Insulin antagonizes epinephrine activation of the membrane transport of fatty acids. Potential site for hormonal suppression of lipid mobilization

Membrane transport of long chain fatty acids in the isolated rat adipocyte can be strongly stimulated by epinephrine (Abumrad, N. A., Perry, P. R., and Whitesell, R. R. (1985) J. Biol. Chem. 260, 9969-9971). We now report that insulin at physiological concentrations can completely block or reverse the epinephrine effect. Insulin was optimally effective at a concentration of about 0.1 nM in inhibiting transport activation by 0.3 and 3 microM epinephrine (0.1 and 1.0 microgram/ml). High concentrations of insulin (above 1 nM) were generally less effective and this was particularly true at the highest dose of epinephrine (1.0 microgram/ml). The insulin effect was shown to be on the transport process since insulin inhibited epinephrine activation of transport in both directions (influx and efflux). No effect of insulin on basal transport was observed over a wide range of concentrations (0.01-10 nM). Insulin's antagonism of transport activation by epinephrine appeared dependent on ATP metabolism since it was abolished by preincubating the cells with dinitrophenol (1 mM). Dinitrophenol, however, could not reverse the insulin effect when exposure to the hormone preceded that to dinitrophenol, consistent with an action of insulin at the transport step. The data indicate that regulation of the membrane transport of fatty acids is a potential site for insulin's action to suppress lipid mobilization.     

Catecholamine activation of the membrane transport of long chain fatty acids in adipocytes is mediated by cyclic AMP and protein kinase

Membrane transport of long chain fatty acids in the isolated adipocyte can be stimulated 5-10-fold by epinephrine (Abumrad, N. A., Perry, P. R., and Whitesell, R. R. (1985) J. Biol. Chem. 260, 9969-9971). This study shows that isoproterenol and norepinephrine are more potent than epinephrine in activating the transport process. The stimulatory effect on transport is mediated by beta-receptor interaction and cAMP. This was shown by the following. alpha-Receptor agonists and antagonists were ineffective; methylisobutylxanthine at low concentration (3 microM) potentiated the effect of a suboptimal dose (0.01 microgram/ml) of epinephrine and was stimulatory at high concentration (100 microM) in the absence of epinephrine; and cAMP analogs were very effective activators. Involvement of the cAMP-dependent protein kinase was indicated by two lines of evidence. 1) Combinations of cAMP analogs which are specific for sites 1 and 2 of the protein kinase, respectively, had synergistic effects on fatty acid transport. Combinations of analogs specific for the same site were only additive in their effects. This is similar to the pattern of protein kinase activation in vitro and to that of lipolysis activation in the intact adipocyte (Beebe, S. J., Holloway, R., Rannels, S. R., and Corbin, J. D. (1984) J. Biol. Chem. 259, 3539-3547). 2) Treatment of cells with various metabolic poisons abolished the stimulatory effect of norepinephrine. The response of fatty acid transport to catecholamines showed multiple parallels with that documented for lipolysis except that it was much more rapid. This suggested that the transport process was a regulatory step in fatty acid mobilization. This interpretation is supported by the observation that basal Vmax for transport is much too slow to accommodate the rate of fatty acid release which is observed following stimulation of intact cells with adrenergic hormones.     

An assessment of the ability of insulin-stimulated cyclic AMP phosphodiesterase to decrease hepatocyte intracellular cyclic AMP concentrations.

Treatment of hepatocytes with either NH4Cl (10mM) or fructose (10mM) blocks insulin's activation of the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of insulin (10 nM) to decrease intracellular cyclic AMP concentrations raised by glucagon (10 nM) was unaffected by pre-treatment with either NH4Cl (10 mM) or fructose (10 mM). It is concluded that the 'dense-vesicle' enzyme does not play a significant role in this action of insulin and that as yet unidentified cyclic AMP phosphodiesterase(s) must be activated by insulin. Treatment of hepatocytes with either NH4Cl or fructose appeared to increase, reversibly, cyclic AMP phosphodiesterase activity. When N6-(phenylisopropyl)adenosine was used to prevent glucagon from blocking insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity, insulin's ability to decrease intracellular cyclic AMP concentrations in glucagon-treated hepatocytes was increased markedly. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase activity can exert a potent effect in decreasing intracellular cyclic AMP concentrations elevated by glucagon.     

