Effects of catecholamines on rat myocardial metabolism. II. Influence of catecholamines on 32p-incorporation into rat myocardial adenylic nucleotides and their turn-over.

1. The influence of catecholamines (adrenaline and noradrenaline) on 32Pi incorporation into intracellular phosphate and adenylic nucleotides has been studied on rat myocardium slices; consequently, the turn-over of nucleotides could be determined and compared under the influence of these two hormones. 2. In order to specify the site of action of these catecholamines, several inhibitors and activators of energetic metabolism were included in the incubation medium: 3'5'-AMP, caffein, ouabain, oligomycin, rotenone + antimycin. 3. Both catecholamines favour Pi exchanges between intra and extracellular spaces; ATP turn-over is greatly increased, while ADP turn-over is slightly decreased, and 32P-incorporation into ADP is increased. 4. 3'5'-AMP and caffein are without effect on Pi penetration; however, caffein increases catecholamine effects on this penetration. ATP turn-over is slightly increased by 3'5'-AMP or caffein. 5. Ouabain decreases ATP turn-over but does not prevent the adrenaline induced acceleration. Inhibitors of oxidative phosphorylation and electron transport decrease ATP-turn-over severely; this inhibition is not released by catecholamines. 6. It is concluded that the catecholamine effects observed are dependent on the oxidative phosphorylations process. The increase of Pi exchange by catecholamines may be related to the increase of extracellular space and cation translocations we observed with the hormones.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes.

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37 degrees C. This increased the concentration of the adenylic nucleotides ATP (+57 per cent, ADP (+39 per cent). Cyclic AMP was increased (+50 per cent) and the intracellular inorganic phosphate decreased (-22 per cent). These changes were accompaned by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bis-phosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD+/NADH ratio along with the increase of the ATP/ADP X PO4 ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD+/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Adenine nucleotide and creatine phosphate levels in rat skeletal muscles and myocardium in myorelaxation

The content of adenylic system components and creatine phosphate was determined in skeletal muscles and myocardium after intraperitoneal injection and short-term action of myorelaxin and arduan. The injected myorelaxin causes no significant changes in macroergic phosphates in skeletal muscles, whereas arduan lowers the ATP amount by 39%. The both myorelaxants have the same effect on the adenylic system components of the myocardium: they lower significantly the level of ATP and enhance that of ADP and AMP. Different variational tendences of variation in the content of creatine phosphate in skeletal muscles (a certain rise) and in the myocardium (a decrease more than by 30%) are observed.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37°C. This increased the concentration of the adenylic nucleotides ATP (+ 57 per cent), ADP (+ 39 per cent). Cyclic AMP was increased (+ 50 per cent) and the intracellular inorganic phosphate decreased (− 22 per cent). These changes were accompanied by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bisphosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD⁺/NADH ratio along with the increase of the ATP/ADP × PO₄ ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD⁺/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Change in ATP and ADP content of fasting and feeding planarians (Polycelis nigra)

The ATP and ADP content of planarians subjected to starvation for two weeks followed by feeding for the same period was investigated. The ATP and ADP content during fasting increased and then, after feeding, returned to normal. The ATP/ADP ratio varied in the same way, which is consistent with the view that the adenylic nucleotide pool is implicated in the regulation of the energy metabolism of the organism.     

Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels

Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production using antimycin A (AA) (complex III inhibition) combined with inhibition of ADP/ATP exchange by bongkrekic acid (BA) (adenine nucleotide translocator (ANT) inhibition) induced a sharp decrease in total cellular ATP in FL5.12 parental cells (to 35% of untreated controls after 24 h of incubation). Within 24 and 48 h, 38% and 75% of the cells had died, respectively. However, in stably transfected FL5.12 Bcl-2 subclones, no cell death occurred under these experimental conditions. Similar results were obtained with Jurkat and Bcl-2 overexpressing Jurkat cells. Total cellular ATP levels were equally affected in FL5.12 Bcl-2 overexpressing cells and FL5.12 parental cells. This indicates that Bcl-2 overexpressing cells are able to survive with very low cellular ATP content. Furthermore, Bcl-2 did not protect against cell death by restoring ATP levels. This suggests that, under these conditions, Bcl-2 acts by inhibiting the signalling cascade triggered by the inhibitors that would normally lead to apoptosis.     

