Energy‐dependent regulation of the steady‐state concentrations of the components of the lactate dehydrogenase reaction in liver

Berry, Michael N.; Grivell, Anthony R.; Wallace, Patricia G.

doi:10.1016/0014-5793(80)80280-8

Cited by 21 publications

(3 citation statements)

References 33 publications

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“…This finding may be related to the observations of Berry et al [32] who reported an increase of the cytosolic lactate/pyruvate ratio in intact rat hepatocytes in response to a lowering of the mitochondrial energy state. However, as can be seen from Fig.4, the lactate/pyruvate ratio had little influence on glucose 6-phosphate formation.…”

Section: Ejfect Of Mitochondria On the Formation Of Glucose 6-phosphatesupporting

confidence: 79%

Gluconeogenesis in vitro

Mörikofer‐Zwez

Stoecklin

Walter

1982

European Journal of Biochemistry

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A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors at near physiological concentrations was shown to form glucose 6-phosphate from malate + 3-phosphoglycerate at a rate of 1.11 0.09 pmol . min-' . g liver-' (mean f SEM, n = 9, 30°C). At least 70% of glucose 6-phosphate formed was derived from malate as calculated from experiments with [U-14C]malate. The indicated rates were measured between 10 min and 30 min incubation time when the system was near steady state with respect to the lactate/pyruvate ratio and to most of the gluconeogenic intermediates.In the absence of mitochondria, the rate of formation of glucose 6-phosphate from malate was about seven times lower than in their presence. A comparison between incubations carried out in presence or absence of mitochondria revealed that mitochondria decreased the lactate/pyruvate ratio and increased the ratio of (ATP + ITP)/(ADP + IDP). It could be shown that under the present incubation conditions, formation of glucose 6-phosphate was closely linked to the ratio of (ATP + ITP)/(ADP + IDP) whereas changing redox ratios had little influence on the gluconeogenic rate.Regulation of gluconeogenesis in liver is known to involve mitochondrial, cytosolic as well as microsomal reactions and has mainly been studied in liver slices, perfused liver or hepatocytes (for a review see [I]). Two early attempts have, however, been made to set up gluconeogenic systems which are readily accessible to low molecular weight effectors in order to obtain more detailed information on regulatory mechanisms. Mendicino and Utter [2] showed that a recombination of purified gluconeogenic enzymes was able to form glucose 6-phosphate from 3-phosphoglycerate or fumarate in the presence of chicken liver mitochondria. Furthermore, the group of Krebs [3,4] described a cell-free system of pigeon liver homogenate which formed glucose from lactate at physiological rates. In both cases, systems were employed where phosphoenolpyruvate carboxykinase is localized within the mitochondria [S,6] which is not the case in rat liver [7]. Attempts of Krebs et al. [3] to obtain gluconeogenesis in liver homogenates from rats and several other species were not successful and this system as well as the one of Mendicino and Utter [2] has not been used for further studies. Beside these investigations on the whole chain of gluconeogenesis in cellfree systems, some studies on partial sequences in subcellular fractions of rat liver have been made by the group of Lardy [8,9], by McDermott and Veneziale [lo] and by ourselves Recently, liver cells rendered permeable to substrates and effectors by treatment with filipin were proposed as a possible [I 1,121. model for studying metabolic regulation by low molecular weight compounds [13]. A different approach to the same problem is described in the present study which demonstrates the conversion of malate to glucose 6-phosphate at rates comparable to those observed in rat hepatocytes in a cell-free system derive...

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Section: Ejfect Of Mitochondria On the Formation Of Glucose 6-phosphatesupporting

confidence: 79%

Gluconeogenesis in vitro

Mörikofer‐Zwez

Stoecklin

Walter

1982

European Journal of Biochemistry

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“…At initial lactate concentrations exceeding 5 mM, the magnitude of the steady-state [lactate]: [pyruvate] ratio was approximately proportional to the lactate concentration, in contrast to data for isolated hepatocytes [26]. This was because the steadystate pyruvate concentration was relatively constant at around 0.4 mM.…”