Insulin-induced subcellular redistribution of insulin-like growth factor II receptors in the rat adipose cell. Counterregulatory effects of isoproterenol, adenosine, and cAMP analogues

Insulin shifts the steady-state subcellular distribution of insulin-like growth factor II (IGF-II) receptors from a large intracellular pool to the plasma membrane in the rat adipose cell (Wardzala, L. J., Simpson, I. A., Rechler, M. M., and Cushman, S. W. (1984) J. Biol. Chem. 259, 8378-8383). In the present study, the counterregulatory effects of adrenergic stimulation, adenosine deaminase, and cAMP on this process were studied. Both isoproterenol (10(-6) M) and adenosine deaminase reduced insulin sensitivity and also rapidly (t1/2 approximately 1.5 min) decreased the effect of a maximal insulin concentration on the number of cell surface IGF-II receptors by 35-50%, and by 70% when added together. The marked reduction in binding was retained in isolated and solubilized plasma membranes. Both isoproterenol and adenosine deaminase alone increased the EC50 for insulin from 0.06 to 0.17 nM and, when combined, to 0.6 nM. N6-Monobutyryl-cAMP and 8-bromo-cAMP were equally potent in reducing IGF-II binding in the absence of insulin and inhibited maximal insulin-stimulated IGF-II binding by 60 and 30%, respectively. However, only the nonhydrolyzable cAMP analogue, N6-monobutyryl-cAMP, reduced the insulin sensitivity (EC50 0.7 nM). An important stimulatory role for Gi (guanine nucleotide-binding regulatory protein that inhibits adenylate cyclase) was indicated by the altered activities of cells from pertussis toxin-treated animals. The results suggest that beta-adrenergic stimulation through a cAMP-dependent mechanism markedly alters the insulin-stimulated redistribution of IGF-II receptors. This effect is additional to the potent antagonistic action of cAMP on insulin's signalling mechanism.     

The action of adenosine in relation to that of insulin on the low-Km cyclic AMP phosphodiesterase in rat adipocytes.

The adenosine-sensitive cyclic AMP phosphodiesterase of rat adipocytes was found to reside in the same subcellular fraction as the enzyme sensitive to insulin. There were several similarities between the action of adenosine and that of insulin on the enzyme. The action of adenosine on the phosphodiesterase is probably like that of insulin, both being receptor-mediated, although different sites or different receptors could be involved. Adenosine analogues with intact ribose but a modified purine moiety elicited a response similar to that of adenosine. Added Ca2+ was also not a requirement for the action of adenosine. The action of adenosine was not synergistic with that of insulin, neither was adenosine essential for insulin action. Insulin stimulated the enzyme even at low cell concentrations and in the presence of adenosine deaminase. Adenosine, however, enhanced the effect of insulin, but only at insulin concentrations that produced submaximal effects. Thus the mechanisms of action could be similar or related. The time-course effect of a suboptimal concentration of insulin was transitory, like that of adenosine, and was influenced by the presence of adenosine, whereas that of a maximally effective concentration of insulin was sustained for at least 20 min and was not affected by the presence of adenosine. Isoprenaline enhanced phosphodiesterase activity stimulated by optimal concentrations of either adenosine or insulin, suggesting that their effects were mediated through different mechanisms of action.     

The antilipolytic effect of insulin in human adipocytes requires activation of the phosphodiesterase

Human fat cells were incubated with two different cAMP analogues, 8-bromocAMP and 6N-monobutyrylcAMP. The former analogue is an excellent substrate for the phosphodiesterase while the latter is resistant to hydrolysis. In the presence of adenosine deaminase, isoproterenol (10(-6)M) stimulated lipolysis 8-10 fold which was similar to the effect exerted by the cAMP analogues. Basal lipolysis and lipolysis activated by 6N-monobutyrylcAMP was not inhibited by insulin even at high concentrations, whereas the effect of 8-bromocAMP was virtually completely inhibited. This effect of insulin was completely prevented by the addition of IBMX. Thus, activation of phosphodiesterase by insulin is necessary to elicit the antilipolytic effect in human adipocytes.     

