Aims/hypothesisObesity 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.MethodsWe 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.ResultsConditional 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/interpretationARC 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3140-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

ObjectivesIt is controversial whether sodium glucose transporter (SGLT) 2 inhibitors increase glucagon secretion via direct inhibition of SGLT2 in pancreatic α cells. The role of SGLT1 in α cells is also unclear. We aimed to elucidate these points that are important not only for basic research but also for clinical insight.MethodsPlasma glucagon levels were assessed in the high-fat, high-sucrose diet (HFHSD) fed C57BL/6J mice treated with dapagliflozin or canagliflozin. RT-PCR, RNA sequence, and immunohistochemistry were conducted to test the expression of SGLT1 and SGLT2 in α cells. We also used αTC1 cells and mouse islets to investigate the molecular mechanism by which SGLT1 modulates glucagon secretion.ResultsDapagliflozin, but not canagliflozin, increased plasma glucagon levels in HFHSD fed mice. SGLT1 and glucose transporter 1 (GLUT1), but not SGLT2, were expressed in αTC1 cells, mouse islets and human islets. A glucose clamp study revealed that the plasma glucagon increase associated with dapagliflozin could be explained as a response to acute declines in blood glucose. Canagliflozin suppressed glucagon secretion by inhibiting SGLT1 in α cells; consequently, plasma glucagon did not increase with canagliflozin, even though blood glucose declined. SGLT1 effect on glucagon secretion depended on glucose transport, but not glucose metabolism. Islets from HFHSD and db/db mice displayed higher SGLT1 mRNA levels and lower GLUT1 mRNA levels than the islets from control mice. These expression levels were associated with higher glucagon secretion. Furthermore, SGLT1 inhibitor and siRNA against SGLT1 suppressed glucagon secretion in isolated islets.ConclusionsThese data suggested that a novel mechanism regulated glucagon secretion through SGLT1 in α cells. This finding possibly explained the distinct effects of dapagliflozin and canagliflozin on plasma glucagon levels in mice.

An understanding of the mechanisms that govern pancreatic endocrine cell ontogeny may offer strategies for their somatic replacement in diabetic patients. During embryogenesis, transcription factor FoxO1 is expressed in pancreatic progenitor cells. Subsequently, it becomes restricted to ␤ cells and to a rare population of insulin-negative juxtaductal cells (FoxO1 Diabetes is characterized by complete or relative deficiency of insulin-producing ␤ cells (1). The growing societal and public health toll of the disease provides impetus to isolate or generate ␤ cells for cellular replacement purposes. Moreover, given that most of the newly found diabetes susceptibility genes appear to affect ␤-cell function rather than insulin action (12,41,43), that the two newest classes of antidiabetic medications are ␤-tropic (2), and that the main therapeutic failures in diabetes are seen in response to ␤-tropic agents (19, 48), studies of ␤-cell biology have wide-ranging implications beyond the replacement issue.Two approaches to ␤-cell generation have been championed: one endeavors to define culture conditions conducive to embryonic stem cell differentiation into ␤ cells (6), while the other is based on the hypothesis that endocrine cell progenitors, often identified with duct epithelial cells, exist in the adult pancreas and can yield functional ␤ cells (4, 42).Lineage-tracing studies indicate that pancreatic endocrine cells arise from a neurogenin 3 (Neurog3)-expressing progenitor pool set aside early in embryonic development (13) and that postnatal ␤-cell turnover is a result of limited ␤-cell replication and apoptosis (8,34,46). These data point to a limited role of pancreatic duct cells in the maintenance of ␤-cell mass through neogenesis from non-␤-cell precursors. Nonetheless, ductal endocrine cell neogenesis can occur following pancreatic duct ligation (17, 49), raising the possibility of generating endocrine cells by commandeering developmental pathways at the genetic level. Along these lines, we made the intriguing observation of a rare population of juxtaductal FoxO1 ϩ cells that do not express insulin. This finding, coupled with the role of FoxOs in governing developmental processes in diverse lineages and in the long-term stability of various tissues (24,32,35), prompted us to examine whether these cells are progenitors of duct-associated endocrine cells. In this study, we used a combination of developmental, genetic, and cell biology analyses to identify, isolate, and functionally characterize these cells. MATERIALS AND METHODS Antibodies and immunohistochemistry.We used the following antibodies: antipancytokeratin (Sigma), antivimentin (Santa Cruz), anti-Nkx2.2 (homeodomain protein) (Hybridoma Bank, University of Iowa), anti-FoxO1 (21), antipancreas and duodenum homeobox protein 1 (anti-Pdx1) (25), antiglucagon (Sigma), anti-insulin (Dako), antisomatostatin (Chemicon), anti-pancreatic polypeptide (anti-Pp) (Linco), antiamylase (Abcam), anti-green fluorescent protein (anti-GFP) (Santa Cruz), and anti-forkhead box-...

