When isolated rat fat cells were incubated with epinephrine or adrenocorticotropin, release of glycerol and nonesterified fatty acids was greater in the presence of 40 ng. per milliliter of insulin than with 2 ng. per milliliter of insulin. Enhanced lipolysis with the higher dose of insulin occurred in the absence of added carbohydrate, did not involve re-esterification of fatty acids, could not be attributed to the presence of glucagon, and was abolished by incubation with anti-insulin gamma globulin or by prior trypsinization of fat cells. Insulin did not augment lipolysis induced by theophylline or added cyclic 3′,5′-adenosine monophosphate (cyclic AMP).

In the presence of 5.5 μM. epinephrine, the initial rate of lipolysis was decreased by insulin in the amount of 2 ng. per milliliter but not by the higher dose, 40 ng. per milliliter. The ability of fat cells to continue accelerated lipolysis was prolonged by 40 ng. per milliliter of insulin, compared with epinephrine alone.

Cycloheximide, and probably puromycin, partially inhibited enhancement of lipolysis by insulin, 40 ng. per milliliter, in the presence of 5.5 μM. epinephrine. This effect of cycloheximide first appeared 120 minutes after exposure of cells to the drug. Responses to epinephrine alone and to 2 ng. per milliliter of insulin were not affected by these drugs.

These results imply that insulin can enhance the lipolytic activity of hormonal stimulators of adenylate cyclase by actions which at least in part require protein synthesis. Indirect evidence suggests that cyclic AMP mediates the lipolytic effects of insulin.