The Experts below are selected from a list of 18855 Experts worldwide ranked by ideXlab platform
Martin D Brand - One of the best experts on this subject based on the ideXlab platform.
-
Uncoupling Protein 3 lowers reactive oxygen species production in isolated mitochondria
Free Radical Biology and Medicine, 2010Co-Authors: Laurence J Toime, Martin D BrandAbstract:Mitochondria are the major cellular producers of reactive oxygen species (ROS), and mitochondrial ROS production increases steeply with increased proton-motive force. The Uncoupling Proteins (UCP1, UCP2, and UCP3) and adenine nucleotide translocase induce proton leak in response to exogenously added fatty acids, superoxide, or lipid peroxidation products. “Mild Uncoupling” by these Proteins may provide a negative feedback loop to decrease proton-motive force and attenuate ROS production. Using wild-type and Ucp3-/- mice, we found that native UCP3 actively lowers the rate of ROS production in isolated energized skeletal muscle mitochondria, in the absence of exogenous activators. The estimated specific activity of UCP3 in lowering ROS production was 90 to 500 times higher than that of the adenine nucleotide translocase. The mild Uncoupling hypothesis was tested by measuring whether the effect of UCP3 on ROS production could be mimicked by chemical Uncoupling. A chemical uncoupler mimicked the effect of UCP3 at early time points after mitochondrial energization, in support of the mild Uncoupling hypothesis. However, at later time points the uncoupler did not mimic UCP3, suggesting that UCP3 can also affect ROS production through a membrane potential-independent mechanism.
-
rapid turnover of mitochondrial Uncoupling Protein 3
Biochemical Journal, 2010Co-Authors: Vian Azzu, Shona A Mookerjee, Martin D BrandAbstract:UCP3 (Uncoupling Protein 3) and its homologues UCP2 and UCP1 are regulators of mitochondrial function. UCP2 is known to have a short half-life of approx. 1 h, owing to its rapid degradation by the cytosolic 26S proteasome, whereas UCP1 is turned over much more slowly by mitochondrial autophagy. In the present study we investigate whether UCP3 also has a short half-life, and whether the proteasome is involved in UCP3 degradation. UCP3 half-life was examined in the mouse C2C12 myoblast cell line by inhibiting Protein synthesis with cycloheximide and monitoring UCP3 Protein levels by immunoblot analysis. We show that UCP3 has a short half-life of 0.5-4 h. Rapid degradation was prevented by a cocktail of proteasome inhibitors, supporting a proteasomal mechanism for turnover. In addition, this phenotype is recapitulated in vitro: UCP3 was degraded in mitochondria isolated from rat skeletal muscle or brown adipose tissue with a half-life of 0.5-4 h, but only in the presence of a purified 26S proteasomal fraction. This in vitro proteolysis was also sensitive to proteasome inhibition. This phenotype is in direct contrast with the related Proteins UCP1 and the adenine nucleotide translocase, which have long half-lives. Therefore UCP3 is turned over rapidly in multiple cell types in a proteasome-dependent manner.
-
Uncoupling Protein 3 protects aconitase against inactivation in isolated skeletal muscle mitochondria
Biochimica et Biophysica Acta, 2005Co-Authors: Darren A Talbot, Martin D BrandAbstract:Mitochondrial Uncoupling Proteins only catalyse proton transport when they are activated. Activators include superoxide and reactive alkenals, suggesting new physiological functions for UCP2 and UCP3: their activation by superoxide when protonmotive force is high causes mild Uncoupling, which lowers protonmotive force and attenuates superoxide generation by the electron transport chain. This feedback loop acts to prevent excessive mitochondrial superoxide production. Superoxide inactivates aconitase in the mitochondrial matrix, so aconitase activity provides a sensitive measure of the effects of UCPs on matrix superoxide. We find that inhibition of UCP3 in isolated skeletal muscle mitochondria by GDP decreases aconitase activity by 25% after 20 min incubation. The GDP effect is absent in skeletal muscle mitochondria from UCP3 knockout mice, showing that it is mediated by UCP3. Protection of aconitase by UCP3 in the absence of nucleotides does not require added fatty acids. The purine nucleoside diphosphates and triphosphates cause aconitase inactivation, but the monophosphates and CDP do not, consistent with the known nucleotide specificity of UCP3. The IC(50) for GDP is about 100 microM. These findings support the proposal that UCP3 attenuates endogenous radical production by the mitochondrial electron transport chain at high protonmotive force.
