Effects of Lipolytic and Antilipolytic Agents on Cyclic 3',5'-Adenosine Monophosphate in Fat Cells

Abstract Isolated fat cells were incubated in Krebs-Ringer phosphate medium. Within 1 min after the addition of 1.1 µm epinephrine, the cyclic AMP content of the cells was elevated. It continued to rise, reaching a peak in 4 to 7 min, and fell thereafter, being only slightly above the control levels after 10 to 15 min. A maximal rate of glycerol production was established within 1 to 2 min, well before the peak in cyclic AMP concentration, and was maintained unchanged while the cyclic AMP level continued to rise and then fell. Both in magnitude and time course, the effect of adrenocorticotropin (ACTH), 0.2 unit per ml, on cyclic AMP levels and on lipolysis was exactly like that of epinephrine (or of both hormones together). Addition of more epinephrine or of ACTH at several times during 1 hour of incubation with 1.1 µm epinephrine did not change cyclic AMP levels. The effects of epinephrine on cyclic AMP levels and on glycerol production were unaltered by the presence in the medium of 4 mm glucose. When propranolol (or insulin) was added several times after epinephrine-simulated lipolysis had begun, the rate of glycerol production fell to zero within 1 to 2 min. In the same period, the cyclic AMP concentration fell to a basal level; i.e. the rate of decrease was much greater than that observed after 4 to 7 min of incubation with epinephrine alone. Furthermore, after incubation of cells for 20 min with epinephrine, the addition of 0.8 mm theophylline produced an 8-fold rise in cyclic AMP content measured 5 min later. This increment was, however, only a fraction of that observed during the first 5 min after addition of epinephrine in the presence of theophylline. Since propranolol inhibits the action of epinephrine but does not alter basal adenyl cyclase activity, and, since 0.8 mm theophylline alone did not alter cyclic AMP levels, it may be concluded that adenyl cyclase was still epinephrine-stimulated during the time when cyclic AMP levels were falling. Whether the rate of formation of cyclic AMP during this period was equal to that during the first minutes after addition of the hormone cannot be established. Attempts to show directly alterations in phosphodiesterase activity during incubation of cells with epinephrine have been unsuccessful. When the effect of epinephrine was terminated after 4 min by the addition of propranolol, introduction of ACTH at several times during the following 20 min caused an immediate resumption of glycerol production at a rate equal to that observed with the initial addition of epinephrine. Cyclic AMP levels, however, rose little if at all in 4 to 5 min after addition of ACTH. The ability of cells to elevate cyclic AMP levels a second time in response to hormonal stimulation was restored when cells that had been incubated for 20 min were washed and suspended in fresh medium. There appears to be something accumulated in the medium during incubation of cells with epinephrine that interferes with the effectiveness of the hormone in causing accumulation of cyclic AMP in fresh cells. Whether this material has its effect on the rate of formation or of degradation of cyclic AMP cannot be decided.