The stimulus-secretion coupling of glucose-induced insulin release. Metabolic effects of menadione in isolated islets.

Pancreatic islets contain an enzyme system which catalyzes the donation of hydrogen from NAD(P)H to menadione (2-methyl-1,4-naphthoquinone). In high concentrations (20 to 50 micrometer), menadione, in addition to lowering the concentration of reduced pyridine nucleotides in the islets, also impairs glycolysis and glucose oxidation, decreases ATP concentration, and inhibits proinsulin biosynthesis. However, at a 10 micrometer concentration, menadione fails to affect the concentration of adenine nucleotides, the utilization of glucose, the production of lactate and pyruvate, the oxidation of [6-14C]glucose and the synthesis of proinsulin; whereas the metabolism of glucose through the pentose shunt is markedly increased. The sole inhibitory effect of menadione 10 micrometer upon metabolic parameters is to reduce the concentration of both NADH and NADPH, such an effect being noticed in islets exposed to glucose 11.1 mM but not in those incubated at a higher glucose level (27.8 mM). Since, in the presence of glucose 11.1 mM, menadione 10 micrometer also severely decreases glucose-stimulated45 calcium net uptake and subsequent insulin release, it is concluded that the availability of reduced pyridine nucleotides may play an essential role in the secretory sequence by coupling metabolic to cationic events. Thus, when insulinotropic nutrients are oxidized in the B-cell, the increased availability of reduced pyridine nucleotides could modify the affinity for cations of native ionophoretic systems, eventually leading to the accumulation of calcium up to a level sufficient to trigger insulin release.     

Regulation of gluconeogenesis during exposure of young rats to hypoxic conditions

1. Two-day-old rats were exposed at constant temperature to atmospheres containing air and nitrogen with the air content varied in steps from 100 to 0%. By using this system of graded hypoxia a comparison was made between rates of gluconeogenesis from lactate, serine and aspartate in the whole animal and the concentrations of several liver metabolites. 2. Gluconeogenesis, expressed as the percentage incorporation of labelled isotope into glucose plus glycogen, proceeds linearly for 30min when the animals are incubated in a normal air atmosphere, but is completely suppressed if the atmosphere is 100% nitrogen. 3. Preincubation of animals for between 5 and 30min under an atmosphere containing 19% air results in the attainment of a new steady state with respect to gluconeogenesis and hepatic concentrations of ATP, ADP, AMP, lactate, pyruvate, beta-hydroxybutyrate and acetoacetate. 4. When lactate (100mumol), aspartate (20mumol) or serine (20mumol) was injected, it was shown that the more severe the hypoxia the greater the depression of gluconeogenesis. Under conditions when gluconeogenesis was markedly inhibited there were no changes in the degree of phosphorylation of hepatic adenine nucleotides, but free [NAD(+)]/[NADH] ratios fell in both cytosol and mitochondrial compartments of the liver cell. 5. Measurements of total liver NAD(+) and NADH showed that the concentrations of these nucleotide coenzymes changed less with anoxia, in comparison with the concentration ratio of free coenzymes. 6. Calculations showed that the difference in NAD(+)-NADH redox potentials between mitochondrial and cytosol compartments increased with the severity of hypoxia. 7. From the constancy of the concentrations of adenine nucleotides it is concluded that liver of hypoxic rats can conserve ATP by lowering the rate of ATP utilization for gluconeogenesis. Gluconeogenesis may be regulated in turn by the changes in mitochondrial and cytosol redox state.     

