Up-regulation of uncoupling proteins by beta-adrenergic stimulation in L6 myotubes

Catecholamine-induced and beta-adrenergic receptor (beta-AR)-mediated thermogenesis in skeletal muscle is a significant component of whole-body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of beta-AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6-24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9-cis retinoic acid, and mimicked by isoproterenol and salbutamol (beta2-AR agonists), but abolished by propranolol and ICI-118,551 (beta2-AR antagonists). Pharmacological and mRNA analyses revealed the existence of beta2-AR, but not beta1- and beta3-ARs, in L6 myotubes. These results suggested that catecholamines up-regulate UCP2 and UCP3 expression through direct action on the beta2-AR in skeletal muscle.     

Effects of Obesity and Stable Weight Reduction on UCP2 and UCP3 Gene Expression in Humans

VIDAL-PUIG, ANTONIO, MICHAEL ROSENBAUM, ROBERT C. CONSIDINE, RUDOLPH L. LEIBEL, G. LYNIS DOHM, AND BRADFORD B. LOWELL. Effects of obesity and stable weight reduction on UCP2 and UCP3 gene expression in humans. Obes Res. Objectives: The molecular determinants of energy expenditure are presently unknown. Recently, two uncoupling protein homologues, UCP2 and UCP3, have been identified. UCP2 is expressed widely, and UCP3 is expressed abundantly in skeletal muscle. Both could be important regulators of energy balance. In this paper, we investigated whether altered UCP2 and UCP3 mRNA levels are associated with obesity or weight reduction. Research Methods and Procedures: UCP2, UCP3 long and short mRNA levels were examined in skeletal muscle and in white adipose tissue of lean, obese, and weight-reduced individuals by RNase protection assay. Results: Expression of UCP2, UCP3S, and UCP3L mRNA in skeletal muscle was similar in lean individuals and in individuals with obesity at stable weight. In contrast, UCP3L and UCP3S mRNAs were decreased by 38% (p < 0.0059) and 48% (p<0.0047), respectively, in 20% weight-reduced patients with obesity at stable weight. In contrast, UCP2 mRNA levels were increased by 30% in skeletal muscle of 20% weight-reduced subjects with obesity. In a different set of patients, mostly lean, UCP3L mRNA in skeletal muscle was decreased by 28% (p = 0.0425) after 10% weight reduction at stable weight. Expression of UCP2 mRNA in subcutaneous adipose tissue was similar in lean individuals and in individuals with obesity, and was increased by 58% during active weight loss. Discussion: Stabilization at reduced body weight in humans is associated with a decrease in UCP3 mRNA in muscle. It is possible that reduced UCP3 expression could contribute to decreased energy expenditure in weight-stable, weight-reduced individuals.     

Rev-erbbeta regulates the expression of genes involved in lipid absorption in skeletal muscle cells: evidence for cross-talk between orphan nuclear receptors and myokines

Rev-erbbeta is an orphan nuclear receptor that selectively blocks trans-activation mediated by the retinoic acid-related orphan receptor-alpha (RORalpha). RORalpha has been implicated in the regulation of high density lipoprotein cholesterol, lipid homeostasis, and inflammation. Reverbbeta and RORalpha are expressed in similar tissues, including skeletal muscle; however, the pathophysiological function of Rev-erbbeta has remained obscure. We hypothesize from the similar expression patterns, target genes, and overlapping cognate sequences of these nuclear receptors that Rev-erbbeta regulates lipid metabolism in skeletal muscle. This lean tissue accounts for >30% of total body weight and 50% of energy expenditure. Moreover, this metabolically demanding tissue is a primary site of glucose disposal, fatty acid oxidation, and cholesterol efflux. Consequently, muscle has a significant role in insulin sensitivity, obesity, and the blood-lipid profile. We utilize ectopic expression in skeletal muscle cells to understand the regulatory role of Rev-erbbeta in this major mass peripheral tissue. Exogenous expression of a dominant negative version of mouse Rev-erbbeta decreases the expression of many genes involved in fatty acid/lipid absorption (including Cd36, and Fabp-3 and -4). Interestingly, we observed a robust induction (>15-fold) in mRNA expression of interleukin-6, an "exercise-induced myokine" that regulates energy expenditure and inflammation. Furthermore, we observed the dramatic repression (>20-fold) of myostatin mRNA, another myokine that is a negative regulator of muscle hypertrophy and hyperplasia that impacts on body fat accumulation. This study implicates Rev-erbbeta in the control of lipid and energy homoeostasis in skeletal muscle. In conclusion, we speculate that selective modulators of Rev-erbbeta may have therapeutic utility in the treatment of dyslipidemia and regulation of muscle growth.     