-
oxidative damage and phospholipid fatty acyl composition in skeletal muscle mitochondria from mice underexpressing or overexpressing Uncoupling Protein 3
Biochemical Journal, 2002Co-Authors: Martin D Brand, John C Clapham, Reinald Pamplona, Manuel Porterootin, Jesus R Requena, Stephen J Roebuck, Julie A Buckingham, Susana CadenasAbstract:Five markers of dierent kinds of oxidative damage to Proteins [glutamic semialdehyde, aminoadipic semialdehyde, N e -(carboxymethyl)lysine, N e -(carboxyethyl)lysine and N e -(malondialdehyde)lysine] and phospholipid fatty acyl composition were identified and measured in skeletal muscle mitochondria isolated from mice genetically engineered to underexpress or overexpress Uncoupling Protein 3 (UCP3). Mitochondria from UCP3underexpressing mice had significantly higher levels of oxidative damage than wild-type controls, suggesting that UCP3 functions in aiao as part of the antioxidant defences of the cell, but mitochondria from UCP3-overexpressing mice had unaltered oxidative damage, suggesting that mild Uncoupling in aiao beyond the normal basal Uncoupling provides little protection against
-
oxidative damage and phospholipid fatty acyl composition in skeletal muscle mitochondria from mice underexpressing or overexpressing Uncoupling Protein 3
Biochemical Journal, 2002Co-Authors: Martin D Brand, John C Clapham, Reinald Pamplona, Manuel Porterootin, Jesus R Requena, Stephen J Roebuck, Julie A Buckingham, Susana CadenasAbstract:Five markers of different kinds of oxidative damage to Proteins [glutamic semialdehyde, aminoadipic semialdehyde, N (epsilon)-(carboxymethyl)lysine, N (epsilon)-(carboxyethyl)lysine and N (epsilon)-(malondialdehyde)lysine] and phospholipid fatty acyl composition were identified and measured in skeletal muscle mitochondria isolated from mice genetically engineered to underexpress or overexpress Uncoupling Protein 3 (UCP3). Mitochondria from UCP3-underexpressing mice had significantly higher levels of oxidative damage than wild-type controls, suggesting that UCP3 functions in vivo as part of the antioxidant defences of the cell, but mitochondria from UCP3-overexpressing mice had unaltered oxidative damage, suggesting that mild Uncoupling in vivo beyond the normal basal Uncoupling provides little protection against oxidative stress. Mitochondria from UCP3-underexpressing mice showed little change, but mitochondria from UCP3-overexpressing mice showed marked changes in mitochondrial phospholipid fatty acyl composition. These changes were very similar to those previously found to correlate with basal proton conductance in mitochondria from a range of species and treatments, suggesting that high Protein expression, or some secondary result of Uncoupling, may cause the observed correlation between basal proton conductance and phospholipid fatty acyl composition.
Francesc Villarroya - One of the best experts on this subject based on the ideXlab platform.
