that oxygen free radical species are produced endogenously by mitochondria, and also nitric oxide (NO) by nitric oxide synthases (NOS) associated to mitochondrial membranes, in consequence these organelles constitute main targets for oxidative damage. The aim of this study was to analyze mitochondrial physiology and NO production in brain cortex mitochondria of streptozotocin (STZ) diabetic rats in an early stage of diabetes and the potential effect of L-arginine administration. The diabetic condition was characterized by a clear hyperglycaemic state with loose of body weight after 4 days of STZ injection. This hyperglycaemic state was associated with mitochondrial dysfunction that was evident by an impairment of the respiratory activity, increased production of superoxide anion and a clear mitochondrial depolarization. In addition, the alteration in mitochondrial physiology was associated with a significant decrease in both NO production and nitric oxide synthase type I (NOS I) expression associated to the mitochondrial membranes. An increased level of thiobarbituric acid-reactive substances (TBARS) in brain cortex homogenates from STZ-diabetic rats indicated the presence of lipid peroxidation. L-arginine treatment to diabetic rats did not change blood glucose levels but significantly ameliorated the oxidative stress evidenced by lower TBARS and a lower level of superoxide anion. This effect was paralleled by improvement of mitochondrial respiratory function and a partial mitochondrial repolarization.In addition, the administration of L-arginine to diabetic rats prevented the decrease in NO production and NOSI expression. These results could indicate that exogenously administered L-arginine may have beneficial effects on mitochondrial function, oxidative stress and NO production in brain cortex mitochondria of STZ-diabetic rats.

  15. Mitochondria, Bioenergetics and Excitotoxicity: New Therapeutic Targets in Perinatal Brain Injury.

    PubMed

    Leaw, Bryan; Nair, Syam; Lim, Rebecca; Thornton, Claire; Mallard, Carina; Hagberg, Henrik

    2017-01-01

    Injury to the fragile immature brain is implicated in the manifestation of long-term neurological disorders, including childhood disability such as cerebral palsy, learning disability and behavioral disorders. Advancements in perinatal practice and improved care mean the majority of infants suffering from perinatal brain injury will survive, with many subtle clinical symptoms going undiagnosed until later in life. Hypoxic-ischemia is the dominant cause of perinatal brain injury, and constitutes a significant socioeconomic burden to both developed and developing countries. Therapeutic hypothermia is the sole validated clinical intervention to perinatal asphyxia; however it is not always neuroprotective and its utility is limited to developed countries. There is an urgent need to better understand the molecular pathways underlying hypoxic-ischemic injury to identify new therapeutic targets in such a small but critical therapeutic window. Mitochondria are highly implicated following ischemic injury due to their roles as the powerhouse and main energy generators of the cell, as well as cell death processes. While the link between impaired mitochondrial bioenergetics and secondary energy failure following loss of high-energy phosphates is well established after hypoxia-ischemia (HI), there is emerging evidence that the roles of mitochondria in disease extend far beyond this. Indeed, mitochondrial turnover, including processes such as mitochondrial biogenesis, fusion, fission and mitophagy, affect recovery of neurons after injury and mitochondria are involved in the regulation of the innate immune response to inflammation. This review article will explore these mitochondrial pathways, and finally will summarize past and current efforts in targeting these pathways after hypoxic-ischemic injury, as a means of identifying new avenues for clinical intervention.

  16. Mitochondria, Bioenergetics and Excitotoxicity: New Therapeutic Targets in Perinatal Brain Injury

    PubMed Central

    Leaw, Bryan; Nair, Syam; Lim, Rebecca; Thornton, Claire; Mallard, Carina; Hagberg, Henrik

    2017-01-01

    Injury to the fragile immature brain is implicated in the manifestation of long-term neurological disorders, including childhood disability such as cerebral palsy, learning disability and behavioral disorders. Advancements in perinatal practice and improved care mean the majority of infants suffering from perinatal brain injury will survive, with many subtle clinical symptoms going undiagnosed until later in life. Hypoxic-ischemia is the dominant cause of perinatal brain injury, and constitutes a significant socioeconomic burden to both developed and developing countries. Therapeutic hypothermia is the sole validated clinical intervention to perinatal asphyxia; however it is not always neuroprotective and its utility is limited to developed countries. There is an urgent need to better understand the molecular pathways underlying hypoxic-ischemic injury to identify new therapeutic targets in such a small but critical therapeutic window. Mitochondria are highly implicated following ischemic injury due to their roles as the powerhouse and main energy generators of the cell, as well as cell death processes. While the link between impaired mitochondrial bioenergetics and secondary energy failure following loss of high-energy phosphates is well established after hypoxia-ischemia (HI), there is emerging evidence that the roles of mitochondria in disease extend far beyond this. Indeed, mitochondrial turnover, including processes such as mitochondrial biogenesis, fusion, fission and mitophagy, affect recovery of neurons after injury and mitochondria are involved in the regulation of the innate immune response to inflammation. This review article will explore these mitochondrial pathways, and finally will summarize past and current efforts in targeting these pathways after hypoxic-ischemic injury, as a means of identifying new avenues for clinical intervention. PMID:28747873

  17. Effect of free malonate on the utilization of glutamate by rat brain mitochondria.

    PubMed

    Koeppen, A H; Riley, K M

    1987-05-01

    Malonate is an effective inhibitor of succinate dehydrogenase in preparations from brain and other organs. This property was reexamined in isolated rat brain mitochondria during incubation with L-glutamate. The biosynthesis of aspartate was determined by a standard spectrofluorometric method and a radiometric technique. The latter was suitable for aspartate assay after very brief incubations of mitochondria with glutamate. At a concentration of 1 mM or higher, malonate totally inhibited aspartate biosynthesis. At 0.2 mM, the inhibitory effect was still present. It is thus possible that the natural concentration of free malonate in adult rat brain of 192 nmol/g wet weight exerts an effect on citric acid cycle reactions in vivo. The inhibition of glutamate utilization by malonate was readily overcome by the addition of malate which provided oxaloacetate for the transamination of glutamate. The reaction was accompanied by the accumulation of 2-oxoglutarate. The metabolism of glutamate was also blocked by inclusion of arsenite and gamma-vinyl-gamma-aminobutyric acid but again added malate allowed transamination to resume. When arsenite and gamma-vinyl-gamma-aminobutyric acid were present, the role of malonate as an inhibitor of malate entry into the mitochondrial interior could be determined without considering the inhibition of succinate dehydrogenase. The apparent Km and Vmax values for uninhibited malate entry were 0.01 mM and 100 nmol/mg protein/min, respectively. Malonate was a competitive inhibitor of malate transport (Ki = 0.75 mM).

  18. Impairment of Mitochondria in Adult Mouse Brain Overexpressing Predominantly Full-Length, N-Terminally Acetylated Human α-Synuclein

    PubMed Central

    Sarafian, Theodore A.; Ryan, Christopher M.; Souda, Puneet; Masliah, Eliezer; Kar, Upendra K.; Vinters, Harry V.; Mathern, Gary W.; Faull, Kym F.; Whitelegge, Julian P.; Watson, Joseph B.

    2013-01-01

    While most forms of Parkinson’s Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice. PMID:23667637

  19. The Cratylia mollis seed lectin induces membrane permeability transition in isolated rat liver mitochondria and a cyclosporine a-insensitive permeability transition in Trypanosoma cruzi mitochondria.

    PubMed

    Fernandes, Mariana P; Leite, Ana C R; Araújo, Flavia F B; Saad, Sara T O; Baratti, M O; Correia, M T S; Coelho, Luana C B B; Gadelha, Fernanda R; Vercesi, Anibal E

    2014-01-01

    Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca(2+) and mitochondrial dysfunction due to matrix Ca(2+) overload. In order to investigate the mechanism of Ca(2+) -induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (∆Ψm ) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca(2+) was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca(2+) this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ∆Ψm disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca(2+) dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA-insensitive MPT in T. cruzi mitochondria. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

  20. Methylene blue improves sensorimotor phenotype and decreases anxiety in parallel with activating brain mitochondria biogenesis in mid-age mice.

    PubMed

    Gureev, Artem P; Syromyatnikov, Mikhail Yu; Gorbacheva, Tatyana M; Starkov, Anatoly A; Popov, Vasily N

    2016-12-01

    Age-related brain dysfunctions are associated with mitochondria malfunctions and increased risk of developing neurodegenerative diseases (ND). Recently, a mitochondria-targeting drug methylene blue has been drawing considerable interest as a potential treatment for ND. We found that aged mice manifested a decrease in physical endurance, spontaneous locomotor activity, and exploration concomitant with an increase in anxiety-related behavior, as compared to adult mice. Treating mice for 60 days with MB slowed down these changes. There were no significant changes in the animals' body weight, oxygen consumption rates, or respiratory quotient index, in adult or aged MB-treated mice. However, MB treatment significantly increased the generation of reactive oxygen species in brain mitochondria. The expression of several genes relevant to mitochondria biogenesis, bioenergetics, and antioxidant defense (NRF1, MTCOX1, TFAM, and SOD2) was greatly suppressed in aged mice; it was restored by MB treatment. It seems plausible that the effects of MB could be mediated by its ability to increase H2O2 production in brain mitochondria, thereby activating Nrf2/ARE signaling pathway and mitochondria biogenesis. Our data and earlier findings support the idea that MB can be an attractive prototype drug for developing safe and efficient gerontoprotective compounds. Copyright © 2016 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  1. Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume.

    PubMed

    Hansson, Magnus J; Morota, Saori; Teilum, Maria; Mattiasson, Gustav; Uchino, Hiroyuki; Elmér, Eskil

    2010-01-01

    Modulation of K(+) conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K(+) channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca(2+) and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K(+) or H(+) conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK(ATP) channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K(+) conductance did not result in augmented DeltapH. The beneficial effect of valinomycin on CRC was not mediated by H(2)O(2)-induced protein kinase Cepsilon activation. Rather, increased K(+) conductance reduced H(2)O(2) generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.

  2. Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume*

    PubMed Central

    Hansson, Magnus J.; Morota, Saori; Teilum, Maria; Mattiasson, Gustav; Uchino, Hiroyuki; Elmér, Eskil

    2010-01-01

    Modulation of K+ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K+ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca2+ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K+ or H+ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoKATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K+ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H2O2-induced protein kinase Cϵ activation. Rather, increased K+ conductance reduced H2O2 generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges. PMID:19880514

  3. Clusianone, a naturally occurring nemorosone regioisomer, uncouples rat liver mitochondria and induces HepG2 cell death.

    PubMed

    Reis, Felippe H Z; Pardo-Andreu, Gilberto L; Nuñez-Figueredo, Yanier; Cuesta-Rubio, Osmany; Marín-Prida, Javier; Uyemura, Sérgio A; Curti, Carlos; Alberici, Luciane C

    2014-04-05

    Clusianone is a member of the polycyclic polyprenylated acylphloroglucinol family of natural products; its cytotoxic mechanism is unknown. Clusianone is a structural isomer of nemorosone, which is a mitochondrial uncoupler and a well-known cytotoxic anti-cancer agent; thus, we addressed clusianone action at the mitochondria and its potential cytotoxic effects on cancer cells. In the HepG2 hepatocarcinoma cell line, clusianone induced mitochondrial membrane potential dissipation, ATP depletion and phosphatidyl serine externalization; this later event is indicative of apoptosis induction. In isolated mitochondria from rat liver, clusianone promoted protonophoric mitochondrial uncoupling. This was evidenced by the dissipation of mitochondrial membrane potential, an increase in resting respiration, an inhibition of Ca(2+) influx, stimulation of Ca(2+) efflux in Ca(2+)-loaded mitochondria, a decrease in ATP and NAD(P)H levels, generation of ROS, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. The cytotoxic and uncoupling actions of clusianone were appreciably less than those of nemorosone, likely due to the presence of an intra-molecular hydrogen bond with the juxtaposed carbonyl group at the C15 position. Therefore, clusianone is capable of pharmacologically increasing the leakage of protons from the mitochondria and with favorable cytotoxicity in relation to nemorosone. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  4. Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria.

    PubMed

    Brailovskaya, I V; Starkov, A A; Mokhova, E N

    2001-08-01

    Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO4 and ascorbate (vitamin C) was studied. FeSO4 (1-4 microM) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca2+. All the effects of FeSO4 + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca2+-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO4 + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO4 and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca2+-dependent pore in the inner mitochondrial membrane.

  5. Advanced In Vivo Heteronuclear MRS Approaches for Studying Brain Bioenergetics Driven by Mitochondria

    PubMed Central

    Zhu, Xiao-Hong; Du, Fei; Zhang, Nanyin; Zhang, Yi; Lei, Hao; Zhang, Xiaoliang; Qiao, Hongyan; Ugurbil, Kamil; Chen, Wei

    2017-01-01

    The greatest merit of in vivo magnetic resonance spectroscopy (MRS) methodology used in biomedical research is its ability for noninvasively measuring a variety of metabolites inside a living organ. It, therefore, provides an invaluable tool for determining metabolites, chemical reaction rates and bioenergetics, as well as their dynamic changes in the human and animal. The capability of in vivo MRS is further enhanced at higher magnetic fields because of significant gain in detection sensitivity and improvement in the spectral resolution. Recent progress of in vivo MRS technology has further demonstrated its great potential in many biomedical research areas, particularly in brain research. Here, we provide a review of new developments for in vivo heteronuclear 31P and 17O MRS approaches and their applications in determining the cerebral metabolic rates of oxygen and ATP inside the mitochondria, in both animal and human brains. PMID:18839099

  6. Acetyl-CoA deficit in brain mitochondria in experimental thiamine deficiency encephalopathy.

    PubMed

    Jankowska-Kulawy, Agnieszka; Bielarczyk, Hanna; Pawełczyk, Tadeusz; Wróblewska, Małgorzata; Szutowicz, Andrzej

    2010-12-01

    Several pathologic conditions are known to cause thiamine deficiency, which induce energy shortages in all tissues, due to impairment of pyruvate decarboxylation. Brain is particularly susceptible to these conditions due to its high rate of glucose to pyruvate-driven energy metabolism. However, cellular compartmentalization of a key energy metabolite, acetyl-CoA, in this pathology remains unknown. Pyrithiamine-evoked thiamine deficiency caused no significant alteration in pyruvate dehydrogenase and 30% inhibition of α-ketoglutarate dehydrogenase activities in rat whole forebrain mitochondria. It also caused 50% reduction of the metabolic flux of pyruvate through pyruvate dehydrogenase, 78% inhibition of its flux through α-ketoglutarate dehydrogenase steps, and nearly 60% decrease of intramitochondrial acetyl-CoA content, irrespective of the metabolic state. State 3 caused a decrease in citrate and an increase in α-ketoglutarate accumulation. These alterations were more evident in thiamine-deficient mitochondria. Simultaneously thiamine deficiency caused no alteration of relative, state 3-induced increases in metabolic fluxes through pyruvate and α-ketoglutarate dehydrogenase steps. These data indicate that a shortage of acetyl-CoA in the mitochondrial compartment may be a primary signal inducing impairment of neuronal and glial cell functions and viability in the thiamine-deficient brain.

  7. Alcohol stimulates Na sup + /Ca sup 2+ exchange in brain mitochondria

    SciTech Connect

    Rottenberg, H.; Marbach, M. )

    1991-01-01

    Ethanol, at low concentrations, specifically stimulates the Na{sup +}-dependent Ca{sup 2+}-efflux in brain mitochondria. In addition, at higher concentrations, ethanol inhibits the Na{sup +}-independent Ca{sup 2+}-efflux. The electrogenic Ca{sup 2+}-uptake system is not affected by ethanol. The specific stimulation of Na{sup +}/Ca{sup 2+} exchange reaches a maximum of 60% stimulation, with half-maximal stimulation at 130 mM ethanol. The inhibition of the Na{sup +}-independent efflux is proportional to the ethanol concentration, becoming significant only above 200 mM, with 50% inhibition at 0.5 M. The inhibition of the Na{sup +}-independent efflux is, in large part, due to an inhibition of the activation of the Cyclosporin-sensitive pore. Long-term ethanol-feeding had no effect on the Ca{sup 2+} transport systems and their sensitivity to acute ethanol treatment. It is suggested that the stimulation of the Na{sup +}-dependent Ca{sup 2+}-efflux, which is the dominant Ca{sup 2+} efflux pathway in brain mitochondria, contributes to the intoxicating effects of ethanol.

  8. Control of the effective P/O ratio of oxidative phosphorylation in liver mitochondria and hepatocytes.

    PubMed

    Brand, M D; Harper, M E; Taylor, H C

    1993-05-01

    The control exerted by substrate oxidation reactions, by ATP turnover and by the proton leak over the oxygen consumption rate, the phosphorylation rate, the proton leak rate and the protonmotive force (delta p) in isolated rat liver mitochondria under a range of conditions between non-phosphorylating (State 4) and maximum phosphorylation (State 3) was investigated by using the top-down approach of metabolic control analysis. The experiments were carried out with saturating concentrations of the substrates succinate, glutamate with malate, or pyruvate with malate. The distribution of control was very similar with each of the three substrates. The effective P/O ratio (i.e. not corrected for leak reactions) was also measured; it varied from zero in State 4 to 80-90% of the maximum theoretical P/O ratio in State 3. Under most conditions control over the effective P/O ratio was shared between proton leak (which had negative control) and the phosphorylating subsystem (which had roughly equal positive control); near State 4, substrate oxidation reactions also acquired some control over this ratio. In resting hepatocytes the effective P/O ratio was only 50% of its maximum theoretical value, corresponding to an effective P/O ratio of only 1.3 for complete oxidation of glucose. The effective P/O ratio for intracellular mitochondrial oxygen consumption was 64% of the maximum value. The control coefficient of the mitochondrial proton leak over the effective P/O ratio in cells was -0.34; the control coefficient of phosphorylation reactions over this ratio was 0.31 and the control coefficient of substrate oxidation reactions over the ratio was 0.03, showing how the coupling efficiency in cells can respond sensitively to agents that change the proton leak or the ATP demand, but not to those that change substrate oxidation.

  9. Long-term calorie restriction reduces proton leak and hydrogen peroxide production in liver mitochondria.

    PubMed

    Hagopian, Kevork; Harper, Mary-Ellen; Ram, Jesmon J; Humble, Stephen J; Weindruch, Richard; Ramsey, Jon J

    2005-04-01

    Calorie restriction (CR) without malnutrition increases maximal life span in diverse species. It has been proposed that reduction in energy expenditure and reactive oxygen species (ROS) production could be a mechanism for life span extension with CR. As a step toward testing this theory, mitochondrial proton leak, H2O2 production, and markers of oxidative stress were measured in liver from FBNF1 rats fed control or 40% CR diets for 12 or 18 mo. CR was initiated at 6 mo of age. Proton leak kinetics curves, generated from simultaneous measures of oxygen consumption and membrane potential, indicated a decrease in proton leak after 18 mo of CR, while only a trend toward a proton leak decrease was observed after 12 mo. Significant shifts in phosphorylation and substrate oxidation curves also occurred with CR; however, these changes occurred in concert with the proton leak changes. Metabolic control analysis indicated no difference in the overall pattern of control of the oxidative phosphorylation system between control and CR animals. At 12 mo, no significant differences were observed between groups for H2O2 production or markers of oxidative stress. However, at 18 mo, protein carbonyl content was lower in CR animals, as was H2O2 production when mitochondria were respiring on either succinate alone or pyruvate plus malate in the presence of rotenone. These results indicate that long-term CR lowers mitochondrial proton leak and H2O2 production, and this is consistent with the idea that CR may act by decreasing energy expenditure and ROS production.

  10. Optic atrophy 1 mediates coenzyme Q-responsive regulation of respiratory complex IV activity in brain mitochondria.

    PubMed

    Takahashi, Kazuhide; Ohsawa, Ikuroh; Shirasawa, Takuji; Takahashi, Mayumi

    2017-11-01

    The oxygen consumption rate (OCR) in brain mitochondria is significantly lower in aged mice than in young mice, and the reduced OCR is rescued by administration of water-solubilized CoQ10 to aged mice via drinking water. However, the mechanism behind this remains unclear. Here, we show that the activity of respiratory complex IV (CIV) in brain mitochondria declined in aged mice than in young mice, with no significant change in individual respiratory complex levels and their supercomplex assembly. Reduced CIV activity in the aged mice coincided with reduced binding of optic atrophy 1 (OPA1) to CIV. Both reduced activity and OPA1 binding of CIV were rescued by water-solubilized CoQ10 administration to aged mice via drinking water. OCR and the activity and OPA1 binding of CIV in isolated brain mitochondria from aged mice were restored by incubation with CoQ10, but not in the presence of 15-deoxy-prostaglandin J2, an inhibitor of a GTPase effector domain-containing GTPase such as OPA1 and DRP1. By contrast, the CoQ10-responsive restoration of OCR in the isolated mitochondria was not inhibited by Mdivi-1, a selective inhibitor of DRP1. Thus, we propose a novel function of OPA1 in regulating the CIV activity in brain mitochondria in response to CoQ10. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Role of Mitochondria in HIV Infection and Associated Metabolic Disorders: Focus on Nonalcoholic Fatty Liver Disease and Lipodystrophy Syndrome

    PubMed Central

    Pérez-Matute, P.; Pérez-Martínez, L.; Blanco, J. R.; Oteo, J. A.

    2013-01-01

    Highly active antiretroviral therapy (HAART) has considerably improved the prognosis of HIV-infected patients. However, prolonged use of HAART has been related to long-term adverse events that can compromise patient health such as HIV-associated lipodystrophy syndrome (HALS) and nonalcoholic fatty liver disease (NAFLD). There is consistent evidence for a central role of mitochondrial dysfunction in these pathologies. Nucleotide reverse transcriptase inhibitors (NRTIs) have been described to be mainly responsible for mitochondrial dysfunction in adipose tissue and liver although nonnucleoside transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs) have also showed mitochondrial toxicity, which is a major concern for the selection and the long-term adherence to a particular therapy. Several mechanisms explain these deleterious effects of HAART on mitochondria, and evidence points to other mechanisms beyond the “Pol-γ hypothesis.” HIV infection has also direct effects on mitochondria. In addition to the negative effects described for HIV itself and/or HAART on mitochondria, HIV-infected patients are more prone to develop a premature aging and, therefore, to present an increased oxidative state that could lead to the development of these metabolic disturbances observed in HIV-infected patients. PMID:23970949

  12. Aluminium toxicity in the rat liver and brain

    NASA Astrophysics Data System (ADS)

    Yumoto, S.; Ohashi, H.; Nagai, H.; Kakimi, S.; Ishikawa, A.; Kobayashi, K.; Ogawa, Y.; Ishii, K.

    1993-04-01

    To investigate the etiology of Alzheimer's disease, we examined the brain and liver tissue uptake of aluminium 5-75 days after aluminium injection into healthy rats. Ten days after the last injection, Al was detected in the brain and the brain cell nuclei by particle-induced X-ray emission (PIXE) analysis. Al was also demonstrated in the liver and the liver cell nuclei by PIXE analysis and electron energy loss spectrometry (EELS). The morphological changes of the rat brain examined 75 days after the injection were similar to those which have been reportedly observed in the brain of patients with Alzheimer's disease. These results support the theory that Alzheimer's disease is caused by irreversible accumulation of aluminium in the brain, as well as in the nuclei of brain cells.

  13. Brain death and marginal grafts in liver transplantation

    PubMed Central

    Jiménez-Castro, M B; Gracia-Sancho, J; Peralta, C

    2015-01-01

    It is well known that most organs for transplantation are currently procured from brain-dead donors; however, the presence of brain death is an important risk factor in liver transplantation. In addition, one of the mechanisms to avoid the shortage of liver grafts for transplant is the use of marginal livers, which may show higher risk of primary non-function or initial poor function. To our knowledge, very few reviews have focused in the field of liver transplantation using brain-dead donors; moreover, reviews that focused on both brain death and marginal grafts in liver transplantation, both being key risk factors in clinical practice, have not been published elsewhere. The present review aims to describe the recent findings and the state-of-the-art knowledge regarding the pathophysiological changes occurring during brain death, their effects on marginal liver grafts and summarize the more controversial topics of this pathology. We also review the therapeutic strategies designed to date to reduce the detrimental effects of brain death in both marginal and optimal livers, attempting to explain why such strategies have not solved the clinical problem of liver transplantation. PMID:26043077

  14. Brain death and marginal grafts in liver transplantation.

    PubMed

    Jiménez-Castro, M B; Gracia-Sancho, J; Peralta, C

    2015-06-04

    It is well known that most organs for transplantation are currently procured from brain-dead donors; however, the presence of brain death is an important risk factor in liver transplantation. In addition, one of the mechanisms to avoid the shortage of liver grafts for transplant is the use of marginal livers, which may show higher risk of primary non-function or initial poor function. To our knowledge, very few reviews have focused in the field of liver transplantation using brain-dead donors; moreover, reviews that focused on both brain death and marginal grafts in liver transplantation, both being key risk factors in clinical practice, have not been published elsewhere. The present review aims to describe the recent findings and the state-of-the-art knowledge regarding the pathophysiological changes occurring during brain death, their effects on marginal liver grafts and summarize the more controversial topics of this pathology. We also review the therapeutic strategies designed to date to reduce the detrimental effects of brain death in both marginal and optimal livers, attempting to explain why such strategies have not solved the clinical problem of liver transplantation.

  15. Ca(2+)-loading modulates potencies of cyclosporin A, Mg2+ and ADP to recouple permeabilized rat liver mitochondria.

    PubMed

    Andreyev AYu; Mikhaylova, L M; Starkov, A A; Kushnareva YuE

    1994-09-01

    We studied the relative potencies of cyclosporin A and endogenous effectors (Mg2+ and ADP) to recouple rat liver mitochondria permeabilized by different Ca(2+)-loading in a P(i)-containing medium. Recoupling efficiency of cyclosporin A dramatically decreased at high Ca(2+)-loading (approx. 100 nM of Ca2+/mg protein and more). Mitochondria permeabilized by high Ca2+ were recoupled with approximately equal efficiency by higher cyclosporin A concentrations or by adding 1-5 mM Mg2+ together with low concentrations of cyclosporin A while potentiating effect of ADP on the cyclosporin A recoupling potency was insignificant. Mg2+ ions at concentrations of 3 mM and higher also prevented the carboxyatractylate-induced reversion of cyclosporin A recoupling effect. The data point to competitive relationships between cyclosporin A and/or Mg2+ ions and Ca2+ ions for the site(s) regulating permeability state of the pore.

  16. O2-dependent hepatotoxicity due to ethylhexanol in the perfused rat liver: mitochondria as a site of action.

    PubMed

    Keller, B J; Yamanaka, H; Liang, D C; Kauffman, F C; Thurman, R G

    1990-03-01

    Toxicity of 2-ethylhexanol, a metabolite of diethylhexyl phthalate, was assessed in the perfused rat liver. Livers from starved rats were perfused with ethylhexanol (3 mM) dissolved in Krebs-Henseleit buffer (pH 7.4, 37 degrees C) saturated with 95% O2-5% CO2 in both the anterograde and retrograde direction. Following infusion of ethylhexanol, O2 uptake and ketone body formation were diminished by 50 and 80%, respectively, and cell damage, as assessed by the appearance of lactate dehydrogenase in the effluent perfusate, was apparent. Both inhibition of O2 uptake by ethylhexanol and the appearance of lactate dehydrogenase in the perfusate were dose-dependent. Only O2-rich upstream regions of the liver lobule were damaged as reflected by trypan blue uptake. Inhibition of O2 uptake by ethylhexanol was also reflected by a 60% decrease in the ATP/ADP ratio. Local rates of O2 uptake, measured using miniature electrodes placed on the liver surface, indicated that ethylhexanol only diminished O2 uptake in O2-rich upstream regions of the liver lobule regardless of the direction of flow. This phenomenon apparently can be explained by a direct effect of ethylhexanol on mitochondria in upstream regions since active state 3 rates of respiration were inhibited by ethylhexanol in isolated mitochondria. Ethylhexanol also caused a dose-dependent decrease in the mitochondrial membrane potential and an increase in the beta-hydroxybutyrate/acetoacetate (B/A) ratio. However, infusion of radical scavengers such as allopurinol, cianidanol and uric acid did not alter lactate dehydrogenase release due to ethylhexanol. Thus, the toxicity of ethylhexanol in the liver is dependent on local O2 tension and mitochondrial are primary targets.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Spermine binding to liver mitochondria deenergized by ruthenium red plus either FCCP or antimycin A.

    PubMed

    Dalla Via, L; Di Noto, V; Toninello, A

    1998-01-23

    Thermodynamic analysis of spermine binding to mitochondria treated with ruthenium red and deenergized with either FCCP or antimycin A confirms the presence of two polyamine binding sites, S1 and S2, both with monocoordination, as previously observed in energized mitochondria [Dalla Via et al., Biochim. Biophys. Acta 1284 (1996) 247-252]. Both sites undergo a marked change in binding capacity and binding affinity upon mitochondrial deenergization. This change is most likely responsible for the incomplete or delayed spermine-mediated inhibition of the permeability transition induced in deenergized mitochondria.

  18. Increased Susceptibility of Gracilinanus microtarsus Liver Mitochondria to Ca2+-Induced Permeability Transition Is Associated with a More Oxidized State of NAD(P)

    PubMed Central

    Ronchi, Juliana A.; Henning, Barbara; Ravagnani, Felipe G.; Figueira, Tiago R.; Castilho, Roger F.; dos Reis, Sergio F.; Vercesi, Anibal E.

    2015-01-01

    In addition to be the cell's powerhouse, mitochondria also contain a cell death machinery that includes highly regulated processes such as the membrane permeability transition pore (PTP) and reactive oxygen species (ROS) production. In this context, the results presented here provide evidence that liver mitochondria isolated from Gracilinanus microtarsus, a small and short life span (one year) marsupial, when compared to mice, are much more susceptible to PTP opening in association with a poor NADPH dependent antioxidant capacity. Liver mitochondria isolated from the marsupial are well coupled and take up Ca2+ but exhibited a much lower Ca2+ retention capacity than mouse mitochondria. Although the known PTP inhibitors cyclosporin A, ADP, and ATP significantly increased the marsupial mitochondria capacity to retain Ca2+, their effects were much larger in mice than in marsupial mitochondria. Both fluorescence and HPLC analysis of mitochondrial nicotinamide nucleotides showed that both content and state of reduction (mainly of NADPH) were lower in the marsupial mitochondria than in mice mitochondria despite the similarity in the activity of the glutathione peroxidase/reductase system. Overall, these data suggest that PTP opening is an important event in processes of Ca2+ signalling to cell death mediated by mitochondrial redox imbalance in G. microtarsus. PMID:26583063

  19. Hydroperoxides can modulate the redox state of pyridine nucleotides and the calcium balance in rat liver mitochondria

    PubMed Central

    Lötscher, Hans Ruedi; Winterhalter, Kaspar H.; Carafoli, Ernesto; Richter, Christoph

    1979-01-01

    When rats are fed a selenium-deficient diet, the glutathione peroxidase activity in liver mitochondria decreases within 5 weeks to 0-6% of that of control animals fed on a diet supplemented with 0.5 ppm of selenium as sodium selenite. Analysis of the temperature dependence of energy-linked Ca2+ uptake by means of Arrhenius plots reveals two breaks (at around 11°C and 24°C) in mitochondria isolated from selenium-supplemented animals, whereas in selenium-deficient rats the break at 11°C is absent. Ca2+-loaded mitochondria of selenium-supplemented rats—i.e., with active glutathione peroxidase in the matrix—lose Ca2+ rapidly, with a concomitant oxidation of endogenous NAD(P)H, when exposed to t-butyl hydroperoxide or H2O2. In contrast, in selenium deficiency, t-butyl hydroperoxide and H2O2 induce neither a release of Ca2+ nor an oxidation of NAD(P)H. The peroxide-induced oxidation of NAD(P)H is reversible in the presence of succinate when no Ca2+ has been taken up. When Ca2+ has previously been accumulated, however, the oxidation of NAD(P)H is irreversible. Enzymatic analysis of mitochondrial pyridine nucleotides reveals that the peroxide-induced oxidation of NAD(P)H in Ca2+-loaded mitochondria leads to a loss of NAD+ and NADP+. It is proposed that the redox state of mitochondrial pyridine nucleotides can be or is in part controlled by glutathione peroxidase and glutathione reductase and is a factor in the balance of Ca2+ between mitochondria and medium. PMID:41241

  20. Effects of methylglyoxal and pyridoxamine in rat brain mitochondria bioenergetics and oxidative status.

    PubMed

    Cardoso, Susana; Carvalho, Cristina; Marinho, Ricardo; Simões, Anabel; Sena, Cristina M; Matafome, Paulo; Santos, Maria S; Seiça, Raquel M; Moreira, Paula I

    2014-10-01

    Advanced glycation end products (AGEs) and methylglyoxal (MG), an important intermediate in AGEs synthesis, are thought to contribute to protein aging and to the pathogenesis of age-and diabetes-associated complications. This study was intended to investigate brain mitochondria bioenergetics and oxidative status of rats previously exposed to chronic treatment with MG and/or with pyridoxamine (PM), a glycation inhibitor. Brain mitochondrial fractions were obtained and several parameters were analyzed: respiratory chain [states 3 and 4 of respiration, respiratory control ratio (RCR), and ADP/O index] and phosphorylation system [transmembrane potential (ΔΨm), ADP-induced depolarization, repolarization lag phase, and ATP levels]; hydrogen peroxide (H2O2) production levels, mitochondrial aconitase activity, and malondialdehyde levels as well as non-enzymatic antioxidant defenses (vitamin E and glutathione levels) and enzymatic antioxidant defenses (glutathione disulfide reductase (GR), glutathione peroxidase (GPx), and manganese superoxide dismutase (MnSOD) activities). MG treatment induced a statistical significant decrease in RCR, aconitase and GR activities, and an increase in H2O2 production levels. The administration of PM did not counteract MG-induced effects and caused a significant decrease in ΔΨm. In mitochondria from control animals, PM caused an adaptive mechanism characterized by a decrease in aconitase and GR activities as well as an increase in both α-tocopherol levels and GPx and MnSOD activities. Altogether our results show that high levels of MG promote brain mitochondrial impairment and PM is not able to reverse MG-induced effects.

  1. The mitochondria permeability transition pore complex in the brain with interacting proteins - promising targets for protection in neurodegenerative diseases.

    PubMed

    Azarashvili, Tamara; Stricker, Rolf; Reiser, Georg

    2010-06-01

    Mitochondria increasingly attract attention as control points within the mechanisms of neuronal death. Mitochondria play a central role in swinging the balance in favor of either survival or death of brain tissue. Cell death in vertebrates proceeds mostly via the mitochondrial pathway of apoptosis. Permeability transition pore (PTP) development in mitochondria is a decisive stage of apoptosis. Therefore, regulation of the permeability of both outer and inner mitochondrial membranes helps to induce neuroprotection. Through PTP control, mitochondria can to a large degree manage the intracellular calcium homeostasis, and thus control the potent death cascade initiated by excess calcium. Here we summarize the evidence for the role of mitochondria in brain cell death. We describe the involvement of the 18-kDa translocator protein (TSPO; previously called peripheral benzodiazepine receptor), and of two new mitochondrial proteins, that is, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and p42(IP4) (also designated centaurin alpha1; ADAP 1), in the control of the PTP. Furthermore, ligands of TSPO, as well as substrates of CNP, are possible modulators of PTP function. This scenario of control and regulation of PTP function might provide multiple important targets, which are suitable for developing protective strategies for neurons and non-neuronal brain cells in therapies of neurodegenerative diseases.

  2. Decreased proteolytic activity of the mitochondrial amyloid-β degrading enzyme, PreP peptidasome, in Alzheimer's disease brain mitochondria.

    PubMed

    Alikhani, Nyosha; Guo, Lan; Yan, Shiqiang; Du, Heng; Pinho, Catarina Moreira; Chen, John Xi; Glaser, Elzbieta; Yan, Shirley ShiDu

    2011-01-01

    Accumulation of amyloid-β peptide (Aβ), the neurotoxic peptide implicated in the pathogenesis of Alzheimer's disease (AD), has been shown in brain mitochondria of AD patients and of AD transgenic mouse models. The presence of Aβ in mitochondria leads to free radical generation and neuronal stress. Recently, we identified the presequence protease, PreP, localized in the mitochondrial matrix in mammalian mitochondria as the novel mitochondrial Aβ-degrading enzyme. In the present study, we examined PreP activity in the mitochondrial matrix of the human brain's temporal lobe, an area of the brain highly susceptible to Aβ accumulation and reactive oxygen species (ROS) production. We found significantly lower hPreP activity in AD brains compared with non-AD age-matched controls. By contrast, in the cerebellum, a brain region typically spared from Aβ accumulation, there was no significant difference in hPreP activity when comparing AD samples to non-AD controls. We also found significantly reduced PreP activity in the mitochondrial matrix of AD transgenic mouse brains (Tg mAβPP and Tg mAβPP/ABAD) when compared to non-transgenic aged-matched mice. Furthermore, mitochondrial fractions isolated from AD brains and Tg mAβPP mice had higher levels of 4-hydroxynonenal, an oxidative product, as compared with those from non-AD and nonTg mice. Accordingly, activity of cytochrome c oxidase was significantly reduced in the AD mitochondria. These findings suggest that decreased PreP proteolytic activity, possibly due to enhanced ROS production, contributes to Aβ accumulation in mitochondria leading to the mitochondrial toxicity and neuronal death that is exacerbated in AD. Clearance of mitochondrial Aβ by PreP may thus be of importance in the pathology of AD.

  3. Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset.

    PubMed

    Karadayian, Analía G; Malanga, Gabriela; Czerniczyniec, Analía; Lombardi, Paulina; Bustamante, Juanita; Lores-Arnaiz, Silvia

    2017-07-01

    Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Oxidant stress, mitochondria, and cell death mechanisms in drug-induced liver injury: lessons learned from acetaminophen hepatotoxicity.

    PubMed

    Jaeschke, Hartmut; McGill, Mitchell R; Ramachandran, Anup

    2012-02-01

    Hepatotoxicity is a serious problem during drug development and for the use of many established drugs. For example, acetaminophen overdose is currently the most frequent cause of acute liver failure in the United States and Great Britain. Evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through the formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and, eventually, to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in a collapse of mitochondrial membrane potential and cessation of adenosine triphosphate synthesis. In addition, the release of intermembrane proteins, such as apoptosis-inducing factor and endonuclease G, and their translocation to the nucleus, leads to nuclear DNA fragmentation. Together, these events trigger necrotic cell death. Alternatively, the release of cytochrome c and other proapoptotic factors from mitochondria can promote caspase activation and apoptotic cell death. Drug toxicity can also induce an inflammatory response with the formation of reactive oxygen species by Kupffer cells and neutrophils. If not properly detoxified, these extracellularly generated oxidants can diffuse into hepatocytes and trigger mitochondrial dysfunction and oxidant stress, which then induces MPT and necrotic cell death. This review addresses the formation of oxidants and the defense mechanisms available for cells and applies this knowledge to better understand mechanisms of drug hepatotoxicity, especially acetaminophen-induced liver injury.

