Insulin-induced activation of NADPH-dependent H2O2 generation in human adipocyte plasma membranes is mediated by Gα(i2)

Abstract

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 GDPβS (guanosine 5'-O-(2-thiodiphosphate)), pertussis toxin and COOH-terminal anti-Gα(i1-2) or the corresponding peptide. Consistently, manganese could be replaced by micromolar concentrations of GTPγS (guanosine 5'-O-(3-thiotriphosphate)), which increased NADPH-dependent H2O2 generation by 20-40%. Insulin shifted the dose response curve for GTPγS to the left (> 10-fold) and increased the maximal response. In the presence of 10 μM GTPγS, 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-Gα1 and anti-insulin receptor β-subunit (anti-IRβ) affinity columns. Partially purified insulin receptor preparations contained Gα(s), Gα(i2), and Gβγ (but no Gα(i1) or Gα(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 Gα(i), but not by Gα0 or Gβγ, indicating that insulin's signal was transduced via Gα(i2). Thus, NADPH oxidase is the first example of an effector system that is coupled to the insulin receptor via a heterotrimeric G protein.