Changes In Adenylate Energy Charge Research Articles

AMP-activated protein kinase and ATP-citrate lyase are two distinct molecular targets for ETC-1002, a novel small molecule regulator of lipid and carbohydrate metabolism

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel investigational drug being developed for the treatment of dyslipidemia and other cardio-metabolic risk factors. The hypolipidemic, anti-atherosclerotic, anti-obesity, and glucose-lowering properties of ETC-1002, characterized in preclinical disease models, are believed to be due to dual inhibition of sterol and fatty acid synthesis and enhanced mitochondrial long-chain fatty acid β-oxidation. However, the molecular mechanism(s) mediating these activities remained undefined. Studies described here show that ETC-1002 free acid activates AMP-activated protein kinase in a Ca(2+)/calmodulin-dependent kinase β-independent and liver kinase β 1-dependent manner, without detectable changes in adenylate energy charge. Furthermore, ETC-1002 is shown to rapidly form a CoA thioester in liver, which directly inhibits ATP-citrate lyase. These distinct molecular mechanisms are complementary in their beneficial effects on lipid and carbohydrate metabolism in vitro and in vivo. Consistent with these mechanisms, ETC-1002 treatment reduced circulating proatherogenic lipoproteins, hepatic lipids, and body weight in a hamster model of hyperlipidemia, and it reduced body weight and improved glycemic control in a mouse model of diet-induced obesity. ETC-1002 offers promise as a novel therapeutic approach to improve multiple risk factors associated with metabolic syndrome and benefit patients with cardiovascular disease.

Energy state and toxin content – experiments on Microcystis aeruginosa (Chroococcales, Cyanophyta)

The correlation between the ratio of microcystin to protein and the energy state of Microcystis aeruginosa NIVA-CYA 228/1 (Chroococcales, Cyanophyta) was examined at three levels of phosphorus and iron in continuous cultures. Phosphorus- and iron-depleted conditions resulted in an increased transparency of the cultures, and together this caused significant changes in adenylate energy charge (EC) and microcystin content of M. aeruginosa. The EC ranged between 0.47 and 0.63. The microcystin content of the cells increased from 3 to 23 ng microcystin μg−1 protein in phosphorus-depleted cultures and from 2 to 12 ng microcystin μg−1 protein in iron-depleted cultures. A positive correlation between EC and microcystin content of the cells in both phosphorus- and iron-depleted cultures indicates that factors that affect EC influence microcystin production. Phosphorus and iron depletion modulated the cellular composition of M. aeruginosa. Carbohydrate constituted 5–8% of dry weight when the organisms were grown in complete O2 medium. During prolonged phosphorus and iron depletion, the carbohydrate content increased to 35–60% of dry weight. The protein content of the cells was on average 65% of dry weight when grown in complete O2 medium and decreased during phosphorus and iron depletion to an average of 35% of dry weight.

Adenine/ribose supply increases adenosine production and protects ATP pool in adenosine kinase-inhibited cardiac cells.

