Regulation of human lipolysis. In vivo observations on the role of adrenergic receptors.

Changes in the plasma free fatty acids of a pancreatectomized subject and in free fatty acids and insulin in 10 normal subjects in response to the in vivo infusion of epinephrine alone, epinephrine plus phentolamine, and epinephrine plus propranolol indicate that both alpha and beta adrenergic receptors are present in human adipose tissue. Under the experimental conditions used, adipose tissue appeared to be more responsive to epinephrine than did the cardiovascular system.

Pharmacological characterizations of adrenergic receptors in human adipocytes.

Three types of adrenergic receptors, beta, alpha-1, and alpha-2, were identified in human adipocytes, isolated from properitoneal adipose tissue, using both the binding of radioactive ligands and the effects of adrenergic agents on receptor-specific biochemical responses. Adrenergic binding studies showed the following results: [(3)H]dihydroalprenolol binding (beta adrenergic) B(max) 280 fmol/mg protein, K(D) 0.38 nM; [(3)H]para-aminoclonidine binding (alpha-2 adrenergic) B(max) 166 fmol/mg protein, K(D) 0.49 nM; [(3)H]WB 4101 binding (alpha-1 adrenergic) B(max) 303 fmol/mg protein, K(D) 0.86 nM. In adipocytes from subcutaneous adipose tissue, [(3)H]dihydroergocryptine binding indicated the presence of alpha-2 but not alpha-1 receptors. Beta and alpha-2 adrenergic receptors appeared to be positively and negatively coupled to adenylate cyclase, respectively. Cells or cell membranes were incubated with epinephrine (10 muM) alone and in combination with the antagonists yohimbine (alpha-2) and prazosin (alpha-1). Epinephrine alone prompted a modest increase in adenylate cyclase activity, cyclic AMP, and glycerol release, an index of lipolysis. Yohimbine (0.1 muM) greatly enhanced these actions whereas prazosin was without effect. The beta agonist, isoproterenol, stimulated glycerol release, whereas the alpha-2 agonist, clonidine, inhibited lipolysis and cyclic AMP accumulation. To assess further alpha-1 receptors, cells were incubated with [(32)P]phosphate and epinephrine (10 muM) alone and in combination with prazosin and yohimbine. Epinephrine alone caused a three- to fourfold increase in (32)P incorporation into phosphatidylinositol. Prazosin (0.1 muM) blocked this action whereas yohimbine (0.1 muM) was without effect. Thus, in a homogeneous cell preparation, the human adipocyte appears to have three different adrenergic receptors, each of which is coupled to a distinct biochemical response.

Insulin inhibition of lipolysis of human adipocytes: the role of cyclic adenosine monophosphate.

To gain information on the manner in which insulin suppresses lipolysis in man, isolated adipocytes, prepared from subcutaneous adipose tissue, were incubated with insulin (100 microunits/ml) alone and in combination with isoproterenol (10(-7) M or 10(-8) M). Cyclic AMP concentration was measured at 60 min; glycerol release, used as an index of lipolysis, was determined at 45 and 75 min. Insulin consistently reduced both basal and stimulated cyclic AMP and glycerol release: the degree of suppression of each was comparable. In subsequent experiments, the ability of insulin to suppress glycerol release stimulated by isoproterenol, theophylline, and dibutyryl cyclic AMP (dbcAMP), respectively, was compared. Insulin substantially reduced the raised levels of cyclic AMP and glycerol release prompted by isoproterenol and theophylline, but it had little effect on increases caused by dbcAMP. These findings support the view that reduction in cyclic AMP is an important component in the regulation of fat mobilization by insulin.

In-vitro observations on isolated adipose tissue cells from hyperobese subjects.

Isolated adipose tissue cells were prepared from subcutaneous samples obtained from nine morbidly obese subjects weighing from 187 to 306% of ideal body weight. The responsiveness of these adipocytes to a number of test substances was determined by measuring cellular cyclic AMP concentration at one-half hour and glycerol release at four hours. Theophylline (10(-3) M) and epinephrine (10(-5) M) stimulated lipolysis; theophylline stimulated an increase in cyclic AMP, while epinephrine failed to prompt a significant change in the nucleotide. Neither the alpha blocker, phentolamine (10(-5) M), nor the beta blocker, propranolol (3 X 10(-5) M), affected lipolysis or cyclic AMP; when these agents were incubated in combination with epinephrine, changes occurred indicative of the presence of both alpha and beta adrenergic receptor sites. Insulin significantly reduced both basal and stimulated lipolysis but failed to affect cyclic AMP. With minor exceptions, adipocytes from hyperobese subjects behaved similarly to cells from unselected donors; at the concentration used, there was no evidence of resistance to insulin.

Normal free fatty acid response to isoproterenol in pseudohypoparathyroidism.