Bimodal effect of insulin on hormone-stimulated lipolysis: relation to intracellular 3',5'-cyclic adenylic acid and free fatty acid levels

The present study was undertaken to determine the relationship between the antilipolytic and lipolytic effects of insulin on hormone-stimulated lipolysis and the mechanisms of these reactions. The dose-response curve of norepinephrine-stimulated lipolysis in rat adipocytes was not sigmoidal but biphasic in nature. Intracellular free fatty acid levels were linearly related to lipolytic rate and also described a biphasic profile in response to increments in norepinephrine concentration. Intracellular 3',5'-cyclic AMP levels measured 10 min after addition of increasing concentrations of norepinephrine showed a rise and a plateau followed by a secondary rise. Insulin was antilipolytic at low concentrations of norepinephrine and distinctly lipolytic at high concentrations. The combined antilipolytic and lipolytic effect of insulin is termed the "bimodal" effect of insulin on hormone-stimulated lipolysis. The bimodal effect of insulin correlated positively with changes in peak intracellular 3',5'-cyclic AMP levels. In the presence of glucose, insulin invariably enhanced lipolysis. It is suggested that the antilipolytic effect of insulin is achieved by both inhibition of adenyl cyclase activity and activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase, the net effect being a low accumulation of 3',5'-cyclic AMP. On the other hand, the lipolytic effect of insulin probably reflects enhancement of adenyl cyclase activity to an extent that overrides any activation of low-K(m) 3',5'-cyclic AMP phosphodiesterase activity, resulting in an increase in peak adipocyte 3',5'-cyclic AMP levels.     

Effects of adenosine deaminase on cyclic adenosine monophosphate accumulation, lipolysis, and glucose metabolism of fat cells.

In fat cells isolated from the parametrial adipose tissue of rats, the addition of purified adenosine deaminase increased lipolysis and cyclic adenosine 3':5'-monophosphate (cyclic AMP) accumulation. Adenosine deaminase markedly potentiated cyclic AMP accumulation due to norepinephrine. The increase in cyclic AMP due to adenosine deaminase was as rapid as that of theophylline with near maximal effects seen after only a 20-sec incubation. The increases in cyclic AMP due to crystalline adenosine deaminase from intestinal mucosa were seen at concentrations as low as 0.05 mug per ml. Further purification of the crystalline enzyme preparation by Sephadex G-100 chromatography increased both adenosine deaminase activity and cyclic AMP accumulation by fat cells. The effects of adenosine deaminase on fat cell metabolism were reversed by the addition of low concentrations of N6-(phenylisopropyl)adenosine, an analog of adenosine which is not deaminated. The effects of adenosine deaminase on cyclic AMP accumulation were blocked by coformycin which is a potent inhibitor of the enzyme. These findings suggest that deamination of adenosine is responsible for the observed effects of adenosine deaminase preparations. Protein kinase activity of fat cell homogenates was unaffected by adenosine or N6-(phenylisopropyl)adenosine. Norepinephrine-activated adenylate cyclase activity of fat cell ghosts was not inhibited by N6-(phenylisopropyl)adenosine. Adenosine deaminase did not alter basal or norepinephrine-activated adenylate cyclase activity. Cyclic AMP phosphodiesterase activity of fat cell ghosts was also unaffected by adenosine deaminase. Basal and insulin-stimulated glucose oxidation were little affected by adenosine deaminase. However, the addition of adenosine deaminase to fat cells incubated with 1.5 muM norepinephrine abolished the antilipolytic action of insulin and markedly reduced the increase in glucose oxidation due to insulin. These effects were reversed by N6-(phenylisopropyl)adenosine. Phenylisopropyl adenosine did not affect insulin action during a 1-hour incubation. If fat cells were incubated for 2 hours with phenylisopropyl adenosine prior to the addition of insulin for 1 hour there was a marked potentiation of insulin action. The potentiation of insulin action by prior incubation with phenylisopropyl adenosine was not unique as prostaglandin E1, and nicotinic acid had similar effects.     

