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Sirtuins post-translationally modulate the function of many cellular proteins that undergo reversible acetylation-deacetylation cycles, affecting physiological responses that have implications for treating diseases of ageing. Potent small-molecule modulators of sirtuins have shown efficacy in preclinical models of metabolic, neurodegenerative and inflammatory diseases, and so hold promise for drug discovery efforts in multiple therapeutic areas. Here, we discuss current knowledge and data that strengthens sirtuins as a druggable set of enzymes for the treatment of age-associated diseases, including activation of SIRT1 in type 2 diabetes.

Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes1,2. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity3–9. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival10–14. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme—peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.

The unabated rise in the prevalence of obesity is a challenge for global health care systems. Efforts to reverse this trend by dietary or behavioral counseling have not been successful, which has stimulated efforts to find a role for pharmacotherapy. Currently only a small number of antiobesity drugs are approved for long-term use and only a few compounds are in clinical development. Despite recent progress in the understanding of the regulation of energy balance, drug discovery has been less productive than expected. In the present review, the clinically available antiobesity agents are discussed. Examples of drug candidates that are currently in development are given and the possible future range of antiobesity agents is illustrated by the targets being addressed in drug discovery. Finally, the efficacy of antiobesity agents and their value in the treatment of obesity are assessed in comparison with other therapeutic approaches, such as surgery and changes in lifestyle.

SummaryThe neural pathways through which central serotonergic systems regulate food intake and body weight remain to be fully elucidated. We report that serotonin, via action at serotonin1B receptors (5-HT1BRs), modulates the endogenous release of both agonists and antagonists of the melanocortin receptors, which are a core component of the central circuitry controlling body weight homeostasis. We also show that serotonin-induced hypophagia requires downstream activation of melanocortin 4, but not melanocortin 3, receptors. These results identify a primary mechanism underlying the serotonergic regulation of energy balance and provide an example of a centrally derived signal that reciprocally regulates melanocortin receptor agonists and antagonists in a similar manner to peripheral adiposity signals.

Activation of melanocortin-4-receptors (MC4Rs) reduces body fat stores by decreasing food intake and increasing energy expenditure. MC4Rs are expressed in multiple CNS sites, any number of which could mediate these effects. To identify the functionally relevant sites of MC4R expression, we generated a loxP-modified, null Mc4r allele (loxTB Mc4r) that can be reactivated by Cre-recombinase. Mice homozygous for the loxTB Mc4r allele do not express MC4Rs and are markedly obese. Restoration of MC4R expression in the paraventricular hypothalamus (PVH) and a subpopulation of amygdala neurons, using Sim1-Cre transgenic mice, prevented 60% of the obesity. Of note, increased food intake, typical of Mc4r null mice, was completely rescued while reduced energy expenditure was unaffected. These findings demonstrate that MC4Rs in the PVH and/or the amygdala control food intake but that MC4Rs elsewhere control energy expenditure. Disassociation of food intake and energy expenditure reveals unexpected divergence in melanocortin pathways controlling energy balance.

Previous reports indicate that the expression and/or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulin-responsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulin-resistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39–50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3′ kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34–57%, and association of p85α with both IRS proteins was reduced by 39–52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and/or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.

Protein-tyrosine phosphatase 1B (PTP-1B) is a major protein-tyrosine phosphatase that has been implicated in the regulation of insulin action, as well as in other signal transduction pathways. To investigate the role of PTP-1B in vivo, we generated homozygotic PTP-1B-null mice by targeted gene disruption. PTP-1B-deficient mice have remarkably low adiposity and are protected from diet-induced obesity. Decreased adiposity is due to a marked reduction in fat cell mass without a decrease in adipocyte number. Leanness in PTP-1B-deficient mice is accompanied by increased basal metabolic rate and total energy expenditure, without marked alteration of uncoupling protein mRNA expression. In addition, insulin-stimulated whole-body glucose disposal is enhanced significantly in PTP-1B-deficient animals, as shown by hyperinsulinemic-euglycemic clamp studies. Remarkably, increased insulin sensitivity in PTP-1B-deficient mice is tissue specific, as insulin-stimulated glucose uptake is elevated in skeletal muscle, whereas adipose tissue is unaffected. Our results identify PTP-1B as a major regulator of energy balance, insulin sensitivity, and body fat stores in vivo.

The physiological significance of changes in uncoupling protein-2 (UCP-2) gene expression is controversial. In this study we investigated the biochemical and functional correlates of UCP-2 gene expression in sc abdominal adipose tissue in humans in vivo. UCP-2 messenger ribonucleic acid expression was quantified by nuclease protection in adipose tissue from lean and obese humans in both the fasting and postprandial states. Plasma fatty acids, insulin, and leptin were all determined in paired samples from the superficial epigastric vein and radial artery, and local production rates were calculated from 133Xe washout. In the fasting state UCP-2 expression correlated inversely with body mass index (r = -0.45; P = 0.026), percent body fat (r = -0.41; P = 0.05), plasma insulin (r = -0.47; P = 0.02), epigastric venous fatty acids (r = -0.45; P = 0.04), and leptin (r = -0.50; P = 0.018). UCP-2 expression remained inversely related with plasma leptin after controlling for percent body (r = -0.45; P = 0.038). At 2 or 4 h postprandially, there were no significant relationships between UCP-2 expression and biochemical parameters. In conclusion, 1) UCP-2 messenger ribonucleic acid expression in sc adipose tissue is inversely related to adiposity and independently linked to local plasma leptin levels; and 2) UCP-2 expression is not acutely regulated by food intake, insulin, or fatty acids. Reduced UCP-2 expression may be a maladaptive response to sustained energy surplus and could contribute to the pathogenesis and maintenance of obesity.

Uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily. Based upon its high homology with UCP1 and its restricted tissue distribution to skeletal muscle and brown adipose tissue, UCP3 has been suggested to play important roles in regulating energy expenditure, body weight, and thermoregulation. Other postulated roles for UCP3 include regulation of fatty acid metabolism, adaptive responses to acute exercise and starvation, and prevention of reactive oxygen species (ROS) formation. To address these questions, we have generated mice lacking UCP3 (UCP3 knockout (KO) mice). Here, we provide evidence that skeletal muscle mitochondria lacking UCP3 are more coupled (i.e. increased state 3/state 4 ratio), indicating that UCP3 has uncoupling activity. In addition, production of ROS is increased in mitochondria lacking UCP3. This study demonstrates that UCP3 has uncoupling activity and that its absence may lead to increased production of ROS. Despite these effects on mitochondrial function, UCP3 does not seem to be required for body weight regulation, exercise tolerance, fatty acid oxidation, or cold-induced thermogenesis. The absence of such phenotypes in UCP3 KO mice could not be attributed to up-regulation of other UCP mRNAs. However, alternative compensatory mechanisms cannot be excluded. The consequence of increased mitochondrial coupling in UCP3 KO mice on metabolism and the possible role of yet unidentified compensatory mechanisms, remains to be determined.

Mitochondria use energy derived from fuel combustion to create a proton electrochemical gradient across the mitochondrial inner membrane. This intermediate form of energy is then used by ATP synthase to synthesize ATP. Uncoupling protein-1 (UCP1) is a brown fat-specific mitochondrial inner membrane protein with proton transport activity. UCP1 catalyzes a highly regulated proton leak, converting energy stored within the mitochondrial proton electrochemical potential gradient to heat. This uncouples fuel oxidation from conversion of ADP to ATP. In rodents, UCP1 activity and brown fat contribute importantly to whole-body energy expenditure. Recently, two additional mitochondrial carriers with high similarity to UCP1 were molecularly cloned. In contrast to UCP1, UCP2 is expressed widely, and UCP3 is expressed preferentially in skeletal muscle. Biochemical studies indicate that UCP2 and UCP3, like UCP1, have uncoupling activity. While UCP1 is known to play an important role in regulating heat production during cold exposure, the biological functions of UCP2 and UCP3 are unknown. Possible functions include 1) control of adaptive thermogenesis in response to cold exposure and diet, 2) control of reactive oxygen species production by mitochondria, 3) regulation of ATP synthesis, and 4) regulation of fatty acid oxidation. This article will survey present knowledge regarding UCP1, UCP2, and UCP3, and review proposed functions for the two new uncoupling proteins.

OBJECTIVE: Examination of the pattern of expression of peroxisome proliferator-activated receptor (PPAR) isoforms alpha and gamma in a model of obesity. DESIGN: Examination of adipose tissue and primary adipocyte cultures from lean and obese Zucker rats at different ages (28 days and 12 weeks). METHODS: mRNA levels were measured by RNase protection assay.RESULTS: The highest levels of PPARalpha and gamma mRNA were present in brown adipose tissue (BAT), followed by liver and white adipose tissue (WAT) for the alpha and gamma subtypes, respectively, at both ages examined. PPARalpha was expressed 100-fold higher in BAT compared with WAT, and PPARgamma mRNA levels were 2-fold higher in the WAT of obese compared with lean rats. PPARalpha and gamma expression was minimal in m. soleus, although higher levels of PPARgamma were found in the diaphragm. In marked contrast to the findings in vivo, virtually no PPARalpha mRNA could be detected in BAT cultures differentiated in vitro. CONCLUSION: PPARalpha and gamma are most highly expressed in BAT in vivo. However, PPARalpha is undetectable in brown adipose cells in vitro, suggesting that the expression of this receptor is induced by some external stimuli. In addition, the expression of PPARgamma was increased in WAT from young obese animals, compatible with an early adaptive phenomenon. Finally, the presence of PPARgamma mRNA is detectable only in particular muscles, such as the diaphragm, suggesting the possibility of an influence of fiber type on its expression, although exercise did not influence the expression of PPARgamma in other skeletal muscles.

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.

The recently discovered uncoupling protein 3 (UCP3) is highly homologous to the mitochondrialinner membrane protein UCP1, which generates heat by uncoupling the respiratory chainfrom oxidative phosphorylation. The thermogenic function of UCP1 protects against cold andregulates the energy balance in rodents. We review in vitro studies investigating the uncouplingactivity of UCP3 and in vivo studies, which address UCP3 gene expression in brown adiposetissue and skeletal muscle under various metabolic conditions. The data presented are, for themost, consistent with an uncoupling role for UCP3 in regulatory thermogenesis. We alsodiscuss mediators of UCP3 regulation and propose a potential role for intracellular fatty acidsin the mechanism of UCP3 modulation. Finally, we hypothesize a role for UCP3 in themetabolic adaptation of the mitochondria to the degradation of fatty acids.

