Targeted mitochondrial uncoupling beyond UCP1 - The fine line between death and metabolic health.

PubMed

Ost, Mario; Keipert, Susanne; Klaus, Susanne

2017-03-01

In the early 1930s, the chemical uncoupling agent 2,4-dinitrophenol (DNP) was promoted for the very first time as a powerful and effective weight loss pill but quickly withdrawn from the market due to its lack of tissue-selectivity with resulting dangerous side effects, including hyperthermia and death. Today, novel mitochondria- or tissue-targeted chemical uncouplers with higher safety and therapeutic values are under investigation in order to tackle obesity, diabetes and fatty liver disease. Moreover, in the past 20 years, transgenic mouse models were generated to understand the molecular and metabolic consequences of targeted uncoupling, expressing functional uncoupling protein 1 (UCP1) ectopically in white adipose tissue or skeletal muscle. Similar to the action of chemical mitochondrial uncouplers, UCP1 protein dissipates the proton gradient across the inner mitochondrial membrane, thus allowing maximum activity of the respiratory chain and compensatory increase in oxygen consumption, uncoupled from ATP synthesis. Consequently, targeted mitochondrial uncoupling in adipose tissue and skeletal muscle of UCP1-transgenic mice increased substrate metabolism and ameliorates obesity, hypertriglyceridemia and insulin resistance. Further, muscle-specific decrease in mitochondrial efficiency promotes a cell-autonomous and cell-non-autonomous adaptive metabolic remodeling with increased oxidative stress tolerance. This review provides an overview of novel chemical uncouplers as well as the metabolic consequences and adaptive processes of targeted mitochondrial uncoupling on metabolic health and survival. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Measuring mitochondrial uncoupling protein-2 level and activity in insulinoma cells.

PubMed

Barlow, Jonathan; Hirschberg, Verena; Brand, Martin D; Affourtit, Charles

2013-01-01

Mitochondrial uncoupling protein-2 (UCP2) regulates glucose-stimulated insulin secretion (GSIS) by pancreatic beta cells-the physiological role of the beta cell UCP2 remains a subject of debate. Experimental studies informing this debate benefit from reliable measurements of UCP2 protein level and activity. In this chapter, we describe how UCP2 protein can be detected in INS-1 insulinoma cells and how it can be knocked down by RNA interference. We demonstrate briefly that UCP2 knockdown lowers glucose-induced rises in mitochondrial respiratory activity, coupling efficiency of oxidative phosphorylation, levels of mitochondrial reactive oxygen species, and insulin secretion. We provide protocols for the detection of the respective UCP2 phenotypes, which are indirect, but invaluable measures of UCP2 activity. We also introduce a convenient method to normalize cellular respiration to cell density allowing measurement of UCP2 effects on specific mitochondrial oxygen consumption. Copyright © 2013 Elsevier Inc. All rights reserved.

Large enhancement of skeletal muscle cell glucose uptake and suppression of hepatocyte glucose-6-phosphatase activity by weak uncouplers of oxidative phosphorylation.

PubMed

Martineau, Louis C

2012-02-01

Perturbation of energy homeostasis in skeletal muscle and liver resulting from a transient inhibition of mitochondrial energy transduction can produce effects of relevance for the control of hyperglycemia through activation of the AMP-activated protein kinase, as exemplified by the antidiabetic drug metformin. The present study focuses on uncoupling of oxidative phosphorylation rather than its inhibition as a trigger for such effects. The reference weak uncoupler 2,4-dinitrophenol, fourteen naturally-occurring phenolic compounds identified as uncouplers in isolated rat liver mitochondria, and fourteen related compounds with little or no uncoupling activity were tested for enhancement of glucose uptake in differentiated C2C12 skeletal muscle cells following 18 h of treatment at 25-100 μM. A subset of compounds were tested for suppression of glucose-6-phosphatase (G6Pase) activity in H4IIE hepatocytes following 16 h at 12.5-25 μM. Metformin (400 μM) was used as a standard in both assays. Dinitrophenol and nine of eleven compounds that induced 50% or more uncoupling at 100 μM in isolated mitochondria enhanced basal glucose uptake by 53 to 269%; the effect of the 4'-hydroxychalcone butein was more than 6-fold that of metformin; negative control compounds increased uptake by no more than 25%. Dinitrophenol and four 4'-hydroxychalconoids also suppressed hepatocyte G6Pase as well as, or more effectively than metformin, whereas the unsubstituted parent compound chalcone, devoid of uncoupling activity, had no effect. Activities key to glycemic control can be induced by a wide range of weak uncouplers, including compounds free of difficult-to-metabolize groups typically associated with uncouplers. Uncoupling represents a valid and possibly more efficient alternative to inhibition for triggering cytoprotective effects of therapeutic relevance to insulin resistance in both muscle and liver. Identification of actives of natural origin and the insights into their structure-activity relationship reported herein may lead to alternatives to metformin. Copyright © 2011 Elsevier B.V. All rights reserved.

Hypercholesterolemia-induced erectile dysfunction: endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase

PubMed Central

Musicki, Biljana; Liu, Tongyun; Lagoda, Gwen A.; Strong, Travis D.; Sezen, Sena F.; Johnson, Justin M.; Burnett, Arthur L.

2010-01-01

INTRODUCTION Hypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood. AIMS We evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED. METHODS Low-density-lipoprotein receptor (LDLR)–null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67phox, p47phox, and gp91phox), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes. MAIN OUTCOME MEASURES Molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED. RESULTS Erectile response was significantly (P<0.05) reduced in hypercholesterolemic LDLR-null mice compared to WT mice. Relative to WT mice, hypercholesterolemia increased (P<0.05) protein expressions of NAD(P)H oxidase subunits p67phox, p47phox and gp91phox, eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P<0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P<0.05) maximal intracavernosal pressure, and reversed (P < 0.05) the abnormalities in protein expressions of gp67phox and gp47phox, 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia. CONCLUSION Activated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED. PMID:20626609

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

PubMed Central

Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

1986-01-01

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

PubMed

Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

2014-07-01

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Uncoupling protein expression in skeletal muscle and adipose tissue in response to in vivo porcine somatotropin treatment

USDA-ARS?s Scientific Manuscript database

The uncoupling proteins are thought to be involved in waste heat production, reducing the energy efficiency of growth in animals. Previous studies have detected their presence in swine and their regulation by the endocrine system. This study attempted to determine whether the uncoupling proteins 2...

Plant uncoupling mitochondrial proteins.

PubMed

Vercesi, Aníbal Eugênio; Borecký, Jiri; Maia, Ivan de Godoy; Arruda, Paulo; Cuccovia, Iolanda Midea; Chaimovich, Hernan

2006-01-01

Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide-sensitive free fatty acid-activated H(+) flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (Deltamu(H)+) and production of reactive oxygen species.

Uncoupling protein homologs may provide a link between mitochondria, metabolism and lifespan

PubMed Central

Wolkow, Catherine A.; Iser, Wendy B.

2008-01-01

Uncoupling proteins (UCPs), which dissipate the mitochondrial proton gradient, have the ability to decouple mitochodrial respiration from ATP production. Since mitochondrial electron transport is a major source of free radical production, it is possible that UCP activity might impact free radical production. Free radicals can react with and damage cellular proteins, DNA and lipids. Accumulated damage from oxidative stress is believed to be a major contributor to cellular decline during aging. If UCP function were to impact mitochondrial free radical production, then one would expect to find a link between UCP activity and aging. This theory has recently been tested in a handful of organisms whose genomes contain UCP1 homologs. Interestingly, these experiments indicate that UCP homologs can affect lifespan, although they do not support a simple relationship between UCP activity and aging. Instead, UCP-like proteins appear to have a variety of effects on lifespan, and on pathways implicated in lifespan regulation. One possible explanation for this complex picture is that UCP homologs may have tissue-specific effects that complicate their effects on aging. Furthermore, the functional analysis of UCP1 homologs is incomplete. Thus, these proteins may perform functions in addition to, or instead of, mitochondrial uncoupling. Although these studies have not revealed a clear picture of UCP effects on aging, they have contributed to the growing knowledge base for these interesting proteins. Future biochemical and genetic investigation of UCP-like proteins will do much to clarify their functions and to identify the regulatory networks in which they are involved. PMID:16707280

Glutathionylation state of uncoupling protein-2 and the control of glucose-stimulated insulin secretion.

PubMed

Mailloux, Ryan J; Fu, Accalia; Robson-Doucette, Christine; Allister, Emma M; Wheeler, Michael B; Screaton, Robert; Harper, Mary-Ellen

2012-11-16

The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H(2)O(2), 10 μM) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS.

Glutathionylation State of Uncoupling Protein-2 and the Control of Glucose-stimulated Insulin Secretion*

PubMed Central

Mailloux, Ryan J.; Fu, Accalia; Robson-Doucette, Christine; Allister, Emma M.; Wheeler, Michael B.; Screaton, Robert; Harper, Mary-Ellen

2012-01-01

The role of reactive oxygen species (ROS) in glucose-stimulated insulin release remains controversial because ROS have been shown to both amplify and impede insulin release. In regard to preventing insulin release, ROS activates uncoupling protein-2 (UCP2), a mitochondrial inner membrane protein that negatively regulates glucose-stimulated insulin secretion (GSIS) by uncoupling oxidative phosphorylation. With our recent discovery that the UCP2-mediated proton leak is modulated by reversible glutathionylation, a process responsive to small changes in ROS levels, we resolved to determine whether glutathionylation is required for UCP2 regulation of GSIS. Using Min6 cells and pancreatic islets, we demonstrate that induction of glutathionylation not only deactivates UCP2-mediated proton leak but also enhances GSIS. Conversely, an increase in mitochondrial matrix ROS was found to deglutathionylate and activate UCP2 leak and impede GSIS. Glucose metabolism also decreased the total amount of cellular glutathionylated proteins and increased the cellular glutathione redox ratio (GSH/GSSG). Intriguingly, the provision of extracellular ROS (H2O2, 10 μm) amplified GSIS and also activated UCP2. Collectively, our findings indicate that the glutathionylation status of UCP2 contributes to the regulation of GSIS, and different cellular sites and inducers of ROS can have opposing effects on GSIS, perhaps explaining some of the controversy surrounding the role of ROS in GSIS. PMID:23035124

TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin.

PubMed

Goralczyk, Anna; van Vijven, Marc; Koch, Mathilde; Badowski, Cedric; Yassin, M Shabeer; Toh, Sue-Anne; Shabbir, Asim; Franco-Obregón, Alfredo; Raghunath, Michael

2017-08-01

Transient receptor potential (TRP) channels are polymodal cell sensors responding to diverse stimuli and widely implicated in the developmental programs of numerous tissues. The evidence for an involvement of TRP family members in adipogenesis, however, is scant. We present the first comprehensive expression profile of all known 27 human TRP genes in mesenchymal progenitors cells during white or brown adipogenesis. Using positive trilineage differentiation as an exclusion criterion, TRP polycystic (P)3, and TPR melastatin (M)8 were found to be uniquely adipospecific. Knockdown of TRPP3 repressed the expression of the brown fat signature genes uncoupling protein (UCP)-1 and peroxisome proliferator-activated receptor γ coactivator (PGC)-1α as well as attenuated forskolin-stimulated uncoupled respiration. However, indices of generalized adipogenesis, such as lipid droplet morphology and fatty acid binding protein (FAPB)-4 expression, were not affected, indicating a principal mitochondrial role of TRPP3. Conversely, activating TRPM8 with menthol up-regulated UCP-1 expression and augmented uncoupled respiration predominantly in white adipocytes (browning), whereas streptomycin antagonized TRPM8-mediated calcium entry, downregulated UCP-1 expression, and mitigated uncoupled respiration; menthol was less capable of augmenting uncoupled respiration (thermogenesis) in brown adipocytes. TRPP3 and TRPM8 hence appear to be involved in the priming of mitochondria to perform uncoupled respiration downstream of adenylate cyclase. Our results also underscore the developmental caveats of using antibiotics in adipogenic studies.-Goralczyk, A., van Vijven, M., Koch, M., Badowski, C., Yassin, M. S., Toh, S.-A., Shabbir, A., Franco-Obregón, A., Raghunath, M. TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin. © FASEB.

Transcriptional upregulation of mitochondrial uncoupling protein 2 protects against oxidative stress-associated neurogenic hypertension.

PubMed

Chan, Samuel H H; Wu, Chiung-Ai; Wu, Kay L H; Ho, Ying-Hao; Chang, Alice Y W; Chan, Julie Y H

2009-10-23

Mitochondrial uncoupling proteins (UCPs) belong to a superfamily of mitochondrial anion transporters that uncouple ATP synthesis from oxidative phosphorylation and mitigates mitochondrial reactive oxygen species production. We assessed the hypothesis that UCP2 participates in central cardiovascular regulation by maintaining reactive oxygen species homeostasis in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons that maintain vasomotor tone located. We also elucidated the molecular mechanisms that underlie transcriptional upregulation of UCP2 in response to oxidative stress in RVLM. In Sprague-Dawley rats, transcriptional upregulation of UCP2 in RVLM by rosiglitazone, an activator of its transcription factor peroxisome proliferator-activated receptor (PPAR)gamma, reduced mitochondrial hydrogen peroxide level in RVLM and systemic arterial pressure. Oxidative stress induced by microinjection of angiotensin II into RVLM augmented UCP2 mRNA or protein expression in RVLM, which was antagonized by comicroinjection of NADPH oxidase inhibitor (diphenyleneiodonium chloride), superoxide dismutase mimetic (tempol), or p38 mitogen-activated protein kinase inhibitor (SB203580) but not by extracellular signal-regulated kinase 1/2 inhibitor (U0126). Angiotensin II also induced phosphorylation of the PPARgamma coactivator, PPARgamma coactivator (PGC)-1alpha, and an increase in formation of PGC-1alpha/PPARgamma complexes in a p38 mitogen-activated protein kinase-dependent manner. Intracerebroventricular infusion of angiotensin II promoted an increase in mitochondrial hydrogen peroxide production in RVLM and chronic pressor response, which was potentiated by gene knockdown of UCP2 but blunted by rosiglitazone. These results suggest that transcriptional upregulation of mitochondrial UCP2 in response to an elevation in superoxide plays an active role in feedback regulation of reactive oxygen species production in RVLM and neurogenic hypertension associated with chronic oxidative stress.

Uncoupling RARA transcriptional activation and degradation clarifies the bases for APL response to therapies

PubMed Central

Ablain, Julien; Leiva, Magdalena; Peres, Laurent; Fonsart, Julien; Anthony, Elodie

2013-01-01

In PML/RARA-driven acute promyelocytic leukemia (APL), retinoic acid (RA) induces leukemia cell differentiation and transiently clears the disease. Molecularly, RA activates PML/RARA-dependent transcription and also initiates its proteasome-mediated degradation. In contrast, arsenic, the other potent anti-APL therapy, only induces PML/RARA degradation by specifically targeting its PML moiety. The respective contributions of RA-triggered transcriptional activation and proteolysis to clinical response remain disputed. Here, we identify synthetic retinoids that potently activate RARA- or PML/RARA-dependent transcription, but fail to down-regulate RARA or PML/RARA protein levels. Similar to RA, these uncoupled retinoids elicit terminal differentiation, but unexpectedly fail to impair leukemia-initiating activity of PML/RARA-transformed cells ex vivo or in vivo. Accordingly, the survival benefit conferred by uncoupled retinoids in APL mice is dramatically lower than the one provided by RA. Differentiated APL blasts sorted from uncoupled retinoid–treated mice retain PML/RARA expression and reinitiate APL in secondary transplants. Thus, differentiation is insufficient for APL eradication, whereas PML/RARA loss is essential. These observations unify the modes of action of RA and arsenic and shed light on the potency of their combination in mice or patients. PMID:23509325

Uncoupling RARA transcriptional activation and degradation clarifies the bases for APL response to therapies.

PubMed

Ablain, Julien; Leiva, Magdalena; Peres, Laurent; Fonsart, Julien; Anthony, Elodie; de Thé, Hugues

2013-04-08

In PML/RARA-driven acute promyelocytic leukemia (APL), retinoic acid (RA) induces leukemia cell differentiation and transiently clears the disease. Molecularly, RA activates PML/RARA-dependent transcription and also initiates its proteasome-mediated degradation. In contrast, arsenic, the other potent anti-APL therapy, only induces PML/RARA degradation by specifically targeting its PML moiety. The respective contributions of RA-triggered transcriptional activation and proteolysis to clinical response remain disputed. Here, we identify synthetic retinoids that potently activate RARA- or PML/RARA-dependent transcription, but fail to down-regulate RARA or PML/RARA protein levels. Similar to RA, these uncoupled retinoids elicit terminal differentiation, but unexpectedly fail to impair leukemia-initiating activity of PML/RARA-transformed cells ex vivo or in vivo. Accordingly, the survival benefit conferred by uncoupled retinoids in APL mice is dramatically lower than the one provided by RA. Differentiated APL blasts sorted from uncoupled retinoid-treated mice retain PML/RARA expression and reinitiate APL in secondary transplants. Thus, differentiation is insufficient for APL eradication, whereas PML/RARA loss is essential. These observations unify the modes of action of RA and arsenic and shed light on the potency of their combination in mice or patients.

Uncoupling proteins (UCP) in unicellular eukaryotes: true UCPs or UCP1-like acting proteins?

PubMed

Luévano-Martínez, Luis Alberto

2012-04-05

Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Thyroid hormone affects secretory activity and uncoupling protein-3 expression in rat harderian gland.

PubMed

Chieffi Baccari, Gabriella; Monteforte, Rossella; de Lange, Pieter; Raucci, Franca; Farina, Paola; Lanni, Antonia

2004-07-01

The effects of T(3) administration on the rat Harderian gland were examined at morphological, biochemical, and molecular levels. T(3) induced hypertrophy of the two cell types (A and B) present in the glandular epithelium. In type A cells, the hypertrophy was mainly due to an increase in the size of the lipid compartment. The acinar lumina were filled with lipoproteic substances, and the cells often showed an olocrine secretory pattern. In type B cells, the hypertrophy largely consisted of a marked proliferation of mitochondria endowed with tightly packed cristae, the mitochondrial number being nearly doubled (from 62 to 101/100 microm(2)). Although the average area of individual mitochondria decreased by about 50%, the total area of the mitochondrial compartment increased by about 80% (from 11 to 19/100 microm(2)). This could be ascribed to T(3)-induced mitochondrial proliferation. The morphological and morphometric data correlated well with our biochemical results, which indicated that mitochondrial respiratory activity is increased in hyperthyroid rats. T(3), by influencing the metabolic function of the mitochondrial compartment, induces lipogenesis and the release of secretory product by type A cells. Mitochondrial uncoupling proteins 2 and 3 were expressed at both mRNA and protein levels in the euthyroid rat Harderian gland. T(3) treatment increased the mRNA levels of both uncoupling protein 2 (UCP2) and UCP3, but the protein level only of UCP3. A possible role for these proteins in the Harderian gland is discussed.

ERK5 signaling gets XIAPed: a role for ubiquitin in the disassembly of a MAPK cascade

PubMed Central

Klein, Aileen M; Cobb, Melanie H

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are tightly controlled through a series of well-characterized phospho-regulatory events. In this issue, Takeda et al (2014) identify the inhibitor of apoptosis protein, XIAP, as a key regulator of ERK5 activation via uncoupling of upstream kinase activity by non-degradative ubiquitination. PMID:25012518

Uncoupling and Turnover in a Cl−/H+ Exchange Transporter

PubMed Central

Walden, Michael; Accardi, Alessio; Wu, Fang; Xu, Chen; Williams, Carole; Miller, Christopher

2007-01-01

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl− for one H+ via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl− ion located near the center of the membrane. Mutations at this position lead to “uncoupling,” such that the H+/Cl− transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl− gradient, but the extent and rate of this H+ pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl− transport that is not linked to counter-movement of H+, i.e., a “leak.” The unitary Cl− transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is ∼4,000 s−1 for wild-type protein, and the uncoupled mutants transport Cl− at similar rates. PMID:17389248

Free fatty acids as inducers and regulators of uncoupling of oxidative phosphorylation in liver mitochondria with participation of ADP/ATP- and aspartate/glutamate-antiporter.

PubMed

Samartsev, V N; Marchik, E I; Shamagulova, L V

2011-02-01

In liver mitochondria fatty acids act as protonophoric uncouplers mainly with participation of internal membrane protein carriers - ADP/ATP and aspartate/glutamate antiporters. In this study the values of recoupling effects of carboxyatractylate and glutamate (or aspartate) were used to assess the degree of participation of ADP/ATP and aspartate/glutamate antiporters in uncoupling activity of fatty acids. These values were determined from the ability of these recoupling agents to suppress the respiration stimulated by fatty acids and to raise the membrane potential reduced by fatty acids. Increase in palmitic and lauric acid concentration was shown to increase the degree of participation of ADP/ATP antiporter and to decrease the degree of participation of aspartate/glutamate antiporter in uncoupling to the same extent. These data suggest that fatty acids are not only inducers of uncoupling of oxidative phosphorylation, but that they also act the regulators of this process. The linear dependence of carboxyatractylate and glutamate recoupling effects ratio on palmitic and lauric acids concentration was established. Comparison of the effects of fatty acids (palmitic, myristic, lauric, capric, and caprylic having 16, 14, 12, 10, and 8 carbon atoms, respectively) has shown that, as the hydrophobicity of fatty acids decreases, the effectiveness decreases to a greater degree than the respective values of their specific uncoupling activity. The action of fatty acids as regulators of uncoupling is supposed to consist of activation of transport of their anions from the internal to the external monolayer of the internal membrane with participation of ADP/ATP antiporter and, at the same time, in inhibition of this process with the participation of aspartate/glutamate antiporter.

Molecular identification and functional characterisation of uncoupling protein 4 in larva and pupa fat body mitochondria from the beetle Zophobas atratus.

PubMed

Slocinska, Malgorzata; Antos-Krzeminska, Nina; Rosinski, Grzegorz; Jarmuszkiewicz, Wieslawa

2012-08-01

Uncoupling protein 4 (UCP4) is a member of the UCP subfamily that mediates mitochondrial uncoupling, and sequence alignment predicts the existence of UCP4 in several insects. The present study demonstrates the first molecular identification of a partial Zophobas atratus UCP4-coding sequence and the functional characterisation of ZaUCP4 in the mitochondria of larval and pupal fat bodies of the beetle. ZaUCP4 shows a high similarity to predicted insect UCP4 isoforms and known mammalian UCP4s, both at the nucleotide and amino acid sequence levels. Bioenergetic studies clearly demonstrate UCP function in mitochondria from larval and pupal fat bodies. In non-phosphorylating mitochondria, ZaUCP activity was stimulated by palmitic acid and inhibited by the purine nucleotide GTP. In phosphorylating mitochondria, ZaUCP4 activity decreased the yield of oxidative phosphorylation. ZaUCP4 was immunodetected with antibodies raised against human UCP4 as a single 36-kDa band. A lower expression of ZaUCP4 at the level of mRNA and protein and a decreased ZaUCP4 activity were observed in the Z. atratus pupal fat body compared with the larval fat body. The different expression patterns and activity of ZaUCP4 during the larval-pupal transformation indicates an important physiological role for UCP4 in insect fat body development and function during insect metamorphosis. Copyright © 2012 Elsevier Inc. All rights reserved.

Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

PubMed Central

Recolin, Bénédicte; Van Der Laan, Siem; Maiorano, Domenico

2012-01-01

Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation. PMID:22187152

MTORC1 Regulates both General Autophagy and Mitophagy Induction after Oxidative Phosphorylation Uncoupling.

PubMed

Bartolomé, Alberto; García-Aguilar, Ana; Asahara, Shun-Ichiro; Kido, Yoshiaki; Guillén, Carlos; Pajvani, Utpal B; Benito, Manuel

2017-09-11

The mechanistic target of rapamycin complex 1 (MTORC1) is a critical negative regulator of general autophagy. We hypothesized that MTORC1 may specifically regulate autophagic clearance of damaged mitochondria. To test this, we used cells lacking tuberous sclerosis complex 2 (TSC2 -/-), which show constitutive MTORC1 activation. TSC2 -/- cells show MTORC1-dependent impaired autophagic flux after chemical uncoupling of mitochondria, increased mitochondrial protein aging and accumulation of p62/SQSTM1 positive mitochondria. Mitochondrial autophagy (mitophagy) was also deficient in cells lacking TSC2, associated with altered expression of PTEN-induced kinase 1 (PINK1) and PARK2 translocation to uncoupled mitochondria, all of which were recovered by MTORC1 inhibition or expression of constitutively active FoxO1. These data prove the necessity of intact MTORC1 signaling to regulate two synergistic processes required for clearance of damaged mitochondria: 1) general autophagy initiation, and 2) PINK1/PARK2-mediated selective targeting of uncoupled mitochondria to the autophagic machinery. Copyright © 2017 American Society for Microbiology.

Stanniocalcin-1 inhibits renal ischemia/reperfusion injury via an AMP-activated protein kinase-dependent pathway

USDA-ARS?s Scientific Manuscript database

AKI is associated with increased morbidity, mortality, and cost of care, and therapeutic options remain limited. Reactive oxygen species are critical for the genesis of ischemic AKI. Stanniocalcin-1 (STC1) suppresses superoxide generation through induction of uncoupling proteins (UCPs), and transgen...

Human 'brite/beige' adipocytes develop from capillary networks, and their implantation improves metabolic homeostasis in mice.

PubMed

Min, So Yun; Kady, Jamie; Nam, Minwoo; Rojas-Rodriguez, Raziel; Berkenwald, Aaron; Kim, Jong Hun; Noh, Hye-Lim; Kim, Jason K; Cooper, Marcus P; Fitzgibbons, Timothy; Brehm, Michael A; Corvera, Silvia

2016-03-01

Uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots, in adipocytes that are termed 'brite' (brown-in-white) or 'beige'. In humans, the presence of brite or beige (brite/beige) adipocytes is correlated with a lean, metabolically healthy phenotype, but whether a causal relationship exists is not clear. Here we report that human brite/beige adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform in response to adenylate cyclase activation from being UCP1 negative to being UCP1 positive, which is a defining feature of the beige/brite phenotype, while displaying uncoupled respiration. When implanted into normal chow-fed, or into high-fat diet (HFD)-fed, glucose-intolerant NOD-scid IL2rg(null) (NSG) mice, brite/beige adipocytes activated in vitro enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Pro-angiogenic conditions therefore drive the proliferation of human beige/brite adipocyte progenitors, and activated beige/brite adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism.

Human ‘brite / beige’ adipocytes develop from capillary networks and their implantation improves metabolic homeostasis in mice

PubMed Central

Min, So Yun; Kady, Jamie; Nam, Minwoo; Rojas-Rodriguez, Raziel; Berkenwald, Aaron; Kim, Jong Hun; Noh, Hye-Lim; Kim, Jason K.; Cooper, Marcus P.; Fitzgibbons, Timothy; Brehm, Michael A.; Corvera, Silvia

2015-01-01

The uncoupling protein 1 (UCP1) is highly expressed in brown adipose tissue, where it generates heat by uncoupling electron transport from ATP production. UCP1 is also found outside classical brown adipose tissue depots1–4, in adipocytes termed ‘brite’ (brown-in-white) or ‘beige’. In humans, the presence of ‘brite/beige’ adipocytes correlates with a lean, metabolically healthy phenotype5–8, but whether a causal relationship exists is not clear. Here we report that human ‘brite/beige’ adipocyte progenitors proliferate in response to pro-angiogenic factors, in association with expanding capillary networks. Adipocytes formed from these progenitors transform from being UCP1-negative to UCP1-positive in response to adenylate cyclase activation, a defining feature of the ‘beige/brite’ phenotype, and display uncoupled respiration. When implanted into normal or high fat diet-fed, glucose intolerant NOD-scid IL2rgnull mice, activated ‘brite/beige’ adipocytes enhance systemic glucose tolerance. These adipocytes express neuroendocrine and secreted factors, including the pro-protein convertase PCSK1, which is strongly associated with human obesity. Thus, pro-angiogenic conditions drive proliferation of human ‘beige/brite’ adipocyte progenitors, and activated ‘beige/brite’ adipocytes can affect systemic glucose homeostasis, potentially through a neuroendocrine mechanism. PMID:26808348

Differential effects of eNOS uncoupling on conduit and small arteries in GTP-cyclohydrolase I-deficient hph-1 mice.

PubMed

d'Uscio, Livius V; Smith, Leslie A; Katusic, Zvonimir S

2011-12-01

In the present study, we used the hph-1 mouse, which displays GTP-cyclohydrolase I (GTPCH I) deficiency, to test the hypothesis that loss of tetrahydrobiopterin (BH(4)) in conduit and small arteries activates compensatory mechanisms designed to protect vascular wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and BH(4) levels were reduced in the aortas and small mesenteric arteries of hph-1 mice. However, the BH(4)-to-7,8-dihydrobiopterin ratio was significantly reduced only in hph-1 aortas. Furthermore, superoxide anion and 3-nitrotyrosine production were significantly enhanced in aortas but not in small mesenteric arteries of hph-1 mice. In contrast to the aorta, protein expression of copper- and zinc-containing superoxide dismutase (CuZnSOD) was significantly increased in small mesenteric arteries of hph-1 mice. Protein expression of catalase was increased in both aortas and small mesenteric arteries of hph-1 mice. Further analysis of endothelial nitric oxide synthase (eNOS)/cyclic guanosine monophosphate (cGMP) signaling demonstrated that protein expression of phosphorylated Ser(1177)-eNOS as well as basal cGMP levels and hydrogen peroxide was increased in hph-1 aortas. Increased production of hydrogen peroxide in hph-1 mice aortas appears to be the most likely mechanism responsible for phosphorylation of eNOS and elevation of cGMP. In contrast, upregulation of CuZnSOD and catalase in resistance arteries is sufficient to protect vascular tissue from increased production of reactive oxygen species generated by uncoupling of eNOS. The results of our study suggest that anatomical origin determines the ability of vessel wall to cope with oxidative stress induced by uncoupling of eNOS.

Uncoupling Mitochondrial Respiration for Diabesity.

PubMed

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

2016-08-01

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

Evidence for involvement of uncoupling proteins in cerebral mitochondrial oxidative phosphorylation deficiency of rats exposed to 5,000 m high altitude.

PubMed

Xu, Yu; Liu, Yuliang; Xia, Chen; Gao, Pan; Liu, Jun-Ze

2013-02-01

The present study aimed to investigate the change of proton leak and discuss the role of cerebral uncoupling proteins (UCPs) and its regulatory molecules non-esterified fatty acid (NEFA) in high altitude mitochondrial oxidative phosphorylation deficiency. The model group animals were exposed to acute high altitude hypoxia, and the mitochondrial respiration, protein leak, UCPs abundance/activity and cerebral NEFA concentration were measured. We found that in the model group, cerebral mitochondrial oxidative phosphorylation was severely impaired with decreased ST3 respiration rate and ATP pool. Proton leak kinetics curves demonstrated an increase in proton leak; GTP binding assay pointed out that total cerebral UCPs activity significantly increased; Q-PCR and western blot showed upregulated expression of UCP4 and UCP5. Moreover, cerebral NEFA concentration increased. In conclusion, UCPs mediated proton leak is closely related to cerebral mitochondria oxidative phosphorylation deficiency during acute high altitude hypoxia and NEFA is involved in this signaling pathway.

Temperature dependence of rat liver mitochondrial respiration with uncoupling of oxidative phosphorylation by fatty acids. Influence of inorganic phosphate.

PubMed

Samartsev, V N; Chezganova, S A; Polishchuk, L S; Paydyganov, A P; Vidyakina, O V; Zeldi, I P

2003-06-01

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22 degrees C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22 degrees C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22 degrees C and from 56 to 67 kJ/mol in the range from 22 to 37 degrees C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells*

PubMed Central

Figarola, James L.; Singhal, Jyotsana; Tompkins, Joshua D.; Rogers, George W.; Warden, Charles; Horne, David; Riggs, Arthur D.; Awasthi, Sanjay; Singhal, Sharad S.

2015-01-01

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. PMID:26534958

Mitochondrial uncoupling agents antagonize rotenone actions in rat substantia nigra dopamine neurons.

PubMed

Wu, Yan-Na; Munhall, Adam C; Johnson, Steven W

2011-06-13

Mild uncoupling of oxidative phosphorylation has been reported to reduce generation of reactive oxygen species (ROS) and therefore may be neuroprotective. We reported previously that the mitochondrial poison rotenone enhanced currents evoked by N-methyl-D-aspartate (NMDA) by a ROS-dependent mechanism in rat midbrain dopamine neurons. Thus, rotenone, which produces a model of Parkinson's disease in rodents, may increase the risk of dopamine neuron excitotoxicity. The purpose of this study was to test the hypothesis that oxidative phosphorylation uncoupling agents would antagonize the effect of rotenone on NMDA current. We used patch pipettes to record whole-cell currents under voltage-clamp (-60 mV) in substantia nigra dopamine neurons in slices of rat brain. Rotenone, NMDA and uncoupling agents were added to the brain slice superfusate. Inward currents evoked by NMDA (30 μM) more than doubled in amplitude after slices were superfused for 30 min with 100 nM rotenone. Continuous superfusion with the uncoupling agent carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (1-3 nM) or 2,4-dinitrophenol (100 nM) significantly antagonized and delayed the ability of rotenone to potentiate NMDA currents. Coenzyme Q₁₀ (1-10 nM), which has been reported to facilitate uncoupling protein activity, also antagonized this action of rotenone. These results suggest that mild uncoupling of oxidative phosphorylation may protect dopamine neurons against injury from mitochondrial poisons such as rotenone. Published by Elsevier B.V.

Physiological uncoupling of mitochondrial oxidative phosphorylation. Studies in different yeast species.

PubMed

Guerrero-Castillo, Sergio; Araiza-Olivera, Daniela; Cabrera-Orefice, Alfredo; Espinasa-Jaramillo, Juan; Gutiérrez-Aguilar, Manuel; Luévano-Martínez, Luís A; Zepeda-Bastida, Armando; Uribe-Carvajal, Salvador

2011-06-01

Under non-phosphorylating conditions a high proton transmembrane gradient inhibits the rate of oxygen consumption mediated by the mitochondrial respiratory chain (state IV). Slow electron transit leads to production of reactive oxygen species (ROS) capable of participating in deleterious side reactions. In order to avoid overproducing ROS, mitochondria maintain a high rate of O(2) consumption by activating different exquisitely controlled uncoupling pathways. Different yeast species possess one or more uncoupling systems that work through one of two possible mechanisms: i) Proton sinks and ii) Non-pumping redox enzymes. Proton sinks are exemplified by mitochondrial unspecific channels (MUC) and by uncoupling proteins (UCP). Saccharomyces. cerevisiae and Debaryomyces hansenii express highly regulated MUCs. Also, a UCP was described in Yarrowia lipolytica which promotes uncoupled O(2) consumption. Non-pumping alternative oxido-reductases may substitute for a pump, as in S. cerevisiae or may coexist with a complete set of pumps as in the branched respiratory chains from Y. lipolytica or D. hansenii. In addition, pumps may suffer intrinsic uncoupling (slipping). Promising models for study are unicellular parasites which can turn off their aerobic metabolism completely. The variety of energy dissipating systems in eukaryote species is probably designed to control ROS production in the different environments where each species lives.

Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish

USDA-ARS?s Scientific Manuscript database

Uncoupling protein 2 is a member of the mitochondrial channel proteins that regulate the flow of hydrogen ions and ATP generation. The relationship between UCP2 and nutrient metabolism has been well-defined in humans but unclear in fish. We hypothesized that increased muscle growth in channel catf...

Anti-obesity efficacy of nanoemulsion oleoresin capsicum in obese rats fed a high-fat diet

PubMed Central

Kim, Joo-Yeon; Lee, Mak-Soon; Jung, Sunyoon; Joo, Hyunjin; Kim, Chong-Tai; Kim, In-Hwan; Seo, Sangjin; Oh, Soojung; Kim, Yangha

2014-01-01

Purpose This study determined the effects of oleoresin capsicum (OC) and nanoemulsion OC (NOC) on obesity in obese rats fed a high-fat diet. Methods The rats were randomly separated into three groups: a high-fat (HF) diet group, HF + OC diet group, and HF + NOC diet group. All groups were fed the diet and water ad libitum for 14 weeks. Results NOC reduced the body weight and adipose tissue mass, whereas OC did not. OC and NOC reduced mRNA levels of adipogenic genes, including peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein-1c, and fatty acid-binding protein in white adipose tissue. The mRNA levels of genes related to β-oxidation or thermogenesis including PPAR-α, palmitoyltransferase-1α, and uncoupling protein-2 were increased by the OC and NOC relative to the HF group. Both OC and NOC clearly stimulated AMP-activated protein kinase (AMPK) activity. In particular, PPAR-α, palmitoyltransferase-1α, uncoupling protein-2 expression, and AMPK activity were significantly increased in the NOC group compared to in the OC group. NOC decreased glycerol-3-phosphate dehydrogenase activity whereas OC did not. Conclusion From these results, NOC could be suggested as a potential anti-obesity agent in obese rats fed a HF diet. The effects of the NOC on obesity were associated with changes of multiple gene expression, activation of AMPK, and inhibition of glycerol-3-phosphate dehydrogenase in white adipose tissue. PMID:24403834

Anti-obesity efficacy of nanoemulsion oleoresin capsicum in obese rats fed a high-fat diet.

PubMed

Kim, Joo-Yeon; Lee, Mak-Soon; Jung, Sunyoon; Joo, Hyunjin; Kim, Chong-Tai; Kim, In-Hwan; Seo, Sangjin; Oh, Soojung; Kim, Yangha

2014-01-01

This study determined the effects of oleoresin capsicum (OC) and nanoemulsion OC (NOC) on obesity in obese rats fed a high-fat diet. THE RATS WERE RANDOMLY SEPARATED INTO THREE GROUPS: a high-fat (HF) diet group, HF + OC diet group, and HF + NOC diet group. All groups were fed the diet and water ad libitum for 14 weeks. NOC reduced the body weight and adipose tissue mass, whereas OC did not. OC and NOC reduced mRNA levels of adipogenic genes, including peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein-1c, and fatty acid-binding protein in white adipose tissue. The mRNA levels of genes related to β-oxidation or thermogenesis including PPAR-α, palmitoyltransferase-1α, and uncoupling protein-2 were increased by the OC and NOC relative to the HF group. Both OC and NOC clearly stimulated AMP-activated protein kinase (AMPK) activity. In particular, PPAR-α, palmitoyltransferase-1α, uncoupling protein-2 expression, and AMPK activity were significantly increased in the NOC group compared to in the OC group. NOC decreased glycerol-3-phosphate dehydrogenase activity whereas OC did not. From these results, NOC could be suggested as a potential anti-obesity agent in obese rats fed a HF diet. The effects of the NOC on obesity were associated with changes of multiple gene expression, activation of AMPK, and inhibition of glycerol-3-phosphate dehydrogenase in white adipose tissue.

Mitochondrial uncoupling proteins in unicellular eukaryotes.

PubMed

Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

2010-01-01

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

Alterations in proton leak, oxidative status and uncoupling protein 3 content in skeletal muscle subsarcolemmal and intermyofibrillar mitochondria in old rats

PubMed Central

2014-01-01

Background We considered of interest to evaluate how aging affects mitochondrial function in skeletal muscle. Methods We measured mitochondrial oxidative capacity and proton leak, together with lipid oxidative damage, superoxide dismutase specific activity and uncoupling protein 3 content, in subsarcolemmal and intermyofibrillar mitochondria from adult (six months) and old (two years) rats. Body composition, resting metabolic rate and plasma non esterified fatty acid levels were also assessed. Results Old rats displayed significantly higher body energy and lipids, while body proteins were significantly lower, compared to adult rats. In addition, plasma non esterified fatty acid levels were significantly higher, while resting metabolic rates were found to be significantly lower, in old rats compared to adult ones. Significantly lower oxidative capacities in whole tissue homogenates and in intermyofibrillar and subsarcolemmal mitochondria were found in old rats compared to adult ones. Subsarcolemmal and intermyofibrillar mitochondria from old rats exhibited a significantly lower proton leak rate, while oxidative damage was found to be significantly higher only in subsarcolemmal mitochondria. Mitochondrial superoxide dismutase specific activity was not significantly affected in old rats, while significantly higher content of uncoupling protein 3 was found in both mitochondrial populations from old rats compared to adult ones, although the magnitude of the increase was lower in subsarcolemmal than in intermyofibrillar mitochondria. Conclusions The decrease in oxidative capacity and proton leak in intermyofibrillar and subsarcolemmal mitochondria could induce a decline in energy expenditure and thus contribute to the reduced resting metabolic rate found in old rats, while oxidative damage is present only in subsarcolemmal mitochondria. PMID:24950599

Mitochondrial Proton Leak Plays a Critical Role in Pathogenesis of Cardiovascular Diseases.

PubMed

Cheng, Jiali; Nanayakkara, Gayani; Shao, Ying; Cueto, Ramon; Wang, Luqiao; Yang, William Y; Tian, Ye; Wang, Hong; Yang, Xiaofeng

2017-01-01

Mitochondrial proton leak is the principal mechanism that incompletely couples substrate oxygen to ATP generation. This chapter briefly addresses the recent progress made in understanding the role of proton leak in the pathogenesis of cardiovascular diseases. Majority of the proton conductance is mediated by uncoupling proteins (UCPs) located in the mitochondrial inner membrane. It is evident that the proton leak and reactive oxygen species (ROS) generated from electron transport chain (ETC) in mitochondria are linked to each other. Increased ROS production has been shown to induce proton conductance, and in return, increased proton conductance suppresses ROS production, suggesting the existence of a positive feedback loop that protects the biological systems from detrimental effects of augmented oxidative stress. There is mounting evidence attributing to proton leak and uncoupling proteins a crucial role in the pathogenesis of cardiovascular disease. We can surmise the role of "uncoupling" in cardiovascular disorders as follows; First, the magnitude of the proton leak and the mechanism involved in mediating the proton leak determine whether there is a protective effect against ischemia-reperfusion (IR) injury. Second, uncoupling by UCP2 preserves vascular function in diet-induced obese mice as well as in diabetes. Third, etiology determines whether the proton conductance is altered or not during hypertension. And fourth, proton leak regulates ATP synthesis-uncoupled mitochondrial ROS generation, which determines pathological activation of endothelial cells for recruitment of inflammatory cells. Continue effort in improving our understanding in the role of proton leak in the pathogenesis of cardiovascular and metabolic diseases would lead to identification of novel therapeutic targets for treatment.

Alternative mitochondrial respiratory chains from two crustaceans: Artemia franciscana nauplii and the white shrimp, Litopenaeus vannamei.

PubMed

Rodriguez-Armenta, Chrystian; Uribe-Carvajal, Salvador; Rosas-Lemus, Monica; Chiquete-Felix, Natalia; Huerta-Ocampo, Jose Angel; Muhlia-Almazan, Adriana

2018-04-01

Mitochondrial ATP is synthesized by coupling between the electron transport chain and complex V. In contrast, physiological uncoupling of these processes allows mitochondria to consume oxygen at high rates without ATP synthesis. Such uncoupling mechanisms prevent reactive oxygen species overproduction. One of these mechanisms are the alternative redox enzymes from the mitochondrial respiratory chain, which may help cells to maintain homeostasis under stress independently of ATP synthesis. To date, no reports have been published on alternative redox enzymes in crustaceans mitochondria. Specific inhibitors were used to identify alternative redox enzymes in mitochondria isolated from Artemia franciscana nauplii, and the white shrimp, Litopenaeus vannamei. We report the presence of two alternative redox enzymes in the respiratory chain of A. franciscana nauplii, whose isolated mitochondria used glycerol-3-phosphate as a substrate, suggesting the existence of a glycerol-3-phosphate dehydrogenase. In addition, cyanide and octyl-gallate were necessary to fully inhibit this species' mitochondrial oxygen consumption, suggesting an alternative oxidase is present. The in-gel activity analysis confirmed that additional mitochondrial redox proteins exist in A. franciscana. A mitochondrial glycerol-3-phosphate dehydrogenase oxidase was identified by protein sequencing as part of a branched respiratory chain, and an alternative oxidase was also identified in this species by western blot. These results indicate different adaptive mechanisms from artemia to face environmental challenges related to the changing levels of oxygen concentration in seawater through their life cycles. No alternative redox enzymes were found in shrimp mitochondria, further efforts will determine the existence of an uncoupling mechanism such as uncoupling proteins.

A critical tyrosine residue determines the uncoupling protein-like activity of the yeast mitochondrial oxaloacetate carrier.

PubMed

Luévano-Martínez, Luis A; Barba-Ostria, Carlos; Araiza-Olivera, Daniela; Chiquete-Félix, Natalia; Guerrero-Castillo, Sergio; Rial, Eduardo; Georgellis, Dimitris; Uribe-Carvajal, Salvador

2012-04-01

The mitochondrial Oac (oxaloacetate carrier) found in some fungi and plants catalyses the uptake of oxaloacetate, malonate and sulfate. Despite their sequence similarity, transport specificity varies considerably between Oacs. Indeed, whereas ScOac (Saccharomyces cerevisiae Oac) is a specific anion-proton symporter, the YlOac (Yarrowia lipolytica Oac) has the added ability to transport protons, behaving as a UCP (uncoupling protein). Significantly, we identified two amino acid changes at the matrix gate of YlOac and ScOac, tyrosine to phenylalanine and methionine to leucine. We studied the role of these amino acids by expressing both wild-type and specifically mutated Oacs in an Oac-null S. cerevisiae strain. No phenotype could be associated with the methionine to leucine substitution, whereas UCP-like activity was dependent on the presence of the tyrosine residue normally expressed in the YlOac, i.e. Tyr-ScOac mediated proton transport, whereas Phe-YlOac lost its protonophoric activity. These findings indicate that the UCP-like activity of YlOac is determined by the tyrosine residue at position 146.

Mitochondrial proticity and ROS signaling: lessons from the uncoupling proteins.

PubMed

Mailloux, Ryan J; Harper, Mary-Ellen

2012-09-01

Fifty years since Peter Mitchell proposed the theory of chemiosmosis, the transformation of cellular redox potential into ATP synthetic capacity is still a widely recognized function of mitochondria. Mitchell used the term 'proticity' to describe the force and flow of the proton circuit across the inner membrane. When the proton gradient is coupled to ATP synthase activity, the conversion of fuel to ATP is efficient. However, uncoupling proteins (UCPs) can cause proton leaks resulting in poor fuel conversion efficiency, and some UCPs might control mitochondrial reactive oxygen species (ROS) production. Once viewed as toxic metabolic waste, ROS are now implicated in cell signaling and regulation. Here, we discuss the role of mitochondrial proticity in the context of ROS production and signaling. Copyright © 2012 Elsevier Ltd. All rights reserved.

Cell Death and Heart Failure in Obesity: Role of Uncoupling Proteins

PubMed Central

Ruiz-Ramírez, Angélica; López-Acosta, Ocarol; Barrios-Maya, Miguel Angel

2016-01-01

Metabolic diseases such as obesity, metabolic syndrome, and type II diabetes are often characterized by increased reactive oxygen species (ROS) generation in mitochondrial respiratory complexes, associated with fat accumulation in cardiomyocytes, skeletal muscle, and hepatocytes. Several rodents studies showed that lipid accumulation in cardiac myocytes produces lipotoxicity that causes apoptosis and leads to heart failure, a dynamic pathological process. Meanwhile, several tissues including cardiac tissue develop an adaptive mechanism against oxidative stress and lipotoxicity by overexpressing uncoupling proteins (UCPs), specific mitochondrial membrane proteins. In heart from rodent and human with obesity, UCP2 and UCP3 may protect cardiomyocytes from death and from a state progressing to heart failure by downregulating programmed cell death. UCP activation may affect cytochrome c and proapoptotic protein release from mitochondria by reducing ROS generation and apoptotic cell death. Therefore the aim of this review is to discuss recent findings regarding the role that UCPs play in cardiomyocyte survival by protecting against ROS generation and maintaining bioenergetic metabolism homeostasis to promote heart protection. PMID:27642497

Overexpression of uncoupling protein-2 in cancer: metabolic and heat changes, inhibition and effects on drug resistance.

PubMed

Pitt, Michael A

2015-12-01

This paper deals with the role of uncoupling protein-2 (UCP2) in cancer. UCP2 is overexpressed in cancer. This overexpression results in uncoupling of mitochondrial oxidative phosphorylation and a shift in production of ATP from mitochondrial oxidative phosphorylation to cytosolic aerobic glycolysis. UCP2 overexpression results in the following changes. Mitochondrial membrane potential (Δψ(m)) is decreased and lactate accumulates. There is a diminished production of reactive oxygen species and apoptosis is inhibited post-exposure to chemotherapeutic agents. There is an increase in heat and entropy production and a departure from the stationary state of non-cancerous tissue. Uncoupling of oxidative phosphorylation may also be caused by protonophores and non-steroidal anti-inflammatory drugs. UCP2 requires activation by superoxide and lipid peroxidation derivatives. As vitamin E inhibits lipid peroxidation, it might be expected that vitamin E would act as a chemotherapeutic agent against cancer. A recent study has shown that vitamin E and another anti-oxidant accelerate cancer progression. UCP2 is inhibited by genipin, chromane compounds and short interfering RNAs (siRNA). Genipin, chromanes and siRNA are taken up by both cancer and non-cancerous cells. Targeting the uptake of these agents by cancer cells by the enhanced permeability and retention effect is considered. Inhibition of UCP2 enhances the action of several anti-cancer agents.

The plant energy-dissipating mitochondrial systems: depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots.

PubMed

Borecky, Jirí; Nogueira, Fábio T S; de Oliveira, Kívia A P; Maia, Ivan G; Vercesi, Aníbal E; Arruda, Paulo

2006-01-01

The simultaneous existence of alternative oxidases and uncoupling proteins in plants has raised the question as to why plants need two energy-dissipating systems with apparently similar physiological functions. A probably complete plant uncoupling protein gene family is described and the expression profiles of this family compared with the multigene family of alternative oxidases in Arabidopsis thaliana and sugarcane (Saccharum sp.) employed as dicot and monocot models, respectively. In total, six uncoupling protein genes, AtPUMP1-6, were recognized within the Arabidopsis genome and five (SsPUMP1-5) in a sugarcane EST database. The recombinant AtPUMP5 protein displayed similar biochemical properties as AtPUMP1. Sugarcane possessed four Arabidopsis AOx1-type orthologues (SsAOx1a-1d); no sugarcane orthologue corresponding to Arabidopsis AOx2-type genes was identified. Phylogenetic and expression analyses suggested that AtAOx1d does not belong to the AOx1-type family but forms a new (AOx3-type) family. Tissue-enriched expression profiling revealed that uncoupling protein genes were expressed more ubiquitously than the alternative oxidase genes. Distinct expression patterns among gene family members were observed between monocots and dicots and during chilling stress. These findings suggest that the members of each energy-dissipating system are subject to different cell or tissue/organ transcriptional regulation. As a result, plants may respond more flexibly to adverse biotic and abiotic conditions, in which oxidative stress is involved.

Beyond desensitization: physiological relevance of arrestin-dependent signaling.

PubMed

Luttrell, Louis M; Gesty-Palmer, Diane

2010-06-01

Heptahelical G protein-coupled receptors are the most diverse and therapeutically important family of receptors in the human genome. Ligand binding activates heterotrimeric G proteins that transmit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by arrestin binding, which uncouples the receptor and G protein and targets the receptor for internalization. It is clear, however, that heptahelical receptor signaling does not end with desensitization. Arrestins bind a host of catalytically active proteins and serve as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into the receptor-arrestin complex. Although many of these arrestin-bound effectors serve to modulate G protein signaling, degrading second messengers and regulating endocytosis and trafficking, other signals seem to extend beyond the receptor-arrestin complex to regulate such processes as protein translation and gene transcription. Although these findings have led to a re-envisioning of heptahelical receptor signaling, little is known about the physiological roles of arrestin-dependent signaling. In vivo, the duality of arrestin function makes it difficult to dissociate the consequences of arrestin-dependent desensitization from those that might be ascribed to arrestin-mediated signaling. Nonetheless, recent evidence generated using arrestin knockouts, G protein-uncoupled receptor mutants, and arrestin pathway-selective "biased agonists" is beginning to reveal that arrestin signaling plays important roles in the retina, central nervous system, cardiovascular system, bone remodeling, immune system, and cancer. Understanding the signaling roles of arrestins may foster the development of pathway-selective drugs that exploit these pathways for therapeutic benefit.

Beyond Desensitization: Physiological Relevance of Arrestin-Dependent Signaling

PubMed Central

Luttrell, Louis M.

2010-01-01

Heptahelical G protein-coupled receptors are the most diverse and therapeutically important family of receptors in the human genome. Ligand binding activates heterotrimeric G proteins that transmit intracellular signals by regulating effector enzymes or ion channels. G protein signaling is terminated, in large part, by arrestin binding, which uncouples the receptor and G protein and targets the receptor for internalization. It is clear, however, that heptahelical receptor signaling does not end with desensitization. Arrestins bind a host of catalytically active proteins and serve as ligand-regulated scaffolds that recruit protein and lipid kinase, phosphatase, phosphodiesterase, and ubiquitin ligase activity into the receptor-arrestin complex. Although many of these arrestin-bound effectors serve to modulate G protein signaling, degrading second messengers and regulating endocytosis and trafficking, other signals seem to extend beyond the receptor-arrestin complex to regulate such processes as protein translation and gene transcription. Although these findings have led to a re-envisioning of heptahelical receptor signaling, little is known about the physiological roles of arrestin-dependent signaling. In vivo, the duality of arrestin function makes it difficult to dissociate the consequences of arrestin-dependent desensitization from those that might be ascribed to arrestin-mediated signaling. Nonetheless, recent evidence generated using arrestin knockouts, G protein-uncoupled receptor mutants, and arrestin pathway-selective “biased agonists” is beginning to reveal that arrestin signaling plays important roles in the retina, central nervous system, cardiovascular system, bone remodeling, immune system, and cancer. Understanding the signaling roles of arrestins may foster the development of pathway-selective drugs that exploit these pathways for therapeutic benefit. PMID:20427692

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells.

PubMed

Figarola, James L; Singhal, Jyotsana; Tompkins, Joshua D; Rogers, George W; Warden, Charles; Horne, David; Riggs, Arthur D; Awasthi, Sanjay; Singhal, Sharad S

2015-12-18

Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Respiratory uncoupling by increased H(+) or K(+) flux is beneficial for heart mitochondrial turnover of reactive oxygen species but not for permeability transition.

PubMed

Morota, Saori; Piel, Sarah; Hansson, Magnus J

2013-09-22

Ischemic preconditioning has been proposed to involve changes in mitochondrial H(+) and K(+) fluxes, in particular through activation of uncoupling proteins and ATP-sensitive K(+) channels (MitoKATP). The objectives of the present study were to explore how increased H(+) and K(+) fluxes influence heart mitochondrial physiology with regard to production and scavenging of reactive oxygen species (ROS), volume changes and resistance to calcium-induced mitochondrial permeability transition (mPT). Isolated rat heart mitochondria were exposed to a wide concentration range of the protonophore CCCP or the potassium ionophore valinomycin to induce increased H(+) and K(+) conductance, respectively. Simultaneous monitoring of mitochondrial respiration and calcium retention capacity (CRC) demonstrated that the relative increase in respiration caused by valinomycin or CCCP correlated with a decrease in CRC, and that no level of respiratory uncoupling was associated with enhanced resistance to mPT. Mitochondria suspended in hyperosmolar buffer demonstrated a dose-dependent reduction in CRC with increasing osmolarity. However, mitochondria in hypoosmolar buffer to increase matrix volume did not display increased CRC. ROS generation was reduced by both K(+)- and H(+)-mediated respiratory uncoupling. The ability of heart mitochondria to detoxify H2O2 was substantially greater than the production rate. The H2O2 detoxification was dependent on respiratory substrates and was dramatically decreased following calcium-induced mPT, but was unaffected by uncoupling via increased K(+) and H(+) conductance. It is concluded that respiratory uncoupling is not directly beneficial to rat heart mitochondrial resistance to calcium overload irrespective of whether H(+) or K(+) conductance is increased. The negative effects of respiratory uncoupling thus probably outweigh the reduction in ROS generation and a potential positive effect by increased matrix volume, resulting in a net sensitization of heart mitochondria to mPT activation.

Uncoupling proteins and the control of mitochondrial reactive oxygen species production.

PubMed

Mailloux, Ryan J; Harper, Mary-Ellen

2011-09-15

Reactive oxygen species (ROS), natural by-products of aerobic respiration, are important cell signaling molecules, which left unchecked can severely impair cellular functions and induce cell death. Hence, cells have developed a series of systems to keep ROS in the nontoxic range. Uncoupling proteins (UCPs) 1-3 are mitochondrial anion carrier proteins that are purported to play important roles in minimizing ROS emission from the electron transport chain. The function of UCP1 in this regard is highly contentious. However, UCPs 2 and 3 are generally thought to be activated by ROS or ROS by-products to induce proton leak, thus providing a negative feedback loop for mitochondrial ROS production. In our laboratory, we have not only confirmed that ROS activate UCP2 and UCP3, but also demonstrated that UCP2 and UCP3 are controlled by covalent modification by glutathione. Furthermore, the reversible glutathionylation is required to activate/inhibit UCP2 and UCP3, but not UCP1. Hence, our findings are consistent with the notion that UCPs 2 and 3 are acutely activated by ROS, which then directly modulate the glutathionylation status of the UCP to decrease ROS emission and participate in cell signaling mechanisms. Copyright © 2011 Elsevier Inc. All rights reserved.

DJ-1 links muscle ROS production with metabolic reprogramming and systemic energy homeostasis in mice.

PubMed

Shi, Sally Yu; Lu, Shun-Yan; Sivasubramaniyam, Tharini; Revelo, Xavier S; Cai, Erica P; Luk, Cynthia T; Schroer, Stephanie A; Patel, Prital; Kim, Raymond H; Bombardier, Eric; Quadrilatero, Joe; Tupling, A Russell; Mak, Tak W; Winer, Daniel A; Woo, Minna

2015-06-16

Reactive oxygen species (ROS) have been linked to a wide variety of pathologies, including obesity and diabetes, but ROS also act as endogenous signalling molecules, regulating numerous biological processes. DJ-1 is one of the most evolutionarily conserved proteins across species, and mutations in DJ-1 have been linked to some cases of Parkinson's disease. Here we show that DJ-1 maintains cellular metabolic homeostasis via modulating ROS levels in murine skeletal muscles, revealing a role of DJ-1 in maintaining efficient fuel utilization. We demonstrate that, in the absence of DJ-1, ROS uncouple mitochondrial respiration and activate AMP-activated protein kinase, which triggers Warburg-like metabolic reprogramming in muscle cells. Accordingly, DJ-1 knockout mice exhibit higher energy expenditure and are protected from obesity, insulin resistance and diabetes in the setting of fuel surplus. Our data suggest that promoting mitochondrial uncoupling may be a potential strategy for the treatment of obesity-associated metabolic disorders.

[Interaction of free fatty acids with mitochondria during uncoupling of oxidative phosphorylation].

PubMed

Samartsev, V N; Rybakova, S R; Dubinin, M V

2013-01-01

The activity of free saturated fatty acids (caprylic, capric, lauric, myristic, palmitic and stearic) as inducers and regulators of uncoupling of oxidative phosphorylation with participation of ADP/ATP antiporter, aspartate/glutamate antiporter and cyclosporin A-sensitive structure was investigated in experiments on rat liver mitochondria. It is established that at equal uncoupling activity of fatty acids the regulatory effect is minimal for caprylic acid and raised with increasing the hydrophobicity of fatty acids reaching the maximum value for stearic acid. There exists the linear dependence of the regulatory effect value of fatty acids on fatty acids content in the hydrophobic region of the inner membrane. The model that describes the interaction of fatty acids with the hydrophobic region of the mitochondrial inner membrane preserving functional activity of organelles is developed. It is established that if molecules of various fatty acids being in the hydrophobic region of the membrane are equally effective as uncoupling regulators, their specific uncoupling activity is different. Caprylic acid, a short-chain fatty acid, possesses the highest uncoupling activity. As the acyl chain length increases, the specific uncoupling activity of fatty acids reduces exponentially. Under these conditions components of the uncoupling activity sensitive to glutamate and carboxyatractylate and glutamate and insensitive to these reagents (but sensitive to cyclosporin A) change approximately equally.

Low Concentrations of Uncouplers of Oxidative Phosphorylation Prevent Inflammatory Activation of Endothelial Cells by Tumor Necrosis Factor.

PubMed

Romaschenko, V P; Zinovkin, R A; Galkin, I I; Zakharova, V V; Panteleeva, A A; Tokarchuk, A V; Lyamzaev, K G; Pletjushkina, O Yu; Chernyak, B V; Popova, E N

2015-05-01

In endothelial cells, mitochondria play an important regulatory role in physiology as well as in pathophysiology related to excessive inflammation. We have studied the effect of low doses of mitochondrial uncouplers on inflammatory activation of endothelial cells using the classic uncouplers 2,4-dinitrophenol and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole, as well as the mitochondria-targeted cationic uncoupler dodecyltriphenylphosphonium (C12TPP). All of these uncouplers suppressed the expression of E-selectin, adhesion molecules ICAM1 and VCAM1, as well as the adhesion of neutrophils to endothelium induced by tumor necrosis factor (TNF). The antiinflammatory action of the uncouplers was at least partially mediated by the inhibition of NFκB activation due to a decrease in phosphorylation of the inhibitory subunit IκBα. The dynamic concentration range for the inhibition of ICAM1 expression by C12TPP was three orders of magnitude higher compared to the classic uncouplers. Probably, the decrease in membrane potential inhibited the accumulation of penetrating cations into mitochondria, thus lowering the uncoupling activity and preventing further loss of mitochondrial potential. Membrane potential recovery after the removal of the uncouplers did not abolish its antiinflammatory action. Thus, mild uncoupling could induce TNF resistance in endothelial cells. We found no significant stimulation of mitochondrial biogenesis or autophagy by the uncouplers. However, we observed a decrease in the relative amount of fragmented mitochondria. The latter may significantly change the signaling properties of mitochondria. Earlier we showed that both classic and mitochondria-targeted antioxidants inhibited the TNF-induced NFκB-dependent activation of endothelium. The present data suggest that the antiinflammatory effect of mild uncoupling is related to its antioxidant action.

Temperature controls oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria.

PubMed

Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Koziel, Agnieszka; Majerczak, Joanna; Zoladz, Jerzy A

2015-06-01

Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling, and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25 °C) and hyperthermia (42 °C) compared to the physiological temperature of resting muscle (35 °C). For nonphosphorylating mitochondria, increasing the temperature from 25 to 42 °C led to a decrease in membrane potential, hydrogen peroxide production, and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. However, the efficiency of oxidative phosphorylation decreased, whereas the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased, with increasing assay temperature. An increase in proton leak, including uncoupling protein-mediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production. Copyright © 2015 Elsevier Inc. All rights reserved.

Lipotoxicity, fatty acid uncoupling and mitochondrial carrier function.

PubMed

Rial, Eduardo; Rodríguez-Sánchez, Leonor; Gallardo-Vara, Eunate; Zaragoza, Pilar; Moyano, Eva; González-Barroso, M Mar

2010-01-01

Diseases like obesity, diabetes or generalized lipodystrophy cause a chronic elevation of circulating fatty acids that can become cytotoxic, a condition known as lipotoxicity. Fatty acids cause oxidative stress and alterations in mitochondrial structure and function. The uncoupling of the oxidative phosphorylation is one of the most recognized deleterious fatty acid effects and several metabolite transporters are known to mediate in their action. The fatty acid interaction with the carriers leads to membrane depolarization and/or the conversion of the carrier into a pore. The result is the opening of the permeability transition pore and the initiation of apoptosis. Unlike the other members of the mitochondrial carrier superfamily, the eutherian uncoupling protein UCP1 has evolved to achieve its heat-generating capacity in the physiological context provided by the brown adipocyte and therefore it is activated by the low fatty acid concentrations generated by the noradrenaline-stimulated lipolysis. Copyright © 2010 Elsevier B.V. All rights reserved.

Olive Leaf Extract from Sicilian Cultivar Reduced Lipid Accumulation by Inducing Thermogenic Pathway during Adipogenesis

PubMed Central

Palmeri, Rosa; Monteleone, Julieta I.; Spagna, Giovanni; Restuccia, Cristina; Raffaele, Marco; Vanella, Luca; Li Volti, Giovanni; Barbagallo, Ignazio

2016-01-01

Olive leaves contain a wide variety of phenolic compounds belonging to phenolic acids, phenolic alcohols, flavonoids, and secoiridoids, and include also many other pharmacological active compounds. They could play an important role in human diet and health because of their ability to lower blood pressure, increase coronary arteries blood flow and decrease the risk of cardiovascular diseases. The aim of this study was to investigate the effect of olive leaf extract (OLE) from Sicilian cultivar on adipogenic differentiation of human adipose derived mesenchymal stem cells and its impact on lipid metabolism. We showed that OLE treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein uncoupling protein 1, sirtuin 1, peroxisome proliferator-activated receptor alpha, and coactivator 1 alpha. Furthermore, OLE significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. Taken together, our results suggest that OLE may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis. PMID:27303302

Activation and function of mitochondrial uncoupling protein in plants.

PubMed

Smith, Anna M O; Ratcliffe, R George; Sweetlove, Lee J

2004-12-10

Plant mitochondrial uncoupling protein (UCP) is activated by superoxide suggesting that it may function to minimize mitochondrial reactive oxygen species (ROS) formation. However, the precise mechanism of superoxide activation and the exact function of UCP in plants are not known. We demonstrate that 4-hydroxy-2-nonenal (HNE), a product of lipid peroxidation, and a structurally related compound, trans-retinal, stimulate a proton conductance in potato mitochondria that is inhibitable by GTP (a characteristic of UCP). Proof that the effects of HNE and trans-retinal are mediated by UCP is provided by examination of proton conductance in transgenic plants overexpressing UCP. These experiments demonstrate that the mechanism of activation of UCP is conserved between animals and plants and imply a conservation of function. Mitochondria from transgenic plants overexpressing UCP were further studied to provide insight into function. Experimental conditions were designed to mimic a bioenergetic state that might be found in vivo (mitochondria were supplied with pyruvate as well as tricarboxylic cycle acids at in vivo cytosolic concentrations and an exogenous ATP sink was established). Under such conditions, an increase in UCP protein content resulted in a modest but significant decrease in the rate of superoxide production. In addition, 13C-labeling experiments revealed an increase in the conversion of pyruvate to citrate as a result of increased UCP protein content. These results demonstrate that under simulated in vivo conditions, UCP is active and suggest that UCP may influence not only mitochondrial ROS production but also tricarboxylic acid cycle flux.

Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction in Caenorhabditis elegans Models of Polyglutamine Diseases

PubMed Central

Shemesh, Netta; Shai, Nadav; Meshnik, Lana; Katalan, Rotem; Ben-Zvi, Anat

2017-01-01

Caenorhabditis elegans somatic protein homeostasis (proteostasis) is actively remodeled at the onset of reproduction. This proteostatic collapse is regulated cell-nonautonomously by signals from the reproductive system that transmit the commitment to reproduction to somatic cells. Here, we asked whether the link between the reproductive system and somatic proteostasis could be uncoupled by activating downstream effectors in the gonadal longevity cascade. Specifically, we examined whether over-expression of lipl-4 (lipl-4(oe)), a target gene of the gonadal longevity pathway, or increase in arachidonic acid (AA) levels, associated with lipl-4(oe), modulated proteostasis and reproduction. We found that lipl-4(oe) rescued somatic proteostasis and postponed the onset of aggregation and toxicity in C. elegans models of polyglutamine (polyQ) diseases. However, lipl-4(oe) also disrupted fatty acid transport into developing oocytes and reduced reproductive success. In contrast, diet supplementation of AA recapitulated lipl-4(oe)-mediated proteostasis enhancement in wild type animals but did not affect the reproductive system. Thus, the gonadal longevity pathway mediates a trade-off between somatic maintenance and reproduction, in part by regulating the expression of genes, such as lipl-4, with inverse effects on somatic maintenance and reproduction. We propose that AA could uncouple such germline to soma crosstalk, with beneficial implications protein misfolding diseases. PMID:28503130

Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins.

PubMed

Talbot, Darren A; Duchamp, Claude; Rey, Benjamin; Hanuise, Nicolas; Rouanet, Jean Louis; Sibille, Brigitte; Brand, Martin D

2004-07-01

Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate).

Renal transplantation induces mitochondrial uncoupling, increased kidney oxygen consumption, and decreased kidney oxygen tension.

PubMed

Papazova, Diana A; Friederich-Persson, Malou; Joles, Jaap A; Verhaar, Marianne C

2015-01-01

Hypoxia is an acknowledged pathway to renal injury and ischemia-reperfusion (I/R) and is known to reduce renal oxygen tension (Po2). We hypothesized that renal I/R increases oxidative damage and induces mitochondrial uncoupling, resulting in increased oxygen consumption and hence kidney hypoxia. Lewis rats underwent syngenic renal transplantation (TX) and contralateral nephrectomy. Controls were uninephrectomized (1K-CON) or left untreated (2K-CON). After 7 days, urinary excretion of protein and thiobarbituric acid-reactive substances were measured, and after 14 days glomerular filtration rate (GFR), renal blood flow, whole kidney Qo2, cortical Po2, kidney cortex mitochondrial uncoupling, renal oxidative damage, and tubulointerstitial injury were assessed. TX, compared with 1K-CON, resulted in mitochondrial uncoupling mediated via uncoupling protein-2 (16 ± 3.3 vs. 0.9 ± 0.4 pmol O2 · s(-1)· mg protein(-1), P < 0.05) and increased whole kidney Qo2 (55 ± 16 vs. 33 ± 10 μmol O2/min, P < 0.05). Corticomedullary Po2 was lower in TX compared with 1K-CON (30 ± 13 vs. 47 ± 4 μM, P < 0.05) whereas no significant difference was observed between 2K-CON and 1K-CON rats. Proteinuria, oxidative damage, and the tubulointerstitial injury score were not significantly different in 1K-CON and TX. Treatment of donors for 5 days with mito-TEMPO reduced mitochondrial uncoupling but did not affect renal hemodynamics, Qo2, Po2, or injury. Collectively, our results demonstrate increased mitochondrial uncoupling as an early event after experimental renal transplantation associated with increased oxygen consumption and kidney hypoxia in the absence of increases in markers of damage. Copyright © 2015 the American Physiological Society.

Ameliorating Endothelial Mitochondrial Dysfunction Restores Coronary Function via Transient Receptor Potential Vanilloid 1-Mediated Protein Kinase A/Uncoupling Protein 2 Pathway.

PubMed

Xiong, Shiqiang; Wang, Peijian; Ma, Liqun; Gao, Peng; Gong, Liuping; Li, Li; Li, Qiang; Sun, Fang; Zhou, Xunmei; He, Hongbo; Chen, Jing; Yan, Zhencheng; Liu, Daoyan; Zhu, Zhiming

2016-02-01

Coronary heart disease arising from atherosclerosis is a leading cause of cardiogenic death worldwide. Mitochondria are the principal source of reactive oxygen species (ROS), and defective oxidative phosphorylation by the mitochondrial respiratory chain contributes to ROS generation. Uncoupling protein 2 (UCP2), an adaptive antioxidant defense factor, protects against mitochondrial ROS-induced endothelial dysfunction in atherosclerosis. The activation of transient receptor potential vanilloid 1 (TRPV1) attenuates vascular dysfunction. Therefore, whether TRPV1 activation antagonizes coronary lesions by alleviating endothelial mitochondrial dysfunction and enhancing the activity of the protein kinase A/UCP2 pathway warrants examination. ApoE(-/-), ApoE(-/-)/TRPV1(-/-), and ApoE(-/-)/UCP2(-/-) mice were fed standard chow, a high-fat diet (HFD), or the HFD plus 0.01% capsaicin. HFD intake profoundly impaired coronary vasodilatation and myocardial perfusion and shortened the survival duration of ApoE(-/-) mice. TRPV1 or UCP2 deficiency exacerbated HFD-induced coronary dysfunction and was associated with increased ROS generation and reduced nitric oxide production in the endothelium. The activation of TRPV1 by capsaicin upregulated UCP2 expression via protein kinase A phosphorylation, thereby alleviating endothelial mitochondrial dysfunction and inhibiting mitochondrial ROS generation. In vivo, dietary capsaicin supplementation enhanced coronary relaxation and prolonged the survival duration of HFD-fed ApoE(-/-) mice. These effects were not observed in ApoE(-/-) mice lacking the TRPV1 or UCP2 gene. The upregulation of protein kinase A /UCP2 via TRPV1 activation ameliorates coronary dysfunction and prolongs the lifespan of atherosclerotic mice by ameliorating endothelial mitochondrial dysfunction. Dietary capsaicin supplementation may represent a promising intervention for the primary prevention of coronary heart disease. © 2015 American Heart Association, Inc.

