Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic ␤-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on ␤-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic ␤ cells (␤TSC2 ؊/؊ mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual ␤ cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the ␤TSC2 ؊/؊ mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of ␤ cells. These results thus indicate that TSC2 regulates pancreatic ␤-cell mass in a biphasic manner.

The total mass of islets of Langerhans is reduced in individuals with type 2 diabetes, possibly contributing to the pathogenesis of this condition. Although the regulation of islet mass is complex, recent studies have suggested the importance of a signaling pathway that includes the insulin or insulin-like growth factor-1 receptors, insulin receptor substrate and phosphatidylinositol (PI) 3-kinase. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a serine-threonine kinase that mediates signaling downstream of PI 3-kinase. Here we show that mice that lack PDK1 specifically in pancreatic beta cells (betaPdk1-/- mice) develop progressive hyperglycemia as a result of a loss of islet mass. The mice show reductions in islet density as well as in the number and size of cells. Haploinsufficiency of the gene for the transcription factor Foxo1 resulted in a marked increase in the number, but not the size, of cells and resulted in the restoration of glucose homeostasis in betaPdk1-/- mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.

Pancreatic β cell failure is thought to underlie the progression from glucose intolerance to overt diabetes, and ER stress is implicated in such β cell dysfunction. We have now shown that the transcription factor CCAAT/ enhancer-binding protein β (C/EBPβ) accumulated in the islets of diabetic animal models as a result of ER stress before the onset of hyperglycemia. Transgenic overexpression of C/EBPβ specifically in β cells of mice reduced β cell mass and lowered plasma insulin levels, resulting in the development of diabetes. Conversely, genetic ablation of C/EBPβ in the β cells of mouse models of diabetes, including Akita mice, which harbor a heterozygous mutation in Ins2 (Ins2 WT/C96Y ), and leptin receptor-deficient (Lepr -/-) mice, resulted in an increase in β cell mass and ameliorated hyperglycemia. The accumulation of C/EBPβ in pancreatic β cells reduced the abundance of the molecular chaperone glucose-regulated protein of 78 kDa (GRP78) as a result of suppression of the transactivation activity of the transcription factor ATF6α, thereby increasing the vulnerability of these cells to excess ER stress. Our results thus indicate that the accumulation of C/EBPβ in pancreatic β cells contributes to β cell failure in mice by enhancing susceptibility to ER stress.

The mechanistic target of rapamycin complex 1 (MTORC1) is a critical negative regulator of general autophagy. We hypothesized that MTORC1 may specifically regulate autophagic clearance of damaged mitochondria. To test this, we used cells lacking tuberous sclerosis complex 2 (TSC2 -/-), which show constitutive MTORC1 activation. TSC2 -/- cells show MTORC1-dependent impaired autophagic flux after chemical uncoupling of mitochondria, increased mitochondrial protein aging and accumulation of p62/SQSTM1 positive mitochondria. Mitochondrial autophagy (mitophagy) was also deficient in cells lacking TSC2, associated with altered expression of PTEN-induced kinase 1 (PINK1) and PARK2 translocation to uncoupled mitochondria, all of which were recovered by MTORC1 inhibition or expression of constitutively active FoxO1. These data prove the necessity of intact MTORC1 signaling to regulate two synergistic processes required for clearance of damaged mitochondria: 1) general autophagy initiation, and 2) PINK1/PARK2-mediated selective targeting of uncoupled mitochondria to the autophagic machinery.

In the liver, signal transducer and activator of transcription 3 (STAT3) plays an important role in the suppression of gluconeogenic enzyme expression. While obesity-associated endoplasmic reticulum (ER) stress has been shown to increase hepatic gluconeogenic enzyme expression, the role of ER stress in STAT3-dependent regulation of such expression is unclear. The current study aimed to elucidate the effect of ER stress on the STAT3-dependent regulation of hepatic gluconeogenic enzyme expression. Genetically obese/diabetic db/db mice and db/db mouse–derived isolated hepatocytes were used as ER stress models. A tyrosine phosphatase inhibitor, a deacetylation inhibitor, and an acetylated mutant of STAT3 were used to examine the effect of ER stress on hepatic STAT3 action. ER stress inhibited STAT3-dependent suppression of gluconeogenic enzyme gene expression by suppressing hepatic Janus kinase (JAK)2 and STAT3 phosphorylation. A tyrosine phosphatase inhibitor restored ER stress–induced suppression of JAK2 phosphorylation but exhibited no improving effect on suppressed STAT3 phosphorylation. STAT3 acetylation is known to correlate with its phosphorylation. ER stress also decreased STAT3 acetylation. An acetylated mutant of STAT3 was resistant to ER stress–induced inhibition of STAT3-phosphorylation and STAT3-dependent suppression of hepatic gluconeogenic enzyme gene expression in vitro and in vivo. Trichostatin A, a histone deacetylase (HDAC) inhibitor, ameliorated ER stress–induced inhibition of STAT3 acetylation and phosphorylation. The current study revealed that ER stress inhibits STAT3-dependent suppression of hepatic gluconeogenic enzymes via JAK2 dephosphorylation and HDAC-dependent STAT3 deacetylation, playing an important role in the increase of hepatic glucose production in obesity and diabetes.

Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) in b-cells is usually found as a consequence of increased metabolic load. Although it plays an essential role in b-cell compensatory mechanisms, mTORC1 negatively regulates autophagy. Using a mouse model with b-cell-specific deletion of Tsc2 (bTsc2 2/2 ) and, consequently, mTORC1 hyperactivation, we focused on the role that chronic mTORC1 hyperactivation might have on b-cell failure. mTORC1 hyperactivation drove an early increase in b-cell mass that later declined, triggering hyperglycemia. Apoptosis and endoplasmic reticulum stress markers were found in islets of older bTsc2 2/2 mice as well as accumulation of p62/SQSTM1 and an impaired autophagic response. Mitochondrial mass was increased in b-cells of bTsc2 2/2 mice, but mitophagy was also impaired under these circumstances. We provide evidence of b-cell autophagy impairment as a link between mTORC1 hyperactivation and mitochondrial dysfunction that probably contributes to b-cell failure.Nutrient overload is one of the main causes of insulin resistance. This triggers the compensatory mechanisms leading to b-cell mass increase and hyperinsulinemia. Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) is elicited under nutrient overload conditions (1-3). mTORC1 plays a positive role in b-cell mass expansion (3-6), and rapamycin treatment impairs b-cell mass adaptation (7,8). On the other hand, mTORC1 hyperactivation is also a cause of insulin resistance (1) and endoplasmic reticulum (ER) stress (9), conditions linked with b-cell dysfunction and diabetes progression (10,11). We previously described how Tsc2 deletion in b-cells (bTsc2 2/2 ) results in chronic mTORC1 hyperactivation leading to a biphasic phenotype with early b-cell mass increase, hyperinsulinemia, and hypoglycemia. This was followed by b-cell failure and hyperglycemia in older mice (3). We also found how the first phase in bTsc2 2/2 mice is characterized by both an increase of mitochondrial mass and enhanced glucose-stimulated insulin secretion (12). Autophagy is a cytoprotective mechanism also found essential for b-cell homeostasis (13,14). Autophagy plays a protective role under stress conditions, such as ER stress (15), and we and others have described its positive role in b-cells under these conditions (16,17). Autophagy is also responsible for the turnover of mitochondria by the specific elimination of defective or damaged organelles. mTORC1 is a critical negative regulator of autophagy (18), and studies have shown how TSC deficiency leads to impaired autophagy in human tumors and cell lines (19,20). In this study, we explored the intriguing possibility that mTORC1 hyperactivity in b-cells, apart from being essential for b-cell compensatory mechanisms, might

Aims/hypothesisThe small G-protein ras-related C3 botulinum toxin substrate 1 (RAC1) plays various roles in mammalian cells, such as in the regulation of cytoskeletal organisation, cell adhesion, migration and morphological changes. The present study examines the effects of RAC1 ablation on pancreatic beta cell function.MethodsIsolated islets from pancreatic beta cell-specific Rac1-knockout (betaRac1−/−) mice and RAC1 knockdown INS-1 insulinoma cells treated with small interfering RNA were used to investigate insulin secretion and cytoskeletal organisation in pancreatic beta cells.ResultsBetaRac1−/− mice showed decreased glucose-stimulated insulin secretion, while there were no apparent differences in islet morphology. Isolated islets from the mice had blunted insulin secretion in response to high glucose levels. In RAC1 knockdown INS-1 cells, insulin secretion was also decreased in response to high glucose levels, consistent with the phenotype of betaRac1−/− mice. Even under high glucose levels, RAC1 knockdown INS-1 cells remained intact with F-actin, which inhibits the recruitment of the insulin granules, resulting in an inhibition of insulin secretion.Conclusions/interpretationIn RAC1-deficient pancreatic beta cells, F-actin acts as a barrier for insulin granules and reduces glucose-stimulated insulin secretion.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-2849-5) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Central insulin action activates hepatic IL-6/STAT3 signaling, which suppresses the gene expression of hepatic gluconeogenic enzymes. The vagus nerve plays an important role in this centrally mediated hepatic response; however, the precise mechanism underlying this brain-liver interaction is unclear. Here, we present our findings that the vagus nerve suppresses hepatic IL-6/STAT3 signaling via α7-nicotinic acetylcholine receptors (α7-nAchR) on Kupffer cells, and that central insulin action activates hepatic IL-6/STAT3 signaling by suppressing vagal activity. Indeed, central insulin-mediated hepatic IL-6/STAT3 activation and gluconeogenic gene suppression were impeded in mice with hepatic vagotomy, pharmacological cholinergic blockade, or α7-nAchR deficiency. In high-fat diet-induced obese and insulin-resistant mice, control of the vagus nerve by central insulin action was disturbed, inducing a persistent increase of inflammatory cytokines. These findings suggest that dysregulation of the α7-nAchR-mediated control of Kupffer cells by central insulin action may affect the pathogenesis of chronic hepatic inflammation in obesity.