Nicotine induces uncoupling protein 1 in white adipose tissue of obese mice.

OBJECTIVE: To test the hypothesis that nicotine not only activates uncoupling protein1 (UCP1) in brown adipose tissue (BAT), but also induces UCP1 in white adipose tissue (WAT), which contributes to the mitigation of obesity in obese mice. DESIGN: Weights of the whole body, the gastrocnemius muscle, interscapular BAT and subcutaneous and retroperitoneal WAT, food intake and the mRNA and protein of UCP1 in these tissues were measured and immunohistochemistry using antiserum against UCP1 was also performed in obese yellow KK mice treated with nicotine for 6 months and control mice treated with physiological saline. RESULTS: Obese mice treated with nicotine for 6 months, compared with those injected with saline, weighed significantly less (P < 0.01) and had smaller subcutaneous and retroperitoneal WAT pads (P < 0.01), while obese mice that received nicotine ate less (P < 0.05) than those injected with saline. In mice treated with nicotine, the mRNA and protein of UCP1 was detected not only in BAT, but also in subcutaneous and retroperitoneal WATs. Immunohistochemically, the BAT of obese mice contained large lipid droplets and appeared rather WAT-like, but changed to typical brown adipocytes after nicotine treatment. The fat pads of nicotine-treated mice contained many multilocular cells that were positive for UCP1. CONCLUSION: Nicotine not only activates UCP1 in BAT, but also induces UCP1 in WAT and decreases food intake, which contributes to the mitigation of obesity.     

Up-regulation of muscle uncoupling protein 3 gene expression in mice following high fat diet, dietary vitamin A supplementation and acute retinoic acid-treatment

OBJECTIVE: To analyse the impact of vitamin A supplementation of both a normal fat (NF) diet and a high fat (HF) diet and of acute retinoic acid (RA)-treatment on the expression of uncoupling protein 3 (UCP3) in mice. DESIGN: C57BL/6J mice were fed for 18 weeks a NF or a HF diet (10 and 45 energy% as fat, respectively), both with the normal vitamin A content or an excess vitamin A (8 mg and 320 mg retinyl palmitate/kg diet, respectively). Body weight and energy intake were recorded periodically. UCP3 mRNA and UCP3 protein levels in skeletal muscle (soleus/gastrocnemius) were analysed, as well as UCP1, UCP2 and UCP3 mRNA levels in interscapular brown adipose tissue (BAT), and UCP2 mRNA, UCP2 protein and leptin mRNA levels in white adipose tissue (WAT) depots. The effect of acute RA-treatment (100 mg/kg/day, 4 days) on UCP3 mRNA levels in skeletal muscle and BAT of NMRI mice was also assessed. RESULTS: Vitamin A supplementation of a NF diet led to increased levels of UCP3 mRNA and UCP3 protein in muscle, UCP1 mRNA in BAT, and UCP2 mRNA in inguinal WAT, but had no impact on body weight or adiposity of B6 mice. HF diet promoted obesity and increased levels of UCP3 mRNA and UCP3 protein in skeletal muscle, and of the mRNAs for all three UCPs in BAT. Supplementing the HF diet with vitamin A had little effect on the final obesity reached and did not lead to further increases of muscle UCP3 mRNA nor BAT UCP1 mRNA over the levels achieved with the non-supplemented HF diet. Adipose leptin mRNA levels were down regulated after vitamin A supplementation, independently of the fat content of the diet. Up-regulation of muscle, but not BAT, UCP3 mRNA levels was also found after acute RA-treatment in NMRI mice. CONCLUSION: The results provide evidence of a stimulatory effect of retinoids on muscle UCP3 expression in vivo, and a differential retinoid-regulation of the UCP3 gene in muscle and BAT.     

Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice

C57BL/6 (B6) mice subjected to a high-fat diet develop metabolic syndrome with obesity, hyperglycemia, and insulin resistance, whereas 129S6/SvEvTac (129) mice are relatively protected from this disorder because of differences in higher basal energy expenditure in 129 mice, leading to lower weight gain. At a molecular level, this difference correlates with a marked higher expression of uncoupling protein 1 (UCP1) and a higher degree of uncoupling in vitro in mitochondria isolated from muscle of 129 versus B6 mice. Detailed histological examination, however, reveals that this UCP1 is in mitochondria of brown adipocytes interspersed between muscle bundles. Indeed, the number of UCP1-positive brown fat cells in intermuscular fat in 129 mice is >700-fold higher than in B6 mice. These brown fat cells are subject to further up-regulation of UCP1 after stimulation with a beta3-adrenergic receptor agonist. Thus, ectopic deposits of brown adipose tissue in intermuscular depots with regulatable expression of UCP1 provide a genetically based mechanism of protection from weight gain and metabolic syndrome between strains of mice.     

