Glycogen metabolism and cyclic AMP levels in isolated islets of lean and genetically obese mice.

The levels of glycogen and cyclic AMP, incorporation of glucose into glycogen and activities of glycogen synthetase and phosphorylase were determined in pancreatic islets isolated from genetically obese mice and their lean litter-mates. Islets from obese mice had elevated glycogen levels, increased phosphorylase activity and an increased amount of glycogen synthetase in the physiologically more effective I-form, indicating an increased turnover of glycogen. There was no significant difference in cyclic AMP levels between islets of lean and obese mice, but inhibition of phosphodiesterase or stimulation of adenyl cyclase increased cyclic AMP levels more in obese than in lean mouse islets, indicating a more rapid turnover of cyclic AMP in the former. It is suggested that cyclic AMP stimulated phosphorolytic breakdown of glycogen may be one of the mechanisms responsible for the increased insulin secretory response to glucose observed in islets from genetically obese mice.     

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.     

Is extracellular calcium required for insulin action?

Isolated hepatocytes and isolated adipocytes incubated in the absence of added calcium ions respond to insulin with a decrease in tissue cyclic AMP levels, and an increase in low Km phosphodiesterase activity. Isolated hepatocytes also showed a diminution of glucagon stimulated glucose output in response to insulin, while adipocytes responded with increased rates of glucose oxidation, lipid synthesis and decreased glycerol output. These responses to insulin are the same as those seen when the cells are incubated in buffers containing physiological concentrations of calcium ions. When extracellular concentrations of calcium ions were made extremely low by using either gelatine or albumin which had been pretreated to remove calcium, and/or the incubation buffers contained EGTA, both the hepatocytes and adipocytes continued to respond to insulin. These results suggest that extracellular calcium ions are not required for insulin action.     

The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release by isolated rat islets of Langerhans

1. Concentrations of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and rates of insulin release were measured in islets of Langerhans isolated from rat pancreas and incubated for various times in the presence of glucose, 3-isobutyl-1-methylxanthine, caffeine, theophylline, adrenaline and diazoxide. 2. Caffeine and theophylline produced small but significant increases in both cyclic AMP and release of insulin when they were incubated in the presence of 10mm-glucose. 3. 3-Isobutyl-1-methylxanthine produced a marked increase in the intracellular concentration of cyclic AMP in the presence of 5mm- and 10mm-glucose. However, insulin release was stimulated only in the presence of 10mm-glucose. 4. In response to rising concentrations of extracellular glucose (5-20mm) there was no detectable increase in the intracellular concentration of cyclic AMP even though there was a marked increase in the rate of insulin release. 5. In response to 10mm-glucose insulin release occurred in two phases and 3-isobutyl-1-methylxanthine potentiated the effect of glucose on both phases. The intracellular concentration of cyclic AMP remained constant with glucose and rose within 10min to its maximum value with 3-isobutyl-1-methylxanthine. 6. Adrenaline and diazoxide inhibited insulin release and lowered the intracellular concentration of cyclic AMP when islets were incubated with glucose or 3-isobutyl-1-methylxanthine. 7. It is suggested that glucose does not stimulate insulin release by increasing the concentration of cyclic AMP in islet cells. However, the concentration of cyclic AMP in islet cells may modulate the effect of glucose on the release process.     

Concentration of adenosine 3':5'-cyclic monophosphate in mouse pancreatic islets measured by a protein-binding radioassay

A protein-binding radioassay for cyclic AMP was modified to detect less than 0.025pmol of the nucleotide. The method was applied to the measurement of cyclic AMP in small numbers of mouse pancreatic islets (as little as 25μg of tissue) by use of barium acetate–H₂SO₄ for deproteinization. The concentration of cyclic AMP in mouse islets incubated in media containing 3.3 or 20mm-glucose was 0.016pmol/10 islets (approx. 1μm in intracellular water). Glucose concentration (3.3 or 20mm) had no detectable effect on islet concentrations of cyclic AMP with periods of incubation or perifusion ranging from 0.5 to 60min, although insulin release rate was rapidly increased by 20mm-glucose. Caffeine (5mm) or 3-isobutyl-1-methylxanthine (1mm), which are known inhibitors of islet cyclic AMP phosphodiesterase, produced marked and rapid increases in islet cyclic AMP concentration at 3.3 or 20mm-glucose, but only enhanced the insulin release rate at the higher glucose concentration. The role of cyclic AMP in insulin release induced by glucose is discussed.     