The uncoupling protein-1 (UCP1), cloned in 1985 (1– 4) and called UCP until 1997, is an inner mitochondrial membrane protein (5) expressed exclusively in the brown adipocyte (6–8). It dissipates the mitochondrial proton (H) gradient generated by the respiratory chain, producing heat instead of ATP (8, 9). In rodents the brown adipose tissue (BAT) contributes to both the maintenance of body temperature in a cold environment through nonshivering thermogenesis and the control of body weight through the regulatory (or facultative) part of diet-induced thermogenesis (10, 11). It has been shown that during cold acclimation the capacity of brown adipocytes to produce heat is determined by the UCP1 content of their mitochondria (12–14). Several observations led to the hypothesis that there could exist uncoupling proteins in tissues other than BAT. First, adult humans, who possess very little active BAT (15, 16), produce heat in their skeletal muscle in response to glucose or catecholamine administration (17–20). Secondly, mitochondrial H leaks have been observed in tissues devoid of UCP1 (21–23). They may account for up to 50% of the oxygen consumption of some tissues (24, 25), and up to 30% of whole body metabolic rate in the rat (24–26). It was suggested that the H leak was related to resting metabolic rate (27, 28). Supporting the previous observations, several groups independently cloned novel UCPs, i.e. UCP2 (29–31) and UCP3 (31, 32). UCP2 is expressed in most tissues studied in humans and rodents, and UCP3 mainly in skeletal muscle in humans, and BAT and skeletal muscle in rodents (31). UCP1, which was thought to be a protein unique to BAT, is in fact a member of an emerging family of uncoupling proteins expressed in humans and animals, and even in plants (33, 34).

Continuous (4 days) intracerebroventricular leptin infusion (12 microg/day) was performed in lean rats, and its hormonometabolic effects were determined. Intracerebroventricular leptin administration did not result in leakage of the hormone into the peripheral circulation. Thus, its effects were elicited by its presence within the central nervous system. Intracerebroventricular leptin infusion produced marked decreases in food intake and body weight gain relative to vehicle-infused fed ad libitum rats. Because decreases in food intake alter hormonometabolic homeostasis, additional control rats pair-fed to the amount of food consumed by leptin-infused ones were included in the study. Intracerebroventricular leptin-infused and vehicle-infused pair-fed rats were characterized, relative to vehicle-infused ad libitum-fed animals, by decreases in body weight and insulinemia and by increases in insulin-stimulated overall glucose utilization and muscle and brown adipose tissue glucose utilization index. Brown adipose tissue uncoupling protein (UCP)1, UCP2, and UCP3 mRNA levels were markedly decreased in pair-fed animals relative to those of fed ad libitum control animals, as were liver and white adipose tissue UCP2 and muscle UCP3 mRNA levels. In marked contrast, intracerebroventricular leptin administration was accompanied by the maintenance of high UCP1, UCP2, and UCP3 expression in all these tissues. Thus, despite analogies between leptin's effects and those of pair-feeding with regard to glucose handling, their respective underlying mechanisms differ. While leptin maintains or favors energy-dissipating mechanisms (UCP1, UCP2, and UCP3), the latter are markedly depressed in pair-fed rats. This effect of leptin may prevent subsequent excessive storage processes, thereby maintaining normal body homeostasis.

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.

Endurance exercise training has been shown to decrease diet-induced thermogenesis (DIT) in rats and humans. In rodents, most thermogenesis is thought to occur in brown adipose tissue via activation of the uncoupling protein-1 (UCP1) and in skeletal muscle. Since the level of UCP1 mRNA in rat BAT was reported to be unmodified by exercise training, the newly described uncoupling proteins UCP2 and UCP3 could be responsible for the decreased DIT in trained rats. UCP3 mRNA levels in endurance-trained rats were found to be reduced by 76% and 59% in tibialis anterior and soleus muscles, respectively. UCP2 mRNA levels were also decreased in tibialis anterior and in heart by 54% and 41%, respectively. Neither white adipose tissue UCP2 nor brown adipose tissue UCP1, UCP2, and UCP3 mRNA levels were modified. The results of this study show that a need for a higher metabolic efficiency is associated with decreased mRNA expression of the uncoupling proteins in skeletal and heart muscles, which would decrease energy dissipation in these tissues. The down-regulation of UCP3 and UCP2 expressions might also contribute to the rapid weight gain known to occur when exercise training ceased.

A new member of the uncoupling protein (UCP) family called UCP3 has recently been cloned and shown to be highly expressed in skeletal muscle of rodents and humans. In the present study, UCP3 was overexpressed in C2C12 myoblasts where it acts as an uncoupling protein. Changes in UCP3 mRNA expression were examined in rodent muscles under conditions known to modulate thermogenesis in brown adipose tissue. In skeletal muscle, UCP3 expression did not change in response to 48 h of cold exposure (6 degrees C), whereas it was decreased by 81% or increased 5.6-fold by 1 week of 50% food restriction or fasting, respectively. It was also decreased by 36% in soleus muscle of obese (fa/fa) as compared with lean Zucker rats. The unexpected rise of UCP3 mRNA level induced by fasting did not change in vitro muscle basal heat production rate but decreased by 31% the capacity to produce heat in response to the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone. This decrease may reflect underlying uncoupling by UCP3. Up-regulation of UCP3 mRNA after a 24-h fast was still observed in mice exposed at thermoneutrality. These results show that the increase in UCP3 expression induced by fasting is associated with the maintenance of thermogenesis measured in muscle in vitro and is not modulated by environmental temperature. The notion that UCP3 expression is modulated by food intake is of importance to better understand the pathophysiology of obesity in humans.