Kitamura T. FoxO1 as a double-edged sword in the pancreas: analysis of pancreas-and ␤-cell-specific FoxO1 knockout mice. Am J Physiol Endocrinol Metab 302: E603-E613, 2012. First published January 3, 2012; doi:10.1152/ajpendo.00469.2011.-Diabetes is characterized by an absolute or relative deficiency of pancreatic ␤-cells. New strategies to accelerate ␤-cell neogenesis or maintain existing ␤-cells are desired for future therapies against diabetes. We previously reported that forkhead box O1 (FoxO1) inhibits ␤-cell growth through a Pdx1-mediated mechanism. However, we also reported that FoxO1 protects against ␤-cell failure via the induction of NeuroD and MafA. Here, we investigate the physiological roles of FoxO1 in the pancreas by generating the mice with deletion of FoxO1 in the domains of the Pdx1 promoter (P-FoxO1-KO) or the insulin 2 promoter (␤-FoxO1-KO) and analyzing the metabolic parameters and pancreatic morphology under two different conditions of increased metabolic demand: high-fat high-sucrose diet (HFHSD) and db/db background. P-FoxO1-KO, but not ␤-FoxO1-KO, showed improved glucose tolerance with HFHSD. Immunohistochemical analysis revealed that P-FoxO1-KO had increased ␤-cell mass due to increased islet number rather than islet size, indicating accelerated ␤-cell neogenesis. Furthermore, insulin-positive pancreatic duct cells were increased in P-FoxO1-KO but not ␤-FoxO1-KO. In contrast, db/db mice crossed with P-FoxO1-KO or ␤-FoxO1-KO showed more severe glucose intolerance than control db/db mice due to decreased glucose-responsive insulin secretion. Electron microscope analysis revealed fewer insulin granules in FoxO1 knockout db/db mice. We conclude that FoxO1 functions as a double-edged sword in the pancreas; FoxO1 essentially inhibits ␤-cell neogenesis from pancreatic duct cells but is required for the maintenance of insulin secretion under metabolic stress.forkhead box O1; pancreatic ␤-cell; diabetes; insulin secretion PANCREATIC ␤-CELLS secrete insulin to maintain plasma glucose levels in an appropriate physiological range. The development of new strategies to accelerate ␤-cell neogenesis or maintain preexisting ␤-cells is desired for future therapies against diabetes.

In liver, glucose utilization and lipid synthesis are inextricably intertwined. When glucose availability exceeds its utilization, lipogenesis increases, leading to increased intrahepatic lipid content and lipoprotein secretion. Although the fate of three-carbon metabolites is largely determined by flux rate through the relevant enzymes, insulin plays a permissive role in this process. But the mechanism integrating insulin receptor signaling to glucose utilization with lipogenesis is unknown. Forkhead box O1 (FoxO1), a downstream effector of insulin signaling, plays a central role in hepatic glucose metabolism through the regulation of hepatic glucose production. In this study, we investigated the mechanism by which FoxO1 integrates hepatic glucose utilization with lipid synthesis. We show that FoxO1 overexpression in hepatocytes reduces activity of carbohydrate response element binding protein (Chrebp), a key regulator of lipogenesis, by suppressing O-linked glycosylation and reducing the protein stability. FoxO1 inhibits high glucose- or O-GlcNAc transferase (OGT)-induced liver-pyruvate kinase (L-PK) promoter activity by decreasing Chrebp recruitment to the L-PK promoter. Conversely, FoxO1 ablation in liver leads to the enhanced O-glycosylation and increased protein level of Chrebp owing to decreased its ubiquitination. We propose that FoxO1 regulation of Chrebp O-glycosylation is a mechanism linking hepatic glucose utilization with lipid synthesis.

