We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.
Chronic alcohol ingestion was accompanied by a mild decrease in insulin binding (from 11.7 to 8.9% per 1 × 106 cells) that was accounted for by changes in the dissociation constant of insulin’s binding sites. The basal rate of 14C-glucose incorporation into glycogen was reduced both in alcoholic and pair-fed animals. Insulin stimulated 14C-glucose incorporation into glycogen in control (72% above basal rate) and pair-fed (76% above basal rate) animals. In contrast, only a minimal stimulation of glucose incorporation into glycogen (30%) induced by insulin was observed in alcoholic animals. Hepatocyte responsiveness to insulin was restored when the animals were switched back to normal dry food diet. When the hepatocytes were incubated with 50 mM alcohol for 1 h at 37 °C (in vitro experiments) insulin binding remained unchanged. There was a mild but significant decrease in insulin’s ability to enhance glucose incorporation into glycogen. The anti-catabolic effect of insulin was unaffected by alcohol. In summary, chronic alcohol ingestion causes significant but reversible changes in post-receptor events of insulin’s action.
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