  5. OXIDANT STRESS, MITOCHONDRIA AND CELL DEATH MECHANISMS IN DRUG-INDUCED LIVER INJURY: LESSONS LEARNED FROM ACETAMINOPHEN HEPATOTOXICITY

    PubMed Central

    Jaeschke, Hartmut; McGill, Mitchell R.; Ramachandran, Anup

    2017-01-01

    Hepatotoxicity is a serious problem during drug development and for the use of many established drugs. For example, acetaminophen overdose is currently the most frequent cause of acute liver failure in the United States and Great Britain. Evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and eventually to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in collapse of the mitochondrial membrane potential and cessation of ATP synthesis. In addition, the release of intermembrane proteins such as apoptosis-inducing factor and endonuclease G and their translocation to the nucleus leads to nuclear DNA fragmentation. Together these events trigger necrotic cell death. Alternatively, release of cytochrome c and other pro-apoptotic factors from mitochondria can promote caspase activation and apoptotic cell death. Drug toxicity can also induce an inflammatory response with formation of reactive oxygen species by Kupffer cells and neutrophils. If not properly detoxified, these extracellularly generated oxidants can diffuse into hepatocytes and trigger mitochondrial dysfunction and oxidant stress, which then induces the MPT and necrotic cell death. This review addresses the formation of oxidants and the defense mechanisms available for the cells and applies this knowledge to better understand mechanisms of drug hepatotoxicity, especially acetaminophen-induced liver injury. PMID:22229890

  6. Microglia-aging: roles of microglial lysosome- and mitochondria-derived reactive oxygen species in brain aging.

    PubMed

    Nakanishi, Hiroshi; Wu, Zhou

    2009-07-19

    The accumulation of lysosome- and mitochondria-derived reactive oxygen species (ROS) are the most important causative factors for aging. Autophagic dysfunction and mitochondrial DNA damage in the central nervous system (CNS) are prominently found in microglia, the resident mononuclear phagocyte population within the CNS. The autophagic dysfunction may induce the defective turnover of mitochondria, which results in the accumulation of ROS-hypergenerating older mitochondria in microglia. ROS activate redox-dependent transduction cascades and transcription factors, including nuclear factor-kappaB, which induce the expression of inflammatory genes. Therefore, "microglia-aging" could function as a major driver for brain aging. Furthermore, the prevention of lysosomal autophagic dysfunction and mitochondrial DNA damage in microglia may therefore be a potentially effective new pharmaceutical intervention against brain aging.

  7. New evidence of mitochondria dysfunction in the female Alzheimer’s brain: deficiency of estrogen receptor-β

    PubMed Central

    Long, Jiangang; He, Ping; Shen, Yong; Li, Rena

    2012-01-01

    Accumulating evidence suggests that mitochondria are important targets for the actions of estrogens and studies indicated that localization of ERβ in neuronal mitochondrial ERβ (mtERβ) might directly affect neuronal mitochondrial function in vitro. However, it is unknown what expression levels and how important of mtERβ in the human brain, particularly in the brain with Alzheimer’s disease (AD). In the present study, by using rapidly autopsied human brain tissue, we found that the frontal cortices of female AD patients exhibited significantly reduced mtERβ, along with reduced mitochondrial cytochrome C oxidase activity, and increased protein carbonylation compared to that in normal controls. The correlation between the mtERβ expression and mitochondrial cytochrome C oxidase activity in the female human brain is significant. To understand the possible mechanisms of mtERβ in AD-related mitochondrial dysfunction, using ERβKO mice as a model, we found that lacking of ERβ enhanced brain reactive oxygen species generation and reduced mitochondrial membrane potential under Aβ peptide insult compared to brain mitochondria from wild-type control mice. Our studies for the first time, demonstrated neuronal mtERβ expression in the human brain and the deficiency of mtERβ in the female AD brain is associated with the dysfunction of mitochondria. Our results from ERβKO mice demonstrated that ERβ depletion-induced mitochondrial dysfunction is mediated through increasing reactive oxygen generation and reduction of mitochondria membrane potential. These results indicate that ERβ depletion has the ability to impair mitochondrial function in mice and reduction of brain mtERβ may significantly contribute to the mitochondrial dysfunction involved in AD pathogenesis in women. PMID:22451324

  8. Proteomic analysis of rat brain mitochondria following exposure to dopamine quinone: implications for Parkinson disease.

    PubMed

    Van Laar, Victor S; Dukes, April A; Cascio, Michael; Hastings, Teresa G

    2008-03-01

    Oxidative stress and mitochondrial dysfunction have been linked to dopaminergic neuron degeneration in Parkinson disease. We have previously shown that dopamine oxidation leads to selective dopaminergic terminal degeneration in vivo and alters mitochondrial function in vitro. In this study, we utilized 2-D difference in-gel electrophoresis to assess changes in the mitochondrial proteome following in vitro exposure to reactive dopamine quinone. A subset of proteins exhibit decreased fluorescence labeling following dopamine oxidation, suggesting a rapid loss of specific proteins. Amongst these proteins are mitochondrial creatine kinase, mitofilin, mortalin, the 75 kDa subunit of NADH dehydrogenase, and superoxide dismutase 2. Western blot analyses for mitochondrial creatine kinase and mitofilin confirmed significant losses in isolated brain mitochondria exposed to dopamine quinone and PC12 cells exposed to dopamine. These results suggest that specific mitochondrial proteins are uniquely susceptible to changes in abundance following dopamine oxidation, and carry implications for mitochondrial stability in Parkinson disease neurodegeneration.

  9. Protective and biogenesis effects of sodium hydrosulfide on brain mitochondria after cardiac arrest and resuscitation.

    PubMed

    Pan, Hao; Xie, Xuemeng; Chen, Di; Zhang, Jincheng; Zhou, Yaguang; Yang, Guangtian

    2014-10-15

    Mitochondrial dysfunction plays a critical role in brain injury after cardiac arrest and cardiopulmonary resuscitation (CPR). Recent studies demonstrated that hydrogen sulfide (H2S) donor compounds preserve mitochondrial morphology and function during ischemia-reperfusion injury. In this study, we sought to explore the effects of sodium hydrosulfide (NaHS) on brain mitochondria 24h after cardiac arrest and resuscitation. Male Sprague-Dawley rats were subjected to 6min cardiac arrest and then resuscitated successfully. Rats received NaHS (0.5mg/kg) or vehicle (0.9% NaCl, 1.67ml/kg) 1min before the start of CPR intravenously, followed by a continuous infusion of NaHS (1.5mg/kg/h) or vehicle (5ml/kg/h) for 3h. Neurological deficit was evaluated 24h after resuscitation and then cortex was collected for assessments. As a result, we found that rats treated with NaHS revealed an improved neurological outcome and cortex mitochondrial morphology 24h after resuscitation. We also observed that NaHS therapy reduced intracellular reactive oxygen species generation and calcium overload, inhibited mitochondrial permeability transition pores, preserved mitochondrial membrane potential, elevated ATP level and ameliorated the cytochrome c abnormal distribution. Further studies indicated that NaHS administration increased mitochondrial biogenesis in cortex at the same time. Our findings suggested that administration of NaHS 1min prior CPR and followed by a continuous infusion ameliorated neurological dysfunction 24h after resuscitation, possibly through mitochondria preservation as well as by promoting mitochondrial biogenesis.

  10. Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria

    PubMed Central

    Caro, Andres A.; Adlong, Luke W.; Crocker, Samuel J.; Gardner, Michael W.; Luikart, Emily F.; Gron, Liz U.

    2012-01-01

    The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125–1000 μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1 μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 1 mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2′,7′ dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants. PMID:22960305

  11. Brain mitochondria as a primary target in the development of treatment strategies for Alzheimer disease.

    PubMed

    Aliev, Gjumrakch; Palacios, Hector H; Walrafen, Brianna; Lipsitt, Amanda E; Obrenovich, Mark E; Morales, Ludis

    2009-10-01

    Alzheimer's disease (AD) and cerebrovascular accidents are two leading causes of age-related dementia. Increasing evidence supports the idea that chronic hypoperfusion is primarily responsible for the pathogenesis that underlies both disease processes. In this regard, hypoperfusion appears to induce oxidative stress (OS), which is largely due to reactive oxygen species (ROS), and over time initiates mitochondrial failure which is known as an initiating factor of AD. Recent evidence indicates that chronic injury stimulus induces hypoperfusion seen in vulnerable brain regions. This reduced regional cerebral blood flow (CBF) then leads to energy failure within the vascular endothelium and associated brain parenchyma, manifested by damaged mitochondrial ultrastructure (the formation of large number of immature, electron-dense "hypoxic" mitochondria) and by overproduction of mitochondrial DNA (mtDNA) deletions. Additionally, these mitochondrial abnormalities co-exist with increased redox metal activity, lipid peroxidation, and RNA oxidation. Interestingly, vulnerable neurons and glial cells show mtDNA deletions and oxidative stress markers only in the regions that are closely associated with damaged vessels, and, moreover, brain vascular wall lesions linearly correlate with the degree of neuronal and glial cell damage. We summarize the large body of evidence which indicates that sporadic, late-onset AD results from a vascular etiology by briefly reviewing mitochondrial damage and vascular risk factors associated with the disease and then we discuss the cerebral microvascular changes reason for the energy failure that occurs in normal aging and, to a much greater extent, AD.

  12. Alteration of SLP2-like immunolabeling in mitochondria signifies early cellular damage in developing and adult mouse brain.

    PubMed

    Morozov, Yury M; Sun, Yu-Yo; Kuan, Chia-Yi; Rakic, Pasko

    2016-01-01

    Mitochondria play a critical role in various pathways of regulated cell death. Here we propose a novel method for detection of initial derangement of mitochondria in degenerating and dying neuronal cells. The method is based on our recent finding that antibodies directed against the cannabinoid type 1 receptor (CB1) also bind the mitochondrial stomatin-like protein 2 (SLP2) that belongs to an inner mitochondrial membrane protein complex. It is well established that SLP2 regulates mitochondrial biogenesis and respiratory functions. We now show that anti-CB1 antibodies recognize conformational epitopes but not the linear amino acid sequence of SLP2. In addition we found that anti-CB1 serum mostly labels swollen mitochondria with early or advanced stages of pathology in mouse brain while other proteins of the complex may mask epitopes of SLP2 in the normal mitochondria. Although neurons and endothelial cells in healthy brains contain occasional immunopositive mitochondria detectable with anti-CB1 serum, their numbers increase significantly after hypoxic insults in parallel with signs of cellular damage. Moreover, use of electron microscopy suggests relocation of SLP2 from its normal functional position in the inner mitochondrial membrane into the mitochondrial matrix in pathological cells. Thus, SLP2-like immunolabeling serves as an in situ histochemical target detecting early derangement of mitochondria. Anti-CB1 serum is crucial for this purpose because available anti-SLP2 antibodies do not provide selective labeling of mitochondria in the fixed tissue. This new method of detecting mitochondrial dysfunction can benefit the in vitro research of human diseases and developmental disorders by enabling analysis in live animal models.

  13. Effect of primycin on the inner membrane permeability of rat liver mitochondria.

    PubMed

    Mészáros, L; König, T; Paróczai, M; Náhm, K; Horváth, I

    1979-02-01

    The effects of primycin on mitochondrial respiration, volume changes, ATPase activity and the acidification following ATP hydrolysis were studied. Primycin in concentrations below 2--3 nmoles/mg mitochondrial protein reacts only with energized mitochondria rendering their inner membrane permeable to K+, Na+, Tris+ but not to TEA+. Above this concentration primycin interacts both with energized and deenergized mitochondria and the inner membrane also becomes permeable for H+, Cl- but not for ATP. In this case mitochondria very probably lose Mg2+. It is concluded that primycin up to concentrations of 2--3 nmoles/mg mitochondrial protein acts like an ionophore, while at higher concentrations it changes the permeability properties of the mitochondrial inner membrane without a drastic alteration of the membrane itself.

  14. Effects of La(III) and Ca(II) on isolated Carassius auratus liver mitochondria: heat production and mitochondrial permeability transition.

    PubMed

    Wu, Man; Gao, Jia-Ling; Sun, Mei-Xiang; Zhang, Ye-Zhong; Liu, Yi; Dai, Jie

    2015-02-01

    The effects of lanthanum and calcium on heat production of mitochondria isolated from Carassius auratus liver were investigated by microcalorimetry, and their effects on mitochondrial swelling and membrane potential (Δψ) were determined by spectroscopic methods. La(3+) showed only inhibitory action on mitochondrial energy turnover with inhibition concentration of 50 % (IC50) being 71.2 μmol L(-1). Similarly, Ca(2+) restrained the heat production of mitochondria, and the IC50 of Ca(2+) was much higher than that of La(3+). In the spectroscopic experiments, La(3+) and Ca(2+) induced fish liver mitochondrial swelling and decreased membrane potential (Δψ), and the induction ability of La(3+) was stronger than that of Ca(2+). It is concluded that the effects of La(3+) and Ca(2+) on fish liver mitochondria differ, and La represents toxic action rather than Ca analogy.

  15. Diamide accelerates opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M; Konovalova, Svetlana A; Brailovskaya, Irina V

    Opening of the mitochondrial permeability transition pore (MPTP) in the inner membrane is due to matrix Ca(2+) overload and matrix glutathione loss. Fixing the 'm' conformation of the adenine nucleotide translocase (ANT) by ADP or N-ethylmaleimide (NEM) inhibits opening of the MPTP. Oxidants (diamide or tert-butylhydroperoxide (tBHP)) fix the ANT in 'c' conformation, and the ability of ADP to inhibit the MPTP is thus attenuated. Earlier we found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria resulted in Tl(+)-induced MPTP opening, which was accompanied by a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration, as well as increased swelling and membrane potential dissipation. These effects, which were increased by diamide and tBHP, were visibly reduced in the presence of the MPTP inhibitors (ADP, NEM, and cyclosporine A). Our data suggest that conformational changes of the ANT and matrix glutathione loss may be directly involved in opening the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria.

  16. The antiestrogen 4-hydroxytamoxifen protects against isotretinoin-induced permeability transition and bioenergetic dysfunction of liver mitochondria: comparison with tamoxifen.

    PubMed

    Silva, Filomena S G; Ribeiro, Mariana P C; Santos, Maria S; Rocha-Pereira, Petronila; Santos-Silva, Alice; Custódio, José B A

    2013-08-01

    The combination of isotretinoin (13-cis-retinoic acid) with antiestrogens seems to be a promising strategy for cancer chemotherapy. The aim of the study was to evaluate the effects of isotretinoin alone or in combination with 4-hydroxytamoxifen (OHTAM) and with its prodrug tamoxifen (TAM), on the functions of rat liver mitochondria, i.e., mitochondrial permeability transition (MPT), bioenergetic functions and adenine nucleotide translocase (ANT). Isotretinoin (5 nmol/mg protein) induced the Ca²⁺-dependent MPT pore opening in mitochondria energized with succinate, which was prevented by OHTAM, cyclosporine A, TAM and ANT ligands. When mitochondria were energized with glutamate/malate and in the absence of added Ca²⁺ isotretinoin decreased the state 3 respiration, the ATP levels, the active ANT content and increased the lag phase of the phosphorylation cycle, demonstrating that isotretinoin decreased the mitochondrial phosphorylation efficiency. These changes of isotretinoin in bioenergetic parameters were not significant in the presence of succinate. The effects of isotretinoin at 5 nmol/mg protein on the Ca²⁺-dependent MPT and phosphorylative efficacy may be related with interactions with the ANT. Above 10 nmol/mg protein isotretinoin strongly diminished the active ANT content, decreased the Δψ, inhibited the complex I and induced proton leak through the Fo fraction of complex V. The combination of OHTAM with isotretinoin only induced significant changes in the energy production systems at concentrations ≥5 nmol isotretinoin/mg protein. Therefore, our results suggest that isotretinoin-associated liver toxicity is possibly related with mitochondrial dysfunctions and that the combination with OHTAM may contribute to decrease its toxicity.

  17. Alzheimer's disease and type 2 diabetes-related alterations in brain mitochondria, autophagy and synaptic markers.

    PubMed

    Carvalho, Cristina; Santos, Maria S; Oliveira, Catarina R; Moreira, Paula I

    2015-08-01

    We aimed to investigate mitochondrial function, biogenesis and autophagy in the brain of type 2 diabetes (T2D) and Alzheimer's disease (AD) mice. Isolated brain mitochondria and homogenates from cerebral cortex and hippocampus of wild-type (WT), triple transgenic AD (3xTg-AD) and T2D mice were used to evaluate mitochondrial functional parameters and protein levels of mitochondrial biogenesis, autophagy and synaptic integrity markers, respectively. A significant decrease in mitochondrial respiration, membrane potential and energy levels was observed in T2D and 3xTg-AD mice. Also, a significant decrease in the levels of autophagy-related protein 7 (ATG7) and glycosylated lysosomal membrane protein 1 (LAMP1) was observed in cerebral cortex and hippocampus of T2D and 3xTg-AD mice. Moreover, both brain regions of 3xTg-AD mice present lower levels of nuclear respiratory factor (NRF) 1 while the levels of NRF2 are lower in both brain regions of T2D and 3xTg-AD mice. A decrease in mitochondrial encoded, nicotinamide adenine dinucleotide dehydrogenase subunit 1 (ND1) was also observed in T2D and 3xTg-AD mice although only statistically significant in T2D cortex. Furthermore, a decrease in the levels of postsynaptic density protein 95 (PSD95) in the cerebral cortex of 3xTg-AD mice and in hippocampus of T2D and 3xTg-AD mice and a decrease in the levels of synaptosomal-associated protein 25 (SNAP 25) in the hippocampus of T2D and 3xTg-AD mice were observed suggesting synaptic integrity loss. These results support the idea that alterations in mitochondrial function, biogenesis and autophagy cause synaptic damage in AD and T2D.

  18. Effect of Synthetic Detergents on the Swelling and the ATPase of Mitochondria Isolated from Rat Liver

    PubMed Central

    Witter, Robert F.; Mink, William

    1958-01-01

    A study was made of the effects of various types of detergents on the swelling of isolated mitochondria and on mitochondrial ATPases which are activated by Mg or DNP respectively. The rate of swelling was measured in the Beckman spectrophotometer by following the decrease in turbidity of dilute suspensions of these organelles. It was found that non-ionic detergents containing a nonyl phenoxy side chain or anionic detergents caused swelling of the mitochondria and activation of Mg-ATPase. On the other hand, cationic detergents promoted the clumping of mitochondria and did not activate Mg-ATPase. DNP-ATPase was inhibited by all of the detergents tested. It would appear from these observations that the inhibition of DNP-ATPase is not related to a gross change in the morphology of the organelles; in contrast, the activation of Mg-ATPase definitely is correlated with swelling of the isolated mitochondria. These data also suggest that the ionic detergents combine with charged sites on the protein moiety of the lipoprotein in the mitochondrial surface, whereas the non-ionic detergents form inclusion compounds with the lipide moiety, thereby altering the mitochondrial structure and permeability. PMID:13502431

  19. Decreased mTOR signalling reduces mitochondrial ROS in brain via accumulation of the telomerase protein TERT within mitochondria

    PubMed Central

    Wan, Tengfei; Bell, Amy; Hill, Kirsten N.; von Zglinicki, Thomas; Saretzki, Gabriele

    2016-01-01

    Telomerase in its canonical function maintains telomeres in dividing cells. In addition, the telomerase protein TERT has non-telomeric functions such as shuttling to mitochondria resulting in a decreased oxidative stress, DNA damage and apoptosis. TERT protein persists in adult neurons and can co-localise to mitochondria under various stress conditions. We show here that TERT expression decreased in mouse brain during aging while release of reactive oxygen species (ROS) from the mitochondrial electron transport chain increased. Dietary restriction (DR) caused accumulation of TERT protein in mouse brain mitochondria correlating to decreased ROS release and improved learning and spatial short-term memory. Decreased mTOR signalling is a mediator of DR. Accordingly, feeding mice with rapamycin increased brain mitochondrial TERT and reduced ROS release. Importantly, the beneficial effects of rapamycin on mitochondrial function were absent in brains and fibroblasts from first generation TERT −/− mice, and when TERT shuttling was inhibited by the Src kinase inhibitor bosutinib. Taken together, our data suggests that the mTOR signalling pathway impinges on the mitochondrial localisation of TERT protein, which might in turn contribute to the protection of the brain by DR or rapamycin against age-associated mitochondrial ROS increase and cognitive decline. PMID:27777385

  20. Determination of boron distribution in rat's brain, kidney and liver.

    PubMed

    Pazirandeh, Ali; Jameie, Behnam; Zargar, Maysam

    2009-07-01

    To determine relative boron distribution in rat's brain, liver and kidney, a mixture of boric acid and borax, was used. After transcardial injection of the solution, the animals were sacrificed and the brain, kidney and liver were removed. The coronal sections of certain areas of the brain were prepared by freezing microtome. The slices were sandwiched within two pieces of CR-39. The samples were bombarded in a thermal neutron field of the TRR pneumatic facility. The alpha tracks are registered on CR-39 after being etched in NaOH. The boron distribution was determined by counting these alpha tracks CR-39 plastics. The distribution showed non-uniformity in brain, liver and kidney.

  1. Cannabinoid-Induced Changes in the Activity of Electron Transport Chain Complexes of Brain Mitochondria.

    PubMed

    Singh, Namrata; Hroudová, Jana; Fišar, Zdeněk

    2015-08-01

    The aim of this study was to investigate changes in the activity of individual mitochondrial respiratory chain complexes (I, II/III, IV) and citrate synthase induced by pharmacologically different cannabinoids. In vitro effects of selected cannabinoids on mitochondrial enzymes were measured in crude mitochondrial fraction isolated from pig brain. Both cannabinoid receptor agonists, Δ(9)-tetrahydrocannabinol, anandamide, and R-(+)-WIN55,212-2, and antagonist/inverse agonists of cannabinoid receptors, AM251, and cannabidiol were examined in pig brain mitochondria. Different effects of these cannabinoids on mitochondrial respiratory chain complexes and citrate synthase were found. Citrate synthase activity was decreased only by Δ(9)-tetrahydrocannabinol and AM251. Significant increase in the complex I activity was induced by anandamide. At micromolar concentration, all the tested cannabinoids inhibited the activity of electron transport chain complexes II/III and IV. Stimulatory effect of anandamide on activity of complex I may participate on distinct physiological effects of endocannabinoids compared to phytocannabinoids or synthetic cannabinoids. Common inhibitory effect of cannabinoids on activity of complex II/III and IV confirmed a non-receptor-mediated mechanism of cannabinoid action on individual components of system of oxidative phosphorylation.

  2. Proteomic profiling of mitochondria: what does it tell us about the ageing brain?

    PubMed

    Ingram, Thomas; Chakrabarti, Lisa

    2016-12-13

    Mitochondrial dysfunction is evident in numerous neurodegenerative and age-related disorders. It has also been linked to cellular ageing, however our current understanding of the mitochondrial changes that occur are unclear. Functional studies have made some progress reporting reduced respiration, dynamic structural modifications and loss of membrane potential, though there are conflicts within these findings. Proteomic analyses, together with functional studies, are required in order to profile the mitochondrial changes that occur with age and can contribute to unravelling the complexity of the ageing phenotype. The emergence of improved protein separation techniques, combined with mass spectrometry analyses has allowed the identification of age and cell-type specific mitochondrial changes in energy metabolism, antioxidants, fusion and fission machinery, chaperones, membrane proteins and biosynthesis pathways. Here, we identify and review recent data from the analyses of mitochondria from rodent brains. It is expected that knowledge gained from understanding age-related mitochondrial changes of the brain should lead to improved biomarkers of normal ageing and also age-related disease progression.

  3. Proteomic profiling of mitochondria: what does it tell us about the ageing brain?

    PubMed Central

    Ingram, Thomas; Chakrabarti, Lisa

    2016-01-01

    Mitochondrial dysfunction is evident in numerous neurodegenerative and age-related disorders. It has also been linked to cellular ageing, however our current understanding of the mitochondrial changes that occur are unclear. Functional studies have made some progress reporting reduced respiration, dynamic structural modifications and loss of membrane potential, though there are conflicts within these findings. Proteomic analyses, together with functional studies, are required in order to profile the mitochondrial changes that occur with age and can contribute to unravelling the complexity of the ageing phenotype. The emergence of improved protein separation techniques, combined with mass spectrometry analyses has allowed the identification of age and cell-type specific mitochondrial changes in energy metabolism, antioxidants, fusion and fission machinery, chaperones, membrane proteins and biosynthesis pathways. Here, we identify and review recent data from the analyses of mitochondria from rodent brains. It is expected that knowledge gained from understanding age-related mitochondrial changes of the brain should lead to improved biomarkers of normal ageing and also age-related disease progression. PMID:27992860

  4. Biapigenin modulates the activity of the adenine nucleotide translocator in isolated rat brain mitochondria.

    PubMed

    Silva, Bruno A; Oliveira, Paulo J; Cristóvão, Armando; Dias, Alberto C P; Malva, João O

    2010-01-01

    In this study, we investigated the effects of biapigenin, a biflavone present in the extracts of Hypericum perforatum, in rat brain mitochondrial bioenergetics and calcium homeostasis. We found that biapigenin significantly decreased adenosine diphosphate (ADP)-induced membrane depolarization and increased repolarization (by 68 and 37%, respectively). These effects were blocked by atractyloside and bongkrekic acid, but not oligomycin. In the presence of biapigenin, an ADP-stimulated state 3 respiration was still noticeable, which did not happen in the presence of adenine nucleotide translocator (ANT) inhibitors. Taking in consideration the relevance of the ANT in the modulation of the mitochondrial permeability transition pore (mPTP), mitochondrial calcium homeostasis was evaluated alone or in the presence of biapigenin. We found that biapigenin reduces mitochondrial calcium retention by increasing calcium efflux, an effect that was blocked by ADP plus oligomycin, an efficient blocker of the mPTP in brain mitochondria. Taken together, the results in this article suggest that biapigenin modulates mPTP opening, possibly by modulating ANT function, contributing for enhanced mitochondrial calcium efflux, thereby reducing calcium burden and contributing for neuroprotection against excitotoxicity.

  5. A Mitofusin-2-dependent inactivating cleavage of Opa1 links changes in mitochondria cristae and ER contacts in the postprandial liver.

    PubMed

    Sood, Aditi; Jeyaraju, Danny Vijey; Prudent, Julien; Caron, Alexandre; Lemieux, Philippe; McBride, Heidi May; Laplante, Mathieu; Tóth, Katalin; Pellegrini, Luca

    2014-11-11

    Hepatic metabolism requires mitochondria to adapt their bioenergetic and biosynthetic output to accompany the ever-changing anabolic/catabolic state of the liver cell, but the wiring of this process is still largely unknown. Using a postprandial mouse liver model and quantitative cryo-EM analysis, we show that when the hepatic mammalian target of rapamycin complex 1 (mTORC1) signaling pathway disengages, the mitochondria network fragments, cristae density drops by 30%, and mitochondrial respiratory capacity decreases by 20%. Instead, mitochondria-ER contacts (MERCs), which mediate calcium and phospholipid fluxes between these organelles, double in length. These events are associated with the transient expression of two previously unidentified C-terminal fragments (CTFs) of Optic atrophy 1 (Opa1), a mitochondrial GTPase that regulates cristae biogenesis and mitochondria dynamics. Expression of Opa1 CTFs in the intermembrane space has no effect on mitochondria morphology, supporting a model in which they are intermediates of an Opa1 degradation program. Using an in vitro assay, we show that these CTFs indeed originate from the cleavage of Opa1 at two evolutionarily conserved consensus sites that map within critical folds of the GTPase. This processing of Opa1, termed C-cleavage, is mediated by the activity of a cysteine protease whose activity is independent from that of Oma1 and presenilin-associated rhomboid-like (PARL), two known Opa1 regulators. However, C-cleavage requires Mitofusin-2 (Mfn2), a key factor in mitochondria-ER tethering, thereby linking cristae remodeling to MERC assembly. Thus, in vivo, mitochondria adapt to metabolic shifts through the parallel remodeling of the cristae and of the MERCs via a mechanism that degrades Opa1 in an Mfn2-dependent pathway.

  6. A simple method for isolating rat brain mitochondria with high metabolic activity: effects of EDTA and EGTA.

    PubMed

    Iglesias-González, Javier; Sánchez-Iglesias, Sofía; Beiras-Iglesias, Andrés; Soto-Otero, Ramón; Méndez-Álvarez, Estefanía

    2013-02-15

    Isolated mitochondria are widely used in metabolic and oxidative stress studies for neurodegenerative diseases. In the present work, the influence of EGTA and EDTA has been tested on a sucrose-based differential centrifugation protocol in order to establish the optimal concentrations to be used in this process. Our results showed alterations in both active and resting respiration, which were dependent on both the addition of EDTA or EGTA to the isolation buffer and the chelator concentration used. However, the addition of chelator to the isolation medium does not modify the mitochondria structure as assessed by both distribution of biological markers and electron micrography in the final pellet. Our results endorse this protocol as the method of choice for metabolic and oxidative stress experiments with fresh isolated rat brain mitochondria. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. The Potential Liver, Brain, and Embryo Toxicity of Titanium Dioxide Nanoparticles on Mice

    NASA Astrophysics Data System (ADS)

    Jia, Xiaochuan; Wang, Shuo; Zhou, Lei; Sun, Li

    2017-08-01

    Nanoscale titanium dioxide (nano-TiO2) has been widely used in industry and medicine. However, the safety of nano-TiO2 exposure remains unclear. In this study, we evaluated the liver, brain, and embryo toxicity and the underlying mechanism of nano-TiO2 using mice models. The results showed that titanium was distributed to and accumulated in the heart, brain, spleen, lung, and kidney of mice after intraperitoneal (i.p.) nano-TiO2 exposure, in a dose-dependent manner. The organ/body weight ratios of the heart, spleen, and kidney were significantly increased, and those of the brain and lung were decreased. High doses of nano-TiO2 significantly damaged the functions of liver and kidney and glucose and lipid metabolism, as showed in the blood biochemistry tests. Nano-TiO2 caused damages in mitochondria and apoptosis of hepatocytes, generation of reactive oxygen species, and expression disorders of protective genes in the liver of mice. We found ruptured and cracked nerve cells and inflammatory cell infiltration in the brain. We also found that the activities of constitutive nitric oxide synthases (cNOS), inducible NOS (iNOS), and acetylcholinesterase, and the levels of nitrous oxide and glutamic acid were changed in the brain after nano-TiO2 exposure. Ex vivo mouse embryo models exhibited developmental and genetic toxicity after high doses of nano-TiO2. The size of nano-TiO2 particles may affect toxicity, larger particles producing higher toxicity. In summary, nano-TiO2 exhibited toxicity in multiple organs in mice after exposure through i.p. injection and gavage. Our study may provide data for the assessment of the risk of nano-TiO2 exposure on human health.

  8. Stimulation of H(2)O(2) generation by calcium in brain mitochondria respiring on alpha-glycerophosphate.

    PubMed

    Tretter, Laszlo; Takacs, Katalin; Kövér, Kinga; Adam-Vizi, Vera

    2007-11-15

    It has been reported recently (Tretter et al., 2007b) that in isolated guinea pig brain mitochondria supported by alpha-glycerophosphate (alpha-GP) reactive oxygen species (ROS) are produced through the reverse electron transport (RET) in the respiratory chain and by alpha-glycerophosphate dehydrogenase (alpha-GPDH). We studied the effect of calcium on the generation of H(2)O(2) as measured by the Amplex Red fluorescent assay in this model. H(2)O(2) production in alpha-GP-supported mitochondria was increased significantly in the presence of 100, 250, and 500 nM Ca(2+), respectively. In addition, Ca(2+) enhanced the membrane potential, the rate of oxygen consumption, and the NAD(P)H autofluorescence in these mitochondria. Direct measurement of alpha-GPDH activity showed that Ca(2+) stimulated the enzyme by decreasing the Km for alpha-GP. In those mitochondria where RET was eliminated by the Complex I inhibitor rotenone (2 microM) or due to depolarization by ADP (1 mM), the rate of H(2)O(2) formation was smaller and the stimulation of H(2)O(2) generation by Ca(2+) was prevented partly, but the stimulatory effect of Ca(2+) was still significant. These data indicate that in alpha-GP-supported mitochondria activation of alpha-GPDH by Ca(2+) leads to an accelerated RET-mediated ROS generation as well as to a stimulated ROS production by alpha-GPDH.

  9. Inhibitory effect of added adenosine diphosphate on palmitate oxidation in mitochondria from rat brain

    SciTech Connect

    Kawamura, N.

    1986-05-01

    It is generally accepted that fatty acids are poor substrates for the oxidation in brain because plasma fatty acids do not traverse the blood-brain barrier. However, a regional difference in the barrier suggests that fatty acids are available for oxidation. Why most of fatty acids are not oxidized is not certain. For this reason, regulation of oxidation of (1-/sup 14/C)palmitate (pal) in rat brain has been studied in nonsynaptic mitochondria (mit) prepared by use of Ficoll/sucrose density gradient. The authors found two contrasting oxidations with respect to ATP concentration; Type A at 2 mM and Type B at 0.5 mM. The rate of Type A was 50% of the level of B. Type A was inhibited by high levels of L-carnitine (car) and Mg/sup 2 +/. Added ADP inhibited Type A, but stimulated B. Addition of carboxyatractyloside was stimulatory for Type A, but inhibitory for B. The rate of Type A showed a downward curvature with increasing protein concentration while that of B showed a linear relationship. Addition of NH/sub 4//sup +/ to Type A stimulated the rate and reduced the inhibitory effects of both added ADP and high levels of car. These results suggest that under the normal level of ATP, the carnitine-dependent transport of pal is inhibited (thereby resulting in the inhibition in pal oxidation) by the transport of ADP into mit mediated by the ATP-ADP translocase, but that the inhibition is not observed under the specified conditions or regions where ATP levels are low or ammonia levels are high.

  10. 1,3-dinitrobenzene induces age- and region-specific oxidation to mitochondria-related proteins in brain.

    PubMed

    Kubik, Laura L; Landis, Rory W; Remmer, Henriette; Bergin, Ingrid L; Philbert, Martin A

    2015-05-01

    Regions of the brain with high energy requirements are especially sensitive to perturbations in mitochondrial function. Hence, neurotoxicant exposures that target mitochondria in regions of high energy demand have the potential to accelerate mitochondrial damage inherently occurring during the aging process. 1,3-Dinitrobenzene (DNB) is a model neurotoxicant that selectively targets mitochondria in brainstem nuclei innervated by the eighth cranial nerve. This study investigates the role of age in the regional susceptibility of brain mitochondria-related proteins (MRPs) to oxidation following exposure to DNB. Male F344 rats (1 month old [young], 3 months old [adult], 18 months old [aged]) were exposed to 10 mg/kg DNB prior to mitochondrial isolation and histopathology experiments. Using a high-throughput proteomic approach, 3 important region- and age-related increases in DNB-induced MRP oxidation were determined: (1) brainstem mitochondria are ×3 more sensitive to DNB-induced oxidation than cortical mitochondria; (2) oxidation of brainstem MRPs is significantly higher than in cortical counterparts; and (3) MRPs from the brainstems of older rats are significantly more oxidized than those from young or adult rats. Furthermore, lower levels of DNB cause signs of intoxication (ataxia, chromodacryorrhea) and vacuolation of the susceptible neuropil in aged animals, while neither is observed in DNB-exposed young rats. Additionally, methemoglobin levels increase significantly in DNB-exposed adult and aged animals, but not young DNB-exposed animals. This suggests that oxidation of key MRPs observed in brainstem of aged animals is necessary for DNB-induced signs of intoxication and lesion formation. These results provide compelling evidence that environmental chemicals such as DNB may aid in the acceleration of injury to specific brain regions by inducing oxidation of sensitive mitochondrial proteins.

  11. Loss of brain function - liver disease

    MedlinePlus

    ... may be made by the body, such as ammonia. Or they may be substances that you take ... MRI EEG Liver function tests Prothrombin time Serum ammonia level Sodium level in the blood Potassium level ...

  12. 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.

  13. Closure of mitochondrial potassium channels favors opening of the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M; Brailovskaya, Irina V; Shumakov, Anton R; Emelyanova, Larisa V

    2015-06-01

    It is known that a closure of ATP sensitive (mitoKATP) or BK-type Ca(2+) activated (mitoKCa) potassium channels triggers opening of the mitochondrial permeability transition pore (MPTP) in cells and isolated mitochondria. We found earlier that the Tl(+)-induced MPTP opening in Ca(2+)-loaded rat liver mitochondria was accompanied by a decrease of 2,4-dinitrophenol-uncoupled respiration and increase of mitochondrial swelling and ΔΨmito dissipation in the medium containing TlNO3 and KNO3. On the other hand, our study showed that the mitoKATP inhibitor, 5-hydroxydecanoate favored the Tl(+)-induced MPTP opening in the inner membrane of Ca(2+)-loaded rat heart mitochondria (Korotkov et al. 2013). Here we showed that 5-hydroxydecanoate increased the Tl(+)-induced MPTP opening in the membrane of rat liver mitochondria regardless of the presence of mitoKATP modulators (diazoxide and pinacidil). This manifested in more pronounced decrease in the uncoupled respiration and acceleration of both the swelling and the ΔΨmito dissipation in isolated rat liver mitochondria, incubated in the medium containing TlNO3, KNO3, and Ca(2+). A slight delay in Ca(2+)-induced swelling of the mitochondria exposed to diazoxide could be result of an inhibition of succinate oxidation by the mitoKATP modulator. Mitochondrial calcium retention capacity (CRC) was markedly decreased in the presence of the mitoKATP inhibitor (5-hydroxydecanoate) or the mitoKCa inhibitor (paxilline). We suggest that the closure of mitoKATP or mitoKCa in calcium loaded mitochondria favors opening of the Tl(+)-induced MPTP in the inner mitochondrial membrane.

  14. 3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

    PubMed Central

    Lynx, Matthew D.; Bentley, Alice T.; McKee, Edward E.

    2006-01-01

    3′-Azido-3′-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5′-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC50) of 7.0 ± 1.0 μM) than AZTTP is of polymerase γ (IC50 of >100 μM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher Vmax and Km than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC50 of 14.4 ± 2.6 μM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria. PMID:16472780

  15. Direct measurement of the initial proton extrusion to oxygen uptake ratio accompanying succinate oxidation by rat liver mitochondria.

    PubMed Central

    Setty, O H; Shrager, R I; Bunow, B; Reynafarje, B; Lehninger, A L; Hendler, R W

    1986-01-01

    The problem of obtaining very early ratios for the H+/O stoichiometry accompanying succinate oxidation by rat liver mitochondria was attacked using new techniques for direct measurement rather than extrapolations based on data obtained after mixing and the recovery of the electrode from initial injection of O2. Respiration was quickly initiated in a thoroughly mixed O2-containing suspension of mitochondria under a CO atmosphere by photolysis of the CO-cytochrome c oxidase complex-. Fast responding O2 and pH electrodes were used to collect data every 10 ms. The response time for each electrode was experimentally measured in each experiment and suitable corrections for electrode relaxations were made. With uncorrected data obtained after 0.8 s, the extrapolation back to zero time on the basis of single-exponential curve fitting confirmed values close to 8.0 as previously reported (Costa et al., 1984). The data directly obtained, however, indicate an initial burst in H+/O ratio that peaked to values of approximately 20 to 30 prior to 50 ms and which was no longer evident after 0.3 s. Newer information and considerations that place all extrapolation methods in question are discussed. PMID:3019443

  16. Evaluation of epirubicin-induced acute oxidative stress toxicity in rat liver cells and mitochondria, and the prevention of toxicity through quercetin administration.

    PubMed

    Kebieche, M; Lakroun, Z; Lahouel, M; Bouayed, J; Meraihi, Z; Soulimani, R

    2009-03-01

    Anticancer therapy with epirubicin (EPI) results in acute hepatotoxicity, likely due to the generation of free radicals. However, the oxidative status of rat liver cells and mitochondria after EPI toxicity has not been investigated. In the present study, we first investigated the pro-oxidant effect of EPI on both hepatic cells and mitochondrial function. Injection of EPI into rats at a dose of 9mg/kg (cumulative dose in human chemotherapy), induced hepatic dysfunction, as revealed by a significant increase in serum glutamate oxaloacetate transaminases (SGOT) and glutamate pyruvate transaminases (SGPT). Oxidative stress in liver cells and mitochondria was provoked by EPI because a statistically significant reduction of catalase (CAT), superoxide dismutase (SOD) and cytosolic glutathione (GSH) levels, and a significant increase in malonedialdehyde (MDA) levels - an indicator of lipid peroxidation that can perforate biological membranes - were observed. Second, the protective effect of quercetin (QE) (0.33mg/kg) against EPI-induced oxidative stress was also investigated. Indeed, the pretreatment of rats with QE protected liver cells and mitochondria from oxidative stress. This treatment prevented hepatic dysfunction by maintaining normal levels of serum transaminases following the inhibition of their hepatic leakage by preventing lipid peroxidation. Thus, QE works through the prevention of cellular membrane perforation and the antioxidant defense system of mitochondria from liver cells, which represent compartments for the permanent production of reactive oxygen species (ROS) through the respiratory chain.

