Role of UCP3 in state 4 respiration during contractile activity-induced mitochondrial biogenesis.

In an effort to better characterize uncoupling protein-3 (UCP3) function in skeletal muscle, we assessed basal UCP3 protein content in rat intermyofibrillar (IMF) and subsarcolemmal (SS) mitochondrial subfractions in conjunction with measurements of state 4 respiration. UCP3 content was 1.3-fold (P < 0.05) greater in IMF compared with SS mitochondria. State 4 respiration was 2.6-fold greater (P < 0.05) in the IMF subfraction than in SS mitochondria. GDP attenuated state 4 respiration by approximately 40% (P < 0.05) in both subfractions. The UCP3 activator oleic acid (OA) significantly increased state 4 respiration in IMF mitochondria only. We used chronic electrical stimulation (3 h/day for 7 days) to investigate the relationship between changes in UCP3 protein expression and alterations in state 4 respiration during contractile activity-induced mitochondrial biogenesis. UCP3 content was increased by 1.9- and 2.3-fold in IMF and SS mitochondria, respectively, which exceeded the concurrent 40% (P < 0.05) increase in cytochrome-c oxidase activity. Chronic contractile activity increased state 4 respiration by 1.4-fold (P < 0.05) in IMF mitochondria, but no effect was observed in the SS subfraction. The uncoupling function of UCP3 accounted for 50-57% of the OA-induced increase in state 4 respiration in IMF mitochondria, which was independent of the induced twofold difference in UCP3 content due to chronic contractile activity. Thus modifications in UCP3 function are more important than changes in UCP3 expression in modifying state 4 respiration. This effect is evident in IMF but not SS mitochondria. We conclude that UCP3 at physiological concentrations accounts for a significant portion of state 4 respiration in both IMF and SS mitochondria, with the contribution being greater in the IMF subfraction. In addition, the contradiction between human and rat training studies with respect to UCP3 protein expression may partly be explained by the greater than twofold difference in mitochondrial UCP3 content between rat and human skeletal muscle.     

Spatially distinct mitochondrial populations exhibit different mitofilin levels

Subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria exhibit unique biochemical and functional properties; however, their association with structural membrane proteins that control mitochondrial morphology and functionality in striated muscle tissue was never reported. In IMF and SS mitochondria isolated from rat heart and gastrocnemius muscle, we analysed the expression levels of mitofilin, a mitochondria-associated protein involved in organelle structure maintenance. The statistically significant higher amounts of mitofilin detected in IMF compared with SS mitochondria, 37-fold in cardiac tissue and 3.8-fold in gastrocnemius, together with the specific energetic requirements of these mitochondrial populations highlight the importance of mitofilin in oxidative phosphorylation functionality and in mitochondrial plasticity in striated muscle. The differential expression levels of mitofilin between IMF and SS also suggest that this protein can be used as a specific molecular marker to comparatively discriminate spatially distant mitochondrial populations.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single ∼33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 °C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 °C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation

A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.     

Upregulation of uncoupling protein-3 in skeletal muscle during exercise: a potential antioxidant function

