Palmitate induces reactive oxygen species production and β-cell dysfunction by activating nicotinamide adenine dinucleotide phosphate oxidase through Src signaling.

[Aims/Introduction]Chronic hyperlipidemia impairs pancreatic β-cell function, referred to as lipotoxicity. We have reported an important role of endogenous reactive oxygen species (ROS) overproduction by activation of Src, a non-receptor tyrosine kinase, in impaired glucose-induced insulin secretion (GIIS) from diabetic rat islets. In the present study, we investigated the role of ROS production by Src signaling in palmitate-induced dysfunction of β-cells. [Materials and Methods]After rat insulinoma INS-1D cells were exposed to 0.6 mmol/L palmitate for 24 h (palmitate exposure); GIIS, ROS production and nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity were examined with or without exposure to10 μmol/L 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), a Src inhibitior, for 30 or 60 min. [Results]Exposure to PP2 recovered impaired GIIS and decreased ROS overproduction as a result of palmitate exposure. Palmitate exposure increased activity of NOX and protein levels of NOX2, a pathological ROS source in β-cells. Palmitate exposure increased the protein level of p47phox, a regulatory protein of NOX2, in membrane fraction compared with control, which was reduced by PP2. Transfection of small interfering ribonucleic acid of p47phox suppressed the augmented p47phox protein level in membrane fraction, decreased augmented ROS production and increased impaired GΙIS by palmitate exposure. In addition, exposure to PP2 ameliorated impaired GIIS and decreased ROS production in isolated islets of KK-Ay mice, an obese diabetic model with hyperlipidemia. [Conclusions]Activation of NOX through Src signaling plays an important role in ROS overproduction and impaired GΙIS caused by chronic exposure to palmitate, suggesting a lipotoxic mechanism of β-cell dysfunction of obese mice

Oral Administration of Apple Procyanidins Ameliorates Insulin Resistance via Suppression of Pro-inflammatory Cytokines Expression in Liver of Diabetic ob/ob 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

Src regulates insulin secretion and glucose metabolism by influencing subcellular localization of glucokinase in pancreatic β-cells

Aims/Introduction: Src, a non-receptor tyrosine kinase, regulates a wide range of cellular functions, and hyperactivity of Src is involved in impaired glucose metabolism in pancreatic β-cells. However, the physiological role of Src in glucose metabolism in normal, unstressed β-cells remains unclear. In the present study, we investigated the role of Src in insulin secretion and glucose metabolism. Materials and Methods: Src was downregulated using small interfering ribonucleic acid in INS-1 cells, and glucose-induced insulin secretion, adenosine triphosphate content, intracellular calcium concentration, glucose utilization and glucokinase activity were measured. Expression levels of messenger ribonucleic acid and protein of glucokinase were examined by semiquantitative real-time polymerase chain reaction and immunoblotting, respectively. Cells were fractionated by digitonin treatment, and subcellular localization of glucokinase was examined by immunoblotting. Interaction between glucokinase and neuronal nitric oxide synthase was estimated by immunoprecipitation. Results: In Src downregulated INS-1 cells, glucose-induced insulin secretion was impaired, whereas insulin secretion induced by high K+ was not affected. Intracellular adenosine triphosphate content and elevation of intracellular calcium concentration by glucose stimulation were suppressed by Src downregulation. Src downregulation reduced glucose utilization in the presence of high glucose, which was accompanied by a reduction in glucokinase activity without affecting its expression. However, Src downregulation reduced glucokinase in soluble, cytoplasmic fraction, and increased it in pellet containing intaracellular organelles. In addition, interaction between glucokinase and neuronal nitric oxide synthase was facilitated by Src downregulation. Conclusions: Src plays an important role in glucose-induced insulin secretion in pancreatic β-cells through maintaining subcellular localization and activity of glucokinase. In INS-1 rat insulinoma cells, down-regulation of Src by small interfering RNA reduced glucose-induced insulin secretion due to impaired glucokinase activity by translocation of the enzyme from cytosol to membrane fraction. Our study indicates that Src plays an important role in maintaining glucokinase activity and glucose-induced insulin secretion in pancreatic β-cells