The lipolytic response to isoproterenol infusion was examined in seven normal subjects and six patients with pseudohypoparathyroidism type I [two had deficiency of the hormone receptor-cyclase coupling protein (N-protein) and four did not]. Despite blunted plasma cAMP responses to isoproterenol in both subgroups of pseudohypoparathyroidism patients, the serum FFA responses were normal. We conclude that changes in plasma cAMP do not reflect the adequacy of the lipolytic response to isoproterenol.

Site of free-fatty-acid inhibition of lipolysis by human adipocytes.

When human adipocytes were incubated in albumin-free buffer, isoproterenol failed to stimulate an increase in either cyclic AMP or glycerol release. Cells incubated for 1/2 hr with 4% albumin and isoproterenol had a striking increase in cyclic AMP; this effect was markedly reduced when FFA concentration was increased by the addition of sodium oleate. When incubation was prolonged to 4 hr, the cyclic AMP concentration of stimulated cells fell towards the basal level. This decline in the level of cyclic AMP was prevented by frequent change in buffer. The ability of epinephrine and sodium fluoride to stimulate the adenylyl cyclase of human adipocyte membranes was not affected by the addition of sodium oleate. However, when intact cells were preincubated for 1 hr with added sodium oleate, the responsiveness to epinephrine of membranes derived from the cells was reduced. No such alternation in responsiveness to sodium fluoride occurred. These results indicate that the inhibitory effect of FFA on lipolysis is associated with a reduced production of cyclic AMP; the latter effect may be the consequence of FFA inhibition of adenylyl cyclase.

The role of free fatty acids in the regulation of lipolysis by human adipose tissue cells.

The effect of added fatty acid on lipolysis and cyclic AMP concentration of human adipose tissue cells was studied. The addition of sodium oleate decreased the lipolytic response of adipocytes to 10(-7) M isoproterenol. Inhibition was detectable with the lowest quantity of oleate added, 0.2 mM, and was progressively greater with increasing quantities of added fatty acid. Palmitic and linoleic acids were as effective as oleic acid in suppressing isoproterenol-stimulated lipolysis. Suppression of cyclic AMP formation was detectable within one minute after the addition of oleate. Cyclic AMP formation, suppressed by accumulated fatty acids, could not be stimulated by the addition of fresh isoproterenol. However, after the accumulated fatty acids were removed by buffer change, cyclic AMP formation was stimulated by fresh isoproterenol. These findings are consistent with the view that fatty acids are physiologically significant regulators of lipolysis.

Studies on desensitization of adrenergic receptors of human adipocytes.

The studies described here were undertaken to determine whether or not desensitization of human adipocyte beta and alpha-2 adrenergic receptors could be demonstrated. Cells, isolated from peritoneal adipose tissue obtained from patients undergoing elective abdominal surgery, were preincubated for 3 hr in buffer alone or in the presence of isoproterenol, 10-5M. Cells in both sets of flasks were then washed and exposed to isoproterenol for 1/2 hr; cyclic AMP was then measured as an end point of beta receptor activation. Cells which had had no prior exposure to isoproterenol responded significantly greater to isoproterenol than did cells that had had prior exposure to the catecholamine, The beta receptor characteristics of cells undergoing beta desensitization were assessed using [3H] dihydroalprenolol. Compared to control cells, adipocytes exposed to isoproterenol had a reduction in Bmax while KD values were the same. Thus desensitization of beta adrenergic receptors of human adipocytes occurs and is associated with down regulation in the number of beta receptors. In comparable studies, preincubation with epinephrine 10-5M did not affect the response of cells to a subsequent exposure to this catecholamine. In alpha-2 receptor binding assays, there was a decreased number of [3H]p-aminoclonidine binding sites, but the level of [3H]yohimbine binding was not altered following the incubation with epinephrine. Thus, desensitization of alpha-2 receptors was not demonstrated.

A simple micromanipulator for multiple uses in freely moving rats: electrophysiology, voltammetry, and simultaneous intracerebral infusions.

An inexpensive, easily fabricated micromanipulator is described that can be used for single-unit recording or voltammetry in freely moving rats. The basic design is configured around the standard coupling system between a plastic syringe and corresponding needle hub. The device can be used with glass or metal microelectrodes for electrophysiology or carbon-fiber or carbon-disk microelectrodes for voltammetry. With either recording technique, the micromanipulator also can accommodate a 33-ga infusion cannula, which allows drugs to be administered directly to the recording site. The entire assembly is lightweight and can be used with a head-mounted amplifier system for relatively noise-free recording.

Alpha-2 adrenergic activation inhibits forskolin-stimulated adenylate cyclase activity and lipolysis in human adipocytes.

Forskolin at 10 muM caused a 100-fold increase in the intracellular concentration of cyclic AMP and a 6-fold increase in glycerol release in the human adipocyte. These responses are comparable to those prompted by 10 muM isoproterenol. The effects of forskolin on cyclic AMP and lipolysis were dose-dependent. Alpha-2 adrenergic activation, achieved with 10 muM epinephrine and 30 muM propranolol, significantly inhibited forskolin-stimulated cyclic AMP accumulation and glycerol release, shifting the dose-response curves to the right. Forskolin at 10 muM caused a 4.5-fold increase in the adenylate cyclase activity of human adipocyte membranes. When either isoproterenol or epinephrine (0.1 mM) was combined with forskolin, the magnitude of response was substantially greater than the sum of responses achieved by each agent incubated alone.