Single nucleotide polymorphisms linked to mitochondrial uncoupling protein genes UCP2 and UCP3 affect mitochondrial metabolism and healthy aging in female nonagenarians.

PubMed

Kim, Sangkyu; Myers, Leann; Ravussin, Eric; Cherry, Katie E; Jazwinski, S Michal

2016-08-01

Energy expenditure decreases with age, but in the oldest-old, energy demand for maintenance of body functions increases with declining health. Uncoupling proteins have profound impact on mitochondrial metabolic processes; therefore, we focused attention on mitochondrial uncoupling protein genes. Alongside resting metabolic rate (RMR), two SNPs in the promoter region of UCP2 were associated with healthy aging. These SNPs mark potential binding sites for several transcription factors; thus, they may affect expression of the gene. A third SNP in the 3'-UTR of UCP3 interacted with RMR. This UCP3 SNP is known to impact UCP3 expression in tissue culture cells, and it has been associated with body weight and mitochondrial energy metabolism. The significant main effects of the UCP2 SNPs and the interaction effect of the UCP3 SNP were also observed after controlling for fat-free mass (FFM) and physical-activity related energy consumption. The association of UCP2/3 with healthy aging was not found in males. Thus, our study provides evidence that the genetic risk factors for healthy aging differ in males and females, as expected from the differences in the phenotypes associated with healthy aging between the two sexes. It also has implications for how mitochondrial function changes during aging.

Single nucleotide polymorphisms linked to mitochondrial uncoupling protein genes UCP2 and UCP3 affect mitochondrial metabolism and healthy aging in female nonagenarians

PubMed Central

Kim, Sangkyu; Myers, Leann; Ravussin, Eric; Cherry, Katie E.; Jazwinski, S. Michal

2016-01-01

Energy expenditure decreases with age, but in the oldest-old, energy demand for maintenance of body functions increases with declining health. Uncoupling proteins have profound impact on mitochondrial metabolic processes; therefore, we focused attention on mitochondrial uncoupling protein genes. Alongside resting metabolic rate (RMR), two SNPs in the promoter region of UCP2 were associated with healthy aging. These SNPs mark potential binding sites for several transcription factors; thus, they may affect expression of the gene. A third SNP in the 3′-UTR of UCP3 interacted with RMR. This UCP3 SNP is known to impact UCP3 expression in tissue culture cells, and it has been associated with body weight and mitochondrial energy metabolism. The significant main effects of the UCP2 SNPs and the interaction effect of the UCP3 SNP were also observed after controlling for fat-free mass (FFM) and physical-activity related energy consumption. The association of UCP2/3 with healthy aging was not found in males. Thus, our study provides evidence that the genetic risk factors for healthy aging differ in males and females, as expected from the differences in the phenotypes associated with healthy aging between the two sexes. It also has implications for how mitochondrial function changes during aging. PMID:26965008

The conserved regulation of mitochondrial uncoupling proteins: From unicellular eukaryotes to mammals.

PubMed

Woyda-Ploszczyca, Andrzej M; Jarmuszkiewicz, Wieslawa

2017-01-01

Uncoupling proteins (UCPs) belong to the mitochondrial anion carrier protein family and mediate regulated proton leak across the inner mitochondrial membrane. Free fatty acids, aldehydes such as hydroxynonenal, and retinoids activate UCPs. However, there are some controversies about the effective action of retinoids and aldehydes alone; thus, only free fatty acids are commonly accepted positive effectors of UCPs. Purine nucleotides such as GTP inhibit UCP-mediated mitochondrial proton leak. In turn, membranous coenzyme Q may play a role as a redox state-dependent metabolic sensor that modulates the complete activation/inhibition of UCPs. Such regulation has been observed for UCPs in microorganisms, plant and animal UCP1 homologues, and UCP1 in mammalian brown adipose tissue. The origin of UCPs is still under debate, but UCP homologues have been identified in all systematic groups of eukaryotes. Despite the differing levels of amino acid/DNA sequence similarities, functional studies in unicellular and multicellular organisms, from amoebae to mammals, suggest that the mechanistic regulation of UCP activity is evolutionarily well conserved. This review focuses on the regulatory feedback loops of UCPs involving free fatty acids, aldehydes, retinoids, purine nucleotides, and coenzyme Q (particularly its reduction level), which may derive from the early stages of evolution as UCP first emerged. Copyright © 2016 Elsevier B.V. All rights reserved.

Repeated immobilization stress increases uncoupling protein 1 expression and activity in Wistar rats.

PubMed

Gao, Bihu; Kikuchi-Utsumi, Kazue; Ohinata, Hiroshi; Hashimoto, Masaaki; Kuroshima, Akihiro

2003-06-01

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

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.

Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

PubMed

Mikitas, Olga V; Ivin, Yuri Y; Golyshev, Sergey A; Povarova, Natalia V; Galkina, Svetlana I; Pletjushkina, Olga Y; Nadezhdina, Elena S; Gmyl, Anatoly P; Agol, Vadim I

2012-05-01

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

Suppression of Injuries Caused by a Lytic RNA Virus (Mengovirus) and Their Uncoupling from Viral Reproduction by Mutual Cell/Virus Disarmament

PubMed Central

Mikitas, Olga V.; Ivin, Yuri Y.; Golyshev, Sergey A.; Povarova, Natalia V.; Galkina, Svetlana I.; Pletjushkina, Olga Y.; Nadezhdina, Elena S.; Gmyl, Anatoly P.

2012-01-01

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive (“security”) viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L− mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases. PMID:22438537

Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis.

PubMed

Musicki, Biljana; Liu, Tongyun; Sezen, Sena F; Burnett, Arthur L

2012-08-01

Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) ), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Relative to hemi mice, SCD increased (P<0.05) protein expression of NADPH oxidase subunits p67(phox) , p47(phox) , and gp91(phox) , 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P<0.05) the abnormalities in protein expressions of p47(phox) , gp91(phox) (but not p67(phox) ) and 4-HNE, but only slightly (P>0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. © 2012 International Society for Sexual Medicine.

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

PubMed Central

Musicki, Biljana; Liu, Tongyun; Sezen, Sena F.; Burnett, Arthur L.

2012-01-01

Introduction Sickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown. Aims We evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis. Methods SCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67phox, p47phox, and gp91phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase-1 [GPx1]) were measured by Western blot in penes. Main Outcome Measures Sources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis. Results Relative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67phox, p47phox, and gp91phox, 4-HNE-modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47phox, gp91phox (but not p67phox) and 4-HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters. Conclusion NADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. PMID:22620981

Uncoupling protein and ATP/ADP carrier increase mitochondrial proton conductance after cold adaptation of king penguins

PubMed Central

Talbot, Darren A; Duchamp, Claude; Rey, Benjamin; Hanuise, Nicolas; Rouanet, Jean Louis; Sibille, Brigitte; Brand, Martin D

2004-01-01

Juvenile king penguins develop adaptive thermogenesis after repeated immersion in cold water. However, the mechanisms of such metabolic adaptation in birds are unknown, as they lack brown adipose tissue and uncoupling protein-1 (UCP1), which mediate adaptive non-shivering thermogenesis in mammals. We used three different groups of juvenile king penguins to investigate the mitochondrial basis of avian adaptive thermogenesis in vitro. Skeletal muscle mitochondria isolated from penguins that had never been immersed in cold water showed no superoxide-stimulated proton conductance, indicating no functional avian UCP. Skeletal muscle mitochondria from penguins that had been either experimentally immersed or naturally adapted to cold water did possess functional avian UCP, demonstrated by a superoxide-stimulated, GDP-inhibitable proton conductance across their inner membrane. This was associated with a markedly greater abundance of avian UCP mRNA. In the presence (but not the absence) of fatty acids, these mitochondria also showed a greater adenine nucleotide translocase-catalysed proton conductance than those from never-immersed penguins. This was due to an increase in the amount of adenine nucleotide translocase. Therefore, adaptive thermogenesis in juvenile king penguins is linked to two separate mechanisms of uncoupling of oxidative phosphorylation in skeletal muscle mitochondria: increased proton transport activity of avian UCP (dependent on superoxide and inhibited by GDP) and increased proton transport activity of the adenine nucleotide translocase (dependent on fatty acids and inhibited by carboxyatractylate). PMID:15146050

26th National Neurotrauma Symposium

DTIC Science & Technology

2008-12-31

Sanchez Alvaro I., Puyana Juan C., Fabio Anthony, Adelson P. David 81 COMPARATIVE NEUROPROTECTIVE EFFECTS OF CYCLOSPORIN A AND NIM811, A NON...Xiangbai, Fabio Anthony, Yuan Zhifang, Niyonkuru Christian, Wagner Amy 99 ACTIVATION OF MITOCHONDRIAL UNCOUPLING PROTEINS BY POLYUNSATURATED FATTY...Metabolism/Physiological Assessments TBI Mitchell Cohen University of California, San Francisco Ferguson Adam, Cohen Mitchell, Morabito Diane

A Brg1 mutation that uncouples ATPase activity from chromatin remodeling reveals an essential role for SWI/SNF-related complexes in β-globin expression and erythroid development

PubMed Central

Bultman, Scott J.; Gebuhr, Thomas C.; Magnuson, Terry

2005-01-01

The Brg1 catalytic subunit of SWI/SNF-related complexes has been implicated in many developmental and physiological processes, but null homozygotes die as blastocysts prior to implantation. To circumvent this early embryonic lethality, we performed an ENU mutagenesis screen and generated a Brg1 hypomorph mutation in the ATPase domain. The mutant Brg1 protein is stable, assembles into SWI/SNF-related complexes, and exhibits normal ATPase activity but is unable to establish DNase I hypersensitivity sites characteristic of open chromatin. Mutant embryos develop normally until midgestation but then exhibit a distinct block in the development of the erythroid lineage, leading to anemia and death. The mutant Brg1 protein is recruited to the β-globin locus, but chromatin remodeling and transcription are perturbed. Histone acetylation and DNA methylation are also affected. To our knowledge, Brg1 is the first chromatin-modifying factor shown to be required for β-globin regulation and erythropoiesis in vivo. Not only does this mutation establish a role for Brg1 during organogenesis, it also demonstrates that ATPase activity can be uncoupled from chromatin remodeling. PMID:16287714

FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2.

PubMed

Steen, Kaylee A; Xu, Hongliang; Bernlohr, David A

2017-01-15

Obesity-linked metabolic disease is mechanistically associated with the accumulation of proinflammatory macrophages in adipose tissue, leading to increased reactive oxygen species (ROS) production and chronic low-grade inflammation. Previous work has demonstrated that deletion of the adipocyte fatty acid-binding protein (FABP4/aP2) uncouples obesity from inflammation via upregulation of the uncoupling protein 2 (UCP2). Here, we demonstrate that ablation of FABP4/aP2 regulates systemic redox capacity and reduces cellular protein sulfhydryl oxidation and, in particular, oxidation of mitochondrial protein cysteine residues. Coincident with the loss of FABP4/aP2 is the upregulation of the antioxidants superoxide dismutase (SOD2), catalase, methionine sulfoxide reductase A, and the 20S proteasome subunits PSMB5 and αβ. Reduced mitochondrial protein oxidation in FABP4/aP2 -/- macrophages attenuates the mitochondrial unfolded-protein response (mtUPR) as measured by expression of heat shock protein 60, Clp protease, and Lon peptidase 1. Consistent with a diminished mtUPR, FABP4/aP2 -/- macrophages exhibit reduced expression of cleaved caspase-1 and NLRP3. Secretion of interleukin 1β (IL-1β), in response to inflammasome activation, is ablated in FABP4/aP2 -/- macrophages, as well as in FABP4/aP2 inhibitor-treated cells, but partially rescued in FABP4/aP2-null macrophages when UCP2 is silenced. Collectively, these data offer a novel pathway whereby FABP4/aP2 regulates macrophage redox signaling and inflammasome activation via control of UCP2 expression. Copyright © 2017 American Society for Microbiology.

Skeletal Muscle Thermogenesis and Its Role in Whole Body Energy Metabolism

PubMed Central

Herrera, Jose Luis; Reis, Felipe C. G.

2017-01-01

Obesity and diabetes has become a major epidemic across the globe. Controlling obesity has been a challenge since this would require either increased physical activity or reduced caloric intake; both are difficult to enforce. There has been renewed interest in exploiting pathways such as uncoupling protein 1 (UCP1)-mediated uncoupling in brown adipose tissue (BAT) and white adipose tissue to increase energy expenditure to control weight gain. However, relying on UCP1-based thermogenesis alone may not be sufficient to control obesity in humans. On the other hand, skeletal muscle is the largest organ and a major contributor to basal metabolic rate and increasing energy expenditure in muscle through nonshivering thermogenic mechanisms, which can substantially affect whole body metabolism and weight gain. In this review we will describe the role of Sarcolipin-mediated uncoupling of Sarcoplasmic Reticulum Calcium ATPase (SERCA) as a potential mechanism for increased energy expenditure both during cold and diet-induced thermogenesis. PMID:29086530

Constitutive NOS uncoupling and NADPH oxidase upregulation in the penis of type 2 diabetic men with erectile dysfunction

PubMed Central

Musicki, Biljana; Burnett, Arthur L.

2016-01-01

Erectile dysfunction (ED) associated with type 2 diabetes mellitus (T2DM) involves dysfunctional nitric oxide (NO) signaling and increased oxidative stress in the penis. However, the mechanisms of endothelial NO synthase (eNOS) and neuronal NO synthase (nNOS) dysregulation, and the sources of oxidative stress, are not well defined, particularly at the human level. The objective of this study was to define whether uncoupled eNOS and nNOS, and NADPH oxidase upregulation, contribute to the pathogenesis of ED in T2DM men. Penile erectile tissue was obtained from 9 T2DM patients with ED who underwent penile prosthesis surgery for ED, and from 6 control patients without T2DM or ED who underwent penectomy for penile cancer. The dimer-to-monomer protein expression ratio, an indicator of uncoupling for both eNOS and nNOS, total protein expressions of eNOS and nNOS, as well as protein expressions of NADPH oxidase catalytic subunit gp91phox (an enzymatic source of oxidative stress) and 4-hydroxy-2-nonenal [4-HNE] and nitrotyrosine (markers of oxidative stress) were measured by Western blot in this tissue. In the erectile tissue of T2DM men, eNOS and nNOS uncoupling and protein expressions of NADPH oxidase subunit gp91phox, 4-HNE- and nitrotyrosine-modified proteins were significantly (p<0.05) increased compared to control values. Total eNOS and nNOS protein expressions were not significantly different between the groups. In conclusion, mechanisms of T2DM-associated ED in the human penis may involve uncoupled eNOS and nNOS and NADPH oxidase upregulation. Our description of molecular factors contributing to the pathogenesis of T2DM-associated ED at the human level is relevant for advancing clinically therapeutic approaches to restore erectile function in T2DM patients. PMID:28076881

Metformin induces oxidative stress in white adipocytes and raises uncoupling protein 2 levels.

PubMed

Anedda, Andrea; Rial, Eduardo; González-Barroso, M Mar

2008-10-01

Metformin is a drug widely used to treat type 2 diabetes. It enhances insulin sensitivity by improving glucose utilization in tissues like liver or muscle. Metformin inhibits respiration, and the decrease in cellular energy activates the AMP-activated protein kinase that in turn switches on catabolic pathways. Moreover, metformin increases lipolysis and beta-oxidation in white adipose tissue, thereby reducing the triglyceride stores. The uncoupling proteins (UCPs) are transporters that lower the efficiency of mitochondrial oxidative phosphorylation. UCP2 is thought to protect against oxidative stress although, alternatively, it could play an energy dissipation role. The aim of this work was to analyse the involvement of UCP2 on the effects of metformin in white adipocytes. We studied the effect of this drug in differentiating 3T3-L1 adipocytes and found that metformin causes oxidative stress since it increases the levels of reactive oxygen species (ROS) and lowers the aconitase activity. Variations in UCP2 protein levels parallel those of ROS. Metformin also increases lipolysis in these cells although only when the levels of ROS and UCP2 have decreased. Hence, UCP2 does not appear to be needed to facilitate fatty acid oxidation. Furthermore, treatment of C57BL/6 mice with metformin also augmented the levels of UCP2 in epididymal white adipose tissue. We conclude that metformin treatment leads to the overexpression of UCP2 in adipocytes to minimize the oxidative stress that is probably due to the inhibition of respiration caused by the drug.

Effect of Zingiber officinale Supplementation on Obesity Management with Respect to the Uncoupling Protein 1 -3826A>G and ß3-adrenergic Receptor Trp64Arg Polymorphism.

PubMed

Ebrahimzadeh Attari, Vahideh; Asghari Jafarabadi, Mohammad; Zemestani, Maryam; Ostadrahimi, Alireza

2015-07-01

The present study aimed to investigate the effect of ginger (Zingiber officinale) supplementation on some obesity-associated parameters, with nutrigenetics approach. Accordingly, 80 eligible obese women (aged 18-45 years) were randomly assigned to receive either ginger (2-g ginger rhizomes powder as two 1-g tablets per day) or placebo supplements (corn starch with the same amount) for 12 weeks. Subjects were tested for changes in body weight, body mass index, waist and hip circumferences, body composition, appetite score, and dietary intake. Moreover, participants were genotyped for the -3826A>G and Trp64Arg polymorphisms of uncoupling protein 1 and ß3-adrenergic receptor genes, respectively. Over 12 weeks, ginger supplementation resulted in a slight but statistically significant decrease in all anthropometric measurements and total appetite score as compared with placebo group, which were more pronounced in subjects with the AA genotype for uncoupling protein 1 and Trp64Trp genotype for ß3-adrenergic receptor gene. However, there was no significant difference in changes of body composition and total energy and macronutrients intake between groups. In conclusion, our findings suggest that ginger consumption has potential in managing obesity, accompanying with an intervention-genotype interaction effect. However, further clinical trials need to explore ginger's efficacy as an anti-obesity agent in the form of powder, extract, or its active components. Copyright © 2015 John Wiley & Sons, Ltd.

Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation

PubMed Central

Aguer, Céline; Fiehn, Oliver; Seifert, Erin L.; Bézaire, Véronic; Meissen, John K.; Daniels, Amanda; Scott, Kyle; Renaud, Jean-Marc; Padilla, Marta; Bickel, David R.; Dysart, Michael; Adams, Sean H.; Harper, Mary-Ellen

2013-01-01

Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (∼40%) and energy expenditure (∼5–10%) and decreased oxidative stress (∼15–20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; ∼30% for WT and ∼70% for UCP3 Tg) and energy expenditure (∼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.—Aguer, C., Fiehn, O., Seifert, E. L., Bézaire, V., Meissen, J. K., Daniels, A., Scott, K., Renaud, J.-M., Padilla, M., Bickel, D. R., Dysart, M., Adams, S. H., Harper, M.-E. Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation. PMID:23825224

Brown fat in a protoendothermic mammal fuels eutherian evolution.

PubMed

Oelkrug, Rebecca; Goetze, Nadja; Exner, Cornelia; Lee, Yang; Ganjam, Goutham K; Kutschke, Maria; Müller, Saskia; Stöhr, Sigrid; Tschöp, Matthias H; Crichton, Paul G; Heldmaier, Gerhard; Jastroch, Martin; Meyer, Carola W

2013-01-01

Endothermy has facilitated mammalian species radiation, but the sequence of events leading to sustained thermogenesis is debated in multiple evolutionary models. Here we study the Lesser hedgehog tenrec (Echinops telfairi), a phylogenetically ancient, 'protoendothermic' eutherian mammal, in which constantly high body temperatures are reported only during reproduction. Evidence for nonshivering thermogenesis is found in vivo during periodic ectothermic-endothermic transitions. Anatomical studies reveal large brown fat-like structures in the proximity of the reproductive organs, suggesting physiological significance for parental care. Biochemical analysis demonstrates high mitochondrial proton leak catalysed by an uncoupling protein 1 ortholog. Strikingly, bioenergetic profiling of tenrec uncoupling protein 1 reveals similar thermogenic potency as modern mouse uncoupling protein 1, despite the large phylogenetic distance. The discovery of functional brown adipose tissue in this 'protoendothermic' mammal links nonshivering thermogenesis directly to the roots of eutherian evolution, suggesting physiological importance prior to sustained body temperatures and migration to the cold.

Brown fat in a protoendothermic mammal fuels eutherian evolution

PubMed Central

Oelkrug, Rebecca; Goetze, Nadja; Exner, Cornelia; Lee, Yang; Ganjam, Goutham K.; Kutschke, Maria; Müller, Saskia; Stöhr, Sigrid; Tschöp, Matthias H.; Crichton, Paul G.; Heldmaier, Gerhard; Jastroch, Martin; Meyer, Carola W.

2013-01-01

Endothermy has facilitated mammalian species radiation, but the sequence of events leading to sustained thermogenesis is debated in multiple evolutionary models. Here we study the Lesser hedgehog tenrec (Echinops telfairi), a phylogenetically ancient, ‘protoendothermic’ eutherian mammal, in which constantly high body temperatures are reported only during reproduction. Evidence for nonshivering thermogenesis is found in vivo during periodic ectothermic–endothermic transitions. Anatomical studies reveal large brown fat-like structures in the proximity of the reproductive organs, suggesting physiological significance for parental care. Biochemical analysis demonstrates high mitochondrial proton leak catalysed by an uncoupling protein 1 ortholog. Strikingly, bioenergetic profiling of tenrec uncoupling protein 1 reveals similar thermogenic potency as modern mouse uncoupling protein 1, despite the large phylogenetic distance. The discovery of functional brown adipose tissue in this ‘protoendothermic’ mammal links nonshivering thermogenesis directly to the roots of eutherian evolution, suggesting physiological importance prior to sustained body temperatures and migration to the cold. PMID:23860571

Mitochondrial uncoupling reduces exercise capacity despite several skeletal muscle metabolic adaptations.

PubMed

Schlagowski, A I; Singh, F; Charles, A L; Gali Ramamoorthy, T; Favret, F; Piquard, F; Geny, B; Zoll, J

2014-02-15

The effects of mitochondrial uncoupling on skeletal muscle mitochondrial adaptation and maximal exercise capacity are unknown. In this study, rats were divided into a control group (CTL, n = 8) and a group treated with 2,4-dinitrophenol, a mitochondrial uncoupler, for 28 days (DNP, 30 mg·kg(-1)·day(-1) in drinking water, n = 8). The DNP group had a significantly lower body mass (P < 0.05) and a higher resting oxygen uptake (Vo2, P < 0.005). The incremental treadmill test showed that maximal running speed and running economy (P < 0.01) were impaired but that maximal Vo2 (Vo2max) was higher in the DNP-treated rats (P < 0.05). In skinned gastrocnemius fibers, basal respiration (V0) was higher (P < 0.01) in the DNP-treated animals, whereas the acceptor control ratio (ACR, Vmax/V0) was significantly lower (P < 0.05), indicating a reduction in OXPHOS efficiency. In skeletal muscle, DNP activated the mitochondrial biogenesis pathway, as indicated by changes in the mRNA expression of PGC1-α and -β, NRF-1 and -2, and TFAM, and increased the mRNA expression of cytochrome oxidase 1 (P < 0.01). The expression of two mitochondrial proteins (prohibitin and Ndufs 3) was higher after DNP treatment. Mitochondrial fission 1 protein (Fis-1) was increased in the DNP group (P < 0.01), but mitofusin-1 and -2 were unchanged. Histochemical staining for NADH dehydrogenase and succinate dehydrogenase activity in the gastrocnemius muscle revealed an increase in the proportion of oxidative fibers after DNP treatment. Our study shows that mitochondrial uncoupling induces several skeletal muscle adaptations, highlighting the role of mitochondrial coupling as a critical factor for maximal exercise capacities. These results emphasize the importance of investigating the qualitative aspects of mitochondrial function in addition to the amount of mitochondria.

Glutathionylation acts as a control switch for uncoupling proteins UCP2 and UCP3.

PubMed

Mailloux, Ryan J; Seifert, Erin L; Bouillaud, Frédéric; Aguer, Céline; Collins, Sheila; Harper, Mary-Ellen

2011-06-17

The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys(25) and Cys(259) as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.

Uncoupling phototoxicity-elicited neural dysmorphology and death by insidious function and selective impairment of Ran-binding protein 2 (Ranbp2).

PubMed

Cho, Kyoung-in; Haney, Victoria; Yoon, Dosuk; Hao, Yin; Ferreira, Paulo A

2015-12-21

Morphological disintegration of neurons is coupled invariably to neural death. In particular, disruption of outer segments of photoreceptor neurons triggers photoreceptor death regardless of the pathological stressors. We show that Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) mice with mutations in SUMO-binding motif (SBM) of cyclophilin-like domain (CLD) of Ran-binding protein 2 (Ranbp2) expressed in a null Ranbp2 background lack untoward effects in photoreceptors in the absence of light-stress. However, compared to wild type photoreceptors, light-stress elicits profound disintegration of outer segments of Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) with paradoxical age-dependent resistance of photoreceptors to death and genotype-independent activation of caspases. Ranbp2(-/-)::Tg-Ranbp2(CLDm-HA) exhibit photoreceptor death-independent changes in ubiquitin-proteasome system (UPS), but death-dependent increase of ubiquitin carrier protein 9(ubc9) levels. Hence, insidious functional impairment of SBM of Ranbp2's CLD promotes neuroprotection and uncoupling of photoreceptor degeneration and death against phototoxicity. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Expression of uncoupling protein 3 is upregulated in skeletal muscle during sepsis.

PubMed

Sun, Xiaoyan; Wray, Curtis; Tian, Xintian; Hasselgren, Per-Olof; Lu, James

2003-09-01

Uncoupling protein 3 (UCP3) is a member of the mitochondrial transporter superfamily that is expressed primarily in skeletal muscle. UCP3 is upregulated in various conditions characterized by skeletal muscle atrophy, including hyperthyroidism, fasting, denervation, diabetes, cancer, lipopolysaccharide (LPS), and treatment with glucocorticoids (GCs). The influence of sepsis, another condition characterized by muscle cachexia, on UCP3 expression and activity is not known. We examined UCP3 gene and protein expression in skeletal muscles from rats after cecal ligation and puncture and from sham-operated control rats. Sepsis resulted in a two- to threefold increase in both mRNA and protein levels of UCP3 in skeletal muscle. Treatment of rats with the glucocorticoid receptor antagonist RU-38486 prevented the sepsis-induced increase in gene and protein expression of UCP3. The UCP3 mRNA and protein levels were increased 2.4- to 3.6-fold when incubated muscles from normal rats were treated with dexamethasone (DEX) and/or free fatty acids (FFA) ex vivo. In addition, UCP3 mRNA and protein levels were significantly increased in normal rat muscles in vivo with treatment of either DEX or FFA. The results suggest that sepsis upregulates the gene and protein expression of UCP3 in skeletal muscle, which may at least in part be mediated by GCs and FFA.

Novel browning agents, mechanisms and therapeutic potentials of brown adipose tissue

USDA-ARS?s Scientific Manuscript database

Non-shivering thermogenesis is the process of biological heat production in mammals and is primarily mediated by brown adipose tissue (BAT). Through ubiquitous expression of uncoupling protein 1 (Ucp1) on the mitochondrial inner membrane, BAT displays uncoupling of fuel combustion and ATP production...

Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus).

PubMed

Talbot, Darren A; Hanuise, Nicolas; Rey, Benjamin; Rouanet, Jean-Louis; Duchamp, Claude; Brand, Martin D

2003-12-26

We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak.

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

Adipocyte Browning and Higher Mitochondrial Function in Periadrenal But Not SC Fat in Pheochromocytoma.

PubMed

Vergnes, Laurent; Davies, Graeme R; Lin, Jason Y; Yeh, Michael W; Livhits, Masha J; Harari, Avital; Symonds, Michael E; Sacks, Harold S; Reue, Karen

2016-11-01

Patients with pheochromocytoma (pheo) show presence of multilocular adipocytes that express uncoupling protein 1 within periadrenal (pADR) and omental (OME) fat depots. It has been hypothesized that this is due to adrenergic stimulation by catecholamines produced by the pheo tumors. To characterize the prevalence and respiratory activity of brown-like adipocytes within pADR, OME, and SC fat depots in human adult pheo patients. This was an observational cohort study. The study took place in a university hospital. We studied 46 patients who underwent surgery for benign adrenal tumors (21 pheos and 25 controls with adrenocortical adenomas). We characterized adipocyte browning in pADR, SC, and OME fat depots for histological and immunohistological features, mitochondrial respiration rate, and gene expression. We also determined circulating levels of catecholamines and other browning-related hormones. Eleven of 21 pheo pADR adipose samples, but only one of 25 pADR samples from control patients exhibited multilocular adipocytes. The pADR browning phenotype was associated with higher plasma catecholamines and raised uncoupling protein 1. Mitochondria from multilocular pADR fat of pheo patients exhibited increased rates of coupled and uncoupled respiration. Global gene expression analysis in pADR fat revealed enrichment in β-oxidation genes in pheo patients with multilocular adipocytes. No SC or OME fat depots exhibited aspects of browning. Browning of the pADR depot occurred in half of pheo patients and was associated with increased catecholamines and mitochondrial activity. No browning was detected in other fat depots, suggesting that other factors are required to promote browning in these depots.

Pentachlorophenol hydroxylase, a poorly functioning enzyme required for degradation of pentachlorophenol by Sphingobium chlorophenolicum

PubMed Central

Hlouchova, Klara; Rudolph, Johannes; Pietari, Jaana M.H.; Behlen, Linda S.; Copley, Shelley D.

2014-01-01

Several strains of Sphingobium chlorophenolicum have been isolated from soil that was heavily contaminated with pentachlorophenol (PCP), a toxic pesticide introduced in the 1930s. S. chlorophenolicum appears to have assembled a poorly functioning pathway for degradation of PCP by patching enzymes recruited via two independent horizontal gene transfer events into an existing metabolic pathway. Flux through the pathway is limited by PCP hydroxylase. PCP hydroxylase is a dimeric protein that belongs to the family of flavin-dependent phenol hydroxylases. In the presence of NADPH, PCP hydroxylase converts PCP to tetrachlorobenzoquinone (TCBQ). The kcat for PCP (0.024 s−1) is very low, suggesting that the enzyme is not well evolved for turnover of this substrate. Structure/activity studies reveal that substrate binding and activity are enhanced by a low pKa for the phenolic proton, increased hydrophobicity, and the presence of a substituent ortho to the hydroxyl group of the phenol. PCP hydroxylase exhibits substantial uncoupling; the C4a-hydroxyflavin intermediate, instead of hydroxylating the substrate, can decompose to produce H2O2 in a futile cycle that consumes NADPH. The extent of uncoupling varies from 0 – 100% with different substrates. Uncoupling is increased by the presence of bulky substituents in the 3-, 4-, or 5-position, and lessened by the presence of a chlorine in the ortho position. The effectiveness of PCP hydroxylase is additionally hindered by its promiscuous activity with TCHQ, a downstream metabolite in the degradation pathway. The conversion of TCHQ to TCBQ reverses flux through the pathway. Substantial uncoupling also occurs during the reaction with TCHQ. PMID:22482720

Endocytosis of the seven-transmembrane RGS1 protein activates G-protein-coupled signalling in Arabidopsis.

PubMed

Urano, Daisuke; Phan, Nguyen; Jones, Janice C; Yang, Jing; Huang, Jirong; Grigston, Jeffrey; Taylor, J Philip; Jones, Alan M

2012-10-01

Signal transduction typically begins by ligand-dependent activation of a concomitant partner that is otherwise in its resting state. However, in cases where signal activation is constitutive by default, the mechanism of regulation is unknown. The Arabidopsis thaliana heterotrimeric Gα protein self-activates without accessory proteins, and is kept in its resting state by the negative regulator, AtRGS1 (regulator of G-protein signalling 1), which is the prototype of a seven-transmembrane receptor fused with an RGS domain. Endocytosis of AtRGS1 by ligand-dependent endocytosis physically uncouples the GTPase-accelerating activity of AtRGS1 from the Gα protein, permitting sustained activation. Phosphorylation of AtRGS1 by AtWNK8 kinase causes AtRGS1 endocytosis, required for both G-protein-mediated sugar signalling and cell proliferation. In animals, receptor endocytosis results in signal desensitization, whereas in plants, endocytosis results in signal activation. These findings reveal how different organisms rearrange a regulatory system to result in opposite outcomes using similar phosphorylation-dependent endocytosis mechanisms.

Uncoupling protein-2 attenuates glucose-stimulated insulin secretion in INS-1E insulinoma cells by lowering mitochondrial reactive oxygen species

PubMed Central

Affourtit, Charles; Jastroch, Martin; Brand, Martin D.

2011-01-01

Glucose-stimulated insulin secretion (GSIS) by pancreatic β cells is regulated by mitochondrial uncoupling protein-2 (UCP2), but opposing phenotypes, GSIS improvement and impairment, have been reported for different Ucp2-ablated mouse models. By measuring mitochondrial bioenergetics in attached INS-1E insulinoma cells with and without UCP2, we show that UCP2 contributes to proton leak and attenuates glucose-induced rises in both respiratory activity and the coupling efficiency of oxidative phosphorylation. Strikingly, the GSIS improvement seen upon UCP2 knockdown in INS-1E cells is annulled completely by the cell-permeative antioxidant MnTMPyP. Consistent with this observation, UCP2 lowers mitochondrial reactive oxygen species at high glucose levels. We conclude that UCP2 plays both regulatory and protective roles in β cells by acutely lowering GSIS and chronically preventing oxidative stress. Our findings thus provide a mechanistic explanation for the apparently discrepant findings in the field. PMID:21172424

Adaptative decrease in expression of the mRNA for uncoupling protein and subunit II of cytochrome c oxidase in rat brown adipose tissue during pregnancy and lactation.

PubMed Central

Martin, I; Giralt, M; Viñas, O; Iglesias, R; Mampel, T; Villarroya, F

1989-01-01

Uncoupling-protein (UCP) mRNA expression is decreased to 15% of virgin control levels between days 10 and 15 of pregnancy, and remains at these low values during late pregnancy and lactation. Abrupt weaning of mid-lactating rats causes a slight but significant increase in UCP mRNA. Expression of mRNA for subunit II of cytochrome c oxidase (COII) decreased to half that of virgin control in late pregnancy and during lactation. Whereas COII mRNA expression is in step with the known modifications of brown-fat mitochondria content during the breeding cycle of the rat, UCP mRNA expression appears to be diminished much earlier than the mitochondrial proton-conductance-pathway activity. On the other hand, the reactivity of brown fat to increase expression of UCP and COII mRNAs in response to acute cold or noradrenaline treatment is not impaired during lactation. Images Fig. 1. Fig. 2. Fig. 3. PMID:2557014

Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced, p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells

PubMed Central

Shi, Yi; Lüscher, Thomas F.; Camici, Giovanni G.

2014-01-01

Background The aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation. Methods and Results Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66Shc at Ser-36, resulting in an enhanced production of superoxide anion (O2 -). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66Shc phosphorylation, suggesting that basal NO production inhibits O2 - production. oxLDL-induced, p66Shc-mediated O2- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66Shc activation and reduced O2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66Shc activation, and reduced O2- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species. Conclusions The present study reports that in primary cultured HAEC treated with oxLDL, p66Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease. PMID:25247687

Dual role of endothelial nitric oxide synthase in oxidized LDL-induced, p66Shc-mediated oxidative stress in cultured human endothelial cells.

PubMed

Shi, Yi; Lüscher, Thomas F; Camici, Giovanni G

2014-01-01

The aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation. Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66Shc at Ser-36, resulting in an enhanced production of superoxide anion (O2-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66Shc phosphorylation, suggesting that basal NO production inhibits O2- production. oxLDL-induced, p66Shc-mediated O2- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66Shc activation and reduced O2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66Shc activation, and reduced O2- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species. The present study reports that in primary cultured HAEC treated with oxLDL, p66Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.

UCP2 and 3 deletion screening and distribution in 15 pig breeds.

PubMed

Li, Yanhua; Li, Hanjie; Zhao, Xingbo; Li, Ning; Wu, Changxin

2007-02-01

The uncoupling protein family is a mitochondrial anion carrier family. It plays an important role in the biological traits of animal body weight, basal metabolic rate and energy conversion. Using PCR and PCR-SSCP, we scanned the porcine uncoupling protein 2 gene (UCP2) and uncoupling protein 3 gene (UCP3) and found seven deletion sites, three in UCP2 and four in UCP3. The deletions in 15 pig breeds showed that deletion influenced weight. The genotype compounding of seven deletion sites in 15 pig breeds had significant effects on performance traits of the pig, such as body weight. We predicted the potential protein factor binding sites using the transcription factor analysis tool TFSearch version 1.3 online. Two deletions (1830 nt and 3219 nt) in UCP3 were found to change the transcriptional factor sites. The 16 bp deletion in 1830 nt added a SP1 site and a 6 bp deletion in 3219 nt removed two MZF1 sites. Seven deletion polymorphisms were covered in introns of linkage genes of UCP2 and UCP3, showing that UCPs have conservation and genetic reliability.

Activation of PPAR{delta} up-regulates fatty acid oxidation and energy uncoupling genes of mitochondria and reduces palmitate-induced apoptosis in pancreatic {beta}-cells

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

Wan, Jun; Jiang, Li; Lue, Qingguo

2010-01-15

Recent evidence indicates that decreased oxidative capacity, lipotoxicity, and mitochondrial aberrations contribute to the development of insulin resistance and type 2 diabetes. The goal of this study was to investigate the effects of peroxisome proliferator-activated receptor {delta} (PPAR{delta}) activation on lipid oxidation, mitochondrial function, and insulin secretion in pancreatic {beta}-cells. After HIT-T15 cells (a {beta}-cell line) were exposed to high concentrations of palmitate and GW501516 (GW; a selective agonist of PPAR{delta}), we found that administration of GW increased the expression of PPAR{delta} mRNA. GW-induced activation of PPAR{delta} up-regulated carnitine palmitoyltransferase 1 (CPT1), long-chain acyl-CoA dehydrogenase (LCAD), pyruvate dehydrogenase kinase 4more » (PDK4), and uncoupling protein 2 (UCP2); alleviated mitochondrial swelling; attenuated apoptosis; and reduced basal insulin secretion induced by increased palmitate in HIT cells. These results suggest that activation of PPAR{delta} plays an important role in protecting pancreatic {beta}-cells against aberrations caused by lipotoxicity in metabolic syndrome and diabetes.« less

Loss of histaminergic modulation of thermoregulation and energy homeostasis in obese mice.

PubMed

Sethi, J; Sanchez-Alavez, M; Tabarean, I V

2012-08-16

Histamine acts centrally to increase energy expenditure and reduce body weight by mechanisms not fully understood. It has been suggested that in the obese state hypothalamic histamine signaling is altered. Previous studies have also shown that histamine acting in the preoptic area controls thermoregulation. We aimed to study the influence of preoptic histamine on body temperature and energy homeostasis in control and obese mice. Activating histamine receptors in the preoptic area by increasing the concentration of endogenous histamine or by local injection of specific agonists induced an elevation of core body temperature and decreased respiratory exchange ratio (RER). In addition, the food intake was significantly decreased. The hyperthermic effect was associated with a rapid increase in mRNA expression of uncoupling proteins in thermogenic tissues, the most pronounced being that of uncoupling protein (UCP) 1 in brown adipose tissue and of UCP2 in white adipose tissue. In diet-induced obese mice histamine had much diminished hyperthermic effects as well as reduced effect on RER. Similarly, the ability of preoptic histamine signaling to increase the expression of uncoupling proteins was abolished. We also found that the expression of mRNA encoding the H1 receptor subtype in the preoptic area was significantly lower in obese animals. These results indicate that histamine signaling in the preoptic area modulates energy homeostasis by regulating body temperature, metabolic parameters and food intake and that the obese state is associated with a decrease in neurotransmitter's influence. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Novel reptilian uncoupling proteins: molecular evolution and gene expression during cold acclimation.

PubMed

Schwartz, Tonia S; Murray, Shauna; Seebacher, Frank

2008-04-22

Many animals upregulate metabolism in response to cold. Uncoupling proteins (UCPs) increase proton conductance across the mitochondrial membrane and can thereby alleviate damage from reactive oxygen species that may form as a result of metabolic upregulation. Our aim in this study was to determine whether reptiles (Crocodylus porosus) possess UCP genes. If so, we aimed to place reptilian UCP genes within a phylogenetic context and to determine whether the expression of UCP genes is increased during cold acclimation. We provide the first evidence that UCP2 and UCP3 genes are present in reptiles. Unlike in other vertebrates, UCP2 and UPC3 are expressed in liver and skeletal muscle of the crocodile, and both are upregulated in liver during cold acclimation but not in muscle. We identified two transcripts of UCP3, one of which produces a truncated protein similar to the UCP3S transcript in humans, and the resulting protein lacks the predicted nucleotide-binding regulatory domain. Our molecular phylogeny suggests that uncoupling protein 1 (UCP1) is ancestral and has been lost in archosaurs. In birds, UCP3 may have assumed a similar function as UCP1 in mammals, which has important ramifications for understanding endothermic heat production.

G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization

PubMed Central

Papkovskaia, Tatiana D.; Chau, Kai-Yin; Inesta-Vaquera, Francisco; Papkovsky, Dmitri B.; Healy, Daniel G.; Nishio, Koji; Staddon, James; Duchen, Michael R.; Hardy, John; Schapira, Anthony H.V.; Cooper, J. Mark

2012-01-01

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD. PMID:22736029

TALEN-engineered AR gene rearrangements reveal endocrine uncoupling of androgen receptor in prostate cancer

PubMed Central

Nyquist, Michael D.; Li, Yingming; Hwang, Tae Hyun; Manlove, Luke S.; Vessella, Robert L.; Silverstein, Kevin A. T.; Voytas, Daniel F.; Dehm, Scott M.

2013-01-01

Androgen receptor (AR) target genes direct development and survival of the prostate epithelial lineage, including prostate cancer (PCa). Thus, endocrine therapies that inhibit the AR ligand-binding domain (LBD) are effective in treating PCa. AR transcriptional reactivation is central to resistance, as evidenced by the efficacy of AR retargeting in castration-resistant PCa (CRPC) with next-generation endocrine therapies abiraterone and enzalutamide. However, resistance to abiraterone and enzalutamide limits this efficacy in most men, and PCa remains the second-leading cause of male cancer deaths. Here we show that AR gene rearrangements in CRPC tissues underlie a completely androgen-independent, yet AR-dependent, resistance mechanism. We discovered intragenic AR gene rearrangements in CRPC tissues, which we modeled using transcription activator-like effector nuclease (TALEN)-mediated genome engineering. This modeling revealed that these AR gene rearrangements blocked full-length AR synthesis, but promoted expression of truncated AR variant proteins lacking the AR ligand-binding domain. Furthermore, these AR variant proteins maintained the constitutive activity of the AR transcriptional program and a CRPC growth phenotype independent of full-length AR or androgens. These findings demonstrate that AR gene rearrangements are a unique resistance mechanism by which AR transcriptional activity can be uncoupled from endocrine regulation in CRPC. PMID:24101480

[Role of thyroid system in adaptation to cold].

PubMed

Maslov, L N; Vychuzhanova, E A; Gorbunov, A S; Tsybul'nikov, S Iu; Khaliulin, I G; Chauski, E

2014-06-01

Adaptation to cold promotes an increase in blood T3 and T4 levels in men and animals. The long-term cold exposure can induce a decrease in concentration of serum total and free T3 in human due to an enhancement of this hormone clearance. Endogenous catecholamines during adaptation to cold raise iodothyronine deiodinase D2 activity in brown fat due to α1-adrenergic receptor stimulation. Triiodothyronine is an inductor of iodothyronine deiodinase expression in brown fat, liver and kidney. Iodothyronine deiodinase D2 plays an important role in adaptation of organism to cold contributing to the high adrenergic reactivity of brown fat. At adaptation to cold T3 interacts with T3Rβ, it is formed T3Rβ-RXR complex, which binds to DNA with following transcription of UCP-1 and UCP-3 genes and UCP-1 and UCP-3 protein synthesis and uncoupling oxidative phosphorylation and an increase in heat production, where T3Rβ is T3-receptor-β, RXR is retinoid X-receptor, UCP is uncoupling protein. Triiodothyronine contributes to normal response to adrenergic agents of brown fat due to T3Rα activation. Sympatho-adrenomedullary and thyroid systems act as synergists in adaptation to cold.

Molecular cloning and functional characterization of the promoter region of the human uncoupling protein-2 gene.

PubMed

Tu, N; Chen, H; Winnikes, U; Reinert, I; Marmann, G; Pirke, K M; Lentes, K U

1999-11-19

As a member of the uncoupling protein family, UCP2 is ubiquitously expressed in rodents and humans, implicating a major role in thermogenesis. To analyze promoter function and regulatory motifs involved in the transcriptional regulation of UCP2 gene expression, 3.3 kb of 5'-flanking region of the human UCP2 (hUCP2) gene have been cloned. Sequence analysis showed that the promoter region of hUCP2 lacks a classical TATA or CAAT box, however, appeared GC-rich resulting in the presence of several Sp-1 motifs and Ap-1/-2 binding sites near the transcription initiation site. Functional characterization of human UCP2 promoter-CAT fusion constructs in transient expression assays showed that minimal promoter activity was observed within 65 bp upstream of the transcriptional start site (+1). 75 bp further upstream (from nt -141 to -66) a strong cis-acting regulatory element (or enhancer) was identified, which significantly enhanced basal promoter activity. The regulation of human UCP2 gene expression involves complex interactions among positive and negative regulatory elements distributed over a minimum of 3.3 kb of the promoter region. Copyright 1999 Academic Press.

Carbon Tetrachloride at Hepatotoxic Levels Blocks Reversibly Gap Junctions between Rat Hepatocytes

NASA Astrophysics Data System (ADS)

Saez, J. C.; Bennett, M. V. L.; Spray, D. C.

1987-05-01

Electrical coupling and dye coupling between pairs of rat hepatocytes were reversibly reduced by brief exposure to halogenated methanes (CBrCl3, CCl4, and CHCl3). The potency of different halomethanes in uncoupling hepatocytes was comparable to their hepatotoxicity in vivo, and the rank order was the same as that of their tendency to form free radicals. The effect of carbon tetrachloride (CCl4) on hepatocytes was substantially reduced by prior treatment with SKF 525A, an inhibitor of cytochrome P-450, and by exposure to the reducing reagent β -mercaptoethanol. Halomethane uncoupling occurred with or without extracellular calcium and did not change intracellular concentrations of calcium and hydrogen ions or the phosphorylation state of the main gap-junctional protein. Thus the uncoupling appears to depend on cytochrome P-450 oxidative metabolism in which free radicals are generated and may result from oxidation of the gap-junctional protein or of a regulatory molecule that leads to closure of gap-junctional channels. Decreases in junctional conductance may be a rapid cellular response to injury that protects healthy cells by uncoupling them from unhealthy ones.

Structural and chemical requirements for hydroxychlorobiphenyls to uncouple rat liver mitochondria and potentiation of uncoupling with aroclor 1254.

PubMed

Ebner, K V; Braselton, W E

1987-01-01

Rat hepatic mitochondrial permeability and succinate + valinomycin-dependent swelling were studied in the presence of hydroxy derivatives of polychlorinated biphenyls (PCBOHs), Aroclor 1254 (ARO) and combinations of both. PCBOHs with two or more chlorines and pKas greater than 8.0 (PCBOH I) induced passive swelling in a potassium acetate-sucrose medium (pH 7.2), maximally stimulated succinate respiration, and suppressed ADP-stimulated H+ uptake. Mono- and certain dichlorinated biphenylols with similar high pKas (PCBOH II) were ineffective. Para-hydroxy PCBs with chlorines substituted in the 3,5 positions and with pKas near 6.8 (PCBOH III) inhibited succinate + valinomycin swelling and ADP-stimulated H+ and oxygen uptake. The efficacy of both PCBOH I and III derivatives required the presence of a hydroxyl moiety and increased directly with the degree of chlorination. Coplanarity was not a determining factor for PCBOH I compounds. ARO activated succinate + valinomycin swelling at low concentrations (3-25 nmol/mg protein) but inhibited at higher concentrations (greater than 40 nmol/mg). Activating concentrations of ARO potentiated the influence of PCBOHs on mitochondria. The uncoupling effects of the PCBOHs and ARO involved permeability changes of the inner membrane, respiratory inhibition, or combinations of both.

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster.

PubMed

Da-Ré, Caterina; De Pittà, Cristiano; Zordan, Mauro A; Teza, Giordano; Nestola, Fabrizio; Zeviani, Massimo; Costa, Rodolfo; Bernardi, Paolo

2014-05-01

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F-ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva-to-adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.

Hyperthyroidism increases the uncoupled ATPase activity and heat production by the sarcoplasmic reticulum Ca2+-ATPase.

PubMed

Arruda, Ana Paula; Da-Silva, Wagner S; Carvalho, Denise P; De Meis, Leopoldo

2003-11-01

The sarcoplasmic reticulum Ca2+-ATPase is able to modulate the distribution of energy released during ATP hydrolysis, so that a portion of energy is used for Ca2+ transport (coupled ATPase activity) and a portion is converted into heat (uncoupled ATPase activity). In this report it is shown that T4 administration to rabbits promotes an increase in the rates of both the uncoupled ATPase activity and heat production in sarcoplasmic reticulum vesicles, and that the degree of activation varies depending on the muscle type used. In white muscles hyperthyroidism promotes a 0.8-fold increase of the uncoupled ATPase activity and in red muscle a 4-fold increase. The yield of vesicles from hyperthyroid muscles is 3-4-fold larger than that obtained from normal muscles; thus the rate of heat production by the Ca2+-ATPase expressed in terms of g of muscle in hyperthyroidism is increased by a factor of 3.6 in white muscles and 12.0 in red muscles. The data presented suggest that the Ca2+-ATPase uncoupled activity may represent one of the heat sources that contributes to the enhanced thermogenesis noted in hyperthyroidism.

Endurance training blocks uncoupling protein 1 up-regulation in brown adipose tissue while increasing uncoupling protein 3 in the muscle tissue of rats fed with a high-sugar diet.

PubMed

de Queiroz, Karina Barbosa; Rodovalho, Gisele Vieira; Guimarães, Juliana Bohnen; de Lima, Daniel Carvalho; Coimbra, Cândido Celso; Evangelista, Elísio Alberto; Guerra-Sá, Renata

2012-09-01

The mitochondrial uncoupling proteins (UCPs) of interscapular brown adipose tissue (iBAT) and of muscles play important roles in energy balance. For instance, the expression of UCP1 and UCP3 are modulated by free fatty acid gradients induced by high-sugar diets and acute exercise that is dependent on sympathetic stimulation. However, the effects of endurance training in animals fed with high-sugar diets are unknown. This study aims to evaluate the long-term effects of diet and exercise on UCP1 and UCP3 levels and energy balance efficiency. Rats fed with standard or high-sugar (HSD) diets were simultaneously subjected to running training over an 8-week period. After the training period, the rats were decapitated, and the iBAT and gastrocnemius muscle tissues were removed for evaluation of the β₃-receptor, Ucp1, and Ucp3 mRNA and protein expression, which were analyzed by quantitative reverse transcriptase polymerase chain reaction and Western blot, respectively. Groups fed with an HSD displayed a higher adiposity index and iBAT weight (P < .05), whereas exhibited an up-regulation of Ucp1 mRNA and protein levels (P < .05). Training increased β₃-receptor mRNA in iBAT and reduced the Ucp3 mRNA in muscle tissues. In association with an HSD, training restored the increasing β₃-receptor mRNA and greatly up-regulated the levels of Ucp3 mRNA. Therefore, training blocked the HSD-induced up-regulation of UCP1 expression in iBAT, whereas it up-regulated the expression of Ucp3 mRNA in muscle. These results suggest that training enhances the relationship between Ucp1/Ucp3 mRNA levels, which could result in higher energy efficiency, but not when HSD-induced elevated sympathetic activity is maintained. Copyright © 2012. Published by Elsevier Inc.

Rat liver uncoupling protein 2: changes induced by a fructose-rich diet.

PubMed

Castro, María C; Massa, María L; Del Zotto, Héctor; Gagliardino, Juan J; Francini, Flavio

2011-10-24

To evaluate the role of uncoupling protein 2 (UCP2) and peroxisome proliferator-activated receptors (PPARs) in the response of liver to glycoxidative stress triggered by administration of a fructose-rich diet (FRD). We assessed blood glucose in the fasting state and after a glucose load (glucose-oxidase method), serum triglyceride (enzymatic measurement), insulin (radioimmunoassay), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels (colorimetric kits) in control and FRD animals. In liver, we measured UCP2, PPARα, PPARδ and PPARγ gene (real-time PCR) and protein (Western blot) expression, fatty acid synthase (FAS) and glycerol-3-phosphate acyltransferase (GPAT) gene expression, as well as triglyceride content. Blood glucose, serum insulin and triglyceride levels, homeostasis model assessment of insulin resistance (HOMA-IR) indexes and impaired glucose tolerance were higher in FRD rats. Whereas UCP2 and PPARδ gene and protein expression increased in these animals; PPARγ levels were lower and those of PPARα remained unchanged. FRD also increased the mRNA expression of PPARδ target genes FAS and GPAT. Our results suggest that a) the increased UCP2 gene and protein expression measured in FRD rats could be part of a compensatory mechanism to reduce reactive oxygen species production induced by the fructose overload, and b) PPARs expression participates actively in the regulation of UCP2 expression, and under the metabolic condition tested, PPARδ played a key role. This knowledge would help to better understand the mechanisms involved in liver adaptation to fructose-induced glycoxidative stress, and to develop appropriate prevention strategies in obesity and type 2 diabetes. Copyright © 2011 Elsevier Inc. All rights reserved.

Arginine de novo and nitric oxide production in disease states

PubMed Central

Luiking, Yvette C.; Ten Have, Gabriella A. M.; Wolfe, Robert R.

2012-01-01

Arginine is derived from dietary protein intake, body protein breakdown, or endogenous de novo arginine production. The latter may be linked to the availability of citrulline, which is the immediate precursor of arginine and limiting factor for de novo arginine production. Arginine metabolism is highly compartmentalized due to the expression of the enzymes involved in arginine metabolism in various organs. A small fraction of arginine enters the NO synthase (NOS) pathway. Tetrahydrobiopterin (BH4) is an essential and rate-limiting cofactor for the production of NO. Depletion of BH4 in oxidative-stressed endothelial cells can result in so-called NOS3 “uncoupling,” resulting in production of superoxide instead of NO. Moreover, distribution of arginine between intracellular transporters and arginine-converting enzymes, as well as between the arginine-converting and arginine-synthesizing enzymes, determines the metabolic fate of arginine. Alternatively, NO can be derived from conversion of nitrite. Reduced arginine availability stemming from reduced de novo production and elevated arginase activity have been reported in various conditions of acute and chronic stress, which are often characterized by increased NOS2 and reduced NOS3 activity. Cardiovascular and pulmonary disorders such as atherosclerosis, diabetes, hypercholesterolemia, ischemic heart disease, and hypertension are characterized by NOS3 uncoupling. Therapeutic applications to influence (de novo) arginine and NO metabolism aim at increasing substrate availability or at influencing the metabolic fate of specific pathways related to NO bioavailability and prevention of NOS3 uncoupling. These include supplementation of arginine or citrulline, provision of NO donors including inhaled NO and nitrite (sources), NOS3 modulating agents, or the targeting of endogenous NOS inhibitors like asymmetric dimethylarginine. PMID:23011059

GTP-Binding Proteins Inhibit cAMP Activation of Chloride Channels in Cystic Fibrosis Airway Epithelial Cells

NASA Astrophysics Data System (ADS)

Schwiebert, Erik M.; Kizer, Neil; Gruenert, Dieter C.; Stanton, Bruce A.

1992-11-01

Cystic fibrosis (CF) is a genetic disease characterized, in part, by defective regulation of Cl^- secretion by airway epithelial cells. In CF, cAMP does not activate Cl^- channels in the apical membrane of airway epithelial cells. We report here whole-cell patch-clamp studies demonstrating that pertussis toxin, which uncouples heterotrimeric GTP-binding proteins (G proteins) from their receptors, and guanosine 5'-[β-thio]diphosphate, which prevents G proteins from interacting with their effectors, increase Cl^- currents and restore cAMP-activated Cl^- currents in airway epithelial cells isolated from CF patients. In contrast, the G protein activators guanosine 5'-[γ-thio]triphosphate and AlF^-_4 reduce Cl^- currents and inhibit cAMP from activating Cl^- currents in normal airway epithelial cells. In CF cells treated with pertussis toxin or guanosine 5'-[β-thio]diphosphate and in normal cells, cAMP activates a Cl^- conductance that has properties similar to CF transmembrane-conductance regulator Cl^- channels. We conclude that heterotrimeric G proteins inhibit cAMP-activated Cl^- currents in airway epithelial cells and that modulation of the inhibitory G protein signaling pathway may have the therapeutic potential for improving cAMP-activated Cl^- secretion in CF.

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

PubMed Central

Eya, Jonathan C.; Ukwuaba, Vitalis O.; Yossa, Rodrigue; Gannam, Ann L.

2015-01-01

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish. PMID:25853266

Gene-gene interactions among genetic variants from obesity candidate genes for nonobese and obese populations in type 2 diabetes.

PubMed

Lin, Eugene; Pei, Dee; Huang, Yi-Jen; Hsieh, Chang-Hsun; Wu, Lawrence Shih-Hsin

2009-08-01

Recent studies indicate that obesity may play a key role in modulating genetic predispositions to type 2 diabetes (T2D). This study examines the main effects of both single-locus and multilocus interactions among genetic variants in Taiwanese obese and nonobese individuals to test the hypothesis that obesity-related genes may contribute to the etiology of T2D independently and/or through such complex interactions. We genotyped 11 single nucleotide polymorphisms for 10 obesity candidate genes including adrenergic beta-2-receptor surface, adrenergic beta-3-receptor surface, angiotensinogen, fat mass and obesity associated gene, guanine nucleotide binding protein beta polypeptide 3 (GNB3), interleukin 6 receptor, proprotein convertase subtilisin/kexin type 1 (PCSK1), uncoupling protein 1, uncoupling protein 2, and uncoupling protein 3. There were 389 patients diagnosed with T2D and 186 age- and sex-matched controls. Single-locus analyses showed significant main effects of the GNB3 and PCSK1 genes on the risk of T2D among the nonobese group (p = 0.002 and 0.047, respectively). Further, interactions involving GNB3 and PCSK1 were suggested among the nonobese population using the generalized multifactor dimensionality reduction method (p = 0.001). In addition, interactions among angiotensinogen, fat mass and obesity associated gene, GNB3, and uncoupling protein 3 genes were found in a significant four-locus generalized multifactor dimensionality reduction model among the obese population (p = 0.001). The results suggest that the single nucleotide polymorphisms from the obesity candidate genes may contribute to the risk of T2D independently and/or in an interactive manner according to the presence or absence of obesity.

Lean phenotype and resistance to diet-induced obesity in vitamin D receptor knockout mice correlates with induction of uncoupling protein-1 in white adipose tissue.

PubMed

Narvaez, Carmen J; Matthews, Donald; Broun, Emily; Chan, Michelle; Welsh, JoEllen

2009-02-01

Increased adiposity is a feature of aging in both mice and humans, but the molecular mechanisms underlying age-related changes in adipose tissue stores remain unclear. In previous studies, we noted that 18-month-old normocalcemic vitamin D receptor (VDR) knockout (VDRKO) mice exhibited atrophy of the mammary adipose compartment relative to wild-type (WT) littermates, suggesting a role for VDR in adiposity. Here we monitored body fat depots, food intake, metabolic factors, and gene expression in WT and VDRKO mice on the C57BL6 and CD1 genetic backgrounds. Regardless of genetic background, both sc and visceral white adipose tissue depots were smaller in VDRKO mice than WT mice. The lean phenotype of VDRKO mice was associated with reduced serum leptin and compensatory increased food intake. Similar effects on adipose tissue, leptin and food intake were observed in mice lacking Cyp27b1, the 1alpha-hydroxylase enzyme that generates 1,25-dihydroxyvitamin D(3), the VDR ligand. Although VDR ablation did not reduce expression of peroxisome proliferator-activated receptor-gamma or fatty acid synthase, PCR array screening identified several differentially expressed genes in white adipose tissue from WT and VDRKO mice. Uncoupling protein-1, which mediates dissociation of cellular respiration from energy production, was greater than 25-fold elevated in VDRKO white adipose tissue. Consistent with elevation in uncoupling protein-1, VDRKO mice were resistant to high-fat diet-induced weight gain. Collectively, these studies identify a novel role for 1,25-dihydroxyvitamin D(3) and the VDR in the control of adipocyte metabolism and lipid storage in vivo.

Absence of uncoupling protein-3 leads to greater activation of an adenine nucleotide translocase-mediated proton conductance in skeletal muscle mitochondria from calorie restricted mice.

PubMed

Bevilacqua, Lisa; Seifert, Erin L; Estey, Carmen; Gerrits, Martin F; Harper, Mary-Ellen

2010-08-01

Calorie restriction (CR), without malnutrition, consistently increases lifespan in all species tested, and reduces age-associated pathologies in mammals. Alterations in mitochondrial content and function are thought to underlie some of the effects of CR. Previously, we reported that rats subjected to variable durations of 40% CR demonstrated a rapid and sustained decrease in maximal leak-dependent respiration in skeletal muscle mitochondria. This was accompanied by decreased mitochondrial reactive oxygen species generation and increased uncoupling protein-3 protein (UCP3) expression. The aim of the present study was to determine the contribution of UCP3, as well as the adenine nucleotide translocase to these functional changes in skeletal muscle mitochondria. Consistent with previous findings in rats, short-term CR (2 weeks) in wild-type (Wt) mice resulted in a lowering of the maximal leak-dependent respiration in skeletal muscle mitochondria, without any change in proton conductance. In contrast, skeletal muscle mitochondria from Ucp3-knockout (KO) mice similarly subjected to short-term CR showed no change in maximal leak-dependent respiration, but displayed an increased proton conductance. Determination of ANT activity (by measurement of inhibitor-sensitive leak) and protein expression revealed that the increased proton conductance in mitochondria from CR Ucp3-KO mice could be entirely attributed to a greater acute activation of ANT. These observations implicate UCP3 in CR-induced mitochondrial remodeling. Specifically, they imply the potential for an interaction, or some degree of functional redundancy, between UCP3 and ANT, and also suggest that UCP3 can minimize the induction of the ANT-mediated 'energy-wasting' process during CR. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Rapamycin negatively impacts insulin signaling, glucose uptake and uncoupling protein-1 in brown adipocytes.

PubMed

García-Casarrubios, Ester; de Moura, Carlos; Arroba, Ana I; Pescador, Nuria; Calderon-Dominguez, María; Garcia, Laura; Herrero, Laura; Serra, Dolors; Cadenas, Susana; Reis, Flavio; Carvalho, Eugenia; Obregon, Maria Jesus; Valverde, Ángela M

2016-12-01

New onset diabetes after transplantation (NODAT) is a metabolic disorder that affects 40% of patients on immunosuppressive agent (IA) treatment, such as rapamycin (also known as sirolimus). IAs negatively modulate insulin action in peripheral tissues including skeletal muscle, liver and white fat. However, the effects of IAs on insulin sensitivity and thermogenesis in brown adipose tissue (BAT) have not been investigated. We have analyzed the impact of rapamycin on insulin signaling, thermogenic gene-expression and mitochondrial respiration in BAT. Treatment of brown adipocytes with rapamycin for 16h significantly decreased insulin receptor substrate 1 (IRS1) protein expression and insulin-mediated protein kinase B (Akt) phosphorylation. Consequently, both insulin-induced glucose transporter 4 (GLUT4) translocation to the plasma membrane and glucose uptake were decreased. Early activation of the N-terminal Janus activated kinase (JNK) was also observed, thereby increasing IRS1 Ser 307 phosphorylation. These effects of rapamycin on insulin signaling in brown adipocytes were partly prevented by a JNK inhibitor. In vivo treatment of rats with rapamycin for three weeks abolished insulin-mediated Akt phosphorylation in BAT. Rapamycin also inhibited norepinephrine (NE)-induced lipolysis, the expression of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and uncoupling protein (UCP)-1 in brown adipocytes. Importantly, basal mitochondrial respiration, proton leak and maximal respiratory capacity were significantly decreased in brown adipocytes treated with rapamycin. In conclusion, we demonstrate, for the first time the important role of brown adipocytes as target cells of rapamycin, suggesting that insulin resistance in BAT might play a major role in NODAT development. Copyright © 2016 Elsevier B.V. All rights reserved.

PSD-95 uncoupling from NMDA receptors by Tat- N-dimer ameliorates neuronal depolarization in cortical spreading depression.

PubMed

Kucharz, Krzysztof; Søndergaard Rasmussen, Ida; Bach, Anders; Strømgaard, Kristian; Lauritzen, Martin

2017-05-01

Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO 2 ) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO 2 . We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

[A correlation between respiration and synthesis of ATP in mitochondria at different degree of uncoupling of oxidative phosphorylation].

PubMed

Samartsev, V N; Kozhina, O V; Polishchuk, L S

2005-01-01

It is known that mitochondrial respiration in state 3 is due to three simultaneous and independent processes: synthesis of ATP (1), endogenous passive proton leakage (2), and proton leakage by protonophoric uncoupler (3). The total rate of processes (2) and (3) is equal to the product of respiration rate in state 4 and coefficient KR, which is defined as the ratio of the deltamuH+ value in state 3 to that in state 4. It is shown that it is possible to calculate both the rates of processes (1), (2) and (3) separately and the protonophoric activity of uncoupler using the coefficient KR and other coefficients, which are determined as the ratio of deltamuH+ values in state 3 or in state 4 to its maximal value. Simple methods of determination of these coefficients were developed, which are based on the study of the dependence of respiration rate in states 3 and 4 on the concentration of protonophoric uncoupler. It was found that the uncoupling action of palmitate, a natural uncoupler of oxidative phosphorylation, unlike classic uncoupler-protonophores DNP and FCCP, depends not only on its protonophoric activity but also on the inhibition of the process (1).

Expression, Purification, and Analysis of G-Protein-Coupled Receptor Kinases

PubMed Central

Sterne-Marr, Rachel; Baillargeon, Alison I.; Michalski, Kevin R.; Tesmer, John J.G.

2015-01-01

G-protein-coupled receptor (GPCR) kinases (GRKs) were first identified based on their ability to specifically phosphorylate activated GPCRs. Although many soluble substrates have since been identified, the chief physiological role of GRKs still remains the uncoupling of GPCRs from heterotrimeric G-proteins by promoting β-arrestin binding through the phosphorylation of the receptor. It is expected that GRKs recognize activated GPCRs through a docking site that not only recognizes the active conformation of the transmembrane domain of the receptor but also stabilizes a more catalytically competent state of the kinase domain. Many of the recent gains in understanding GRK-receptor interactions have been gleaned through biochemical and structural analysis of recombinantly expressed GRKs. Described herein are current techniques and procedures being used to express, purify, and assay GRKs in both in vitro and living cells. PMID:23351749

Peroxisome proliferator-activated receptors γ/mitochondrial uncoupling protein 2 signaling protects against seizure-induced neuronal cell death in the hippocampus following experimental status epilepticus

PubMed Central

2012-01-01

Background Status epilepticus induces subcellular changes that may lead to neuronal cell death in the hippocampus. However, the mechanism of seizure-induced neuronal cell death remains unclear. The mitochondrial uncoupling protein 2 (UCP2) is expressed in selected regions of the brain and is emerged as an endogenous neuroprotective molecule in many neurological disorders. We evaluated the neuroprotective role of UCP2 against seizure-induced hippocampal neuronal cell death under experimental status epilepticus. Methods In Sprague–Dawley rats, kainic acid (KA) was microinjected unilaterally into the hippocampal CA3 subfield to induce prolonged bilateral seizure activity. Oxidized protein level, translocation of Bcl-2, Bax and cytochrome c between cytosol and mitochondria, and expression of peroxisome proliferator-activated receptors γ (PPARγ) and UCP2 were examined in the hippocampal CA3 subfield following KA-induced status epilepticus. The effects of microinjection bilaterally into CA3 area of a PPARγ agonist, rosiglitazone or a PPARγ antagonist, GW9662 on UCP2 expression, induced superoxide anion (O2· -) production, oxidized protein level, mitochondrial respiratory chain enzyme activities, translocation of Bcl-2, Bax and cytochrome c, and DNA fragmentation in bilateral CA3 subfields were examined. Results Increased oxidized proteins and mitochondrial or cytosol translocation of Bax or cytochrome c in the hippocampal CA3 subfield was observed 3–48 h after experimental status epilepticus. Expression of PPARγ and UCP2 increased 12–48 h after KA-induced status epilepticus. Pretreatment with rosiglitazone increased UCP2 expression, reduced protein oxidation, O2· - overproduction and dysfunction of mitochondrial Complex I, hindered the translocation of Bax and cytochrome c, and reduced DNA fragmentation in the CA3 subfield. Pretreatment with GW9662 produced opposite effects. Conclusions Activation of PPARγ upregulated mitochondrial UCP2 expression, which decreased overproduction of reactive oxygen species, improved mitochondrial Complex I dysfunction, inhibited mitochondrial translocation of Bax and prevented cytosolic release of cytochrome c by stabilizing the mitochondrial transmembrane potential, leading to amelioration of apoptotic neuronal cell death in the hippocampus following status epilepticus. PMID:22849356

Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin

PubMed Central

Lee, Yang; Willers, Chrissie; Kunji, Edmund R. S.; Crichton, Paul G.

2015-01-01

Uncoupling protein 1 (UCP1) catalyzes fatty acid-activated, purine nucleotide-sensitive proton leak across the mitochondrial inner membrane of brown adipose tissue to produce heat, and could help combat obesity and metabolic disease in humans. Studies over the last 30 years conclude that the protein is a dimer, binding one nucleotide molecule per two proteins, and unlike the related mitochondrial ADP/ATP carrier, does not bind cardiolipin. Here, we have developed novel methods to purify milligram amounts of UCP1 from native sources by using covalent chromatography that, unlike past methods, allows the protein to be prepared in defined conditions, free of excess detergent and lipid. Assessment of purified preparations by TLC reveal that UCP1 retains tightly bound cardiolipin, with a lipid phosphorus content equating to three molecules per protein, like the ADP/ATP carrier. Cardiolipin stabilizes UCP1, as demonstrated by reconstitution experiments and thermostability assays, indicating that the lipid has an integral role in the functioning of the protein, similar to other mitochondrial carriers. Furthermore, we find that UCP1 is not dimeric but monomeric, as indicated by size exclusion analysis, and has a ligand titration profile in isothermal calorimetric measurements that clearly shows that one nucleotide binds per monomer. These findings reveal the fundamental composition of UCP1, which is essential for understanding the mechanism of the protein. Our assessment of the properties of UCP1 indicate that it is not unique among mitochondrial carriers and so is likely to use a common exchange mechanism in its primary function in brown adipose tissue mitochondria. PMID:26038550

Effects of 2 G on adiposity, leptin, lipoprotein lipase, and uncoupling protein-1 in lean and obese Zucker rats

NASA Technical Reports Server (NTRS)

Warren, L. E.; Horwitz, B. A.; Hamilton, J. S.; Fuller, C. A.