Uncoupling protein-3 (UCP3) expression has been shown to increase dramatically in response to muscular contraction, but the physiological significance of UCP3 upregulation is still elusive. In this study, UCP3 mRNA and protein expression were investigated along with mitochondrial respiratory function, reactive oxygen species (ROS) generation, and antioxidant defense in rat skeletal muscle during and after an acute bout of prolonged exercise. UCP3 mRNA expression was elevated sharply at 45 min of exercise, reaching 7- to 8-fold above resting level at 150 min. The increase in UCP3 protein content showed a latent response but was elevated approximately 1.9-fold at 120 min of exercise. Both UCP3 mRNA and UCP3 protein gradually returned to resting levels 24 h postexercise. Mitochondrial ROS production was progressively increased during exercise. However, ROS showed a dramatic drop at 150 min although their levels remained severalfold higher during the recovery. Mitochondrial State 4 respiration rate was increased by 46 and 58% (p < 0.05) at 90 and 120 min, respectively, but returned to resting rate at 150 min, when State 3 respiration and respiratory control index (RCI) were suppressed. ADP-to-oxygen consumption (P/O) ratio and ATP synthase activity were lowered at 3 h postexercise, whereas proton motive force and mitochondrial malondialdehyde content were unchanged. Manganese superoxide dismutase gene expression was not affected by exercise except for an increase in mRNA abundance at 3 h postexercise. These data demonstrate that UCP3 expression in rat skeletal muscle can be rapidly upregulated during prolonged exercise, possibly owing to increased ROS generation. Increased UCP3 may partially alleviate the proton gradient across the inner membrane, thereby reducing further ROS production by the electron transport chain. However, prolonged exercise caused a decrease in energy coupling efficiency in muscle mitochondria revealed by an increased respiration rate due to proton leak (State 4/State 3 ratio) and decreased RCI. We thus propose that the compromise of the oxidative phosphorylation efficiency due to UCP3 upregulation may serve an antioxidant function to protect the muscle mitochondria from exercise-induced oxidative stress     

Regulation of UCP1 and UCP3 in arctic ground squirrels and relation with mitochondrial proton leak.

Uncoupling protein (UCP) 1 (UCP1) catalyzes a proton leak in brown adipose tissue (BAT) mitochondria that results in nonshivering thermogenesis (NST), but the extent to which UCP homologs mediate NST in other tissues is controversial. To clarify the role of UCP3 in mediating NST in a hibernating species, we measured Ucp3 expression in skeletal muscle of arctic ground squirrels in one of three activity states (not hibernating, not hibernating and fasted for 48 h, or hibernating) and housed at 5 degrees C or -10 degrees C. We then compared Ucp3 mRNA levels in skeletal muscle with Ucp1 mRNA and UCP1 protein levels in BAT in the same animals. Ucp1 mRNA and UCP1 protein levels were increased on cold exposure and decreased with fasting, with the highest UCP1 levels in thermogenic hibernators. In contrast, Ucp3 mRNA levels were not affected by temperature but were increased 10-fold during fasting and >3-fold during hibernation. UCP3 protein levels were increased nearly fivefold in skeletal muscle mitochondria isolated from fasted squirrels compared with nonhibernators, but proton leak kinetics in the presence of BSA were unchanged. Proton leak in BAT mitochondria also did not differ between fed and fasted animals but did show classical inhibition by the purine nucleotide GDP. Levels of nonesterified fatty acids were highest during hibernation, and tissue temperatures during hibernation were related to Ucp1, but not Ucp3, expression. Taken together, these results do not support a role for UCP3 as a physiologically relevant mediator of NST in muscle.     

Overexpression of GLUT4 in mice causes up-regulation of UCP3 mRNA in skeletal muscle

Mitochondrial uncoupling protein 3 (UCP3) is expressed in skeletal muscles. We have hypothesized that increased glucose flux in skeletal muscles may lead to increased UCP3 expression. Male transgenic mice harboring insulin-responsive glucose transporter (GLUT4) minigenes with differing lengths of 5'-flanking sequence (-3237, -2000, -1000 and -442 bp) express different levels of GLUT4 protein in various skeletal muscles. Expression of the GLUT4 transgenes caused an increase in UCP3 mRNA that paralleled the increase of GLUT4 protein in gastrocnemius muscle. The effects of increased intracellular GLUT4 level on the expression of UCP1, UCP2 and UCP3 were compared in several tissues of male 4 month-old mice harboring the -1000 GLUT4 minigene transgene. In the -1000 GLUT4 transgenic mice, expression of GLUT4 mRNA and protein in skeletal muscles, brown adipose tissue (BAT), and white adipose tissue (WAT) was increased by 1.4 to 4.0-fold. Compared with non-transgenic littermates, the -1000 GLUT4 mice exhibited about 4- and 1.8-fold increases of UCP3 mRNA in skeletal muscle and WAT, respectively, and a 38% decrease of UCP1 mRNA in BAT. The transgenic mice had a 16% increase in oxygen consumption and a 14% decrease in blood glucose and a 68% increase in blood lactate, but no change in FFA or beta-OHB levels. T3 and leptin concentrations were decreased in transgenic mice. Expression of UCP1 in BAT of the -442 GLUT4 mice, which did not overexpress GLUT4 in this tissue, was not altered. These findings indicate that overexpression of GLUT4 up-regulates UCP3 expression in skeletal muscle and down-regulates UCP1 expression in BAT, possibly by increasing the rate of glucose uptake into these tissues.     

