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Association of NAD(P)H Oxidase with Glucose-Induced Insulin Secretion by Pancreatic β-Cells

Department of Physiology and Biophysics (D.M., E.R., F.A., M.F.R.G., M.S.R., S.B., R.C., A.R.C.), Institute of Biomedical Sciences, University of São Paulo, 05508-900 São Paulo, Brazil; Department of General Biology (H.R.O.-E.), State University of Ponta Grossa, 84010-919 Ponta Grossa, Brazil; and Department of Physiology (A.E.H.), Federal University of São Paulo, 04023-900 São Paulo, Brazil

Address all correspondence and requests for reprints to: Angelo Rafael Carpinelli, Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, Avenue Prof. Lineu Prestes 1524, 05508-900 São Paulo, Brazil. E-mail: angelo{at}icb.usp.br.

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H]oxidase components in pancreatic β-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic β-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47^PHOX during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-¹⁴C]glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47^PHOX expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic β-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the β-cell metabolism and the machinery involved in insulin secretion were also shown.