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 (ßTSC2(-/-)) mice. The present study examines the effects of TSC2 ablation on insulin secretion from pancreatic beta cells. METHODS: Isolated islets from ßTSC2(-/-) 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 ßTSC2(-/-) 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 ßTSC2(-/-) 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, ßTSC2(-/-) 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.

Ablation of C/EBPbeta alleviates ER stress and pancreatic beta 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.

Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice.

Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic beta-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 beta-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic beta cells (betaTSC2(-/-) 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 beta cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the betaTSC2(-/-) mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of beta cells. These results thus indicate that TSC2 regulates pancreatic beta-cell mass in a biphasic manner.

Ablation of PDK1 in pancreatic beta cells induces diabetes as a result of loss of beta 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 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.

A series of maturity onset diabetes of the young, type 2 (MODY2) mouse models generated by a large-scale ENU mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.