The relationships between cyclic AMP (cAMP) levels and glycerol release (lipolysis) were determined for isoproterenol and forskolin under varied conditions in the isolated fat cell of the rat. Prevention of the inhibitory action of endogenous adenosine [by adenosine deaminase (100 mU/ml) or theophylline (3.3 X 10(-4) M)] resulted in increased levels of cAMP and increased rates of lipolysis with forskolin. However, the relationship between cAMP levels and rates of lipolysis remained the same under all conditions. N6-phenylisopropyladenosine (PIA; an analog of adenosine) abolished the increase in cAMP level produced by isoproterenol (10(-7) M) or forskolin (10(-6) M) and the lipolytic response to forskolin. However, PIA failed to inhibit completely the lipolytic response to isoproterenol. Dose-response curves to isoproterenol were determined in the presence and absence of adenosine deaminase. PIA (10(-6) M) inhibited the increase in cAMP levels under both conditions. PIA also inhibited the lipolytic responses that were associated with increases in cAMP levels, i.e., high concentrations of isoproterenol alone and isoproterenol with adenosine deaminase. A plot of cAMP levels against corresponding rates of lipolysis for all conditions agreed with previous observations that the relationship for isoproterenol differs from that for forskolin. At any concentration of cAMP the corresponding lipolytic response was greater for isoproterenol than for forskolin. The possibility of a cAMP-independent lipolytic response was discussed.

Using the flask-incubated fat cell system, alterations in glycerol release (lipolysis) and cAMP accumulation were determined after incubation with isoproterenol or forskolin. These agents caused concentration-dependent increases in both cAMP accumulation and lipolysis. The maximum responses to forskolin for each variable were greater than the corresponding responses to isoproterenol. The maximum responses to isoproterenol for both cAMP accumulation and glycerol release were increased by the presence of either adenosine deaminase or theophylline. Under these conditions, high concentrations of isoproterenol continued to increase cAMP accumulation while having no further effect on lipolysis. These results support the concept that the maximum response to isoproterenol alone was limited by the accumulation of cAMP within the cells. The maximum response to isoproterenol in the presence of either theophylline or adenosine deaminase (and to forskolin) was limited by some step in the lipolytic process distal to cAMP accumulation. The relationships between cAMP levels and lipolysis for isoproterenol and forskolin were found to be different. A 6-fold increase in cAMP levels was sufficient to maximally increase lipolysis with isoproterenol, whereas the maximum lipolytic response to forskolin was associated with a 20-fold increase in cAMP levels. A plot of log cAMP vs. glycerol release resulted in linear relationships for both drugs. The slope of the line for isoproterenol was significantly greater than that for forskolin. At any given concentration of cAMP the corresponding lipolytic response was greater for isoproterenol than for forskolin.

Using the flask-incubated fat cell system, the effects of Ca2+ removal from the incubation medium on the lipolytic system were studied. The removal of Ca2+ resulted in a total abolition of the lipolytic response and the increased cyclic AMP accumulation produced by ACTH. The lipolytic response to isoproterenol and forskolin were reduced approximately 40% by Ca2+ removal, but cyclic AMP accumulation was not altered in the presence of either of these agents using a Ca2+-free medium. The lipolytic response to the dibutyryl analog of cyclic AMP was also reduced by omission of Ca2+ from the incubation medium. It is concluded the Ca2+ is required for the interaction of ACTH with its receptor and the resultant activation of adenylate cyclase. Ca2+ also is required at some step in the lipolytic process distal to cyclic AMP.

