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Aims/hypothesis: Obesity is associated with ageing and increased energy intake, while restriction of energy intake improves health and longevity in multiple organisms; the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) is implicated in this process. Pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical for energy balance regulation, and the level of SIRT1 protein decreases with age in the ARC. In the current study we tested whether conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevents age-associated weight gain and diet-induced obesity., Methods: We targeted Sirt1 cDNA sequence into the Rosa26 locus and generated conditional Sirt1 knock-in mice. These mice were crossed with mice harbouring either Pomc-Cre or Agrp-Cre and the metabolic variables, food intake, energy expenditure and sympathetic activity in adipose tissue of the resultant mice were analysed. We also used a hypothalamic cell line to investigate the molecular mechanism by which Sirt1 overexpression modulates leptin signalling., Results: Conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevented age-associated weight gain; overexpression in POMC neurons stimulated energy expenditure via increased sympathetic activity in adipose tissue, whereas overexpression in AgRP neurons suppressed food intake. SIRT1 improved leptin sensitivity in hypothalamic neurons in vitro and in vivo by downregulating protein-tyrosine phosphatase 1B, T cell protein-tyrosine phosphatase and suppressor of cytokine signalling 3. However, these phenotypes were absent in mice consuming a high-fat, high-sucrose diet due to decreases in ARC SIRT1 protein and hypothalamic NAD(+) levels., Conclusions/interpretation: ARC SIRT1 is a negative regulator of energy balance, and decline in ARC SIRT1 function contributes to disruption of energy homeostasis by ageing and diet-induced obesity.

Aims/hypothesis: The pancreas and hypothalamus are critical for maintaining nutrient and energy homeostasis, and combined disorders in these organs account for the onset of the metabolic syndrome. Activating transcription factor 3 (ATF3) is an adaptive response transcription factor. The physiological role of ATF3 in the pancreas has been controversial, and its role in the hypothalamus remains unknown. To elucidate the roles of ATF3 in these organs, we generated pancreas- and hypothalamus-specific Atf3 knockout (PHT-Atf3-KO) mice in this study., Methods: We crossed mice bearing floxed Atf3 alleles with Pdx1-cre mice, in which cre is specifically expressed in the pancreas and hypothalamus, and analysed metabolic variables, pancreatic morphology, food intake, energy expenditure and sympathetic activity in adipose tissue. We also used a hypothalamic cell line to investigate the molecular mechanism by which ATF3 regulates transcription of the gene encoding agouti-related protein (Agrp)., Results: Although PHT-Atf3-KO mice displayed better glucose tolerance, neither plasma glucagon nor insulin level was altered in these mice. However, these mice exhibited higher insulin sensitivity, which was accompanied by a leaner phenotype due to decreased food intake and increased energy expenditure. We also observed decreased hypothalamic Agrp expression in PHT-Atf3-KO mice. Importantly, an increase in ATF3 levels is induced by fasting or low glucose in the hypothalamus. We also showed that ATF3 interacts with forkhead box-containing protein, O subfamily 1 (FoxO1) on the Agrp promoter and activates Agrp transcription., Conclusions/interpretation: Our results suggest that ATF3 plays an important role in the control of glucose and energy metabolism by regulating Agrp.

Recent studies have revealed that insulin signaling in pancreatic β-cells and the hypothalamus is critical for maintaining nutrient and energy homeostasis, the failure of which are hallmarks of metabolic syndrome. We previously reported that forkhead transcription factor forkhead box-containing protein of the O subfamily (FoxO)1, a downstream effector of insulin signaling, plays important roles in β-cells and the hypothalamus when we investigated the roles of FoxO1 independently in the pancreas and hypothalamus. However, because metabolic syndrome is caused by the combined disorders in hypothalamus and pancreas, to elucidate the combined implications of FoxO1 in these organs, we generated constitutively active FoxO1 knockin (KI) mice with specific activation in both the hypothalamus and pancreas. The KI mice developed obesity, insulin resistance, glucose intolerance, and hypertriglyceridemia due to increased food intake, decreased energy expenditure, and impaired insulin secretion, which characterize metabolic syndrome. The KI mice also had increased hypothalamic Agouti-related protein and neuropeptide Y levels and decreased uncoupling protein 1 and peroxisome proliferator-activated receptor γ coactivator 1α levels in adipose tissue and skeletal muscle. Impaired insulin secretion was associated with decreased expression of pancreatic and duodenum homeobox 1 (Pdx1), muscyloaponeurotic fibrosarcoma oncogene homolog A (MafA), and neurogenic differentiation 1 (NeuroD) in islets, although β-cell mass was paradoxically increased in KI mice. Based on these results, we propose that uncontrolled FoxO1 activation in the hypothalamus and pancreas accounts for the development of obesity and glucose intolerance, hallmarks of metabolic syndrome.

Silent information regulator (SIR)2 is an nicotinamide adenine dinucleotide dependent deacetylase implicated in the regulation of life span in species as diverse as yeast, worms, and flies. Mammalian Sirt1 is the most closely related homolog of the SIR2 gene. Pharmacological activators of Sirt1 have been reported to increase the life span and improve the health of mice fed a high-fat diet and to reverse diabetes in rodents. Sirt1 links the energy availability status with cellular metabolism in peripheral organs including liver, pancreas, muscle, and white adipose tissue. Insulin and leptin signaling regulate food intake by controlling the expression of orexigenic and anorexigenic neuropeptides in the arcuate nucleus of the hypothalamus via Forkhead box O (Foxo)-1 and signal transducer and activator of transcription-3. Sirt1 has been reported to improve insulin sensitivity in vitro, but the role of hypothalamic Sirt1 in regulating feeding has not been addressed. We found that hypothalamic Sirt1 protein levels increase on feeding, and this induction is abrogated in diet-induced obese mice and db/db mice. We also demonstrate for the first time that Sirt1 protein turnover is regulated by the proteasome and ubiquitination in a hypothalamic cell line and in vivo by feeding, and this regulation is not seen in a pituitary cell line AtT20. Forced expression of wild-type Sirt1 in the mediobasal hypothalamus by adenovirus microinjection suppressed Foxo1-induced hyperphagia, a model for central insulin resistance. Moreover, Sirt1 suppressed Foxo1-dependent expression of the orexigenic neuropeptide Agouti-related peptide in vitro. We propose that on feeding, Sirt1 protein is stabilized in the hypothalamus, leading to decreased Foxo1-dependent expression of orexigenic neuropeptide Agouti-related peptide and cessation of feeding.