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.     

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.     

Expression of an uncoupling protein gene homolog in chickens

An avian uncoupling protein (UCP) gene homolog was recently sequenced from skeletal muscle and was proposed to have a role in thermogenesis in chickens, ducks and hummingbirds. Since mammalian UCP 2 and UCP 3 also appear to have functions associated with energy and substrate partitioning and body weight regulation, the purpose of this study was to further characterize chicken UCP under conditions of nutritional stress and/or leptin administration. Male 3-week-old chickens were starved for 24 or 48 h and then half of each group was refed for an additional 24 h. In a follow-up experiment, chickens were fed or starved for 48 h with or without leptin administration. Feed deprivation increased UCP mRNA expression in skeletal muscle by up to 260% (P<0.001), and in a time-dependent manner in pectoralis muscle. Refeeding for 24 h normalized muscle UCP mRNA levels. Leptin administration had no effect on muscle UCP. Chicken muscle UCP mRNA levels were highly correlated with plasma triglyceride and non-esterified fatty acid (NEFA) concentrations, and with circulating levels of insulin, insulin-like growth factor (IGF)-I and IGF-II. These results suggest that, as in mammals, avian UCP is up-regulated during feed deprivation and is highly correlated with increased fatty acid oxidation and flux into skeletal muscle.     

Nur77 regulates lipolysis in skeletal muscle cells. Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway

Skeletal muscle is a major mass peripheral tissue that accounts for approximately 40% of total body weight and 50% of energy expenditure and is a primary site of glucose disposal and fatty acid oxidation. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. Excessive caloric intake is sensed by the brain and induces beta-adrenergic receptor (beta-AR)-mediated adaptive thermogenesis. Beta-AR null mice develop severe obesity on a high fat diet. However, the target gene(s), target tissues(s), and molecular mechanism involved remain obscure. We observed that 30-60 min of beta-AR agonist (isoprenaline) treatment of C2C12 skeletal muscle cells strikingly activated (>100-fold) the expression of the mRNA encoding the nuclear hormone receptor, Nur77. In contrast, the expression of other nuclear receptors that regulate lipid and carbohydrate metabolism was not induced. Stable transfection of Nur77-specific small interfering RNAs (siNur77) into skeletal muscle cells repressed endogenous Nur77 mRNA expression. Moreover, we observed attenuation of gene and protein expression associated with the regulation of energy expenditure and lipid homeostasis, for example AMP-activated protein kinase gamma3, UCP3, CD36, adiponectin receptor 2, GLUT4, and caveolin-3. Attenuation of Nur77 expression resulted in decreased lipolysis. Finally, in concordance with the cell culture model, injection and electrotransfer of siNur77 into mouse tibialis cranialis muscle resulted in the repression of UCP3 mRNA expression. This study demonstrates regulatory cross-talk between the nuclear hormone receptor and beta-AR signaling pathways. Moreover, it suggests Nur77 modulates the expression of genes that are key regulators of skeletal muscle lipid and energy homeostasis. In conclusion, we speculate that Nur77 agonists would stimulate lipolysis and increase energy expenditure in skeletal muscle and suggest selective activators of Nur77 may have therapeutic utility in the treatment of obesity.     

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.     

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.     

Changes in UCP mRNA expression levels in brown adipose tissue and skeletal muscle after feeding a high-energy diet and relationships with leptin, glucose and PPARgamma

Brown adipose tissue and skeletal muscle are known to be important sites for nonshivering thermogenesis. In this context, it is accepted that uncoupling proteins (UCPs) are involved in such process, but little is known about the physiological regulation of these proteins as affected by the intake of a high-energy (cafeteria) diet inducing fat deposition. In this study, the UCP messenger RNA (mRNA) expression in interscapular brown adipose tissue (iBAT) and skeletal muscle was assessed to evaluate the influence of a dietary manipulation on energy homeostasis regulation. We report a statistically significant increase in mRNA levels of iBAT UCP1 and UCP3 and a statistical marginal rise in skeletal muscle UCP3 mRNA expression after feeding a high-energy diet, whereas no changes in UCP2 expression were found in either tissue. Furthermore, significant positive associations between iBAT UCP1 and UCP3 mRNA levels with serum leptin were found. Although the expression of the beta(3) adrenoceptor (beta(3)AR) was about 50% in the lean controls compared with the obese group in iBAT, no statistically significant changes were observed concerning peroxisome proliferator-activated receptor gamma2 (PPARgamma2) mRNA levels in muscle or iBAT. We conclude that feeding a diet inducing weight and fat gain produces different outcomes on iBAT and skeletal muscle UCP mRNA expression, revealing a tissue-dependent response for the three UCPs. Results suggest that the regulation of UCP expression in both tissues under these specific dietary conditions may be related to leptin circulating levels.     