Hyperoxia-mediated oxidative stress increases expression of UCP3 mRNA and protein in skeletal muscle

The uncoupling protein-3 (UCP3) is a mitochondrial protein expressed mainly in skeletal muscle. Among several hypotheses for its physiological function, UCP3 has been proposed to prevent excessive production of reactive oxygen species. In the present study, we evaluated the effect of an oxidative stress induced by hyperoxia on UCP3 expression in mouse skeletal muscle and C2C12 myotubes. We found that the hyperoxia-mediated oxidative stress was associated with a 5-fold and 3-fold increase of UCP3 mRNA and protein levels, respectively, in mouse muscle. Hyperoxia also enhanced reactive oxygen species production and UCP3 mRNA expression in C2C12 myotubes. Our findings support the view that both in vivo and in vitro UCP3 may modulate reactive oxygen species production in response to an oxidative stress.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5-fold and 4-fold, respectively, and UCP3 protein levels by 2-fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.     

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.     

Downregulation of uncoupling protein-3 in vivo is linked to changes in muscle mitochondrial energy metabolism as a result of capsiate administration

Although it has been suggested that the skeletal muscle mitochondrial uncoupling protein-3 (UCP3) is involved in regulating energy expenditure, its role is still poorly understood. In the present study, we aimed at investigating noninvasively, using magnetic resonance techniques, metabolic changes occurring in exercising muscle as a result of capsiate treatment, which has been previously linked to UCP3 upregulation. We showed that capsiate ingestion strongly reduced UCP3 gene expression in rat gastrocnemius muscle. This large underexpression was accompanied by a significant increase in the rate of mitochondrial ATP production and phosphocreatine level both at rest and during muscle stimulation. Similarly, the stimulation-induced ATP fall and ADP accumulation were significantly less after capsiate administration than in untreated rats. The larger oxidative ATP production rate could not be explained by a proportional decrease in the anaerobic component, i.e., glycolysis and phosphocreatine breakdown. In addition, the mechanical performance was not affected by capsiate administration. Finally, the plasma free fatty acid (FFA) level increased in capsiate-treated rats, whereas no significant change was observed after muscle stimulation in the control group. Considering the corresponding enhanced UCP3 mRNA expression occurring in the control group after muscle stimulation, one can suggest that changes in FFA level and UCP3 mRNA expression are not mechanistically correlated. Overall, we have shown that capsiate administration induced a UCP3 downregulation coupled with an increased mitochondrial ATP synthesis, whereas the muscle force-generating capacity was unchanged. This suggests that a decrease in muscle efficiency and/or additional noncontractile ATP-consuming mechanisms result from UCP3 downregulation.     

Hyperlipemia: a role in regulating UCP3 gene expression in skeletal muscle during cancer cachexia?

Rats bearing the Yoshida AH-130 ascites hepatoma showed an increased expression of both uncoupling protein-2 (UCP2) (two-fold) and UCP3 (three- to four-fold) in skeletal muscle (both soleus and gastrocnemius). The increase in mRNA content was associated with increased circulating concentrations of fatty acids (two-fold), triglyceride (two-fold) and cholesterol (1.9-fold). Administration of nicotinic acid to tumor-bearing rats abolishes the hyperlipidemic increase associated with tumor burden. The vitamin treatment also resulted in a decreased UCP3 gene expression in soleus muscle but not in gastrocnemius. It is concluded that circulating fatty acids may be involved in the regulation of UCP3 gene expression in aerobic muscles during experimental cancer cachexia. Since the UCP3 protein could have a role in energy expenditure, it may be suggested that hypolipidemic agents may have a beneficial role in the treatment of the cachectic syndrome.     

