Hydrogen Peroxide Generated during Cellular Insulin Stimulation Is Integral to Activation of the Distal Insulin Signaling Cascade in 3T3-L1 Adipocytes

Kalyankar Mahadev,Xiangdong Wu,Assaf Zilbering,Li Zhu,J Todd R Lawrence,Barry J Goldstein

doi:10.1074/jbc.m105061200

Abstract

In a variety of cell types, insulin stimulation elicits the rapid production of H(2)O(2), 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 H(2)O(2) generation on downstream insulin signaling using diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H(2)O(2) production. DPI completely inhibited the activation of phosphatidylinositol (PI) 3'-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Akt by up to 49%; these activities were restored when H(2)O(2) was added back to cells that had been pretreated with DPI. Interestingly, the H(2)O(2)-induced activation of Akt was entirely mediated by upstream stimulation of PI 3'-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3'-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Akt by exogenous H(2)O(2). 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 H(2)O(2) generation by other growth factors, such as platelet-derived growth factor, we also found that insulin-stimulated cellular production of H(2)O(2) 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 H(2)O(2) 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.

Highlights

Major advances in our understanding of the regulation of the insulin action pathway have focused on the key role of tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins [1]
The Insulin-induced Burst of Intracellular Oxidant Is Abolished by diphenyleneiodonium chloride (DPI)—To visualize the intracellular generation of reactive oxygen species in response to insulin, 3T3-L1 adipocytes were incubated with CM-DCF, a sensitive oxidant indicator dye that readily diffuses into cells and is trapped after cleavage by cellular esterases
Elaboration of H2O2 in response to insulin signal transduction has been known to occur in adipose tissue for many years, the significance of reactive oxygen species for the insulin action cascade has not been identified [32, 42, 43]

Summary

Introduction

Major advances in our understanding of the regulation of the insulin action pathway have focused on the key role of tyrosine phosphorylation of the insulin receptor and its cellular substrate proteins [1]. Following treatment of 3T3-L1 adipocytes with 100 nM insulin, using the pNPP assay, there was a significant reduction in PTPase enzyme activity of 32 and 62% of the basal level observed in the control cell lysates after 1 or 5 min, respectively (Fig. 2A).

Results

Conclusion