Human fat cells possess a plasma membrane-bound H2O2-generating system that is activated by insulin via a mechanism bypassing the receptor kinase.

Insulin caused a transient increase in H202 accumulation in human fat cell suspensions that was observed only in the presence of an inhibitor of catalase and heme-containing peroxidases, such as azide, and reached peak levels of30gM within 5 min. The cells contained a plasma membrane-bound NADPH oxidase, producing 1 mol H202/mol ofNADPH oxidation, that was activated on exposure of intact cells to insulin at contrations that are physiologically relevant (0.1-10 nM). The hormone effect was rapid and was due to a selective increase in substrate affinity. The enzyme was magnesium dependent, required a flavine nucleotide for optimal activity, and was most active at pH 5.0-6.5. In contrast to all other hormone- or cytokine-sensitive NADPH oxidases that have been characterized in sufficient detail, the human fat cell oxidase retained its hormone responsiveness after cell disruption, and only Mn2', but no ATP, was required for a ligand-induced activation in crude plasma membranes. The results demonstrate that insulin utilizes tyrosine kinase-independent pathways for receptor signaling and strongly support the view that H202 contributes to the intracellular propagation of the insulin signal. (J. Clin. Invest.