Responsiveness to glucagon by isolated rat hepatocytes controlled by the redox state of the cytosolic nicotinamide--adenine dinucleotide couple acting on adenosine 3'':5''-cyclic monophosphate phosphodiesterase.

1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.     

Regulation of lipogenesis in adipocytes. Independent effects of thyroid hormones, cyclic AMP and insulin on the uptake of deoxy-D-glucose.

Thyroidectomy is known to enhance fat cell phosphodiesterase activity; as a result, the response to lipolytic hormones is markedly reduced. Thyroidectomy also stimulates overall lipogenesis and the uptake of glucose: the present experiments investigated whether there was a correlation between cyclic AMP and glucose uptake. The parameter measured was the transport and phosphorylation (uptake) of deoxy-D-glucose in the presence of two modifiers of the cyclic AMP pool: phosphodiesterase inhibitors and the analogue, dibutyryl cyclic AMP. The inhibition by methylxanthines and dibutyryl cyclic AMP of deoxy-D-glucose uptake observed, was the same in fat cells from normal and thyroidectomized rats: the latter nonetheless still maintained their enhanced glucose uptake. It was therefore concluded that thyroid hormones and cyclic AMP control this step by different, separate pathways. Insulin, well known for its lipogenic effect, enhanced deoxy-D-glucose uptake in fat cells from both normal and thyroidectomized rats to the same extent (about 40%). An additive effect of thyroidectomy and insulin on glucose uptake was thus demonstrated. These results imply that glucose uptake in the adipocyte is controlled by at least three factors: thyroid hormones, cyclic AMP and insulin, each of which can act independently. Maximum glucose uptake is achieved in the presence of a combination of low concentrations of cyclic AMP, of insulin, and in the absence of thyroid hormones.     

Effect of glucose on adenosine 3', 5'-monophosphate levels in rat pancreatic islets.

Time course of the changes in insulin release and cyclic AMP levels in isolated rat islets incubated in media containing 5 or 16.7 mM of glucose were followed. The higher glucose concentration caused a slight but significant increase of cyclic AMP levels after 10 min incubation, but not 5 min incubation, whereas the stimulation of insulin release by 16.7 mM of glucose was apparent in both incubation times. Theophylline increased cyclic AMP levels markedly but did not stimulate insulin release when the glucose concentration was 5 mM. A slight augmentation by theophylline of insulin release was observed in the incubation medium containing 16.7 mM glucose. All these findings suggest that the elevation of cyclic AMP in islets may not play a role for the initiation of the insulin release induced by glucose, though it may act to modulate the glucose effect.     

The effect of manganese on lipogenesis, lipolysis, adenosine 3',5' cyclic monophosphate and cyclic nucleotide phosphodiesterase activity in adipocytes from NZY mice

The insulin-like action of Mn2+ was investigated in adipocytes isolated from male mice of the NZY strain. In agreement with previous reports Mn2+ was found to stimulate both the oxidation of [U-14C]glucose to CO2 and the incorporation of [U-14C]glucose into total lipid and fatty acid, and to inhibit lipolysis stimulated by epinephrine, cyclic AMP or theophylline. The maximum effect of Mn2+ was greater than that of a maximal concentration of insulin and when both agents were present in these concentrations the effect was similar to that observed with Mn2+ alone. Mn2+ lowered the level of cyclic AMP in adipocytes incubated with isoproterenol. The effect was seen as early as 1 minute and it was greater than a maximal concentration of insulin. When Mn2+ was added to suspensions of adipocytes it increased the activity of the membrane-bound low Km cyclic nucleotide phosphodiesterase in subsequently prepared homogenates. The enzyme was stimulated 1.8-fold by Mn2+ compared with a 1.7-fold stimulation by insulin and a 2-fold stimulation in the presence of both Mn2+ and insulin.     