  17. 3′-AZIDO-3′-DEOXYTHYMIDINE (AZT) IS A COMPETITIVE INHIBITOR OF THYMIDINE PHOSPHORYLATION IN ISOLATED RAT HEART AND LIVER MITOCHONDRIA

    PubMed Central

    Lynx, Matthew D.; McKee, Edward E.

    2006-01-01

    Long-term use of 3′-azido-3′deoxythymidine (AZT) is associated with various tissue toxicities, including hepatotoxicity and cardiomyopathy, and with mitochondrial DNA depletion. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial DNA polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart and liver mitochondria, AZT itself was shown to be a more potent inhibitor of thymidine phosphorylation (IC50 of 7.0 ± 1.0 μM AZT in heart mitochondria and of 14.4 ± 2.6 μM AZT in liver mitochondria) than AZTTP is of polymerase γ (IC50 of >100 μM AZTTP), suggesting that depletion of mitochondrial stores of TTP may limit replication and could be the cause of the mitochondrial DNA depletion observed in tissues affected by AZT toxicity. The purpose of this work is to characterize the nature of AZT inhibition of thymidine phosphorylation in isolated rat heart and rat liver mitochondria. In both of these tissues, AZT was found to be a competitive inhibitor of the phosphorylation of thymidine to TMP, catalyzed by thymidine kinase 2. The inhibition constant (Ki) for heart mitochondria is 10.6 ± 4.5 μM AZT, and for liver mitochondria Ki is 14.0 ± 2.5 μM AZT. Since AZT is functioning as a competitive inhibitor, increasing thymidine concentrations may be one mechanism to overcome the inhibition and decrease AZT-related toxicity in these tissues. PMID:16720018

  18. Changes of total water and sucrose space accompanying induced ion uptake or phosphate swelling of rat liver mitochondria

    PubMed Central

    Harris, E. J.; Van Dam, K.

    1968-01-01

    1. Total water exchangeable with tritiated water and sucrose space were measured in rat liver mitochondria during the uptake of K+ induced by valinomycin and the release caused by nigericin. The K+ content and the sucrose-inaccessible water rose and fell together. 2. Swelling resulting from phosphate addition in a medium of high K+ concentration was associated mainly with increased sucrose-accessible water, which carried dissolved K+. This change was reversed by addition of ATP. 3. The response of the sucrose-inaccessible space to changed osmolarity was qualitatively that expected if the mitochondrial K+ is assumed to be present in this space with a univalent anion. 4. It is brought out that the light-scattering method fails to distinguish between changes in sucrose space and in sucrose-inaccessible space, which in the present experiments could be altered respectively by phosphate (in high K+ solution) and by cation uptake induced by antibiotic. PMID:5639931

  19. Role of mitochondria in apoptotic and necroptotic cell death in the developing brain

    PubMed Central

    Thornton, Claire; Hagberg, Henrik

    2015-01-01

    Hypoxic–ischemic encephalopathy induces secondary brain injury characterized by delayed energy failure. Currently, therapeutic hypothermia is the sole treatment available after severe intrapartum asphyxia in babies and acts to attenuate secondary loss of high energy phosphates improving both short- and long-term outcome. In order to develop the next generation of neuroprotective therapies, we urgently need to understand the underlying molecular mechanisms leading to cell death. Hypoxia–ischemia creates a toxic intracellular environment including accumulation of reactive oxygen/nitrosative species and intracellular calcium after the insult, inducing mitochondrial impairment. More specifically mitochondrial respiration is suppressed and calcium signaling is dysregulated. At a certain threshold, Bax-dependent mitochondrial permeabilization will occur leading to activation of caspase-dependent and apoptosis-inducing factor-dependent apoptotic cell death. In addition, hypoxia–ischemia induces inflammation, which leads to the release of TNF-α, TRAIL, TWEAK, FasL and Toll-like receptor agonists that will activate death receptors on neurons and oligodendroglia. Death receptors trigger apoptotic death via caspase-8 and necroptotic cell death through formation of the necrosome (composed of RIP1, RIP3 and MLKL), both of which converge at the mitochondria. PMID:25661091

  20. Study of the mechanism of permeabilization of lecithin liposomes and rat liver mitochondria by the antimicrobial drug triclosan.

    PubMed

    Belosludtsev, Konstantin N; Belosludtseva, Natalia V; Tenkov, Kirill S; Penkov, Nikita V; Agafonov, Alexey V; Pavlik, Lyubov L; Yashin, Valery A; Samartsev, Victor N; Dubinin, Mikhail V

    2017-09-19

    The effect of the antimicrobial compound triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol;) on the permeability of lecithin liposomes and rat liver mitochondria was studied. It was found that triclosan was able to increase nonspecific permeability of liposomes in a dose-dependent manner, which was detected by the release of the fluorescent probe sulforhodamine B (SRB) from vesicles. A partial release of SRB occurs instantly at the moment of triclosan addition, which is followed by a slow leakage of the dye. The triclosan-induced release of SRB from liposomes grew as pH of the medium was decreased from 9.5 to 7.5. As revealed by the laurdan generalized polarization (GP) technique, triclosan increased laurdan GP in lecithin liposomes, indicating a decrease in membrane fluidity. Measurements of GP as a function of fluorescence excitation wavelength gave an ascending line for triclosan-containing liposomes, which can be interpreted as phase heterogeneity of the lipid/triclosan system. Dynamic light scattering experiments also showed that at a high triclosan-to-lipid molar ratio (~0.5), a population of smaller light-scattering particles (~0.4 of the size of liposomes) appear in the system. Experiments with rat liver mitochondria demonstrated that triclosan (10-70μM) induced a high-amplitude cyclosporin А-insensitive swelling of the organelles accompanied the release of cytochrome c. On the basis of the results obtained, possible mechanisms of the toxic effect of triclosan in eukaryotic cells are discussed. Copyright © 2017. Published by Elsevier B.V.

  1. On the mechanism by which hormones induce the release of Ca2+ from mitochondria in the liver cell.

    PubMed Central

    Whiting, J A; Barritt, G J

    1982-01-01

    1. The abilities of dinitrophenol, NaCl, Ruthenium Red and the Ca2+-selective ionophore A23187 to release 45 Ca2+ from isolated hepatocytes and liver mitochondria (incubated at 37 degrees C in the presence of 0.1 microM-free Ca2+, Mg2+, ATP and phosphate ions) were compared with the action of adrenaline on 45Ca2+ release from isolated hepatocytes. The effects of adrenaline were most closely described by those of the ionophore A23187. 2. In isolated hepatocytes, a release of 45Ca2+ and stimulation of O2 utilization similar to that induced by adrenaline was observed in the presence of 500 and 20 microM-arachidonic acid respectively. The effect of arachidonic acid on 45Ca2+ release was not specific for this unsaturated fatty acid. 3. Inhibitors of arachidonic acid metabolism, including indomethacin and eicosa-5,811,14-tetraynoic acid, did not block the effects of adrenaline on 45Ca2+ or glucose release from isolated hepatocytes. 4. The ability of adrenaline to stimulate 45Ca2+ release from isolated hepatocytes was rapidly reversed after the subsequent addition of phenoxybenzamine to the cell suspension, and was completely blocked by 0.5 mM-dibucaine. 5. The results are consistent with the action of a Ca2+-selective ionophore in the mechanism by which adrenaline induces the release of Ca2+ from mitochondria in the liver cell and indicate that it is unlikely that arachidonic acid or a metabolite of arachidonic acid is involved in this process. PMID:6812571

  2. High Ca2+ load promotes hydrogen peroxide generation via activation of α-glycerophosphate dehydrogenase in brain mitochondria.

    PubMed

    Tretter, Laszlo; Adam-Vizi, Vera

    2012-12-01

    H(2)O(2) generation associated with α-glycerophosphate (α-GP) oxidation was addressed in guinea pig brain mitochondria challenged with high Ca(2+) load (10 μM). Exposure to 10 μM Ca(2+) induced an abrupt 2.5-fold increase in H(2)O(2) release compared to that measured in the presence of a physiological cytosolic Ca(2+) concentration (100 nM) from mitochondria respiring on 5 mM α-GP in the presence of ADP (2 mM). The Ca(2+)-induced stimulation of H(2)O(2) generation was reversible and unaltered by the uniporter blocker Ru 360, indicating that it did not require Ca(2+) uptake into mitochondria. Enhanced H(2)O(2) generation by Ca(2+) was also observed in the absence of ADP when mitochondria exhibited permeability transition pore opening with a decrease in the NAD(P)H level, dissipation of membrane potential, and mitochondrial swelling. Furthermore, mitochondria treated with the pore-forming peptide alamethicin also responded with an elevated H(2)O(2) generation to a challenge with 10 μM Ca(2+). Ca(2+)-induced promotion of H(2)O(2) formation was further enhanced by the complex III inhibitor myxothiazol. With 20 mM α-GP concentration, stimulation of H(2)O(2) formation by Ca(2+) was detected only in the presence, not in the absence, of ADP. It is concluded that α-glycerophosphate dehydrogenase, which is accessible to and could be activated by a rise in the level of cytosolic Ca(2+), makes a major contribution to Ca(2+)-stimulated H(2)O(2) generation. This work highlights a unique high-Ca(2+)-stimulated reactive oxygen species-forming mechanism in association with oxidation of α-GP, which is largely independent of the bioenergetic state and can proceed even in damaged, functionally incompetent mitochondria.

  3. Amburana cearensis seed extract protects brain mitochondria from oxidative stress and cerebellar cells from excitotoxicity induced by glutamate.

    PubMed

    Lima Pereira, Érica Patrícia; Santos Souza, Cleide; Amparo, Jessika; Short Ferreira, Rafael; Nuñez-Figueredo, Yanier; Gonzaga Fernandez, Luzimar; Ribeiro, Paulo Roberto; Braga-de-Souza, Suzana; Amaral da Silva, Victor Diogenes; Lima Costa, Silvia

    2017-09-14

    Amburana cearensis (Allemao) A.C.Sm. is a medicinal plant of the Brazilian Caatinga reported to present antioxidant and anti-inflammatory activity. This study aimed to evaluate the neuroprotective effect of the extracts obtained from the seeds of A. cearensis in primary cultures of cerebellar cells subjected to excitotoxicity induced by glutamate and brain mitochondria submitted to oxidative stress. and methods: Primary cultures of cerebellar cells were treated with the ethanol (ETAC), hexane (EHAC), dichloromethane (EDAC) and ethyl acetate (EAAC) extracts of the seeds of A.cearensis and subjected to excitotoxicity induced by glutamate (10µM). Mitochondria isolated from rat brains were submitted to oxidative stress and treated with ETAC. Only the EHAC extract reduced cell viability by 30% after 72h of treatment. Morphological analyses by Immunofluorescence showed positive staining for glutamine synthetase, β-III tubulin, GFAP and IBA1 similar to control cultures, indicating a better preservation of astrocytes, neurons and microglia, after excitotoxic damage induced by glutamate in cerebellar cultures treated with the extracts. The ETAC extract also protected mitochondria isolated from rat brains from oxidative stress, reducing the swelling, dissipation of the membrane potential, ROS production and calcium influx. Thus, this study suggests that the seed extracts from A. Cearensis exhibit neuroprotective potential against oxidative stress and excitotoxicity induced by glutamate and can be considered a potential therapeutic agent in the treatment of neurodegenerative diseases. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  4. Neuroprotective Effects of Mitochondria-Targeted Plastoquinone and Thymoquinone in a Rat Model of Brain Ischemia/Reperfusion Injury.

    PubMed

    Silachev, Denis N; Plotnikov, Egor Y; Zorova, Ljubava D; Pevzner, Irina B; Sumbatyan, Natalia V; Korshunova, Galina A; Gulyaev, Mikhail V; Pirogov, Yury A; Skulachev, Vladimir P; Zorov, Dmitry B

    2015-08-11

    We explored the neuroprotective properties of natural plant-derived antioxidants plastoquinone and thymoquinone (2-demethylplastoquinone derivative) modified to be specifically accumulated in mitochondria. The modification was performed through chemical conjugation of the quinones with penetrating cations: Rhodamine 19 or tetraphenylphosphonium. Neuroprotective properties were evaluated in a model of middle cerebral artery occlusion. We demonstrate that the mitochondria-targeted compounds, introduced immediately after reperfusion, possess various neuroprotective potencies as judged by the lower brain damage and higher neurological status. Plastoquinone derivatives conjugated with rhodamine were the most efficient, and the least efficiency was shown by antioxidants conjugated with tetraphenylphosphonium. Antioxidants were administered intraperitoneally or intranasally with the latter demonstrating a high level of penetration into the brain tissue. The therapeutic effects of both ways of administration were similar. Long-term administration of antioxidants in low doses reduced the neurological deficit, but had no effect on the volume of brain damage. At present, cationic decylrhodamine derivatives of plastoquinone appear to be the most promising anti-ischemic mitochondria-targeted drugs of the quinone family. We suggest these antioxidants could be potentially used for a stroke treatment.

  5. Mitochondrial toxicity of diclofenac and its metabolites via inhibition of oxidative phosphorylation (ATP synthesis) in rat liver mitochondria: Possible role in drug induced liver injury (DILI).

    PubMed

    Syed, Muzeeb; Skonberg, Christian; Hansen, Steen Honoré

    2016-03-01

    Diclofenac is a widely prescribed NSAID, which by itself and its reactive metabolites (Phase-I and Phase-II) may be involved in serious idiosyncratic hepatotoxicity. Mitochondrial injury is one of the mechanisms of drug induced liver injury (DILI). In the present work, an investigation of the inhibitory effects of diclofenac (Dic) and its phase I [4-hydroxy diclofenac (4'-OH-Dic) and 5-hydroxy diclofenac (5-OH-dic)] and Phase-II [diclofenac acyl glucuronide (DicGluA) and diclofenac glutathione thioester (DicSG)] metabolites, on ATP synthesis in rat liver mitochondria was carried out. A mechanism based inhibition of ATP synthesis is exerted by diclofenac and its metabolites. Phase-I metabolite (4'-OH-Dic) and Phase-II metabolites (DicGluA and DicSG) showed potent inhibition (2-5 fold) of ATP synthesis, where as 5-OH-Dic, one of the Phase-I metabolite, was a less potent inhibitor as compared to Dic. The calculated kinetic constants of mechanism based inhibition of ATP synthesis by Dic showed maximal rate of inactivation (Kinact) of 2.64 ± 0.15 min(-1) and half maximal rate of inactivation (KI) of 7.69 ± 2.48 μM with Kinact/KI ratio of 0.343 min(-1) μM(-1). Co-incubation of mitochondria with Dic and reduced GSH exhibited a protective effect on Dic mediated inhibition of ATP synthesis. Our data from this study strongly indicate that Dic as well as its metabolites could be involved in the hepato-toxic action through inhibition of ATP synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Brain region-specific altered expression and association of mitochondria-related genes in autism

    PubMed Central

    2012-01-01

    Background Mitochondrial dysfunction (MtD) has been observed in approximately five percent of children with autism spectrum disorders (ASD). MtD could impair highly energy-dependent processes such as neurodevelopment, thereby contributing to autism. Most of the previous studies of MtD in autism have been restricted to the biomarkers of energy metabolism, while most of the genetic studies have been based on mutations in the mitochondrial DNA (mtDNA). Despite the mtDNA, most of the proteins essential for mitochondrial replication and function are encoded by the genomic DNA; so far, there have been very few studies of those genes. Therefore, we carried out a detailed study involving gene expression and genetic association studies of genes related to diverse mitochondrial functions. Methods For gene expression analysis, postmortem brain tissues (anterior cingulate gyrus (ACG), motor cortex (MC) and thalamus (THL)) from autism patients (n=8) and controls (n=10) were obtained from the Autism Tissue Program (Princeton, NJ, USA). Quantitative real-time PCR arrays were used to quantify the expression of 84 genes related to diverse functions of mitochondria, including biogenesis, transport, translocation and apoptosis. We used the delta delta Ct (∆∆Ct) method for quantification of gene expression. DNA samples from 841 Caucasian and 188 Japanese families were used in the association study of genes selected from the gene expression analysis. FBAT was used to examine genetic association with autism. Results Several genes showed brain region-specific expression alterations in autism patients compared to controls. Metaxin 2 (MTX2), neurofilament, light polypeptide (NEFL) and solute carrier family 25, member 27 (SLC25A27) showed consistently reduced expression in the ACG, MC and THL of autism patients. NEFL (P = 0.038; Z-score 2.066) and SLC25A27 (P = 0.046; Z-score 1.990) showed genetic association with autism in Caucasian and Japanese samples, respectively. The expression of

  7. H sup + -ATP synthase from rat liver mitochondria. A simple, rapid purification method of the functional complex and its characterization

    SciTech Connect

    Yoshihara, Yutaka; Nagase, Hideki; Yamane, Takeshi; Oka, Hideki; Tani, Isamu; Higuti, Tomihiko )

    1991-07-16

    A novel, simple, and rapid preparative method for purification of rat liver H{sup +}-ATP synthase by anion-exchange HPLC was developed. The H{sup +}-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H{sup +}-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-P{sub i} exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H{sup +}-ATP synthase. An immunochemical study and a labeling experiment with N,N{prime}-({sup 14}C)dicyclohexylcarbondiimide (({sup 14}C)DCCD) demonstrated the presence of chargerin II (a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the {sigma} subunit in F{sub 1} were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit {sigma}, and subunit e. This is the first report of the existence of subunit b, factor 6, and chargerin II in K{sup +}-ATP synthase purified from rat liver mitochondria.

  8. Oxidative Inactivation of Liver Mitochondria in High Fructose Diet-Induced Metabolic Syndrome in Rats: Effect of Glycyrrhizin Treatment.

    PubMed

    Sil, Rajarshi; Chakraborti, Abhay Sankar

    2016-09-01

    Metabolic syndrome is a serious health problem in the present world. Glycyrrhizin, a triterpenoid saponin of licorice (Glycyrrhiza glabra) root, has been reported to ameliorate the primary complications and hepatocellular damage in rats with the syndrome. In this study, we have explored metabolic syndrome-induced changes in liver mitochondrial function and effect of glycyrrhizin against the changes. Metabolic syndrome was induced in rats by high fructose (60%) diet for 6 weeks. The rats were then treated with glycyrrhizin (50 mg/kg body weight) by single intra-peritoneal injection. After 2 weeks of the treatment, the rats were sacrificed to collect liver tissue. Elevated mitochondrial ROS, lipid peroxidation and protein carbonyl, and decreased reduced glutathione content indicated oxidative stress in metabolic syndrome. Loss of mitochondrial inner membrane cardiolipin was observed. Mitochondrial complex I activity did not change but complex IV activity decreased significantly. Mitochondrial MTT reduction ability, membrane potential, phosphate utilisation and oxygen consumption decreased in metabolic syndrome. Reduced mitochondrial aconitase activity and increased aconitase carbonyl content suggested oxidative damage of the enzyme. Elevated Fe(2+) ion level in mitochondria might be associated with increased ROS generation in metabolic syndrome. Glycyrrhizin effectively attenuated mitochondrial oxidative stress and aconitase degradation, and improved electron transport chain activity. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Dysfunction of mouse liver mitochondria induced by 2,2'-azobis-(2-amidinopropane) dihydrochloride, a radical initiator, in vitro and in vivo.

    PubMed

    Kanno, T; Utsumi, T; Ide, A; Takehara, Y; Saibara, T; Akiyama, J; Yoshioka, T; Utsumi, K

    1994-09-01

    Mouse liver mitochondria were uncoupled in a time dependent by intraperitoneal injection of a radical initiator, 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH) (100 mg/kg). State 3 respiration, ADP/O ratio and respiratory control ratio (RCR) were decreased 30 min after injection but there was no effect on state 4 respiration. Lipid peroxidation was increased and oxidative phosphorylation was uncoupled at one hr after drug injection but gradually recovered to normal levels after 14 hr in vivo. State 3 respiration, RCR and ADP/O ratio but not state 4 respiration of isolated mouse mitochondria were inhibited by short term incubation with AAPH in vitro. This inhibitory action was concentration dependent (ID50 = 5 mM) but was not prevented by alpha-tocopherol. AAPH had no effect on electron transport or the membrane potential of these isolated mitochondria. However, mitochondria were uncoupled via lipid peroxidation and swelling by long term incubation with AAPH. These inhibitory effects of AAPH were reduced by its spontaneous degradation not only in vitro but also in vivo. Thus AAPH induces mitochondrial dysfunction by direct action in the early period of treatment and free radicals produced from AAPH mediate mitochondrial swelling via lipid peroxidation in the late period. From these findings, it is concluded that mitochondrial phosphorylation plays an important role in the pathogenesis of liver injury induced by AAPH and that radicals generated by AAPH might be a source of liver injury and mitochondrial dysfunction in vivo.

  10. Topological probes of monoamine oxidases A and B in rat liver mitochondria: inhibition by TEMPO-substituted pargyline analogues and inactivation by proteolysis.

    PubMed

    Wang, Jin; Edmondson, Dale E

    2011-04-05

    TEMPO-substituted pargyline analogues differentially inhibit recombinant human monoamine oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria, suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane [Upadhyay, A., and Edmondson, D. E. (2009) Biochemistry 48, 3928]. This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the m- and p-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an inhibition pattern opposite to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast upon trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardioprotectants or neuroprotectants.

  11. Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis†

    PubMed Central

    Wang, Jin; Edmondson, Dale E.

    2011-01-01

    TEMPO-substituted pargyline analogues differentially inhibit recombinant human Monoamine Oxidase A (MAO A) and B (MAO B) in intact yeast mitochondria suggesting these membrane-bound enzymes are located on differing faces of the mitochondrial outer membrane (Upadhyay, A. and Edmondson, D.E., Biochemistry 48, 3928, 2009). This approach is extended to the recombinant rat enzymes and to rat liver mitochondria. The differential specificities exhibited for human MAO A and MAO B by the meta- and para-amido TEMPO pargylines are not as absolute with the rat enzymes. Similar patterns of reactivity are observed for rat MAO A and B in mitochondrial outer membrane preparations expressed in Pichia pastoris or isolated from rat liver. In intact yeast mitochondria, recombinant rat MAO B is inhibited by the pargyline analogue whereas MAO A activity shows no inhibition. Intact rat liver mitochondria exhibit an opposite inhibition pattern to that observed in yeast where MAO A is inhibited and MAO B activity is unaffected. Protease inactivation studies show specificity in that MAO A is sensitive to trypsin whereas MAO B is sensitive to β-chymotrypsin. In intact mitochondrial preparations, MAO A is readily inactivated in rat liver but not in yeast on trypsin treatment and MAO B is readily inactivated by β-chymotrypsin in yeast but not in rat liver. These data show MAO A is oriented on the cytosolic face and MAO B is situated on the surface facing the intermembrane space of the mitochondrial outer membrane in rat liver. The differential mitochondrial outer membrane topology of MAO A and MAO B is relevant to their inhibition by drugs designed to be cardio-protectants or neuro-protectants. PMID:21341713

  12. Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling

    PubMed Central

    Bao, Li; Avshalumov, Marat V.; Patel, Jyoti C.; Lee, Christian R.; Miller, Evan W.; Chang, Christopher J.; Rice, Margaret E.

    2010-01-01

    Hydrogen peroxide (H2O2) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H2O2 signaling, which activates ATP-sensitive potassium (KATP) channels to inhibit dopamine release. However, the origin of this modulatory H2O2 has been elusive. Here we addressed three possible sources of H2O2 produced for rapid neuronal signaling in striatum: mitochondrial respiration; monoamine oxidase (MAO); and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H2O2 and tissue analysis of ATP, we found that co-application of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H2O2 production, but maintained tissue ATP content. Strikingly, co-application of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H2O2 in release modulation. By contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H2O2 production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H2O2 generation for dynamic neuronal signaling. PMID:19605638

  13. Mitochondria are the source of hydrogen peroxide for dynamic brain-cell signaling.

    PubMed

    Bao, Li; Avshalumov, Marat V; Patel, Jyoti C; Lee, Christian R; Miller, Evan W; Chang, Christopher J; Rice, Margaret E

    2009-07-15

    Hydrogen peroxide (H(2)O(2)) is emerging as a ubiquitous small-molecule messenger in biology, particularly in the brain, but underlying mechanisms of peroxide signaling remain an open frontier for study. For example, dynamic dopamine transmission in dorsolateral striatum is regulated on a subsecond timescale by glutamate via H(2)O(2) signaling, which activates ATP-sensitive potassium (K(ATP)) channels to inhibit dopamine release. However, the origin of this modulatory H(2)O(2) has been elusive. Here we addressed three possible sources of H(2)O(2) produced for rapid neuronal signaling in striatum: mitochondrial respiration, monoamine oxidase (MAO), and NADPH oxidase (Nox). Evoked dopamine release in guinea-pig striatal slices was monitored with carbon-fiber microelectrodes and fast-scan cyclic voltammetry. Using direct fluorescence imaging of H(2)O(2) and tissue analysis of ATP, we found that coapplication of rotenone (50 nM), a mitochondrial complex I inhibitor, and succinate (5 mM), a complex II substrate, limited H(2)O(2) production, but maintained tissue ATP content. Strikingly, coapplication of rotenone and succinate also prevented glutamate-dependent regulation of dopamine release, implicating mitochondrial H(2)O(2) in release modulation. In contrast, inhibitors of MAO or Nox had no effect on dopamine release, suggesting a limited role for these metabolic enzymes in rapid H(2)O(2) production in the striatum. These data provide the first demonstration that respiring mitochondria are the primary source of H(2)O(2) generation for dynamic neuronal signaling.

  14. Pathological characteristics of liver allografts from donation after brain death followed by cardiac death in pigs.

    PubMed

    Ye, Hui; Wang, Dong-Ping; Zhang, Chuan-Zhao; Zhang, Long-Juan; Wang, Hao-Chen; Li, Zhuo-Hui; Chen, Zhen; Zhang, Tao; Cai, Chang-Jie; Ju, Wei-Qiang; Ma, Yi; Guo, Zhi-Yong; He, Xiao-Shun

    2014-10-01

    Donation after brain death followed by circulatory death (DBCD) is a unique practice in China. The aim of this study was to define the pathologic characteristics of DBCD liver allografts in a porcine model. Fifteen male pigs (25-30 kg) were allocated randomly into donation after brain death (DBD), donation after circulatory death (DCD) and DBCD groups. Brain death was induced by augmenting intracranial pressure. Circulatory death was induced by withdrawal of life support in DBCD group and by venous injection of 40 mL 10% potassium chloride in DCD group. The donor livers were perfused in situ and kept in cold storage for 4 h. Liver tissue and common bile duct samples were collected for hematoxylin and eosin staining, TUNEL testing and electron microscopic examination. Spot necrosis was found in hepatic parenchyma of DBD and DBCD groups, while a large area of necrosis was shown in DCD group. The apoptosis rate of hepatocytes in DBD [(0.56±0.30)%] and DBCD [(0.50 ± 0.11)%] groups was much lower than that in DCD group [(3.78±0.33)%] (P<0.05). And there was no significant difference between DBD group and DBCD group (P>0.05)). The structures of bile duct were intact in both DBD and DBCD groups, while the biliary epithelium was totally damaged in DCD group. Under electron microscope, the DBD hepatocytes were characterized by intact cell membrane, well-organized endoplasmic reticulum, mild mitochondria edema and abundant glycogens. Broken cell membrane, mild inflammatory cell infiltration and sinusoidal epithelium edema, as well as reduced glycogen volume, were found in the DBCD hepatocytes. The DCD hepatocytes had more profound cell organelle injury and much less glycogen storage. In conclusion, the preservation injury of DBCD liver allografts is much less severe than that of un-controlled DCD, but more severe than that of DBD liver allografts under electron microscope, which might reflect post-transplant liver function to some extent.

  15. Effective protection of Terminalia catappa L. leaves from damage induced by carbon tetrachloride in liver mitochondria.

    PubMed

    Tang, Xinhui; Gao, Jing; Wang, Yanping; Fan, Yi-Mei; Xu, Li-Zhi; Zhao, Xiao-Ning; Xu, Qiang; Qian, Zhong Ming

    2006-03-01

    The protective effects of chloroform extracts of Terminalia catappa L. leaves (TCCE) on carbon tetrachloride (CCl4)-induced liver damage and the possible mechanisms involved in the protection were investigated in mice. We found that increases in the activity of serum aspartate aminotransferase and alanine aminotransferase and the level of liver lipid peroxidation (2.0-fold, 5.7-fold and 2.8-fold) induced by CCl4 were significantly inhibited by oral pretreatment with 20, 50 or 100 mg/kg of TCCE. Morphological observation further confirmed the hepatoprotective effects of TCCE. In addition, the disruption of mitochondrial membrane potential (14.8%), intramitochondrial Ca2+ overload (2.1-fold) and suppression of mitochondrial Ca2+-ATPase activity (42.0%) in the liver of CCl4-insulted mice were effectively prevented by pretreatment with TCCE. It can be concluded that TCCE have protective activities against liver mitochondrial damage induced by CCl4, which suggests a new mechanism of the hepatoprotective effects of TCCE.

  16. Homocysteine, Liver Function Derangement and Brain Atrophy in Alcoholics.

    PubMed

    Fernández-Rodríguez, Camino; González-Reimers, Emilio; Quintero-Platt, Geraldine; de la Vega-Prieto, María José; Pérez-Hernández, Onán; Martín-González, Candelaria; Espelosín-Ortega, Elisa; Romero-Acevedo, Lucía; Santolaria-Fernández, Francisco

    2016-11-01

    Hyperhomocysteinemia may be involved in the development of brain atrophy in alcoholics. Its pathogenesis is multifactorial. In the present study, we analyse the relationship between homocysteine levels and brain atrophy, and the relative weight of co-existing factors such as liver function impairment, the amount of ethanol consumed, serum vitamin B12, B6, and folic acid levels on homocysteine levels and brain alterations in alcoholic patients. We included 59 patients admitted to this hospital for major withdrawal symptoms and 24 controls. The mini-mental state examination test and a brain computed tomography (CT) scan were performed and several indices were calculated. Serum levels of homocysteine, folic acid, vitamin B6 and vitamin B12 were determined. Liver function was assessed by Child-Pugh score. The daily consumption of ethanol in grams per day and years of addiction were recorded. A total of 83.6% and 80% of the patients showed cerebellar or frontal atrophy, respectively. Patients showed altered values of brain indices, higher levels of homocysteine and vitamin B12, but lower levels of folic acid, compared with controls. Homocysteine, B12 and liver function variables showed significant correlations with brain CT indices. Multivariate analyses disclosed that Pugh's score, albumin and bilirubin were independently related to cerebellar atrophy, frontal atrophy, cella index or ventricular index. Serum vitamin B12 was the only factor independently related to Evans index. It was also related to cella index, but after bilirubin. Homocysteine levels were independently related to ventricular index, but after bilirubin. Vitamin B12 and homocysteine levels are higher among alcoholics. Liver function derangement, vitamin B12 and homocysteine are all independently related to brain atrophy, although not to cognitive alterations. Hyperhomocysteinemia has been described in alcoholics and may be related to brain atrophy, a reversible condition with an obscure pathogenesis

  17. Rotenone exerts similar stimulatory effects on H2O2 production by isolated brain mitochondria from young-adult and old rats.

    PubMed

    Michelini, Luiz G B; Figueira, Tiago R; Siqueira-Santos, Edilene S; Castilho, Roger F

    2015-03-04

    Chronic and systemic treatment of rodents with rotenone, a classical inhibitor of mitochondrial respiratory complex I, results in neurochemical, behavioral, and neuropathological features of Parkinson's disease. The aim of the present study was to evaluate whether brain mitochondria from old rats (24 months old) would be more susceptible to rotenone-induced inhibition of oxygen consumption and increased generation of H2O2 than mitochondria from young-adult rats (3-4 months old). Isolated brain mitochondria were incubated in the presence of different rotenone concentrations (5, 10, and 100nM), and oxygen consumption and H2O2 production were measured during respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration). Respiratory state 3 and citrate synthase activity were significantly lower in mitochondria from old rats. Mitochondria from young-adult and old rats showed similar sensitivity to rotenone-induced inhibition of oxygen consumption. Similarly, H2O2 production rates by both types of mitochondria were dose-dependently stimulated to the same extent by increasing concentrations of rotenone. We conclude that rotenone exerts similar effects on oxygen consumption and H2O2 production by isolated brain mitochondria from young-adult and old rats. Therefore, aging does not increase the mitochondrial H2O2 generation in response to complex I inhibition. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Cinnamon intake alleviates the combined effects of dietary-induced insulin resistance and acute stress on brain mitochondria.

    PubMed

    Couturier, Karine; Hininger, Isabelle; Poulet, Laurent; Anderson, Richard A; Roussel, Anne-Marie; Canini, Frédéric; Batandier, Cécile

    2016-02-01

    Insulin resistance (IR), which is a leading cause of the metabolic syndrome, results in early brain function alterations which may alter brain mitochondrial functioning. Previously, we demonstrated that rats fed a control diet and submitted to an acute restraint stress exhibited a delayed mitochondrial permeability transition pore (mPTP) opening. In this study, we evaluated the combined effects of dietary and emotional stressors as found in western way of life. We studied, in rats submitted or not to an acute stress, the effects of diet-induced IR on brain mitochondria, using a high fat/high fructose diet (HF(2)), as an IR inducer, with addition or not of cinnamon as an insulin sensitizer. We measured Ca(2+) retention capacity, respiration, ROS production, enzymatic activities and cell signaling activation. Under stress, HF(2) diet dramatically decreased the amount of Ca(2+) required to open the mPTP (13%) suggesting an adverse effect on mitochondrial survival. Cinnamon added to the diet corrected this negative effect and resulted in a partial recovery (30%). The effects related to cinnamon addition to the diet could be due to its antioxidant properties or to the observed modulation of PI3K-AKT-GSK3β and MAPK-P38 pathways or to a combination of both. These data suggest a protective effect of cinnamon on brain mitochondria against the negative impact of an HF(2) diet. Cinnamon could be beneficial to counteract deleterious dietary effects in stressed conditions.

  19. Rat MYH, a glycosylase for repair of oxidatively damaged DNA, has brain-specific isoforms that localize to neuronal mitochondria.

    PubMed

    Englander, Ella W; Hu, Zhaoyong; Sharma, Abha; Lee, Heung-Man; Wu, Zhao-Hui; Greeley, George H

    2002-12-01

    Mitochondrial genomes are exposed to a heavy load of reactive oxygen species (ROS) that damage DNA. Since in neurons, mitochondrial DNA integrity must be maintained over the entire mammalian life span, neuronal mitochondria most likely repair oxidatively damaged DNA. We show that the Escherichia coli MutY DNA glycosylase homolog (MYH) in rat (rMYH) involved in repair of oxidative damage is abundantly expressed in the rat brain, with isoforms that are exclusive to brain tissue. Confocal microscopy and western analyses reveal localization of rMYH in neuronal mitochondria. To assess involvement of MYH in the neuronal response to oxidative DNA damage, we used a rat model of respiratory hypoxia, in which acutely reduced blood oxygenation leads to generation of superoxide, and formation and subsequent removal of 8-hydroxy-2'-deoxyguanosine (8OHdG). Removal of 8OHdG is accompanied by a spatial increase in rMYH immunoreactivity in the brain and an increase in levels of one of the three mitochondrial MYH isoforms, suggesting that inducible and non-inducible MYH isoforms exist in the brain. The mitochondrial localization of oxidative DNA damage repair enzymes in neurons may represent a specialized neuronal mechanism that safeguards mitochondrial genomes in the face of routine and accidental exposures to heavy loads of injurious ROS.

  20. On the mechanism by which dicyclohexylcarbodiimide and quinine inhibit K+ transport in rat liver mitochondria.

    PubMed

    Garlid, K D; DiResta, D J; Beavis, A D; Martin, W H

    1986-02-05

    Passive uptake of potassium acetate into the mitochondrial matrix can be induced by nigericin, a K+/H+ antiporter, or by A23187, a Mg2+/2H+ antiporter. The latter process is thought to reflect operation of the Mg2+-dependent, endogenous K+/H+ antiporter, but there is ambiguity with respect to the mechanism of K+ transport in this assay (Nakashima, R.A., and Garlid, K.D. (1982) J. Biol. Chem. 257, 9252-9254). Kinetic analysis of potassium acetate transport provides verification that Mg2+ depletion 1) unmasks the K+/H+ antiporter, 2) opens up an intrinsic anion uniporter, 3) has no effect on acetic acid transport, and 4) does not induce high K+ uniport conductance. Mg2+-dependent uptake of potassium acetate is thereby shown to be mediated specifically by operation of the endogenous K+/H+ antiporter, as previously proposed. An extension of this analysis confirms that N,N'-dicyclohexylcarbodiimide and quinine block potassium acetate uptake via specific action on the K+/H+ antiporter. These findings support those of a previous study (Martin, W.H., Beavis, A.D., and Garlid, K.D. (1984) J. Biol. Chem. 259, 2062-2065) in which binding of [14C]N,N'-dicyclohexylcarbodiimide to membrane proteins under selective conditions was used to identify an 82,000-dalton band as the protein responsible for K+/H+ antiport in mitochondria.

  1. Effect of exercise on mouse liver and brain bioenergetic infrastructures.

    PubMed

    E, Lezi; Lu, Jianghua; Burns, Jeffrey M; Swerdlow, Russell H

    2013-01-01

    To assess the effects of exercise on liver and brain bioenergetic infrastructures, we exposed C57BL/6 mice to 6 weeks of moderate-intensity treadmill exercise. During the training period, fasting blood glucose was lower in exercised mice than in sedentary mice, but serum insulin levels were not reduced. At week 6, trained mice showed a paradoxical decrease in plasma lactate during exercise, which was accompanied by an increase in the liver monocarboxylate transporter 2 protein level (∼30%, P < 0.05). Exercise increased liver peroxisomal proliferator-activated receptor-γ coactivator 1α expression (approximately twofold, P < 0.001), NAD-dependent deacetylase sirtuin-1 protein (∼30%, P < 0.05), p38 protein (∼15%, P < 0.05), cytochrome c oxidase subunit 4 isoform 1 protein (∼50%, P < 0.05) and AMP-activated protein kinase phosphorylation (∼40%, P < 0.05). Despite this, liver mitochondrial DNA copy number (∼30%, P = 0.05), mitochondrial transcription factor A expression (∼15%, P < 0.05), cytochrome c oxidase subunit 2 expression (∼10%, P < 0.05), cAMP-response element binding protein phosphorylation (∼60%, P < 0.05) and brain-derived neurotrophic factor expression (∼40%, P < 0.05) were all reduced, while cytochrome oxidase and citrate synthase activities were unchanged. The only altered brain parameter observed was a reduction in tumour necrosis factor α expression (∼35%, P < 0.05); tumour necrosis factor α expression was unchanged in liver. Our data suggest that lactate produced by exercising muscle modifies the liver bioenergetic infrastructure, and enhanced liver uptake may in turn limit the ability of exercise-generated lactate to modify brain bioenergetics. Also, it appears that, at least in the liver, a dissociated mitochondrial biogenesis, in which some components are strategically enhanced while others are minimized, can occur.

