Abstract
Genistein, an isoflavone putative tyrosine kinase inhibitor, was used to investigate the coupling of insulin receptor tyrosine kinase activation to four metabolic effects of insulin in the isolated rat adipocyte. Genistein inhibited insulin-stimulated glucose oxidation in a concentration-dependent manner with an ID50 of 25 micrograms/ml and complete inhibition at 100 micrograms/ml. Genistein also prevented insulin's (10(-9) M) inhibition of isoproterenol-stimulated lipolysis with an ID50 of 15 micrograms/ml and a complete effect at 50 micrograms/ml. The effect of genistein (25 micrograms/ml) was not reversed by supraphysiological (10(-7) M) insulin levels. In contrast, genistein up to 100 micrograms/ml had no effect on insulin's (10(-9) M) stimulation of either pyruvate dehydrogenase or glycogen synthase activity. We determined whether genistein influenced insulin receptor beta-subunit autophosphorylation or tyrosine kinase substrate phosphorylation either in vivo or in vitro by anti-phosphotyrosine immunoblotting. Genistein at 100 micrograms/ml did not inhibit insulin's (10(-7) M) stimulation of insulin receptor tyrosine autophosphorylation or tyrosine phosphorylation of the cellular substrates pp185 and pp60. Also, genistein did not prevent insulin-stimulated autophosphorylation of partially purified human insulin receptors from NIH 3T3/HIR 3.5 cells or the phosphorylation of histones by the activated receptor tyrosine kinase. In control experiments using either NIH 3T3 fibroblasts or partially purified membranes from these cells, genistein did inhibit platelet-derived growth factor's stimulation of its receptor autophosphorylation. These findings indicate the following: (a) Genistein can inhibit certain responses to insulin without blocking insulin's stimulation of its receptor tyrosine autophosphorylation or of the receptor kinase substrate tyrosine phosphorylation. (b) In adipocytes genistein must block the stimulation of glucose oxidation and the antilipolytic effects of insulin at site(s) downstream from the insulin receptor tyrosine kinase. (c) The inhibitory effects of genistein on hormonal signal transduction cannot necessarily be attributed to inhibition of tyrosine kinase activity, unless specifically demonstrated.
Highlights
Genistein, an isoflavone putative tyrosine kinase in- The predominant theory of insulin action proposes that the hibitor, wasused to investigate thecoupling of insulin interaction of insulin with its receptor causes tyrosine autoreceptor tyrosine kinase activation to four metabolic phosphorylation of the receptor /3-subunit resulting in actieffects of insulin in the isolated rat adipocyte
The assumption was made that the recipient cell lines expressed the signal transduction machinery necessary to couple an occupied insulin receptor to a cellular response, it is known that these cell lines do not express insulin-sensitive glucosetransporter (GLUT4),which is a molecular marker of muscle and adipose tissue [15]
The Effect of Genisteinon Insulin Action-Experiments were designed to determine if genistein, a known tyrosine kinase inhibitor [18, 19], blocked insulin action in isolated adipocytes
Summary
Dept. of Genetics, University of Illinois College of Medicine, 808 S. The identification and synthesis of inhibitors of protein tyrosine kinases [16,17,18] allow a new biochemical approach to study the role of tyrosine phosphorylation in insulin action This approachpermits assessment of the potentialanti-insulin effects of these inhibitors inphysiologically meaningful target cells such as the rat adipocyte. We undertook such a study with the antibiotic genistein, which inhibits both EGF' [18] and PDGF [19] receptor tyrosine kinase activity. Receptor prepared from 3 X lo cells was incubated for 10 min at 30"C in 30 p1 containing a final concentration of 33.3 mM HEPES, pH 7.4, 10 mM MgC12,3 mM MnCIZ,100 ~ L MNa3V0,, 1% MezSO, mM N-acetyl-D-glucosamine, mM NaCl, 0.017%Triton X-100, 0.017%BSA, and in the presence or absence of 25mgof histone H2b, lo-? SDS-PAGE and immunoblotting with anti-phosphotyrosine antibodies were carried out as described above
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