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

Brown adipose tissue (BAT) has been recognized for more than 20 years to play a key role in cold-induced non-shivering thermogenesis (CIT, NST), and body weight homeostasis in animals. BAT is a flexible tissue that can be recruited by stimuli (including small molecules in animals), and atrophies in the absence of a stimulus. In fact, the contribution of BAT (and UCP1) to resting metabolic rate and healthy body weight homeostasis in animals (rodents) is now well established. Many investigations have shown that resistance to obesity and associated disorders in various rodent models is due to increased BAT mass and the number of brown adipocytes or UCP1 expression in various depots. The recent discovery of active BAT in adult humans has rekindled the notion that BAT is a therapeutic target for combating obesity-related metabolic disorders. In this review, we highlight investigations performed in rodents that support the contention that activation of BAT formation and/or function in obese individuals is therapeutically powerful. We also propose that enhancement of brown adipocyte functions in white adipose tissue (WAT) will also regulate energy balance as well as reduce insulin resistance in obesity-associated inflammation in WAT.

The sterol regulatory element-binding protein (SREBP) transcription factor family is a critical regulator of lipid and sterol homeostasis in eukaryotes. In mammals, SREBPs are highly active in the fed state to promote the expression of lipogenic and cholesterogenic genes and facilitate fat storage. During fasting, SREBP-dependent lipid/cholesterol synthesis is rapidly diminished in the mouse liver; however, the mechanism has remained incompletely understood. Moreover, the evolutionary conservation of fasting regulation of SREBP-dependent programs of gene expression and control of lipid homeostasis has been unclear. We demonstrate here a conserved role for orthologs of the NAD+-dependent deacetylase SIRT1 in metazoans in down-regulation of SREBP orthologs during fasting, resulting in inhibition of lipid synthesis and fat storage. Our data reveal that SIRT1 can directly deacetylate SREBP, and modulation of SIRT1 activity results in changes in SREBP ubiquitination, protein stability, and target gene expression. In addition, chemical activators of SIRT1 inhibit SREBP target gene expression in vitro and in vivo, correlating with decreased hepatic lipid and cholesterol levels and attenuated liver steatosis in diet-induced and genetically obese mice. We conclude that SIRT1 orthologs play a critical role in controlling SREBP-dependent gene regulation governing lipid/cholesterol homeostasis in metazoans in response to fasting cues. These findings may have important biomedical implications for the treatment of metabolic disorders associated with aberrant lipid/cholesterol homeostasis, including metabolic syndrome and atherosclerosis.

Background Calorie restriction (CR) produces a number of health benefits and ameliorates diseases of aging such as type 2 diabetes. The components of the pathways downstream of CR may provide intervention points for developing therapeutics for treating diseases of aging. The NAD+-dependent protein deacetylase SIRT1 has been implicated as one of the key downstream regulators of CR in yeast, rodents, and humans. Small molecule activators of SIRT1 have been identified that exhibit efficacy in animal models of diseases typically associated with aging including type 2 diabetes. To identify molecular processes induced in the liver of mice treated with two structurally distinct SIRT1 activators, SIRT501 (formulated resveratrol) and SRT1720, for three days, we utilized a systems biology approach and applied Causal Network Modeling (CNM) on gene expression data to elucidate downstream effects of SIRT1 activation. Results Here we demonstrate that SIRT1 activators recapitulate many of the molecular events downstream of CR in vivo, such as enhancing mitochondrial biogenesis, improving metabolic signaling pathways, and blunting pro-inflammatory pathways in mice fed a high fat, high calorie diet. Conclusion CNM of gene expression data from mice treated with SRT501 or SRT1720 in combination with supporting in vitro and in vivo data demonstrates that SRT501 and SRT1720 produce a signaling profile that mirrors CR, improves glucose and insulin homeostasis, and acts via SIRT1 activation in vivo. Taken together these results are encouraging regarding the use of small molecule activators of SIRT1 for therapeutic intervention into type 2 diabetes, a strategy which is currently being investigated in multiple clinical trials.

The prevalence of Type 2 diabetes is increasing at an alarming rate in most parts of the world. Effective therapeutic drugs are urgently needed, not only to control the disease but also to prevent or delay its progression. Therapies that target the underlying pathogenesis could, in theory, hold such potential. Recent evidence strongly suggests that impaired mitochondrial function is part of the underlying pathogenesis of insulin resistance and Type 2 diabetes. Peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1alpha) is a transcription co-activator that plays a key role in regulating mitochondrial biogenesis and energy metabolism in multiple tissues. Thus, improvement and restoration of mitochondrial function and oxidative capacity through activation of PGC-1alpha could provide new treatments for metabolic diseases. A diverse array of proteins has been shown to regulate PGC-1alpha transcription and/or activity, some of which represent promising targets for pharmaceutical intervention.

An enhanced metabolic efficiency for accelerating the recovery of fat mass (or catch-up fat) is a characteristic feature of body weight regulation after weight loss or growth retardation and is the outcome of an "adipose-specific" suppression of thermogenesis, i.e., a feedback control system in which signals from the depleted adipose tissue fat stores exert a suppressive effect on thermogenesis. Using a previously described rat model of semistarvation-refeeding in which catch-up fat results from suppressed thermogenesis per se, we report here that the gene expression of stearoyl-coenzyme A desaturase 1 (SCD1) is elevated in skeletal muscle after 2 wk of semistarvation and remains elevated in parallel to the phase of suppressed thermogenesis favoring catch-up fat during refeeding. These elevations in the SCD1 transcript are skeletal muscle specific and are associated with elevations in microsomal Delta9 desaturase enzyme activity, in the Delta9 desaturation index, and in the relative content of SCD1-derived monounsaturates in several lipid fractions extracted from skeletal muscle. An elevated skeletal muscle SCD1, by desaturating the products of de novo lipogenesis and diverting them away from mitochondrial oxidation, would inhibit substrate cycling between de novo lipogenesis and lipid oxidation, thereby leading to a state of suppressed thermogenesis that regulates the body's fat stores.

