We have investigated the theory that the insulininduced loss of insulin binding from adipocytes is due to internalization of insulin receptors. Cell-surface receptors were assessed by the binding capacity of intact cells at 160C. Total (i.e., cell-surface plus intracellular) receptors were assessed by solubilizing the cells in 1% Triton X-100 and then measuring binding by the solubilized extract. Intracellular receptors were measured by treating the cells with trypsin before solubilizing them. The trypsin treatment removed >90% of the cell-surface binding, so that any significant binding by soluble extracts ofthese cells must represent intracellular receptors. Adipocytes were incubated with insulin (100 ng/ml) with or without chloroquine (0.2 mM) for 4 hr. Insulin alone resulted in a 62% loss of cell-surface receptors, but only a 46% loss of total receptors, and a 170% increase in intracellular receptors, suggesting that the lost cell-surface receptors were internalized, where some were degraded. Insulin in the presence of chloroquine resulted in a 34% loss of cell-surface receptors, but no loss of total receptors, and a 300% increase in intracellular receptors. Thus, in the presence of chloroquine receptors were internalized but not degraded. The loss ofcell-surface receptors and appearance of intracellular receptors were time and dose dependent and were linearly related. These results demonstrate that insulin causes translocation of insulin receptors from the cell surface to the cell interior, where they can be degraded (or inactivated) by a chloroquine-sensitive process.In many physiological and pathological conditions involving hyperinsulinemia, tissues possess a markedly reduced number of insulin receptors per cell (1-5). In vitro evidence supports the theory that it is the hyperinsulinemia that causes the loss of receptors, and this process has been termed "down-regulation" (6). Thus, insulin-induced receptor loss has been demonstrated in cultured human lymphocytes (6, 7), adipose tissue (8, 9), isolated adipocytes (10), cultured fibroblasts (11), and cultured hepatocytes (12). However, the fate of the lost receptors is unknown. After binding to its receptor the hormone is taken up into the cell and degraded (13-19). Some authors have suggested that the receptor is internalized along with its ligand (20)(21)(22) and that this is one mechanism underlying the eventual decrease in cellular insulin receptors. However, the data to support this hypothesis are circumstantial, and direct evidence to validate this idea is lacking. Therefore, to test this hypothesis, we have assessed the translocation ofinsulin receptors from the cell surface to the cell interior.MATERIALS AND METHODS Materials. Porcine monocomponent insulin was generously supplied by Ronald Chance of Eli Lilly; Na'25I was purchased from New England Nuclear; bovine serum albumin (fraction V), from Armour Pharmaceuticals (Chicago, IL); collagenase, from Worthington; polyethylene glycol (approximate molecular weight 6000), bovine gamma globul...