Endogenous phosphorylation of microsomal proteins in bovine corpus luteum. Tenfold activation by adenosine 3′:5′-cyclic monophosphate

Free ribosomes and a smooth-microsomal fraction were prepared from bovine corpus luteum. Both preparations will self-phosphorylate when incubated with Mg(2+) and ATP, but at low concentrations of Mg(2+) and ATP the self-phosphorylation of the smooth-microsomal fraction was much more dependent on cyclic AMP than was that of free ribosomes, stimulation by the nucleotide being up to 10-fold in the former case. The self-phosphorylation of the smooth-microsomal fraction was studied further. The reaction bears similarities to that brought about by soluble cyclic AMP-dependent protein kinase, being inhibited by Ca(2+) and the heat-stable inhibitor protein from skeletal muscle. Cyclic GMP will activate the reaction at concentrations higher than those required for full activation by cyclic AMP. In the presence of cyclic AMP, phosphate bound to protein is found almost exclusively as phosphoserine. Several proteins are phosphorylated, as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the phosphorylation of all of them is markedly stimulated by cyclic AMP. If the reaction is carried out at high concentrations of Mg(2+) and ATP, a distinct cyclic AMP-independent phosphorylation is observed. This activity is not inhibited by the heat-stable inhibitor protein, and phosphate is found esterified with both threonine and serine residues.     

Involvement of adenosine 3′:5′-cyclic monophosphate in lipid mobilization in Locusta migratoria

The rôle of cyclic AMP in hormone-induced lipid mobilization in Locusta migratoria was investigated. Injection of a corpus cardiacum extract into adult female locusts resulted in an increased level of cyclic AMP in the fat body. The cAMP concentration is maximal at about 5 min of incubation and returns to the resting level after about 10 min. The dose-response curve is linear up to about 0.01 corpus cardiacum pair equivalents.Dibutyryl-cyclic AMP mimics the lipid mobilizing effect of corpus cardiacum extract. After flight the cyclic AMP concentration in fat body increased. Injection of corpus cardiacum extract had no effect on flight muscle cyclic AMP concentration.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Steroidogenesis in isolated adrenocortical cells. Correlation with receptor-bound adenosine 3′:5′-cyclic monophosphate

Because several groups have recently questioned a mediating role for cyclic AMP in adrenocortical steroidogenesis, we analysed the problem in more detail by measuring three different cyclic AMP pools in cells isolated from decapsulated rat adrenals. Extra-cellular, total intracellular and bound intracellular cyclic AMP were determined by radioimmunoassay in comparison with corticosterone production induced by low corticotropin concentrations. The increase in extracellular and total intracellular cyclic AMP with low corticotropin concentrations was dependent on the presence of a phosphodiesterase inhibitor and short incubation times. Bound intracellular cyclic AMP was less dependent on these two parameters. In unstimulated cells cyclic AMP bound to its receptor represents only a small fraction of the total intracellular cyclic AMP. After stimulation by a concentration of corticotropin around the threshold for corticosterone production, an increase in bound cyclic AMP was observed which correlated very well with steroidogenesis both temporally and with respect to corticotropin concentration. This finding was complemented by measuring a concomitant decrease in free receptor sites. Full occupancy of the receptors was not necessary for maximal steroidogenesis. Binding kinetics of cyclic [(3)H]AMP in concentrations equivalent to the intracellular cyclic AMP concentration suggest the presence of at least three different intracellular cyclic AMP pools. These observations are in agreement with a possible role for cyclic AMP as a mediator of acute steroidogenesis induced by low corticotropin concentrations.     

An effect of insulin on adipose-tissue adenosine 3′:5′-cyclic monophosphate phosphodiesterase

1. 3':5'-Cyclic nucleotide phosphodiesterase activity was measured in homogenates prepared from epididymal fat-pads and isolated fat-cells incubated in the absence and presence of insulin. 2. Homogenates of insulin-treated tissues showed an increase in phosphodiesterase activity compared with controls. No effect of insulin was observed when the hormone was added directly to homogenates. 3. There was kinetic evidence for the presence of two 3':5'-cyclic nucleotide phosphodiesterases in adipose tissue. Insulin raised the maximal velocity of the low-K(m) enzyme and lowered the K(m) of the higher-K(m) enzyme. 4. It is suggested that the effect of insulin on adipose tissue phosphodiesterase accounts for the ability of this hormone to lower cyclic-AMP concentration in the tissue.     