2001-01-01

Male Zucker rats were exposed to 2 G for 8 wk to test the hypothesis that the leptin regulatory pathway contributes to recovery from effects of 2 G on feeding, growth, and nutrient partitioning. After initial hypophagia, body mass-independent food intake of the lean rats exposed to 2 G surpassed that of the lean rats maintained at 1 G, but food intake of the obese rats exposed to 2 G remained low. After 8 wk at 2 G, body mass and carcass fat were less in both genotypes. Leptin and percent fat were lower in lean rats exposed to 2 G vs. 1 G but did not differ in obese rats exposed to 2 G vs. 1 G. Although exposure to 2 G did not alter uncoupling protein-1 levels, it did elicit white fat pad-specific changes in lipoprotein lipase activity in obese but not lean rats. We conclude that 2 G affects both genotypes but that the lean Zucker rats recover their food intake and growth rate and retain "normal" lipoprotein lipase activity to a greater degree than do the obese rats, emphasizing the importance of a functional leptin regulatory pathway in this acclimation.

Pig has no uncoupling protein 1.

PubMed

Hou, Lianjie; Shi, Jia; Cao, Lingbo; Xu, Guli; Hu, Chingyuan; Wang, Chong

2017-06-10

Brown adipose tissue (BAT) is critical for mammal's survival in the cold environment. Uncoupling protein 1 (UCP1) is responsible for the non-shivering thermogenesis in the BAT. Pig is important economically as a meat-producing livestock. However, whether BAT or more precisely UCP1 protein exists in pig remains a controversy. The objective of this study was to ascertain whether pig has UCP1 protein. In this study, we used rapid amplification of cDNA ends (RACE) technique to obtain the UCP1 mRNA 3' end sequence, confirmed only exons 1 and 2 of the UCP1 gene are transcribed in the pig. Then we cloned the pig UCP1 gene exons 1 and 2, and expressed the UCP1 protein from the truncated pig gene using E. coli BL21. We used the expressed pig UCP1 protein as antigen for antibody production in a rabbit. We could not detect any UCP1 protein expression in different pig adipose tissues by the specific pig UCP1 antibody, while our antibody can detect the cloned pig UCP1 as well as the mice adipose UCP1 protein. This result shows although exons 1 and 2 of the pig UCP1 gene were transcribed but not translated in the pig adipose tissue. Furthermore, we detected no uncoupled respiration in the isolated pig adipocytes. Thus, these results unequivocally demonstrate that pig has no UCP1 protein. Our results have resolved the controversy of whether pigs have the brown adipose tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

A novel amino acid and metabolomics signature in mice overexpressing muscle uncoupling protein 3.

PubMed

Aguer, Céline; Piccolo, Brian D; Fiehn, Oliver; Adams, Sean H; Harper, Mary-Ellen

2017-02-01

Uncoupling protein 3 (UCP3) is highly selectively expressed in skeletal muscle and is known to lower mitochondrial reactive oxygen species and promote fatty acid oxidation; however, the global impact of UCP3 activity on skeletal muscle and whole-body metabolism have not been extensively studied. We utilized untargeted metabolomics to identify novel metabolites that distinguish mice overexpressing UCP3 in muscle, both at rest and after exercise regimens that challenged muscle metabolism, to potentially unmask subtle phenotypes. Male wild-type (WT) and muscle-specific UCP3-overexpressing transgenic (UCP3 Tg) C57BL/6J mice were compared with or without a 5 wk endurance training protocol at rest or after an acute exercise bout (EB). Skeletal muscle, liver, and plasma samples were analyzed by gas chromatography time-of-flight mass spectrometry. Discriminant metabolites were considered if within the top 99th percentile of variable importance measurements obtained from partial least-squares discriminant analysis models. A total of 80 metabolites accurately discriminated UCP3 Tg mice from WT when modeled within a specific exercise condition (i.e., untrained/rested, endurance trained/rested, untrained/EB, and endurance trained/EB). Results revealed that several amino acids and amino acid derivatives in skeletal muscle and plasma of UCP3 Tg mice (e.g., Asp, Glu, Lys, Tyr, Ser, Met) were significantly reduced after an EB; that metabolites associated with skeletal muscle glutathione/Met/Cys metabolism (2-hydroxybutanoic acid, oxoproline, Gly, and Glu) were altered in UCP3 Tg mice across all training and exercise conditions; and that muscle metabolite indices of dehydrogenase activity were increased in UCP3 Tg mice, suggestive of a shift in tissue NADH/NAD + ratio. The results indicate that mitochondrial UCP3 activity affects metabolism well beyond fatty acid oxidation, regulating biochemical pathways associated with amino acid metabolism and redox status. That select metabolites were altered in liver of UCP3 Tg mice highlights that changes in muscle UCP3 activity can also affect other organ systems, presumably through changes in systemic metabolite trafficking.-Aguer, C., Piccolo, B. D., Fiehn, O., Adams, S. H., Harper, M.-E. A novel amino acid and metabolomics signature in mice overexpressing muscle uncoupling protein 3. © FASEB.

Mechanisms of inverse agonist action at D2 dopamine receptors

PubMed Central

Roberts, David J; Strange, Philip G

2005-01-01

Mechanisms of inverse agonist action at the D2(short) dopamine receptor have been examined. Discrimination of G-protein-coupled and -uncoupled forms of the receptor by inverse agonists was examined in competition ligand-binding studies versus the agonist [3H]NPA at a concentration labelling both G-protein-coupled and -uncoupled receptors. Competition of inverse agonists versus [3H]NPA gave data that were fitted best by a two-binding site model in the absence of GTP but by a one-binding site model in the presence of GTP. Ki values were derived from the competition data for binding of the inverse agonists to G-protein-uncoupled and -coupled receptors. Kcoupled and Kuncoupled were statistically different for the set of compounds tested (ANOVA) but the individual values were different in a post hoc test only for (+)-butaclamol. These observations were supported by simulations of these competition experiments according to the extended ternary complex model. Inverse agonist efficacy of the ligands was assessed from their ability to reduce agonist-independent [35S]GTPγS binding to varying degrees in concentration–response curves. Inverse agonism by (+)-butaclamol and spiperone occurred at higher potency when GDP was added to assays, whereas the potency of (−)-sulpiride was unaffected. These data show that some inverse agonists ((+)-butaclamol, spiperone) achieve inverse agonism by stabilising the uncoupled form of the receptor at the expense of the coupled form. For other compounds tested, we were unable to define the mechanism. PMID:15735658

Sarcolipin and uncoupling protein 1 play distinct roles in diet-induced thermogenesis and do not compensate for one another.

PubMed

Rowland, Leslie A; Maurya, Santosh K; Bal, Naresh C; Kozak, Leslie; Periasamy, Muthu

2016-07-01

It is well known that uncoupling protein 1 (UCP1) in brown adipose tissue plays an important role in diet-induced thermogenesis. In this study, whether sarcolipin (SLN), a regulator of sarco/endoplasmic reticulum Ca(2+) -ATPase pump in muscle, is also an important player of diet-induced thermogenesis was investigated, as well as whether loss of SLN could be compensated by increased UCP1 expression and vice versa. Age- and sex-matched UCP1(-/-) , SLN(-/-) , and double knockout for both UCP1 and SLN mice maintained in C57Bl/6J background were challenged to high-fat diet for 12 weeks and then analyzed for weight gain, alterations in serum metabolites, and changes in thermogenic protein expression. Loss of either SLN or UCP1 alone was sufficient to cause diet-induced obesity. No compensatory upregulation of UCP1 in SLN(-/-) mice or vice versa was found. Paradoxically, loss of both mechanisms failed to exacerbate the obesity phenotype. Data suggest that both SLN- and UCP1-based adaptive thermogenic mechanisms were essential for achieving maximal diet-induced thermogenesis. When both mechanisms were absent, less efficient thermogenic mechanisms were activated to counter energy imbalance. © 2016 The Obesity Society.

AKI after Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

PubMed Central

Huang, Luping; Belousova, Tatiana; Pan, Jenny Szu-Chin; Du, Jie; Ju, Huiming; Lu, Lianghao; Zhang, Pumin; Truong, Luan D.; Nuotio-Antar, Alli

2014-01-01

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre–transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions. PMID:24700878

Measurement of Single Channel Currents from Cardiac Gap Junctions

NASA Astrophysics Data System (ADS)

Veenstra, Richard D.; Dehaan, Robert L.

1986-08-01

Cardiac gap junctions consist of arrays of integral membrane proteins joined across the intercellular cleft at points of cell-to-cell contact. These junctional proteins are thought to form pores through which ions can diffuse from cytosol to cytosol. By monitoring whole-cell currents in pairs of embryonic heart cells with two independent patch-clamp circuits, the properties of single gap junction channels have been investigated. These channels had a conductance of about 165 picosiemens and underwent spontaneous openings and closings that were independent of voltage. Channel activity and macroscopic junctional conductance were both decreased by the uncoupling agent 1-octanol.

Protective effect of hydroxytyrosol in arsenic-induced mitochondrial dysfunction in rat brain.

PubMed

Soni, Manisha; Prakash, Chandra; Sehwag, Sfurti; Kumar, Vijay

2017-07-01

The present study was planned to investigate the protective effect of hydroxytyrosol (HT) against arsenic (As)-induced mitochondrial dysfunction in rat brain. Rats exposed to sodium arsenite (25 ppm for 8 weeks) showed decreased mitochondrial complexes (I, II, IV) activities, mitochondrial superoxide dismutase (MnSOD), and catalase activities in brain mitochondria. As-treated rats showed reduced mRNA expression of complex I (ND-1, ND-2), IV (COX-1, COX-4) subunits, and uncoupling protein-2 (UCP-2). In addition to this, As exposure downregulated the protein expression of MnSOD. Administration of HT with As restored the enzymatic activities of mitochondrial complexes, MnSOD and catalase, increased the mRNA levels of complexes subunits and UCP-2 as well as proteins level of MnSOD. These results suggest that HT efficiently restores mitochondrial dysfunction in As neurotoxicity and might be used as potential mitoprotective agent in future. © 2017 Wiley Periodicals, Inc.

Short-term consumption of a high-fat diet impairs whole-body efficiency and cognitive function in sedentary men.

PubMed

Edwards, Lindsay M; Murray, Andrew J; Holloway, Cameron J; Carter, Emma E; Kemp, Graham J; Codreanu, Ion; Brooker, Helen; Tyler, Damian J; Robbins, Peter A; Clarke, Kieran

2011-03-01

We recently showed that a short-term high-fat diet blunted exercise performance in rats, accompanied by increased uncoupling protein levels and greater respiratory uncoupling. In this study, we investigated the effects of a similar diet on physical and cognitive performance in humans. Twenty sedentary men were assessed when consuming a standardized, nutritionally balanced diet (control) and after 7 d of consuming a diet comprising 74% kcal from fat. Efficiency was measured during a standardized exercise task, and cognition was assessed using a computerized assessment battery. Skeletal muscle mitochondrial function was measured using (31)P magnetic resonance spectroscopy. The diet increased mean ± se plasma free fatty acids by 44% (0.32±0.03 vs. 0.46±0.05 mM; P<0.05) and decreased whole-body efficiency by 3% (21±1 vs. 18±1%; P<0.05), although muscle uncoupling protein (UCP3) content and maximal mitochondrial function were unchanged. High-fat diet consumption also increased subjects' simple reaction times (P<0.01) and decreased power of attention (P<0.01). Thus, we have shown that a high-fat diet blunts whole-body efficiency and cognition in sedentary men. We suggest that this effect may be due to increased respiratory uncoupling.

Distinct regions of the Pseudomonas syringae coiled-coil effector AvrRps4 are required for activation of immunity

PubMed Central

Sohn, Kee Hoon; Hughes, Richard K.; Piquerez, Sophie J.; Jones, Jonathan D. G.; Banfield, Mark J.

2012-01-01

Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding–leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4-dependent immunity in resistant accessions of Arabidopsis. To better understand the molecular basis of AvrRps4-triggered immunity, we determined the crystal structure of processed AvrRps4 (AvrRps4C, residues 134–221), revealing that it forms an antiparallel α-helical coiled coil. Structure-informed mutagenesis reveals an electronegative surface patch in AvrRps4C required for recognition by RPS4; mutations in this region can also uncouple triggering of the hypersensitive response from disease resistance. This uncoupling may result from a lower level of defense activation, sufficient for avirulence but not for triggering a hypersensitive response. Natural variation in AvrRps4 reveals distinct recognition specificities that involve a surface-exposed residue. Recently, a direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 has been implicated in activation of immunity. However, we were unable to detect direct interaction between AvrRps4 and Enhanced Disease Susceptibility 1 after coexpression in Nicotiana benthamiana or in yeast cells. How intracellular plant immune receptors activate defense upon effector perception remains an unsolved problem. The structure of AvrRps4C, and identification of functionally important residues for its activation of plant immunity, advances our understanding of these processes in a well-defined model pathosystem. PMID:22988101

Regulation of thermogenesis in plants: the interaction of alternative oxidase and plant uncoupling mitochondrial protein.

PubMed

Zhu, Yan; Lu, Jianfei; Wang, Jing; Chen, Fu; Leng, Feifan; Li, Hongyu

2011-01-01

Thermogenesis is a process of heat production in living organisms. It is rare in plants, but it does occur in some species of angiosperm. The heat is generated via plant mitochondrial respiration. As possible involvement in thermogenesis of mitochondrial factors, alternative oxidases (AOXs) and plant uncoupling mitochondrial proteins (PUMPs) have been well studied. AOXs and PUMPs are ubiquitously present in the inner membrane of plant mitochondria. They serve as two major energy dissipation systems that balance mitochondrial respiration and uncoupled phosphorylation by dissipating the H+ redox energy and proton electrochemical gradient (ΔμH+) as heat, respectively. AOXs and PUMPs exert similar physiological functions during homeothermic heat production in thermogenic plants. AOXs have five isoforms, while PUMPs have six. Both AOXs and PUMPs are encoded by small nuclear multigene families. Multiple isoforms are expressed in different tissues or organs. Extensive studies have been done in the area of thermogenesis in higher plants. In this review, we focus on the involvement and regulation of AOXs and PUMPs in thermogenesis.

Control of Mitochondrial pH by Uncoupling Protein 4 in Astrocytes Promotes Neuronal Survival*

PubMed Central

Perreten Lambert, Hélène; Zenger, Manuel; Azarias, Guillaume; Chatton, Jean-Yves; Magistretti, Pierre J.; Lengacher, Sylvain

2014-01-01

Brain activity is energetically costly and requires a steady and highly regulated flow of energy equivalents between neural cells. It is believed that a substantial share of cerebral glucose, the major source of energy of the brain, will preferentially be metabolized in astrocytes via aerobic glycolysis. The aim of this study was to evaluate whether uncoupling proteins (UCPs), located in the inner membrane of mitochondria, play a role in setting up the metabolic response pattern of astrocytes. UCPs are believed to mediate the transmembrane transfer of protons, resulting in the uncoupling of oxidative phosphorylation from ATP production. UCPs are therefore potentially important regulators of energy fluxes. The main UCP isoforms expressed in the brain are UCP2, UCP4, and UCP5. We examined in particular the role of UCP4 in neuron-astrocyte metabolic coupling and measured a range of functional metabolic parameters including mitochondrial electrical potential and pH, reactive oxygen species production, NAD/NADH ratio, ATP/ADP ratio, CO2 and lactate production, and oxygen consumption rate. In brief, we found that UCP4 regulates the intramitochondrial pH of astrocytes, which acidifies as a consequence of glutamate uptake, with the main consequence of reducing efficiency of mitochondrial ATP production. The diminished ATP production is effectively compensated by enhancement of glycolysis. This nonoxidative production of energy is not associated with deleterious H2O2 production. We show that astrocytes expressing more UCP4 produced more lactate, which is used as an energy source by neurons, and had the ability to enhance neuronal survival. PMID:25237189

Defective Expression of the Mitochondrial-tRNA Modifying Enzyme GTPBP3 Triggers AMPK-Mediated Adaptive Responses Involving Complex I Assembly Factors, Uncoupling Protein 2, and the Mitochondrial Pyruvate Carrier.

PubMed

Martínez-Zamora, Ana; Meseguer, Salvador; Esteve, Juan M; Villarroya, Magda; Aguado, Carmen; Enríquez, J Antonio; Knecht, Erwin; Armengod, M-Eugenia

2015-01-01

GTPBP3 is an evolutionary conserved protein presumably involved in mitochondrial tRNA (mt-tRNA) modification. In humans, GTPBP3 mutations cause hypertrophic cardiomyopathy with lactic acidosis, and have been associated with a defect in mitochondrial translation, yet the pathomechanism remains unclear. Here we use a GTPBP3 stable-silencing model (shGTPBP3 cells) for a further characterization of the phenotype conferred by the GTPBP3 defect. We experimentally show for the first time that GTPBP3 depletion is associated with an mt-tRNA hypomodification status, as mt-tRNAs from shGTPBP3 cells were more sensitive to digestion by angiogenin than tRNAs from control cells. Despite the effect of stable silencing of GTPBP3 on global mitochondrial translation being rather mild, the steady-state levels and activity of Complex I, and cellular ATP levels were 50% of those found in the controls. Notably, the ATPase activity of Complex V increased by about 40% in GTPBP3 depleted cells suggesting that mitochondria consume ATP to maintain the membrane potential. Moreover, shGTPBP3 cells exhibited enhanced antioxidant capacity and a nearly 2-fold increase in the uncoupling protein UCP2 levels. Our data indicate that stable silencing of GTPBP3 triggers an AMPK-dependent retrograde signaling pathway that down-regulates the expression of the NDUFAF3 and NDUFAF4 Complex I assembly factors and the mitochondrial pyruvate carrier (MPC), while up-regulating the expression of UCP2. We also found that genes involved in glycolysis and oxidation of fatty acids are up-regulated. These data are compatible with a model in which high UCP2 levels, together with a reduction in pyruvate transport due to the down-regulation of MPC, promote a shift from pyruvate to fatty acid oxidation, and to an uncoupling of glycolysis and oxidative phosphorylation. These metabolic alterations, and the low ATP levels, may negatively affect heart function.

Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria.

PubMed

Samartsev, V N; Belosludtsev, K N; Chezganova, S A; Zeldi, I P

2002-11-01

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg2+ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl2 and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg2+ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg2+ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.

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

Jung, D.W.; Laties, G.G.

Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg/sup 2 +/, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg/sup 2 +/-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60/sup 0/C for 2 minutesmore » also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.« less

Trends in Thermostability Provide Information on the Nature of Substrate, Inhibitor, and Lipid Interactions with Mitochondrial Carriers*

PubMed Central

Crichton, Paul G.; Lee, Yang; Ruprecht, Jonathan J.; Cerson, Elizabeth; Thangaratnarajah, Chancievan; King, Martin S.; Kunji, Edmund R. S.

2015-01-01

Mitochondrial carriers, including uncoupling proteins, are unstable in detergents, which hampers structural and mechanistic studies. To investigate carrier stability, we have purified ligand-free carriers and assessed their stability with a fluorescence-based thermostability assay that monitors protein unfolding with a thiol-reactive dye. We find that mitochondrial carriers from both mesophilic and thermophilic organisms exhibit poor stability in mild detergents, indicating that instability is inherent to the protein family. Trends in the thermostability of yeast ADP/ATP carrier AAC2 and ovine uncoupling protein UCP1 allow optimal conditions for stability in detergents to be established but also provide mechanistic insights into the interactions of lipids, substrates, and inhibitors with these proteins. Both proteins exhibit similar stability profiles across various detergents, where stability increases with the size of the associated detergent micelle. Detailed analysis shows that lipids stabilize carriers indirectly by increasing the associated detergent micelle size, but cardiolipin stabilizes by direct interactions as well. Cardiolipin reverses destabilizing effects of ADP and bongkrekic acid on AAC2 and enhances large stabilizing effects of carboxyatractyloside, revealing that this lipid interacts in the m-state and possibly other states of the transport cycle, despite being in a dynamic interface. Fatty acid activators destabilize UCP1 in a similar way, which can also be prevented by cardiolipin, indicating that they interact like transport substrates. Our controls show that carriers can be soluble but unfolded in some commonly used detergents, such as the zwitterionic Fos-choline-12, which emphasizes the need for simple validation assays like the one used here. PMID:25653283

Role of positively charged residues of the second transmembrane domain in the ion transport activity and conformation of human uncoupling protein-2.

PubMed

Hoang, Tuan; Matovic, Tijana; Parker, James; Smith, Matthew D; Jelokhani-Niaraki, Masoud

2015-04-14

Residing at the inner mitochondrial membrane, uncoupling protein-2 (UCP2) mediates proton transport from the intermembrane space (IMS) to the mitochondrial matrix and consequently reduces the rate of ATP synthesis in the mitochondria. The ubiquitous expression of UCP2 in humans can be attributed to the protein's multiple physiological roles in tissues, including its involvement in protective mechanisms against oxidative stress, as well as glucose and lipid metabolisms. Currently, the structural properties and ion transport mechanism of UCP2 and other UCP homologues remain poorly understood. UCP2-mediated proton transport is activated by fatty acids and inhibited by di- and triphosphate purine nucleotides. UCP2 also transports chloride and some other small anions. Identification of key amino acid residues of UCP2 in its ion transport pathway can shed light on the protein's ion transport function. On the basis of our previous studies, the second transmembrane helix segment (TM2) of UCP2 exhibited chloride channel activity. In addition, it was suggested that the positively charged residues on TM2 domains of UCPs 1 and 2 were important for their chloride transport activity. On this basis, to further understand the role of these positively charged residues on the ion transport activity of UCP2, we recombinantly expressed four TM2 mutants: R76Q, R88Q, R96Q, and K104Q. The wild type UCP2 and its mutants were purified and reconstituted into liposomes, and their conformation and ion (proton and chloride) transport activity were studied. TM2 Arg residues at the matrix interface of UCP2 proved to be crucial for the protein's anion transport function, and their absence resulted in highly diminished Cl(-) transport rates. On the other hand, the two other positively charged residues of TM2, located at the UCP2-IMS interface, could participate in the salt-bridge formation in the protein and promote the interhelical tight packing in the UCP2. Absence of these residues did not influence Cl(-) transport rates, but disturbed the dense packing in UCP2 and resulted in higher UCP2-mediated proton transport rates in the presence of long chain fatty acids. Overall, the outcome of this study provides a deeper and more detailed molecular image of UCP2's ion transport mechanism.

Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli.

PubMed

Borecký, J; Maia, I G; Costa, A D; Jezek, P; Chaimovich, H; de Andrade, P B; Vercesi, A E; Arruda, P

2001-09-14

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid-induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K(i) (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH-dependent. Kinetics of AtPUMP1-mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.

The Hydroxyl at Position C1 of Genipin Is the Active Inhibitory Group that Affects Mitochondrial Uncoupling Protein 2 in Panc-1 Cells

PubMed Central

Hou, Jianwei; Ding, Yue; Zhang, Tong; Zhang, Yong; Wang, Jianying; Shi, Chenchen; Fu, Wenwei; Cai, Zhenzhen

2016-01-01

Genipin (GNP) effectively inhibits uncoupling protein 2 (UCP2), which regulates the leakage of protons across the inner mitochondrial membrane. UCP2 inhibition may induce pancreatic adenocarcinoma cell death by increasing reactive oxygen species (ROS) levels. In this study, the hydroxyls at positions C10 (10-OH) and C1 (1-OH) of GNP were hypothesized to be the active groups that cause these inhibitory effects. Four GNP derivatives in which the hydroxyl at position C10 or C1 was replaced with other chemical groups were synthesized and isolated. Differences in the inhibitory effects of GNP and its four derivatives on pancreatic carcinoma cell (Panc-1) proliferation were assessed. The effects of GNP and its derivatives on apoptosis, UCP2 inhibition and ROS production were also studied to explore the relationship between GNP’s activity and its structure. The derivatives with 1-OH substitutions, geniposide (1-GNP1) and 1-ethyl-genipin (1-GNP2) lacked cytotoxic effects, while the other derivatives that retained 1-OH, 10-piv-genipin (10-GNP1) and 10-acetic acid-genipin (10-GNP2) exerted biological effects similar to those of GNP, even in the absence of 10-OH. Thus, 1-OH is the key functional group in the structure of GNP that is responsible for GNP’s apoptotic effects. These cytotoxic effects involve the induction of Panc-1 cell apoptosis through UCP2 inhibition and subsequent ROS production. PMID:26771380

The Hydroxyl at Position C1 of Genipin Is the Active Inhibitory Group that Affects Mitochondrial Uncoupling Protein 2 in Panc-1 Cells.

PubMed

Yang, Yang; Yang, Yifu; Hou, Jianwei; Ding, Yue; Zhang, Tong; Zhang, Yong; Wang, Jianying; Shi, Chenchen; Fu, Wenwei; Cai, Zhenzhen

2016-01-01

Genipin (GNP) effectively inhibits uncoupling protein 2 (UCP2), which regulates the leakage of protons across the inner mitochondrial membrane. UCP2 inhibition may induce pancreatic adenocarcinoma cell death by increasing reactive oxygen species (ROS) levels. In this study, the hydroxyls at positions C10 (10-OH) and C1 (1-OH) of GNP were hypothesized to be the active groups that cause these inhibitory effects. Four GNP derivatives in which the hydroxyl at position C10 or C1 was replaced with other chemical groups were synthesized and isolated. Differences in the inhibitory effects of GNP and its four derivatives on pancreatic carcinoma cell (Panc-1) proliferation were assessed. The effects of GNP and its derivatives on apoptosis, UCP2 inhibition and ROS production were also studied to explore the relationship between GNP's activity and its structure. The derivatives with 1-OH substitutions, geniposide (1-GNP1) and 1-ethyl-genipin (1-GNP2) lacked cytotoxic effects, while the other derivatives that retained 1-OH, 10-piv-genipin (10-GNP1) and 10-acetic acid-genipin (10-GNP2) exerted biological effects similar to those of GNP, even in the absence of 10-OH. Thus, 1-OH is the key functional group in the structure of GNP that is responsible for GNP's apoptotic effects. These cytotoxic effects involve the induction of Panc-1 cell apoptosis through UCP2 inhibition and subsequent ROS production.

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria.

PubMed

Rey, Benjamin; Roussel, Damien; Romestaing, Caroline; Belouze, Maud; Rouanet, Jean-Louis; Desplanches, Dominique; Sibille, Brigitte; Servais, Stéphane; Duchamp, Claude

2010-04-28

Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle. The abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase). This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity.

Up-regulation of avian uncoupling protein in cold-acclimated and hyperthyroid ducklings prevents reactive oxygen species production by skeletal muscle mitochondria

PubMed Central

2010-01-01

Background Although identified in several bird species, the biological role of the avian homolog of mammalian uncoupling proteins (avUCP) remains extensively debated. In the present study, the functional properties of isolated mitochondria were examined in physiological or pharmacological situations that induce large changes in avUCP expression in duckling skeletal muscle. Results The abundance of avUCP mRNA, as detected by RT-PCR in gastrocnemius muscle but not in the liver, was markedly increased by cold acclimation (CA) or pharmacological hyperthyroidism but was down-regulated by hypothyroidism. Activators of UCPs, such as superoxide with low doses of fatty acids, stimulated a GDP-sensitive proton conductance across the inner membrane of muscle mitochondria from CA or hyperthyroid ducklings. The stimulation was much weaker in controls and not observed in hypothyroid ducklings or in any liver mitochondrial preparations. The production of endogenous mitochondrial reactive oxygen species (ROS) was much lower in muscle mitochondria from CA and hyperthyroid ducklings than in the control or hypothyroid groups. The addition of GDP markedly increased the mitochondrial ROS production of CA or hyperthyroid birds up to, or above, the level of control or hypothyroid ducklings. Differences in ROS production among groups could not be attributed to changes in antioxidant enzyme activities (superoxide dismutase or glutathione peroxidase). Conclusion This work provides the first functional in vitro evidence that avian UCP regulates mitochondrial ROS production in situations of enhanced metabolic activity. PMID:20426850

Tuning the hydrophobicity overcomes unfavorable deprotonation making octylamino-substituted 7-nitrobenz-2-oxa-1,3-diazole (n-octylamino-NBD) a protonophore and uncoupler of oxidative phosphorylation in mitochondria.

PubMed

Denisov, Stepan S; Kotova, Elena A; Khailova, Ljudmila S; Korshunova, Galina A; Antonenko, Yuri N

2014-08-01

The environmentally sensitive fluorescent probe 7-nitrobenz-2-oxa-1,3-diazole (NBD) is generally utilized to monitor dynamic properties of membrane lipids and proteins. Here we studied the behavior of a homologous series of 4-n-alkylamino-substituted NBD derivatives (NBD-Cn; n=4, 6, 8, 9, 10, 12) in planar lipid bilayers, liposomes and isolated mitochondria. NBD-C10 induced proton conductivity in planar lipid membranes, while NBD-C4 was ineffective. The NBD-Cn compounds readily provoked proton permeability of neutral liposomes being less effective in negatively charged liposomes. NBD-Cn increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Remarkably, the bell-shaped dependence of the uncoupling activity of NBD-Cn on the alkyl chain length was found in mitochondria in contrast to the monotonous dependence in liposomes. The effect of NBD-Cn on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by mitochondria swelling. Binding of NBD-Cn to mitochondria increased with n, as shown by fluorescence correlation spectroscopy. It was concluded that despite a pKa value of the amino group in NBD-Cn being about 10, i.e. far from the physiological pH range, the expected hindering of the uncoupling activity could be overcome by inserting the alkyl chain of a certain length. Copyright © 2014 Elsevier B.V. All rights reserved.

Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature.

PubMed

Charlton, R R; Wenner, C E

1978-03-15

1. The interaction of intact Ehrlich ascites-tumour cells with Ca2+ at 37 degrees C consists of Ca2+ uptake followed by efflux from the cells. Under optimum conditions, two or three cycles of uptake and efflux are observed in the first 15 min after Ca2+ addition. 2. The respiratory substrates malate, succinate and ascorbate plus p-phenylenediamine support Ca2+ uptake. Ca2+ uptake at 37 degrees C is sensitive to the respiratory inhibitors rotenone and antimycin A when appropriate substrates are present. Ca2+ uptake and retention are inhibited by the uncoupler S-13. 3. Increasing extracellular Pi (12 to 30 mM) stimulates uncoupler-sensitive Ca2+ uptake, which reaches a maximum extent of 15 nmol/mg of protein when supported by succinate respiration. Ca2+ efflux is partially inhibited at 30 mM-Pi. 4. Optimum Ca2+ uptake occurs in the presence of succinate and Pi, suggesting that availability of substrate and Pi are rate-limiting. K. Ca2+ uptake occurs at 4 degrees C and is sensitive to uncouplers and oligomycin. Ca2+ efflux at this temperature is minimal. These data are consistent with a model in which passive diffusion of Ca2+ through the plasma membrane is followed by active uptake by the mitochondria. Ca2+ uptake is supported by substrates entering respiration at all three energy-coupling sites. Ca2+ efflux appears to be an active process with a high temperature coefficient.

Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1

PubMed Central

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

2017-01-01

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

Thyroid hormone effects on mitochondrial energetics.

PubMed

Harper, Mary-Ellen; Seifert, Erin L

2008-02-01

Thyroid hormones are the major endocrine regulators of metabolic rate, and their hypermetabolic effects are widely recognized. The cellular mechanisms underlying these metabolic effects have been the subject of much research. Thyroid hormone status has a profound impact on mitochondria, the organelles responsible for the majority of cellular adenosine triphosphate (ATP) production. However, mechanisms are not well understood. We review the effects of thyroid hormones on mitochondrial energetics and principally oxidative phosphorylation. Genomic and nongenomic mechanisms have been studied. Through the former, thyroid hormones stimulate mitochondriogenesis and thereby augment cellular oxidative capacity. Thyroid hormones induce substantial modifications in mitochondrial inner membrane protein and lipid compositions. Results are consistent with the idea that thyroid hormones activate the uncoupling of oxidative phosphorylation through various mechanisms involving inner membrane proteins and lipids. Increased uncoupling appears to be responsible for some of the hypermetabolic effects of thyroid hormones. ATP synthesis and turnover reactions are also affected. There appear to be complex relationships between mitochondrial proton leak mechanisms, reactive oxygen species production, and thyroid status. As the majority of studies have focused on the effects of thyroid status on rat liver preparations, there is still a need to address fundamental questions regarding thyroid hormone effects in other tissues and species.

UCP2 muscle gene transfer modifies mitochondrial membrane potential.

PubMed

Marti, A; Larrarte, E; Novo, F J; Garcia, M; Martinez, J A

2001-01-01

The aim of this work was to evaluate the effect of uncoupling protein 2 (UCP2) muscle gene transfer on mitochondrial activity. Five week-old male Wistar rats received an intramuscular injection of plasmid pXU1 containing UCP2 cDNA in the right tibialis anterior muscles. Left tibialis anterior muscles were injected with vehicle as control. Ten days after DNA injection, tibialis anterior muscles were dissected and muscle mitochondria isolated and analyzed. There were two mitochondrial populations in the muscle after UCP2 gene transfer, one of low fluorescence and complexity and the other, showing high fluorescence and complexity. UCP2 gene transfer resulted in a 3.6 fold increase in muscle UCP2 protein levels compared to control muscles assessed by Western blotting. Furthermore, a significant reduction in mitochondria membrane potential assessed by spectrofluorometry and flow cytometry was observed. The mitochondria membrane potential reduction might account for a decrease in fluorescence of the low fluorescence mitochondrial subpopulation. It has been demonstrated that UCP2 muscle gene transfer in vivo is associated with a lower mitochondria membrane potential. Our results suggest the potential involvement of UCP2 in uncoupling respiration. International Journal of Obesity (2001) 25, 68-74

An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

DOE PAGES

Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; ...

2015-03-23

N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producingmore » more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.« less

Molecular properties of muscarinic acetylcholine receptors

PubMed Central

HAGA, Tatsuya

2013-01-01

Muscarinic acetylcholine receptors, which comprise five subtypes (M1-M5 receptors), are expressed in both the CNS and PNS (particularly the target organs of parasympathetic neurons). M1-M5 receptors are integral membrane proteins with seven transmembrane segments, bind with acetylcholine (ACh) in the extracellular phase, and thereafter interact with and activate GTP-binding regulatory proteins (G proteins) in the intracellular phase: M1, M3, and M5 receptors interact with Gq-type G proteins, and M2 and M4 receptors with Gi/Go-type G proteins. Activated G proteins initiate a number of intracellular signal transduction systems. Agonist-bound muscarinic receptors are phosphorylated by G protein-coupled receptor kinases, which initiate their desensitization through uncoupling from G proteins, receptor internalization, and receptor breakdown (down regulation). Recently the crystal structures of M2 and M3 receptors were determined and are expected to contribute to the development of drugs targeted to muscarinic receptors. This paper summarizes the molecular properties of muscarinic receptors with reference to the historical background and bias to studies performed in our laboratories. PMID:23759942

Muscle uncoupling protein 3 overexpression mimics endurance training and reduces circulating biomarkers of incomplete β-oxidation.

PubMed

Aguer, Céline; Fiehn, Oliver; Seifert, Erin L; Bézaire, Véronic; Meissen, John K; Daniels, Amanda; Scott, Kyle; Renaud, Jean-Marc; Padilla, Marta; Bickel, David R; Dysart, Michael; Adams, Sean H; Harper, Mary-Ellen

2013-10-01

Exercise substantially improves metabolic health, making the elicited mechanisms important targets for novel therapeutic strategies. Uncoupling protein 3 (UCP3) is a mitochondrial inner membrane protein highly selectively expressed in skeletal muscle. Here we report that moderate UCP3 overexpression (roughly 3-fold) in muscles of UCP3 transgenic (UCP3 Tg) mice acts as an exercise mimetic in many ways. UCP3 overexpression increased spontaneous activity (∼40%) and energy expenditure (∼5-10%) and decreased oxidative stress (∼15-20%), similar to exercise training in wild-type (WT) mice. The increase in complete fatty acid oxidation (FAO; ∼30% for WT and ∼70% for UCP3 Tg) and energy expenditure (∼8% for WT and 15% for UCP3 Tg) in response to endurance training was higher in UCP3 Tg than in WT mice, showing an additive effect of UCP3 and endurance training on these two parameters. Moreover, increases in circulating short-chain acylcarnitines in response to acute exercise in untrained WT mice were absent with training or in UCP3 Tg mice. UCP3 overexpression had the same effect as training in decreasing long-chain acylcarnitines. Outcomes coincided with a reduction in muscle carnitine acetyltransferase activity that catalyzes the formation of acylcarnitines. Overall, results are consistent with the conclusions that circulating acylcarnitines could be used as a marker of incomplete muscle FAO and that UCP3 is a potential target for the treatment of prevalent metabolic diseases in which muscle FAO is affected.

Mitochondrial energy-dissipating systems (alternative oxidase, uncoupling proteins, and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings.

PubMed

Grabelnych, O I; Borovik, O A; Tauson, E L; Pobezhimova, T P; Katyshev, A I; Pavlovskaya, N S; Koroleva, N A; Lyubushkina, I V; Bashmakov, V Yu; Popov, V N; Borovskii, G B; Voinikov, V K

2014-06-01

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (-2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.

Chemically induced and light-independent cryptochrome photoreceptor activation.

PubMed

Rosenfeldt, Gesa; Viana, Rafael Muñoz; Mootz, Henning D; von Arnim, Albrecht G; Batschauer, Alfred

2008-01-01

The cryptochrome photoreceptors of higher plants are dimeric proteins. Their N-terminal photosensory domain mediates dimerization, and the unique C-terminal extension (CCT) mediates signaling. We made use of the human FK506-binding protein (FKBP) that binds with high affinity to rapamycin or rapamycin analogs (rapalogs). The FKBP-rapamycin complex is recognized by another protein, FRB, thus allowing rapamycin-induced dimerization of two target proteins. Here we demonstrate by bioluminescence resonance energy transfer (BRET) assays the applicability of this regulated dimerization system to plants. Furthermore, we show that fusion proteins consisting of the C-terminal domain of Arabidopsis cryptochrome 2 fused to FKBP and FRB and coexpressed in Arabidopsis cells specifically induce the expression of cryptochrome-controlled reporter and endogenous genes in darkness upon incubation with the rapalog. These results demonstrate that the activation of cryptochrome signal transduction can be chemically induced in a dose-dependent fashion and uncoupled from the light signal, and provide the groundwork for gain-of-function experiments to study specifically the role of photoreceptors in darkness or in signaling cross-talk even under light conditions that activate members of all photoreceptor families.

Uncoupling proteins of invertebrates: A review.

PubMed

Slocinska, Malgorzata; Barylski, Jakub; Jarmuszkiewicz, Wieslawa

2016-09-01

Uncoupling proteins (UCPs) mediate inducible proton conductance in the mitochondrial inner membrane. Herein, we summarize our knowledge regarding UCPs in invertebrates. Since 2001, the presence of UCPs has been demonstrated in nematodes, mollusks, amphioxi, and insects. We discuss the following important issues concerning invertebrate UCPs: their evolutionary relationships, molecular and functional properties, and physiological impact. Evolutionary analysis indicates that the branch of vertebrate and invertebrate UCP4-5 diverged early in the evolutionary process prior to the divergence of the animal groups. Several proposed physiological roles of invertebrate UCPs are energy control, metabolic balance, and preventive action against oxidative stress. © 2016 IUBMB Life, 68(9):691-699, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

Potential roles for uncoupling proteins in HIV lipodystrophy.

PubMed

Nolan, David; Pace, Craig

2004-07-01

The 'HIV lipodystrophy syndrome' consists of several distinct components, including lipoatrophy (pathological subcutaneous fat loss), lipohypertrophy (abdominal/visceral adiposity), and metabolic complications including insulin resistance and dyslipidemia. Lipoatrophy appears to represent an adipose tissue-specific form of mitochondrial toxicity associated strongly with stavudine NRTI therapy, whilst the 'metabolic syndrome' phenotype is associated with HIV protease inhibitor therapy. In this context, the role of uncoupling proteins (UCPs) in modulating resting energy expenditure in response to elevated fatty acid flux associated with the 'metabolic syndrome' is supported by clinical data as well as findings of elevated adipose tissue UCP expression. The role of UCPs in this syndrome therefore exemplifies the multifactorial nature of these antiretroviral therapy complications.

Expression profiling analysis: Uncoupling protein 2 deficiency improves hepatic glucose, lipid profiles and insulin sensitivity in high-fat diet-fed mice by modulating expression of genes in peroxisome proliferator-activated receptor signaling pathway.

PubMed

Zhou, Mei-Cen; Yu, Ping; Sun, Qi; Li, Yu-Xiu

2016-03-01

Uncoupling protein 2 (UCP2), which was an important mitochondrial inner membrane protein associated with glucose and lipid metabolism, widely expresses in all kinds of tissues including hepatocytes. The present study aimed to explore the impact of UCP2 deficiency on glucose and lipid metabolism, insulin sensitivity and its effect on the liver-associated signaling pathway by expression profiling analysis. Four-week-old male UCP2-/- mice and UCP2+/+ mice were randomly assigned to four groups: UCP2-/- on a high-fat diet, UCP2-/- on a normal chow diet, UCP2+/+ on a high-fat diet and UCP2+/+ on a normal chow diet. The differentially expressed genes in the four groups on the 16th week were identified by Affymetrix gene array. The results of intraperitoneal glucose tolerance test and insulin tolerance showed that blood glucose and β-cell function were improved in the UCP2-/- group on high-fat diet. Enhanced insulin sensitivity was observed in the UCP2-/- group. The differentially expressed genes were mapped to 23 pathways (P < 0.05). We concentrated on the 'peroxisome proliferator-activated receptor (PPAR) signaling pathway' (P = 3.19 × 10(-11)), because it is closely associated with the regulation of glucose and lipid profiles. In the PPAR signaling pathway, seven genes (PPARγ, Dbi, Acsl3, Lpl, Me1, Scd1, Fads2) in the UCP2-/- mice were significantly upregulated. The present study used gene arrays to show that activity of the PPAR signaling pathway involved in the improvement of glucose and lipid metabolism in the liver of UCP2-deficient mice on a long-term high-fat diet. The upregulation of genes in the PPAR signaling pathway could explain our finding that UCP2 deficiency ameliorated insulin sensitivity. The manipulation of UCP2 protein expression could represent a new strategy for the prevention and treatment of diabetes.

Salvianolic acid A inhibits calpain activation and eNOS uncoupling during focal cerebral ischemia in mice.

PubMed

Mahmood, Qaisar; Wang, Guang-Fa; Wu, Gang; Wang, Huan; Zhou, Chang-Xin; Yang, Hong-Yu; Liu, Zhi-Rong; Han, Feng; Zhao, Kui

2017-02-15

Salvianolic acid A (SAA) is obtained from Chinese herb Salviae Miltiorrhizae Bunge (Labiatae), has been reported to have the protective effects against cardiovascular and neurovascular diseases. The aim of present study was to investigate the relationship between the effectiveness of SAA against neurovascular injury and its effects on calpain activation and endothelial nitric oxide synthase (eNOS) uncoupling. SAA or vehicle was given to C57BL/6 male mice for seven days before the occlusion of middle cerebral artery (MCAO) for 60min. High-resolution positron emission tomography scanner (micro-PET) was used for small animal imaging to examine glucose metabolism. Rota-rod time and neurological deficit scores were calculated after 24h of reperfusion. The volume of infarction was determined by Nissl-staining. The calpain proteolytic activity and eNOS uncoupling were determined by western blot analysis. SAA administration increased glucose metabolism and ameliorated neuronal damage after brain ischemia, paralleled with decreased neurological deficit and volume of infarction. In addition, SAA pretreatment inhibited eNOS uncoupling and calpain proteolytic activity. Furthermore, SAA inhibited peroxynitrite (ONOO - ) generation and upregulates AKT, FKHR and ERK phosphorylation. These findings strongly suggest that SAA elicits a neurovascular protective role through the inhibition of eNOS uncoupling and ONOO - formation. Moreover, SAA attenuates spectrin and calcineurin breakdown and therefore protects the brain against ischemic/reperfusion injury. Copyright © 2016 Elsevier GmbH. All rights reserved.

Effects of mutations in the human uncoupling protein 3 gene on the respiratory quotient and fat oxidation in severe obesity and type 2 diabetes.

PubMed Central

Argyropoulos, G; Brown, A M; Willi, S M; Zhu, J; He, Y; Reitman, M; Gevao, S M; Spruill, I; Garvey, W T

1998-01-01

Human uncoupling protein 3 (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative ATP phosphorylation. With the capacity to participate in thermogenesis and energy balance, UCP3 is an important obesity candidate gene. A missense polymorphism in exon 3 (V102I) was identified in an obese and diabetic proband. A mutation introducing a stop codon in exon 4 (R143X) and a terminal polymorphism in the splice donor junction of exon 6 were also identified in a compound heterozygote that was morbidly obese and diabetic. Allele frequencies of the exon 3 and exon 6 splice junction polymorphisms were determined and found to be similar in Gullah-speaking African Americans and the Mende tribe of Sierra Leone, but absent in Caucasians. Moreover, in exon 6-splice donor heterozygotes, basal fat oxidation rates were reduced by 50%, and the respiratory quotient was markedly increased compared with wild-type individuals, implicating a role for UCP3 in metabolic fuel partitioning. PMID:9769326

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

PubMed Central

Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Houlden, Henry

2017-01-01

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. PMID:28360103

pH-dependent modulation of connexin-based gap junctional uncouplers

PubMed Central

Skeberdis, Vytenis A; Rimkute, Lina; Skeberdyte, Aiste; Paulauskas, Nerijus; Bukauskas, Feliksas F

2011-01-01

Abstract Gap junction (GJ) channels formed from connexin (Cx) proteins provide a direct pathway for electrical and metabolic cell–cell communication exhibiting high sensitivity to intracellular pH (pHi). We examined pHi-dependent modulation of junctional conductance (gj) of GJs formed of Cx26, mCx30.2, Cx36, Cx40, Cx43, Cx45, Cx46, Cx47 and Cx50 by reagents representing several distinct groups of uncouplers, such as long carbon chain alkanols (LCCAs), arachidonic acid, carbenoxolone, isoflurane, flufenamic acid and mefloquine. We demonstrate that alkalization by NH4Cl to pH ∼8 increased gj in cells expressing mCx30.2 and Cx45, yet did not affect gj of Cx26, Cx40, Cx46, Cx47 and Cx50 and decreased it in Cx43 and Cx36 GJs. Unexpectedly, cells expressing Cx45, but not other Cxs, exhibited full coupling recovery after alkalization with NH4Cl under the continuous presence of LCCAs, isoflurane and mefloquine. There was no coupling recovery by alkalization in the presence of arachidonic acid, carbenoxolone and flufenamic acid. In cells expressing Cx45, IC50 for octanol was 0.1, 0.25 and 2.68 mm at pHi values of 6.9, 7.2 and 8.1, respectively. Histidine modification of Cx45 protein by N-bromosuccinimide reduced the coupling-promoting effect of NH4Cl as well as the uncoupling effect of octanol. This suggests that LCCAs and some other uncouplers may act through the formation of hydrogen bonds with the as-of-yet unidentified histidine/s of the Cx45 GJ channel protein. PMID:21606109

Control of mitochondrial pH by uncoupling protein 4 in astrocytes promotes neuronal survival.

PubMed

Perreten Lambert, Hélène; Zenger, Manuel; Azarias, Guillaume; Chatton, Jean-Yves; Magistretti, Pierre J; Lengacher, Sylvain

2014-11-07

Brain activity is energetically costly and requires a steady and highly regulated flow of energy equivalents between neural cells. It is believed that a substantial share of cerebral glucose, the major source of energy of the brain, will preferentially be metabolized in astrocytes via aerobic glycolysis. The aim of this study was to evaluate whether uncoupling proteins (UCPs), located in the inner membrane of mitochondria, play a role in setting up the metabolic response pattern of astrocytes. UCPs are believed to mediate the transmembrane transfer of protons, resulting in the uncoupling of oxidative phosphorylation from ATP production. UCPs are therefore potentially important regulators of energy fluxes. The main UCP isoforms expressed in the brain are UCP2, UCP4, and UCP5. We examined in particular the role of UCP4 in neuron-astrocyte metabolic coupling and measured a range of functional metabolic parameters including mitochondrial electrical potential and pH, reactive oxygen species production, NAD/NADH ratio, ATP/ADP ratio, CO2 and lactate production, and oxygen consumption rate. In brief, we found that UCP4 regulates the intramitochondrial pH of astrocytes, which acidifies as a consequence of glutamate uptake, with the main consequence of reducing efficiency of mitochondrial ATP production. The diminished ATP production is effectively compensated by enhancement of glycolysis. This nonoxidative production of energy is not associated with deleterious H2O2 production. We show that astrocytes expressing more UCP4 produced more lactate, which is used as an energy source by neurons, and had the ability to enhance neuronal survival. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The mRNA expression levels of uncoupling proteins 1 and 2 in mononuclear cells from patients with metabolic disorders: obesity and type 2 diabetes mellitus.

PubMed

Margaryan, Sona; Witkowicz, Agata; Partyka, Anna; Yepiskoposyan, Levon; Manukyan, Gayane; Karabon, Lidia

2017-10-19

Type 2 diabetes mellitus (T2DM) and obesity are metabolic disorders whose major hallmark is insulin resistance. Impaired mitochondrial activity, such as reduced ratio of energy production to respiration, has been implicated in the development of insulin resistance. Uncoupling proteins (UCPs) are proton carriers, expressed in the mitochondrial inner membrane, that uncouple oxygen consumption by the respiratory chain from ATP synthesis. The aim of the study was to determine transcriptional levels of UCP1 and UCP2 in peripheral blood mononuclear cells (PBMCs) from patients with metabolic disorders: T2DM, obesity and from healthy individuals. The mRNA levels of UCP1, UCP2 were determined by Real-Time PCR method using Applied Biosystems assays. The UCP1 mRNA expression level was not detectable in the majority of studied samples, while very low expression was found in PBMCs from 3 obese persons. UCP2 mRNA expression level was detectable in all samples. The median mRNA expression of UCP2 was lower in all patients with metabolic disorders as compared to the controls (0.20+0.14 vs. 0.010+0.009, p=0.05). When compared separately, the differences of medians UCP2 mRNA expression level between the obese individuals and the controls as well as between the T2DM patients and the controls did not reach statistical significance. Decreased UCP2 gene expression in mononuclear cells from obese and diabetic patients might contribute to the immunological abnormalities in these metabolic disorders and suggests its role as a candidate gene in future studies of obesity and diabetes.

Uncoupling thermotolerance from the induction of heat shock proteins.

PubMed Central

Smith, B J; Yaffe, M P

1991-01-01

Exposure of cells to elevated temperatures causes a rapid increase in the synthesis of heat shock proteins (hsps) and induces thermotolerance, the increased ability of cells to survive exposure to lethal temperatures; however, the connection between hsp induction and the acquisition of thermotolerance is unclear. hsp induction in the yeast Saccharomyces cerevisiae is mediated by the activation of heat-shock transcription factor, and recently we have described a mutation, hsf1-m3, in heat-shock transcription factor that prevents the factor's activation. We now demonstrate that this mutation results in a general block in heat-shock induction but does not affect the acquisition of thermotolerance. Our results indicate that high-level induction of the major hsps is not required for cells to acquire thermotolerance. Images PMID:1763024

AKI after conditional and kidney-specific knockdown of Stanniocalcin-1

USDA-ARS?s Scientific Manuscript database

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overex...

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

PubMed

Reis, M; Farage, M; de Souza, A C; de Meis, L

2001-11-16

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.

Activation of cGMP/Protein Kinase G Pathway in Postconditioned Myocardium Depends on Reduced Oxidative Stress and Preserved Endothelial Nitric Oxide Synthase Coupling

PubMed Central

Inserte, Javier; Hernando, Victor; Vilardosa, Úrsula; Abad, Elena; Poncelas‐Nozal, Marcos; Garcia‐Dorado, David

2013-01-01

Background The cGMP/protein kinase G (PKG) pathway is involved in the cardioprotective effects of postconditioning (PoCo). Although PKG signaling in PoCo has been proposed to depend on the activation of the phosphatidylinositol 3‐kinase (PI3K)/Akt cascade, recent data bring into question a causal role of reperfusion injury signaling kinase (RISK) in PoCo protection. We hypothesized that PoCo increases PKG activity by reducing oxidative stress–induced endothelial nitric oxide synthase (NOS) uncoupling at the onset of reperfusion. Methods and Results Isolated rat hearts were submitted to 40 minutes of ischemia and reperfusion with and without a PoCo protocol. PoCo reduced infarct size by 48% and cGMP depletion. Blockade of cGMP synthesis (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one) and inhibition of PKG (KT5823) or NOS (l‐NAME) abolished protection, but inhibition of PI3K/Akt cascade (LY294002) did not (n=5 to 7 per group). Phosphorylation of the RISK pathway was higher in PoCo hearts. However, this difference is due to increased cell death in control hearts because in hearts reperfused with the contractile inhibitor blebbistatin, a drug effective in preventing cell death at the onset of reperfusion, RISK phosphorylation increased during reperfusion without differences between control and PoCo groups. In these hearts, PoCo reduced the production of superoxide (O2−) and protein nitrotyrosylation and increased nitrate/nitrite levels in parallel with a significant decrease in the oxidation of tetrahydrobiopterin (BH4) and in the monomeric form of endothelial NOS. Conclusions These results demonstrate that PoCo activates the cGMP/PKG pathway via a mechanism independent of the PI3K/Akt cascade and dependent on the reduction of O2− production at the onset of reperfusion, resulting in attenuated oxidation of BH4 and reduced NOS uncoupling. PMID:23525447

Brown adipose tissue dynamics in wild-type and UCP1-knockout mice: in vivo insights with magnetic resonance[S

PubMed Central

Grimpo, Kirsten; Völker, Maximilian N.; Heppe, Eva N.; Braun, Steve; Heverhagen, Johannes T.; Heldmaier, Gerhard

2014-01-01

We used noninvasive magnetic resonance imaging (MRI) and magnetic resonance spectroscopy to compare interscapular brown adipose tissue (iBAT) of wild-type (WT) and uncoupling protein 1 (UCP1)-knockout mice lacking UCP1-mediated nonshivering thermogenesis (NST). Mice were sequentially acclimated to an ambient temperature of 30°C, 18°C, and 5°C. We detected a remodeling of iBAT and a decrease in its lipid content in all mice during cold exposure. Ratios of energy-rich phosphates (ATP/ADP, phosphocreatine/ATP) in iBAT were maintained stable during noradrenergic stimulation of thermogenesis in cold- and warm-adapted mice and no difference between the genotypes was observed. As free fatty acids (FFAs) serve as fuel for thermogenesis and activate UCP1 for uncoupling of oxidative phosphorylation, brown adipose tissue is considered to be a main acceptor and consumer of FFAs. We measured a major loss of FFAs from iBAT during noradrenergic stimulation of thermogenesis. This mobilization of FFAs was observed in iBAT of WT mice as well as in mice lacking UCP1. The high turnover and the release of FFAs from iBAT suggests an enhancement of lipid metabolism, which in itself contributes to the sympathetically activated NST and which is independent from uncoupled respiration mediated by UCP1. Our study demonstrates that MRI, besides its potential for visualizing and quantification of fat tissue, is a valuable tool for monitoring functional in vivo processes like lipid and phosphate metabolism during NST. PMID:24343897

Uncoupling Lipid Metabolism from Inflammation through Fatty Acid Binding Protein-Dependent Expression of UCP2

PubMed Central

Xu, Hongliang; Hertzel, Ann V.; Steen, Kaylee A.; Wang, Qigui; Suttles, Jill

2015-01-01

Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2. PMID:25582199

Intrinsic and extrinsic uncoupling of oxidative phosphorylation.

PubMed

Kadenbach, Bernhard

2003-06-05

This article reviews parameters of extrinsic uncoupling of oxidative phosphorylation (OxPhos) in mitochondria, based on induction of a proton leak across the inner membrane. The effects of classical uncouplers, fatty acids, uncoupling proteins (UCP1-UCP5) and thyroid hormones on the efficiency of OxPhos are described. Furthermore, the present knowledge on intrinsic uncoupling of cytochrome c oxidase (decrease of H(+)/e(-) stoichiometry=slip) is reviewed. Among the three proton pumps of the respiratory chain of mitochondria and bacteria, only cytochrome c oxidase is known to exhibit a slip of proton pumping. Intrinsic uncoupling was shown after chemical modification, by site-directed mutagenesis of the bacterial enzyme, at high membrane potential DeltaPsi, and in a tissue-specific manner to increase thermogenesis in heart and skeletal muscle by high ATP/ADP ratios, and in non-skeletal muscle tissues by palmitate. In addition, two mechanisms of respiratory control are described. The first occurs through the membrane potential DeltaPsi and maintains high DeltaPsi values (150-200 mV). The second occurs only in mitochondria, is suggested to keep DeltaPsi at low levels (100-150 mV) through the potential dependence of the ATP synthase and the allosteric ATP inhibition of cytochrome c oxidase at high ATP/ADP ratios, and is reversibly switched on by cAMP-dependent phosphorylation. Finally, the regulation of DeltaPsi and the production of reactive oxygen species (ROS) in mitochondria at high DeltaPsi values (150-200 mV) are discussed.

Ursolic Acid-enriched herba cynomorii extract induces mitochondrial uncoupling and glutathione redox cycling through mitochondrial reactive oxygen species generation: protection against menadione cytotoxicity in h9c2 cells.

PubMed

Chen, Jihang; Wong, Hoi Shan; Ko, Kam Ming

2014-01-27

Herba Cynomorii (Cynomorium songaricum Rupr., Cynomoriaceae) is one of the most commonly used 'Yang-invigorating' tonic herbs in Traditional Chinese Medicine (TCM). An earlier study in our laboratory has demonstrated that HCY2, an ursolic acid-enriched fraction derived from Herba Cynomorii, increased mitochondrial ATP generation capacity (ATP-GC) and induced mitochondrial uncoupling as well as a cellular glutathione response, thereby protecting against oxidant injury in H9c2 cells. In this study, we demonstrated that pre-incubation of H9c2 cells with HCY2 increased mitochondrial reactive oxygen species (ROS) generation in these cells, which is likely an event secondary to the stimulation of the mitochondrial electron transport chain. The suppression of mitochondrial ROS by the antioxidant dimethylthiourea abrogated the HCY2-induced enhancement of mitochondrial uncoupling and glutathione reductase (GR)-mediated glutathione redox cycling, and also protected against menadione-induced cytotoxicity. Studies using specific inhibitors of uncoupling protein and GR suggested that the HCY2-induced mitochondrial uncoupling and glutathione redox cycling play a determining role in the cytoprotection against menadione-induced oxidant injury in H9c2 cells. Experimental evidence obtained thus far supports the causal role of HCY2-induced mitochondrial ROS production in eliciting mitochondrial uncoupling and glutathione antioxidant responses, which offer cytoprotection against oxidant injury in H9c2 cells.

The development of structure-activity relationships for mitochondrial dysfunction: uncoupling of oxidative phosphorylation.

PubMed

Naven, Russell T; Swiss, Rachel; Klug-McLeod, Jacquelyn; Will, Yvonne; Greene, Nigel

2013-01-01

Mitochondrial dysfunction has been implicated as an important factor in the development of idiosyncratic organ toxicity. An ability to predict mitochondrial dysfunction early in the drug development process enables the deselection of those drug candidates with potential safety liabilities, allowing resources to be focused on those compounds with the highest chance of success to the market. A database of greater than 2000 compounds was analyzed to identify structural and physicochemical features associated with the uncoupling of oxidative phosphorylation (herein defined as an increase in basal respiration). Many toxicophores associated with potent uncoupling activity were identified, and these could be divided into two main mechanistic classes, protonophores and redox cyclers. For the protonophores, potent uncoupling activity was often promoted by high lipophilicity and apparent stabilization of the anionic charge resulting from deprotonation of the protonophore. The potency of redox cyclers did not appear to be prone to variations in lipophilicity. Only 11 toxicophores were of sufficient predictive performance that they could be incorporated into a structural-alert model. Each alert was associated with one of three confidence levels (high, medium, and low) depending upon the lipophilicity-activity profile of the structural class. The final model identified over 68% of those compounds with potent uncoupling activity and with a value for specificity above 99%. We discuss the advantages and limitations of this approach and conclude that although structural alert methodology is useful for identifying toxicophores associated with mitochondrial dysfunction, they are not a replacement for the mitochondrial dysfunction assays in early screening paradigms.

RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans.

PubMed

Steiner, Florian A; Okihara, Kristy L; Hoogstrate, Suzanne W; Sijen, Titia; Ketting, René F

2009-02-01

RNA interference (RNAi) is a process in which double-stranded RNA is cleaved into small interfering RNAs (siRNAs) that induce the destruction of homologous single-stranded mRNAs. Argonaute proteins are essential components of this silencing process; they bind siRNAs directly and can cleave RNA targets using a conserved RNase H motif. In Caenorhabditis elegans, the Argonaute protein RDE-1 has a central role in RNAi. In animals lacking RDE-1, the introduction of double-stranded RNA does not trigger any detectable level of RNAi. Here we show that RNase H activity of RDE-1 is required only for efficient removal of the passenger strand of the siRNA duplex and not for triggering the silencing response at the target-mRNA level. These results uncouple the role of the RDE-1 RNase H activity in small RNA maturation from its role in target-mRNA silencing in vivo.

Arginase up-regulation and eNOS uncoupling contribute to impaired endothelium-dependent vasodilation in a rat model of intrauterine growth restriction.

PubMed

Grandvuillemin, Isabelle; Buffat, Christophe; Boubred, Farid; Lamy, Edouard; Fromonot, Julien; Charpiot, Philippe; Simoncini, Stephanie; Sabatier, Florence; Dignat-George, Françoise; Peyter, Anne-Christine; Simeoni, Umberto; Yzydorczyk, Catherine

2018-05-09

Individuals born after intrauterine growth restriction (IUGR) are at increased risk of developing cardiovascular diseases in adulthood, notably hypertension (HTN). Alterations in the vascular system, particularly impaired endothelium-dependent vasodilation, may play an important role in long-term effects of IUGR. Whether such vascular dysfunction precedes HTN has not been fully established in individuals born after IUGR. Moreover, the intimate mechanisms of altered endothelium-dependent vasodilation remain incompletely elucidated. We therefore investigated, using a rat model of IUGR, whether impaired endothelium-dependent relaxation precedes the development of HTN and whether key components of the L-Arginine-nitric oxide (NO) pathway are involved in its pathogenesis. Pregnant rats were fed with a control (CTRL, 23% casein) or low-protein diet (LP, 9% casein) to induce IUGR. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography in 5- and 8-week-old male offspring. Aortic rings were isolated to investigate relaxation to acetylcholine, NO production, eNOS protein content, arginase activity, and superoxide anion production. SBP was not different at 5 weeks, but significantly increased in 8-week-old LP vs. CRTL offspring. In 5-week-old LP vs. CRTL males, endothelium-dependent vasorelaxation was significantly impaired, but restored by pre-incubation with L-Arginine or the arginase inhibitor BEC; NO production was significantly reduced, but restored by L-Arginine pretreatment; total eNOS protein, dimer/monomer ratio, and arginase activity were significantly increased; superoxide anion production was significantly enhanced, but normalized by pretreatment with the NOS inhibitor L-NNA. In this model, IUGR leads to early-impaired endothelium-dependent vasorelaxation, resulting from arginase up-regulation and eNOS uncoupling, which precedes the development of HTN.

Impaired Expression of Uncoupling Protein 2 (UCP2) Causes Defective Post-ischemic Angiogenesis in Mice Deficient in AMP-activated Protein Kinase α Subunits

PubMed Central

Xu, Ming-Jiang; Song, Ping; Shirwany, Najeeb; Liang, Bin; Xing, Junjie; Viollet, Benoit; Wang, Xian; Zhu, Yi; Zou, Ming-Hui

2011-01-01

Objective The aim of the present study was to determine whether mitochondrial uncoupling protein (UCP)-2 is required for AMPK-dependent angiogenesis in ischemia in vivo. Methods and Results Angiogenesis was assayed by monitoring endothelial tube formation (a surrogate for angiogenesis) in human umbilical vein endothelial cells (HUVECs), isolated mouse aortic endothelial cells (MAECs), and pulmomary microvascular endothelial cells (PMECs), or in ischemic thigh adductor muscles from wild-type (WT) mice or mice deficient in either AMPKα1 or AMPKα2. AMPK inhibition with pharmacological inhibitor (compound C) or genetic means (transfection of AMPKα-specific siRNA) significantly lowered the tube formation in HUVECs. Consistently, compared with WT mice, tube formation in MAECs isolated from either AMPKα1−/− or AMPKα2−/− mice, which exhibited oxidative stress and reduced expression of UCP2, were significantly impaired. In addition, adenoviral overexpression of UCP2, but not adenoviruses encoding green florescence protein (GFP), normalized tube formation in MAECs from either AMPKα1−/− or AMPKα2−/− mice. Similarly, supplementation with sodium nitroprusside (SNP), a nitric oxide (NO) donor, restored tube formation. Furthermore, ischemia significantly increased angiogenesis, serine 1177 phosphorylation of endothelial NO synthase (eNOS), and UCP2 in ischemic thigh adductor muscles from WT mice, but not from either AMPKα1−/− or AMPKα2−/− mice. Conclusion We conclude that AMPK-dependent UCP2 expression in endothelial cells promotes angiogenesis in vivo. PMID:21597006

Nitric oxide, PKC-ε, and connexin43 are crucial for ischemic preconditioning-induced chemical gap junction uncoupling.

PubMed

Rong, Bing; Xie, Fei; Sun, Tao; Hao, Li; Lin, Ming-Jie; Zhong, Jing-Quan

2016-10-25

Ischemic preconditioning (IPC) maintains connexin43 (Cx43) phosphorylation and reduces chemical gap junction (GJ) coupling in cardiomyocytes to protect against ischemic damage. However, the signal transduction pathways underlying these effects are not fully understood. Here, we investigated whether nitric oxide (NO) and protein kinase C-ε (PKC-ε) contribute to IPC-induced cardioprotection by maintaining Cx43 phosphorylation and inhibiting chemical GJ coupling. IPC reduced ischemia-induced myocardial infarction and increased cardiomyocyte survival; phosphorylated Cx43, eNOS, and PKC-ε levels; and chemical GJ uncoupling. Administration of the NO donor SNAP mimicked the effects of IPC both in vivo and in vitro, maintaining Cx43 phosphorylation, promoting chemical GJ uncoupling, and reducing myocardial infarction. Preincubation with the NO synthase inhibitor L-NAME or PKC-ε translocation inhibitory peptide (PKC-ε-TIP) abolished these effects of IPC. Additionally, by inducing NO production, IPC induced translocation of PKC-ε, but not PKC-δ, from the cytosolic to the membrane fraction in primary cardiac myocytes. IPC-induced cardioprotection thus involves increased NO production, PKC-ε translocation, Cx43 phosphorylation, and chemical GJ uncoupling.

Skeletal muscle respiratory uncoupling prevents diet-induced obesity and insulin resistance in mice.

PubMed

Li, B; Nolte, L A; Ju, J S; Han, D H; Coleman, T; Holloszy, J O; Semenkovich, C F

2000-10-01

To determine whether uncoupling respiration from oxidative phosphorylation in skeletal muscle is a suitable treatment for obesity and type 2 diabetes, we generated transgenic mice expressing the mitochondrial uncoupling protein (Ucp) in skeletal muscle. Skeletal muscle oxygen consumption was 98% higher in Ucp-L mice (with low expression) and 246% higher in Ucp-H mice (with high expression) than in wild-type mice. Ucp mice fed a chow diet had the same food intake as wild-type mice, but weighed less and had lower levels of glucose and triglycerides and better glucose tolerance than did control mice. Ucp-L mice were resistant to obesity induced by two different high-fat diets. Ucp-L mice fed a high-fat diet had less adiposity, lower levels of glucose, insulin and cholesterol, and an increased metabolic rate at rest and with exercise. They were also more responsive to insulin, and had enhanced glucose transport in skeletal muscle in the setting of increased muscle triglyceride content. These data suggest that manipulating respiratory uncoupling in muscle is a viable treatment for obesity and its metabolic sequelae.

Muscle Mitochondrial Uncoupling Dismantles Neuromuscular Junction and Triggers Distal Degeneration of Motor Neurons

PubMed Central

Dupuis, Luc; Gonzalez de Aguilar, Jose-Luis; Echaniz-Laguna, Andoni; Eschbach, Judith; Rene, Frédérique; Oudart, Hugues; Halter, Benoit; Huze, Caroline; Schaeffer, Laurent; Bouillaud, Frédéric; Loeffler, Jean-Philippe

2009-01-01

Background Amyotrophic lateral sclerosis (ALS), the most frequent adult onset motor neuron disease, is associated with hypermetabolism linked to defects in muscle mitochondrial energy metabolism such as ATP depletion and increased oxygen consumption. It remains unknown whether muscle abnormalities in energy metabolism are causally involved in the destruction of neuromuscular junction (NMJ) and subsequent motor neuron degeneration during ALS. Methodology/Principal Findings We studied transgenic mice with muscular overexpression of uncoupling protein 1 (UCP1), a potent mitochondrial uncoupler, as a model of muscle restricted hypermetabolism. These animals displayed age-dependent deterioration of the NMJ that correlated with progressive signs of denervation and a mild late-onset motor neuron pathology. NMJ regeneration and functional recovery were profoundly delayed following injury of the sciatic nerve and muscle mitochondrial uncoupling exacerbated the pathology of an ALS animal model. Conclusions/Significance These findings provide the proof of principle that a muscle restricted mitochondrial defect is sufficient to generate motor neuron degeneration and suggest that therapeutic strategies targeted at muscle metabolism might prove useful for motor neuron diseases. PMID:19404401

Changes in the hydrogen-bonding strength of internal water molecules and cysteine residues in the conductive state of channelrhodopsin-1

NASA Astrophysics Data System (ADS)

Lórenz-Fonfría, Víctor A.; Muders, Vera; Schlesinger, Ramona; Heberle, Joachim

2014-12-01

Water plays an essential role in the structure and function of proteins, particularly in the less understood class of membrane proteins. As the first of its kind, channelrhodopsin is a light-gated cation channel and paved the way for the new and vibrant field of optogenetics, where nerve cells are activated by light. Still, the molecular mechanism of channelrhodopsin is not understood. Here, we applied time-resolved FT-IR difference spectroscopy to channelrhodopsin-1 from Chlamydomonas augustae. It is shown that the (conductive) P2380 intermediate decays with τ ≈ 40 ms and 200 ms after pulsed excitation. The vibrational changes between the closed and the conductive states were analyzed in the X-H stretching region (X = O, S, N), comprising vibrational changes of water molecules, sulfhydryl groups of cysteine side chains and changes of the amide A of the protein backbone. The O-H stretching vibrations of "dangling" water molecules were detected in two different states of the protein using H218O exchange. Uncoupling experiments with a 1:1 mixture of H2O:D2O provided the natural uncoupled frequencies of the four O-H (and O-D) stretches of these water molecules, each with a very weakly hydrogen-bonded O-H group (3639 and 3628 cm-1) and with the other O-H group medium (3440 cm-1) to moderately strongly (3300 cm-1) hydrogen-bonded. Changes in amide A and thiol vibrations report on global and local changes, respectively, associated with the formation of the conductive state. Future studies will aim at assigning the respective cysteine group(s) and at localizing the "dangling" water molecules within the protein, providing a better understanding of their functional relevance in CaChR1.

The mechanism of uncoupling by picrate in Escherichia coli K-12 membrane systems.

PubMed

Michels, M; Bakker, E P

1981-06-01

The mechanism of action of the uncoupler picrate on intact cells and everted membrane vesicles of Escherichia coli K-12 was investigated. Like in mitochondria [Hanstein, W. G. and Hatefi, Y. (1974) Proc. Natl Acad. Sci. USA, 71, 288-292], it was observed that picrate uncoupled energy-linked functions only in everted, but not in intact membrane systems. In the vesicles picrate also decreased the magnitude of the transmembrane proton-motive force at concentrations similar to those at which it caused uncoupling. Experiments with 14C-labelled picrate showed that this compound bound both to deenergized intact cells and everted vesicles. However, upon energization of the membrane, picrate was extruded from the intact cell and taken up to a larger extent by the vesicles. These energy-dependent changes in picrate uptake correlated with the magnitude of the transmembrane electrical potential, delta psi. It is therefore proposed that picrate is a permeant uncoupler, that delta psi is the driving force for picrate movement across biological membranes, and that the uncoupling activity of picrate in everted membrane systems is due to its protonophoric action.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

PubMed

Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

2017-05-26

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Mitochondrial uncoupling protein may participate in futile cycling of pyruvate and other monocarboxylates.

