The mechanism by which adenosine decreases gluconeogenesis from lactate in isolated rat hepatocytes.
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ABSTRACT:
SUBMITTER: Lavoinne A
PROVIDER: S-EPMC1148295 | biostudies-other | 1987 Sep
REPOSITORIES: biostudies-other
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Project description:1. In freshly prepared isolated rat liver cells there is a lag in gluconeogenesis from lactate. The magnitude of the lag increases with increasing lactate concentration. 2. The lag is virtually abolished by lysine. 3. A few other amino acids (tyrosine, arginine, asparagine, ornithine) and NH(4)Cl had effects similar to, but less pronounced than, lysine during the early stage of incubation. Lysine was unique in accelerating gluconeogenesis beyond the lag period. 4. The effects of the accelerators are not additive. 5. Glycine, serine, threonine, cysteine, tryptophan and histidine at 2mm markedly inhibit (>20%) gluconeogenesis from lactate. 6. Oleate, which promotes gluconeogenesis from lactate by supplying acetyl-CoA required for the pyruvate carboxylase reaction, had no effect on the lag, yet oleate oxidation showed no lag. 7. Preincubation of cells decreased the lag and decreased the magnitude of the lysine effect. 8. Pyruvate (added at 1mm to give an initial [lactate]/[pyruvate] ratio of 10) also abolished the lag and decreased the lysine effect by about 50%. 9. Lysine reversed the inhibition by ethanol of gluconeogenesis from lactate. 10. All accelerators increased the rate of re-oxidation of cytosolic NADH as shown by a rapid re-adjustment of the [lactate]/[pyruvate] ratio on addition of 10mm-lactate. 11. The accelerated rates of gluconeogenesis are associated with an increased formation of aspartate and glutamate and especially alanine. 12. The existence of the lag period can be explained on the basis of the fact that the accumulation of pyruvate during the lag diverts oxaloacetate from gluconeogenesis to malate formation, i.e. that the re-oxidation of cytosolic NADH takes precedence over gluconeogenesis. This means that much oxaloacetate formed by the pyruvate carboxylase reaction has to be transferred twice from the mitochondria to the cytosol by the aspartate shuttle. Under these conditions the operation of the shuttle limits the rate of gluconeogenesis from lactate. Lysine and other accelerators may increase the effectiveness of the shuttle by providing components of the aspartate aminotransferases involved. The question of why lysine specifically accelerates gluconeogenesis beyond the lag period is discussed.
| S-EPMC1168283 | biostudies-other
Project description:1. Hepatocytes isolated from starved rats and incubated without other substrates oxidized ethanol at a rate of 0.8-0.9mumol/min per g wet wt. of cells. Addition of 10mm-lactate increased this rate 2-fold. 2. Quinolinate (5mm) or tryptophan (1mm) decreased the rate of gluconeogenesis with 10mm-lactate and 8mm-ethanol from 0.39 to 0.04-0.08mumol/min per g wet wt. of cells, but rates of ethanol oxidation were not decreased. From these results it appears that acceleration of ethanol oxidation by lactate is not dependent upon the stimulation of gluconeogenesis and the consequent increased demand for ATP. 3. As another test of the relationship between ethanol oxidation and gluconeogenesis, the initial lactate concentration was varied from 0.5mm to 10mm and pyruvate was added to give an initial [lactate]/[pyruvate] ratio of 10. This substrate combination gave a large stimulation of ethanol oxidation (from 0.8 to 2.6mumol/min per g wet wt. of cells) at low lactate concentrations (0.5-2.0mm), but rates remained nearly constant (2.6-3.0mumol/min per g wet wt. of cells) at higher lactate concentrations (2.0-10mm). 4. In contrast, owing to the presence of ethanol, the rate of glucose synthesis was only slightly increased (from 0.08 to 0.12mumol/min per g wet wt. of cells) between 0.5mm- and 2.0mm-lactate and continued to increase (from 0.12 to 0.65mumol/min per g wet wt. of cells) with lactate concentrations between 2 and 10mm. 5. In the presence of ethanol, O(2) uptake increased with increasing substrate concentration over the entire range. 6. Changes in concentrations of glutamate and 2-oxoglutarate closely paralleled changes in the rate of ethanol oxidation. 7. In isolated hepatocytes, rates of ethanol oxidation are lower than those in vivo apparently because of depletion of malate-aspartate shuttle intermediates during cell preparation. Rates are returned to those observed in vivo by substrates that increase the intracellular concentration of shuttle metabolites.
