Pertussis toxin reversal of the antilipolytic action of insulin in rat adipocytes in the presence of forskolin does not involve cyclic AMP

Insulin inhibition of lipolysis in the presence of forskolin was reversed by a four hour exposure of adipocytes to pertussis toxin. In contrast, the antilipolytic action of insulin against lipolysis due to theophylline was unaffected by pertussis toxin as was the ability of insulin to lower cyclic AMP in the presence of either forskolin or theophylline. The stimulation of adenylate cyclase by norepinephrine in crude plasma membranes obtained from rat adipocytes was inhibited by N6-(Phenylisopropyl)adenosine (PIA) and abolished by pretreating rat adipocytes with pertussis toxin. The stimulation of glucose metabolism by insulin was not altered by pertussis toxin pretreatment of rat adipocytes. These findings suggest that pertussis toxin selectively abolishes the antilipolytic effect of insulin in the presence of forskolin through a cyclic AMP independent mechanism.     

Effects of pertussis toxin treatment on the metabolism of rat adipocytes

The protein toxin present in Bordetella pertussis vaccine blocks the inhibition of adenylate cyclase by prostaglandins and adenosine which may be secondary to ADP-ribosylation of an inhibitory guanine nucleotide-binding protein. The stimulatory effects of alpha 1-catecholamine agonists on 32P uptake into phosphatidic acid and phosphatidylinositol in isolated rat adipocytes were virtually abolished by pertussis toxin treatment. In contrast, the stimulatory effects of insulin were increased in adipocytes after pertussis toxin treatment. Pertussis toxin treatment did not alter insulin stimulation of glucose oxidation and actually increased glucose conversion to lipid. Basal lipolysis was elevated in adipocytes by pertussis toxin treatment but not basal cyclic AMP. However, the increases in cyclic AMP and lipolysis due to low concentrations of catecholamines and forskolin were markedly potentiated by pertussis toxin treatment. The inhibitory effects of adenosine on cyclic AMP stimulation due to catecholamines were abolished by pertussis toxin. These data indicate that pertussis toxin selectively interferes with inhibition of cyclic AMP accumulation in rat adipocytes by adenosine, potentiates the increases in cyclic AMP due to catecholamines, increases the stimulatory effects of insulin on adipocyte metabolism, and interferes with alpha 1-catecholamine stimulation of phosphatidylinositol turnover.     

Effect of thyroid status on alpha- and beta-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased beta-catecholamine responsiveness in hyperthyroid animals is due to a decrease in alpha-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the alpha 2-adrenergic inhibition of adenylate cyclase or the alpha 1-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamsters. No effect of hyperthyroidism was found on the Kd for binding of [3H]dihydroergocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to beta-catecholamines was enhanced in adipocytes from hyperthyroid hamsters, as was lipolysis. However, in adipocytes from hyperthyroid hamsters the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on alpha-catecholamine action by enhances the activation of lipolysis by beta-catecholamine agonists.     

Fat cell adenylate cyclase system. Enhanced inhibition by adenosine and GTP in the hypothyroid rat

Hypothyroidism is associated with an enhanced sensitivity of rat fat cells to the inhibitory action of adenosine and adenosine agonists. The sensitivity of the forskolin-stimulated cyclic AMP response of rat fat cells to the adenosine agonist N6-phenylisopropyladenosine is amplified 3-fold by hypothyroidism. Forskolin-stimulated adenylate cyclase activity is more sensitive to inhibition by this adenosine agonist in membranes of fat cells isolated from hypothyroid as compared to euthyroid rats. Hypothyroidism does not significantly alter the number of affinity of binding sites for N6-cyclohexyl[3H]adenosine or N6-phenylisopropyladenosine in membranes of rat fat cells. GTP-induced inhibition of forskolin-stimulated adenylate cyclase was markedly enhanced in the hypothyroid state, suggesting an alteration in the inhibitory regulatory component (Ni)-mediated control of adenylate cyclase. Incubating membranes with [alpha-32P]NAD+ and preactivated pertussis toxin results in the radiolabeling of two peptides with Mr = 40,000 and 41,000 as visualized in autoradiograms of polyacrylamide gels run in sodium dodecyl sulfate. The amount of label incorporated by pertussis toxin into these two peptides (putative subunits of Ni) per mg of protein of membrane is increased 2-3-fold in the hypothyroid state. The amount of the stimulatory regulatory component, Ns, in fat cell membranes is not altered by hypothyroidism (Malbon, C. C., Graziano, M. P., and Johnson, G. L. (1984) J. Biol. Chem. 259, 3254-3260). The amplified response of hypothyroid rat fat cells to the inhibitory action of adenosine appears to reflect a specific increase in the activity and abundance of Ni.     