-
sirt1 is involved in glucocorticoid mediated control of Uncoupling Protein 3 gene transcription
Journal of Biological Chemistry, 2007Co-Authors: Ramon Amat, Gemma Solanes, Marta Giralt, Francesc VillarroyaAbstract:UCP3 (Uncoupling Protein-3) is a mitochondrial membrane transporter expressed preferentially in skeletal muscle. UCP3 lowers mitochondrial membrane potential and protects muscle cells against an overload of fatty acids, and it probably reduces excessive production of reactive oxygen species. Accordingly, ucp3 gene transcription is highly sensitive to fatty acid-dependent stimulation and also to other unrelated stress signals. In this study, glucocorticoids are identified as major inducers of ucp3 gene transcription in muscle. Glucocorticoids activate the transcription of the ucp3 gene through a glucocorticoid receptor-binding site in the promoter region. Glucocorticoids are capable of inducing ucp3 gene transcription independently from the myogenic regulatory factor MyoD, in contrast with the transcriptional activation of the ucp3 gene through other nuclear hormone receptors. An interplay of regulatory factors modulates positively (p300) or negatively (histone deacetylases) the action of glucocorticoids on ucp3 gene transcription via histone acetylation or deacetylation processes, respectively. Among them, SIRT1 acts as a major repressor of ucp3 gene expression in response to glucocorticoids. The action of SIRT1 requires its deacetylase activity and results in histone deacetylation in the ucp3 promoter. Moreover, it involves a specific impairment of association of p300 with the glucocorticoid receptor. Agents activating SIRT1, such as resveratrol, repress ucp3 gene expression. The control of SIRT1 activity via the metabolic redox status of the cell points to a novel regulatory pathway of ucp3 gene transcription in response to metabolic and stress signaling in muscle cells.
-
Overexpression of mitochondrial Uncoupling Protein-3 does not decrease production of the reactive oxygen species, elevated by palmitate in skeletal muscle cells.
FEBS Letters, 2007Co-Authors: Carine Duval, Yolanda Cámara, Elayne Hondares, Brigitte Sibille, Francesc VillarroyaAbstract:Fatty acids induced an increase in reactive oxygen species (ROS) and enhanced NF-kappaB activation in L6 myotubes differentiated in culture. Palmitate proved more effective than oleate in eliciting these effects. The induction of Uncoupling Protein-3 (UCP3) at levels similar to those occurring in vivo, attained through the use of an adenoviral vector, led to a reduction of mitochondrial membrane potential in L6 myotubes. However, the capacity of palmitate to increase ROS was not reduced but, quite the opposite, it was moderately enhanced due to the presence of UCP3. The presence of UCP3 in mitochondria did not modify the expression of genes encoding ROS-related enzymes, either in basal conditions or in the presence of palmitate. However, in the presence of UCP3, UCP2 mRNA expression was down-regulated in response to palmitate. We conclude that UCP3 does not act as a protective agent against palmitate-dependent induction of ROS production in differentiated skeletal muscle cells.
-
developmental and tissue specific involvement of peroxisome proliferator activated receptor α in the control of mouse Uncoupling Protein 3 gene expression
Endocrinology, 2006Co-Authors: Neus Pedraza, Gemma Solanes, Roser Iglesias, Meritxell Rosell, Joan Villarroya, Frank J Gonzalez, Francesc VillarroyaAbstract:Uncoupling Protein-3 (UCP3) is a member of the mitochondrial carrier family expressed preferentially in skeletal muscle and heart. It appears to be involved in metabolic handling of fatty acids in a way that minimizes excessive production of reactive oxygen species. Fatty acids are powerful regulators of UCP3 gene transcription. We have found that the role of peroxisome proliferator-activated receptor- (PPAR )o n the control of UCP3 gene expression depends on the tissue and developmental stage. In adults, UCP3 mRNA expression is unaltered in skeletal muscle from PPAR-null mice both in basal conditions and under the stimulus of starvation. In contrast,UCP3mRNAisdown-regulatedinadultheartbothinfed and fasted PPAR-null mice. This occurs despite the increased levels of free fatty acids caused by fasting in PPARnull mice. In neonates, PPAR-null mice show impaired UCP3 mRNA expression in skeletal muscle in response to milk intake, and this is not a result of reduced free fatty acid levels. ThemurineUCP3promoterisactivatedbyfattyacidsthrough either PPAR or PPAR but not by PPAR or retinoid X receptor alone. PPAR-dependent activation could be a potential compensatory mechanism to ensure appropriate expression of UCP3 gene in adult skeletal muscle in the absence of PPAR. However, among transcripts from other PPAR and PPAR target genes, only those acutely induced by milk intake in wild-type neonates were altered in muscle or heart from PPAR-null neonates. Thus, PPAR-dependent regulation is required for appropriate gene regulation of UCP3 as part of the subset of fatty-acid-responsive genes in neonatal muscle and heart. (Endocrinology 147: 4695–4704, 2006)
-
thyroid hormones directly activate the expression of the human and mouse Uncoupling Protein 3 genes through a thyroid response element in the proximal promoter region
Biochemical Journal, 2005Co-Authors: Gemma Solanes, Bradford B. Lowell, Antonio Vidalpuig, Neus Pedraza, Veronica Calvo, Francesc VillarroyaAbstract:The transcription of the human UCP3 (Uncoupling Protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo. The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo, and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle.