Involvement of phosphoinositide metabolism in potentiation by adrenaline of ADP-induced aggregation of rabbit platelets.

Changes in phosphoinositide metabolism were examined in washed rabbit platelets stimulated with 0.5 microM-ADP, 50 microM-adrenaline, or ADP and adrenaline in combination. Adrenaline does not stimulate platelet aggregation when used alone, but does potentiate aggregation stimulated by ADP. In platelets prelabelled with [32P]Pi and [3H]glycerol, adrenaline was found to potentiate the ADP-induced changes in platelet phospholipids, causing larger increases in the amount and labelling of phosphatidylinositol 4-phosphate (PIP) and phosphatidic acid than was observed with ADP alone. The combination of ADP and adrenaline did not produce a greater decrease in phosphatidylinositol 4,5-bisphosphate (PIP2) than was produced by ADP alone. In platelets prelabelled with [3H]inositol, adrenaline potentiated the increases in labelling of inositol phosphate and inositol bisphosphate stimulated by ADP; no increase in inositol trisphosphate labelling was detected with ADP alone or with the combination of ADP and adrenaline. Phentolamine, an alpha-adrenergic-receptor antagonist, blocked potentiation by adrenaline of ADP-induced changes in phosphoinositide metabolism. Propranolol and sotalol, beta-adrenergic-receptor antagonists, augmented the potentiation; this is consistent with the concept that the effect of adrenaline is mediated by beta-adrenergic receptors. The effect of adrenaline on phosphoinositide metabolism appears to be to potentiate the mechanisms by which ADP causes turnover of PIP and possibly degradation of PI, rather than the mechanism by which PIP2 is decreased.     

Effect of ATP translocation on citrulline and oxaloacetate synthesis by isolated rat liver mitochondria.

The possibility that the availability of ATP may affect the rate of synthesis of carbamoyl phosphate (measured as citrulline) by carbamoyl phosphate synthase (ammonia) was studied using respiring isolated rat liver mitochondria incubated with added ADP, with hexokinase, glucose, and ATP, or with atractylate, in order to enhance or prevent the efflux of mitochondrial ATP. The effects of these agents were compared with those on oxaloacetate synthesis from pyruvate. Addition of hexokinase, glucose, and ATP to isolated mitochondria resulted in an inhibition of citrulline synthesis which was proportional to the amounts of glucose 6-phosphate formed; under these conditions, matrix ATP and ATP/ADP tended to decrease. The addition of increasing amounts of ADP also resulted in proportional inhibition of citrulline synthesis, but in this case the matrix content of ATP and ADP increased, and ATP/ADP decreased very slightly. In the presence of atractylate, citrulline synthesis was maximal despite a 30% decrease in matrix ATP and ATP/ADP. These effects were observed whether pyruvate, succinate, glutamate, or β-OH-butyrate was used as the respiratory substrate. ADP, the hexokinase system, and atractylate had qualitatively similar but much less pronounced effects on oxaloacetate synthesis from pyruvate. Within the limits of variation observed in these experiments, the rate of synthesis of citrulline appears not to be affected by the matrix content of total ATP, total ADP, or by ATP/ADP. It is affected, however, by the velocity of translocation of ATP into the extramitochondrial medium. These findings suggest that carbamoyl phosphate synthase (ammonia) may be loosely associated with the mitochondrial inner membrane, and may compete for ATP with the ATP-ADP translocator to an extent determined by the extramitochondrial demands for ATP.     

Frog liver glycogen synthase. In vitro and in vivo interconversions between I and D forms.

1. Frog liver has enzymatic systems able to interconvert glycogen synthase. 2. D to I conversion is achieved in vitro by incubation at 30 degrees C. ATP, ADP, inorganic phosphate and glycogen are inhibitors of this conversion, whereas glucose-6-P and Mg2+ stimulate it. 3. I to D conversion in vitro depends on ATP-Mg2+. Cyclic-AMP activates this conversion, while glucose-6-P inhibits it. 4. Injection of glucose, ribose, mannose, fructose, galactose, and cortisone into frogs increase liver percentage of I activity. 5. Glucagon and adrenaline decrease percentage of I activity.     