Perfusion cultures require optimum respiratory ATP supply to maximize cell-specific and volumetric productivities

Perfusion processes are an emerging alternative to common fed-batch processes in the growing biopharmaceutical industry. However, the challenge of maintaining high cell-specific productivities remains. In this study, glucose limitation was applied to two perfusion steady states and compared with a third steady state without any detectable limitation. The metabolic phenotype was enhanced under glucose limitation with a decrease of 30% in glucose uptake and 75% in lactate formation. Cell-specific productivities were substantially improved by 50%. Remarkably, the productivities showed a strong correlation to respiratory adenosine triphosphate (ATP) supply. As less reduced nicotinamide adenine dinucleotide (NADH) remained in the cytosol, the ATP generation from oxidative phosphorylation was increased by almost 30%. Consequently, the efficiency of carbon metabolism and the resulting respiratory ATP supply was crucial for maintaining the highly productive cellular state. This study highlights that glucose limitation can be used for process intensification in perfusion cultures as ATP generation via respiration is significantly increased, leading to elevated productivities.     

Regulation of adenine nucleotide concentration at endothelium-fluid interface by viscous shear flow.

The action of adenine nucleotides on vascular endothelial cells is apparently mediated by the local flow conditions. Because nucleotides are sequentially degraded from ATP-->ADP-->AMP-->adenosine by ecto-enzymes at the endothelial surface, it has been hypothesized that the observed flow effect is caused by the flow-dependent change of nucleotide concentration at the cell surface. In this study, we have calculated the concentration profiles of adenine nucleotides at the cell surface under flow conditions encountered in an in vitro parallel-plate flow system, as has been used in several related experimental studies. When medium containing uniformly distributed ATP is perfused over endothelial monolayers, our results show that ATP concentration in the cell vicinity gradually decreases in the streamwise direction as a result of enzymatic degradation. This hydrolysis of ATP results in the generation of ADP, and ADP concentration in turn gradually increases at the cell surface. The concentration profiles of nucleotides are dependent on the levels of applied wall shear rate. As the corresponding shear stress increases from 0.1 to 30 dynes/cm2, ATP concentration at the cell surface at the center of coverslip increases from 0.66 to 0.93. Under no-flow conditions, our model predicts a steady decline of ATP concentration and a transient increase of ATP-derived ADP, comparable to the published results of previous experiments. These numerical results, combined with our recent experimental data, provide insights into the cellular mechanisms by which hemodynamic flow modulates the effects of vasoactive agents on endothelium.     

Rickettsial permeability. An ADP-ATP transport system.

The obligate intracellular parasitic bacterium, Rickettsia prowazeki, has a carrier-mediated transport system for ADP and ATP. The transport of nucleotides was measured by membrane filtration assays; the assay was shown not to harm the relatively labile rickettsiae. The nucleotide transport system was shown to reside in the rickettsiae, not in the contaminating yolk sac mitochondria of the preparation. The influx of nucleotide had an activation energy of 12 to 13 kcal above 22 deg-rees (an apparent transition temperature), and 30 kcal below this value. The uptake of nucleotide was independent of the Mg2+ concentration, but was markedly stimulated by the phosphate concentration. The pH optimum of the influx of nucleotide was pH 7. The specificity of the transport system was remarkable in that it required a specific moiety in each portion of the nucleotide, i.e. an adenine base, a ribose sugar, and two or three, but not one, phosphates. Of the wide variety of compounds tested, the system could transport only ADP, ATP, and (beta, gamma-methylene) adenosine 5'-triphosphate. The influx of nucleotide was a saturable process; half-maximum velocity was achieved at a nucleotide concentration of about 75 muM. ADP and ATP were competitive inhibitors of each other's transport. Although at least 95% of the labeled intracellular nucleotide was exchangeable, efflux of labeled nucleotide was observed only in the presence of unlabeled nucleotide in the medium. Half-maximum efflux was achieved at a concentration of about 75 muM. A large intracellular to extracellular concentration gradient of labeled nucleotide was maintained in the presence of metabolic inhibitors and uncouplers, which completely abolished rickettsial hemolysis. While having no effect on the steady state, KCN and DNP accelerated both influx and efflux. Measurements of the endogenous pool of adenine nucleotides in isolated rickettsiae show that is was large (5 mM), and that these unlabeled nucleotides exchanged, on approximately a 1/1 basis, with exogenously added nucleotide. These studies support the proposal that rickettsiae are not "leaky" to adenine nucleotides or to small molecules in general, and that they have a carrier-mediated transport system which allows an exchange of host and parasite ADP and ATP.     