  2. Complement Component C1q Mediates Mitochondria-Driven Oxidative Stress in Neonatal Hypoxic–Ischemic Brain Injury

    PubMed Central

    Ten, Vadim S.; Yao, Jun; Ratner, Veniamin; Sosunov, Sergey; Fraser, Deborah A.; Botto, Marina; Baalasubramanian, Sivasankar; Morgan, B. Paul; Silverstein, Samuel; Stark, Raymond; Polin, Richard; Vannucci, Susan J.; Pinsky, David; Starkov, Anatoly A.

    2010-01-01

    Hypoxic–ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia–ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia–ischemia. Only C1q−/− mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q−/− brain mitochondria and preserved activity of the respiratory chain. Compared with C1q+/+ neurons, cortical C1q−/− neurons exhibited resistance to oxygen– glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q−/− neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation. PMID:20147536

  3. Complement component c1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury.

    PubMed

    Ten, Vadim S; Yao, Jun; Ratner, Veniamin; Sosunov, Sergey; Fraser, Deborah A; Botto, Marina; Sivasankar, Baalasubramanian; Morgan, B Paul; Silverstein, Samuel; Stark, Raymond; Polin, Richard; Vannucci, Susan J; Pinsky, David; Starkov, Anatoly A

    2010-02-10

    Hypoxic-ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia-ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia-ischemia. Only C1q(-/-) mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q(-/-) brain mitochondria and preserved activity of the respiratory chain. Compared with C1q(+/+) neurons, cortical C1q(-/-) neurons exhibited resistance to oxygen-glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q(-/-) neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation.

  4. A comparison of Zn2+- and Ca2+- triggered depolarization of liver mitochondria reveals no evidence of Zn2+-induced permeability transition

    PubMed Central

    Devinney, Michael J.; Malaiyandi, Latha M.; Vergun, Olga; DeFranco, Donald B.; Hastings, Teresa G.; Dineley, Kirk E.

    2013-01-01

    Intracellular Zn2+ toxicity is associated with mitochondrial dysfunction. Zn2+ depolarizes mitochondria in assays using isolated organelles as well as cultured cells. Some reports suggest that Zn2+-induced depolarization results from the opening of the mitochondrial permeability transition pore (mPTP). For a more detailed analysis of this relationship, we compared Zn2+-induced depolarization with the effects of Ca2+ in single isolated rat liver mitochondria monitored with the potentiometric probe Rhodamine123. Consistent with previous work, we found that relatively low levels of Ca2+ caused rapid, complete and irreversible loss of mitochondrial membrane potential, an effect that was diminished by classic inhibitors of mPT, including high Mg2+, ADP and cyclosporine A. Zn2+ also depolarized mitochondria, but only at relatively high concentrations. Furthermore Zn2+-induced depolarization was slower, partial and sometimes reversible, and was not affected by inhibitors of mPT. We also compared the effects of Ca2+ and Zn2+ in a calcein-retention assay. Consistent with the well-documented ability of Ca2+ to induce mPT, we found that it caused rapid and substantial loss of matrix calcein. In contrast, calcein remained in Zn2+-treated mitochondria. Considered together, our results suggest that Ca2+ and Zn2+ depolarize mitochondria by considerably different mechanisms, that opening of the mPTP is not a direct consequence of Zn2+-induced depolarization, and that Zn2+ is not a particularly potent mitochondrial inhibitor. PMID:19349076

  5. Adverse effects of high doses of intravenous alpha lipoic Acid on liver mitochondria.

    PubMed

    Vigil, Michael; Berkson, Burton M; Garcia, Ana Patricia

    2014-01-01

    Alpha lipoic acid (ALA, thioctic acid), among other actions, is an essential coenzyme in the conversion of pyruvate to acetyl co-enzyme A. Therefore, it is necessary for the production of energy for aerobic organisms. Scientists have found that it can be used medically to help regenerate liver tissue, reverse the complications of diabetes mellitus, slow or stop the growth of cancer cells, and chelate heavy metals, among other actions. In this article, the authors describe the cellular mitochondrial damage from excessively high doses of this beneficial agent.

  6. The role of ADP in the modulation of the calcium-efflux pathway in rat brain mitochondria.

    PubMed Central

    Vitorica, J; Satrústegui, J

    1985-01-01

    The role of ADP in the regulation of Ca2+ efflux in rat brain mitochondria was investigated. ADP was shown to inhibit Ruthenium-Red-insensitive H+- and Na+-dependent Ca2+-efflux rates if Pi was present, but had no effect in the absence of Pi. The primary effect of ADP is an inhibition of Pi efflux, and therefore it allows the formation of a matrix Ca2+-Pi complex at concentrations above 0.2 mM-Pi and 25 nmol of Ca2+/mg of protein, which maintains a constant free matrix Ca2+ concentration. ADP inhibition of Pi and Ca2+ efflux is nucleotide-specific, since in the presence of oligomycin and an inhibitor of adenylate kinase ATP does not substitute for ADP, is dependent on the amount of ADP present, and requires ADP concentrations in excess of the concentrations of translocase binding sites. Brain mitochondria incubated with 0.2 mM-Pi and ADP showed Ca2+-efflux rates dependent on Ca2+ loads at Ca2+ concentrations below those required for the formation of a Pi-Ca2+ complex, and behaved as perfect cytosolic buffers exclusively at high Ca2+ loads. The possible role of brain mitochondrial Ca2+ in the regulation of the tricarboxylic acid-cycle enzymes and in buffering cytosolic Ca2+ is discussed. PMID:3977831

  7. Inflammation decreases the level of alpha7 nicotinic acetylcholine receptors in the brain mitochondria and makes them more susceptible to apoptosis induction.

    PubMed

    Lykhmus, Olena; Gergalova, Galyna; Zouridakis, Marios; Tzartos, Socrates; Komisarenko, Sergiy; Skok, Maryna

    2015-11-01

    α7 nicotinic acetylcholine receptors (α7 nAChRs) are involved in regulating inflammatory reactions, as well as the cell viability. They are expressed in both the plasma membrane and mitochondria of eukaryotic cells. Previously we found that neuroinflammation resulted in the decrease of α7 nAChR density in the brain of mice and was accompanied by accumulation of amyloid-beta (Aβ) peptides and memory impairment. In the present paper, it is shown that inflammation induced by either regular bacterial lipopolysaccharide (LPS) injections or immunizations with α7 nAChR extracellular domain (1-208) affected also the brain cell mitochondria. Using various modifications of sandwich ELISA, we observed the decrease of α7 nAChRs and accumulation of Aβ(1-40) and Aβ(1-42) in mitochondria of immunized or LPS-treated mice compared to control ones. Mitochondria of treated mice responded with cytochrome c release to lower Ca(2+) concentrations than mitochondria of control mice and were less sensitive to its attenuation with α7 nAChR agonist PNU282987. It is concluded that inflammation decreases α7 nAChR expression in both mitochondria and cell plasma membrane and makes mitochondria more susceptible to apoptosis induction.

  8. The anti-apoptotic effect of fluid mechanics preconditioning by cells membrane and mitochondria in rats brain microvascular endothelial cells.

    PubMed

    Tian, Shan; Zhu, Fengping; Hu, Ruiping; Tian, Song; Chen, Xingxing; Lou, Dan; Cao, Bing; Chen, Qiulei; Li, Bai; Li, Fang; Bai, Yulong; Wu, Yi; Zhu, Yulian

    2017-10-05

    Exercise preconditioning is a simple and effective way to prevent ischemia. This paper further provided the mechanism in hemodynamic aspects at the cellular level. To study the anti-apoptotic effects of fluid mechanics preconditioning, Cultured rats brain microvascular endothelial cells were given fluid intervention in a parallel plate flow chamber before oxygen glucose deprivation. It showed that fluid mechanics preconditioning could inhibit the apoptosis of endothelial cells, and this process might be mediated by the shear stress activation of Tie-2 on cells membrane surface and Bcl-2 on the mitochondria surface. Copyright © 2017. Published by Elsevier B.V.

  9. Effect of plasma exchange on hepatocyte oxidative stress, mitochondria function, and apoptosis in patients with acute fatty liver of pregnancy.

    PubMed

    Tang, Wanxin; Huang, Zhongying; Wang, Yufang; Bo, Hong; Fu, Ping

    2012-03-01

    Acute fatty liver of pregnancy (AFLP) is an uncommon but clinically severe hepatopathy, and reactive oxygen species (ROS)-mediated mitochondrial apoptosis may be its key pathogenesis. Traditional therapy is inadequate for patients with severe conditions so the application of plasma exchange (PE) has been attempted. The present study aims to determine whether or not PE can lessen injuries to hepatocytes by ameliorating ROS and mitochondrial functions. Thirteen patients with AFLP were included in the experimental group, while fifteen patients made up the case-control group. PE was applied to patients in the PE group once a day for 1-3 days. Cultured hepatocytes were treated with serum or replacement fluid from patients and controls, respectively. Malondialdehyde, superoxide dismutase (SOD), mitochondrial membrane potential (MMP), caspase-3, caspase-9, and apoptosis of hepatocytes were measured. The clinical details and prognoses were also assessed. Patients in the experimental group had shorter durations of hepatic function recovery, intensive care unit (ICU) stay, and hospitalization than those in the case-control group, although both groups showed the same mortality. PE could induce the production of SOD, inhibit the production of malondialdehyde, and recover MMP. The upregulation of caspase-3 and caspase-9 expression, as well as increase in apoptosis rate in the AFLP group, could be inhibited by PE. Moreover, PE also appeared to have a dose-dependent effect. PE protects hepatocytes by reducing damage to the mitochondria caused by oxidative stress; thus, it could be beneficial in the treatment of patients with severe AFLP and induce liver function recovery.

  10. Polyphenolic fraction of Algerian propolis reverses doxorubicin induced oxidative stress in liver cells and mitochondria.

    PubMed

    Wided, Kebsa; Hassiba, Rouibah; Mesbah, Lahouel

    2014-11-01

    Doxorubicin (DOX) is a potent anticancer drug; its use has been limited by its hepatotoxicity, which is due to free radicals generation. Propolis, a honeybee product very rich in flavonoids and therapeutic possibilities, has gained popularity as a food and alternative medicine. The present study treats DOX pro-oxidant effect on hepatic cells and mitochondrial functions. The prophylactic effect of propolis ethanolic extract (EEP) against DOX induced mitochondrial oxidative stress has also been investigated. We find that doxorubicin at the amount of 10mg/Kg altered liver mitochondrial functions as attested by the overproduction of superoxide anion (O2(2-)) by mitochondrial respiratory chain complex III. The hepatic tissue from DOX treated rats showed also a marked depletion in reduced GSH contents and an inhibition in (Mn-SOD), (Cu-Zn SOD) and (CAT) enzymatic activities. DOX increase cytosolic and mitochondrial lipid peroxidation as attested by the MDA content. These results are reversed after one mount per os pretreatment by EEP at the amount of 100mg/kg. Propolis polyphenolic fraction protects liver tissue from oxidative stress by protecting mitochondrial functions and reinforcement of enzymatic and non enzymatic antioxidants.

  11. Studies on the Mechanism of Action of the in vitro PGB(x) Effect. III. The Interaction between PGB(x) and Rat Liver Mitochondria

    DTIC Science & Technology

    1981-09-17

    previously reported (2, 3 ). Note: Abbreviations used in this report are: RIM, rat liver mitochondria; Pi, inorganic phosphate ; PG-, prostaglandin; PGBx... dimethyl POPOP, 1,4-BIS (2-( 4 -methyl-5-phenyloxazoly 1)) benzene ’ar NADC-81243-60 EXPERIMENTAL The bast method for the measurement of the PGBx-RLM...DOCUMENTATION PAGE READ INSTRUCTIONS I BEFORE COMPLETING FORM A. REPORT NUMBER 2. GOVT ACCESSION NO. 3 . FIECIPIEN~r5 CATALOG NUMBER NADC-8 1243-60

  12. The effect of artichoke (Cynara scolymus L.) extract on respiratory chain system activity in rat liver mitochondria.

    PubMed

    Juzyszyn, Z; Czerny, B; Myśliwiec, Z; Pawlik, A; Droździk, M

    2010-06-01

    The effect of artichoke extract on mitochondrial respiratory chain (MRC) activity in isolated rat liver mitochondria (including reaction kinetics) was studied. The effect of the extract on the activity of isolated cytochrome oxidase was also studied. Extract in the range of 0.68-2.72 microg/ml demonstrated potent and concentration-dependent inhibitory activity. Concentrations > or =5.4 microg/ml entirely inhibited MRC activity. The succinate oxidase system (MRC complexes II-IV) was the most potently inhibited, its activity at an extract concentration of 1.36 microg/ml being reduced by 63.3% compared with the control (p < 0.05). The results suggest a complex inhibitory mechanism of the extract. Inhibition of the succinate oxidase system was competitive (K(i) = 0.23 microg/ml), whereas isolated cytochrome oxidase was inhibited noncompetitively (K(i) = 126 microg/ml). The results of this study suggest that the salubrious effects of artichoke extracts may rely in part on the effects of their active compounds on the activity of the mitochondrial respiratory chain system.

  13. Tl(+) showed negligible interaction with inner membrane sulfhydryl groups of rat liver mitochondria, but formed complexes with matrix proteins.

    PubMed

    Korotkov, Sergey M; Brailovskaya, Irina V; Kormilitsyn, Boris N; Furaev, Viktor V

    2014-04-01

    The effects of Tl(+) on protein sulfhydryl (SH) groups, swelling, and respiration of rat liver mitochondria (RLM) were studied in a medium containing TlNO3 and sucrose, or TlNO3 and KNO3 as well as glutamate plus malate, or succinate plus rotenone. Detected with Ellman's reagent, an increase in the content of the SH groups was found in the inner membrane fraction, and a simultaneous decline was found in the content of the matrix-soluble fraction for RLM, incubated and frozen in 25-75 mM TlNO3 . This increase was greater in the medium containing KNO3 regardless of the presence of Ca(2+) . It was eliminated completely for RLM injected in the medium containing TlNO3 and then washed and frozen in the medium containing KNO3 . Calcium-loaded RLM showed increased swelling and decreased respiration. These results suggest that a ligand interaction of Tl(+) with protein SH groups, regardless of the presence of calcium, may underlie the mechanism of thallium toxicity.

  14. A novel PGC-1α isoform in brain localizes to mitochondria and associates with PINK1 and VDAC

    SciTech Connect

    Choi, Joungil; Batchu, Vera Venkatanaresh Kumar; Schubert, Manfred; Castellani, Rudolph J.; Russell, James W.

    2013-06-14

    Highlights: •Novel 35 kDa PGC-1α localizes to mitochondrial inner membrane and matrix in brain. •Mitochondrial localization of 35 kDa PGC-1α depends on VDAC protein. •Mitochondrial localization of 35 kDa PGC-1α depends on membrane potential. •The 35 kDa PGC-1α associates and colocalizes with PINK in brain mitochondria. -- Abstract: Peroxisome proliferator-activated receptor-gamma co-activator 1α (PGC-1α) and PTEN-induced putative kinase 1 (PINK1) are powerful regulators of mitochondrial function. Here, we report that a previously unrecognized, novel 35 kDa PGC-1α isoform localizes to the mitochondrial inner membrane and matrix in brain as determined by protease protection and carbonate extraction assays, as well as by immunoelectron microscopy. Immunoelectron microscopy and import experiments in vitro revealed that 35 kDa PGC-1α colocalizes and interacts with the voltage-dependent anion channel (VDAC), and that its import depends on VDAC. Valinomycin treatment which depolarizes the membrane potential, abolished mitochondrial localization of the 35 kDa PGC-1α. Using blue native-PAGE, co-immunoprecipitation, and immunoelectron microscopy analyses, we found that the 35 kDa PGC-1α binds and colocalizes with PINK1 in brain mitochondria. This is the first report regarding mitochondrial localization of a novel 35 kDa PGC-1α isoform and its association with PINK1, suggesting possible regulatory roles for mitochondrial function in the brain.

  15. Dual coenzyme activities of high-Km aldehyde dehydrogenase from rat liver mitochondria.

    PubMed

    Tsai, C S; Senior, D J

    1990-04-01

    Various kinetic approaches were carried out to investigate kinetic attributes for the dual coenzyme activities of mitochondrial aldehyde dehydrogenase from rat liver. The enzyme catalyses NAD(+)- and NADP(+)-dependent oxidations of ethanal by an ordered bi-bi mechanism with NAD(P)+ as the first reactant bound and NAD(P)H as the last product released. The two coenzymes presumably interact with the kinetically identical site. NAD+ forms the dynamic binary complex with the enzyme, while the enzyme-NAD(P)H complex formation is associated with conformation change(s). A stopped-flow burst of NAD(P)H formation, followed by a slower steady-state turnover, suggests that either the deacylation or the release of NAD(P)H is rate limiting. Although NADP+ is reduced by a faster burst rate, NAD+ is slightly favored as the coenzyme by virtue of its marginally faster turnover rate.

  16. Ca2+ Handling in Isolated Brain Mitochondria and Cultured Neurons Derived from the YAC128 Mouse Model of Huntington’s Disease

    PubMed Central

    Pellman, Jessica J.; Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay

    2015-01-01

    We investigated Ca2+ handling in isolated brain synaptic and nonsynaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington’s disease (HD). Both synaptic and nonsynaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca2+ uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca2+ capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca2+ uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca2+-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100μM glutamate produced transient elevations in cytosolic Ca2+ followed by recovery to near resting levels. Following recovery of cytosolic Ca2+, mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca2+, suggesting similar Ca2+ release and, consequently, Ca2+ loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca2+ handling in brain mitochondria of YAC128 mice. PMID:25963273

  17. BAX insertion, oligomerization, and outer membrane permeabilization in brain mitochondria: role of permeability transition and SH-redox regulation

    PubMed Central

    Brustovetsky, Tatiana; Li, Tsyregma; Yang, Youyun; Zhang, Jiang-Ting; Antonsson, Bruno; Brustovetsky, Nickolay

    2010-01-01

    BAX cooperates with truncated BID (tBID) and Ca2+ in permeabilizing the outer mitochondrial membrane (OMM) and releasing mitochondrial apoptogenic proteins. The mechanisms of this cooperation are still unclear. Here we show that in isolated brain mitochondria, recombinant BAX readily self-integrates/oligomerizes in the OMM but produces only a minuscule release of cytochrome c, indicating that BAX insertion/oligomerization in the OMM does not always lead to massive OMM permeabilization. Ca2+ in a mitochondrial permeability transition (mPT)-dependent and recombinant tBID in an mPT-independent manner promoted BAX insertion/oligomerization in the OMM and augmented cytochrome c release. Neither tBID nor Ca2+ induced BAX oligomerization in the solution without mitochondria, suggesting that BAX oligomerization required interaction with the organelles and followed rather than preceded BAX insertion in the OMM. Recombinant Bcl-xL failed to prevent BAX insertion/oligomerization in the OMM but strongly attenuated cytochrome c release. On the other hand, a reducing agent, dithiothreitol (DTT), inhibited BAX insertion/oligomerization augmented by tBID or Ca2+ and suppressed the BAX-mediated release of cytochrome c and Smac/DIABLO but failed to inhibit Ca2+-induced swelling. Altogether, these data suggest that in brain mitochondria, BAX insertion/oligomerization can be dissociated from OMM permeabilization and that tBID and Ca2+ stimulate BAX insertion/oligomerization and BAX-mediated OMM permeabilization by different mechanisms involving mPT induction and modulation of the SH-redox state. PMID:20655869

  18. The use of choline acetyltransferase for measuring the synthesis of acetyl-coenzyme A and its release from brain mitochondria.

    PubMed

    Tucek, S

    1967-09-01

    1. A method for measuring small amounts of acetyl-CoA synthesized in subcellular fractions of the brain from pyruvate and released from particles into the incubation medium has been developed by using placental choline acetyltransferase and choline in the incubation medium to transform acetyl-CoA into acetylcholine. Acetylcholine is measured by biological assay. Optimum conditions of incubation are described. 2. With fresh mitochondria, a decrease of acetyl-CoA output into the medium is observed in the presence of ATP or ADP, and an increase in the presence of calcium chloride or 2,4-dinitrophenol. Fluorocitrate and malonate have little or no effect. 3. After the mitochondria had been treated with ether, the release of acetyl-CoA into the medium is much larger; presumably, nearly all acetyl-CoA synthesized is then released and transformed into acetylcholine under the conditions used. The release of acetyl-CoA is diminished in the presence of Krebs-cycle intermediates and ADP. 4. Of all subcellular fractions, the highest acetyl-CoA production from pyruvate is found in the crude mitochondria; rates up to 51 mumoles of acetyl-CoA/g. of original tissue/hr. are observed in ether-treated samples. 5. The activities of acetyl-CoA synthetase and ATP citrate lyase found in homogenates and nerve-ending fractions of brain tissue are considerably lower than those of pyruvate oxidase complex and choline acetyltransferase. 6. The bearing of some of the findings on the question of the source of acetyl radicals for the synthesis of acetylcholine in vivo is discussed.

  19. Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice.

    PubMed

    Joseph, Ajay; Lee, Taewon; Moland, Carrie L; Branham, William S; Fuscoe, James C; Leakey, Julian E A; Allaben, William T; Lewis, Sherry M; Ali, Akhtar A; Desai, Varsha G

    2009-04-01

    Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F(1) female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I-IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.

  20. The effect of chronic tianeptine administration on the brain mitochondria: direct links with an animal model of depression.

    PubMed

    Głombik, Katarzyna; Stachowicz, Aneta; Olszanecki, Rafał; Ślusarczyk, Joanna; Trojan, Ewa; Lasoń, Władysław; Kubera, Marta; Budziszewska, Bogusława; Spedding, Michael; Basta-Kaim, Agnieszka

    2016-12-01

    A growing body of evidence has focused on the impact of mitochondrial disturbances in the development of depression, but little data exist regarding the effects of chronic administration of antidepressant drugs on the brain's mitochondrial protein profile. The aim of this study was to investigate the impact of chronic treatment with an atypical antidepressant drug-tianeptine-on the mitochondria-enriched subproteome profile in the hippocampus and the frontal cortex of 3-month-old male rats following a prenatal stress procedure. Rats that were exposed to a prenatal stress procedure displayed depressive- and anxiety-like disturbances based on the elevated plus-maze and Porsolt tests. Moreover, two-dimensional electrophoresis coupled with mass spectrometry showed structure-dependent mitoproteome changes in brains of prenatally stressed rats after chronic tianeptine administration. A component of 2-oxoglutarate and succinate flavoprotein subunit dehydrogenases, isocitrate subunit alpha, was upregulated in the hippocampus. In the frontal cortex, there was a striking increase in the expression of glutamate dehydrogenase and cytochrome bc1 complex subunit 2. These findings suggest that mitochondria are underappreciated targets for therapeutic interventions, and mitochondrial function may be crucial for the effective treatment of stress-related diseases.

  1. Protections of SMND-309, a novel derivate of salvianolic acid B, on brain mitochondria contribute to injury amelioration in cerebral ischemia rats.

    PubMed

    Tian, Jingwei; Fu, Fenghua; Li, Guisheng; Gao, Yubai; Zhang, Yunjuan; Meng, Qingsheng; Li, Changlu; Liu, Fu

    2009-08-01

    SMND-309, a novel compound named (2E)-2-{6-[(E)-2-carboxylvinyl]-2,3-dihydroxyphenyl}-3-(3,4-dihydroxyphenyl) propenoic acid, is a new derivate of salvianolic acid B. The present study was conducted to investigate whether SMND-309 has a protective effect on brain injury after focal cerebral ischemia, and if it did so, to investigate its effects on brain mitochondria. Adult male SD rats were subjected to middle cerebral artery occlusion (MCAO) by bipolar electro-coagulation. Behavioral tests and brain patho-physiological tests were used to evaluate the damage to central nervous system. Origin targets including mitochondria production of reactive oxygen species, antioxidant potentia, membrane potential, energy metabolism, mitochondrial respiratory enzymes activities and mitochondria swelling degree were evaluated. The results showed that SMND-309 decreased neurological deficit scores, reduced the number of dead hippocampal neuronal cells in accordance with its depression on mitochondria swelling degree, reactive oxygen species production, improvements on mitochondria swelling, energy metabolism, membrane potential level and mitochondrial respiratory chain complex activities. All of these findings indicate that SMND-309 exerted potent neuroprotective effects in the model of permanent cerebral ischemia, contributed to its protections on brain mitochondrial structure and function.

  2. Effects of Treating Old Rats with an Aqueous Agaricus blazei Extract on Oxidative and Functional Parameters of the Brain Tissue and Brain Mitochondria

    PubMed Central

    de Sá-Nakanishi, Anacharis B.; Soares, Andréia A.; de Oliveira, Andrea Luiza; Fernando Comar, Jurandir; Peralta, Rosane M.; Bracht, Adelar

    2014-01-01

    Dysfunction of the mitochondrial respiratory chain and increased oxidative stress is a striking phenomenon in the brain of aged individuals. For this reason there has been a constant search for drugs and natural products able to prevent or at least to mitigate these problems. In the present study the effects of an aqueous extract of Agaricus blazei, a medicinal mushroom, on the oxidative state and on the functionality of mitochondria from the brain of old rats (21 months) were conducted. The extract was administered intragastrically during 21 days at doses of 200 mg/kg. The administration of the A. blazei extract was protective to the brain of old rats against oxidative stress by decreasing the lipid peroxidation levels and the reactive oxygen species content and by increasing the nonenzymic and enzymic antioxidant capacities. Administration of the A. blazei extract also increased the activity of several mitochondrial respiratory enzymes and, depending on the substrate, the mitochondrial coupled respiration. PMID:24876914

  3. Effects of treating old rats with an aqueous Agaricus blazei extract on oxidative and functional parameters of the brain tissue and brain mitochondria.

    PubMed

    de Sá-Nakanishi, Anacharis B; Soares, Andréia A; de Oliveira, Andrea Luiza; Comar, Jurandir Fernando; Peralta, Rosane M; Bracht, Adelar

    2014-01-01

    Dysfunction of the mitochondrial respiratory chain and increased oxidative stress is a striking phenomenon in the brain of aged individuals. For this reason there has been a constant search for drugs and natural products able to prevent or at least to mitigate these problems. In the present study the effects of an aqueous extract of Agaricus blazei, a medicinal mushroom, on the oxidative state and on the functionality of mitochondria from the brain of old rats (21 months) were conducted. The extract was administered intragastrically during 21 days at doses of 200 mg/kg. The administration of the A. blazei extract was protective to the brain of old rats against oxidative stress by decreasing the lipid peroxidation levels and the reactive oxygen species content and by increasing the nonenzymic and enzymic antioxidant capacities. Administration of the A. blazei extract also increased the activity of several mitochondrial respiratory enzymes and, depending on the substrate, the mitochondrial coupled respiration.

  4. Mitochondrial superoxide mediates labile iron level: evidence from Mn-SOD-transgenic mice and heterozygous knockout mice and isolated rat liver mitochondria.

    PubMed

    Ibrahim, Wissam H; Habib, Hosam M; Kamal, Hina; St Clair, Daret K; Chow, Ching K

    2013-12-01

    Superoxide is the main reactive oxygen species (ROS) generated by aerobic cells primarily in mitochondria. It is also capable of producing other ROS and reactive nitrogen species (RNS). Moreover, superoxide has the potential to release iron from its protein complexes. Unbound or loosely bound cellular iron, known as labile iron, can catalyze the formation of the highly reactive hydroxyl radical. ROS/RNS can cause mitochondrial dysfunction and damage. Manganese superoxide dismutase (Mn-SOD) is the chief ROS-scavenging enzyme and thereby the primary antioxidant involved in protecting mitochondria from oxidative damage. To investigate whether mitochondrial superoxide mediates labile iron in vivo, the levels of labile iron were determined in the tissues of mice overexpressing Mn-SOD and heterozygous Mn-SOD-knockout mice. Furthermore, the effect of increased mitochondrial superoxide generation on labile iron levels was determined in isolated rat liver mitochondria exposed to various electron transport inhibitors. The results clearly showed that increased expression of Mn-SOD significantly lowered the levels of labile iron in heart, liver, kidney, and skeletal muscle, whereas decreased expression of Mn-SOD significantly increased the levels of labile iron in the same organs. In addition, the data showed that peroxidative damage to membrane lipids closely correlated with the levels of labile iron in various tissues and that altering the status of Mn-SOD did not alter the status of other antioxidant systems. Results also showed that increased ROS production in isolated liver mitochondria significantly increased the levels of mitochondrial labile iron. These findings constitute the first evidence suggesting that mitochondrial superoxide is capable of releasing iron from its protein complexes in vivo and that it could also release iron from protein complexes contained within the organelle.

  5. Oxidative stress is involved in the permeabilization of the inner membrane of brain mitochondria exposed to hypoxia/reoxygenation and low micromolar Ca2+.

    PubMed

    Schild, Lorenz; Reiser, Georg

    2005-07-01

    From in vivo models of stroke it is known that ischemia/reperfusion induces oxidative stress that is accompanied by deterioration of brain mitochondria. Previously, we reported that the increase in Ca2+ induces functional breakdown and morphological disintegration in brain mitochondria subjected to hypoxia/reoxygenation (H/R). Protection by ADP indicated the involvement of the mitochondrial permeability transition pore in the mechanism of membrane permeabilization. Until now it has been unclear how reactive oxygen species (ROS) contribute to this process. We now report that brain mitochondria which had been subjected to H/R in the presence of low micromolar Ca2+ display low state 3 respiration (20% of control), loss of cytochrome c, and reduced glutathione levels (75% of control). During reoxygenation, significant mitochondrial generation of hydrogen peroxide (H2O2) was detected. The addition of the membrane permeant superoxide anion scavenger TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) suppressed the production of H2O2 by brain mitochondria metabolizing glutamate plus malate by 80% under normoxic conditions. TEMPOL partially protected brain mitochondria exposed to H/R and low micromolar Ca2+ from decrease in state 3 respiration (from 25% of control to 60% of control with TEMPOL) and permeabilization of the inner membrane. Membrane permeabilization was obvious, because state 3 respiration could be stimulated by extramitochondrial NADH. Our data suggest that ROS and Ca2+ synergistically induce permeabilization of the inner membrane of brain mitochondria exposed to H/R. However, permeabilization can only partially be prevented by suppressing mitochondrial generation of ROS. We conclude that transient deprivation of oxygen and glucose during temporary ischemia coupled with elevation in cytosolic Ca2+ concentration triggers ROS generation and mitochondrial permeabilization, resulting in neural cell death.

  6. Papyriferic acid, an antifeedant triterpene from birch trees, inhibits succinate dehydrogenase from liver mitochondria.

    PubMed

    McLean, Stuart; Richards, Stephen M; Cover, Siow-Leng; Brandon, Sue; Davies, Noel W; Bryant, John P; Clausen, Thomas P

    2009-10-01

    Papyriferic acid (PA) is a triterpene that is secreted by glands on twigs of the juvenile ontogenetic phase of resin producing tree birches (e.g., Betula neoalaskana, B. pendula) and that deters browsing by mammals such as the snowshoe hare (Lepus americanus). We investigated the pharmacology of PA as a first step in understanding its antifeedant effect. After oral administration to rats, PA and several metabolites were found in feces but not urine, indicating that little was absorbed systemically. Metabolism involved various combinations of hydrolysis of its acetyl and malonyl ester groups, and hydroxylation of the terpene moiety. The presence of a malonyl group suggested a possible interaction with succinate dehydrogenase (SDH), a mitochondrial enzyme known to be competitively inhibited by malonic acid. The effect of PA on the oxidation of succinate by SDH was examined in mitochondrial preparations from livers of ox, rabbit, and rat. In all three species, PA was a potent inhibitor of SDH. Kinetic analysis indicated that, unlike malonate, PA acted by an uncompetitive mechanism, meaning that it binds to the enzyme-substrate complex. The hydrolysis product of PA, betulafolienetriol oxide, was inactive on SDH. Overall, the evidence suggests that PA acts as the intact molecule and interacts at a site other than the succinate binding site, possibly binding to the ubiquinone sites on complex II. Papyriferic acid was potent (K(iEIS) ranged from 25 to 45 microM in the three species) and selective, as malate dehydrogenase was unaffected. Although rigorous proof will require further experiments, we have a plausible mechanism for the antifeedant effect of PA: inhibition of SDH in gastrointestinal cells decreases mitochondrial energy production resulting in a noxious stimulus, 5-HT release, and sensations of nausea and discomfort. There is evidence that the co-evolution of birches and hares over a large and geographically-diverse area in Northern Europe and America has

  7. [Peculiarities of functioning of liver mitochondria of river lamprey Lampetra fluviatilis and common frog Rana temporaria at periods of suppression and activation of energy metabolism].

    PubMed

    Emel'ianova, L V; Savina, M V; Beliaeva, E A; Braĭlovskaia, I V

    2007-01-01

    The work dealt with study of mitochondria in reversible metabolic suppression of hepatocytes of the river lamprey Lampetra fluviatilis in the course of prespawning starvation and of liver mitochondria of the common frog Rana temporaria during hibernation and activity. In winter the metabolic depression of lamprey hepatocytes, unlike that of frog hepatocytes, has been found to be due to deactivation of complex I of the electron transport mitochondrial chain, a low rate of NAD-dependent substrate oxidation, a low content of adenine nucleotide content, and a high degree of mitochondrial membrane permeability to H+ and other monovalent ions (KCl-, K+). The mitochondrial membrane permeability decreases in the presence of ethyleneglycoldiamineethyltetraacetic acid (EGTA), cyclosporine A (CsA), adenosine-5'-diphosphate (ADP), and Mg+. These facts indicate the presence in these mitochondria of the Ca2+ -dependent unspecific pore in the low-conductance state. Histological studies showed the lamprey and the frog to have principal differences in use of energy substrates at the period of metabolic depression. Lampreys utilize predominantly lipids, whereas frogs--glycogen. The clearly pronounces activation of lipid consumption is observed at the spring period before spawning and death of lamprey. Possible causes of metabolic depression are discussed as well as similarity and difference in behavior of mitochondria of cyclostomes and amphibians throughout metabolic depression and activity.

  8. Mitochondria modify exercise-induced development of stem cell-derived neurons in the adult brain.

    PubMed

    Steib, Kathrin; Schäffner, Iris; Jagasia, Ravi; Ebert, Birgit; Lie, D Chichung

    2014-05-07

    Neural stem cells in the adult mammalian hippocampus continuously generate new functional neurons, which modify the hippocampal network and significantly contribute to cognitive processes and mood regulation. Here, we show that the development of new neurons from stem cells in adult mice is paralleled by extensive changes to mitochondrial mass, distribution, and shape. Moreover, exercise-a strong modifier of adult hippocampal neurogenesis-accelerates neuronal maturation and induces a profound increase in mitochondrial content and the presence of mitochondria in dendritic segments. Genetic inhibition of the activity of the mitochondrial fission factor dynamin-related protein 1 (Drp1) inhibits neurogenesis under basal and exercise conditions. Conversely, enhanced Drp1 activity furthers exercise-induced acceleration of neuronal maturation. Collectively, these results indicate that adult hippocampal neurogenesis requires adaptation of the mitochondrial compartment and suggest that mitochondria are targets for enhancing neurogenesis-dependent hippocampal plasticity.

  9. Nucleoside reverse transcriptase inhibitors (NRTIs)-induced expression profile of mitochondria-related genes in the mouse liver.

    PubMed

    Desai, Varsha G; Lee, Taewon; Delongchamp, Robert R; Leakey, Julian E A; Lewis, Sherry M; Lee, Fei; Moland, Carrie L; Branham, William S; Fuscoe, James C

    2008-03-01

    Mitochondrial dysfunction has been implicated in the adverse effects of nucleoside reverse transcriptase inhibitors (NRTIs) used to treat HIV-1 infections. To gain insight into the mechanism by which NRTIs alter mitochondrial function, the expression level of 542 genes associated with mitochondrial structure and functions was determined in the livers of p53 haplodeficient (+/-) C3B6F1 female mouse pups using mouse mitochondria-specific oligonucleotide microarray. The pups were transplacentally exposed to zidovudine (AZT) at 240 mg/kg bw/day or a combination of AZT and lamivudine (3TC) at 160 and 100mg/kg bw/day, respectively, from gestation day 12 through 18, followed by continuous treatment by oral administration from postnatal day 1-28. In addition, AZT/3TC effect was investigated in wild-type (+/+) C3B6F1 female mice. The genotype did not significantly affect the gene expression profile induced by AZT/3TC treatment. However, the transcriptional level of several genes associated with oxidative phosphorylation, mitochondrial tRNAs, fatty acid oxidation, steroid biosynthesis, and a few transport proteins were significantly altered in pups treated with AZT and AZT/3TC compared to their vehicle counterparts. Interestingly, AZT/3TC altered the expression level of 153 genes with false discovery rate of less than 0.05, in contrast to only 20 genes by AZT alone. These results suggest that NRTI-related effect on expression level of genes associated with mitochondrial functions was much greater in response to AZT/3TC combination treatment than AZT alone.

  10. Coenzyme Q10 remarkably improves the bio-energetic function of rat liver mitochondria treated with statins.

    PubMed

    Mohammadi-Bardbori, Afshin; Najibi, Asma; Amirzadegan, Najmeh; Gharibi, Raziyeh; Dashti, Ayat; Omidi, Mahmoud; Saeedi, Arastoo; Ghafarian-Bahreman, Ali; Niknahad, Hossein

    2015-09-05

    CoQ10 shares a biosynthetic pathway with cholesterol therefore it can be a potential target of the widely available lipid-lowering agents such as statins. Statins are the most widely prescribed cholesterol-lowering drugs with the ability to inhibit HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase. Preclinical and clinical safety data have shown that statins do not cause serious adverse effects in humans. However, their long-term administration is associated with a variety of myopatic complaints. The aim of this study was to investigate whether CoQ10 supplementation of animals under high fat diet (HFD) treated with statins is able to bypass the mitochondrial metabolic defects or not? Animals were divided into 7 groups and fed with either regular (RD) or HFD during experiments. The first group considered as regular control and fed with a RD. Groups 2-7 including HFD control, CoQ10 (10mg/kg), simvastatin (30mg/kg), atorvastatin (30mg/kg), simvastatin+CoQ10 or atorvastatin+CoQ10 treated orally for 30 days and fed with HFD. At the end of treatments, the animals were killed and blood samples were collected for biochemical examinations. The rat liver mitochondria were isolated and several mitochondrial indices including succinate dehydrogenase activity (SDA), ATP levels, mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (MPP) were determined. We found that triglyceride (Tg), cholesterol (Chol) and low-density lipoprotein (LDL) were augmented with HFD compared to RD and treatment with statins remarkably lowered the Tg, Chol and LDL levels. Mitochondrial parameters including, SDA, ATP levels, MMP and MPP were reduced with statin treatment and improved by co-administration with CoQ10.

