We have examined the ability of insulin to regulate insulin action in primary cultured adipocytes, and found that insulin induces progressive insulin resistance in this target tissue. To assess effects at both receptor and postreceptor sites, we cultured cells in the absence (control) and presence of 100 ng/ml insulin, and, after various times, measured the dose response of insulin's ability to bind cell-surface receptors and stimulate 2-deoxyglucose transport. In control cells, insulin binding (0.2 ng/ml) was increased 10–13% due to an apparent increase in receptor affinity (6–24 h). A comparable increase in affinity was also observed in treated cells; however, concomitantly, insulin decreased the number of cell-surface receptors causing a slowly progressive net decrease in binding after a 6– 10-h lag (maximal 30% at 24 h). When insulin action was assessed in control cells, the functional consequence of increased receptor binding was hypersensitization (i.e., increased insulin sensitivity) manifested by a leftward shift in the 2-deoxyglucose dose-response curve. On the other hand, in the treated cells, insulin produced insulin resistance initially by decreasing insulin sensitivity. The ED50 for insulin stimulation of glucose transport increased 84% from 0.31 to 0.57 ng/ml at 6 h without a net change in insulin binding; this was the result of a decrease in coupling efficiency between occupied receptors and the insulin effect. Receptor uncoupling progressively increased in severity, but before the full effect was reached insulin also caused a rapid decline in maximally stimulated glucose transport rates (between 6 and 10 h). This decrease in insulin responsiveness (maximal 52%) exacerbated overall insulin resistance, and was indicative of a postreceptor defect in the glucose transport system. Finally, insulin-induced receptor downregulation contributed, along with uncoupling, to a further decrease in insulin sensitivity, and constituted a more long-term regulatory mechanism. We also observed that insulin could regulate the basal glucose transport system by preventing a progressive rise in basal transport observed in control cells.

In conclusion, primary cultured adipocytes can be used to study long-term regulation of insulin action. We found that insulin induces progressive insulin resistance with sequential effects at multiple sites in the insulin action pathway, including decreased coupling efficiency between occupied receptors and stimulated glucose transport, a postreceptor defect in insulin responsiveness of the glucose transport system, and receptor downregulation. These mechanisms may be relevant to the cellular defects in insulin action present in clinical states of insulin resistance and hyperinsulinemia.

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