PubMed

Jezek, P; Borecký, J

1998-08-01

The physiological role of monocarboxylate transport in brown adipose tissue mitochondria has been reevaluated. We studied pyruvate, alpha-ketoisovalerate, alpha-ketoisocaproate, and phenylpyruvate uniport via the uncoupling protein (UCP1) as a GDP-sensitive swelling in K+ salts induced by valinomycin or by monensin and carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone in Na+ salts. We have demonstrated that this uniport is inhibited by fatty acids. GDP inhibition in K+ salts was not abolished by an uncoupler, indicating a negligible monocarboxylic acid penetration via the lipid bilayer. In contrast, the electroneutral pyruvate uptake (swelling in ammonium pyruvate or potassium pyruvate induced by change in pH) mediated by the pyruvate carrier was inhibited by its specific inhibitor alpha-cyano-4-hydroxycinnamate but not by fatty acids. Moreover, alpha-cyano-4-hydroxycinnamate enhanced the energization of brown adipose tissue mitochondria, which was monitored fluorometrically by 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide and safranin O. Consequently, we suggest that UCP1 might participate in futile cycling of unipolar ketocarboxylates under certain physiological conditions while expelling these anions from the matrix. The cycle is completed on their return via the pyruvate carrier in an H+ symport mode.

Dissociation Between Brown Adipose Tissue 18F-FDG Uptake and Thermogenesis in Uncoupling Protein 1-Deficient Mice.

PubMed

Hankir, Mohammed K; Kranz, Mathias; Keipert, Susanne; Weiner, Juliane; Andreasen, Sille G; Kern, Matthias; Patt, Marianne; Klöting, Nora; Heiker, John T; Brust, Peter; Hesse, Swen; Jastroch, Martin; Fenske, Wiebke K

2017-07-01

18 F-FDG PET imaging is routinely used to investigate brown adipose tissue (BAT) thermogenesis, which requires mitochondrial uncoupling protein 1 (UCP1). It remains uncertain, however, whether BAT 18 F-FDG uptake is a reliable surrogate measure of UCP1-mediated heat production. Methods: UCP1 knockout (KO) and wild-type (WT) mice housed at thermoneutrality were treated with the selective β3 adrenergic receptor agonist CL 316, 243 and underwent metabolic cage, infrared thermal imaging and 18 F-FDG PET/MRI experiments. Primary brown adipocytes were additionally examined for their bioenergetics by extracellular flux analysis as well as their uptake of 2-deoxy- 3 H-glucose. Results: In response to CL 316, 243 treatments, oxygen consumption, and BAT thermogenesis were diminished in UCP1 KO mice, but BAT 18 F-FDG uptake was fully retained. Isolated UCP1 KO brown adipocytes exhibited defective induction of uncoupled respiration whereas their glycolytic flux and 2-deoxy- 3 H-glucose uptake rates were largely unaffected. Conclusion: Adrenergic stimulation can increase BAT 18 F-FDG uptake independently of UCP1 thermogenic function. © 2017 by the Society of Nuclear Medicine and Molecular Imaging.

Drug-activated multiple pathways of defensin mRNA regulation in HL-60 cells are defined by reversed roles of participating protein kinases.

PubMed

Herwig, S; Su, Q; Tempst, P

1998-10-01

Defensin transcription in HL-60 promyelocytic leukemia cells is greatly enhanced during retinoic acid (RA)-induced differentiation. We have probed this regulatory pathway by selective modulation of various kinase activities. Induction was potentiated by elevated cAMP and attenuated by protein kinase C inhibition, entirely correlated to enhanced or blocked morphological differentiation, respectively. Yet, defensin mRNA was also induced in undifferentiated HL-60 cells, but not in others, by cAMP alone. By contrast, modulators that cooperated with RA had adverse effects on the normal capacity of dimethyl sulfoxide to up regulate these transcripts as well. Thus, defensin mRNA accumulation can be selectively uncoupled from maturation stage; and transcript levels may be regulated by multiple pathways, each independently acted upon by different chemical inducers.

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

PubMed

Cavalheiro, R A; Fortes, F; Borecký, J; Faustinoni, V C; Schreiber, A Z; Vercesi, A E

2004-10-01

The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

The genetic association study between polymorphisms in uncoupling protein 2 and uncoupling protein 3 and metabolic data in dogs.

PubMed

Udagawa, Chihiro; Tada, Naomi; Asano, Junzo; Ishioka, Katsumi; Ochiai, Kazuhiko; Bonkobara, Makoto; Tsuchida, Shuichi; Omi, Toshinori

2014-12-11

The uncoupling proteins (UCPs) in the mitochondrial inner membrane are members of the mitochondrial anion carrier protein family that play an important role in energy homeostasis. Genetic association studies have shown that human UCP2 and UCP3 variants (SNPs and indels) are associated with obesity, insulin resistance, type 2 diabetes mellitus, and metabolic syndrome. The aim of this study was to examine the genetic association between polymorphisms in UCP2 and UCP3 and metabolic data in dogs. We identified 10 SNPs (9 intronic and 1 exonic) and 4 indels (intronic) in UCP2, and 13 SNPs (11 intronic and 2 exonic) and one indel (exonic) in UCP3, by DNA sequence analysis of 11 different dog breeds (n=119). An association study between these UCP2 and UCP3 variants and the biochemical parameters of glucose, total cholesterol, lactate dehydrogenase and triglyceride in Labrador Retrievers (n=50) showed that none of the UCP2 polymorphisms were significantly associated with the levels of these parameters. However, four UCP3 SNPs (intron 1) were significantly associated with total cholesterol levels. In addition, the allele frequencies of two of the four SNPs associated with higher total cholesterol levels in a breed that is susceptible to hypercholesterolemia (Shetland Sheepdogs, n=30), compared with the control breed (Shiba, n=30). The results obtained from a limited number of individuals suggest that the UCP3 gene in dogs may be associated with total cholesterol levels. The examination of larger sample sizes and further analysis will lead to increased precision of these results.

Exploring Uncoupling Proteins and Antioxidant Mechanisms under Acute Cold Exposure in Brains of Fish

PubMed Central

Lucassen, Magnus; Schmidt, Maike M.; Dringen, Ralf; Abele, Doris; Hwang, Pung-Pung

2011-01-01

Exposure to fluctuating temperatures accelerates the mitochondrial respiration and increases the formation of mitochondrial reactive oxygen species (ROS) in ectothermic vertebrates including fish. To date, little is known on potential oxidative damage and on protective antioxidative defense mechanisms in the brain of fish under cold shock. In this study, the concentration of cellular protein carbonyls in brain was significantly increased by 38% within 1 h after cold exposure (from 28°C to 18°C) of zebrafish (Danio rerio). In addition, the specific activity of superoxide dismutase (SOD) and the mRNA level of catalase (CAT) were increased after cold exposure by about 60% (6 h) and by 60%–90% (1 and 24 h), respectively, while the specific glutathione content as well as the ratio of glutathione disulfide to glutathione remained constant and at a very low level. In addition, cold exposure increased the protein level of hypoxia-inducible factor (HIF) by about 50% and the mRNA level of the glucose transporter zglut3 in brain by 50%–100%. To test for an involvement of uncoupling proteins (UCPs) in the cold adaptation of zebrafish, five UCP members were annotated and identified (zucp1-5). With the exception of zucp1, the mRNA levels of the other four zucps were significantly increased after cold exposure. In addition, the mRNA levels of four of the fish homologs (zppar) of the peroxisome proliferator-activated receptor (PPAR) were increased after cold exposure. These data suggest that PPARs and UCPs are involved in the alterations observed in zebrafish brain after exposure to 18°C. The observed stimulation of the PPAR-UCP axis may help to prevent oxidative damage and to maintain metabolic balance and cellular homeostasis in the brains of ectothermic zebrafish upon cold exposure. PMID:21464954

BAIBA Does Not Regulate UCP-3 Expression in Human Skeletal Muscle as a Response to Aerobic Exercise.

PubMed

Morales, Flor E; Forsse, Jeffrey S; Andre, Thomas L; McKinley-Barnard, Sarah K; Hwang, Paul S; Anthony, Ian G; Tinsley, Grant M; Spillane, Mike; Grandjean, Peter W; Ramirez, Alejandro; Willoughby, Darryn S

2017-01-01

β-Aminoisobutyric acid (BAIBA) has shown to modulate uncoupling protein (UCP)-1 expression, which is mainly expressed in white adipose tissue; however, no studies to date have analyzed its potential effect on the main uncoupling protein of skeletal muscle, UCP-3. The main goal of this study was to assess the potential effect of acute aerobic exercise on serum BAIBA and skeletal muscle UCP-3. The secondary goal was to assess the potential involvement of the transcription factors proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and peroxisome proliferator-activated receptor alpha (PPARα), as well as free fatty acids (FFAs) in UCP-3 expression. A tertiary goal of the study was to evaluate the potential effect of consuming a preexercise meal on the outcome of the first 2 objectives. In a randomized crossover design, untrained participants performed 2 acute cycling sessions (350 kcal at 70% of their VO 2peak ) after 2 experimental conditions: (1) consumption of a multi-macronutrient shake and (2) a fasting period of 8 hours. Blood samples were taken at baseline, preexercise, postexercise, 1 hour, and 4 hours postexercise, and muscle biopsies were taken at the last 4 time points. UCP-3 protein concentration and expression, as well as the mRNA expression of PGC-1α and PPARα, were measured in muscle, and BAIBA, glucose, and FFA were measured in serum. Aerobic exercise failed to induce a significant effect on serum BAIBA, PGC-1α, and PPARα regardless on the feeding condition. Despite the lack of effect of exercise on the previous variables, UCP-3 expression and protein concentration significantly increased in the shake condition. The expression of human skeletal muscle UCP-3 as a result of exercise might be controlled by factors other than BAIBA.

Activation of the AMP-Activated Protein Kinase by Eicosapentaenoic Acid (EPA, 20:5 n-3) Improves Endothelial Function In Vivo

PubMed Central

Wu, Yong; Zhang, Cheng; Dong, Yunzhou; Wang, Shuangxi; Song, Ping; Viollet, Benoit; Zou, Ming-Hui

2012-01-01

The aim of the present study was to test the hypothesis that the cardiovascular-protective effects of eicosapentaenoic acid (EPA) may be due, in part, to its ability to stimulate the AMP-activated protein kinase (AMPK)-induced endothelial nitric oxide synthase (eNOS) activation. The role of AMPK in EPA-induced eNOS phosphorylation was investigated in bovine aortic endothelial cells (BAEC), in mice deficient of either AMPKα1 or AMPKα2, in eNOS knockout (KO) mice, or in Apo-E/AMPKα1 dual KO mice. EPA-treatment of BAEC increased both AMPK-Thr172 phosphorylation and AMPK activity, which was accompanied by increased eNOS phosphorylation, NO release, and upregulation of mitochondrial uncoupling protein-2 (UCP-2). Pharmacologic or genetic inhibition of AMPK abolished EPA-enhanced NO release and eNOS phosphorylation in HUVEC. This effect of EPA was absent in the aortas isolated from either eNOS KO mice or AMPKα1 KO mice fed a high-fat, high-cholesterol (HFHC) diet. EPA via upregulation of UCP-2 activates AMPKα1 resulting in increased eNOS phosphorylation and consequent improvement of endothelial function in vivo. PMID:22532857

Bardoxolone Methyl Prevents Mesenteric Fat Deposition and Inflammation in High-Fat Diet Mice.

PubMed

Dinh, Chi H L; Szabo, Alexander; Yu, Yinghua; Camer, Danielle; Wang, Hongqin; Huang, Xu-Feng

2015-01-01

Mesenteric fat belongs to visceral fat. An increased deposition of mesenteric fat contributes to obesity associated complications such as type 2 diabetes and cardiovascular diseases. We have investigated the therapeutic effects of bardoxolone methyl (BARD) on mesenteric adipose tissue of mice fed a high-fat diet (HFD). Male C57BL/6J mice were administered oral BARD during HFD feeding (HFD/BARD), only fed a high-fat diet (HFD), or fed low-fat diet (LFD) for 21 weeks. Histology and immunohistochemistry were used to analyse mesenteric morphology and macrophages, while Western blot was used to assess the expression of inflammatory, oxidative stress, and energy expenditure proteins. Supplementation of drinking water with BARD prevented mesenteric fat deposition, as determined by a reduction in large adipocytes. BARD prevented inflammation as there were fewer inflammatory macrophages and reduced proinflammatory cytokines (interleukin-1 beta and tumour necrosis factor alpha). BARD reduced the activation of extracellular signal-regulated kinase (ERK) and Akt, suggesting an antioxidative stress effect. BARD upregulates energy expenditure proteins, judged by the increased activity of tyrosine hydroxylase (TH) and AMP-activated protein kinase (AMPK) and increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and uncoupling protein 2 (UCP2) proteins. Overall, BARD induces preventive effect in HFD mice through regulation of mesenteric adipose tissue.

Frequent Extreme Cold Exposure and Brown Fat and Cold-Induced Thermogenesis: A Study in a Monozygotic Twin

PubMed Central

Vosselman, Maarten J.; Vijgen, Guy H. E. J.; Kingma, Boris R. M.; Brans, Boudewijn; van Marken Lichtenbelt, Wouter D.

2014-01-01

Introduction Mild cold acclimation is known to increase brown adipose tissue (BAT) activity and cold-induced thermogenesis (CIT) in humans. We here tested the effect of a lifestyle with frequent exposure to extreme cold on BAT and CIT in a Dutch man known as ‘the Iceman’, who has multiple world records in withstanding extreme cold challenges. Furthermore, his monozygotic twin brother who has a ‘normal’ sedentary lifestyle without extreme cold exposures was measured. Methods The Iceman (subject A) and his brother (subject B) were studied during mild cold (13°C) and thermoneutral conditions (31°C). Measurements included BAT activity and respiratory muscle activity by [18F]FDG-PET/CT imaging and energy expenditure through indirect calorimetry. In addition, body temperatures, cardiovascular parameters, skin perfusion, and thermal sensation and comfort were measured. Finally, we determined polymorphisms for uncoupling protein-1 and β3-adrenergic receptor. Results Subjects had comparable BAT activity (A: 1144 SUVtotal and B: 1325 SUVtotal), within the range previously observed in young adult men. They were genotyped with the polymorphism for uncoupling protein-1 (G/G). CIT was relatively high (A: 40.1% and B: 41.9%), but unlike during our previous cold exposure tests in young adult men, here both subjects practiced a g-Tummo like breathing technique, which involves vigorous respiratory muscle activity. This was confirmed by high [18F]FDG-uptake in respiratory muscle. Conclusion No significant differences were found between the two subjects, indicating that a lifestyle with frequent exposures to extreme cold does not seem to affect BAT activity and CIT. In both subjects, BAT was not higher compared to earlier observations, whereas CIT was very high, suggesting that g-Tummo like breathing during cold exposure may cause additional heat production by vigorous isometric respiratory muscle contraction. The results must be interpreted with caution given the low subject number and the fact that both participants practised the g-Tummo like breathing technique. PMID:25014028

Long-chain 3-hydroxy fatty acids accumulating in long-chain 3-hydroxyacyl-CoA dehydrogenase and mitochondrial trifunctional protein deficiencies uncouple oxidative phosphorylation in heart mitochondria.

PubMed

Tonin, Anelise M; Amaral, Alexandre U; Busanello, Estela N B; Grings, Mateus; Castilho, Roger F; Wajner, Moacir

2013-02-01

Cardiomyopathy is a common clinical feature of some inherited disorders of mitochondrial fatty acid β-oxidation including mitochondrial trifunctional protein (MTP) and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiencies. Since individuals affected by these disorders present tissue accumulation of various fatty acids, including long-chain 3-hydroxy fatty acids, in the present study we investigated the effect of 3-hydroxydecanoic (3 HDCA), 3-hydroxydodecanoic (3 HDDA), 3-hydroxytetradecanoic (3 HTA) and 3-hydroxypalmitic (3 HPA) acids on mitochondrial oxidative metabolism, estimated by oximetry, NAD(P)H content, hydrogen peroxide production, membrane potential (ΔΨ) and swelling in rat heart mitochondrial preparations. We observed that 3 HTA and 3 HPA increased resting respiration and diminished the respiratory control and ADP/O ratios using glutamate/malate or succinate as substrates. Furthermore, 3 HDDA, 3 HTA and 3 HPA decreased ΔΨ, the matrix NAD(P)H pool and hydrogen peroxide production. These data indicate that these fatty acids behave as uncouplers of oxidative phosphorylation. We also verified that 3 HTA-induced uncoupling-effect was not mediated by the adenine nucleotide translocator and that this fatty acid induced the mitochondrial permeability transition pore opening in calcium-loaded organelles since cyclosporin A prevented the reduction of mitochondrial ΔΨ and swelling provoked by 3 HTA. The present data indicate that major 3-hydroxylated fatty acids accumulating in MTP and LCHAD deficiencies behave as strong uncouplers of oxidative phosphorylation potentially impairing heart energy homeostasis.

H2O2-Activated Mitochondrial Phospholipase iPLA2γ Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein–Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic β-Cells

PubMed Central

Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin

2015-01-01

Abstract Aims: Pancreatic β-cell chronic lipotoxicity evolves from acute free fatty acid (FA)–mediated oxidative stress, unprotected by antioxidant mechanisms. Since mitochondrial uncoupling protein-2 (UCP2) plays antioxidant and insulin-regulating roles in pancreatic β-cells, we tested our hypothesis, that UCP2-mediated uncoupling attenuating mitochondrial superoxide production is initiated by FA release due to a direct H2O2-induced activation of mitochondrial phospholipase iPLA2γ. Results: Pro-oxidant tert-butylhydroperoxide increased respiration, decreased membrane potential and mitochondrial matrix superoxide release rates of control but not UCP2- or iPLA2γ-silenced INS-1E cells. iPLA2γ/UCP2-mediated uncoupling was alternatively activated by an H2O2 burst, resulting from palmitic acid (PA) β-oxidation, and it was prevented by antioxidants or catalase overexpression. Exclusively, nascent FAs that cleaved off phospholipids by iPLA2γ were capable of activating UCP2, indicating that the previously reported direct redox UCP2 activation is actually indirect. Glucose-stimulated insulin release was not affected by UCP2 or iPLA2γ silencing, unless pro-oxidant activation had taken place. PA augmented insulin secretion via G-protein–coupled receptor 40 (GPR40), stimulated by iPLA2γ-cleaved FAs (absent after GPR40 silencing). Innovation and Conclusion: The iPLA2γ/UCP2 synergy provides a feedback antioxidant mechanism preventing oxidative stress by physiological FA intake in pancreatic β-cells, regulating glucose-, FA-, and redox-stimulated insulin secretion. iPLA2γ is regulated by exogenous FA via β-oxidation causing H2O2 signaling, while FAs are cleaved off phospholipids, subsequently acting as amplifying messengers for GPR40. Hence, iPLA2γ acts in eminent physiological redox signaling, the impairment of which results in the lack of antilipotoxic defense and contributes to chronic lipotoxicity. Antioxid. Redox Signal. 23, 958–972. PMID:25925080

Exposure of embryos to cyclically cold incubation temperatures durably affects energy metabolism and antioxidant pathways in broiler chickens.

PubMed

Loyau, T; Collin, A; Yenisey, C; Crochet, S; Siegel, P B; Akşit, M; Yalçin, S

2014-08-01

Cyclically cold incubation temperatures have been suggested as a means to improve resistance of broiler chickens to ascites; however, the underlying mechanisms are not known. Nine hundred eggs obtained from 48 wk Ross broiler breeders were randomly assigned to 2 incubation treatments: control I eggs were incubated at 37.6°C throughout, whereas for cold I eggs the incubation temperature was reduced by 1°C for 6 h daily from 10 to 18 d of incubation. Thereafter, chickens were reared at standard temperatures or under cold exposure that was associated or not with a postnatal cold acclimation at d 5 posthatch. At hatch, hepatic catalase activity and malondialdehyde content were measured. Serum thyroid hormone and triglyceride concentrations, and muscle expression of several genes involved in the regulation of energy metabolism and oxidative stress were also measured at hatch and 5 and 25 d posthatch. Cold incubation induced modifications in antioxidant pathways with higher catalase activity, but lower expression of avian uncoupling protein 3 at hatch. However, long-term enhancement in the expression of avian uncoupling protein 3 was observed, probably caused by an increase in the expression of the transcription factor peroxisome proliferator activated receptor-γ coactivator-1α. These effects were not systematically associated with an increase in serum triiodothyronine concentrations that were observed only in chickens exposed to both cold incubation and later acclimation at 5 d with cold rearing. Our results suggest that these conditions of cyclically cold incubation resulted in the long-term in changes in antioxidant pathways and energy metabolism, which could enhance the health of chickens reared under cold conditions. © Poultry Science Association Inc.

Genistein modulates oxidative stress in breast cancer cell lines according to ERα/ERβ ratio: effects on mitochondrial functionality, sirtuins, uncoupling protein 2 and antioxidant enzymes.

PubMed

Nadal-Serrano, Mercedes; Pons, Daniel Gabriel; Sastre-Serra, Jorge; Blanquer-Rosselló, M del Mar; Roca, Pilar; Oliver, Jordi

2013-09-01

Genistein is a biologically active isoflavone with estrogenic activity and can be found in a variety of soy products. This natural compound displays a wide array of biological activities, but it is best known for its ability to inhibit cancer progression, especially for hormone-related ones such as breast cancer. Genistein has been shown to bind both the estrogen receptor alpha (ERα) and the estrogen receptor beta (ERβ), although it has a higher affinity for the ERβ. The ERα/ERβ ratio is a prognostic marker for breast tumors, and ERβ expression could indicate the presence of tumors more benign in state, whereas ERα indicates malignant tumors. The objective of the present study was to investigate the effects of genistein on oxidative stress and mitochondrial functionality through its interaction with the estrogen receptor in breast cancer cell lines with different ERα/ERβ ratios. The lower ERα/ERβ ratio T47D cell line showed lower oxidative stress and greater mitochondrial functionality, along with an up-regulation of uncoupling protein 2 and sirtuins. On the other hand, genistein-treated MCF-7 cell line, with the highest ERα/ERβ ratio, reported no changes for the control situation. On the whole, our results show different genistein effects depending on ERα/ERβ ratio for oxidative stress regulation, mitochondrial functionality, and modulation of UCPs, antioxidant enzymes and sirtuins in breast cancer cell lines. Effects of genistein on oxidative stress and mitochondria could be due at least in part, to a higher ERβ presence, but could also be due to up-regulation of ERβ caused by the genistein treatment. Copyright © 2013 Elsevier Ltd. All rights reserved.

Renal mitochondrial impairment is attenuated by AT1 blockade in experimental Type I diabetes.

PubMed

de Cavanagh, Elena M V; Ferder, León; Toblli, Jorge E; Piotrkowski, Bárbara; Stella, Inés; Fraga, Cesar G; Inserra, Felipe

2008-01-01

To investigate whether ANG II type 1 (AT(1)) receptor blockade could protect kidney mitochondria in streptozotocin-induced Type 1 diabetes, we treated 8-wk-old male Sprague-Dawley rats with a single streptozotocin injection (65 mg/kg ip; STZ group), streptozotocin and drinking water containing either losartan (30 mg.kg(-1).day(-1); STZ+Los group) or amlodipine (3 mg.kg(-1).day(-1); STZ+Amlo group), or saline (intraperitoneally) and pure water (control group). Four-month-long losartan or amlodipine treatments started 30 days before streptozotocin injection to improve the antioxidant defenses. The number of renal lesions, plasma glucose and lipid levels, and proteinuria were higher and creatinine clearance was lower in STZ and STZ+Amlo compared with STZ+Los and control groups. Glycemia was higher in STZ+Los compared with control. Blood pressure, basal mitochondrial membrane potential and mitochondrial pyruvate content, and renal oxidized glutathione levels were higher and NADH/cytochrome c oxidoreductase activity was lower in STZ compared with the other groups. In STZ and STZ+Amlo groups, mitochondrial H(2)O(2) production rate was higher and uncoupling protein-2 content, cytochrome c oxidase activity, and renal glutathione level were lower than in STZ+Los and control groups. Mitochondrial nitric oxide synthase activity was higher in STZ+Amlo compared with the other groups. Mitochondrial pyruvate content and H(2)O(2) production rate negatively contributed to electron transfer capacity and positively contributed to renal lesions. Uncoupling protein-2 content negatively contributed to mitochondrial H(2)O(2) production rate and renal lesions. Renal glutathione reduction potential positively contributed to mitochondria electron transfer capacity. In conclusion, AT(1) blockade protects kidney mitochondria and kidney structure in streptozotocin-induced diabetes independently of blood pressure and glycemia.

Uncoupling protein 2 deficiency mimics the effects of hypoxia and endoplasmic reticulum stress on mitochondria and triggers pseudohypoxic pulmonary vascular remodeling and pulmonary hypertension.

PubMed

Dromparis, Peter; Paulin, Roxane; Sutendra, Gopinath; Qi, Andrew C; Bonnet, Sébastien; Michelakis, Evangelos D

2013-07-05

Mitochondrial signaling regulates both the acute and the chronic response of the pulmonary circulation to hypoxia, and suppressed mitochondrial glucose oxidation contributes to the apoptosis-resistance and proliferative diathesis in the vascular remodeling in pulmonary hypertension. Hypoxia directly inhibits glucose oxidation, whereas endoplasmic reticulum (ER)-stress can indirectly inhibit glucose oxidation by decreasing mitochondrial calcium (Ca²⁺m levels). Both hypoxia and ER stress promote proliferative pulmonary vascular remodeling. Uncoupling protein 2 (UCP2) has been shown to conduct calcium from the ER to mitochondria and suppress mitochondrial function. We hypothesized that UCP2 deficiency reduces Ca²⁺m in pulmonary artery smooth muscle cells (PASMCs), mimicking the effects of hypoxia and ER stress on mitochondria in vitro and in vivo, promoting normoxic hypoxia inducible factor-1α activation and pulmonary hypertension. Ucp2 knockout (KO)-PASMCs had lower mitochondrial calcium than Ucp2 wildtype (WT)-PASMCs at baseline and during histamine-stimulated ER-Ca²⁺ release. Normoxic Ucp2KO-PASMCs had mitochondrial hyperpolarization, lower Ca²⁺-sensitive mitochondrial enzyme activity, reduced levels of mitochondrial reactive oxygen species and Krebs' cycle intermediates, and increased resistance to apoptosis, mimicking the hypoxia-induced changes in Ucp2WT-PASMC. Ucp2KO mice spontaneously developed pulmonary vascular remodeling and pulmonary hypertension and exhibited a pseudohypoxic state with pulmonary vascular and systemic hypoxia inducible factor-1α activation (increased hematocrit), not exacerbated further by chronic hypoxia. This first description of the role of UCP2 in oxygen sensing and in pulmonary hypertension vascular remodeling may open a new window in biomarker and therapeutic strategies.

Structure and dynamics of a constitutively active neurotensin receptor

DOE PAGES

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; ...

2016-12-07

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

Structure and dynamics of a constitutively active neurotensin receptor

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

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecularmore » dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. In conclusion, the loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.« less

Structure and dynamics of a constitutively active neurotensin receptor

PubMed Central

Krumm, Brian E.; Lee, Sangbae; Bhattacharya, Supriyo; Botos, Istvan; White, Courtney F.; Du, Haijuan; Vaidehi, Nagarajan; Grisshammer, Reinhard

2016-01-01

Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist. PMID:27924846

Short-Chain Fatty Acid Acetate Stimulates Adipogenesis and Mitochondrial Biogenesis via GPR43 in Brown Adipocytes.

PubMed

Hu, Jiamiao; Kyrou, Ioannis; Tan, Bee K; Dimitriadis, Georgios K; Ramanjaneya, Manjunath; Tripathi, Gyanendra; Patel, Vanlata; James, Sean; Kawan, Mohamed; Chen, Jing; Randeva, Harpal S

2016-05-01

Short-chain fatty acids play crucial roles in a range of physiological functions. However, the effects of short-chain fatty acids on brown adipose tissue have not been fully investigated. We examined the role of acetate, a short-chain fatty acid formed by fermentation in the gut, in the regulation of brown adipocyte metabolism. Our results show that acetate up-regulates adipocyte protein 2, peroxisomal proliferator-activated receptor-γ coactivator-1α, and uncoupling protein-1 expression and affects the morphological changes of brown adipocytes during adipogenesis. Moreover, an increase in mitochondrial biogenesis was observed after acetate treatment. Acetate also elicited the activation of ERK and cAMP response element-binding protein, and these responses were sensitive to G(i/o)-type G protein inactivator, Gβγ-subunit inhibitor, phospholipase C inhibitor, and MAPK kinase inhibitor, indicating a role for the G(i/o)βγ/phospholipase C/protein kinase C/MAPK kinase signaling pathway in these responses. These effects of acetate were mimicked by treatment with 4-chloro-α-(1-methylethyl)-N-2-thiazolylbenzeneacetamide, a synthetic G protein-coupled receptor 43 (GPR43) agonist and were impaired in GPR43 knockdown cells. Taken together, our results indicate that acetate may have important physiological roles in brown adipocytes through the activation of GPR43.

Uncoupling and oxidative stress in liver mitochondria isolated from rats with acute iron overload.

PubMed

Pardo Andreu, G L; Inada, N M; Vercesi, A E; Curti, C

2009-01-01

One hypothesis for the etiology of cell damage arising from iron overload is that its excess selectively affects mitochondria. Here we tested the effects of acute iron overload on liver mitochondria isolated from rats subjected to a single dose of i.p. 500 mg/kg iron-dextran. The treatment increased the levels of iron in mitochondria (from 21 +/- 4 to 130 +/- 7 nmol/mg protein) and caused both lipid peroxidation and glutathione oxidation. The mitochondria of iron-treated rats showed lower respiratory control ratio in association with higher resting respiration. The mitochondrial uncoupling elicited by iron-treatment did not affect the phosphorylation efficiency or the ATP levels, suggesting that uncoupling is a mitochondrial protective mechanism against acute iron overload. Therefore, the reactive oxygen species (ROS)/H+ leak couple, functioning as a mitochondrial redox homeostatic mechanism could play a protective role in the acutely iron-loaded mitochondria.

Effect of Brood Pheromone on Survival and Nutrient Intake of African Honey Bees (Apis mellifera scutellata) under Controlled Conditions.

PubMed

Démares, Fabien J; Yusuf, Abdullahi A; Nicolson, Susan W; Pirk, Christian W W

2017-05-01

The influence of pheromones on insect physiology and behavior has been thoroughly reported for numerous aspects, such as attraction, gland development, aggregation, mate and kin recognition. Brood pheromone (BP) is released by honey bee larvae to indicate their protein requirements to the colony. Although BP is known to modulate pollen and protein consumption, which in turn can affect physiological and morphological parameters, such as hypopharyngeal gland (HPG) development and ovarian activation, few studies have focused on the effect of BP on nutritional balance. In this study, we exposed newly emerged worker bees for 14 d and found that BP exposure increased protein intake during the first few days, with a peak in consumption at day four following exposure. BP exposure decreased survival of caged honey bees, but did not affect either the size of the HPG acini or ovarian activation stage. The uncoupling of the BP releaser effect, facilitated by working under controlled conditions, and the presence of larvae as stimulating cues are discussed.

Capsaicin in Metabolic Syndrome

PubMed Central

Bliss, Edward

2018-01-01

Capsaicin, the major active constituent of chilli, is an agonist on transient receptor potential vanilloid channel 1 (TRPV1). TRPV1 is present on many metabolically active tissues, making it a potentially relevant target for metabolic interventions. Insulin resistance and obesity, being the major components of metabolic syndrome, increase the risk for the development of cardiovascular disease, type 2 diabetes, and non-alcoholic fatty liver disease. In vitro and pre-clinical studies have established the effectiveness of low-dose dietary capsaicin in attenuating metabolic disorders. These responses of capsaicin are mediated through activation of TRPV1, which can then modulate processes such as browning of adipocytes, and activation of metabolic modulators including AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor α (PPARα), uncoupling protein 1 (UCP1), and glucagon-like peptide 1 (GLP-1). Modulation of these pathways by capsaicin can increase fat oxidation, improve insulin sensitivity, decrease body fat, and improve heart and liver function. Identifying suitable ways of administering capsaicin at an effective dose would warrant its clinical use through the activation of TRPV1. This review highlights the mechanistic options to improve metabolic syndrome with capsaicin. PMID:29772784

Capsaicin in Metabolic Syndrome.

PubMed

Panchal, Sunil K; Bliss, Edward; Brown, Lindsay

2018-05-17

Capsaicin, the major active constituent of chilli, is an agonist on transient receptor potential vanilloid channel 1 (TRPV1). TRPV1 is present on many metabolically active tissues, making it a potentially relevant target for metabolic interventions. Insulin resistance and obesity, being the major components of metabolic syndrome, increase the risk for the development of cardiovascular disease, type 2 diabetes, and non-alcoholic fatty liver disease. In vitro and pre-clinical studies have established the effectiveness of low-dose dietary capsaicin in attenuating metabolic disorders. These responses of capsaicin are mediated through activation of TRPV1, which can then modulate processes such as browning of adipocytes, and activation of metabolic modulators including AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor α (PPARα), uncoupling protein 1 (UCP1), and glucagon-like peptide 1 (GLP-1). Modulation of these pathways by capsaicin can increase fat oxidation, improve insulin sensitivity, decrease body fat, and improve heart and liver function. Identifying suitable ways of administering capsaicin at an effective dose would warrant its clinical use through the activation of TRPV1. This review highlights the mechanistic options to improve metabolic syndrome with capsaicin.

Cellular and subcellular localization of uncoupling protein 2 in the human kidney.

PubMed

Nigro, Michelangelo; De Sanctis, Claudia; Formisano, Pietro; Stanzione, Rosita; Forte, Maurizio; Capasso, Giovambattista; Gigliotti, Giuseppe; Rubattu, Speranza; Viggiano, Davide

2018-06-23

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.

Upregulation of uncoupling proteins by oral administration of capsiate, a nonpungent capsaicin analog.

PubMed

Masuda, Yoriko; Haramizu, Satoshi; Oki, Kasumi; Ohnuki, Koichiro; Watanabe, Tatsuo; Yazawa, Susumu; Kawada, Teruo; Hashizume, Shu-ichi; Fushiki, Tohru

2003-12-01

Capsiate is a nonpungent capsaicin analog, a recently identified principle of the nonpungent red pepper cultivar CH-19 Sweet. In the present study, we report that 2-wk treatment of capsiate increased metabolic rate and promoted fat oxidation at rest, suggesting that capsiate may prevent obesity. To explain these effects, at least in part, we examined uncoupling proteins (UCPs) and thyroid hormones. UCPs and thyroid hormones play important roles in energy expenditure, the maintenance of body weight, and thermoregulation. Two-week treatment of capsiate increased the levels of UCP1 protein and mRNA in brown adipose tissue and UCP2 mRNA in white adipose tissue. This dose of capsiate did not change serum triiodothyronine or thyroxine levels. A single dose of capsiate temporarily raised both UCP1 mRNA in brown adipose tissue and UCP3 mRNA in skeletal muscle. These results suggest that UCP1 and UCP2 may contribute to the promotion of energy metabolism by capsiate, but that thyroid hormones do not.

Nitric oxide, PKC-ε, and connexin43 are crucial for ischemic preconditioning-induced chemical gap junction uncoupling

PubMed Central

Sun, Tao; Hao, Li; Lin, Ming-Jie; Zhong, Jing-Quan

2016-01-01

Ischemic preconditioning (IPC) maintains connexin43 (Cx43) phosphorylation and reduces chemical gap junction (GJ) coupling in cardiomyocytes to protect against ischemic damage. However, the signal transduction pathways underlying these effects are not fully understood. Here, we investigated whether nitric oxide (NO) and protein kinase C-ε (PKC-ε) contribute to IPC-induced cardioprotection by maintaining Cx43 phosphorylation and inhibiting chemical GJ coupling. IPC reduced ischemia-induced myocardial infarction and increased cardiomyocyte survival; phosphorylated Cx43, eNOS, and PKC-ε levels; and chemical GJ uncoupling. Administration of the NO donor SNAP mimicked the effects of IPC both in vivo and in vitro, maintaining Cx43 phosphorylation, promoting chemical GJ uncoupling, and reducing myocardial infarction. Preincubation with the NO synthase inhibitor L-NAME or PKC-ε translocation inhibitory peptide (PKC-ε-TIP) abolished these effects of IPC. Additionally, by inducing NO production, IPC induced translocation of PKC-ε, but not PKC-δ, from the cytosolic to the membrane fraction in primary cardiac myocytes. IPC-induced cardioprotection thus involves increased NO production, PKC-ε translocation, Cx43 phosphorylation, and chemical GJ uncoupling. PMID:27655723

Destabilization of mitochondrial functions as a target against breast cancer progression: Role of TPP{sup +}-linked-polyhydroxybenzoates

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

Sandoval-Acuña, Cristian

Mitochondrion is an accepted molecular target in cancer treatment since it exhibits a higher transmembrane potential in cancer cells, making it susceptible to be targeted by lipophilic-delocalized cations of triphenylphosphonium (TPP{sup +}). Thus, we evaluated five TPP{sup +}-linked decyl polyhydroxybenzoates as potential cytotoxic agents in several human breast cancer cell lines that differ in estrogen receptor and HER2/neu expression, and in metabolic profile. Results showed that all cell lines tested were sensitive to the cytotoxic action of these compounds. The mechanism underlying the cytotoxicity would be triggered by their weak uncoupling effect on the oxidative phosphorylation system, while having amore » wider and safer therapeutic range than other uncouplers and a significant lowering in transmembrane potential. Noteworthy, while the TPP{sup +}-derivatives alone led to almost negligible losses of ATP, when these were added in the presence of an AMP-activated protein kinase inhibitor, the levels of ATP fell greatly. Overall, data presented suggest that decyl polyhydroxybenzoates-TPP{sup +} and its derivatives warrant future investigation as potential anti-tumor agents. - Highlights: • TPP{sup +}-polyhydroxybenzoates are cytotoxic to various subtypes of breast cancer cells. • Cytotoxicity is not-dependent on the expression of estrogen/growth factor receptors. • Cytotoxicity appears to be triggered by a weak mitochondrial uncoupling effect. • Effects include loss of transmembrane potential and apoptosis was detected. • TPP{sup +}-polyhydroxybenzoates inhibit migration of highly metastatic cells.« less

Human papillomavirus 16E6 and NFX1-123 potentiate notch signaling and differentiation without activating cellular arrest

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

Vliet-Gregg, Portia A.; Hamilton, Jennifer R.; Katzenellenbogen, Rachel A., E-mail: rkatzen@uw.edu

High-risk human papillomavirus (HR HPV) oncoproteins bind host cell proteins to dysregulate and uncouple apoptosis, senescence, differentiation, and growth. These pathways are important for both the viral life cycle and cancer development. HR HPV16 E6 (16E6) interacts with the cellular protein NFX1-123, and they collaboratively increase the growth and differentiation master regulator, Notch1. In 16E6 expressing keratinocytes (16E6 HFKs), the Notch canonical pathway genes Hes1 and Hes5 were increased with overexpression of NFX1-123, and their expression was directly linked to the activation or blockade of the Notch1 receptor. Keratinocyte differentiation genes Keratin 1 and Keratin 10 were also increased, butmore » in contrast their upregulation was only indirectly associated with Notch1 receptor stimulation and was fully unlinked to growth arrest, increased p21{sup Waf1/CIP1}, or decreased proliferative factor Ki67. This leads to a model of 16E6, NFX1-123, and Notch1 differently regulating canonical and differentiation pathways and entirely uncoupling cellular arrest from increased differentiation. - Highlights: • 16E6 and NFX1-123 increased the Notch canonical pathway through Notch1. • 16E6 and NFX1-123 increased the differentiation pathway indirectly through Notch1. • 16E6 and NFX1-123 increased differentiation gene expression without growth arrest. • Increased NFX1-123 with 16E6 may create an ideal cellular phenotype for HPV.« less

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

PubMed

Krebber, H; Taura, T; Lee, M S; Silver, P A

1999-08-01

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

Test Systems to Study the Structure and Function of Uncoupling Protein 1: A Critical Overview

PubMed Central

Hirschberg, Verena; Fromme, Tobias; Klingenspor, Martin

2011-01-01

The discovery of active brown adipose tissue (BAT) in healthy adult humans has renewed interest in the biology of this organ. BAT is capable of distributing nutrient energy in the form of heat allowing small mammals to efficiently defend their body temperature when acutely exposed to the cold. On the other hand BAT might be a target for the treatment of obesity and related diseases, as its pharmacological activation could allow release of excess energy stored in white adipose tissue depots. Energy dissipation in BAT depends on the activity of uncoupling protein 1 (UCP1), therefore a BAT-based obesity therapy requires a detailed understanding of structure and function of UCP1. Although UCP1 has been in the focus of research since its discovery, central questions concerning its mechanistic function and regulation are not yet resolved. They have been addressed in native mitochondria but also in several test systems, which are generally used to lower inter-experimental variability and to simplify analysis conditions. Different test systems have contributed to our current knowledge about UCP1 but of course all of them have certain limitations. We here provide an overview about research on UCP1 structure and function in test systems. So far, these have nearly exclusively been employed to study rodent and not human UCP1. Considering that the amino acid sequence of mouse and human UCP1 is only 79% identical, it will be essential to test whether the human version has a similarly high catalytic activity, allowing a relevant amount of energy dissipation in human BAT. Besides the issue of comparable mechanistic function a sufficiently high expression level of human UCP1 is a further prerequisite for anti-obesity therapeutic potential. Treatments which induce BAT hyperplasia and UCP1 expression in humans might therefore be equally important to discover as mere activators of the thermogenic process. PMID:22654819

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-07-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. © 2016 The Authors. BioEssays published by WILEY Periodicals, Inc.

Mitochondrial uncoupling proteins regulate angiotensin‐converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies

PubMed Central

Maubaret, Cecilia; Pedersen‐Bjergaard, Ulrik; Brull, David J.; Gohlke, Peter; Payne, John R.; World, Michael; Thorsteinsson, Birger; Humphries, Steve E.; Montgomery, Hugh E.

2015-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin‐converting enzyme (ACE) is the central component of endocrine and local tissue renin–angiotensin systems (RAS), which also regulate diverse aspects of whole‐body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations (healthy young UK men and Scandinavian diabetic patients) serum ACE (sACE) activity was significantly higher amongst UCP3‐55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold (P < 0·01) whilst increasing ACE expression within a physiological range (<1·8‐fold at 48 h; P < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role. PMID:27347560

Mitochondrial uncoupling proteins regulate angiotensin-converting enzyme expression: crosstalk between cellular and endocrine metabolic regulators suggested by RNA interference and genetic studies.

PubMed

Dhamrait, Sukhbir S; Maubaret, Cecilia; Pedersen-Bjergaard, Ulrik; Brull, David J; Gohlke, Peter; Payne, John R; World, Michael; Thorsteinsson, Birger; Humphries, Steve E; Montgomery, Hugh E

2016-01-01

Uncoupling proteins (UCPs) regulate mitochondrial function, and thus cellular metabolism. Angiotensin-converting enzyme (ACE) is the central component of endocrine and local tissue renin-angiotensin systems (RAS), which also regulate diverse aspects of whole-body metabolism and mitochondrial function (partly through altering mitochondrial UCP expression). We show that ACE expression also appears to be regulated by mitochondrial UCPs. In genetic analysis of two unrelated populations ( healthy young UK men and Scandinavian diabetic patients ) serum ACE (sACE) activity was significantly higher amongst UCP3-55C (rather than T) and UCP2 I (rather than D) allele carriers. RNA interference against UCP2 in human umbilical vein endothelial cells reduced UCP2 mRNA sixfold ( P  < 0·01) whilst increasing ACE expression within a physiological range (<1·8-fold at 48 h; P  < 0·01). Our findings suggest novel hypotheses. Firstly, cellular feedback regulation may occur between UCPs and ACE. Secondly, cellular UCP regulation of sACE suggests a novel means of crosstalk between (and mutual regulation of) cellular and endocrine metabolism. This might partly explain the reduced risk of developing diabetes and metabolic syndrome with RAS antagonists and offer insight into the origins of cardiovascular disease in which UCPs and ACE both play a role.

A SNP uncoupling Mina expression from the TGFβ signaling pathway.

PubMed

Lian, Shang L; Mihi, Belgacem; Koyanagi, Madoka; Nakayama, Toshinori; Bix, Mark

2018-03-01

Mina is a JmjC family 2-oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell-type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1-region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1-region SNPs perturbs a Mina cis-regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus-spanning 26-kilobase genomic interval. We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c-but not C57Bl/6 allele-abolished both Smad3 binding and transforming growth factor beta (TGFβ) responsiveness. Our results demonstrate the TGFβ signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFβ signaling pathway. © 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

A SNP uncoupling Mina expression from the TGFβ signaling pathway

PubMed Central

Lian, Shang L.; Mihi, Belgacem; Koyanagi, Madoka; Nakayama, Toshinori

2017-01-01

Abstract Introduction Mina is a JmjC family 2‐oxoglutarate oxygenase with pleiotropic roles in cell proliferation, cancer, T cell differentiation, pulmonary inflammation, and intestinal parasite expulsion. Although Mina expression varies according to cell‐type, developmental stage and activation state, its transcriptional regulation is poorly understood. Across inbred mouse strains, Mina protein level exhibits a bimodal distribution, correlating with inheritance of a biallelic haplotype block comprising 21 promoter/intron 1‐region SNPs. We previously showed that heritable differences in Mina protein level are transcriptionally regulated. Methods Accordingly, we decided to test the hypothesis that at least one of the promoter/intron 1‐region SNPs perturbs a Mina cis‐regulatory element (CRE). Here, we have comprehensively scanned for CREs across a Mina locus‐spanning 26‐kilobase genomic interval. Results We discovered 8 potential CREs and functionally validated 4 of these, the strongest of which (E2), residing in intron 1, contained a SNP whose BALB/c—but not C57Bl/6 allele—abolished both Smad3 binding and transforming growth factor beta (TGFβ) responsiveness. Conclusions Our results demonstrate the TGFβ signaling pathway plays a critical role in regulating Mina expression and SNP rs4191790 controls heritable variation in Mina expression level, raising important questions regarding the evolution of an allele that uncouples Mina expression from the TGFβ signaling pathway. PMID:28967702

Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction.

PubMed

Ramsden, David B; Ho, Philip W-L; Ho, Jessica W-M; Liu, Hui-Fang; So, Danny H-F; Tse, Ho-Man; Chan, Koon-Ho; Ho, Shu-Leong

2012-07-01

Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I-V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed "mild uncoupling." UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed.

Cyanide-induced death of dopaminergic cells is mediated by uncoupling protein-2 up-regulation and reduced Bcl-2 expression

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

Zhang, X.; Li, L.; Zhang, L.

Cyanide is a potent inhibitor of mitochondrial oxidative metabolism and produces mitochondria-mediated death of dopaminergic neurons and sublethal intoxications that are associated with a Parkinson-like syndrome. Cyanide toxicity is enhanced when mitochondrial uncoupling is stimulated following up-regulation of uncoupling protein-2 (UCP-2). In this study, the role of a pro-survival protein, Bcl-2, in cyanide-mediated cell death was determined in a rat dopaminergic immortalized mesencephalic cell line (N27 cells). Following pharmacological up-regulation of UCP-2 by treatment with Wy14,643, cyanide reduced cellular Bcl-2 expression by increasing proteasomal degradation of the protein. The increased turnover of Bcl-2 was mediated by an increase of oxidativemore » stress following UCP-2 up-regulation. The oxidative stress involved depletion of mitochondrial glutathione (mtGSH) and increased H{sub 2}O{sub 2} generation. Repletion of mtGSH by loading cells with glutathione ethyl ester reduced H{sub 2}O{sub 2} generation and in turn blocked the cyanide-induced decrease of Bcl-2. To determine if UCP-2 mediated the response, RNAi knock down was conducted. The RNAi decreased cyanide-induced depletion of mtGSH, reduced H{sub 2}O{sub 2} accumulation, and inhibited down-regulation of Bcl-2, thus blocking cell death. To confirm the role of Bcl-2 down-regulation in the cell death, it was shown that over-expression of Bcl-2 by cDNA transfection attenuated the enhancement of cyanide toxicity after UCP-2 up-regulation. It was concluded that UCP-2 up-regulation sensitizes cells to cyanide by increasing cellular oxidative stress, leading to an increase of Bcl-2 degradation. Then the reduced Bcl-2 levels sensitize the cells to cyanide-mediated cell death.« less

Whole-Body Vibration Partially Reverses Aging-Induced Increases in Visceral Adiposity and Hepatic Lipid Storage in Mice

PubMed Central

van Dijk, Theo H.; Havinga, Rick; van der Zee, Eddy A.; Groen, Albert K.; Reijngoud, Dirk-Jan; Bakker, Barbara M.; van Dijk, Gertjan

2016-01-01

At old age, humans generally have declining muscle mass and increased fat deposition, which can increase the risk of developing cardiometabolic diseases. While regular physical activity postpones these age-related derangements, this is not always possible in the elderly because of disabilities or risk of injury. Whole-body vibration (WBV) training may be considered as an alternative to physical activity particularly in the frail population. To explore this possibility, we characterized whole-body and organ-specific metabolic processes in 6-month and 25-month old mice, over a period of 14 weeks of WBV versus sham training. WBV training tended to increase blood glucose turnover rates and stimulated hepatic glycogen utilization during fasting irrespective of age. WBV was effective in reducing white fat mass and hepatic triglyceride content only in old but not in young mice and these reductions were related to upregulation of hepatic mitochondrial uncoupling of metabolism (assessed by high-resolution respirometry) and increased expression of uncoupling protein 2. Because these changes occurred independent of changes in food intake and whole-body metabolic rate (assessed by indirect calorimetry), the liver-specific effects of WBV may be a primary mechanism to improve metabolic health during aging, rather than that it is a consequence of alterations in energy balance. PMID:26886917

Mitochondrial uncoupling, ROS generation and cardioprotection.

PubMed

Cadenas, Susana

2018-05-31

Mitochondrial oxidative phosphorylation is incompletely coupled, since protons translocated to the intermembrane space by specific respiratory complexes of the electron transport chain can return to the mitochondrial matrix independently of the ATP synthase -a process known as proton leak- generating heat instead of ATP. Proton leak across the inner mitochondrial membrane increases the respiration rate and decreases the electrochemical proton gradient (Δp), and is an important mechanism for energy dissipation that accounts for up to 25% of the basal metabolic rate. It is well established that mitochondrial superoxide production is steeply dependent on Δp in isolated mitochondria and, correspondingly, mitochondrial uncoupling has been identified as a cytoprotective strategy under conditions of oxidative stress, including diabetes, drug-resistance in tumor cells, ischemia-reperfusion (IR) injury or aging. Mitochondrial uncoupling proteins (UCPs) are able to lower the efficiency of oxidative phosphorylation and are involved in the control of mitochondrial reactive oxygen species (ROS) production. There is strong evidence that UCP2 and UCP3, the UCP1 homologues expressed in the heart, protect against mitochondrial oxidative damage by reducing the production of ROS. This review first analyzes the relationship between mitochondrial proton leak and ROS generation, and then focuses on the cardioprotective role of chemical uncoupling and uncoupling mediated by UCPs. This includes their protective effects against cardiac IR, a condition known to increase ROS production, and their roles in modulating cardiovascular risk factors such as obesity, diabetes and atherosclerosis. Copyright © 2018. Published by Elsevier B.V.

Brown Adipose YY1 Deficiency Activates Expression of Secreted Proteins Linked to Energy Expenditure and Prevents Diet-Induced Obesity

PubMed Central

Verdeguer, Francisco; Soustek, Meghan S.; Hatting, Maximilian; Blättler, Sharon M.; McDonald, Devin; Barrow, Joeva J.

2015-01-01

Mitochondrial oxidative and thermogenic functions in brown and beige adipose tissues modulate rates of energy expenditure. It is unclear, however, how beige or white adipose tissue contributes to brown fat thermogenic function or compensates for partial deficiencies in this tissue and protects against obesity. Here, we show that the transcription factor Yin Yang 1 (YY1) in brown adipose tissue activates the canonical thermogenic and uncoupling gene expression program. In contrast, YY1 represses a series of secreted proteins, including fibroblast growth factor 21 (FGF21), bone morphogenetic protein 8b (BMP8b), growth differentiation factor 15 (GDF15), angiopoietin-like 6 (Angptl6), neuromedin B, and nesfatin, linked to energy expenditure. Despite substantial decreases in mitochondrial thermogenic proteins in brown fat, mice lacking YY1 in this tissue are strongly protected against diet-induced obesity and exhibit increased energy expenditure and oxygen consumption in beige and white fat depots. The increased expression of secreted proteins correlates with elevation of energy expenditure and promotion of beige and white fat activation. These results indicate that YY1 in brown adipose tissue controls antagonistic gene expression programs associated with energy balance and maintenance of body weight. PMID:26503783

Adaptive responses of GLUT-4 and citrate synthase in fast-twitch muscle of voluntary running rats

NASA Technical Reports Server (NTRS)

Henriksen, E. J.; Halseth, A. E.

1995-01-01

Glucose transporter (GLUT-4) protein, hexokinase, and citrate synthase (proteins involved in oxidative energy production from blood glucose catabolism) increase in response to chronically elevated neuromuscular activity. It is currently unclear whether these proteins increase in a coordinated manner in response to this stimulus. Therefore, voluntary wheel running (WR) was used to chronically overload the fast-twitch rat plantaris muscle and the myocardium, and the early time courses of adaptative responses of GLUT-4 protein and the activities of hexokinase and citrate synthase were characterized and compared. Plantaris hexokinase activity increased 51% after just 1 wk of WR, whereas GLUT-4 and citrate synthase were increased by 51 and 40%, respectively, only after 2 wk of WR. All three variables remained comparably elevated (+50-64%) through 4 wk of WR. Despite the overload of the myocardium with this protocol, no substantial elevations in these variables were observed. These findings are consistent with a coordinated upregulation of GLUT-4 and citrate synthase in the fast-twitch plantaris, but not in the myocardium, in response to this increased neuromuscular activity. Regulation of hexokinase in fast-twitch muscle appears to be uncoupled from regulation of GLUT-4 and citrate synthase, as increases in the former are detectable well before increases in the latter.

Structural basis for activation of trimeric Gi proteins by multiple growth factor receptors via GIV/Girdin

PubMed Central

Lin, Changsheng; Ear, Jason; Midde, Krishna; Lopez-Sanchez, Inmaculada; Aznar, Nicolas; Garcia-Marcos, Mikel; Kufareva, Irina; Abagyan, Ruben; Ghosh, Pradipta

2014-01-01

A long-standing issue in the field of signal transduction is to understand the cross-talk between receptor tyrosine kinases (RTKs) and heterotrimeric G proteins, two major and distinct signaling hubs that control eukaryotic cell behavior. Although stimulation of many RTKs leads to activation of trimeric G proteins, the molecular mechanisms behind this phenomenon remain elusive. We discovered a unifying mechanism that allows GIV/Girdin, a bona fide metastasis-related protein and a guanine-nucleotide exchange factor (GEF) for Gαi, to serve as a direct platform for multiple RTKs to activate Gαi proteins. Using a combination of homology modeling, protein–protein interaction, and kinase assays, we demonstrate that a stretch of ∼110 amino acids within GIV C-terminus displays structural plasticity that allows folding into a SH2-like domain in the presence of phosphotyrosine ligands. Using protein–protein interaction assays, we demonstrated that both SH2 and GEF domains of GIV are required for the formation of a ligand-activated ternary complex between GIV, Gαi, and growth factor receptors and for activation of Gαi after growth factor stimulation. Expression of a SH2-deficient GIV mutant (Arg 1745→Leu) that cannot bind RTKs impaired all previously demonstrated functions of GIV—Akt enhancement, actin remodeling, and cell migration. The mechanistic and structural insights gained here shed light on the long-standing questions surrounding RTK/G protein cross-talk, set a novel paradigm, and characterize a unique pharmacological target for uncoupling GIV-dependent signaling downstream of multiple oncogenic RTKs. PMID:25187647

[Human brown adipose tissue].

PubMed

Virtanen, Kirsi A; Nuutila, Pirjo

2015-01-01

Adult humans have heat-producing and energy-consuming brown adipose tissue in the clavicular region of the neck. There are two types of brown adipose cells, the so-called classic and beige adipose cells. Brown adipose cells produce heat by means of uncoupler protein 1 (UCP1) from fatty acids and sugar. By applying positron emission tomography (PET) measuring the utilization of sugar, the metabolism of brown fat has been shown to multiply in the cold, presumably influencing energy consumption. Active brown fat is most likely present in young adults, persons of normal weight and women, least likely in obese persons.

Chronic mitochondrial uncoupling treatment prevents acute cold-induced oxidative stress in birds.

PubMed

Stier, Antoine; Massemin, Sylvie; Criscuolo, François

2014-12-01

Endotherms have evolved two major types of thermogenesis that allow them to actively produce heat in response to cold exposure, either through muscular activity (i.e. shivering thermogenesis) or through futile electro-chemical cycles (i.e. non-shivering thermogenesis). Amongst the latter, mitochondrial uncoupling is of key importance because it is suggested to drive heat production at a low cost in terms of oxidative stress. While this has been experimentally shown in mammals, the oxidative stress consequences of cold exposure and mitochondrial uncoupling are clearly less understood in the other class of endotherms, the birds. We compared metabolic and oxidative stress responses of zebra finches chronically treated with or without a chemical mitochondrial uncoupler (2,4-dinitrophenol: DNP), undergoing an acute (24 h) and a chronic (4 weeks) cold exposure (12 °C). We predicted that control birds should present at least a transient elevation of oxidative stress levels in response to cold exposure. This oxidative stress cost should be more pronounced in control birds than in DNP-treated birds, due to their lower basal uncoupling state. Despite similar increase in metabolism, control birds presented elevated levels of DNA oxidative damage in response to acute (but not chronic) cold exposure, while DNP-treated birds did not. Plasma antioxidant capacity decreased overall in response to chronic cold exposure. These results show that acute cold exposure increases oxidative stress in birds. However, uncoupling mitochondrial functioning appears as a putative compensatory mechanism preventing cold-induced oxidative stress. This result confirms previous observations in mice and underlines non-shivering thermogenesis as a putative key mechanism for endotherms in mounting a response to cold at a low oxidative cost.

Low concentration of uncouplers of oxidative phosphorylation decreases the TNF-induced endothelial permeability and lethality in mice.

PubMed

Zakharova, Vlada V; Pletjushkina, Olga Yu; Galkin, Ivan I; Zinovkin, Roman A; Chernyak, Boris V; Krysko, Dmitri V; Bachert, Claus; Krysko, Olga; Skulachev, Vladimir P; Popova, Ekaterina N

2017-04-01

Mitochondrial dysfunctions occur in many diseases linked to the systemic inflammatory response syndrome (SIRS). Mild uncoupling of oxidative phosphorylation is known to rescue model animals from pathologies related to mitochondrial dysfunctions and overproduction of reactive oxygen species (ROS). To study the potential of SIRS therapy by uncoupling, we tested protonophore dinitrophenol (DNP) and a free fatty acid (FFA) anion carrier, lipophilic cation dodecyltriphenylphosphonium (C 12 TPP) in mice and in vitro models of SIRS. DNP and C 12 TPP prevented the body temperature drop and lethality in mice injected with high doses of a SIRS inducer, tumor necrosis factor (TNF). The mitochondria-targeted antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) which also catalyzes FFA-dependent uncoupling revealed similar protective effects and downregulated expression of the NFκB-regulated genes (VCAM1, ICAM1, MCP1, and IL-6) involved in the inflammatory response of endothelium in aortas of the TNF-treated mice. In vitro mild uncoupling rescued from TNF-induced endothelial permeability, disassembly of cell contacts and VE-cadherin cleavage by the matrix metalloprotease 9 (ММР9). The uncouplers prevented TNF-induced expression of MMP9 via inhibition of NFκB signaling. Water-soluble antioxidant Trolox also prevented TNF-induced activation and permeability of endothelium in vitro via inhibition of NFκB signaling, suggesting that the protective action of the uncouplers is linked to their antioxidant potential. Copyright © 2017 Elsevier B.V. All rights reserved.

Bardoxolone Methyl Prevents Mesenteric Fat Deposition and Inflammation in High-Fat Diet Mice

PubMed Central

Dinh, Chi H. L.; Szabo, Alexander; Yu, Yinghua; Wang, Hongqin; Huang, Xu-Feng

2015-01-01

Mesenteric fat belongs to visceral fat. An increased deposition of mesenteric fat contributes to obesity associated complications such as type 2 diabetes and cardiovascular diseases. We have investigated the therapeutic effects of bardoxolone methyl (BARD) on mesenteric adipose tissue of mice fed a high-fat diet (HFD). Male C57BL/6J mice were administered oral BARD during HFD feeding (HFD/BARD), only fed a high-fat diet (HFD), or fed low-fat diet (LFD) for 21 weeks. Histology and immunohistochemistry were used to analyse mesenteric morphology and macrophages, while Western blot was used to assess the expression of inflammatory, oxidative stress, and energy expenditure proteins. Supplementation of drinking water with BARD prevented mesenteric fat deposition, as determined by a reduction in large adipocytes. BARD prevented inflammation as there were fewer inflammatory macrophages and reduced proinflammatory cytokines (interleukin-1 beta and tumour necrosis factor alpha). BARD reduced the activation of extracellular signal-regulated kinase (ERK) and Akt, suggesting an antioxidative stress effect. BARD upregulates energy expenditure proteins, judged by the increased activity of tyrosine hydroxylase (TH) and AMP-activated protein kinase (AMPK) and increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and uncoupling protein 2 (UCP2) proteins. Overall, BARD induces preventive effect in HFD mice through regulation of mesenteric adipose tissue. PMID:26618193

Resveratrol up-regulates hepatic uncoupling protein 2 and prevents development of nonalcoholic fatty liver disease in rats fed a high-fat diet.

PubMed

Poulsen, Morten Møller; Larsen, Jens Ø; Hamilton-Dutoit, Stephen; Clasen, Berthil F; Jessen, Niels; Paulsen, Søren K; Kjær, Thomas N; Richelsen, Bjørn; Pedersen, Steen B

2012-09-01

Obesity is associated with a markedly increased risk of nonalcoholic fatty liver disease. The anti-inflammatory polyphenol resveratrol possess promising properties in preventing this metabolic condition by dampening the pathological inflammatory reaction in the hepatic tissue. However, in the current study, we hypothesize that the beneficial effect of resveratrol is not solely attributable to its anti-inflammatory potential. Eight-week-old male Wistar rats were randomly distributed into 3 groups of 12 animals each: control diet (C), high-fat diet (HF), and HF supplemented with 100 mg resveratrol daily (HFR). After 8 weeks of dietary treatment, the rats were euthanized and relevant tissues were prepared for subsequent analysis. Resveratrol prevented the high fat-induced steatosis assessed by semiquantitative grading, which furthermore corresponded with a complete normalization of the hepatic triglyceride content (P < .001), despite no change in total body fat. In HFR, the hepatic uncoupling protein 2 expression was significantly increased by 76% and 298% as compared with HF and C, respectively. Moreover, the hepatic mitochondria content in HFR was significantly higher as compared with both C and HF (P < .001 and P = .004, respectively). We found no signs of hepatic inflammation, hereby demonstrating that resveratrol protects against fatty liver disease independently of its proposed anti-inflammatory potential. Our data might indicate that an increased number of mitochondria and, particularly, an increase in hepatic uncoupling protein 2 expression are involved in normalizing the hepatic fat content due to resveratrol supplementation in rodents fed a high-fat diet. Copyright © 2012 Elsevier Inc. All rights reserved.

The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation

PubMed Central

Riley, Christopher L.; Dao, Christine; Kenaston, M. Alexander; Muto, Luigina; Kohno, Shohei; Nowinski, Sara M.; Solmonson, Ashley D.; Pfeiffer, Matthew; Sack, Michael N.; Lu, Zhongping; Fiermonte, Giuseppe; Sprague, Jon E.

2016-01-01

Key points Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation.UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively.Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1.Acute noradrenaline‐induced hyperthermia requires UCP1 but not UCP3.Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. Abstract Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue‐dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine‐induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle‐specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology. PMID:27647490

Extracellular annexins and dynamin are important for sequential steps in myoblast fusion

PubMed Central

Leikina, Evgenia; Melikov, Kamran; Sanyal, Sarmistha; Verma, Santosh K.; Eun, Bokkee; Gebert, Claudia; Pfeifer, Karl; Lizunov, Vladimir A.; Kozlov, Michael M.

2013-01-01

Myoblast fusion into multinucleated myotubes is a crucial step in skeletal muscle development and regeneration. Here, we accumulated murine myoblasts at the ready-to-fuse stage by blocking formation of early fusion intermediates with lysophosphatidylcholine. Lifting the block allowed us to explore a largely synchronized fusion. We found that initial merger of two cell membranes detected as lipid mixing involved extracellular annexins A1 and A5 acting in a functionally redundant manner. Subsequent stages of myoblast fusion depended on dynamin activity, phosphatidylinositol(4,5)bisphosphate content, and cell metabolism. Uncoupling fusion from preceding stages of myogenesis will help in the analysis of the interplay between protein machines that initiate and complete cell unification and in the identification of additional protein players controlling different fusion stages. PMID:23277424

Raised intracellular [Ca2+] abolishes excitation-contraction coupling in skeletal muscle fibres of rat and toad.

PubMed Central

Lamb, G D; Junankar, P R; Stephenson, D G

1995-01-01

1. Raising the intracellular [Ca2+] for 10 s at 23 degrees C abolished depolarization-induced force responses in mechanically skinned muscle fibres of toad and rat (half-maximal effect at 10 and 23 microM, respectively), without affecting the ability of caffeine or low [Mg2+] to open the ryanodine receptor (RyR)/Ca2+ release channels. Thus, excitation-contraction coupling was lost, even though the Ca2+ release channels were still functional. Coupling could not be restored in the duration of an experiment (up to 1 h). 2. The Ca(2+)-dependent uncoupling had a Q10 > 3.5, and was three times slower at pH 5.8 than at pH 7.1. Sr2+ caused similar uncoupling at twenty times higher concentration, but Mg2+, even at 10 mM, was ineffective. Uncoupling was not noticeably affected by removal of ATP or application of protein kinase or phosphatase inhibitors. 3. Confocal laser scanning microscopy showed that the transverse tubular system was sealed in its entirety in mechanically skinned fibres and that its integrity was maintained in uncoupled fibres. Electron microscopy revealed distorted or severed triad junctions and Z-line aberrations in uncoupled fibres. 4. Only when uncoupling was induced at a relatively slow rate (e.g. over 60 s with 2.5 microM Ca2+) could it be prevented by the protease inhibitor leupeptin (1 mM). Immunostaining of Western blots showed no evidence of proteolysis of the RyR, the alpha 1-subunit of dihydropyridine receptor (DHPR) or triadin in uncoupled fibres. 5. Fibres which, whilst intact, were stimulated repeatedly by potassium depolarization with simultaneous application of 30 mM caffeine showed reduced responsiveness after skinning to depolarization but not to caffeine. Rapid release of endogenous Ca2+, or raised [Ca2+] under conditions which minimized the loss of endogenous diffusible myoplasmic molecules from the skinned fibre, caused complete uncoupling. Taken together, these results suggest that Ca(2+)-dependent uncoupling can also occur in intact fibres. 6. This Ca(2+)-dependent loss of depolarization-induced Ca2+ release may play an important feedback role in muscle by stopping Ca2+ release in localized areas where it is excessive and may be responsible for long-lasting muscle fatigue after severe exercise, as well as contributing to muscle weakness in various dystrophies. Images Figure 1 Figure 7 Figure 9 PMID:8847631

Derivatives of Rhodamine 19 as Mild Mitochondria-targeted Cationic Uncouplers*

PubMed Central

Antonenko, Yuri N.; Avetisyan, Armine V.; Cherepanov, Dmitry A.; Knorre, Dmitry A.; Korshunova, Galina A.; Markova, Olga V.; Ojovan, Silvia M.; Perevoshchikova, Irina V.; Pustovidko, Antonina V.; Rokitskaya, Tatyana I.; Severina, Inna I.; Simonyan, Ruben A.; Smirnova, Ekaterina A.; Sobko, Alexander A.; Sumbatyan, Natalia V.; Severin, Fedor F.; Skulachev, Vladimir P.

2011-01-01

A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C12R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H+ ions was generated in the presence of C12R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C12R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C12R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C12R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease. PMID:21454507

Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins

NASA Astrophysics Data System (ADS)

Faure, Guilhem; Koonin, Eugene V.

2015-05-01

Robustness to destabilizing effects of mutations is thought of as a key factor of protein evolution. The connections between two measures of robustness, the relative core size and the computationally estimated effect of mutations on protein stability (ΔΔG), protein abundance and the selection pressure on protein-coding genes (dN/dS) were analyzed for the organisms with a large number of available protein structures including four eukaryotes, two bacteria and one archaeon. The distribution of the effects of mutations in the core on protein stability is universal and indistinguishable in eukaryotes and bacteria, centered at slightly destabilizing amino acid replacements, and with a heavy tail of more strongly destabilizing replacements. The distribution of mutational effects in the hyperthermophilic archaeon Thermococcus gammatolerans is significantly shifted toward strongly destabilizing replacements which is indicative of stronger constraints that are imposed on proteins in hyperthermophiles. The median effect of mutations is strongly, positively correlated with the relative core size, in evidence of the congruence between the two measures of protein robustness. However, both measures show only limited correlations to the expression level and selection pressure on protein-coding genes. Thus, the degree of robustness reflected in the universal distribution of mutational effects appears to be a fundamental, ancient feature of globular protein folds whereas the observed variations are largely neutral and uncoupled from short term protein evolution. A weak anticorrelation between protein core size and selection pressure is observed only for surface residues in prokaryotes but a stronger anticorrelation is observed for all residues in eukaryotic proteins. This substantial difference between proteins of prokaryotes and eukaryotes is likely to stem from the demonstrable higher compactness of prokaryotic proteins.

Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

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

Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

1988-01-01

The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated /sup 32/P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingestedmore » ethanol in a liquid diet. /sup 32/P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice.« less

Arrestin–dependent but G–protein coupled receptor kinase–independent uncoupling of D2–dopamine receptors

PubMed Central

Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Octeau, J. Christopher; Kovoor, Abraham

2016-01-01

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (Kir3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-Kir3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. PMID:23815307

Arrestin-dependent but G-protein coupled receptor kinase-independent uncoupling of D2-dopamine receptors.

PubMed

Celver, Jeremy; Sharma, Meenakshi; Thanawala, Vaidehi; Christopher Octeau, J; Kovoor, Abraham

2013-10-01

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor. © 2013 International Society for Neurochemistry.

Complementary Roles of Estrogen-Related Receptors in Brown Adipocyte Thermogenic Function

PubMed Central

Gantner, Marin L.; Hazen, Bethany C.; Eury, Elodie; Brown, Erin L.

2016-01-01

Brown adipose tissue (BAT) thermogenesis relies on a high abundance of mitochondria and the unique expression of the mitochondrial Uncoupling Protein 1 (UCP1), which uncouples substrate oxidation from ATP synthesis. Adrenergic stimulation of brown adipocytes activates UCP1-mediated thermogenesis; it also induces the expression of Ucp1 and other genes important for thermogenesis, thereby endowing adipocytes with higher oxidative and uncoupling capacities. Adipocyte mitochondrial biogenesis and oxidative capacity are controlled by multiple transcription factors, including the estrogen-related receptor (ERR)α. Whole-body ERRα knockout mice show decreased BAT mitochondrial content and oxidative function but normal induction of Ucp1 in response to cold. In addition to ERRα, brown adipocytes express ERRβ and ERRγ, 2 nuclear receptors that are highly similar to ERRα and whose function in adipocytes is largely unknown. To gain insights into the roles of all 3 ERRs, we assessed mitochondrial function and adrenergic responses in primary brown adipocytes lacking combinations of ERRs. We show that adipocytes lacking just ERRα, the most abundant ERR, show only mild mitochondrial defects. Adipocytes lacking ERRβ and ERRγ also show just mild defects. In contrast, adipocytes lacking all 3 ERRs have severe reductions in mitochondrial content and oxidative capacity. Moreover, adipocytes lacking all 3 ERRs have defects in the transcriptional and metabolic response to adrenergic stimulation, suggesting a wider role of ERRs in BAT function than previously appreciated. Our study shows that ERRs have a great capacity to compensate for each other in protecting mitochondrial function and the metabolic response to adrenergic signaling, processes vital to BAT function. PMID:27763777

Mapping the nuclear localization signal in the matrix protein of potato yellow dwarf virus.