  11. Compromised incision of oxidized pyrimidines in liver mitochondria of mice deficient in NTH1 and OGG1 glycosylases.

    PubMed

    Karahalil, Bensu; de Souza-Pinto, Nadja C; Parsons, Jason L; Elder, Rhoderick H; Bohr, Vilhelm A

    2003-09-05

    Mitochondrial DNA is constantly exposed to high levels of endogenously produced reactive oxygen species, resulting in elevated levels of oxidative damaged DNA bases. A large spectrum of DNA base alterations can be detected after oxidative stress, and many of these are highly mutagenic. Thus, an efficient repair of these is necessary for survival. Some of the DNA repair pathways involved have been characterized, but others are not yet determined. A DNA repair activity for thymine glycol and other oxidized pyrimidines has been described in mammalian mitochondria, but the nature of the glycosylases involved in this pathway remains unclear. The generation of mouse strains lacking murine thymine glycol-DNA glycosylase (mNTH1) and/or murine 8-oxoguanine-DNA glycosylase (mOGG1), the two major DNA N-glycosylase/apurinic/apyrimidinic (AP) lyases involved in the repair of oxidative base damage in the nucleus, has provided very useful biological model systems for the study of the function of these and other glycosylases in mitochondrial DNA repair. In this study, mouse liver mitochondrial extracts were generated from mNTH1-, mOGG1-, and [mNTH1, mOGG1]-deficient mice to ascertain the role of each of these glycosylases in the repair of oxidized pyrimidine base damage. We also characterized for the first time the incision of various modified bases in mitochondrial extracts from a double-knock-out [mNTH1, mOGG1]-deficient mouse. We show that mNTH1 is responsible for the repair of thymine glycols in mitochondrial DNA, whereas other glycosylase/AP lyases also participate in removing other oxidized pyrimidines, such as 5-hydroxycytosine and 5-hydroxyuracil. We did not detect a backup glycosylase or glycosylase/AP lyase activity for thymine glycol in the mitochondrial mouse extracts.

  12. Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures.

    PubMed

    Battaglia, Valentina; Compagnone, Alessandra; Bandino, Andrea; Bragadin, Marcantonio; Rossi, Carlo Alberto; Zanetti, Filippo; Colombatto, Sebastiano; Grillo, Maria Angelica; Toninello, Antonio

    2009-03-01

    It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.

  13. [Activity of oil isolated from Amaranth seeds on energetic functions of rat liver mitochondria after adrenaline introduction].

    PubMed

    Sirota, T V; Eliseeva, O P; Khunderiakova, N V; Kaminskiĭ, D V; Makhotina, O A; Kondrashova, M N

    2007-01-01

    It has been shown that a three-week feeding of rats with oil derived from seeds of amaranth (Amaranthus cruentus L.) leads to a moderate activation of respiration of coupled and uncoupled rat liver mitochondria (MCh) that oxidize succinate and succinate + glutamate, as well as alpha-ketoglutarate and alpha-ketoglutarate + malonate. In animals receiving the amaranth oil, the injection of adrenaline did not affect the oil-activated respiration of MCh during succinate oxidation; i. e., animals prepared by an oil-enriched diet were resistant to the action of adrenaline, which prevented from possible hyperactivation of mitochondrial functions. In the group of control animals, which received no oil, the injection of adrenaline activated the rate of phosphorylating respiration of MCh during oxidation of succinate or succinate + glutamate: the rate of oxygen uptake in state 3 respiration (by Chance) increased, and the phosphorylation time decreased. The injection of adrenaline did not affect the parameters of respiration of MCh that oxidize a-ketoglutarate; however, in the presence of malonate, the oxidation of alpha-ketoglutarate in state 3 and uncoupled respiration have shown mild but significant increase in response to adrenaline. In animals receiving the amaranth oil, the oil-induced activation of respiration of MCh in response to adrenaline retained but did not increase; however, the phosphorylation time significantly decreased. Thus, concentrated oil of seeds activates the respiration of MCh. In addition, it enhances an energetic function of MCh, which prevents from the hyper-activation of mitochondrial respiration by adrenaline. Therefore an activation of energetic function of MCh by amaranth oil could explain its adaptogenic effect on rats.

  14. Mitochondria in White, Brown, and Beige Adipocytes

    PubMed Central

    Cedikova, Miroslava; Kripnerová, Michaela; Dvorakova, Jana; Pitule, Pavel; Grundmanova, Martina; Babuska, Vaclav; Mullerova, Dana; Kuncova, Jitka

    2016-01-01

    Mitochondria play a key role in energy metabolism in many tissues, including cardiac and skeletal muscle, brain, liver, and adipose tissue. Three types of adipose depots can be identified in mammals, commonly classified according to their colour appearance: the white (WAT), the brown (BAT), and the beige/brite/brown-like (bAT) adipose tissues. WAT is mainly involved in the storage and mobilization of energy and BAT is predominantly responsible for nonshivering thermogenesis. Recent data suggest that adipocyte mitochondria might play an important role in the development of obesity through defects in mitochondrial lipogenesis and lipolysis, regulation of adipocyte differentiation, apoptosis, production of oxygen radicals, efficiency of oxidative phosphorylation, and regulation of conversion of white adipocytes into brown-like adipocytes. This review summarizes the main characteristics of each adipose tissue subtype and describes morphological and functional modifications focusing on mitochondria and their activity in healthy and unhealthy adipocytes. PMID:27073398

  15. Effect of salicylic acid and diclofenac on the medium-chain and long-chain acyl-CoA formation in the liver and brain of mouse.

    PubMed

    Kasuya, Fumiyo; Kazumi, Maya; Tatsuki, Takao; Suzuki, Risa

    2009-07-01

    Medium-chain and long-chain acyl-CoA esters are key metabolites in fatty acid metabolism. Effects of salicylic acid on the in vivo formation of acyl-CoAs in mouse liver and brain were investigated. Further, inhibition of the medium-chain and long-chain acyl-CoA synthetases by salicylic acid and diclofenac was determined in mouse liver and brain mitochondria. Acyl-CoA esters were analyzed by liquid chromatography-tandem mass spectrometry. The amounts of medium-chain acyl-CoAs (C(6), C(8) and C(10)) were less than long-chain acyl-CoAs (C(16:0), C(18:0), C(18:1) and C(20:4)) in both liver and brain. The administration of salicylic acid decreased the levels of both the medium-chain (C(6), C(8) and C(10)) and long-chain acyl-CoAs (C(16:0), C(18:0), C(18:1) and C(20:4)) in liver. In brain, however, only long-chain acyl-CoAs were decreased. The level of salicylyl-CoA detected in brain was about 12% of that in liver. Salicylic acid had a strong inhibitory activity (IC(50) = 0.1 mm) for the liver mitochondrial formation of hexanoyl-CoA from hexanoic acid, whereas diclofenac was weak (IC(50) = 4.4 mm). In contrast, diclofenac (IC(50) = 1.4 mm) inhibited the liver mitochondrial long-chain acyl-CoA synthetases more potently than salicylic acid (IC(50) = 25.5 mm). Similar inhibitory activities for the acyl-CoA synthetases were obtained in the case of the brain and liver mitochondria, except for the weak inhibition of brain medium-chain acyl-CoA synthetases by salicylic acid (IC(50) = 1.8 mm). These findings suggest that salicylic acid and diclofenac exhibit different mechanisms of inhibition of fatty acid metabolism depending on the length of the acyl chain and tissues, and they may contribute to the further understanding of the toxic effects associated with these drugs.

  16. Imaging Plasmodium Immunobiology in Liver, Brain, and Lung

    PubMed Central

    Frevert, Ute; Nacer, Adéla; Cabrera, Mynthia; Movila, Alexandru; Leberl, Maike

    2013-01-01

    Plasmodium falciparum malaria is responsible for the deaths of over half a million African children annually. Until a decade ago, dynamic analysis of the malaria parasite was limited to in vitro systems with the typical limitations associated with 2D monocultures or entirely artificial surfaces. Due to extremely low parasite densities, the liver was considered a black box in terms of Plasmodium sporozoite invasion, liver stage development, and merozoite release into the blood. Further, nothing was known about the behavior of blood stage parasites in organs such as brain where clinical signs manifest and the ensuing immune response of the host that may ultimately result in a fatal outcome. The advent of fluorescent parasites, advances in imaging technology, and availability of an ever-increasing number of cellular and molecular probes have helped illuminate many steps along the pathogenetic cascade of this deadly tropical parasite. PMID:24076429

  17. Imaging Plasmodium immunobiology in the liver, brain, and lung.

    PubMed

    Frevert, Ute; Nacer, Adéla; Cabrera, Mynthia; Movila, Alexandru; Leberl, Maike

    2014-02-01

    Plasmodium falciparum malaria is responsible for the deaths of over half a million African children annually. Until a decade ago, dynamic analysis of the malaria parasite was limited to in vitro systems with the typical limitations associated with 2D monocultures or entirely artificial surfaces. Due to extremely low parasite densities, the liver was considered a black box in terms of Plasmodium sporozoite invasion, liver stage development, and merozoite release into the blood. Further, nothing was known about the behavior of blood stage parasites in organs such as the brain where clinical signs manifest and the ensuing immune response of the host that may ultimately result in a fatal outcome. The advent of fluorescent parasites, advances in imaging technology, and availability of an ever-increasing number of cellular and molecular probes have helped illuminate many steps along the pathogenetic cascade of this deadly tropical parasite.

  18. Developmental programming by high fructose decreases phosphorylation efficiency in aging offspring brain mitochondria, correlating with enhanced UCP5 expression

    PubMed Central

    Mortensen, Ole H; Larsen, Lea H; Ørstrup, Laura KH; Hansen, Lillian HL; Grunnet, Niels; Quistorff, Bjørn

    2014-01-01

    Fructose has recently been observed to affect brain metabolism and cognitive function in adults. Yet, possible late-onset effects by gestational fructose exposure have not been examined. We evaluated mitochondrial function in the brain of aging (15 months) male offspring of Fischer F344 rat dams fed a high-fructose diet (50% energy from fructose) during gestation and lactation. Maternal fructose exposure caused a significantly lower body weight of the offspring throughout life after weaning, while birth weight, litter size, and body fat percentage were unaffected. Isolated brain mitochondria displayed a significantly increased state 3 respiration of 8%, with the substrate combinations malate/pyruvate, malate/pyruvate/succinate, and malate/pyruvate/succinate/rotenone, as well as a significant decrease in the P/O2 ratio, compared with the control. Uncoupling protein 5 (UCP5) protein levels increased in the fructose group compared with the control (P=0.03) and both UCP5 mRNA and protein levels were inversely correlated with the P/O2 ratio (P=0.008 and 0.03, respectively), suggesting that UCP5 may have a role in the observed decreased phosphorylation efficiency. In conclusion, maternal high-fructose diet during gestation and lactation has long-term effects (fetal programming) on brain mitochondrial function in aging rats, which appears to be linked to an increase in UCP5 protein levels. PMID:24756078

  19. Depletion of mitochondrial enzyme system in liver, lung, brain, stomach and kidney induced by benzo(a)pyrene.

    PubMed

    Ji, Xiaoying; Li, Yongfei; He, Jianlong; Shah, Walayat; Xue, Xiaochang; Feng, Guodong; Zhang, Huqin; Gao, Meili

    2016-04-01

    Mitochondrial dysfunction has recently received considerable attention as it plays an important role in adult human pathology caused by various drugs, endogenous agents and environmental agents. Benzo(a)pyrene (BaP), is a ubiquitous environmental contaminant mainly derived from anthropogenic activity during incomplete combustion of organic materials from various sources. The present study aimed to evaluate the effects of benzo(a)pyrene (BaP) on mitochondrial enzymes in the multiple organs including liver, lung, brain, stomach and kidney. ICR mice were exposed to different doses of BaP (2.5, 5 and 10mg/kg body weight) through oral gavage and intraperitoneal injection treatment for 13 weeks consecutively. The induced mitochondrial damage in the examined organs was assayed in terms of significant increase in lipid peroxidation (LPO) and prominent decrease in antioxidant enzymes. Non enzymatic antioxidants and Krebs cycle's enzymes were also significantly decreased in mitochondria. Additionally, BaP induced the body growth retardation and decrease in relative liver weight, increase in relative lung, stomach, kidney and brain weights, and this was further certified through histopathological lesions. Liver and lungs were more prominently damaged by BaP. The mitochondrial depletion increased in BaP dose-dependent manner.

  20. In brain mitochondria the branched-chain fatty acid phytanic acid impairs energy transduction and sensitizes for permeability transition.

    PubMed

    Schönfeld, Peter; Kahlert, Stefan; Reiser, Georg

    2004-10-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) accumulates at high levels throughout the body in the adult form of Refsum disease, a peroxisomal genetic disorder. However, it is still unclear why increased levels of phytanic acid have cytotoxic effects. In the present study, we examined the influence of non-esterified phytanic acid on energy-related functions of mitochondria from adult rat brain. Phytanic acid at low concentrations (5-20 microM, i.e. 5-20 nmol/mg of mitochondrial protein) de-energized mitochondria, as indicated by depolarization, stimulation of non-phosphorylating oxygen uptake and inhibition of the reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. The unbranched homologue palmitic acid exerted much smaller effects. In addition, phytanic acid reduced state 3 respiration, which was partly due to inhibition of the ADP/ATP carrier. Phytanic acid decreased the rate of adenine nucleotide exchange and increased the degree of control, which the ADP/ATP carrier has on state 3 respiration. Important for functional consequences is the finding that mitochondria, which are preloaded with small amounts of Ca2+ (100 nmol/mg of protein), became highly sensitized to rapid permeability transition even when only low concentrations of phytanic acid (below 5 microM) were applied. In conclusion, the incorporation of phytanic acid into the inner mitochondrial membrane increases the membrane H+ conductance and disturbs the protein-linked functions in energy coupling. This is most probably essential for the short-term toxicity of phytanic acid. Thus in neural tissue, which becomes enriched with phytanic acid, the reduction in mitochondrial ATP supply and the facilitation of the opening of the permeability transition pore are two major mechanisms by which the branched-chain fatty acid phytanic acid induces the onset of degenerative processes.

  1. Biochemical mechanism of GSH depletion induced by 1,2-dibromoethane in isolated rat liver mitochondria. Evidence of a GSH conjugation process.

    PubMed

    Botti, B; Ceccarelli, D; Tomasi, A; Vannini, V; Muscatello, U; Masini, A

    1989-09-15

    HPLC measurements of GSH and GSSG levels in isolated rat liver mitochondria, on addition of 1,2-dibromoethane (DBE), revealed the presence of a glutathione (GSH)-conjugating pathway of DBE. This process required the structural integrity of the mitochondrial matrix and inner membrane complex and was inhibited by the uncouplers of oxidative phosphorylation, particularly 2,4-dinitrophenol. On the other hand it was not affected by the energetic state of the mitochondria, since other mitochondrial inhibitors like KCN and oligomycin did not have any effect on it. This process also did not require the involvement of mitochondrial inner membrane transport systems, based on the measurement of the mitochondrial transmembrane potential. The involvement of mitochondrial GSH-S-transferases, located either in the matrix or in the intermembrane space, is discussed.

  2. Selective Toxicity of Persian Gulf Sea Cucumber (Holothuria parva) and Sponge (Haliclona oculata) Methanolic Extracts on Liver Mitochondria Isolated from an Animal Model of Hepatocellular Carcinoma

    PubMed Central

    Seydi, Enayatollah; Motallebi, Abbasali; Dastbaz, Maryam; Dehghan, Sahar; Salimi, Ahmad; Nazemi, Melika; Pourahmad, Jalal

    2015-01-01

    Background: Natural products isolated from marine environments are well known for their pharmacodynamic potential in diverse disease treatments, such as for cancer or inflammatory conditions. Sea cucumbers are marine animals of the phylum Echinoderm and the class Holothuroidea, with leathery skin and gelatinous bodies. Sponges are important components of Persian Gulf animal communities, and the marine sponges of the genus Haliclona have been known to display broad-spectrum biological activity. Many studies have shown that sea cucumbers and sponges contain antioxidants and anti-cancer compounds. Objectives: This study was designed to determine the selective toxicity of Persian Gulf sea cucumber (Holothuria parva) and sponge (Haliclona oculata) methanolic extracts on liver mitochondria isolated from an animal model of hepatocellular carcinoma, as part of a national project that hopes to identify novel potential anticancer candidates among Iranian Persian Gulf flora and fauna. Materials and Methods: To induce hepatocarcinogenesis, rats were given diethylnitrosamine (DEN) injections (200 mg/kg i.p. by a single dose), and then the cancer was promoted with 2-acetylaminofluorene (2-AAF) (0.02 w/w) for two weeks. Histopathological evaluations were performed, and levels of liver injury markers and a specific liver cancer marker (alpha-fetoprotein), were determined for confirmation of hepatocellular carcinoma induction. Finally, mitochondria were isolated from cancerous and non-cancerous hepatocytes. Results: Our results showed that H. parva methanolic extracts (250, 500, and 1000 µg/mL) and H. oculata methanolic extracts (200, 400, and 800 µg/mL) increased reactive oxygen species (ROS) formation, mitochondrial membrane potential (MMP), mitochondrial swelling, and cytochrome c release in the mitochondria obtained from cancerous hepatocytes, but not in mitochondria obtained from non-cancerous liver hepatocytes. These extracts also induced caspase-3 activation, which is

  3. Effect of thyroid state on enzymatic and non-enzymatic processes in H2O2 removal by liver mitochondria of male rats.

    PubMed

    Venditti, P; Napolitano, G; Barone, D; Coppola, I; Di Meo, S

    2015-03-05

    We investigated thyroid state effect on capacity of rat liver mitochondria to remove exogenously produced H2O2, determining their ability to decrease fluorescence generated by an H2O2 detector system. The rate of H2O2 removal by both non respiring and respiring mitochondria was increased by hyperthyroidism and decreased by hypothyroidism. However, the rate was higher in the presence of respiratory substrates, in particular pyruvate/malate, indicating a respiration-dependent process. Generally, the changes in H2O2 removal rates mirrored those in H2O2 release rates excluding the possibility that endogenous and exogenous H2O2 competed for the removing system. Pharmacological inhibition revealed thyroid state-linked differences in antioxidant enzyme contribution to H2O2 removal which were consistent with those in antioxidant system activities. The H2O2 removal was only in part due to enzymatic systems and that imputable to non-enzymatic processes was higher in hyperthyroid and lower in hypothyroid mitochondria. The levels of cytochrome c and the light emissions, due to luminol oxidation catalyzed by cytochrome/H2O2, exhibited similar changes with thyroid state supporting the idea that non-enzymatic scavenging was mainly due to hemoprotein action, which produces hydroxyl radicals. Further support was obtained showing that the whole antioxidant capacity, which provides an evaluation of capacity of the systems, different from cytochromes, assigned to H2O2 scavenging, was lower in hyperthyroid than in hypothyroid state. In conclusion, our results show that mitochondria from hyperthyroid liver have a high capacity for H2O2 removal, which, however, leading in great part to more reactive oxygen species, results harmful for such organelles.

  4. EFFECTS OF AGE, DIETARY AND BEHAVIORAL ENRICHMENT ON BRAIN MITOCHONDRIA IN A CANINE MODEL OF HUMAN AGING

    PubMed Central

    Head, E.; Nukala, V. N.; Fenoglio, K.A.; Muggenburg, B. A.; Cotman, C. W.; Sullivan, P. G.

    2009-01-01

    Dogs develop cognitive decline and a progressive accumulation of oxidative damage. In a previous longitudinal study, we demonstrated that aged dogs treated with either an antioxidant diet or with behavioral enrichment show cognitive improvement. The antioxidant diet included cellular antioxidants (Vitamins E, C, fruits and vegetables) and mitochondrial co-factors (lipoic acid and carnitine). Behavioral enrichment consisted of physical exercise, social enrichment and cognitive training. We hypothesized that the antioxidant treatment improved neuronal function through increased mitochondrial function. Thus, we measured reactive oxygen species (ROS) production and bioenergetics in mitochondria isolated from young, aged and treated aged animals. Aged canine brain mitochondria show significant increases in ROS production and a reduction in NADH-linked respiration. Mitochondrial function (ROS and NADH-linked respiration) was improved selectively in aged dogs treated with an antioxidant diet. In contrast behavioral enrichment had no effect on any mitochondrial parameters. These results suggest that an antioxidant diet improves cognition by maintaining mitochondrial homeostasis, which may be an independent molecular pathway not engaged by behavioral enrichment. PMID:19703441

  5. Resveratrol Directly Binds to Mitochondrial Complex I and Increases Oxidative Stress in Brain Mitochondria of Aged Mice

    PubMed Central

    Chupin, Stéphanie; Baron, Stéphanie; Nivet-Antoine, Valérie; Vessières, Emilie; Ayer, Audrey; Henrion, Daniel; Lenaers, Guy; Reynier, Pascal; Procaccio, Vincent

    2015-01-01

    Resveratrol is often described as a promising therapeutic molecule for numerous diseases, especially in metabolic and neurodegenerative disorders. While the mechanism of action is still debated, an increasing literature reports that resveratrol regulates the mitochondrial respiratory chain function. In a recent study we have identified mitochondrial complex I as a direct target of this molecule. Nevertheless, the mechanisms and consequences of such an interaction still require further investigation. In this study, we identified in silico by docking study a binding site for resveratrol at the nucleotide pocket of complex I. In vitro, using solubilized complex I, we demonstrated a competition between NAD+ and resveratrol. At low doses (<5μM), resveratrol stimulated complex I activity, whereas at high dose (50 μM) it rather decreased it. In vivo, in brain mitochondria from resveratrol treated young mice, we showed that complex I activity was increased, whereas the respiration rate was not improved. Moreover, in old mice with low antioxidant defenses, we demonstrated that complex I activation by resveratrol led to oxidative stress. These results bring new insights into the mechanism of action of resveratrol on mitochondria and highlight the importance of the balance between pro- and antioxidant effects of resveratrol depending on its dose and age. These parameters should be taken into account when clinical trials using resveratrol or analogues have to be designed. PMID:26684010

  6. Resveratrol Directly Binds to Mitochondrial Complex I and Increases Oxidative Stress in Brain Mitochondria of Aged Mice.

    PubMed

    Gueguen, Naïg; Desquiret-Dumas, Valérie; Leman, Géraldine; Chupin, Stéphanie; Baron, Stéphanie; Nivet-Antoine, Valérie; Vessières, Emilie; Ayer, Audrey; Henrion, Daniel; Lenaers, Guy; Reynier, Pascal; Procaccio, Vincent

    2015-01-01

    Resveratrol is often described as a promising therapeutic molecule for numerous diseases, especially in metabolic and neurodegenerative disorders. While the mechanism of action is still debated, an increasing literature reports that resveratrol regulates the mitochondrial respiratory chain function. In a recent study we have identified mitochondrial complex I as a direct target of this molecule. Nevertheless, the mechanisms and consequences of such an interaction still require further investigation. In this study, we identified in silico by docking study a binding site for resveratrol at the nucleotide pocket of complex I. In vitro, using solubilized complex I, we demonstrated a competition between NAD+ and resveratrol. At low doses (<5μM), resveratrol stimulated complex I activity, whereas at high dose (50 μM) it rather decreased it. In vivo, in brain mitochondria from resveratrol treated young mice, we showed that complex I activity was increased, whereas the respiration rate was not improved. Moreover, in old mice with low antioxidant defenses, we demonstrated that complex I activation by resveratrol led to oxidative stress. These results bring new insights into the mechanism of action of resveratrol on mitochondria and highlight the importance of the balance between pro- and antioxidant effects of resveratrol depending on its dose and age. These parameters should be taken into account when clinical trials using resveratrol or analogues have to be designed.

  7. cis-4-Decenoic and decanoic acids impair mitochondrial energy, redox and Ca(2+) homeostasis and induce mitochondrial permeability transition pore opening in rat brain and liver: Possible implications for the pathogenesis of MCAD deficiency.

    PubMed

    Amaral, Alexandre Umpierrez; Cecatto, Cristiane; da Silva, Janaína Camacho; Wajner, Alessandro; Godoy, Kálita Dos Santos; Ribeiro, Rafael Teixeira; Wajner, Moacir

    2016-09-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is biochemically characterized by tissue accumulation of octanoic (OA), decanoic (DA) and cis-4-decenoic (cDA) acids, as well as by their carnitine by-products. Untreated patients present episodic encephalopathic crises and biochemical liver alterations, whose pathophysiology is poorly known. We investigated the effects of OA, DA, cDA, octanoylcarnitine (OC) and decanoylcarnitine (DC) on critical mitochondrial functions in rat brain and liver. DA and cDA increased resting respiration and diminished ADP- and CCCP-stimulated respiration and complexes II-III and IV activities in both tissues. The data indicate that these compounds behave as uncouplers and metabolic inhibitors of oxidative phosphorylation. Noteworthy, metabolic inhibition was more evident in brain as compared to liver. DA and cDA also markedly decreased mitochondrial membrane potential, NAD(P)H content and Ca(2+) retention capacity in Ca(2+)-loaded brain and liver mitochondria. The reduction of Ca(2+) retention capacity was more pronounced in liver and totally prevented by cyclosporine A and ADP, as well as by ruthenium red, demonstrating the involvement of mitochondrial permeability transition (mPT) and Ca(2+). Furthermore, cDA induced lipid peroxidation in brain and liver mitochondria and increased hydrogen peroxide formation in brain, suggesting the participation of oxidative damage in cDA-induced alterations. Interestingly, OA, OC and DC did not alter the evaluated parameters, implying lower toxicity for these compounds. Our results suggest that DA and cDA, in contrast to OA and medium-chain acylcarnitines, disturb important mitochondrial functions in brain and liver by multiple mechanisms that are possibly involved in the neuropathology and liver alterations observed in MCAD deficiency.

  8. Cardiolipin-mediated procoagulant activity of mitochondria contributes to traumatic brain injury-associated coagulopathy in mice.

    PubMed

    Zhao, Zilong; Wang, Min; Tian, Ye; Hilton, Tristan; Salsbery, Breia; Zhou, Eric Z; Wu, Xiaoping; Thiagarajan, Perumal; Boilard, Eric; Li, Min; Zhang, Jianning; Dong, Jing-Fei

    2016-06-02

    Cardiolipin (CL) is an anionic phospholipid located exclusively in the mitochondrial inner membrane. Its presence in blood indicates mitochondrial damage and release from injured cells. Here, we report the detection of CL-exposed brain-derived mitochondrial microparticles (mtMPs) at 17 547 ± 2677/μL in the peripheral blood of mice subjected to fluid percussion injury to the brain. These mtMPs accounted for 55.2% ± 12.6% of all plasma annexin V-binding microparticles found in the acute phase of injury. They were also released from cultured neuronal and glial cells undergoing apoptosis. The mtMPs synergized with platelets to facilitate vascular leakage by disrupting the endothelial barrier. The disrupted endothelial barrier allowed the release of mtMPs into the systemic circulation to promote coagulation in both traumatically injured and mtMP- or CL-injected mice, leading to enhanced fibrinolysis, vascular fibrin deposition, and thrombosis. This mtMP-induced coagulation was mediated by CL transported from the inner to the outer mitochondrial membrane and was blocked by the scavenging molecule lactadherin. The mtMP-bound CL was ∼1600 times as active as purified CL in promoting coagulation. This study uncovered a novel procoagulant activity of CL and CL-exposed mitochondria that may contribute to traumatic brain injury-associated coagulopathy and identified potential pathways to block this activity.

  9. Effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in brain mitochondria from rats and dogs.

    PubMed

    Sugiyama, Y; Fujita, T; Matsumoto, M; Okamoto, K; Imada, I

    1985-12-01

    The effects of idebenone (CV-2619) and its metabolites on respiratory activity and lipid peroxidation in isolated brain mitochondria from rats and dogs were studied. CV-2619 was easily reduced by canine brain mitochondria in the presence of respiratory substrates. Reduced CV-2619 (2H-CV-2619) was rapidly oxidized through the cytochrome b chain, indicating that the compound functioned simply as an electron carrier of mitochondrial respiratory system. Both nicotinamide adenine dinucleotide (NADH)- and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent lipid peroxidations were examined in canine brain mitochondria in the presence of adenosine diphosphate (ADP) and Fe3+. NADH-cytochrome c reductase activity was sensitive to NADPH-dependent lipid peroxidation. CV-2619 (10(-5)M) strongly inhibited both types of the lipid peroxidation reactions and protected the resultant inactivation of the NADH-cytochrome c reductase activity. Activities of succinate oxidase in rat and canine brain mitochondria were virtually unaffected by CV-2619 and its metabolites (10(-5)-10(-6) M). On the other hand, CV-2619 markedly suppressed the state 3 respiration in glutamate oxidation in a dose dependent manner without any effect on the state 4 respiration and the ADP/O ratio in intact rat brain mitochondria. The inhibitory effect of CV-2619 was also observed in NADH-cytochrome c reductase, but not in NADH-2,6-dichlorophenolindophenol (DCIP) and NADH-ubiquinone reductases in canine brain mitochondria. These facts and results of inhibitor analysis suggest that the action site of CV-2619 is NADH-linked complex I in the mitochondrial respiratory chain and is different from that of inhibitors of oxidative phosphorylation such as rotenone, oligomycin and 2,4-dinitrophenol. Finally, the above findings suggest that CV-2619 acts as an electron carrier in respiratory chains and functions as an antioxidant against membrane damage caused by lipid peroxidation in brain mitochondria. It appears

  10. Antcin H Protects Against Acute Liver Injury Through Disruption of the Interaction of c-Jun-N-Terminal Kinase with Mitochondria.

    PubMed

    Huo, Yazhen; Win, Sanda; Than, Tin Aung; Yin, Shutao; Ye, Min; Hu, Hongbo; Kaplowitz, Neil

    2017-02-10

    Antrodia Camphorate (AC) is a mushroom that is widely used in Asian countries to prevent and treat various diseases, including liver diseases. However, the active ingredients that contribute to the biological functions remain elusive. The purpose of the present study is to test the hepatoprotective effect of Antcin H, a major triterpenoid chemical isolated from AC, in murine models of acute liver injury. We found that Antcin H pretreatment protected against liver injury in both acetaminophen (APAP) and galactosamine/tumor necrosis factor (TNF)α models. More importantly, Antcin H also offered a significant protection against acetaminophen-induced liver injury when it was given 1 h after acetaminophen. The protection was verified in primary mouse hepatocytes. Antcin H prevented sustained c-Jun-N-terminal kinase (JNK) activation in both models. We excluded an effect of Antcin H on acetaminophen metabolism and TNF receptor signaling and excluded a direct effect as a free radical scavenger or JNK inhibitor. Since the sustained JNK activation through its interaction with mitochondrial Sab, leading to increased mitochondrial reactive oxygen species (ROS), is pivotal in both models, we examined the effect of Antcin H on p-JNK binding to mitochondria and impairment of mitochondrial respiration. Antcin H inhibited the direct effect of p-JNK on isolated mitochondrial function and binding to isolated mitochondria. Innovation and Conclusion: Our study has identified Antcin H as a novel active ingredient that contributes to the hepatoprotective effect of AC, and Antcin H protects against liver injury through disruption of the binding of p-JNK to Sab, which interferes with the ROS-dependent self-sustaining activation of MAPK cascade. Antioxid. Redox Signal. 26, 207-220.

  11. Clinical science workshop: targeting the gut-liver-brain axis.

    PubMed

    Patel, Vishal C; White, Helen; Støy, Sidsel; Bajaj, Jasmohan S; Shawcross, Debbie L

    2016-12-01

    A clinical science workshop was held at the ISHEN meeting in London on Friday 11th September 2014 with the aim of thrashing out how we might translate what we know about the central role of the gut-liver-brain axis into targets which we can use in the treatment of hepatic encephalopathy (HE). This review summarises the integral role that inter-organ ammonia metabolism plays in the pathogenesis of HE with specific discussion of the roles that the small and large intestine, liver, brain, kidney and muscle assume in ammonia and glutamine metabolism. Most recently, the salivary and gut microbiome have been shown to underpin the pathophysiological changes which culminate in HE and patients with advanced cirrhosis present with enteric dysbiosis with small bowel bacterial overgrowth and translocation of bacteria and their products across a leaky gut epithelial barrier. Resident macrophages within the liver are able to sense bacterial degradation products initiating a pro-inflammatory response within the hepatic parenchyma and release of cytokines such as tumour necrosis factor alpha (TNF-α) and interleukin-8 into the systemic circulation. The endotoxemia and systemic inflammatory response that are generated predispose both to the development of infection as well as the manifestation of covert and overt HE. Co-morbidities such as diabetes and insulin resistance, which commonly accompany cirrhosis, may promote slow gut transit, promote bacterial overgrowth and increase glutaminase activity and may need to be acknowledged in HE risk stratification assessments and therapeutic regimens. Therapies are discussed which target ammonia production, utilisation or excretion at an individual organ level, or which reduce systemic inflammation and endotoxemia which are known to exacerbate the cerebral effects of ammonia in HE. The ideal therapeutic strategy would be to use an agent that can reduce hyperammonemia and reduce systemic inflammation or perhaps to adopt a combination of

  12. Sulfurtransferases and cyanide detoxification in mouse liver, kidney, and brain.

    PubMed

    Wróbel, M; Jurkowska, H; Sliwa, L; Srebro, Z

    2004-01-01

    The activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and cystathionase in mouse liver, kidney, and four brain regions: tele-, meso-, di- and rhombencephalon was studied 30 min and 2 h following a sublethal dose of cyanide (4 mg/ kg body weight) intraperitoneal injection. Simultaneously, sulfane sulfur levels and total sulfur content, a direct or indirect source of sulfur for CN(-) conversion to SCN(-), were also investigated in these tissues. In the liver this dose of cyanide seemed to impair the process of cyanide detoxification by MPST, as well as rhodanese inhibition. The effects of cyanide administration to mice proved to be totally different in the liver and kidney. In the kidney, a significant increase in the rhodanese activity was observed as early as 30 min following cyanide intoxication, and an elevated cystathionase activity after 2 h was detected. This suggests the involvement of cystathionase in cyanide detoxification in the kidney. The activity of MPST remained at the same level as in the control group. In the rhombencephalon, similarly as in the kidney, L-cysteine desulfuration pathways, which generate sulfane sulfur and sulfurtransferases that transfer sulfane sulfur atoms to CN(-), seemed to play an important role as a defense system against cyanide. The stable level of sulfane sulfur and total sulfur content was accompanied in the rhombencephalon by an increased activity of MPST, cystathionase and rhodanese. In other brain regions the role of these three sulfurtransferases was not so clear and it seemed that in the telencephalon, where the total sulfur content, but not the sulfane sulfur level, was significantly increased, some sulfur-containing compounds, such as GSH and/or cysteine, appeared in response to cyanide.

  13. Decline in cytochrome c oxidase activity in rat-brain mitochondria with aging. Role of peroxidized cardiolipin and beneficial effect of melatonin.

    PubMed

    Petrosillo, Giuseppe; De Benedictis, Valentina; Ruggiero, Francesca M; Paradies, Giuseppe

    2013-10-01

    Reactive oxygen species (ROS) are considered a key factor in mitochondrial dysfunction associated with brain aging process. Mitochondrial respiration is an important source of ROS and hence a potential contributor to brain functional changes with aging. In this study, we examined the effect of aging on cytochrome c oxidase activity and other bioenergetic processes such as oxygen consumption, membrane potential and ROS production in rat brain mitochondria. We found a significant age-dependent decline in the cytochrome c oxidase activity which was associated with parallel changes in state 3 respiration, membrane potential and with an increase in H2O2 generation. The cytochrome aa3 content was practically unchanged in mitochondria from young and aged animals. The age-dependent decline of cytochrome c oxidase activity could be restored, in situ, to the level of young animals, by exogenously added cardiolipin. In addition, exposure of brain mitochondria to peroxidized cardiolipin resulted in an inactivation of this enzyme complex. It is suggested that oxidation/depletion of cardiolipin could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunction in brain aging. Melatonin treatment of old animals largely prevented the age-associated alterations of mitochondrial bioenergetic parameters. These results may prove useful in elucidating the molecular mechanisms underlying mitochondrial dysfunction associated with brain aging process, and may have implications in etiopathology of age-associated neurodegenerative disorders and in the development of potential treatment strategies.

  14. Cocaine and mitochondria-related signaling in the brain: A mechanistic view and future directions.

    PubMed

    de Oliveira, Marcos Roberto; Jardim, Fernanda Rafaela

    2016-01-01

    Cocaine is extensively used as a psychostimulant among subjects at different ages worldwide. Cocaine causes neuronal dysfunction and, consequently, negatively affects human behavior and decreases life quality severely. Cocaine acts through diverse mechanisms, including mitochondrial impairment and activation of cell signaling pathways associated to stress response. There is some controversy regarding the effect of cocaine in inducing cell death through apoptosis in different experimental models. The aim of the present work is to discuss data associated to the mitochondrial consequences of cocaine exposure of mammalian cells in several experimental models from in vitro to in vivo, including postmortem human tissue analyses. Furthermore, future directions are proposed in order to serve as a suggestive guide in relation to the next steps towards the complete elucidation of the mechanisms of toxicity elicited by cocaine upon mitochondria of neuronal cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Decrease of rotenone inhibition is a sensitive parameter of complex I damage in brain non-synaptic mitochondria of aged rats.

    PubMed

    Genova, M L; Bovina, C; Marchetti, M; Pallotti, F; Tietz, C; Biagini, G; Pugnaloni, A; Viticchi, C; Gorini, A; Villa, R F; Lenaz, G

    1997-06-30

    We investigated NADH oxidation in non-synaptic and synaptic mitochondria from brain cortex of 4- and 24-month-old rats. The NADH oxidase activity was significantly lower in non-synaptic mitochondria from aged rats; we also found a significant decrease of sensitivity of NADH oxidation to the specific Complex I inhibitor, rotenone. Since the rotenone-binding site encompasses Complex I subunits encoded by mtDNA, these results are in accordance with the mitochondrial theory of aging, whereby somatic mtDNA mutations are at the basis of cellular senescence. Accordingly, a 5 kb deletion was detected only in the cortex of the aged animals.

  16. Evolution of energy metabolism. Proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile.

    PubMed

    Brand, M D; Couture, P; Else, P L; Withers, K W; Hulbert, A J

    1991-04-01

    Standard metabolic rate is 7-fold greater in the rat (a typical mammal) than in the bearded dragon, Amphibolurus vitticeps (a reptile with the same body mass and temperature). Rat hepatocytes respire 4-fold faster than do hepatocytes from the lizard. The inner membrane of isolated rat liver mitochondrial has a proton permeability that is 4-5-fold greater than the proton permeability of the lizard liver mitochondrial membrane per mg of mitochondrial protein. The greater permeability of rat mitochondria is not caused by differences in the surface area of the mitochondrial inner membrane, but differences in the fatty acid composition of the mitochondrial phospholipids may be involved in the permeability differences. Greater proton permeability of the mitochondrial inner membrane may contribute to the greater standard metabolic rate of mammals.

  17. Two dimensional gel electrophoretic resolution of the polypeptides of mitochondria isolated from different rat organs

    SciTech Connect

    Haldar, D.; Osorno, H.; Diaz, J.; Lipfert, L.; Sheehan, D.

    1987-05-01

    A comparative study of rat liver, kidney, heart, spleen, and brain mitochondria were made by resolving their polypeptides by two dimensional gel electrophoresis and staining with silver. The mitochondria were purified using either a sucrose or a ficoll gradient and the purity monitored by enzyme markers. Mitochondria from each organ exhibited a characteristic 2-D gel pattern. There were many similarities and differences between the mitochondria isolated from different organs. Under optimal conditions, the liver mitochondria (40 ..mu..g) produced 200 to 250 spots. The total number of spots decreased in the order: liver > brain > heart, kidney > spleen. The liver mitochondria showed no spots in the pI <5 and there was a predominant spot of carbamyl phosphate synthetase (CPS). The other mitochondrial preparations showed hardly any CPS but showed some other major spots. Identification of some of the membrane proteins were made by different methods, e.g., VDAC and MAO by parallel migration of the pure proteins, MAO by labeling with /sup 3/H-pargyline, and the alpha- and beta-subunits of the F/sub 1/ ATPase by comparing with published data.

  18. Effect of the general anaesthetics, alphaxalone, hexobarbitone and halothane on calcium uptake into rat brain mitochondria in vitro and in vivo.