Direct effects of adenosine 3′,5′-cyclic monophosphate and adenosine 5′-monophosphate upon tyrosinase activity

1. The direct actions of cAMP and 5′-AMP upon goldfish integumental and mushroom tyrosine activity were examined.2. cAMP and 5′-AMP produce similar alterations in goldfish enzyme activity, being stimulatory at low concentrations (5′-AMP being more effective than cAMP) and inhibitory at high concentrations.3. Theophylline stimulates goldfish tyrosinase activity.4. Mushroom tyrosinase activity is inhibited by cAMP and by theophylline.5. cAMP and 5′-AMP stimulation of goldfish tyrosinase activity may result from the direct action of these nucleotides on the enzyme in addition to cAMP stimulation of cAMP-dependent protein kinase.6. 5′-AMP stimulation of goldfish tyrosinase activity may indicate a key regulatory role of this nucleotide in cAMP-mediated events.     

Protein phosphokinase reactions in mammalian testis. Stimulatory effects of adenosine 3′:5′-cyclic monophosphate on the phosphorylation of basic proteins

The soluble fraction of rat testis homogenates is rich in enzymes catalysing the phosphorylation by ATP of many different proteins. The phosphorylation of certain histones, and also of a homogeneous basic protein from guinea-pig seminal-vesicle secretion, is markedly enhanced by 3':5'-cyclic AMP. Various factors affecting these reactions as catalysed by partially purified testicular enzyme preparations are described, and their possible physiological significance is discussed.     

Metabolic control mechanisms in mammalian systems. Involvement of adenosine 3′:5′-cyclic monophosphate in androgen action

1. The ability of exogenously administered cyclic AMP (adenosine 3':5'-monophosphate) to exert andromimetic action on certain carbohydrate-metabolizing enzymes was investigated in the rat prostate gland and seminal vesicles. 2. Cyclic AMP, when injected concurrently with theophylline, produced marked increases in hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, pyruvate kinase, and two hexose monophosphate-shunt enzymes, as well as alpha-glycerophosphate dehydrogenase activity in accessory sexual tissues of castrated rats. The 6-N,2'-O-dibutyryl analogue of cyclic AMP caused increases of enzyme activity that were greater than those induced by the parent compound. 3. Time-course studies demonstrated that, whereas significant increases in the activities of most enzymes occurred within 4h after the injection of cyclic AMP, maximal increases were attained at 16-24h. 4. Increase in the activity of the various prostatic and vesicular enzymes was dependent on the dose of cyclic AMP; in most instances, 2.5mg of the cyclic nucleotide/rat was sufficient to elicit a statistically significant response. 5. Administration of cyclic AMP and theophylline also produced stimulation of enzyme activities in secondary sexual tissues of immature rats. 6. Cyclic AMP and theophylline did not affect significantly any of the enzymes studied in hepatic tissue. 7. Stimulation of various carbohydrate-metabolizing enzymes in the prostate gland and seminal vesicles by cyclic AMP was independent of adrenal function. 8. Concurrent treatment with actinomycin or cycloheximide prevented the cyclic AMP- and theophylline-induced increases in enzyme activities in both castrated and adrenalectomized-castrated animals. 9. Administration of a single dose of testosterone propionate (5.0mg/100g) to castrated rats caused a significant increase in cyclic AMP concentration in both accessory sexual tissues. 10. In addition, treatment with theophylline potentiated the effects of a submaximal dose of testosterone (1.0mg/100g) on all those prostatic and seminal-vesicular enzymes that are increased by exogenous cyclic AMP. 11. The evidence indicates that cyclic AMP may be involved in triggering the known metabolic actions of androgens on secondary sexual tissues of the rat.     

Anti-anabolic effects of adenosine 3′:5′-cyclic monophosphate. Inhibition of protein synthesis

1. Evidence is presented that cyclic AMP inhibits the incorporation of l-[4,5-(3)H]leucine into protein in a cell-free system from rat liver. This inhibition occurs after aminoacyl-tRNA formation. 2. Microsomal fractions, isolated after the incubation of postmitochondrial supernatant with cyclic AMP and ATP, show a diminished ability to synthesize protein. Both cyclic AMP and ATP are required for this effect. 3. A possible physiological role for the anti-anabolic action of cyclic AMP is discussed in terms of the control of gluconeogenesis.     

