INSULIN STIMULATES BOTH THE ALPHA-1 AND THE ALPHA-2 ISOFORMS OF THE RAT ADIPOCYTE (NA+,K+) ATPASE - 2 MECHANISMS OF STIMULATION

Results obtained with adipocyte ghosts indicated that the relative pumping activities of the alpha-1 and alpha-2 isoforms of the (Na+,K+) pump depend strongly on intracellular sodium concentration, [Na+]i (McGill, D. (1991) J. Biol. Chem. 266, 15817-15823). Accordingly, [Na+]i was determined in rat adipocytes as a function of ouabain concentration and found to increase gradually as the concentration of ouabain increased. Incubation conditions were therefore designed such that the [Na+]i at 0 M and 10(-5) M ouabain were identical, in order to study the activities of both forms of the pump under identical conditions. Under these conditions, the alpha-2 isozyme accounts for 42% of the total pumping activity; these data prove that the activity of the alpha-2 isozyme is suppressed to a much greater extent than that of the alpha-1 isozyme, in relation to maximally obtainable activities measured in plasma membranes (Lytton J., Lin, J. C., and Guidotti, G. (1985) J. Biol. Chem. 260, 1177-1184). Furthermore, insulin stimulation of Rb-86(+)/K+ uptake in adipocytes results from a 58 and a 128% increase in the activities of the alpha-1 and alpha-2 isozymes (Na+,K+) pump, respectively. In addition, it is shown that under the conditions used to determine the [Na+]i dependence of Rb-86+/K+ uptake into adipocytes (0 mM KCl, various [NaCl]), [Na+]i decreases rapidly upon the addition of KCl/(RbCl)-Rb-86 for the initiation of the uptake measurement. By making uptake measurements quickly after the addition of KCl to eliminate the effect of a decreasing [Na+]i, we demonstrate that the stimulation of the alpha-1 isozyme is due to a small decrease in the K0.5Na+ whereas the stimulation of the alpha-2 isozyme results from a decrease in the K0.5Na+ and an increase in the V(max).