Objectives: It is controversial whether sodium glucose transporter (SGLT) 2 inhibitors increase glucagon secretion via direct inhibition of SGLT2 in pancreatic a cells. The role of SGLT1 in a cells is also unclear. We aimed to elucidate these points that are important not only for basic research but also for clinical insight. Methods: Plasma glucagon levels were assessed in the high-fat, high-sucrose diet (HFHSD) fed C57BL/6J mice treated with dapagliflozin or canagliflozin. RT-PCR, RNA sequence, and immunohistochemistry were conducted to test the expression of SGLT1 and SGLT2 in a cells. We also used aTC1 cells and mouse islets to investigate the molecular mechanism by which SGLT1 modulates glucagon secretion.Results: Dapagliflozin, but not canagliflozin, increased plasma glucagon levels in HFHSD fed mice. SGLT1 and glucose transporter 1 (GLUT1), but not SGLT2, were expressed in aTC1 cells, mouse islets and human islets. A glucose clamp study revealed that the plasma glucagon increase associated with dapagliflozin could be explained as a response to acute declines in blood glucose. Canagliflozin suppressed glucagon secretion by inhibiting SGLT1 in a cells; consequently, plasma glucagon did not increase with canagliflozin, even though blood glucose declined. SGLT1 effect on glucagon secretion depended on glucose transport, but not glucose metabolism. Islets from HFHSD and db/db mice displayed higher SGLT1 mRNA levels and lower GLUT1 mRNA levels than the islets from control mice. These expression levels were associated with higher glucagon secretion. Furthermore, SGLT1 inhibitor and siRNA against SGLT1 suppressed glucagon secretion in isolated islets.Conclusions: These data suggested that a novel mechanism regulated glucagon secretion through SGLT1 in a cells. This finding possibly explained the distinct effects of dapagliflozin and canagliflozin on plasma glucagon levels in mice.

Diet affects health through ingested calories and macronutrients, and macronutrient balance affects health span. The mechanisms regulating macronutrient-based diet choices are poorly understood. Previous studies had shown that NAD-dependent deacetylase sirtuin-1 (SIRT1) in part influences the health-promoting effects of caloric restriction by boosting fat use in peripheral tissues. Here, we show that neuronal SIRT1 shifts diet choice from sucrose to fat in mice, matching the peripheral metabolic shift. SIRT1-mediated suppression of simple sugar preference requires oxytocin signalling, and SIRT1 in oxytocin neurons drives this effect. The hepatokine FGF21 acts as an endocrine signal to oxytocin neurons, promoting neuronal activation and Oxt transcription and suppressing the simple sugar preference. SIRT1 promotes FGF21 signalling in oxytocin neurons and stimulates Oxt transcription through NRF2. Thus, neuronal SIRT1 contributes to the homeostatic regulation of macronutrient-based diet selection in mice.

During prolonged fasting, fatty acid (FA) released from adipose tissue is a major energy source for peripheral tissues, including the heart, skeletal muscle and liver. We recently showed that FA binding protein 4 (FABP4) and FABP5, which are abundantly expressed in adipocytes and macrophages, are prominently expressed in capillary endothelial cells in the heart and skeletal muscle. In addition, mice deficient for both FABP4 and FABP5 (FABP4/5 DKO mice) exhibited defective uptake of FA with compensatory up-regulation of glucose consumption in these tissues during fasting. Here we showed that deletion of FABP4/5 resulted in a marked perturbation of metabolism in response to prolonged fasting, including hyperketotic hypoglycemia and hepatic steatosis. Blood glucose levels were reduced, whereas the levels of non-esterified FA (NEFA) and ketone bodies were markedly increased during fasting. In addition, the uptake of the 125I-BMIPP FA analogue in the DKO livers was markedly increased after fasting. Consistent with an increased influx of NEFA into the liver, DKO mice showed marked hepatic steatosis after a 48-hr fast. Although gluconeogenesis was observed shortly after fasting, the substrates for gluconeogenesis were reduced during prolonged fasting, resulting in insufficient gluconeogenesis and enhanced hypoglycemia. These metabolic responses to prolonged fasting in DKO mice were readily reversed by re-feeding. Taken together, these data strongly suggested that a maladaptive response to fasting in DKO mice occurred as a result of an increased influx of NEFA into the liver and pronounced hypoglycemia. Together with our previous study, the metabolic consequence found in the present study is likely to be attributed to an impairment of FA uptake in the heart and skeletal muscle. Thus, our data provided evidence that peripheral uptake of FA via capillary endothelial FABP4/5 is crucial for systemic metabolism and may establish FABP4/5 as potentially novel targets for the modulation of energy homeostasis.