Dietary vitamin A supplementation in rats: suppression of leptin and induction of UCP1 mRNA

All-trans-retinoic acid (RA), an active metabolite of vitamin A, induces the gene expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and suppresses leptin gene expression in white adipose tissue (WAT) when given as an acute dose. These contrasting effects of RA leave in doubt the overall effect of chronic RA or vitamin A supplementation on energy homeostasis. To investigate the effects of dietary vitamin A supplementation on leptin and UCP1 gene expression, rats were fed either a normal diet (2.6 retinol/kg diet) or a vitamin A-supplemented diet (129 mg retinol/kg diet) for 8 weeks, and adiposity, serum leptin levels, leptin mRNA levels in perirenal WAT, UCP1 and UCP2 mRNA levels in BAT, and beta3-adrenergic receptor mRNA levels in BAT and WAT were examined. Rats on both diets gained a similar amount of weight, but there was a small 9% decrease in the adiposity index in the vitamin A-supplemented rats. Dietary vitamin A supplementation increased UCP1 gene expression in BAT by 31%, but suppressed leptin gene expression by 44% and serum leptin levels by 65%. UCP2 and beta3-adrenergic receptor gene expression in BAT and perirenal WAT were unchanged by the vitamin A diet. These data suggest that dietary vitamin A has a role in regulating energy homeostasis by enhancing UCP1 gene expression and decreasing serum leptin levels.     

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     

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.     

Effect of high-fat diet, surrounding temperature, and enterostatin on uncoupling protein gene expression

Nonshivering thermogenesis induced in brown adipose tissue (BAT) during high-fat feeding is mediated through uncoupling protein 1 (UCP1). UCP2 is a recently identified homologue found in many tissues. To determine the role of UCP1 and UCP2 in thermoregulation and energy balance, we investigated the long-term effect of high-fat feeding on mRNA levels in mice at two different ambient temperatures. We also treated mice with the anorectic peptide enterostatin and compared mRNA levels in BAT, white adipose tissue (WAT), stomach, and duodenum. Here, we report that high-fat feeding at 23 degrees C increased UCP1 and UCP2 levels in BAT four- and threefold, respectively, and increased UCP2 levels fourfold in WAT. However, at 29 degrees C, UCP1 decreased, whereas UCP2 remained unchanged in BAT and increased twofold in WAT. Enterostatin increased UCP1 and decreased UCP2 mRNA in BAT. In stomach and duodenum, high-fat feeding decreased UCP2 mRNA, whereas enterostatin increased it. Our results suggest that the regulation of uncoupling protein mRNA levels by high-fat feeding is dependent on ambient temperature and that enterostatin is able to modulate it.     

Fucoxanthin from edible seaweed, Undaria pinnatifida, shows antiobesity effect through UCP1 expression in white adipose tissues

Mitochondrial uncoupling protein 1 (UCP1) is usually expressed only in brown adipose tissue (BAT) and a key molecule for metabolic thermogenesis to avoid an excess of fat accumulation. However, there is little BAT in adult humans. Therefore, UCP1 expression in tissues other than BAT is expected to reduce abdominal fat. Here, we show reduction of abdominal white adipose tissue (WAT) weights in rats and mice by feeding lipids from edible seaweed, Undaria pinnatifida. Clear signals of UCP1 protein and mRNA were detected in WAT of mice fed the Undaria lipids, although there is little expression of UCP1 in WAT of mice fed control diet. The Undaria lipids mainly consisted of glycolipids and seaweed carotenoid, fucoxanthin. In the fucoxanthin-fed mice, WAT weight significantly decreased and UCP1 was clearly expressed in the WAT, while there was no difference in WAT weight and little expression of UCP1 in the glycolipids-fed mice. This result indicates that fucoxanthin upregulates the expression of UCP1 in WAT, which may contribute to reducing WAT weight.     