    PubMed

    Sweetman, A J; Esmail, A F

    1982-01-01

    In vitro studies of the effect of alphaxalone on ADP-stimulated respiration and Ca2+ uptake in rat brain mitochondria showed that the anesthetic was a potent inhibitor of both reactions. In vivo measurements showed that although alphaxalone did not inhibit oxidative phosphorylation during anaesthesia, the uptake of Ca2+ into the mitochondrial fraction was strongly inhibited. The degree of inhibition was dependent on the dose of anaesthetic administered and was reversed by the CNS stimulant, bemigride. Halothane and hexobarbitone also inhibited mitochondrial Ca2+ uptake in vivo. The levels of anaesthetic in the brain were determined after the administration of 14C-alphaxalone. There was no significant difference between the brain regions examined, but the levels reached were equal to those required to block CA2+ transport in isolated mitochondria.

  19. The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria*

    PubMed Central

    Ronchi, Juliana Aparecida; Francisco, Annelise; Passos, Luiz Augusto Correa; Figueira, Tiago Rezende; Castilho, Roger Frigério

    2016-01-01

    The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP+ at the expense of NADH oxidation and H+ movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP+ ratio severalfold higher than the NADH/NAD+ ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated NntC57BL/6J allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt+/+, Nnt+/−, and Nnt−/− mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt+/− and Nnt−/− exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt−/− mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt+/− mitochondria matched that in the Nnt+/+ mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism. PMID:27474736

  20. The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria.

    PubMed

    Ronchi, Juliana Aparecida; Francisco, Annelise; Passos, Luiz Augusto Correa; Figueira, Tiago Rezende; Castilho, Roger Frigério

    2016-09-16

    The forward reaction of nicotinamide nucleotide transhydrogenase (NNT) reduces NADP(+) at the expense of NADH oxidation and H(+) movement down the electrochemical potential across the inner mitochondrial membrane, establishing an NADPH/NADP(+) ratio severalfold higher than the NADH/NAD(+) ratio in the matrix. In turn, NADPH drives processes, such as peroxide detoxification and reductive biosynthesis. In this study, we generated a congenic mouse model carrying a mutated Nnt(C57BL/6J) allele from the C57BL/6J substrain. Suspensions of isolated mitochondria from Nnt(+/+), Nnt(+/-), and Nnt(-/-) mouse liver were biochemically evaluated and challenged with exogenous peroxide under different respiratory states. The respiratory substrates were also varied, and the participation of concurrent NADPH sources (i.e. isocitrate dehydrogenase-2, malic enzymes, and glutamate dehydrogenase) was assessed. The principal findings include the following: Nnt(+/-) and Nnt(-/-) exhibit ∼50% and absent NNT activity, respectively, but the activities of concurrent NADPH sources are unchanged. The lack of NNT activity in Nnt(-/-) mice impairs peroxide metabolism in intact mitochondria. The contribution of NNT to peroxide metabolism is decreased during ADP phosphorylation compared with the non-phosphorylating state; however, it is accompanied by increased contributions of concurrent NADPH sources, especially glutamate dehydrogenase. NNT makes a major contribution to peroxide metabolism during the blockage of mitochondrial electron transport. Interestingly, peroxide metabolism in the Nnt(+/-) mitochondria matched that in the Nnt(+/+) mitochondria. Overall, this study demonstrates that the respiratory state and/or substrates that sustain energy metabolism markedly influence the relative contribution of NNT (i.e. varies between nearly 0 and 100%) to NADPH-dependent mitochondrial peroxide metabolism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Copper(II) and iron(III) ions inhibit respiration and increase free radical-mediated phospholipid peroxidation in rat liver mitochondria: Effect of antioxidants.

    PubMed

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

    2017-07-01

    Rat liver mitochondria (1.5-2.1mg protein·mL(-1)) supplemented with either 25 and 100μM Cu(2+) or 100 and 500μM Fe(3+) show inhibition of active respiration (O2 consumption in state 3) and increased phospholipid peroxidation . Liver mitochondria were supplemented with the antioxidants reduced glutathione, N-acetylcysteine or butylated hydroxitoluene, to evaluate their effects on the above-mentioned alterations. Although the mitochondrial dysfunction is clearly associated to phospholipid peroxidation, the different responses to antioxidant supplementation indicate that the metal ions have differences in their mechanisms of toxicity. Mitochondrial phospholipid peroxidation through the formation of hydroxyl radical by a Fenton/Haber-Weiss mechanism seems to precede the respiratory inhibition and to be the main fact in Fe-induced mitochondrial dysfunction. In the case of Cu(2+), it seems that the ion oxidizes glutathione, and low molecular weight protein thiol groups in a direct reaction, as part of its intracellular redox cycling. The processes involving phospholipid peroxidation, protein oxidation and mitochondrial respiratory inhibition characterize a redox dyshomeostatic situation that ultimately leads to cell death. However, Cu(2+) exposure involves an additional, yet unidentified, toxic event as previous reduction of the metal with N-acetylcysteine has only a minor effect in preventing the mitochondrial damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. To involvement the conformation of the adenine nucleotide translocase in opening the Tl(+)-induced permeability transition pore in Ca(2+)-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey M; Konovalova, Svetlana A; Brailovskaya, Irina V; Saris, Nils-Erik L

    2016-04-01

    The conformation of adenine nucleotide translocase (ANT) has a profound impact in opening the mitochondrial permeability transition pore (MPTP) in the inner membrane. Fixing the ANT in 'c' conformation by phenylarsine oxide (PAO), tert-butylhydroperoxide (tBHP), and carboxyatractyloside as well as the interaction of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) with mitochondrial thiols markedly attenuated the ability of ADP to inhibit the MPTP opening. We earlier found (Korotkov and Saris, 2011) that calcium load of rat liver mitochondria in medium containing TlNO3 and KNO3 stimulated the Tl(+)-induced MPTP opening in the inner mitochondrial membrane. The MPTP opening as well as followed increase in swelling, a drop in membrane potential (ΔΨmito), and a decrease in state 3, state 4, and 2,4-dinitrophenol-uncoupled respiration were visibly enhanced in the presence of PAO, tBHP, DIDS, and carboxyatractyloside. However, these effects were markedly inhibited by ADP and membrane-penetrant hydrophobic thiol reagent, N-ethylmaleimide (NEM) which fix the ANT in 'm' conformation. Cyclosporine A additionally potentiated these effects of ADP and NEM. Our data suggest that conformational changes of the ANT may be directly involved in the opening of the Tl(+)-induced MPTP in the inner membrane of Ca(2+)-loaded rat liver mitochondria. Using the Tl(+)-induced MPTP model is discussed in terms finding new transition pore inhibitors and inducers among different chemical and natural compounds.

  3. Effect of high-intensity exercise on aged mouse brain mitochondria, neurogenesis, and inflammation.

    PubMed

    E, Lezi; Burns, Jeffrey M; Swerdlow, Russell H

    2014-11-01

    In aged mice, we assessed how intensive exercise affects brain bioenergetics, inflammation, and neurogenesis-relevant parameters. After 8 weeks of a supra-lactate threshold treadmill exercise intervention, 21-month-old C57BL/6 mice showed increased brain peroxisome proliferator-activated receptor gamma coactivator-1α protein, mammalian target of rapamycin and phospho-mammalian target of rapamycin protein, citrate synthase messenger RNA, and mitochondrial DNA copy number. Hippocampal vascular endothelial growth factor A (VEGF-A) gene expression trended higher, and a positive correlation between VEGF-A and PRC messenger RNA levels was observed. Brain doublecortin, brain-derived neurotrophic factor, tumor necrosis factor-α, and CCL11 gene expression, as well as plasma CCL11 protein levels, were unchanged. Despite these apparent negative findings, a negative correlation between plasma CCL11 protein levels and hippocampal doublecortin gene expression was observed; further analysis indicated exercise may mitigate this relationship. Overall, our data suggest supra-lactate threshold exercise activates a partial mitochondrial biogenesis in aged mice, and a gene (VEGF-A) known to support neurogenesis. Our data are consistent with another study that found systemic inflammation in general, and CCL11 protein specifically, suppresses hippocampal neurogenesis. Our study supports the view that intense exercise above the lactate threshold may benefit the aging brain; future studies to address the extent to which exercise-generated lactate mediates the observed effects are warranted.

  4. Effects of polyphenols on brain ageing and Alzheimer's disease: focus on mitochondria.

    PubMed

    Schaffer, Sebastian; Asseburg, Heike; Kuntz, Sabine; Muller, Walter E; Eckert, Gunter P

    2012-08-01

    The global trend of the phenomenon of population ageing has dramatic consequences on public health and the incidence of neurodegenerative diseases. Physiological changes that occur during normal ageing of the brain may exacerbate and initiate pathological processes that may lead to neurodegenerative disorders, especially Alzheimer's disease (AD). Hence, the risk of AD rises exponentially with age. While there is no cure currently available, sufficient intake of certain micronutrients and secondary plant metabolites may prevent disease onset. Polyphenols are highly abundant in the human diet, and several experimental and epidemiological evidences indicate that these secondary plant products have beneficial effects on AD risks. This study reviews current knowledge on the potential of polyphenols and selected polyphenol-rich diets on memory and cognition in human subjects, focusing on recent data showing in vivo efficacy of polyphenols in preventing neurodegenerative events during brain ageing and in dementia. Concentrations of polyphenols in animal brains following oral administration have been consistently reported to be very low, thus eliciting controversial discussion on their neuroprotective effects and potential mechanisms. Whether polyphenols exert any direct antioxidant effects in the brain or rather act by evoking alterations in regulatory systems of the brain or even the body periphery is still unclear. To understand the mechanisms behind the protective abilities of polyphenol-rich foods, an overall understanding of the biotransformation of polyphenols and identification of the various metabolites arising in the human body is also urgently needed.

  5. Increase in the ADP/ATP exchange in rat liver mitochondria irradiated in vitro by helium-neon laser

    SciTech Connect

    Passarella, S.; Ostuni, A.; Atlante, A.; Quagliariello, E.

    1988-10-31

    To gain some insight into the mechanism of cell photostimulation by laser light, measurements were made of the rate of ADP/ATP exchange in mitochondria irradiated with the low power continuous wave Helium Neon laser (energy dose 5 Joules/cm2). To do this a method has been developed to continuously monitor ATP efflux from phosphorylating mitochondria caused by externally added ADP, by photometrically following the NADP+ reduction which occurs in the presence of glucose, hexokinase, glucose-6-phosphate dehydrogenase and effluxed ATP. The NADP+ reduction rate shows hyperbolic dependence on ADP concentration (Km and Vmax values 8.5 +/- 0.87 microM and 20.7 +/- 0.49 nmoles NADP+ reduced/min x mg mitochondrial protein, respectively), and proves to measure the activity of the ADP/ATP translocator as shown by inhibition experiments using atracyloside, powerful inhibitor of this carrier. Irradiation was found to enhance the rate of ADP/ATP antiport, with externally added ADP ranging between 5 and 100 microM. As a result of experiments carried out with mitochondria loaded with either ATP or ADP, the increase in the activity of the ADP/ATP translocator is here proposed to depend on the increase in the electrochemical proton gradient which occurs owing to irradiation of mitochondria.

  6. The influence of thyroxine administered in vivo on the transmembrane protonic electrochemical potential difference in rat liver mitochondria.

    PubMed

    Shears, S B; Bronk, J R

    1979-02-15

    When mitochondria from normal and thyroxine-treated rats were energized by incubation with succinate, phosphate and MgCl2, it was found that the hormone treatment increased the transmembrane protonic electrochemical potential difference by 16mV and the respiration rate by 46%. Other experiments show these changes to be associated with increases in the intramitochondrial K+ and phosphate concentrations.

  7. The effect of dianabol on certain cell energy processes in postirradiation disease. III. The effect of chronic administration of dianabol and irradiation on oxidative phosphorylation in rat liver mitochondria.

    PubMed

    Sierakowski, S; Mackowiak, J

    1979-10-01

    Chronic administration of Dianabol did not prevent the radiation-induced changes of oxidative phosphorylation in rat liver mitochondria. Irradiation of rats with X-rays, in a dose of 600 R for the whole body, causes damage of oxidative phosphorylation in rat liver mitochondria. Progressive reducation of oxygen consumption, decrease of high-energy bond formation and a drop of the P/O coefficient were observed in irradiated animals. The presumable role of the postirradiation damage of oxidative phosphorylation and the effects of radioprotective compounds on this process are discussed.

  8. Neuronal mitochondrial toxicity of malondialdehyde: inhibitory effects on respiratory function and enzyme activities in rat brain mitochondria.

    PubMed

    Long, Jiangang; Liu, Changsheng; Sun, Lijuan; Gao, Hongxiang; Liu, Jiankang

    2009-04-01

    Malondialdehyde (MDA) is a product of oxidative damage to lipids, amino acids and DNA, and accumulates with aging and diseases. MDA can possibly react with amines so as to modify proteins and inactivate enzymes; it can also modify nucleosides so as to cause mutagenicity. Brain mitochondrial dysfunction is a major contributor to aging and neurodegenerative diseases. We hypothesize that MDA accumulated during aging targets mitochondrial enzymes so as to cause further mitochondrial dysfunction and additional contributions to aging and neurodegeneration. Herein, we investigated the neuronal mitochondrial toxic effects of MDA on mitochondrial respiration and activities of enzymes (mitochondrial complexes I-V, alpha-ketoglutarate dehydrogenase (KGDH) and pyruvate dehydrogenase (PDH)), in isolated rat brain mitochondria. MDA depressed mitochondrial membrane potential, and also showed a dose-dependent inhibition of mitochondrial complex I- and complex II-linked respiration. Complex I and II, and PDH activities were depressed by MDA at >or=0.2 micromol/mg; KGDH and complex V were inhibited by >or=0.4 and >or=1.6 micromol MDA/mg, respectively. However, MDA did not have any toxic effects on complex III and IV activities over the range 0-2 micromol/mg. MDA significantly elevated mitochondrial reactive oxygen species (ROS) and protein carbonyls at 0.2 and 0.002 micromol/mg, respectively. As for the antioxidant defense system, a high dose of MDA slightly decreased mitochondrial GSH and superoxide dismutase. These results demonstrate that MDA causes neuronal mitochondrial dysfunction by directly promoting generation of ROS and modifying mitochondrial proteins. The results suggest that MDA-induced neuronal mitochondrial toxicity may be an important contributing factor to brain aging and neurodegenerative diseases.

  9. Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

    SciTech Connect

    Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

    2008-10-01

    Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore.

  10. Protective effects of taurine in traumatic brain injury via mitochondria and cerebral blood flow.

    PubMed

    Wang, Qin; Fan, Weijia; Cai, Ying; Wu, Qiaoli; Mo, Lidong; Huang, Zhenwu; Huang, Huiling

    2016-09-01

    In mammalian tissues, taurine is an important natural component and the most abundant free amino acid in the heart, retina, skeletal muscle, brain, and leukocytes. This study is to examine the taurine's protective effects on neuronal ultrastructure, the function of the mitochondrial respiratory chain complex, and on cerebral blood flow (CBF). The model of traumatic brain injury (TBI) was made for SD rats by a fluid percussion device, with taurine (200 mg/kg) administered by tail intravenous injection once daily for 7 days after TBI. It was found that CBF was improved for both left and right brain at 30 min and 7 days post-injury by taurine. Reaction time was prolonged relative to the TBI-only group. Neuronal damage was prevented by 7 days taurine. Mitochondrial electron transport chain complexes I and II showed greater activity with the taurine group. The improvement by taurine of CBF may alleviate edema and elevation in intracranial pressure. Importantly taurine improved the hypercoagulable state.

  11. In situ rat brain and liver spontaneous chemiluminescence after acute ethanol intake.

    PubMed

    Boveris, A; Llesuy, S; Azzalis, L A; Giavarotti, L; Simon, K A; Junqueira, V B; Porta, E A; Videla, L A; Lissi, E A

    1997-09-19

    The influence of acute ethanol administration on the oxidative stress status of rat brain and liver was assessed by in situ spontaneous organ chemiluminescence (CL). Brain and liver CL was significantly increased after acute ethanol administration to fed rats, a response that is time-dependent and evidenced at doses higher than 1 g/kg. Ethanol-induced CL development is faster in liver compared with brain probably due to the greater ethanol metabolic capacity of the liver, whereas the net enhancement in brain light emission at 3 h after ethanol treatment is higher than that of the liver, which could reflect the greater susceptibility of brain to oxidative stress. The effect of ethanol on brain and liver CL seems to be mediated by acetaldehyde, due to its abolishment by the alcohol dehydrogenase inhibitor 4-methylpyrazole and exacerbation by the aldehyde dehydrogenase inhibitor disulfiram. In brain, these findings were observed in the absence of changes in the activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase. However, the content of brain glutathione was significantly decreased by 31%, by ethanol, thus establishing an enhanced oxidative stress in this tissue.

  12. Study of the Effects of ATP Suppliers and Thiol Reductants on Toxicity of Pioglitazone in Isolated Rat Liver Mitochondria

    PubMed Central

    Rezaiean Mehrabadi, Abbas; Jamshidzadeh, Akram; Rashedinia, Marzieh; Niknahad, Hossein

    2015-01-01

    Pioglitazone (PG) is one of thiazolidinediones used for the treatment of type II diabetes mellitus. Some reports of its hepatotoxicity exist, but the mechanism of its hepatotoxicity is not well known. In the present study, the protective effect of some ATP suppliers are investigated against mitochondrial toxicity of PG in isolated rat mitochondria. Mitochondrial viability was investigated by MTT assay. The effects of PG on superoxide dismutase activity, ATP production, mitochondrial swelling and oxidative stress were also investigated. PG reduced mitochondrial viability with an LC50 of 880±32 µM. It reduced ATP production and superoxide dismutase activity in mitochondria and increased mitochondrial swelling, but no oxidant effect was present as measured by TBARS formation. Fructose, dihydroxyacetone, dithioteritol, and N-acetylcysteine reduced mitochondrial toxicity of PG. Therefore, PG toxicity may be due to its mitochondrial toxicity and energy depletion, and ATP suppliers could be effective in preventing its toxicity. PMID:26330870

  13. A Fast Method for the Segmentation of Synaptic Junctions and Mitochondria in Serial Electron Microscopic Images of the Brain.

    PubMed

    Márquez Neila, Pablo; Baumela, Luis; González-Soriano, Juncal; Rodríguez, Jose-Rodrigo; DeFelipe, Javier; Merchán-Pérez, Ángel

    2016-04-01

    Recent electron microscopy (EM) imaging techniques permit the automatic acquisition of a large number of serial sections from brain samples. Manual segmentation of these images is tedious, time-consuming and requires a high degree of user expertise. Therefore, there is considerable interest in developing automatic segmentation methods. However, currently available methods are computationally demanding in terms of computer time and memory usage, and to work properly many of them require image stacks to be isotropic, that is, voxels must have the same size in the X, Y and Z axes. We present a method that works with anisotropic voxels and that is computationally efficient allowing the segmentation of large image stacks. Our approach involves anisotropy-aware regularization via conditional random field inference and surface smoothing techniques to improve the segmentation and visualization. We have focused on the segmentation of mitochondria and synaptic junctions in EM stacks from the cerebral cortex, and have compared the results to those obtained by other methods. Our method is faster than other methods with similar segmentation results. Our image regularization procedure introduces high-level knowledge about the structure of labels. We have also reduced memory requirements with the introduction of energy optimization in overlapping partitions, which permits the regularization of very large image stacks. Finally, the surface smoothing step improves the appearance of three-dimensional renderings of the segmented volumes.

  14. Studies on the effects of the narcotic alkaloids, cocaine, morphine, and codeine on nonenzymatic lipid peroxidation in rat brain mitochondria.

    PubMed

    Das, N P; Ratty, A K

    1987-04-01

    This paper presents evidence of studies on the effects of the narcotic alkaloids, cocaine hydrochloride, morphine sulfate, and codeine phosphate, on nonenzymatic lipid peroxidation in rat brain mitochondria. These organelles abound in polyunsaturated fatty acids and are thus susceptible to oxidative attack. Lipid peroxidation was indexed mainly by assaying the extent of malonaldehyle (MDA) production and also the formation of fluorescent products in the course of the reaction. We found that morphine sulfate lowered fluorescence while the other two alkaloids showed no effect on lipid peroxidation in the absence of the inducers, 1.0 mM ascorbic acid or 0.1 mM ferrous sulfate. The apparent antioxidative nature of morphine sulfate was also observed in its effects on induced and noninduced MDA production, both cocaine hydrochloride and codeine phosphate stimulated MDA production by about 20% in the absence of any inducers. This paper also attempts to draw a structure-activity relationship for the antioxidative action of opium alkaloids, which we postulated to be due to the chelating capability of the alkaloid molecule.

  15. Cytochrome c release from isolated rat liver mitochondria can occur independently of outer-membrane rupture: possible role of contact sites.

    PubMed Central

    Doran, E; Halestrap, A P

    2000-01-01

    Percoll-purified rat liver mitochondria were shown to contain BAX dimer and rapidly (<2 min) release 5-10% of their cytochrome c when incubated in a standard KCl incubation medium under energized conditions. This release was not accompanied by release of adenylate kinase (AK), another intermembrane protein, and was not inhibited by Mg(2+), dATP, inhibitors of the permeability transition or ligands of the peripheral benzodiazepine receptor. However, release was greatly reduced by the presence of 5% (w/v) dextran (40 kDa), which caused a decrease in the light scattering (A(520)) of mitochondrial suspensions. Dextran also inhibited both mitochondrial oxidation of exogenous ferrocytochrome c in the presence of rotenone and antimycin, and respiratory-chain-driven reduction of exogenous ferricytochrome c. Hypo-osmotic medium or digitonin treatment of mitochondria caused a large additional release of both cytochrome c and AK that was not blocked by dextran. Polyaspartate, which stabilizes the low conductance state of the voltage-dependent anion channel (VDAC), increased cytochrome c release. VDAC and BAX are both found at the contact sites between the inner and outer membranes and dextran is known to stabilize these contact sites in isolated mitochondria. Thus our data suggest that regulation of a specific permeability pathway for cytochrome c may be mediated by changes in protein-protein interactions within contact sites. The adenine nucleotide translocase is known to bind to VDAC and thus provides an additional link between the specific cytochrome c release pathway and the permeability transition. PMID:10816428

  16. Uncoupling, metabolic inhibition and induction of mitochondrial permeability transition in rat liver mitochondria caused by the major long-chain hydroxyl monocarboxylic fatty acids accumulating in LCHAD deficiency.

    PubMed

    Hickmann, Fernanda Hermes; Cecatto, Cristiane; Kleemann, Daniele; Monteiro, Wagner Oliveira; Castilho, Roger Frigério; Amaral, Alexandre Umpierrez; Wajner, Moacir

    2015-01-01

    Patients with long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency commonly present liver dysfunction whose pathogenesis is unknown. We studied the effects of long-chain 3-hydroxylated fatty acids (LCHFA) that accumulate in LCHAD deficiency on liver bioenergetics using mitochondrial preparations from young rats. We provide strong evidence that 3-hydroxytetradecanoic (3HTA) and 3-hydroxypalmitic (3HPA) acids, the monocarboxylic acids that are found at the highest tissue concentrations in this disorder, act as metabolic inhibitors and uncouplers of oxidative phosphorylation. These conclusions are based on the findings that these fatty acids decreased ADP-stimulated (state 3) and uncoupled respiration, mitochondrial membrane potential and NAD(P)H content, and, in contrast, increased resting (state 4) respiration. We also verified that 3HTA and 3HPA markedly reduced Ca2+ retention capacity and induced swelling in Ca2+-loaded mitochondria. These effects were mediated by mitochondrial permeability transition (MPT) induction since they were totally prevented by the classical MPT inhibitors cyclosporin A and ADP, as well as by ruthenium red, a Ca2+ uptake blocker. Taken together, our data demonstrate that the major monocarboxylic LCHFA accumulating in LCHAD deficiency disrupt energy mitochondrial homeostasis in the liver. It is proposed that this pathomechanism may explain at least in part the hepatic alterations characteristic of the affected patients.

  17. Supplementation of coconut oil from different sources to the diet induces cellular damage and rapid changes in fatty acid composition of chick liver and hepatic mitochondria.

    PubMed

    Gil-Villarino, A; Torres, M I; Zafra, M F; García-Peregrín, E

    1997-07-01

    Supplementation of 20% coconut oil from two commercial sources pharmaceutical ("Pharmacy") and cooking ("Pastry") use, to the chick diet for 14 days produced a clear damage to the hepatic mitochondria, accompanied by an accumulation of glycogen and lipid droplets in the hepatocyte cytoplasm. These effects may be accounted for the high proportion of fat supplemented to the diets (20%). Pharmacy coconut oil induced a high percentage of cellular death when administered for 14 days. Fatty acid profiles in liver and hepatic mitochondria rapidly changed (24 hr) after both coconut oils supplementation to the diet. The accumulation of shorter chain fatty acids (12:0 and 14:0) was always higher after Pharmacy than after Pastry diet feeding. This fact may contribute, at least in part, to the cellular damage mentioned above especially after Pharmacy diet feeding. Mitochondrial ratios of saturated/unsaturated and saturated/polyunsaturated fatty acids rapidly changed in parallel to these ratios in both diets. Most of the mitochondrial parameters measured tend to recuperate the control values when diets were supplied for 5-14 days. Nevertheless, the maintenance of the mentioned ratios after 14-days Pharmacy diet feeding at significantly higher levels than those observed in control, seems to suggest the lack of the homeostatic mechanism in these membranes and could be also related with the high percentage of cellular death observed after this dietary manipulation.

  18. Modulation of oxidative phosphorylation machinery signifies a prime mode of anti-ageing mechanism of calorie restriction in male rat liver mitochondria.

    PubMed

    Dani, Diksha; Shimokawa, Isao; Komatsu, Toshimitsu; Higami, Yoshikazu; Warnken, Uwe; Schokraie, Elham; Schnölzer, Martina; Krause, Frank; Sugawa, Michiru D; Dencher, Norbert A

    2010-06-01

    Mitochondria being the major source and target of reactive oxygen species (ROS) play a crucial role during ageing. We analyzed ageing and calorie restriction (CR)-induced changes in abundance of rat liver mitochondrial proteins to understand key aspects behind the age-retarding mechanism of CR. The combination of blue-native (BN) gel system with fluorescence Difference Gel Electrophoresis (DIGE) facilitated an efficient analysis of soluble and membrane proteins, existing as monomers or multi-protein assemblies. Changes in abundance of specific key subunits of respiratory chain complexes I, IV and V, critical for activity and/or assembly of the complexes were identified. CR lowered complex I assembly and complex IV activity, which is discussed as a molecular mechanism to minimize ROS production at mitochondria. Notably, the antioxidant system was found to be least affected. The GSH:GSSG couple could be depicted as a rapid mean to handle the fluctuations in ROS levels led by reversible metabolic shifts. We evaluated the relative significance of ROS generation against quenching. We also observed parallel and unidirectional changes as effect of ageing and CR, in subunits of ATP synthase, cytochrome P450 and glutathione S-transferase. This is the first report on such 'putatively hormetic' ageing-analogous effects of CR, besides the age-retarding ones.

  19. Liver transplant outcomes using ideal donation after circulatory death livers are superior to using older donation after brain death donor livers.

    PubMed

    Scalea, Joseph R; Redfield, Robert R; Foley, David P

    2016-09-01

    Multiple reports have demonstrated that liver transplantation following donation after circulatory death (DCD) is associated with poorer outcomes when compared with liver transplantation from donation after brain death (DBD) donors. We hypothesized that carefully selected, underutilized DCD livers recovered from younger donors have excellent outcomes. We performed a retrospective study of the United Network for Organ Sharing database to determine graft survivals for patients who received liver transplants from DBD donors of age ≥ 60 years, DBD donors < 60 years, and DCD donors < 50 years of age. Between January 2002 and December 2014, 52,271 liver transplants were performed in the United States. Of these, 41,181 (78.8%) underwent transplantation with livers from DBD donors of age < 60 years, 8905 (17.0%) from DBD donors ≥ 60 years old, and 2195 (4.2%) livers from DCD donors < 50 years of age. DCD livers of age < 50 years with < 6 hours of cold ischemia time (CIT) had superior graft survival when compared with DBD livers ≥ age 60 years (P < 0.001). In 2014, there were 133 discarded DCD livers; of these, 111 (83.4%) were from donors < age 50 years old. Young DCD donor livers (age < 50 years old) with short CITs yield results better than that seen with DBD livers > 60 years old. Careful donor organ and recipient selection can lead to excellent results, despite previous reports suggesting otherwise. Increased acceptance of these DCD livers would lead to shorter wait list times and increased national liver transplant rates. Liver Transplantation 22 1197-1204 2016 AASLD. © 2016 by the American Association for the Study of Liver Diseases.

  20. Epigenomic Landscape of Human Fetal Brain, Heart, and Liver.

    PubMed

    Yan, Liying; Guo, Hongshan; Hu, Boqiang; Li, Rong; Yong, Jun; Zhao, Yangyu; Zhi, Xu; Fan, Xiaoying; Guo, Fan; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Wang, Yan; Wen, Lu; Qiao, Jie; Tang, Fuchou

    2016-02-26

    The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Binding of exogenous brain protein kinase C to liver nuclei

    SciTech Connect

    Misra, U.K.; Wolf, M.; Besterman, J.; Cuatrecasas, P.; Sahyoun, N.

    1986-05-01

    Protein kinase C is found both in the cytosol and bound to membranes. Binding of the enzyme to plasma membranes is controlled by calcium whereas enzyme activators regulate both its membrane binding and enzyme catalysis. Activation of protein kinase C has been implicated in several regulatory processes including gene expression. Accordingly, the possibility of direct interaction of protein kinase C with the nucleus was examined utilizing /sup 3/H-PDBu binding to detect the enzyme. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4/sup 0/C or at 21/sup 0/C, and the reaction was completed by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca/sup 2 +/ with a K/sub m/sub app// of 0.5 ..mu..M. Chelation of Ca/sup 2 +/ with EGTA resulted in a rapid dissociation of protein kinase C from the nuclei. Differential extraction experiments suggested that about 50% of the enzyme was bound to chromatin and 25% was associated with the nuclear matrix. Moreover, protein kinase C bound to nuclei was able to phosphorylate several endogenous nuclear substrates, including chromatin proteins, in a Ca/sup 2 +/ phosphatidyl serine dependent reaction.

  2. Epigenomic Landscape of Human Fetal Brain, Heart, and Liver*

    PubMed Central

    Yan, Liying; Guo, Hongshan; Hu, Boqiang; Li, Rong; Yong, Jun; Zhao, Yangyu; Zhi, Xu; Fan, Xiaoying; Guo, Fan; Wang, Xiaoye; Wang, Wei; Wei, Yuan; Wang, Yan; Wen, Lu; Qiao, Jie; Tang, Fuchou

    2016-01-01

    The epigenetic regulation of spatiotemporal gene expression is crucial for human development. Here, we present whole-genome chromatin immunoprecipitation followed by high throughput DNA sequencing (ChIP-seq) analyses of a wide variety of histone markers in the brain, heart, and liver of early human embryos shortly after their formation. We identified 40,181 active enhancers, with a large portion showing tissue-specific and developmental stage-specific patterns, pointing to their roles in controlling the ordered spatiotemporal expression of the developmental genes in early human embryos. Moreover, using sequential ChIP-seq, we showed that all three organs have hundreds to thousands of bivalent domains that are marked by both H3K4me3 and H3K27me3, probably to keep the progenitor cells in these organs ready for immediate differentiation into diverse cell types during subsequent developmental processes. Our work illustrates the potentially critical roles of tissue-specific and developmental stage-specific epigenomes in regulating the spatiotemporal expression of developmental genes during early human embryonic development. PMID:26719341

  3. Y3+, La3+, and some bivalent metals inhibited the opening of the Tl+-induced permeability transition pore in Ca2+-loaded rat liver mitochondria.

    PubMed

    Korotkov, Sergey; Konovalova, Svetlana; Emelyanova, Larisa; Brailovskaya, Irina

    2014-12-01

    We showed earlier that diminution of 2,4-dinitrophenol (DNP)-stimulated respiration and increase of both mitochondrial swelling and electrochemical potential (ΔΨmito) dissipation in medium containing TlNO3 and KNO3 were caused by opening of Tl(+)-induced mitochondrial permeability transition pore (MPTP) in the inner membrane of Ca(2+)-loaded rat liver mitochondria. The MPTP opening was studied in the presence of bivalent metal ions (Sr(2+), Ba(2+), Mn(2+), Co(2+) and Ni(2+)), trivalent metal ions (Y(3+) and La(3+)), and ruthenium red. We found that these metal ions (except Ba(2+) and Co(2+)) as well as ruthenium red inhibited to the MPTP opening that manifested in preventing both diminution of the DNP-stimulated respiration and increase of the swelling and of the ΔΨmito dissipation in medium containing TlNO3, KNO3, and Ca(2+). Inhibition of the MPTP opening by Sr(2+) and Mn(2+) is suggested because of their interaction with high affinity Ca(2+) sites, facing the matrix side and participating in the MPTP opening. The inhibitory effects of metal ions (Y(3+), La(3+), and Ni(2+)), and ruthenium red are accordingly discussed in regard to competitive and noncompetitive inhibition of the mitochondrial Ca(2+)-uniporter. High concentrations (50μM) of Y(3+) and La(3+) favored of MPTP opening in the inner membrane of rat liver mitochondria in Ca(2+) free medium containing TlNO3. The latter MPTP opening was markedly eliminated by MPTP inhibitors (cyclosporine A and ADP).

  4. Quercetin accumulation by chronic administration causes the caspase-3 activation in liver and brain of mice.

    PubMed

    Choi, Eun Jeong; Kim, Gun-Hee

    2010-01-01

    Quercetin is an excellent antioxidant that has a variety of side effects. This study investigated whether the chronic administration of quercetin in mice induces apoptosis. Mice were divided randomly into three treatment groups. Quercetin was administered orally to two of three groups at 100 and 250 mg/kg body weight (BW) for 18 days. The serum quercetin level increased in a dose-dependent manner, although the quercetin levels in the liver and brain were lower than in serum. Nevertheless, quercetin induced apoptosis in both the liver and brain, as evidenced by increased caspase-3 expression and activity. Quercetin-induced apoptosis seems to be associated with quercetin accumulation. Moreover, with quercetin accumulation, the brain was more susceptible to apoptosis than the liver. In conclusion, quercetin administration at a high dose may lead to apoptosis in the liver and brain of mouse.

  5. Sex-specific divergence of antioxidant pathways in fetal brain, liver, and skeletal muscles.

    PubMed

    Al-Gubory, Kaïs H; Garrel, Catherine

    2016-01-01

    The sex-specific divergence of antioxidant pathways in fetal organs of opposite-sex twin is unknown and remains urgently in need of investigation. Such study faces many challenges, mainly the ethical impossibility of obtaining human fetal organs. Opposite-sex sheep twins represent a unique model for studying a sex dimorphism for antioxidant systems. The activity of total superoxide dismutase (SOD), SOD1, SOD2, glutathione peroxidase (GPX), glutathione reductase (GR) and catalase (CAT), the content of total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were measured in brain, lung, liver, kidney, and skeletal muscles of female and male fetuses collected from sheep twin pregnancies at day 65 of gestation. Lipid peroxidation was assessed by measuring melondialdehyde (MDA) tissue content. Male brain has greater total SOD and SOD1 activities than female brain. Female liver has greater SOD2 activity than male liver. Male liver has greater GR activity than female liver. Male liver has higher total GSH and GSSG content than female liver. Male skeletal muscles have higher total GSH, GSH, and GSSG content than female skeletal muscles. Female brain and liver have higher MDA content than male brain and liver. This is the first report of a sex dimorphism for fetal organ antioxidative pathways. Brain, liver, and skeletal muscles of male and female fetuses display distinct antioxidant pathways. Such sexually dimorphic responses to early life oxidative stress might be involved in the sex-related difference in fetal development that may have a long-term effect on offspring. Our study urges researchers to take into consideration the importance of sex as a biologic variable in their investigations.

  6. Alleviation of Ischemia-Reperfusion Injury in Liver Steatosis by Augmenter of Liver Regeneration Is Attributed to Antioxidation and Preservation of Mitochondria.

    PubMed

    Weng, Junhua; Li, Wen; Jia, Xiaowei; An, Wei

    2017-10-01

    Fatty liver is one of the major impediments to liver surgery and liver transplantation because steatotic hepatocytes are more susceptible to ischemia-reperfusion injury (IRI). In this study, the effects of augmenter of liver regeneration (ALR) on hepatic IRI in steatotic mice were investigated. In vivo, liver steatosis of mice was induced by feeding a methionine-choline-deficient diet for 2 weeks. Three days before hepatic partial warm IRI, mice were transfected with the ALR-containing adenovirus. In an in vitro study, the protective effect of ALR on steatotic HepG2 cells was analyzed after hypoxia/reoxygenation (HR) treatment. The transfection of the ALR gene into steatotic mice attenuated liver injury, inhibiting hepatic oxidative stress, increasing antioxidation capacities, promoting liver regeneration, and consequently suppressing cell apoptosis/death. Furthermore, resistance to HR injury was notably increased in ALR-transfected cells compared with the vector-transfected cells. The HR-induced rise in the mitochondrial reactive oxygen species was reduced, and cellular antioxidant activities were enhanced. The ALR transfection prevented cells from apoptosis, which can be attributed to the preservation of the mitochondrial membrane potential, enhancement of oxygen consumption rate and production of adenosine triphosphate. ALR protects steatotic hepatocytes from IRI by attenuating oxidative stress and mitochondrial dysfunction, as well as improving antioxidant effect. ALR may be used as a potential therapeutic agent when performing surgery and transplantation of steatotic liver.

  7. Ageing and inflammation - A central role for mitochondria in brain health and disease.

    PubMed

    Currais, Antonio

    2015-05-01

    To develop successful therapies that prevent or treat neurodegenerative diseases requires an understanding of the upstream events. Ageing is by far the greatest risk factor for most of these diseases, and to clarify their causes will require an understanding of the process of ageing itself. Starting with the question Why do we age as individual organisms, but the line of pluripotent embryonic stem cells and germ cells carried by individuals and transmitted to descendants is immortal? this review discusses how the process of cellular differentiation leads to the accumulation of biological imperfections with ageing, and how these imperfections may be the cause of chronic inflammatory responses to stress that undermine cellular function. Both differentiation and inflammation involve drastic metabolic changes associated with alterations in mitochondrial dynamics that shift the balance between aerobic glycolysis and oxidative phosphorylation. With ageing, mitochondrial dysfunction can be both the cause and consequence of inflammatory processes and elicit metabolic adaptations that might be either protective or become progressively detrimental. It is argued here that an understanding of the relationship between metabolism, differentiation and inflammation is essential to understand the pathological mechanisms governing brain health and disease during ageing.

  8. Safety evaluation of mercury based Ayurvedic formulation (Sidh Makardhwaj) on brain cerebrum, liver & kidney in rats.

    PubMed

    Kumar, Gajendra; Srivastava, Amita; Sharma, Surinder Kumar; Gupta, Yogendra Kumar

    2014-04-01

    Sidh Makardhwaj (SM) is a mercury based Ayurvedic formulation used in rheumatoid arthritis and neurological disorders. However, toxicity concerns due to mercury content are often raised. Therefore, the present study was carried out to evaluate the effect of SM on brain cerebrum, liver and kidney in rats. Graded doses of SM (10, 50, 100 mg/kg), mercuric chloride (1 mg/kg) and normal saline were administered orally to male Wistar rats for 28 days. Behavioural parameters were assessed on days 1, 7, 14 and 28 using Morris water maze, passive avoidance, elevated plus maze and rota rod. Liver and kidney function tests were done on day 28. Animals were sacrificed and brain cerebrum acetylcholinesterase activity, levels of malondialdehyde (MDA), reduced glutathione (GSH) in brain cerebrum, liver, kidney were estimated. The levels of mercury in brain cerebrum, liver and kidney were estimated and histopathology of these tissues was also performed. SM in the doses used did not cause significant change in neurobehavioural parameters, brain cerebrum AChE activity, liver (ALT, AST, ALP bilirubin) and kidney (serum urea and creatinine) function tests as compared to control. The levels of mercury in brain cerebrum, liver, and kidney were found to be raised in dose dependent manner. However, the levels of MDA and GSH in these tissues did not show significant changes at doses of 10 and 50 mg/kg. Also, there was no histopathological change in cytoarchitecture of brain cerebrum, liver, and kidney tissues at doses of 10 and 50 mg/kg. The findings of the present study suggest that Sidh Makardhwaj upto five times the equivalent human dose administered for 28 days did not show any toxicological effects on rat brain cerebrum, liver and kidney.