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.     

Subcellular localization and hormone sensitivity of adipocyte cyclic AMP phosphodiesterase.

Treatment of intact adipocytes with either or both insulin and adrenaline stimulated membrane cyclic AMP phosphodiesterase activity only in the endoplasmic reticulum subfraction. The cyclic GMP-inhibited cyclic AMP phosphodiesterase activity was also found in this fraction. Quantitative Western blotting using a specific polyclonal antibody, raised against the homogeneous 'dense-vesicle' cyclic AMP phosphodiesterase from rat liver, identified a single 63 kDa species which was localized in the adipocyte endoplasmic reticulum fraction. The ability of adrenaline to stimulate adipocyte membrane cyclic AMP phosphodiesterase was shown to be mediated via beta-adrenoceptors and not alpha 1-adrenoceptors. Membrane cyclic AMP phosphodiesterase was stimulated by glucagon but not by vasopressin, A23187 or 12-O-tetradecanoylphorbol 13-acetate (TPA). Treatment of adipocytes with either chloroquine or dansyl cadaverine failed to affect the ability of insulin to stimulate cyclic AMP phosphodiesterase activity. Treatment of an isolated adipocyte endoplasmic reticulum membrane fraction with purified protein kinase A increased its cyclic AMP phosphodiesterase activity some 2-fold. When this fraction was treated with purified protein kinase A and [32P]ATP, label was incorporated into a 63 kDa protein which was specifically immunoprecipitated with the antiserum against the liver 'dense-vesicle' cyclic AMP phosphodiesterase.     

Studies of phosphodiesterase effects on adipose tissue metabolism in obese subjects by the microdialysis technique.

The effect of non-selective (theophylline) inhibition of cyclic AMP breakdown on norepinephrine stimulated lipolysis rate was investigated in subcutaneous adipose tissue of obese subjects. In addition, changes in interstitial glucose and lactate concentration were assessed by means of the microdialysis technique. The interaction of endogenous released insulin and theophylline on adipocyte metabolism was determined. Theophylline and norepinephrine alone increased glycerol outflow significantly. When both agents were perfused in combination, interstitial glycerol concentration increased further. The enhanced glycerol level due to theophylline application was slightly decreased by insulin. In the presence of theophylline, extracellular glucose concentration increased, in contrast to the catecholamine. Norepinephrine decreased interstitial glucose level. When both drugs were added in combination, the level of interstitial glucose increased to about 1 mM, greater than with theophylline alone. With each intervention, lactate was synthesized. Local adipose tissue blood flow was increased by theophylline and theophylline plus norepinephrine. In conclusion, post-receptor mechanisms increased norepinephrine maximal stimulated lipolysis rate in subcutaneous adipose tissue. Glucose uptake was inhibited by the non-specific inhibitor of phosphodiesterase. The effect of insulin on inhibition of lipolysis was modest but sustained in the presence of high theophylline (10(-4) M) concentration. Phosphodiesterase activity may be relatively low in obese subjects in comparison with lean subjects. In lean subjects theophylline caused a transient reversal of the antilipolytic effect of insulin.     

Adenosine 3′:5′-cyclic monophosphate concentrations and phosphodiesterase activities during axenic growth and differentiation of cells of the cellular slime mould Dictyostelium discoideum

During growth of myxamoebae of Dictyostelium discoideum (strain Ax-2) in axenic medium, the myxamoebae secrete cyclic AMP. As the cells leave the exponential phase of growth and enter the stationary phase, there is an approximate doubling of the intracellular cyclic AMP content, but the amount of extracellular cyclic AMP remains proportional, at all times, to the number of myxamoebae present. During development of axenically grown myxamoebae, there is first a rise in the intracellular concentration of cyclic AMP, followed by a rise in the amount of extracellular cyclic AMP, which reaches a peak at the time of aggregation and then declines. There is a second peak in the amount of extracellular cyclic AMP found at the time of fruiting-body formation, but this second peak is not associated with a rise in the intracellular cyclic AMP concentration. Controls thus exist over the synthesis and secretion of cyclic AMP. Evidence is presented for the belief that the activity of the adenylate cyclase enzyme controls the amount of cyclic AMP synthesized rather than the activity or amount of cyclic AMP phosphodiesterase present. Similar changes occur in extracellular cyclic AMP and phosphodiesterase concentrations during incubation of myxamoebae in buffered suspensions to those occuring during the first few hours of development of such cells on solid media, but the timing of these changes is different.     

