Murine 3T3-L1 fibroblasts develop, under certain stimuli, many characteristics of mature adipocytes. We have studied the insulin receptors and insulin responsiveness of 2- deoxyglucose transport and glucose oxidation of these cells at three different stages of growth and differentiation: 1) sparse, growing fibroblasts; 2) confluent, undifferentiated fibroblasts; and 3) fatty fibroblasts. Sparse, growing 3T3-L1 cells had the lowest number of insulin receptors (about 9000/cell). As the growing cells reached confluence, insulin binding increased 4-fold, and with differentiation into fatty fibroblasts, a further 3-fold increase occurred. Analysis of binding data indicated that these changes were accounted for by increases in insulin receptor number with no change of receptor affinity. Growth of the cells and fatty differentiation were also accompanied by changes in glucose metabolism and insulin responsiveness. Insulin had only a minimal effect on glucose metabolism in sparse and confluent fibroblasts but had a marked effect in fatty fibroblasts. In the latter stage, the cells exhibited a 2.5- to 3.5-fold increase in glucose transport and a 4- fold increase in oxidation in the presence of insulin. Even more striking were the changes in coupling between insulin binding and biological response. Insulin binding and response in glucose oxidation were linearly coupled in sparse, growing cell+s, whereas confluent, undifferentiated cells displayed a nonlinear relationship, such that 15% receptor occupancy caused 50% of the biological response. In fatty fibroblasts, the degree of nonlinear coupling was more pronounced and, in addition, spare receptors were present for the first time. Thus, a maximal biological response required only 40% receptor occupancy. Exposure of the cells to insulin (10-6 M) for up to 20 h had no effect on insulin binding and only a minimal effect on glucose metabolism. Inhibition of protein synthesis with cycloheximide caused a reduction of both insulin binding and glucose metabolism. Insulin receptor number and 2-deoxyglycose transport decayed with a similar t1/2 (20—24 h), whereas a more rapid and parallel drop of basal and insulin-stimulated glucose oxidation was found (t1/2 = 4.5 h). The 3T3-L1 cell provides a useful model for studies of insulin binding and action in the developing fat cell. Changes occur not only in the number of receptors and magnitude of biological response, but also in the nature of the coupling between receptor and the biological effect. © 1979 by The Endocrine Society.