Rat fat-cells have three types of adenosine receptors (Ra, Ri and P). Differential effects of pertussis toxin.

Activation of rat adipocyte R1 adenosine receptors by phenylisopropyladenosine (PIA) decreased cyclic AMP and lipolysis; this effect was blocked in cells from pertussis-toxin-treated rats. In contrast, the ability of 2',5'-dideoxyadenosine to decrease cyclic AMP was not affected by pertussis-toxin treatment. Addition of adenosine deaminase to the medium in which adipocytes from control animals were incubated resulted in activation of lipolysis. Interestingly, adipocytes from toxin-treated rats (which had an already increased basal lipolysis) responded in an opposite fashion to the addition of adenosine deaminase, i.e. the enzyme decreased lipolysis, which suggested that adenosine might be increasing lipolysis in these cells. Studies with the selective agonists N-ethylcarboxamidoadenosine (NECA) and PIA indicated that adenosine increases lipolysis and cyclic AMP accumulation in these cells and that these actions are mediated through Ra adenosine receptors. Adenosine-mediated accumulation of cyclic AMP was also observed in cells preincubated with pertussis toxin (2 micrograms/ml) for 3 h. In these studies NECA was also more effective than PIA. Our results indicate that there are three types of adenosine receptors in fat-cells, whose actions are affected differently by pertussis toxin, i.e. Ri-mediated actions are abolished, Ra-mediated actions are revealed and P-mediated actions are not affected.     

Effect of thyroid status on α- and β-catecholamine responsiveness of hamster adipocytes

It has been suggested that part of the increased β-catecholamine responsiveness in hyperthyroid animals is due to a decrease in α-catecholamine action. The present results indicate that neither hyperthyroidism nor hypothyroidism altered the α₂-adrenergic inhibition of adenylate cyclase or the α₁-adrenergic stimulation of phosphatidylinositol turnover in adipocytes from the white adipose tissue of hamster. No effect of hyperthyroidism was found on the K_d of [³H]dihydroegocryptine or the number of binding sites in membranes prepared from hamster adipocyte tissue. The stimulation of cyclic AMP due to β-catecholamines was enhanced in adipocytes from hyperthyroid hamster, as was lipolysis. However, in adipocytes from hyperthyroid hamster the maximal stimulation of cyclic AMP due to isoproterenol, ACTH or epinephrine plus yohimbine, as seen in the presence of adenosine deaminase and theophylline, was less than in adipocytes from euthyroid hamsters. The activation of adenylate cyclase by isoproterenol was the same in membranes from hyperthyroid as compared to those from euthyroid hamsters in the absence or presence of guanine nucleotides. These data suggest that thyroid status has little effect on α-catecholamine action but enhances the activation of lipolysis by β-catecholamine agonists.     