Mice lacking β-adrenoceptors, which mediate the thermogenic effects of norepinephrine and epinephrine, show diminished thermogenesis and high susceptibility to obesity, whereas mice lacking stearoyl-CoA desaturase 1 (SCD1), which catalyzes the synthesis of monounsaturated fatty acids, show enhanced thermogenesis and high resistance to obesity. In testing whether β-adrenergic control of thermogenesis might be mediated via repression of the SCD1 gene, we found that in mice lacking β-adrenoceptors, the gene expression of SCD1 is elevated in liver, skeletal muscle and white adipose tissue. In none of these tissues/organs, however, could a link be found between increased sympathetic nervous system activity and diminished SCD1 gene expression when thermogenesis is increased in response to diet or cold, nor is the SCD1 transcript repressed by the administration of epinephrine. Taken together, these studies suggest that the elevated SCD1 transcript in tissues of mice lacking β-adrenoceptors is not a direct effect of blunted β-adrenergic signalling, and that β-adrenergic control of SCD1 repression is unlikely to be a primary effector mechanism in sympathoadrenal regulation of thermogenesis. Whether approaches that target both SCD1 and molecular effectors of thermogenesis under β-adrenergic control might be more effective than targeting SCD1 alone are potential avenues for future research in obesity management.

The prevalence of obesity is increasing rapidly in most parts of the world and effective therapeutic drugs are urgently needed. The discovery of leptin in 1994 initiated a new understanding of adipose tissue function, and adipose tissue is now known to not only store and release fatty acids, but also to produce a wealth of factors that have an impact on the regulation of body weight and blood glucose homeostasis. Also, adipocytes express proteins that engage signalling pathways playing important roles in fuel substrate and energy metabolism. These proteins constitute a diverse array of adipose target candidates for the development of drugs to treat obesity. Some of these potential targets have been validated and are now in drug development stages, providing hope that the current obesity epidemic can be addressed by effective drug treatments in the near future.

6 páginas, 4 figuras, 1 tabla.-- et al., Resveratrol (3,5,4'-trihydroxystilbene) extends the lifespan of diverse species including Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster. In these organisms, lifespan extension is dependent on Sir2, a conserved deacetylase proposed to underlie the beneficial effects of caloric restriction. Here we show that resveratrol shifts the physiology of middle-aged mice on a high-calorie diet towards that of mice on a standard diet and significantly increases their survival. Resveratrol produces changes associated with longer lifespan, including increased insulin sensitivity, reduced insulin-like growth factor-1 (IGF-I) levels, increased AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor- coactivator 1(PGC-1) activity, increased mitochondrial number, and improved motor function. Parametric analysis of gene set enrichment revealed that resveratrol opposed the effects of the high-calorie diet in 144 out of 153 significantly altered pathways. These data show that improving general health in mammals using small molecules is an attainable goal, and point to new approaches for treating obesity-related disorders and diseases of ageing., This research was supported (in part) by the Intramural Research Program of the NIH, NIA, a Spanish MCyT grant to P.N., NIH grants to D.A.S., and by the support of P. F. Glenn and The Paul F. Glenn Laboratories for the Biological Mechanisms of Aging.

A critical defect in type 2 diabetes is impaired insulin-stimulated glucose transport and metabolism in muscle and adipocytes. To understand the metabolic adaptations this elicits, we generated mice with targeted disruption of the GLUT4 glucose transporter in both adipocytes and muscle (AMG4KO). In contrast to total body GLUT4-null mice, AMG4KO mice exhibit normal growth, development, adipose mass, and longevity. They develop fasting hyperglycemia and glucose intolerance and are at risk for greater insulin resistance than mice lacking GLUT4 in only one tissue. Hyperinsulinemic-euglycemic clamp studies showed a 75% decrease in glucose infusion rate and markedly reduced 2-deoxyglucose uptake into skeletal muscle (85–90%) and white adipose tissue (65%). However, AMG4KO mice adapt by preferentially utilizing lipid fuels, as evidenced by a lower respiratory quotient and increased clearance of lipids from serum after oral lipid gavage. While insulin action on hepatic glucose production and gluconeogenic enzymes is impaired, hepatic glucokinase expression, incorporation of 14C-glucose into lipids, and hepatic VLDL-triglyceride release are increased. The lipogenic activity may be mediated by increased hepatic expression of SREBP-1c and acetyl-CoA carboxylase. Thus, inter-tissue communication results in adaptations to impaired glucose transport in muscle and adipocytes that involve increased hepatic glucose uptake and lipid synthesis, while muscle adapts by preferentially utilizing lipid fuels. Genetic determinants limiting this “metabolic flexibility” may contribute to insulin resistance and type 2 diabetes in humans.

The biochemical basis for the regulation of fibre-type determination in skeletal muscle is not well understood. In addition to the expression of particular myofibrillar proteins, type I (slow-twitch) fibres are much higher in mitochondrial content and are more dependent on oxidative metabolism than type II (fast-twitch) fibres. We have previously identified a transcriptional co-activator, peroxisome-proliferator-activated receptor-gamma co-activator-1 (PGC-1 alpha), which is expressed in several tissues including brown fat and skeletal muscle, and that activates mitochondrial biogenesis and oxidative metabolism. We show here that PGC-1 alpha is expressed preferentially in muscle enriched in type I fibres. When PGC-1 alpha is expressed at physiological levels in transgenic mice driven by a muscle creatine kinase (MCK) promoter, a fibre type conversion is observed: muscles normally rich in type II fibres are redder and activate genes of mitochondrial oxidative metabolism. Notably, putative type II muscles from PGC-1 alpha transgenic mice also express proteins characteristic of type I fibres, such as troponin I (slow) and myoglobin, and show a much greater resistance to electrically stimulated fatigue. Using fibre-type-specific promoters, we show in cultured muscle cells that PGC-1 alpha activates transcription in cooperation with Mef2 proteins and serves as a target for calcineurin signalling, which has been implicated in slow fibre gene expression. These data indicate that PGC-1 alpha is a principal factor regulating muscle fibre type determination.