Effect of wounding on nucleotide pools inBidens pilosa L.

Wounding both cotyledons ofBidens pilosa (var.radiatus) induces the inhibition of hypocotyl growth. The wound signal is transmitted very rapidly from cotyledon to hypocotyl and can be visualized by the change in nucleotide pools. First we have shown that the irradiance of the plant can change the ATP level without plant wounding. Therefore, plants were harvested at the start of the light period. Under these conditions, we have determined in hypocotyl the levels of adenosine triphosphate (ATP), guanosine triphosphate (GTP) and non adenylic triphosphates (NTP), and adenylate energy charge (AEC) after wounding. We have observed a transient (2 min) increase in the ATP level followed by a decrease 5 to 30 min later. A similar result was obtained for the GTP level but with some delay. The GTP level increased in 5 min and then decreased after 60 min. For the NTP level the decrease is effective from 5 to 60 min after wounding. The calculation of AEC has shown that a very tight control in the level of ATP may be involved in response to wounding.     

Adenosine triphosphate catabolism in homogenates of rat secretory enamel organs incubated in histochemical lead media.

To investigate how lead, when used as trapping agent, influences the ATP hydrolysis and to study how ATP is catalyzed in histochemical systems, homogenized secretory enamel organs were incubated in histochemical [3H]-ATP media. Aliquots from the media were taken after 3, 10, 20 and 30 min, the ATP and formed metabolites were separated by electrophoresis and radiometrically quantitated. In media lacking both lead and homogenate 2% of the ATP was spontaneously hydrolyzed during 30 min incubation at room temperature. The presence of lead caused an additional 8% hydrolysis at pH 7.2 and an additional 20% hydrolysis at pH 9.4. In the presence of homogenate, however, lead caused a net decrease of the hydrolysis of ATP as well as of ADP and AMP. This enzyme inhibition varied from around zero to some 80%, depending on pH and substrated involved. In homogenate-containing lead media, at both pH 7.2 AND 9.4, ATP was rapidly hydrolyzed primarily to ADP and subsequently to AMP and adenosine and/or inosine. After 5--10 min ADP constituted the predominant substrate at both pH:s. At pH 7.2 ADP remained so for the rest of the incubation, whereas at pH 9.4 AMP was predominant substrate at the end of the incubation. AMP was the finan catabolic product in experiments at pH 7.2, and adenosine and/or inosine at pH 9.4. Inorganic phosphate was liberated almost linearly during the whole incubation period. The results indicate that histochemical studies of substrate specific ATP-ases should be performed with short incubation times and, when high specific activities are present, in large quantities of incubation media to reduce interference by ADP and AMP hydrolyzing enzymes.     

An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.     

Adenosine triphosphate catabolism in homogenates of rat secretory enamel organs incubated in histochemical lead media

Summary To investigate how lead, when used as trapping agent, influences the ATP hydrolysis and to study how ATP is catalyzed in histochemical systems, homogenized secretory enamel organs were incubated in histochemical [³H]-ATP media. Aliquots from the media were taken after 3, 10, 20 and 30 min, and ATP and formed metabolites were separated by electrophoresis and radiometrically quantitated.In media lacking both lead and homogenate 2% of the ATP was spontaneously hydrolyzed during 30 min incubation at room temperature. The presence of lead caused an additional 8% hydrolysis at pH 7.2 and an additional 20% hydrolysis at pH 9.4. In the presence of homogenate, however, lead caused a net decrease of the hydrolysis of ATP as well as of ADP and AMP. This enzyme inhibition varied from around zero to some 80%, depending on pH and substrates involved.In homogenate-containing lead media, at both pH 7.2 and 9.4, ATP was rapidly hydrolyzed primarily to ADP and subsequently to AMP and adenosine and/or inosine. After 5–10 min ADP constituted the predominant substrate at both pH:s. At pH 7.2 ADP remained so for the rest of the incubation, whereas at pH 9.4 AMP was the predominant substrate at the end of the incubation. AMP was the final catabolic product in experiments at pH 7.2, and adenosine and/or inosine at pH 9.4. Inorganic phosphate was liberated almost linearly during the whole incubation period.The results indicate that histochemical studies of substrate specific ATP-ases should be performed with short incubation times and, when high specific activities are present, in large quantities of incubation media to reduce interference by ADP and AMP hydrolyzing enzymes.     

