Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta

Summary 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 mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total ⁸⁶Rb⁺ (K⁺) uptake from 0.8 ± 0.04 to 1.02 ± 0.05 nmol · mg^–1· protein^–1· min^–1 (p < 0.005). These changes in K⁺ flux, though small, can alter the membrane potential. 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 prior to insulin action, no increase in ⁸⁶Rb⁺ uptake via the Na⁺/K⁺-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50–200 nmol/l) and LY294 002 (50 μmol/l) attenuated both total insulin-stimulated ⁸⁶Rb⁺ uptake as well as uptake via the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter. Neither the inhibitor of p70 S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098 059 (50 μmol/l), had any effect on insulin's stimulation of K⁺ influx. A 10 μmol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 μmol/l it was ineffective, suggesting involvement of the atypical PKC-ζ isoform. We conclude that insulin-stimulated K⁺ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na⁺/K⁺-ATPase and Na⁺/K⁺/2Cl^– cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-ζ. [Diabetologia (1998) 41: 1199–1204] Received: 20 March 1998 and in revised form: 2 June 1998