Energetics of vacuolar compartmentation of arginine in Neurospora crassa

The energy requirements for the uptake and retention of arginine by vacuoles of Neurospora crassa have been studied. Exponentially growing mycelial cultures were treated with inhibitors of respiration or glycolysis or an uncoupler of respiration. Catabolism of arginine was monitored as urea production in urease-less strains. The rationale was that the rate and extent of such catabolism was indicative of the cytosolic arginine concentration. No catabolism was observed in cultures treated with an inhibitor or an uncoupler of respiration, but cultures treated with inhibitors of glycolysis rapidly degraded arginine. These differences could not be accounted for by alterations in the level or activity of arginase. Mycelia growing in arginine-supplemented medium and treated with an inhibitor or uncoupler of respiration degraded an amount of arginine equivalent to the cytosolic fraction of the arginine pool. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge. The inhibitors of glycolysis reduced the ATP pool but did not affect the energy charge. The results suggest that metabolic energy is required for the transport of arginine into the vacuoles but not for its retention. The latter is affected by inhibitors of glycolysis. The form of energy and the nature of the vacuolar transport mechanism(s) are discussed.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Characterization of Vacuolar Arginine Uptake and Amino Acid Efflux inNeurospora crassaUsing Cupric Ion to Permeabilize the Plasma Membrane

Treatment ofNeurospora crassamycelia with cupric ion has been shown to permeabilize the plasma and mitochondrial membranes. Permeabilized mycelia were shown to take up arginine into the vacuoles. Uptake was ATP-independent and appeared to be driven by an existing K⁺-gradient. The kinetic characteristics of the observed uptake were similar to those observed using vacuolar membrane vesicles: theK_mfor arginine uptake was found to be 4.2–4.5 mM. Permeabilized mycelia were used to study the regulation of arginine uptake into vacuoles. The results suggest that uptake is relatively indifferent to the contents of the vacuoles and is not affected by growth of mycelia in amino acid-supplemented medium. Efflux of arginine, lysine, and ornithine from vacuoles was also measured using mycelia permeabilized with cupric ion. Arginine release was shown to be specifically enhanced by cytosolic ornithine and/or increases in the vacuolar pool of arginine or ornithine. Lysine efflux was shown be indifferent to the presence of other amino acids. These observations emphasize the importance of vacuolar compartmentation in controlling arginine and ornithine metabolism and suggest that vacuolar compartmentation may play an important role in nitrogen homeostasis of filamentous fungi.     

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes

It was previously shown that cells die with increased cytosolic ATP after stimulation with apoptotic inducers including staurosporine (STS). To identify the source of apoptotic ATP elevation, we monitored, in real time, the cytosolic ATP level in luciferase-expressing HeLa cells. A mitochondrial uncoupler or a respiration chain inhibitor was found to decrease cytosolic ATP by about 50%. However, even when mitochondrial ATP synthesis was suppressed, STS induced a profound elevation of intracellular ATP. In contrast, the STS-induced ATP increase was prevented by any of three inhibitors of the glycolytic pathway: 2-deoxyglucose, iodoacetamide, and NaF. The STS effect strongly depended on intracellular calcium and was mimicked by a calcium ionophore. We conclude that Ca(2+)-dependent activation of anaerobic glycolysis, but not aerobic mitochondrial oxidative phosphorylation, is responsible for the STS-induced elevation of ATP in apoptotic HeLa cells.     

Mobilization of vacuolar arginine in Neurospora crassa. Mechanism and role of glutamine

Nitrogen starvation has been shown to increase the cytosolic arginine concentration and to accelerate protein turnover in mycelia of Neurospora crassa. The cytosolic arginine is derived from a metabolically inactive vacuolar pool. Redistribution of arginine between cytosolic and vacuolar compartments is the result of mobilization of this metabolite in response to nitrogen starvation. Mobilization of arginine (and purines) also occurred in response to glutamine limitation, but arginine accumulated upon proline starvation. These observations indicate that mobilization is a consequence of glutamine limitation rather than a general response to amino acid starvation (or limitation). Analysis of the amino acid pools in mycelia subjected to starvation or limitation suggests that glutamine (or a metabolite derived from glutamine) provides a signal which determines the metabolic fate of vacuolar arginine. The results are consistent with the hypothesis that vacuolar compartmentation provides a readily available store of nitrogen-rich compounds to be utilized during differentiation or under conditions of nutritional stress.     