Glucose 1,6-bisphosphate-overloaded erythrocytes: a strategy to investigate the metabolic role of the bisphosphate in red blood cells.

Human erythrocytes overloaded with glucose 1,6-bisphosphate were prepared in order to establish the metabolic significance of this phosphorylated sugar in the intact red cell. The intracellular glucose 1,6-bisphosphate concentration was increased six- and twofold over the normal level by encapsulating (i) the commercially available compound and (ii) the glucose 1,6-bisphosphate synthase obtained from rabbit skeletal muscle, respectively. In both experimental conditions, a reduction of glucose utilization by the loaded cells was observed after reequilibration to the steady state. At the steady state, the concentrations of the glycolytic intermediates and of the adenine nucleotides appeared substantially unmodified when compared with those of controls, with the exception of a 50% reduction of glucose and fructose 6-phosphate measured in erythrocytes encapsulated with exogenous glucose 1,6-bisphosphate. Under the considered experimental conditions, the elevated intracellular glucose 1,6-bisphosphate appears to display an inhibitory effect on hexokinase that overcomes the possible activation of phosphofructokinase or pyruvate kinase.     

腺嘌呤对大肠杆菌DH5α和其耐乙酸突变株DA19 代谢流分布的影响

【目的】本文通过分析在基本培养基中添加腺嘌呤对大肠杆菌DH5α和其耐乙酸突变株DA19代谢流分布的影响,从而进一步了解二者在代谢调控方面的差异。【方法】对2个菌株分别在氮源限制基本培养基及添加腺嘌呤的氮源限制基本培养基中进行连续培养,分析两者代谢流变化差异,并与酶活测定结果进行比较。【结果】添加腺嘌呤降低了DH5α的葡萄糖比消耗速率和乙酸的比生成速率,提高了菌体关于葡萄糖的得率,而丙酮酸比生成速率变化不明显。与MN培养基相比,添加腺嘌呤后DH5α降低了乙酸分流比,提高了分泌丙酮酸和三羧酸循环分流比,同时明显改变了磷酸果糖激酶、6-磷酸葡萄糖脱氢酶和乙酸激酶酶活。与DH5α不同,添加腺嘌呤使得DA19的丙酮酸比生成速率增加了近57%,而其它参数无明显改变。与MN培养基相比,DA19在添加腺嘌呤后降低了三羧酸循环分流比,大大提高了分泌丙酮酸分流比,而关键酶活未发生明显改变。酶活变化与代谢流结果基本一致。【结论】由于大肠杆菌DH5α和DA19嘌呤核苷酸从头合成途径能力存在差异,因此添加腺嘌呤对两个菌株的代谢流分布产生了完全不同的影响。     

Studies on the functional significance of mitochondrial bound hexokinase in rabbit reticulocytes

Mitochondria from rabbit reticulocytes contain about 50% of the total reticulocyte hexokinases. The proportion of mitochondrial hexokinases may be changed under different metabolic conditions. Mitochondrial bound and soluble hexokinases exhibit different kinetic properties (KMATP and glucose-6-phosphate inhibition). The respiratory rate of isolated reticulocyte mitochondria in the presence of glucose depends on the glucose-6-phosphate concentration, as the ADP generation by the endogenous hexokinases is strongly inhibited by glucose-6-phosphate. In the experimental system all intermediary states of mitochondrial respiration can be adjusted between the state of maximal activity (state 3 or active state) and the controlled or resting state (state 4) by different glucose-6-phosphate levels. The stationary levels of the extramitochondrial adenine nucleotides in this experimental system have been measured. The rate of mitochondrial respiration and ATP formation depends on the extramitochondrial ATP/ADP ratio. At ratios of about 10 and lower the mitochondria are in their maximum phosphorylation state, at higher ratios the mitochondrial ATP formation is controlled by the extramitochondrial ATP/ADP ratio. It is postulated that the close intercounnection between the mitochondrial hexokinase and the mitochondrial ATP forming system in reticulocytes is of funcitonal significance for mitochondrial-cytosolic interactions in rabbit reticulocytes and probably in other types of cells with mitochondrial hexokinases, too.     

