Stimulation of insulin-like growth factor II receptor binding and insulin receptor kinase activity in rat adipocytes. Effects of vanadate and H2O2.
Autophosphorylation of the insulin receptor on tyrosine residues and activation of the endogenous insulin receptor kinase is postulated to be a critical step in the mechanism of action of insulin. To investigate this hypothesis, the insulin-mimicking effects of vanadate (sodium orthovanadate) and H2O2 (hydrogen peroxide) alone and in combination were examined in freshly isolated rat adipocytes. Vanadate and H2O2 stimulated the translocation of insulin-like growth factor II (IGF-II) receptors to the plasma membrane of rat adipocytes in a manner analogous to insulin. IGF-II binding was increased by maximally effective doses of vanadate (1 mM), H2O2 (1 mM), and insulin (10 ng/ml) to 172 +/- 10, 138 +/- 12, and 289 +/- 16% of control, respectively. Previously (Kadota, S., Fantus, I. G., Hersh, B., and Posner, B. I. (1986) Biochem. Biophys. Res. Commun. 138, 174-178), we showed that the combination of these concentrations of vanadate plus insulin was not more potent than insulin alone. In this study, similar results were found with H2O2 plus insulin. In contrast, the combination of vanadate plus H2O2 was synergistic, effecting an increase of IGF-II binding to 488 +/- 23% of control. Amiloride inhibited the effects of vanadate, H2O2, and insulin. Adipocyte insulin receptors purified by wheat germ agglutinin chromatography were assayed for tyrosine kinase activity using the synthetic substrate poly(Glu,Tyr) (4:1). Basal activity (no in vitro insulin) was stimulated by exposure of intact cells to vanadate, H2O2, insulin, and vanadate + H2O2 to 147.7 +/- 4.3, 178.2 +/- 43.4, 495.0 +/- 67.1, and 913.2 +/- 92.0% of control, respectively. The stimulation of tyrosine kinase activity by these agents was accounted for by the insulin receptor as the augmented activity was completely immunoprecipitated with insulin receptor antibody. In these studies, the increase in IGF-II binding correlated significantly with the activation of the insulin receptor-tyrosine kinase (r = 0.927, p less than 0.001). These data support the hypothesis that activation of the insulin receptor kinase is linked to insulin action.
D000273:Adipose Tissue / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology; D007335:Insulin-Like Growth Factor II / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011972:Receptor, Insulin / Q000187:drug effects / Q000378:metabolism*; D017451:Receptors, Somatomedin; D013002:Somatomedins / Q000378:metabolism*; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology*
D007328:Insulin; D017451:Receptors, Somatomedin; D013002:Somatomedins; D014639:Vanadium; D014638:Vanadates; D007335:Insulin-Like Growth Factor II; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D011972:Receptor, Insulin