Potential Role of NOX3 and Superoxide Production in the Mechanism by Which Inflammatory Cytokines Cause Tyrosine Nitration of Insulin Signalling Proteins and Insulin Resistance in Insulin Target Cells

Abstract

Obesity-linked type 2 diabetes is well recognized to be associated with a chronic inflammatory state. However the mechanisms that link inflammation with obesity and development of type 2 diabetes are still poorly understood. We have already established that iNOS expression in metabolic tissues plays a major role in promoting insulin resistance in the context of obesity and metabolic endotoxemia. One of the mechanisms by which iNOS can mediate insulin resistance is through tyrosine nitration of insulin signalling proteins through the formation of peroxynitrite (ONOO–) from nitric oxide (NO) and superoxide anion (O2 –). To clarify the mechanism underlying tyrosine nitration of insulin signalling proteins, we hypothesize that superoxide (O2–) generation is a major contributor to ONOO– production and tyrosine nitration in insulin target cells treated with cytokines. We found that NOX3, a critical component of NADPH oxidase, can be markedly induced by inflammatory cytokines in 3T3 L1 adipocytes and primary hepatocytes. We have confirmed that NOX3 induction leads to O2– production in 3T3L1 cells as measured by the nitrotetrazolium reduction assay (NBT assay), and in FAO cells as measured by electro paramagnetic resonance and that this can be blunted by treatment with diphenyliodonium (DPI), a NADPH oxidase inhibitor. NOX3 induction and O2–generation are tightly coupled with iNOS-linked NO production and tyrosine nitration upon cytokine activation. These initial studies show that O2– is generated by key insulin target cells upon treatment with cytokines, and that NOX3 may be a key NADPH oxidase system responsible for this O2– production in inflammatory settings.