Uncoupling proteins 2 and 3 with age: regulation by fasting and beta3-adrenergic agonist treatment

In rodents, adaptive thermogenesis in brown adipose tissue (BAT) serves both to regulate body mass after hyperphagia and to conserve energy during food deprivation. In addition to uncoupling protein 1 (UCP1), UCP3 and possibly UCP2 may have a role in energy homeostasis in BAT. We examined basal levels of UCP2 and UCP3 mRNA with age and regulation of UCP1, UCP2, and UCP3 mRNA by two conditions known to modulate energy homeostasis: fasting and beta3-adrenergic agonists. UCP1, UCP2, and UCP3 mRNA levels were unchanged between 3, 24, and 31 months of age in BAT, and UCP2 and UCP3 mRNA levels were unchanged between 6 and 24 months of age in retroperitoneal white adipose tissue (RTWAT). Following a 2-day fast, there were sizable reductions in BAT UCP1 and UCP3 mRNA, but these decreases with fasting were significantly less in the older compared with the young rats. Fasting had no effect on UCP2 mRNA levels at any age. The beta3-adrenergic agonist, CL316,243, increased BAT UCP1 and UCP3 mRNA equally in both young and old rats. The beta3-adrenergic agonist did not increase UCP2 mRNA in BAT but did increase expression in RTWAT of both young and old rats. In summary, these data indicate that the expression of the three uncoupling proteins is unchanged with age. Although the upregulation of these uncoupling proteins by beta3-adrenergic agonist treatment is maintained with age, the downregulation by fasting is diminished with age. The parallel regulation of UCP1 and UCP3 expression in BAT suggests that UCP3, like UCP1, may have a role in energy homeostasis in BAT. The diminished downregulation of UCP1 and UCP3 expression in BAT by fasting suggests that energy conservation in response to food deprivation is impaired with age, and this may contribute to an inability of older animals to maintain body mass during periods when food is limited.     

Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding,adiposity, and uncoupling protein expression in mice

To examine the functional role of CRH in the regulation of energy homeostasis by leptin, we measured the effects of the CRH antagonist, alpha-helical CRH 8-41 (alphaCRH) on a number of factors affected by leptin activity. These included food intake, body weight, hypothalamic c-fos-like immunoreactivity (c-FLI), weight and histological characterization of white adipose tissue, and mRNA expressions of uncoupling protein (UCP) in brown adipose tissue (BAT) in C57Bl/6 mice. Central infusion of leptin into the lateral cerebroventricle (icv) caused significant induction of c-FLI in the paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus, and arcuate nucleus. In all these nuclei, the effect of leptin on expression of cFLI in the PVN and VMH was decreased by treatment with alphaCRH. Administration of leptin markedly decreased cumulative food intake and body weight with this effect being attenuated by pretreatment with alphaCRH. In peripheral tissue, leptin up-regulated BAT UCP1 mRNA expression and reduced fat depositions in this tissue. Those changes in BAT were also decreased by treatment with alphaCRH. As a consequence of the effects on food intake or energy expenditure, treatment with alphaCRH attenuated the leptin-induced reduction of body adiposity, fat cell size, triglyceride contents, and ob mRNA expression in white adipose tissue. Taken together, these results indicate that CRH neurons in the PVN and VMH may be an important mediator for leptin that contribute to regulation of feeding, adiposity, and UCP expression.     

