Expression of the adipocyte phenotype by differentiating 3T3-L1 preadipocytes occurs upon exposure of the cells to insulin. Differentiation-linked changes in "25I-labeled insulin binding to 3T3-L1 cells were monitored and compared with those in nondifferentiating 3T3-C2 controls treated similarly. Without chronic insulin treatment, 3T3-L1 cells failed to express the adipocyte phenotype but maintained a level of 25,000-35,000 insulin-binding sites per cell. Treatment of 3T3-L1 cells with insulin resulted in an initial suppression of insulin binding followed by a 12-fold increase that paralleled the appearance of differentiated cells. A maximum of 170,000 insulin-binding sites per cell was attained for a population in which >75% of the cells had differentiated. The increase of insulin receptor level appears to be differentiation-dependent and is not a general response of cells to the culture conditions. 3T3-C2 cells maintained in the presence of insulin for 30 days exhibited the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The bindin capacity of 3T3-L1 cells for epidermal growth factor remained unchanged between 25,000 and 40,000 sites per cell and was independent of the state of differentiation. Thus, induction by insulin of differentiation-linked expression in 3T3-L1 cells results in receptor-specific changes. Insulin receptors increase in number but epidermal growth factor receptors remain constant.3T3-L1 preadipocytes, a subline of mouse 3T3 fibroblasts cloned by Green and associates (1, 2), undergo differentiation in culture to cells with morphological and biochemical characteristics of adipocytes. Accompanying adipocyte conversion are marked increases in the synthesis and deposition of cytoplasmic fat (3), as well as a coordinate increase in the activities of enzymes of the lipogenic pathway (4, 5). The increase in activity of one of the enzymes of this pathway, acetyl-CoA carboxylase, is due to an increase in the amount of enzyme per cell rather than to a change in its specific activity (4).Under usual culture conditions, expression of the adipocyte phenotype in 3T3-L1 cells occurs only after cell division has ceased at confluency (6). The time required for confluent cultures to express the adipocyte phenotype can be modified by agents or hormones known to affect the lipogenic or lipolytic process (2). Expression is accelerated by chronic exposure of the cells to insulin and may be further enhanced by brief treatment of the cells at confluence with prostaglandin F2a or methylisobutylxanthine (7). Epinephrine and dibutyryl cyclic AMP have the opposing effect (2). The mechanisms by which these agents alter the differentiation process are unknown.Preadipocyte differentiation also leads to changes in the responsiveness of 3T3-L1 cells to hormones known to regulate the metabolism of adipocytes. For example, adenylate cyclase of differentiated 3T3-L1 cells is stimulated by isoproterenol or corticotropin (ACTH), but in undifferentiated cells the response is ...

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