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Insulin-mediated glucose transport and utilisation are decreased in skeletal muscle from type 2 diabetic and glucose-intolerant individuals because of alterations in insulin receptor signalling, GLUT4 translocation to the plasma membrane and microvascular blood flow. Catalytic activity of the muscle-specific isoform of neuronal nitric oxide synthase (nNOS) also participates in the regulation of glucose transport and appears to be decreased in a relevant animal model of drastic insulin resistance, the obese Zucker fa/fa rat. Our objective was to determine the molecular mechanisms involved in this defect.Isolated rat muscles and primary cultures of myocytes were used for western blot analysis of protein expression, immunohistochemistry, glucose uptake measurements and GLUT4 translocation assays.nNOS expression was reduced in skeletal muscle from fa/fa rats. This was caused by increased ubiquitination of the enzyme and subsequent degradation by the ubiquitin proteasome pathway. The degradation occurred through a greater interaction of nNOS with the chaperone heat-shock protein 70 and the co-chaperone, carboxyl terminus of Hsc70-interacting protein (CHIP). In addition, an alteration in nNOS sarcolemmal localisation was observed. We confirmed the implication of nNOS breakdown in defective insulin-induced glucose transport by demonstrating that blockade of proteasomal degradation or overexpression of nNOS improved basal and/or insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of insulin-resistant myocytes.Recovery of nNOS in insulin-resistant muscles should be considered a potential new approach to address insulin resistance.

Pancreatic beta cell hyperactivity is known to occur in obesity, particularly in insulin-resistant states. Our aim was to investigate whether changes in neuronal nitric oxide synthase (nNOS) function affect beta cell compensation in two relevant models: the Zucker fa/fa rats and pancreatic islets from obese humans.Glucose-induced insulin response was evaluated in the isolated perfused rat pancreas and in human pancreatic islets from obese individuals. Expression of nNOS (also known as NOS1) and subcellular localisation of nNOS were studied by quantitative RT-PCR, immunoblotting, immunofluorescence and electron microscopy.Pancreatic beta cells from Zucker fa/fa rats and obese individuals were found to be hyper-responsive to glucose. Pharmacological blockade of nNOS was unable to modify beta cell response to glucose in fa/fa rats and in islets from obese individuals, suggesting an abnormal control of insulin secretion by the enzyme. In both cases, nNOS activity in islet cell extracts remained unchanged, despite a drastic increase in nNOS protein and an enhancement in the dimer/monomer ratio, pointing to the presence of high amounts of catalytically inactive enzyme. This relative decrease in activity could be mainly related to increases in islet asymmetric dimethyl-arginine content, an endogenous inhibitor of nNOS activity. In addition, mitochondrial nNOS level was decreased, which contrasts with a strongly increased association with insulin granules.Increased nNOS production and dimerisation, together with a relative decrease in catalytic activity and relocalisation, are involved in beta cell hyperactivity in insulin-resistant rats but also in human islets isolated from obese individuals.