UCP3 gene expression does not correlate with muscle oxidation rates in troglitazone-treated Zucker fatty rats

Uncoupling protein-3 (UCP3), a mitochondrial carrier protein predominantly expressed in muscle, has been suggested to release stored energy as heat. The insulin-sensitizing thiazolidinediones enhance glucose disposal in skeletal muscle and have been reported to increase the expression of uncoupling proteins in various experimental systems. We therefore studied the effect of troglitazone treatment on UCP3 gene expression in muscles from lean and obese Zucker rats. In comparison with obese littermates, basal UCP3 mRNA levels in lean Zucker rats tended to be higher in white and red gastrocnemius muscles, but were lower in soleus (P<0.001) muscle and heart (P<0.01). In lean rats, troglitazone significantly increased UCP3 gene expression in white and red gastrocnemius and heart muscles (all P<0.01). In contrast, the drug reduced UCP3 mRNA expression in red gastrocnemius and soleus muscles of obese littermates (all P<0.001). The troglitazone-dependent decrease in UCP3 gene expression was accompanied by an increased weight gain in obese rats, while no such effect was observed in lean rats. In obese rats, improvement of insulin resistance by troglitazone was associated with increased rates of basal and insulin-stimulated CO(2) production from glucose measured in soleus muscle. These studies demonstrate that effects of troglitazone on UCP3 gene expression depend on the phenotype of Zucker rats and that troglitazone-induced metabolic improvements are not related to increased uncoupling resulting from upregulation of UCP3 mRNA expression in muscle.     

Effect of chronic contractile activity on SS and IMF mitochondrial apoptotic susceptibility in skeletal muscle

Chronic contractile activity of skeletal muscle induces an increase in mitochondria located in proximity to the sarcolemma [subsarcolemmal (SS)] and in mitochondria interspersed between the myofibrils [intermyofibrillar (IMF)]. These are energetically favorable metabolic adaptations, but because mitochondria are also involved in apoptosis, we investigated the effect of chronic contractile activity on mitochondrially mediated apoptotic signaling in muscle. We hypothesized that chronic contractile activity would provide protection against mitochondrially mediated apoptosis despite an elevation in the expression of proapoptotic proteins. To induce mitochondrial biogenesis, we chronically stimulated (10 Hz; 3 h/day) rat muscle for 7 days. Chronic contractile activity did not alter the Bax/Bcl-2 ratio, an index of apoptotic susceptibility, and did not affect manganese superoxide dismutase levels. However, contractile activity increased antiapoptotic 70-kDa heat shock protein and apoptosis repressor with a caspase recruitment domain by 1.3- and 1.4-fold (P<0.05), respectively. Contractile activity elevated SS mitochondrial reactive oxygen species (ROS) production 1.4- and 1.9-fold (P<0.05) during states IV and III respiration, respectively, whereas IMF mitochondrial state IV ROS production was suppressed by 28% (P<0.05) and was unaffected during state III respiration. Following stimulation, exogenous ROS treatment produced less cytochrome c release (25-40%) from SS and IMF mitochondria, and also reduced apoptosis-inducing factor release (approximately 30%) from IMF mitochondria, despite higher inherent cytochrome c and apoptosis-inducing factor expression. Chronic contractile activity did not alter mitochondrial permeability transition pore (mtPTP) components in either subfraction. However, SS mitochondria exhibited a significant increase in the time to Vmax of mtPTP opening. Thus, chronic contractile activity induces predominantly antiapoptotic adaptations in both mitochondrial subfractions. Our data suggest the possibility that chronic contractile activity can exert a protective effect on mitochondrially mediated apoptosis in muscle.     

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.     

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

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.