scholarly journals Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes

1993 ◽  
Vol 289 (3) ◽  
pp. 821-828 ◽  
Author(s):  
C G Jones ◽  
S K Hothi ◽  
M A Titheradge
Keyword(s):  
Pyruvate Kinase ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Acute Effect ◽  
Isolated Hepatocytes ◽  
Pyruvate Oxidation ◽  
Parallel Increase ◽  
Glucose Output

Treatment of 18 h-starved rats with dexamethasone and subsequent isolation and incubation of the hepatocytes in the presence of the steroid increased gluconeogenic flux with both 1.0 mM pyruvate and 1.0 mM lactate plus 0.2 mM pyruvate as the substrate. The magnitude of stimulation was comparable with both substrates. The increase in glucose output was accompanied by an increased flux through pyruvate carboxylase, although the absolute flux and magnitude were considerably less in the presence of the more reduced substrate. The effect of the steroid on the flux through pyruvate dehydrogenase was substrate-dependent, an inhibition occurring with the more oxidized substrate. There was no effect of steroid treatment on [1-14C]lactate or pyruvate oxidation or on tricarboxylic-acid-cycle flux as measured by [3-14C]pyruvate oxidation. Dexamethasone treatment resulted in a parallel increase in both pyruvate kinase flux and glucose synthesis with both substrates employed, indicating that the steroid had no effect on the partitioning of phosphoenolpyruvate between pyruvate and lactate formation and gluconeogenesis. Similarly there was no effect of the steroid on either the activity ratio or the total pyruvate kinase activity in the cells. It is suggested that the acute effect of the dexamethasone to increase gluconeogenesis resides at the level of phosphoenolpyruvate formation, i.e. pyruvate carboxylase and possibly phosphoenolpyruvate carboxykinase.

scholarly journals The effect of treatment of the rat with bacterial endotoxin on gluconeogenesis and pyruvate metabolism in subsequently isolated hepatocytes

1993 ◽  
Vol 289 (1) ◽  
pp. 169-172 ◽  
Author(s):  
C G Jones ◽  
M A Titheradge
Keyword(s):  
Pyruvate Kinase ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Feedback Inhibition ◽  
Isolated Hepatocytes ◽  
Bacterial Endotoxin ◽  
Pyruvate Metabolism ◽  
Pyruvate Kinase Activity ◽  
Glucose Synthesis ◽  
Effect Of Treatment

The effect of treatment of rats with bacterial endotoxin on gluconeogenesis and the flux through pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), pyruvate carboxylase and pyruvate dehydrogenase (PDH) was measured in isolated hepatocytes, prepared from animals starved for 18 h, incubated in the presence of 1 mM pyruvate. The lipopolysaccharide reduced gluconeogenesis by 50% and lowered the flux through pyruvate kinase, PEPCK and pyruvate carboxylase by comparable amounts. There was no effect of endotoxaemia on PDH flux, indicating that the lowered rate of gluconeogenesis is not the result of a redistribution of pyruvate metabolism between oxidation and carboxylation. The results confirm that a stimulation of pyruvate kinase activity following treatment with lipopolysaccharide is not involved in the inhibition of gluconeogenesis, but that the effect resides at the level of phosphoenolpyruvate formation. The most favoured mechanism for the inhibition of glucose synthesis is via an inhibition of PEPCK and subsequent feedback inhibition of pyruvate carboxylase, although a secondary effect at the level of the mitochondria and pyruvate carboxylase cannot be excluded.

Use of labeling pattern of liver glutamate to calculate rates of citric acid cycle and gluconeogenesis

1997 ◽  
Vol 272 (1) ◽  
pp. E51-E58 ◽  
Author(s):  
V. Large ◽  
H. Brunengraber ◽  
M. Odeon ◽  
M. Beylot
Keyword(s):  
Citric Acid ◽  
Pyruvate Kinase ◽  
Pyruvate Dehydrogenase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Citric Acid Cycle ◽  
Glucose Production ◽  
Acid Cycle ◽  
Glucose Output ◽  
Labeling Pattern ◽  
Calculated Flux