Urinary and plasma cyclic AMP levels during short term starvation in obese man: response to glucagon stimulation.

Glucagon is known to elevate the intracellular concentration of cyclic AMP in the hepatocyte. The increase in intracellular cyclic AMP is reflected by an increase in the plasma concentration of the nucleotide. Intravenous glucagon stimulation was performed on six obese non-diabetic human subjects before and after a three day fast. All patients responded to starvation by a lowering of plasma immunoreactive insulin and blood glucose. Whereas the plasma immunoreactive glucagon concentration increased over the three day period, the plasma and urinary cyclic AMP did not significantly change. Intravenous glucagon promoted qualitatively similar increases in the blood glucose and plasma concentrations of insulin and cyclic AMP before and after three days starvation.     

The effect of metabolites,papaverine, and probenecid on cyclic AMP efflux from isolated rat islets of Langerhans

The process of cyclic AMP efflux from rat islets of Langerhans has been studied. The dynamics of glucose-induced cyclic AMP efflux closely resembled the pattern of glucose-induced insulin release. Thus, both processes were dose-dependent for glucose having the same threshold concentrations (4–8 mmol/l glucose), with the time course of cyclic AMP efflux and insulin release from 0–60 min being very similar. Galactose did not affect insulin release, cyclic AMP efflux and intra-islet cyclic AMP accumulation. On the other hand, inosine, N-acetylglucosamine, α-ketoisocaproic acid, L-leucine and xylitol all promoted insulin release and cyclic AMP efflux. Except for L-leucine, all these substances enhanced the intracellular accumulation of cyclic AMP. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, greatly augmented all these parameters in the presence of glucose whereas in the absence of glucose, insulin release was not enhanced, while both cyclic AMP efflux and cyclic AMP accumulation were elevated. The drug, probenecid, did not alter either insulin release or intra-islet cyclic AMP levels, while cyclic AMP efflux was markedly reduced (though not abolished). Papaverine inhibited both insulin release and cyclic AMP efflux, but was found to augment the intra-islet cyclic AMP levels. The efflux of cyclic AMP correlates more closely with insulin release than with the cyclic AMP accumulation in most instances. The efflux is independent of either insulin secretory granule extrusion or intracellular fluctuations of the nucleotide, though it is not yet known whether cyclic AMP efflux may have some regulatory significance in insulin release.     

Insulin secretion. Interrelationships of glucose, cyclic adenosine 3:5-monophosphate, and calcium.

Glucose elevates both cyclic adenosine 3:5-monophosphate (cyclic AMP) and insulin secretion rapidly and in a parallel dose-dependent fashion in perifused rat islets. Theophylline stimulates cyclic AMP much more than glucose, yet secretion is much less. When the two agents are combined, cyclic AMP is similar to theophylline alone yet secretion is augmented synergistically. Glucose-induced cyclic AMP generation and insulin secretion are dependent on extracellular calcium. Theophylline-induced insulin secretion is also extracellular calcium-dependent; however, theophylline-induced cyclic AMP elevation is independent of extracellular calcium. Thus, extracellular calcium has multiple effects on insulin secretion, some of which appear unrelated to a terminal secretory process. When glucose is combined with theophylline at physiologic levels of extracellular calcium, both the first and second phases of secretion are prominent. At extracellular calcium levels of 0.05 mM, only the second phase is prominent whereas at 10 nM extracellular calcium (ethylene glycol bis(beta-aminoethyl ether)-N,-tetraacetic acid) only the first phase is prominent. A divalent cation ionophore (a23187, Eli Lilly), which transports calcium and magnesium ions across biological membranes, was used to elucidate further the role of calcium and magnesium. If the ionophore (10 muM) is perifused for 5 min at low extracellular calcium and magnesium, and physiologic calcium is then added, a sudden spike of insulin release occurs in the absence of cyclic AMP generation. Similar results were obtained with magnesium. When the ionophore is perifused for 30 min at low calcium and magnesium, insulin secretion again occurs in the absence of cyclic AMP generation. Electron microscopic examination of the B cells following perifusion with the ionophore shows no specific alterations. These observations suggest that: (a) glucose elevates cyclic AMP, but the latter acts primarily as a positive feed-forward modulator of glucose-induced insulin release; and (b) extracellular calcium has multiple effects on insulin secretion both upon, and independent of, the cyclic AMP system.     