Effect of pertussis toxin on the hormonal regulation of cyclic AMP levels in hamster fat cells

Pertussis toxin was purified approx. 1800-fold from pertussis vaccine. Administration of as little as 1 microgram of toxin/100 g body weight to hamsters markedly decreased the sensitivity of their adipocytes to agents that inhibit adenylate cyclase through receptor-mediated, GTP-dependent mechanisms such as alpha 2-adrenergic amines, prostaglandins, phenylisopropyladenosine and nicotinic acid. On the contrary, the inhibitory effect of 2',5'-dideoxyadenosine on cyclic AMP accumulation was not affected by the toxin. Activation of adenylate cyclase by isoproterenol, ACTH or forskolin was not diminished by the toxin but the maximum cyclic AMP accumulation was consistently increased. Furthermore, the dose-response curves for ACTH and forskolin were clearly shifted to the left in adipocytes from toxin-treated hamsters as compared to control adipocytes. It is concluded that pertussis toxin blocks the transfer of inhibitory information from the receptors to adenylate cyclase.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Activation of lipolysis and cyclic AMP accumulation in rabbit adipocytes by isoproterenol in the presence of forskolin or pertussis toxin

Adipocytes from rabbits are relatively insensitive to catecholamines or forskolin. However, the combination of catecholamines plus forskolin increased cyclic AMP accumulation and lipolysis much more than either agent alone. Pertussis toxin treatment also restored sensitivity to catecholamines. No defect in activation by catecholamines of adenylate cyclase was seen in isolated membranes incubated in the presence of GTP. Rabbit adipocytes appear to have an excess of the inhibitory guanine nucleotide binding protein (Ni). However, in plasma membranes this protein appeared to be relatively inactive as there was an activation of adenylate cyclase activity by catecholamines in the presence of GTP. These data suggest that in intact rabbit adipocytes catecholamines and forskolin are ineffective as stimulators of adenylate cyclase due to an excess of inhibitory guanine nucleotide binding proteins.     

Effect of adenosine deaminase, N6-phenylisopropyladenosine and hypothyroidism on the responsiveness of rat brown adipocytes to noradrenaline.

Adenosine deaminase (1 unit/ml) potentiated the lipolytic action of noradrenaline in adipocytes isolated from brown adipose tissue of 1- and 6-week-old rats by decreasing the EC50 (concn. giving 50% of maximal effect) for noradrenaline by 3-4-fold. With cells from neonatal rabbit tissue, adenosine deaminase only had a small, non-significant, effect on the EC50 for noradrenaline. Lipolysis in rat brown adipocytes was inhibited by low concentrations of N6-phenylisopropyladenosine (PIA). Rabbit cells were far less sensitive to PIA. PIA, prostaglandin E1 and nicotinate all inhibited noradrenaline-stimulated respiration in rat brown adipocytes. Hypothyroidism diminished the maximum response of respiration and lipolysis to noradrenaline in rat cells and increased the EC50 for noradrenaline. Responsiveness of lipolysis to noradrenaline was particularly decreased in hypothyroidism and was partially restored by addition of adenosine deaminase. Lipolysis in cells from hypothyroid rats was more sensitive to the anti-lipolytic action of PIA. Bordetella pertussis toxin increased lipolysis in the presence of PIA, suggesting an involvement of the Ni guanine-nucleotide-binding protein in the control of brown-adipocyte metabolism.     

Increased sensitivity of fat cell adenylate cyclase to stimulatory agonists during fasting is not related to impaired inhibitory coupling system

In rat adipocytes, inhibition of the forskolin-stimulated cyclic AMP response by nicotinic acid and N6-phenylisopropyladenosine was unaltered by a 72 h fasting. Under assay conditions favouring inhibition, basal and forskolin-stimulated adenylate cyclase responses to inhibition by GTP and nicotinic acid were also unimpaired by fasting. Under the same conditions, however, low GTP concentrations elicited a clear activatory effect in membranes from fasted but not from fed rats. Fasting failed to alter the incorporation of [32P]ADP ribose into the alpha i-subunit of Ni and the attenuation of nicotinic acid inhibitory action that are both induced by pertussis toxin. These results, demonstrating unimpaired inhibitory control of adenylate cyclase in starved rat adipocytes, suggest that the permissive effect of fasting on the action of stimulatory receptor agonists in fat cells reflects a specific increase in the activity of the adenylate cyclase stimulatory coupling system.     