The objective of the present study was to establish the optimal combination of inhibitors of adenosine metabolism and nucleotide precursors resulting in long-term increase in endogenous adenosine concentration without adverse metabolic consequences in non-ischemic cardiomyocytes and endothelial cells. Cardiomyocytes and endothelial cells were isolated after collagenase digestion of the rat heart. Freshly isolated cardiac myocytes or cultured endothelial cells were incubated for up to 8 h with no inhibitors or substrates or with various combinations of adenosine deaminase inhibitor: 5 micron M erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), adenosine kinase inhibitors: 10 micro M 5'-iodotubercidin (ITu) or 10 micro M 5'-aminoadenosine (AA) and nucleotide precursors: 100 micro M adenine, 2.5 mm ribose and 5 mm inorganic phosphate. Nucleotide, nucleoside and base concentrations were evaluated at the end of the incubation by HPLC in cardiomyocyte or endothelial cells extracts and in incubation media. Adenosine content in cardiomyocyte suspension was enhanced after 3 h incubation in the presence of ITu+EHNA as compared to EHNA alone (2.8+/-0.2 v 0.9+/-0.2 nmol/mg protein, respectively). ATP decreased from an initial value of 22.7+/-0.7 nmol/mg protein to 18.9+/-0.7 in the presence of ITu+EHNA, while ATP was maintained at 21.8+/-0.7 nmol/mg protein with EHNA. With adenine+ITu+EHNA, the changes were similar to those observed with ITu+EHNA. However, with ribose+adenine+ITu+EHNA, ATP increased to 25. 8+/-1.2 nmol/mg protein and adenosine concentration was elevated to 3.9+/-0.3 nmol/mg protein. Similar results were observed if AA was used instead of ITu to inhibit adenosine kinase. All the changes were maintained after 8 h of incubation. Adenosine content was increased in endothelial cells incubated with ITu+EHNA to 3.1+/-0.4 nmol/mg protein as compared to 1.1+/-0.2 nmol/mg protein with EHNA alone after 3 h, while ATP decreased (18.1+/-1.1 v 22.0+/-1.4 nmol/mg protein with EHNA+ITu or EHNA, respectively). In the presence of adenine+ITu+EHNA, adenosine content increased after 3 h to 6.5+/-0.9 nmol/mg protein while ATP was elevated to 26.1+/-0.8 nmol/mg protein. Additional presence of ribose was without effect. No changes in adenylate energy charge were observed in cardiomyocytes or endothelium under any conditions studied. Inhibition of adenosine kinase and adenosine deaminase caused a decrease in ATP together with increased adenosine content both in endothelial cells and cardiomyocytes. However, the addition of adenine (endothelial cells) or adenine with ribose (cardiomyocytes) together with inhibitors of adenosine metabolism protected cells from ATP depletion and further increased adenosine concentration.

Effect of Storage Temperature on Stress Levels in Live Clams (Paphies donacina; tuatua)

Live New Zealand subtidal clams (Paphies donacina; tuatua) were stored in polyethylene-lined polystyrene boxes in an air chiller at 4oC and 0oC. Stress levels were monitored by measuring the accumulation of two glycolytic metabolites, D-lactate and octopine, and by measuring changes in adenylate energy charge (AEC). Tuatua mortalities were higher during storage at 0°C. Storage temperature affected the rate of D-lactate production and the ability to maintain AEC. D-lactate and/or AEC levels are useful indices of live quality during post harvest handling when product is stored out of sea water.

Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on Cape Cod.

Changes in adenylate energy charge (ECA) and in total adenine nucleotides (A(T) and DNA content (both normalized to the abundance of free-living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. ECAS (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated ECAS (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although ECA and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired energy metabolism of lymphocytes in cirrhotics after hepatectomy

To clarify the mechanism of high susceptibility to infection in cirrhotics, the changes in adenylate energy charge and MTT assay in peripheral blood lymphocytes were studied in cirrhotic and noncirrhotic patients in the early postoperative period after hepatectomy. The adenylate energy charge measured by radioactive labeling of the lymphocyte adenine nucleotide pool showed no significant difference preoperatively between cirrhotics and noncirrhotics, but a significant difference was observed in the pre- and postoperative distribution of adenine nucleotide metabolites ( P < 0.01). In the cirrhotic group, the adenylate energy charge of lymphocytes decreased significantly to 0.807 ± 0.011 on the third postoperative day compared with preoperative value (0.891 ± 0.006, P < 0.01) and was restored to the normal range on the fifth and tenth postoperative days (0.886 ± 0.006, 0.899 ± 0.014), while no significant decrease was observed in the noncirrhotic group. MTT assay revealed that lymphocyte cell function decreased significantly in cirrhotics after hepatectomy. These results indicate that, in cirrhotic patients, the energy metabolism of lymphocytes is already impaired to some extent preoperatively, and that it undergoes further deterioration when surgical stress is applied. It is suggested that the decreased energy metabolism in the lymphocyte may be responsible for the increased susceptibility to infection in postoperative cirrhotics.