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.     

Regulation of glutamine metabolism in Chlorella pyrenoidosa. Regulation of glutamine synthetase activity by adenylic system components]

A decrease of glutamine synthetase (E. C. 6.3.1.2.) activity was observed under the assimilation of ammonium nitrogen in Chlorella. At the same time a decrease of ATP content in Chlorella cells took place. The ATP content was 7-fold decreased, while ADP and AMP contents were 4-fold and 3-fold increased respectively, after 15 min. of Chlorella incubation on "ammonium" medium. Further incubation for 45 min, resulted in gradual increase of ATP content and in decrease of ADP and AMP contents. The value of energy charge in ammonium assimilating Chlorella cells sharply decreased for first 15 min. of incubation and then it normalized gradually. The experiments with glutamine synthetase preparation, isolated from ammonium assimilating cells, have shown that ADP and AMP are strong inhibitors of the enzyme in the presence of Mg2+, and only ADP produces the inhibitory effect in the presence of Mn2+. No enzyme reactivation was observed after the transfer of ammonium assimilating cells into nitrogen-free medium or nitrate medium, the enzyme activity increasing at the expense of enzyme protein synthesis denovo.     

Investigation of the dependence of the intramitochondrial [ATP]/[ADP] ratio on the respiration rate

The relationship between the respiration rate and the intra- and extramito-chondrial adenine nucleotides was investigated in isolated rat liver mitochondria.

For the determination of adenine nucleotide patterns in both compartments a new procedure was developed, based on the evaluation of these metabolites from incubation of various amounts of mitochondria under identical stationary states of oxidative phosphorylation. These identical states were adjusted by addition of appropriate amounts of hexokinase to a glucose-containing incubation mixture.

Adenine nucleotides were measured in aliquots of the total extract of the incubation mixture without any separation. The concentrations of the adenine nucleotides in both compartments were obtained from a plot of the total concentration of these species versus mitochondrial protein. Disturbances of this method by unspecific efflux of adenine nucleotides could be excluded.

The results obtained for the total adenine nucleotide content (12 nmol · mg⁻¹ protein) and the intramitochondrial [ATP]/[ADP] ratio (about 4 in the resting state) are in good agreement with data obtained by other methods.

Strong evidence is provided for a decrease of the intramitochondrial [ATP]/[ADP] ratio with increasing rate of oxygen consumption. Therefore it is not necessary to assume a microcompartmentation of the intramitochondrial adenine nucleotide pool in respect to the ATPase reaction and the adenine nucleotide translocation.     

Adenine nucleotide metabolism by chondrocytes in vitro: Role of ATP in chondrocyte maturation and matrix mineralization

