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Clear cell renal cell carcinoma (ccRCC) is characterized by the constitutive up-regulation of the hypoxia inducible factor-1. One of its target enzymes, pyruvate dehydrogenase (PDH) kinase 1 (PDHK1) showed increased protein expression in tumor as compared to patient-matched normal tissues. PDHK1 phosphorylated and inhibited PDH whose enzymatic activity was severely diminished, depriving the TCA cycle of acetylCoA. We and others have shown a decrease in the protein expressions of all respiratory complexes alluding to a compromise in oxidative phosphorylation (OXPHOS). On the contrary, we found that key parameters of OXPHOS, namely ATP biosynthesis and membrane potential were consistently measurable in mitochondria isolated from ccRCC tumor tissues. Interestingly, an endogenous mitochondrial membrane potential (MMP) was evident when ADP was added to mitochondria isolated from ccRCC but not in normal tissues. In addition, the MMP elicited in the presence of ADP by respiratory substrates namely malate/glutamate, succinate, α-ketoglutarate and isocitrate was invariably higher in ccRCC. Two additional hallmarks of ccRCC include a loss of uncoupling protein (UCP)-2 and an increase in UCP-3. Based on our data, we proposed that inhibition of UCP3 by ADP could contribute to the endogenous MMP observed in ccRCC and other cancer cells.

Estrogen is a hormone that regulates physiological processes and its dysregulation may relate to muscle disorders particularly in female, although the mechanism remains to be elucidated. We here show that estrogen deficiency repressed exercise endurance in female mice whereas the administration of estrogen to ovariectomized mice recovered it. Microarray analysis of mouse muscles showed that mitochondrial uncoupling protein 3 (UCP3) is upregulated by ovariectomy and downregulated by estrogen administration. Intriguingly, ectopic expression of constitutively active estrogen receptor α decreased UCP3 level and increased cellular ATP content in differentiated myoblastic C2C12 cells. Overall, the present study suggests that estrogen plays a critical role in the regulation of energy expenditure and exercise endurance in female.

Mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3) are postulated to contribute to antioxidant defense, nutrient partitioning, and energy efficiency in the heart. To distinguish isotype function in response to metabolic stress we measured cardiac mitochondrial function and cardiac UCP gene expression following chronic hypobaric hypoxia. Isolated mitochondrial O(2) consumption and ATP synthesis rate were reduced but respiratory coupling was unchanged compared to normoxic groups. Concurrently, left ventricular UCP3 mRNA levels were significantly decreased with hypoxia (p

Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

Until very recently, the uncoupling protein-1 (UCP1), present only in brown adipose tissue (BAT), was considered to be the only mitochondrial carrier protein that stimulated heat production by dissipating the proton gradient generated during respiration across the inner mitochondrial membrane and therefore uncoupling respiration from ATP synthesis. Recently, new uncoupling proteins, UCP2, UCP3, and UCP4, and brain mitochondrial carrier protein-1 (BMCP-1) have been described in mammalian tissues. The present review deals with the possible role of these proteins in different pathological conditions involving alterations in energy balance such as obesity or cachexia. In conclusion, the emergence of the UCP family has altered the approaches to bioenergetics and stressed the importance of uncoupling respiration in different pathophysiological conditions. An extensive qualitative and quantitative characterization of the new members of the UCP family in mammalian tissues will allow a better understanding of the molecular and regulatory mechanisms of thermogenesis and energy metabolism. At this point, we hope that the knowledge presented in the present review will not only stimulate a debate about the role of the UCP family in disease but also lead to applications beneficial for human health.