The use of the labeling pattern of hepatic glutamate during infusion of L-[3-13C]- or [3-14C]lactate to calculate rates of citric acid cycle activity and gluconeogenesis has been proposed. We tested the validity of this approach by perfusing isolated rat livers (48 h starved) with pyruvate and lactate (10% enriched with [3-13C]lactate) without (control) or with infusion of glucagon (to inhibit pyruvate kinase), mercaptopicolinate (to inhibit phosphoenolpyruvate carboxykinase), or dichloroacetate (to stimulate pyruvate dehydrogenase). Compared with control experiments, glucagon increased glucose output (P < 0.05) and decreased the calculated flux through pyruvate kinase (P < 0.05). Mercaptopicolinate almost totally suppressed glucose production and dramatically reduced the calculated gluconeogenic rate and flux through phosphoenolpyruvate carboxykinase (P < 0.001). Dichloroacetate moderately increased the calculated flux through pyruvate dehydrogenase (P < 0.05). In experiments with perfused livers from fed rats, the calculated gluconeogenic rate and flux through phosphoenolpyruvate carboxykinase were very low compared with control experiments (P < 0.001), whereas the pyruvate dehydrogenase flux was increased (P < 0.05). Therefore, the expected modifications of the citric acid cycle activity and gluconeogenic rate were clearly detected using the labeling pattern of glutamate to calculate these metabolic rates. Except for the perfusions with mercaptopicolinate, the dilution by isotopic exchange in the oxaloacetate pool calculated from the model agreed with the actual dilution of enrichment between liver pyruvate and phosphoenolpyruvate. The present results support the validity of this approach to trace liver metabolism.

scholarly journals E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity

2016 ◽  
Vol 113 (39) ◽  
pp. 10998-11003 ◽  
Author(s):  
Matthieu Lacroix ◽  
Geneviève Rodier ◽  
Olivier Kirsh ◽  
Thibault Houles ◽  
Hélène Delpech ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Energy Demand ◽  
Clinical Symptoms ◽  
Tricarboxylic Acid Cycle ◽  
Striated Muscles ◽  
Pyruvate Oxidation ◽  
Mitochondrial Pyruvate Carrier ◽  
Lactic Acidemia ◽  
Transcription Factor 1 ◽  
Dihydrolipoyl Dehydrogenase

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

Ontogeny of pyruvate dehydrogenase complex and key enzymes involved in glycolysis and tricarboxylic acid cycle in rabbit fetal lung, heart, and liver

1990 ◽  
Vol 68 (10) ◽  
pp. 1210-1217 ◽  
Author(s):  
Bhagu R. Bhavnani ◽  
Duncan G. Wallace
Keyword(s):  
Pyruvate Kinase ◽  
Pyruvate Dehydrogenase ◽  
Fetal Liver ◽  
Tricarboxylic Acid Cycle ◽  
Pyruvate Dehydrogenase Complex ◽  
Fetal Lung ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Key Enzymes ◽  
Dehydrogenase Complex

The metabolic pathways by which the glycogen is utilized by fetal tissues is not well established. In the present study the ontogeny of seven key enzymes involved in glycolysis and the tricarboxylic acid cycle has been established for rabbit fetal lung, heart, and liver. In the fetal lung the activities of phosphofructokinase, pyruvate kinase, lactic dehydrogenase, citrate synthase, and malate dehydrogenase increase from day 21 to 25. Thereafter the levels either drop to day 19 levels or do not change. The isocitrate dehydrogenase activity continues to increase from day 19 of gestation to maximum level on day 31 of gestation. In fetal heart the pattern of activity is similar, but in fetal liver most of the enzymes reach maximum levels earlier and, with the exception of pyruvate kinase, do not show a significant fall in activity near term. The pattern of development of pyruvate dehydrogenase complex is different; maximum activity is observed on day 27 in fetal lung and heart and on day 21 in fetal liver. These results indicate that all three fetal tissues can oxidize glucose. Also, the accumulation of glycogen, particularly in fetal lung, appears to ensure that at specific times during gestation adequate quantities of energy (ATP) and substrates, required for surfactant phospholipid synthesis, are available independent of maternal supply of glucose or during brief episodes of hypoxia.Key words: glycogen, glycolysis, tricarboxylic acid cycle, pyruvate dehydrogenase, surfactant.