The objective of the investigation was to explore the notion that chondrocytes in the growth plate secrete nucleotides and that these compounds are used to regulate cell maturation and matrix mineralization. Chondrocytes were isolated from the cephalic region of chick embryo sterna and maintained in culture until confluent. To promote expression of the mature phenotype, cultures were then treated with retinoic acid. During the culture period, medium was removed and analyzed for nucleotides using a modified reverse-phase high-performance liquid chromatography (HPLC) procedure. We found that culture medium, conditioned by the chondrocytes, contained significant quantities of nucleotides. Moreover, the nucleotide concentrations were similar in magnitude to levels reported for media conditioned by other cell types. In terms of species, adenosine diphosphate (ADP) was the major nucleotide present in the conditioned medium; adenosine monophosphate (AMP) was present, but at a lower concentration than ADP. To examine the possibility that adenosine triphosphate (ATP) was released by the cultured chondrocytes, but was rapidly degraded into ADP and AMP, we examined the kinetics of ATP breakdown by chondrocytes. We found that chondrocytes degraded over 70% of exogenous ATP within 15 minutes. Similar experiments performed with ADP and AMP indicated that these nucleotides were also degraded by the cells, but at a slower rate than ATP. To determine whether the extracellular nucleotides modulate cartilage development, we examined the effect of exogenous ATP on four major determinants of chondrocyte function: alkaline phosphatase activity, cell proliferation rate, anaerobic metabolism, and mineral deposition. We found that ATP caused only minimum alterations in cell number and alkaline phosphatase activity; however, it increased the lactate content of the medium probably by stimulating anaerobic glycolysis. We noted that ATP had a significant effect on the amount and type of mineral deposited into chondrocyte cultures. Compared with untreated controls, ATP stimulated formation of a small amount of poorly crystallized calcium phosphate. The results of the study show for the first time that chondrocytes release nucleotides into the extracellular milieu. Although they are rapidly degraded, they serve to regulate both mineral formation and energy metabolism. © 1995 Wiley-Liss Inc.     

Physiological Studies on Pea Tendrils. II. The Role of Light and ATP in Contact Coiling

Excised pea (Pisum sativum L.) tendrils incubated in the light coil more than those incubated in the dark. This light effect, which displays spectral responses characteristic of chlorophyll-mediated mechanisms, is increased by at least 8 hours of prior dark incubation of plants from which the tendrils were derived. Considerable evidence indicates a major role of ATP in coiling. For example, inhibitors of ATP production decrease contact coiling. Exogenous ATP increases curvature in the dark, whereas exogenous adenosine, AMP and ADP are practically without effect. The ATP effect can be reversed by the addition of sucrose to the bathing solution. Tendrils of plants placed in the dark overnight have lower ATP levels than those held in the light. One half hour after stimulation, the endogenous ATP level of tendrils on plants kept in the light decreased fourfold. In the same period, the endogenous inorganic phosphate level increased markedly, indicating high adenosine triphosphatase activity.     

The responses of rat hepatocytes to glucagon and adrenaline. Application of quantified elasticity analysis.

The internal control of hepatocyte metabolism has been previously analysed using metabolic control analysis. The aim of this paper is to extend this analysis to include the responses of the cells to hormonal stimulus. Hepatocyte metabolism was divided into nine reaction blocks: glycogen breakdown, glucose release, glycolysis, lactate production, NADH oxidation, pyruvate oxidation, proton leak, mitochondrial phosphorylation and ATP consumption, linked by five intermediates: mitochondrial membrane potential, cytoplasmic NADH/NAD and total cellular ATP, glucose 6-phosphate and pyruvate. The kinetic responses of the reaction blocks to the intermediates were determined previously in the absence of added hormones. In this study, the changes in flux and intermediate levels that occurred upon addition of either glucagon or adrenaline were measured. From comparison of the fractional changes in fluxes and intermediate levels with the known kinetics of the system, it was possible to determine the primary sites of action of the hormones. The results show that the majority of processes in the cell are responsive to the hormones. The notable exception to this is the failure of adrenaline to have a direct effect on glycolysis. The activity change of each metabolic block observed in the presence of either hormone was quantified and compared to the indirect effects on each block caused by changes in metabolite levels. The second stage of the analysis was to use the calculated activity changes and the known control pattern of the system to give a semiquantitative analysis of the regulatory pathways employed by the hormones to achieve the changes in fluxes and metabolite levels. This was instructive in analysing, for example, how glucagon caused a decrease in flux through glycolysis and an increase in oxidative phosphorylation without large changes in metabolite levels (homeostasis). Conversely, it could be seen that the failure of adrenaline to maintain a constant glucose 6-phosphate concentration was due to the stimulation of glycogen breakdown and inhibition of glucose release.     

Opposite effects of d-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells

d-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM d-glucose. Even in the absence of d-glucose, d-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of d-fructose could be attributed to an increase in the ATP content of islet cells. After 30-60 min incubation in the presence of d-glucose and/or d-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). d-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM d-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, d-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of d-glucose and/or d-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either ⁸⁶Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.