Actions of Insulin in Fat Cells

When isolated rat epididymal fat cells were incubated with [*2511iodoinsulin for 5 min at 37”, radioactivity accumulated in the plasma membrane fraction (Peak 1) and an unidentified particulate fraction (Peak 2) as reported previously (Kono, T., Robinson, F. W., and Sarver, J. A. (1975)5. Biol. Chem. 250, 7826-7835). This accumulation of radioactivity in Peak 2 (but not that in Peak 1) was greatly impaired when cells were incubated with iodoinsulin in the presence of a variety of metabolic inhibitors that reduce the cellular content of ATP. The reduction in the ATP level coincided with a disappearance of the stimulatory effects of insulin on sugar transport and the hormone-sensitive phosphodiesterase. In contrast, ATP depletion had no significant effects, at least during a 5to 15-min incubation, on the intracellular water space and on the basal sugar transport and phosphodiesterase activities. When cells once depleted of ATP by treatment with 2,4dinitrophenol(1 mM; 10 min) were washed and suspended in fresh buffer, the ATP level was recovered almost fully in 10 min. This recovery coincided with the restoration of responsiveness to insulin. When cells were incubated with [12511iodoinsulin or insulin for 5 min at 15” instead of 37”, a negligible quantity of radioactivity accumulated in Peak 2 and insulin failed to activate sugar transport. In contrast, under the same conditions, radioactivity accumulated in Peak 1 and insulin stimulated phosphodiesterase considerably. These results suggest that ATP, or some other compound metabolically related to ATP, may be necessary for the actions of insulin on sugar transport and phosphodiesterase. ATP, or some other related compound, may also be necessary in the formation of the radioactive Peak 2, although the physiological function and cellular location of this peak are yet to be ascertained.