scholarly journals [14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate

1985 ◽  
Vol 227 (3) ◽  
pp. 851-865 ◽  
Author(s):  
D F Heath ◽  
J G Rose
Keyword(s):  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Co2 Exchange ◽  
Published Data ◽  
Citrate Cycle ◽  
Incomplete Mixing ◽  
Metabolic Channelling ◽  
Glucose Output

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Metabolic control of hepatic gluconeogenesis during exercise

1983 ◽  
Vol 212 (3) ◽  
pp. 633-639 ◽  
Author(s):  
G L Dohm ◽  
E A Newsholme
Keyword(s):  
Metabolic Control ◽  
Pyruvate Kinase ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Prolonged Exercise ◽  
Allosteric Effectors ◽  
Fructose 1,6 Bisphosphatase ◽  
Metabolite Concentrations ◽  
Fructose 1,6 Bisphosphate ◽  
Hexose Phosphates

Prolonged exercise increased the concentrations of the hexose phosphates and phosphoenolpyruvate and depressed those of fructose 1,6-bisphosphate, triose phosphates and pyruvate in the liver of the rat. Since exercise increases gluconeogenic flux, these changes in metabolite concentrations suggest that metabolic control is exerted, at least, at the fructose 6-phosphate/fructose 1,6-bisphosphate and phosphoenolpyruvate/pyruvate substrate cycles. Exercise increased the maximal activities of glucose 6-phosphatase, fructose 1,6-bisphosphatase, pyruvate kinase and pyruvate carboxylase in the liver, but there were no changes in those of glucokinase, 6-phosphofructokinase and phosphoenolpyruvate carboxykinase. Exercise changed the concentrations of several allosteric effectors of the glycolytic or gluconeogenic enzymes in liver; the concentrations of acetyl-CoA, ADP and AMP were increased, whereas those of ATP, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate were decreased. The effect of exercise on the phosphorylation-dephosphorylation state of pyruvate kinase was investigated by measuring the activities under conditions of saturating and subsaturating concentrations of substrate. The submaximal activity of pyruvate kinase (0.5 mM-phosphoenolpyruvate), expressed as percentage of Vmax., decreased in the exercised animals to less than half that found in the controls. These changes suggest that hepatic pyruvate kinase is less active during exercise, possibly owing to phosphorylation of the enzyme, and this may play a role in increasing the rate of gluconeogenesis.

scholarly journals Effect of treatment of rats with dexamethasone in vivo on gluconeogenesis and metabolite compartmentation in subsequently isolated hepatocytes

1984 ◽  
Vol 219 (1) ◽  
pp. 117-123 ◽  
Author(s):  
E H Allan ◽  
M A Titheradge
Keyword(s):  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Intact Cell ◽  
Isolated Hepatocytes ◽  
Dexamethasone Treatment ◽  
Acetyl Coa ◽  
General Activation ◽  
Effect Of Treatment ◽  
Stimulation Of

Hepatocytes prepared from rats treated with dexamethasone for 2 or 3h and maintained in the presence of 10 microM-dexamethasone in the preparation and incubation buffers showed significantly elevated rates of gluconeogenesis compared with those prepared from control animals. Dexamethasone treatment also increased the sensitivity of the cells to glucagon and the catecholamines. Analysis of the concentrations of metabolites in the gluconeogenic pathway indicated that dexamethasone decreased the intracellular concentration of pyruvate and increased those of phosphoenolpyruvate, acetyl-CoA and citrate, suggesting a stimulation of the reaction(s) converting pyruvate into phosphoenolpyruvate. This was substantiated by analysis of the pattern of metabolites found in the mitochondrial compartment after digitonin fractionation of the cells. Inclusion of 3-mercaptopicolinate in the incubation enhanced the effect of the hormone on the distribution of metabolites. Thus, in the absence of an effect of the steroid at the level of phosphoenolpyruvate carboxykinase or pyruvate kinase, dexamethasone treatment still increased the formation of malate, aspartate and citrate from pyruvate, indicating a stimulation in the intact cell of pyruvate carboxylase. It is suggested that the stimulation of pyruvate carboxylase is a result of a general activation of mitochondrial function, with an increase in the intramitochondrial concentrations of acetyl-CoA and ATP, a decrease in glutamate and an enhanced intramitochondrial [ATP]/[ADP] ratio.