PubMed

Anderson, Gavin; Jang, Chanyong; Wang, Renyuan; Goodin, Michael

2018-05-01

The ability of the matrix (M) protein of potato yellow dwarf virus (PYDV) to remodel nuclear membranes is controlled by a di-leucine motif located at residues 223 and 224 of its primary structure. This function can be uncoupled from that of its nuclear localization signal (NLS), which is controlled primarily by lysine and arginine residues immediately downstream of the LL motif. In planta localization of green fluorescent protein fusions, bimolecular fluorescence complementation assays with nuclear import receptor importin-α1 and yeast-based nuclear import assays provided three independent experimental approaches to validate the authenticity of the M-NLS. The carboxy terminus of M is predicted to contain a nuclear export signal, which is belived to be functional, given the ability of M to bind the Arabidopsis nuclear export receptor 1 (XPO1). The nuclear shuttle activity of M has implications for the cell-to-cell movement of PYDV nucleocapsids, based upon its interaction with the N and Y proteins.

Renal mitochondrial dysfunction in spontaneously hypertensive rats is attenuated by losartan but not by amlodipine.

PubMed

de Cavanagh, Elena M V; Toblli, Jorge E; Ferder, León; Piotrkowski, Bárbara; Stella, Inés; Inserra, Felipe

2006-06-01

Mitochondrial dysfunction is associated with cardiovascular damage; however, data on a possible association with kidney damage are scarce. Here, we aimed at investigating whether 1) kidney impairment is related to mitochondrial dysfunction; and 2) ANG II blockade, compared with Ca2+ channel blockade, can reverse potential mitochondrial changes in hypertension. Eight-week-old male spontaneously hypertensive rats (SHR) received water containing losartan (40 mg.kg-1.day-1, SHR+Los), amlodipine (3 mg.kg-1.day-1, SHR+Amlo), or no additions (SHR) for 6 mo. Wistar-Kyoto rats (WKY) were normotensive controls. Glomerular and tubulointerstitial damage, systolic blood pressure, and proteinuria were higher, and creatinine clearance was lower in SHR vs. SHR+Los and WKY. In SHR+Amlo, blood pressure was similar to WKY, kidney function was similar to SHR, and renal lesions were lower than in SHR, but higher than in SHR+Los. In kidney mitochondria from SHR and SHR+Amlo, membrane potential, nitric oxide synthase, manganese-superoxide dismutase and cytochrome oxidase activities, and uncoupling protein-2 content were lower than in SHR+Los and WKY. In SHR and SHR+Amlo, mitochondrial H2O2 production was higher than in SHR+Los and WKY. Renal glutathione content was lower in SHR+Amlo relative to SHR, SHR+Los, and WKY. In SHR and SHR+Amlo, glutathione was relatively more oxidized than in SHR+Los and WKY. Tubulointerstitial alpha-smooth muscle actin labeling was inversely related to manganese-superoxide dismutase activity and uncoupling protein-2 content. These findings suggest that oxidant stress is associated with renal mitochondrial dysfunction in SHR. The mitochondrial-antioxidant actions of losartan may be an additional or alternative way to explain some of the beneficial effects of AT1-receptor antagonists.

Maternal nutritional programming of fetal adipose tissue development: differential effects on messenger ribonucleic acid abundance for uncoupling proteins and peroxisome proliferator-activated and prolactin receptors.

PubMed

Bispham, J; Gardner, D S; Gnanalingham, M G; Stephenson, T; Symonds, M E; Budge, H

2005-09-01

Maternal nutrient restriction at specific stages of gestation has differential effects on fetal development such that the offspring are programmed to be at increased risk of a range of adult diseases, including obesity. We investigated the effect of maternal nutritional manipulation through gestation on fetal adipose tissue deposition in conjunction with mRNA abundance for uncoupling protein (UCP)1 and 2, peroxisome proliferator-activated receptors (PPAR)alpha and gamma, together with long and short forms of the prolactin receptor (PRLR). Singleton-bearing ewes were either nutrient restricted (3.2-3.8 MJ day(-1) metabolizable energy) or fed to appetite (8.7-9.9 MJ day(-1)) over the period of maximal placental growth, i.e. between 28 and 80 d gestation. After 80 d gestation, ewes were either fed to calculated requirements, (6.7-7.5 MJ day(-1)), or to appetite (8.0-10.9 MJ day(-1)). At term, offspring of nutrient-restricted ewes possessed more adipose tissue, an adaptation that was greatest in those born to mothers that fed to requirements in late gestation. This was accompanied by an increased mRNA abundance for UCP2 and PPARalpha, an adaptation not seen in mothers re-fed to appetite. Maternal nutrition had no effect on mRNA abundance for UCP1, PPARgamma, or PRLR. Irrespective of maternal nutrition, mRNA abundance for UCP1 was positively correlated with PPARgamma and the long and short forms of PRLR, indicating that these factors may act together to ensure that UCP1 abundance is maximized in the newborn. In conclusion, we have shown, for the first time, differential effects of maternal nutrition on key regulatory components of fetal fat metabolism.

PPARδ agonist GW501516 prevents uncoupling of endothelial nitric oxide synthase in cerebral microvessels of hph-1 mice

PubMed Central

Santhanam, Anantha Vijay R.; d’Uscio, Livius V.; He, Tongrong; Katusic, Zvonimir S.

2012-01-01

Peroxisome proliferator-activated receptor delta (PPARδ) is ubiquitously expressed in the vasculature, including cerebral circulation. The role of PPARδ in metabolism of tetrahydrobiopterin (BH4) has not been studied in the cerebral microvasculature. In the present study, the effects of PPARδ agonist GW501516 on uncoupling of endothelial nitric oxide synthase (eNOS) were determined in cerebral microvessels of BH4-deficient hph-1 mice. Wild-type (B6CBA) and hph-1 mice were orally gavaged with a selective PPARδ activator, GW501516 (2 mg/kg/day) for 14 days, and thereafter, cerebral microvessels were isolated and studied. Treatment of hph-1 mice with GW501516 significantly reduced oxidation of BH4 and increased the ratio of BH4 to 7,8-BH2 (P<0.05, n=6–9). Attenuation of L-NAME-inhibitable superoxide anion levels by GW501516 demonstrated that activation of PPARδ might prevent uncoupling of endothelial nitric oxide synthase (eNOS, P<0.05, n=6–9). Western blotting studies demonstrated that GW501516 selectively increased the endothelial expressions of CuZn superoxide dismutase (P<0.05, n=6–9) and catalase (P<0.05, n=6–8). PPARδ activation increased the total nitrite and nitrate (NO2 + NO3) content in cerebral microvessels (P<0.05, n=6). Obtained results suggest that in vivo activation of PPARδ prevents eNOS uncoupling, restores bioavailability of NO and may help preserve endothelial function in the BH4-deficient cerebral circulation. PMID:22982594

PPARδ agonist GW501516 prevents uncoupling of endothelial nitric oxide synthase in cerebral microvessels of hph-1 mice.

PubMed

Santhanam, Anantha Vijay R; d'Uscio, Livius V; He, Tongrong; Katusic, Zvonimir S

2012-11-05

Peroxisome proliferator-activated receptor delta (PPARδ) is ubiquitously expressed in the vasculature, including cerebral circulation. The role of PPARδ in metabolism of tetrahydrobiopterin (BH₄) has not been studied in the cerebral microvasculature. In the present study, the effects of PPARδ agonist GW501516 on uncoupling of endothelial nitric oxide synthase (eNOS) were determined in cerebral microvessels of BH₄-deficient hph-1 mice. Wild-type (B6CBA) and hph-1 mice were orally gavaged with a selective PPARδ activator, GW501516 (2 mg/kg/day) for 14 days, and thereafter, cerebral microvessels were isolated and studied. Treatment of hph-1 mice with GW501516 significantly reduced oxidation of BH₄ and increased the ratio of BH₄ to 7,8-BH₂ (P<0.05, n=6-9). Attenuation of L-NAME-inhibitable superoxide anion levels by GW501516 demonstrated that activation of PPARδ might prevent uncoupling of endothelial nitric oxide synthase (eNOS, P<0.05, n=6-9). Western blotting studies demonstrated that GW501516 selectively increased the endothelial expressions of CuZn superoxide dismutase (P<0.05, n=6-9) and catalase (P<0.05, n=6-8). PPARδ activation increased the total nitrite and nitrate (NO₂+NO₃) content in cerebral microvessels (P<0.05, n=6). Obtained results suggest that in vivo activation of PPARδ prevents eNOS uncoupling, restores bioavailability of NO and may help preserve endothelial function in the BH₄-deficient cerebral circulation. Copyright © 2012 Elsevier B.V. All rights reserved.

Brk/PTK6 Sustains Activated EGFR Signaling through Inhibiting EGFR Degradation and Transactivating EGFR

PubMed Central

Li, X; Lu, Y; Liang, K; Hsu, J -M.; Albarracin, C; Mills, G B; Hung, M-C; Fan, Z

2011-01-01

Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from Cbl-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy. PMID:22231447

Ethylene Glycol Quenching of Nitrogenase Catalysis: An Electron Paramagnetic Resonance Spectroscopic Study of Nitrogenase Turnover States and CO Bonding.

PubMed

Hales, Brian J

2015-07-14

Most hydrophilic organic solvents inhibit enzymatic activity. Nitrogenase is shown to be approximately 3 times more sensitive to organic inhibition than most other soluble enzymes. Ethylene glycol (EG) is demonstrated to rapidly inhibit nitrogenase activity without uncoupling ATP hydrolysis. Our data suggest the mechanism of inhibition is EG's blocking of binding of MgATP to the nitrogenase Fe protein. EG quenching allows, for the first time, the observation of the relaxation of the intermediate reaction states at room temperature. Electron paramagnetic resonance (EPR) spectroscopy is used to monitor the room-temperature decay of the nitrogenase turnover states following EG quenching of catalytic activity. The return of the intermediate states to the resting state occurs in multiple phases over 2 h. During the initial stage, nitrogenase still possesses the ability to generate CO-induced EPR signals even though catalytic activity has ceased. During the last phase of relaxation, the one-electron reduced state of the MoFe protein (E1) relaxes to the resting state (E0) in a slow first-order reaction.

Dynamic control of Hsf1 during heat shock by a chaperone switch and phosphorylation

PubMed Central

Zheng, Xu; Krakowiak, Joanna; Patel, Nikit; Beyzavi, Ali; Ezike, Jideofor; Khalil, Ahmad S; Pincus, David

2016-01-01

Heat shock factor (Hsf1) regulates the expression of molecular chaperones to maintain protein homeostasis. Despite its central role in stress resistance, disease and aging, the mechanisms that control Hsf1 activity remain unresolved. Here we show that in budding yeast, Hsf1 basally associates with the chaperone Hsp70 and this association is transiently disrupted by heat shock, providing the first evidence that a chaperone repressor directly regulates Hsf1 activity. We develop and experimentally validate a mathematical model of Hsf1 activation by heat shock in which unfolded proteins compete with Hsf1 for binding to Hsp70. Surprisingly, we find that Hsf1 phosphorylation, previously thought to be required for activation, in fact only positively tunes Hsf1 and does so without affecting Hsp70 binding. Our work reveals two uncoupled forms of regulation - an ON/OFF chaperone switch and a tunable phosphorylation gain - that allow Hsf1 to flexibly integrate signals from the proteostasis network and cell signaling pathways. DOI: http://dx.doi.org/10.7554/eLife.18638.001 PMID:27831465

Uncoupling proteins and sleep deprivation.

PubMed

Cirelli, C; Tononi, G

2004-07-01

In both humans and animals sleep deprivation (SD) produces an increase in food intake and in energy expenditure (EE). The increase in EE is a core element of the SD syndrome and, in rats, is negatively correlated with survival rate. However, the mechanisms involved are not understood. A large component of resting EE is accounted for by the mitochondrial proton leak, which is mediated by uncoupling proteins (UCPs). We measured UCP2, UCP3, and UCP5 mRNA levels in rats during the spontaneous sleep/waking cycle and after short (8 hours) and long (7 days) SD. During spontaneous sleep and waking there was no change in the level of mitochondrial uncoupling as measured by UCPs expression, either in the brain or in peripheral tissues. During SD, by contrast, UCP3 expression in skeletal muscle was elevated, but the increase was similar, compared to sleep, after both short-term and long-term SD. UCP2 expression, on the other hand, was strongly increased in the liver and skeletal muscle of long-term sleep deprived animals and much less so, or not at all, in yoked controls or in rats that lost only 8 hours of sleep. Since the skeletal muscle is the largest tissue in the body, an elevated muscular expression of UCP2 is likely to affect the overall resting EE and may thus contribute to its increase after SD.

Feedback repression is required for mammalian circadian clock function.

PubMed

Sato, Trey K; Yamada, Rikuhiro G; Ukai, Hideki; Baggs, Julie E; Miraglia, Loren J; Kobayashi, Tetsuya J; Welsh, David K; Kay, Steve A; Ueda, Hiroki R; Hogenesch, John B

2006-03-01

Direct evidence for the requirement of transcriptional feedback repression in circadian clock function has been elusive. Here, we developed a molecular genetic screen in mammalian cells to identify mutants of the circadian transcriptional activators CLOCK and BMAL1, which were uncoupled from CRYPTOCHROME (CRY)-mediated transcriptional repression. Notably, mutations in the PER-ARNT-SIM domain of CLOCK and the C terminus of BMAL1 resulted in synergistic insensitivity through reduced physical interactions with CRY. Coexpression of these mutant proteins in cultured fibroblasts caused arrhythmic phenotypes in population and single-cell assays. These data demonstrate that CRY-mediated repression of the CLOCK/BMAL1 complex activity is required for maintenance of circadian rhythmicity and provide formal proof that transcriptional feedback is required for mammalian clock function.

Phosphorylation of GENOMES UNCOUPLED 4 Alters Stimulation of Mg Chelatase Activity in Angiosperms1[OPEN

PubMed Central

Hochheuser, Caroline; Fufezan, Christian; Heinze, Laura

2016-01-01

GENOMES UNCOUPLED 4 (GUN4) is a positive regulator of light-dependent chlorophyll biosynthesis. GUN4 activates Mg chelatase (MgCh) that catalyzes the insertion of an Mg2+ ion into protoporphyrin IX. We show that Arabidopsis (Arabidopsis thaliana) GUN4 is phosphorylated at Ser 264 (S264), the penultimate amino acid residue at the C terminus. While GUN4 is preferentially phosphorylated in darkness, phosphorylation is reduced upon accumulation of Mg porphyrins. Expression of a phosphomimicking GUN4(S264D) results in an incomplete complementation of the white gun4-2 null mutant and a chlorotic phenotype comparable to gun4 knockdown mutants. Phosphorylated GUN4 has a reduced stimulatory effect on MgCh in vitro and in vivo but retains its protein stability and tetrapyrrole binding capacity. Analysis of GUN4 found in oxygenic photosynthetic organisms reveals the evolution of a C-terminal extension, which harbors the phosphorylation site of GUN4 expressed in angiosperms. Homologs of GUN4 from Synechocystis and Chlamydomonas lack the conserved phosphorylation site found in a C-terminal extension of angiosperm GUN4. Biochemical studies proved the importance of the C-terminal extension for MgCh stimulation and inactivation of GUN4 by phosphorylation in angiosperms. An additional mechanism regulating MgCh activity is proposed. In conjunction with the dark repression of 5-aminolevulinic acid synthesis, GUN4 phosphorylation minimizes the flow of intermediates into the Mg branch of the tetrapyrrole metabolic pathway for chlorophyll biosynthesis. PMID:27688621

Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2

PubMed Central

Gavrish, Ekaterina; Sit, Clarissa S.; Cao, Shugeng; Kandror, Olga; Spoering, Amy; Peoples, Aaron; Ling, Losee; Fetterman, Ashley; Hughes, Dallas; Bissell, Anthony; Torrey, Heather; Akopian, Tatos; Mueller, Andreas; Epstein, Slava; Goldberg, Alfred; Clardy, Jon; Lewis, Kim

2014-01-01

Summary Languishing antibiotic discovery and flourishing antibiotic resistance have prompted development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against M. tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally-encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2 catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for lassomycin's bacteriocidal activity. PMID:24684906

Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

PubMed Central

Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

2015-01-01

Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

AMP-activated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1.

PubMed

Anilkumar, Ujval; Weisová, Petronela; Düssmann, Heiko; Concannon, Caoimhín G; König, Hans-Georg; Prehn, Jochen H M

2013-03-01

Neuronal preconditioning is a phenomenon where a previous exposure to a sub-lethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischaemic and mitochondrial uncoupling-induced preconditioning in neurons, however, the molecular basis of AMPK-mediated preconditioning has been less well characterized. We investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. Analysing potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein myeloid cell leukaemia sequence 1 (MCL-1) in AICAR-preconditioned neurons. Interestingly, over-expression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitored intracellular Ca²⁺ levels during NMDA excitation revealed that MCL-1 over-expressing neurons exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection. This study identifies MCL-1 as a key effector of AMPK-induced preconditioning in neurons. © 2012 International Society for Neurochemistry.

Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein–protein interaction

PubMed Central

Steiner, Samuel; Lori, Christian; Boehm, Alex; Jenal, Urs

2013-01-01

In many bacterial pathogens, the second messenger c-di-GMP stimulates the production of an exopolysaccharide (EPS) matrix to shield bacteria from assaults of the immune system. How c-di-GMP induces EPS biogenesis is largely unknown. Here, we show that c-di-GMP allosterically activates the synthesis of poly-β-1,6-N-acetylglucosamine (poly-GlcNAc), a major extracellular matrix component of Escherichia coli biofilms. C-di-GMP binds directly to both PgaC and PgaD, the two inner membrane components of the poly-GlcNAc synthesis machinery to stimulate their glycosyltransferase activity. We demonstrate that the PgaCD machinery is a novel type c-di-GMP receptor, where ligand binding to two proteins stabilizes their interaction and promotes enzyme activity. This is the first example of a c-di-GMP-mediated process that relies on protein–protein interaction. At low c-di-GMP concentrations, PgaD fails to interact with PgaC and is rapidly degraded. Thus, when cells experience a c-di-GMP trough, PgaD turnover facilitates the irreversible inactivation of the Pga machinery, thereby temporarily uncoupling it from c-di-GMP signalling. These data uncover a mechanism of c-di-GMP-mediated EPS control and provide a frame for c-di-GMP signalling specificity in pathogenic bacteria. PMID:23202856

Leucine deprivation stimulates fat loss via increasing CRH expression in the hypothalamus and activating the sympathetic nervous system.

PubMed

Cheng, Ying; Zhang, Qian; Meng, Qingshu; Xia, Tingting; Huang, Zhiying; Wang, Chunxia; Liu, Bin; Chen, Shanghai; Xiao, Fei; Du, Ying; Guo, Feifan

2011-09-01

We previously showed that leucine deprivation decreases abdominal fat mass largely by increasing energy expenditure, as demonstrated by increased lipolysis in white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). The goal of the present study was to investigate the possible involvement of central nervous system (CNS) in this regulation and elucidate underlying molecular mechanisms. For this purpose, levels of genes and proteins related to lipolysis in WAT and UCP1 expression in BAT were analyzed in wild-type mice after intracerebroventricular administration of leucine or corticotrophin-releasing hormone antibodies, or in mice deleted for three β-adrenergic receptors, after being maintained on a leucine-deficient diet for 7 d. Here, we show that intracerebroventricular administration of leucine significantly attenuates abdominal fat loss and blocks activation of hormone sensitive lipase in WAT and induction of UCP1 in BAT in leucine-deprived mice. Furthermore, we provide evidence that leucine deprivation stimulates fat loss by increasing expression of corticotrophin-releasing hormone in the hypothalamus via activation of stimulatory G protein/cAMP/protein kinase A/cAMP response element-binding protein pathway. Finally, we show that the effect of leucine deprivation on fat loss is mediated by activation of the sympathetic nervous system. These results suggest that CNS plays an important role in regulating fat loss under leucine deprivation and thereby provide novel and important insights concerning the importance of CNS leucine in the regulation of energy homeostasis.

Fatty acid homeostasis and induction of lipid regulatory genes in skeletal muscles of peroxisome proliferator-activated receptor (PPAR) alpha knock-out mice. Evidence for compensatory regulation by PPAR delta.

PubMed

Muoio, Deborah M; MacLean, Paul S; Lang, David B; Li, Shi; Houmard, Joseph A; Way, James M; Winegar, Deborah A; Corton, J Christopher; Dohm, G Lynis; Kraus, William E

2002-07-19

Ablation of peroxisome proliferator activated receptor (PPAR) alpha, a lipid-activated transcription factor that regulates expression of beta-oxidative genes, results in profound metabolic abnormalities in liver and heart. In the present study we used PPAR alpha knockout (KO) mice to determine whether this transcription factor is essential for regulating fuel metabolism in skeletal muscle. When animals were challenged with exhaustive exercise or starvation, KO mice exhibited lower serum levels of glucose, lactate, and ketones and higher nonesterified fatty acids than wild type (WT) littermates. During exercise, KO mice exhausted earlier than WT and exhibited greater rates of glycogen depletion in liver but not skeletal muscle. Fatty acid oxidative capacity was similar between muscles of WT and KO when animals were fed and only 28% lower in KO muscles when animals were starved. Exercise-induced regulation and starvation-induced regulation of pyruvate-dehydrogenase kinase 4 and uncoupling protein 3, two classical and robustly responsive PPAR alpha target genes, were similar between WT and KO in skeletal muscle but markedly different between genotypes in heart. Real time quantitative PCR analyses showed that unlike in liver and heart, in mouse skeletal muscle PPAR delta is severalfold more abundant than either PPAR alpha or PPAR gamma. In both human and rodent myocytes, the highly selective PPAR delta agonist GW742 increased fatty acid oxidation about 2-fold and induced expression of several lipid regulatory genes, including pyruvate-dehydrogenase kinase 4 and uncoupling protein 3, responses that were similar to those elicited by the PPAR alpha agonist GW647. These results show redundancy in the functions of PPARs alpha and delta as transcriptional regulators of fatty acid homeostasis and suggest that in skeletal muscle high levels of the delta-subtype can compensate for deficiency of PPAR alpha.

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).

PubMed

Chan, Chun-Yuan; Prudom, Catherine; Raines, Summer M; Charkhzarrin, Sahba; Melman, Sandra D; De Haro, Leyma P; Allen, Chris; Lee, Samuel A; Sklar, Larry A; Parra, Karlett J

2012-03-23

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.

Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

PubMed

Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

2017-07-28

The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

The brain and brown fat

PubMed Central

Gonzalez, Francisco; Fernø, Johan; Diéguez, Carlos; Rahmouni, Kamal; Nogueiras, Rubén

2015-01-01

Brown adipose tissue (BAT) is a specialized organ responsible for thermogenesis, a process required for maintaining body temperature. BAT is regulated by the sympathetic nervous system (SNS), which activates lipolysis and mitochondrial uncoupling in brown adipocytes. For many years, BAT was considered to be important only in small mammals and newborn humans, but recent data have shown that BAT is also functional in adult humans. On the basis of this evidence, extensive research has been focused on BAT function, where new molecules, such as irisin and bone morphogenetic proteins, particularly BMP7 and BMP8B, as well as novel central factors and new regulatory mechanisms, such as orexins and the canonical ventomedial nucleus of the hypothalamus (VMH) AMP- activated protein kinase (AMPK)–SNS–BAT axis, have been discovered and emerged as potential drug targets to combat obesity. In this review we provide an overview of the complex central regulation of BAT and how different neuronal cell populations co-ordinately work to maintain energy homeostasis. PMID:24915455

The pyruvate kinase of Stigmatella aurantiaca is an indole binding protein and essential for development.

PubMed

Stamm, Irmela; Lottspeich, Friedrich; Plaga, Wulf

2005-06-01

Myxospore formation of the myxobacterium Stigmatella aurantiaca can be uncoupled from the cooperative development i.e. fruiting body formation, by low concentrations of indole. Two putative indole receptor proteins were isolated by their capacity to bind indole and identified as pyruvate kinase (PK) and aldehyde dehydrogenase. The PK activity of Stigmatella crude extracts was stimulated by indole. Cloning of the PK gene (pykA) and the construction of a pykA disruption mutant strikingly revealed that PK is essential for multicellular development: Fruiting body formation was abolished in the mutant strain and indole-induced spore formation was delayed. The developmental defects could be complemented by insertion of the pykA gene at the mtaB locus of the Stigmatella genome excluding any polar effects of the pykA disruption.

Human neuronal uncoupling proteins 4 and 5 (UCP4 and UCP5): structural properties, regulation, and physiological role in protection against oxidative stress and mitochondrial dysfunction

PubMed Central

Ramsden, David B; Ho, Philip W-L; Ho, Jessica W-M; Liu, Hui-Fang; So, Danny H-F; Tse, Ho-Man; Chan, Koon-Ho; Ho, Shu-Leong

2012-01-01

Uncoupling proteins (UCPs) belong to a large family of mitochondrial solute carriers 25 (SLC25s) localized at the inner mitochondrial membrane. UCPs transport protons directly from the intermembrane space to the matrix. Of five structural homologues (UCP1 to 5), UCP4 and 5 are principally expressed in the central nervous system (CNS). Neurons derived their energy in the form of ATP that is generated through oxidative phosphorylation carried out by five multiprotein complexes (Complexes I–V) embedded in the inner mitochondrial membrane. In oxidative phosphorylation, the flow of electrons generated by the oxidation of substrates through the electron transport chain to molecular oxygen at Complex IV leads to the transport of protons from the matrix to the intermembrane space by Complex I, III, and IV. This movement of protons to the intermembrane space generates a proton gradient (mitochondrial membrane potential; MMP) across the inner membrane. Complex V (ATP synthase) uses this MMP to drive the conversion of ADP to ATP. Some electrons escape to oxygen-forming harmful reactive oxygen species (ROS). Proton leakage back to the matrix which bypasses Complex V resulting in a major reduction in ROS formation while having a minimal effect on MMP and hence, ATP synthesis; a process termed “mild uncoupling.” UCPs act to promote this proton leakage as means to prevent excessive build up of MMP and ROS formation. In this review, we discuss the structure and function of mitochondrial UCPs 4 and 5 and factors influencing their expression. Hypotheses concerning the evolution of the two proteins are examined. The protective mechanisms of the two proteins against neurotoxins and their possible role in regulating intracellular calcium movement, particularly with regard to the pathogenesis of Parkinson's disease are discussed. PMID:22950050

The mitochondrial uncoupling protein-2 promotes chemoresistance in cancer cells

PubMed Central

Derdak, Zoltan; Mark, Nicholas M.; Beldi, Guido; Robson, Simon C.; Wands, Jack R.; Baffy, György

2008-01-01

Cancer cells acquire drug resistance as a result of selection pressure dictated by unfavorable microenvironments. This survival process is facilitated through efficient control of oxidative stress originating from mitochondria that typically initiates programmed cell death. We show this critical adaptive response in cancer cells to be linked to uncoupling protein-2 (UCP2), a mitochondrial suppressor of reactive oxygen species (ROS). UCP2 is present in drug-resistant lines of various cancer cells and in human colon cancer. Overexpression of UCP2 in HCT116 human colon cancer cells inhibits ROS accumulation and apoptosis post-exposure to chemotherapeutic agents. Tumor xenografts of UCP2-overexpressing HCT116 cells retain growth in nude mice receiving chemotherapy. Augmented cancer cell survival is accompanied by altered N-terminal phosphorylation of the pivotal tumor suppressor p53 and induction of the glycolytic phenotype (Warburg effect). These findings link UCP2 with molecular mechanisms of chemoresistance. Targeting UCP2 may be considered a novel treatment strategy for cancer. PMID:18413749

The evolutionary capacitor HSP90 buffers the regulatory effects of mammalian endogenous retroviruses.

PubMed

Hummel, Barbara; Hansen, Erik C; Yoveva, Aneliya; Aprile-Garcia, Fernando; Hussong, Rebecca; Sawarkar, Ritwick

2017-03-01

Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.

Mitochondrial efficiency and exercise economy following heat stress: a potential role of uncoupling protein 3.

PubMed

Salgado, Roy M; Sheard, Ailish C; Vaughan, Roger A; Parker, Daryl L; Schneider, Suzanne M; Kenefick, Robert W; McCormick, James J; Gannon, Nicholas P; Van Dusseldorp, Trisha A; Kravitz, Len R; Mermier, Christine M

2017-02-01

Heat stress has been reported to reduce uncoupling proteins (UCP) expression, which in turn should improve mitochondrial efficiency. Such an improvement in efficiency may translate to the systemic level as greater exercise economy. However, neither the heat-induced improvement in mitochondrial efficiency (due to decrease in UCP), nor its potential to improve economy has been studied. Determine: (i) if heat stress in vitro lowers UCP3 thereby improving mitochondrial efficiency in C2C12 myocytes; (ii) whether heat acclimation (HA) in vivo improves exercise economy in trained individuals; and (iii) the potential improved economy during exercise at altitude. In vitro, myocytes were heat stressed for 24 h (40°C), followed by measurements of UCP3, mitochondrial uncoupling, and efficiency. In vivo, eight trained males completed: (i) pre-HA testing; (ii) 10 days of HA (40°C, 20% RH); and (iii) post-HA testing. Pre- and posttesting consisted of maximal exercise test and submaximal exercise at two intensities to assess exercise economy at 1600 m (Albuquerque, NM) and 4350 m. Heat-stressed myocytes displayed significantly reduced UCP3 mRNA expression and, mitochondrial uncoupling (77.1 ± 1.2%, P < 0.0001) and improved mitochondrial efficiency (62.9 ± 4.1%, P < 0.0001) compared to control. In humans, at both 1600 m and 4350 m, following HA, submaximal exercise economy did not change at low and moderate exercise intensities. Our findings indicate that while heat-induced reduction in UCP3 improves mitochondrial efficiency in vitro, this is not translated to in vivo improvement of exercise economy at 1600 m or 4350 m. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Regulation of Heat Stress by HSF1 and GR

DTIC Science & Technology

2016-09-01

Geiger PC. (2009). Heat treatment improves glucose tolerance and prevents skeletal muscle insulin resistance in rats fed a high -fat diet . Diabetes...appear to have different effects on the mitochondrial morphology and fission protein in skeletal muscle cells. The signaling pathways involving HSF1...preliminary results show that mitochondrial uncoupling proteins 2 and 3 (UCP2, UCP3) were down-regulated in in the gastrocnemius muscles of INT mice

Serum levels of uncoupling proteins in patients with differential insulin resistance

PubMed Central

Pan, Heng-Chih; Lee, Chin-Chan; Chou, Kuei-Mei; Lu, Shang-Chieh; Sun, Chiao-Yin

2017-01-01

Abstract The uncoupling protein (UCP) belongs to a family of energy-dissipating proteins in mitochondria. Increasing evidences have indicated that UCPs have immense impact on glucose homeostasis and are key proteins in metabolic syndrome. For applying the findings to clinical practice, we designed a study to explore the association between serum UCPs 1–3 and insulin resistance. This investigation prospectively recorded demographical parameter and collected blood samples of 1071 participants from 4 districts in Northeastern Taiwan during the period from August 2013 to July 2014. Propensity score matching by age and sex in patients with top and bottom third homeostasis model assessment of insulin resistance (HOMA-IR) levels was performed, and 326 subjects were enrolled for further studies. The mean age of the patients was 59.4 years and the majority of them (65.5%) were females. The prevalence of metabolic syndrome was 35.5%. Our results demonstrated that serum UCPs 1–3 were significantly associated with differences in HOMA-IR levels. Multiple logistic regression analysis indicated that low UCP 1 and features of metabolic syndrome, namely hypertension, diabetes, body mass index, and high-density lipoprotein, were independent determinants for high HOMA-IR levels. We thus determined that low serum UCP 1 is a predictor for high resistance to insulin. PMID:28984759

Uncoupling protein-2 mediates DPP-4 inhibitor-induced restoration of endothelial function in hypertension through reducing oxidative stress.

PubMed

Liu, Limei; Liu, Jian; Tian, Xiao Yu; Wong, Wing Tak; Lau, Chi Wai; Xu, Aimin; Xu, Gang; Ng, Chi Fai; Yao, Xiaoqiang; Gao, Yuansheng; Huang, Yu

2014-10-10

Although uncoupling protein 2 (UCP2) negatively regulates intracellular reactive oxygen species (ROS) production and protects vascular function, its participation in vascular benefits of drugs used to treat cardiometabolic diseases is largely unknown. This study investigated whether UCP2 and associated oxidative stress reduction contribute to the improvement of endothelial function by a dipeptidyl peptidase-4 inhibitor, sitagliptin, in hypertension. Pharmacological inhibition of cyclooxygenase-2 (COX-2) but not COX-1 prevented endothelial dysfunction, and ROS scavengers reduced COX-2 mRNA and protein expression in spontaneously hypertensive rats (SHR) renal arteries. Angiotensin II (Ang II) evoked endothelium-dependent contractions (EDCs) in C57BL/6 and UCP2 knockout (UCP2KO) mouse aortae. Chronic sitagliptin administration attenuated EDCs in SHR arteries and Ang II-infused C57BL/6 mouse aortae and eliminated ROS overproduction in SHR arteries, which were reversed by glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin 9-39, AMP-activated protein kinase (AMPK)α inhibitor compound C, and UCP2 inhibitor genipin. By contrast, sitagliptin unaffected EDCs in Ang II-infused UCP2KO mice. Sitagliptin increased AMPKα phosphorylation, upregulated UCP2, and downregulated COX-2 expression in arteries from SHR and Ang II-infused C57BL/6 mice. Importantly, exendin 9-39, compound C, and genipin reversed the inhibitory effect of GLP-1R agonist exendin-4 on Ang II-stimulated mitochondrial ROS rises in SHR endothelial cells. Moreover, exendin-4 improved the endothelial function of renal arteries from SHR and hypertensive patients. We elucidate for the first time that UCP2 serves as an important signal molecule in endothelial protection conferred by GLP-1-related agents. UCP2 could be a useful target in treating hypertension-related vascular events. UCP2 inhibits oxidative stress and downregulates COX-2 expression through GLP-1/GLP-1R/AMPKα cascade.

Protein kinase C is involved in cyclic adenosine monophosphate formation due to PGF2 alpha desensitization in bovine iris sphincter.

PubMed

Tachado, S D; Zhang, Y; Abdel-Latif, A A

1993-05-01

To examine the mechanisms underlying the effects of PGF2 alpha receptor desensitization on agonist-induced second messenger formation and contraction in bovine iris sphincter. Short-term PGF2 alpha receptor desensitization of the bovine iris sphincter was carried out by incubating the tissue in Krebs-Ringer bicarbonate buffer containing 25 microM PGF2 alpha for 45 min at 37 degrees C. The effects of PGF2 alpha and other pharmacologic agents on inositol 1,4,5-triphosphate (IP3) production and cyclic adenosine monophosphate (cAMP) formation in desensitized and nondesensitized tissues were monitored by anion-exchange chromatography and radioimmunoassay. In the isolated bovine iris sphincter, protein kinase C (PKC) is involved in the activation of adenylate cyclase and the desensitization of prostaglandin F2 alpha receptor-mediated responses supported by these findings. (A) Exposure of the tissue to phorbol 12,13-dibutyrate, used to activate PKC, enhanced basal cAMP formation in a dose (EC50 = 8.8 x 10(-8) M) and time (t1/2 = 7.5 min) dependent manner. Phorbol 12,13-dibutyrate increased cAMP levels by twofold and it potentiated the isoproterenol-induced cAMP formation. The biologically inactive phorbol ester, 4 alpha-phorbol had no effect. Staurosporine, a potent PKC inhibitor, inhibited phorbol 12,13-dibutyrate-induced cAMP formation in a dose-dependent manner (IC50 of 0.25 microM). The increase in cAMP levels by phorbol 12,13-dibutyrate results from stimulation of adenylate cyclase, rather than from inhibition of cAMP phosphodiesterase, and it is not mediated through Ca2+ mobilization. Pretreatment of the tissue with phorbol 12,13-dibutyrate inhibited IP3 production in response to PGF2 alpha. (B) Desensitization of the sphincter with PGF2 alpha for 45 min increased cAMP formation and attenuated IP3 production and contraction. The effects of PGF2 alpha desensitization were reversed by pretreatment of the tissue with staurosporine. Down-regulation of PKC prevented the PGF2 alpha-stimulated increase in cAMP formation. In the desensitized tissue, diacylglycerol, the endogenous activator of PKC, may arise from phosphatidylcholine, via phospholipase D. (A) Activation of PKC in the bovine iris sphincter leads to stimulation of adenylate cyclase and to an increase in cAMP formation. The cAMP formed inhibits IP3 production and muscle contraction. (B) PGF2 alpha desensitization results in adenylate cyclase activation, mediated through PKC. (C) PGF2 alpha desensitization could uncouple the receptor from the Gq and Gi proteins and enhance PG stimulation of adenylate cyclase activity through the Gs protein. (D) Uncoupling of the G proteins from the PG receptor and activation of PKC, both of which result in enhanced cAMP formation, may underlie the mechanism of PGF2 alpha desensitization. (E) These observations demonstrate "cross talk" between the two second messenger systems and their physiologic consequences.

Early Decrease in Respiration and Uncoupling Event Independent of Cytochrome c Release in PC12 Cells Undergoing Apoptosis

PubMed Central

Berghella, Libera; Ferraro, Elisabetta

2012-01-01

Cytochrome c is a key molecule in mitochondria-mediated apoptosis. It also plays a pivotal role in cell respiration. The switch between these two functions occurs at the moment of its release from mitochondria. This process is therefore extremely relevant for the fate of the cell. Since cytochrome c mediates respiration, we studied the changes in respiratory chain activity during the early stages of apoptosis in order to contribute to unravel the mechanisms of cytochrome c release. We found that, during staurosporine (STS)- induced apoptosis in PC12 cells, respiration is affected before the release of cytochrome c, as shown by a decrease in the endogenous uncoupled respiration and an uncoupling event, both occurring independently of cytochrome c release. The decline in the uncoupled respiration occurs also upon Bcl-2 overexpression (which inhibits cytochrome c release), while the uncoupling event is inhibited by Bcl-2. We also observed that the first stage of nuclear condensation during STS-induced apoptosis does not depend on the release of cytochrome c into the cytosol and is a reversibile event. These findings may contribute to understand the mechanisms affecting mitochondria during the early stages of apoptosis and priming them for the release of apoptogenic factors. PMID:22666257

Identification of Critical Residues Involved in Ligand Binding and G Protein Signaling in Human Somatostatin Receptor Subtype 2

PubMed Central

Parry, Jesse J.; Chen, Ronald; Andrews, Rebecca; Lears, Kimberly A.

2012-01-01

G protein signaling through human somatostatin receptor subtype 2 (SSTR2) is well known, but the amino acids involved in stimulation of intracellular responses upon ligand binding have not been characterized. We constructed a series of point mutants in SSTR2 at amino acid positions 89, 139, and 140 in attempts to disrupt G protein signaling upon ligand binding. The aspartic acid changes at position 89 to either Ala, Leu, or Arg generated mutant receptors with varying expression profiles and a complete inability to bind somatostatin-14 (SST). Mutations to Asp 139 and Arg 140 also led to varying expression profiles with some mutants maintaining their affinity for SST. Mutation of Arg 140 to Ala resulted in a mutated receptor that had a Bmax and dissociation constant (Kd) similar to wild-type receptor but was still coupled to the G protein as determined in both a cAMP assay and a calcium-release assay. In contrast, mutation of Asp 139 to Asn resulted in a mutated receptor with Bmax and Kd values that were similar to wild type but was uncoupled from G protein-mediated cAMP signaling, but not calcium release. Thus, we identified mutations in SSTR2 that result in either receptor expression levels that are similar to wild type but is completely ablated for ligand binding or a receptor that maintains affinity for SST and is uncoupled from G protein-mediated cAMP signaling. PMID:22495673

Hedgehog regulates Norrie disease protein to drive neural progenitor self-renewal.

PubMed

McNeill, Brian; Mazerolle, Chantal; Bassett, Erin A; Mears, Alan J; Ringuette, Randy; Lagali, Pamela; Picketts, David J; Paes, Kim; Rice, Dennis; Wallace, Valerie A

2013-03-01

Norrie disease (ND) is a congenital disorder characterized by retinal hypovascularization and cognitive delay. ND has been linked to mutations in 'Norrie Disease Protein' (Ndp), which encodes the secreted protein Norrin. Norrin functions as a secreted angiogenic factor, although its role in neural development has not been assessed. Here, we show that Ndp expression is initiated in retinal progenitors in response to Hedgehog (Hh) signaling, which induces Gli2 binding to the Ndp promoter. Using a combination of genetic epistasis and acute RNAi-knockdown approaches, we show that Ndp is required downstream of Hh activation to induce retinal progenitor proliferation in the retina. Strikingly, Ndp regulates the rate of cell-cycle re-entry and not cell-cycle kinetics, thereby uncoupling the self-renewal and cell-cycle progression functions of Hh. Taken together, we have uncovered a cell autonomous function for Ndp in retinal progenitor proliferation that is independent of its function in the retinal vasculature, which could explain the neural defects associated with ND.

Measuring Interference of Drug-Like Molecules with the Respiratory Chain: Toward the Early Identification of Mitochondrial Uncouplers in Lead Finding

PubMed Central

Matter, Hans; Diekert, Kerstin; Dörner, Wolfgang; Dröse, Stefan; Licher, Thomas

2013-01-01

Abstract The electron transport chain (ETC) couples electron transfer between donors and acceptors with proton transport across the inner mitochondrial membrane. The resulting electrochemical proton gradient is used to generate chemical energy in the form of adenosine triphosphate (ATP). Proton transfer is based on the activity of complex I–V proteins in the ETC. The overall electrical activity of these proteins can be measured by proton transfer using Solid Supported Membrane technology. We tested the activity of complexes I, III, and V in a combined assay, called oxidative phosphorylation assay (oxphos assay), by activating each complex with the corresponding substrate. The oxphos assay was used to test in-house substances from different projects and several drugs currently available on the market that have reported effects on mitochondrial functions. The resulting data were compared to the influence of the respective compounds on mitochondria as determined by oxygen consumption and to data generated with an ATP depletion assay. The comparison shows that the oxidative phosphorylation assay provides both a rapid approach for detecting interaction of compounds with respiratory chain proteins and information on their mode of interaction. Therefore, the oxphos assay is a useful tool to support structure activity relationship studies by allowing early identification of mitotoxicity and for analyzing the outcome of phenotypic screens that are susceptible to the generation of mitotoxicity-related artifacts. PMID:23992120

Anti-obesity effects of Arctii Fructus (Arctium lappa) in white/brown adipocytes and high-fat diet-induced obese mice.

PubMed

Han, Yo-Han; Kee, Ji-Ye; Kim, Dae-Seung; Park, Jinbong; Jeong, Mi-Young; Mun, Jung-Geon; Park, Sung-Joo; Lee, Jong-Hyun; Um, Jae-Young; Hong, Seung-Heon

2016-12-07

Arctii Fructus is traditionally used in oriental pharmacies as an anti-inflammatory medicine. Although several studies have shown its anti-inflammatory effects, there have been no reports on its use in obesity related studies. In this study, the anti-obesity effect of Arctii Fructus was investigated in high-fat diet (HFD)-induced obese mice, and the effect was confirmed in white and primary cultured brown adipocytes. Arctii Fructus inhibited weight gain and reduced the mass of white adipose tissue in HFD-induced obese mice. Serum levels of triglyceride and LDL-cholesterol were reduced, and HDL-cholesterol was increased in the Arctii Fructus treated group. In 3T3-L1 cells, a water extract (WAF) and 70% EtOH extract (EtAF) of Arctii Fructus significantly inhibited adipogenesis and suppressed the expression of proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha. In particular, EtAF activated the phosphorylation of AMP-activated protein kinase. On the other hand, uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator 1-alpha, known as brown adipocytes specific genes, were increased in primary cultured brown adipocytes by WAF and EtAF. This study shows that Arctii Fructus prevents the development of obesity through the inhibition of white adipocyte differentiation and activation of brown adipocyte differentiation which suggests that Arctii Fructus could be an effective therapeutic for treating or preventing obesity.

Fluorescent recovery after photobleaching (FRAP) analysis of nuclear export rates identifies intrinsic features of nucleocytoplasmic transport.

PubMed

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-02-17

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level.

Electrophysiological and Structural Remodeling in Heart Failure Modulate Arrhythmogenesis. 2D Simulation Study

PubMed Central

Gomez, Juan F.; Cardona, Karen; Martinez, Laura; Saiz, Javier; Trenor, Beatriz

2014-01-01

Background Heart failure is operationally defined as the inability of the heart to maintain blood flow to meet the needs of the body and it is the final common pathway of various cardiac pathologies. Electrophysiological remodeling, intercellular uncoupling and a pro-fibrotic response have been identified as major arrhythmogenic factors in heart failure. Objective In this study we investigate vulnerability to reentry under heart failure conditions by incorporating established electrophysiological and anatomical remodeling using computer simulations. Methods The electrical activity of human transmural ventricular tissue (5 cm×5 cm) was simulated using the human ventricular action potential model Grandi et al. under control and heart failure conditions. The MacCannell et al. model was used to model fibroblast electrical activity, and their electrotonic interactions with myocytes. Selected degrees of diffuse fibrosis and variations in intercellular coupling were considered and the vulnerable window (VW) for reentry was evaluated following cross-field stimulation. Results No reentry was observed in normal conditions or in the presence of HF ionic remodeling. However, defined amount of fibrosis and/or cellular uncoupling were sufficient to elicit reentrant activity. Under conditions where reentry was generated, HF electrophysiological remodeling did not alter the width of the VW. However, intermediate fibrosis and cellular uncoupling significantly widened the VW. In addition, biphasic behavior was observed, as very high fibrotic content or very low tissue conductivity hampered the development of reentry. Detailed phase analysis of reentry dynamics revealed an increase of phase singularities with progressive fibrotic components. Conclusion Structural remodeling is a key factor in the genesis of vulnerability to reentry. A range of intermediate levels of fibrosis and intercellular uncoupling can combine to favor reentrant activity. PMID:25054335

Marked over expression of uncoupling protein-2 in beta cells exerts minor effects on mitochondrial metabolism

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

Hals, Ingrid K., E-mail: ingrid.hals@ntnu.no; Ogata, Hirotaka; Pettersen, Elin

2012-06-29

Highlights: Black-Right-Pointing-Pointer The impact of UCP-2 over expression on mitochondrial function is controversial. Black-Right-Pointing-Pointer We tested mitochondrial functions at defined levels of overexpression. Black-Right-Pointing-Pointer We find minor increases of fatty acid oxidation and uncoupling. Black-Right-Pointing-Pointer Effects were seen only at high level (fourfold) of over expression. Black-Right-Pointing-Pointer Hence it is doubtful whether these effects are of importance in diabetes. -- Abstract: Evidence is conflicting as to the impact of elevated levels of uncoupling protein-2 (UCP-2) on insulin-producing beta cells. Here we investigated effects of a fourfold induction of UCP-2 protein primarily on mitochondrial parameters and tested for replication of positivemore » findings at a lower level of induction. We transfected INS-1 cells to obtain a tet-on inducible cell line. A 48 h exposure to 1 {mu}g/ml of doxycycline (dox) induced UCP-2 fourfold (424 {+-} 113%, mean {+-} SEM) and 0.1 {mu}g/ml twofold (178 {+-} 29%, n = 3). Fourfold induced cells displayed normal viability (MTT, apoptosis), normal cellular insulin contents and, glucose-induced insulin secretion (+27 {+-} 11%) as well as D-[U-{sup 14}C]-glucose oxidation (+5 {+-} 9% at 11 mM glucose). Oxidation of [1-{sup 14}C]-oleate was increased from 4088 to 5797 fmol/{mu}g prot/2 h at 3.3 mM glucose, p < 0.03. Oxidation of L-[{sup 14}C(U)]-glutamine was unaffected. Induction of UCP-2 did not significantly affect measures of mitochondrial membrane potential (Rhodamine 123) or mitochondrial mass (Mitotracker Green) and did not affect ATP levels. Oligomycin-inhibited oxygen consumption (a measure of mitochondrial uncoupling) was marginally increased, the effect being significant in comparison with dox-only treated cells, p < 0.05. Oxygen radicals, assessed by dichlorofluorescin diacetate, were decreased by 30%, p < 0.025. Testing for the lower level of UCP-2 induction did not reproduce any of the positive findings. A fourfold induction of UCP-2 was required to exert minor metabolic effects. These findings question an impact of moderately elevated UCP-2 levels in beta cells as seen in diabetes.« less

Ca2+-induced uncoupling of Aplysia bag cell neurons.

PubMed

Dargaei, Zahra; Standage, Dominic; Groten, Christopher J; Blohm, Gunnar; Magoski, Neil S

2015-02-01

Electrical transmission is a dynamically regulated form of communication and key to synchronizing neuronal activity. The bag cell neurons of Aplysia are a group of electrically coupled neuroendocrine cells that initiate ovulation by secreting egg-laying hormone during a prolonged period of synchronous firing called the afterdischarge. Accompanying the afterdischarge is an increase in intracellular Ca2+ and the activation of protein kinase C (PKC). We used whole cell recording from paired cultured bag cell neurons to demonstrate that electrical coupling is regulated by both Ca2+ and PKC. Elevating Ca2+ with a train of voltage steps, mimicking the onset of the afterdischarge, decreased junctional current for up to 30 min. Inhibition was most effective when Ca2+ entry occurred in both neurons. Depletion of Ca2+ from the mitochondria, but not the endoplasmic reticulum, also attenuated the electrical synapse. Buffering Ca2+ with high intracellular EGTA or inhibiting calmodulin kinase prevented uncoupling. Furthermore, activating PKC produced a small but clear decrease in junctional current, while triggering both Ca2+ influx and PKC inhibited the electrical synapse to a greater extent than Ca2+ alone. Finally, the amplitude and time course of the postsynaptic electrotonic response were attenuated after Ca2+ influx. A mathematical model of electrically connected neurons showed that excessive coupling reduced recruitment of the cells to fire, whereas less coupling led to spiking of essentially all neurons. Thus a decrease in electrical synapses could promote the afterdischarge by ensuring prompt recovery of electrotonic potentials or making the neurons more responsive to current spreading through the network. Copyright © 2015 the American Physiological Society.

RISK FACTORS FOR NEURAL TUBE DEFECTS: ASSOCIATIONS BETWEEN UNCOUPLING PROTEIN 2 POLYMORPHISMS AND SPINA BIFIDA. (R828292)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

PubMed Central

Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

2010-01-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

Developmental programming, adiposity, and reproduction in ruminants.

PubMed

Symonds, M E; Dellschaft, N; Pope, M; Birtwistle, M; Alagal, R; Keisler, D; Budge, H

2016-07-01

Although sheep have been widely adopted as an animal model for examining the timing of nutritional interventions through pregnancy on the short- and long-term outcomes, only modest programming effects have been seen. This is due in part to the mismatch in numbers of twins and singletons between study groups as well as unequal numbers of males and females. Placental growth differs between singleton and twin pregnancies which can result in different body composition in the offspring. One tissue that is especially affected is adipose tissue which in the sheep fetus is primarily located around the kidneys and heart plus the sternal/neck region. Its main role is the rapid generation of heat due to activation of the brown adipose tissue-specific uncoupling protein 1 at birth. The fetal adipose tissue response to suboptimal maternal food intake at defined stages of development differs between the perirenal abdominal and pericardial depots, with the latter being more sensitive. Fetal adipose tissue growth may be mediated in part by changes in leptin status of the mother which are paralleled in the fetus. Then, over the first month of life plasma leptin is higher in females than males despite similar adiposity, when fat is the fastest growing tissue with the sternal/neck depot retaining uncoupling protein 1, whereas other depots do not. Future studies should take into account the respective effects of fetal number and sex to provide more detailed insights into the mechanisms by which adipose and related tissues can be programmed in utero. Copyright © 2016 Elsevier Inc. All rights reserved.

Vitamin D deficiency decreases adiposity in rats and causes altered expression of uncoupling proteins and steroid receptor coactivator3.

PubMed

Bhat, Mehrajuddin; Noolu, Bindu; Qadri, Syed S Y H; Ismail, Ayesha

2014-10-01

The vitamin D endocrine system is functional in the adipose tissue, as demonstrated in vitro, in cultured adipocytes, and in vivo in mutant mice that developed altered lipid metabolism and fat storage in the absence of either 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or the vitamin D receptor. The aim of the present study was to examine the role of vitamin D and calcium on body adiposity in a diet-induced vitamin D deficient rat model. Vitamin D-deficient rats gained less weight and had lower amounts of visceral fat. Consistent with reduced adipose tissue mass, the vitamin D-deficient rats had low circulating levels of leptin, which reflects body fat stores. Expression of vitamin D and calcium sensing receptors, and that of genes involved in adipogenesis such as peroxisome proliferator-activated receptor, fatty acid synthase and leptin were significantly reduced in white adipose tissue of deficient rats compared to vitamin D-sufficient rats. Furthermore, the expression of uncoupling proteins (Ucp1 and Ucp2) was elevated in the white adipose tissue of the deficient rat indicative of higher energy expenditure, thereby leading to a lean phenotype. Expression of the p160 steroid receptor coactivator3 (SRC3), a key regulator of adipogenesis in white adipose tissue was decreased in vitamin D-deficient state. Interestingly, most of the changes observed in vitamin D deficient rats were corrected by calcium supplementation alone. Our data demonstrates that dietary vitamin D and calcium regulate adipose tissue function and metabolism. Copyright © 2014 Elsevier Ltd. All rights reserved.

Oxidative damage mediated iNOS and UCP-2 upregulation in rat brain after sub-acute cyanide exposure: dose and time-dependent effects.

PubMed

Bhattacharya, Rahul; Singh, Poonam; John, Jebin Jacob; Gujar, Niranjan L

2018-04-03

Cyanide-induced chemical hypoxia is responsible for pronounced oxidative damage in the central nervous system. The disruption of mitochondrial oxidative metabolism has been associated with upregulation of uncoupling proteins (UCPs). The present study addresses the dose- and time-dependent effect of sub-acute cyanide exposure on various non-enzymatic and enzymatic oxidative stress markers and their correlation with inducible-nitric oxide synthase (iNOS) and uncoupling protein-2 (UCP-2) expression. Animals received (oral) triple distilled water (vehicle control), 0.25 LD50 potassium cyanide (KCN) or 0.50 LD50 KCN daily for 21 d. Animals were sacrificed on 7, 14 and 21 d post-exposure to measure serum cyanide and nitrite, and brain malondialdehyde (MDA), reduced glutathione (GSH), glutathione disulfide (GSSG), cytochrome c oxidase (CCO), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CA) levels, together with iNOS and UCP-2 expression, and DNA damage. The study revealed that a dose- and time-dependent increase in cyanide concentration was accompanied by corresponding CCO inhibition and elevated MDA levels. Decrease in GSH levels was not followed by reciprocal change in GSSG levels. Diminution of SOD, GPx, GR and CA activity was congruent with elevated nitrite levels and upregulation of iNOS and UCP-2 expression, without any DNA damage. It was concluded that long-term cyanide exposure caused oxidative stress, accompanied by upregulation of iNOS. The upregulation of UCP-2 further sensitized the cells to cyanide and accentuated the oxidative stress, which was independent of DNA damage.

Meat-type chickens have a higher efficiency of mitochondrial oxidative phosphorylation than laying-type chickens.

PubMed

Toyomizu, Masaaki; Kikusato, Motoi; Kawabata, Yusuke; Azad, Md Abul Kalam; Inui, Eriko; Amo, Taku

2011-05-01

Meat-type chickens show high feed efficiency and have a very rapid growth rate compared with laying-type chickens. To clarify whether the type-specific difference in feed conversion efficiency is involved in mitochondrial bioenergetics, modular kinetic analysis was applied to oxidative phosphorylation in skeletal muscle mitochondria of both type chickens. Mitochondria from skeletal muscle of meat-type chickens showed greater substrate oxidation and phosphorylating activities, and less proton leak than those of the laying-type, resulting in a higher efficiency of oxidative phosphorylation. Gene expression and protein content of uncoupling protein (avUCP) but not adenine nucleotide translocase (avANT) gene expression were lower in skeletal muscle mitochondria of meat-type chickens than the laying-type. The current results regarding a higher efficiency of oxidative phosphorylation and UCP content may partially support the high feed efficiency of meat-type chickens. Copyright © 2011 Elsevier Inc. All rights reserved.

Structural and mechanistic basis of proton-coupled metal ion transport in the SLC11/NRAMP family

PubMed Central

Ehrnstorfer, Ines A.; Manatschal, Cristina; Arnold, Fabian M.; Laederach, Juerg; Dutzler, Raimund

2017-01-01

Secondary active transporters of the SLC11/NRAMP family catalyse the uptake of iron and manganese into cells. These proteins are highly conserved across all kingdoms of life and thus likely share a common transport mechanism. Here we describe the structural and functional properties of the prokaryotic SLC11 transporter EcoDMT. Its crystal structure reveals a previously unknown outward-facing state of the protein family. In proteoliposomes EcoDMT mediates proton-coupled uptake of manganese at low micromolar concentrations. Mutants of residues in the transition-metal ion-binding site severely affect transport, whereas a mutation of a conserved histidine located near this site results in metal ion transport that appears uncoupled to proton transport. Combined with previous results, our study defines the conformational changes underlying transition-metal ion transport in the SLC11 family and it provides molecular insight to its coupling to protons. PMID:28059071

Inhibition of nuclear factor-κB signal by pyrrolidine dithiocarbamate alleviates lipopolysaccharide-induced acute lung injury

PubMed Central

Yang, Hongfu; Sun, Rongqing; Ma, Ning; Liu, Qilong; Sun, Xiaoge; Zi, Panpan; Wang, Junsheng; Chao, Ke; Yu, Lei

2017-01-01

This study mainly studied the effect of inhibition of nuclear factor-κB (NF-κB) signal by pyrrolidine dithiocarbamate (PDTC) on lipopolysaccharide (LPS)-induced inflammatory response, oxidative stress, and mitochondrial dysfunction in a murine acute lung injury model. The results showed that LPS exposure activated NF-κB and its upstream proteins and caused lung inflammation, oxidative stress, and mitochondrial dysfunction in mice. While inhibition of NF-κB by PDTC adminstration alleviated LPS-induced generation of lymphocytes, IL-1β, and TNF-α. Malondialdehyde, a common oxidative product, was markedly reduced after PDTC treatment in LPS-challenged mice. Furthermore, PDTC alleviated LPS-induced mitochondrial dysfunction via improving ATP synthesis and uncoupling protein 2 expression. In conclusion, inhibition of NF-κB by PDTC alleviated LPS-induced acute lung injury via maintaining inflammatory status, oxidative balance, and mitochondrial function in mice. PMID:28521300

Role of NF-κB in oxidative stress-induced defective dopamine D1 receptor signaling in the renal proximal tubules of Sprague Dawley rats

PubMed Central

Fardoun, Riham Zein; Asghar, Mohammad; Lokhandwala, Mustafa

2009-01-01

Dopamine promotes sodium excretion, in part, via activation of D1 receptors in renal proximal tubules (PT) and subsequent inhibition of Na, K-ATPase. Recently, we have reported that oxidative stress causes D1 receptors-G-protein uncoupling via mechanisms involving Protein Kinase C (PKC) and G-protein Coupled Receptor Kinase 2 (GRK2) in the primary culture of renal PT of Sprague Dawley (SD) rats. There are reports suggesting that redox-sensitive nuclear transcription factor, NF-κB, is activated in conditions associated with oxidative stress. This study was designed to identify the role of NF-κB in oxidative stress–induced defective renal D1 receptor –G-protein coupling and function. Treatment of the PT with hydrogen peroxide (H2O2, 50 μM/20 min) induced the nuclear translocation of NF-κB, increased PKC activity, and triggered the translocation of GRK2 to the proximal tubular membranes. This was accompanied by hyperphosphorylation of D1 receptors and defective D1 receptor-G-protein coupling. The functional consequence of these changes was decreased D1 receptor activation-mediated inhibition of Na, K-ATPase activity. Interestingly, pre-treatment with pyrrolidine dithiocarbamate (PDTC, 25 μM/10min), an NF-κB inhibitor, blocked the H2O2-induced nuclear translocation of NF-κB, increase in PKC activity, as well as GRK2 translocation and hyperphosphorylation of D1 receptors in the proximal tubular membranes. Furthermore, PDTC restored D1 receptor G-protein coupling and D1 receptor agonist-mediated inhibition of the Na, KATPase activity. Therefore, we suggest that oxidative stress causes nuclear translocation of NF-κB in the renal proximal tubules, which contributes to defective D1-receptor-G-protein coupling and function via mechanism involving PKC, membranous translocation of GRK 2, and subsequent phosphorylation of dopamine D1 receptors. PMID:17320758

Loss of Intralipid®- but Not Sevoflurane-Mediated Cardioprotection in Early Type-2 Diabetic Hearts of Fructose-Fed Rats: Importance of ROS Signaling

PubMed Central

Zhang, Liyan; Affolter, Andreas; Gandhi, Manoj; Hersberger, Martin; Warren, Blair E.; Lemieux, Hélène; Sobhi, Hany F.; Clanachan, Alexander S.; Zaugg, Michael

2014-01-01

Background Insulin resistance and early type-2 diabetes are highly prevalent. However, it is unknown whether Intralipid® and sevoflurane protect the early diabetic heart against ischemia-reperfusion injury. Methods Early type-2 diabetic hearts from Sprague-Dawley rats fed for 6 weeks with fructose were exposed to 15 min of ischemia and 30 min of reperfusion. Intralipid® (1%) was administered at the onset of reperfusion. Peri-ischemic sevoflurane (2 vol.-%) served as alternative protection strategy. Recovery of left ventricular function was recorded and the activation of Akt and ERK 1/2 was monitored. Mitochondrial function was assessed by high-resolution respirometry and mitochondrial ROS production was measured by Amplex Red and aconitase activity assays. Acylcarnitine tissue content was measured and concentration-response curves of complex IV inhibition by palmitoylcarnitine were obtained. Results Intralipid® did not exert protection in early diabetic hearts, while sevoflurane improved functional recovery. Sevoflurane protection was abolished by concomitant administration of the ROS scavenger N-2-mercaptopropionyl glycine. Sevoflurane, but not Intralipid® produced protective ROS during reperfusion, which activated Akt. Intralipid® failed to inhibit respiratory complex IV, while sevoflurane inhibited complex I. Early diabetic hearts exhibited reduced carnitine-palmitoyl-transferase-1 activity, but palmitoylcarnitine could not rescue protection and enhance postischemic functional recovery. Cardiac mitochondria from early diabetic rats exhibited an increased content of subunit IV-2 of respiratory complex IV and of uncoupling protein-3. Conclusions Early type-2 diabetic hearts lose complex IV-mediated protection by Intralipid® potentially due to a switch in complex IV subunit expression and increased mitochondrial uncoupling, but are amenable to complex I-mediated sevoflurane protection. PMID:25127027

Klebsiella aerogenes UreF: Identification of the UreG Binding Site and Role in Enhancing the Fidelity of Urease Activation†

PubMed Central

Boer, Jodi L.; Hausinger, Robert P.

2012-01-01

The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a complex of UreD—UreF—UreG bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. The present study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein:protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in co-purification of UreD and urease apoprotein; whereas UreG bound to only a subset of the species. Notably, reduced interaction with UreG correlated with the low activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD—UreF—UreG—urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the non-cognate metal Zn. PMID:22369361

[Obesity studies in candidate genes].

PubMed

Ochoa, María del Carmen; Martí, Amelia; Martínez, J Alfredo

2004-04-17

There are more than 430 chromosomic regions with gene variants involved in body weight regulation and obesity development. Polymorphisms in genes related to energy expenditure--uncoupling proteins (UCPs), related to adipogenesis and insulin resistance--hormone-sensitive lipase (HLS), peroxisome proliferator-activated receptor gamma (PPAR gamma), beta adrenergic receptors (ADRB2,3), and alfa tumor necrosis factor (TNF-alpha), and related to food intake--ghrelin (GHRL)--appear to be associated with obesity phenotypes. Obesity risk depends on two factors: a) genetic variants in candidate genes, and b) biographical exposure to environmental risk factors. It is necessary to perform new studies, with appropriate control groups and designs, in order to reach relevant conclusions with regard to gene/environmental (diet, lifestyle) interactions.

Leucine Deprivation Stimulates Fat Loss via Increasing CRH Expression in the Hypothalamus and Activating The Sympathetic Nervous System

PubMed Central

Cheng, Ying; Zhang, Qian; Meng, Qingshu; Xia, Tingting; Huang, Zhiying; Wang, Chunxia; Liu, Bin; Chen, Shanghai; Xiao, Fei; Du, Ying

2011-01-01

We previously showed that leucine deprivation decreases abdominal fat mass largely by increasing energy expenditure, as demonstrated by increased lipolysis in white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT). The goal of the present study was to investigate the possible involvement of central nervous system (CNS) in this regulation and elucidate underlying molecular mechanisms. For this purpose, levels of genes and proteins related to lipolysis in WAT and UCP1 expression in BAT were analyzed in wild-type mice after intracerebroventricular administration of leucine or corticotrophin-releasing hormone antibodies, or in mice deleted for three β-adrenergic receptors, after being maintained on a leucine-deficient diet for 7 d. Here, we show that intracerebroventricular administration of leucine significantly attenuates abdominal fat loss and blocks activation of hormone sensitive lipase in WAT and induction of UCP1 in BAT in leucine-deprived mice. Furthermore, we provide evidence that leucine deprivation stimulates fat loss by increasing expression of corticotrophin-releasing hormone in the hypothalamus via activation of stimulatory G protein/cAMP/protein kinase A/cAMP response element-binding protein pathway. Finally, we show that the effect of leucine deprivation on fat loss is mediated by activation of the sympathetic nervous system. These results suggest that CNS plays an important role in regulating fat loss under leucine deprivation and thereby provide novel and important insights concerning the importance of CNS leucine in the regulation of energy homeostasis. PMID:21719534

Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.

PubMed

Karavaeva, Iuliia E; Golyshev, Sergey A; Smirnova, Ekaterina A; Sokolov, Svyatoslav S; Severin, Fedor F; Knorre, Dmitry A

2017-04-01

Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast Saccharomyces cerevisiae cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes ATG8 , ATG32 or ATG33 , implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes. © 2017. Published by The Company of Biologists Ltd.

Dose Dependent Effects of Reactive Oxygen and Nitrogen Species on the Function of Neuronal Nitric Oxide Synthase

PubMed Central

Sun, Jian; Druhan, Lawrence J.; Zweier, Jay L.

2014-01-01

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dosedependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (≤ 50 μM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (≤ 50 μM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme. PMID:18201545

The role of mitochondria in protection of the heart by preconditioning

PubMed Central

Halestrap, Andrew P.; Clarke, Samantha J.; Khaliulin, Igor

2007-01-01

A prolonged period of ischaemia followed by reperfusion irreversibly damages the heart. Such reperfusion injury (RI) involves opening of the mitochondrial permeability transition pore (MPTP) under the conditions of calcium overload and oxidative stress that accompany reperfusion. Protection from MPTP opening and hence RI can be mediated by ischaemic preconditioning (IP) where the prolonged ischaemic period is preceded by one or more brief (2–5 min) cycles of ischaemia and reperfusion. Following a brief overview of the molecular characterisation and regulation of the MPTP, the proposed mechanisms by which IP reduces pore opening are reviewed including the potential roles for reactive oxygen species (ROS), protein kinase cascades, and mitochondrial potassium channels. It is proposed that IP-mediated inhibition of MPTP opening at reperfusion does not involve direct phosphorylation of mitochondrial proteins, but rather reflects diminished oxidative stress during prolonged ischaemia and reperfusion. This causes less oxidation of critical thiol groups on the MPTP that are known to sensitise pore opening to calcium. The mechanisms by which ROS levels are decreased in the IP hearts during prolonged ischaemia and reperfusion are not known, but appear to require activation of protein kinase Cε, either by receptor-mediated events or through transient increases in ROS during the IP protocol. Other signalling pathways may show cross-talk with this primary mechanism, but we suggest that a role for mitochondrial potassium channels is unlikely. The evidence for their activity in isolated mitochondria and cardiac myocytes is reviewed and the lack of specificity of the pharmacological agents used to implicate them in IP is noted. Some K+ channel openers uncouple mitochondria and others inhibit respiratory chain complexes, and their ability to produce ROS and precondition hearts is mimicked by bona fide uncouplers and respiratory chain inhibitors. IP may also provide continuing protection during reperfusion by preventing a cascade of MPTP-induced ROS production followed by further MPTP opening. This phase of protection may involve survival kinase pathways such as Akt and glycogen synthase kinase 3 (GSK3) either increasing ROS removal or reducing mitochondrial ROS production. PMID:17631856

Fluorescent Recovery after Photobleaching (FRAP) Analysis of Nuclear Export Rates Identifies Intrinsic Features of Nucleocytoplasmic Transport*

PubMed Central

Cardarelli, Francesco; Tosti, Luca; Serresi, Michela; Beltram, Fabio; Bizzarri, Ranieri

2012-01-01

A quantitative description of carrier-mediated nuclear export in live cells is presented. To this end, we fused a prototypical leucine-rich nuclear export signal (NES) to GFP as a cargo model and expressed the fluorescent chimera in live CHO-K1 cells. By modeling FRAP data, we calculate the NES affinity for the export machinery and the maximum rate of nuclear export achievable at saturation of endogenous carriers. The measured active-export time through the Nuclear Pore Complex (NPC) is 18 ms, remarkably similar to the previously determined active-import rate. Also, our results reveal that active export/import and active export/passive diffusion fluxes are uncoupled, thus complementing previous reports on active import/passive diffusion uncoupling. These findings suggest differential gating at the NPC level. PMID:22190681

Transport Pathways—Proton Motive Force Interrelationship in Durum Wheat Mitochondria

PubMed Central

Trono, Daniela; Laus, Maura N.; Soccio, Mario; Pastore, Donato

2014-01-01

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoKATP) and the plant uncoupling protein (PUCP) are able to strongly reduce the proton motive force (pmf) to control mitochondrial production of reactive oxygen species; under these conditions, mitochondrial carriers lack the driving force for transport and should be inactive. However, unexpectedly, DWM uncoupling by PmitoKATP neither impairs the exchange of ADP for ATP nor blocks the inward transport of Pi and succinate. This uptake may occur via the plant inner membrane anion channel (PIMAC), which is physiologically inhibited by membrane potential, but unlocks its activity in de-energized mitochondria. Probably, cooperation between PIMAC and carriers may accomplish metabolite movement across the inner membrane under both energized and de-energized conditions. PIMAC may also cooperate with PmitoKATP to transport ammonium salts in DWM. Interestingly, this finding may trouble classical interpretation of in vitro mitochondrial swelling; instead of free passage of ammonia through the inner membrane and proton symport with Pi, that trigger metabolite movements via carriers, transport of ammonium via PmitoKATP and that of the counteranion via PIMAC may occur. Here, we review properties, modulation and function of the above reported DWM channels and carriers to shed new light on the control that they exert on pmf and vice-versa. PMID:24821541

PPARdelta activator GW-501516 has no acute effect on glucose transport in skeletal muscle.

PubMed

Terada, Shin; Wicke, Scott; Holloszy, John O; Han, Dong-Ho

2006-04-01

It has been reported that treatment of cultured human skeletal muscle myotubes with the peroxisome proliferator-activated receptor-delta (PPARdelta) activator GW-501516 directly stimulates glucose transport and enhances insulin action. Cultured myotubes are minimally responsive to insulin stimulation of glucose transport and are not a good model for studying skeletal muscle glucose transport. The purpose of this study was to evaluate the effect of GW-501516 on glucose transport to determine whether the findings on cultured myotubes have relevance to skeletal muscle. Rat epitrochlearis and soleus muscles were treated for 6 h with 10, 100, or 500 nM GW-501516, followed by measurement of 2-deoxyglucose uptake. GW-501516 had no effect on glucose uptake. There was no effect on insulin sensitivity or responsiveness. Also, in contrast to findings on myotubes, treatment of muscles with GW-501516 did not result in increased phosphorylation or increased expression of AMP-activated protein kinase (AMPK) or p38 mitogen-activated protein kinase (MAPK). Treatment of epitrochlearis muscles with GW-501516 for 24 h induced a threefold increase in uncoupling protein-3 mRNA, providing evidence that the GW-501516 compound that we used gets into and is active in skeletal muscle. In conclusion, our results show that, in contrast to myotubes in culture, skeletal muscle does not respond to GW-501516 with 1) an increase in AMPK or p38 MAPK phosphorylation or expression or 2) direct stimulation of glucose transport or enhanced insulin action.

