Prevention by Protein Kinase C Inhibitors of Glucose-Induced Insulin-Receptor Tyrosine Kinase Resistance in Rat Fat Cells

Abstract

Hyperglycemia causes insulin-receptor kinase (IRK) resistance in fat cells. We characterized the mechanism of IRK inhibition and studied whether it is the consequence of a glucose-induced stimulation of protein kinase C (PKC). Fat cells were incubated for 1 or 12 h in culture medium containing either a low- (5-mM) or high- (25-mM) glucose concentration. IRK was isolated, insulin binding was determined, and autophosphorylation was studied in vitro with [γ-32P]ATP or was determined by Western blotting with anti-phosphotyrosine antibodies. Substrate phosphorylation was investigated with the artificial substrate poly(Glu80-Tyr20). Partially purified insulin receptor from rat fat cells, which were cultured under high-glucose conditions for 1 or 12 h, showed no alteration of insulin binding but a reduced insulin effecton autophosphorylation (30 ± 7% of control) and poly(Glu80-Tyr20) phosphorylation (55.5 ± 9% of control). Lineweaver-Burk plots of the enzyme kinetics revealed, beside a reduced Vmax, and increased KM (from 30 μM to 80 μM) for ATP of IRK from high-glucose–treated cells. Because a similar inhibition pattern was earlier found for IRK from fat cells afteracute phorbol ester stimulation, we investigated whether activation of PKC might be the cause of the reduced IRK activity. We isolated PKC from the cytosol and the membrane fraction of high- and low-glucose fat cells and determined the diacylglycerol- and phospholipid-stimulated PKC activity toward the substrate histone. There was no significant change of cytosolic PKC; however, membrane-associated PKC activity was increased in high-glucose–treated cells. To evaluate whether the activation of PKC causes the inhibitors (H 7, staurosrine, and polymyxin B) and tested whether the effect of hyperglycemia was stillpresent when fat cells were pretreated with phorbolester (tetradecanoylphorbol acetate) for 24 h. Indeed, H 7, staurosporine, and polymyxin B blocked the inhibitory effect of hyperglycemia on IRK. Furthermore, in cells treated for 24 h with tetradecanoylphorhol acetate, no inhibitory effect of hyperglycemia was observed. This and the effect of the PKC inhibitors are consistent with a causal relationship between IRK inhibition and PKC activation