scholarly journals Phosphoenolpyruvate Carboxykinase as the Sole Anaplerotic Enzyme in Saccharomyces cerevisiae

2010 ◽  
Vol 76 (16) ◽  
pp. 5383-5389 ◽  
Author(s):  
Rintze M. Zelle ◽  
Josh Trueheart ◽  
Jacob C. Harrison ◽  
Jack T. Pronk ◽  
Antonius J. A. van Maris
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pyruvate Kinase ◽  
Pyruvate Carboxylase ◽  
Sole Carbon Source ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Single Point ◽  
Point Mutations ◽  
Equilibrium Analysis ◽  
Evolutionary Engineering

ABSTRACT Pyruvate carboxylase is the sole anaplerotic enzyme in glucose-grown cultures of wild-type Saccharomyces cerevisiae. Pyruvate carboxylase-negative (Pyc−) S. cerevisiae strains cannot grow on glucose unless media are supplemented with C4 compounds, such as aspartic acid. In several succinate-producing prokaryotes, phosphoenolpyruvate carboxykinase (PEPCK) fulfills this anaplerotic role. However, the S. cerevisiae PEPCK encoded by PCK1 is repressed by glucose and is considered to have a purely decarboxylating and gluconeogenic function. This study investigates whether and under which conditions PEPCK can replace the anaplerotic function of pyruvate carboxylase in S. cerevisiae. Pyc− S. cerevisiae strains constitutively overexpressing the PEPCK either from S. cerevisiae or from Actinobacillus succinogenes did not grow on glucose as the sole carbon source. However, evolutionary engineering yielded mutants able to grow on glucose as the sole carbon source at a maximum specific growth rate of ca. 0.14 h−1, one-half that of the (pyruvate carboxylase-positive) reference strain grown under the same conditions. Growth was dependent on high carbon dioxide concentrations, indicating that the reaction catalyzed by PEPCK operates near thermodynamic equilibrium. Analysis and reverse engineering of two independently evolved strains showed that single point mutations in pyruvate kinase, which competes with PEPCK for phosphoenolpyruvate, were sufficient to enable the use of PEPCK as the sole anaplerotic enzyme. The PEPCK reaction produces one ATP per carboxylation event, whereas the original route through pyruvate kinase and pyruvate carboxylase is ATP neutral. This increased ATP yield may prove crucial for engineering of efficient and low-cost anaerobic production of C4 dicarboxylic acids in S. cerevisiae.

scholarly journals [13C]bicarbonate labelled from hyperpolarized [1-13C]pyruvate is an in vivo marker of hepatic gluconeogenesis in fasted state

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Emine Can ◽  
Jessica A. M. Bastiaansen ◽  
Dominique-Laurent Couturier ◽  
Rolf Gruetter ◽  
Hikari A. I. Yoshihara ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Hepatic Gluconeogenesis ◽  
Fasted State ◽  
Basal Plasma ◽  
Fasted Rats ◽  
In Vivo Assessment

AbstractHyperpolarized [1-13C]pyruvate enables direct in vivo assessment of real-time liver enzymatic activities by 13C magnetic resonance. However, the technique usually requires the injection of a highly supraphysiological dose of pyruvate. We herein demonstrate that liver metabolism can be measured in vivo with hyperpolarized [1-13C]pyruvate administered at two- to three-fold the basal plasma concentration. The flux through pyruvate dehydrogenase, assessed by 13C-labeling of bicarbonate in the fed condition, was found to be saturated or partially inhibited by supraphysiological doses of hyperpolarized [1-13C]pyruvate. The [13C]bicarbonate signal detected in the liver of fasted rats nearly vanished after treatment with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, indicating that the signal originates from the flux through PEPCK. In addition, the normalized [13C]bicarbonate signal in fasted untreated animals is dose independent across a 10-fold range, highlighting that PEPCK and pyruvate carboxylase are not saturated and that hepatic gluconeogenesis can be directly probed in vivo with hyperpolarized [1-13C]pyruvate.

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