IRS-1 Mediates Inhibition of Ca2+ Mobilization by Insulin via the Inhibitory G-protein Gi

Abstract

Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein Gq, thereby raising cytosolic Ca2+, [Ca2+]i. The rise in [Ca 2+]i is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, Gi. Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca2+]i (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y12 receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during Gs-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N G-monomethyl-L-arginine). Hence, insulin might interfere with G i. Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and Giα2; (ii) co-precipitation of IRS-1 with Giα2 but not with other Gα subunits. Despite persistent receptor activation, the association of IRS-1 with G iα2 is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca2+ signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca2+ signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca 2+]i, tyrosine phosphorylation of Giα 2, and aggregation by interfering with the phosphorylation of the insulin receptor β subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and Gi.