Synthesis and release of adenosine 3′: 5′-cyclic monophosphate by Chlamydomonas reinhardtii

One of the labeled compounds synthesized by Chlamydomonas reinhardtii when ³²Pi was supplied was isolated from both the cells and the medium in which the cells had grown. This compound copurified with authentic [8-³H]cAMP by TLC to a constant ratio of ³²P/³H. The compound was degraded by beef heart cyclic nucleotide phosphodiesterase to a product which cochromatographed with authentic 5′AMP, at the same rate as the hydrolysis of authentic cAMP-[³H] to 5′AMP-[³H]. In both cases, 1-Me-3-isoBu-xanthine, a specific inhibitor of the phosphodiesterase, totally blocked the reaction. It is concluded that the compound synthesized by C. reinhardtii was cAMP, 85% of which was released into the medium.     

Regulation of renal gluconeogenesis by calcium ions, hormones and adenosine 3′:5′-cyclic monophosphate

1. The effect of Ca(2+), glucagon, adrenaline and adenosine 3':5'-cyclic monophosphate on gluconeogenesis by rat kidney-cortex slices was studied. 2. Glucose formation from a range of substrates, with the exception of glycerol, was increased by an increase in extracellular Ca(2+) concentration. 3. Hormones and adenosine 3':5'-cyclic monophosphate, at low Ca(2+) concentrations, stimulated glucose production from several substrates, but not from glycerol, fructose, malate or fumarate. 4. Hormonal stimulation was not detected in the absence of Ca(2+) or at 2.5mm-Ca(2+). 5. Ca(2+), hormones and adenosine 3':5'-cyclic monophosphate had no effect on phosphoenolpyruvate carboxylase activity. 6. It is proposed that Ca(2+) and adenosine 3':5'-cyclic monophosphate-mediated hormone action activate the same rate-limiting step in gluconeogenesis: this step is tentatively identified as the rate of transfer of substrates across the mitochondrial membrane.     

Adenosine 3′:5′-cyclic monophosphate and catabolite repression in Escherichia coli

1. Both permanent and transient catabolite repression of beta-galactosidase synthesis in Escherichia coli are abolished by 5mm-3':5'-cyclic-AMP when elicited by glucose, but not when caused by a mixture of glucose, glucose 6-phosphate, gluconate and casein hydrolysate (casamino acids). 2. Glucose uptake is slightly increased by 3':5'-cyclic-AMP. 3. No significant effects of the nucleotide were found on the synthesis of protein and RNA, either in exponential growth on one substrate, or during a growth shift from glycerol to glycerol plus glucose. 4. Marked changes in the soluble-protein profiles of cells growing in glycerol and glucose were caused by the presence of 3':5'-cyclic-AMP. 5. Measurements of (14)CO(2) release from specifically-labelled glucose showed that 3':5'-cyclic-AMP greatly stimulated glycolytic activity while having a minor depressing effect on the metabolic flow through the pentose phosphate cycle. 6. The concentrations of several metabolic intermediates, particularly fructose 1,6-diphosphate, were greatly affected by the presence of 3':5'-cyclic-AMP. 7. Several metabolites partially relieved glucose repression of beta-galactosidase synthesis in EDTA-treated cells; three out of five of these metabolites reversed the effect more effectively than did 3':5'-cyclic-AMP. 8. The evidence for and against a direct role for 3':5'-cyclic-AMP is discussed. It is concluded that the evidence for indirect action is at least as strong as that for direct action.     

The influence on platelet aggregation of drugs that affect the accumulation of adenosine 3′:5′-cyclic monophosphate in platelets