-
functional relationship between myod and peroxisome proliferator activated receptor dependent regulatory pathways in the control of the human Uncoupling Protein 3 gene transcription
Molecular Endocrinology, 2003Co-Authors: Gemma Solanes, Marta Giralt, Roser Iglesias, Neus Pedraza, Francesc VillarroyaAbstract:Uncoupling Protein-3 (UCP3) gene is a member of the mitochondrial carrier superfamily preferentially expressed in skeletal muscle and up-regulated by fatty acids. Peroxisome proliferator-activated receptor (PPAR)alpha and PPARdelta (also known as PPARbeta) mediate human UCP3 gene regulation by fatty acids through a direct-repeat (DR-1) element in the promoter. DR-1 mutation renders UCP3 promoter unresponsive to PPAR ligand in vitro and consistently blocks gene induction by fatty acids in vivo. Although they act through separate sites in the promoter, MyoD and PPAR-dependent regulatory pathways are functionally connected: only in the presence of MyoD, does UCP3 become sensitive to PPAR ligand-dependent regulation. MyoD controls UCP3 promoter activity through a noncanonical Ebox site located in the proximal region, close to transcription initiation site. Moreover, acetylation processes play a crucial role in the control of UCP3 gene regulation. The coactivator p300 Protein enhances PPAR ligand-mediated regulation whereas a mutant form devoid of histone acetylase activity blocks the response of the promoter to fatty acids. Conversely, histone deacetylase-1 blunts MyoD-dependent expression of the UCP3 promoter and reduces PPAR-dependent responsiveness. A mutated form of MyoD unable to be acetylated has a lower transactivation capacity on the human UCP3 promoter with respect to wild-type MyoD. It is concluded that MyoD and PPAR-dependent pathways mediate human UCP3 gene regulation and that acetylase activity elicited by coregulators is implicated in the functional interaction between these regulatory pathways. Therefore the convergence of MyoD and PPAR-dependent pathways provides a molecular mechanism for skeletal muscle specificity and fatty acid regulation of human UCP3 gene.
Patrick Schrauwen - One of the best experts on this subject based on the ideXlab platform.
-
uncoupled respiration ros production acute lipotoxicity and oxidative damage in isolated skeletal muscle mitochondria from ucp3 ablated mice
Biochimica et Biophysica Acta, 2011Co-Authors: Patrick Schrauwen, Esther Moonenkornips, Miranda Nabben, Irina G Shabalina, Denis Van Beurden, Barbara Cannon, Jan Nedergaard, Joris HoeksAbstract:The function of Uncoupling Protein 3 (UCP3) is still not established. Mitochondrial Uncoupling, control of ROS production, protection against lipotoxicity and protection against oxidative stress ar ...
