ACTION OF INSULIN ON NA+-K+-ATPASE AND THE NA+-K+-2CL(-) COTRANSPORTER IN 3T3-L1 ADIPOCYTES

The Na+-K+-ATPase presents several different isoforms of its alpha- and beta-subunits. We detected alpha(1)- and beta(1)-mRNA transcripts and polypeptides in 3T3-L1 fibroblasts; during differentiation into adipocytes, alpha(1)-mRNA decreased, alpha(2)-mRNA was induced, beta(1)-mRNA dropped to undetectable levels, and beta(2)-mRNA was never expressed, suggesting that 3T3-L1 adipocytes may express an unidentified Na+-K+-ATPase beta-subunit isoform. Insulin rapidly increased ion pump activity [ouabain-sensitive Rb-86(+) (K+) uptake] in 3T3-L1 fibroblasts and adipocytes without changing the plasma membrane concentration of alpha(1) or alpha(2)-subunits as determined by subcellular membrane fractionation and immunoblotting or by [H-3]ouabain binding to intact cells. Monensin, which raises the concentration of intracellular Na+, increased Na+-K+ pump activity, and no further stimulation was achieved with insulin. The stimulation of the pump by insulin was reduced by bumetanide, an inhibitor of the Na+-K+-2Cl(-) cotransporter, and was prevented by omission of extracellular Cl-. Insulin increased both ouabain-sensitive and bumetanide-sensitive Rb-86(+)(K+) uptake. These results suggest that insulin activation of the Na+-K+-ATPase in 3T3-L1 adipocytes is mediated by an elevation in intracellular Na+ that is likely the consequence of Na+-K+-2Cl(-) cotransporter activation.