Correction by dexamethasone treatment of the altered lipolytic cascade induced by adrenalectomy in rat adipocytes

The ability of glucocorticoid-treatment to reverse the metabolic alterations caused by adrenalectomy in rat adipocytes was studied. Correction of the enhanced adenosine antilipolytic effect and of the defect in lipolysis, cyclic AMP and adenylate cyclase responsiveness to guanine nucleotides were all achieved after a 24 h dexamethasone treatment, whereas correction of the defect in beta-adrenoceptor-density required a 48 h treatment. The latter treatment, however, failed to reverse the defect in both the adenylate cyclase catalytic activity and protein content per fat cell. These different kinetics of restoration indicate that correction by dexamethasone of the defective cyclic AMP and lipolytic responses on one hand and of the guanine nucleotide control of adenylate cyclase on the other one are two related phenomenoms.     

Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni

Adenosine, acting via A1 adenosine receptors, can inhibit adenylate cyclase activity in adipocytes. To assess the effects of chronic adenosine agonist exposure on the A1 adenosine receptor system of adipocytes, rats were infused with (-)-phenylisopropyladenosine or vehicle for 6 days and membranes were prepared. Basal as well as isoproterenol-, sodium fluoride-, and forskolin-stimulated adenylate cyclase activities were significantly increased (approximately 2-fold) in membranes from treated animals. (-)-Phenylisopropyladenosine-mediated inhibition of forskolin-stimulated adenylate cyclase activity was significantly (p = 0.0001) attenuated in membranes from treated rats (20.1 +/- 2.1% inhibition) versus controls (31.6 +/- 2.3% inhibition). Prostaglandin E1-induced inhibition of forskolin-stimulated adenylate cyclase activity was also attenuated: 11.7 +/- 3.6 versus 23.2 +/- 4.6% (p = 0.001). Using the A1 adenosine receptor agonist radioligand (-)-N6-(3-[125I]iodo-4-hydroxyphenylisopropyl)adenosine, 32% fewer high affinity binding sites were detected in membranes from treated animals (p less than 0.04). Photoaffinity labeling with N6-2-(3-[125I]iodo-4-azidophenyl)ethyladenosine revealed no gross difference in receptor structure. The number of beta-adrenergic receptors as well as the percentage of receptors in the high affinity state as assessed by (-)-3-[125I]iodocyanopindolol binding were the same in both groups. In membranes from treated rats, the amount of [alpha-32P]NAD incorporated by pertussis toxin into the alpha subunit of the inhibitory guanine nucleotide regulatory protein (Ni) was decreased by 37 +/- 11%. Concurrently, the quantity of label incorporated by cholera toxin into the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Ns) was increased by 44 +/- 14% in treated membranes. Finally, the capacity of Ns solubilized from treated membranes to stimulate adenylate cyclase activity when reconstituted into cyc- S49 lymphoma cell membranes was enhanced by approximately 50% compared to control. Thus, heterologous desensitization, manifested by a diminished capacity to inhibit adenylate cyclase and an enhanced responsiveness to stimulatory effectors, can be induced in the A1 adenosine receptor-adenylate cyclase system of adipocytes. A decrease in Ni alpha subunit concomitant with an increase in Ns alpha subunit quantity and activity may represent the biochemical mechanism of desensitization in this system.     

