Development of insulin responsiveness of the glucose transporter and the (Na+,K+)-adenosine triphosphatase during in vitro adipocyte differentiation.

The development of insulin responsiveness of the transport systems for glucose (2-deoxyglucose) and potassium (Rb+) was compared during in vitro adipocyte differentiation of 3T3-L1 cells. Growing cells exhibited minimal increases in 2-deoxyglucose and Rb+ transport rates in response to insulin. Low levels of insulin stimulation of both transport processes became apparent when cultures attained confluence, and these levels were maintained in uninduced cells which retained their fibroblast-like morphology. Following a 48-h induction treatment with methylisobutylxanthine and dexamethasone, a dramatic and simultaneous increase in insulin sensitivity of both deoxyglucose and Rb+ uptake was observed. In fully differentiated 3T3-L1 adipocytes, insulin caused a 6-10-fold increase above the basal rate for deoxyglucose uptake and a 1.50-1.60-fold increase of Rb+ uptake. The insulin dose response relationships were identical for both deoxyglucose uptake and Rb+ uptake and half-maximal stimulation occurred at insulin concentrations of 2-3 nM in 3T3-L1 fibroblasts, 550 pM in 3T3-L1 adipocytes, and 100 pM in mature rat adipocytes. Basal rates of deoxyglucose and Rb+ uptakes were 2-9-fold higher in growing cells than in confluent cells, and fatty cells exhibited lower transport rates relative to nonfatty cells. The number of active Na+ pumps on the cell surface was determined by quantitation of the covalent phosphorylated intermediate of the (Na+,K+)-ATPase. Plasma membranes of growing cells contained a larger number of (Na+,K+)-ATPase specific phosphorylation sites and higher (Na+,K+)-ATPase activity than those of confluent cells. Although (Na+,K+)-ATPase and Rb+ transport activities were greater in uninduced cells than in induced cells, both cultures exhibited the same number of phosphorylation sites, implying that the Na+ pump in 3T3-L1 adipocytes was operating at a reduced efficiency.