Inducible brown adipogenesis of supraclavicular fat in adult humans

Brown adipose tissue (BAT) plays key roles in thermogenesis and energy homeostasis in rodents. Metabolic imaging using positron emission tomography (PET)-computer tomography has identified significant depots of BAT in the supraclavicular fossa of adult humans. Whether supraclavicular fat contains precursor brown adipocytes is unknown. The aim of the present study was to determine the adipogenic potential of precursor cells in human supraclavicular fat. We obtained fat biopsies from the supraclavicular fossa of six individuals, as guided by PET-computer tomography, with paired sc fat biopsies as negative controls. Each piece of fat tissue was divided and processed for histology, gene analysis, and primary culture. Cells were examined for morphological changes in culture and harvested for RNA and protein upon full differentiation for analysis of UCP1 level. Histological/molecular analysis of supraclavicular fat revealed higher abundance of BAT in PET-positive than PET-negative individuals. In all subjects, fibroblast-like cells isolated from supraclavicular fat differentiated in vitro and uniformly into adipocytes containing multilobulated lipid droplets, expressing high level of UCP1. The total duration required from inoculation to emergence of fibroblast-like cells was 32-34 and 40-42 d for PET-positive- and PET-negative-derived samples, respectively, whereas the time required to achieve full differentiation was 7 d, regardless of PET status. Precursor cells from sc fat failed to proliferate or express UCP1. In summary, preadipocytes isolated from supraclavicular fat are capable of differentiating into brown adipocytes in vitro, regardless of PET status. This study provides the first evidence of inducible brown adipogenesis in the supraclavicular region in adult humans.     

Acute effects of brain-derived neurotrophic factor on energy expenditure in obese diabetic mice

OBJECTIVE: We recently demonstrated that chronic treatment with brain-derived neurotrophic factor (BDNF) regulates energy expenditure in obese diabetic C57BL/KsJ-db/db mice. In this study, we investigated the acute effects of BDNF on energy expenditure. DESIGN: After BDNF was singly administered to male db/db mice (aged 10-12 weeks), their body temperature and whole body glucose oxidation were measured. Their norepinephrine (NE) turnover and uncoupling protein (UCP) 1 expression in interscapular brown adipose tissue (BAT) were also analyzed. RESULTS: Even though the body temperatures of hyperphagic db/db mice dropped remarkably in a 24 h period after food deprivation, only a single subcutaneous administration of BDNF significantly prevented the reduction of body temperature. BDNF was also observed to have similar efficacy in cold exposure experiments at 15 degrees C. Respiratory excretion of (14)CO(2) after intravenous injection of D-[(14)C(U)]-glucose was significantly increased by BDNF administration, indicating that BDNF increases whole-body glucose oxidation. BDNF administered intracerebroventricularly was also able to prevent the reduction of body temperature of db/db mice. To clarify the BDNF action mechanism we examined NE turnover in BAT. Four hours after a single administration, BDNF reduced NE content in the presence of the tyrosine hydroxylase inhibitor, alpha-methyl-P-tyrosine methyl ester, indicating enhanced NE turnover in BAT. BDNF also increased the expression of the UCP1 mRNA and protein in BAT. CONCLUSION: These data indicate that BDNF rapidly regulates energy metabolism in obese diabetic animals, partly through activating the sympathetic nervous system and inducing UCP1 gene expression in BAT.     

Uncoupling proteins: gender dependence and their relation to body weight control

Members of the uncoupling protein (UCP) family have different purported functions, which can be either directly or indirectly related to the control of body weight. In this sense, most studies on this topic have been carried out using male subjects, although different works with males and females have shown important sex-associated differences in the regulation of these proteins; for instance, sex differences have been shown in the cold-, diet- and overweight-induced expression of brown adipose tissue UCP1 and also in the correlation of muscle UCP3 with overweight. In these kinds of studies, models of obesity such as cafeteria diet feeding and postcafeteria have been very useful. Moreover, sex hormones have been shown to modulate UCP1 expression in brown adipocytes in vitro. All these sex-dependent differences, as well as sex differences in body weight gain under a hypercaloric diet, could be related to the different respective biological functions of males and females, taking into account the fact that the gender effect in future studies on obesity could be of interest.     

Apelin, an APJ receptor ligand, regulates body adiposity and favors the messenger ribonucleic acid expression of uncoupling proteins in mice

Apelin, the endogenous ligand of the APJ receptor, has been identified in a variety of tissues, including stomach, heart, skeletal muscle, and white adipose tissue. We sought to clarify the effects of apelin on body adiposity and the expression of uncoupling proteins (UCPs) in C57BL/6 mice. Treatment with ip apelin at a dose of 0.1 mumol/kg.d for 14 d decreased the weight of white adipose tissue and serum levels of insulin and triglycerides, compared with controls, without influencing food intake. Apelin treatment also decreased body adiposity and serum levels of insulin and triglycerides in obese mice fed a high-fat diet. Apelin increased the serum adiponectin level and decreased that of leptin. Additionally, apelin treatment increased mRNA expression of UCP1, a marker of peripheral energy expenditure, in brown adipose tissue (BAT) and of UCP3, a regulator of fatty acid export, in skeletal muscle. In addition, immunoblot bands and relative densities of UCP1 content in BAT were also higher in the apelin group than controls. Furthermore, apelin treatment increased body temperature and O(2) consumption and decreased the respiratory quotient. In conclusion, apelin appears to regulate adiposity and lipid metabolism in both lean and obese mice. In addition, apelin regulates insulin resistance by influencing the circulating adiponectin level, the expression of BAT UCP1, and energy expenditure in mice.     

Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes

The pituitary hormone prolactin (PRL) exerts pleiotropic effects, which are mediated by a membrane receptor (PRLR) present in numerous cell types including adipocytes. Brown adipose tissue (BAT) expresses uncoupling proteins (UCPs), involved in thermogenesis, but also secretes leptin, a key hormone involved in the control of body weight. To investigate PRL effects on BAT, we used the T37i brown adipose cell line, and demonstrated that PRLRs are expressed as a function of cell differentiation. Addition of PRL leads to activation of the JAK/STAT and MAP kinase signaling pathways, demonstrating that PRLRs are functional in these cells. Basal and catecholamine-induced UCP1 expression were not affected by PRL. However, PRL combined with insulin significantly increases leptin expression and release, indicating that PRL potentiates the stimulatory effect of insulin as revealed by the recruitment of insulin receptor substrates and the activation of phosphatidylinositol 3-kinase. To explore the in vivo physiological relevance of PRL action in BAT, we showed that leptin content was significantly increased in BAT of PRLR-null mice compared with wild-type mice, highlighting the involvement of PRL in the leptin secretion process. This study provides the first evidence for a functional link between PRL and energy balance via a cross-talk between insulin and PRL signaling pathways in brown adipocytes.     

Norepinephrine is required for leptin effects on gene expression in brown and white adipose tissue.

Exogenous leptin enhances energy utilization in ob/ob mice by binding its hypothalamic receptor and selectively increasing peripheral fat oxidation. Leptin also increases uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT), but the neurotransmitter that mediates this effect has not been established. The present experiments sought to determine whether leptin regulates UCP1 expression in BAT and its own expression in white adipose tissue (WAT) through the long or short forms of leptin receptor and modulation of norepinephrine release. Mice lacking dopamine beta-hydroxylase (Dbh-/-), the enzyme responsible for synthesizing norepinephrine and epinephrine from dopamine, were treated with leptin (20 microg/g body weight/day) for 3 days before they were euthanized. UCP1 messenger RNA (mRNA) and protein expression were 5-fold higher in BAT from control (Dbh+/-) compared with Dbh-/- mice. Leptin produced a 4-fold increase in UCP1 mRNA levels in Dbh+/- mice but had no effect on UCP1 expression in Dbh-/-. The beta3-adrenergic agonist, CL-316,243 increased UCP1 expression and established that BAT from both groups of mice was capable of responding to beta-adrenergic stimulation. Similarly, exogenous leptin reduced leptin mRNA in WAT from Dbh+/- but not Dbh-/- mice. In separate experiments, leptin produced comparable reductions in food intake in both Dbh+/- and Dbh-/- mice, illustrating that norepinephrine is not required for leptin's effect on food intake. Lastly, db/db mice lacking the long form of the leptin receptor failed to increase UCP1 mRNA in response to exogenous leptin but increased UCP1 mRNA in response to CL-316,243. These studies establish that norepinephrine is required for leptin to regulate its own expression in WAT and UCP1 expression in BAT and indicate that these effects are likely mediated through the centrally expressed long form of the leptin receptor.     

Uncoupling proteins: gender-dependence and their relation to body weight control

The members of the uncoupling protein family have different purported functions, which can be either directly or indirectly related to the control of body weight. In this sense, a great part of the studies carried out on this topic have been made using male subjects, although different works with male and female subjects have shown important sex-associated differences in the regulation of these proteins; for instance, sex differences have been shown in the cold-, diet- and overweight-induced expression of brown adipose tissue UCP1 and also in the correlation of muscle UCP3 with overweight. In these kinds of studies, models of obesity such as the cafeteria diet feeding and postcafeteria have been very useful. Moreover, sex hormones have been shown to modulate UCP1 expression in brown adipocytes in vitro. All of these sex-dependent differences, as well as sex differences in body weight gain under a hypercaloric diet, could be related to the different respective biological functions of male and female subjects and taking into account the gender effect in future studies on obesity could be of interest.     