Activation of hepatocyte glycogen synthase by metabolic inhibitors

Incubation of isolated rat hepatocytes with metabolic inhibitors causes an increase in the -glucose 6-P/+glucose 6-P activity ratio of glycogen synthase after decreasing ATP and increasing AMP levels. Concomitantly, the activity of phosphorylase is increased six-fold by the same treatment. This activation of both enzymes remains after gel filtration of the hepatocyte extracts. Addition of metabolic inhibitors to cells pretreated with an inhibitor of AMP-deaminase results in an accumulation of AMP and, simultaneously, in a further increase in the activation state of glycogen synthase. The correlation coefficient between the intracellular concentration of AMP and glycogen synthase activity is r = 0.93. It is proposed that the covalent activation of glycogen synthase by metabolic inhibitors can be triggered by changes in the level of the intracellular concentrations of adenine nucleotides.     

Activation of pyruvate dehydrogenase by insulin in isolated brown fat cells.

Isolated fat cells from rat brown adipose tissue in vitro respond to insulin with an increase of pyruvate dehydrogenase (EC 1.2.4.1) activity due to conversion of the inactive form of the enzyme (PDHb) to the active form (PDHa). Like in white adipocytes this effect depends on the presence of glucose or 2-deoxyglucose in the medium. The interrelationship between the steady state of the PDH-system and the phosphorylation state of the adenine nucleotides was studied in white adipose tissue. While insulin in the presence of 2-deoxyglucose caused a large fall of the tissue ATP/ADP ratio which could explain the increase of PDHa activity, the ATP/ADP ratio remained unchanged during incubations with insulin and glucose. Thus it appears that other factors than the ATP/ADP ratio are involved in the regulation of PDH activity by insulin the nature of which remains to be elucidated.     

Adenine nucleotide regulation in pancreatic beta-cells: modeling of ATP/ADP-Ca2+ interactions

Glucose metabolism stimulates insulin secretion in pancreatic beta-cells. A consequence of metabolism is an increase in the ratio of ATP to ADP ([ATP]/[ADP]) that contributes to depolarization of the plasma membrane via inhibition of ATP-sensitive K+ (K(ATP)) channels. The subsequent activation of calcium channels and increased intracellular calcium leads to insulin exocytosis. Here we evaluate new data and review the literature on nucleotide pool regulation to determine the utility and predictive value of a new mathematical model of ion and metabolic flux regulation in beta-cells. The model relates glucose consumption, nucleotide pool concentration, respiration, Ca2+ flux, and K(ATP) channel activity. The results support the hypothesis that beta-cells maintain a relatively high [ATP]/[ADP] value even in low glucose and that dramatically decreased free ADP with only modestly increased ATP follows from glucose metabolism. We suggest that the mechanism in beta-cells that leads to this result can simply involve keeping the total adenine nucleotide concentration unchanged during a glucose elevation if a high [ATP]/[ADP] ratio exits even at low glucose levels. Furthermore, modeling shows that independent glucose-induced oscillations of intracellular calcium can lead to slow oscillations in nucleotide concentrations, further predicting an influence of calcium flux on other metabolic oscillations. The results demonstrate the utility of comprehensive mathematical modeling in understanding the ramifications of potential defects in beta-cell function in diabetes.     

The contribution of adenine nucleotide loss to ischemia-induced impairment of rat kidney cortex mitochondria.

