Aims/hypothesis Sirtuin-1 (SIRT1) is a potential therapeutic target to combat insulin resistance and type 2 diabetes. This study aims to identify a microRNA (miRNA) targeting SIRT1 to regulate hepatic insulin sensitivity. Methods Luciferase assay combined with mutation and immunoblotting was used to screen and verify the bioinformatically predicted miRNAs. miRNA and mRNA levels were measured by real-time PCR. Insulin signalling was detected by immunoblotting and glycogen synthesis. Involvement of SIRT1 was studied with adenovirus, inhibitor and SIRT1-deficient hepatocytes. The role of miR-181a in vivo was explored with adenovirus and locked nucleic acid antisense oligonucleotides. Results miR-181a targets the 3′ untranslated region (3′UTR) of Sirt1 mRNA through a miR-181a binding site, and downregulates SIRT1 protein abundance at the translational level. miR-181a is increased in insulin-resistant cultured hepatocytes and liver, and in the serum of diabetic patients. Overexpression of miR-181a decreases SIRT1 protein levels and activity, and causes insulin resistance in hepatic cells. Inhibition of miR-181a by antisense oligonucleotides increases SIRT1 protein levels and activity, and improves insulin sensitivity in hepatocytes. Ectopic expression of SIRT1 abrogates the effect of miR-181a on insulin sensitivity, and inhibition of SIRT1 activity or SIRT1 deficiency markedly attenuated the improvement in insulin sensitivity induced by antisense miR-181a. In addition, overexpression of miR181a by adenovirus impairs hepatic insulin signalling, and intraperitoneal injection of locked nucleic acid antisense oligonucleotides for miR-181a improves glucose homeostasis in diet-induced obesity mice. Conclusions/interpretation miR-181a regulates SIRT1 and improves hepatic insulin sensitivity. Inhibition of miR-181a might be a potential new strategy for treating insulin resistance and type 2 diabetes.

Aims/hypothesis: The role of gamma-aminobutyric acid (GABA) and A-type GABA receptors (GABA A Rs) in modulating islet endocrine function has been actively investigated since the identification of GABA and GABA A Rs in the pancreatic islets. However, the reported effects of GABA A R activation on insulin secretion from islet beta cells have been controversial. Methods: This study examined the hypothesis that the effect of GABA on beta cell insulin secretion is dependent on glucose concentration. Results: Perforated patchclamp recordings in INS-1 cells demonstrated that GABA, at concentrations ranging from 1 to 1,000 μmol/l, induced a transmembrane current (I GABA ) which was sensitive to the GABA A R antagonist bicuculline. The current-voltage relationship revealed that I GABA reversed at −42±2.2 mV, independently of glucose concentration. Nevertheless, the glucose concentration critically controlled the membrane potential (V M ), i.e., at low glucose (0 or 2.8 mmol/l) the endogenous V M of INS-1 cells was below the I GABA reversal potential and at high glucose (16.7 or 28 mmol/l), the endogenous V M of INS-1 cells was above the I GABA reversal potential. Therefore, GABA dose-dependently induced membrane depolarisation at a low glucose concentration, but hyperpolarisation at a high glucose concentration. Consistent with electrophysiological findings, insulin secretion assays demonstrated that at 2.8 mmol/l glucose, GABA increased insulin secretion in a dose-dependent fashion (p<0.05, n=7). This enhancement was blocked by bicuculline (p<0.05, n=4). In contrast, in the presence of 28 mmol/l glucose, GABA suppressed the secretion of insulin (p<0.05, n=5). Conclusions/interpretation: These findings indicate that activation of GABA A Rs in beta cells regulates insulin secretion in concert with changes in glucose levels.

Aims/hypothesis: The antioxidant compound α-lipoic acid (α-LA) possesses antidiabetic and anti-obesity properties. In the hypothalamus, α-LA suppresses appetite and prevents obesity by inhibiting AMP-activated protein kinase (AMPK). Given the therapeutic potential of α-LA for the treatment of type 2 diabetes and obesity, and the importance of AMPK in beta cells, we examined the effect of α-LA on pancreatic beta cell function. Materials and methods: Isolated rat islets and MIN6 beta cells were treated acutely (15-90 min) or chronically (18-24 h) with α-LA or the known AMPK-activating compounds 5′-amino-imidazole-4-carboxamide ribonucleoside (AICAR) and metformin. Insulin secretion, the AMPK-signalling pathway, mitochondrial function and cell growth were assessed. Results: Acute or chronic treatment of islets and MIN6 cells with α-LA led to dose-dependent rises in phosphorylation of the AMPK α-subunit and acetyl CoA carboxylase. Chronic exposure to α-LA, AICAR or metformin caused a reduction in insulin secretion. α-LA inhibited the p70 s6 kinase translational control pathway, and inhibited MIN6 growth in a manner similar to rapamycin. Unlike AICAR and metformin, α-LA also acutely inhibited insulin secretion. Examination of the effect of α-LA on mitochondrial function showed that acute treatment with this compound elevated reactive oxygen species (ROS) production and enhanced mitochondrial depolarisation induced by Ca 2+ . Conclusions/ interpretation: This study is the first to demonstrate that α-LA directly affects beta cell function. The chronic effects of α-LA include AMPK activation and reductions in insulin secretion and content, and cell growth. Acutely, α-LA also inhibits insulin secretion, an effect probably involving the ROS-induced impairment of mitochondrial function.