| S-EPMC1185658 | biostudies-other
Project description:1. Gluconeogenesis from lactate or pyruvate was studied in perfused livers from starved rats at perfusate pH7.4 or under conditions simulating uncompensated metabolic acidosis (perfusate pH6.7-6.8). 2. In 'acidotic' perfusions gluconeogenesis and uptake of lactate or pyruvate were decreased. 3. Measurement of hepatic intermediate metabolites suggested that the effect of acidosis was exerted at a stage preceding phosphoenolpyruvate. 4. Total intracellular oxaloacetate concentration was significantly decreased in the acidotic livers perfused with lactate. 5. It is suggested that decreased gluconeogenesis in acidosis is due to substrate limitation of phosphoenolypyruvate carboxykinase. 6. The possible reasons for the fall in oxaloacetate concentration in acidotic livers are discussed; two of the more likely mechanisms are inhibition of the pyruvate carboxylase system and a change in the [malate]/[oxaloacetate] ratio due to the fall in intracellular pH.
| S-EPMC1164773 | biostudies-other
Project description:The sensitivity of glucose production from L-lactate by isolated liver cells from starved rats to inhibition by alpha-cyano-4-hydroxycinnamate was studied. A small percentage of the maximal rate of gluconeogenesis was insensitive to inhibition by alpha-cyano-4-hydroxycinnamate, and evidence is presented to show that this is due to pyruvate entry into the mitochondria as alanine. After subtraction of this rate, Dixon plots of the reciprocal of the rate of gluconeogenesis against inhibitor concentration were linear both in the absence and presence of glucagon, phenylephrine or valinomycin, each of which stimulated gluconeogenesis by 30-50%. Pyruvate kinase activity was decreased by glucagon, but not by phenylephrine or valinomycin. Inhibition of gluconeogenesis by quinolinate (inhibitor of phosphoenolpyruvate carboxykinase) or monochloroacetate (probably inhibiting pyruvate carboxylation) caused a significant deviation from linearity of the Dixon plot obtained with alpha-cyano-4-hydroxycinnamate. Amytal, however, inhibited gluconeogenesis without affecting the linearity of this plot. These data, coupled with a computer simulation study, suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain. An additional role for pyruvate kinase and pyruvate carboxylase is quite compatible with the data presented.
| S-EPMC1163301 | biostudies-other
Project description:Gluconeogenesis was studied in hepatocytes isolated from phenobarbital-pretreated rats fasted for 24 h. In closed vial incubations, glucose production from lactate (20 mmol/l) and pyruvate (2 mmol/l), alanine (20 mmol/l) or glutamine (20 mmol/l) was suppressed by about 30-45%, although glycerol metabolism was not affected. In hepatocytes perifused with lactate and pyruvate (ratio 10:1), glucose production was inhibited by 50%, even at low gluconeogenic flux. From the determination of gluconeogenic intermediates at several steady states of gluconeogenic flux, we have found a single relationship between phosphoenolpyruvate and the rate of glucose production (Jglucose), and two different curves between cytosolic oxaloacetate and Jglucose in controls and in phenobarbital-pretreated hepatocytes. By using 3-mercaptopicolinate to determine the flux control coefficient of phosphoenolpyruvate carboxykinase we found that phenobarbital pretreatment led to an increase in this coefficient from 0.3 (controls) to 0.8 (phenobarbital group). These observations were confirmed by the finding that the activity of phosphoenolpyruvate carboxykinase was decreased by 50% after phenobarbital treatment. Hence we conclude that the inhibitory effect of phenobarbital on gluconeogenesis is due, at least partly, to a decrease in the flux through phosphoenolpyruvate carboxykinase.