-
reduced skeletal muscle Uncoupling Protein 3 content in prediabetic subjects and type 2 diabetic patients restoration by rosiglitazone treatment
The Journal of Clinical Endocrinology and Metabolism, 2006Co-Authors: Patrick Schrauwen, Gert Schaart, Esther Moonenkornips, Ellen E Blaak, Marco Mensink, J P J E Sels, Aaron P Russell, Matthijs K. C. HesselinkAbstract:Context: The mitochondrial Uncoupling Protein-3 (UCP3) has been implicated in the protection of the mitochondrial matrix against lipid-induced mitochondrial damage. Recent evidence points toward mitochondrial aberrations as a major contributor to the development of insulin resistance and diabetes, and UCP3 is reduced in diabetes. Objective: We compared skeletal muscle UCP3 Protein levels in prediabetic subjects [i.e. impaired glucose tolerance (IGT)], diabetic patients, and healthy controls and examined whether rosiglitazone treatment was able to restore UCP3. Patients, Design, Intervention: Ten middle-aged obese men with type 2 diabetes mellitus [age, 61.4 ± 3.1 yr; body mass index (BMI), 29.8 ± 2.9 kg/m2], nine IGT subjects (age, 59.0 ± 6.6 yr; BMI, 29.7 ± 3.0 kg/m2), and 10 age- and BMI-matched healthy controls (age, 57.3 ± 7.4 yr; BMI, 30.1 ± 3.9 kg/m2) participated in this study. After baseline comparisons, diabetic patients received rosiglitazone (2 × 4 mg/d) for 8 wk. Main Outcome Measures: Muscle ...
-
exercise training restores Uncoupling Protein 3 content in limb muscles of patients with chronic obstructive pulmonary disease
American Journal of Physiology-endocrinology and Metabolism, 2006Co-Authors: Harry R Gosker, Patrick Schrauwen, Matthijs K. C. Hesselink, R Broekhuizen, Esther Moonenkornips, Kimberly A Ward, Frits M E Franssen, Emiel F M Wouters, Annemie M W J ScholsAbstract:Oxidative capacity and Uncoupling Protein-3 (UCP3) content are reduced in limb muscles of patients with chronic obstructive pulmonary disease (COPD). It has been hypothesized that the physiological...
-
putative function and physiological relevance of the mitochondrial Uncoupling Protein 3 involvement in fatty acid metabolism
Progress in Lipid Research, 2006Co-Authors: Patrick Schrauwen, Joris Hoeks, Matthijs K. C. HesselinkAbstract:The discovery of the human homologue of the thermogenic Protein UCP1, named Uncoupling Protein 3 (UCP3), boosted research on the role of this skeletal muscle Protein in energy metabolism and body weight regulation. Nowadays, 9 years after its discovery emerging data indicate that the primary physiological role of UCP3 may be the mitochondrial handling of fatty acids rather than regulating energy expenditure via thermogenesis. UCP3 has been proposed to export fatty acid anions or fatty acid peroxides away from the matrix-side of the mitochondrial inner membrane to prevent their deleterious accumulation. In this way, UCP3 could protect mitochondria against lipid-induced oxidative mitochondrial damage, a function especially important under conditions of high fatty acid supply to skeletal muscle mitochondria. Such function may be clinically relevant in the development of type 2 diabetes mellitus, a condition characterized by muscular fat accumulation, mitochondrial damage and low levels of UCP3.
