Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by G alpha(12)

Human fat cells possess a multireceptor-linked H2O2-generating system that is activated by insulin. Previous studies revealed that manganese was the sole cofactor required for a hormonal regulation of NADPH-dependent H2O2 generation in vitro. In this report it is shown that the synergistic activation of NADPH-dependent H2O2 generation by Mn2+ and insulin was blocked by GDP beta S (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-G alpha(i1-2) or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTP gamma S to the left (>10-fold) and increased the maximal response. In the presence of 10 mu M GTP gamma S, the hormone was active at picomolar concentrations, indicating that insulin acted via its cognate receptor. The insulin receptor and G(i) were co-adsorbed on anti-Ga-i and anti-insulin receptor beta-subunit (anti-IR beta) affinity columns. Partially purified insulin receptor preparations contained G alpha(s), G alpha(i2), and G beta gamma (but no G alpha(i1), or Ga alpha(i3)). The functional nature of the insulin receptor-G(i2) complex was made evident by insulin's ability to modulate labeling of G(i) by bacterial toxins. Insulin action was mimicked by activated G alpha(i), but not by G alpha(o), or G beta gamma, indicating that insulin's signal was transduced via G alpha(i2). Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.