Study of the regulation of oxidation and CO2 assimilation in intact Nitrobacter winogradskyi cells.

1. Changes of the adenine nucleotides in resting and growing Nitrobacter winogradskyi cells were measured in connection with regulating processes during nitrite oxidation and endogenous respiration. 2. After the addition of nitrite to endogenously respiring cells the ATP pool increased strongly during the first 60 sec at the expense of the ADP pool. At this point the energy charge was approx. 0.55. After the first 90 sec the ATP pool dropped, oscillating, to a lower level. The CO2 assimilation began at this point. 3. Under a nitrogen atmosphere the AMP pool increased and the ATP pool decreased. With a value of approx. 0.17 the energy charge was extremely low. When oxygen was added the Nitrobacter cells began to oxidize stored NADH. The ATP pool increased in a few seconds whereas the AMP pool decreased. The P/O ratio of endogenously respiring cells equaled 0.6 under these conditions. 4. During the changeover from anaerobic to aerobic conditions and in the presence of nitrite the nitrite oxidation and CO2 assimilation, opposed to aerobic conditions, were inhibited at first after the nitrite addition. The changeover of the respiratory chain enzymes from a reduced to an oxidized charge and the ATP increase were delayed in comparison with experiments without nitrite. According to these findings the endogenous respiration must be almost nil while nitrite oxidizing cells are growing.     

Energy metabolism of isolated hepatocytes at various levels of oxidative phosphorylation uncoupling]

The energy metabolism changes in isolated hepatocytes at different levels of proton conductivity of cellular membranes were studied. The low doses of the uncoupler which increased hepatocyte respiration rate but did not markedly affect the mitochondrial potential caused: the reduction in total adenine nucleotide contents (ATP + ADP + AMP), the oxidation of mitochondrial NADH, the increase in the rates of glycogenolysis and net flux via phosphofructokinase without any changes in the rates of glucose, lactate and pyruvate accumulation. High doses of the uncoupler which eliminated completely oxidative phosphorylation decreased Atkinson's energy charge down to 0.5, reduced cytoplasmic NADH, induced a further increase in the glycogenolysis rate, increased the rates of glucose and lactate accumulation, heightened glucose-6-phosphate content and lowered contents of 3-phosphoglycerate and 2-oxoglutarate.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Relationship between ATP and energy charge during lethal metabolic stress of the marine isopod Cirolana borealis

The relationship between ATP and energy charge was studied in individuals of Cirolana borealis under heavy metabolic stress caused by anoxia or exposure to toluene. Prolonged anoxia led to a lowering of the ATP content to about 10% after 4 days, with a simultaneous decrease in energy charge to about 0.25. A lowering of the total adenylate pool reduced the fall in energy charge somewhat, but this effect was marked only in late anoxia when the individuals had become inactive. Exposure to 0.14 mM toluene for 8 days led to a similar decrease in ATP and energy charge. Exposure to 1.4 mM toluene for 24 h led to only slight changes in the adenine nucleotide pool, although the individuals became narcotized within a few hours. The energy charge associated with moribund individuals thus varied much. The mechanism of energy charge stabilization through reduction of the adenine nucleotide pool as ATP declined seemed to be of little significance for the survival of the individuals.     

Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.     

