The accumulation of aspartate in the presence of ethanol in rat liver.
ABSTRACT: 1. Isolated hepatocytes were used to establish the reasons for the accumulation of aspartate, previously observed when the isolated rat liver was perfused with ethanol in the presence of alanine or ammonium lactate. 2. The isolated cells did not form aspartate when incubated with alanine and ethanol, but much aspartate was formed on incubation with ammonium lactate and ethanol. 3. Urea was the main nitrogenous product on incubation with alanine, in contrast with the perfused liver, where major quantities of NH4+ are also formed. When the formation of urea was nullified by the addition of urease, alanine plus ethanol caused aspartate formation, indicating that aspartate formation depends on the presence of critical concentrations of NH4+. 4. The accumulated aspartate was present in the cytosol. Ethanol halved the content of 2-oxoglutarate in the cytosol and more than trebled that of glutamate in the mitochondria. 5. The findings support the assumption that 2-oxoglutarate formed by the mitochondrial aspartate aminotransferase is not translocated to the cytosol in the presence of ethanol and NH4+, because it is rapidly converted into glutamate, the dehydrogenation of ethanol providing the required NADH. Aspartate, however, is translocated to the cytosol and accumulates there because of the lack of stoicheiometric amounts of oxoglutarate.
Project description:1. The rate of gluconeogenesis from alanine in the perfused rat liver is affected by the presence of other metabolizable substances, especially fatty acids, ornithine and ethanol. Gluconeogenesis is accelerated by oleate and by ornithine. When both oleate and ornithine were present the acceleration was greater than expected on the basis of mere additive effects. 2. Much NH(3) and some urea were formed from alanine when no ornithine was added. With ornithine almost all the nitrogen released from alanine appeared as urea. 3. Lactate was a major product of alanine metabolism. Addition of oleate, and especially of oleate plus ornithine, decreased lactate formation. 4. Ethanol had no major effect on gluconeogenesis from alanine when this was the sole added precursor. Gluconeogenesis was strongly inhibited (87%) when oleate was also added, but ethanol greatly accelerated gluconeogenesis when ornithine was added together with alanine. 5. In the absence of ethanol the alanine carbon and alanine nitrogen removed were essentially recovered in the form of glucose, lactate, pyruvate, NH(3) and urea. 6. In the presence of ethanol the balance of both alanine carbon and alanine nitrogen showed substantial deficits. These deficits were largely accounted for by the formation of aspartate and glutamine, the formation of which was increased two- to three-fold. 7. When alanine was replaced by lactate plus NH(4)Cl, ethanol also caused a major accumulation of amino acids, especially of aspartate and alanine. 8. Earlier apparently discrepant results on the effects of ethanol on gluconeogenesis from alanine are explained by the fact that under well defined conditions ethanol can inhibit, or accelerate, or be without major effect on the rate of gluconeogenesis. 9. It is pointed out that in the synthesis of urea through the ornithine cycle half of the nitrogen must be supplied in the form of asparate and half in the form of carbamoyl phosphate. The accumulation of aspartate and other amino acids suggests that ethanol interferes with the control mechanisms which regulate the stoicheiometric formation of aspartate and carbamoyl phosphate.
Project description:1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.
Project description:1. The time-course of the changes in the concentrations of hepatic metabolites in response to a non-toxic load of NH(4)Cl were measured in fed and starved rats. 2. There was a rapid increase (after 2min) in [alanine] and [aspartate] which remained high for 10-15min; the absolute increase in [alanine] was smaller in starved rats. 3. These changes were accompanied by a decrease in [oxoglutarate] and in the [3-hydroxybutyrate]/[acetoacetate] ratio. 4. Prior administration of l-arginine to fed rats resulted in smaller increases in [alanine] and [aspartate] after the ammonia load. This is presumably due to stimulation of the urea cycle. 5. Increased formation of alanine in starved rats occurred after prior administration of dihydroxyacetone to increase the availability of pyruvate. 6. Administration of l-cycloserine, an inhibitor of glutamate-alanine aminotransferase, completely prevented the increase in [alanine] after the ammonia load; in this case the absolute increase in [aspartate] was higher. 7. [Oxoglutarate], [citrate] and [isocitrate] at 25min after the ammonia load were higher than the initial concentrations, but returned to normal by 50min. It is suggested that this ;overshoot' may be due to temporary compartmentation of oxoglutarate. 8. The mechanisms and physiological significance of alanine and aspartate formation in these experiments are discussed.
