Insulin-receptor interaction in isolated fat cells.

Suspensions of fat cells were prepared from rat epididymal adipose tissue by digestion of the tissue with crude bacterial collagenase. One portion of each suspension was exposed to p-chloromercuribenzenesulfonic acid (CMS), washed, and then incubated in Krebs-Ringer-bicarbonate buffer. A second portion, the control, was not exposed to CMS, but it was otherwise treated identically. Acceleration of glucose transport was estimated from the rate of conversion of glucoseJ4C to “CO2 and 14C-labeled lipids. Inhibition of the lipolytic action of adrenocorticotropic hormone (ACTH) was evaluated by measuring (a) the cyclic 3’,5’-AMP concentration of the cell suspensions and (b) the rate of release of glycerol into the incubation medium. The optimal concentration of CMS was 10m3 M and the optimal exposure time was 30 min. Glucose utilization by cells exposed to CMS was 5 to 20 times that of the control, but only 50 to 80% that of cells maximally stimulated by insulin. Most of the increased glucose utilization was due to increased glucose entry via a speciiic transport system since glucose utilization, when stimulated by CMS, was inhibited by j-o-methyl glucose. Cells exposed to CMS showed a slight increase in nonspecsc permeability, however, since (a) there was a slight increase in the amount of malic dehydrogenase appearing in the incubation medium, and (b) stimulation by CMS plus insulin exceeded that of insulin alone when the glucose concentration of the incubation medium was <20 mu. Incubation of CMS exposed cells in 2,3-dimercaptopropanol resulted in partial reversal of the stimulatory effect of CMS. Exposure of cells to CMS inhibited lipolysis both in the basal state and when lipolysis was stimulated by ACTH. This inhibition was overcome either by a IO-fold increase in the concentration of ACTH or by the addition of theophylline, showing that the CMS exposure had not irreversibly inhibited the lipolytic enzymes of the cell. The antilipolytic action of CMS was probably mediated through an effect on the adenyl cyclase system since the increase in cyclic 3’,5’-AMP produced by ACTH was inhibited by the CMS exposure. This inhibitory effect of CMS was likewise overcome by a IO-fold increase in the ACTH concentration.