1. The involvement of intracellular 3':5'-cyclic AMP in the inhibition of platelet aggregation by prostaglandin E(1), isoprenaline and adenosine has been examined by a radiochemical technique. Platelet-rich plasma was incubated with radioactive adenine to incorporate (14)C radioactivity into platelet nucleotides. Pairs of identically treated samples were taken, one for the photometric measurement of platelet aggregation induced by ADP, the other for estimation of the radioactivity of 3':5'-cyclic AMP. 2. Theophylline, papaverine, dipyridamole and 2,6-bis-(diethanolamino)-4-piperidinopyrimido[5,4d]pyrimidine (compound RA233) were found to inhibit 3':5'-cyclic AMP phosphodiesterase from platelets. At concentrations of 3':5'-cyclic AMP greater than 50mum the most active inhibitor was dipyridamole; at 3':5'-cyclic AMP concentrations less than 19mum, papaverine and compound RA233 were more active than dipyridamole. 3. In the presence of compound RA233 (50mum), the effectiveness of prostaglandin E(1) as an inhibitor of platelet aggregation was increased tenfold. Compound RA233 also increased the stimulation by prostaglandin E(1) of the incorporation of radioactivity into 3':5'-cyclic AMP. 4. Compound RA233 (50mum) increased the effectiveness of both adenosine and 2-chloroadenosine as inhibitors of aggregation by 70-100-fold, and in the presence of compound RA233 both adenosine and 2-chloroadenosine stimulated the incorporation of radioactivity into 3':5'-cyclic AMP; the extent of the stimulation was proportional to the logarithm of the nucleoside concentration. 5. Compound RA233 (100-500mum) inhibited platelet aggregation by itself and caused small increases in the radioactivity of 3':5'-cyclic AMP. Partial positive correlations were found between the radioactivity of 3':5'-cyclic AMP in platelets measured at the time of addition of the aggregating agent (ADP) and the extent to which the aggregation was inhibited. 6. The results are interpreted as indicating that adenosine, 2-chloroadenosine, isoprenaline, prostaglandin E(1) and drugs that inhibit platelet 3':5'-cyclic AMP phosphodiesterase all inhibit aggregation by a common mechanism involving intracellular 3':5'-cyclic AMP.     

Protein kinase activity in membrane preparations from ox brain. Stimulation of intrinsic activity by adenosine 3′:5′-cyclic monophosphate

When Ac(2)-l-Lys-d-Ala-d-Ala and either meso-diaminopimelic acid or Gly-l-Ala are exposed to the exocellular dd-carboxypeptidase-transpeptidase of Streptomyces R61, transpeptidation reactions yielding Ac(2)-l-Lys-d-Ala-(d)-meso- diaminopimelic acid and Ac(2)-l-Lys-d-Ala-Gly-l-Ala occur concomitantly with the hydrolysis of the tripeptide into Ac(2)-l-Lys-d-Ala. The proportion of the enzyme activity which can be channelled in the transpeptidation and the hydrolysis pathways depends upon the pH and the polarity of the environment. Transpeptidation is favoured both by increasing the pH and by decreasing the water content of the reaction mixtures. Kinetics suggest that the reactions proceed through an ordered mechanism in which the acceptor molecule (meso-diaminopimelic acid or Gly-l-Ala) binds first to the enzyme. Both acceptors behave as non-competitive inhibitors of the hydrolysis pathway. Transpeptidation is inhibited by high concentrations of Gly-l-Ala but not by high concentrations of meso-diaminopimelic acid. The occurrence on the enzyme of an additional inhibitory binding site for Gly-l-Ala is suggested.     

The action of local anaesthetics on lipolysis and on adenosine 3′:5′-cyclic monophosphate content in isolated rat fat-cells

1. Local anaesthetics inhibited hormone-stimulated lipolysis in isolated rat fat-cells. The most potent anaesthetic was dibucaine, which inhibited adrenaline-stimulated lipolysis by 50% at a concentration of 0.16mm. 2. The amount of inhibition produced by a given concentration of anaesthetic was very similar with adrenaline, theophylline and dibutyryl cyclic AMP, at submaximal and maximal concentrations. 3. The inhibitory effect of dibucaine on lipolysis was apparent within 5 min and was constant over 1h. 4. Dibucaine inhibited basal, adrenaline-stimulated and insulin-stimulated glucose uptake at concentrations 6-10-fold higher than those inhibiting lipolysis. 5. The effects of dibucaine on lipolysis and glucose uptake were reversed after removal of anaesthetic and washing of cells. 6. Dibucaine further elevated the concentration of cyclic AMP in the presence of adrenaline or adrenaline plus theophylline. 7. Dibucaine had no effect on ATP content at concentrations causing 80% inhibition of lipolysis, but lowered ATP content at higher concentrations. 8. The relative potency of different local anaesthetics as inhibitors of hormone-stimulated lipolysis paralleled their potency as inhibitors of ion movements in other systems. 9. The possibility is discussed that Ca(2+) ions are involved in the regulation of lipolysis, and that local anaesthetics inhibit lipolysis by interfering with Ca(2+) translocation.     

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.     

