- ELSEVIER SCIENCE INC
It has been reported that pertussis toxin (PTX) suppresses the function of trimeric guanine nucleotide binding protein (G-protein). We examined the effect of PTX on insulin-induced glucose uptake, diacylglycerol (DG)-protein kinase C (PKC) signalling, phosphatidylinositol (PI) 3-kinase and PKC zeta activation and insulin-induced tyrosine phosphorylation of Gi alpha to clarify the role of G-protein for insulin-mediated signal transduction mechanism in rat adipocytes and soleus muscles. Isolated adipocytes and soleus muscles were preincubated with 0.01 similar to 1 ng/ml PTX for 2 hours, followed by stimulation with 10-100 nM insulin or 1 mu M tetradecanoyl phorbol-13-acetate (TPA). Pretreatment with PTX resulted in dose-responsive decreases in insulin-stimulated [H-3]2-deoxyglucose (DOG) uptake, and unchanged TPA-stimulated [H-3]2-DOG uptake, without affecting basal [3H]2-DOG uptake. In adipocytes, insulin-induced DG-PKC signalling, PI 3-kinase activation and PKC zeta translocation from cytosol to the membrane were suppressed when treated with PTX, despite no changes in [I-125]insulin-specific binding and insulin receptor tyrosine kinase activity. Moreover, to elucidate insulin-stimulated tyrosine phosphorylation of 40 kDa alpha-subunit of G-protein (Gi alpha-2), adipocytes were stimulated with 10 nM insulin for 10 minutes, homogenized, immunoprecipitated with anti-phosphotyrosine antibody, and immunoblotted with anti-Gi alpha-2 antibody. Insulin-induced tyrosine phosphorylation of Gi alpha-2 was found by immunoblot analysis with anti-Gi alpha-2 antibody. These results suggest that G-protein regulates DG-PKC signalling by binding of Gi alpha-2 with GTP and PI 3-kinase-PKC zeta signalling by releasing of G beta gamma via dissociation of trimeric G-protein after insulin receptor tyrosine phosphorylation in insulin-sensitive tissues. (C) 2000 Elsevier Science Inc. All rights reserved.
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