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.     

Links between fatty acids and expression of UCP2 and UCP3 mRNAs

Physiological and pathological states that are associated with elevated plasma fatty acids (FAs) increase uncoupling protein 2 (UCP2) mRNA in white adipose tissue and UCP3 mRNA in skeletal muscle and heart. A direct effect of unsaturated fatty acids from all classes has been shown in various cultured cells. There is evidence that FAs could induce expression of UCPs by acting as ligands for peroxisome proliferator-activated receptors, influencing the function of sterol responsive element binding protein or activating 5'-AMP-activated protein kinase. Oleic acid has been shown to stimulate the activity of the promoter regions of UCP2 and UCP3 genes and the FA responsive regions are beginning to be characterised.     

Human Uncoupling Protein‐3 and Obesity: An Update

The cloning of the uncoupling protein (UCP)1 homologs UCP2 and UCP3 has raised considerable interest in the mechanism. The expression of UCP3 mainly in skeletal muscle mitochondria and the potency of the skeletal muscle as a thermogenic organ made UCP3 an attractive target for studies toward manipulation of energy expenditure to fight disorders such as obesity and type 2 diabetes. Overexpressing UCP3 in mice resulted in lean, hyperphagic mice. However, the lack of an apparent phenotype in mice lacking UCP3 triggered the search for alternative functions of UCP3. The observation that fatty acid levels significantly affect UCP3 expression has given UCP3 a position in fatty acid handling and/or oxidation. Emerging data indicate that the primary physiological role of UCP3 may be the mitochondrial handling of fatty acids rather than the regulation of energy expenditure through thermogenesis. It has been proposed that UCP3 functions to export fatty acid anions away from the mitochondrial matrix. In doing so, fatty acids are exchanged with protons, explaining the uncoupling activity of UCP3. The exported fatty acid anions may originate from hydrolysis of fatty acid esters by a mitochondrial thioesterase, or they may have entered the mitochondria as nonesterified fatty acids by incorporating into and flip‐flopping across the mitochondrial inner membrane. Regardless of the origin of the fatty acid anions, this putative function of UCP3 might be of great importance in protecting mitochondria against fatty acid accumulation and may help to maintain muscular fat oxidative capacity.     

Dietary conjugated linoleic acid reduces body fat mass and affects gene expression of proteins regulating energy metabolism in mice

ICR and C57BL/6J mice were fed experimental diets containing either a 2% fatty acid preparation rich in conjugated linoleic acid (CLA) or a preparation rich in linoleic acid and free of CLA for 21 days. CLA greatly decreased weights of white adipose tissue and interscapular brown adipose tissue in the two strains. CLA reduced mRNA levels of glucose transporter 4 (Glut 4) in white and brown adipose tissue of both strains. A CLA-dependent decrease in mRNA levels of peroxisome proliferator activated receptor (PPAR) gamma was seen in interscapular brown adipose tissue of both strains and in white adipose tissue of C57BL/6J but not ICR mice. Dietary CLA was found to cause a decrease in the mRNA levels of uncoupling protein (UCP) 1 in brown adipose tissue when the value was corrected for the expression of a house-keeping gene (beta-actin) in the two strains. Uncorrected values were, however, indistinguishable between the animals fed the CLA diet and CLA-free diet. UCP 3 expression in brown adipose tissue was much lower in mice fed the CLA diet than in those fed the control diet in both strains. In contrast, CLA greatly up-regulated the gene expression of UCP 2 in brown adipose tissue. Dietary CLA also increased UCP 2 mRNA level in skeletal muscle. It is apparent that dietary CLA decreases white and brown adipose tissue mass, accompanying changes in the gene expression of proteins regulating energy metabolism in white and brown adipose tissues, and skeletal muscle of mice.     