Among other actions, leptin has been suggested to increase energy expenditure and to modulate the menstrual cycle. In fact, the main effect of leptin seems to be modulating the sympathetic nervous system and gonadotropin-releasing hormone pulsatility. We investigated whether changes in the plasma steroid concentrations during the estrous cycle and after ovariectomy and steroid replacement can modulate plasma leptin levels, adipose tissue leptin mRNA expression, and some of the candidates for mediating energy expenditure (uncoupling proteins (UCP) 1, 2, and 3 mRNA) in white and brown adipose tissue. Rats in estrous cycle or ovariectomized rats with or without estradiol or progesterone replacement therapy for 18 days were studied. Plasma leptin, insulin, estradiol and progesterone were measured with radioimmunoassays. Leptin mRNA expression was measured in subcutaneous, periovarian and mesenteric white adipose tissue and in interscapular brown adipose tissue. Expression of UCP 1, 2, and 3 mRNA in periovarian white and brown adipose tissue was analyzed. Plasma leptin levels were significantly decreased in the estrous (1.1 +/- 0.4 ng/ml) compared with the pro-estrous (1.7 +/- 0.4 ng/ml, F = 3.0, p = 0.046) phase of cycle. UCP1 mRNA levels in brown adipose tissue were more elevated during pro-estrus than during metestrus (F = 3.17, p = 0.039). Gene expressions of leptin, UCP2 or UCP3 mRNA did not change significantly during the cycle. In ovariectomized rats, estradiol and/or progesterone treatment had no effect on plasma leptin levels. Gene expression analysis of leptin and UCP1, 2 and 3 in adipose tissue was not affected by steroid replacement. In conclusion, the estrous cycle appears to have a minor effect on modulation of leptin and uncoupling proteins. Only plasma leptin levels and expression of UCP1 mRNA are modestly elevated during the estrous cycle in the rat. Since estrogen and/or progesterone substitution in ovariectomized rats does not affect circulating leptin concentration or expression of leptin and UCPs in adipose tissue, it is unlikely that steroids play a major role in their regulation.

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5' and 3' ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being approximately 72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3',3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room's temperature to 12-14 degrees C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

The earliest defect in developing type 2 diabetes is insulin resistance, characterized by decreased glucose transport and metabolism in muscle and adipocytes. The glucose transporter GLUT4 mediates insulin-stimulated glucose uptake in adipocytes and muscle by rapidly moving from intracellular storage sites to the plasma membrane. In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression is decreased in adipose tissue but preserved in muscle. Because skeletal muscle is the main site of insulin-stimulated glucose uptake, the role of adipose tissue GLUT4 downregulation in the pathogenesis of insulin resistance and diabetes is unclear. To determine the role of adipose GLUT4 in glucose homeostasis, we used Cre/loxP DNA recombination to generate mice with adipose-selective reduction of GLUT4 (G4A-/-). Here we show that these mice have normal growth and adipose mass despite markedly impaired insulin-stimulated glucose uptake in adipocytes. Although GLUT4 expression is preserved in muscle, these mice develop insulin resistance in muscle and liver, manifested by decreased biological responses and impaired activation of phosphoinositide-3-OH kinase. G4A-/- mice develop glucose intolerance and hyperinsulinaemia. Thus, downregulation of GLUT4 and glucose transport selectively in adipose tissue can cause insulin resistance and thereby increase the risk of developing diabetes.

OBJECTIVE: To investigate whether the region of chromosome 11 (11q13) containing the genes UCP2 and UCP3 could be excluded for linkage with a variety of obesity-related phenotypes in humans. DESIGN: Exclusion mapping using a variance component approach in extended pedigrees. SUBJECTS: Four-hundred and fifty eight individuals (195 females, 263 males) distributed in 10 Mexican American families of probands randomly ascertained with respect to any disease state and who are participating in the San Antonio Family Heart Study. Ages range from 18 to 87 (mean age 35 y). MEASUREMENTS: Serum leptin levels, fat mass (FM), body mass index (BMI), and waist circumference. RESULTS: We were able to exclude the chromosomal region containing UCP2/UCP3 as having an effect on this set of obesity-related phenotypes at relative effect sizes of 10% or greater (P-values < 0.05). CONCLUSIONS: These results suggest that variation in these genes is unlikely to have a substantial effect on the expression of obesity-related phenotypes in the Mexican American population.

[reaction: see text] (1R,2S,3S,4R,5R)-4-Amino-5-(hydroxymethyl)cyclopentane-1,2,3-triol 1, prepared from D-glucose, inhibits beta-glucosidases from Caldocellum saccharolyticum (Ki = 1.8 x 10(-7) M) and from almonds (Ki = 3.4 x 10(-6) M). Inhibition is not influenced by N-ethylation (--15) but is strongly reduced upon N-acetylation (--12). Inversion of stereochemistry at C(5) (--14) has little effect on inhibition of beta-glucosidases. These experiments suggest that 1 acts as an analogue of a protonated beta-glucoside.