Project description:1. Transient and steady-state changes caused by acetate utilization were studied in perfused rat heart. The transient period occupied 6min and steady-state changes were followed in a further 6min of perfusion. 2. In control perfusions glucose oxidation accounted for 75% of oxygen utilization; the remaining 25% was assumed to represent oxidation of glyceride fatty acids. With acetate in the steady state, acetate oxidation accounted for 80% of oxygen utilization, which increased by 20%; glucose oxidation was almost totally suppressed. The rate of tricarboxylate-cycle turnover increased by 67% with acetate perfusion. The net yield of ATP in the steady state was not altered by acetate. 3. Acetate oxidation increased muscle concentrations of acetyl-CoA, citrate, isocitrate, 2-oxoglutarate, glutamate, alanine, AMP and glucose 6-phosphate, and lowered those of CoA and aspartate; the concentrations of pyruvate, ATP and ADP showed no detectable change. The times for maximum changes were 1min, acetyl-CoA, CoA, alanine and AMP; 6min, citrate, isocitrate, glutamate and aspartate; 2-4min, 2-oxoglutarate. Malate concentration fell in the first minute and rose to a value somewhat greater than in the control by 6min. There was a transient and rapid rise in glucose 6-phosphate concentration in the first minute superimposed on the slower rise over 6min. 4. Acetate perfusion decreased the output of lactate, the muscle concentration of lactate and the [lactate]/[pyruvate] ratio in perfusion medium and muscle in the first minute; these returned to control values by 6min. 5. During the first minute acetate decreased oxygen consumption and lowered the net yield of ATP by 30% without any significant change in muscle ATP or ADP concentrations. 6. The specific radioactivities of cycle metabolites were measured during and after a 1min pulse of [1-(14)C]acetate delivered in the first and twelfth minutes of acetate perfusion. A model based on the known flow rates and concentrations of cycle metabolites was analysed by computer simulation. The model, which assumed single pools of cycle metabolites, fitted the data well with the inclusion of an isotope-exchange reaction between isocitrate and 2-oxoglutarate+bicarbonate. The exchange was verified by perfusions with [(14)C]bicarbonate. There was no evidence for isotope exchange between citrate and acetyl-CoA or between 2-oxoglutarate and malate. There was rapid isotope equilibration between 2-oxoglutarate and glutamate, but relatively poor isotope equilibration between malate and aspartate. 7. It is concluded that the citrate synthase reaction is displaced from equilibrium in rat heart, that isocitrate dehydrogenase and aconitate hydratase may approximate to equilibrium, that alanine aminotransferase is close to equilibrium, but that aspartate transamination is slow for reasons that have yet to be investigated. 8. The slow rise in citrate concentration as compared with the rapid rise in that of acetyl-CoA is attributed to the slow generation of oxaloacetate by aspartate aminotransferase. 9. It is proposed that the tricarboxylate cycle may operate as two spans: acetyl-CoA-->2-oxoglutarate, controlled by citrate synthase, and 2-oxoglutarate-->oxaloacetate, controlled by 2-oxoglutarate dehydrogenase; a scheme for cycle control during acetate oxidation is outlined. The initiating factors are considered to be changes in acetyl-CoA, CoA and AMP concentrations brought about by acetyl-CoA synthetase. 10. Evidence is presented for a transient inhibition of phosphofructokinase during the first minute of acetate perfusion that was not due to a rise in whole-tissue citrate concentration. The probable importance of metabolite compartmentation is stressed.
Project description:1. The activities of the mitochondrial and cytosol isoenzyme forms of l-alanine-glyoxylate and l-alanine-2-oxoglutarate aminotransferases were determined in rat liver during foetal and neonatal development. 2. The mitochondrial glyoxylate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. The cytosol glyoxylate aminotransferase and the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities first appear prenatally, increase further after birth and then rise to the adult values during weaning. 4. In foetal liver the mitochondrial glyoxylate aminotransferase and the cytosol 2-oxoglutarate aminotransferase activities are increased after injection in utero of glucagon, dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) or thyroxine. The cytosol glyoxylate aminotransferase and the mitochondrial 2-oxoglutarate aminotransferase activities are increased after injection in utero of cortisol or thyroxine. 5. After birth the further normal increases in the mitochondrial and cytosol 2-oxoglutarate aminotransferase activities can be hastened by cortisol injection, whereas the increase in cytosol glyoxylate aminotransferase activity requires cortisol treatment together with the intragastric administration of casein. 6. The results are discussed with reference to the metabolic patterns and the changes in regulatory stimuli (hormonal and dietary) that occur during the period of development.
Project description:Nitrate (NO3-) and ammonium (NH4+) are major inorganic nitrogen (N) supplies for plants, but NH4+ as the sole or dominant N source causes growth inhibition in many plants, known as ammonium toxicity. Small amounts of NO3- can significantly mitigate ammonium toxicity, and the anion channel SLAC1 homolog 3 (SLAH3) is involved in this process, but the mechanistic detail of how SLAH3 regulates nitrate-dependent alleviation of ammonium toxicity is still largely unknown. In this study, we identified SnRK1.1, a central regulator involved in energy homeostasis, and various stress responses, as a SLAH3 interactor in Arabidopsis (Arabidopsis thaliana). Our results suggest that SNF1-related protein kinase 1 (SnRK1.1) functions as a negative regulator of SLAH3. Kinase assays indicate SnRK1.1 strongly phosphorylates the C-terminal of SLAH3 at the site S601. Under high-NH4+/low-pH condition, phospho-mimetic and phospho-dead mutations in SLAH3 S601 result in barely rescued phenotypes and fully complemented phenotypes in slah3. Furthermore, SnRK1.1 migrates from cytoplasm to nucleus under high-NH4+/low-pH conditions. The translocation of SnRK1.1 from cytosol to nucleus under high-ammonium stress releases the inhibition on SLAH3, which allows SLAH3-mediated NO3- efflux leading to alleviation of high-NH4+/low-pH stress. Our study reveals that the C-terminal phosphorylation also plays important role in SLAH3 regulation and provides additional insights into nitrate-dependent alleviation of ammonium toxicity in plants.
