A Comparison of the Effects of Lipolytic and Antilipolytic Agents on Adenosine 3′,5′-Monophosphate Levels in Adipose Cells as Determined by Prior Labeling with Adenine-8-14C

Abstract

A simple and direct method for assaying cyclic 3′,5′-adenosine monophosphate in isolated adipose cells is described. The method consists in measuring the formation and accumulation of cyclic AMP-8-14C by cells which have been previously labeled with adenine-8-14C. By use of the described assay, the known effects of a variety of hormones on adipose cell cyclic AMP levels have been confirmed. Thus, it was shown that (a) cyclic AMP formation, stimulated by norepinephrine or corticotropin, was evident before lipolysis providing theophylline was also present; (b) in the absence of theophylline, norepinephrine induced lipolysis without elevating cyclic AMP levels; (c) in the absence of norepinephrine, theophylline also induced lipolysis without elevating cyclic AMP levels; and (d) the synergistic effect of theophylline (or puromycin) and norepinephrine on induction of lipolysis was less marked than the corresponding synergistic effect of theophylline (or puromycin) and norepinephrine on elevating cyclic AMP levels. The data are consistent with the hypothesis that cyclic AMP exists in different intracellular compartments, only one of which, comprising a small fraction of total cellular cyclic AMP, is involved in activation of the hormone-sensitive lipase. By use of the new assay, the decrease in cyclic AMP levels in adipocytes induced by insulin and prostaglandin E1 has also been confirmed. On the other hand, several proteases which mimic insulin action in isolated adipose cells have been shown in the present studies to be without effect on cyclic AMP levels. Further, polyene antibiotics, such as Filipin and pimaricin, which also mimic insulin action in vitro, lowered cellular cyclic AMP levels and markedly increased the levels observed in the medium. The effects of Filipin on (a) facilitating cyclic AMP leakage, (b) stimulating glucose utilization, and (c) inhibiting hormone-induced lipolysis were all counteracted by the presence of cholesterol in the incubation medium.