Adenine nucleotides and respiration were assayed with rat kidney mitochondria depleted of adenine nucleotides by pyrophosphate treatment and by normothermic ischemia, respectively, with the aim of identifying net uptake of ATP as well as elucidating the contribution of adenine nucleotide loss to the ischemic impairment of oxidative phosphorylation. Treatment of rat kidney mitochondria with pyrophosphate caused a loss of adenine nucleotides as well as a decrease of state 3 respiration. After incubation of pyrophosphate-treated mitochondria with ATP, Mg2+ and phosphate, the content of adenine nucleotides increased. We propose that kidney mitochondria possess a mechanism for net uptake of ATP. Restoration of a normal content of matrix adenine nucleotides was related to full recovery of the rate of state 3 respiration. A hyperbolic relationship between the matrix content of adenine nucleotides and the rate of state 3 respiration was observed. Mitochondria isolated from kidneys exposed to normothermic ischemia were characterized by a decrease in the content of adenine nucleotides as well as in state 3 respiration. Incubation of ischemic mitochondria with ATP, Mg2+ and phosphate restored the content of adenine nucleotides to values measured in freshly-isolated mitochondria. State 3 respiration of ischemic mitochondria reloaded with ATP recovered only partially. The rate of state 3 respiration increased by ATP-reloading approached that of uncoupler-stimulated respiration measured with ischemic mitochondria. These findings suggest that the decrease of matrix adenine nucleotides contributes to the impairment of ischemic mitochondria as well as underlining the occurrence of additional molecular changes of respiratory chain limiting the oxidative phosphorylation.     

Dynamics of energy charge and adenine nucleotides during uncoupling of catabolism and anabolism in Penicillium ochrochloron

Filamentous fungi are able to spill energy when exposed to energy excess by uncoupling catabolism from anabolism, e.g. via overflow metabolism. In current study we tested the hypothesis that overflow metabolism is regulated via the energetic status of the hyphae (i.e. energy charge, ATP concentration). This hypothesis was studied in Penicillium ochrochloron during the steady state of glucose- or ammonium-limited chemostat cultures as well as during three transient states ((i) glucose pulse to a glucose-limited chemostat, (ii) shift from glucose-limited to ammonium-limited conditions in a chemostat, and (iii) ammonium exhaustion in batch culture). Organic acids were excreted under all conditions, even during exponential growth in batch culture as well as under glucose-limited conditions in a chemostat. Partial uncoupling of catabolism and anabolism via overflow metabolism was thus constitutively present. Under all tested conditions, overflow metabolism was independent of the energy charge or the ATP concentration of the hyphae. There was a reciprocal correlation between glucose uptake rate and intracellular adenine nucleotide content. During all transients states a rapid decrease in energy charge and the concentrations of nucleotides was observed shortly after a change in glycolytic flux (“ATP paradoxon”). A possible connection between the change in adenine nucleotide concentrations and the purine salvage pathway is discussed.     

A reassessment of decreased amino acid accumulation by ehrlich ascites tumor cells in the presence of metabolic inhibitors

This study was undertaken to examine the mechanism by which metabolic inhibition reduces amino acid active transport in ehrlich ascites tumor cells. At 37 degrees C the metabolic inhibitor combination 0.1 mM 2,4-dinitrophenol (DNP) + 10 mM 2- deoxy-D-glucose (DOG) reduced the cell ATP concentration to 0.10- 0.15 mM in less than 5 min. This inhibition was associated with a 20.6 percent +/- 6.4 percent (SD) decrease in the initial influx of α-aminoisobutyric acid (AIB), and a two- to fourfold increase in the unidirectional efflux. These effects could be dissociated from changes in cell Na(+) or K(+) concentrations. Cells incubated to the steady state in 1.0-1.5 mM AIB showed an increased steady-state flux in the presence of DNP + DOG. Steady- state fluxes were consistent with trans-inhibition of AIB influx and trans-stimulation of efflux in control cells, but trans- stimulation of both fluxes in inhibited cells. In spite of the reduction of the cell ATP concentration to less than 0.15 mM and greatly reduced transmembrane concentration gradients of Na(+) and K(+), cells incubated to the steady state in the presence of the inhibitors still established an AIB distribution ration 13.8 +/- 2.6. The results are interpreted to indicate that a component of the reduction of AIB transport produced by metabolic inhibition is attributable to other actions in addition to the reduction of cation concentration gradients. Reduction of cell ATP alone is not responsible for the effects of metabolic inhibition, and both the transmembrane voltage and direct coupling to substrate oxidation via plasma-membrane-bound enzymes must be considered as possible energy sources for amino acid active transport.     

