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 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.

scholarly journals Evidence that the stimulation of lipogenesis in the mammary glands of starved lactating rats re-fed with a chow diet is dependent on continued hepatic gluconeogenesis during the absorptive period. Effects of a gluconeogenic inhibitory, mercaptopicolinic acid, in vivo

1985 ◽  
Vol 228 (3) ◽  
pp. 727-733 ◽  
Author(s):  
D H Williamson ◽  
V Ilic ◽  
R G Jones
Keyword(s):  
Mammary Gland ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Mammary Glands ◽  
Hepatic Glycogen ◽  
Chow Diet ◽  
Hepatic Gluconeogenesis ◽  
Rapid Stimulation ◽  
Stimulation Of

The rapid stimulation of lipogenesis in mammary gland that occurs on re-feeding starved lactating rats with a chow diet was decreased (60%) by injection of mercaptopicolinic acid, an inhibitor of hepatic gluconeogenesis at the phosphoenolpyruvate carboxykinase step. Mercaptopicolinate had no effect on lipogenesis in mammary glands of fed lactating rats. The inhibition of lipogenesis persisted in vitro when acini from mammary glands of re-fed rats treated with mercaptopicolinate were incubated with [1-14C]glucose. Mercaptopicolinate added in vitro had no significant effect on lipogenesis in acini from starved-re-fed lactating rats. Mercaptopicolinate prevented the deposition of glycogen and increased the rate of lipogenesis in livers of starved-re-fed lactating rats, whereas it had no significant effect on livers of fed lactating rats. Administration of intraperitoneal glucose restored the rate of mammary-gland lipogenesis in re-fed rats treated with mercaptopicolinate to the values for re-fed rats. Hepatic glycogen deposition was also restored, and the rate of hepatic lipogenesis was stimulated 5-fold. It is concluded that stimulation of mammary-gland lipogenesis on re-feeding with a chow diet after a period of starvation is in part dependent on continued hepatic gluconeogenesis during the absorptive period. Possible sources of the glucose precursors are discussed.

scholarly journals Effect of treatment in vivo of rats with bacterial endotoxin on fructose 2,6-bisphosphate metabolism and L-pyruvate kinase activity and flux in isolated liver cells

1992 ◽  
Vol 284 (3) ◽  
pp. 761-766 ◽  
Author(s):  
E D Ceppi ◽  
R G Knowles ◽  
K M Carpenter ◽  
M A Titheradge
Keyword(s):  
Pyruvate Kinase ◽  
Intact Cell ◽  
Total Activity ◽  
Liver Cells ◽  
Bacterial Endotoxin ◽  
Phosphorylation State ◽  
Isolated Liver Cells ◽  
Effect Of Treatment ◽  
Isolated Liver ◽  
Stimulation Of

The effect of treatment of rats with bacterial endotoxin on fructose 2,6-bisphosphate (Fru-2,6-P2) metabolism was investigated in isolated liver cells prepared from 18 h-starved animals. The results obtained support the hypothesis that a stimulation of 6-phosphofructo-1-kinase (PFK-1) activity and an inhibition of fructose-1,6-bisphosphatase (Fru-1,6-P2ase) may be one mechanism underlying the inhibition of gluconeogenesis from lactate and pyruvate by endotoxin. We suggest that the stimulation of PFK-1 and inhibition of Fru-1,6-P2ase activity is the result of a 2-3-fold increase in Fru-2,6-P2. The latter is not due to changes in the total activity or phosphorylation state of the bifunctional 6-phosphofructo-2-kinase (PFK-2)/fructose-2,6-bisphosphatase, but appears to be the result of a decrease in the cytosolic concentration of phosphoenolpyruvate (PEP), an inhibitor of PFK-2 activity. The effect of endotoxin is resistant to the presence of glucagon, which has comparable effects in cells prepared from both control and endotoxin-treated animals. The mechanism by which endotoxin treatment of the rat decreases PEP and gluconeogenesis remains to be established. However, it does not involve alterations in either the total activity or the phosphorylation state of pyruvate kinase, nor does it involve increased flux through this enzyme in the intact cell, which is in fact decreased in this model of septic shock. It is suggested that the decreased flux may result from a lower rate of formation of PEP, suggesting that the prime lesion in sepsis is an inhibition of one or more of the steps leading to PEP formation.

scholarly journals The characteristics and site of inhibition of gluconeogenesis in rat liver cells by bacterial endotoxin. Stimulation of phosphofructokinase-1

1987 ◽  
Vol 242 (3) ◽  
pp. 721-728 ◽  
Author(s):  
R G Knowles ◽  
J P McCabe ◽  
S J Beevers ◽  
C I Pogson
Keyword(s):  
Enzyme Activities ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Liver Cells ◽  
Atp Content ◽  
Rat Liver Cells ◽  
Endogenous Substrates ◽  
And Control ◽  
Control Coefficients ◽  
Stimulation Of

The characteristics and site of inhibition of gluconeogenesis by endotoxin were investigated in liver cells isolated from control and endotoxin-treated rats. Endotoxin treatment was associated with inhibition (40-50%) of gluconeogenesis from lactate plus pyruvate over a range of concentrations of substrate and of oleate and with or without glucose or glucagon. Similar inhibition was observed with asparagine, proline, glutamine, alanine and a substrate mixture, but not with glycerol, glyceraldehyde, dihydroxyacetone or endogenous substrates. There was no change in cellular ATP content or in the rates of ketogenesis or ureogenesis from asparagine, proline or glutamine. Other effects on isotopic fluxes, metabolite contents, enzyme activities and control coefficients were consistent with the suggestion that the effects of endotoxin on gluconeogenesis are exerted at the level of phosphofructokinase-1, and not at phosphoenolpyruvate carboxykinase, pyruvate kinase, pyruvate carboxylase or glucokinase.

