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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2454.)
© 2006 American Heart Association, Inc.

Vascular Biology

S-Glutathiolation of p21ras by Peroxynitrite Mediates Endothelial Insulin Resistance Caused by Oxidized Low-Density Lipoprotein

Nicolas Clavreul; Markus M. Bachschmid; Xiuyun Hou; Chaomei Shi; Azra Idrizovic; Yasuo Ido; David Pimentel; Richard A. Cohen

From the Vascular Biology Unit (N.C., M.M.B., X.H., C.S., A.I., R.A.C.), Diabetes and Metabolism Unit (Y.I.), and Myocardial Biology Unit Evans Department of Medicine (D.P.), Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Mass.

Correspondence to Richard A. Cohen, Director, Vascular Biology Unit, 650 Albany Street, X704, Boston, MA 02118. E-mail racohen{at}bu.edu

Objectives— To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite.

Methods and Results— OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2- to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 µmol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant.

Conclusions— This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling.

Bovine aortic endothelial cells were exposed to oxLDL. OxLDL induced peroxynitrite production and triggered redox-activation of p21ras via S-glutathiolation of its cysteine-118. Oxidant activation of p21ras was responsible for Erk-dependent phosphorylation of IRS-1 serine-616, which promoted endothelial insulin resistance.

Key Words: p21ras • peroxynitrite • S-glutathiolation • glutathione • insulin resistance • oxidized LDL

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