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     

Impaired response of UCP family to cold exposure in diabetic (db/db) mice

Impaired activity of the uncoupling protein (UCP) family has been proposed to promote obesity development. The present study examined differences in UCP responses to cold exposure between leptin-resistance obese (db/db) mice and their lean (C57Ksj) littermates. Basal UCP1 and UCP3 mRNA expression in brown adipose tissue was lower in obese mice compared with lean mice, but UCP2 expression in white adipose tissue (WAT) was higher. Basal skeletal muscle UCP3 did not change remarkably. The UCP family mRNAs, which were upregulated 12 and 24 h after cold exposure (4 degrees C), were returned to prior levels 12 h after rewarming exposure (21 degrees C) in lean mice. The accelerating effects of cold exposure on the UCP family were impaired in db/db obese mice. Together with these changes, WAT lipoprotein lipase mRNA was downregulated, and the concentration of serum free fatty acid was increased in response to cold exposure in the lean mice but not in db/db obese littermates. The impaired function of the UCP family and diminished lipolysis in response to cold exposure indicate that the reduced lipolytic activity may contribute to the inactivation of the UCP family in db/db obese mice.     

Insulin-like growth factor-I-induced androgen receptor activation is mediated by the PI3K/Akt pathway in C2C12 skeletal muscle cells

Although insulin-like growth factor-I (IGF-I) and androgen receptor (AR) are well known effectors of skeletal muscle, the molecular mechanism by which signaling pathways integrating AR and IGF-I in skeletal muscle cells has not been previously examined. In this study, the role of PI3K/Akt on IGF-I-induced gene expression and activation of AR in skeletal muscle cells was investigated. C2C12 cells were treated with IGF-I in the absence or presence of inhibitors of PI3K/Akt pathway (LY294002 and Wortmannin). Inhibition of the PI3K/Akt pathway with LY294002 or Wortmannin led to a significant decrease in IGF-I-induced AR phosphorylation and total AR protein expression. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by LY294002 or Wortmannin. Confocal images showed that IGF-I-induced AR translocation from cytosol to nucleus was inhibited significantly in response to treatment with LY294002 or Wortmannin. The present results suggest that modulating effect of IGF-I on AR gene expression and activation in C2C12 mouse skeletal muscle cells is mediated at least in part by the PI3K/Akt pathway.     

Human Uncoupling Proteins and Obesity

SCHRAUWEN, PATRICK, KEN WALDER, AND ERIC RAVUSSIN. Human coupling proteins and obesity. Obes. Res. 1999;7:97–105. Uncoupling protein (UCP) 2 and UCP3 are newly discovered proteins that can uncouple ATP production from mitochondrial respiration, thereby dissipating energy as heat and affecting energy metabolism efficiency. In contrast to UCP1, which is only present in brown adipose tissue, UCP2 has a wide tissue distribution, whereas UCP3 is expressed predominantly in skeletal muscle. Some evidence of a role for UCPs in modulating metabolic rate was provided by linkage and association studies. Furthermore, UCP3 gene expression was found to correlate negatively with body mass index and positively with sleeping metabolic rate in Pima Indians. Treatment with thyroid hormone increases expression of the UCP2 and UCP3 genes. Other regulators of UCP2 and UCP3 gene expression are β₃-adrenergic agonists and glucocorticoids. Surprisingly, fasting has a stimulatory effect on UCP2 and UCP3 mRNA levels, possibly explained by the effects of free fatty acid on UCP2 and UCP3 gene expression.     

Overexpression of UCP-3 in skeletal muscle of mice results in increased expression of mitochondrial thioesterase mRNA

Mice overexpressing human UCP-3 in skeletal muscle (UCP-3tg) are lean despite overeating, have increased metabolic rate, and their skeletal muscle mitochondria show increased proton conductance. The true function of UCP-3 however, has yet to be determined. It is assumed that UCP-3tg mice have increased fatty acid beta-oxidation to fuel their increased metabolic rate. In this study we have quantified skeletal muscle mRNA levels of a number of genes involved in fatty acid metabolism. mRNA levels of uncoupling protein-2, carnitine palmitoyl transferase-1beta and fatty acid binding proteins, and transporters were unchanged when compared to wild-type mice. Lipoprotein lipase mRNA was slightly, but significantly, increased by 50%. The most notable change in gene expression was a threefold increase in mitochondrial thioesterase (MTE-1) expression. In the face of a chronic increase in mitochondrial uncoupling these changes suggest that increased flux of fatty acids through the beta-oxidation pathway does not necessarily require marked changes in expression of genes involved in fatty acid metabolism. The large increase in MTE-1 both confirms the importance of this gene in situations where mitochondrial beta-oxidation is increased and supports the hypothesis that UCP-3 exports fatty acids generated by MTE-1 in the mitochondrion.     

