A study of insulin-receptor internalization and recycling was undertaken in primary cultures of rat hepatocytes and a human hepatoma cell line (HepG2). Receptors were quantitated by measuring 125I-insulin binding to partially purified soluble receptor preparations from untreated cells (total receptors) and trypsinized cells (intracellular receptors). In resting HepG2 cells, exposure to insulin results in internalization of insulin receptors, the rate and extent of which is dependent on the insulin concentration. However, receptors do not accumulate inside the cell in proportion to the higher rates of internalization at high concentrations of insulin. This lack of accumulation is explained by much higher recycling rates after exposure to high concentrations of insulin. Similar results were noted for primary cultures of rat hepatocytes. These results imply qualitatively different fates for receptors internalized after exposure to different concentrations of insulin. To further investigate the possibility of different pathways for insulin-receptor internalization and processing, cells in low (1 ng/ml) or high (100 ng/ml) concentrations of insulin were exposed to drugs or treatments known to affect receptor metabolism. Hypotonic shock and hypokalemia, which arrest coated-pit formation, blocked internalization of insulin and insulin receptors at low concentrations of insulin but allowed internalization in response to high concentrations of insulin. The lysosomotropic drugs monensin and chloroquine caused intracellular accumulation of insulin and its receptors internalized at low concentrations of insulin but had a relatively smaller effect on receptors internalized at high concentrations of insulin. All internalization is blocked by 2,4-dinitrophenol. We conclude that high doses of insulin lead to insulin-receptor internalization and recycling through a pathway that is functionally distinct from the pathway taken by receptors internalized by low (physiologic) concentrations of insulin. The pharmacologic experiments raise the possibility that the high-dose pathway, unlike the low-dose pathway, may proceed independently of coated pits and endosomal acidification.

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