-
the role of Uncoupling Protein 3 in fatty acid metabolism protection against lipotoxicity
Proceedings of the Nutrition Society, 2004Co-Authors: Patrick Schrauwen, Matthijs K. C. HesselinkAbstract:The physiological function of the mitochondrial Uncoupling Protein (UCP), UCP3, is still under debate. There is, however, ample evidence to indicate that, in contrast to UCP1, the primary function of UCP3 is not the dissipation of energy. Rather, several lines of evidence suggest that UCP3 is associated with cellular fatty acid metabolism. The highest levels of expression of UCP3 have been found in type 2 glycolytic muscle fibres, and fasting and high-fat diets up regulate UCP3. This up-regulation is most pronounced in muscle with a low fat oxidative capacity. Acute exercise also up regulates UCP3, and this effect has been shown to be a result of the exercise-induced increase in plasma fatty acid levels. In contrast, regular physical activity, which increases fat oxidative capacity, reduces UCP3 content. Based on these data it has been postulated that UCP3 functions to export those fatty acids that cannot be oxidized from the mitochondrial matrix, in order to prevent fatty acid accumulation inside the matrix. Several experiments have been conducted to test this hypothesis. Blocking carnitine palmitoyltransferase 1, thereby reducing fat oxidative capacity, rapidly induces UCP3. High-fat diets, which increase the mitochondrial supply of fatty acids, also up regulate UCP. However, feeding a similar amount of medium-chain fatty acids, which can be oxidized inside the mitochondrial matrix and therefore does not need to be exported from the matrix, does not affect UCP3 Protein levels. In addition, UCP3 is increased in patients with defective beta-oxidation and is reduced after restoring oxidative capacity. In conclusion, it is suggested that UCP3 has an important physiological function in facilitating outward transport from the mitochondrial matrix of fatty acid anions that cannot be oxidized, thereby protecting against lipid-induced mitochondrial damage.
Bradford B. Lowell - One of the best experts on this subject based on the ideXlab platform.
-
thyroid hormones directly activate the expression of the human and mouse Uncoupling Protein 3 genes through a thyroid response element in the proximal promoter region
Biochemical Journal, 2005Co-Authors: Gemma Solanes, Bradford B. Lowell, Antonio Vidalpuig, Neus Pedraza, Veronica Calvo, Francesc VillarroyaAbstract:The transcription of the human UCP3 (Uncoupling Protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo. The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo, and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle.
-
in vivo effects of Uncoupling Protein 3 gene disruption on mitochondrial energy metabolism
Journal of Biological Chemistry, 2001Co-Authors: Gary W Cline, Bradford B. Lowell, Antonio Vidalpuig, Sylvie Dufour, Kevin S Cadman, Gerald I ShulmanAbstract:To clarify the role of Uncoupling Protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using 31P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of 13C enrichment in C-4 and C-2 of glutamate during an infusion of [2-13C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from Pi more than 4-fold under fasting conditions (wild type, 2.2 ± 0.6versus knockout, 9.1 ± 1.4 μmol/g of muscle/min,p
-
Assessment of Uncoupling activity of the human Uncoupling Protein 3 short form and three mutants of the Uncoupling Protein gene using a yeast heterologous expression system.
FEBS letters, 1999Co-Authors: Thilo Hagen, Chen-yu Zhang, Lawrence J. Slieker, Wendy K. Chung, Rudolph L. Leibel, Bradford B. LowellAbstract:The human Uncoupling Protein 3 gene generates two mRNA transcripts, Uncoupling Protein 3L and Uncoupling Protein 3S, which are predicted to encode long and short forms of the Uncoupling Protein 3 Protein, respectively. While Uncoupling Protein 3L is similar in length to the other known Uncoupling Proteins, Uncoupling Protein 3S lacks the last 37 C-terminal residues. A splice site mutation in the human Uncoupling Protein 3 gene, resulting in the exclusive expression of Uncoupling Protein 3S, and a number of point mutations in the Uncoupling Protein 3 gene have been described. This study compares the biochemical activity of Uncoupling Protein 3S as well as three mutants of the Uncoupling Protein 3 gene (V9M, V1021, R282C) with that of Uncoupling Protein 3L utilizing a yeast expression system. All Proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the Uncoupling function. Both Uncoupling Protein 3S and Uncoupling Protein 3L decreased the yeast growth rate by 3S and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide. In isolated mitochondria, Uncoupling Protein 3S and Uncoupling Protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to Uncoupling Protein 1 (102% increase). A truncated version of Uncoupling Protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced Uncoupling activity. The two naturally occurring Uncoupling Protein 3 mutants, V9M and V1021, were similar to Uncoupling Protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption. The R282C mutant had a reduced effect compared to Uncoupling Protein 3L. In summary, Uncoupling Protein 3S and the three mutants of Uncoupling Protein 3 appear to be functional Proteins with biochemical activities similar to Uncoupling Protein 3L, although Uncoupling Protein 3S and the R282C mutant have a modestly reduced function.