AMP-activated protein kinase: a cellular energy sensor with a key role in metabolic disorders and in cancer

It is essential to life that a balance is maintained between processes that produce ATP and those that consume it. An obvious way to do this would be to have systems that monitor the levels of ATP and ADP, although because of the adenylate kinase reaction (2ADP?ATP+AMP), AMP is actually a more sensitive indicator of energy stress than ADP. Following the discoveries that glycogen phosphorylase and phosphofructokinase were regulated by AMP and ATP, Daniel Atkinson proposed that all enzymes at branch points between biosynthesis and degradation would be regulated by adenine nucleotides. This turned out to be correct, but what Atkinson did not anticipate was that sensing of nucleotides would, in most cases, be performed not by the metabolic enzymes themselves, but by a signalling protein, AMPK (AMP-activated protein kinase). AMPK occurs in essentially all eukaryotes and consists of heterotrimeric complexes comprising catalytic α subunits and regulatory β and γ subunits, of which the latter carries the nucleotide-binding sites. Once activated by a metabolic stress, it phosphorylates numerous targets that alter enzyme activity and gene expression to initiate corrective responses. In lower eukaryotes, it is critically involved in the responses to starvation for a carbon source. Because of its ability to switch cellular metabolism from anabolic to catabolic mode, AMPK has become a key drug target to combat metabolic disorders associated with overnutrition such as Type?2 diabetes, and some existing anti-diabetic drugs (e.g. metformin) and many 'nutraceuticals' work by activating AMPK, usually via inhibition of mitochondrial ATP production. AMPK activators also potentially have anticancer effects, and there is already evidence that metformin provides protection against the initiation of cancer. Whether AMPK activators can be used to treat existing cancer is less clear, because many tumour cells appear to have been selected for mutations that inactivate the AMPK system. However, if we can identify the various mechanisms by which this occurs, we may be able to find ways of overcoming it.     

通过改换大肠杆菌腺苷酸代谢途径来增加胞内能荷水平

原核生物细胞能量和物质代谢的途径是一个很复杂的网络,改变代谢途径中的基因会对能量和物质流产生怎样的影响,仍然不是很清楚.以往的文献和研究已经将大肠杆菌的腺嘌呤核苷酸补救合成途径研究的很透彻.使用HPLC对删除了add基因的大肠杆菌细胞内腺苷类核苷酸分析表明,在腺嘌呤核苷酸补救途径中单一基因的途径操作不能有效改变腺嘌呤类核苷酸的代谢流向.实验中通过删除大肠杆菌JM83株中的add基因(编码腺苷脱氨酶[EC:3.5.4.4][1,2]),deoD基因(编码嘌呤核苷磷酸酶[EC:2.4.2.1][3,4]),amn基因(编码AMP核苷酶[EC:3.2.2.4][5])并引入外源ado1基因(来自酵母编码腺苷激酶[EC:2.7.1.20][6,7,8]),构建了菌株J991 (add-,deoD-,amn-,ado1 ,JM83),将其在含腺苷的LB培养基培养,使用HPLC分析其胞内腺苷类能量物质发现,ATP,ADP,AMP胞内含量都有所增加,分别都比对照JM83菌株提高一倍左右,大大加强了腺苷转化AMP的代谢流量,实现了改变物质代谢流向并使ATP积累的目的.该菌种实现了高产ATP代谢通路的构建,为下游生物工程发酵提供了较野生菌更高效的菌种,有望通过发酵工程优化培养,大幅提高ATP产量.同时,"尝试改变AMP的浓度而非直接针对ATP调节代谢途径,达到ATP积累的目"这一思路为同类研究提供参考.最后也表明在腺嘌呤核苷酸补救代谢途径中,为达到物质代谢流改变的目的,多基因联合操作较之单基因敲除更为有效.     