Variola virus E3L Zα domain, but not its Z-DNA binding activity, is required for PKR inhibition.

PubMed

Thakur, Meghna; Seo, Eun Joo; Dever, Thomas E

2014-02-01

Responding to viral infection, the interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase PKR phosphorylates translation initiation factor eIF2α to inhibit cellular and viral protein synthesis. To overcome this host defense mechanism, many poxviruses express the protein E3L, containing an N-terminal Z-DNA binding (Zα) domain and a C-terminal dsRNA-binding domain (dsRBD). While E3L is thought to inhibit PKR activation by sequestering dsRNA activators and by directly binding the kinase, the role of the Zα domain in PKR inhibition remains unclear. Here, we show that the E3L Zα domain is required to suppress the growth-inhibitory properties associated with expression of human PKR in yeast, to inhibit PKR kinase activity in vitro, and to reverse the inhibitory effects of PKR on reporter gene expression in mammalian cells treated with dsRNA. Whereas previous studies revealed that the Z-DNA binding activity of E3L is critical for viral pathogenesis, we identified point mutations in E3L that functionally uncouple Z-DNA binding and PKR inhibition. Thus, our studies reveal a molecular distinction between the nucleic acid binding and PKR inhibitory functions of the E3L Zα domain, and they support the notion that E3L contributes to viral pathogenesis by targeting PKR and other components of the cellular anti-viral defense pathway.

The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia

NASA Astrophysics Data System (ADS)

Nikawa, Takeshi; Choi, Inho; Haruna, Marie; Hirasaka, Katsuya; Maita Ohno, Ayako; Kondo Teshima, Shigetada

Overloaded mitochondrial calcium concentration contributes to progression of mitochondrial dysfunction in aged muscle, leading to sarcopenia. Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. Recently, it has been reported that UCP3 is associated with calcium uptake into mitochondria. However, the mechanisms by which UCP3 regulates mitochondrial calcium uptake are not well understood. Here we report that UCP3 interacts with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that is localized in mitochondria, which is involved in cellular responses to calcium ion. The hydrophilic sequences within the loop 2, matrix-localized hydrophilic domain of mouse UCP3 are necessary for binding to Hax-1 of the C-terminal domain in adjacent to mitochondrial innermembrane. Interestingly, these proteins interaction occur the calcium-dependent manner. Indeed, overexpression of UCP3 significantly enhanced calcium uptake into mitochondria on Hax-1 endogenously expressing C2C12 myoblasts. In addition, Hax-1 knock-down enhanced calcium uptake into mitochondria on both UCP3 and Hax-1 endogenously expressing C2C12 myotubes, but not myoblasts. Finally, the dissociation of UCP3 and Hax-1 enhances calcium uptake into mitochondria in aged muscle. These studies identify a novel UCP3-Hax-1 complex regulates the influx of calcium ion into mitochondria in muscle. Thus, the efficacy of UCP3-Hax-1 in mitochondrial calcium regulation may provide a novel therapeutic approach against mitochondrial dysfunction-related disease containing sarcopenia.

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline.

PubMed

Yang, Long; Bashaw, Greg J

2006-11-22

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Here, using genetics, biochemistry, and cell biology, we demonstrate that Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion.

Lassomycin, a ribosomally synthesized cyclic peptide, kills mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2.

PubMed

Gavrish, Ekaterina; Sit, Clarissa S; Cao, Shugeng; Kandror, Olga; Spoering, Amy; Peoples, Aaron; Ling, Losee; Fetterman, Ashley; Hughes, Dallas; Bissell, Anthony; Torrey, Heather; Akopian, Tatos; Mueller, Andreas; Epstein, Slava; Goldberg, Alfred; Clardy, Jon; Lewis, Kim

2014-04-24

Languishing antibiotic discovery and flourishing antibiotic resistance have prompted the development of alternative untapped sources for antibiotic discovery, including previously uncultured bacteria. Here, we screen extracts from uncultured species against Mycobacterium tuberculosis and identify lassomycin, an antibiotic that exhibits potent bactericidal activity against both growing and dormant mycobacteria, including drug-resistant forms of M. tuberculosis, but little activity against other bacteria or mammalian cells. Lassomycin is a highly basic, ribosomally encoded cyclic peptide with an unusual structural fold that only partially resembles that of other lasso peptides. We show that lassomycin binds to a highly acidic region of the ClpC1 ATPase complex and markedly stimulates its ATPase activity without stimulating ClpP1P2-catalyzed protein breakdown, which is essential for viability of mycobacteria. This mechanism, uncoupling ATPase from proteolytic activity, accounts for the bactericidal activity of lassomycin. Copyright © 2014 Elsevier Ltd. All rights reserved.

Quantitative reduction of the TCR adapter protein SLP-76 unbalances immunity and immune regulation.

PubMed

Siggs, Owen M; Miosge, Lisa A; Daley, Stephen R; Asquith, Kelly; Foster, Paul S; Liston, Adrian; Goodnow, Christopher C

2015-03-15

Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies. Copyright © 2015 by The American Association of Immunologists, Inc.

Uncoupling protein-2 deficient mice are not protected against warm ischemia/reperfusion injury of the liver.

PubMed

Le Minh, Khoi; Berger, Andreas; Eipel, Christian; Kuhla, Angela; Minor, Thomas; Stegemann, Judith; Vollmar, Brigitte

2011-12-01

Uncoupling protein-2 (UCP2) might play an important role in mediating ischemia/reperfusion (I/R) injury due to its function in uncoupling of oxidative phosphorylation and in the proton leak-associated increase of reactive oxygen species (ROS) production. The aim of this study was to elucidate the role of UCP2 in hepatic I/R injury. UCP2 wild type and UCP2 deficient mice were subjected to I/R of the left liver lobe. Sham-operated animals without I/R served as controls. Intravital fluorescence microscopy was used for assessing postischemic microcirculatory dysfunction. Indicators of hepatic inflammatory response, oxidative stress, and bioenergetic status as well as histomorphology were investigated. Under sham conditions UCP2-/-mice presented slightly but not significantly higher levels of hepatic ATP and energy charge than wild type mice. In addition, they exhibited higher systemic IL-6 levels and intrahepatic leukocyte adherence. After exposure to I/R, the extent of reperfusion injury did not differ between UCP2+/+ and UCP2-/-mice, as indicated by a comparable loss of sinusoidal perfusion, hepatic ATP, and energy charge levels, as well as rise of transaminases and disintegration of liver structures. Intrahepatic leukocyte adherence and plasma IL-6 levels of postischemic UCP2-/-mice still exceeded those of UCP2+/+mice. UCP2 appears to be of minor relevance for the manifestation and extent of postischemic reperfusion injury in nondiseased livers with the increased ATP availability being counteracted by the higher pro-inflammatory IL-6 levels in UCP2 deficient mice. Copyright © 2011 Elsevier Inc. All rights reserved.

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

PubMed

Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

2018-08-01

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

Radix Stellariae extract prevents high-fat-diet-induced obesity in C57BL/6 mice by accelerating energy metabolism

PubMed Central

Li, Yin; Liu, Xin; Fan, Yu

2017-01-01

Stellaria dichotoma L. is widely distributed in Ningxia and surrounding areas in northwestern China. Its root, Radix Stellariae (RS), has been used in herbal formulae for treating asthenic-fever, infection, malaria, dyspepsia in children and several other symptoms. This study investigated whether the RS extract (RSE) alleviates metabolic disorders. The results indicated that RSE significantly inhibited body weight gain in high-fat (HF)-diet-fed C57BL/6 mice, reduced fasting glucose levels, and improved insulin tolerance. Moreover, RSE increased the body temperature of the mice and the expression of uncoupling proteins and peroxisome proliferator-activated receptors in the white adipose tissue. Thus, RSE alleviated metabolic disorders in HF-diet-fed C57BL/6 mice by potentially activating UCP and PPAR signaling. PMID:28507819

Analysis of differential secondary effects of novel rexinoids: select rexinoid X receptor ligands demonstrate differentiated side effect profiles

PubMed Central

Marshall, Pamela A; Jurutka, Peter W; Wagner, Carl E; van der Vaart, Arjan; Kaneko, Ichiro; Chavez, Pedro I; Ma, Ning; Bhogal, Jaskaran S; Shahani, Pritika; Swierski, Johnathon C; MacNeill, Mairi

2015-01-01

In order to determine the feasibility of utilizing novel rexinoids for chemotherapeutics and as potential treatments for neurological conditions, we undertook an assessment of the side effect profile of select rexinoid X receptor (RXR) analogs that we reported previously. We assessed pharmacokinetic profiles, lipid and thyroid-stimulating hormone (TSH) levels in rats, and cell culture activity of rexinoids in sterol regulatory element-binding protein (SREBP) induction and thyroid hormone inhibition assays. We also performed RNA sequencing of the brain tissues of rats that had been dosed with the compounds. We show here for the first time that potent rexinoid activity can be uncoupled from drastic lipid changes and thyroid axis variations, and we propose that rexinoids can be developed with improved side effect profiles than the parent compound, bexarotene (1). PMID:26038698

Disrupting nNOS-PSD-95 coupling in the hippocampal dentate gyrus promotes extinction memory retrieval.

PubMed

Li, Jun; Han, Zhou; Cao, Bo; Cai, Cheng-Yun; Lin, Yu-Hui; Li, Fei; Wu, Hai-Ying; Chang, Lei; Luo, Chun-Xia; Zhu, Dong-Ya

2017-11-04

Granule cells in the dentate gyrus regenerate constantly in adult hippocampus and then integrate into neural circuits in the hippocampus thereby providing the neural basis for learning and memory. Promoting the neurogenesis in the hippocampus facilitates learning and memory such as spatial learning, object identification, and extinction learning. The interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) is reported to negatively regulate neurogenesis in brain, so we hypothesized that disrupting this interaction might facilitate the neurogenesis in the dentate gyrus (DG) and thus enhance the extinction memory retrieval of fear learning. We found that uncoupling the nNOS-PSD-95 complex in remote contextual fear condition promoted both neuronal proliferation and survival in the DG, contributing to an enhanced retrieval of the extinction memory. Moreover, the nNOS-PSD-95 uncoupling-induced neurogenesis may be mediated by the extracellular signal-regulated kinase (ERK) as the phosphorylation level of ERK1/2 was increased after uncoupling. These findings suggest that the nNOS-PSD-95 complex may serve as a novel target for the treatment of post-traumatic stress disorder (PTSD). Copyright © 2017 Elsevier Inc. All rights reserved.

Mechanisms of Mitochondrial Defects in Gulf War Syndrome

DTIC Science & Technology

2014-10-01

parameters: uncoupling ratio, net routine flux control ratio, respiratory control ratio, leak flux control ratio, phosphorylation respiratory... oxidative phosphorylation subunit) Quantitative analysis of individual mitochondrial proteins. The technique has been established and validated for muscle...Blue Native and Clear Native Analyses (non-denatured analysis of supercomplex formation and monomeric oxidative phosphorylation enzyme assembly

MELATONIN-INDUCED SUPPRESSION OF PC12 CELL GROWTH IS MEDIATED BY ITS GI COUPLED TRANSMEMBRANE RECEPTORS. (R826248)

EPA Science Inventory

The effects of pertussis toxin, an uncoupler of Gi protein from adenylate cyclase, and luzindole, a competitive inhibitor of melatonin receptor binding, were examined for their ability to inhibit melatonin-induced suppression of PC12 cell growth. Both agents inhibited the mela...

Hypoxia-induced decrease of UCP3 gene expression in rat heart parallels metabolic gene switching but fails to affect mitochondrial respiratory coupling.

PubMed

Essop, M Faadiel; Razeghi, Peter; McLeod, Chris; Young, Martin E; Taegtmeyer, Heinrich; Sack, Michael N

2004-02-06

Mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3) are postulated to contribute to antioxidant defense, nutrient partitioning, and energy efficiency in the heart. To distinguish isotype function in response to metabolic stress we measured cardiac mitochondrial function and cardiac UCP gene expression following chronic hypobaric hypoxia. Isolated mitochondrial O(2) consumption and ATP synthesis rate were reduced but respiratory coupling was unchanged compared to normoxic groups. Concurrently, left ventricular UCP3 mRNA levels were significantly decreased with hypoxia (p<0.05) while UCP2 levels remained unchanged versus controls. Diminished UCP3 expression was associated with coordinate regulation of counter-regulatory metabolic genes. From these data, we propose a role for UCP3 in the regulation of fatty acid oxidation in the heart as opposed to uncoupling of mitochondria. Moreover, the divergent hypoxia-induced regulation of UCP2 and UCP3 supports distinct mitochondrial regulatory functions of these inner mitochondrial membrane proteins in the heart in response to metabolic stress.

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

PubMed Central

Ross, S R; Choy, L; Graves, R A; Fox, N; Solevjeva, V; Klaus, S; Ricquier, D; Spiegelman, B M

1992-01-01

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line. Images PMID:1323843

Mitophagy controls beige adipocyte maintenance through a Parkin-dependent and UCP1-independent mechanism.

PubMed

Lu, Xiaodan; Altshuler-Keylin, Svetlana; Wang, Qiang; Chen, Yong; Henrique Sponton, Carlos; Ikeda, Kenji; Maretich, Pema; Yoneshiro, Takeshi; Kajimura, Shingo

2018-04-24

Beige adipocytes are an inducible form of mitochondria-enriched thermogenic adipocytes that emerge in response to external stimuli, such as chronic cold exposure. We have previously shown that after the withdrawal of external stimuli, beige adipocytes directly acquire a white fat-like phenotype through autophagy-mediated mitochondrial degradation. We investigated the upstream pathway that mediates mitochondrial clearance and report that Parkin-mediated mitophagy plays a key role in the beige-to-white adipocyte transition. Mice genetically deficient in Park2 showed reduced mitochondrial degradation and retained thermogenic beige adipocytes even after the withdrawal of external stimuli. Norepinephrine signaling through the PKA pathway inhibited the recruitment of Parkin protein to mitochondria in beige adipocytes. However, mitochondrial proton uncoupling by uncoupling protein 1 (UCP1) was dispensable for Parkin recruitment and beige adipocyte maintenance. These results suggest a physiological mechanism by which external cues control mitochondrial homeostasis in thermogenic fat cells through mitophagy. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Arginase-I enhances vascular endothelial inflammation and senescence through eNOS-uncoupling.

PubMed

Zhu, Cuicui; Yu, Yi; Montani, Jean-Pierre; Ming, Xiu-Fen; Yang, Zhihong

2017-02-02

Augmented arginase-II (Arg-II) is implicated in endothelial senescence and inflammation through a mutual positive regulatory circuit with S6K1. This study was conducted to investigate whether Arg-I, another isoform of arginase that has been also reported to play a role in vascular endothelial dysfunction, promotes endothelial senescence through similar mechanisms. The non-senescent human endothelial cells from umbilical veins (passage 2 to 4) were transduced with empty recombinant adenovirus vector (rAd/CMV) as control or rAd/CMV-Arg-I to overexpress Arg-I. Overexpressing Arg-I promoted eNOS-uncoupling, enhanced senescence markers including p53-S15, p21 and senescence-associated β-galactosidase (SA-β-gal) staining, and increased inflammatory vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) as well as monocyte adhesion to endothelial cells without activating S6K1. All the effects of Arg-I were inhibited by the anti-oxidant N-acetylcysteine (NAC). Our study demonstrates that Arg-I promotes endothelial senescence and inflammatory responses through eNOS-uncoupling unrelated to activation of the S6K1 pathway.

Both retinoic-acid-receptor- and retinoid-X-receptor-dependent signalling pathways mediate the induction of the brown-adipose-tissue-uncoupling-protein-1 gene by retinoids.

PubMed Central

Alvarez, R; Checa, M; Brun, S; Viñas, O; Mampel, T; Iglesias, R; Giralt, M; Villarroya, F

2000-01-01

The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte. PMID:10600643

Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes.

PubMed

Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K; McGraw, Timothy E

2016-12-20

Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin's effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Sildenafil Citrate-Restored eNOS and PDE5 Regulation in Sickle Cell Mouse Penis Prevents Priapism Via Control of Oxidative/Nitrosative Stress

PubMed Central

Hsu, Lewis L.; Berkowitz, Dan E.; Champion, Hunter C.; Burnett, Arthur L.

2013-01-01

Sildenafil citrate revolutionized the practice of sexual medicine upon its federal regulatory agency approval approximately 15 years ago as the prototypical phosphodiesterase type 5 inhibitor indicated for the treatment of male erectile dysfunction. We now provide scientific support for its alternative use in the management of priapism, a clinical disorder of prolonged and uncontrolled penile erection. Sildenafil administered continuously to sickle cell mice, which show a priapism phenotype, reverses oxidative/nitrosative stress effects in the penis, mainly via reversion of uncoupled endothelial nitric oxide synthase to the functional coupled state of the enzyme, which in turn corrects aberrant signaling and function of the nitric oxide/cyclic GMP/protein kinase G/phosphodiesterase type 5 cascade. Priapism tendencies in these mice are reverted partially toward normal neurostimulated erection frequencies and durations after sildenafil treatment in association with normalized cyclic GMP concentration, protein kinase G activity and phosphodiesterase type 5 activity in the penis. Thus, sildenafil exerts pleiotropic effects in the penis that extend to diverse erection disorders. PMID:23844149

Dendritic cells control fibroblastic reticular network tension and lymph node expansion.

PubMed

Acton, Sophie E; Farrugia, Aaron J; Astarita, Jillian L; Mourão-Sá, Diego; Jenkins, Robert P; Nye, Emma; Hooper, Steven; van Blijswijk, Janneke; Rogers, Neil C; Snelgrove, Kathryn J; Rosewell, Ian; Moita, Luis F; Stamp, Gordon; Turley, Shannon J; Sahai, Erik; Reis e Sousa, Caetano

2014-10-23

After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion--driven by lymphocyte influx and proliferation--that is the critical hallmark of adaptive immunity.

Sildenafil citrate-restored eNOS and PDE5 regulation in sickle cell mouse penis prevents priapism via control of oxidative/nitrosative stress.

PubMed

Bivalacqua, Trinity J; Musicki, Biljana; Hsu, Lewis L; Berkowitz, Dan E; Champion, Hunter C; Burnett, Arthur L

2013-01-01

Sildenafil citrate revolutionized the practice of sexual medicine upon its federal regulatory agency approval approximately 15 years ago as the prototypical phosphodiesterase type 5 inhibitor indicated for the treatment of male erectile dysfunction. We now provide scientific support for its alternative use in the management of priapism, a clinical disorder of prolonged and uncontrolled penile erection. Sildenafil administered continuously to sickle cell mice, which show a priapism phenotype, reverses oxidative/nitrosative stress effects in the penis, mainly via reversion of uncoupled endothelial nitric oxide synthase to the functional coupled state of the enzyme, which in turn corrects aberrant signaling and function of the nitric oxide/cyclic GMP/protein kinase G/phosphodiesterase type 5 cascade. Priapism tendencies in these mice are reverted partially toward normal neurostimulated erection frequencies and durations after sildenafil treatment in association with normalized cyclic GMP concentration, protein kinase G activity and phosphodiesterase type 5 activity in the penis. Thus, sildenafil exerts pleiotropic effects in the penis that extend to diverse erection disorders.

Reduced heart rate variability: an indicator of cardiac uncoupling and diminished physiologic reserve in 1,425 trauma patients.

PubMed

Morris, John A; Norris, Patrick R; Ozdas, Asli; Waitman, Lemuel R; Harrell, Frank E; Williams, Anna E; Cao, Hanqing; Jenkins, Judith M

2006-06-01

Measurements of a patient's physiologic reserve (age, injury severity, admission lactic acidosis, transfusion requirements, and coagulopathy) reflect robustness of response to surgical insult. We have previously shown that cardiac uncoupling (reduced heart rate variability, HRV) in the first 24 hours after injury correlates with mortality and autonomic nervous system failure. We hypothesized: Deteriorating physiologic reserve correlates with reduced HRV and cardiac uncoupling. There were 1,425 trauma ICU patients that satisfied the inclusion criteria. Differences in mortality across categorical measurements of the domains of physiologic reserve were assessed using the chi test. The relationship of cardiac uncoupling and physiologic reserve was examined using multivariate logistic regression models for various levels of cardiac uncoupling (>0 through 28% reduced HRV in the first 24 hours). Of these, 797 (55.9%) patients exhibited cardiac uncoupling. Deteriorating measures of physiologic reserve reflected increased risk of death. Measures of acidosis (admission lactate, time to lactate normalization, and lactate deterioration over the first 24 hours), coagulopathy, age, and injury severity contributed significantly to the risk of cardiac uncoupling (area under receiver operator curve, ROC=0.73). The association between deteriorating reserve and cardiac uncoupling increases with the threshold for uncoupling (ROC=0.78). Reduced heart rate variability is a new biomarker reflecting the loss of command and control of the heart (cardiac uncoupling). Risk of cardiac uncoupling increases significantly as a patient's physiologic reserve deteriorates and physiologic exhaustion approaches. Cardiac uncoupling provides a noninvasive, overall measure of a patient's clinical trajectory over the first 24 hours of ICU stay.

Inhibition of Uncoupling Protein 2 Attenuates Cardiac Hypertrophy Induced by Transverse Aortic Constriction in Mice.

PubMed

Ji, Xiao-Bing; Li, Xiu-Rong; Hao-Ding; Sun, Qi; Zhou, Yang; Wen, Ping; Dai, Chun-Sun; Yang, Jun-Wei

2015-01-01

Uncoupling protein 2 (UCP2) is critical in regulating energy metabolism. Due to the significant change in energy metabolism of myocardium upon pressure overload, we hypothesize that UCP2 could contribute to the etiology of cardiac hypertrophy. Adult male C57BL/6J mice were subjected to pressure overload by using transverse aortic constriction (TAC), and then received genipin (a UCP2 selective inhibitor; 25 mg/kg/d, ip) or vehicle for three weeks prior to histologic assessment of myocardial hypertrophy. ATP concentration, ROS level, and myocardial apoptosis were also examined. A parallel set of experiments was also conducted in UCP2-/- mice. TAC induced left ventricular hypertrophy, as reflected by increased ventricular weight/thickness and increased size of myocardial cell (vs. sham controls). ATP concentration was decreased; ROS level was increased. Apoptosis and fibrosis markers were increased. TAC increased mitochondrial UCP2 expression in the myocardium at both mRNA and protein levels. Genipin treatment attenuated cardiac hypertrophy and the histologic/biochemical changes described above. Hypertrophy and associated changes induced by TAC in UCP2-/- mice were much less pronounced than in WT mice. Blocking UCP2 expression attenuates cardiac hypertrophy induced by pressure overload. © 2015 S. Karger AG, Basel.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study.

PubMed

Chen, Yanyan; Xu, Yuanyuan; Zheng, Hongzhi; Fu, Jingqi; Hou, Yongyong; Wang, Huihui; Zhang, Qiang; Yamamoto, Masayuki; Pi, Jingbo

2016-09-09

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-double knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. Copyright © 2016 Elsevier Inc. All rights reserved.

Glutaredoxin-2 is required to control proton leak through uncoupling protein-3.

PubMed

Mailloux, Ryan J; Xuan, Jian Ying; Beauchamp, Brittany; Jui, Linda; Lou, Marjorie; Harper, Mary-Ellen

2013-03-22

Glutathionylation has emerged as a key modification required for controlling protein function in response to changes in cell redox status. Recently, we showed that the glutathionylation state of uncoupling protein-3 (UCP3) modulates the leak of protons back into the mitochondrial matrix, thus controlling reactive oxygen species production. However, whether or not UCP3 glutathionylation is mediated enzymatically has remained unknown because previous work relied on the use of pharmacological agents, such as diamide, to alter the UCP3 glutathionylation state. Here, we demonstrate that glutaredoxin-2 (Grx2), a matrix oxidoreductase, is required to glutathionylate and inhibit UCP3. Analysis of bioenergetics in skeletal muscle mitochondria revealed that knock-out of Grx2 (Grx2(-/-)) increased proton leak in a UCP3-dependent manner. These effects were reversed using diamide, a glutathionylation catalyst. Importantly, the increased leak did not compromise coupled respiration. Knockdown of Grx2 augmented proton leak-dependent respiration in primary myotubes from wild type mice, an effect that was absent in UCP3(-/-) cells. These results confirm that Grx2 deactivates UCP3 by glutathionylation. To our knowledge, this is the first enzyme identified to regulate UCP3 by glutathionylation and is the first study on the role of Grx2 in the regulation of energy metabolism.

Effect of Overproduction of Mitochondrial Uncoupling Protein 2 on Cos7 Cells: Induction of Senescent-like Morphology and Oncotic Cell Death.

PubMed

Nishio, Koji; Ma, Qian

2016-01-01

The maintenance of mitochondrial membrane potential is essential for cell growth and survival. Mitochondrial uncoupling protein 2 plays the most important roles in uncoupling oxidative phosphorylation and decreasing mitochondrial O2- production by regulating the mitochondrial membrane potential. We propose that mouse UCP2 has two glycine-rich motifs, motif 1: EGIRGLWKG (170-178) and a known Walker A-like motif 2: EGPRAFYKG (264-272). These motifs seem to be important for the function of UCP2. We investigated the biological effects of overproduced-UCP2 and its physiological consequence in Cos7 cells. We introduced several amino acid changes in the motif 1. The expression vectors of the green fluorescent protein (GFP)-fused UCP2 and mutant UCP2 were constructed and expressed in Cos7 cells. The UCP2-GFP-expressed cells significantly down-regulated the mitochondrial membrane potentials and induced the enlarged cell shapes. Next we generated the stably UCP2-GFP-expressed Cos7 cells by selection with the antibiotic Genecitin (G418). Within the first few weeks following G418-selection, the stably UCP2-GFP-expressed cells could not divide well and gradually manifested the irregular and enlarged senescent-like cell morphology. The UCP2/K177E- or UCP2/G174L-expressed cells did not induce the enlarged cell shapes. Hence, UCP2/K177E and UCP2/G174L produced the functional incompetence of the glycine-rich motif 1. The senescent-like cells significantly decreased the mitochondrial membrane potentials and finally died nearly one month. Overproduction of UCP2 irreversibly reduces the mitochondrial membrane potentials and induces the senescent-like morphology and finally oncotic cell death in Cos7 cells. These changes seem to occur from the irreversible metabolic changes following total loss of cellular ATP.

Eradication of acute promyelocytic leukemia-initiating cells through PML-RARA degradation.

PubMed

Nasr, Rihab; Guillemin, Marie-Claude; Ferhi, Omar; Soilihi, Hassan; Peres, Laurent; Berthier, Caroline; Rousselot, Philippe; Robledo-Sarmiento, Macarena; Lallemand-Breitenbach, Valérie; Gourmel, Bernard; Vitoux, Dominique; Pandolfi, Pier Paolo; Rochette-Egly, Cécile; Zhu, Jun; de Thé, Hugues

2008-12-01

Retinoic acid and arsenic trioxide target the protein stability and transcriptional repression activity of the fusion oncoprotein PML-RARA, resulting in regression of acute promyelocytic leukemia (APL). Phenotypically, retinoic acid induces differentiation of APL cells. Here we show that retinoic acid also triggers growth arrest of leukemia-initiating cells (LICs) ex vivo and their clearance in PML-RARA mouse APL in vivo. Retinoic acid treatment of mouse APLs expressing the fusion protein PLZF-RARA triggers full differentiation, but not LIC loss or disease remission, establishing that differentiation and LIC loss can be uncoupled. Although retinoic acid and arsenic synergize to clear LICs through cooperative PML-RARA degradation, this combination does not enhance differentiation. A cyclic AMP (cAMP)-dependent phosphorylation site in PML-RARA is crucial for retinoic acid-induced PML-RARA degradation and LIC clearance. Moreover, activation of cAMP signaling enhances LIC loss by retinoic acid, identifying cAMP as another potential APL therapy. Thus, whereas transcriptional activation of PML-RARA is likely to control differentiation, its catabolism triggers LIC eradication and long-term remission of mouse APL. Therapy-triggered degradation of oncoproteins could be a general strategy to eradicate cancer stem cells.

Coordination changes and auto-hydroxylation of FIH-1: uncoupled O2-activation in a human hypoxia sensor

PubMed Central

Chen, Yuan-Han; Comeaux, Lindsay M.; Herbst, Robert W.; Saban, Evren; Kennedy, David C.; Maroney, Michael J.; Knapp, Michael J.

2008-01-01

Hypoxia sensing is the generic term for pO2-sensing in humans and other higher organisms. These cellular responses to pO2 are largely controlled by enzymes that belong to the Fe(II) α-ketoglutarate (αKG) dependent dioxygenase superfamily, including the human enzyme called the Factor Inhibiting HIF (FIH-1), which couples O2-activation to the hydroxylation of the Hypoxia Inducible Factor α (HIFα). Uncoupled O2-activation by human FIH-1 was studied by exposing the resting form of FIH-1, (αKG+Fe)FIH-1, to air in the absence of HIFα. Uncoupling lead to two distinct enzyme oxidations, one a purple chromophore (λmax = 583 nm) arising from enzyme auto-hydroxylation of Trp296, forming an Fe(III)–O–Trp296 chromophore (Y.-H. Chen, L. M. Comeaux, S. J. Eyles, M. J. Knapp, Chem. Commun. (2008) DOI:10.1039/B809099H); the other a yellow chromophore due to Fe(III) in the active site, which under some conditions also contained variable levels of an oxygenated surface residue, (oxo)Met275. The kinetics of purple FIH-1 formation were independent of Fe(II) and αKG concentrations, however product yield was saturable with increasing [αKG] and required excess Fe(II). Yellow FIH-1 was formed from (succinate+Fe)FIH-1, or by glycerol addition to (αKG+Fe)FIH-1, suggesting that glycerol could intercept the active oxidant from the FIH-1 active site and prevent hydroxylation. Both purple and yellow FIH-1 contained high-spin, rhombic Fe(III) centers, as shown by low temperature EPR. XAS indicated distorted octahedral Fe(III) geometries, with subtle differences in inner-shell ligands for yellow and purple FIH-1. EPR of Co(II)-substituted FIH-1, (αKG+Co)FIH-1, indicated a mixture of 5-coordinate and 6-coordinate enzyme forms, suggesting that resting FIH-1 can readily undergo uncoupled O2-activation by loss of an H2O ligand from the metal center. PMID:18805587

Pleiotropic effects of apolipoprotein C3 on HDL functionality and adipose tissue metabolic activity.

PubMed

Zvintzou, Evangelia; Lhomme, Marie; Chasapi, Stella; Filou, Serafoula; Theodoropoulos, Vassilis; Xapapadaki, Eva; Kontush, Anatol; Spyroulias, George; Tellis, Constantinos C; Tselepis, Alexandros D; Constantinou, Caterina; Kypreos, Kyriakos E

2017-09-01

APOC3 is produced mainly by the liver and intestine and approximately half of plasma APOC3 associates with HDL. Though it was believed that APOC3 associates with HDL by simple binding to preexisting particles, recent data support that biogenesis of APOC3-containing HDL (APOC3-HDL) requires Abca1. Moreover, APOC3-HDL contributes to plasma triglyceride homeostasis by preventing APOC3 association with triglyceride-rich lipoproteins. Interestingly, APOC3-HDL also shows positive correlation with the morbidly obese phenotype. However, the roles of APOC3 in HDL functionality and adipose tissue metabolic activity remain unknown. Therefore, here we investigated the direct effects of APOC3 expression on HDL structure and function, as well as white adipose tissue (WAT) and brown adipose tissue (BAT) metabolic activity. C57BL/6 mice were infected with an adenovirus expressing human APOC3 or a recombinant attenuated control adenovirus expressing green fluorescent protein and blood and tissue samples were collected at 5 days postinfection. HDL was then analyzed for its apolipoprotein and lipid composition and particle functionality. Additionally, purified mitochondria from BAT and WAT were analyzed for uncoupling protein 1, cytochrome c (Cytc), and Cytc oxidase subunit 4 protein levels as an indirect measure of their metabolic activity. Serum metabolomic analysis was performed by NMR. Combined, our data show that APOC3 modulates HDL structure and function, while it selectively promotes BAT metabolic activation. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Relationship between expression of muscle-specific uncoupling protein 2 messenger RNA and genetic selection toward growth in channel catfish.

PubMed

Kobayashi, Y; Peterson, B C; Waldbieser, G C

2015-04-01

This study tested the hypothesis that increased growth in channel catfish is associated with expression of the genes that code for uncoupling proteins (UCP) 2 and 3, members of the mitochondrial channel proteins involved in nutrient sensing and metabolism. The specific objective was to contrast the levels of UCP2 messenger RNA (mRNA) in fast vs slow growing catfish as well as in fed vs fasted catfish. Two distinct UCP2 transcripts were identified and named UCP2a and UCP2b, respectively. Nucleotide and amino acid sequence of catfish UCP2s were highly similar to UCP2 and other UCPs from other fish and mammals (>75%). Expression of UCP2a mRNA was detectable at very low levels in various metabolically active tissues, whereas the expression of UCP2b mRNA was readily detectable in the muscle and heart. In a 21-wk feeding study, fish that grew faster had a greater percent body fat at the end of the study (P < 0.01). Expression of UCP2b mRNA tended to be lower (P < 0.10) in fast growing fish in the middle of the study although levels were similar at the beginning and the end of the study. In the fed vs fasted study, expression of UCP2b mRNA in muscle was increased (P < 0.05) in fish assigned to 30 d of fasting. Our results suggest that, based on the nucleotide and amino acid sequence similarities and tissue mRNA distribution, catfish UCP2b may be the analog to UCP3. Moreover, our results suggest selection toward growth and associated fat accumulation appears to be independent of muscle UCP2b mRNA expression and UCP2b-mediated mechanisms. Copyright © 2015 Elsevier Inc. All rights reserved.

Spectroscopic and DFT Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Covalent and Non-Local Electrostatic Contributions to Reduction Potentials

PubMed Central

Hadt, Ryan G.; Sun, Ning; Marshall, Nicholas M.; Hodgson, Keith O.; Hedman, Britt; Lu, Yi; Solomon, Edward I.

2012-01-01

The reduction potentials (E0) of type 1 (T1) or blue copper (BC) sites in proteins and enzymes with identical first coordination spheres around the redox active copper ion can vary by ~400 mV. Here, we use a combination of low temperature electronic absorption and magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, and S K-edge X-ray absorption spectroscopies to investigate a series of second sphere variants—F114P, N47S, and F114N in Pseudomonas aeruginosa azurin (Az)—which modulate hydrogen bonding to and protein derived dipoles nearby the Cu-S(Cys) bond. Density functional theory (DFT) calculations correlated to the experimental data allow for the fractionation of the contributions to tuning E0 into covalent and non-local electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly non-local electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from non-local electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long range protein/active interactions, while affording further insight into the second sphere mechanisms available to the protein to tune the E0 of electron transfer sites in biology. PMID:22985400

Escherichia coli FtsH (HflB) degrades a membrane-associated TolAI-II-beta-lactamase fusion protein under highly denaturing conditions.

PubMed

Cooper, K W; Baneyx, F

2001-03-01

TolAI--II--beta-lactamase, a fusion protein consisting of the inner membrane and transperiplasmic domains of TolA followed by TEM--beta-lactamase associated with the inner membrane but remained confined to the cytoplasm when expressed at high level in Escherichia coli. Although the fusion protein was resistant to proteolysis in vivo, it was hydrolyzed during preparative SDS-polyacrylamide electrophoresis and when insoluble cellular fractions unfolded with 5 M urea were subjected to microdialysis. Inhibitor profiling studies revealed that both a metallo- and serine protease were involved in TolAI--II--beta-lactamase degradation under denaturing conditions. The in vitro degradation rates of the fusion protein were not affected when insoluble fractions were harvested from a strain lacking protease IV, but were significantly reduced when microdialysis experiments were conducted with material isolated from an isogenic ftsH1 mutant. Adenine nucleotides were not required for degradation, and ATP supplementation did not accelerate the apparent rate of TolAI--II--beta-lactamase hydrolysis under denaturing conditions. Our results indicate that the metalloprotease active site of FtsH remains functional in the presence of 3--5 M urea and suggest that the ATPase and proteolytic activities of FtsH can be uncoupled if the substrate is sufficiently unstructured. Thus, a key role of the FtsH AAA module appears to be the net unfolding of bound substrates so that they can be efficiently engaged by the protease active site. Copyright 2001 Academic Press.

Treatment with geraniol ameliorates methionine-choline-deficient diet-induced non-alcoholic steatohepatitis in rats.

PubMed

Chen, Jun; Fan, Xiaoxia; Zhou, Lin; Gao, Xiaogang

2016-07-01

Non-alcoholic steatohepatitis (NASH) is one of the most common causes of chronic liver disease and is considered to be a causative factor of cryptogenic cirrhosis and hepatocellular carcinoma. The aim of this work was to investigate whether treatment with geraniol (a monoterpene) attenuated NASH induced by methionine-choline-deficient (MCD) diet in rats. Rats were fed with MCD diet to induce NASH and treated with geraniol (200 mg/kg/day) for 10 weeks. Treatment with geraniol reduced histological scores, fibrosis, and apoptosis in livers, lowered activities of alanine aminotransferase and aspartate aminotransferase in serum, and attenuated hepatic fat accumulation in rats fed with MCD diet. Treatment with geraniol preserved hepatic mitochondrial function, evidenced by reduced mitochondrial reactive oxygen species formation, enhanced adenosine triphosphate formation and membrane integrity, restored mitochondrial electron transport chain enzyme activity, and increased mitochondrial DNA content in rats fed with MCD diet. Treatment with geraniol reduced uncoupling protein 2 protein expression, and enhanced protein expression of prohibitin, mRNA expression of peroxisome proliferator-activated receptor α, and activity of mitochondrial carnitine palmitoyl transferase-I in livers of rats fed with MCD diet. Treatment with geraniol abated oxidative stress, evidenced by reduced malondialdehyde and 3-nitrotyrosine formation, enhanced activity of glutathione S-epoxide transferase, and down-regulated expression of inducible nitric oxide synthase and cytochrome P450 2E1 in livers of rats fed with MCD diet. Treatment with geraniol reduced myeloperoxidase activity and protein expression of tumor necrosis factor alpha and IL-6 in livers of rats fed with MCD diet. Treatment with geraniol attenuated MCD-induced NASH in rats. © 2015 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Synchronizing noisy nonidentical oscillators by transient uncoupling

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

Tandon, Aditya, E-mail: adityat@iitk.ac.in; Mannattil, Manu, E-mail: mmanu@iitk.ac.in; Schröder, Malte, E-mail: malte@nld.ds.mpg.de

2016-09-15

Synchronization is the process of achieving identical dynamics among coupled identical units. If the units are different from each other, their dynamics cannot become identical; yet, after transients, there may emerge a functional relationship between them—a phenomenon termed “generalized synchronization.” Here, we show that the concept of transient uncoupling, recently introduced for synchronizing identical units, also supports generalized synchronization among nonidentical chaotic units. Generalized synchronization can be achieved by transient uncoupling even when it is impossible by regular coupling. We furthermore demonstrate that transient uncoupling stabilizes synchronization in the presence of common noise. Transient uncoupling works best if the unitsmore » stay uncoupled whenever the driven orbit visits regions that are locally diverging in its phase space. Thus, to select a favorable uncoupling region, we propose an intuitive method that measures the local divergence at the phase points of the driven unit's trajectory by linearizing the flow and subsequently suppresses the divergence by uncoupling.« less

Stability, chromatin association and functional activity of mammalian pre-replication complex proteins during the cell cycle

PubMed Central

Okuno, Yukiko; McNairn, Adrian J.; den Elzen, Nicole; Pines, Jonathon; Gilbert, David M.

2001-01-01

We have examined the behavior of pre-replication complex (pre-RC) proteins in relation to key cell cycle transitions in Chinese Hamster Ovary (CHO) cells. ORC1, ORC4 and Cdc6 were stable (T1/2 >2 h) and associated with a chromatin-containing fraction throughout the cell cycle. Green fluorescent protein-tagged ORC1 associated with chromatin throughout mitosis in living cells and co-localized with ORC4 in metaphase spreads. Association of Mcm proteins with chromatin took place during telophase, ∼30 min after the destruction of geminin and cyclins A and B, and was coincident with the licensing of chromatin to replicate in geminin-supplemented Xenopus egg extracts. Neither Mcm recruitment nor licensing required protein synthesis throughout mitosis. Moreover, licensing could be uncoupled from origin specification in geminin-supplemented extracts; site-specific initiation within the dihydrofolate reductase locus required nuclei from cells that had passed through the origin decision point (ODP). These results demonstrate that mammalian pre-RC assembly takes place during telophase, mediated by post-translational modifications of pre-existing proteins, and is not sufficient to select specific origin sites. A subsequent, as yet undefined, step selects which pre-RCs will function as replication origins. PMID:11483529

Effect of LKB1 deficiency on mitochondrial content, fiber type, and muscle performance in the mouse diaphragm

PubMed Central

Brown, Jacob D.; Hancock, Chad R.; Mongillo, Anthony D.; Barton, J. Benjamin; DiGiovanni, Ryan A.; Parcell, Allen C.; Winder, William W.; Thomson, David M.

2010-01-01

Aim The Liver Kinase B1 (LKB1)/AMP-Activated Protein Kinase (AMPK) signaling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1-mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been fully characterized in the diaphragm. Methods Two methods of AMPK activation were used to characterize LKB1/AMPK signaling in diaphragms from muscle-specific LKB1 knockout (KO) and littermate control mice: (1) acute injection of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and (2) 5-min direct electrical stimulation of the diaphragm. Diaphragms were excised 60 minutes post-AICAR injection and immediately after electrical stimulation. Results AMPK phosphorylation increased with AICAR and electrical stimulation in control but not KO mice. Acetyl CoA carboxylase phosphorylation increased with AICAR in control but not KO mice, but increased in both genotypes with electrical stimulation. While the majority of mitochondrial protein levels were lower in KO diaphragms, uncoupling protein 3, complex I, and cytochrome oxidase IV protein levels were not different between genotypes. KO diaphragms have a lower percentage of IIx fibers and an elevated percentage of IIb fibers when compared to control diaphragms. While in vitro peak force generation was similar between genotypes, KO diaphragms fatigued more quickly and had an impaired ability to recover. Conclusion LKB1 regulates AMPK phosphorylation, mitochondrial protein expression, fiber type distribution, as well as recovery of the diaphragm from fatigue. PMID:21073663

Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

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

B McCray; E Skordalakes; J Taylor

2011-12-31

Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Throughmore » extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.« less

Nebivolol ameliorated kidney damage in Zucker diabetic fatty rats by regulation of oxidative stress/NO pathway: comparison with captopril.

PubMed

Wang, Yan; An, Wenjing; Zhang, Fei; Niu, Mengzhen; Liu, Yu; Shi, Ruizan

2018-06-23

The aim was to evaluate the effects and mechanisms of nebivolol on renal damage in Zucker diabetic fatty (ZDF) rats, in comparison with those of atenolol and captopril. Animals were divided into: control lean Zucker rats, ZDF rats, ZDF rats orally treated with nebivolol (10 mg/kg), atenolol (100 mg/kg) or captopril (40 mg/kg) for 6 months. Systolic blood pressure (SBP), blood glucose, kidney structure and function, plasma and kidney levels of nitric oxide (NO) and asymmetric dimethylarginine (ADMA), and oxidant status were evaluated. Kidney expressions of AMP-activated protein kinase (AMPK), NADPH oxidase (NOX) isoforms 2 and 4 and subunit p22 phox , nitric oxide synthase (NOS) isoforms, eNOS uncoupling, protein arginine N-methyltransferase (PRMT) 1, and dimethylarginine dimethylaminohydrolase (DDAH) 1 and 2 were tested. All drugs induced a similar control of SBP. Nebivolol did not affect the increased plasma glucose. Unlike atenolol, nebivolol prevented the decrease in plasma insulin, and, like captopril, it reduced plasma lipid contents. Nebivolol ameliorated, to a greater extent than captopril, damages to renal structure and function, which were associated with an improvement in interlobular artery dysfunction. Nebivolol elevated kidney phosphorylation of AMPK, attenuated NOX4 and p22 phox expression and oxidative stress marker levels. Nebivolol increased plasma and renal NO, enhanced expressions of eNOS, p-eNOS and nNOS, and suppressed eNOS uncoupling and iNOS expression. High ADMA in plasma and kidney were decreased by nebivolol through increasing DDAH2 and decreasing PRMT1. Long-term treatment of nebivolol ameliorated diabetic nephropathy, at least in part, via regulation of renal oxidative stress/NO pathway. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Uncoupling protein-2 mediates the protective action of berberine against oxidative stress in rat insulinoma INS-1E cells and in diabetic mouse islets

PubMed Central

Liu, Limei; Liu, Jian; Gao, Yuansheng; Yu, Xiaoxing; Xu, Gang; Huang, Yu

2014-01-01

BACKGROUND AND PURPOSE Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets. EXPERIMENTAL APPROACH Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by elisa. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy. KEY RESULTS Incubation of INS-1E cells and rat islets with HG (30 mmol·L−1; 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m+ mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine. CONCLUSIONS AND IMPLICATIONS Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine. PMID:24588674

Uncoupling protein-2 mediates the protective action of berberine against oxidative stress in rat insulinoma INS-1E cells and in diabetic mouse islets.

PubMed

Liu, Limei; Liu, Jian; Gao, Yuansheng; Yu, Xiaoxing; Xu, Gang; Huang, Yu

2014-07-01

Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets. Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by ELISA. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy. Incubation of INS-1E cells and rat islets with HG (30 mmol·L(-1); 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m(+) mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine. Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine. © 2014 The British Pharmacological Society.

A Novel Approach to the Nutrigenetics and Nutrigenomics of Obesity and Weight Management.

PubMed

Joffe, Yael T; Houghton, Christine A

2016-07-01

Nutrigenetics and nutrigenomics, as well as diet and exercise, play an important role in the development and treatment of obesity and its comorbidities. If an individual's susceptibility to becoming obese and their responsiveness to weight loss interventions are to be understood, then it needs to be addressed at a molecular and metabolic level, including genetic interaction. This review proposes a three-pillar approach to more fully comprehend the complexity of diet-gene interactions in obesity. Peroxisomal proliferating-activated receptor gamma (PPARG) and mitochondrial uncoupling protein-1 (UCP-1) are explored in detail. Illustrating how an understanding of nutritional biochemistry, nutrigenomics, and nutrigenetics may be the key to understanding differences observed in the obese phenotype that vary both within and across populations.

Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment*

PubMed Central

Labriola, Jonathan M.; Pandhare, Akash; Jansen, Michaela; Blanton, Michael P.; Corringer, Pierre-Jean; Baenziger, John E.

2013-01-01

Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state. PMID:23463505

An evaluation of upper-body muscle activation during coupled and uncoupled instability resistance training.

PubMed

Campbell, Brian M; Kutz, Matt R; Morgan, Amy L; Fullenkamp, Adam M; Ballenger, Ryan

2014-07-01

Recently, there has been a growth in the popularity of resistance exercises performed on unstable surfaces. However, the relationship between unstable surface training and load coupling on muscle activation is unclear. The purpose of this study was to evaluate changes in muscle activation during a barbell (BB) (coupled) and dumbbell (DB) (uncoupled) chest press exercise performed on an unstable surface. The 3 specific chest press conditions included 50% 1 repetition maximum (RM) with BB (50% BB), 50% 1RM with DBs (50% DB), and 25% 1RM with DBs (25% DB). Ten male subjects participated in the study (age, 23.9 ± 2.6 years; body weight, 82.8 ± 10.2 kg). During testing, mean electromyographic activity was assessed for pectoralis major (PM), triceps brachii, anterior deltoid (AD), and rectus abdominis (RA) and was presented as a percent change across the lifting conditions. It was observed that muscle activation increased by 15% in both the PM and RA from the 50% BB condition to the 50% DB condition. Also, the greatest percent difference in muscle activation between the 50 and 25% DB conditions occurred for PM and AD (+54% during 50% DB). These results suggest that demands on the core musculature to provide stability are increased with the use of DBs (uncoupled) as opposed to a BB (coupled). Where instability training provides a sufficient hypertrophy stimulus in prime mover muscle groups, there may be the added benefit of core stability training. Specifically, this type of training may benefit both untrained persons and those engaged in active rehabilitation.

Atypical mitochondrial fission upon bacterial infection

PubMed Central

Stavru, Fabrizia; Palmer, Amy E.; Wang, Chunxin; Youle, Richard J.; Cossart, Pascale

2013-01-01

We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted pore-forming toxin listeriolysin O (LLO). Here, we examine factors involved in canonical fusion and fission. Strikingly, LLO-induced mitochondrial fragmentation does not require the traditional fission machinery, as Drp1 oligomers are absent from fragmented mitochondria following Listeria infection or LLO treatment, as the dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and as fragmentation proceeds efficiently in cells with impaired Drp1 function. LLO does not cause processing of the fusion protein optic atrophy protein 1 (Opa1), despite inducing a decrease in the mitochondrial membrane potential, suggesting a unique Drp1- and Opa1-independent fission mechanism distinct from that triggered by uncouplers or the apoptosis inducer staurosporine. We show that the ER marks LLO-induced mitochondrial fragmentation sites even in the absence of functional Drp1, demonstrating that the ER activity in regulating mitochondrial fission can be induced by exogenous agents and that the ER appears to regulate fission by a mechanism independent of the canonical mitochondrial fission machinery. PMID:24043775

Poliovirus replication requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1.

PubMed

Belov, George A; Kovtunovych, Gennadiy; Jackson, Catherine L; Ehrenfeld, Ellie

2010-10-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. © Published 2010. This article is a US Government work and is in the public domain in the USA.

Indomethacin Enhances Brown Fat Activity.

PubMed

Hao, Lei; Kearns, Jamie; Scott, Sheyenne; Wu, Dayong; Kodani, Sean D; Morisseau, Christophe; Hammock, Bruce D; Sun, Xiaocun; Zhao, Ling; Wang, Shu

2018-06-01

Indomethacin, a nonsteroidal anti-inflammatory drug, has been shown to induce white adipocyte differentiation; however, its roles in brown adipocyte differentiation and activation in brown adipose tissue (BAT) and obesity are unknown. To address this issue, we treated mouse brown preadipocytes with different doses of indomethacin, and delivered indomethacin to interscapular BAT (iBAT) of obese mice using implanted osmotic pumps. Indomethacin dose dependently increased brown preadipocyte differentiation and upregulated both mRNA and protein expression of uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor (PPAR) γ coactivator 1-alpha. The mechanistic study showed that indomethacin significantly activated the reporter driven by the PPAR response element, indicating that indomethacin may work as a PPAR γ agonist in this cell line. Consistently, indomethacin significantly decreased iBAT mass and fasting blood glucose levels in high-fat diet-induced obesity (DIO) mice. Histologic analysis showed that brown adipocytes of indomethacin-treated mice contained smaller lipid droplets compared with control mice, suggesting that indomethacin alleviated the whitening of BAT induced by the high-fat diet. Moreover, indomethacin significantly increased UCP1 mRNA expression in iBAT. Taken together, this study indicates that indomethacin can promote mouse brown adipocyte differentiation, and might increase brown fat and glucose oxidation capacity in DIO mice. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

30 CFR 57.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Coupling or uncoupling cars. 57.14215 Section... and Equipment Safety Practices and Operational Procedures § 57.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then...

30 CFR 57.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Coupling or uncoupling cars. 57.14215 Section... and Equipment Safety Practices and Operational Procedures § 57.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then...

30 CFR 56.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Coupling or uncoupling cars. 56.14215 Section... Equipment Safety Practices and Operational Procedures § 56.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then moved at minimum...

30 CFR 56.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Coupling or uncoupling cars. 56.14215 Section... Equipment Safety Practices and Operational Procedures § 56.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then moved at minimum...

30 CFR 56.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Coupling or uncoupling cars. 56.14215 Section... Equipment Safety Practices and Operational Procedures § 56.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then moved at minimum...

30 CFR 56.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Coupling or uncoupling cars. 56.14215 Section... Equipment Safety Practices and Operational Procedures § 56.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then moved at minimum...

30 CFR 57.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Coupling or uncoupling cars. 57.14215 Section... and Equipment Safety Practices and Operational Procedures § 57.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then...

30 CFR 57.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Coupling or uncoupling cars. 57.14215 Section... and Equipment Safety Practices and Operational Procedures § 57.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then...

30 CFR 57.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 1 2010-07-01 2010-07-01 false Coupling or uncoupling cars. 57.14215 Section... and Equipment Safety Practices and Operational Procedures § 57.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then...

30 CFR 56.14215 - Coupling or uncoupling cars.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Coupling or uncoupling cars. 56.14215 Section... Equipment Safety Practices and Operational Procedures § 56.14215 Coupling or uncoupling cars. Prior to coupling or uncoupling cars manually, trains shall be brought to a complete stop, and then moved at minimum...

Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity

PubMed Central

Zheng, Qiantao; Lin, Jun; Huang, Jiaojiao; Zhang, Hongyong; Zhang, Rui; Zhang, Xueying; Cao, Chunwei; Hambly, Catherine; Qin, Guosong; Yao, Jing; Song, Ruigao; Jia, Qitao; Wang, Xiao; Li, Yongshun; Zhang, Nan; Piao, Zhengyu; Ye, Rongcai; Speakman, John R.; Wang, Hongmei; Zhou, Qi; Wang, Yanfang; Jin, Wanzhu

2017-01-01

Uncoupling protein 1 (UCP1) is localized on the inner mitochondrial membrane and generates heat by uncoupling ATP synthesis from proton transit across the inner membrane. UCP1 is a key element of nonshivering thermogenesis and is most likely important in the regulation of body adiposity. Pigs (Artiodactyl family Suidae) lack a functional UCP1 gene, resulting in poor thermoregulation and susceptibility to cold, which is an economic and pig welfare issue owing to neonatal mortality. Pigs also have a tendency toward fat accumulation, which may be linked to their lack of UCP1, and thus influences the efficiency of pig production. Here, we report application of a CRISPR/Cas9-mediated, homologous recombination (HR)-independent approach to efficiently insert mouse adiponectin-UCP1 into the porcine endogenous UCP1 locus. The resultant UCP1 knock-in (KI) pigs showed an improved ability to maintain body temperature during acute cold exposure, but they did not have alterations in physical activity levels or total daily energy expenditure (DEE). Furthermore, ectopic UCP1 expression in white adipose tissue (WAT) dramatically decreased fat deposition by 4.89% (P < 0.01), consequently increasing carcass lean percentage (CLP; P < 0.05). Mechanism studies indicated that the loss of fat upon UCP1 activation in WAT was linked to elevated lipolysis. UCP1 KI pigs are a potentially valuable resource for agricultural production through their combination of cold adaptation, which improves pig welfare and reduces economic losses, with reduced fat deposition and increased lean meat production. PMID:29078316

Pomegranate vinegar attenuates adiposity in obese rats through coordinated control of AMPK signaling in the liver and adipose tissue

PubMed Central

2013-01-01

Background The effect of pomegranate vinegar (PV) on adiposity was investigated in high-fat diet (HF)-induced obese rats. Methods The rats were divided into 5 groups and treated with HF with PV or acetic acid (0, 6.5 or 13% w/w) for 16 weeks. Statistical analyses were performed by the Statistical Analysis Systems package, version 9.2. Results Compared to control, PV supplementation increased phosphorylation of AMP-activated protein kinase (AMPK), leading to changes in mRNA expressions: increases for hormone sensitive lipase and mitochondrial uncoupling protein 2 and decreases for sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptorγ (PPARγ) in adipose tissue; increases for PPARα and carnitinepalmitoyltransferase-1a (CPT-1a) and decrease for SREBP-1c in the liver. Concomitantly, PV reduced increases of body weight (p = 0.048), fat mass (p = 0.033), hepatic triglycerides (p = 0.005), and plasma triglycerides (p = 0.001). Conclusions These results suggest that PV attenuates adiposity through the coordinated control of AMPK, which leads to promotion of lipolysis in adipose tissue and stimulation of fatty acid oxidation in the liver. PMID:24180378

Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme.

PubMed

Avila, Frank W; Cohen, Allie B; Ameerudeen, Fatima S; Duneau, David; Suresh, Shruthi; Mattei, Alexandra L; Wolfner, Mariana F

2015-08-01

Within the mated reproductive tracts of females of many taxa, seminal fluid proteins (SFPs) coagulate into a structure known as the mating plug (MP). MPs have diverse roles, including preventing female remating, altering female receptivity postmating, and being necessary for mated females to successfully store sperm. The Drosophila melanogaster MP, which is maintained in the mated female for several hours postmating, is comprised of a posterior MP (PMP) that forms quickly after mating begins and an anterior MP (AMP) that forms later. The PMP is composed of seminal proteins from the ejaculatory bulb (EB) of the male reproductive tract. To examine the role of the PMP protein PEBme in D. melanogaster reproduction, we identified an EB GAL4 driver and used it to target PEBme for RNA interference (RNAi) knockdown. PEBme knockdown in males compromised PMP coagulation in their mates and resulted in a significant reduction in female fertility, adversely affecting postmating uterine conformation, sperm storage, mating refractoriness, egg laying, and progeny generation. These defects resulted from the inability of females to retain the ejaculate in their reproductive tracts after mating. The uncoagulated MP impaired uncoupling by the knockdown male, and when he ultimately uncoupled, the ejaculate was often pulled out of the female. Thus, PEBme and MP coagulation are required for optimal fertility in D. melanogaster. Given the importance of the PMP for fertility, we identified additional MP proteins by mass spectrometry and found fertility functions for two of them. Our results highlight the importance of the MP and the proteins that comprise it in reproduction and suggest that in Drosophila the PMP is required to retain the ejaculate within the female reproductive tract, ensuring the storage of sperm by mated females. Copyright © 2015 by the Genetics Society of America.

Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα

PubMed Central

Yan, Ming; Audet-Walsh, Étienne; Manteghi, Sanaz; Dufour, Catherine Rosa; Walker, Benjamin; Baba, Masaya; St-Pierre, Julie; Giguère, Vincent; Pause, Arnim

2016-01-01

The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat. PMID:27151976

Human deafness mutation E385D disrupts the mechanochemical coupling and subcellular targeting of myosin-1a.

PubMed

Yengo, Christopher M; Ananthanarayanan, Shobana K; Brosey, Chris A; Mao, Suli; Tyska, Matthew J

2008-01-15

Missense mutations in the membrane-binding actin-based motor protein, myosin-1a (Myo1a), have recently been linked to sensorineural deafness in humans. One of these mutations, E385D, impacts a residue in the switch II region of the motor domain that is present in virtually all members of the myosin superfamily. We sought to examine the impact of E385D on the function of Myo1a, both in terms of mechanochemical activity and ability to target to actin-rich microvilli in polarized epithelial cells. While E385D-Myo1a demonstrated actin-activated ATPase activity, the V(MAX) was reduced threefold relative to wild-type. Despite maintaining an active mechanochemical cycle, E385D-Myo1a was unable to move actin in the sliding filament assay. Intriguingly, when an enhanced-green-fluorescent-protein-tagged form of E385D-Myo1a was stably expressed in polarized epithelial cells, this mutation abolished the microvillar targeting normally demonstrated by wild-type Myo1a. Notably, these data are the first to suggest that mechanical activity is essential for proper localization of Myo1a in microvilli. These studies also provide a unique example of how even the most mild substitution of invariant switch II residues can effectively uncouple enzymatic and mechanical activity of the myosin motor domain.

Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A

PubMed Central

Taylor, Emily J. A.; Pantazaka, Evangelia; Shelley, Kathryn L.

2017-01-01

In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2. Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3. The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through “hyperactive signaling junctions,” wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals. PMID:28877931

Presence of brown adipocytes in retroperitoneal fat from patients with benign adrenal tumors: relationship with outdoor temperature.

PubMed

Betz, Matthias Johannes; Slawik, Marc; Lidell, Martin E; Osswald, Andrea; Heglind, Mikael; Nilsson, Daniel; Lichtenauer, Urs Daniel; Mauracher, Brigitte; Mussack, Thomas; Beuschlein, Felix; Enerbäck, Sven

2013-10-01

Brown adipose tissue (BAT) is a metabolically highly active organ with increased thermogenic activity in rodents exposed to cold temperature. Recently its presence in the cervical adipose tissue of human adults and its association with a favorable metabolic phenotype have been reported. The objective of the study was to determine the prevalence of retroperitoneal BAT in human adults. This was an observational cohort study. The study was conducted at a tertiary referral hospital. Fifty-seven patients who underwent surgery for benign adrenal tumors were included in this study. Prevalence of retroperitoneal BAT adjacent to the removed adrenal tumor as determined by uncoupling protein 1 (UCP1) protein and mRNA expression was measured. Using protein and mRNA expression analysis, we detected UCP1 protein in 26 of 57 patients (45.6%) as well as high mRNA expression of genes characteristic for brown adipocytes, independent of the adrenal tumor type. The presence of brown adipocytes within the retroperitoneal fat was associated with a significantly lower outdoor temperature during the month prior to surgery. Importantly, UCP1 expression on both mRNA and protein level was inversely correlated to outdoor temperature, whereas body mass index, sex, age, and diabetes status were not. These findings suggest that human retroperitoneal adipose tissue can acquire a BAT phenotype, thereby adapting to environmental challenges. These adaptive processes might provide a valuable therapeutic target in the treatment of obesity and insulin resistance.

Cell death and cell lysis are separable events during pyroptosis

PubMed Central

DiPeso, Lucian; Ji, Daisy X; Vance, Russell E; Price, Jordan V

2017-01-01

Although much insight has been gained into the mechanisms by which activation of the inflammasome can trigger pyroptosis in mammalian cells, the precise kinetics of the end stages of pyroptosis have not been well characterized. Using time-lapse fluorescent imaging to analyze the kinetics of pyroptosis in individual murine macrophages, we observed distinct stages of cell death and cell lysis. Our data demonstrate that cell membrane permeability resulting from gasdermin D pore formation is coincident with the cessation of cell movement, loss of mitochondrial activity, and cell swelling, events that can be uncoupled from cell lysis. We propose a model of pyroptosis in which cell death can occur independently of cell lysis. The uncoupling of cell death from cell lysis may allow for better control of cytosolic contents upon activation of the inflammasome. PMID:29147575

Mitochondrial uncoupler exerts a synthetic lethal effect against β-catenin mutant tumor cells.

PubMed

Shikata, Yuki; Kiga, Masaki; Futamura, Yushi; Aono, Harumi; Inoue, Hiroyuki; Kawada, Manabu; Osada, Hiroyuki; Imoto, Masaya

2017-04-01

The wingless/int-1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When β-catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As β-catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in-house natural product library for compounds that exhibited synthetic lethality towards β-catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in β-catenin mutated tumor cells. Significant tumor regression was observed in the β-catenin mutant HCT 116 xenograft model, but not in the β-catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of β-catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated β-catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor β-catenin mutations. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Mitochondrial uncoupling in skeletal muscle by UCP1 augments energy expenditure and glutathione content while mitigating ROS production.

PubMed

Adjeitey, Cyril Nii-Klu; Mailloux, Ryan J; Dekemp, Robert A; Harper, Mary-Ellen

2013-08-01

Enhancement of proton leaks in muscle tissue represents a potential target for obesity treatment. In this study, we examined the bioenergetic and physiological implications of increased proton leak in skeletal muscle. To induce muscle-specific increases in proton leak, we used mice that selectively express uncoupling protein-1 (UCP1) in skeletal muscle tissue. UCP1 expression in muscle mitochondria was ∼13% of levels in brown adipose tissue (BAT) mitochondria and caused increased GDP-sensitive proton leak. This was associated with an increase in whole body energy expenditure and a decrease in white adipose tissue content. Muscle UCP1 activity had divergent effects on mitochondrial ROS emission and glutathione levels compared with BAT. UCP1 in muscle increased total mitochondrial glutathione levels ∼7.6 fold. Intriguingly, unlike in BAT mitochondria, leak through UCP1 in muscle controlled mitochondrial ROS emission. Inhibition of UCP1 with GDP in muscle mitochondria increased ROS emission ∼2.8-fold relative to WT muscle mitochondria. GDP had no impact on ROS emission from BAT mitochondria from either genotype. Collectively, these findings indicate that selective induction of UCP1-mediated proton leak in muscle can increase whole body energy expenditure and decrease adiposity. Moreover, ectopic UCP1 expression in skeletal muscle can control mitochondrial ROS emission, while it apparently plays no such role in its endogenous tissue, brown fat.

Mitochondrial uncoupling in skeletal muscle by UCP1 augments energy expenditure and glutathione content while mitigating ROS production

PubMed Central

Adjeitey, Cyril Nii-Klu; Mailloux, Ryan J.; deKemp, Robert A.

2013-01-01

Enhancement of proton leaks in muscle tissue represents a potential target for obesity treatment. In this study, we examined the bioenergetic and physiological implications of increased proton leak in skeletal muscle. To induce muscle-specific increases in proton leak, we used mice that selectively express uncoupling protein-1 (UCP1) in skeletal muscle tissue. UCP1 expression in muscle mitochondria was ∼13% of levels in brown adipose tissue (BAT) mitochondria and caused increased GDP-sensitive proton leak. This was associated with an increase in whole body energy expenditure and a decrease in white adipose tissue content. Muscle UCP1 activity had divergent effects on mitochondrial ROS emission and glutathione levels compared with BAT. UCP1 in muscle increased total mitochondrial glutathione levels ∼7.6 fold. Intriguingly, unlike in BAT mitochondria, leak through UCP1 in muscle controlled mitochondrial ROS emission. Inhibition of UCP1 with GDP in muscle mitochondria increased ROS emission ∼2.8-fold relative to WT muscle mitochondria. GDP had no impact on ROS emission from BAT mitochondria from either genotype. Collectively, these findings indicate that selective induction of UCP1-mediated proton leak in muscle can increase whole body energy expenditure and decrease adiposity. Moreover, ectopic UCP1 expression in skeletal muscle can control mitochondrial ROS emission, while it apparently plays no such role in its endogenous tissue, brown fat. PMID:23757405

Parkin-mediated mitophagy is downregulated in browning of white adipose tissue.

PubMed

Taylor, David; Gottlieb, Roberta A

2017-04-01

Browning of white adipose tissue (WAT) promotes increased energy expenditure through the action of uncoupling protein 1 (UCP1) and is an attractive target to promote weight loss in obesity. Lowering of mitochondrial membrane potential by UCP1 is uniquely beneficial in this context; in other tissues, reduced membrane potential promotes mitochondrial clearance via mitophagy. It is unknown how parkin-mediated mitophagy is regulated in beige adipocytes. The relationship between parkin expression and WAT browning was investigated in 3T3-L1 adipocytes and parkin-deficient male C57BL/6 mice in response to pharmacological browning stimuli. Rosiglitazone treatment in 3T3-L1 adipocytes promoted mitochondrial biogenesis, UCP1 expression, and mitochondrial uncoupling. Parkin expression was decreased and reduced mitochondrial-associated parkin, and p62 indicated a reduction in mitophagy activity. Parkin overexpression prevented mitochondrial remodeling in response to rosiglitazone. In CL 316,243-treated wild-type mice, decreased parkin expression was observed in subcutaneous inguinal WAT, where UCP1 was strongly induced. CL 316,243 treatment weakly induced UCP1 expression in the gonadal depot, where parkin expression was unchanged. In contrast, parkin-deficient mice exhibited robust UCP1 expression in gonadal WAT following CL 316,243 treatment. WAT browning was associated with a decrease in parkin-mediated mitophagy, and parkin expression antagonized browning of WAT. © 2017 The Obesity Society.

Deterioration of physical performance and cognitive function in rats with short-term high-fat feeding.

PubMed

Murray, Andrew J; Knight, Nicholas S; Cochlin, Lowri E; McAleese, Sara; Deacon, Robert M J; Rawlins, J Nicholas P; Clarke, Kieran

2009-12-01

Efficiency, defined as the amount of work produced for a given amount of oxygen consumed, is a key determinant of endurance capacity, and can be altered by metabolic substrate supply, in that fatty acid oxidation is less efficient than glucose oxidation. It is unclear, however, whether consumption of a high-fat diet would be detrimental or beneficial for endurance capacity, due to purported glycogen-sparing properties. In addition, a high-fat diet over several months leads to cognitive impairment. Here, we tested the hypothesis that short-term ingestion of a high-fat diet (55% kcal from fat) would impair exercise capacity and cognitive function in rats, compared with a control chow diet (7.5% kcal from fat) via mitochondrial uncoupling and energy deprivation. We found that rats ran 35% less far on a treadmill and showed cognitive impairment in a maze test with 9 d of high-fat feeding, with respiratory uncoupling in skeletal muscle mitochondria, associated with increased uncoupling protein (UCP3) levels. Our results suggest that high-fat feeding, even over short periods of time, alters skeletal muscle UCP3 expression, affecting energy production and physical performance. Optimization of nutrition to maximize the efficiency of mitochondrial ATP production could improve energetics in athletes and patients with metabolic abnormalities.

Mild uncoupling of respiration and phosphorylation as a mechanism providing nephro- and neuroprotective effects of penetrating cations of the SkQ family.

PubMed

Plotnikov, E Y; Silachev, D N; Jankauskas, S S; Rokitskaya, T I; Chupyrkina, A A; Pevzner, I B; Zorova, L D; Isaev, N K; Antonenko, Y N; Skulachev, V P; Zorov, D B

2012-09-01

It is generally accepted that mitochondrial production of reactive oxygen species is nonlinearly related to the value of the mitochondrial membrane potential with significant increment at values exceeding 150 mV. Due to this, high values of the membrane potential are highly dangerous, specifically under pathological conditions associated with oxidative stress. Mild uncoupling of oxidative phosphorylation is an approach to preventing hyperpolarization of the mitochondrial membrane. We confirmed data obtained earlier in our group that dodecylrhodamine 19 (C(12)R1) (a penetrating cation from SkQ family not possessing a plastoquinone group) has uncoupling properties, this fact making it highly potent for use in prevention of pathologies associated with oxidative stress induced by mitochondrial hyperpolarization. Further experiments showed that C(12)R1 provided nephroprotection under ischemia/reperfusion of the kidney as well as under rhabdomyolysis through diminishing of renal dysfunction manifested by elevated level of blood creatinine and urea. Similar nephroprotective properties were observed for low doses (275 nmol/kg) of the conventional uncoupler 2,4-dinitrophenol. Another penetrating cation that did not demonstrate protonophorous activity (SkQR4) had no effect on renal dysfunction. In experiments with induced ischemic stroke, C(12)R1 did not have any effect on the area of ischemic damage, but it significantly lowered neurological deficit. We conclude that beneficial effects of penetrating cation derivatives of rhodamine 19 in renal pathologies and brain ischemia may be at least partially explained by uncoupling of oxidation and phosphorylation.

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling.

PubMed

Zhdanov, Alexander V; Waters, Alicia H C; Golubeva, Anna V; Dmitriev, Ruslan I; Papkovsky, Dmitri B

2014-01-01

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O2 consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca(2+) or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment. © 2013.

Regulatory effects of GRK2 on GPCRs and non-GPCRs and possible use as a drug target (Review).

PubMed

Han, Chen-Chen; Ma, Yang; Li, Yifan; Wang, Yang; Wei, Wei

2016-10-01

G protein-coupled receptor kinase 2 (GRK2) is a key member of the G protein-coupled receptor kinase (GRK) family. GRK2 activity is regulated by the C-terminus of GRK2 which contains a plekstrin homology domain and the N-terminus of GRK2 which contains the RGS homology domain with binding sites for several proteins and lipids such as G protein-coupled receptors (GPCRs), G protein, phospholipase C, phosphatidylinositol 4,5-bisphosphate, extracellular signal‑regulated kinase, protein kinase A and Gβγ. GRK2 phosphorylates the GPCR and enhances the affinity of binding to arrestins, which uncouple the receptors from G proteins, and target the receptors for desensitization and internalization. GRK2 also regulates non‑GPCR desensitization and internalization by phosphorylation, and is important in maintaining the balance between the receptors and signal transduction. Previous findings have indicated that the upregulation of GRK2 in heart failure enhances dysfunctional adrenergic signaling and myocyte death. Collagen-induced arthritis induces the upregulation of GRK2 expression in fibroblast-like synoviocytes. In this review, we discussed the evidence for the association between altered GRK2 levels and various diseases, which suggests that GRK2 may be an effective drug target for preventing and treating heart failure, hypertension and inflammatory disease.