Absence of UCP3 in brown adipose tissue does not impair nonshivering thermogenesis

We report on a novel Djungarian hamster mutant lineage that exhibits a loss of uncoupling protein (UCP) 3 mRNA and protein in brown adipose tissue (BAT), whereas UCP3 expression in skeletal muscle is only mildly diminished. In response to 2 d of cold exposure, UCP3 mRNA was 4.5-fold elevated in BAT of wild-type hamsters but remained undetectable in mutant hamsters. Notably, in BAT of warm- and cold-exposed mutant hamsters, UCP1 and UCP2 mRNA levels were increased. The tissue specificity of UCP3 deficiency suggests that the underlying unknown mutation impairs a factor controlling UCP3 gene expression selectively in brown adipocytes. In wild-type but not mutant primary brown adipocytes, UCP3 gene expression was stimulated by treatment with peroxisome proliferator activated receptor (PPAR) ligands. This implies that the underlying mutation causing UCP3 deficiency is expressed within brown adipocytes and disrupts PPAR-dependent transactivation of the UCP3 gene. On the functional level, we found no direct phenotypic consequences of altered UCP expression in BAT. The absence of UCP3 in BAT of cold-acclimated mutant hamsters affected neither maximal nonshivering thermogenesis elicited by noradrenaline nor the uncoupled respiration of isolated mitochondria in the presence of oligomycin and in response to palmitate.     

Increased uncoupling protein-2 and -3 gene expressions in skeletal muscle of STZ-induced diabetic rats

Streptozotocin (STZ)-induced diabetic animals are vulnerable to cold stress. Uncoupling proteins (UCPs) play an important role in regulating thermogenesis. We investigated the gene expressions of UCPs in brown adipose tissue (BAT), white adipose tissue (WAT), liver and gastrocnemius muscle of STZ-diabetic rats using Northern blot. UCP-1, -2 and -3 mRNA expressions in BAT were all remarkably lower in STZ-diabetic rats than those in control rats. Both UCP-2 and -3 gene expressions in gastrocnemius muscle were substantially elevated in STZ-diabetic rats and insulin treatment restored UCP gene expressions to normal levels. These results suggest that in STZ-diabetic rats, the overexpression of UCP-2 and UCP-3 in skeletal muscle provides a defense against hypothermogenesis caused by decreased UCPs in BAT.     

Orexin-A regulates body temperature in coordination with arousal status

Orexins, hypothalamic neuropeptides, are involved in modulation of food intake and arousal status. To further examine their physiological roles in brain function, the effect of centrally administered orexin-A on body temperature was investigated in rats. Assessed by a telemetry sensor system implanted into the abdominal cavity, infusion of orexin-A into the third cerebroventricle (i3vt) increased body temperature in a dose-responsive manner. Expression of uncoupling protein 1 (UCP1) mRNA in brown adipose tissue (BAT), as a marker for peripheral thermogenesis, failed to increase after the infusion. Expression of UCP3 mRNA in skeletal muscle was up-regulated, whereas UCP2 in white adipose tissue was unchanged after the infusion. The resulting information indicates that orexin neurons regulate body temperature in coordination with arousal status independently of peripheral thermogenesis, which is regulated by BAT UCP1.     

Role of adipogenic and thermogenic genes in susceptibility or resistance to develop diet-induced obesity in rats

The aim of the present work was to assess whether changes in adipose tissue gene expression related with adipogenesis and/or thermogenesis could be involved in the mechanism conferring susceptibility or resistance to develop obesity in high-fat fed outbreed rats. For this purpose, male Wistar rats were fed with standard laboratory diet (control group) or high fat diet. After 15 days, two groups of rats with significant differences on body weight gain in response to the high fat diet were characterized and identified as diet-induced obesity (DIO) and diet resistant (DR) rats. A significant increase in visceral white adipose tissue (WAT) PPARgamma and aP2 (p < 0.05) mRNA levels associated to a decrease in RARgamma expression (p < 0.05) was observed in DIO rats, suggesting an increase of adipogenesis. Furthermore, our data showed a marked increase in brown adipose tissue (BAT) of UCP1 mRNA in DIO animals (p < 0.01) (without affecting PGC-1alpha gene expression), whereas no changes were found in WAT UCP2 gene expression. All these data suggest that the variations found in the expression pattern of PPARgamma, aP2 and RARgamma by high-fat diet could be involved, at least in part, in the differences in body weight gain and adiposity observed between DR and DIO animals. The compensatory adaptations through the increase in energy expenditure by changes on the expression levels of UCP1 seem not to be enough to avoid the obesity onset in the DIO group.     