Comparison of cadmium cytotoxicity in human versus rat nasal epithelial cells in vitro

Differences in the sensitivity of human and Fischer 344 rat tissues to cadmium sulphate (CdSO 4) toxicity were investigated in an in vitro model using human and rat nasal turbinate epithelial (NTE) cells. Both rat and human NTE cells were obtained from fresh, normal tissue. Methods were developed for isolating and culturing NTE cells from rat and human tissue using identical procedures, and for measuring the cellular nucleotides by high-performance liquid chromatography. Changes in adenylate energy charge and nucleotide levels were used as toxicity endpoints. Cellular Cd levels were measured by graphite-furnace atomic absorption spectrometry and expressed per unit DNA. Cd uptake was significantly greater in human NTE cells than in rat cells, particularly at the highest exposure concentration (4.8 m m-CdSO 4). The effects of CdSO 4 on the adenylate energy charge of human and rat NTE cells were similar except at high exposure concentrations and after long exposure times; after a 2–4-hr exposure to 4.8 m m CdSO 4 the adenylate energy charge of human cells was significantly less than that of the rat cells.

Substrate specificity and regulation of the maize (Zea mays) leaf ADP: protein phosphotransferase catalysing phosphorylation/inactivation of pyruvate, orthophosphate dikinase.

The protein substrate specificity of the maize (Zea mays) leaf ADP: protein phosphotransferase (regulatory protein, RP) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, orthophosphate dikinase from Zea mays and the non-sulphur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf RP via phosphorylation, with a stoichiometry of approximately 1 mol of phosphate incorporated/mol of 92.7-kDa protomer. Inactivation required both ADP and ATP, with ADP being the specific donor for regulatory phosphorylation. The requirements for inactivation/phosphorylation in this heterologous system were identical with those previously established for the tetrameric maize leaf dikinase. The ADP-dependent maize leaf RP did not phosphorylate alternative protein substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca2+ or calmodulin. The regulation of the maize leaf ADP: protein phosphotransferase was studied in terms of changes in adenylate energy charge and pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation of maize leaf dikinase was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of regulatory phosphorylation (Ki = 80 microM), consistent with its interaction with the catalytic phosphorylated intermediate of dikinase, the true protein substrate for ADP-dependent phosphorylation/inactivation.

Sequential changes in blood concentrations of fuels in relation to arterial blood ketone body ratio after 70% hepatectomy in rabbits.

The changes in arterial blood levels of glucose, free fatty acids, ketone bodies, and amino acids were investigated after 70% hepatectomy in rabbits in relation to the changes in adenylate energy charge of the remnant liver and ketone body ratio of arterial blood. Hepatic energy charge decreased to 0.767 +/- 0.008 within 24 h (p less than 0.001), and arterial blood ketone body ratio decreased to 0.415 +/- 0.041 within 12 h (p less than 0.001). Plasma free fatty acid concentrations increased to about twice the original values within 12 h (p less than 0.01), total amino acid concentrations also doubled within 24 h (p less than 0.01), and blood glucose levels decreased to below 100 mg/dl at 12 and 24 h (p less than 0.05). Afterward, at 96 h postoperatively (p.o.), blood ketone body ratio and energy charge levels were restored to near normal levels with the normalization of blood glucose, plasma free fatty acids, and plasma amino acids. These findings suggest that the decreased energy charge of the remnant liver concomitant with a fall in blood ketone body ratio may be the basis of the sequential changes in the blood concentration of fuels after massive hepatectomy.

Changes in adenylate energy charge and total adenine nucleotide concentration in mouse testis induced by cadmium administration

Subcutaneous administration of CdCl 2 (3 mg/kg body weight) caused a decrease in adenylate energy charge, (ATP + 0.5ADP)/(ATP + ADP + AMP), and a depletion of the adenine nucleotide pool in the testis of mice. This preceded a decrease in LDH-X activity in the testicular extracts. These results biochemically support the hypothesis that the testicular damage induced by cadmium is secondary to anoxia caused by vascular lesion.