Effect of beta1- and beta2-adrenergic stimulation on energy expenditure,substrate oxidation,and UCP3 expression in humans

In humans, beta-adrenergic stimulation increases energy and fat metabolism. In the case of beta1-adrenergic stimulation, it is fueled by an increased lipolysis. We examined the effect of beta2-adrenergic stimulation, with and without a blocker of lipolysis, on thermogenesis and substrate oxidation. Furthermore, the effect of beta1-and beta2-adrenergic stimulation on uncoupling protein 3 (UCP3) mRNA expression was studied. Nine lean males received a 3-h infusion of dobutamine (DOB, beta1) or salbutamol (SAL, beta2). Also, we combined SAL with acipimox to block lipolysis (SAL+ACI). Energy and substrate metabolism were measured continuously, blood was sampled every 30 min, and muscle biopsies were taken before and after infusion. Energy expenditure significantly increased approximately 13% in all conditions. Fat oxidation increased 47 +/- 7% in the DOB group and 19 +/- 7% in the SAL group but remained unchanged in the SAL+ACI condition. Glucose oxidation decreased 40 +/- 9% upon DOB, remained unchanged during SAL, and increased 27 +/- 11% upon SAL+ACI. Plasma free fatty acid (FFA) levels were increased by SAL (57 +/- 11%) and DOB (47 +/- 16%), whereas SAL+ACI caused about fourfold lower FFA levels compared with basal levels. No change in UCP3 was found after DOB or SAL, whereas SAL+ACI downregulated skeletal muscle UCP3 mRNA levels 38 +/- 13%. In conclusion, beta2-adrenergic stimulation directly increased energy expenditure independently of plasma FFA levels. Furthermore, this is the first study to demonstrate a downregulation of skeletal muscle UCP3 mRNA expression after the lowering of plasma FFA concentrations in humans, despite an increase in energy expenditure upon beta2-adrenergic stimulation.     

Exercise training reverses impaired skeletal muscle metabolism induced by artificial selection for low aerobic capacity

We have used a novel model of genetically imparted endurance exercise capacity and metabolic health to study the genetic and environmental contributions to skeletal muscle glucose and lipid metabolism. We hypothesized that metabolic abnormalities associated with low intrinsic running capacity would be ameliorated by exercise training. Selective breeding for 22 generations resulted in rat models with a fivefold difference in intrinsic aerobic capacity. Low (LCR)- and high (HCR)-capacity runners remained sedentary (SED) or underwent 6 wk of exercise training (EXT). Insulin-stimulated glucose transport, insulin signal transduction, and rates of palmitate oxidation were lower in LCR SED vs. HCR SED (P < 0.05). Decreases in glucose and lipid metabolism were associated with decreased β?-adrenergic receptor (β?-AR), and reduced expression of Nur77 target proteins that are critical regulators of muscle glucose and lipid metabolism [uncoupling protein-3 (UCP3), fatty acid transporter (FAT)/CD36; P < 0.01 and P < 0.05, respectively]. EXT reversed the impairments to glucose and lipid metabolism observed in the skeletal muscle of LCR, while increasing the expression of β?-AR, Nur77, GLUT4, UCP3, and FAT/CD36 (P < 0.05) in this tissue. However, no metabolic improvements were observed following exercise training in HCR. Our results demonstrate that metabolic impairments resulting from genetic factors (low intrinsic aerobic capacity) can be overcome by an environmental intervention (exercise training). Furthermore, we identify Nur77 as a potential mechanism for improved skeletal muscle metabolism in response to EXT.     

