AIM/HYPOTHESIS: Oxidative stress was shown to selectively induce impaired metabolic response to insulin, raising the possible involvement of alterations in Insulin-Receptor-Substrate (IRS) proteins. This study was conducted to assess whether oxidative stress induced IRS protein degradation and enhanced serine phosphorylation, and to assess their functional importance. METHODS: 3T3-L1 adipocytes and rat hepatoma cells (FAO) were exposed to micro-molar H(2)O(2) by adding glucose oxidase to the culture medium, and IRS1 content, its serine phosphorylation and downstream metabolic insulin effects were measured. RESULTS: Cells exposed to oxidative stress exhibited decreased IRS1 (but not IRS2) content, and increased serine phosphorylation of both proteins. Total protein ubiquitination was increased in oxidized cells, but not in cells exposed to prolonged insulin treatment. Yet, lactacystin and MG132, two unrelated proteasome inhibitors, prevented IRS1 degradation induced by prolonged insulin but not by oxidative stress. The PI 3-kinase inhibitor LY294002 and the mTOR inhibitor rapamycin, but not the MEK1 inhibitor PD98059, could prevent IRS1 changes in oxidized cells. Rapamycin, which protected against IRS1 degradation and serine phosphorylation was not associated with improved response to acute insulin stimulation. Moreover, the antioxidant alpha lipoic acid, while protecting against oxidative stress-induced insulin resistance in 3T3-L1 adipocytes, could not prevent IRS1 degradation and serine phosphorylation. CONCLUSION/INTERPRETATION: Oxidative stress induces serine phosphorylation of IRS1 and increases its degradation by a proteasome-independent pathway; yet, these changes do not correlate with the induction of impaired metabolic response to insulin.