The effect of glucose, insulin and noradrenaline on lipolysis and on the concentrations of adenosine 3′:5′-cyclic monophosphate and adenosine 5′-triphosphate in adipose tissue

1. The deoxyfluoro-d-glucopyranose 6-phosphates were prepared from the corresponding deoxyfluoro-d-glucoses and ATP by using hexokinase. 2. 3-Deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-glucose 6-phosphate were substrates for glucose phosphate isomerase, and in addition the products of this reaction, 3-deoxy-3-fluoro- and 4-deoxy-4-fluoro-d-fructose 6-phosphate respectively, were good substrates for phosphofructokinase. 3. Some C-2-substituted derivatives of d-glucose 6-phosphate were found to be competitive inhibitors of glucose phosphate isomerase. 4. The possible role of the hydroxyl groups in the binding of d-glucose 6-phopshate to glucose phosphate isomerase is discussed.     

Implication of guanosine 3′,5′-cyclic monophosphate,adenosine 3′,5′-cyclic monophosphate,adenosine 5′-mono-, di- and triphosphate and fructose-2,6-bisphosphate in the regulation of the glycolytic pathway in hypoxic/anoxic mussel,Mytilus galloprovincialis

The change in the content of cyclic GMP, cyclic AMP, ATP, ADP, AMP and fructose-2,6-bisphosphate that occurred in the mantle of the mussel Mytilus galloprovincialis Lmk when specimens of this mollusk were subjected to a hypoxia/anoxia situation were assessed. After the early 24 h in anaerobiosis, a clear decrease was observed in the ATP content, which remained close to that value for the rest of the time. AMP content doubled during the early 24 h in anaerobiosis and, from that time on, it remained close to that value. Fructose-2,6-bisphoshate and cyclic GMP showed a similar behavior. The levels of these compounds rose significantly during the early hours in anaerobiosis, and then fell to values similar to those of aerobiosis, remaining constant for the rest of the time. Neither ADP nor cAMP showed significant variations.     

The effect of starvation on phosphodiesterase activity and the content of adenosine 3′:5′-cyclic monophosphate in isolated mouse pancreatic islets

1. The concentration of cyclic AMP and the activity of phosphodiesterase were measured in isolated pancreatic islets from fed or 48h-starved mice. 2. Two different phosphodiesterases were detected. Neither the maximum activity nor the K(m) values of these enzymes were changed by starvation. 3. The concentration of cyclic AMP in non-incubated islets was the same in islets from fed and starved mice. 4. Incubation with 3.3mm-glucose for 5-30min had no effect on the concentration of cyclic AMP, irrespective of the nutritional state of the mice. Incubation with 16.7mm-glucose for 5-30min raised the concentration of cyclic AMP by about 30% in islets from fed mice. This rise was prevented by addition of mannoheptulose (3mg/ml). Incubation with 16.7mm-glucose had no effect on the cyclic AMP content in islets from starved mice. 5. In islets from fed mice 10min incubation with 5mm-caffeine had no effect on the concentration of cyclic AMP in the presence of 3.3 or 16.7mm-glucose, whereas the cyclic AMP content was increased approx. 150% in islets from starved mice. 6. After 10min incubation with 1mm-3-isobutyl-1-methylxanthine in the presence of 3.3 or 16.7mm-glucose the concentration of cyclic AMP was raised by 250% in islets from fed mice and by 400% in islets from starved mice. 7. A threefold function of glucose in the insulin-secretory process is suggested, according to which the decreased islet glucose metabolism is the primary defect in the insulin-secretory mechanism during starvation.     

The relationship between the concentration of adenosine 3′:5′-cyclic monophosphate and the anti-lipolytic action of insulin in isolated rat fat-cells

The relationship between cyclic AMP content and lipolysis, as measured by glycerol formation, was studied in isolated rat fat-cells. Inhibition of lipolysis by insulin in the presence of a low concentration of adrenaline was accompanied by little or no lowering of cyclic AMP content, measured after 15min incubation. The time-course of cyclic AMP content after addition of adrenaline showed that the effect of insulin in lowering cyclic AMP content measured after 2-5min was gradually lost over the next hour, mainly because of the fall in cyclic AMP content after an early peak in the presence of adrenaline alone. There was a 44% loss of immunoreactive insulin, from an initial concentration of 0.3nm, during a 1h incubation with fat-cells. Insulin did not affect partitioning of cyclic AMP between cells and incubation medium. When the correlation between cyclic AMP content and rate of lipolysis was investigated for a wide range of adrenaline concentrations, it was found that the lowering of cyclic AMP content by insulin was much less than that required to account for the amount of inhibition of lipolysis. It is concluded that inhibition of adrenaline-stimulated lipolysis by insulin involves factors in addition to a decrease in intracellular cyclic AMP concentration.