The antilipolytic effect of insulin does not require adenylate cyclase or phosphodiesterase action

Insulin antagonized the lipolytic actions of epinephrine in rat epididymal adipocytes when the phosphodiesterase inhibitor, Ro 20-1724, was present. Adipocytes were depleted of functional cAMP by inhibiting adenylate cyclase with N6-phenylisopropyladenosine in the presence of adenosine deaminase such that Ro 20-1724 no longer stimulated lipolysis. The cAMP analogs 8-thioisopropyl-cAMP or 8-thiomethyl-cAMP, which are resistant to phosphodiesterase hydrolysis, were subsequently added to bypass adenylate cyclase and phosphodiesterase action. Under these conditions, insulin antagonized the lipolytic effects of these analogs, even in the presence of Ro 20-1724.     

N6-(Phenylisopropyl)adenosine prevents glucagon both blocking insulin''s activation of the plasma-membrane cyclic AMP phosphodiesterase and uncoupling hormonal stimulation of adenylate cyclase activity in hepatocytes.

Glucagon (10nM) prevented insulin (10nM) from activating the plasma-membrane cyclic AMP phosphodiesterase. This effect of glucagon was abolished by either PIA [N6-(phenylisopropyl)adenosine] (100nM) or adenosine (10 microM). Neither PIA nor adenosine exerted any effect on the plasma-membrane cyclic AMP phosphodiesterase activity either alone or in combination with glucagon. Furthermore, PIA and adenosine did not potentiate the action of insulin in activating this enzyme. 2-Deoxy-adenosine (10 microM) was ineffective in mimicking the action of adenosine. The effect of PIA in preventing the blockade by glucagon of insulin's action was inhibited by low concentrations of theophylline. Half-maximal effects of PIA were elicited at around 6nM-PIA. It is suggested that adenosine is exerting its effects on this system through an R-type receptor. This receptor does not appear to be directly coupled to adenylate cyclase, however, as PIA did not affect either the activity of adenylate cyclase or intracellular cyclic AMP concentrations. Insulin's activation of the plasma-membrane cyclic AMP phosphodiesterase, in the presence of both glucagon and PIA, was augmented by increasing intracellular cyclic AMP concentrations with either dibutyryl cyclic AMP or the cyclic AMP phosphodiesterase inhibitor Ro-20-1724. PIA also inhibited the ability of glucagon to uncouple (desensitize) adenylate cyclase activity in intact hepatocytes. This occurred at a half-maximal concentration of around 3 microM-PIA. However, if insulin (10 nM) was also present in the incubation medium, PIA exerted its action at a much lower concentration, with a half-maximal effect occurring at around 4 nM.     

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.     

Impaired insulin-mediated inhibition of lipolysis and glucose transport with aging

Regulation of hormone action with aging has been extensively studied; adipocytes provide an interesting model for some of these questions. We have compared the ability of insulin to stimulate glucose uptake and suppress lipolysis in adipocytes isolated from two month and twelve month-old rats. The ability of insulin to stimulate maximal glucose transport was decreased in adipocytes from the older rats (P less than 0.001); as well, insulin's EC50 was also higher (P less than 0.01) in these cells. Furthermore, these defects were present when insulin-stimulated glucose transport was measured in the presence or absence of adenosine deaminase which metabolizes endogenously released adenosine. Endogenously released adenosine is a stimulator of glucose transport and an inhibitor of lipolysis. Maximal suppression of isoproterenol-induced lipolysis by insulin was similar when adipocytes isolated from the two age groups were incubated in the absence of adenosine deaminase. However, maximal insulin-mediated suppression of lipolysis was found to be significantly decreased (P less than 0.001) in adipocytes isolated from older rats when the experiments were done in the presence of adenosine deaminase; also, insulin's EC50 was increased in these cells under these conditions (P less than 0.001). These results emphasize the importance of the adenosine receptor in modulating the response of isolated adipocytes to insulin, particularly for lipolysis, and document the presence of age-associated defects in insulin regulation of both glucose transport and lipolysis.     

Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3′:5′-cyclic monophosphate

Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-(14)C]glucose into (14)CO(2). Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into (14)CO(2). Apparent rates of glucose transport were significantly increased by insulin, H(2)O(2), adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H(2)O(2), adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H(2)O(2), adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.     

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.     

Impaired insulin action in subcutaneous adipocytes from women with visceral obesity

Visceral obesity is associated with resistance to the antilipolytic effect of insulin in vivo. We investigated whether subcutaneous abdominal and gluteal adipocytes from viscerally obese women exhibit insulin resistance in vitro. Subjects were obese black and white premenopausal nondiabetic women matched for visceral adipose tissue (VAT), total adiposity, and age. Independently of race and adipocyte size, increased VAT was associated with decreased sensitivity to insulin's antilipolytic effect in subcutaneous abdominal and gluteal adipocytes. Absolute lipolytic rates at physiologically relevant concentrations of insulin or the adenosine receptor agonist N(6)-(phenylisopropyl)adenosine were higher in subjects with the highest vs. lowest VAT area. Independently of cell size, abdominal adipocytes were less sensitive to the antilipolytic effect of insulin than gluteal adipocytes, which may partly explain increased nonesterified fatty acid fluxes in upper vs. lower body obese women. Moreover, increased VAT was associated with decreased responsiveness, but not decreased sensitivity, to insulin's stimulatory effect on glucose transport in abdominal adipocytes. These data suggest that insulin resistance of subcutaneous abdominal and, to a lesser extent, gluteal adipocytes may contribute to increased systemic lipolysis in both black and white viscerally obese women.     

Effect of adenosine on insulin activation of rat adipocyte pyruvate dehydrogenase

Adenosine and its analogue N6-phenylisopropyladenosine stimulated pyruvate dehydrogenase activity of isolated rat adipocytes. Maximal stimulation was obtained with concentrations between 50 and 100 mu M, with the effect decreasing at higher concentrations. The effects of insulin on this enzyme was modified by adenosine. The concentration of insulin (10 mu units/ml) that produced almost half-maximal stimulation, had little or no effect, when adenosine deaminase was present. Adenosine also enhanced the effect of suboptimal but not optimal concentrations of insulin. Thus, the mechanism of adenosine action on adipocyte pyruvate dehydrogenase could in some way be similar or related to that of insulin.     

The interaction between the adenylate cyclase system and insulin-stimulated glucose transport. Evidence for the importance of both cyclic-AMP-dependent and -independent mechanisms.

The counter-regulatory effect of adenosine, isoprenaline and selected cyclic AMP analogues on insulin-stimulated 3-O-methylglucose transport and insulin binding were studied in rat fat-cells. Isoprenaline alone had no consistent effect on glucose transport in the presence of maximally effective insulin concentrations. However, it decreased insulin binding by approx. 20% and increased EC50 (concn. giving 50% of maximal stimulation) for insulin from 8 +/- 1 to 17 +/- 2 mu units/ml. Adenosine deaminase (ADA) alone only exerted a slight effect, whereas isoprenaline and ADA in combination consistently decreased the maximal effect of insulin on glucose transport, decreased insulin binding by approx. 30% and markedly decreased insulin-sensitivity (EC50 61 +/- 8 mu units/ml). In cells from pertussis-toxin-treated animals, isoprenaline alone decreased the insulin response by approx. 75%, decreased insulin binding by approx. 45% and caused a marked rightward shift in the dose-response curve for insulin (EC50 103 +/- 34 mu units/ml). The importance of cyclic AMP for these effects was evaluated with the analogue N6-monobutyryl cyclic AMP, which is resistant to hydrolysis by the phosphodiesterase. The importance of phosphodiesterase activation by insulin was studied with 8-bromo cyclic AMP, which is an excellent substrate for this enzyme. N6-Monobutyryl cyclic AMP, in contrast with 8-bromo cyclic AMP, markedly impaired insulin-sensitivity (EC50 approx. 100 mu units/ml). However, the maximal effect of insulin was only slightly attenuated. In conclusion: (1) beta-adrenergic stimulation and cyclic AMP markedly alter insulin-sensitivity, but not responsiveness, mainly through post-receptor perturbations; (2) when cyclic AMP is increased phosphodiesterase activation by insulin is a critical step to elicit insulin action; (3) adenosine modulates the insulin-antagonistic effect of beta-adrenergic stimulation via Ni (inhibitory nucleotide-binding protein) through both cyclic-AMP-dependent and -independent mechanisms.     

