Expression of insulin receptors during preadipocyte differentiation

Abstract

3T3-L1 preadipocytes, cloned from mouse embryo 3T3 fibroblasts, differentiate in monolayer culture into cells with morphological and biochemical characteristics of adipocytes. Differentiation-linked changes in 125I-insulin binding to 3T3-L1 cells were monitored and compared with those in non-differentiating 3T3-C2 controls treated similarly. In the absence of the appropriate inducing agents, e.g. insulin, methylisobutylxanthine and dexamethasone, 3T3-L1 cells fail to express the adipocyte phenotype while maintaining a level of 25,000–35,000 insulin binding sites per cell. Induction of 3T3-L1 cells with insulin present results in an initial suppression of insulin binding followed by a 12-fold increase which parallels the appearance of differentiated cells. A maximum of 170,000 insulin binding sites per cell is attained for a population in which >75% of the cells have differentiated. The rise 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 exhibit the undifferentiated phenotype and suppressed levels of insulin binding (35,000 sites per cell). The binding capacity of 3T3-L1 cells for epidermal growth factor remains unchanged between 25,000 to 40,000 sites per cell, and is 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 rise in number, while epidermal growth factor receptors remain constant. A density-shift method is described for analysing insulin receptor synthesis and turnover in cultured cells labeled with heavy ( 2H, 13C and 15N) amino acids. Solubilized newly-synthesized “heavy” and old “light” receptors are separated by isopycnic banding on cesium chloride gradients and are then quantitated. Insulin receptor synthesis and turnover was studied by this technique in 3T3-L1 preadipocytes which undergo an increase in insulin binding capacity during differentiation. The results indicate that the rise in insulin binding capacity results from new receptor synthesis, that the insulin receptor has a relatively short half-life of 6.7 hr, and that an increased rate of receptor synthesis contributes to the elevation of insulin receptor level during differentiation.