Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes

In a variety of cell types, insulin stimulation elicits the rapid production of H2O2, which causes the oxidative inhibition of protein-tyrosine phosphatases and enhances the tyrosine phosphorylation of proteins in the early insulin action cascade (Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001) J. Biol. Chem. 276, 21938-21942). In the present work, we explored the potential role of insulin-induced 11,02 generation on downstream insulin signaling using diphenyleneiodonium (I)PI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H2O2 production. DPI completely inhibited the activation of phosphatidylinositol (PI) T-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Alit by up to 49%; these activities were restored when H2O2 was added back to cells that had been pretreated with DPI. Interestingly, the H2O2-induced activation of Akt was entirely mediated by upstream stimulation of PI T-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3'-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Alit by exogenous H2O2. Preventing oxidant generation with DPI also blocked insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane, providing further evidence for an oxidant signal in the regulation of the distal insulin-signaling cascade. Finally, in contrast to the cellular mechanism of H2O2 generation by other growth factors, such as platelet-derived growth factor, we also found that insulin-stimulated cellular production of H2O2 may occur through a unique pathway, independent of cellular PI 3'-kinase activity. Overall, these data provide insight into the physiological role of insulin-dependent H2O2 generation, which is not only involved in the regulation of tyrosine phosphorylation events in the early insulin signaling cascade but also has important effects on the regulation of downstream insulin signaling, involving the activation of PI 3'-kinase, Akt, and ultimately cellular glucose transport in response to insulin.