Insulin Receptor Regulation and Desensitization in Rat Hepatoma Cells: Concomitant Changes in Receptor Number and in Binding Affinity

Abstract

We have studied the effects of chronic exposure to insulin on the binding and the biologic activity of the hormone using a well-differentiated cell line (Fao) derived from the Reuber H35 rat hepatoma. Prolonged incubation (24 h) with 10−6 M insulin produced a 20–25% decrease in binding of tracer concentrations (2 × 10−11 M) of125I-insulin, and a leftward shift of the curve for inhibition by unlabeled insulin. Scatchard analysis of the binding data revealed that a 75–80% decrease in the number of binding sites had occurred in the insulin-treated cells, but was accompanied by an increase in apparent receptor affinity. Kinetic studies suggested negative cooperativity in insulin binding and indicated that the change in affinity was accounted for by a decrease in the rate of dissociation. Both the decrease in receptor number and the increase in affinity were dependent on time, temperature, and the insulin concentration during the treatment period. Both effects were also blocked by cycloheximide, suggesting that they required new protein synthesis. Plasma membranes isolated from downregulated cells retained both the change in receptor number and affinity. Anti-receptor antibodies present in two human sera (B-2 and B-9) inhibited 125I-insulin binding in downregulated cells with equal or slightly greater sensitivity than in control cells. The changes in insulin binding were accompanied by changes in insulin's biologic effects in these cells. Induction of tyrosine aminotransferase (TAT) activity by insulin, an effect that is observed in control cells within 4–6 h and is half-maximal with 5 × 10−10 M insulin, was totally abolished in downregulated cells. In addition, insulin increased two- to threefold the I-form of glycogen synthase in control cells, but failed to stimulate the enzyme in downregulated cells. These results indicate that prolonged exposure of Fao hepatoma cells to insulin produces a type of downregulation characterized by a decrease in insulin receptor number and a concomitant increase in receptor affinity. This is also accompanied by a total desensitization of the cells to insulin's effects on both tyrosine aminotransferase and glycogen synthase.