IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi.
Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). 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 [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-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 G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i).
Ferreira
Irlando Andrade
IA
Thrombosis and Haemostasis Laboratory, Department of Hematology, University Medical Center Utrecht, and Institute for Biomembranes, Utrecht University, 3584 CH Utrecht, The Netherlands. i.a.ferreira@lab.azu.nl
Eybrechts
Kurt L
KL
Mocking
Astrid I M
AI
Kroner
Christine
C
Akkerman
Jan-Willem N
JW
D000244:Adenosine Diphosphate / Q000737:chemistry; D001792:Blood Platelets / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004837:Epinephrine / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D009569:Nitric Oxide / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D011233:Precipitin Tests; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism
C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D009569:Nitric Oxide; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D011972:Receptor, Insulin; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D002118:Calcium; D004837:Epinephrine