High‐Fat Diet Feeding Elevates Skeletal Muscle Uncoupling Protein 3 Levels But Not Its Activity in Rats

Objective: The objective of this study is to test the impact of high‐fat diet (HFD) feeding on skeletal muscle (SM) uncoupling protein 3 (UCP3) expression and its association with mitochondrial ion permeability and whole‐body energy homeostasis. Research Methods and Procedures: Sprague–Dawley rats were fed ad libitum either a HFD (60% of energy from fat, n = 6) or a low‐fat diet (12% of energy from fat, n = 6) for 4 weeks. Twenty‐four‐hour energy expenditure was measured by indirect calorimetry in the last week of the dietary treatment. Blood samples were collected for plasma leptin and free fatty acid assays, and mitochondria were isolated from hindlimb SM for subsequent determinations of UCP3 levels and mitochondrial ion permeability. Results: Plasma leptin levels were higher in rats fed the HFD despite the same body weight in two groups. The same dietary treatment also rendered a 2‐fold increase in plasma free fatty acid and SM UCP3 protein levels (Western blot) compared with the group fed the low‐fat diet. However, the elevated UCP3 protein levels did not correlate with mitochondrial swelling rates, a measure of mitochondrial chloride, and proton permeability, or with 24‐hour energy expenditure. Discussion: The high correlation between the levels of plasma free fatty acid levels and SM UCP3 suggests that circulating free fatty acid may play an important role in UCP3 expression during the HFD feeding. However, the dissociation between the UCP3 protein levels and 24‐hour energy expenditure as well as mitochondrial ion permeability suggests that mitochondrial proton leak mediated by muscle UCP3 may not be a major contributor in energy balance in HFD feeding, and other regulatory mechanisms independent of gene regulation may be responsible for the control of UCP3‐mediated uncoupling activity.     

禁食及胰岛素水平对大鼠解偶联蛋白-1、2、3基因表达的影响

 为探讨禁食和胰岛素对解偶联蛋白 - 1、2、3基因 (UCP1 ,2 ,3)表达的影响 ,应用 RT- PCR方法观察了在不同禁食时间和应用胰岛素条件下大鼠白色脂肪组织、棕色脂肪组织和骨骼肌中 UCP1 ,2 ,3m RNA水平的变化 .UCP1基因只在大鼠棕色脂肪组织中表达 .UCP2 ,3基因在三种组织中均有表达 ,在白色脂肪组织中以 UCP2表达为主 ;在骨骼肌中以 UCP3表达为主 .过夜禁食使棕色脂肪组织 UCP1 ,3m RNA水平明显下降 (P<0 .0 1 ) ;UCP2 m RNA水平在三种组织中均呈上升反应 ,以白色脂肪组织中表现最为明显 (P<0 .0 5) ;而对白色脂肪组织和骨骼肌中 UCP3基因表达无明显影响 .禁食时间延长至 48h,除棕色脂肪组织中 UCP2 ,3基因有明显下降外 ,各组织中UCPs基因表达基本调节至正常或高于对照组水平 .胰岛素对 UCPs基因表达水平有一定的上调作用 ,这一作用对棕色脂肪组织 UCPs各基因及骨骼肌中 UCP3基因表现得尤为明显 (P<0 .0 5) .大鼠 UCPs基因表达有一定的组织特异性 ;禁食时间对三种组织中 UCPs各成员基因表达的影响有时相上的区别 ;胰岛素可以调 UCPs各成员基因的表达 .结果反映了 UCPs各成员在能量代谢调节上的不同作用 ,这为理解膳食 -产热与体重调节的关系 ,及其能量代谢平衡与疾病关系提供了实验依据