Using the flask-incubated fat cell system, effects of isoproterenol and forskolin on glycerol release, cyclic AMP levels and protein kinase were studied. Isoproterenol increased cyclic AMP levels, protein kinase activity and glycerol release over the same concentration range (10(-9) M to 10(-6) M). Forskolin also increased all three variables in a concentration-dependent manner (10(-7) M to 10(-4) M). The maximum response for each variable was significantly greater with forskolin than with isoproterenol. A combination of isoproterenol and forskolin resulted in an additional increase in cyclic AMP over forskolin alone, but no significant increase in protein kinase activity or glycerol release. These results support the concepts that the maximum lipolytic response to isoproterenol is limited by the accumulation of cyclic AMP and the maximum lipolytic response to forskolin is limited by some step distal to cyclic AMP production, possibly activation of protein kinase. At high concentrations of forskolin or with a combination of forskolin and isoproterenol, cyclic AMP levels were in excess of those needed to maximally activate protein kinase and lipolysis.

The time and dose-relationships of isoproterenol and lipolysis, force of contraction, cyclic AMP levels, protein kinase activity and phosphorylase activity were studied in perfused rat hearts. All five parameters were increased by isoproterenol over very similar concentration ranges. Dibutyryl cyclic AMP also increased phosphorylase activity, force of contraction and lipolysis. The study supports a role for cyclic AMP and protein kinase in myocardial lipolysis but suggests that a cyclic AMP-independent mechanism also exists.

A rapid, sustained lipolytic response to growth hormone (GH; 20 microgram/ml) was observed in experiments using the perifused fat cell system. No lipolytic response to this agent was observed when fat cells were incubated by the traditional flask incubation method, although isoproterenol stimulated lipolysis in this preparation. In experiments using flask incubated fat cells, isoproterenol increased cyclic AMP content while GH had no effect. However, in the presence of theophylline, isoproterenol and GH significantly increased cyclic AMP levels. In perifused fat cells, both isoproterenol and GH significantly increased cyclic AMP levels in the absence of theophylline. The presence of adenosine deaminase resulted in significant increases in the lipolytic response to isoproterenol and unmasked a lipolytic response to GH when the flask-incubated fat cell system was used. The antilipolytic action of adenosine was determined in perifused fat cells. It was found that the lipolytic response to GH was at least 10 times as sensitive to the inhibitory action of adenosine as was the lipolytic response to isoproterenol. It is concluded that the lipolytic response to GH in the flask-incubation method is prevented by the accumulation of adenosine. This rapid lipolytic response is unmasked in the perifused fat cell system because adenosine fails to accumulate as it is washed from the cell population by the constantly flowing buffer.

By using perfused fat cells the effect of isoproterenol on adenosine 3':5'-monophosphate (cAMP) levels, cAMP-dependent protein kinase activity and lipolysis was studied. An infusion of isoproterenol (10(-7) M) resulted in a time-dependent increase in cAMP levels and protein kinase activity in the fat cells. Both parameters reached maximum values after 5 min of drug infusion, then declined to steady-state values by 10 min. At 60 min, cAMP levels were still significantly (P less than .05) elevated over basal. Dose-response curves were determined for isoproterenol on cAMP levels, protein kinase activity and glycerol release. All three parameters were increased by isoproterenol over the same concentration range (10(-9)--10(-7) M). A plot of cAMP levels or protein kinase activity ratios vs. glycerol release resulted in linear relationship with high degrees of correlation (r = 0.98). The rates at which cAMP levels and glycerol release decline after termination of isoproterenol infusion were studied. Half-life values of 5.8 and 6.9 min were obtained for cAMP levels and glycerol release, respectively. These results support the hypotheses that cAMP, acting through protein kinase, is an intracellular mediator of the lipolytic response to isoproterenol. It is concluded that cAMP is not formed in great excess of that necessary to maximally increase lipolysis.

The antilipolytic action of insulin was investigated using the perifused fat cell system. Epinephrine (10−5 M) and glucagon (5 × 10−6 M) both stimulated lipolysis by at least 6-fold, and insulin inhibited both responses. The time course and the magnitude of the antilipolytic action of insulin were determined in the perifused fat cell system. At a concentration of 100 μU/ml, insulin inhibited epinephrine-stimulated lipolysis approximately 50 percent, with a half-time response of 4–5 min. The antilipolytic action of insulin persisted for at least 45 min. following the termination of insulin infusion. This prolonged phase of the action of insulin could be terminated by treatment of the cells with trypsin. These results suggest that only small percentage of insulin receptors is occupied during maximum lipolytic activity and that in adipose tissue many spare receptors exist for insulin.