The effects of branched-chain amino acid granules on the accumulation of tissue triglycerides and uncoupling proteins in diet-induced obese mice.

PubMed

Arakawa, Mie; Masaki, Takayuki; Nishimura, Junko; Seike, Masataka; Yoshimatsu, Hironobu

2011-01-01

It has been demonstrated the involvement of branched-chain amino acids (BCAA) on obesity and related metabolic disorder. We investigated the effects of branched-chain amino acids (BCAA) on obesity and on glucose/fat homeostasis in mice fed on a high-fat (45%) diet. BCAA was dissolved in 0.5% methylcellulose and added to the drinking water (BCAA-treated group). A high-fat diet was provided for 6 weeks and BCAA was given for 2 weeks. The BCAA-treated group gained almost 7% less body weight and had less epididymal adipose tissue (WAT) mass than the control group (p<0.05). BCAA supplementation also reduced the hepatic and skeletal muscle triglyceride (TG) concentrations (p<0.05). The hepatic levels of PPAR-alpha and uncoupling protein (UCP) 2, and the level of PPAR-alpha and UCP3 in the skeletal muscle were greater in the BCAA-treated group than in the control mice (p<0.05). These results demonstrate that the liver and muscle TG concentration are less in BCAA-treated group. BCAA affects PPAR-alpha and UCP expression in muscle and liver tissue.

Genetic variants of uncoupling proteins-2 and -3 in relation to maximal oxygen uptake in different sports.

PubMed

Holdys, Joanna; Gronek, Piotr; Kryściak, Jakub; Stanisławski, Daniel

2013-01-01

Uncoupling proteins 2 and 3 (UCP2 and UCP3) as mitochondrial electron transporters are involved in regulation of ATP production and energy dissipation as heat. Energy efficiency plays an important role in physical performance, especially in aerobic fitness. The aim of this study was to examine the association between maximal oxygen uptake and genetic variants of the UCP2 and UCP3 genes. The studies were carried out in a group of 154 men and 85 women, professional athletes representing various sports and fitness levels and students of the University of Physical Education in Poznań. Physiological and molecular procedures were used, i.e. direct measurement of maximum oxygen uptake (VO₂max) and analysis of an insertion/deletion (I/D) polymorphism in the 3'untranslated region of exon 8 of the UCP2 gene and a C>T substitution in exon 5 (Y210Y) of the UCP3 gene. No statistically significant associations were found, only certain trends. Insertion allele (I) of the I/D UCP2 and the T allele of the UCP3 gene were favourable in obtaining higher VO₂max level and might be considered as endurance-related alleles.

Muscle mitohormesis promotes cellular survival via serine/glycine pathway flux.

PubMed

Ost, Mario; Keipert, Susanne; van Schothorst, Evert M; Donner, Verena; van der Stelt, Inge; Kipp, Anna P; Petzke, Klaus-Jürgen; Jove, Mariona; Pamplona, Reinald; Portero-Otin, Manuel; Keijer, Jaap; Klaus, Susanne

2015-04-01

Recent studies on mouse and human skeletal muscle (SM) demonstrated the important link between mitochondrial function and the cellular metabolic adaptation. To identify key compensatory molecular mechanisms in response to chronic mitochondrial distress, we analyzed mice with ectopic SM respiratory uncoupling in uncoupling protein 1 transgenic (UCP1-TG) mice as model of muscle-specific compromised mitochondrial function. Here we describe a detailed metabolic reprogramming profile associated with mitochondrial perturbations in SM, triggering an increased protein turnover and amino acid metabolism with induced biosynthetic serine/1-carbon/glycine pathway and the longevity-promoting polyamine spermidine as well as the trans-sulfuration pathway. This is related to an induction of NADPH-generating pathways and glutathione metabolism as an adaptive mitohormetic response and defense against increased oxidative stress. Strikingly, consistent muscle retrograde signaling profiles were observed in acute stress states such as muscle cell starvation and lipid overload, muscle regeneration, and heart muscle inflammation, but not in response to exercise. We provide conclusive evidence for a key compensatory stress-signaling network that preserves cellular function, oxidative stress tolerance, and survival during conditions of increased SM mitochondrial distress, a metabolic reprogramming profile so far only demonstrated for cancer cells and heart muscle. © FASEB.

Structural biology of intrinsically disordered proteins: Revisiting unsolved mysteries.

PubMed

Sigalov, Alexander B

2016-06-01

The emergence of intrinsically disordered proteins (IDPs) has challenged the classical protein structure-function paradigm by introducing a new paradigm of "coupled binding and folding". This paradigm suggests that IDPs fold upon binding to their partners. Further studies, however, revealed a novel and previously unrecognized phenomenon of "uncoupled binding and folding" suggesting that IDPs do not necessarily fold upon interaction with their lipid and protein partners. The complex and often unusual biophysics of IDPs makes structural characterization of these proteins and their complexes not only challenging but often resulting in opposite conclusions. For this reason, some crucial questions in this field remain unsolved for well over a decade. Considering an important role of IDPs in cellular regulation, signaling and control in health and disease, more efforts are needed to solve these mysteries. Here, I focus on two long-standing contradictions in the literature concerning dimerization and membrane-binding activities of IDPs. Molecular explanation of these discrepancies is provided. I also demonstrate how resolution of these critical issues in the field of IDPs results in our expanded understanding of cell function and has multiple applications in biology and medicine. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Superresolution microscopy reveals spatial separation of UCP4 and F0F1-ATP synthase in neuronal mitochondria

PubMed Central

Klotzsch, Enrico; Smorodchenko, Alina; Löfler, Lukas; Moldzio, Rudolf; Parkinson, Elena; Schütz, Gerhard J.; Pohl, Elena E.

2015-01-01

Because different proteins compete for the proton gradient across the inner mitochondrial membrane, an efficient mechanism is required for allocation of associated chemical potential to the distinct demands, such as ATP production, thermogenesis, regulation of reactive oxygen species (ROS), etc. Here, we used the superresolution technique dSTORM (direct stochastic optical reconstruction microscopy) to visualize several mitochondrial proteins in primary mouse neurons and test the hypothesis that uncoupling protein 4 (UCP4) and F0F1-ATP synthase are spatially separated to eliminate competition for the proton motive force. We found that UCP4, F0F1-ATP synthase, and the mitochondrial marker voltage-dependent anion channel (VDAC) have various expression levels in different mitochondria, supporting the hypothesis of mitochondrial heterogeneity. Our experimental results further revealed that UCP4 is preferentially localized in close vicinity to VDAC, presumably at the inner boundary membrane, whereas F0F1-ATP synthase is more centrally located at the cristae membrane. The data suggest that UCP4 cannot compete for protons because of its spatial separation from both the proton pumps and the ATP synthase. Thus, mitochondrial morphology precludes UCP4 from acting as an uncoupler of oxidative phosphorylation but is consistent with the view that UCP4 may dissipate the excessive proton gradient, which is usually associated with ROS production. PMID:25535394

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

PubMed Central

Mochizuki, Nobuyoshi; Brusslan, Judy A.; Larkin, Robert; Nagatani, Akira; Chory, Joanne

2001-01-01

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway. PMID:11172074

Constitutive Uncoupling of Pathways of Gene Expression That Control Growth and Differentiation in Myeloid Leukemia: A Model for the Origin and Progression of Malignancy

NASA Astrophysics Data System (ADS)

Sachs, Leo

1980-10-01

Chemical carcinogens and tumor promoters have pleiotropic effects. Tumor initiators can produce a variety of mutations and tumor promoters can regulate a variety of physiological molecules that control growth and differentiation. The appropriate mutation and the regulation of the appropriate molecules to induce cell growth can initiate and promote the sequence of changes required for transformation of normal cells into malignant cells. After this sequence of changes, some tumors can still be induced to revert with a high frequency from a malignant phenotype to a nonmalignant phenotype. Results obtained from analysis of regulation of growth and differentiation in normal and leukemic myeloid cells, the phenotypic reversion of malignancy by induction of normal differentiation in myeloid leukemia, and the blocks in differentiation-defective leukemic cell mutants have been used to propose a general model for the origin and progression of malignancy. The model states that malignancy originates by changing specific pathways of gene expression required for growth from inducible to constitutive in cells that can still be induced to differentiate normally by the physiological inducer of differentiation. The malignant cells, unlike the normal cells, then no longer require the physiological inducer for growth. This changes the requirements for growth and uncouples growth from differentiation. Constitutive expression of other specific pathways can uncouple other controls, which then causes blocks in differentiation and the further progression of malignancy. The existence of specific constitutive pathways of gene expression that uncouple controls in malignant cells can also explain the expression of fetal proteins, hormones, and some other specialized products of normal development in various types of tumors.

The Succinated Proteome

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

Merkley, Eric D.; Metz, Thomas O.; Smith, Richard D.

Succination is a chemical modification of cysteine in protein by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes. Increased succination of proteins is also detected in the kidney of a fumarase conditional knock-out mouse which develops renal tumors. Keap1, the gatekeeper of the antioxidant response, was identified as a major succinated protein in renal cancer cells, suggesting that succinationmore » may play a role in activation of the antioxidant response. A wide range of proteins is subject to succination, including enzymes, adipokines, cytoskeletal proteins and ER chaperones with functional cysteine residues. There is also significant overlap between succinated and glutathionylated proteins, and with proteins containing cysteine residues that are readily oxidized to the sulfenic (cysteic) acid. Succination of adipocyte proteins is inhibited by uncouplers, which discharge the mitochondrial membrane potential (Δψm) and by ER stress inhibitors. 2SC serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic post-translational modification of proteins by proteomics approaches.« less

Dnmt1-dependent Chk1 pathway suppression is protective against neuron division.

PubMed

Oshikawa, Mio; Okada, Kei; Tabata, Hidenori; Nagata, Koh-Ichi; Ajioka, Itsuki

2017-09-15

Neuronal differentiation and cell-cycle exit are tightly coordinated, even in pathological situations. When pathological neurons re-enter the cell cycle and progress through the S phase, they undergo cell death instead of division. However, the mechanisms underlying mitotic resistance are mostly unknown. Here, we have found that acute inactivation of retinoblastoma (Rb) family proteins (Rb, p107 and p130) in mouse postmitotic neurons leads to cell death after S-phase progression. Checkpoint kinase 1 (Chk1) pathway activation during the S phase prevented the cell death, and allowed the division of cortical neurons that had undergone acute Rb family inactivation, oxygen-glucose deprivation (OGD) or in vivo hypoxia-ischemia. During neurogenesis, cortical neurons became protected from S-phase Chk1 pathway activation by the DNA methyltransferase Dnmt1, and underwent cell death after S-phase progression. Our results indicate that Chk1 pathway activation overrides mitotic safeguards and uncouples neuronal differentiation from mitotic resistance. © 2017. Published by The Company of Biologists Ltd.

Hydro-ethanolic extract of cashew tree (Anacardium occidentale) nut and its principal compound, anacardic acid, stimulate glucose uptake in C2C12 muscle cells.

PubMed

Tedong, Leonard; Madiraju, Padma; Martineau, Louis C; Vallerand, Diane; Arnason, John T; Desire, Dzeufiet D P; Lavoie, Louis; Kamtchouing, Pierre; Haddad, Pierre S

2010-12-01

Products of cashew tree (Anacardium occidentale) are used in traditional medicine for various ailments, including diabetes. The anti-diabetic properties of cashew plant parts were studied using differentiated C2C12 myoblasts (myotubes) and rat liver mitochondria. Hydroethanolic extract of cashew seed (CSE) and its active component, anacardic acid (AA), stimulated glucose transport into C2C12 myotubes in a concentration-dependent manner. Extracts of other parts (leaves, bark and apple) of cashew plant were inactive. Significant synergistic effect on glucose uptake with insulin was noticed at 100 μg/mL CSE. CSE and AA caused activation of adenosine monophosphate-activated protein kinase in C2C12 myotubes after 6 h of incubation. No significant effect was noticed on Akt and insulin receptor phosphorylation. Both CSE and AA exerted significant uncoupling of succinate-stimulated respiration in rat liver mitochondria. Activation of adenosine monophosphate-activated protein kinase by CSE and AA likely increases plasma membrane glucose transporters, resulting in elevated glucose uptake. In addition, the dysfunction of mitochondrial oxidative phosphorylation may enhance glycolysis and contribute to increased glucose uptake. These results collectively suggest that CSE may be a potential anti-diabetic nutraceutical. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The efficiency of cellular energy transduction and its implications for obesity.

PubMed

Harper, Mary-Ellen; Green, Katherine; Brand, Martin D

2008-01-01

We assess the existence, mechanism, and functions of less-than-maximal coupling efficiency of mitochondrial oxidative phosphorylation and its potential as a target for future antiobesity interventions. Coupling efficiency is the proportion of oxygen consumption used to make adenosine triphosphate (ATP) and do useful work. High coupling efficiency may lead to fat deposition; low coupling efficiency to a decrease in fat stores. We review obligatory and facultative energy expenditure and the role of a futile cycle of proton pumping and proton leak across the mitochondrial inner membrane in dissipating energy. Basal proton conductance is catalyzed primarily by the adenine nucleotide translocase but can be mimicked by chemical uncouplers. Inducible proton conductance is catalyzed by specific uncoupling proteins. We discuss the opportunities and pitfalls of targeting these processes as a treatment for obesity by decreasing coupling efficiency and increasing energy expenditure, either directly or through central mechanisms of energy homeostasis.

Role of arachidonic acid and protein kinase C during maturation-inducing hormone-dependent meiotic resumption and ovulation in ovarian follicles of Atlantic croaker

USGS Publications Warehouse

Patino, R.; Yoshizaki, G.; Bolamba, D.; Thomas, P.

2003-01-01

The roles of arachidonic acid (AA) and protein kinase C (PKC) during in vitro maturation-inducing hormone (MIH)-dependent meiotic resumption (maturation) and ovulation were studied in ovarian follicles of Atlantic croaker (Micropogonias undulatus). The requirement for cyclooxygenase (COX) metabolites of AA was examined using a nonspecific COX inhibitor, indomethacin (IM), as well as two COX products, prostaglandin (PG) F2?? and PGE2, whereas the role of lipoxygenase (LOX) was investigated using a specific LOX inhibitor, nordihydroguaiaretic acid (NDGA). The involvement of PKC was examined using phorbol 12-myristate 13-acetate (PMA), a PKC activator, as well as GF109203X (GF), a specific inhibitor of PKC and 1-(5-isoquin- olinesulfonyl)-2-methylpiperazine (H7), nonspecific inhibitor of protein kinases. Genomic mechanisms were examined with the transcription-inhibitor actinomycin D (ActD) and the functionality of heterologous (oocyte-granulosa) gap junctions (GJ) with a dye transfer assay. The AA (100 ??M) and PGF2?? (5 ??M) did not induce maturation, and NDGA (10 ??M) did not affect MIH-dependent maturation. However, IM (100 ??M) partially inhibited MIH-dependent maturation. Conversely, AA and both PGs induced, and IM and NDGA inhibited, MIH-dependent ovulation in matured follicles. The PMA (1 ??g/ml) did not induce maturation but caused ovulation in matured follicles, whereas PKC inhibitors (GF, 5 ??M; H7, 50??M) did not affect MIH-dependent maturation but inhibited MIH- and PMA-dependent ovulation. The PMA-dependent ovulation was inhibited by IM but not by NDGA. In addition, ActD (5 ??M) blocked MIH-dependent, but not PMA-dependent, ovulation, and PGF2?? restored MIH-dependent ovulation in ActD-blocked follicles. The AA and PGs did not induce, and GF did not inhibit, MIH-dependent heterologous GJ uncoupling. In conclusion, AA and PKC mediate MIH-dependent ovulation but not meiotic resumption or heterologous GJ uncoupling in croaker follicles, but a permissive role of COX products of AA during maturation is possible. A novel model of MIH-dependent ovulation is proposed in which 1) LOX and COX metabolites of AA are both required for ovulation, but at upstream and downstream sites of the pathway, respectively, relative to PKC, and 2) PKC is downstream of genomic activation.

Novel insights of dietary polyphenols and obesity

PubMed Central

Wang, Shu; Moustaid-Moussa, Naima; Chen, Lixia; Mo, Huanbiao; Shastri, Anuradha; Su, Rui; Bapat, Priyanka; Kwun, InSook; Shen, Chwan-Li

2013-01-01

Prevalence of obesity has steadily increased over the past three decades both in the United States and worldwide. Recent studies have shown the role of dietary polyphenols in the prevention of obesity and obesity-related chronic diseases. Here we evaluated the impact of commonly consumed polyphenols, including green tea catechins and epigallocatechin gallates, resveratrol, and curcumin, on obesity and obesity-related-inflammation. Cellular studies demonstrated that these dietary polyphenols reduce viability of adipocytes and proliferation of preadipocytes, suppress adipocyte differentiation and triglyceride accumulation, stimulate lipolysis and fatty acid β-oxidation, and reduce inflammation. Concomitantly, the polyphenols modulate signaling pathways including the AMP-activated protein kinase, peroxisome proliferator activated receptor γ, CCAAT/enhancer binding protein α, PPAR gamma activator 1-alpha, sirtuin 1, sterol regulatory element binding protein-1c, uncoupling proteins 1 and 2, and nuclear factor kappa B that regulate adipogenesis, antioxidant and anti-inflammatory responses. Animal studies strongly suggest that commonly consumed polyphenols described in this review have a pronounced effect on obesity as shown by lower body weight, fat mass, and triglycerides through enhancing energy expenditure and fat utilization, and modulating glucose hemostasis. Limited human studies have been conducted in this area, and are inconsistent about the anti-obesity impact of dietary polyphenols, probably due to the various study designs and lengths, variation among subjects (age, gender, ethnicity), chemical forms of the dietary polyphenols used and confounding factors such as other weight reducing agents. Future randomized controlled trials are warranted to reconcile the discrepancies between preclinical efficacies and inconclusive clinic outcomes of these polyphenols. PMID:24314860

Anticancer Properties of PPARα-Effects on Cellular Metabolism and Inflammation

PubMed Central

Grabacka, Maja; Reiss, Krzysztof

2008-01-01

Peroxisome proliferator-activated receptors (PPARs) have lately attracted much attention as therapeutic targets. Previously, PPAR ligands were associated with the treatment of diabetes, hyperlipidemia and cardiovascular diseases, as they modulate the expression of genes regulating glucose and lipid metabolism. Recently, PPAR ligands have been also considered as potential anticancer agents, with relatively low systemic toxicity. The emerging evidence for antiproliferative, proapoptotic, antiinflammatory and potential antimetastatic properties of PPARα ligands prompted us to discuss possible roles of PPARα in tumor suppression. PPARα activation can target cancer cells energy balance by blocking fatty acid synthesis and by promoting fatty acid β-oxidation. In the state of limited nutrient availability, frequently presents in the tumor microenvironment, PPARα cooperates with AMP-dependent protein kinase in: (i) repressing oncogenic Akt activity, (ii) inhibiting cell proliferation, and (iii) forcing glycolysis-dependent cancer cells into “metabolic catastrophe.” Other potential anticancer effects of PPARα include suppression of inflammation, and upregulation of uncoupling proteins (UCPs), which attenuates mitochondrial reactive oxygen species production and cell proliferation. In conclusion, there are strong premises that the low-toxic and well-tolerated PPAR ligands should be considered as new therapeutic agents to fight disseminating cancer, which represents the major challenge for modern medicine and basic research. PMID:18509489

Lycopene and Apo-10′-lycopenoic Acid Have Differential Mechanisms of Protection against Hepatic Steatosis in β-Carotene-9′,10′-oxygenase Knockout Male Mice123

PubMed Central

Ip, Blanche C; Liu, Chun; Lichtenstein, Alice H; von Lintig, Johannes; Wang, Xiang-Dong

2015-01-01

Background: Nonalcoholic fatty liver disease is positively associated with obesity and cardiovascular disease risk. Apo-10′-lycopenoic acid (APO10LA), a potential oxidation product of apo-10′-lycopenal that is generated endogenously by β-carotene-9′,10′-oxygenase (BCO2) cleavage of lycopene, inhibited hepatic steatosis in BCO2-expressing mice. Objective: The present study evaluated lycopene and APO10LA effects on hepatic steatosis in mice without BCO2 expression. Methods: Male and female BCO2-knockout (BCO2-KO) mice were fed a high saturated fat diet (HSFD) with or without APO10LA (10 mg/kg diet) or lycopene (100 mg/kg diet) for 12 wk. Results: Lycopene or APO10LA supplementation reduced hepatic steatosis incidence (78% and 72%, respectively) and severity in BCO2-KO male mice. Female mice did not develop steatosis, had greater hepatic total cholesterol (3.06 vs. 2.31 mg/g tissue) and cholesteryl ester (1.58 vs. 0.86 mg/g tissue), but had lower plasma triglyceride (TG) (229 vs. 282 mg/dL) and cholesterol (97.1 vs. 119 mg/dL) than male mice. APO10LA-mitigated steatosis in males was associated with reduced hepatic total cholesterol (18%) and activated sirtuin 1 signaling, which resulted in reduced fatty acids (FAs) and TG synthesis markers [stearoyl-coenzyme A (CoA) desaturase protein, 71%; acetyl-CoA carboxylase phosphorylation, 79%; AMP-activated protein kinase phosphorylation, 67%], and elevated cholesterol efflux genes (cytochrome P450 family 7A1, 65%; ATP-binding cassette transporter G5/8, 11%). These APO10LA-mediated effects were not mimicked by lycopene supplementation. Intriguingly, steatosis inhibition by lycopene induced peroxisome proliferator–activated receptor (PPAR)α- and PPARγ-related genes in mesenteric adipose tissue (MAT) that increases mitochondrial uncoupling [cell death–inducing DNA fragmentation factor, α subunit-like effector a, 55%; PR domain-containing 16, 47%; uncoupling protein 3 (Ucp3), 55%], FA β-oxidation (PPARα, 53%; very long chain acyl-CoA dehydrogenase, 38%), and uptake (FA transport protein 4, 29%; lipoprotein lipase 43%). Expressions of 10 MAT PPAR-related genes were inversely correlated with steatosis score, suggesting that lycopene reduced steatosis by increasing MAT FA utilization. Conclusions: Our data suggest that lycopene and APO10LA inhibit HSFD-induced steatosis in BCO2-KO male mice through differential mechanisms. Sex disparity of BCO2-KO mice was observed in the outcomes of HSFD-induced liver steatosis and plasma lipids. PMID:25644347

The Split Personality of Glutamate Transporters: A Chloride Channel and a Transporter.

PubMed

Cater, Rosemary J; Ryan, Renae M; Vandenberg, Robert J

2016-03-01

Transporters and ion channels are conventionally categorised into distinct classes of membrane proteins. However, some membrane proteins have a split personality and can function as both transporters and ion channels. The excitatory amino acid transporters (EAATs) in particular, function as both glutamate transporters and chloride (Cl(-)) channels. The EAATs couple the transport of glutamate to the co-transport of three Na(+) ions and one H(+) ion into the cell, and the counter-transport of one K(+) ion out of the cell. The EAAT Cl(-) channel is activated by the binding of glutamate and Na(+), but is thermodynamically uncoupled from glutamate transport and involves molecular determinants distinct from those responsible for glutamate transport. Several crystal structures of an EAAT archaeal homologue, GltPh, at different stages of the transport cycle, alongside numerous functional studies and molecular dynamics simulations, have provided extensive insights into the mechanism of substrate transport via these transporters. However, the molecular determinants involved in Cl(-) permeation, and the mechanism by which this channel is activated are not entirely understood. Here we will discuss what is currently known about the molecular determinants involved in EAAT-mediated Cl(-) permeation and the mechanisms that underlie their split personality.

Uncoupling effect of palmitate is exacerbated in skeletal muscle mitochondria of sea-acclimatized king penguins (Aptenodytes patagonicus).

PubMed

Rey, Benjamin; Duchamp, Claude; Roussel, Damien

2017-09-01

In king penguin juveniles, the environmental transition from a terrestrial to a marine habitat, occurring at fledging, drastically stimulates lipid catabolism and the remodelling of muscle mitochondria to sustain extensive swimming activity and thermoregulation in the cold circumpolar oceans. However, the exact nature of these mechanisms remains only partially resolved. Here we investigated, in vitro, the uncoupling effect of increasing doses of fatty acids in pectoralis muscle intermyofibrillar mitochondria isolated, either from terrestrial never-immersed or experimentally cold water immersed pre-fledging king penguins or from sea-acclimatized fledged penguins. Mitochondria exhibited much greater palmitate-induced uncoupling respiration and higher maximal oxidative capacity after acclimatization to marine life. Such effects were not reproduced experimentally after repeated immersions in cold water, suggesting that the plasticity of mitochondrial characteristics may not be primarily driven by cold exposure per se but by other aspects of sea acclimatization. Copyright © 2017. Published by Elsevier Inc.

Mitochondrial uncouplers inhibit clathrin-mediated endocytosis largely through cytoplasmic acidification

PubMed Central

Dejonghe, Wim; Kuenen, Sabine; Mylle, Evelien; Vasileva, Mina; Keech, Olivier; Viotti, Corrado; Swerts, Jef; Fendrych, Matyáš; Ortiz-Morea, Fausto Andres; Mishev, Kiril; Delang, Simon; Scholl, Stefan; Zarza, Xavier; Heilmann, Mareike; Kourelis, Jiorgos; Kasprowicz, Jaroslaw; Nguyen, Le Son Long; Drozdzecki, Andrzej; Van Houtte, Isabelle; Szatmári, Anna-Mária; Majda, Mateusz; Baisa, Gary; Bednarek, Sebastian York; Robert, Stéphanie; Audenaert, Dominique; Testerink, Christa; Munnik, Teun; Van Damme, Daniël; Heilmann, Ingo; Schumacher, Karin; Winne, Johan; Friml, Jiří; Verstreken, Patrik; Russinova, Eugenia

2016-01-01

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane. PMID:27271794

Effect of surfactants on apparent oxygen consumption of photosystem I isolated from Arthrospira platensis.

PubMed

Yu, Daoyong; Huang, Guihong; Xu, Fengxi; Ge, Baosheng; Liu, Shuang; Xu, Hai; Huang, Fang

2014-11-01

Surfactants play a significant role in solubilization of photosystem I (PSI) in vitro. Triton X-100 (TX), n-Dodecyl-β-D-maltoside (DDM), and sodium dodecyl sulfate (SDS) were employed to solubilize PSI particles in MES buffer to compare the effect of surfactant and its dosage on the apparent oxygen consumption rate of PSI. Through a combined assessment of sucrose density gradient centrifugation, Native PAGE and 77 K fluorescence with the apparent oxygen consumption, the nature of the enhancement of the apparent oxygen consumption activity of PSI by surfactants has been analyzed. Aggregated PSI particles can be dispersed by surfactant molecules into micelles, and the apparent oxygen consumption rate is higher for surfactant-solubilized PSI than for integral PSI particles. For DDM, PSI particles are solubilized mostly as the integral trimeric form. For TX, PSI particles are solubilized as incomplete trimeric and some monomeric forms. For the much harsher surfactant, SDS, PSI particles are completely solubilized as monomeric and its subunit forms. The enhancement of the oxygen consumption rate cannot be explained only by the effects of surfactant on the equilibrium between monomeric and trimeric forms of solubililized PSI. Care must be taken when the electron transfer activity of PSI is evaluated by methods based on oxygen consumption because the apparent oxygen consumption rate is influenced by uncoupled chlorophyll (Chl) from PSI, i.e., the larger the amount of uncoupled Chl, the higher the rate of apparent oxygen consumption. 77 K fluorescence spectra can be used to ensure that there is no uncoupled Chl present in the system. In order to eliminate the effect of trace uncoupled Chl, an efficient physical quencher of (1)O2, such as 1 mM NaN3, may be added into the mixture.

Insulin alleviates mitochondrial oxidative stress involving upregulation of superoxide dismutase 2 and uncoupling protein 2 in septic acute kidney injury

PubMed Central

Chen, Guang-Dao; Zhang, Jun-Liang; Chen, Yi-Ting; Zhang, Ju-Xing; Wang, Tao; Zeng, Qi-Yi

2018-01-01

The aim of the present study was to explore the effects and mechanisms of insulin on mitochondrial oxidative stress in septic acute kidney injury (AKI). Male Sprague Dawley rats were divided randomly into four groups: Control group, sham surgery group, cecal ligation and puncture (CLP) group, and CLP plus insulin group. Blood specimens and kidney tissues were obtained at 12 and 24 h after surgery as separate experiments. Analyses of histology and indicators of renal injury [blood urea nitrogen (BUN) and serum creatinine (CRE) and neutrophil gelatinase-associated lipocalin (NGAL)], mitochondrial function [adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP)], oxidative stress [inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS) and nitric oxide (NO)], endogenous antioxidant systems [superoxide dismutase (SOD) and glutathione (GSH)] as well as the expression of uncoupling protein (UCP), PINK1 protein (a major mediator of mitophagy), PGC1α protein (a major regulator of mitochondrial biogenesis) were performed. Compared with CLP group, the CLP plus insulin group had milder histological damage, higher levels of ATP and MMP as well as lower levels of BUN, serum CRE and NGAL, intrarenal iNOS, mitochondrial ROS and total NO. Moreover, the CLP plus insulin group demonstrated increased expression of SOD2 and UCP2. In contrast, insulin administration suppressed mitophagy meanwhile did not upregulate total GSH and induce mitochondrial biogenesis following CLP. These findings indicated that the upregulation of SOD2 and UCP2 may be involved in insulin protecting against mitochondrial oxidative stress in septic AKI. PMID:29563990

Insulin alleviates mitochondrial oxidative stress involving upregulation of superoxide dismutase 2 and uncoupling protein 2 in septic acute kidney injury.

PubMed

Chen, Guang-Dao; Zhang, Jun-Liang; Chen, Yi-Ting; Zhang, Ju-Xing; Wang, Tao; Zeng, Qi-Yi

2018-04-01

The aim of the present study was to explore the effects and mechanisms of insulin on mitochondrial oxidative stress in septic acute kidney injury (AKI). Male Sprague Dawley rats were divided randomly into four groups: Control group, sham surgery group, cecal ligation and puncture (CLP) group, and CLP plus insulin group. Blood specimens and kidney tissues were obtained at 12 and 24 h after surgery as separate experiments. Analyses of histology and indicators of renal injury [blood urea nitrogen (BUN) and serum creatinine (CRE) and neutrophil gelatinase-associated lipocalin (NGAL)], mitochondrial function [adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP)], oxidative stress [inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS) and nitric oxide (NO)], endogenous antioxidant systems [superoxide dismutase (SOD) and glutathione (GSH)] as well as the expression of uncoupling protein (UCP), PINK1 protein (a major mediator of mitophagy), PGC1α protein (a major regulator of mitochondrial biogenesis) were performed. Compared with CLP group, the CLP plus insulin group had milder histological damage, higher levels of ATP and MMP as well as lower levels of BUN, serum CRE and NGAL, intrarenal iNOS, mitochondrial ROS and total NO. Moreover, the CLP plus insulin group demonstrated increased expression of SOD2 and UCP2. In contrast, insulin administration suppressed mitophagy meanwhile did not upregulate total GSH and induce mitochondrial biogenesis following CLP. These findings indicated that the upregulation of SOD2 and UCP2 may be involved in insulin protecting against mitochondrial oxidative stress in septic AKI.

Nordihydroguaiaretic acid protects against high-fat diet-induced fatty liver by activating AMP-activated protein kinase in obese mice

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

Lee, Myoung-Su; Kim, Daeyoung; Jo, Keunae

Research highlights: {yields} NDGA decreases high-fat diet-induced body weight gain and adiposity. {yields} NDGA reduces high-fat diet-induced triglyceride accumulation in liver. {yields} NDGA improves lipid storage in vitro through altering lipid regulatory proteins. {yields} Inhibition of lipid storage in vivo and in vitro is mediated by AMPK activation. -- Abstract: Nonalcoholic fatty liver disease, one of the most common causes of chronic liver disease, is strongly associated with metabolic syndrome. Nordihydroguaiaretic acid (NDGA) has been reported to inhibit lipoprotein lipase; however, the effect of NDGA on hepatic lipid metabolism remains unclear. We evaluated body weight, adiposity, liver histology, and hepaticmore » triglyceride content in high-fat diet (HFD)-fed C57BL/6J mice treated with NDGA. In addition, we characterized the underlying mechanism of NDGA's effects in HepG2 hepatocytes by Western blot and RT-PCR analysis. NDGA (100 or 200 mg/kg/day) reduced weight gain, fat pad mass, and hepatic triglyceride accumulation, and improved serum lipid parameters in mice fed a HFD for 8 weeks. NDGA significantly increased AMP-activated protein kinase (AMPK) phosphorylation in the liver and in HepG2 hepatocytes. NDGA downregulated the level of mature SREBP-1 and its target genes (acetyl-CoA carboxylase and fatty acid synthase), but, it upregulated expression of genes involved in fatty acid oxidation, such as peroxisome proliferator-activated receptor (PPAR){alpha}, PPAR{gamma} coactivator-1, carnitine palmitoyl transferase-1, and uncoupling protein-2. The specific AMPK inhibitor compound C attenuated the effects of NDGA on expression of lipid metabolism-related proteins in HepG2 hepatocytes. The beneficial effects of NDGA on HFD-induced hepatic triglyceride accumulation are mediated through AMPK signaling pathways, suggesting a potential target for preventing NAFLD.« less

A controlled-release mitochondrial protonophore reverses hypertriglyceridemia, nonalcoholic steatohepatitis, and diabetes in lipodystrophic mice.

PubMed

Abulizi, Abudukadier; Perry, Rachel J; Camporez, João Paulo G; Jurczak, Michael J; Petersen, Kitt Falk; Aspichueta, Patricia; Shulman, Gerald I

2017-07-01

Lipodystrophy is a rare disorder characterized by complete or partial loss of adipose tissue. Patients with lipodystrophy exhibit hypertriglyceridemia, severe insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). Efforts to ameliorate NASH in lipodystrophies with pharmacologic agents have met with limited success. We examined whether a controlled-release mitochondrial protonophore (CRMP) that produces mild liver-targeted mitochondrial uncoupling could decrease hypertriglyceridemia and reverse NASH and diabetes in a mouse model (fatless AZIP/F-1 mice) of severe lipodystrophy and diabetes. After 4 wk of oral CRMP (2 mg/kg body weight per day) or vehicle treatment, mice underwent hyperinsulinemic-euglycemic clamps combined with radiolabeled glucose to assess liver and muscle insulin responsiveness and tissue lipid measurements. CRMP treatment reversed hypertriglyceridemia and insulin resistance in liver and skeletal muscle. Reversal of insulin resistance could be attributed to reductions in diacylglycerol content and reduced PKC-ε and PKC-θ activity in liver and muscle respectively. CRMP treatment also reversed NASH as reflected by reductions in plasma aspartate aminotransferase and alanine aminotransferase concentrations; hepatic steatosis; and hepatic expression of IL-1α, -β, -2, -4, -6, -10, -12, CD69, and caspase 3 and attenuated activation of the IRE-1α branch of the unfolded protein response. Taken together, these results provide proof of concept for the development of liver-targeted mitochondrial uncoupling agents as a potential novel therapy for lipodystrophy-associated hypertriglyceridemia, NASH and diabetes.-Abulizi, A., Perry, R. J., Camporez, J. P. G., Jurczak, M. J., Petersen, K. F., Aspichueta, P., Shulman, G. I. A controlled-release mitochondrial protonophore reverses hypertriglyceridemia, nonalcoholic steatohepatitis, and diabetes in lipodystrophic mice. © FASEB.

Myocardial architecture and patient variability in clinical patterns of atrial fibrillation

NASA Astrophysics Data System (ADS)

Manani, Kishan A.; Christensen, Kim; Peters, Nicholas S.

2016-10-01

Atrial fibrillation (AF) increases the risk of stroke by a factor of 4-5 and is the most common abnormal heart rhythm. The progression of AF with age, from short self-terminating episodes to persistence, varies between individuals and is poorly understood. An inability to understand and predict variation in AF progression has resulted in less patient-specific therapy. Likewise, it has been a challenge to relate the microstructural features of heart muscle tissue (myocardial architecture) with the emergent temporal clinical patterns of AF. We use a simple model of activation wave-front propagation on an anisotropic structure, mimicking heart muscle tissue, to show how variation in AF behavior arises naturally from microstructural differences between individuals. We show that the stochastic nature of progressive transversal uncoupling of muscle strands (e.g., due to fibrosis or gap junctional remodeling), as occurs with age, results in variability in AF episode onset time, frequency, duration, burden, and progression between individuals. This is consistent with clinical observations. The uncoupling of muscle strands can cause critical architectural patterns in the myocardium. These critical patterns anchor microreentrant wave fronts and thereby trigger AF. It is the number of local critical patterns of uncoupling as opposed to global uncoupling that determines AF progression. This insight may eventually lead to patient-specific therapy when it becomes possible to observe the cellular structure of a patient's heart.

Heat Shock Factor 1 Deficiency Affects Systemic Body Temperature Regulation.

PubMed

Ingenwerth, Marc; Noichl, Erik; Stahr, Anna; Korf, Horst-Werner; Reinke, Hans; von Gall, Charlotte

2016-01-01

Heat shock factor 1 (HSF1) is a ubiquitous heat-sensitive transcription factor that mediates heat shock protein transcription in response to cellular stress, such as increased temperature, in order to protect the organism against misfolded proteins. In this study, we analysed the effect of HSF1 deficiency on core body temperature regulation. Body temperature, locomotor activity, and food consumption of wild-type mice and HSF1-deficient mice were recorded. Prolactin and thyroid-stimulating hormone levels were measured by ELISA. Gene expression in brown adipose tissue was analysed by quantitative real-time PCR. Hypothalamic HSF1 and its co-localisation with tyrosine hydroxylase was analysed using confocal laser scanning microscopy. HSF1-deficient mice showed an increase in core body temperature (hyperthermia), decreased overall locomotor activity, and decreased levels of prolactin in pituitary and blood plasma reminiscent of cold adaptation. HSF1 could be detected in various hypothalamic regions involved in temperature regulation, suggesting a potential role of HSF1 in hypothalamic thermoregulation. Moreover, HSF1 co-localises with tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, suggesting a potential role of HSF1 in the hypothalamic control of prolactin release. In brown adipose tissue, levels of prolactin receptor and uncoupled protein 1 were increased in HSF1-deficient mice, consistent with an up-regulation of heat production. Our data suggest a role of HSF1 in systemic thermoregulation. © 2015 S. Karger AG, Basel.

Control of Retrograde Signaling by Rapid Turnover of GENOMES UNCOUPLED11[OPEN

PubMed Central

Chalvin, Camille; Wu, Xu Na

2018-01-01

The exchange of signals between cellular compartments coordinates development and differentiation, modulates metabolic pathways, and triggers responses to environmental conditions. The proposed central regulator of plastid-to-nucleus retrograde signaling, GENOMES UNCOUPLED1 (GUN1), is present at very low levels, which has hampered the discovery of its precise molecular function. Here, we show that the Arabidopsis (Arabidopsis thaliana) GUN1 protein accumulates to detectable levels only at very early stages of leaf development, where it functions in the regulation of chloroplast biogenesis. GUN1 mRNA is present at high levels in all tissues, but GUN1 protein undergoes rapid degradation (with an estimated half-life of ∼4 h) in all tissues where chloroplast biogenesis has been completed. The rapid turnover of GUN1 is controlled mainly by the chaperone ClpC1, suggesting degradation of GUN1 by the Clp protease. Degradation of GUN1 slows under stress conditions that alter retrograde signaling, thus ensuring that the plant has sufficient GUN1 protein. We also find that the pentatricopeptide repeat motifs of GUN1 are important determinants of GUN1 stability. Moreover, overexpression of GUN1 causes an early flowering phenotype, suggesting a function of GUN1 in developmental phase transitions beyond chloroplast biogenesis. Taken together, our results provide new insight into the regulation of GUN1 by proteolytic degradation, uncover its function in early chloroplast biogenesis, and suggest a role in developmental phase transitions. PMID:29367233

Uncoupling binding of substrate CO from turnover by vanadium nitrogenase.

PubMed

Lee, Chi Chung; Fay, Aaron W; Weng, Tsu-Chien; Krest, Courtney M; Hedman, Britt; Hodgson, Keith O; Hu, Yilin; Ribbe, Markus W

2015-11-10

Biocatalysis by nitrogenase, particularly the reduction of N2 and CO by this enzyme, has tremendous significance in environment- and energy-related areas. Elucidation of the detailed mechanism of nitrogenase has been hampered by the inability to trap substrates or intermediates in a well-defined state. Here, we report the capture of substrate CO on the resting-state vanadium-nitrogenase in a catalytically competent conformation. The close resemblance of this active CO-bound conformation to the recently described structure of CO-inhibited molybdenum-nitrogenase points to the mechanistic relevance of sulfur displacement to the activation of iron sites in the cofactor for CO binding. Moreover, the ability of vanadium-nitrogenase to bind substrate in the resting-state uncouples substrate binding from subsequent turnover, providing a platform for generation of defined intermediate(s) of both CO and N2 reduction.

Increased hepatic mitochondrial FA oxidation reduces plasma and liver TG levels and is associated with regulation of UCPs and APOC-III in rats

PubMed Central

Lindquist, Carine; Bjørndal, Bodil; Rossmann, Christine Renate; Tusubira, Deusdedit; Svardal, Asbjørn; Røsland, Gro Vatne; Tronstad, Karl Johan; Hallström, Seth; Berge, Rolf Kristian

2017-01-01

Hepatic mitochondrial function, APOC-III, and LPL are potential targets for triglyceride (TG)-lowering drugs. After 3 weeks of dietary treatment with the compound 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), the hepatic mitochondrial FA oxidation increased more than 5-fold in male Wistar rats. Gene expression analysis in liver showed significant downregulation of APOC-III and upregulation of LPL and the VLDL receptor. This led to lower hepatic (53%) and plasma (73%) TG levels. Concomitantly, liver-specific biomarkers related to mitochondrial biogenesis and function (mitochondrial DNA, citrate synthase activity, and cytochrome c and TFAM gene expression) were elevated. Interestingly, 1-triple TTA lowered plasma acetylcarnitine levels, whereas the concentration of β-hydroxybutyrate was increased. The hepatic energy state was reduced in 1-triple TTA-treated rats, as reflected by increased AMP/ATP and decreased ATP/ADP ratios, whereas the energy state remained unchanged in muscle and heart. The 1-triple TTA administration induced gene expression of uncoupling protein (UCP)2 and UCP3 in liver. In conclusion, the 1-triple TTA-mediated clearance of blood TG may result from lowered APOC-III production, increased hepatic LPL gene expression, mitochondrial FA oxidation, and (re)uptake of VLDL facilitating drainage of FAs to the liver for β-oxidation and production of ketone bodies as extrahepatic fuel. The possibility that UCP2 and UCP3 mediate a moderate degree of mitochondrial uncoupling should be considered. PMID:28473603

Development of a Computational Assay for the Estrogen Receptor

DTIC Science & Technology

2006-07-01

University Ashley Deline, Senior Thesis in chemistry, " Molecular Dynamic Simulations of a Glycoform and its Constituent Parts Related to Rheumatoid Arthritis...involves running a long molecular dynamics (MD) simulation of the uncoupled receptor in order to sample the protein’s unique conformations. The second...Receptor binding domain. * Performed several long molecular dynamics simulations (800 ps - 3 ns) on the ligand-ER system using ligands with known

Transcriptome response signatures associated with the overexpression of a mitochondrial uncoupling protein (AtUCP1) in tobacco.

PubMed

Laitz, Alessandra Vasconcellos Nunes; Acencio, Marcio Luis; Budzinski, Ilara G F; Labate, Mônica T V; Lemke, Ney; Ribolla, Paulo Eduardo Martins; Maia, Ivan G

2015-01-01

Mitochondrial inner membrane uncoupling proteins (UCP) dissipate the proton electrochemical gradient established by the respiratory chain, thus affecting the yield of ATP synthesis. UCP overexpression in plants has been correlated with oxidative stress tolerance, improved photosynthetic efficiency and increased mitochondrial biogenesis. This study reports the main transcriptomic responses associated with the overexpression of an UCP (AtUCP1) in tobacco seedlings. Compared to wild-type (WT), AtUCP1 transgenic seedlings showed unaltered ATP levels and higher accumulation of serine. By using RNA-sequencing, a total of 816 differentially expressed genes between the investigated overexpressor lines and the untransformed WT control were identified. Among them, 239 were up-regulated and 577 were down-regulated. As a general response to AtUCP1 overexpression, noticeable changes in the expression of genes involved in energy metabolism and redox homeostasis were detected. A substantial set of differentially expressed genes code for products targeted to the chloroplast and mainly involved in photosynthesis. The overall results demonstrate that the alterations in mitochondrial function provoked by AtUCP1 overexpression require important transcriptomic adjustments to maintain cell homeostasis. Moreover, the occurrence of an important cross-talk between chloroplast and mitochondria, which culminates in the transcriptional regulation of several genes involved in different pathways, was evidenced.

Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

NASA Astrophysics Data System (ADS)

Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

2017-06-01

MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

Low-level lasers affect uncoupling protein gene expression in skin and skeletal muscle tissues

NASA Astrophysics Data System (ADS)

Canuto, K. S.; Sergio, L. P. S.; Paoli, F.; Mencalha, A. L.; Fonseca, A. S.

2016-03-01

Wavelength, frequency, power, fluence, and emission mode determine the photophysical, photochemical, and photobiological responses of biological tissues to low-level lasers. Free radicals are involved in these responses acting as second messengers in intracellular signaling processes. Irradiated cells present defenses against these chemical species to avoid unwanted effects, such as uncoupling proteins (UCPs), which are part of protective mechanisms and minimize the effects of free radical generation in mitochondria. In this work UCP2 and UCP3 mRNA gene relative expression in the skin and skeletal muscle tissues of Wistar rats exposed to low-level red and infrared lasers was evaluated. Samples of the skin and skeletal muscle tissue of Wistar rats exposed to low-level red and infrared lasers were withdrawn for total RNA extraction, cDNA synthesis, and the evaluation of gene expression by quantitative polymerase chain reaction. UCP2 and UCP3 mRNA expression was differently altered in skin and skeletal muscle tissues exposed to lasers in a wavelength-dependent effect, with the UCP3 mRNA expression dose-dependent. Alteration on UCP gene expression could be part of the biostimulation effect and is necessary to make cells exposed to red and infrared low-level lasers more resistant or capable of adapting in damaged tissues or diseases.

Systems Analysis of the Response of Photosynthesis, Metabolism, and Growth to an Increase in Irradiance in the Photosynthetic Model Organism Chlamydomonas reinhardtii[C][W][OPEN

PubMed Central

Mettler, Tabea; Mühlhaus, Timo; Hemme, Dorothea; Schöttler, Mark-Aurel; Rupprecht, Jens; Idoine, Adam; Veyel, Daniel; Pal, Sunil Kumar; Yaneva-Roder, Liliya; Winck, Flavia Vischi; Sommer, Frederik; Vosloh, Daniel; Seiwert, Bettina; Erban, Alexander; Burgos, Asdrubal; Arvidsson, Samuel; Schönfelder, Stephanie; Arnold, Anne; Günther, Manuela; Krause, Ursula; Lohse, Marc; Kopka, Joachim; Nikoloski, Zoran; Mueller-Roeber, Bernd; Willmitzer, Lothar; Bock, Ralph; Schroda, Michael; Stitt, Mark

2014-01-01

We investigated the systems response of metabolism and growth after an increase in irradiance in the nonsaturating range in the algal model Chlamydomonas reinhardtii. In a three-step process, photosynthesis and the levels of metabolites increased immediately, growth increased after 10 to 15 min, and transcript and protein abundance responded by 40 and 120 to 240 min, respectively. In the first phase, starch and metabolites provided a transient buffer for carbon until growth increased. This uncouples photosynthesis from growth in a fluctuating light environment. In the first and second phases, rising metabolite levels and increased polysome loading drove an increase in fluxes. Most Calvin-Benson cycle (CBC) enzymes were substrate-limited in vivo, and strikingly, many were present at higher concentrations than their substrates, explaining how rising metabolite levels stimulate CBC flux. Rubisco, fructose-1,6-biosphosphatase, and seduheptulose-1,7-bisphosphatase were close to substrate saturation in vivo, and flux was increased by posttranslational activation. In the third phase, changes in abundance of particular proteins, including increases in plastidial ATP synthase and some CBC enzymes, relieved potential bottlenecks and readjusted protein allocation between different processes. Despite reasonable overall agreement between changes in transcript and protein abundance (R2 = 0.24), many proteins, including those in photosynthesis, changed independently of transcript abundance. PMID:24894045

PSD-95 interacts with NBCn1 and enhances channel-like activity without affecting Na/HCO(3) cotransport.

PubMed

Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R Kyle; Smith, Yoland; Yun, C Chris; Choi, Inyeong

2012-01-01

The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO(3)(-) movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO(3) cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO(3) cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. Copyright © 2012 S. Karger AG, Basel.

PSD-95 Interacts with NBCn1 and Enhances Channel-like Activity without Affecting Na/HCO3 Cotransport

PubMed Central

Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R. Kyle; Smith, Yoland; Yun, C. Chris; Choi, Inyeong

2013-01-01

Background/Aims The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. PMID:23183381

Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

PubMed Central

Beck, Ilse M.; Drebert, Zuzanna J.; Hoya-Arias, Ruben; Bahar, Ali A.; Devos, Michael; Clarisse, Dorien; Desmet, Sofie; Bougarne, Nadia; Ruttens, Bart; Gossye, Valerie; Denecker, Geertrui; Lievens, Sam; Bracke, Marc; Tavernier, Jan; Declercq, Wim; Gevaert, Kris; Berghe, Wim Vanden; Haegeman, Guy; De Bosscher, Karolien

2013-01-01

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA’s anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells. PMID:23935933

Active management of plant canopy temperature as a tool for modifying plant metabolic activity

USDA-ARS?s Scientific Manuscript database

The relationship between a plant and its thermal environment is a major determiner of its growth and development. Since plants grow and develop within continuously variable thermal environments, they are subjected to continuous thermal variation over their life cycle. Transpiration serves to uncoupl...

Visual information underpinning skilled anticipation: The effect of blur on a coupled and uncoupled in situ anticipatory response.

PubMed

Mann, David L; Abernethy, Bruce; Farrow, Damian

2010-07-01

Coupled interceptive actions are understood to be the result of neural processing-and visual information-which is distinct from that used for uncoupled perceptual responses. To examine the visual information used for action and perception, skilled cricket batters anticipated the direction of balls bowled toward them using a coupled movement (an interceptive action that preserved the natural coupling between perception and action) or an uncoupled (verbal) response, in each of four different visual blur conditions (plano, +1.00, +2.00, +3.00). Coupled responses were found to be better than uncoupled ones, with the blurring of vision found to result in different effects for the coupled and uncoupled response conditions. Low levels of visual blur did not affect coupled anticipation, a finding consistent with the comparatively poorer visual information on which online interceptive actions are proposed to rely. In contrast, some evidence was found to suggest that low levels of blur may enhance the uncoupled verbal perception of movement.

A cell death assay for assessing the mitochondrial targeting of proteins.

PubMed

Camara Teixeira, Daniel; Cordonier, Elizabeth L; Wijeratne, Subhashinee S K; Huebbe, Patricia; Jamin, Augusta; Jarecke, Sarah; Wiebe, Matthew; Zempleni, Janos

2018-06-01

The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

Adipose tissue uncoupling protein 1 levels and function are increased in a mouse model of developmental obesity induced by maternal exposure to high-fat diet.

PubMed

Bytautiene Prewit, E; Porter, C; La Rosa, M; Bhattarai, N; Yin, H; Gamble, P; Kechichian, T; Sidossis, L S

2018-05-17

With brown adipose tissue (BAT) becoming a possible therapeutic target to counteract obesity, the prenatal environment could represent a critical window to modify BAT function and browning of white AT. We investigated if levels of uncoupling protein 1 (UCP1) and UCP1-mediated thermogenesis are altered in offspring exposed to prenatal obesity. Female CD-1 mice were fed a high-fat (HF) or standard-fat (SF) diet for 3 months before breeding. After weaning, all pups were placed on SF. UCP1 mRNA and protein levels were quantified using quantitative real-time PCR and Western blot analysis, respectively, in brown (BAT), subcutaneous (SAT) and visceral (VAT) adipose tissues at 6 months of age. Total and UCP1-dependent mitochondrial respiration were determined by high-resolution respirometry. A Student's t-test and Mann-Whitney test were used (significance: P<0.05). UCP1 mRNA levels were not different between the HF and SF offspring. UCP1 protein levels, total mitochondrial respiration and UCP1-dependent respiration were significantly higher in BAT from HF males (P=0.02, P=0.04, P=0.005, respectively) and females (P=0.01, P=0.04, P=0.02, respectively). In SAT, the UCP1 protein was significantly lower in HF females (P=0.03), and the UCP1-dependent thermogenesis was significantly lower from HF males (P=0.04). In VAT, UCP1 protein levels and UCP1-dependent respiration were significantly lower only in HF females (P=0.03, P=0.04, respectively). There were no differences in total respiration in SAT and VAT. Prenatal exposure to maternal obesity leads to significant increases in UCP1 levels and function in BAT in offspring with little impact on UCP1 levels and function in SAT and VAT.

Protection against the Metabolic Syndrome by Guar Gum-Derived Short-Chain Fatty Acids Depends on Peroxisome Proliferator-Activated Receptor γ and Glucagon-Like Peptide-1

PubMed Central

den Besten, Gijs; Gerding, Albert; van Dijk, Theo H.; Ciapaite, Jolita; Bleeker, Aycha; van Eunen, Karen; Havinga, Rick; Groen, Albert K.; Reijngoud, Dirk-Jan; Bakker, Barbara M.

2015-01-01

The dietary fiber guar gum has beneficial effects on obesity, hyperglycemia and hypercholesterolemia in both humans and rodents. The major products of colonic fermentation of dietary fiber, the short-chain fatty acids (SCFAs), have been suggested to play an important role. Recently, we showed that SCFAs protect against the metabolic syndrome via a signaling cascade that involves peroxisome proliferator-activated receptor (PPAR) γ repression and AMP-activated protein kinase (AMPK) activation. In this study we investigated the molecular mechanism via which the dietary fiber guar gum protects against the metabolic syndrome. C57Bl/6J mice were fed a high-fat diet supplemented with 0% or 10% of the fiber guar gum for 12 weeks and effects on lipid and glucose metabolism were studied. We demonstrate that, like SCFAs, also guar gum protects against high-fat diet-induced metabolic abnormalities by PPARγ repression, subsequently increasing mitochondrial uncoupling protein 2 expression and AMP/ATP ratio, leading to the activation of AMPK and culminating in enhanced oxidative metabolism in both liver and adipose tissue. Moreover, guar gum markedly increased peripheral glucose clearance, possibly mediated by the SCFA-induced colonic hormone glucagon-like peptide-1. Overall, this study provides novel molecular insights into the beneficial effects of guar gum on the metabolic syndrome and strengthens the potential role of guar gum as a dietary-fiber intervention. PMID:26292284

Factor XI and Contact Activation as Targets for Antithrombotic Therapy

PubMed Central

Gailani, David; Bane, Charles E.; Gruber, Andras

2015-01-01

Summary The most commonly used anticoagulants produce therapeutic antithrombotic effects either by inhibiting thrombin or factor Xa, or by lowering the plasma levels of the precursors of these key enzymes, prothrombin and factor X. These drugs do not distinguish between thrombin generation contributing to thrombosis from thrombin generation required for hemostasis. Thus, anticoagulants increase bleeding risk, and many patients who would benefit from therapy go untreated because of comorbidities that place them at unacceptable risk for hemorrhage. Studies in animals demonstrate that components of the plasma contact activation system contribute to experimentally-induced thrombosis, despite playing little or no role in hemostasis. Attention has focused on factor XII, the zymogen of a protease (factor XIIa) that initiates contact activation when blood is exposed to foreign surfaces; and factor XI, the zymogen of the protease factor XIa, which links contact activation to the thrombin generation mechanism. In the case of factor XI, epidemiologic data indicate this protein contributes to stroke and venous thromboembolism, and perhaps myocardial infarction, in humans. A phase 2 trial showing that reduction of factor XI may be more effective than low-molecular-weight heparin at preventing venous thrombosis during knee replacement surgery provides proof of concept for the premise that an antithrombotic effect can be uncoupled from an anticoagulant effect in humans by targeting components of contact activation. Here we review data on the role of factor XI and factor XII in thrombosis, and results of pre-clinical and human trials for therapies targeting these proteins. PMID:25976012

Moringa oleifera Lam. improves lipid metabolism during adipogenic differentiation of human stem cells.

PubMed

Barbagallo, I; Vanella, L; Distefano, A; Nicolosi, D; Maravigna, A; Lazzarino, G; Di Rosa, M; Tibullo, D; Acquaviva, R; Li Volti, G

2016-12-01

Moringa oleifera Lam., a multipurpose tree, is used traditionally for its nutritional and medicinal properties. It has been used for the treatment of a variety of conditions, including inflammation, cancer and metabolic disorders. We investigated the effect of Moringa oleifera Lam. on adipogenic differentiation of human adipose-derived mesenchymal stem cells and its impact on lipid metabolism and cellular antioxidant systems. We showed that Moringa oleifera Lam. treatment during adipogenic differentiation reduces inflammation, lipid accumulation and induces thermogenesis by activation of uncoupling protein 1 (UCP1), sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor alpha (PPARα), and coactivator 1 alpha (PGC1α). In addition, Moringa oleifera Lam. induces heme oxygenase-1 (HO-1), a well established protective and antioxidant enzyme. Finally Moringa oleifera Lam. significantly decreases the expression of molecules involved in adipogenesis and upregulates the expression of mediators involved in thermogenesis and lipid metabolism. Our results suggest that Moringa oleifera Lam. may promote the brown remodeling of white adipose tissue inducing thermogenesis and improving metabolic homeostasis.

Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics

PubMed Central

Tan, Dun-Xian; Manchester, Lucien C.; Qin, Lilan; Reiter, Russel J.

2016-01-01

Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT) is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s). Melatonin protects mitochondria by scavenging reactive oxygen species (ROS), inhibiting the mitochondrial permeability transition pore (MPTP), and activating uncoupling proteins (UCPs). Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria. PMID:27999288

Fatty Acids Change the Conformation of Uncoupling Protein 1 (UCP1)*

PubMed Central

Divakaruni, Ajit S.; Humphrey, Dickon M.; Brand, Martin D.

2012-01-01

UCP1 catalyzes proton leak across the mitochondrial inner membrane to disengage substrate oxidation from ATP production. It is well established that UCP1 is activated by fatty acids and inhibited by purine nucleotides, but precisely how this regulation occurs remains unsettled. Although fatty acids can competitively overcome nucleotide inhibition in functional assays, fatty acids have little effect on purine nucleotide binding. Here, we present the first demonstration that fatty acids induce a conformational change in UCP1. Palmitate dramatically changed the binding kinetics of 2′/3′-O-(N-methylanthraniloyl)-GDP, a fluorescently labeled nucleotide analog, for UCP1. Furthermore, palmitate accelerated the rate of enzymatic proteolysis of UCP1. The altered kinetics of both processes indicate that fatty acids change the conformation of UCP1, reconciling the apparent discrepancy between existing functional and ligand binding data. Our results provide a framework for how fatty acids and nucleotides compete to regulate the activity of UCP1. PMID:22952235

49 CFR 215.125 - Defective uncoupling device.

Code of Federal Regulations, 2014 CFR

2014-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Freight Car Components Draft System § 215.125 Defective uncoupling device. A railroad may not place or continue in service a car, if the car has an uncoupling device without sufficient vertical and lateral clearance to prevent— (a...

49 CFR 215.125 - Defective uncoupling device.

Code of Federal Regulations, 2011 CFR

2011-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Freight Car Components Draft System § 215.125 Defective uncoupling device. A railroad may not place or continue in service a car, if the car has an uncoupling device without sufficient vertical and lateral clearance to prevent— (a...

49 CFR 215.125 - Defective uncoupling device.

Code of Federal Regulations, 2010 CFR

2010-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Freight Car Components Draft System § 215.125 Defective uncoupling device. A railroad may not place or continue in service a car, if the car has an uncoupling device without sufficient vertical and lateral clearance to prevent— (a...

49 CFR 215.125 - Defective uncoupling device.

Code of Federal Regulations, 2013 CFR

2013-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Freight Car Components Draft System § 215.125 Defective uncoupling device. A railroad may not place or continue in service a car, if the car has an uncoupling device without sufficient vertical and lateral clearance to prevent— (a...

49 CFR 215.125 - Defective uncoupling device.

Code of Federal Regulations, 2012 CFR

2012-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION RAILROAD FREIGHT CAR SAFETY STANDARDS Freight Car Components Draft System § 215.125 Defective uncoupling device. A railroad may not place or continue in service a car, if the car has an uncoupling device without sufficient vertical and lateral clearance to prevent— (a...

Depressed mitochondrial function and electron transport Complex II-mediated H2O2 production in the cortex of type 1 diabetic rodents.

PubMed

Chowdhury, Subir Roy; Djordjevic, Jelena; Thomson, Ella; Smith, Darrell R; Albensi, Benedict C; Fernyhough, Paul

2018-05-23

Abnormalities in mitochondrial function under diabetic conditions can lead to deficits in function of cortical neurons and their support cells exhibiting a pivotal role in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. We aimed to assess simultaneously mitochondrial respiration rates and membrane potential or H 2 O 2 generation and proteins involved in mitochondrial dynamics, antioxidants and AMPK/SIRT/PGC-1α pathway activity in cortex under diabetic conditions. Cortical mitochondria from streptozotocin (STZ)-induced type 1 diabetic rats or mice, and aged-match controls were used for simultaneous measurements of mitochondrial respiration rates and mitochondrial membrane potential (mtMP) or H 2 O 2 using OROBOROS oxygraph and measurements of enzymatic activities by a spectrophotometer. Protein levels in cortical mitochondria and homogenates were determined by Western blotting. Mitochondrial coupled respiration rates and FCCP-induced uncoupled respiration rates were significantly decreased in mitochondria of STZ-diabetic cortical rats compared to controls. The mtMP in the presence of ADP was significantly depolarized and succinate-dependent respiration rates and H 2 O 2 were significantly diminished in mitochondria of diabetic animals compared to controls, accompanied with reduced expression of CuZn- and Mn-superoxide dismutase. The enzymatic activities of Complex I, II, and IV and protein levels of certain components of Complex I and II, mitofusin 2 (Mfn2), dynamin-related protein 1 (DRP1), P-AMPK, SIRT2 and PGC-1α were significantly diminished in diabetic cortex. Deficits in mitochondrial function, dynamics, and antioxidant capabilities putatively mediated through sub-optimal AMPK/SIRT/PGC-1α signaling, are involved in the development of early sub-clinical neurodegeneration in the cortex under diabetic conditions. Copyright © 2017. Published by Elsevier Inc.

Ononitol monohydrate enhances PRDM16 & UCP-1 expression, mitochondrial biogenesis and insulin sensitivity via STAT6 and LTB4R in maturing adipocytes.

PubMed

Subash-Babu, P; Alshatwi, Ali A

2018-03-01

Ononitol monohydrate (OMH), a glycoside was originally isolated from Cassia tora (Linn.). Glycosides regulate lipid metabolism but scientific validation desired. Hence, we aimed to evaluate the effect of OMH on enhancing mitochondrial potential, mitochondrial biogenesis, upregulate the expression of brown adipogenesis specific genes in maturing adipocytes. In addition, we observed the inter-relation between adipocyte and T-lymphocyte; whether, OMH treated adipocyte-condition medium stimulate T-cell chemokine linked with insulin resistance. In a dose dependent manner OMH treated to preadipocyte significantly inhibited maturation and enhanced mitochondrial biogenesis, it was confirmed by Oil red 'O and Nile red stain without inducing cytotoxicity. The mRNA levels of adipocyte browning related genes such as, PR domain containing 16 (PRDM16), peroxisome proliferator activated receptor gamma coactivator 1 alpha (PPARγC1α) and uncoupling protein-1 (UCP-1) have been significantly upregulated. In addition, adipogenic transcription factors [such as proliferator activated receptor γ (PPARγ), CCAAT/enhancer binding protein (C/EBPα) and sterol regulatory element binding protein-1c (SREBP-1c)] and adipogenic genes were significantly down-regulated by treatment with OMH when compared to control cells. Protein expression levels of adiponectin have been increased; leptin, C/EBPα and leukotriene B4 receptor (LTB4R) were down regulated by OMH in mature adipocytes. In addition, adipocyte condition medium and OMH treated T-lymphocyte, significantly increased insulin signaling pathway related mRNAs, such as interlukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT 6 ) and decreased leukotriene B4 (LTB 4 ). The present findings suggest that OMH increased browning factors in differentiating and maturing preadipocyte also decreased adipose tissue inflammation as well as the enhanced insulin signaling. Copyright © 2018. Published by Elsevier Masson SAS.

Creating leptin-like biofunctions by active immunization against chicken leptin receptor in growing chickens.

PubMed

Lei, M M; Wu, S Q; Shao, X B; Li, X W; Chen, Z; Ying, S J; Shi, Z D

2015-01-01

In this study, immunization against chicken leptin receptor (cLEPR) extracellular domain (ECD) was applied to investigate leptin regulation and LEPR biofunction in growing chicken pullets. A recombinant protein (cLEPR ECD) based on the cLEPR complemenary DNA sequence corresponding to the 582nd to 796th amino acid residues of cLEPR mature peptide was prepared and used as antigen. Immunization against cLEPR ECD in growing chickens increased anti-cLEPR ECD antibody titers in blood, enhanced proportions of phosphorylated janus kinase 2 (JAK2) and served as signal transducer and activator of transcription 3 (STAT3) protein in liver tissue. Chicken live weight gain and abdominal fat mass were significantly decreased (P < 0.05), but feed intake was stimulated by cLEPR ECD immunization (P < 0.05). The treatment also upregulated the gene expression levels of lepR, AMP-activated protein kinase (AMPK), acetyl CoA carboxylase-2 (ACC2), and uncoupling protein 3 (UCP3) in liver, abdominal fat, and breast muscle (P < 0.05) but decreased fasn expression levels (P < 0.01). Apart from that of lepR, the expression of appetite-regulating genes, such as orexigenic genes, agouti-related peptide (AgRP) and neuropeptide Y (NPY), were upregulated (P < 0.01), whereas the anorexigenic gene proopiomelanocortin (POMC) was downregulated in the hypothalamic tissue of cLEPR-immunized pullets (P < 0.01). Blood concentrations of metabolic molecules, such as glucose, triglycerides, and very-low-density lipoprotein, were significantly decreased in cLEPR-immunized pullets but those of cholesterol, high-density lipoprotein, and low-density lipoprotein increased. These results demonstrate that antibodies to membrane proximal cLEPR ECD enhance cLEPR signal transduction, which stimulates metabolism and reduces fat deposition in chickens. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial neuronal uncoupling proteins: a target for potential disease-modification in Parkinson's disease

PubMed Central

2012-01-01

This review gives a brief insight into the role of mitochondrial dysfunction and oxidative stress in the converging pathogenic processes involved in Parkinson's disease (PD). Mitochondria provide cellular energy in the form of ATP via oxidative phosphorylation, but as an integral part of this process, superoxides and other reactive oxygen species are also produced. Excessive free radical production contributes to oxidative stress. Cells have evolved to handle such stress via various endogenous anti-oxidant proteins. One such family of proteins is the mitochondrial uncoupling proteins (UCPs), which are anion carriers located in the mitochondrial inner membrane. There are five known homologues (UCP1 to 5), of which UCP4 and 5 are predominantly expressed in neural cells. In a series of previous publications, we have shown how these neuronal UCPs respond to 1-methyl-4-phenylpyridinium (MPP+; toxic metabolite of MPTP) and dopamine-induced toxicity to alleviate neuronal cell death by preserving ATP levels and mitochondrial membrane potential, and reducing oxidative stress. We also showed how their expression can be influenced by nuclear factor kappa-B (NF-κB) signaling pathway specifically in UCP4. Furthermore, we previously reported an interesting link between PD and metabolic processes through the protective effects of leptin (hormone produced by adipocytes) acting via UCP2 against MPP+-induced toxicity. There is increasing evidence that these endogenous neuronal UCPs can play a vital role to protect neurons against various pathogenic stresses including those associated with PD. Their expression, which can be induced, may well be a potential therapeutic target for various drugs to alleviate the harmful effects of pathogenic processes in PD and hence modify the progression of this disease. PMID:23210978

Genipin-induced inhibition of uncoupling protein-2 sensitizes drug-resistant cancer cells to cytotoxic agents.

PubMed

Mailloux, Ryan J; Adjeitey, Cyril Nii-Klu; Harper, Mary-Ellen

2010-10-13

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

The antioxidant uncoupling protein 2 stimulates hnRNPA2/B1, GLUT1 and PKM2 expression and sensitizes pancreas cancer cells to glycolysis inhibition.

PubMed

Brandi, Jessica; Cecconi, Daniela; Cordani, Marco; Torrens-Mas, Margalida; Pacchiana, Raffaella; Dalla Pozza, Elisa; Butera, Giovanna; Manfredi, Marcello; Marengo, Emilio; Oliver, Jordi; Roca, Pilar; Dando, Ilaria; Donadelli, Massimo

2016-12-01

Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

Vomiting and gastric electrical dysrhythmia in dogs.

PubMed

Ueno, T; Chen, J D Z

2004-04-01

The correlation between gastric myoelectrical activity (GMA) and gastrointestinal symptoms such as nausea and vomiting is poorly understood. The aim of this study was to assess the association of GMA with vomiting induced by retrograde gastric electrical stimulation or duodenal balloon distention. Ten dogs were involved in this study. Vomiting was induced by retrograde gastric electrical stimulation in 6 dogs and by duodenal balloon distention in 4 dogs. Computerized spectral analysis and visual analysis were applied to detect the GMA change during various periods before and after vomiting. Gastric dysrhythmia preceded vomiting but was of brief duration. The major pattern of dysrhythmia immediately before vomiting was tachyarrhythmia and gastric slow wave was completely uncoupled before vomiting. Gastric dysrhythmia and slow wave uncoupling were also noticed immediately after vomiting but the dogs recovered quickly. The major pattern of dysrhythmia after vomiting was arrhythmia. GMA was normal during the periods other than 5 min before and during vomiting and 5 min after vomiting. Gastric dysrhythmia seems to be the cause of vomiting induced by retrograde gastric electrical stimulation or duodenal balloon distention. It is brief and characterized with tachyarrhythmia and uncoupling.

Thermogenic Blend Alone or in Combination with Whey Protein Supplement Stimulates Fat Metabolism and Improves Body Composition in Mice.

