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Abstract
Glucocorticoids are known to rapidly inhibit glucose transport when added to isolated rat adipocytes. To determine whether this inhibition of transport persists following isolation of the plasma membranes, adipocytes were incubated in the absence or presence of a maximally inhibitory concentration of dexamethasone, a synthetic glucocorticoid, and plasma membrane vesicles were prepared. D-Glucose uptake into vesicles from steroid-treated cells was inhibited by an average of 40%. The ability of dexamethasone to inhibit transport depended upon pretreatment of cells with hormone prior to membrane isolation. Furthermore, the decreased rate of transport was prevented by the simultaneous addition to the cell of actinomycin D or cycloheximide with dexamethasone, indicating a requirement for RNA and protein synthesis. The effect of dexamethasone on glucose transport was further investigated using our recently developed cytochalasin B affinity-labeling protocol to identify the transporter on sodium dodecyl sulfate-polyacrylamide gels. A peak of radioactivity having Mr = 54,000 was identified which exhibited the properties expected for the glucose transporter, in that label incorporation was prevented by D-glucose and unlabeled cytochalasin B, but not by D-sorbitol or unlabeled cytochalasins A, D, or E. Dexamethasone was found to cause a significant (average 33%) decrease in the amount of labeled transporter in the plasma membrane which was prevented by the simultaneous addition of actinomycin D with dexamethasone to the cells. A similar percentage decrease was not found in a microsomal membrane fraction nor in a total cellular membrane fraction. These results suggest that glucocorticoids may decrease glucose transport in rat adipocytes by selectively decreasing the number of transporters in the plasma membrane.
Highlights
Glucocorticoids are known to rapidly inhibigtlucose hepatoma tissue culturceells
The abilityof dexamethasone to inhibittrans- glucocorticoids toinhibitnutrienttransport.The glucose port depended upon pretreatment of cells with hor- transport system inadipocytes has beenwell characterized in mone prior to membrane isolation
We report that the inhiprevented by D-glucose and unlabeled cytochalasin B, bition of glucose transport caused by dexamethasone added but not by D-sorbitol or unlabeled cytochalasins A, D, to intactcells persists throughout membraneisolation proceor E
Summary
Protein from each experimental treatment (based on a Lowry protein assay, Ref. 28) were used, regardless of the total protein yield. Cells were centrifuged and washed with 0.25 M sucrose, 1 mM EDTA, and 10 mM Tris-HC1,pH 7.4 (sucrose-Tris buffer) at room temperature The same for both D- and L-glucose and for membranes of all experimental conditions, including membranes from cells treated with dexamethasone or insulin. The resultingpellet was resuspended in ice-coldbuffer containing 0.25 M sucrose, 1mM EDTA, and 10 mM sodium phosphate, pH 7.4 (sucrose-phosphate buffer) These membranes exhibited as much as 8-fold enrichment of 5' nucleotidase specific activity over the startinghomogenate. All samples were quick frozen using a dry ice-acetone bath and stored a t -70'C Note that for both thetransportand affinitylabeling experiments described below, equal amounts of membrane transporter was affinity labeled with [3H]cytochalasinB using modifications of the technique we developed to label the hexose transporter in isolated human erythrocytes [17]. The average degree of tose:N-acetylglucosamine galactosyltransferase was determined by transport inhibition in 8 different experiments was found to the method of Bergeron et al [31]
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