Effect of Cytochalasin B and D on Groups of Insulin Receptors and on Insulin Action in Rat Adipocytes

Abstract

A B S T R A C T The possible physiological imiiportainee of the groups of insulin receptors on rat adipocytes and the relationship of these groups to insulin action were investigated. The effect of cytochalasin B aind D on l)iological actions of insulin was measured aind coimipared with the effect of these agents on the ultrastructural distribution of groups of insulin receptors. Cytochalasin B had no effect on epinephrine-stimulated lipolysis, insulin inhibition of epinephrine-stimulated lipolysis, or insulin stimulation of protein synthesis. Cytochalasin B, over a concentration rainge of 50 nM to 5 ,uM, progressively inhibited the basal glucose transport system, as measured by glucose oxidation, 2-deoxyglucose transport, and 3-O-methylglucose transport. Insulin was capable of fully stimulating remaining basal transport at submaximal concentrations of cytochalasin B. Insulin pretreatment of adipocytes partially protected the glucose transport system from inhibition by cytochalasin B. Cytochalasin B markedly altered the distribution pattern of insulin receptors, which caused an increase in the number of single receptor molecules by decreasing the number of larger groups. A significant correlation (r = 0.964; P < 0.001) was found between the percent increase in single receptors and the percent decrease in glucose transport. Ferritin-insulin pretreatment of adipocytes prevented disruption of the groups of insulin receptors by cytochalasin B. Cytochalasin D had no effect on the biological actions of insulin or on the groups of insulin receptors. These data suggest that the ability of insulin to affect adlipoeyte metabolism is independent of the hormonie occupying adjacent, groupedl receptor sites. The marked contrast in effects of cytochalasin B and D on groups of insulini receptors ancd glucose transport suggests that the microfilaimenit system is not involved in insulin actioni or in holding the groups of insulin receptors together, as both agenits are known disrupters of microfilamenits and inhibitors of actin gelation. The correlation betweeni the effects of cytochalasini B on insulin receptor distribution and glucose transport leads to the speculation that the glycoprotein molecules containiing the insulin receptor are funcetionally linked with the glucose transport systemii.

In vitro effects of a sulfonylurea on insulin action in adipocytes. Potentiation of insulin-stimulated hexose transport.

“…Several reports also suggest that the internalization process may be of little significance in mediating insulin action in adipose tissue. For example, Jarett and Smith (25) did not find coated pits on adipocyte plasma membranes when assayed by electron microscopy, and have also reported that clustering of receptors in fat cells is not related to biologic response (26). Taken together with our data, these studies suggest that tissue-specific differences may exist for potentiation of insulin action by sulfonylureas.…”

Partial disruption of naturally occurring groups of insulin receptors on adipocyte plasma membranes by dithiothreitol and N-ethylmaleimide: the role of disulfide bonds.

“…In contrast, the organizational pattern found on liver plasma membranes showed that the receptors occurred predominantly as single receptor sites (3). Cytochalasin B, but not cytochalasin D, was found to partially disrupt the groups of receptors on adipocytes as determined by both visual and computer-assisted analytical methods (2,4). These findings indicated that neither microfilaments nor actin is involved in maintaining the grouped receptors and suggested that cytochalasin B, due to its thiol binding properties (5), may prevent the formation of disulfide bonds that, in part, hold together the groups of insulin receptors.…”

“…Morphological studies of the distribution of insulin receptors on rat adipocyte plasma membranes using monomeric ferritininsulin have shown that the occupied insulin receptors occurred predominantly in natural groups of two to six receptors (1,2). In contrast, the organizational pattern found on liver plasma membranes showed that the receptors occurred predominantly as single receptor sites (3).…”

“…The findings on liver plasma membranes were confirmed by Jacobs and Cuatrecasas (9), who also found that dithiothreitol treatment ofplacental membranes decreases insulin binding to these membranes. These biochemical findings, coupled with the different organizational patterns of insulin receptors on adipocyte (1,2), liver (3,10), and placental (11) membranes suggested that, not only are disulfide bonds involved with the insulin receptors but also, some form of tissue-specific heterogeneity of insulin receptor complexing occurs.…”

Ultrastructural basis for chloroquine-induced increase in intracellular insulin in adipocytes: alteration of lysosomal function.

“…Adipocytes were prepared from epididymal fat pads of 120-g Sprague-Dawley rats by collagenase digestion as described (16 (21). Fm-I, like 125I-insulin, will identify only occupied receptor sites and, in the event that the label, the hormone, or the receptor is degraded, the localization of the label relative to the hormone receptor is unknown.…”