Insulin and phorbol esters affect the maximum velocity rather than the half-saturation constant of 3-O-methylglucose transport in rat adipocytes.

The kinetics of the equilibrium exchange flux of 3-O-methylglucose (MeGlc) were examined in isolated rat adipocytes using a recently described technique (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899) in which the cells, under basal conditions, were reported to exhibit a high Km (35 mM) that was reduced (to 3 mM) upon treatment with insulin. When this technique was employed in the present study, the Km observed in basal adipocytes was 6.4 +/- 0.4 mM; insulin treatment did not affect this parameter (6.3 +/- 0.5 mM), although it increased the maximum velocity (phi max) 21-fold (from 3.0 +/- 0.3 to 63.7 +/- 1.1 nmol X min-1 X microliter of intracellular water-1). The large discrepancy in the basal Km values observed in the previous (35 mM) and the present (6.4 mM) studies is shown to be associated with relatively minor differences in basal MeGlc flux; these minor differences may reflect insufficient mixing of labeled MeGlc in the flux measurements of the previous study. In addition, the active phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, at a concentration of 0.3 microM, caused a 2.8-fold elevation of phi max, with no modulation of Km. These results indicate that phi max, not Km, is the major kinetic parameter of hexose transport affected by insulin and phorbol esters, leading to enhancement of hexose uptake by the isolated rat adipocyte.