Acute and long-term amphetamine treatments alter extracellular ascorbate in neostriatum but not nucleus accumbens of freely moving rats.

The ability of amphetamine to alter the extracellular level of ascorbate, an apparent modulator of neostriatal function, was assessed voltammetrically in the neostriatum and nucleus accumbens of awake, behaving rats. Whereas acute administration (1.0 and 5.0 mg/kg d-amphetamine) produced a dose-dependent rise in neostriatal ascorbate, there was no change in the nucleus accumbens. Vehicle injections had no significant effect on ascorbate levels in either location. Administration of 5.0 mg/kg d-amphetamine for one week enhanced neostriatal ascorbate release even further, but this effect returned to acute levels when treatment continued for a second week. Multiple amphetamine injections for up to two weeks failed to alter extracellular ascorbate in the nucleus accumbens. The results of these experiments confirm a site-specific action of amphetamine on ascorbate release and suggest complex changes in the extracellular level of this substance in the neostriatum with long-term treatment.

Comparative effects of forskolin and isoproterenol on the cyclic AMP content of human adipocytes.

Alterations in adipocyte cyclic AMP concentrations in response to 100 microM forskolin and 10 microM isoproterenol over a 4 hour period were found to be similar; with each agent, a peak response was noted within 30 minutes. In general, the greater the magnitude of peak response, the more rapid the decline of cyclic AMP concentration during the ensuing 3 1/2 hours. Alpha-2 adrenergic activation, achieved with 10 microM clonidine or 10 microM epinephrine, substantially reduced the cyclic AMP concentrations in cells stimulated by 100 microM forskolin or 10 microM isoproterenol. Isoproterenol-stimulated cells appeared to be more sensitive to alpha adrenergic inhibition than did forskolin-stimulated cells. Cells preincubated for 3 hours with 100 microM forskolin were markedly less responsive to a second exposure to the diterpine. Cells exposed to forskolin for 3 hours also had a reduced response when incubated with isoproterenol; thus, desensitization to forskolin appears to be heterologous. Forskolin desensitization did not appear to be dependent on cellular ATP depletion since cells mildly stimulated during preincubation were as severely desensitized as those adipocytes strongly stimulated. Maximum desensitization required a preincubation time of 1-2 hours with either isoproterenol or forskolin.

The effect of alpha and beta adrenergic receptor stimulation on the adenylate cyclase activity of human adipocytes.

The effects of the mixed agonist epinephrine and the beta agonist isoproterenol, each alone and in combination with the alpha adrenergic blocker phentolamine and the beta blocker propranolol on the adenylate cyclase activity of human adipocyte membrane fragments were determined in a calcium free buffer. Neither phentolamine (10 muM) nor propranolol (32 muM) affected basal adenylate cyclase activity. Epinephrine (10 muM) stimulated adenylate cyclase activity and this effect was slightly enhanced by phentolamine. The combination of epinephrine plus propranolol depressed adenylate cyclase below the basal level. Isoproterenol (10 muM) markedly stimulated adenylate cyclase; the addition of phentolamine caused an equivocal further increase while the addition of propranolol depressed adenylate cyclase activity to, but not below, the basal level. These findings are consistent with the hypothesis that human adipocytes have both alpha and beta adrenergic receptors and that these receptors are associated with the cell membrane adenylate cyclase system.

The effect of fasting on the adrenergic receptor activity of human adipocytes.

To assess the influence of fasting of the alpha- and beta-adrenergic receptor activities of human adipocytes, subcutaneous adipose tissue was obtained from normal obese subjects in the fed state and after 3 days of fasting. Isolated cells prepared from the adipose tissue were incubated with epinephrine, 10(-6)M, and in the presence of the alpha-blocker phentolamine, 10(-5)M, and the beta-blocker propranolol, 10(-5)M, respectively; intracellular cyclic AMP levels and glycerol released into the buffer were measured. Consistent with past observations, epinephrine stimulated cyclic AMP and glycerol release when incubated with cells from fed subjects. Phentolamine enhanced this action and propranolol reduced it below the basal level. In contrast, epinephrine suppressed cyclic AMP and lipolysis when incubated with adipocytes from fasted individuals. This reversal in the effect of epinephrine on adipocy.se in alpha-activity. Additional studies suggest that this change in alpha- and beta-adrenergic receptor balance occurred after 1 day of fast and was not significantly exaggerated when fast was prolonged to 8 days. These findings are consistent with the view that in the fasting state the sympathetic nervous system and circulating catecholamines act to conserve adipose tissue triglyceride.