We have examined the relationship between insulin-induced receptor downregulation and the induction of a postreceptor defect in the insulin-stimulated glucose transport system in rat adipocytes, and found that downregulation was linked to the expression of the postreceptor defect. When recycling of insulin receptors was inhibited by 20 mM Tris, insulin pretreatment (100 ng/ml) for 4 h at 37 degrees C induced both net loss (65%) of cell-surface receptors and a 63% decrease in maximal insulin responsiveness. In contrast, when cells were treated with insulin alone for 4 h at 37 degrees C so that receptors could recycle, or treated at 16 degrees C with Tris plus insulin to inhibit receptor internalization, neither receptor downregulation nor a postreceptor defect was observed. Induction of the postreceptor defect was specific for insulin under conditions when downregulation would occur, since treatment of cells with Tris and the insulin mimicker spermine did not result in receptor loss or the postreceptor defect. Other experiments revealed that receptor downregulation occurred first without loss of insulin responsiveness, but, once the postreceptor defect appeared, its severity was correlated to the degree of further receptor loss, as a function of insulin dose and exposure time. Tris (20 mM) alone acutely decreased maximally stimulated glucose transport rates slightly (22%), but this effect was rapidly reversible after Tris removal and could not have been directly responsible for the lasting and profound postreceptor defect seen after pretreatment with insulin plus Tris. Taken together, these data suggest that insulin-induced receptor loss is linked to the induction of the postreceptor defect. The postreceptor defect was due to an inability to maximally increase the maximum velocity of glucose transport. Furthermore, the expression of the postreceptor defect depended upon the extent to which the glucose transport system was allowed to deactivate; maintaining the glucose transport system in an activated state prevented its expression. Thus, the mechanism could involve rapid inactivation or sequestration of glucose transporters during deactivation such that they become refractory to the subsequent stimulatory effects of insulin. In conclusion, (a) insulin does not acutely induce a postreceptor defect in the glucose transport system of adipocytes without loss of cell-surface insulin receptors; (b) the defect in stimulated glucose transport has been induced distal to the insulin receptor via a mechanism linked to receptor loss; and (c) the postreceptor lesion is due to decreased number of intrinsic activity of glucose transporters on the cell-surface in the presence of a maximally effective insulin concentration. These data suggest that insulin receptor downregulation and postreceptor defects in insulin action, which frequently co-exist both in vivo and in vitro, may be linked mechanistically.
W T Garvey, J M Olefsky, S Marshall