-
Assessment of Uncoupling activity of Uncoupling Protein 3 using a yeast heterologous expression system.
FEBS letters, 1999Co-Authors: Chen-yu Zhang, Thilo Hagen, Lawrence J. Slieker, V K Mootha, Bradford B. LowellAbstract:Uncoupling Protein 3L, Uncoupling Protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both Uncoupling Protein 3L and Uncoupling Protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, Uncoupling Protein 1 increased the state 4 respiration by 1.8-fold, while Uncoupling Protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant Uncoupling Protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of Uncoupling Protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type Uncoupling Protein 1. Thus, absence of this histidine pair in Uncoupling Protein 2 and Uncoupling Protein 3 does not by itself rule out the possibility that these carriers have an Uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of Uncoupling Protein 3L and Uncoupling Protein 1 in whole yeast versus isolated mitochondria, with Uncoupling Protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that Uncoupling Protein 3L, like Uncoupling Protein 1, has an Uncoupling activity. However, the mechanism of action and/or regulation of the activity of Uncoupling Protein 3L is likely to be different.
-
the human Uncoupling Protein 3 gene genomic structure chromosomal localization and genetic basis for short and long form transcripts
Journal of Biological Chemistry, 1997Co-Authors: Gemma Solanes, Danica Grujic, Antonio Vidalpuig, Jeffrey S Flier, Bradford B. LowellAbstract:Abstract Uncoupling Protein-3 (UCP3) is a recently identified candidate mediator of adaptive thermogenesis in humans. Unlike UCP1 and UCP2, UCP3is expressed preferentially and at high levels in human skeletal muscle and exists as short and long form transcripts,UCP3 S and UCP3 L.UCP3 S is predicted to encode a Protein which lacks the last 37 C-terminal residues of UCP3 L. In the present study, we have defined the intron-exon structure for the human UCP3 gene and determined thatUCP3 S is generated when a cleavage and polyadenylation signal (AATAAA) located in the last intron prematurely terminates message elongation. In addition we have mappedUCP3 to the distal segment of human chromosome 11q13 (between framework markers D11S916 and D11S911), adjacent toUCP2. Of note, UCP2 and UCP3 in both mice and humans colocalize in P1 and BAC genomic clones indicating that these two UCPs are located within 75–150 kilobases of each other and most likely resulted from a gene duplication event. Previous studies have noted that mouse UCP2 maps to a region of chromosome 7 which is coincident with three independently mapped quantitative trait loci for obesity. Our study shows thatUCP3 is also coincident with these quantitative trait loci raising the possibility that abnormalities in UCP3 are responsible for obesity in these models.
Mary-ellen Harper - One of the best experts on this subject based on the ideXlab platform.
