Effects of Insulin on the Levels of Adenosine 3′:5′-Monophosphate and Lipolysis in Isolated Rat Epididymal Fat Cells

Abstract Effects of insulin on the level of adenosine 3':5'-monophosphate (cyclic AMP) and the rate of lipolysis in isolated rat epididymal fat cells were determined under various conditions. When cells were incubated with 0.01 to 100 nm insulin, it gave: (a) a biphasic inhibitory effect that was maximal between 0.1 and 1 nm and submaximal at either lower or higher insulin concentrations on lipolysis stimulated by 0.5 mm dibutyryl cyclic AMP, 10 nm adrenocorticotropic hormone (ACTH), or 1 to 10 µm norepinephrine; (b) a monophasic inhibitory effect that was maximal at 1 to 100 nm on lipolysis induced by 5 or 20 mm cyclic AMP or 1 to 4 mm caffeine; and (c) a monophasic stimulatory effect that was maximal at 10 to 100 nm on lipolysis induced by 1 mm dibutyryl cyclic AMP or 0.1 to 1 mm norepinephrine. Under the above conditions, lipolysis was either inhibited or stimulated by either 10 times recrystallized Novo insulin or a highly purified single component insulin, but not by modified insulin preparations that were inactive in stimulating glucose utilization in fat cells. On the other hand, neither the inhibitory nor stimulatory effect of insulin was observed when the cellular insulin receptor or receptors were modified by exposure of cells to trypsin (1 mg per ml) for 15 min. Insulin at either 0.2 or 10 nm significantly decreased the cellular level of cyclic AMP in the presence or absence of various lipolytic agents tested, except in the presence of high concentrations (0.1 to 1 mm) of catecholamines (see below). The effect of insulin to lower the cellular level of cyclic AMP was seemingly large enough to account for the antilipolytic effect of this hormone when the cyclic AMP level was low (or when the determinations were made in the presence of 2 to 5 mm caffeine, 10 to 100 nm glucagon, or less than 10 nm ACTH) but not when the nucleotide level was high (or when the determinations were made in the presence of 1 µm norepinephrine or 10 to 100 nm ACTH). Insulin at 10 nm further increased the levels of cyclic AMP and lipolysis that were highly elevated by 0.1 to 1 mm norepinephrine, whereas insulin at 0.2 nm had no significant effects on the two parameters under these conditions. On the other hand, either in the absence of any lipolytic agent or in the presence of 2 mm caffeine or 10 nm ACTH, insulin at 10 nm lowered the level of cyclic AMP to a larger degree than it did at 0.2 nm. Consequently, the effect of insulin on the cyclic AMP level was monophasic while its effect on lipolysis was biphasic (see above) in the presence of 10 nm ACTH. The effects of the insulin-like action of trypsin on the levels of cyclic AMP and lipolysis were similar to those of insulin at 0.2 nm. Incidentally, all the effects of norepinephrine noted above were observed with epinephrine. The above mentioned insulin effects do not necessarily indicate the physiological effects of this hormone since they were observed in the presence of wide concentration ranges of insulin and certain lipolytic agents. Thus, the above data suggest that, under the given in vitro conditions, insulin (rather than its possible contaminants) induces multiple effects on the levels of cyclic AMP and lipolysis depending on its concentration and on the nature and concentrations of the lipolytic agent used. Part of the difference in the effects of lipolytic agents might be explained by the difference in the cyclic AMP levels elevated by these agents. Since all the insulin effects tested seem to be mediated by the cellular insulin receptor or receptors, it is suggested that the insulin receptor system of fat cells can respond to a wide concentration range (approximately from 0.01 to 100 nm) of this hormone.