Metabolic regulation via intracellular pH.

Despite earlier notions that intracellular pH (pHi) was invariant with time, recent studies have documented pHi changes of from 0.1 to 1.6 U during metabolic and developmental transitions in a variety of cells. Here we review the evidence for pHi-mediated regulation of gamete activation, cellular dormancy, the cell cycle, and stimulus-response coupling. Intracellular Ca2+ level changes also accompany many of these same transitions, and mounting evidence suggests that pHi and Ca2+ changes can be interdependent, both in their mechanisms and their effects. Although the significance of such interactions is still largely unclear, one example--the pronounced pH dependence of Ca2+ binding by calmodulin--suggests their potential importance in metabolic regulation. Similar evidence suggests that pHi changes also influence intracellular adenosine 3',5'-cyclic monophosphate levels, and vice versa. Finally we show that changes in adenylate energy charge can significantly alter pHi. In light of these interactions--and because pHi, unlike most other effectors, does not require specialized receptors--we suggest that pHi functions as a synergistic messenger, providing a metabolic context within and through which the actions of other effectors are integrated.

Variations in Adenylates and Adenylate Energy Charge During Phosphate-mediated Inhibition of Ethylene Biosynthesis in Penicillium digitatum

Summary Changes in adenine nucleotides and adenylate energy charge in shake cultures of Penicillium digitatum were determined under conditions when ethylene biosynthesis was either activated or inhibited. Activation of ethylene biosynthesis under phosphate-limiting growth conditions was accompanied by a 28% decrease in the ATP level of the mycelia. Under these growth conditions, changes in the adenylate energy charge were inversely related to the concentration of AMP but did not correlate with changes in the rate of ethylene biosynthesis or in total adenylate nucleotides. Adenine and AMP, when added individually, partially prevented the inhibitory effect of 0.01 mM orthophosphate on ethylene biosynthesis. On addition of orthophosphate, orthophosphate and adenine, or orthophosphate and AMP a rapid decrease within 2 hours in ethylene biosynthesis occurred without a striking increase in the ATP level of the cultures. In non-growing mycelia of P. digitatum , there was considerable modulation in the concentration of individual adenine nucleotides but only small changes in the adenylate energy charge were observed. The data indicated that levels of adenylates, glutamate, or the adenylate energy charge do not mediate or limit phosphate inhibition of ethylene biosynthesis. However, it is suggested that an orthophosphate-repressible phosphatase and/or a protein kinase may be involved in this process.

Changes in Adenylate Energy Charge of the Liver After an Oral Glucose Load

Changes in the adenine nucleotide metabolism after an oral glucose load were studied in the liver of normal and alloxan-diabetic rats. Changes in the energy charge were positively correlated with those in the blood glucose and plasma immunoreactive insulin levels. One hour after an oral glucose load when the plasma immunoreactive insulin levels increased maximally, the energy charge increased maximally from 0.846 to 0.867 (P less than 0.001). The increase in the energy charge was accompanied by a concomitant decrease in the ADP levels (P less than 0.05). The respiratory control ration, state 3 respiration per unit of cytochrome a (+a3), and DNP-induced ATPase activity per unit of cytochrome a (+a3) increased significantly. The adenylate kinase and pyruvate kinase activities in the liver remained unchanged. On the other hand, the energy charge in the liver of alloxan-diabetic rats did not increase significantly after an oral glucose load. It was suggested that an increase in the energy charge of the liver is attributable to the more rapid flux of intermediary metabolism in the enhanced ADP-phosphorylating reactions by mitochondria, owing to an elevated level of insulin available to the hepatic cells.