-
skeletal muscle mitoflashes ph and the role of Uncoupling Protein 3
Archives of Biochemistry and Biophysics, 2019Co-Authors: Skye Mcbride, Lan Weilapierre, Fiona Mcmurray, Megan Macfarlane, X Qiu, David A Patten, Robert T Dirksen, Mary-ellen HarperAbstract:Mitochondrial reactive oxygen species (ROS) are important cellular signaling molecules, but can cause oxidative damage if not kept within tolerable limits. An important proximal form of ROS in mitochondria is superoxide. Its production is thought to occur in regulated stochastic bursts, but current methods using mitochondrial targeted cpYFP to assess superoxide flashes are confounded by changes in pH. Accordingly, these flashes are generally referred to as 'mitoflashes'. Here we provide regulatory insights into mitoflashes and pH fluctuations in skeletal muscle, and the role of Uncoupling Protein-3 (UCP3). Using quantitative confocal microscopy of mitoflashes in intact muscle fibers, we show that the mitoflash magnitude significantly correlates with the degree of mitochondrial inner membrane depolarization and ablation of UCP3 did not affect this correlation. We assessed the effects of the absence of UCP3 on mitoflash activity in intact skeletal muscle fibers, and found no effects on mitoflash frequency, amplitude or duration, with a slight reduction in the average size of mitoflashes. We further investigated the regulation of pH flashes (pHlashes, presumably a component of mitoflash) by UCP3 using mitochondrial targeted SypHer (mt-SypHer) in skeletal muscle fibers. The frequency of pHlashes was significantly reduced in the absence of UCP3, without changes in other flash properties. ROS scavenger, tiron, did not alter pHlash frequency in either WT or UCP3KO mice. High resolution respirometry revealed that in the absence of UCP3 there is impaired proton leak and Complex I-driven respiration and maximal coupled respiration. Total cellular production of hydrogen peroxide (H2O2) as detected by Amplex-UltraRed was unaffected. Altogether, we demonstrate a correlation between mitochondrial membrane potential and mitoflash magnitude in skeletal muscle fibers that is independent of UCP3, and a role for UCP3 in the control of pHlash frequency and of proton leak- and Complex I coupled-respiration in skeletal muscle fibers. The differential regulation of mitoflashes and pHlashes by UCP3 and tiron also indicate that the two events, though may be related, are not identical events.
-
a novel amino acid and metabolomics signature in mice overexpressing muscle Uncoupling Protein 3
The FASEB Journal, 2017Co-Authors: Celine Aguer, Brian D Piccolo, Oliver Fiehn, Sean H Adams, Mary-ellen HarperAbstract: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.
-
glutaredoxin 2 is required to control proton leak through Uncoupling Protein 3
Journal of Biological Chemistry, 2013Co-Authors: Ryan J Mailloux, Jian Ying Xuan, Brittany Beauchamp, Linda Jui, Marjorie F Lou, Mary-ellen HarperAbstract:Abstract 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.
-
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
Biochimica et Biophysica Acta, 2010Co-Authors: Lisa Bevilacqua, Erin L. Seifert, Carmen Estey, Martin F Gerrits, Mary-ellen HarperAbstract: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.
-
essential role for Uncoupling Protein 3 in mitochondrial adaptation to fasting but not in fatty acid oxidation or fatty acid anion export
Journal of Biological Chemistry, 2008Co-Authors: Erin L. Seifert, Véronic Bézaire, Carmen Estey, Mary-ellen HarperAbstract:Abstract Uncoupling Protein-3 (UCP3) is a mitochondrial inner membrane Protein expressed most abundantly in skeletal muscle and to a lesser extent in heart and brown adipose tissue. Evidence supports a role for UCP3 in fatty acid oxidation (FAO); however, the underlying mechanism has not been explored. In 2001 we proposed a role for UCP3 in fatty acid export, leading to higher FAO rates (Himms-Hagen, J., and Harper, M. E. (2001) Exp. Biol. Med. (Maywood) 226, 78–84). Specifically, this widely held hypothesis states that during elevated FAO rates, UCP3 exports fatty acid anions, thereby maintaining mitochondrial co-enzyme A availability; reactivation of exported fatty acid anions would ultimately enable increased FAO. Here we tested mechanistic aspects of this hypothesis as well as its functional implications, namely increased FAO rates. Using complementary mechanistic approaches in mitochondria from wild-type and Ucp3–/– mice, we find that UCP3 is not required for FAO regardless of substrate type or supply rate covering a 20-fold range. Fatty acid anion export and reoxidation during elevated FAO, although present in skeletal muscle mitochondria, are independent of UCP3 abundance. Interestingly, UCP3 was found to be necessary for the fasting-induced enhancement of FAO rate and capacity, possibly via mitigated mitochondrial oxidative stress. Thus, although our observations indicate that UCP3 can impact FAO rates, the mechanistic basis is not via an integral function for UCP3 in the FAO machinery. Overall our data indicate a function for UCP3 in mitochondrial adaptation to perturbed cellular energy balance and integrate previous observations that have linked UCP3 to reduced oxidative stress and FAO.