The effects of ovariectomy (OVX) and estrogen substitution on body weight, body composition, food intake, weight gain, and expression of uncoupling proteins (UCPs) in brown adipose tissue (BAT), white adipose tissue (WAT), and skeletal muscle were studied in four groups of rats: (1) Sham-operated rats (N = 8), (2) ovariectomized rats (OVX - E) (N = 8), (3) estrogen-treated OVX rats (OVX + E) (N = 8), and (4) OVX rats on energy restriction (OVX - E + D) (N = 8). OVX was associated with an increase in food intake and body weight gain during a 5-week study period compared to sham-operated rats. The estrogen-substituted rats had a significantly lower food intake and weight gain during the 5 weeks compared to the sham-operated group. However, we also included a nontreated OVX group that was allowed to eat only enough chow to match the weight gain of the sham-operated group. To match the weight gain in the two groups, the OVX group had to consume 16% less chow than the sham-operated group. In BAT, the UCP1 expression was significantly lower in estrogen-deficient rats compared to either intact rats or estrogen-substituted rats, whereas UCP2 and UCP3 mRNA expression was similar in BAT from all four groups. In WAT, both estrogen-deficient groups had significantly lower UCP2 mRNA expression compared to the control rats and estrogen-treated rats; In contrast, the UCP3 mRNA expression in WAT was similar in all four groups. Finally, in skeletal muscle the OVX group on mild energy restriction had reduced UCP3 mRNA expression compared to control, OVX, and estrogen-treated rats. In contrast, the UCP2 mRNA expression in skeletal muscle was similar in all four groups. Thus, the findings that estrogen deficiency is followed by reduced UCP1 expression in BAT and reduced UCP2 expression in WAT in association with weight gain probably caused by a decrease in energy expenditure might indicate that UCPs play a role for the estrogen-mediated changes in body weight and energy expenditure.

To study the regulation of the mitochondrial uncoupling protein 2 and 3 (UCP2 and UCP3), we studied the effect of insulin and muscle contraction on UCP mRNA expression in rat skeletal muscle in vitro. Insulin dose-dependently increased skeletal muscle UCP2 and UCP3 mRNA expression in m. extensor digitorum longus (EDL) with maximal stimulation obtained at around 0.6-6 nM. The concentration of insulin giving half-maximal stimulation was 60 pM for the UCP2 and 48 pM for the UCP3 mRNA expression. The effect of insulin was maximal after 2 h and the effect was sustained during the whole study period (6 h). The insulin-induced increase in UCP mRNA was independent of the glucose uptake (as UCP mRNA was stimulated even in incubations without glucose). In addition, electrically induced contractions (in vitro) increased UCP2 and UCP3 mRNA expression 60-120 min after a single bout of contraction (for 10 min). Both the increment of UCP2 and UCP3 mRNA were sustained throughout the study period (4 h) (153 +/- 62 and 216 +/- 71% above basal, P0.05 respectively). Finally, 5-aminoimidazole-4-carboxamid-ribosid (AICAR), an activator of the AMP-activated protein kinase (AMPK), that is activated during exercise, was able to mimic the increase in UCP2 and UCP3 mRNA expression. In conclusion, UCP2 and UCP3 mRNA expression in skeletal muscle are stimulated rapidly by insulin and contraction in vitro, thus the stimulation is direct and not caused by changes in other hormones or metabolites. Even a brief bout of contraction induces an increase in UCP2 and UCP3 expression, an effect that could be mimicked by activation of the AMP-activated protein kinase by AICAR.

Previously it was concluded (1) that, differently from UCP1, on expression in Saccharomyces cerevisiae, UCP3, and UCP3 short (UCP3s) are in a deranged state, allowing for unregulated uncoupling. Here we show that the bulk of UCP3 and UCP3s is in extramitochondrial aggregates whether expressed with high or medium expression vectors. The evidence is based on the insolubility of most UCP3 and UCP3s in nonionic detergents such as Triton X100, in contrast to UCP1. Using very high expression vector, macroscopic evidence for extramitochondrial UCP3 containing particles is a viscous white sediment surrounding the mitochondrial fraction which contains UCP3 as inclusion body type aggregate. Together with the previous data it is concluded that uncoupling due to small amounts of incorporated, deranged, and nucleotide insensitive UCP3 prevents incorporation of the bulk of UCP3 into mitochondria. This finding also provides a simple and stringent assay for the state of heterologously expressed in mitochondrial membrane proteins.

The effect of long-term hindlimb unloading (2 or 5 week) on the expression of uncoupling protein-3 (UCP3) gene was investigated in rat skeletal muscles. The interaction of hindlimb unloading and thyroid status was also investigated at 2 weeks. Whatever the duration, mechanical unloading induced a similar increase in UCP3 mRNA relative abundance in the slow-twitch soleus (SOL) muscle (+80%, P < 0.05), whereas no effect was observed in the fast-twitch extensor digitorum longus (EDL) muscle. Hypothyroidism down-regulated while hyperthyroidism up-regulated UCP3 mRNA relative abundance in both SOL and EDL muscles, but thyroid status did not prevent the up-regulation of UCP3 induced by 2 weeks of suspension. These data therefore indicate for the first time that long-term hindlimb unloading up-regulates muscle UCP3 gene expression in a muscle-specific manner which is independent of thyroid status.