In Vivo Evidence that Lithium Inactivates Gi Modulation of Adenylate Cyclase in Brain

In vivo microdialysis of cyclic AMP from prefrontal cortex complemented by ex vivo measures was used to investigate the possibility that lithium produces functional changes in G proteins that could account for its effects on adenylate cyclase activity. Four weeks of lithium administration (serum lithium concentration of 0.85 +/- 0.05 mM; n = 11) significantly increased the basal cyclic AMP content in dialysate from prefrontal cortex of anesthetized rats. Forskolin infused through the probe increased dialysate cyclic AMP, but the magnitude of this increase was unaffected by chronic lithium administration. Inactivation of the inhibitory guanine nucleotide binding protein Gi with pertussis toxin increased dialysate cyclic AMP in control rats, as did stimulation with cholera toxin (which activates the stimulatory guanine nucleotide binding protein Gs). The effect of pertussis toxin was abolished following chronic lithium, whereas the increase in cyclic AMP after cholera toxin was enhanced. In vitro pertussis toxin-catalyzed ADP ribosylation of alpha i (and alpha o) was increased by 20% in prefrontal cortex from lithium-treated rats, but the alpha i and alpha s contents (as determined by immunoblot) as well as the cholera toxin-catalyzed ADP ribosylation of alpha s were unchanged. Taken together, these results suggest that chronic lithium administration may interfere with the dissociation of Gi into its active components and thereby remove a tonic inhibitory influence on adenylate cyclase, with resultant enhanced basal and cholera toxin-stimulated adenylate cyclase activity.     

Cyclic nucleotide phosphodiesterases and thyroid hormones.

Evidence is presented that modulation of the maximum velocity of a particulate low K-m cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase by thyroid hormones is one mechanism for the regulation of the responsiveness of rat epididymal adipocytes to lipolytic agents such as epinephrine and glucagon. Fat cells of propylthiouracil-induced hypothyroid rats are unresponsive to lipolytic agents and the V-max of particulate low K-m cyclic AMP phosphodiesterase of these cells is elevated above normal. In vivo treatment of hypothyroid rats with triiodothyronine restores to control values both the lipolytic response of the fat cells to epinephrine and the V-max of the particulate bound low K-m cyclic AMP phosphodiesterase. No similar correlation is found with the soluble high K-m cyclic AMP phosphodiesterase. The phosphodiesterases of fat cells from normal and hypothyroid rats respond identically in vitro to propylthiouracil, triiodothyronine, methylisobutylxanthine, or theophylline, although the particulate low K-m cyclic AMP phosphodiesterase is inhibited to a greater extent than soluble cyclic guanosine 3':5'-monophosphate phosphodiesterase activity. Protein kinase of fat cells from hypothyroid rats can be stimulated by cyclic AMP to the same total activity as observed in fat cells of normal rats. However, less of the protein kinase in fat cells from hypothyroid rats was in the cyclic AMP-independent form. This shift in the equilibrium of protein kinase forms is consistent with an increased activity of low K-m cyclic AMP phosphodiesterase and probably results from a lowering of the lipolytically significant pool of cyclic AMP.     

Pertussis toxin stimulates cholecystokinin-induced cyclic AMP formation but is without effect on secretagogue-induced calcium mobilization in exocrine pancreas

