S-glutathiolation of p21ras by peroxynitrite mediates endothelial insulin resistance caused by oxidized low-density lipoprotein

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 mu mol/ 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.