Administration of beta-adrenergic receptor (beta-AR) agonists, especially beta(3)-AR agonists, is well known to increase thermogenesis in rodents and humans. In this work we studied the role of the beta(3)-AR in regulating mRNA expression of genes involved in thermogenesis, i.e., mitochondrial uncoupling proteins UCP2 and UCP3, and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1), in mouse skeletal muscle. For this purpose, different beta(3)-AR agonists were administered acutely to both wild type mice and mice whose beta(3)-AR gene has been disrupted (beta(3)-AR KO mice). CL 316243 increased the expression of UCP2, UCP3 and PGC-1 in wild type mice only. By contrast, BRL 37344 and CGP 12177 increased the expression of UCP2 and UCP3 in both wild type and beta(3)-AR KO mice, whereas they increased the expression of PGC-1 in wild type mice only. Finally, acute (3 h) cold exposure increased the expression of UCP2 and UCP3, but not PGC-1, in skeletal muscle of both wild type and beta(3)-AR KO mice. These results show that selective stimulation of the beta(3)-AR affects the expression of UCP2, UCP3 and PGC-1 in skeletal muscle. This effect is probably indirect, as muscle does not seem to express beta(3)-AR. In addition, our data suggest that BRL 37344 and CGP 12177 act, in part, through an as yet unidentified receptor, possibly a beta(4)-AR.

Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple oxidative phosphorylation by dissipating the proton gradient across the membrane. We have investigated regulation of the UCP3 gene in skeletal muscle and C2C12 muscle cells. UCP3 mRNA in mouse skeletal muscle is markedly increased by fasting and rapidly (within 4 h) decreased by re-feeding. Methyl palmoxirate, which inhibits fatty acid uptake by mitochondria and increases blood free fatty acids, prevents the fall in UCP3 message level induced by re-feeding. These findings suggest that fatty acid or a metabolite thereof, activates the UCP3 gene. Proof that fatty acid per se up-regulates UCP3 mRNA was obtained with C2C12 muscle cells in culture. Thus, oleic acid activated expression of UCP3 mRNA in differentiated C2C12 myotubes in a time and concentration-dependent manner. Moreover, BRL49653, a ligand for the nuclear hormone receptor PPARγ induces expression of UCP3 mRNA suggesting that PPARγ may regulate transcription of the UCP3 gene.

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 β-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.

Uncoupling protein-1 (UCP1) dissipates the transmitochondrial proton gradient as heat. UCP2 and UCP3 are two recently discovered homologues that also have uncoupling activity and thus presumably have a role in energy homeostasis. We now report the genomic structure of murine UCP3 (7 exons) and UCP2 (8 exons). UCP3 is approximately 8 kilobases upstream of UCP2. An UCP3 variant mRNA, UCP3S, was also found and characterized. The effect of a high fat diet (45% versus 10%) on UCP3 and UCP2 mRNA levels was measured. Eating the 45% fat diet for eight weeks caused greater weight gain in AKR and C57BL/6J mice than in the obesity-resistant A/J mice. The high fat diet increased muscle UCP3 expression twofold in C57BL/6J animals. UCP2 expression increased slightly on the 45% fat diet in white adipose of AKR mice, but not in A/J or C57BL/6J mice. In skeletal muscle, UCP2 expression showed little variation with diet. Thus, UCP2 and UCP3 expression levels change in response to diet-induced obesity, but the changes are modest and depend on the tissue and genotype. The data suggest that it is not a reduction in UCP2 or UCP3 expression that causes obesity in the susceptible mice.

Pioglitazone is a thiazolidinedione drug (TZD) which potently and specifically stimulates peroxisome proliferator-activated receptor gamma (PPAR gamma) and sensitizes cells to insulin. Since TZDs are thought to increase energy expenditure, changes in mitochondrial thermogenesis uncoupling protein-2 and -3 mRNA levels in response to pioglitazone treatment were measured in mouse skeletal muscle. Normally hyperglycemic and hyperinsulinemic KK/Ta mice were given pioglitazone for 2 weeks to treat this non-insulin dependent diabetes-like condition. During treatment, UCP2 mRNA levels increased to 185% of normal untreated control levels in soleus muscle. In contrast, UCP3 mRNA levels significantly decreased, up to 67% of normal untreated control levels. Interestingly, UCP3 mRNA levels correlated quite strongly with blood glucose levels, with r = 0.82 for gastrocnemius tissue and r = 0.92 for soleus tissue. These results may indicate that pioglitazone increases glucose catabolism by direct upregulation of muscle UCP2 gene expression in vivo. Therefore, UCP3 gene expression is controlled by a different mechanism than UCP2 expression.