Individual severity of dietary obesity in unselected Wistar rats: relationship with hyperphagia

We investigated the relative importance of overeating, thermogenesis, and uncoupling protein (UCP) expression in determining the severity of obesity in male Wistar rats fed a highly palatable diet. After 2 wk of feeding, body weight did not differ significantly from controls (248 +/- 4 vs. 229 +/- 3 g; P > 0.3), but rectal temperature, brown adipose tissue (BAT) mass, UCP3 expression in gastrocnemius muscle, and UCP2 expression in white adipose tissue (WAT) were all elevated in diet-fed animals. In a further study, rats fed a palatable diet for 8 wk exhibited higher energy intake and rectal temperature than controls. Dietary-obese rats were divided into high (427-490 g; n = 8) and low (313-410 g; n = 10) weight gainers. The high gainers ate significantly more than the low gainers, and energy intake was positively correlated with weight gain (r(2) = 0.72, P < 0.01). UCP2 and UCP3 mRNA levels in gastrocnemius muscle were significantly increased above lean controls in all diet-fed animals, whereas UCPs in WAT and BAT did not differ significantly from controls. Whereas rats fed palatable food exhibited a thermogenic response, there was no significant difference in core temperature between high and low gain groups (37. 5 +/- 0.1 vs. 37.6 +/- 0.1 degrees C; P > 0.5). We conclude that a higher energy intake is the critical factor determining susceptibility to dietary obesity in unselected Wistar rats.     

Tissue-specific expression and cold-induced mRNA levels of uncoupling proteins in the Djungarian hamster

The uncoupling protein 1 (UCP1), a mitochondrial transmembrane protein, is responsible for adaptive thermogenesis in brown adipose tissue (BAT). Two UCP1 homologues, UCP2 and UCP3, were recently discovered, but it is controversial whether they also play a role in energy homeostasis. Djungarian hamster UCPs were found to exhibit high similarity with homologues known in other species. UCP1 mRNA was restricted to BAT, UCP2 mRNA was expressed in multiple tissues, and UCP3 mRNA was detected mainly in BAT and skeletal muscles. We examined the cold-induced regulation of hamster UCP mRNA levels and tested their correlation with serum free fatty acid (FFA) concentrations. In BAT UCP1, UCP2, and UCP3 expression was upregulated in the cold, but the increase and time course of increase differed. In skeletal muscle, UCP2 and UCP3 mRNA levels were not altered. Cold-induced changes of serum FFA levels correlated with the stimulation of UCP1 mRNA in BAT but not with UCP2 and UCP3.     

Intrauterine food restriction alters the expression of uncoupling proteins in brown adipose tissue of rat newborns

A previous study from our laboratory showed that maternal food restriction (MFR) delays thermoregulation in newborn rats. In neonates brown adipose tissue (BAT) is essential for thermogenesis due to the presence of uncoupling proteins (UCPs). The aim of this study was to evaluate the influence of MFR on the UCPs mRNA and protein expression in BAT and skeletal muscle (SM) of the newborn rat. Female Wistar EPM-1 control rats (CON) received chow ad libitum during pregnancy, whereas food-restricted dams (RES) received 50% of the amount ingested by CON. Fifteen hours after birth, the litters were weighed and sacrificed. Blood was collected for hormonal analysis. BAT and SM were used for determination of UCPs mRNA and protein expression, and Ca²⁺-ATPase sarcoplasmic reticulum (SERCA1). RES pups showed a significant reduction in body weight and fat content at birth. MFR caused a significant increase in the expression of UCP1 and UCP2 in BAT, without changes in UCP3 and SERCA1 expression in BAT and SM. No differences between groups were found for leptin, T4 and glucose levels. RES pups showed increased insulin and decreased T3 levels. The delay in development of thermoregulation previously described in RES animals appears not to result from impairment in thermogenesis, but from an increase in heat loss, since MFR caused low birth weight in pups, leading to greater surface/volume ratio. The higher expression of UCP1 and UCP2 in BAT suggests a compensatory mechanism to increased thermogenesis.     