It has been claimed that factors favoring the development or maintenance of animal or human obesity may include increases in glucocorticoid production or hyperresponsiveness of the hypothalamic-pituitary-adrenal axis. In normal rats, glucocorticoids have been shown to be necessary for chronic intracerebroventricular infusion of neuropeptide Y to produce obesity and related abnormalities. Conversely, glucocorticoids inhibited the body weight-lowering effect of leptin. Such dual action of glucocorticoids may occur within the central nervous system, since both neuropeptide Y and leptin act within the hypothalamus. The aim of this study was to determine the effects of glucocorticoids (dexamethasone) given intracerebroventricularly to normal rats on body weight homeostasis and hypothalamic levels of neuropeptide Y and corticotropin-releasing hormone. Continuous central glucocorticoid infusion for 3 days resulted in marked sustained increases in food intake and body weight relative to saline-infused controls. The infusion abolished endogenous corticosterone output and produced hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia, three salient abnormalities of obesity syndromes. Central glucocorticoid infusion also produced a marked decrease in the expression of uncoupling protein (UCP)-1 and UCP-3 in brown adipose tissue and UCP-3 in muscle. Finally, chronic central glucocorticoid administration increased the hypothalamic levels of neuropeptide Y and decreased those of corticotropin-releasing hormone. When the same dose of glucocorticoids was administered peripherally, it resulted in decreases in food intake and body weight, in keeping with the decrease in hypothalamic neuropeptide Y levels. These results suggest that glucocorticoids induce an obesity syndrome in rodents by acting centrally and not peripherally.

The newly identified mitochondrial uncoupling proteins UCP2 and UCP3, by contrast with UCP1, are highly expressed in human beings, UCP2 being ubiquitous and UCP3 specific to skeletal muscle. Because of its tissue distribution, UCP3 might have an important role in wholebody energy homoeostasis in human beings. Variations in its degree of expression might contribute to the interindividual variability in resting energy expenditure and in the energybalance dysregulation found in obesity and type 2 diabetes. We investigated whether mRNA expression of UCP2 and UCP3 in human vastus lateralis muscle was correlated with variables related to fat or glucose metabolism and with resting energy expenditure in a mixed population of obese patients and obese patients with type 2 diabetes. Nine obese, 10 h fasted patients, including three with type 2 diabetes (untreated but dietetically controlled, fasting plasma glucose of 8·1 to 10·5 mmol/L, table), volunteered, with written consent, to participate, which was accepted by the local Human Investigation Committee. We measured lean body mass with bioelectrical impedance. Plasma glucose concentrations were determined with a Beckman Glucose Analyzer II (Beckman Instruments, CA, USA), and plasma free fatty acids and insulin concentrations with commercial kits (Wako Chemicals GmbH, Germany, and Abbott, IL). Glucose uptake was measured with a euglycaemic hyperinsulinaemic clamp, and glucose and lipid oxidation rates, as well as resting energy expenditure, were determined by indirect calorimetry. The concentrations of UCP2 and UCP3 mRNA in vastus lateralis biopsy fragments were measured by northern blot. The expression of UCP2 and UCP3 mRNA normalised to the respective concentrations of 18S rRNA were compared with 12 physical, biological, and metabolic variables and analysed (Simple Pearson ProductMoment correlations). The mRNA expression of UCP3 was positively and linearly correlated with circulating free fatty acids (r=0·83; p=0·005), whereas that of UCP2 was not (r=0·40). When adjustments were made for age, percentage of fat mass, body-mass index, lean body mass and bodyweight, the correlation between UCP3 mRNA and concentrations of free fatty acids was stronger (r=0·99; p 0·05) was seen between UCP2 or UCP3 mRNA expression and age, percentage fat mass, body-mass index, lean body mass, resting energy expenditure per kg lean body mass, bodyweight, fasting plasma glucose, insulin concentrations, insulin-induced glucose uptake, glucose oxidation, and lipid oxidation. Free fatty acids might control muscle UCP3 expression. A 10-day severe calorie restriction increases muscle UCP2 and UCP3 mRNA expression, and in rodents infusion of free fatty acids increases muscle UCP3 mRNA. Our data suggest a role for muscle UCP3 in the metabolic adaptations to increases in fatty-acid supply, and, therefore, the involvement of UCP3 in a compensatory mechanism linking obesity to increased muscle thermogenesis.

Levels of expression of the leptin receptor (OB-R) splice variants have been studied in human omental white and perirenal brown adipose tissues by reverse transcription-PCR. The level of mRNA expression of the full length form (OB-Rb) was approximately 15% of that of the sum of all splice variants in white or brown adipose tissue. In an attempt to quantify the gene expression of OB-Rb in human white adipose tissue, a quantitative competitive PCR technique was developed, using oligonucleotide primers designed for OB-Rb and an internal standard for a “MIMIC” competition strategy. The levels of expression of OB-Rb mRNA in the omental fat of lean and obese patients were compared and no difference could be observed between the two groups. The quantitative RT-PCR technique allows for a fast and accurate measurement of the expression of the OB-Rb mRNA in small tissue samples.