| S-EPMC1130505 | biostudies-other
Project description:1. Measurements were made of the activities of the four key enzymes involved in gluconeogenesis, pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxylase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), of serine dehydratase (EC 4.2.1.13) and of the four enzymes unique to glycolysis, glucokinase (EC 2.7.1.2), hexokinase (EC 2.7.1.1), phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), in livers from starved rats perfused with glucose, fructose or lactate. Changes in perfusate concentrations of glucose, fructose, lactate, pyruvate, urea and amino acid were monitored for each perfusion. 2. Addition of 15mm-glucose at the start of perfusion decreased the activity of pyruvate carboxylase. Constant infusion of glucose to maintain the concentration also decreased the activities of phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and serine dehydratase. Addition of 2.2mm-glucose initially to give a perfusate sugar concentration similar to the blood sugar concentration of starved animals had no effect on the activities of the enzymes compared with zero-time controls. 3. Addition of 15mm-fructose initially decreased glucokinase activity. Constant infusion of fructose decreased activities of glucokinase, phosphofructokinase, pyruvate carboxylase, phosphoenolpyruvate carboxylase, glucose 6-phosphatase and serine dehydratase. 4. Addition of 7mm-lactate initially elevated the activity of pyruvate carboxylase, as also did constant infusion; maintenance of a perfusate lactate concentration of 18mm induced both pyruvate carboxylase and phosphoenolpyruvate carboxylase activities. 5. Addition of cycloheximide had no effect on the activities of the enzymes after 4h of perfusion at either low or high concentrations of glucose or at high lactate concentration. Cycloheximide also prevented the loss or induction of pyruvate carboxylase and phosphoenolpyruvate carboxylase activities with high substrate concentrations. 6. Significant amounts of glycogen were deposited in all perfusions, except for those containing cycloheximide at the lowest glucose concentration. Lipid was found to increase only in the experiments with high fructose concentrations. 7. Perfusion with either fructose or glucose decreased the rates of ureogenesis; addition of cycloheximide increased urea efflux from the liver.
| S-EPMC1177795 | biostudies-literature
Project description:Gluconeogenesis from pyruvate, alanine, lactate and propionate was inhibited by methylmalonate in both rat and guinea-pig hepatocytes. The effect was dose-dependent. Gluconeogenesis from glycerol and xylitol was not affected.
| S-EPMC1161861 | biostudies-other
Project description:1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40-50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD(+)]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP(+)]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD(+)]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.
| S-EPMC1164999 | biostudies-other
Project description:AimsTo understand the mechanism by which imeglimin (a new oral hypoglycemic agent whose phase 3 development program in Japan has now been completed) decreases hepatic glucose production.Materials and methodsWe compared the effect of imeglimin and metformin on glucose production, ATP/ADP ratio, oxygen consumption rate, mitochondrial redox potential and membrane potential in primary rat hepatocytes.ResultsWe found that both imeglimin and metformin dose-dependently decreased glucose production and the ATP/ADP ratio. Moreover, they both increased mitochondrial redox potential (assessed by mitochondrial NAD(P)H fluorescence) and decreased membrane potential (assessed by TMRM fluorescence). However, contrary to metformin, which inhibits mitochondrial Complex I, imeglimin did not decrease the oxygen consumption rate in intact cells. By measuring the oxygen consumption of in situ respiratory chain as a function of the concentration of NADH, we observed that imeglimin decreased the affinity of NADH for the respiratory chain but did not affect its Vmax (ie competitive inhibition) whereas metformin decreased both the Vmax and the affinity (ie uncompetitive inhibition).ConclusionsWe conclude that imeglimin induces a kinetic constraint on the respiratory chain that does not affect its maximal activity. This kinetic constraint is offset by a decrease in the mitochondrial membrane potential, which induces a thermodynamic constraint on the ATPase responsible for a decrease in the ATP/ADP ratio.
| S-EPMC8029524 | biostudies-literature
Project description:Binding of 4-methyl-2-oxo[1-14C]valerate to defatted bovine serum albumin inhibited the utilization of this 2-oxo acid by fed-rat hepatocytes in vitro. With 0-50g of albumin/l in the presence of 0.05mM 2-oxo acid or on increasing the 2-oxo acid concentration from 0 to 2mM in the presence of 26g of albumin/l, the extent of inhibition was essentially dependent on the change in the free 2-oxo acid concentration. Intrahepatocyte 4-methyl-2-oxo[1-14C]valerate concentrations were similar to extracellular free 2-oxo acid concentrations, suggesting equilibration so that the plasma membrane appears not to be rate-limiting for the utilization of this substrate by the isolated liver cells.
| S-EPMC1153140 | biostudies-other