Shun-ichiro Asahara

Ablation of PDK1 in pancreatic β cells induces diabetes as a result of loss of β cell mass

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 β cells (βPdk1−/− 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 βPdk1−/− mice. These results suggest that PDK1 is important in maintenance of pancreatic cell mass and glucose homeostasis.

Biphasic Response of Pancreatic β-Cell Mass to Ablation of Tuberous Sclerosis Complex 2 in Mice

ABSTRACT Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic β-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 β-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic β cells (βTSC2−/− 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 β cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the βTSC2−/− mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of β cells. These results thus indicate that TSC2 regulates pancreatic β-cell mass in a biphasic manner.

Ablation of C/EBPβ alleviates ER stress and pancreatic β cell failure through the GRP78 chaperone in mice

Pancreatic beta cell failure is thought to underlie the progression from glucose intolerance to overt diabetes, and ER stress is implicated in such beta cell dysfunction. We have now shown that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) accumulated in the islets of diabetic animal models as a result of ER stress before the onset of hyperglycemia. Transgenic overexpression of C/EBPbeta specifically in beta cells of mice reduced beta cell mass and lowered plasma insulin levels, resulting in the development of diabetes. Conversely, genetic ablation of C/EBPbeta in the beta cells of mouse models of diabetes, including Akita mice, which harbor a heterozygous mutation in Ins2 (Ins2WT/C96Y), and leptin receptor-deficient (Lepr-/-) mice, resulted in an increase in beta cell mass and ameliorated hyperglycemia. The accumulation of C/EBPbeta in pancreatic beta 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 ATF6alpha, thereby increasing the vulnerability of these cells to excess ER stress. Our results thus indicate that the accumulation of C/EBPbeta in pancreatic beta cells contributes to beta cell failure in mice by enhancing susceptibility to ER stress.

Endoplasmic Reticulum Stress Inhibits STAT3-Dependent Suppression of Hepatic Gluconeogenesis via Dephosphorylation and Deacetylation

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.

Pancreatic β-Cell Failure Mediated by mTORC1 Hyperactivity and Autophagic Impairment

Hyperactivation of the mammalian target of rapamycin complex 1 (mTORC1) in β-cells is usually found as a consequence of increased metabolic load. Although it plays an essential role in β-cell compensatory mechanisms, mTORC1 negatively regulates autophagy. Using a mouse model with β-cell–specific deletion of Tsc2 (βTsc2−/−) and, consequently, mTORC1 hyperactivation, we focused on the role that chronic mTORC1 hyperactivation might have on β-cell failure. mTORC1 hyperactivation drove an early increase in β-cell mass that later declined, triggering hyperglycemia. Apoptosis and endoplasmic reticulum stress markers were found in islets of older βTsc2−/− mice as well as accumulation of p62/SQSTM1 and an impaired autophagic response. Mitochondrial mass was increased in β-cells of βTsc2−/− mice, but mitophagy was also impaired under these circumstances. We provide evidence of β-cell autophagy impairment as a link between mTORC1 hyperactivation and mitochondrial dysfunction that probably contributes to β-cell failure.

Ablation of TSC2 Enhances Insulin Secretion by Increasing the Number of Mitochondria through Activation of mTORC1

Aim We previously found that chronic tuberous sclerosis protein 2 (TSC2) deletion induces activation of mammalian target of rapamycin Complex 1 (mTORC1) and leads to hypertrophy of pancreatic beta cells from pancreatic beta cell-specific TSC2 knockout (βTSC2−/−) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells. Methods Isolated islets from βTSC2−/− mice and TSC2 knockdown insulin 1 (INS-1) insulinoma cells treated with small interfering ribonucleic acid were used to investigate insulin secretion, ATP content and the expression of mitochondrial genes. Results Activation of mTORC1 increased mitochondrial DNA expression, mitochondrial density and ATP production in pancreatic beta cells of βTSC2−/− mice. In TSC2 knockdown INS-1 cells, mitochondrial DNA expression, mitochondrial density and ATP production were increased compared with those in control INS-1 cells, consistent with the phenotype of βTSC2−/− mice. TSC2 knockdown INS-1 cells also exhibited augmented insulin secretory response to glucose. Rapamycin inhibited mitochondrial DNA expression and ATP production as well as insulin secretion in response to glucose. Thus, βTSC2−/− mice exhibit hyperinsulinemia due to an increase in the number of mitochondria as well as enlargement of individual beta cells via activation of mTORC1. Conclusion Activation of mTORC1 by TSC2 ablation increases mitochondrial biogenesis and enhances insulin secretion from pancreatic beta cells.

