Synthesis, Characterization, and Trapping of a Cyclic Diborylcarbene, an Electrophilic Carbene.

A carbene bearing two geminal boryl substituents, called diborylcarbene (DBC), has been predicted to be highly Lewis acidic in sharp contrast to the well-studied persistent carbenes stabilized by π-donating substituents. Studies on DBC have been limited to either the base-trapping or theoretical calculations. Herein, we developed chemical equivalents for DBC, namely, K/X-diborylcarbenoids 2X (X = F or Cl). Treatment of 2F with Al(C6F5)3 yielded [AlF(C6F5)3]--stabilized DBC 1-FAl, which showed a significant low-field shift of the carbenoid carbon from 169 ppm (doublet, coupling with 19F) to 242 ppm (singlet). The loss of halogen was also detected through electrospray ionization time-of-flight mass spectrometry analysis of 2X only in the presence of Al(C6F5)3. Generated DBC 1 from 1-FAl or 2Cl was successfully trapped with excess amounts of trialkylphosphines (PR3, R = Me or Et), which afforded the corresponding DBC-PR3 adducts. In addition, the Lewis acidity of DBC 1 was evaluated both experimentally and theoretically to reveal that 1 is one of the most Lewis acidic species among neutral molecules.

Synthesis and reactivity of PC(sp3)P-pincer iridium complexes bearing a diborylmethyl anion.

Novel PCP-pincer iridium complexes bearing a diborylmethyl anion were synthesized. Strong σ-electron-donation to the metal and significant π-backdonation from the metal to boron atoms at the ß-position were observed both experimentally and computationally. H/D exchange of the aromatic C-H bond proceeded smoothly and, in addition, the α-methine-hydrogen between boron atoms was found to be replaced with deuterium in benzene-d6 solution possibly through diborylcarbene metal complexes as intermediates.

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

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 ß-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 ß-cell mass and that genomic imprinting may be a determinant of the onset of diabetes mellitus.

Constitutive activation of Rac1 in pancreatic ß cells facilitates F-actin depolymerization but exerts no influence on the increase of pancreatic ß cell mass and facilitation of insulin secretion.

Insulin secretion from pancreatic ß cells has an important role in the onset of type 2 diabetes. Insulin secretion from pancreatic ß cells is regulated by pancreatic ß cell mass and their insulin secretory function. By using pancreatic ß cell-specific Rac1-knockout mice, we recently showed that Rac1 deletion, even with no reduction in pancreatic ß cell mass, inhibits F-actin depolymerization, which causes insulin secretion to decline. However, the effect of Rac1 deficiency on the growth and apoptosis of pancreatic ß cells was not clarified. Further, the effect of constitutive Rac1 activation on the secretion of insulin from pancreatic ß cells has not been studied. Here, we used pancreatic islets isolated from pancreatic ß cell-specific Rac1-knockout mice to evaluate the growth and apoptosis of pancreatic ß cells. We found that Rac1 deficiency does not influence the growth or apoptosis of pancreatic ß cells. Further, when a constitutively activated form of Rac1 (G12V) is expressed, F-actin depolymerization was increased in the pancreatic ß cell lines, which had no effect on pancreatic ß cell growth or glucose-stimulated insulin secretion. These findings indicate that excessive Rac1 expression or activation in pancreatic ß cells facilitates F-actin depolymerization, but has no effect on insulin secretion.

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.

Effect of intrauterine undernutrition during late gestation on pancreatic beta cell mass.

We analyzed the effect of low birth weight on pancreatic beta cell mass. We used pregnant C57BL6J mice, and we reduced their food supply by 30% during the late gestational period and examined the changes in the metabolism and pancreatic beta cell mass. Pancreatic beta cell mass at birth was greatly decreased in the mice of the food restriction group (RG) as compared to the mice of the control group (CG). The body weight of RG mice exhibited a "catch-up growth" pattern and became equivalent to that of CG mice 7 days after birth, and thereafter exceeded that of CG mice; however, the pancreatic beta cell mass in RG mice remained lower than that in CG mice at the age of 4 weeks. A high-fat diet significantly increased the pancreatic beta cell mass in RG mice as compared to that in CG mice at 12 weeks of age. However, RG mice fed on high-fat diets tended to exhibit a decrease in the pancreatic beta cell mass at approximately 20 weeks of age. The plasma insulin concentrations also tended to be decreased in RG mice after 24 weeks of age as compared to those of CG mice. These results thus indicate that the growth of pancreatic beta cells is insufficient in RG mice, and pancreatic beta cell failure can easily develop as a consequence of insulin resistance.

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.