The role of a pertussis toxin sensitive GTP-binding protein in mediating between cholecystokinin receptors and phosphatidylinositol 4,5-bisphosphate phosphodiesterase as well as in preventing cholecystokinin from increasing cellular cyclic AMP has been investigated using dispersed acini from rabbit pancreas. Pertussis toxin pretreatment (500 ng/ml, 2 h) did not affect cholecystokinin(octapeptide) (CCK-8)-induced increases in cytosolic free Ca2+ as judged from changes in fluorescence obtained from quin2-loaded acini. Although pretreatment with pertussis toxin was also without effect on resting acinar cell cyclic AMP levels, adenylate cyclase activity was increased, since inhibition of cyclic AMP phosphodiesterase activity by isobutylmethylxanthine (IBMX) resulted in an additional increase in cyclic AMP levels in toxin-treated acini, indicating that acinar cell adenylate cyclase activity is under some tonic inhibitory control by the pertussis toxin-sensitive inhibitory GTP-binding protein (Gi) of the adenylate cyclase system. CCK-8 gave an increase in cyclic AMP levels in both control (1.6-fold) and toxin-treated (2.3-fold) acini, leading to cyclic AMP levels in the toxin-treated acini 2-times as high as those in control acini. In the presence of IBMX, the cyclic AMP response to CCK-8 was again markedly enhanced in acini pretreated with the toxin (3.2- vs. 1.8-fold), resulting in cAMP levels in the toxin-treated acini 3.7-times those in the absence of IBMX, 2.5-times those in control acini in the presence of IBMX and 7.0-times those in control acini in the absence of IBMX. Neither the pretreatment with pertussis toxin, nor the presence of IBMX alone, nor the combination had an effect on basal amylase secretion. However, all three treatments potentiated the stimulatory effect of CCK-8 on amylase secretion and the amount of potentiation was proportional to the cyclic AMP levels reached. Our findings suggest that in the intact pancreatic acinar cell Gi inhibition of the catalytic subunit of the adenylate cyclase may largely be responsible for preventing cholecystokinin from increasing cellular cyclic AMP. They moreover show that cyclic AMP is a modulatory agent in rabbit pancreatic enzyme secretion, not able to stimulate secretion itself, but potentiating effects mediated by the phosphatidylinositol-calcium pathway.     

Pertussis toxin reverses Gpp(NH)p inhibition of basal and forskolin activated adipocyte adenylate cyclase

Inhibition of basal adenylate cyclase by GTP or guanyl-5'-yl imidodiphosphate was abolished in membranes isolated from rat adipocytes previously incubated with pertussis toxin. Forskolin (0.1 microM) stimulated adenylate cyclase about 4-fold and inhibition of cyclase by GTP or guanyl-5'-yl imidodiphosphate was also abolished by pertussis toxin treatment of rat adipocytes. Forskolin (1 microM) increased adenylate cyclase activity at least ten-fold and the inhibitory effect of GppNHp was reduced but not abolished by pertussis toxin. In rabbit adipocytes, pertussis toxin reversed the inhibition of adenylate cyclase activity by GppNHp to the same extent as that by GTP in the presence of 1 microM forskolin. The present results indicate that pertussis toxin can reverse the inhibition of adipocyte adenylate cyclase by nonhydrolyzable GTP analogs as well as that by GTP.     

Inhibition of lipolysis and cyclic AMP accumulation by adenosine analogues in hamster epididymal adipocytes exposed to cholera toxin

The effects of adenosine, N6-phenylisopropyl adenosine and 2',5'-dideoxyadenosine on lipolysis and cyclic AMP accumulation, in hamster adipocytes treated with cholera toxin, were studied. Cholera toxin caused an increase in lipolysis and cyclic AMP accumulation that was dependent upon the concentration of toxin and the length of time cells were exposed to the toxin. When N6-phenylisopropyl adenosine or 2',5'-dideoxyadenosine were present, the lipolytic and cyclic AMP responses to cholera toxin were inhibited. The adenosine analogues were equally effective inhibitors of lipolysis and cyclic AMP accumulation, when they were added 1 or 2 h after exposure to the toxin. Enzymatic removal of endogenously produced adenosine with adenosine deaminase potentiated both the lipolytic and cyclic AMP responses to cholera toxin. In addition, the inhibitory effects of N6-phenylisopropyl adenosine, 2'5'-dideoxyadenosine and clonidine on lipolysis and cyclic AMP were enhanced consequent to enzymatic removal of adenosine. These data show responses of intact fat cells to N6-phenylisopropyl adenosine, 2',5'-dideoxyadenosine or removal of endogenous adenosine and provide evidence for an adenosine sensitivity of fat cells exposed to cholera toxin.     