Ras-related C3 botulinum toxin substrate 1 (RAC1) regulates glucose-stimulated insulin secretion via modulation of F-actin.

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.

Histidine augments the suppression of hepatic glucose production by central insulin action

Glucose intolerance in type 2 diabetes is related to enhanced hepatic glucose production (HGP) due to the increased expression of hepatic gluconeogenic enzymes. Previously, we revealed that hepatic STAT3 decreases the expression of hepatic gluconeogenic enzymes and suppresses HGP. Here, we show that increased plasma histidine results in hepatic STAT3 activation. Intravenous and intracerebroventricular (ICV) administration of histidine-activated hepatic STAT3 reduced G6Pase protein and mRNA levels and augmented HGP suppression by insulin. This suppression of hepatic gluconeogenesis by histidine was abolished by hepatic STAT3 deficiency or hepatic Kupffer cell depletion. Inhibition of HGP by histidine was also blocked by ICV administration of a histamine H1 receptor antagonist. Therefore, histidine activates hepatic STAT3 and suppresses HGP via central histamine action. Hepatic STAT3 phosphorylation after histidine ICV administration was attenuated in histamine H1 receptor knockout (Hrh1KO) mice but not in neuron-specific insulin receptor knockout (NIRKO) mice. Conversely, hepatic STAT3 phosphorylation after insulin ICV administration was attenuated in NIRKO but not in Hrh1KO mice. These findings suggest that central histidine action is independent of central insulin action, while both have additive effects on HGP suppression. Our results indicate that central histidine/histamine-mediated suppression of HGP is a potential target for the treatment of type 2 diabetes.

Central Insulin Action Activates Kupffer Cells by Suppressing Hepatic Vagal Activation via the Nicotinic Alpha 7 Acetylcholine Receptor

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.

Paternal allelic mutation at the Kcnq1 locus reduces pancreatic β-cell mass by epigenetic modification of Cdkn1c

Significance Recently, the potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1) gene has received much attention as a candidate susceptibility gene for type 2 diabetes in Asian, European, and other populations. The molecular mechanism underlying the association of KCNQ1 with the onset of type 2 diabetes has remained unclear; however, we have now found that a paternal allelic mutation of Kcnq1 results in the up-regulation of the neighboring imprinted gene cyclin-dependent kinase inhibitor 1C (Cdkn1c), a cell cycle inhibitor, in pancreatic β-cells of mice, with this effect being mediated by epigenetic modification of the Cdkn1c promoter. These changes seem to be responsible for the reduced pancreatic β-cell mass and impaired glucose tolerance characteristics of Kcnq1 mutant mice. Genetic factors are important determinants of the onset and progression of diabetes mellitus. Numerous susceptibility genes for type 2 diabetes, including potassium voltage-gated channel, KQT-like subfamily Q, member1 (KCNQ1), have been identified in humans by genome-wide analyses and other studies. Experiments with genetically modified mice have also implicated various genes in the pathogenesis of diabetes. However, the possible effects of the parent of origin for diabetes susceptibility alleles on disease onset have remained unclear. Here, we show that a mutation at the Kcnq1 locus reduces pancreatic β-cell mass in mice by epigenetic modulation only when it is inherited from the father. The noncoding RNA KCNQ1 overlapping transcript1 (Kcnq1ot1) is expressed from the Kcnq1 locus and regulates the expression of neighboring genes on the paternal allele. We found that disruption of Kcnq1 results in reduced Kcnq1ot1 expression as well as the increased expression of cyclin-dependent kinase inhibitor 1C (Cdkn1c), an imprinted gene that encodes a cell cycle inhibitor, only when the mutation is on the paternal allele. Furthermore, histone modification at the Cdkn1c promoter region in pancreatic islets was found to contribute to this phenomenon. Our observations suggest that the Kcnq1 genomic region directly regulates pancreatic β-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.