In skeletal muscles, increased utilization of lipids and carbohydrates accompanied with increased energy expenditure has been observed during and after exercise. UCP3, mitochondrial uncoupling protein, is expressed in skeletal muscles. We investigated UCP3 mRNA levels in exercise training mice which increased energy expenditure and in sciatic nerve-denervated mice which decreased energy expenditure. Mice exercised by 2 wk swimming had 14- to 18-fold increases of UCP3 mRNA in skeletal muscles 3 h after the last swimming, but no increases of UCP1 mRNA in BAT and of UCP2 mRNA in WAT. However, 22 h after exercise, UCP3 mRNA increases observed in skeletal muscles 3 h after exercise returned to sedentary levels. Similar transient increases of UCP3 mRNA were observed in 1 wk treadmill running training or a single exercise bout. In denerved gastrocnemius, GLUT4 and UCP3 mRNA decreased by 58 and 45%, respectively. These data indicate that UCP3 may have a role for fine adjustments of energy expenditure and that up-regulation of UCP3 mRNA may be a defense mechanism against extra energy supply to consume extra energy in skeletal muscles.

The expressions of uncoupling proteins 2 and 3 (UCP2; UCP3) mRNA were studied in obese (fa/fa) Zucker rats treated with two weight gain reducing agents for three weeks. The specific beta 3-adrenoceptor agonist BRL 35135 (0.5 mg/kg/day orally) increased the expression of UCP3 mRNA by 3.8-fold (P0.0001; two-way ANOVA) and that of UCP1 mRNA by 2.6-fold (P = 0.014) in brown adipose tissue, but had no effect on expression of UCP3 mRNA in white fat or in the soleus muscle, or on UCP2 mRNA expression in brown or white fat. The antihyperglycemic metformin (300 mg/kg/day orally) had no effect on expressions of UCP1, UCP2 or UCP3 in any tissue studied. Concentrations of plasma insulin were significantly correlated with the levels of white fat UCP2 mRNA (in the control group: r = 0.89, P = 0.0015) and UCP3 mRNA (in the control group: r = 0.80, P = 0.009) suggesting that insulin may play a role in the control of UCP2 and UCP3 mRNA expressions in white adipose tissue.

Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. UCP1 is expressed exclusively in brown adipocytes while UCP2 is expressed widely. We now report the molecular cloning of a third uncoupling protein homologue, designated UCP3. At the amino acid level, hUCP3 is 71% identical to hUCP2 and 57% identical to hUCP1. UCP3 is distinguished from UCP1 and UCP2 by its abundant and preferential expression in skeletal muscle in humans, and brown adipose tissue and skeletal muscle in rodents. Since skeletal muscle and brown adipose tissue are believed to be important sites for regulated energy expenditure in humans and rodents, respectively, UCP3 may be an important mediator of adaptive thermogenesis. Since UCP3 is minimally expressed in human heart and other critical organs, it is a promising target for anti-obesity drug development aimed at increasing thermogenesis.

The uncoupling protein (UCP) family has been suggested as a possible determinant affecting obesity risk given their function in the regulation of energy metabolism. In an effort to elucidate the effects of UCP family polymorphisms on obesity phenotypes, we genotyped 10 polymorphisms in UCP2 and UCP3 among overweight female subjects (n = 458), and genetic effects on BMI and changes after a very low calorie diet (VLCD) were examined. Analyses of VLCD-induced changes among the subjects who had finished one month-weight control program (n = 301) revealed that several polymorphisms in UCP2-3 gene cluster showed associations with changes of BMI and fat mass, however not of protein mass. One of the major haplotypes of UCP2-3 gene cluster, ht1 (GGCdelCGTACC), and UCP2−866G>A showed significant associations with VLCD-induced fat reduction (P = 0.002 and 0.004; Pcorr = 0.03 and 0.01, respectively), and these results suggested that UCP2-3 polymorphisms were important genetic factor for the VLCD-induced reduction of body fat mass.