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.     

Ghrelin regulates adiposity in white adipose tissue and UCP1 mRNA expression in brown adipose tissue in mice

To examine the involvement of ghrelin in obesity, we investigated the effects of treatment with peripherally administered ghrelin on food intake, adiposity, and expression of uncoupling protein (UCP) mRNA in brown (BAT) and white (WAT) adipose tissue in mice. Acute bolus administration of ghrelin at a dose of 120 nmol/kg increased cumulative food intake over 4 and 24 h as compared to controls (p<0.05 for each), whereas 12 nmol/kg/day ghrelin showed no remarkable effect (p>0.1). Chronic repeated treatment with 12 nmol/kg/day ghrelin for 7 days increased body weight and adiposity assessed by the weight of adipose tissue, triglyceride content in WAT (p<0.05 for each versus control). In addition, the same treatment decreased and increased mRNA expression of BAT UCP1 and WAT UCP2, respectively (p<0.05 for each). In conclusion, ghrelin can regulate body weight, adiposity and UCPs mRNA expression in mice. The present results provide evidence for a new regulatory loop involving ghrelin and UCP, and add novel insights into the regulatory mechanisms of obesity.     

Sex‐Dependent Dietary Obesity,Induction of UCPs,and Leptin Expression in Rat Adipose Tissues

Objective: The aim of this study was to determine the sex‐dependent differences in the response of key parameters involved in thermogenesis and control of body weight in brown adipose tissue (BAT) and white adipose tissue (WAT) in postcafeteria‐fed rats, a model of dietary obesity. Research Methods and Procedures: BAT and WAT were obtained from male and female control and postcafeteria‐fed Wistar rats. Postcafeteria‐fed rats were initially fed with cafeteria diet from day 10 of life until day 110 (cafeteria period) and with standard chow diet from then until day 180 of life (postcafeteria period). Body mass and energy intake were evaluated. Biometric parameters were analyzed in interscapular BAT (IBAT). Levels of uncoupling protein 1 (UCP1), α₂‐adrenergic receptor (AR), and β₃‐AR proteins and UCP1, UCP2, UCP3, β₃‐AR, and leptin mRNAs, in IBAT or WAT, were studied by Western blot and Northern blot analyses, respectively. Results: Rats attained 59% (females) and 39% (males) increase in body weight at the end of the cafeteria period. During the postcafeteria period, the rats showed a loss of body weight, which was higher in females. Postcafeteria‐fed female rats also presented higher activation of thermogenic parameters in IBAT, including UCP1, UCP2, and UCP3 mRNAs. Female control rats showed lower levels of both α2 and β₃‐ARs in BAT compared with male rats, but these levels in postcafeteria‐fed female and male rats were the same, because males tended to down‐regulate them. Levels of leptin mRNA in response to the postcafeteria state depended on gender and the specific WAT depot studied. Discussion: It is suggested that in postcafeteria‐fed female rats, BAT thermogenic capacity becomes more efficiently activated than in males. Female rats also showed a bigger weight loss. The parallel regulation of the levels of UCP2 and UCP3 mRNAs, with respect to UCP1 mRNA, with higher activation in female postcafeteria‐fed rats, suggests a possible role of both UCP2 and UCP3 in the regulation of energy expenditure and in the control of body weight. The distinct responses to overweight of α2 and β₃‐ARs—which were sex dependent—and leptin mRNA—which depended on both sex and WAT depot—also support the different response of thermogenesis‐related parameters between overweight males and females.     

