Insulin receptor synthesis and turnover in differentiating 3T3-L1 preadipocytes

3T3-L1 "preadipocytes" can be induced to differentiate in culture into cells having the morphological and biochemical characteristics of adipocytes. The binding of 125I-insulin to the cell-surface of differentiated and undifferentiated 3T3-L1 cells and nondifferentiating 3T3-C2 cells was compared. In the absence of agents which induce adipocyte conversion, ie, insulin or insulin plus methylisobutylxanthine (MIX) and dexamethasone (DEX), 3T3-L1 cells fail to express the adipocyte phenotype and maintain a constant number of insulin binding sites. Induction of adipocyte conversion with 3T3-L1 cells in the presence of insulin causes apparent down-regulation of insulin receptors followed by a 12--15-fold increase in receptor number which parallels differentiation. Approximately 170,000 insulin binding sites per cell are expressed when greater than 75% of the cells have differentiated. The rise of insulin receptor level is differentiation-dependent. 3T3-C2 cells, which do not differentiate in the presence of insulin or insulin plus MIX and DEX, exhibit only insulin-induced down-regulation of insulin receptors. The increase of insulin receptor level in 3T3-L1 cells in receptor-specific since the levels of epidermal growth factor receptor or choleragen receptor, respectively, remain constant or decrease substantially. A heavy isotope, density-shift technique was used to analyze insulin receptor synthesis and turnover in cells labeled with "heavy" (2H, 13C, and 15N) amino acids. Solubilized newly-synthesized "heavy" and old "light" receptors were separated by isopycnic banding on CsCl gradients and quantitated. The size of the soluble receptor isolated after isopycnic banding in CsCl gradients is approximately 400,000 daltons. Mixing of "light" and "heavy" membranes prior to extraction of receptor revealed no change in "light" or "heavy" receptor isopycnic banding densities. Thus, no detectable interchange of subunits occurs between receptor molecules during extraction or equilibrium centrifugation. Insulin receptor synthesis and turnover, studied by the density-shift technique showed that the rise of receptor level during differentiation results primarily from an increased rate of receptor synthesis. The rate of insulin receptor degradation was not significantly altered. The t1/2 for degradation of the insulin receptor in differentiated 3T3-L1 cells in culture was 6--7 hours in the presence of insulin. Removal of insulin from the medium did not materially affect the rate of receptor degradation. Inhibition of protein synthesis with cycloheximide causes a lengthening of the t1/2 for insulin receptor degradation to 26 hours. Thus, the synthesis of a short-lived protein appears to be required for a critical step in the pathway of insulin receptor degradation.