Cell-free synthesis of active ribulose-1,5-bisphosphate carboxylase in the mesophyll chloroplasts of Sorghum vulgare

In the presence of either methyl xanthines or adenosine deaminase, isoproterenol elicited large dramatic increases in accumulation of cyclic AMPP. In contrast, cyclic AMP accumulation in response to epinephrine or norepinephrine was not potentiated by either methyl xanthines or by adenosine deaminase. Blocking the alpha adrenergic activity of norepinephrine and epinephrine with phentolamine established synergism between these catecholamines and methyl xanthines and adenosine deaminase. The activity of the particulate phosphodiesterase was not influenced by norepinephrine suggesting that the lack of synergism between the catecholamines norepinephrine and epinephrine and methyl xanthines is unrelated to this enzyme. The data are interpreted to suggest that the alpha adrenergic activity of catecholamines prevents the potentiation of cyclic AMP accumulation that occurs when the action of endogenously produced adenosine is interfered with, either by its degradation with adenosine deaminase or by receptor blockade with methyl xanthine. Because a major action of adenosine on fat cells is to inhibit adenylate cyclase it is suggested that alpha adrenergic receptor activation limits the extent to which the enzyme adenylate cyclase can be activated in a fashion similar to that of adenosine.     

Alterations in response of rat white adipocytes to insulin, noradrenaline, corticotropin and glucagon after adrenalectomy. Correction of these changes by adenosine deaminase.

1. Adipocytes isolated from rats 6--9 days after adrenalectomy had significantly increased sensitivity to insulin action against noradrenaline-stimulated lipolysis. In the presence of adenosine deaminase there was no significant difference in insulin sensitivity between cells from adrenalectomized and sham-operated rats. 2. Adipocytes from adrenalectomized rats had decreased lipolytic responses to all concentrations of noradrenaline and glucagon tested and a decreased lipolytic response to low but not high concentrations of corticotropin. There was no difference in lipolytic response to theophylline after adrenalectomy. Adenosine deaminase corrected the differences in response to noradrenaline and glucagon resulting from adrenalectomy. 3. In the presence of adenosine deaminase rates of lipolysis, after stimulation by high concentrations of noradrenaline, glucagon, corticotropin or theophylline, were the same in cells from adrenalectomized or sham-operated rats. 4. These findings and previously reported effects of adenosine and adrenalectomy on adipocyte function are discussed. It is proposed that changes in adipocyte hormone responsiveness after adrenalectomy may result from changes in adenosine metabolism or release.     

Interactions between catecholamines,methyl xanthines and adenosine in regulation of cyclic AMP accumulation in hamster adipocytes

In the presence of either methyl xanthines or adenosine deaminase, isoproterenol elicited large dramatic increases in accumulation of cyclic AMP. In contrast, cyclic AMP accumulation in response to epinephrine or norepinephrine was not potentiated by either methyl xanthines or by adenosine deaminase. Blocking the alpha adrenergic activity of norepinephrine and epinephrine with phentolamine established synergism between these catecholamines and methyl xanthines and adenosine deaminase. The activity of the particulate phosphodiesterase was not influenced by norepinephrine suggesting that the lack of synergism between the catecholamines norepinephrine and epinephrine and methyl xanthines is unrelated to this enzyme. The data are interpreted to suggest that the alpha adrenergic activity of catecholamines prevents the potentiation of cyclic AMP accumulation that occurs when the action of endogenously produced adenosine is interfered with, either by its degradation with adenosine deaminase or by receptor blockade with methyl xanthine. Because a major action of adenosine on fat cells is to inhibit adenylate cyclase it is suggested that alpha adrenergic receptor activation limits the extent to which the enzyme adenylate cyclase can be activated in a fashion similar to that of adenosine.