Cyclic AMP increases the concentration of insulin receptors in cultured fibroblasts and lymphocytes.

Incubation of SV40 transformed fibroblasts with dibutyryl cyclic AMP, 8-bromo-cyclic AMP, or 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, produced a two-fold increase in insulin receptor concentration without an effect on receptor affinity. The increase was dose-dependent, was observed after 8 hrs of treatment, and reached a maximum level by 12 to 24 hours. Upon removal of the nucleotide, receptor number decreased towards basal level.Incubation of cultured human lymphocytes (IM-9 line) with cyclic AMP derivatives or MIX also increased the number of insulin receptors without an alteration in receptor affinity. This effect was partially blocked by inhibition of protein synthesis and was independent of changes in cell cycle. The increase in insulin receptors was a specific response to cyclic AMP as the number of receptors for human growth hormone was unaltered. Incubation with 8-bromo-cyclic GMP did not alter the level of insulin binding.     

Effects of probenecid on transport and metabolism of cyclic AMP by isolated rabbit renal tubules

The effects of probenecid on the transport and metabolism of cyclic [14C]-AMP were studied in isolated rabbit kidney cortex tubules. Incubation in a medium with 10-400 microM probenecid for 30 min caused a 30-70% decrease in the tubular uptake of labeled material from a medium containing 0.1 mM cyclic [14C]AMP. The radioactivity in the tubules, after 30 min incubation, with or without probenecid, was mostly in the form of inosine and hypoxanthine. The disappearance of external cyclic [14C]AMP was retarded by probenecid and the concentration ratio of cyclic AMP to inosine + hypoxanthine was increased. Cyclic AMP phosphodiesterase activities, from both the soluble and particulate fractions of the kidney, were inhibited by probenecid. These findings indicate that the changes caused by probenecid on the renal disposal of extracellular cyclic AMP can be accounted for by a decrease in the accumulation of the products of cyclic AMP metabolism secondary to inhibition of extracellular cyclic AMP phosphodiesterase activity.     

Attenuation of epinephrine-induced increase in liver cyclic AMP by endogeneous insulin in vivo.

1. Epinephrine-induced increase in rat liver cyclic AMP in vivo was potentiated when the circulating insulin was suppressed by injection of anti-insulin serum or by induction of diabetes. Consequently, phosphorylase was activated, glycogen synthetase was inactivated and glycogen accumulation induced by glucose load was prevented by epinephrine in the insulin-deficient rats to a much larger extent than in normal rats. 2. Insulin lack was effective in potentiating epinephrine-induced increase in liver and muscule cyclic AMP even after the treatment of rats with theophylline; the potentiation could not be solely accounted for by the inhibition of cyclic AMP phosphodiesterase. Thus, it is likely that insulin lack enhaces epinephrine activation of adenylate cyclase. 3. Unlike epinephrine, glucagon increased liver cyclic AMP to essentially the same extent whether the rat was treated with anti-insulin serum or not. 4. Based on the difference in dose-response curves between normal and insulin-deficient rats, a possibility is discussed that there are two adenylate cylase in the liver with higher and lower affinities for epinephrine and that circulating insulin blocks the high affinity enzyme selectively.     

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.