Insulin Generates Free Radicals by an NAD(P)H, Phosphatidylinositol 3′-Kinase-Dependent Mechanism in Human Skin Fibroblasts Ex Vivo

• DOI: 10.2337/diabetes.53.5.1344
• Publication Date: 2007-03-06T19:04:22Z
• Authors: Giulio Ceolotto; Michela Bevilacqua; Italia Papparella; Elisabetta Baritono; Lorenzo Franco; Carlo Corvaja; Martina Mazzoni; Andrea Semplicini; Angelo Avogaro
• Keywords: Mitogen-Activated Protein Kinase 1; 0303 health sciences; Mitogen-Activated Protein Kinase 3; Free Radicals; NADPH Oxidases; Biological Transport; Fibroblasts; Phosphoproteins; 3. Good health; Oxidative Stress; Phosphatidylinositol 3-Kinases; 03 medical and health sciences; Hyperinsulinism; Humans; Insulin; Mitogen-Activated Protein Kinases; Phosphorylation; Cells, Cultured; NADP; Skin

Oxidative stress may be involved in the development of vascular complications associated with diabetes; however, the molecular mechanism responsible for increased production of free radicals in diabetes remains uncertain. Therefore, we examined whether acute hyperinsulinemia increases the production of free radicals and whether this condition affects proliferative extracellular signal-regulated kinase (ERK-1 and -2) signaling in human fibroblasts in vitro. Insulin treatment significantly increased intracellular superoxide anion (O2−) production, an effect completely abolished by Tiron, a cell-permeable superoxide dismutase (SOD) mimetic and by polyethylene glycol (PEG)-SOD, but not by PEG catalase. Furthermore, insulin-induced O2− production was attenuated by the NAD(P)H inhibitor apocynin, but not by rotenone or oxypurinol. Inhibition of the phosphatidylinositol 3′-kinase (PI 3′-kinase) pathway with LY294002 blocked insulin-stimulated O2− production, suggesting a direct involvement of PI 3′-kinase in the activation of NAD(P)H oxidase. The insulin-induced free radical production led to membranous translocation of p47phox and markedly enhanced ERK-1 and -2 activation in human fibroblasts. In conclusion, these findings provided direct evidence that elevated insulin levels generate O2− by an NAD(P)H-dependent mechanism that involves the activation of PI 3′-kinase and stimulates ERK-1- and ERK-2-dependent pathways. This effect of insulin may contribute to the pathogenesis and progression of cardiovascular disease in the insulin resistance syndrome.