Leptin is required for uncoupling protein-1-independent thermogenesis during cold stress

We investigated the role of leptin in regulating energy metabolism through induction of uncoupling protein (UCP)-1-based brown fat thermogenesis by comparing phenotypes of energy balance in ob/ob and double-mutant ob/ob.Ucp1(-/-) mice. Measurements of adiposity and lean body mass (nuclear magnetic resonance), energy expenditure (indirect calorimetry), body weight, food intake, and core body temperature were determined in the two mutant stocks of 3-month-old mice maintained at an initial ambient temperature of 28 C for 21 d and then at 21 C for 16 d, and finally with leptin administration for 8 d at 21 C. No phenotypic differences between ob/ob and ob/ob.Ucp1(-/-) mice were detected, suggesting that UCP1-based thermogenesis is not essential for the regulation of adiposity in ob/ob mice at temperatures between 21 and 28 C. Although both Ucp1(-/-) and ob/ob mice can survive in extreme cold at 4 C, provided they are adapted to the cold by gradually lowering ambient temperature, ob/ob.Ucp1(-/-) mice could not adapt and survive at temperatures lower than 12 C unless they were administered leptin. As the ambient temperature was reduced from 20 to 16 C, ob/ob.Ucp1(-/-) mice treated with leptin have elevated levels of circulating T(3) that correlate with elevated sarcoendoplasmic reticulum Ca(2+) ATPase 2a mRNA levels in gastrocnemius muscle. Furthermore, ob/ob.Ucp1(-/-) mice, treated with T(3), were able to maintain body temperature and stimulate sarcoendoplasmic reticulum Ca(2+) ATPase 2a expression when the ambient temperature was gradually reduced to 4 C. Thus, in the absence of UCP1, leptin-induced thermogenesis protects body temperature in part through its action on the thyroid hormone axis.     

Adiponectin reduces thermogenesis by inhibiting brown adipose tissue activation in mice

Aims/hypothesis

Adiponectin is an adipocyte-derived hormone that plays an important role in energy homeostasis. The main objective of this study was to investigate whether or not adiponectin regulates brown adipose tissue (BAT) activation and thermogenesis.

Methods

Core body temperatures (CBTs) of genetic mouse models were monitored at room temperature and during cold exposure. Cultured brown adipocytes and viral vector-mediated gene transduction were used to study the regulatory effects of adiponectin on Ucp1 gene expression and the underlying mechanisms.

Results

The CBTs of adiponectin knockout mice (Adipoq ^?/?) were significantly higher than those of wild type (WT) mice both at room temperature and during the cold (4°C) challenge. Conversely, reconstitution of adiponectin in Adipoq ^?/? mice significantly blunted β adrenergic receptor agonist-induced thermogenesis of interscapular BAT. After 10 days of intermittent cold exposure, Adipoq ^?/? mice exhibited higher UCP1 expression and more brown-like structure in inguinal fat than WT mice. Paradoxically, we found that the anti-thermogenic effect of adiponectin requires neither AdipoR1 nor AdipoR2, two well-known adiponectin receptors. In sharp contrast to the anti-thermogenic effects of adiponectin, AdipoR1 and especially AdipoR2 promote BAT activation. Mechanistically, adiponectin was found to inhibit Ucp1 gene expression by suppressing β₃-adrenergic receptor expression in brown adipocytes.

Conclusions/interpretation

This study demonstrates that adiponectin suppresses thermogenesis, which is likely to be a mechanism whereby adiponectin reduces energy expenditure.     

Hibernoma formation in transgenic mice and isolation of a brown adipocyte cell line expressing the uncoupling protein gene.

Transgenic mice were produced containing the adipocyte-specific regulatory region from the adipocyte P2 (aP2) gene linked to the simian virus 40 transforming genes. Most of the transgenic mice developed brown fat tumors (hibernomas) in their interscapular brown adipose tissue. Hibernoma formation was noticeable in some of the mice as early as 1 day after birth and most of the mice developed very large tumors by 1 month of age. All of the tumor tissue expressed the brown fat-specific uncoupling protein (UCP) gene as well as the aP2 gene. Several of the tumors have been used to establish cultured cell lines and at least one of these lines can be induced to differentiate into brown adipocytes. The cultured adipocytes express mRNA for UCP upon stimulation with N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate, norepinephrine, isoproterenol or D7114, a beta 3 adrenergic agonist. Thus, regulation of the key thermogenic gene UCP can now be studied in an established cell line.