We sought a correlation between rat skeletal muscle triiodothyronine (T3)-mediated regulation of uncoupling protein-3 (UCP3) expression and mitochondrial activity. UCP3 mRNA expression increased strongly during the hypothyroid-hyperthyroid transition. The rank order of mitochondrial State 3 and State 4 respiration rates was hypothyroid

The control of uncoupling protein-2 (UCP2) mRNA expression in rat brown adipose tissue (BAT), heart and skeletal muscles was examined. Cold exposure (48 h) increased UCP2 mRNA in BAT, heart and soleus muscle by 2.4-, 4.3- and 2.6-fold, respectively. Fasting (48 h) had no effect on UCP2 mRNA expression neither in BAT nor in heart, but markedly increased it in skeletal muscles. While the upregulation of UCP2 mRNA in response to cold exposure is in line with a putative uncoupling role for this protein in thermoregulatory thermogenesis, the unexpected upregulation of UCP2 in skeletal muscles in response to fasting seems inconsistent with its role as an uncoupling protein involved in dietary regulation of thermogenesis.

The ob gene product is known to control food intake and energy expenditure. To determine whether thermogenic agents directly control ob gene expression, the effects of beta-adrenoceptor agonists on the level of the ob gene messenger RNA (mRNA) and on leptin secretion have been studied in mouse brown adipocytes differentiated in culture. These cells highly expressed the beta 3-adrenoceptor, the uncoupling protein, and the ob gene mRNAs. The ob gene was expressed in mouse brown adipocytes earlier than in mouse white adipocytes under the same culture conditions and to a similar level. The beta 3-, beta L-, and beta 2-adrenoceptor agonists BRL 37344, dobutamine, and terbutaline inhibited ob gene expression in mouse brown adipocytes differentiated in culture with EC50 values of 0.3, 1.0, and 85 nM, respectively. Leptin secretion by the cells under basal conditions was 78 +/- 10 pg/microgram DNA-4 h and was decreased by exposure to the beta-adrenoceptor agonists. The ob gene mRNA half-life was 9.4 h and was decreased to 2.4 h by 1 nM BRL 37344, indicating that the inhibitory effect of the beta 3-agonist might be due to destabilization of ob gene mRNA. (Bu)2cAMP (10-100 microM) and forskolin (20 microM) mimicked the effect of the beta-adrenoceptor agonists. FFA (150-800 microM) had only a small inhibitory effect on ob gene mRNA expression. The results suggest the existence in brown adipose tissue of a retroregulatory pathway by which leptin production in inhibited when the sympathetic nervous system is stimulated.

Targeted disruption of mouse beta3-adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the beta3-adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null (-/-) mice. In brown adipose tissue, beta3-adrenoceptor disruption induced a 66% decrease (P < 0.005) in beta1-adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in -/- mice, body fat accumulation was favored (+41%, P < 0.01), with a slight increase in food intake (+6%, NS). These effects were accentuated by high fat feeding: -/- mice showed increased total body fat (+56%, P < 0.025) and food intake (+12%, P < 0.01), and a decrease in the fat-free dry mass (-10%, P < 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in -/- and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed -/- mice suggests that the threshold of body fat content inducing leptin secretion is higher in -/- than in control mice. Taken together, these studies demonstrate that beta3-adrenoceptor disruption creates conditions which predispose to the development of obesity.

1. The possible existence of a beta 3-adrenoceptor in human brown and white adipose tissues was investigated by mRNA expression and binding studies. 2. The relative amounts of beta 1-, beta 2- and beta 3-adrenoceptor mRNA, as determined by total RNA Northern blot analysis in newborn brown adipose tissue, were 28, 63 and 9% respectively of the total beta-adrenoceptor mRNA. 3. The beta 1/beta 2-adrenoceptors of human brown adipose tissue plasma membranes were characterized using [3H]-CGP 12177 as a ligand. Their Kd and Bmax values were 1.9 nM and 156 fmol mg-1 of membrane proteins, respectively. The beta 3-adrenoceptor was characterized by use of the new specific radioligand [3H]-SB 206606. The binding of this ligand was stereospecifically displaced by the active R,R- or the inactive S,S-enantiomer of BRL 37344 up to a concentration of about 10 microM. The Kd and Bmax values of the brown adipose tissue membrane beta 3-adrenoceptors were 87 nM and 167 fmol mg-1 of proteins, respectively. A low affinity [3H]-CGP 12177 binding site population was also detected in these membranes. 4. In human omental white adipose tissue, no beta 3-adrenoceptor mRNA could be detected in total RNA Northern blots and the beta 1-and beta 2-adrenoceptor mRNAs represented 9 and 91%, respectively of the total beta-adrenoceptor mRNA, and no specific binding of [3H]-SB 206606 could be measured.

Nature Reviews Drug Discovery 7, 841–853 (2008) | doi:10.1038/nrd2665 The structure of suramin on page 844 is incorrect; the correct structure is shown below: The following paragraph and associated reference were mistakenly omitted from the discussion of the role of sirtuins in cancer on page 850: “In a recent effort to identify activators of p53 in a mammalian cell-based screen, a reporter construct was placed under the regulation of a p53 target promoter, and active compounds that increased reporter activity were selected from the library screen.

Brown adipose tissue (BAT) and skeletal muscle are important sites of nonshivering thermogenesis. The uncoupling protein-1 (UCP1) is the main effector of nonshivering thermogenesis in BAT and the recently described ubiquitous UCP2 [1]has been implicated in energy balance. In an attempt to better understand the biochemical events underlying nonshivering thermogenesis in muscle, we screened a human skeletal muscle cDNA library and isolated three clones: UCP2, UCP3L and UCP3S. The novel UCP3 was 57% and 73% identical to human UCP1 and UCP2, respectively, highly skeletal muscle-specific and its expression was unaffected by cold acclimation. This new member of the UCP family is a candidate protein for the modulation of the respiratory control in skeletal muscle.