We found opposite regulation of uncoupling protein 3 (UCP3) in slow-twitch soleus and fast-twitch gastrocnemius muscles of rats during cold exposure. Namely, the UCP3 mRNA level was downregulated in the soleus muscles, but upregulated in the gastrocnemius muscles after a 24-h cold exposure. In the analysis of UCP3 protein, we first succeeded in detecting UCP3 short-form as well as the long-form in vivo, which levels were decreased markedly in the cold-exposed soleus muscles. However, the levels of UCP3 and cytochrome oxidase subunit IV were well maintained in the cold-exposed gastrocnemius muscles with a rise in the total mitochondrial protein level, suggesting an increase of total oxidative ability. The fast-twitch muscle rather than the slow-twitch one may play an important role in adaptive responses, including thermogenesis under acute cold exposure.

We examined the genetic contribution of single nucleotide polymorphisms (SNPs) of the energy metabolism-related genes, including beta 3 adrenergic receptor (beta3AR), apolipoprotein E (apo-E), promoter of uncoupling protein 3 (UCP3-p), peroxisome proliferator-activated receptor gamma 2 (PPARgamma2) and leptin receptor (LEPR) to metabolic disorders, in 118 inhabitants of Palau. The data were statistically analyzed and ethnically compared to correlate SNPs and their metabolic parameters. UCP3-p (P0.01) and PPARgamma2 (p = 0.05) correlated with plasma HbA1c, and UCP3-p correlated with fasting blood glucose (P0.01) in males, but not in females. UCP3-p correlated with body fat (%) (P0.01) in females, but not in males. Plasma leptin levels and apo-E were correlated in both groups. The frequency of SNPs for PPARgamma2, LEPR, and UCP3-p are significantly different between Palauans and Caucasians.

We have previously reported that systemic epidermal growth factor (EGF) treatment in rats reduces the amount of adipose tissue despite an unaltered food intake. The mitochondrial uncoupling proteins (UCP2 and UCP3) are thought to uncouple the respiratory chain and thus to increase energy expenditure. In order to find out whether the UCP system was involved in the EGF-induced weight loss, the effects of EGF on UCP2 and UCP3 in adipose tissue and skeletal muscle were investigated in the present study. Eight rats were treated with placebo or EGF (150 microg/kg/day) for seven days via mini-osmotic pumps. The EGF-treated rats gained significantly less body weight during the study period than the placebo-treated animals and had significantly less adipose tissue despite a similar food intake. The placebo group and the EGF group had similar UCP2 mRNA expression (in both adipose tissue and skeletal muscle), whereas the EGF-treated group compared to the placebo group had significantly higher UCP3 mRNA expression in both skeletal muscle (3.76 +/- 0.90 vs 8.41 +/- 0.87, P0.05) and in adipose tissue (6.38 +/- 0.71 vs 12.48 +/- 1.79, P0.05). In vitro studies with adipose tissue fragments indicated that the EGF effect probably is mediated indirectly as incubations with EGF (10 microM) were unable to affect adipose tissue UCP expression, whereas incubations with bromopalmitate stimulated both UCP2 and UCP3 mRNA expression twofold. Thus, EGF treatment in vivo was found to enhance UCP3 mRNA expression in both adipose tissue and skeletal muscle, which may indicate that the EGF effect on body composition might involve up-regulation of UCP3 in skeletal muscle and adipose tissue.

Brown adipose tissue (BAT) is the specific site for metabolic heat production in mammals. To establish a novel immortal brown adipocyte cell line, the stromal-vascular fraction containing preadipocytes was obtained from interscapular BAT of mice deficient of a tumor-suppressor gene p53. The p53-deficient cells, tentatively named as HB2 cells, could be cultured in vitro after repeated passages and differentiated into adipocytes in the presence of insulin, T3 and/or troglitazone, expressing some adipocyte-specific genes and accumulating intracellular lipid droplets. The mRNA level of uncoupling protein 1 (UCP1), a mitochondrial protein specifically present in brown adipocytes, was undetectable in HB2 preadipocytes, but increased after adipose differentiation. In HB2 adipocytes, UCP1 mRNA expression was markedly activated after stimulation of the beta-adrenergic receptor pathway. The mRNA of UCP2 and UCP3, recently cloned isoforms of UCP1, were also detected in HB2 adipocytes, but their levels were not influenced by adrenergic stimulation. Thus HB2 cells seem useful for in vitro studies of BAT and UCP functions.

Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters that dissipate the proton gradient, releasing stored energy as heat, without coupling to other energy-consuming processes. Therefore, the UCPs are thought to be important determinants of the metabolic efficiency. To elucidate relationships between the UCPs expressions and insulin sensitivity improvement, we treated KK-A y mice with β 3 adrenergic receptor agonist for 21 days and examined the changes of the UCPs mRNA expressions in various tissues. Chronic treatment of a specific β 3 adrenergic receptor agonist, CL316,243 (0.2mg/kg body weight/day s.c.) markedly increased the expressions of uncoupling protein 1 (UCP1), uncoupling protein 2 (UCP2), and uncoupling protein 3 (UCP3) by 14-fold, 6-fold, and 16-fold, respectively, in the brown adipose tissue (BAT). The UCP1 and UCP3 mRNA expressions in the white adipose tissue (WAT) were also increased by 12-fold and 9-fold, respectively, but the UCP2 mRNA expression was not changed in this tissue. Interestingly, the UCP2 and UCP3 mRNA expressions were strikingly decreased in the skeletal muscle and heart. Particularly, the UCP3 mRNA expression level in the skeletal muscle was dropped to 10% of that of the saline-treated control mice, indicating that the UCPs mRNA expressions are regulated in tissue-specific ways. The concentrations of plasma insulin and circulating free fatty acid (FFA) were significantly decreased, suggesting that they correlate with the reductions of the UCP2 and UCP3 mRNA expressions in the skeletal muscle and heart. It has been thought that the UCP1 and UCP3 mRNA expressions in the BAT and WAT are mainly controlled by the hypothalamus via the sympathetic nervous system, while the levels of insulin, FFA or both may play important roles in the control of the UCP2 and UCP3 mRNA expressions in the skeletal muscle and heart.

A rapid, highly sensitive method to determine the mRNA level of muscle-specific markers using TaqMan PCR analysis was developed and used to study sequential gene regulation of myoblasts during induced differentiation of C2C12cells. mRNA levels of muscle regulatory factor (MRF) myogenin, α-actin, thermoregulatory uncoupling protein UCP2 and glucose transporter isotype glut4 increased rapidly during early stage differentiation. In contrast, myf5, β-actin, UCP1 and glut1 mRNA levels gradually decreased during 8 days. However, the mRNA level of other genes such as MyoD, glyceraldehyde-3-phosphate dehydrogenase and hexokinase II changed only slightly in comparison. Muscle-specific uncoupling protein UCP3 mRNA was detected during differentiation and increased rapidly within 8 days. These data clearly show the sequential and the differential regulation of muscle-specific genes in C2C12cells during multinucleate myotube formation.

The expression of the two novel uncoupling proteins genes, UCP2 and UCP3, is differentially regulated during prenatal maturation of brown adipose tissue in the mouse. UCP2 gene is expressed early in prenatal development, when neither UCP1 nor UCP3 gene expression yet occurs. UCP3 mRNA is absent at any stage of fetal life; it appears suddenly at birth and reaches adult levels in a few hours. UCP2 mRNA increased after birth but more slowly than UCP3 and UCP1 mRNA. Short-time exposure of adult mice to cold caused a rise in UCP2 or UCP1 mRNA levels but not in that of UCP3. The postnatal rise in UCP2 gene expression appears to be a response to the thermic stress associated with birth, similarly to UCP1, whereas different biological signals may be responsible for the surge in UCP3 gene expression at birth.

Uncoupling protein (UCP1) is a transmembrane proton transporter present in the mitochondria of brown adipose tissue (BAT), a specialized tissue which functions in temperature homeostasis and energy balance (Nicholls, D. G., and Locke, R. M. (1984) Physiol. Rev. 64, 2-40; Lowell, D. D., and Flier, J. S. (1997) Annu. Rev. Med.). UCP1 mediates the thermogenesis that is characteristic of BAT by uncoupling mitochondrial oxidation of substrates from ATP synthesis. Recently, two proteins related to UCP1 have been identified and designated UCP2 (Fleury, C., et al. (1997) Nature Genetics 15, 269-272) or UCP homolog (UCPH) (Gimeno, R. E., et al. (1997) Diabetes 46, 900-906) and UCP3 (Boss, O., et al. (1997) FEBS Lett. 408, 39-42; Vidal-Puig, A., et al. (1997) Biochem. Biophys. Res. Commun. 235, 79-82). We investigated the regulation in rats of UCP3, which is expressed primarily in skeletal muscle and BAT. Expression of rat UCP3 mRNA in BAT was upregulated by in vivo treatment with triiodothyronine (T3) and by exposure to cold, suggesting that UCP3 is active in thermogenesis and energy expenditure. In skeletal muscle, UCP3 mRNA was also upregulated by T3 but, surprisingly, not by cold exposure. A hypothesis is proposed to account for this differential regulation.