Project description:Some aspects of tricarboxylic acid-cycle activity during differentiation and aging in Dictyostelium discoideum were examined. The concentrations of glutamate, aspartate, alanine, citrate, 2-oxoglutarate, succinate, fumarate, malate, oxaloacetate, pyruvate and acetyl-CoA were determined at four stages over the course of differentiation. The rate of O2 utilization was also determined over differentiation. In addition, experiments are described in which the specific radioactivities of citrate, 2-oxoglutarate, succinate, fumarate and malate were determined during a 30 min labelling of cells from the preculmination stage of development with [14C]glutamate, [14C]aspartate or [14C]alanine. A similar experiment was also performed with cells from the aggregation stage of development using [14C]glutamate.
Project description:1. In confirmation of previous work, administration of d(+)-galactosamine (0.5-0.75g/kg body wt.) to rats caused a hepatitis with histological evidence of liver damage and a 9-fold rise in aspartate aminotransferase activity in serum. 2. There was a significant elevation of blood lactate and pyruvate concentrations in 24h-starved rats treated with galactosamine but no change in the [lactate]/[pyruvate] ratio. 3-Hydroxybutyrate and acetoacetate concentrations in blood were decreased. 3. The changes in the concentrations of lactate, pyruvate and ketone bodies in the freeze-clamped liver were parallel to those observed in the blood. 4. In the livers of 24h-starved galactosamine-treated rats there were large increases in the concentrations of alanine (3-fold), citrate (5-fold), 2-oxoglutarate (4-fold), with smaller increases in malate, glutamate and aspartate. There was a 4-fold rise in the value of the mass-action ratio of the alanine aminotransferase system in the livers of galactosamine-treated rats when compared to controls. 5. There was a significant decrease in the activities of aspartate and alanine aminotransferases in the cytoplasm and the soluble fraction of sonicated homogenates of the livers of rats treated with galactosamine. The activity of phosphoenolpyruvate carboxylase was decreased by 75% of the control value. 6. Glucose synthesis from lactate in perfused livers from galactosamine-treated rats was inhibited 39% when compared with controls. 7. The results indicate that the conversion of lactate into glucose is decreased in the livers of galactosamine-treated rats and that this decrease may be due to the loss of phosphoenolpyruvate carboxylase from damaged hepatocytes.
Project description:1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.
Project description:BACKGROUND:Nitrogen (N) metabolism plays an important role in plant drought tolerance. 2-(3,4-Dichlorophenoxy) triethylamine (DCPTA) regulates many aspects of plant development; however, the effects of DCPTA on soil drought tolerance are poorly understood, and the possible role of DCPTA on nitrogen metabolism has not yet been explored. RESULTS:In the present study, the effects of DCPTA on N metabolism in maize (Zea mays L.) under soil drought and rewatering conditions during the pre-female inflorescence emergence stage were investigated in 2016 and 2017. The results demonstrated that the foliar application of DCPTA (25?mg/L) significantly alleviated drought-induced decreases in maize yield, shoot and root relative growth rate (RGR), leaf relative water content (RWC), net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr), and nitrate (NO3-), nitrite (NO2-), soluble protein contents, and nitrate reductase (NR), nitrite reductase (NiR), isocitrate dehydrogenase (ICDH), alanine aminotransferase (AlaAT) and aspartate aminotransferase (AspAT) activities. In addition, the foliar application of DCPTA suppressed the increases of intercellular CO2 concentration (Ci), ammonium (NH4+) and free amino acid contents, and the glutamate dehydrogenase (GDH) and protease activities of the maize. Simultaneously, under drought conditions, the DCPTA application improved the spatial and temporal distribution of roots, increased the root hydraulic conductivity (Lp), flow rate of root-bleeding sap and NO3- delivery rates of the maize. Moreover, the DCPTA application protected the chloroplast structure from drought injury. CONCLUSIONS:The data show, exogenous DCPTA mitigates the repressive effects of drought on N metabolism by maintained a stabilized supply of 2-oxoglutarate (2-OG) and reducing equivalents provided by photosynthesis via favorable leaf water status and chloroplast structure, and NO3- uptake and long-distance transportation from the roots to the leaves via the production of excess roots, as a result, DCPTA application enhances drought tolerance during the pre-female inflorescence emergence stage of maize.