  9. Safety evaluation of mercury based Ayurvedic formulation (Sidh Makardhwaj) on brain cerebrum, liver & kidney in rats

    PubMed Central

    Kumar, Gajendra; Srivastava, Amita; Sharma, Surinder Kumar; Gupta, Yogendra Kumar

    2014-01-01

    Background & objectives: Sidh Makardhwaj (SM) is a mercury based Ayurvedic formulation used in rheumatoid arthritis and neurological disorders. However, toxicity concerns due to mercury content are often raised. Therefore, the present study was carried out to evaluate the effect of SM on brain cerebrum, liver and kidney in rats. Methods: Graded doses of SM (10, 50, 100 mg/kg), mercuric chloride (1 mg/kg) and normal saline were administered orally to male Wistar rats for 28 days. Behavioural parameters were assessed on days 1, 7, 14 and 28 using Morris water maze, passive avoidance, elevated plus maze and rota rod. Liver and kidney function tests were done on day 28. Animals were sacrificed and brain cerebrum acetylcholinesterase activity, levels of malondialdehyde (MDA), reduced glutathione (GSH) in brain cerebrum, liver, kidney were estimated. The levels of mercury in brain cerebrum, liver and kidney were estimated and histopathology of these tissues was also performed. Results: SM in the doses used did not cause significant change in neurobehavioural parameters, brain cerebrum AChE activity, liver (ALT, AST, ALP bilirubin) and kidney (serum urea and creatinine) function tests as compared to control. The levels of mercury in brain cerebrum, liver, and kidney were found to be raised in dose dependent manner. However, the levels of MDA and GSH in these tissues did not show significant changes at doses of 10 and 50 mg/kg. Also, there was no histopathological change in cytoarchitecture of brain cerebrum, liver, and kidney tissues at doses of 10 and 50 mg/kg. Interpretation & conclusions: The findings of the present study suggest that Sidh Makardhwaj upto five times the equivalent human dose administered for 28 days did not show any toxicological effects on rat brain cerebrum, liver and kidney. PMID:24927349

  10. Differential metabolism of 4-hydroxynonenal in liver, lung and brain of mice and rats

    SciTech Connect

    Zheng, Ruijin; Dragomir, Ana-Cristina; Mishin, Vladimir; Richardson, Jason R.; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-08-15

    The lipid peroxidation end-product 4-hydroxynonenal (4-HNE) is generated in tissues during oxidative stress. As a reactive aldehyde, it forms Michael adducts with nucleophiles, a process that disrupts cellular functioning. Liver, lung and brain are highly sensitive to xenobiotic-induced oxidative stress and readily generate 4-HNE. In the present studies, we compared 4-HNE metabolism in these tissues, a process that protects against tissue injury. 4-HNE was degraded slowly in total homogenates and S9 fractions of mouse liver, lung and brain. In liver, but not lung or brain, NAD(P)+ and NAD(P)H markedly stimulated 4-HNE metabolism. Similar results were observed in rat S9 fractions from these tissues. In liver, lung and brain S9 fractions, 4-HNE formed protein adducts. When NADH was used to stimulate 4-HNE metabolism, the formation of protein adducts was suppressed in liver, but not lung or brain. In both mouse and rat tissues, 4-HNE was also metabolized by glutathione S-transferases. The greatest activity was noted in livers of mice and in lungs of rats; relatively low glutathione S-transferase activity was detected in brain. In mouse hepatocytes, 4-HNE was rapidly taken up and metabolized. Simultaneously, 4-HNE-protein adducts were formed, suggesting that 4-HNE metabolism in intact cells does not prevent protein modifications. These data demonstrate that, in contrast to liver, lung and brain have a limited capacity to metabolize 4-HNE. The persistence of 4-HNE in these tissues may increase the likelihood of tissue injury during oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a highly reactive aldehyde. • Rodent liver, but not lung or brain, is efficient in degrading 4-hydroxynonenal. • 4-hydroxynonenal persists in tissues with low metabolism, causing tissue damage.

  11. Protective effects of dietary avocado oil on impaired electron transport chain function and exacerbated oxidative stress in liver mitochondria from diabetic rats.

    PubMed

    Ortiz-Avila, Omar; Gallegos-Corona, Marco Alonso; Sánchez-Briones, Luis Alberto; Calderón-Cortés, Elizabeth; Montoya-Pérez, Rocío; Rodriguez-Orozco, Alain R; Campos-García, Jesús; Saavedra-Molina, Alfredo; Mejía-Zepeda, Ricardo; Cortés-Rojo, Christian

    2015-08-01

    Electron transport chain (ETC) dysfunction, excessive ROS generation and lipid peroxidation are hallmarks of mitochondrial injury in the diabetic liver, with these alterations also playing a role in the development of non-alcoholic fatty liver disease (NAFLD). Enhanced mitochondrial sensitivity to lipid peroxidation during diabetes has been also associated to augmented content of C22:6 in membrane phospholipids. Thus, we aimed to test whether avocado oil, a rich source of C18:1 and antioxidants, attenuates the deleterious effects of diabetes on oxidative status of liver mitochondria by decreasing unsaturation of acyl chains of membrane lipids and/or by improving ETC functionality and decreasing ROS generation. Streptozocin-induced diabetes elicited a noticeable increase in the content of C22:6, leading to augmented mitochondrial peroxidizability index and higher levels of lipid peroxidation. Mitochondrial respiration and complex I activity were impaired in diabetic rats with a concomitant increase in ROS generation using a complex I substrate. This was associated to a more oxidized state of glutathione, All these alterations were prevented by avocado oil except by the changes in mitochondrial fatty acid composition. Avocado oil did not prevented hyperglycemia and polyphagia although did normalized hyperlipidemia. Neither diabetes nor avocado oil induced steatosis. These results suggest that avocado oil improves mitochondrial ETC function by attenuating the deleterious effects of oxidative stress in the liver of diabetic rats independently of a hypoglycemic effect or by modifying the fatty acid composition of mitochondrial membranes. These findings might have also significant implications in the progression of NAFLD in experimental models of steatosis.

  12. Rhein Elicits In Vitro Cytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

    PubMed Central

    Bounda, Guy-Armel; Zhou, Wang; Wang, Dan-dan; Yu, Feng

    2015-01-01

    Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM) for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01). Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism. PMID:26221172

  13. Brain death is associated with endoplasmic reticulum stress and apoptosis in rat liver.

    PubMed

    Cao, S; Wang, T; Yan, B; Lu, Y; Zhao, Y; Zhang, S

    2014-12-01

    Cell death pathways initiated by stress on the endoplasmic reticulum (ER) have been implicated in a variety of common diseases, such as ischemia/reperfusion injury, diabetes, heart disease, and neurodegenerative disorders. However, the contribution of ER stress to apoptosis and liver injury after brain death is not known. In the present study, we found that brain death induces a variety of signature ER stress markers, including ER stress-specific X box-binding protein 1 and up-regulation of glucose-regulated protein 78. Furthermore, brain death causes up-regulation of C/EBP homologous protein and caspase-12. Consistent with this, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling assay and transmission electron microscopy confirmed apoptosis in the liver after brain death. Taken together, the present study provides strong evidence supporting the presence and importance of ER stress and response in mediating brain death-induced apoptosis and liver injury.

  14. Toxicity of cuprizone a Cu(2+) chelating agent on isolated mouse brain mitochondria: a justification for demyelination and subsequent behavioral dysfunction.

    PubMed

    Faizi, Mehrdad; Salimi, Ahmad; Seydi, Enayatolla; Naserzadeh, Parvaneh; Kouhnavard, Mehdi; Rahimi, Atena; Pourahmad, Jalal

    2016-05-01

    Multiple Sclerosis (MS) is a complex disease with an unknown etiology and no effective cure, despite decades of extensive research that led to the development of several partially effective treatments. In this study we aimed to investigate brain mitochondrial dysfunction in demyelination induced by cuprizone in mice. Cuprizone was used for induction of demyelination in mice through a diet containing 0.2% w/w cuprizone for 5 weeks. Behavioral tests for proving of MS was performed and then mitochondria from brain of animals were isolated and afterwards parameters of mitochondrial dysfunction examined. Results of mitochondrial dysfunction parameters such as mitochondrial swelling, production ROS, collapse of the membrane potential showed that isolated mitochondria from cuprizone treated mice have been damaged compared to those of untreated control mice. It is likely that demyelination induced mitochondrial damage led to increased mitochondrial ROS formation and progression of oxidative damages in neurons. It is suggested that cuprizone which is a Cu(2+) chelating agent causes impairment of electron transport chain (complex IV) and antioxidant system (SOD) in mitochondria leading to decreased ATP production and increased ROS formation.

  15. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

    PubMed

    Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

    2015-06-01

    Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

  16. Effects of lysophospholipids on Ca2+ transport in rat liver mitochondria incubated at physiological Ca2+ concentrations in the presence of Mg2+, phosphate and ATP at 37 degrees C.

    PubMed Central

    Dalton, S; Hughes, B P; Barritt, G J

    1984-01-01

    Lysophospholipids caused the release of 45Ca2+ from isolated rat liver mitochondria incubated at 37 degrees C in the presence of low concentrations of free Ca2+, ATP, Mg2+, and phosphate ions. The concentrations of lysophosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidic acid and lysophosphatidylinositol which gave half-maximal effects were 5, 26, 40 and 56 microM, respectively. The effects of lysophosphatidylethanolamine were not associated with a significant impairment of the integrity of the mitochondria as monitored by measurement of membrane potential and the rate of respiration. Lysophosphatidylethanolamine did not induce the release of Ca2+ from a microsomal fraction, or enhance Ca2+ inflow across the plasma membrane of intact cells, but did release Ca2+ from an homogenate prepared from isolated hepatocytes and incubated under the same conditions as isolated mitochondria. The proportion of mitochondrial 45Ca2+ released by lysophosphatidylethanolamine was not markedly affected by altering the total amount of Ca2+ in the mitochondria, the concentration of extramitochondrial Mg2+, by the addition of Ruthenium Red, or when oleoyl lysophosphatidylethanolamine was employed instead of the palmitoyl derivative. The effects of 5 microM-lysophosphatidylethanolamine were reversed by washing the mitochondria. The possibility that lysophosphatidylethanolamine acts to release Ca2+ from mitochondria in intact hepatocytes following the binding of Ca2+-dependent hormones to the plasma membrane is briefly discussed. PMID:6517860

  17. Long-chain α,ω-dioic acids as inducers of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria loaded with calcium or strontium ions.

    PubMed

    Dubinin, M V; Adakeeva, S I; Samartsev, V N

    2013-04-01

    Long-chain saturated monocarboxylic fatty acids can induce nonspecific permeability of the inner membrane (open pores) of liver mitochondria loaded with Ca2+ or Sr(2+) by the mechanism insensitive to cyclosporin A. In this work we investigated the effect of their metabolites - α,ω-dioic (dicarboxylic) acids - as potential inducers of pore opening by a similar mechanism. It was established that the addition of α,ω-hexadecanedioic acid (HDA) at a concentration of 10-30 µM to liver mitochondria loaded with Ca2+ or Sr(2+) leads to swelling of the organelles and release of these ions from the matrix. The maximum effect of HDA is observed at 50 µM Ca2+ concentration. Cyclosporin A at a concentration of 1 µM, previously added to the mitochondria, did not inhibit the observed processes. The calcium uniporter inhibitor ruthenium red, which blocks influx of Ca2+ and Sr(2+) to the matrix of mitochondria, prevented HDA-induced swelling. The effect of HDA as inducer of swelling of mitochondria was compared with similar effects of α,ω-tetradecanedioic and α,ω-dodecanedioic acids whose acyl chains are two and four carbon atoms shorter than HDA, respectively. It was found that the efficiency of these α,ω-dioic acids decreases with reducing number of carbon atoms in their acyl chains. It was concluded that in the presence of Ca2+ or Sr(2+) long-chain saturated α,ω-dioic acids can induce a cyclosporin A-insensitive permeability of the inner membrane (open pores) of liver mitochondria as well as their monocarboxylic analogs.

  18. Alteration in glutathione content and associated enzyme activities in the synaptic terminals but not in the non-synaptic mitochondria from the frontal cortex of Parkinson's disease brains.

    PubMed

    Harish, G; Mahadevan, Anita; Srinivas Bharath, M M; Shankar, S K

    2013-01-01

    Altered redox dynamics contribute to physiological aging and Parkinson's disease (PD). This is reflected in the substantia nigra (SN) of PD patients as lowered antioxidant levels and elevated oxidative damage. Contrary to this observation, we previously reported that non-SN regions such as caudate nucleus and frontal cortex (FC) exhibited elevated antioxidants and lowered mitochondrial and oxidative damage indicating constitutive protective mechanisms in PD brains. To investigate whether the sub-cellular distribution of antioxidants could contribute to these protective effects, we examined the distribution of antioxidant/oxidant markers in the neuropil fractions [synaptosomes, non-synaptic mitochondria and cytosol] of FC from PD (n = 9) and controls (n = 8). In the control FC, all the antioxidant activities [Superoxide dismutase (SOD), glutathione (GSH), GSH peroxidase (GPx), GSH-S-transferase (GST)] except glutathione reductase (GR) were the highest in cytosol, but several fold lower in mitochondria and much lower in synaptosomes. However, FC synaptosomes from PD brains had significantly higher levels of GSH (p = 0.01) and related enzymes [GPx (p = 0.02), GR (p = 0.06), GST (p = 0.0001)] compared to controls. Conversely, mitochondria from the FC of PD cases displayed elevated SOD activity (p = 0.02) while the GSH and related enzymes were relatively unaltered. These changes in the neuropil fractions were associated with unchanged or lowered oxidative damage. Further, the mitochondrial content in the synaptosomes of both PD and control brains was ≥five-fold lower compared to the non-synaptic mitochondrial fraction. Altered distribution of oxidant/antioxidant markers in the neuropil fractions of the human brain during aging and PD has implications for (1) degenerative and protective mechanisms (2) distinct antioxidant mechanisms in synaptic terminals compared to other compartments.

  19. Effect of chronic heroin and cocaine administration on global DNA methylation in brain and liver.

    PubMed

    Fragou, Domniki; Zanos, Panos; Kouidou, Sofia; Njau, Samuel; Kitchen, Ian; Bailey, Alexis; Kovatsi, Leda

    2013-04-26

    Drug abuse is associated with epigenetic changes, such as histone modifications and DNA methylation. The purpose of the present study was to examine the effect of chronic cocaine and heroin administration on global DNA methylation in brain and liver. Male, 8 week old, C57BL/6J mice received heroin in a chronic 'intermittent' escalating dose paradigm, or cocaine in a chronic escalating dose 'binge' paradigm, which mimic the human pattern of opioid or cocaine abuse respectively. Following sacrifice, livers and brains were removed and DNA was extracted from them. The extracted DNA was hydrolyzed and 2'-deoxycytidine and 5-methyl-2'-deoxycytidine were determined by HPLC-UV. The % 5-methyl-2'-deoxycytidine content of DNA was significantly higher in the brain compared to the liver. There were no differences between the control animals and the cocaine or heroin treated animals in neither of the tissues examined, which is surprising since cocaine administration induced gross morphological changes in the liver. Moreover, there was no difference in the % 5-methyl-2'-deoxycytidine content of DNA between the cocaine and the heroin treated animals. The global DNA methylation status in the brain and liver of mice chronically treated with cocaine or heroin remains unaffected, but this finding cannot exclude the existence of anatomical region or gene-specific methylation differences. This is the first time that global DNA methylation in the liver and whole brain has been studied following chronic cocaine or heroin treatment.

  20. Exendin-4 attenuates brain death-induced liver damage in the rat.

    PubMed

    Carlessi, Rodrigo; Lemos, Natalia E; Dias, Ana L; Brondani, Leticia A; Oliveira, Jarbas R; Bauer, Andrea C; Leitão, Cristiane B; Crispim, Daisy

    2015-11-01

    The majority of liver grafts destined for transplantation originate from brain dead donors. However, significantly better posttransplantation outcomes are achieved when organs from living donors are used, suggesting that brain death (BD) causes irreversible damage to the liver tissue. Recently, glucagon-like peptide-1 (GLP1) analogues were shown to possess interesting hepatic protection effects in different liver disease models. We hypothesized that donor treatment with the GLP1 analogue exendin-4 (Ex-4) could alleviate BD-induced liver damage. A rat model of BD was employed in order to estimate BD-induced liver damage and Ex-4's potential protective effects. Liver damage was assessed by biochemical determination of circulating hepatic markers. Apoptosis in the hepatic tissue was assessed by immunoblot and immunohistochemistry using an antibody that only recognizes the active form of caspase-3. Gene expression changes in inflammation and stress response genes were monitored by quantitative real-time polymerase chain reaction. Here, we show that Ex-4 administration to the brain dead liver donors significantly reduces levels of circulating aspartate aminotransferase and lactate dehydrogenase. This was accompanied by a remarkable reduction in hepatocyte apoptosis. In this model, BD caused up-regulation of tumor necrosis factor and stress-related genes, confirming previous findings in clinical and animal studies. In conclusion, treatment of brain dead rats with Ex-4 reduced BD-induced liver damage. Further investigation is needed to determine the molecular basis of the observed liver protection. After testing in a randomized clinical trial, the inclusion of GLP1 analogues in organ donor management might help to improve organ quality, maximize organ donation, and possibly increase liver transplantation success rates.

  1. Liver irradiation causes distal bystander effects in the rat brain and affects animal behaviour.

    PubMed

    Kovalchuk, Anna; Mychasiuk, Richelle; Muhammad, Arif; Hossain, Shakhawat; Ilnytskyy, Slava; Ghose, Abhijit; Kirkby, Charles; Ghasroddashti, Esmaeel; Kovalchuk, Olga; Kolb, Bryan

    2016-01-26

    Radiation therapy can not only produce effects on targeted organs, but can also influence shielded bystander organs, such as the brain in targeted liver irradiation. The brain is sensitive to radiation exposure, and irradiation causes significant neuro-cognitive deficits, including deficits in attention, concentration, memory, and executive and visuospatial functions. The mechanisms of their occurrence are not understood, although they may be related to the bystander effects.We analyzed the induction, mechanisms, and behavioural repercussions of bystander effects in the brain upon liver irradiation in a well-established rat model.Here, we show for the first time that bystander effects occur in the prefrontal cortex and hippocampus regions upon liver irradiation, where they manifest as altered gene expression and somewhat increased levels of γH2AX. We also report that bystander effects in the brain are associated with neuroanatomical and behavioural changes, and are more pronounced in females than in males.

  2. Liver irradiation causes distal bystander effects in the rat brain and affects animal behaviour

    PubMed Central

    Kovalchuk, Anna; Mychasiuk, Richelle; Muhammad, Arif; Hossain, Shakhawat; Ilnytskyy, Slava; Ghose, Abhijit; Kirkby, Charles; Ghasroddashti, Esmaeel; Kovalchuk, Olga; Kolb, Bryan

    2016-01-01

    Radiation therapy can not only produce effects on targeted organs, but can also influence shielded bystander organs, such as the brain in targeted liver irradiation. The brain is sensitive to radiation exposure, and irradiation causes significant neuro-cognitive deficits, including deficits in attention, concentration, memory, and executive and visuospatial functions. The mechanisms of their occurrence are not understood, although they may be related to the bystander effects. We analyzed the induction, mechanisms, and behavioural repercussions of bystander effects in the brain upon liver irradiation in a well-established rat model. Here, we show for the first time that bystander effects occur in the prefrontal cortex and hippocampus regions upon liver irradiation, where they manifest as altered gene expression and somewhat increased levels of γH2AX. We also report that bystander effects in the brain are associated with neuroanatomical and behavioural changes, and are more pronounced in females than in males. PMID:26678032

  3. Evaluation of the inhibitory effects of quercetin-related flavonoids and tea catechins on the monoamine oxidase-A reaction in mouse brain mitochondria.

    PubMed

    Bandaruk, Yauhen; Mukai, Rie; Kawamura, Tomoyuki; Nemoto, Hisao; Terao, Junji

    2012-10-17

    Quercetin, a typical dietary flavonoid, is thought to exert antidepressant effects by inhibiting the monoamine oxidase-A (MAO-A) reaction, which is responsible for regulation of the metabolism of the neurotransmitter 5-hydroxytryptamine (5-HT) in the brain. This study compared the MAO-A inhibitory activity of quercetin with those of O-methylated quercetin (isorhamnetin, tamarixetin), luteolin, and green tea catechins ((-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, and (-)-epigallocatechin gallate) by measuring the formation of the oxidative deamination product of 5-HT, 5-hydroxyindole aldehyde (5-HIAL), in mouse brain mitochondria. Quercetin was inferior to luteolin in the inhibition of MAO-A activity, whereas isorhamnetin, tamarixetin, and tea catechins scarcely exerted inhibitory activity. Quercetin did not affect MAO-A activity in mouse intestinal mitochondria, indicating that it does not evoke side effects on the metabolism of dietary monoamines in the gut. These data suggest that quercetin is a weak (but safe) MAO-A inhibitor in the modulation of 5-HT levels in the brain.

  4. Comprehensive analysis of phospholipids in the brain, heart, kidney, and liver: brain phospholipids are least enriched with polyunsaturated fatty acids.

    PubMed

    Choi, Jaewoo; Yin, Tai; Shinozaki, Koichiro; Lampe, Joshua W; Stevens, Jan F; Becker, Lance B; Kim, Junhwan

    2017-10-09

    It is commonly accepted that brain phospholipids are highly enriched with long-chain polyunsaturated fatty acids (PUFAs). However, the evidence for this remains unclear. We used HPLC-MS to analyze the content and composition of phospholipids in rat brain and compared it to the heart, kidney, and liver. Phospholipids typically contain one PUFA, such as 18:2, 20:4, or 22:6, and one saturated fatty acid, such as 16:0 or 18:0. However, we found that brain phospholipids containing monounsaturated fatty acids in the place of PUFAs are highly elevated compared to phospholipids in the heart, kidney, and liver. The relative content of phospholipid containing PUFAs is ~ 60% in the brain, whereas it is over 90% in other tissues. The most abundant species of phosphatidylcholine (PC) is PC(16:0/18:1) in the brain, whereas PC(18:0/20:4) and PC(16:0/20:4) are predominated in other tissues. Moreover, several major species of plasmanyl and plasmenyl phosphatidylethanolamine are found to contain monounsaturated fatty acid in the brain only. Overall, our data clearly show that brain phospholipids are the least enriched with PUFAs of the four major organs, challenging the common belief that the brain is highly enriched with PUFAs.

  5. Effect of progesterone and its synthetic analogues on the activity of mitochondrial permeability transition pore in isolated rat liver mitochondria.

    PubMed

    Fedotcheva, Nadezhda I; Teplova, Vera V; Fedotcheva, Tatiana A; Rzheznikov, Vladimir M; Shimanovskii, Nikolai L

    2009-10-15

    The influence of progesterone and its synthetic analogues on the induction of the Ca(2+)-dependent mitochondrial permeability transition pore (MPTP) has been studied. The novel synthetic analogue of progesterone 17a-acetoxy-3b-butanoyloxy-6-methyl-pregna-4,6-diene-20-on (buterol) was compared with progesterone and medroxyprogesterone acetate (MPA). It was found that progesterone and buterol have opposite effects on the induction of MPTP opening by calcium ions. By contrast to progesterone, which decreased the calcium ion concentration necessary for pore opening, and MPA, which also, although at a lesser extent, activated the pore induction, buterol at a concentration of 20-100 microM blocked the pore opening and increased the calcium retention capacity of mitochondria more than twofold. The action of buterol is specific to the pore since it did not affect the respiration, whereas progesterone completely inhibited NAD-dependent respiration. MPA acted similar to progesterone but less effectively. The inhibitory effect of buterol was eliminated in the presence of carboxyatractyloside, which selectively binds the thiol groups of adenylate translocase and prevents the adenine nucleotide binding. These data indicate that buterol interacts with thiol groups, which explains its inhibitory effect not only on the mitochondrial pore but also on the transport system of xenobiotics in tumor cells in which buterol reduces the multidrug resistance.

  6. Extremely low-frequency magnetic field induces manganese accumulation in brain, kidney and liver of rats.

    PubMed

    Çelik, Mustafa Salih; Güven, Kemal; Akpolat, Veysi; Akdağ, Mehmet Zulkuf; Nazıroğlu, Mustafa; Gül-Güven, Reyhan; Çelik, M Yusuf; Erdoğan, Sait

    2015-06-01

    The aim of the present study was to determine the effects of extremely low-frequency magnetic field (ELF-MF) on accumulation of manganese (Mn) in the kidney, liver and brain of rats. A total of 40 rats were randomly divided into eight groups. Four control groups received 0, 3.75, 15 and 60 mg Mn per kg body weight orally every 2 days for 45 days, respectively. The remaining four groups received same concentrations of Mn and were also exposed to ELF-MF (1.5 mT; 50 Hz) for 4 h for 5 days a week during 45 days. Following the last exposure, kidney, liver and brain were taken from all rats and they were analyzed for Mn accumulation levels using an inductively coupled plasma-optical emission spectrometer. In result of the current study, we observed that Mn levels in brain, kidney and liver were higher in Mn groups than in control groups. Mn levels in brain, kidney and liver were also higher in Mn plus ELF-MF groups than in Mn groups. In conclusion, result of the current study showed that the ELF-MF induced manganese accumulation in kidney, liver and brain of rats.

  7. Isolation of Peroxisomes from Mouse Brain Using a Continuous Nycodenz Gradient: A Comparison to the Isolation of Liver and Kidney Peroxisomes.

    PubMed

    Schönenberger, Miriam J; Kovacs, Werner J

    2017-01-01

    In the central nervous system (CNS) peroxisomes are present in all cell types, namely neurons, oligodendrocytes, astrocytes, microglia, and endothelial cells. Brain peroxisomes are smaller in size compared to peroxisomes from other tissues and are therefore referred to as microperoxisomes. We have established a purification procedure to isolate highly purified peroxisomes from the central nervous system that are well separated from the endoplasmic reticulum and mitochondria and are free of myelin contamination. The major difficulty in purification of brain peroxisomes compared to peroxisomes from liver or kidney is the presence of large amounts of myelin in the CNS, which results in contamination of the subcellular fractions. Hence, the crucial step of the isolation procedure is the elimination of myelin by the use of a sucrose gradient, since without the elimination of myelin no significant enrichment of purified peroxisomes can be achieved. Another difficulty is that in brain tissue the abundance of peroxisomes decreases significantly during postnatal development. We provide a detailed protocol for the isolation of peroxisomes from mouse central nervous system as well as a protocol for the isolation of peroxisomes from the liver and kidney using a continuous Nycodenz gradient.

  8. In vitro effects of cholesterol β-D-glucoside, cholesterol and cycad phytosterol glucosides on respiration and reactive oxygen species generation in brain mitochondria.

    PubMed

    Panov, Alexander; Kubalik, Nataliya; Brooks, Benjamin R; Shaw, Christopher A

    2010-10-01

    The cluster of neurodegenerative disorders in the western Pacific termed amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) has been repeatedly linked to the use of seeds of various species of cycad. Identification and chemical synthesis of the most toxic compounds in the washed cycad seeds, a variant phytosteryl glucosides, and even more toxic cholesterol β-D-glucoside (CG), which is produced by the human parasite Helicobacter pylori, provide a possibility to study in vitro the mechanisms of toxicity of these compounds. We studied in detail the effects of CG on the respiratory activities and generation of reactive oxygen species (ROS) by nonsynaptic brain and heart mitochondria oxidizing various substrates. The stimulatory effects of CG on respiration and ROS generation showed strong substrate dependence, suggesting involvement of succinate dehydrogenase (complex II). Maximal effects on ROS production were observed with 1 μmol CG/1 mg mitochondria. At this concentration the cycad toxins β-sitosterol-β-D-glucoside and stigmasterol-β-D-glucoside had effects on respiration and ROS production similar to CG. However, poor solubility precluded full concentration analysis of these toxins. Cholesterol, stigmasterol and β-sitosterol had no effect on mitochondrial functions studied at concentrations up to 100 μmol/mg protein. Our results suggest that CG may influence mitochondrial functions through changes in the packing of the bulk membrane lipids, as was shown earlier by Deliconstantinos et al. (Biochem Cell Biol 67:16-24, 1989). The neurotoxic effects of phytosteryl glucosides and CG may be associated with increased oxidative damage of neurons. Unlike heart mitochondria, in activated neurons mitochondria specifically increase ROS production associated with succinate oxidation (Panov et al., J Biol Chem 284:14448-14456, 2009).

  9. Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate.

    PubMed Central

    Kim, J J; Wang, M; Paschke, R

    1993-01-01

    The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme. Images Fig. 1 Fig. 2 Fig. 3 PMID:8356049

  10. Brain death induces the alteration of liver protein expression profiles in rabbits.

    PubMed

    Du, Bing; Li, Ling; Zhong, Zhibiao; Fan, Xiaoli; Qiao, Bingbing; He, Chongxiang; Fu, Zhen; Wang, Yanfeng; Ye, Qifa

    2014-08-01

    At present, there is no accurate method for evaluating the quality of liver transplant from a brain-dead donor. Proteomics are used to investigate the mechanisms involved in brain death‑induced liver injury and to identify sensitive biomarkers. In the present study, age‑ and gender‑matched rabbits were randomly divided into the brain death and sham groups. The sham served as the control. A brain‑death model was established using an intracranial progressive pressurized method. The differentially expressed proteins extracted from the liver tissues of rabbits that were brain‑dead for 6 h in the two groups were determined by two‑dimensional gel electrophoresis and matrix‑assisted laser desorption ionization time of flight mass spectrometry. Although there was no obvious functional and morphological difference in 2, 4 and 6 h after brain death, results of the proteomics analysis revealed 973±34 and 987±38 protein spots in the control and brain death groups, respectively. Ten proteins exhibited a ≥2‑fold alteration. The downregulated proteins were: aldehyde dehydrogenase, runt‑related transcription factor 1 (RUNX1), inorganic pyrophosphatase, glutamate‑cysteine ligase regulatory subunit and microsomal cytochrome B5. By contrast, the expression of dihydropyrimidinase-related protein 4, peroxiredoxin‑6, 3‑phosphoinositide‑dependent protein kinase‑1, 3-mercaptopyruvate and alcohol dehydrogenase were clearly upregulated. Immunohistochemistry and western blot analysis results revealed that the expression of RUNX1 was gradually increased in a time‑dependent manner in 2, 4, and 6 h after brain death. In conclusion, alteration of the liver protein expression profile induced by brain death indicated the occurrence of complex pathological changes even if no functional or morphological difference was identified. Thus, RUNX1 may be a sensitive predict factor for evaluating the quality of brain death donated liver.

  11. Purification of the high-Km aldehyde reductase from rat brain and liver and from ox brain.

    PubMed Central

    Rivett, A J; Smith, I L; Tipton, K F

    1981-01-01

    A procedure is described that yields an apparently homogeneous preparation of the high-Km aldehyde reductase from rat brain. This procedure is also applicable to the purification of this enzyme from rat liver and ox brain. In the latter case, however, the purified preparation could be resolved into two protein bands, both of which had enzyme activity, by polyacrylamide-gel electrophoresis. Since a sample of the ox brain enzyme from an earlier step in the purification procedure only showed the presence of a single band of activity after electrophoresis, this apparent multiplicity probably results from modification of the enzyme, possibly by oxidation, during the final step of the purification. A number of properties of the rat brain enzyme were determined and these were compared with those of the enzyme from rat liver. The two preparations were similar in their stabilities, behaviour during purification, kinetic properties, electrophoretic mobilities and amino acid compositions. Antibodies to the rat liver enzyme cross-reacted with that from brain and the inhibition of both these preparations by the antiserum was similar, further supporting the view that the enzymes from these two sources were closely similar if not identical. PMID:6798966

  12. Salsalate (Salicylate) Uncouples Mitochondria, Improves Glucose Homeostasis, and Reduces Liver Lipids Independent of AMPK-β1

    PubMed Central

    Smith, Brennan K.; Ford, Rebecca J.; Desjardins, Eric M.; Green, Alex E.; Hughes, Meghan C.; Houde, Vanessa P.; Day, Emily A.; Marcinko, Katarina; Crane, Justin D.; Mottillo, Emilio P.; Perry, Christopher G.R.; Kemp, Bruce E.; Tarnopolsky, Mark A.; Steinberg, Gregory R.

    2017-01-01

    Salsalate is a prodrug of salicylate that lowers blood glucose in patients with type 2 diabetes (T2D) and reduces nonalcoholic fatty liver disease (NAFLD) in animal models; however, the mechanism mediating these effects is unclear. Salicylate directly activates AMPK via the β1 subunit, but whether salsalate requires AMPK-β1 to improve T2D and NAFLD has not been examined. Therefore, wild-type (WT) and AMPK-β1–knockout (AMPK-β1KO) mice were treated with a salsalate dose resulting in clinically relevant serum salicylate concentrations (~1 mmol/L). Salsalate treatment increased VO2, lowered fasting glucose, improved glucose tolerance, and led to an ~55% reduction in liver lipid content. These effects were observed in both WT and AMPK-β1KO mice. To explain these AMPK-independent effects, we found that salicylate increases oligomycin-insensitive respiration (state 4o) and directly increases mitochondrial proton conductance at clinical concentrations. This uncoupling effect is tightly correlated with the suppression of de novo lipogenesis. Salicylate is also able to stimulate brown adipose tissue respiration independent of uncoupling protein 1. These data indicate that the primary mechanism by which salsalate improves glucose homeostasis and NAFLD is via salicylate-driven mitochondrial uncoupling. PMID:27554471

  13. Antioxidant effects of JM-20 on rat brain mitochondria and synaptosomes: mitoprotection against Ca²⁺-induced mitochondrial impairment.

    PubMed

    Nuñez-Figueredo, Yanier; Pardo-Andreu, Gilberto L; Ramírez-Sánchez, Jeney; Delgado-Hernández, René; Ochoa-Rodríguez, Estael; Verdecia-Reyes, Yamila; Naal, Zeki; Muller, Alexandre Pastoris; Portela, Luis Valmor; Souza, Diogo O

    2014-10-01

    Because mitochondrial oxidative stress and impairment are important mediators of neuronal damage in neurodegenerative diseases and in brain ischemia/reperfusion, in the present study, we evaluated the antioxidant and mitoprotective effect of a new promising neuroprotective molecule, JM-20, in mitochondria and synaptosomes isolated from rat brains. JM-20 inhibited succinate-mediated H₂O₂ generation in both mitochondria and synaptosomes incubated in depolarized (high K(+)) medium at extremely low micromolar concentration and with identical IC₅₀ values of 0.91 μM. JM-20 also repressed glucose-induced H₂O₂ generation stimulated by rotenone or by antimycin A in synaptosomes incubated in high sodium-polarized medium at extremely low IC₅₀ values of 0.395 μM and 2.452 μM, respectively. JM-20 was unable to react directly with H₂O₂ or with superoxide anion radicals but displayed a cathodic reduction peak at -0.71V, which is close to that of oxygen (-0.8V), indicating high electron affinity. JM-20 also inhibited uncoupled respiration in mitochondria or synaptosomes and was a more effective inhibitor in the presence of the respiratory substrates glutamate/malate than in the presence of succinate. JM-20 also prevented Ca(2+)-induced mitochondrial permeability transition pore opening, membrane potential dissipation and cytochrome c release, which are key pathogenic events during stroke. This molecule also prevented Ca(2+) influx into synaptosomes and mitochondria; the former effect was a consequence of the latter because JM-20 inhibition followed the patterns of carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP), which is a classic mitochondrial uncoupler. Because the mitochondrion is considered an important source and target of neuronal cell death signaling after an ischemic insult, the antioxidant and protective effects of JM-20 against the deleterious effects of Ca(2+) observed at the mitochondrial level in this study may endow this molecule

  14. Acetaminophen from liver to brain: New insights into drug pharmacological action and toxicity.

    PubMed

    Ghanem, Carolina I; Pérez, María J; Manautou, José E; Mottino, Aldo D

    2016-07-01

    Acetaminophen (APAP) is a well-known analgesic and antipyretic drug. It is considered to be safe when administered within its therapeutic range, but in cases of acute intoxication, hepatotoxicity can occur. APAP overdose is the leading cause of acute liver failure in the northern hemisphere. Historically, studies on APAP toxicity have been focused on liver, with alterations in brain function attributed to secondary effects of acute liver failure. However, in the last decade the pharmacological mechanism of APAP as a cannabinoid system modulator has been documented and some articles have reported "in situ" toxicity by APAP in brain tissue at high doses. Paradoxically, low doses of APAP have been reported to produce the opposite, neuroprotective effects. In this paper we present a comprehensive, up-to-date overview of hepatic toxicity as well as a thorough review of both toxic and beneficial effects of APAP in brain. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. An eudesman derivative from Verbesina persicifolia D.C. as a natural mild uncoupler in liver mitochondria. A new potential anti-obesity agent?

    PubMed

    Dalla Via, Lisa; García-Argáez, Aída N; Braga, Alessandra; Martínez-Vázquez, Mariano; Grancara, Silvia; Martinis, Pamela; Agostinelli, Enzo; Toninello, Antonio

    2014-01-01

    4β-cinnamoyloxy,1β,3α-dihydroxyeudesm-7,8-ene (CDE) extracted from Verbesina persicifolia induces bioenergetic collapse in rat liver mitochondria (RLM), monitored as a fall in the respiratory control index and ADP/O values. This fall in energy is accompanied by a protonophore effect and membrane potential (Δψ) collapse, demonstrating that CDE behaves as a typical uncoupling agent. However, when examining the effect of CDE in detail, we found that it acts as a "mild" uncoupler because it drops Δψ and increases respiratory state 4. The proposed mechanism is based on the interaction of CDE with membrane protein cytochrome C oxidase, which is implicated in proton permeability, and with the respiratory chain for the generation of reactive oxygen species which mediate and regulate the activity of the above membrane protein. Considering the energy collapse, "mild" uncoupling, and the fact that CDE is largely used in folk medicines, this extract may be viewed as a potentially effective anti-obesity drug and a natural lead compound for developing new natural uncouplers against obesity.

  16. The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria: electrical nature and coupling of the exchange reaction with H+ translocation.

    PubMed Central

    Indiveri, C; Tonazzi, A; Stipani, I; Palmieri, F

    1997-01-01

    The mechanism and the electrical nature of ornithine/citrulline exchange has been investigated in proteoliposomes reconstituted with the ornithine/citrulline carrier purified from rat liver mitochondria. The stoichiometry of the exchanging substrates was close to 1:1. The exchange was not affected by inducing electrogenic flux of K+ with valinomycin. In contrast, the pH gradient generated by the K+/H+ exchanger nigericin in the presence of an outwardly directed K+ gradient stimulated the ornithineout/citrullinein exchange, but not the ornithine/ornithine homoexchange. Experiments in which either the internal or the external pH was varied, while keeping constant the pH in the other compartment, indicated that maximal exchange rates are found at pH 6 in the compartment containing citrulline and at pH 8 in the compartment containing ornithine. Changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the exchanges ornithineout/citrullinein and citrullineout/ornithinein are accompanied by translocation of H+ in the same direction as citrulline. It is concluded that the mitochondrial ornithine/citrulline carrier catalyses an electroneutral exchange of ornithine+ for citrulline plus an H+. A reasonable model is one in which ornithine binds to a deprotonated carrier and citrulline to a protonated carrier and both substrate-carrier complexes are neutral. The physiological implications of this transport process are discussed. PMID:9359400

  17. The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria catalyses a second transport mode: ornithine+/H+ exchange.