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 An in Vitro Model of Post-Exercise Hepatic Gluconeogenesis in the Gulf Toadfish Opsanus Beta

1989 ◽  
Vol 147 (1) ◽  
pp. 393-406 ◽  
Author(s):  
PATRICK J. WALSH
Keyword(s):  
Isolated Hepatocytes ◽  
Blood Parameters ◽  
Strenuous Exercise ◽  
Hepatic Gluconeogenesis ◽  
Post Exercise ◽  
Opsanus Beta ◽  
Stimulation Of ◽  
Gulf Toadfish

During strenuous exercise, fish develop substantial proton and lactate loads. Although acidosis is usually rapidly corrected during recovery (1–2 h), lactate levels often remain elevated for up to 8–12 h. The quantitative role of the liver in clearance of the lactate load during recovery from exercise in fish has received little direct examination. The purposes of this study were (1) to attempt to quantify hepatic contribution to lactate clearance, and (2) to identify factors that regulate hepatic gluconeogenesis during recovery from exercise in fish. Both in vivo and in vitro (isolated hepatocytes) approaches were used. Important blood parameters (pHe, Ccoco2, [lactate], [glucose], [epinephrine] and [norepinephrine]) were measured in the gulf toadfish (Opsanus beta Goode and Bean) during recovery from strenuous exercise, and they conformed to the general patterns for sluggish benthic species noted in earlier studies. When toadfish hepatocytes wereexposed to simulated post-exercise conditions in vitro, gluconeogenesis from lactate was stimulated by over 2.5-fold in ‘0–1 h-’ and ‘l-2h-post-exercise periods’. Variation of the extracellular parameters in controlled combinations indicated that exercise-induced changes in [glucose], [epinephrine], [norepinephrine], Pcoco2 and [HCO3−] had no significant effects on rates of gluconeogenesis.The observed stimulation of gluconeogenesis could be induced independently byeither decreased pH (which lowered Km for lactate) or increased [lactate] (bysimple hyperbolic kinetic effects), but the effects were not additive. Despite thispotentially adaptive stimulation of gluconeogenesis, I estimate, based on observedin vitro rates and in vivo estimates of lactate load, that hepatic gluconeogenesisaccounts for less than 2% of the lactate load clearance in toadfish.

scholarly journals Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 833-846 ◽  
Author(s):  
M C Scrutton ◽  
I Beis
Keyword(s):  
Rat Liver ◽  
Pyruvate Carboxylase ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Specific Activity ◽  
Competitive Inhibitor ◽  
Product Inhibition ◽  
Detectable Effect ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Hydrolysis Of

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7–0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.

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 Lactate-stimulated ethanol oxidation in isolated hepatocytes

1978 ◽  
Vol 172 (1) ◽  
pp. 29-36 ◽  
Author(s):  
Kathryn E. Crow ◽  
Neal W. Cornell ◽  
Richard L. Veech
Keyword(s):  
Ethanol Oxidation ◽  
Lactate Concentration ◽  
Isolated Hepatocytes ◽  
Cell Preparation ◽  
O2 Uptake ◽  
Glucose Synthesis ◽  
The Relationship ◽  
Pyruvate Ratio ◽  
Stimulation Of

1. Hepatocytes isolated from starved rats and incubated without other substrates oxidized ethanol at a rate of 0.8–0.9μmol/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 0.04–0.08μmol/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 2.6μmol/min per g wet wt. of cells) at low lactate concentrations (0.5–2.0mm), but rates remained nearly constant (2.6–3.0μmol/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 0.12μmol/min per g wet wt. of cells) between 0.5mm- and 2.0mm-lactate and continued to increase (from 0.12 to 0.65μmol/min per g wet wt. of cells) with lactate concentrations between 2 and 10mm. 5. In the presence of ethanol, O2 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.

scholarly journals Metabolic effects of platelet-activating factor in rats in vivo. Stimulation of hepatic glycogenolysis and lipogenesis

1990 ◽  
Vol 269 (1) ◽  
pp. 269-272 ◽  
Author(s):  
R D Evans ◽  
V Ilic ◽  
D H Williamson
Keyword(s):  
Platelet Activating Factor ◽  
Hepatic Metabolism ◽  
Isolated Hepatocytes ◽  
Metabolic Effects ◽  
Hepatic Glycogen ◽  
Dose Dependent Fashion ◽  
Paf Receptor ◽  
High Doses ◽  
Stimulation Of

1. The effects of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine; PAF) on hepatic metabolism in vivo in rats were studied. 2. PAF stimulated synthesis of hepatic lipid (saponified and non-saponified) in a dose-dependent fashion and caused hypertriglyceridaemia. There was no effect of PAF on lipogenesis in isolated hepatocytes. 3. High doses of PAF also decreased hepatic glycogen. 4. All doses of PAF decreased plasma insulin, and this was accompanied by hyperglycaemia, except at the lowest dose. 5. The selective PAF-receptor antagonist L659.989 prevented the stimulation of lipogenesis, but indomethacin did not.

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