Up-regulation of liver uncoupling protein-2 mRNA by either fish oil feeding or fibrate administration in mice

Fish oil feeding showed less obesity in rodents, relative to other dietary oils. N-3 fatty acids rich in fish oil and fibrate compounds are peroxisome proliferator-activated receptor alpha (PPARalpha) ligands that stimulate beta-oxidation of fatty acids in liver and are used for treatment of hypertriglycemic patients. Since UCP-2, a member of an uncoupling protein family, has been shown to express in hepatocytes, the effects of these agents on the expression of UCP2 mRNA were investigated. C57BL/6J mice were divided into three groups; the first group was given a high-carbohydrate diet, and the other two groups were given a high-fat diet (60% of total energy) as safflower oil or fish oil for 5 months. Safflower oil diet fed mice developed obesity, but those fed fish oil diet did not. Therefore, the effects of fish oil feeding on the expression of UCP1, UCP2 and UCP3 in liver, skeletal muscle (gastrocnemius), white adipose tissue (WAT) and brown adipose tissue (BAT) were assessed by Northern blotting. Compared with safflower oil feeding, fish oil feeding up-regulated liver UCP2, BAT UCP2 and skeletal muscle UCP3 mRNA, while down-regulated WAT UCP2 and BAT UCP3 mRNA. Among these alterations, 5-fold up-regulation of liver UCP2 mRNA, relative to carbohydrate feeding, was noteworthy. Fenofibrate administration (about 500 mg/kg BW/d) for 2 wks also induced liver UCP2 expression by 9-fold. These data indicated that fish oil feeding and fibrate administration each up-regulated UCP2 mRNA expression in liver possibly via PPARalpha and hence each has the potential of increasing energy expenditure for prevention of obesity.     

Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase

Strategies to prevent and treat obesity aim to decrease energy intake and/or increase energy expenditure. Regarding the increase of energy expenditure, two key intracellular targets may be considered (1) mitochondrial oxidative phosphorylation, the major site of ATP production, and (2) AMP-activated protein kinase (AMPK), the master regulator of cellular energy homeostasis. Experiments performed mainly in transgenic mice revealed a possibility to ameliorate obesity and associated disorders by mitochondrial uncoupling in metabolically relevant tissues, especially in white adipose tissue (WAT), skeletal muscle (SM), and liver. Thus, ectopic expression of brown fat-specific mitochondrial uncoupling protein 1 (UCP1) elicited major metabolic effects both at the cellular/tissue level and at the whole-body level. In addition to expected increases in energy expenditure, surprisingly complex phenotypic effects were detected. The consequences of mitochondrial uncoupling in WAT and SM are not identical, showing robust and stable obesity resistance accompanied by improvement of lipid metabolism in the case of ectopic UCP1 in WAT, while preservation of insulin sensitivity in the context of high-fat feeding represents the major outcome of muscle UCP1 expression. These complex responses could be largely explained by tissue-specific activation of AMPK, triggered by a depression of cellular energy charge. Experimental data support the idea that (1) while being always activated in response to mitochondrial uncoupling and compromised intracellular energy status in general, AMPK could augment energy expenditure and mediate local as well as whole-body effects; and (2) activation of AMPK alone does not lead to induction of energy expenditure and weight reduction.     

Acute central infusion of leptin modulates fatty acid mobilization by affecting lipolysis and mRNA expression for uncoupling proteins

Chronic administration of leptin has been shown to reduce adiposity through energy intake and expenditure. The present study aims to examine how acute central infusion of leptin regulates peripheral lipid metabolism, as assessed by markers indicative of their mobilization and utilization. A bolus infusion of 1 microg/rat leptin into the third cerebroventricle increased the expression of mRNA for hormone-sensitive lipase (HSL), an indicator of lipolysis, in white adipose tissue (WAT). This was accompanied by elevation of plasma levels of glycerol, but not of free fatty acids, as compared to the saline control (P < 0.03). The same treatment with leptin decreased plasma insulin levels but did not affect the plasma glucose level (P < 0.05 for insulin). Among the major regulators of the transportation or utilization of energy substrates, leptin treatment increased expression of mRNA for uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), UCP2 in WAT, and UCP3 in quadriceps skeletal muscle, but not those for fatty acid-binding protein in WAT, carnitine phosphate transferase-1, a marker for beta oxidation of fatty acids in muscle, nor glucose transporter 4 in WAT and muscle (P < 0.01 for HSL, P < 0.05 for UCP1, and P < 0.005 for UCP2 and UCP3). These results indicate that, even in a single bolus, leptin may regulate the mobilization and/or utilization of energy substrates such as fatty acids by affecting lipolytic activity in WAT and by increasing the expression of UCPs in BAT, WAT, and muscle.     

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.     

Role of the beta(3)-adrenergic receptor and/or a putative beta(4)-adrenergic receptor on the expression of uncoupling proteins and peroxisome proliferator-activated receptor-gamma coactivator-1.