    PubMed

    Indiveri, C; Tonazzi, A; Stipani, I; Palmieri, F

    1999-08-01

    The mechanism of unidirectional transport of ornithine (i.e. in the absence of a counter-metabolite) has been investigated in proteoliposomes reconstituted with the ornithine carrier purified from rat liver mitochondria. The efflux of [(3)H]ornithine from proteoliposomes was stimulated by the addition of H(+) (but not of other cations) to the incubation medium. On keeping the pH in the compartment containing ornithine constant at 8.0, the flux of ornithine into or out of the proteoliposomes increased on decreasing the pH in the opposite compartment from 8.0 to 6.0. Ornithine influx was also stimulated when a higher H(+) concentration was generated inside the vesicles relative to the outside by the K(+)/H(+) exchanger nigericin in the presence of an outwardly directed K(+) gradient. A valinomycin-induced electrogenic flux of K(+) did not affect ornithine transport in the absence of a counter-metabolite. Furthermore, changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the flux of ornithine is accompanied by translocation of H(+) in the opposite direction. It is concluded that the mitochondrial ornithine carrier catalyses an electroneutral exchange of ornithine(+) for H(+), in addition to the well-known 1:1 exchange of metabolites. Lysine(+), but not citrulline, can also be exchanged for H(+) by the ornithine carrier. The ornithine(+)/H(+) transport mode of the exchanger is an essential step in the catabolism of excess arginine.

  18. Toxicity of the Flame-Retardant BDE-49 on Brain Mitochondria and Neuronal Progenitor Striatal Cells Enhanced by a PTEN-Deficient Background

    PubMed Central

    Giulivi, Cecilia

    2013-01-01

    Polybrominated diphenyl ethers (PBDEs) represent an important group of flame retardants extensively used, tonnage of which in the environment has been steadily increasing over the past 25 years. PBDEs or metabolites can induce neurotoxicity and mitochondrial dysfunction (MD) through a variety of mechanisms. Recently, PBDEs with < 5 Br substitutions (i.e., 2,2′,4,4′-tetrabromodiphenyl ether [BDE-47] and 2,2′,4,5′-tetrabromodiphenyl ether [BDE-49]) have gained interest because of their high bioaccumulation. In particular, congeners such as BDE-49 arise as one of the most biologically active, with concentrations typically lower than those observed for BDE-47 in biological tissues; however, its potential to cause MD at biologically relevant concentrations is unknown. To this end, the effect of BDE-49 was studied in brain mitochondria and neuronal progenitor striatal cells (NPC). BDE-49 uncoupled mitochondria at concentrations < 0.1 nM, whereas at > 1 nM, it inhibited the electron transport at Complex V (mixed type inhibition; IC50 = 6 nM) and Complex IV (noncompetitive inhibition; IC50 = 40 nM). These concentrations are easily achieved in plasma concentrations considering that BDE-49 (this study, 400-fold) and other PBDEs accumulate 1–3 orders of magnitude in the cells, particularly in mitochondria and microsomes. Similar effects were observed in NPC and exacerbated with PTEN (negative modulator of the PI3K/Akt pathway) deficiency, background associated with autism-like behavior, schizophrenia, and epilepsy. PBDE-mediated MD per se or enhanced by a background that confers susceptibility to this exposure may have profound implications in the energy balance of brain. PMID:23288049

  19. Case report: brain and liver abscesses caused by oral infection with Streptococcus intermedius.

    PubMed

    Wagner, Kai Wolfgang; Schön, Ralf; Schumacher, Martin; Schmelzeisen, Rainer; Schulze, Dirk

    2006-10-01

    Organ abscesses are a rare and life-threatening complication mostly of hematogenously disseminated infections. We report a case of brain and liver abscesses. Identification of the lesions was made by contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI), respectively. An oral examination comprised an oral focus of infection. Streptococcus intermedius was isolated from oral smear, liver and ventricular drainage, and blood sample. After the commencement of antibiotic therapy, drainage of abscesses and oral rehabilitation, complete recovery was noted.

  20. Liver and brain tryptophan metabolism following hydrocortisone administration to rats and gerbils.

    PubMed

    Green, A R; Sourkes, T L; Young, S N

    1975-02-01

    1 Liver tryptophan pyrrolase activity is low in the mongolian gerbil (Meriones unguiculatus) and is not induced by hydrocortisone (5 mg/kg). In contrast, there is measurable activity in the rat liver and this is induced by hydrocortisone. In vivo measurements confirmed the absence of induction in gerbils but suggested that they were able to metabolize tryptophan. However no detectable pyrrolase activity was found in any other tissues either before or after hydrocortisone. 2 In agreement with previous observations hydrocortisone decreased rat brain 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) 6 h after administration. Brain tryptophan concentrations were also decreased at this time. In contrast, hydrocortisone did not alter gerbil brain 5-HT, 5-HIAA or trytophan. alpha-Methyltryptophan activated hepatic tryptophan pyrrolase and decreased brain 5-HT and 5-HIAA in both animals. 3 Results suggest that the decrease in rat brain 5-HT and 5-HIAA following hydrocortisone may be associated with the rise in liver tryptophan pyrrolase and that the brain amine changes are mediated through the decrease in brain tryptophan concentration.

  1. [Thiamine triphosphatase activity in mammalian mitochondria].

    PubMed

    Rusina, I M; Makarchikov, A F

    2003-01-01

    Mitochondrial preparations isolated from bovine kidney and brain as well as the liver and the brain of rat show thiamine triphosphatase (ThTPase) activity. The activity was determined from the particles by freezing-thawing suggesting that a soluble enzyme is involved. The liberation patterns of ThTPase and marker enzyme activities from mitochondria under osmotic shock or treatment with increasing Triton X-100 concentrations indicate the presence of ThTPase both in the matrix and intermembrane space. It was found, basing on gel filtration behavior, that the mitochondrial ThTPase has the same molecular mass as specific cytosolic ThTPase (EC 3.6.1.28). The enzymes, however, were clearly distinguishable in Km values, the mitochondrial one showing a higher apparent affinity for substrate. These results imply the existence of ThTPase multiple forms in mammalian cells.

  2. Anti-oxidant defences and peroxidation in liver and brain of aged rats.

    PubMed Central

    Barja de Quiroga, G; Pérez-Campo, R; López Torres, M

    1990-01-01

    Old rats (28 months), when compared with young adults (9 months), did not show differences in activities of superoxide dismutase (SOD) or selenium-dependent and -independent glutathione peroxidases (GPx), or in levels of GSH, GSSG, GSSG/GSH and endogenous peroxidation in liver and brain. Rates of stimulated peroxidation in vitro were decreased in the livers of old rats. Old animals showed decreased levels of hepatic catalase and glutathione reductase. Nevertheless, when enzyme activities were referred to cytochrome oxidase activity these decreases disappeared, and GPx and SOD (brain) were even increased in old rats. PMID:2176082

  3. Lactate administration reproduces specific brain and liver exercise-related changes.

    PubMed

    E, Lezi; Lu, Jianghua; Selfridge, J Eva; Burns, Jeffrey M; Swerdlow, Russell H

    2013-10-01

    The effects of exercise are not limited to muscle, and its ability to mitigate some chronic diseases is under study. A more complete understanding of how exercise impacts non-muscle tissues might facilitate design of clinical trials and exercise mimetics. Here, we focused on lactate's ability to mediate changes in liver and brain bioenergetic-associated parameters. In one group of experiments, C57BL/6 mice underwent 7 weeks of treadmill exercise sessions at intensities intended to exceed the lactate threshold. Over time, the mice dramatically increased their lactate threshold. To ensure that plasma lactate accumulated during the final week, the mice were run to exhaustion. In the liver, mRNA levels of gluconeogenesis-promoting genes increased. While peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α) expression increased, there was a decrease in PGC-1β expression, and overall gene expression changes favored respiratory chain down-regulation. In the brain, PGC-1α and PGC-1β were unchanged, but PGC-1-related co-activator expression and mitochondrial DNA copy number increased. Brain tumor necrosis factor alpha expression fell, whereas vascular endothelial growth factor A expression rose. In another group of experiments, exogenously administered lactate was found to reproduce some but not all of these observed liver and brain changes. Our data suggest that lactate, an exercise byproduct, could mediate some of the effects exercise has on the liver and the brain, and that lactate itself can act as a partial exercise mimetic.

  4. Concurrent Low Brain and High Liver Uptake on FDG PET Are Associated with Cardiovascular Risk Factors

    PubMed Central

    Nam, Hyun-Yeol; Jun, Sungmin; Pak, Kyoungjune

    2017-01-01

    Objective Concurrent low brain and high liver uptake are sometimes observed on fluorine-18-labeled fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). We investigated the potential clinical significance of this uptake pattern related to metabolic syndrome (MS). Materials and Methods We retrospectively reviewed data from 264 consecutive males who had undergone general health check-ups, including FDG PET/CT scans. After an overnight fast, the men had their peripheral blood drawn and the levels of various laboratory parameters measured; an FDG PET/CT scan was performed on the same day. We measured the maximum standardized uptake values of the brain and liver from regions of interest manually placed over the frontal cortex at the level of the centrum semiovale and the right lobe of the liver parenchyma, respectively. Results Fasting blood glucose (FBG; odds ratio [OR] = 1.063, p < 0.001) and glycated hemoglobin (HbA1c; OR = 3.634, p = 0.010) were the strongest predictive factors for low brain FDG uptake, whereas waist circumference (OR = 1.200, p < 0.001) and γ-glutamyl transpeptidase (OR = 1.012, p = 0.001) were the strongest predictive factors for high liver uptake. Eleven subjects (4.2%) showed concurrent low brain and high liver FDG uptake, and all but one of these subjects (90.9%) had MS. Systolic blood pressure, waist circumference, FBG, triglyceride, alanine aminotransferase, insulin resistance (measured by homeostasis model assessment), insulin, HbA1c, and body mass index were higher in subjects with this FDG uptake pattern than in those without (all, p < 0.001). Conclusion Concurrent low brain and high liver FDG uptake were closely associated with MS. Moreover, subjects with this pattern had higher values for various cardiovascular risk factors than did those without. PMID:28246520

  5. Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease.

    PubMed

    Sunny, Nishanth E; Kalavalapalli, Srilaxmi; Bril, Fernando; Garrett, Timothy J; Nautiyal, Manisha; Mathew, Justin T; Williams, Caroline M; Cusi, Kenneth

    2015-08-15

    Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD.

  6. Cross-talk between branched-chain amino acids and hepatic mitochondria is compromised in nonalcoholic fatty liver disease

    PubMed Central

    Kalavalapalli, Srilaxmi; Bril, Fernando; Garrett, Timothy J.; Nautiyal, Manisha; Mathew, Justin T.; Williams, Caroline M.; Cusi, Kenneth

    2015-01-01

    Elevated plasma branched-chain amino acids (BCAA) in the setting of insulin resistance have been relevant in predicting type 2 diabetes mellitus (T2DM) onset, but their role in the etiology of hepatic insulin resistance remains uncertain. We determined the link between BCAA and dysfunctional hepatic tricarboxylic acid (TCA) cycle, which is a central feature of hepatic insulin resistance and nonalcoholic fatty liver disease (NAFLD). Plasma metabolites under basal fasting and euglycemic hyperinsulinemic clamps (insulin stimulation) were measured in 94 human subjects with varying degrees of insulin sensitivity to identify their relationships with insulin resistance. Furthermore, the impact of elevated BCAA on hepatic TCA cycle was determined in a diet-induced mouse model of NAFLD, utilizing targeted metabolomics and nuclear magnetic resonance (NMR)-based metabolic flux analysis. Insulin stimulation revealed robust relationships between human plasma BCAA and indices of insulin resistance, indicating chronic metabolic overload from BCAA. Human plasma BCAA and long-chain acylcarnitines also showed a positive correlation, suggesting modulation of mitochondrial metabolism by BCAA. Concurrently, mice with NAFLD failed to optimally induce hepatic mTORC1, plasma ketones, and hepatic long-chain acylcarnitines, following acute elevation of plasma BCAA. Furthermore, elevated BCAA failed to induce multiple fluxes through hepatic TCA cycle in mice with NAFLD. Our data suggest that BCAA are essential to mediate efficient channeling of carbon substrates for oxidation through mitochondrial TCA cycle. Impairment of BCAA-mediated upregulation of the TCA cycle could be a significant contributor to mitochondrial dysfunction in NAFLD. PMID:26058864

  7. [Study on corresponding areas the liver and lung channels in brain with fMRI].

    PubMed

    Xu, Fang-Ming; Xie, Peng; Lü, Fa-Jin; Mou, Jun; Li, Yong-Mei; Zhao, Jian-Nong; Chen, Wei-Juan; Gong, Qi-Yong; Zhao, Li-Bo; Liu, Qing-Jun; Shen, Lin; Zhai, Hong; Yang, De-Yu

    2007-10-01

    To explore distribution of the Liver and Lung Channels in the brain so as to provide imaging basis for construction of channel theory in the brain. Sixty healthy student volunteers were randomly divided into a Liver Channel group (I) and a Lung Channel group (II), and the each group was further divided into five subgroups with 6 volunteers in each subgroup, based on five-shu-point principles which, were Dadun (LR 1, I 1), Xingjian (LR 2, I 2), Taichong (LR 3, I 3), Zhongfeng (LR 4, I 4), Ququan (LR 8, I 5), Shaoshang (LU 11, II 1), Yuji (LU 10, II 2), Taiyuan (LU 9, II 3), Jingqu (LU 8, II 4), and Chize (LU 5, II 5), respectively. In order to observe the brain activating patterns during acupuncture at the different acupoints, functional magnetic resonance imaging (fMRI) technique was adopted. All image data were then analyzed with SPM 2 software. The statistical parameter gram was composed of the pixel P < 0.01, and anatomic location was made according to Talairach coordinate, attaining experimentally activated areas, and the commonly activated area of five-shu-point of each channel was considered as the brain distribution of the Liver and Lung Channels. The common areas activated by the five-shu-points of the Liver Channel were homolateral Brodmann area (BA) 34, BA 47, red nucleus, contralateral BA 19, BA 30, BA 39, the superior parietal lobule, cerebellum decline, and bilateral BA 3 and culmen. The common areas activated by the five-shu-points of the Lung Channels included homolateral BA 2, BA 18, BA 35, and contralateral BA 9 and substania nigra. There are relatively specific corresponding brain areas for the Liver and Lung Channels, indicating that there is possible relatively specific connection between channels and the brain.

  8. Melatonin Alleviates Intracerebral Hemorrhage-Induced Secondary Brain Injury in Rats via Suppressing Apoptosis, Inflammation, Oxidative Stress, DNA Damage, and Mitochondria Injury.

    PubMed

    Wang, Zhong; Zhou, Feng; Dou, Yang; Tian, Xiaodi; Liu, Chenglin; Li, Haiying; Shen, Haitao; Chen, Gang

    2017-08-01

    Intracerebral hemorrhage (ICH) is a cerebrovascular disease with high mortality and morbidity, and the effective treatment is still lacking. We designed this study to investigate the therapeutic effects and mechanisms of melatonin on the secondary brain injury (SBI) after ICH. An in vivo ICH model was induced via autologous whole blood injection into the right basal ganglia in Sprague-Dawley (SD) rats. Primary rat cortical neurons were treated with oxygen hemoglobin (OxyHb) as an in vitro ICH model. The results of the in vivo study showed that melatonin alleviated severe brain edema and behavior disorders induced by ICH. Indicators of blood-brain barrier (BBB) integrity, DNA damage, inflammation, oxidative stress, apoptosis, and mitochondria damage showed a significant increase after ICH, while melatonin reduced their levels. Meanwhile, melatonin promoted further increasing of expression levels of antioxidant indicators induced by ICH. Microscopically, TUNEL and Nissl staining showed that melatonin reduced the numbers of ICH-induced apoptotic cells. Inflammation and DNA damage indicators exhibited an identical pattern compared to those above. Additionally, the in vitro study demonstrated that melatonin reduced the apoptotic neurons induced by OxyHb and protected the mitochondrial membrane potential. Collectively, our investigation showed that melatonin ameliorated ICH-induced SBI by impacting apoptosis, inflammation, oxidative stress, DNA damage, brain edema, and BBB damage and reducing mitochondrial membrane permeability transition pore opening, and melatonin may be a potential therapeutic agent of ICH.

  9. Morpho-pathological and physiological changes of the brain and liver after ozone exposure.

    PubMed

    Creţu, Denisa Ioana; Sovrea, Alina; Ignat, R M; Filip, Adriana; Bidian, Cristina; Creţu, Aurica

    2010-01-01

    This paper presents the consequences of long exposure to ozone in order to draw attention to this matter as far as the brain and liver are concerned. The material used was represented by two batches of 10 rats each that were daily exposed to ozone for 10 minutes at 0.5 ppm O3. From the first group blood was collected after two weeks to determine the indicators of oxidative stress and samples of brain and liver were drawn for histological studies. Tissue changes were highlighted using Hematoxylin-Eosin and argentic impregnation. In addition, the brain and liver samples taken from study subjects were turned into homogeneous preparations in order to determine the intensity of oxidative stress occurred in these organs compared with the witness group. The second batch was exposed for a further two weeks, after which the same sampling techniques and determining methods as for the first group were applied. The results show a correlation between the values of malondialdehyde (MDA) and glutathione (GSH), obtained both in blood and in the homogeneous preparations, and the microscopic changes that implicate a pathological state. Therefore, cerebral edema was discovered in the brain hemispheres and the cerebellum indicating necrotic signs accompanied by a reduction in the molecular layer and Purkinje cells with pale core. The liver presented hepatocellular necrosis, extended from the port area to the centrolobular vein.

  10. Brain and liver fatty acid composition changes upon consumption of Lactobacillus rhamnosus LA68.

    PubMed

    Ivanovic, Nevena; Minic, Rajna; Djuricic, Ivana; Dimitrijevic, Ljiljana; Sobajic, Sladjana; Zivkovic, Irena; Djordjevic, Brizita

    2015-02-01

    Recent reports suggest that the metabolic activity of the enteric microbiota may influence the fatty acid composition of the host tissue. There are many studies dealing with the influence of lactobacilli on various pathological conditions, and some of the effects are strain-specific. This study was designed to test the effects of a particular Lactobacillus strain, Lactobacillus rhamnosus LA68 on fatty acid composition of the liver and the brain of C57BL/6 mice in the absence of an underlying pathological condition. Female mice were supplemented with live L. rhamnosus LA68 bacteria for the duration of 1 month. Serum biochemistry was analyzed and liver and brain fatty acid composition was assessed by gas-liquid chromatography. Significant changes in liver and brain fatty acid composition were detected. In the liver tissue we detected an increase in palmitoleic acid (p = 0.038), while in the brain compartment we found an increase in palmitic (p = 0.042), stearic (p = 0.017), arachidonic acid (p = 0.009) and docosahexaenoic acid (p = 0.004) for control versus experimental group. These results show discrete changes caused by LA68 strain consumption. Even short duration of administration of LA68 influences the fatty acid composition of the host which adds to the existing knowledge about Lactobacillus host interaction, and adds to the growing knowledge of metabolic intervention possibilities.

  11. Converging actions of alcohol on liver and brain immune signaling.

    PubMed

    Szabo, Gyongyi; Lippai, Dora

    2014-01-01

    Chronic excessive alcohol consumption results in inflammation in multiple organs, including the brain. While the contribution of neuroinflammation to alcohol-related cognitive dysfunction and behavioral alterations is established, the mechanisms by which alcohol triggers inflammation in the brain are only partially understood. There are acute and long-term alterations in brain function due to intercellular and intracellular changes of different cell types as a result of alcohol consumption. This review focuses on the alcohol-induced proinflammatory cellular and molecular changes in the central nervous system. Alcohol passes through the blood-brain barrier and alters neurotransmission. Alcohol use activates microglia and astrocyte, contributing to neurodegeneration and impaired regeneration. Alcohol-induced cell injury in the brain results in release of damage-associated molecular patterns, such as high mobility group box 1, that trigger inflammatory changes through activation of pattern recognition receptors. In addition, alcohol consumption increases intestinal permeability and results in increased levels of pathogen-associated molecular pattern such as endotoxin in the systemic circulation that triggers PRRs and inflammation. The Toll-like receptor-4 pathway that activates nuclear factor-κB and secretion of proinflammatory cytokines, tumor necrosis factor-α, interleukin-1-beta, and chemokines, including monocyte chemotactic protein-1, has been suggested to contribute to alcohol-induced neuroinflammation. Alcohol-induced IL-1β secretion also requires Nod-like receptor-mediated inflammasome and caspase-1 activation, and, consistent with this, disruption of IL-1/IL-1-receptor signaling prevents alcohol-induced neuroinflammation. Delicate regulators of inflammatory gene expressions are micro-RNAs (miRs) that have recently been identified in alcohol-related neuroinflammation. Alcohol induces miR155, a regulator of inflammation in the brain, and deficiency in mi

  12. Changes relevant to catecholamine metabolism in liver and brain of ascorbic acid deficient guinea-pigs.

    PubMed

    Deana, R; Bharaj, B S; Verjee, Z H; Galzigna, L

    1975-01-01

    A chronic deficiency of ascorbic acid was induced in guinea pig. The level of catecholamines, copper and the activities of ceruplasmin, catecholamine oxidase, monoamineoxidase and acetylcholinesterase were checked in brain, liver and serum. Also the levels of ascorbic acid and glutathione were measured in the organs of ascorbic acid-deficient animals. The most important changes due to the ascorbic acid deficiency were observed in the brain were monoamineoxidase, catecholamineoxidase, acetylcholinesterase and the concentration of catecholamines were altered. The statement that brain is the organ most affected by the ascorbic acid deficiency is discussed.

  13. Detection of Labile Low-Molecular-Mass Transition Metal Complexes in Mitochondria

    PubMed Central

    McCormick, Sean P.; Moore, Michael J.; Lindahl, Paul A.

    2015-01-01

    Liquid chromatography was used with an on-line inductively coupled plasma mass spectrometer to detect low-molecular-mass (LMM) transition metal complexes in mitochondria isolated from fermenting yeast cells, human Jurkat cells, and mouse brain and liver. These complexes constituted 20 – 40% of total mitochondrial Mn, Fe, Zn, and Cu ions. The major LMM Mn complex in yeast mitochondria had a mass of ca. 1100 Da and a concentration of ~ 2 μM. Mammalian mitochondria contained a second Mn species with a mass of ca. 2000 Da at a comparable concentration. The major Fe complex in mitochondria isolated from exponentially growing yeast cells had a mass of ca. 580 Da; the concentration of Fe580 in mitochondria was ca. 100 μM. When mitochondria were isolated from fermenting cells in post-exponential phase, the mass of the dominant LMM Fe complex was ca. 1100 Da. Upon incubation, the intensity of Fe1100 declined and Fe580 increased, suggesting that the two are interrelated. Mammalian mitochondria contained Fe580 and 2 other Fe species (Fe2000 and Fe1100) at concentrations of ca. 50 μM each. The dominant LMM Zn species in mitochondria had a mass of ca. 1200 Da and a concentration of ca. 110 μM. Mammalian mitochondria contained a second major LMM Zn species at 1500 Da. The dominant LMM Cu species in yeast mitochondria had a mass of ca. 5000 Da and a concentration in yeast mitochondria of ca. 16 μM; Cu5000 was not observed in mammalian mitochondria. The dominant Co species in mitochondria, Co1200, had a concentration of 20 nM and was probably a cobalamin. Mammalian but not yeast mitochondria contained a LMM Mo species, Mo730, at ca. 1 μM concentration. Increasing Mn, Fe, Cu, and Zn concentrations 10 fold in the medium increased the concentration of the same element in the corresponding isolated mitochondria. Treatment with metal chelators confirmed that these LMM species were labile. The dominant S species at 1100 Da was not free GSH or GSSG. PMID:26018429

  14. Distribution of labeled products from (1-/sup 14/C), (U-/sup 14/C) and (16-/sup 14/C)-palmitate in isolated rat hepatocytes and liver mitochondria

    SciTech Connect

    Chatzidakis, C.; Otto, D.A.

    1986-05-01

    Fatty acids (FA) labeled in different carbon positions are used to study the distribution of labeled oxidation products. With rat hepatocytes (Hep) the authors observed significant differences in the distribution of label into products from (1-/sup 14/C) and (U-/sup 14/C)-palmitate (P). The total recovery of label in products (/sup 14/CO/sub 2/ + acid soluble fraction (ASF)) was identical between the two labeled FA. However, /sup 14/CO/sub 2/ production from (U-/sup 14/C)-P was only 40% of that from (1-/sup 14/C)-P. A recent report showed that approximately = 95% of succinate (Suc) utilized by Hep does not complete one full turn through the citric acid cycle. The authors observed that /sup 14/CO/sub 2/ evolution from (2,3-/sup 14/C)-Suc was approximately = 9% of that from (1,4-/sup 14/C)-Suc, indicating that the differences in label distribution between (1-/sup 14/C) and (U-/sup 14/C)-P are partially due to less /sup 14/CO/sub 2/ production from label in the even carbon positions of the FA with consequently more label remaining in the ASF. The /sup 14/CO/sub 2/ production from (16-/sup 14/C)-P was only 4% of that from (1-/sup 14/C)-P a value less than expected from the Suc experiments. Ketone bodies (KB) comprised 78% of total labeled products from (16-/sup 14/C)-P as compared to 28% from (1-/sup 14/C)-P and 41% from (U-/sup 14/C)-P, giving support to the previously reported preferential use of the omega-C/sub 2/ unit for KB synthesis without entry into the acetyl-CoA pool. Studies with isolated rat liver mitochondria gave results similar to those with Hep, indicating minimal involvement of perioxisomal ..beta..-oxidation.

  15. Citric Acid Effects on Brain and Liver Oxidative Stress in Lipopolysaccharide-Treated Mice

    PubMed Central

    Youness, Eman R.; Mohammed, Nadia A.; Morsy, Safaa M. Youssef; Omara, Enayat A.; Sleem, Amany A.

    2014-01-01

    Abstract Citric acid is a weak organic acid found in the greatest amounts in citrus fruits. This study examined the effect of citric acid on endotoxin-induced oxidative stress of the brain and liver. Mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 μg/kg). Citric acid was given orally at 1, 2, or 4 g/kg at time of endotoxin injection and mice were euthanized 4 h later. LPS induced oxidative stress in the brain and liver tissue, resulting in marked increase in lipid peroxidation (malondialdehyde [MDA]) and nitrite, while significantly decreasing reduced glutathione, glutathione peroxidase (GPx), and paraoxonase 1 (PON1) activity. Tumor necrosis factor-alpha (TNF-α) showed a pronounced increase in brain tissue after endotoxin injection. The administration of citric acid (1–2 g/kg) attenuated LPS-induced elevations in brain MDA, nitrite, TNF-α, GPx, and PON1 activity. In the liver, nitrite was decreased by 1 g/kg citric acid. GPx activity was increased, while PON1 activity was decreased by citric acid. The LPS-induced liver injury, DNA fragmentation, serum transaminase elevations, caspase-3, and inducible nitric oxide synthase expression were attenuated by 1–2 g/kg citric acid. DNA fragmentation, however, increased after 4 g/kg citric acid. Thus in this model of systemic inflammation, citric acid (1–2 g/kg) decreased brain lipid peroxidation and inflammation, liver damage, and DNA fragmentation. PMID:24433072

  16. Citric acid effects on brain and liver oxidative stress in lipopolysaccharide-treated mice.

    PubMed

    Abdel-Salam, Omar M E; Youness, Eman R; Mohammed, Nadia A; Morsy, Safaa M Youssef; Omara, Enayat A; Sleem, Amany A

    2014-05-01

    Citric acid is a weak organic acid found in the greatest amounts in citrus fruits. This study examined the effect of citric acid on endotoxin-induced oxidative stress of the brain and liver. Mice were challenged with a single intraperitoneal dose of lipopolysaccharide (LPS; 200 μg/kg). Citric acid was given orally at 1, 2, or 4 g/kg at time of endotoxin injection and mice were euthanized 4 h later. LPS induced oxidative stress in the brain and liver tissue, resulting in marked increase in lipid peroxidation (malondialdehyde [MDA]) and nitrite, while significantly decreasing reduced glutathione, glutathione peroxidase (GPx), and paraoxonase 1 (PON1) activity. Tumor necrosis factor-alpha (TNF-α) showed a pronounced increase in brain tissue after endotoxin injection. The administration of citric acid (1-2 g/kg) attenuated LPS-induced elevations in brain MDA, nitrite, TNF-α, GPx, and PON1 activity. In the liver, nitrite was decreased by 1 g/kg citric acid. GPx activity was increased, while PON1 activity was decreased by citric acid. The LPS-induced liver injury, DNA fragmentation, serum transaminase elevations, caspase-3, and inducible nitric oxide synthase expression were attenuated by 1-2 g/kg citric acid. DNA fragmentation, however, increased after 4 g/kg citric acid. Thus in this model of systemic inflammation, citric acid (1-2 g/kg) decreased brain lipid peroxidation and inflammation, liver damage, and DNA fragmentation.

  17. High resolution proton magnetic resonance spectroscopy of human brain and liver

    SciTech Connect

    Barany, M.; Spigos, D.G.; Mok, E.; Venkatasubramanian, P.N.; Wilbur, A.C.; Langer, B.G.

    1987-01-01

    Water-suppressed and slice-selective proton spectra of live human brain exhibited several resonances that were tentatively assigned to metabolites such as N-acetylaspartate, glutamate, phosphocreatine and creatine, choline derivatives, and taurine. In the liver spectrum of a healthy volunteer, the major resonance was tentatively assigned to a fatty acyl methylene and the minor resonances to protons in carnitine, taurine, glutamate, and glutamine. In the spectrum of a cancerous liver, resonances in addition to those present in the normal liver were seen. Protein degradation in the liver with cancer was indicated by resonances from urea and from the ring protons in tryptophan, tyrosine, and phenylalanine. Furthermore, increased nucleic acid synthesis was indicated by resonances from nucleotide protons.

  18. Pituitary and Brain Dopamine D2 Receptors Regulate Liver Gene Sexual Dimorphism

    PubMed Central

    Ramirez, Maria Cecilia; Ornstein, Ana Maria; Luque, Guillermina Maria; Perez Millan, Maria Ines; Garcia-Tornadu, Isabel; Rubinstein, Marcelo

    2015-01-01

    Liver sexual gene dimorphism, which depends mainly on specific patterns of GH secretion, may underlie differential susceptibility to some liver diseases. Because GH and prolactin secretion are regulated by dopaminergic pathways, we studied the participation of brain and lactotrope dopamine 2 receptors (D2Rs) on liver gene sexual dimorphism, to explore a link between the brain and liver gene expression. We used global D2R knockout mice (Drd2−/−) and conducted a functional dissection strategy based on cell-specific Drd2 inactivation in neurons (neuroDrd2KO) or pituitary lactotropes. Disruption of neuronal D2Rs (which impaired the GH axis) decreased most of male or female-predominant class I liver genes and increased female–predominant class II genes in males, consistent with the positive (class I) or negative (class II) regulation of these genes by GH. Notably, sexual dimorphism was lost for class I and II genes in neuroDrd2KO mice. Disruption of lactotrope D2Rs did not modify class I or II genes in either sex, because GH axis was preserved. But surprisingly, 1 class II gene (Prlr) and female-predominant class I genes were markedly up-regulated in lacDrd2KO females, pointing to direct or indirect effects of prolactin in the regulation of selected female-predominant liver genes. This suggestion was strengthened in the hyperprolactinemic Drd2−/− female mouse, in which increased expression of the same 4 liver genes was observed, despite a decreased GH axis. We hereby demonstrate endocrine-mediated D2R actions on sexual dimorphic liver gene expression, which may be relevant during chronic dopaminergic medications in psychiatric disease. PMID:25545383

  19. Pituitary and brain dopamine D2 receptors regulate liver gene sexual dimorphism.

    PubMed

    Ramirez, Maria Cecilia; Ornstein, Ana Maria; Luque, Guillermina Maria; Perez Millan, Maria Ines; Garcia-Tornadu, Isabel; Rubinstein, Marcelo; Becu-Villalobos, Damasia

    2015-03-01

    Liver sexual gene dimorphism, which depends mainly on specific patterns of GH secretion, may underlie differential susceptibility to some liver diseases. Because GH and prolactin secretion are regulated by dopaminergic pathways, we studied the participation of brain and lactotrope dopamine 2 receptors (D2Rs) on liver gene sexual dimorphism, to explore a link between the brain and liver gene expression. We used global D2R knockout mice (Drd2(-/-)) and conducted a functional dissection strategy based on cell-specific Drd2 inactivation in neurons (neuroDrd2KO) or pituitary lactotropes. Disruption of neuronal D2Rs (which impaired the GH axis) decreased most of male or female-predominant class I liver genes and increased female-predominant class II genes in males, consistent with the positive (class I) or negative (class II) regulation of these genes by GH. Notably, sexual dimorphism was lost for class I and II genes in neuroDrd2KO mice. Disruption of lactotrope D2Rs did not modify class I or II genes in either sex, because GH axis was preserved. But surprisingly, 1 class II gene (Prlr) and female-predominant class I genes were markedly up-regulated in lacDrd2KO females, pointing to direct or indirect effects of prolactin in the regulation of selected female-predominant liver genes. This suggestion was strengthened in the hyperprolactinemic Drd2(-/-) female mouse, in which increased expression of the same 4 liver genes was observed, despite a decreased GH axis. We hereby demonstrate endocrine-mediated D2R actions on sexual dimorphic liver gene expression, which may be relevant during chronic dopaminergic medications in psychiatric disease.

  20. Effects of electroacupuncture of different intensities on energy metabolism of mitochondria of brain cells in rats with cerebral ischemia-reperfusion injury.

    PubMed

    Tian, Wei-qian; Peng, Yong G; Cui, Su-yang; Yao, Feng-zhen; Li, Bao-gui

    2015-08-01

    To observe the effects of electroacupuncture (EA) of different intensities on lactate dehydrogernase (LDH), succinate dehydrogenase (SDH) and ATPase in brain tissue of rats with cerebral ischemia-reperfusion injury (CI/R). Forty male SD rats were uniformly randomized into sham operation group (group A), CI/R group (group B), CI/R+5 mA EA (group C), CI/R+3 mA EA (group D) and CI/R+1 mA EA (group E) groups with eight rats in each group. Transient general brain ischemia was induced by four-vessel occlusion and reperfusion. The rats in group C, group D and group E were punctured and stimulated at Baihui (GV20), Mingmen (GV4) and Zusanli (ST36) with the same intermittent and rarefaction-dense wave (30 to 50 Hz) and different electric current intensities: 5 mA, 3 mA and 1 mA for 20 min after CI/R. Then the activities of Na(+)-K(+)-ATPase, SDH and LDH in mitochondria of brain tissue were measured by spectrophotometry. The ischemic cerebral cortex tissue was taken for observing the ultrastructure changes of impaired nerve cells. Compared with group A, the activities of LDH, SDH and Na(+)-K(+)-ATPase were lowerer in the group B (P<0.05 or P<0.01). However, the activities of LDH, SDH and Na(+)-K(+)-ATPase were higher in the group D than those in the group B (P<0.05 orP<0.01). In group A, the anatomical structure of the cerebral cortex cells was basically normal; in group B, the neuronal cellular structures were severely damaged, the neuronal mitochondria got swelling, the mitochondrial cristae were broken, the medullated nerve fifibers were not integrated. In group C, group D and group E, the ultrastructure of impaired neuron were improved. Group D was the best among three groups above. EA of 3 mA intensity could strengthen aerobic metabolism by elevating the activities of SDH and LDH, meanwhile maintaining the ionic equilibrium in the exterior and interior brain cell and relieving the cellular edema by reinforcing the activities of Na(+)-K(+)-ATPase.

  1. Waterborne manganese exposure alters plasma, brain, and liver metabolites accompanied by changes in stereotypic behaviors

    PubMed Central

    Fordahl, Steve; Cooney, Paula; Qiu, Yunping; Xie, Guoxiang; Jia, Wei; Erikson, Keith M.

    2011-01-01

    Overexposure to waterborne manganese (Mn) is linked with cognitive impairment in children and neurochemical abnormalities in other experimental models. In order to characterize the threshold between Mn-exposure and altered neurochemistry, it is important to identify biomarkers that positively correspond with brain Mn-accumulation. The objective of this study was to identify Mn-induced alterations in plasma, liver, and brain metabolites using liquid/gas chromatography-time of flight-mass spectrometry metabolomic analyses; and to monitor corresponding Mn-induced behavior changes. Weanling Sprague-Dawley rats had access to deionized drinking water either Mn-free or containing 1g Mn/L for six weeks. Behaviors were monitored during the sixth week for a continuous 24h period while in a home cage environment using video surveillance. Mn-exposure significantly increased liver, plasma, and brain Mn concentrations compared to control, specifically targeting the globus pallidus (GP). Mn significantly altered 98 metabolites in the brain, liver, and plasma; notably shifting cholesterol and fatty acid metabolism in the brain (increased oleic and palmitic acid; 12.57 and 15.48 fold change (FC), respectively), and liver (increased oleic acid, 14.51 FC; decreased hydroxybutyric acid, −14.29 FC). Additionally, Mn-altered plasma metabolites homogentisic acid, chenodeoxycholic acid, and aspartic acid correlated significantly with GP and striatal Mn. Total distance traveled was significantly increased and positively correlated with Mn-exposure, while nocturnal stereotypic and exploratory behaviors were reduced with Mn-exposure and performed largely during the light cycle compared to unexposed rats. These data provide putative biomarkers for Mn-neurotoxicity and suggest that Mn disrupts the circadian cycle in rats. PMID:22056924

  2. Inhibition of rat brain tryptophan metabolism by ethanol withdrawal and possible involvement of the enhanced liver tryptophan pyrrolase activity.

    PubMed Central

    Badawy, A A; Punjani, N F; Evans, C M; Evans, M

    1980-01-01

    1. Chronic ethanol administration to rats was previously shown to enhance brain 5-hydroxytryptamine synthesis by increasing the availability of circulating tryptophan to the brain secondarily to the NAD(P)H-mediated inhibition of liver tryptophan pyrrolase activity. 2. At 24h after ethanol withdrawal, all the above effects were observed because liver [NAD(P)H] was still increased. By contrast, all aspects of liver and brain tryptophan metabolism were normal at 12 days after withdrawal. 3. At 7--9 days after withdrawal, brain 5-hydroxytryptamine synthesis was decreased, as was tryptophan availability to the brain. Liver tryptophan pyrrolase activity at these time-intervals was maximally enhanced. 4. Administration of nicotinamide during the withdrawal phase not only abolished the withdrawal-induced enhancement of tryptophan pyrrolase activity on day 8, but also maintained the inhibition previously caused by ethanol. Under these conditions, the withdrawal-induced decreases in brain 5-hydroxytryptamine synthesis and tryptophan availability to the brain were abolished, and both functions were enhanced. Nicotinamide alone exerted similar effects in control rats. 5. It is suggested that ethanol withdrawal inhibits brain 5-hydroxytryptamine synthesis by decreasing tryptophan availability to the brain secondarily to the enhanced liver tryptophan pyrrolase activity. 6. The results are discussed in relation to the possible involvement of 5-hydroxytryptamine in dependence on ethanol and other drugs. PMID:7195200