Evidence for new factors in the coordinate regulation of energy metabolism in Escherichia coli. Effects of hypoxia, chloramphenicol succinate, and 2,4-dinitrophenol on glucose utilization, glycogen synthesis, adenylate energy charge, and hexose phosphates during the first two periods of nitrogen starvation.

We studied the effects of decreased aeration, chloramphenicol succinate, and 2,4-dinitrophenol on the cellular rates of glycogen synthesis and glucose utilization and on the cellular concentrations of adenine nucleotides, glucose 6-phosphate, fructose 1,6-diphosphate, and phosphoenolpyruvate during the first two periods of nitrogen starvation of Escherichia coli W4597(K). A quantitative relationship between the changes in the rates and the accompanying changes in the hexose phosphates is demonstrated. However, the relationship for glycogen synthesis is different in different sets of metabolic conditions. We suggest that this difference reflects a change in the steady state level of a previously unknown effector of ADP-glucose synthetase (glucose 1-phosphate adenylyltransferase, EC 2.7.7.27) the rate-limiting enzyme of bacterial glycogen synthesis. We show that the properties of the hypothetical in vivo effector are consistent with the inhibitory effects of ppGpp (guanosine 3'-diphosphate 5'-diphosphate) and pppGpp (guanosine 3'-diphosphate 5'-triphosphate) on this enzyme in vitro. In addition, tetracycline, an inhibitor of the synthesis of these nucleotides, apparently prevents the change in the quantitative relationship. The relationship between glucose utilization and the hexose phosphates is altered at the transition to Period II of nitrogen starvation. We propose that this change reflects the alteration of the cellular steady state level of an unknown effector of the glucose phosphotransferase system. In contrast to the ATP-hexose phosphate system of shared regulatory effects, the specific effects of the unknown effectors allow the rates of glucose utilization and glycogen synthesis to be altered independently of each other and independently of changes in the rate of glycolysis. This independence allows a greater latitude of response for the individual pathways in more severe metabolic stress or in accommodating the metabolic changes necessary for long term survival.     

Cytosolic NAD content strictly depends on ATP concentration in isolated liver cells

By focusing on the question of the thermodynamic relationships involved in the regulation of biological energy conversion, bioenergetic studies usually consider the free pyridine and adenine nucleotide rather than their total pools, in either cytosol or mitochondria. In this study, we report a new observation that, at steady state, nicotinamide nucleotide content is increased by a rise in the ATP content of the whole cell under physiological conditions. It is a straight line relationship when only NAD⁺ and ATP are considered. When regarding the compartmentation of this phenomenon, it appears that the linear relationship between [NAD⁺] and [ATP] occurs only in the cytosol. Such a dependence could be a supplementary mechanism of regulation between various metabolic pathways in the liver cell.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

Decrease of hepatic mono and oligo adenosine diphosphoribose content and augmentation of [14C]ribose incorporation during induction of growth by bovine growth hormone in hypophysectomized rats

Following $\text{in}\text{vivo}$ pulse labeling with [¹⁴C]ribose the specific radioactivities of mono- and polyadenosine diphosphoribose, NAD and adenine nucleotides were determined in livers of hypophysectomized Long Evans rats. These analyses were also performed after induction of growth with four successive daily injections of bovine growth hormone. As a consequence of brief treatment with growth hormone polyadenosine diphosphoribose content markedly diminished whereas its specific radioactivity increased. NAD concentration did not vary but its specific radioactivity increased similarly to that of the homopolymer. The steady state concentration of adenine nucleotides remained unchanged, except for ATP which decreased, and their specific radioactivities uniformly decreased.