Freezing and cellular metabolism in the gall fly larva,Eurosta solidaginis

Summary The effects of extracellular freezing on intracellular metabolism were monitored over both a short (9 h) and long (12 weeks) time course using the freeze tolerant larvae of the gall fly,Eurosta solidaginis.The process of freezing, monitored over the short time course, had no effect upon cellular energy levels (adenylates, arginine phosphate) but initiated a rise in glucose-6-P and lactate levels. This suggests that freezing initiates a shift towards glycolysis as the predominant mode of energy production. The process of thawing at 3°C (after 24 h at –16°C) also had no effect, even transient, on cellular energy levels demonstrating that thawing and the rapid redistribution of water and solutes which must accompany it does not disrupt cellular metabolism. During thawing accumulated lactate was quickly cleared with a t 1/2 of 20–30 min.Long term freezing at –16°C had dramatic effects on energy metabolism. Freezing for up to 1 week had minimal effects with only a small drop in arginine phosphate reserves and an increase in lactate content noted. Between 1 and 2 weeks of freezing, however, larvae showed strong signs of energy stress. The arginine phosphate pool fell from 75% to 30% of control levels, ATP content dropped by 50% and energy charge dropped to 0.75. This state, with continued lactate accumulation, was maintained through 4 weeks of freezing. Between 6 and 12 weeks of freezing energy stress became even greater. Phosphagen and ATP contents dropped to 5 and 25% of control values and energy charge decreased to about 0.50. Despite this stress, however, 94% of larvae survived 12 weeks of freezing with an 86% hatch rate of adults. The data demonstrate that the larvae can survive prolonged periods of winter freezing drawing upon glycolysis and phosphagen reserves to supply the continued basal energy demands of the cell.     

Bioenergetic properties of human sarcoma cells help define sensitivity to metabolic inhibitors

Sarcomas represent a diverse group of malignancies with distinct molecular and pathological features. A better understanding of the alterations associated with specific sarcoma subtypes is critically important to improve sarcoma treatment. Renewed interest in the metabolic properties of cancer cells has led to an exploration of targeting metabolic dependencies as a therapeutic strategy. In this study, we have characterized key bioenergetic properties of human sarcoma cells in order to identify metabolic vulnerabilities between sarcoma subtypes. We have also investigated the effects of compounds that inhibit glycolysis or mitochondrial respiration, either alone or in combination, and examined relationships between bioenergetic parameters and sensitivity to metabolic inhibitors. Using 2-deoxy-D-glucose (2-DG), a competitive inhibitor of glycolysis, oligomycin, an inhibitor of mitochondrial ATP synthase, and metformin, a widely used anti-diabetes drug and inhibitor of complex I of the mitochondrial respiratory chain, we evaluated the effects of metabolic inhibition on sarcoma cell growth and bioenergetic function. Inhibition of glycolysis by 2-DG effectively reduced the viability of alveolar rhabdomyosarcoma cells vs. embryonal rhabdomyosarcoma, osteosarcoma, and normal cells. Interestingly, inhibitors of mitochondrial respiration did not significantly affect viability, but were able to increase sensitivity of sarcomas to inhibition of glycolysis. Additionally, inhibition of glycolysis significantly reduced intracellular ATP levels, and sensitivity to 2-DG-induced growth inhibition was related to respiratory rates and glycolytic dependency. Our findings demonstrate novel relationships between sarcoma bioenergetics and sensitivity to metabolic inhibitors, and suggest that inhibition of metabolic pathways in sarcomas should be further investigated as a potential therapeutic strategy.     

A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.     

Stabilization of adenylate energy charge and its relation to human sperm motility.

The adenylate energy charge of human ejaculated spermatozoa was studied when the sperm motility was perturbed by varying pH, prolonged incubation, and caffeine. Between pH 8 and 9, which was optimal for the sperm motility, the energy charge was in the physiological range of 0.8 to 0.9. Above pH 9, the mobility, ATP content, and adenine nucleotide pool declined rapidly but the energy charge was maintained slightly below 0.8. Below pH 8, the motility also dropped drastically, but the ATP, nucleotide pool, and energy charge fell only slightly. Prolonged incubations of the spermatozoa decreased the motility, ATP, and nucleotide pool. However, the energy charge would remain above 0.6. Caffeine stimulation of the motility caused a rapid fall of ATP and the reduction of the physiological energy charge by 0.2 unit, unless glucose was added. Imidazole which reduced the caffeine-stimulated motility did not alter the physiological energy charge of the spermatozoa. The study showed that the spermatozoa could maintain the energy charge above 0.6 under stress.     