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.     

Nr4a1 siRNA expression attenuates α-MSH regulated gene expression in 3T3-L1 adipocytes

Several recent investigations have underscored the growing role of melanocortin signaling in the peripheral regulation of lipid, glucose, and energy homeostasis. In addition, the melanocortins play a critical role in the central control of satiety. These observations, and the latest reports highlighting the emerging role of the nuclear hormone receptor (NR) 4A subgroup in metabolism, have prompted us to investigate the cross talk between [Nle(4), d-Phe(7)] (NDP)-α-MSH and Nr4a signaling in adipose. We have shown that NDP-MSH strikingly and preferentially induces the expression of the NR4A subgroup (but not any other members of the NR superfamily) in differentiated 3T3-L1 adipocytes. Utilization of quantitative PCR on custom-designed metabolic TaqMan low-density arrays identified the concomitant and marked induction of the mRNAs encoding Il-6, Cox2, Pdk4, and Pck-1 after NDP-MSH treatment. Similar experiments demonstrated that the mRNA expression profile induced by cAMP and NDP-MSH treatment displayed unique but also overlapping properties and suggested that melanocortin-mediated induction of gene expression involves cAMP-dependent and -independent signaling. Nr4a1/Nur77 small interfering RNA (siRNA) expression suppressed NDP-MSH-mediated induction of Nr4a1/Nur77 and Nr4a3/Nor-1 (but not Nr4a2/Nurr1). Moreover, expression of the siRNA-attenuated NDP-MSH mediated induction of the mRNAs encoding Il-6, Cox2/Ptgs2, and Pck-1 expression. In addition, Nur77 siRNA expression attenuated NDP-MSH-mediated glucose uptake. In vivo, ip administration of NDP-MSH to C57 BL/6J (male) mice significantly induced the expression of the mRNA encoding Nur77 and increased IL-6, Cox2, Pck1, and Pdk4 mRNA expression in (inguinal) adipose tissue. We conclude that Nur77 expression is necessary for MSH-mediated induction of gene expression in differentiated adipocytes. Furthermore, this study demonstrates cross talk between MSH and Nr4a signaling in adipocytes.     

Genomic organization and regulation by dietary fat of the uncoupling protein 3 and 2 genes

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.     

Nur77 Decreases Atherosclerosis Progression in apoE?/? Mice Fed a High-Fat/High-Cholesterol Diet

Rationale

It is clear that lipid disorder and inflammation are associated with cardiovascular diseases and underlying atherosclerosis. Nur77 has been shown to be involved in inflammatory response and lipid metabolism.

Objective

Here, we explored the role of Nur77 in atherosclerotic plaque progression in apoE^−/− mice fed a high-fat/high cholesterol diet.

Methods and Results

The Nur77 gene, a nuclear hormone receptor, was highly induced by treatment with Cytosporone B (Csn-B, specific Nur77 agonist), recombinant plasmid over-expressing Nur77 (pcDNA-Nur77), while inhibited by treatment with siRNAs against Nur77 (si-Nur77) in THP-1 macrophage-derived foam cells, HepG2 cells and Caco-2 cells, respectively. In addition, the expression of Nur77 was highly induced by Nur77 agonist Csn-B, lentivirus encoding Nur77 (LV-Nur77), while silenced by lentivirus encoding siRNA against Nur77 (si-Nur77) in apoE^−/− mice fed a high-fat/high cholesterol diet, respectively. We found that increased expression of Nur77 reduced macrophage-derived foam cells formation and hepatic lipid deposition, downregulated gene levels of inflammatory molecules, adhesion molecules and intestinal lipid absorption, and decreases atherosclerotic plaque formation.

Conclusion

These observations provide direct evidence that Nur77 is an important nuclear hormone receptor in regulation of atherosclerotic plaque formation and thus represents a promising target for the treatment of atherosclerosis.     

Systemic administration of epidermal growth factor increases UCP3 mRNA levels in skeletal muscle and adipose tissue in rats

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, P < 0.05) and in adipose tissue (6.38 +/- 0.71 vs 12.48 +/- 1.79, P < 0.05). 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.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Regulation of UCP1, UCP2, and UCP3 mRNA expression in brown adipose tissue, white adipose tissue, and skeletal muscle in rats by estrogen

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.