Presence of a functional inhibitory GTP-binding regulatory component, Gi, linked to adenylate cyclase in adipocytes of ob/ob mice

It has been reported recently (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) that adipocytes from the obese mouse strain (ob/ob), unlike normal mice (+/+), lack functional Gi, a GTP-regulated protein complex that mediates inhibition of adenylate cyclase. In contrast, we have found functional Gi linked to inhibition of adenylate cyclase in adipocyte membranes from both ob/ob and +/+ mice. This conclusion is based on observation of: 1) GTP-dependent inhibition of adenylate cyclase by antilipolytic agents, such as prostaglandin E2, nicotinic acid, and the adenosine receptor agonist, phenylisopropyladenosine (PIA); 2) classical biphasic GTP kinetics, with stimulation by low and inhibition by high concentrations of GTP; and 3) elimination of cyclase inhibition by antilipolytic agents upon treatment of ob/ob adipocytes with pertussis toxin. Upon treatment with pertussis toxin and [32P] NAD, purified adipocyte membranes from ob/ob mice incorporated twice as much radioactivity per unit membrane protein than those from +/+ mice in the 40,000-42,000 region. The inhibitory actions of PIA on adenylate cyclase were blocked by the adenosine receptor antagonists, theophylline and isobutylmethylxanthine. However, in contrast to other known inhibitory adenosine receptors, relatively high (100 nM) PIA concentrations were required for half-maximal inhibition of adenylate cyclases from both +/+ and ob/ob adipocytes. The adipocyte adenylate cyclase from both mouse strains were approximately equally susceptible to inhibition by nicotinic acid and prostaglandin E2. However, the ob/ob cyclase was inhibited by 47% with PIA, whereas the enzyme from the +/+ mouse was inhibited by only 27% (p less than 0.01). This greater inhibition by adenosine may contribute to abnormal fat metabolism in adipocytes from ob/ob mice.     

Mechanisms altered beta-adrenergic responsiveness in the hyperthyroid and hypothyroid turkey erythrocyte

Studies on the relationship between thyroid hormone and the beta-adrenergic catecholamines have been carried out in the turkey erythrocyte. Conditions of thyroid hormone excess and deficiency were examined with respect to their effects on the beta receptor itself, as well as to their effects on associated biochemical and physiological indices of beta receptor function, including agonist stimulated adenylate cyclase activity, cellular cyclic AMP generation, and catecholamine-induced stimulation of potassium ion influx. Erythrocytes obtained from hypothyroid turkeys showed a marked (approximately 50%) reduction in beta receptor number without any change in receptor affinity for agonists or antagonists. Catecholamine-sensitive adenylate cyclase activity and cellular cyclic AMP levels were similarly reduced. The sensitivity of these cells to agonist-stimulated potassium influx was significantly decreased, but maximal agonist-stimulated transport rate was unchanged. Analysis of the quantitative relationship between beta receptor number, agonist concentration, and level of catecholamine-stimulated potassium influx indicates that, at any given absolute level of receptor occupancy, the level of agonist-stimulated potassium influx is identical in hypothyroid and normal erythrocytes, and that the diminished physiological sensitivity of the hypothyroid cell is attributable in its entirety to a reduction in beta receptor number per se. The results obtained in the hyperthyroid turkey erythrocyte were strikingly different. Here, beta receptor number, binding affinity for agonists and antagonists, catecholamine-sensitive adenylate cyclase activity, and maximal cyclic AMP levels were all unchanged. In contrast, maximal agonist-stimulated potassium ion transport was markedly reduced, while the concentration of isoproterenol required for half-maximal stimulation was only slightly increased. Analysis of the relationship between beta receptor number, agonist concentration, and catecholamine-stimulated potassium influx rate indicates that, at all absolute levels of beta receptor occupancy, the stimulation of monovalent cation influx is markedly blunted in the hyperthyroid cell. In contrast to the findings in the hypothyroid cell, where decreased physiologic sensitivity to catecholamines is directly attributable to a reduction in beta receptor number, the primary abnormality responsible for diminished catecholamine responsiveness in the hyperthyroid cell would appear to be located at a point "distal" to the beta receptor itself.