Effects of nitric oxide donor, isosorbide dinitrate, on energy metabolism of rat reticulocytes

Since nitric oxide (NO) in many cells is involved in energy metabolism, the aim of this study was to evaluate the role of isosorbide dinitrate (ISDN), a NO donor, in energy metabolism of rat reticulocytes, particularly due to their high content of hemoglobin--an effective scavenger of NO. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated in the absence (control) or in the presence of different concentrations of ISDN. ISDN decreased total and coupled oxygen consumption (p<0.05) while increased uncoupled oxygen consumption (p<0.05) in a dose- and time-dependent manner. This was followed by enhancement of glycolysis, as measured by increased glucose consumption and lactate accumulation (p<0.05). Levels of all glycolytic intermediates in the presence of ISDN indicate only stimulation of pyruvate kinase activity. ISDN did not alter the concentration of ATP, while increased ADP and AMP levels (p>0.05). In rat reticulocytes under steady-state conditions, 95.4% of overall energy was produced by oxidative phosphorylation but only 4.6% by glycolysis. Due to a reduced coupled oxygen consumption in the presence of ISDN, ATP production via oxidative phosphorylation was significantly diminished. A simultaneous increase of glycolytic ATP production is not enough to ensure constant ATP production. The calculated mean ATP turnover time was prolonged by 199% in the presence of 1.5 mmol/l ISDN. In conclusion, ISDN a) inhibited total and coupled respiration but enhanced uncoupled respiration, b) stimulated glycolysis, c) decreased ATP production and d) prolonged ATP turnover time in rat reticulocytes. These effects were mediated by NO as the effector molecule.     

Characteristics and energy requirements of an alpha-aminoisobutyric acid transport system in Streptococcus lactis.

Galactose-grown cells of Streptococcus lactis ML3 acculated alpha-aminoisobutyric acid (AIB) by using energy derived from glycolysis and arginine catabolism. The transport system displayed low-affinity Michaelis-Menten saturation kinetics. Using galactose or arginine as energy sources, similar V max and K m values for AIB entry were obtained, but on prolonged incubation the intracellular steady-state concentration of AIB in cells metabolizing arginine was only 65 to 70% that attained by glycolyzing cells. Efflux of AIB FROM PRELOADED CElls was temperature dependent and exhibited the characteristics of a first-order reaction. The rate of AIB exit was accelerated two- to threefold in the presence of metabolizable energy sources. Metabolic inhibitors including p-chloromercuribenzoate, dinitrophenol, azide, arsentate, and N, N'-dicyclohexylcarbodiimide either prevented or greatly reduced AIB uptake. Fluoride, iodoacetate and N-ethylmaleimide abolished galactose-dependent, but not arginine-energized, AIB uptake. K+ and Rb+ reduced the steady-state intracellular AIB concentration by approximately 40%, and these cations also induced rapid efflux of solute from actively transporting cells. Equivalent concentrations (10 mM) of Na+, Li+, or NH4+ were much less inhibitory. The proton-conducting ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenlyhydrazone abolished uptake and induced AIB efflux even though glycolysis and arginine catabolism continued at 60 and 140%, respectively, of control rates. A proton motive force is most likely involved in the active transport of AIB, whereas data from efflux studies suggest that energy is coupled to AIB exit in cells of S. lactis ML3.     

