INSULIN STIMULATION OF K-L1 FIBROBLASTS INVOLVES PHOSPHATIDYLINOSITOL3-KINASE AND PROTEIN-KINASE C-ZETA( UPTAKE IN 3T3)

Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na+/K+-ATPase and Na+/ K+/2Cl(-) cotransporter, the intracellular signalling molecules mediat ing this effect of insulin have not been elucidated. Treatment of 3T3- L1 fibroblasts with insulin increased total Rb-86(+) (K+) uptake from 0.8 +/- 0.04 to 1.02 +/- 0.05 nmol.mg(-1).protein(-1).min(-1) (p < 0.0 05). These changes in K+ flux, though small, can alter the membrane po tential. Uptake occurred through both the Na+/K+-ATPase and Na+/K+/2Cl (-) cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na+/K+/2Cl(-) cotransporter pr ior to insulin action, no increase in Rb-86(+) uptake via the Na+/K+-A TPase was observed. The structurally distinct phosphatidylinositol 3-k inase inhibitors wortmannin (50-200 nmol/l) and LY294002 (50 mu mol/l) attenuated both total insulin-stimulated Rb-86(+) uptake as well as u ptake via the Na+/K+-ATPase and Na+/K+/2Cl(-) cotransporter. Neither t he inhibitor of p70 S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098059 (50 mu mo l/l), had any effect on insulin's stimulation of K+ influx. A 10 mu mo l/l concentration of the protein kinase C (PKC) inhibitor bisindolylma leimide attenuated insulin action but at 1 mu mol/l it was ineffective , suggesting involvement of the atypical PKC-zeta isoform. We conclude that insulin-stimulated K+ uptake in 3T3-L1 fibroblasts appears to in volve concerted regulation of both the Na+/K+-ATPase and Na+/K+/2Cl(-) cotransporter and we show for the first time that this process is sig nalled via a pathway involving phosphatidylinositol 3-kinase and PKC-z eta.