Regulation of ternary [Met-tRNA f · GTP · eukaryotic initiation factor 2] protein synthesis initiation complex formation by the adenylate energy charge

Formation of the ternary [Met-tRNA f · GTP · eukaryotic initiation factor 2] protein synthesis initiation complex in rabbit reticulocyte ribosomal eluates is dependent on the GTP: GDP ratio and on the adenylate energy charge. The elements controlling ternary initiation complex formation have been studied in a reconstituted system containing eukaryotic initiation factor 2 and nucleoside diphosphate kinase purified from the ribosomal eluate. The concentration of GTP required for half maximal formation of the ternary initiation complex is 2.5 · 10 −6 M; GDP is a potent competitive inhibitor with K i = 3.4 · 10 −7 M . Sensitive control of ternary initiation complex formation by the adenylate energy charge occurs through nucleoside diphosphate kinase regulation of the GTP : GDP ratio. Over a wide range of GTP : GDP ratios, 50% of maximal ternary initiation complex formation is observed at an adenylate energy charge of 0.85–0.90 resembling that seen in the unfractionated system. Small changes in adenylate energy charge near this value result in significant changes in the extent of ternary initiation complex formation. Since GTP is continually hydrolyzed to GDP during protein synthesis and since GDP is a competitive inhibitor of GTP binding to several of the protein factors necessary for mRNA translation, the synthetic process provides sensitive control by product inhibition. Ribosome-associated nucleoside diphosphate kinase control of GTP regeneration in response to the adenylate energy charge provides one mechanism for linking protein synthesis to the nutrient state and energy charge of the cell.

Concentration of adenylates and energy charge values in cotton roots infected with Meloidogyne incognita

Infection of resistant and susceptible cotton seedlings by Meloidogyne incognita resulted in appreciable increases in the levels of adenosine monophosphate, adenosine diphosphate and adenosine triphosphate in roots within 1 day after inoculation. In spite of marked increases in the adenylate content of the infected tissues, there were no appreciable changes in adenylate energy charge of either susceptible or resistant plants. The results of this study suggest that in Meloidogyne-infected tissues, oxidative phosphorylation is not uncoupled and, therefore, the machinery for adenosine triphosphate production remains highly functional. Moreover, the stability of energy charge in infected tissues indicates that infection does not upset the energy metabolism of the host cells.

Changes in adenylate energy charge in Ehrlich ascites tumor cells deprived of serum, glucose, or amino acids.

The adenylate energy charge in Ehrlich ascites tumor cells increases when cells are cultivated in serum-limiting medium and decreases when they are incubated in glucose- or amino acid-limited media. Protein synthetic rates decrease in cells deprived of serum, glucose, or amino acids. Supplementation of deprived cells with respective nutrients restores normal protein synthetic rates and adenylate energy charge values. Serum-deprived cells incubated in depleted serum media do not increase their rates of protein synthesis and their adenylate energy charge remains elevated. These results suggest that serum factors regulate protein synthetic rates by mechanisms other than those regulating the availability in cells of glucose or of amino acids. The increased rates of utilization of glucose and of amino acids following the addition of serum are probably due to increased biosynthetic requirements.

Additional Preliminary Dental Examiners

Changes in adenine nucleotides and adenylate energy charge in shake cultures of Penicillium digitatum were determined under conditions when ethylene biosynthesis was either activated or inhibited. Activation of ethylene biosynthesis under phosphate-limiting growth conditions was accompanied by a 28% decrease in the ATP level of the mycelia. Under these growth conditions, changes in the adenylate energy charge were inversely related to the concentration of AMP but did not correlate with changes in the rate of ethylene biosynthesis or in total adenylate nucleotides. Adenine and AMP, when added individually, partially prevented the inhibitory effect of 0.01 mM orthophosphate on ethylene biosynthesis. On addition of orthophosphate, orthophosphate and adenine, or orthophosphate and AMP a rapid decrease within 2 hours in ethylene biosynthesis occurred without a striking increase in the ATP level of the cultures. In non-growing mycelia of P. digitatum, there was considerable modulation in the concentration of individual adenine nucleotides but only small changes in the adenylate energy charge were observed. The data indicated that levels of adenylates, glutamate, or the adenylate energy charge do not mediate or limit phosphate inhibition of ethylene biosynthesis. However, it is suggested that an orthophosphate-repressible phosphatase and/or a protein kinase may be involved in this process.