Effects of running training on in vitro brown adipose tissue thermogenesis in rats

Brown adipose tissue (BAT) is a major site of nonshivering thermogenesis (NST) during cold acclimation for most mammals. Repetitive nonthermal stress such as immobilization has been shown to enhance the capacity of NST as cold acclimation. In the present study, the effects of running training, another type of nonthermal stress, were investigated on in vitro thermogenesis and the cellularity of interscapular BAT in rats. The rats were subjected to treadmill running for 30 min daily at 30 m/min under 8° inclination for 4–5 weeks. In vitro thermogenesis was then measured in minced tissue blocks incubated in a Krebs-Ringer phosphate buffer containing glucose and albumin at 37° C, using a Clark type oxygen electrode. The trained rats showed less body weight gain during the experiment. The weights of BAT and epididymal white adipose tissue were smaller in the trained rats. Noradrenaline- and glucagon-stimulated oxygen consumption were also significantly smaller in the trained rats. The tissue DNA level was greater in the trained rats, but the DNA content per tissue pad did not significantly differ. The results indicate that running training reduces BAT thermogenesis, possibly as an adaptation to conserve energy substrates for physical work.     

The Effects of Rimonabant on Brown Adipose Tissue in Rat: Implications for Energy Expenditure

The cannabinoid CB1 receptor antagonist rimonabant (SR 141716) produces a sustained decrease in body weight on a background of a transient reduction in food intake. An increase in energy expenditure has been implicated, possibly mediated via peripheral endocannabinoid system; however, the role of the central endocannabinoid system is unclear. The present study investigates this role. Rimonabant (10 mg/kg IP) was administered for 21 days to rats surgically implanted with biotelemetry devices to measure temperature in the interscapular brown adipose tissue (BAT). BAT temperature as a putative measure of thermogenesis in the BAT, physical activity, body weight, food intake, as well as changes in UCP1 messenger RNA (mRNA) and protein were measured. In addition, role of the CNS in mediating these actions of rimonabant was determined in rats where the BAT was sympathetically denervated. As expected, chronic administration of rimonabant significantly reduced body weight for the entire treatment period despite only a transient decrease in food intake. There was a profound increase in BAT temperature, particularly during the dark phase of each circadian cycle throughout the treatment period. A corresponding increase in uncoupling protein (UCP1) was also observed following chronic rimonabant treatment. The rimonabant‐induced elevation in BAT temperature and decrease in body weight were significantly attenuated following denervation, indicating an involvement of the CNS. These findings suggest that the long‐term weight loss associated with rimonabant treatment is due at least in part to an elevation in energy expenditure, represented here by elevated temperature recorded in the BAT, which is mediated primarily by the central endocannabinoid system.     

Opposite regulation of uncoupling protein 1 and uncoupling protein 3 in vivo in brown adipose tissue of cold-exposed rats

Earlier we reported a 14-fold increase of glycogen in the brown adipose tissue (BAT) in rats when the animals were placed back from cold to neutral temperature. To elucidate the mechanism, here we compared the level of glucose transporter 4 (GLUT4) protein, uncoupling protein (UCP) 1 and UCP3 mRNA and protein expressions in the BAT under the same conditions. We found that the increased GLUT4 level in cold was maintained during the reacclimation. After 1 week cold exposure the mRNA and protein content of UCP1 increased parallel, while the protein level of UCP3 decreased, contrary to its own mRNA level.     

Interaction between heat acclimation and exogenous insulin in brown adipose tissue of rats

Seventy-one male Wistar strain rats (7 weeks old) were kept at 5, 25, or 34° C, respectively, for 2 weeks with or without insulin administration. Insulin (Novo Lente MC) was given subcutaneously in a dose of 3.62 nmol/125 µl saline per 100 g body weight. An apparent effect of insulin treatment was noted only in heat-exposed rats, resulting in a remarkable gain in inter-scapular brown adipose tissue (BAT) mass of heat-acclimated, insulin-treated rats in terms of weight or weight per unit body weight. The BAT from heat-acclimated, insulin-treated rats had significantly higher levels of protein, DNA, RNA, and triglyceride than BAT from heat-acclimated, saline-treated rats. Therefore, it seems likely that the growth of BAT in heat-acclimated, insulin-treated rats was mostly due to the anabolic effects of insulin. The uncoupling protein mRNA was, however, present in BAT of heat-acclimated, insulin-treated rats at rather a depressed level, explaining a corresponding decrease in cold tolerance. On the other hand, the expression of insulin receptor mRNA was attenuated in BAT of rats from all the insulin-treated groups, possibly due to the down-regulation of insulin. Thus, there appeared to be some linkage among BAT, heat acclimation, and insulin.