Utilisation of glycogen,ATP and arginine phosphate in exercise and recovery in terrestrial red crabs,Gecarcoidea natalis

Intermittent locomotion by terrestrial crustaceans may under specific circumstances increase walking distance and may allow partial re-oxidization of anaerobic products, and replenishment of ATP and arginine phosphate. The Christmas Island red crab G. natalis undertakes a substantial breeding migration each year. The leg muscles of G. natalis subjected to bouts of 2.5 min walking and 2.5 min rest were severely anaerobic. Adenylate energy charge and the large arginine phosphate stores were greatly reduced. Walking for 4 min with pauses of only 1 min exacerbated the anaerobiosis and utilised 50% of the endogenous muscle glycogen. Post-exercise, the adenylate energy charge recovered before the arginine phosphate charge and a large and persistent hyperglycaemia accompanied the restoration of glycogen. Arginine phosphate functioned as a large, longer term, energy reservoir-almost as part of the adenylate pool. Gluconeogenesis is yet to be generally substantiated in decapod crustaceans but G. natalis appears to remove lactate slowly and to reincorporate exogenous glucose into muscle glycogen in the same time frame as lactate removal from the haemolymph. The 4:1 exercise/pause regimen facilitated access to energy stores and increased walking distance, and it allowed L-lactate and H(+) efflux from the muscle during pausing. These responses are similar to those of G. natalis in the field, except during the migration when walking was entirely aerobic. Determinations of adenylate, fuel and arginine phosphate reserves and usage during the migration are required together with more detailed behavioral analysis to resolve the dichotomy in metabolic response.     

Isolation and nitrogenase activity of vesicles from Frankia sp. strain EAN1pec.

Vesicles, specialized cell structures thought to be the site of nitrogen fixation in the actinorhizal bacteria, were isolated from Frankia sp. strain EAN1pec by using French pressure disruption of mycelia followed by differential and isopycnic gradient centrifugation. The isolated vesicles reduced acetylene when incubated anaerobically with Mg2+ ions, ATP, and dithionite. No nitrogenase activity was detected in the disrupted mycelial fractions. Vesicles permeabilized by freeze-thaw or detergents showed increased rates of acetylene reduction due to increased permeability of dithionite. The effect on nitrogenase activity of different ATP concentrations was the same in normal and permeabilized vesicles. The endogenous respiratory rate of vesicles was significantly lower than that of mycelia, and the respiration rate of vesicles did not increase following the addition of succinate. The low respiratory activity of vesicles and their apparent dependence on externally supplied ATP for acetylene reduction suggest that the energy and reducing power for nitrogen fixation may be supplied from the mycelia to which they are attached.     

Regulation of lipolysis and cyclic AMP synthesis through energy supply in isolated human fat cells.

The effects of glucose and of various inhibitors of glycolysis or of oxidative phosphorylation on stimulated lipolysis and on intracellular cyclic AMP and ATP levels were investigated in isolated human fat cells. The glycolysis inhibitors, NaF and monoiodoacetate, inhibited epinephrine or theophylline-stimulated lipolysis and parallely reduced the intracellular cyclic AMP and ATP levels; however, neither NaF nor monoidoacetate significantly affected dibutyryl cyclic AMP-induced lipolysis. Removal of glucose from the medium also reduced the rate of epinephrine-stimulated lipolysis and the intracellular cyclic AMP and ATP levels but failed to modify the lipolytic activity of dibutyryl cyclic AMP. The oxidative phosphorylation inhibitors, antimycin A and, under fixed conditions, 2,4-dinitrophenol also strongly decreased the adipocyte cyclic AMP and ATP levels but inhibited as well the rate of epinephrine- and of dibutyryl cyclic AMP-induced lipolysis. N-Ethylmaleimide, a mixed glycolysis and oxidative phosphorylation inhibitor, not only reduced the intracellular cyclic AMP and ATP levels and epinephrine- or theophylline-induced lipolysis, but also that stimulated by dibutyryl cyclic AMP. When glycolysis was almost fully inhibited, human fat cells were insensitive to epinephrine but remained fully responsive to dibutyryl cyclic AMP. These results, showing a relationship between ATP availability, cyclic AMP synthesis and lipolysis, suggest a different ATP requirement for cyclic AMP synthesis and triacylglycerol lipase activation, a difference which could explain why ATP issued from glucose breakdown appears to be a determinant factor for cyclic AMP synthesis, but not for triacylglycerol lipase activation in human fat cells.