PubMed

Vieira-Brock, Paula de Lima; Vaughan, Brent M; Vollmer, David L

2018-01-01

Certain food ingredients promote thermogenesis and fat loss. Similarly, whey protein improves body composition. Due to this potential synergistic effect, a blend of thermogenic food ingredients containing African mango, citrus fruit extract, Coleus forskohlii , dihydrocapsiate, and red pepper was tested alone and in combination with a whey protein supplement for its effects on body composition in sedentary mice during high-fat diet. The objective of this study was to evaluate the interaction of thermogenic foods on improving body composition during consumption of an unhealthy diet. C57BL/6J young adult male mice ( n = 12) were placed on a 60% high-fat diet for 4 weeks and subsequently randomly assigned to receive daily dosing by oral gavage of vehicle, the novel blend alone or with whey protein supplement for another 4 weeks. Body composition, thermal imaging of brown adipose tissue (BAT), mitochondrial BAT uncoupling protein 1 (UCP1), and plasma levels of leptin were assessed. Novel blend alone and in combination with protein supplement attenuated body weight gain, fat, and increased surface BAT temperature in comparison to vehicle control and to baseline ( P < 0.5). The combination of novel blend and whey protein supplement also significantly increased UCP1 protein expression in BAT mitochondria in comparison to vehicle control and novel blend alone ( P < 0.5). These data indicate that this novel blend stimulates thermogenesis and attenuates the gain in body weight and fat in response to high-fat diet in mice and these effects were improved when administered in combination with whey protein supplement. 30 days oral administration to mice of a novel blend containing African mango seed extract, citrus fruits extract, Coleus forskohlii root extract, dihydrocapsiate and red pepper fruit extract reduced body weight and fat gain in response to high-fat diet without impairing muscle mass.The novel blend stimulated thermogenesis as shown by the increased thermal imaging and UCP1 protein expression in brown adipose tissue, indicating that improvement in body composition potentially occurred due to a fat-burning effect.The positive effects on body weight, fat, and thermogenesis were improved when the novel blend was administered in combination with a whey protein supplement suggesting that protein provides a synergistic fat-burning effect. Abbreviations Used: BAT: Brown adipose tissue, UCP1: Uncoupling protein 1, DEXA: Dual-energy X-ray absorptiometry.

[On specific properties of the mitochondrial oxidative phosphorylation system operating as a supercomplex].

PubMed

Nesterov, S V; Skorobogatova, Iu A; Iaguzhinskiĭ, L S

2014-01-01

This paper represents the study of endogenous and exogenous fatty acids affecting the mitochondrial phosphorylation system effectiveness depending on temperature. The experiment was set up under conditions in which the oxidative phosphorylation system operates as a supercomplex. Rat liver mitochondria were isolated without purposive fatty acids removal from membranes, then studied in hypotonic medium (120 mOsm). We managed to detect a very narrow interval 19 ± 1°C where the fatty acid uncoupling effect is weak up to disappearing. At the same small temperature range, a structural rearrangement that takes place in the enzyme system is accompanied with denser packing of membrane protein complexes. Thus, at the temperatures close to 19°C the supercomplex works in the specific regime protected (or partially protected) from the uncoupling effect of fatty acids. Here we also discuss a physiological significance of the increased ATP-synthesis effectiveness at lower temperatures and the most probable character of structural rearrangement taking place at 19°C in the enzymes in the mitochondrial membrane.

ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs.

PubMed

Lee, Michelle H; Goralczyk, Anna G; Kriszt, Rókus; Ang, Xiu Min; Badowski, Cedric; Li, Ying; Summers, Scott A; Toh, Sue-Anne; Yassin, M Shabeer; Shabbir, Asim; Sheppard, Allan; Raghunath, Michael

2016-02-17

Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced 'browning' in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow.

Impaired Adaptability of in Vivo Mitochondrial Energetics to Acute Oxidative Insult in Aged Skeletal Muscle

PubMed Central

Siegel, Michael P.; Wilbur, Tim; Mathis, Mark; Shankland, Eric; Trieu, Atlas; Harper, Mary-Ellen; Marcinek, David J.

2012-01-01

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires 31P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O2 concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration. PMID:22935551

Differences in mitochondrial gene expression profiles, enzyme activities and myosin heavy chain types in yak versus bovine skeletal muscles.

PubMed

Lin, Y Q; Xu, Y O; Yue, Y; Jin, S Y; Qu, Y; Dong, F; Li, Y P; Zheng, Y C

2012-08-29

Hypoxia can affect energy metabolism. We examined gene expression and enzyme activity related to mitochondrial energy metabolism, as well as myosin heavy chain (MyHC) types in yaks (Bos grunniens) living at high altitudes. Real-time quantitative PCR assays indicated that the yak has significantly lower levels of carnitine palmitoyltransferase (CPT) mRNA in the biceps femoris and lower levels of uncoupling protein 3 (UCP3) mRNA in both biceps femoris and longissimus dorsi than in Yellow cattle. No significant differences between yak and Yellow cattle were observed in the activities of mitochondrial β-hydroxyacyl-CoA dehydrogenase, isocitrate dehydrogenase and cytochrome oxidase in the same muscles. Semi-quantitative RT-PCR analysis showed that the MyHC 1 mRNA levels in yak biceps femoris was lower than in Yellow cattle. We conclude that the yak has significantly lower mRNA levels of CPT, UCP3, and MyHC 1 in biceps femoris than in Yellow cattle, suggesting that the yak biceps femoris has lower fatty acid oxidation capacity and greater glycolytic metabolic potential.

Central serotonergic neurons activate and recruit thermogenic brown and beige fat and regulate glucose and lipid homeostasis.

PubMed

McGlashon, Jacob M; Gorecki, Michelle C; Kozlowski, Amanda E; Thirnbeck, Caitlin K; Markan, Kathleen R; Leslie, Kirstie L; Kotas, Maya E; Potthoff, Matthew J; Richerson, George B; Gillum, Matthew P

2015-05-05

Thermogenic brown and beige adipocytes convert chemical energy to heat by metabolizing glucose and lipids. Serotonin (5-HT) neurons in the CNS are essential for thermoregulation and accordingly may control metabolic activity of thermogenic fat. To test this, we generated mice in which the human diphtheria toxin receptor (DTR) was selectively expressed in central 5-HT neurons. Treatment with diphtheria toxin (DT) eliminated 5-HT neurons and caused loss of thermoregulation, brown adipose tissue (BAT) steatosis, and a >50% decrease in uncoupling protein 1 (Ucp1) expression in BAT and inguinal white adipose tissue (WAT). In parallel, blood glucose increased 3.5-fold, free fatty acids 13.4-fold, and triglycerides 6.5-fold. Similar BAT and beige fat defects occurred in Lmx1b(f/f)ePet1(Cre) mice in which 5-HT neurons fail to develop in utero. We conclude 5-HT neurons play a major role in regulating glucose and lipid homeostasis, in part through recruitment and metabolic activation of brown and beige adipocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial thermogenesis and obesity.

PubMed

Gambert, Ségolène; Ricquier, Daniel

2007-11-01

Thermogenesis is activated at the expense of carbon molecules. Mitochondria play a dominant role in oxidation and parallel heat production since the recovery of oxidation energy is less than perfect. Recent data of mitochondriogenesis and mitochondrial thermogenesis may boost research into certain aspects of obesity. Recent studies have outlined the unexpected decreased thermogenesis that limits fat loss during prolonged food restriction. Activation of fat oxidation in skeletal muscle remains a strategy against fat accumulation, however. Certain adipose depots have the potential to promote thermogenesis, either using mitochondrial uncoupling protein or independently. Peroxisome proliferator-activated receptor gamma coactivators alpha and ss are important regulators of mitochondria thermogenesis. Brain mitochondria are involved in the control of refeeding after starvation. This dual action of mitochondria inform their role in thermogenesis and energy partitioning. The importance of thyroid hormones in mitochondria thermogenesis is also confirmed. The clinical and research implications of these findings are that the mechanisms inhibiting adaptive thermogenesis during diet restriction should be investigated. An important field of research is the contribution of transcriptional coactivators to adipocyte plasticity since adipocytes have an underestimated ability to oxidise fatty acids in addition to their role in triglyceride storage.

MKK6 controls T3-mediated browning of white adipose tissue.

PubMed

Matesanz, Nuria; Bernardo, Edgar; Acín-Pérez, Rebeca; Manieri, Elisa; Pérez-Sieira, Sonia; Hernández-Cosido, Lourdes; Montalvo-Romeral, Valle; Mora, Alfonso; Rodríguez, Elena; Leiva-Vega, Luis; Lechuga-Vieco, Ana Victoria; Ruiz-Cabello, Jesús; Torres, Jorge L; Crespo-Ruiz, Maria; Centeno, Francisco; Álvarez, Clara V; Marcos, Miguel; Enríquez, Jose Antonio; Nogueiras, Ruben; Sabio, Guadalupe

2017-10-11

Increasing the thermogenic capacity of adipose tissue to enhance organismal energy expenditure is considered a promising therapeutic strategy to combat obesity. Here, we report that expression of the p38 MAPK activator MKK6 is elevated in white adipose tissue of obese individuals. Using knockout animals and shRNA, we show that Mkk6 deletion increases energy expenditure and thermogenic capacity of white adipose tissue, protecting mice against diet-induced obesity and the development of diabetes. Deletion of Mkk6 increases T3-stimulated UCP1 expression in adipocytes, thereby increasing their thermogenic capacity. Mechanistically, we demonstrate that, in white adipose tissue, p38 is activated by an alternative pathway involving AMPK, TAK, and TAB. Our results identify MKK6 in adipocytes as a potential therapeutic target to reduce obesity.Brown and beige adipose tissues dissipate heat via uncoupling protein 1 (UCP1). Here the authors show that the stress activated kinase MKK6 acts as a repressor of UCP1 expression, suggesting that its inhibition promotes adipose tissue browning and increases organismal energy expenditure.

eNOS uncoupling in cardiovascular diseases--the role of oxidative stress and inflammation.

PubMed

Karbach, Susanne; Wenzel, Philip; Waisman, Ari; Munzel, Thomas; Daiber, Andreas

2014-01-01

Many cardiovascular diseases and drug-induced complications are associated with - or even based on - an imbalance between the formation of reactive oxygen and nitrogen species (RONS) and antioxidant enzymes catalyzing the break-down of these harmful oxidants. According to the "kindling radical" hypothesis, the formation of RONS may trigger in certain conditions the activation of additional sources of RONS. According to recent reports, vascular dysfunction in general and cardiovascular complications such as hypertension, atherosclerosis and coronary artery diseases may be connected to inflammatory processes. The present review is focusing on the uncoupling of endothelial nitric oxide synthase (eNOS) by different mechanisms involving so-called "redox switches". The oxidative depletion of tetrahydrobiopterin (BH4), oxidative disruption of the dimeric eNOS complex, S-glutathionylation and adverse phosphorylation as well as RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) will be discussed. But also new concepts of eNOS uncoupling and state of the art detection of this process will be described. Another part of this review article will address pharmaceutical interventions preventing or reversing eNOS uncoupling and thereby normalize vascular function in a given disease setting. We finally turn our attention to the inflammatory mechanisms that are also involved in the development of endothelial dysfunction and cardiovascular disease. Inflammatory cell and cytokine profiles as well as their interactions, which are among the kindling mechanisms for the development of vascular dysfunction will be discussed on the basis of the current literature.

Inappropriate Expression of an NLP Effector in Colletotrichum orbiculare Impairs Infection on Cucurbitaceae Cultivars via Plant Recognition of the C-Terminal Region.

PubMed

Azmi, Nur Sabrina Ahmad; Singkaravanit-Ogawa, Suthitar; Ikeda, Kyoko; Kitakura, Saeko; Inoue, Yoshihiro; Narusaka, Yoshihiro; Shirasu, Ken; Kaido, Masanori; Mise, Kazuyuki; Takano, Yoshitaka

2018-01-01

The hemibiotrophic pathogen Colletotrichum orbiculare preferentially expresses a necrosis and ethylene-inducing peptide 1 (Nep1)-like protein named NLP1 during the switch to necrotrophy. Here, we report that the constitutive expression of NLP1 in C. orbiculare blocks pathogen infection in multiple Cucurbitaceae cultivars via their enhanced defense responses. NLP1 has a cytotoxic activity that induces cell death in Nicotiana benthamiana. However, C. orbiculare transgenic lines constitutively expressing a mutant NLP1 lacking the cytotoxic activity still failed to infect cucumber, indicating no clear relationship between cytotoxic activity and the NLP1-dependent enhanced defense. NLP1 also possesses the microbe-associated molecular pattern (MAMP) sequence called nlp24, recognized by Arabidopsis thaliana at its central region, similar to NLPs of other pathogens. Surprisingly, inappropriate expression of a mutant NLP1 lacking the MAMP signature is also effective for blocking pathogen infection, uncoupling the infection block from the corresponding MAMP. Notably, the deletion analyses of NLP1 suggested that the C-terminal region of NLP1 is critical to enhance defense in cucumber. The expression of mCherry fused with the C-terminal 32 amino acids of NLP1 was enough to trigger the defense of cucurbits, revealing that the C-terminal region of the NLP1 protein is recognized by cucurbits and, then, terminates C. orbiculare infection.

Novel Proteins Required for Meiotic Silencing by Unpaired DNA and siRNA Generation in Neurospora crassa

PubMed Central

Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Decker, Logan M.; Lee, Seung A.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

2013-01-01

During meiosis in the filamentous fungus Neurospora crassa, unpaired genes are identified and silenced by a process known as meiotic silencing by unpaired DNA (MSUD). Previous work has uncovered six proteins required for MSUD, all of which are also essential for meiotic progression. Additionally, they all localize in the perinuclear region, suggesting that it is a center of MSUD activity. Nevertheless, at least a subset of MSUD proteins must be present inside the nucleus, as unpaired DNA recognition undoubtedly takes place there. In this study, we identified and characterized two new proteins required for MSUD, namely SAD-4 and SAD-5. Both are previously uncharacterized proteins specific to Ascomycetes, with SAD-4 having a range that spans several fungal classes and SAD-5 seemingly restricted to a single order. Both genes appear to be predominantly expressed in the sexual phase, as molecular study combined with analysis of publicly available mRNA-seq datasets failed to detect significant expression of them in the vegetative tissue. SAD-4, like all known MSUD proteins, localizes in the perinuclear region of the meiotic cell. SAD-5, on the other hand, is found in the nucleus (as the first of its kind). Both proteins are unique compared to previously identified MSUD proteins in that neither is required for sexual sporulation. This homozygous-fertile phenotype uncouples MSUD from sexual development and allows us to demonstrate that both SAD-4 and SAD-5 are important for the production of masiRNAs, which are the small RNA molecules associated with meiotic silencing. PMID:23502675

Dietary soya protein improves intra-myocardial lipid deposition and altered glucose metabolism in a hypertensive, dyslipidaemic, insulin-resistant rat model.

PubMed

Oliva, María E; Creus, Agustina; Ferreira, María R; Chicco, Adriana; Lombardo, Yolanda B

2018-01-01

This study investigates the effects of replacing dietary casein by soya protein on the underlying mechanisms involved in the impaired metabolic fate of glucose and lipid metabolisms in the heart of dyslipidaemic rats chronically fed (8 months) a sucrose-rich (62·5 %) diet (SRD). To test this hypothesis, Wistar rats were fed an SRD for 4 months. From months 4 to 8, half the animals continued with the SRD and the other half were fed an SRD in which casein was substituted by soya. The control group received a diet with maize starch as the carbohydrate source. Compared with the SRD-fed group, the following results were obtained. First, soya protein significantly (P<0·001) reduced the plasma NEFA levels and normalised dyslipidaemia and glucose homoeostasis, improving insulin resistance. The protein levels of fatty acid translocase at basal state and under insulin stimulation and the protein levels and activity of muscle-type carnitine palmitoyltransferase 1 were normalised. Second, a significant (P<0·001) reduction of TAG, long-chain acyl CoA and diacylglycerol levels was observed in the heart muscle. Third, soya protein significantly increased (P<0·01) GLUT4 protein level under insulin stimulation and normalised glucose phosphorylation and oxidation. A reduction of phosphorylated AMP protein kinase protein level was recorded without changes in uncoupling protein 2 and PPARα. Fourth, hydroxyproline concentration decreased in the left ventricle and hypertension was normalised. The new information provided shows the beneficial effects of soya protein upon the altered pathways of glucose and lipid metabolism in the heart muscle of this rat model.

An improved procedure for reconstitution of the uncoupling protein and in-depth analysis of H+/OH- transport.

PubMed

Winkler, E; Klingenberg, M

1992-07-01

An improved procedure for reincorporation of isolated uncoupling protein (UCP) from brown adipose tissue into phospholipid vesicles is reported and H+ uptake in K(+)-driven exchange diffusion quantitatively analyzed. UCP is isolated and reconstituted with medium-length linear-chain alkyl polyoxyethylene. In the critical step of vesicle formation, the stepwise removal of the detergent by polystyrene beads is applied. Vesicles are generated in the presence of solutes and buffers to be internalized which are then removed by gel filtration. The internal volume is about 4 microliters/mg phospholipid with a vesicle diameter of 100 nm. One vesicle contains, on average, six molecules UCP. The best results are obtained with purified egg yolk phosphatidylcholine. Addition of PtdEtn, PtdSer decreases the vesicle size and, still more, H(+)-transport activity by UCP. Asolectin completely inactivates UCP. K(+)-gradient-driven H+ uptake is 80% inhibited by external GTP and 95% by internal plus external GTP. When H+ transport is recorded externally by a pH electrode and internally by pyranine, the kinetics show no delay resulting from intervening membrane-bound H+ pools. Total H+ uptake after addition of carbonylcyanine m-chlorophenylhydrazone (CCCP) and valinomycin corresponds to the diffusion between H+ and K+ and is unchanged by GTP. The linear correlation of H(+)-transport inhibition to GTP binding demonstrates that all UCP molecules incorporated are equally active. The exchange diffusion between H+ uptake and K+ efflux is demonstrated using a K+ electrode and 86Rb measurements. Recording delta psi using 3,3'-diispropylthiadicarbocyanine shows a rapid generation of delta psi on valinomycin addition, which decreases only slightly with H+ uptake, even after addition of CCCP or gramicidin. The delta psi collapses only after addition of external K+. By demonstrating that valinomycin-induced K+ and H+ fluxes reflect relaxation into the diffusion equilibrium state, the transport rate of UCP can be evaluated as a first-order rate, VH+/CH+, in which the rate, VH+, is related to H(+)-uptake capacity, CH+. This allows quantitative comparison of transport rates independently of the variable CH+. The dependence on delta psi of H+ transport is measured by varying external K+ concentration. A virtually linear relation of the rate to the K(+)-diffusion potential is observed, although the capacity is only slightly changed. The linear VH+/delta psi relationship resembles an open-channel type of transport, but is discussed in terms of a low-activation-barrier type of carrier mechanism, in contrast to the log (VH+/delta psi) relation found for the ADP/ATP carrier with high activation barriers.

Mitochondrial targeted HSP90 inhibitor Gamitrinib-TPP (G-TPP) induces PINK1/Parkin-dependent mitophagy.

PubMed

Fiesel, Fabienne C; James, Elle D; Hudec, Roman; Springer, Wolfdieter

2017-12-05

Loss-of-function mutations in PINK1 or PARKIN are associated with early-onset Parkinson's disease. Upon mitochondrial stress, PINK1 and Parkin together mediate a response that protects cells from the accumulation of harmful, damaged mitochondria. PINK1, the upstream kinase accumulates on the mitochondrial surface and recruits the E3 ubiquitin ligase Parkin on site to ubiquitylate substrate proteins. The joint activity of both to generate phosphorylated poly-ubiquitin chains on the mitochondrial surface induces the recruitment of autophagy receptors and eventually whole organelles are cleared by autophagy. While this pathway is generally accepted to occur upon chemical uncoupling of mitochondria, the (patho-) physiologic relevance has been questioned. However, few studies have indicated that PINK1 and Parkin are also activated upon accumulation of misfolded proteins in the mitochondrial lumen upon overexpression of ΔOTC (Ornithine transcarbamylase). Here, we used the mitochondrial targeted HSP90 inhibitor Gamitrinib-triphenylphosphonium (G-TPP), an anti-cancer agent, to chemically interfere with mitochondrial protein folding. G-TPP treatment induced PINK1 accumulation, ubiquitin phosphorylation at Ser65, Parkin activation and its recruitment to mitochondria was specific for mitochondrial HSP90 inhibition and largely independent of mitochondrial membrane depolarization. Mitophagy induction was observed by monitoring autophagy receptor recruitment and the mitoKeima reporter. Importantly, mitophagy was not only induced in cancer cells but also in primary human fibroblasts and thereof converted neurons. G-TPP treatment might represent a novel strategy to study PINK1 and Parkin-mediated mitochondrial quality control using a more physiologically relevant stress.

Regulators of Mitochondrial Quality Control Differs in Subcutaneous Fat of Metabolically Healthy and Unhealthy Obese Monkeys

PubMed Central

Kavanagh, Kylie; Davis, Ashley T; Peters, Diane E; Le Grand, Andre; Bharadwaj, Manish S; Molina, Anthony JA

2016-01-01

Objectives Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity respectively (MHO). Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess is emerging as a key pathway. We aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. Methods Vervet monkeys (Chlorocebus aethiops; n=171) were screened for Metabolic Syndrome. A subset of MHO and MUO monkeys (n=6/group) had SQ fat biopsies collected for histologic evaluations and examination of key mitochondrial proteins. Results Obesity was seen in 20% of monkeys, and within this population, 31% were healthy which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators, and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. Conclusions We characterized a novel and translatable spontaneously obese animal model of healthy and unhealthy obesity, occurring independently of dietary factors. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health. PMID:28236433

Regulators of mitochondrial quality control differ in subcutaneous fat of metabolically healthy and unhealthy obese monkeys.

PubMed

Kavanagh, Kylie; Davis, Ashley T; Peters, Diane E; LeGrand, Andre C; Bharadwaj, Manish S; Molina, Anthony J A

2017-04-01

Obesity exists with and without accompanying cardiometabolic disease, termed metabolically unhealthy obesity (MUO) and healthy obesity (MHO), respectively. Underlying differences in the ability of subcutaneous (SQ) fat to respond to nutrient excess are emerging as a key pathway. This study aimed to document the first spontaneous animal model of MHO and MUO and differences in SQ adipose tissue. Vervet monkeys (Chlorocebus aethiops; N = 171) were screened for metabolic syndrome. A subset of MHO and MUO monkeys (n = 6/group) had SQ fat biopsies collected for histological evaluations and examination of key mitochondrial proteins. Obesity was seen in 20% of monkeys, and within this population, 31% were healthy, which mirrors human prevalence estimates. MUO monkeys had more than 60% lower adiponectin concentrations despite similar fat cell size, uncoupling protein 3, and activated macrophage abundance. However, alternatively activated/anti-inflammatory macrophages were 70% lower. Deficiencies of 50% or more in mitochondrial quality control regulators and selected mitochondrial fission and fusion markers were observed in the SQ fat of MUO monkeys despite comparable mitochondrial content. A novel and translatable spontaneously obese animal model of MHO and MUO, occurring independently of dietary factors, was characterized. Differences in mitochondrial quality and inflammatory cell populations of subcutaneous fat may underpin divergent metabolic health. © 2017 The Obesity Society.

Sperm Mitochondrial Integrity Is Not Required for Hyperactivated Motility, Zona Binding, or Acrosome Reaction in the Rhesus Macaque1

PubMed Central

Hung, Pei-hsuan; Miller, Marion G.; Meyers, Stuart A.; VandeVoort, Catherine A.

2008-01-01

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 μM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque. PMID:18480469

Two active molecular phenotypes of the tachykinin NK1 receptor revealed by G-protein fusions and mutagenesis.

PubMed

Holst, B; Hastrup, H; Raffetseder, U; Martini, L; Schwartz, T W

2001-06-08

The NK1 neurokinin receptor presents two non-ideal binding phenomena, two-component binding curves for all agonists and significant differences between agonist affinity determined by homologous versus heterologous competition binding. Whole cell binding with fusion proteins constructed between either Galpha(s) or Galpha(q) and the NK1 receptor with a truncated tail, which secured non-promiscuous G-protein interaction, demonstrated monocomponent agonist binding closely corresponding to either of the two affinity states found in the wild-type receptor. High affinity binding of both substance P and neurokinin A was observed in the tail-truncated Galpha(s) fusion construct, whereas the lower affinity component was displayed by the tail-truncated Galpha(q) fusion. The elusive difference between the affinity determined in heterologous versus homologous binding assays for substance P and especially for neurokinin A was eliminated in the G-protein fusions. An NK1 receptor mutant with a single substitution at the extracellular end of TM-III-(F111S), which totally uncoupled the receptor from Galpha(s) signaling, showed binding properties that were monocomponent and otherwise very similar to those observed in the tail-truncated Galpha(q) fusion construct. Thus, the heterogenous pharmacological phenotype displayed by the NK1 receptor is a reflection of the occurrence of two active conformations or molecular phenotypes representing complexes with the Galpha(s) and Galpha(q) species, respectively. We propose that these molecular forms do not interchange readily, conceivably because of the occurrence of microdomains or "signal-transductosomes" within the cell membrane.

Multiple Transduction Pathways Mediate Thyrotropin Receptor Signaling in Preosteoblast-Like Cells

PubMed Central

Boutin, Alisa; Neumann, Susanne

2016-01-01

It has been shown that the TSH receptor (TSHR) couples to a number of different signaling pathways, although the Gs-cAMP pathway has been considered primary. Here, we measured the effects of TSH on bone marker mRNA and protein expression in preosteoblast-like U2OS cells stably expressing TSHRs. We determined which signaling cascades are involved in the regulation of IL-11, osteopontin (OPN), and alkaline phosphatase (ALPL). We demonstrated that TSH-induced up-regulation of IL-11 is primarily mediated via the Gs pathway as IL-11 was up-regulated by forskolin (FSK), an adenylyl cyclase activator, and inhibited by protein kinase A inhibitor H-89 and by silencing of Gαs by small interfering RNA. OPN levels were not affected by FSK, but its up-regulation was inhibited by TSHR/Gi-uncoupling by pertussis toxin. Pertussis toxin decreased p38 MAPK kinase phosphorylation, and a p38 inhibitor and small interfering RNA knockdown of p38α inhibited OPN induction by TSH. Up-regulation of ALPL expression required high doses of TSH (EC50 = 395nM), whereas low doses (EC50 = 19nM) were inhibitory. FSK-stimulated cAMP production decreased basal ALPL expression, whereas protein kinase A inhibition by H-89 and silencing of Gαs increased basal levels of ALPL. Knockdown of Gαq/11 and a protein kinase C inhibitor decreased TSH-stimulated up-regulation of ALPL, whereas a protein kinase C activator increased ALPL levels. A MAPK inhibitor and silencing of ERK1/2 inhibited TSH-stimulated ALPL expression. We conclude that TSH regulates expression of different bone markers via distinct signaling pathways. PMID:26950201

11β-HSD1 Modulates the Set Point of Brown Adipose Tissue Response to Glucocorticoids in Male Mice

PubMed Central

Doig, Craig L.; Fletcher, Rachel S.; Morgan, Stuart A.; McCabe, Emma L.; Larner, Dean P.; Tomlinson, Jeremy W.; Stewart, Paul M.; Philp, Andrew

2017-01-01

Glucocorticoids (GCs) are potent regulators of energy metabolism. Chronic GC exposure suppresses brown adipose tissue (BAT) thermogenic capacity in mice, with evidence for a similar effect in humans. Intracellular GC levels are regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity, which can amplify circulating GC concentrations. Therefore, 11β-HSD1 could modulate the impact of GCs on BAT function. This study investigated how 11β-HSD1 regulates the molecular architecture of BAT in the context of GC excess and aging. Circulating GC excess was induced in 11β-HSD1 knockout (KO) and wild-type mice by supplementing drinking water with 100 μg/mL corticosterone, and the effects on molecular markers of BAT function and mitochondrial activity were assessed. Brown adipocyte primary cultures were used to examine cell autonomous consequences of 11β-HSD1 deficiency. Molecular markers of BAT function were also examined in aged 11β-HSD1 KO mice to model lifetime GC exposure. BAT 11β-HSD1 expression and activity were elevated in response to GC excess and with aging. 11β-HSD1 KO BAT resisted the suppression of uncoupling protein 1 (UCP1) and mitochondrial respiratory chain subunit proteins normally imposed by GC excess. Furthermore, brown adipocytes from 11β-HSD1 KO mice had elevated basal mitochondrial function and were able to resist GC-mediated repression of activity. BAT from aged 11β-HSD1 KO mice showed elevated UCP1 protein and mitochondrial content, and a favorable profile of BAT function. These data reveal a novel mechanism in which increased 11β-HSD1 expression, in the context of GC excess and aging, impairs the molecular and metabolic function of BAT. PMID:28368470

A single oral dose of flavan-3-ols enhances energy expenditure by sympathetic nerve stimulation in mice.

PubMed

Kamio, Naoya; Suzuki, Takuma; Watanabe, Yuto; Suhara, Yoshitomo; Osakabe, Naomi

2016-02-01

Numerous clinical studies have found that ingestion of chocolate reduces the risk of metabolic syndrome, however, the mechanisms were remain unclear. We have reported that a single dose of a flavan-3-ol fraction derived from cocoa (FL) enhanced energy expenditure (EE) and increased the mRNA expression levels of uncoupling proteins (UCPs) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), and the protein level of phosphorylated AMP-activated protein kinase (AMPK)α in tissues, along with plasma adrenaline level. In the present study, we examined whether the EE enhancing activity of FL is mediated by adrenergic effect using several adrenalin receptor (AR) blockers. In the first study, mice were butoxamine, as β2AR blocker, with vehicle or 10mg/kg FL orally. We found that pretreatment with butoxamine prevented the increases of EE, the mRNA expression of UCP-3, and phosphorylated AMPKα that were induced in the gastrocnemius muscle of mice by 10mg/kg FL. Secondly, mice were given SR52930, as β3AR blocker. Pretreatment with SR52930 prevented the increases of EE, the mRNA expression of UCP-3, and phosphorylated AMPKα that were induced in the gastrocnemius muscle of mice by 10mg/kg FL. Pretreatment with a combination of both blockers also reduced the increments in mRNA expression levels of UCPs and PGC-1α, however, phosphorylated AMPKα in skeletal muscle was rather increased. These results suggest that the ability of a single oral dose of FL to enhance metabolic activity is mediated by sympathetic nerve system (SNS). Copyright © 2015. Published by Elsevier Inc.

Understanding the conformation transition in the activation pathway of β2 adrenergic receptor via a targeted molecular dynamics simulation.

PubMed

Xiao, Xiuchan; Zeng, Xiaojun; Yuan, Yuan; Gao, Nan; Guo, Yanzhi; Pu, Xuemei; Li, Menglong

2015-01-28

G protein coupled receptors (GPCRs) play a crucial role in regulating signal recognition and transduction through their activation. The conformation transition in the activation pathway is of particular importance for their function. However, it has been poorly elucidated due to experimental difficulties in determining the conformations and the time limitation of conventional molecular dynamics (CMD) simulation. Thus, in this work, we employed a targeted molecular dynamic (TMD) simulation to study the activation process from an inactive structure to a fully active one for β2 adrenergic receptor (β2AR). As a reference, 110 ns CMD simulations on wild β2AR and its D130N mutant were also carried out. TMD results show that there is at least an intermediate conformation cluster in the activation process, evidenced by the principal component analysis and the structural and dynamic differences of some important motifs. It is noteworthy that the activation of the ligand binding site lags the G-protein binding site, displaying uncoupled correlation. Comparisons between the CMD and TMD results show that the D130N mutation significantly speeds up ICL2 and key ionic lock to enter into the intermediate state, which to some extent facilitates the activation involved in the NPxxY, DRY region and the separation between TM3 and TM6. However, the contribution from the D130N mutation to the activation of the ligand binding site could not be observed within the scale of 110 ns time. These observations could provide novel insights into previous studies for better understanding of the activation mechanism for β2AR.

Examining a role for PKG Iα oxidation in the pathogenesis of cardiovascular dysfunction during diet-induced obesity.

PubMed

Rudyk, Olena; Eaton, Philip

2017-09-01

Protein kinase G (PKG) Iα is the end-effector kinase that mediates nitric oxide (NO)-dependent and oxidant-dependent vasorelaxation to maintain blood pressure during health. A hallmark of cardiovascular disease is attenuated NO production, which in part is caused by NO Synthase (NOS) uncoupling, which in turn increases oxidative stress because of superoxide generation. NOS uncoupling promotes PKG Iα oxidation to the interprotein disulfide state, likely mediated by superoxide-derived hydrogen peroxide, and because the NO-cyclic guanosine monophosphate (cGMP) pathway otherwise negatively regulates oxidation of the kinase to its active disulfide dimeric state. Diet-induced obesity is associated with NOS uncoupling, which may in part contribute to the associated cardiovascular dysfunction due to exacerbated PKG Iα disulfide oxidation to the disulfide state. This is a rational hypothesis because PKG Iα oxidation is known to significantly contribute to heart failure that arises from chronic myocardial oxidative stress. Bovine arterial endothelial cells (BAECs) or smooth muscle cells (SMCs) were exposed to drugs that uncouple NOS. These included 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) which promotes its S-glutathiolation, 4-diamino-6-hydroxy-pyrimidine (DAHP) which inhibits guanosine-5'-triphosphate-cyclohydrolase 2 to prevent BH 4 synthesis or methotrexate (MTX) which inhibits the regeneration of BH 4 from BH 2 by dihydrofolate reductase. While all the drugs mentioned above induced robust PKG Iα disulfide dimerization in cells, exposure of BAECs to NOS inhibitor L-NMMA did not. Increased PKG Iα disulfide formation occurred in hearts and aortae from mice treated in vivo with DAHP (10mM in a drinking water for 3 weeks). Redox-dead C42S PKG Iα knock-in (KI) mice developed less pronounced cardiac posterior wall hypertrophy and did not develop cardiac dysfunction, assessed by echocardiography, compared to the wild-type (WT) mice after chronic DAHP treatment. WT or KI mice were then subjected to a diet-induced obesity protocol by feeding them with a high fat Western-type diet (RM 60% AFE) for 27 weeks, which increased body mass, adiposity, plasma leptin, resistin and glucagon levels comparably in each genotype. Obesity-induced hypertension, assessed by radiotelemetry, was mild and transient in the WT, while the basally hypertensive KI mice were resistant to further increases in blood pressure following high fat feeding. Although the obesogenic diet caused mild cardiac dysfunction in the WT but not the KI mice, gross changes in myocardial structure monitored by echocardiography were not apparent in either genotype. The level of cyclic guanosine monophosphate (cGMP) was decreased in the aortae of WT and KI mice following high fat feeding. PKG Iα oxidation was not evident in the hearts of WT mice fed a high fat diet. Despite robust evidence for PKG Iα oxidation during NOS uncoupling in cell models, it is unlikely that PKG Iα oxidation occurs to a significant extent in vivo during diet-induced obesity and so is unlikely to mediate the associated cardiovascular dysfunction. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells

PubMed Central

Xie, Liping; Liu, Zhen; Lu, Hui; Zhang, Wen; Mi, Qiongyu; Li, Xiaozhen; Tang, Yan; Chen, Qi; Ferro, Albert; Ji, Yong

2012-01-01

BACKGROUND AND PURPOSE One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O2•−) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism. EXPERIMENTAL APPROACH HUVECs were incubated with ox-LDL with/without pyridoxine, NG-nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O2•− was measured using lucigenin chemiluminescence, and O2•−-sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot. KEY RESULTS Ox-LDL significantly increased O2•− production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O2•− generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments. CONCLUSIONS AND IMPLICATIONS Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction. PMID:21797845

Gap junction communications influence upon fibroblast synthesis of Type I collagen and fibronectin.

PubMed

Ehrlich, H Paul; Sun, Bonnie; Saggers, Gregory C; Kromath, Fatuma

2006-07-01

In rats polyvinyl alcohol sponge subcutaneous implants treated with gap junctional intercellular communications (GJIC) uncouplers showed reduced deposition of connective tissue. Do uncouplers inhibit the synthesis and deposition of a new connective tissue by fibroblasts? Confluent human dermal fibroblasts in serum-free medium received either endosulfan or oleamide, GJIC uncouplers. Collected media were subjected to Dot Blot analysis for native Type I collagen and fibronectin. Uncoupler-treated fibroblasts released less Type I collagen, while there was no change in fibronectin release. Collagen synthesis was restored to normal, when the uncouplers were removed, showing that these uncouplers were reversible and not toxic to cells. Northern blot analysis revealed procollagen alpha1 (I) mRNA was minimally affected by endosulfan. Oleamide-treated 17-day chick embryo calvaria explants were incubated with Type I collagen antibody, frozen, cryosectioned, and then subjected to rhodamine (Rh) tagged anti-mouse-IgG antibody, to detect newly deposited Type I collagen. Fluorescent antibody-collagen complexes were localized on the periphery of cells in control calvaria, but absent around cells in oleamide-treated calvaria. GJIC optimize collagen synthesis but not fibronectin synthesis. The lack of connective tissue deposited in granulation tissues treated with uncouplers appears related to the inhibition of collagen synthesis. These findings suggest that altering GJIC might control collagen deposition in scarring. 2006 Wiley-Liss, Inc.

Impaired adaptability of in vivo mitochondrial energetics to acute oxidative insult in aged skeletal muscle.

PubMed

Siegel, Michael P; Wilbur, Tim; Mathis, Mark; Shankland, Eric G; Trieu, Atlas; Harper, Mary-Ellen; Marcinek, David J

2012-01-01

Periods of elevated reactive oxygen species (ROS) production are a normal part of mitochondrial physiology. However, little is known about age-related changes in the mitochondrial response to elevated ROS in vivo. Significantly, ROS-induced uncoupling of oxidative phosphorylation has received attention as a negative feedback mechanism to reduce mitochondrial superoxide production. Here we use a novel in vivo spectroscopy system to test the hypothesis that ROS-induced uncoupling is diminished in aged mitochondria. This system simultaneously acquires (31)P magnetic resonance and near-infrared optical spectra to non-invasively measure phosphometabolite and O(2) concentrations in mouse skeletal muscle. Using low dose paraquat to elevate intracellular ROS we assess in vivo mitochondrial function in young, middle aged, and old mice. Oxidative phosphorylation was uncoupled to the same degree in response to ROS at each age, but this uncoupling was associated with loss of phosphorylation capacity and total ATP in old mice only. Using mice lacking UCP3 we demonstrate that this in vivo uncoupling is independent of this putative uncoupler of skeletal muscle mitochondria. These data indicate that ROS-induced uncoupling persists throughout life, but that oxidative stress leads to mitochondrial deficits and loss of ATP in aged organisms that may contribute to impaired function and degeneration. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

The role of nuclear factor E2-Related factor 2 and uncoupling protein 2 in glutathione metabolism: Evidence from an in vivo gene knockout study

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

Chen, Yanyan; The Hamner Institutes for Health Sciences, Research Triangle Park, NC; Xu, Yuanyuan, E-mail: yyxu@cmu.edu.cn

Nuclear factor E2-related factor 2 (NRF2) and uncoupling protein 2 (UCP2) are indicated to protect from oxidative stress. They also play roles in the homeostasis of glutathione. However, the detailed mechanisms are not well understood. In the present study, we found Nrf2-knockout (Nrf2-KO) mice exhibited altered glutathione homeostasis and reduced expression of various genes involved in GSH biosynthesis, regeneration, utilization and transport in the liver. Ucp2-knockout (Ucp2-KO) mice exhibited altered glutathione homeostasis in the liver, spleen and blood, as well as increased transcript of cystic fibrosis transmembrane conductance regulator in the liver, a protein capable of mediating glutathione efflux. Nrf2-Ucp2-doublemore » knockout (DKO) mice showed characteristics of both Nrf2-KO and Ucp2-KO mice. But no significant difference was observed in DKO mice when compared with Nrf2-KO or Ucp2-KO mice, except in blood glutathione levels. These data suggest that ablation of Nrf2 and Ucp2 leads to disrupted GSH balance, which could result from altered expression of genes involved in GSH metabolism. DKO may not evoke more severe oxidative stress than the single gene knockout. - Highlights: • Nrf2/Ucp2 deficiency leads to alteration of glutathione homeostasis. • Nrf2 regulates expression of genes in glutathione generation and utilization. • Ucp2 affects glutathione metabolism by regulating hepatic efflux of glutathione. • Nrf2 deficiency may not aggravate oxidative stress in Ucp2-deficient mice.« less

Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

PubMed Central

Mitsopoulos, Panagiotis; Chang, Yu-Han; Wai, Timothy; König, Tim; Dunn, Stanley D.; Langer, Thomas

2015-01-01

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III2 and I-III2-IV1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function. PMID:25776552

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

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

McLaughlin, K. J.; Nash, R. P.; Redinbo, M. R.

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. Inmore » this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.« less

Phyllodulcin, a Natural Sweetener, Regulates Obesity-Related Metabolic Changes and Fat Browning-Related Genes of Subcutaneous White Adipose Tissue in High-Fat Diet-Induced Obese Mice.

PubMed

Kim, Eunju; Lim, Soo-Min; Kim, Min-Soo; Yoo, Sang-Ho; Kim, Yuri

2017-09-21

Phyllodulcin is a natural sweetener found in Hydrangea macrophylla var. thunbergii . This study investigated whether phyllodulcin could improve metabolic abnormalities in high-fat diet (HFD)-induced obese mice. Animals were fed a 60% HFD for 6 weeks to induce obesity, followed by 7 weeks of supplementation with phyllodulcin (20 or 40 mg/kg body weight (b.w.)/day). Stevioside (40 mg/kg b.w./day) was used as a positive control. Phyllodulcin supplementation reduced subcutaneous fat mass, levels of plasma lipids, triglycerides, total cholesterol, and low-density lipoprotein cholesterol and improved the levels of leptin, adiponectin, and fasting blood glucose. In subcutaneous fat tissues, supplementation with stevioside or phyllodulcin significantly decreased mRNA expression of lipogenesis-related genes, including CCAAT/enhancer-binding protein α ( C/EBPα ), peroxisome proliferator activated receptor γ ( PPARγ ), and sterol regulatory element-binding protein-1C ( SREBP-1c ) compared to the high-fat group. Phyllodulcin supplementation significantly increased the expression of fat browning-related genes, including PR domain containing 16 ( Prdm16 ), uncoupling protein 1 ( UCP1 ), and peroxisome proliferator-activated receptor γ coactivator 1-α ( PGC-1α ), compared to the high-fat group. Hypothalamic brain-derived neurotrophic factor-tropomyosin receptor kinase B (BDNF-TrkB) signaling was upregulated by phyllodulcin supplementation. In conclusion, phyllodulcin is a potential sweetener that could be used to combat obesity by regulating levels of leptin, fat browning-related genes, and hypothalamic BDNF-TrkB signaling.

Regulatory motifs for CREB-binding protein and Nfe2l2 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene.

PubMed

Rim, Jong S; Kozak, Leslie P

2002-09-13

Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.

RNAseq analysis of fast skeletal muscle in restriction-fed transgenic coho salmon (Oncorhynchus kisutch): an experimental model uncoupling the growth hormone and nutritional signals regulating growth.

PubMed

Garcia de la Serrana, Daniel; Devlin, Robert H; Johnston, Ian A

2015-07-31

Coho salmon (Oncorhynchus kisutch) transgenic for growth hormone (Gh) express Gh in multiple tissues which results in increased appetite and continuous high growth with satiation feeding. Restricting Gh-transgenics to the same lower ration (TR) as wild-type fish (WT) results in similar growth, but with the recruitment of fewer, larger diameter, muscle skeletal fibres to reach a given body size. In order to better understand the genetic mechanisms behind these different patterns of muscle growth and to investigate how the decoupling of Gh and nutritional signals affects gene regulation we used RNA-seq to compare the fast skeletal muscle transcriptome in TR and WT coho salmon. Illumina sequencing of individually barcoded libraries from 6 WT and 6 TR coho salmon yielded 704,550,985 paired end reads which were used to construct 323,115 contigs containing 19,093 unique genes of which >10,000 contained >90 % of the coding sequence. Transcripts coding for 31 genes required for myoblast fusion were identified with 22 significantly downregulated in TR relative to WT fish, including 10 (vaspa, cdh15, graf1, crk, crkl, dock1, trio, plekho1a, cdc42a and dock5) associated with signaling through the cell surface protein cadherin. Nineteen out of 44 (43 %) translation initiation factors and 14 of 47 (30 %) protein chaperones were upregulated in TR relative to WT fish. TR coho salmon showed increased growth hormone transcripts and gene expression associated with protein synthesis and folding than WT fish even though net rates of protein accretion were similar. The uncoupling of Gh and amino acid signals likely results in additional costs of transcription associated with protein turnover in TR fish. The predicted reduction in the ionic costs of homeostasis in TR fish associated with increased fibre size were shown to involve multiple pathways regulating myotube fusion, particularly cadherin signaling.

The plasticity of the association between mu-opioid receptor and glutamate ionotropic receptor N in opioid analgesic tolerance and neuropathic pain.

PubMed

Sánchez-Blázquez, Pilar; Rodríguez-Muñoz, Maria; Berrocoso, Esther; Garzón, Javier

2013-09-15

Multiple groups have reported the functional cross-regulation between mu-opioid (MOP) receptor and glutamate ionotropic receptor N (GluN), and the post-synaptic association of these receptors has been implicated in the transmission and modulation of nociceptive signals. Opioids, such as morphine, disrupt the MOP receptor-GluN receptor complex to stimulate the activity of GluN receptors via protein kinase C (PKC)/Src. This increased GluN receptor activity opposes MOP receptor signalling, and via neural nitric oxide synthase (nNOS) and calcium and calmodulin regulated kinase II (CaMKII) induces the phosphorylation and uncoupling of the opioid receptor, which results in the development of morphine analgesic tolerance. Both experimental in vivo activation of GluN receptors and neuropathic pain separate the MOP receptor-GluN receptor complex via protein kinase A (PKA) and reduce the analgesic capacity of morphine. The histidine triad nucleotide-binding protein 1 (HINT1) associates with the MOP receptor C-terminus and connects the activities of MOP receptor and GluN receptor. In HINT1⁻/⁻ mice, morphine promotes enhanced analgesia and produces tolerance that is not related to GluN receptor activity. In these mice, the GluN receptor agonist N-methyl-D-aspartate acid (NMDA) does not antagonise the analgesic effects of morphine. Treatments that rescue morphine from analgesic tolerance, such as GluN receptor antagonism or PKC, nNOS and CaMKII inhibitors, all induce MOP receptor-GluN receptor re-association and reduce GluN receptor/CaMKII activity. In mice treated with NMDA or suffering from neuropathic pain (induced by chronic constriction injury, CCI), GluN receptor antagonists, PKA inhibitors or certain antidepressants also diminish CaMKII activity and restore the MOP receptor-GluN receptor association. Thus, the HINT1 protein stabilises the association between MOP receptor and GluN receptor, necessary for the analgesic efficacy of morphine, and this coupling is reduced following the activation of GluN receptors, similar to what is observed in neuropathic pain. © 2013 Elsevier B.V. All rights reserved.

Uncoupling of sgRNAs from their associated barcodes during PCR amplification of combinatorial CRISPR screens

PubMed Central

2018-01-01

Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling. PMID:29799876

De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis.

PubMed

Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

2012-04-01

Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified.

Bardoxolone methyl prevents fat deposition and inflammation in the visceral fat of mice fed a high-fat diet.

PubMed

Dinh, Chi H L; Szabo, Alexander; Camer, Danielle; Yu, Yinghua; Wang, Hongqin; Huang, Xu-Feng

2015-03-05

Key features of diet-induced obesity are visceral fat deposition, macrophage infiltration and inflammation that can lead to metabolic disorders. This study examined the effects of bardoxolone methyl (BARD) in preventing obesity and inflammation in the visceral fat of mice fed high-fat diet. Male C57BL/6J mice were fed a high-fat diet (HFD), a low-fat diet (LFD, i.e., lab chow diet) or a high-fat diet supplemented with BARD (HFD/BARD) for 21weeks. BARD at a dosage of 10mg/kg body weight was administered orally in drinking water. Histology, immunohistochemistry and Western blot were used for the analysis of epididymal adipose tissue. Morphological results demonstrated that HFD fed mice treated with BARD had smaller adipocytes and fewer macrophages present in epididymal adipose tissue than the HFD group. Furthermore, BARD administration reduced the inflammatory profile in this tissue by increasing the expression of nuclear factor of kappa-light-polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α) protein and decreasing the protein expression of tumour necrosis factor alpha (TNF-α). BARD also prevented oxidative stress reflected by a reduction in stress activated proteins, including signal transducer and activator of transcription 3 (STAT3), protein kinase B (Akt), extracellular-signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). BARD administration activated the sympathetic nervous system in epididymal adipose tissue assessed by the increased synthesis of tyrosine hydroxylase (TH) and uncoupling protein 2 (UCP2). The expression of inflammatory and sympathetic nervous system proteins in BARD mice fed a HFD was equivalent to that of the LFD control mice, indicating the anti-inflammatory and anti-obesity properties of this drug. In conclusion, the oral administration of BARD in HFD mice prevented fat deposition, inflammation and oxidative stress, and improved sympathetic activity in visceral fat. This study suggests a potential therapeutic role of BARD in preventing the development of obesity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Brown adipose tissue

PubMed Central

Townsend, Kristy; Tseng, Yu-Hua

2012-01-01

Obesity is currently a global pandemic, and is associated with increased mortality and co-morbidities including many metabolic diseases. Obesity is characterized by an increase in adipose mass due to increased energy intake, decreased energy expenditure, or both. While white adipose tissue is specialized for energy storage, brown adipose tissue has a high concentration of mitochondria and uniquely expresses uncoupling protein 1, enabling it to be specialized for energy expenditure and thermogenesis. Although brown fat was once considered only necessary in babies, recent morphological and imaging studies have provided evidence that, contrary to prior belief, this tissue is present and active in adult humans. In recent years, the topic of brown adipose tissue has been reinvigorated with many new studies regarding brown adipose tissue differentiation, function and therapeutic promise. This review summarizes the recent advances, discusses the emerging questions and offers perspective on the potential therapeutic applications targeting this tissue. PMID:23700507

Thyroid hormone-induced oxidative stress.

PubMed

Venditti, P; Di Meo, S

2006-02-01

Hypermetabolic state in hyperthyroidism is associated with tissue oxidative injury. Available data indicate that hyperthyroid tissues exhibit an increased ROS and RNS production. The increased mitochondrial ROS generation is a side effect of the enhanced level of electron carriers, by which hyperthyroid tissues increase their metabolic capacity. Investigations of antioxidant defence system have returned controversial results. Moreover, other thyroid hormone-linked biochemical changes increase tissue susceptibility to oxidative challenge, which exacerbates the injury and dysfunction they suffer under stressful conditions. Mitochondria, as a primary target for oxidative stress, might account for hyperthyroidism linked tissue dysfunction. This is consistent with the inverse relationship found between functional recovery of ischemic hyperthyroid hearts and mitochondrial oxidative damage and respiration impairment. However, thyroid hormone-activated mitochondrial mechanisms provide protection against excessive tissue dysfunction, including increased expression of uncoupling proteins, proteolytic enzymes and transcriptional coactivator PGC-1, and stimulate opening of permeability transition pores.

A Ketone Ester Diet Increases Brain Malonyl-CoA and Uncoupling Proteins 4 and 5 while Decreasing Food Intake in the Normal Wistar Rat*

PubMed Central

Kashiwaya, Yoshihiro; Pawlosky, Robert; Markis, William; King, M. Todd; Bergman, Christian; Srivastava, Shireesh; Murray, Andrew; Clarke, Kieran; Veech, Richard L.

2010-01-01

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD+]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain l-glutamate by 15–20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain. PMID:20529850

A ketone ester diet increases brain malonyl-CoA and Uncoupling proteins 4 and 5 while decreasing food intake in the normal Wistar Rat.

PubMed

Kashiwaya, Yoshihiro; Pawlosky, Robert; Markis, William; King, M Todd; Bergman, Christian; Srivastava, Shireesh; Murray, Andrew; Clarke, Kieran; Veech, Richard L

2010-08-20

Three groups of male Wistar rats were pair fed NIH-31 diets for 14 days to which were added 30% of calories as corn starch, palm oil, or R-3-hydroxybutyrate-R-1,3-butanediol monoester (3HB-BD ester). On the 14th day, animal brains were removed by freeze-blowing, and brain metabolites measured. Animals fed the ketone ester diet had elevated mean blood ketone bodies of 3.5 mm and lowered plasma glucose, insulin, and leptin. Despite the decreased plasma leptin, feeding the ketone ester diet ad lib decreased voluntary food intake 2-fold for 6 days while brain malonyl-CoA was increased by about 25% in ketone-fed group but not in the palm oil fed group. Unlike the acute effects of ketone body metabolism in the perfused working heart, there was no increased reduction in brain free mitochondrial [NAD(+)]/[NADH] ratio nor in the free energy of ATP hydrolysis, which was compatible with the observed 1.5-fold increase in brain uncoupling proteins 4 and 5. Feeding ketone ester or palm oil supplemented diets decreased brain L-glutamate by 15-20% and GABA by about 34% supporting the view that fatty acids as well as ketone bodies can be metabolized by the brain.

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways.

PubMed

Wijesuriya, Tishani Methsala; De Ceuninck, Leentje; Masschaele, Delphine; Sanderson, Matthea R; Carias, Karin Vanessa; Tavernier, Jan; Wevrick, Rachel

2017-11-01

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Progesterone Directly and Rapidly Inhibits GnRH Neuronal Activity via Progesterone Receptor Membrane Component 1

PubMed Central

Bashour, Nicholas Michael

2012-01-01

GnRH neurons are essential for reproduction, being an integral component of the hypothalamic-pituitary-gonadal axis. Progesterone (P4), a steroid hormone, modulates reproductive behavior and is associated with rapid changes in GnRH secretion. However, a direct action of P4 on GnRH neurons has not been previously described. Receptors in the progestin/adipoQ receptor family (PAQR), as well as progesterone receptor membrane component 1 (PgRMC1) and its partner serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1) mRNA binding protein 1 (SERBP1), have been shown to mediate rapid progestin actions in various tissues, including the brain. This study shows that PgRMC1 and SERBP1, but not PAQR, are expressed in prenatal GnRH neurons. Expression of PgRMC1 and SERBP1 was verified in adult mouse GnRH neurons. To investigate the effect of P4 on GnRH neuronal activity, calcium imaging was used on primary GnRH neurons maintained in explants. Application of P4 significantly decreased the activity of GnRH neurons, independent of secretion of gamma-aminobutyric acidergic and glutamatergic input, suggesting a direct action of P4 on GnRH neurons. Inhibition was not blocked by RU486, an antagonist of the classic nuclear P4 receptor. Inhibition was also maintained after uncoupling of the inhibitory regulative G protein (Gi/o), the signal transduction pathway used by PAQR. However, AG-205, a PgRMC1 ligand and inhibitor, blocked the rapid P4-mediated inhibition, and inhibition of protein kinase G, thought to be activated downstream of PgRMC1, also blocked the inhibitory activity of P4. These data show for the first time that P4 can act directly on GnRH neurons through PgRMC1 to inhibit neuronal activity. PMID:22822163

A high-throughput screen for mitochondrial function reveals known and novel mitochondrial toxicants in a library of environmental agents

PubMed Central

Datta, Sandipan; Sahdeo, Sunil; Gray, Jennifer A.; Morriseau, Christophe; Hammock, Bruce D.; Cortopassi, Gino

2016-01-01

Mitochondrial toxicity is emerging as a major mechanism underlying serious human health consequences. This work performs a high-throughput screen (HTS) of 176 environmental chemicals for mitochondrial toxicity utilizing a previously reported biosensor platform. This established HTS confirmed known mitochondrial toxins and identified novel mitotochondrial uncouplers such as 2, 2′-Methylenebis(4-chlorophenol) and pentachlorophenol. It also identified a mitochondrial ‘structure activity relationship’ (SAR) in the sense that multiple environmental chlorophenols are mitochondrial inhibitors and uncouplers. This study demonstrates proof-of-concept that a mitochondrial HTS assay detects known and novel environmental mitotoxicants, and could be used to quickly evaluate human health risks from mitotoxicants in the environment. PMID:27717841

The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation.

PubMed

Dauber, Bianca; Poon, David; Dos Santos, Theodore; Duguay, Brett A; Mehta, Ninad; Saffran, Holly A; Smiley, James R

2016-07-01

The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects of RNA metabolism. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

PPARγ activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1α and D2

PubMed Central

Festuccia, William T.; Blanchard, Pierre-Gilles; Oliveira, Thiago B.; Magdalon, Juliana; Paschoal, Vivian A.; Richard, Denis

2012-01-01

Here, we investigated whether pharmacological PPARγ activation modulates key early events in brown adipose tissue (BAT) recruitment induced by acute cold exposure with the aim of unraveling the interrelationships between sympathetic and PPARγ signaling. Sprague-Dawley rats treated or not with the PPARγ ligand rosiglitazone (15 mg·kg−1·day−1, 7 days) were kept at 23°C or exposed to cold (5°C) for 24 h and evaluated for BAT gene expression, sympathetic activity, thyroid status, and adrenergic signaling. Rosiglitazone did not affect the reduction in body weight gain and the increase in feed efficiency, V̇o2, and BAT sympathetic activity induced by 24-h cold exposure. Rosiglitazone strongly attenuated the increase in serum total and free T4 and T3 levels and BAT iodothyronine deiodinase type 2 (D2) and PGC-1α mRNA levels and potentiated the reduction in BAT thyroid hormone receptor (THR) β mRNA levels induced by cold. Administration of T3 to rosiglitazone-treated rats exacerbated the cold-induced increase in energy expenditure but did not restore a proper activation of D2 and PGC-1α, nor further increased uncoupling protein 1 expression. Regarding adrenergic signaling, rosiglitazone did not affect the changes in BAT cAMP content and PKA activity induced by cold. Rosiglitazone alone or in combination with cold increased CREB binding to DNA, but it markedly reduced the expression of one of its major coactivators, CREB binding protein. In conclusion, pharmacological PPARγ activation impairs short-term cold elicitation of BAT adrenergic and thyroid signaling, which may result in abnormal tissue recruitment and thermogenic activity. PMID:23100029

[Systemic and local mechanisms leading to cachexia in cancer].

PubMed

Grabiec, Kamil; Burchert, Marta; Milewska, Marta; Błaszczyk, Maciej; Grzelkowska-Kowalczyk, Katarzyna

2013-12-31

Cachexia is a multifactorial syndrome of atrophy of skeletal muscle and adipose tissue, resulting in progressive loss of body weight associated with low quality of life and poor prognosis in cancer. Studies on experimental animal models and observations on patients have shown that the soluble factors secreted by tumor cells and tissues of the patient can participate in regulation of the wasting process. Cachexia is often accompanied by anorexia, which is caused by predominance of signals inhibiting appetite in the hypothalamus, such as release of proopiomelanocortin and anorexigenic action of proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α). Cachexia is also accompanied by extensive metabolic changes consisting of increase of resting energy expenditure and disturbance of carbohydrate, protein and lipid metabolism. Increased expression of protein uncoupling phosphorylation leads to increased thermogenesis in skeletal muscle. Tumor tissue hypoxia caused by its growth beyond blood vessels activates the transcription factor HIF-1, which results in increase in glycolysis, and leads to lactic acid accumulation and activation of the energy inefficient Cori cycle. Loss of fat tissue is caused by increase of lipolysis induced by lipid-mobilizing factor (LMF) and proinflammatory cytokines. Skeletal muscle wasting in cachexia is caused by a reduction of protein synthesis at the stage of initiation and elongation of translation and the simultaneous increase of protein degradation via ubiquitin-dependent and lysosomal pathways. The main mediators of skeletal muscle wasting in cancer are proteolysis-inducing factor (PIF), proinflammatory cytokines, and angiotensin II acting through increased levels of reactive oxygen species (ROS) and nuclear factor NF-κB activation, as well as glucocorticoid activated FOXO transcription factors and myostatin. Understanding of the complexity of the interaction of factors produced by the tumor and the patient's body may form the basis for the development of effective treatments for cachexia in cancer and other pathological conditions.

Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells.

PubMed

Stöckl, Petra; Zankl, Christina; Hütter, Eveline; Unterluggauer, Hermann; Laun, Peter; Heeren, Gino; Bogengruber, Edith; Herndler-Brandstetter, Dietmar; Breitenbach, Michael; Jansen-Dürr, Pidder

2007-09-15

The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.

The cytotoxic effects of brown Cuban propolis depend on the nemorosone content and may be mediated by mitochondrial uncoupling.

PubMed

Pardo Andreu, Gilberto L; Reis, Felippe H Z; Dalalio, Felipe M; Nuñez Figueredo, Yanier; Cuesta Rubio, Osmany; Uyemura, Sergio A; Curti, Carlos; Alberici, Luciane C

2015-02-25

Three main types of Cuban propolis directly related to their secondary metabolite composition have been identified: brown, red and yellow propolis; the former is majoritarian and is characterized by the presence of nemorosone. In this study, brown Cuban propolis extracts were found cytotoxic against HepG2 cells and primary rat hepatocytes, in close association with the nemorosone contents. In mitochondria isolated from rat liver the extracts displayed uncoupling activity, which was demonstrated by the increase in succinate-supported state 4 respiration rates, dissipation of mitochondrial membrane potential, Ca(2+) release from Ca(2+)-loaded mitochondria, and a marked ATP depletion. As in cells, the degree of such mitotoxic events was closely correlated to the nemorosone content. The propolis extracts that do not contain nemorosone were neither cytotoxic nor mitotoxic, except R-29, whose detrimental effect upon cells and mitochondria could be mediated by its isoflavonoids and chalcones components, well known mitochondrial uncouplers. Our results at least partly unravel the cytotoxic mechanism of Cuban propolis, particularly regarding brown propolis, and raise concerns about the toxicological implication of Cuban propolis consumption. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions.

PubMed

Toranzo, G Sánchez; Oterino, J; Zelarayán, L; Bonilla, F; Bühler, M I

2007-02-01

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP(3). The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell-cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP(3) or Ca(2+) through the gap junctions, which would increase the Ca(2+) level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.

Sulfhydryl modification of V449C in the glutamate transporter EAAT1 abolishes substrate transport but not the substrate-gated anion conductance

PubMed Central

Seal, Rebecca P.; Shigeri, Yasushi; Eliasof, Scott; Leighton, Barbara H.; Amara, Susan G.

2001-01-01

Excitatory amino acid transporters (EAATs) buffer and remove synaptically released l-glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. Here, we demonstrate that modification of a cysteine substituted within a C-terminal domain of EAAT1 abolishes transport in both the forward and reverse directions without affecting activation of the anion conductance. EC50s for l-glutamate and sodium are significantly lower after modification, consistent with kinetic models of the transport cycle that link anion channel gating to an early step in substrate translocation. Also, decreasing the pH from 7.5 to 6.5 decreases the EC50 for l-glutamate to activate the anion conductance, without affecting the EC50 for the entire transport cycle. These findings demonstrate for the first time a structural separation of transport and the uncoupled anion flux. Moreover, they shed light on some controversial aspects of the EAAT transport cycle, including the kinetics of proton binding and anion conductance activation. PMID:11752470

PI3K/Akt is involved in brown adipogenesis mediated by growth differentiation factor-5 in association with activation of the Smad pathway

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

Hinoi, Eiichi; Iezaki, Takashi; Fujita, Hiroyuki

2014-07-18

Highlights: • Akt is preferentially phosphorylated in BAT and sWAT of aP2-GDF5 mice. • PI3K/Akt signaling is involved in GDF5-induced brown adipogenesis. • PI3K/Akt signaling regulates GDF5-induced Smad5 phosphorylation. - Abstract: We have previously demonstrated promotion by growth differentiation factor-5 (GDF5) of brown adipogenesis for systemic energy expenditure through a mechanism relevant to activating the bone morphological protein (BMP) receptor/mothers against decapentaplegic homolog (Smad)/peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) pathway. Here, we show the involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in brown adipogenesis mediated by GDF5. Overexpression of GDF5 in cells expressing adipocyte protein-2 markedly accelerated the phosphorylationmore » of Smad1/5/8 and Akt in white and brown adipose tissues. In brown adipose tissue from heterozygous GDF5{sup Rgsc451} mutant mice expressing a dominant-negative (DN) GDF5 under obesogenic conditions, the basal phosphorylation of Smad1/5/8 and Akt was significantly attenuated. Exposure to GDF5 not only promoted the phosphorylation of both Smad1/5/8 and Akt in cultured brown pre-adipocytes, but also up-regulated Pgc1a and uncoupling protein-1 expression in a manner sensitive to the PI3K/Akt inhibitor Ly294002 as well as retroviral infection with DN-Akt. GDF5 drastically promoted BMP-responsive luciferase reporter activity in a Ly294002-sensitive fashion. Both Ly294002 and DN-Akt markedly inhibited phosphorylation of Smad5 in the nuclei of brown pre-adipocytes. These results suggest that PI3K/Akt signals play a role in the GDF5-mediated brown adipogenesis through a mechanism related to activation of the Smad pathway.« less

Thermogenic Blend Alone or in Combination with Whey Protein Supplement Stimulates Fat Metabolism and Improves Body Composition in Mice

PubMed Central

Vieira-Brock, Paula de Lima; Vaughan, Brent M.; Vollmer, David L.

2018-01-01

Background: Certain food ingredients promote thermogenesis and fat loss. Similarly, whey protein improves body composition. Due to this potential synergistic effect, a blend of thermogenic food ingredients containing African mango, citrus fruit extract, Coleus forskohlii, dihydrocapsiate, and red pepper was tested alone and in combination with a whey protein supplement for its effects on body composition in sedentary mice during high-fat diet. Objective: The objective of this study was to evaluate the interaction of thermogenic foods on improving body composition during consumption of an unhealthy diet. Materials and Methods: C57BL/6J young adult male mice (n = 12) were placed on a 60% high-fat diet for 4 weeks and subsequently randomly assigned to receive daily dosing by oral gavage of vehicle, the novel blend alone or with whey protein supplement for another 4 weeks. Body composition, thermal imaging of brown adipose tissue (BAT), mitochondrial BAT uncoupling protein 1 (UCP1), and plasma levels of leptin were assessed. Results: Novel blend alone and in combination with protein supplement attenuated body weight gain, fat, and increased surface BAT temperature in comparison to vehicle control and to baseline (P < 0.5). The combination of novel blend and whey protein supplement also significantly increased UCP1 protein expression in BAT mitochondria in comparison to vehicle control and novel blend alone (P < 0.5). Conclusions: These data indicate that this novel blend stimulates thermogenesis and attenuates the gain in body weight and fat in response to high-fat diet in mice and these effects were improved when administered in combination with whey protein supplement. SUMMARY 30 days oral administration to mice of a novel blend containing African mango seed extract, citrus fruits extract, Coleus forskohlii root extract, dihydrocapsiate and red pepper fruit extract reduced body weight and fat gain in response to high-fat diet without impairing muscle mass.The novel blend stimulated thermogenesis as shown by the increased thermal imaging and UCP1 protein expression in brown adipose tissue, indicating that improvement in body composition potentially occurred due to a fat-burning effect.The positive effects on body weight, fat, and thermogenesis were improved when the novel blend was administered in combination with a whey protein supplement suggesting that protein provides a synergistic fat-burning effect. Abbreviations Used: BAT: Brown adipose tissue, UCP1: Uncoupling protein 1, DEXA: Dual-energy X-ray absorptiometry PMID:29568185

Transcriptomic and macroevolutionary evidence for phenotypic uncoupling between frog life history phases

PubMed Central

Wollenberg Valero, Katharina C.; Garcia-Porta, Joan; Rodríguez, Ariel; Arias, Mónica; Shah, Abhijeet; Randrianiaina, Roger Daniel; Brown, Jason L.; Glaw, Frank; Amat, Felix; Künzel, Sven; Metzler, Dirk; Isokpehi, Raphael D.; Vences, Miguel

2017-01-01

Anuran amphibians undergo major morphological transitions during development, but the contribution of their markedly different life-history phases to macroevolution has rarely been analysed. Here we generate testable predictions for coupling versus uncoupling of phenotypic evolution of tadpole and adult life-history phases, and for the underlying expression of genes related to morphological feature formation. We test these predictions by combining evidence from gene expression in two distantly related frogs, Xenopus laevis and Mantidactylus betsileanus, with patterns of morphological evolution in the entire radiation of Madagascan mantellid frogs. Genes linked to morphological structure formation are expressed in a highly phase-specific pattern, suggesting uncoupling of phenotypic evolution across life-history phases. This gene expression pattern agrees with uncoupled rates of trait evolution among life-history phases in the mantellids, which we show to have undergone an adaptive radiation. Our results validate a prevalence of uncoupling in the evolution of tadpole and adult phenotypes of frogs. PMID:28504275

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain...consistent with viral TM fusion proteins [9,10]. GPC con- tains a 58 residue hydrophobic N-terminal signal peptide (SP), which directs the precursor to the...including GPC, GP1, and GP2. Various signal peptides , purification tags, and modifications to internal domains were employed for the generation and

Immunological Properties of Hepatitis B Core Antigen Fusion Proteins

NASA Astrophysics Data System (ADS)

Francis, Michael J.; Hastings, Gillian Z.; Brown, Alan L.; Grace, Ken G.; Rowlands, David J.; Brown, Fred; Clarke, Berwyn E.

1990-04-01

The immunogenicity of a 19 amino acid peptide from foot-and-mouth disease virus has previously been shown to approach that of the inactivated virus from which it was derived after multimeric particulate presentation as an N-terminal fusion with hepatitis B core antigen. In this report we demonstrate that rhinovirus peptide-hepatitis B core antigen fusion proteins are 10-fold more immunogenic than peptide coupled to keyhole limpet hemocyanin and 100-fold more immunogenic than uncoupled peptide with an added helper T-cell epitope. The fusion proteins can be readily administered without adjuvant or with adjuvants acceptable for human and veterinary application and can elicit a response after nasal or oral dosing. The fusion proteins can also act as T-cell-independent antigens. These properties provide further support for their suitability as presentation systems for "foreign" epitopes in the development of vaccines.

eNOS-uncoupling in age-related erectile dysfunction

PubMed Central

Johnson, JM; Bivalacqua, TJ; Lagoda, GA; Burnett, AL; Musicki, B

2011-01-01

Aging is associated with ED. Although age-related ED is attributed largely to increased oxidative stress and endothelial dysfunction in the penis, the molecular mechanisms underlying this effect are not fully defined. We evaluated whether endothelial nitric oxide synthase (eNOS) uncoupling in the aged rat penis is a contributing mechanism. Correlatively, we evaluated the effect of replacement with eNOS cofactor tetrahydrobiopterin (BH4) on erectile function in the aged rats. Male Fischer 344 ‘young’ (4-month-old) and ‘aged’ (19-month-old) rats were treated with a BH4 precursor sepiapterin (10 mg/kg intraperitoneally) or vehicle for 4 days. After 1-day washout, erectile function was assessed in response to electrical stimulation of the cavernous nerve. Endothelial dysfunction (eNOS uncoupling) and oxidative stress (thiobarbituric acid reactive substances, TBARS) were measured by conducting western blot in penes samples. Erectile response was significantly reduced in aged rats, whereas eNOS uncoupling and TBARS production were significantly increased in the aged rat penis compared with young rats. Sepiapterin significantly improved erectile response in aged rats and prevented increase in TBARS production, but did not affect eNOS uncoupling in the penis of aged rats. These findings suggest that aging induces eNOS uncoupling in the penis, resulting in increased oxidative stress and ED. PMID:21289638

Triclosan is a Mitochondrial Uncoupler in Live Zebrafish

PubMed Central

Shim, Juyoung; Weatherly, Lisa M.; Luc, Richard H.; Dorman, Maxwell T.; Neilson, Andy; Ng, Ryan; Kim, Carol H.; Millard, Paul J.; Gosse, Julie A.

2016-01-01

Triclosan (TCS) is a synthetic antimicrobial agent used in many consumer goods at millimolar concentrations. As a result of exposure, TCS has been detected widely in humans. We have recently discovered that TCS is a proton ionophore mitochondrial uncoupler in multiple types of living cells. Here we present novel data indicating that TCS is also a mitochondrial uncoupler in a living organism: 24 hour post fertilization zebrafish embryos. These experiments were conducted using a Seahorse Bioscience XFe 96 Extracellular Flux Analyzer modified for bidirectional temperature control, using the XF96 spheroid plate to position and measure one zebrafish embryo per well. Using this method, following acute exposure to TCS, basal oxygen consumption rate (OCR) increases, without a decrease in survival or heartbeat rate. TCS also decreases ATP-linked respiration and spare respiratory capacity and increases proton leak: all indicators of mitochondrial uncoupling. Our data indicate, that TCS is a mitochondrial uncoupler in vivo, which should be taken into consideration when assessing the toxicity and/or pharmaceutical uses of TCS. This is the first example of usage of a Seahorse Extracellular Flux Analyzer to measure bioenergetic flux of a single zebrafish embryo per well in a 96 well assay format. The method developed in this study provides a high-throughput tool to identify previously-unknown mitochondrial uncouplers in a living organism. PMID:27111768