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.

A multiple-chamber perifused fat cell system is described. Six chambers containing fat cells were perifused in parallel with buffer. Perifusate was collected for assay of glycerol as an index of lipolytic rates and cells in each chamber can be taken for analysis of biochemical intermediates. The system is so designed that drugs can be infused into the buffer and equally distributed in each chamber or can be individually infused into the buffer to one chamber, allowing for six different conditions to be tested in the same population of fat cells. The time and distribution characteristics of infused material are described. Time relationships are described for isoproterenol and glycerol release and for cyclic AMP levels in the fat cells, and the dose-response relationship between isoproterenol and glycerol release is shown.

The addition of epinephrine (10−6 M) to isolated fat cells resulted in increased lipolytic activity. In the presence of 1, 2 and 4% bovine serum albumin (BSA), lipolytic rates were linear for a 60 min period of time. Rates of lipolysis were increased by increasing the BSA concentration. In other experiments, the effect of glucose (10 mg/ml) was tested on basal, epinephrine-stimulated, and theophylline-stimulated lipolytic activity. In the absence of BSA, glucose resulted in a nearly 3-fold increase in lipolytic rates under all three conditions. In the presence of BSA (4%), the addition of glucose resulted in a further increase in lipolytic activity. The time course of cyclic AMP accumulation following the addition of epinephrine (3 × 10−6M) was determined in the absence and presence of BSA (4%). In the presence of BSA, accumulation of the cyclic nucleotide continued for a longer period of time and reached a value nearly twice that in the absence of BSA. In another experiment, cyclic AMP accumulation at 5 min following the addition of epinephrine was increased by the addition of 1% BSA. An additional increase was observed by the addition of 2% BSA. When the BSA concentration was increased to 4%, no additional increase was observed. BSA (4%) was also shown to increase the lipolytic response to dibutytyl cyclic AMP. It was concluded that the presence of albumin promoted lipolysis by preventing a negative feedback effect of free fatty acids on cyclic AMP accumulations at some point distal to the production of cyclic AMP. Additionally, it was suggested that albumin facilitates the lipolytic process by some mechanism in addition to reducing intracellular free fatty acid levels.

Denervated frog sartorius muscles showed an approximately 2--3 fold increase of cyclic GMP in their end-plate rich regions which did not appear up to 5 weeks after denervation in the normally end-plate-free pelvic region. No increase in cyclic AMP was seen in these preparations. The results suggest that the increase of cyclic GMP is related to processes specific to the region in which end plates are normally present.

The perifused fat cell system is a system with which lipolytic activity can be monitored on a minute-to-minute basis. Thus, the rate at which lipolysis changes following the addition and removal of hormones can be followed. Catecholamines and other lipolytic agents produced a time-dependent increase in lipolysis following addition of agents, and a time-dependent decrease in lipolysis occurred following removal of the agent. ACTH also produced an increase in lipolysis. However, on termination of ACTH infusion, the lipolytic rate did not return to basal level but remained elevated for at least an additional 30 min (persistent phase). The persistent phase could be terminated by removal of Ca2+. Readdition of Ca2+ in the absence of additional ACTH resulted in a rapid increase in glycerol release. No persistant phase occurred following ACTH if the adipocytes were perifused in a Ca2+-free buffer. However, if Ca2+ was added to the system 20 min after termination of ACTH infusion, lipolysis increased to a rate greater than that obtained initially by infusing ACTH in a Ca2+-free buffer. It is concluded that ACTH is bound to some component of the fat cell in a Ca2+ independent, tenacious manner, and the full manifestation of that binding is dependent on the presence of Ca2+.