Section: Discussioncontrasting

confidence: 57%

Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells

Grivell

Korpelainen

Williams

et al. 1995

Biochemical Journal

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The rate of transfer of reducing equivalents from cytoplasm to mitochondria has been examined in Ehrlich ascites tumour cells incubated in the presence of lactate. The flux of reducing equivalents was determined from the rate of metabolism of reduced intermediates that are oxidized within the cytosol. The magnitude of the flux of reducing equivalents was dependent on both the concentration of added lactate and the presence of carbohydrate. The rate of flux was twice as great in the presence of glucose and four times as high when glucose and lactate were added together as when lactate was the only added substrate. Fructose was less effective than glucose in stimulating reducing equivalent flux. In the presence of glucose or fructose, there was a substantial accumulation of hexose phosphates, dihydroxyacetone phosphate and glycerol 3-phosphate. Rotenone, an inhibitor of NADH dehydrogenase, and amino-oxyacetate, which inhibits the malate/aspartate shuttle, were powerful suppressors of reducing equivalent flux from lactate as sole substrate, but were much less potent in the presence of carbohydrate. Antimycin substantially inhibited reducing equivalent flux from all combinations of added substrates, consistent with its ability to block oxidation of reducing equivalents transferred by both the malate/aspartate and glycerol 3-phosphate shuttles. The glycerol 3-phosphate shuttle represents around 80% of the maximum total observed activity but is active only while glycolytic intermediates are present to provide the necessary substrates of the shuttle. This Ehrlich ascites cell line has an essentially similar total reducing equivalent shuttle capacity to that of isolated hepatocytes.

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“…The results of the steady-state kinetic analysis indicate that incubation of hepatocytes with antimycin A plus oligomycin does not significantly affect Ca2+ transport across the plasma membrane under the experimental conditions used. Although a decrease in the cellular ATP concentrations was observed in the presence of the inhibitors, other studies (Whiting & Barritt, 1982) have shown that Ca2+ outflow induced by ionophore A23 187 at 0.1 mM extracellular Ca2+ is not impeded by treatment of cells with dinitrophenol, an agent which also decreases the intracellular concentration of ATP (Berry et al, 1980;Blackmore et al, 1982). The absence of an effect of adrenaline on flux R21 and fractional transfer rate k21 for the transport of Ca2+ from the medium to the cell in cells incubated with antimycin A plus oligomycin indicates that normal mitochondrial function is required for this action of adrenaline on the liver cell [compare with the requirement for mitochondrial function in other actions of a-adrenergic agonists on the liver (Reinhart et al, 1 982a) and the inhibition by antimycin A of agonist-induced 45Ca2+ influx in the parotid gland (Poggioli et al, 1983)].…”

Section: Kinetics Ofintracellular Ca2+-transportprocessesmentioning

confidence: 95%

A kinetic investigation of the effects of adrenaline on 45Ca2+ exchange in isolated hepatocytes at different Ca2+ concentrations, at 20° C and in the presence of inhibitors of mitochondrial Ca2+ transport

Parker¹,

Barritt²,

Wadsworth³

1983

Biochemical Journal

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The effects of adrenaline on 45Ca2+-exchange curves for isolated hepatocytes incubated under various steady-state conditions were investigated. Kinetic analysis showed that the simplest compartment configuration consistent with each set of data was a series configuration of a three-compartment closed system comprising compartment 1 (C1), the extracellular medium, and two kinetically distinct compartments of cellular exchangeable Ca2+, C2 and C3 (C1 = C2 = C3). Subcellular fractionation of hepatocytes labelled with 45Ca2+ at 0.1 mM-Ca2+ indicated that C3 includes exchangeable Ca2+ in the mitochondria and endoplasmic reticulum. The following results were obtained from experiments conducted at 37 degrees C at five different extracellular Ca2+ concentrations. For both untreated and adrenaline-treated cells, plots of the flux from C1 to C2 as a function of the extracellular Ca2+ concentration were best described by straight lines consistent with Ca2+ influx across the plasma membrane being a diffusion process. Adrenaline increased the value of the permeability constant for Ca2+ influx by 40%. For untreated cells, plots of the flux between C2 and C3 as a function of the concentrations of Ca2+ in these compartments approached a plateau at high Ca2+ concentrations. Adrenaline caused a 3-fold increase in the concentration of Ca2+ that gives half-maximal rate of Ca2+ transport from C2 to C3. At 1.3 mM extracellular Ca2+, a decrease in incubation temperature from 37 degrees C to 20 degrees C decreased the quantity of Ca2+ in C3 and the flux and fractional transfer rates for the transport of Ca2+ between C2 and C3. At 20 degrees C adrenaline increased the quantity of Ca2+ in C3 and the fractional transfer rates for the transfer of Ca2+ from C1 to C2, and from C2 to C3. At 37 degrees C and 2.4 mM extracellular Ca2+, antimycin A plus oligomycin decreased the quantity of Ca2+ in C3 and increased the fractional transfer rate for the transport of Ca2+ from C3 to C2. In the presence of antimycin A and oligomycin, adrenaline did not increase the quantity of Ca2+ in C2 or the flux and fractional transfer rate for the transport of Ca2+ from C1 to C2, whereas these parameters were increased in the absence of the inhibitors.

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Energy‐dependent regulation of the steady‐state concentrations of the components of the lactate dehydrogenase reaction in liver

Cited by 21 publications

References 33 publications

Gluconeogenesis in vitro

Gluconeogenesis in vitro

Substrate-dependent utilization of the glycerol 3-phosphate or malate/aspartate redox shuttles by Ehrlich ascites cells

A kinetic investigation of the effects of adrenaline on 45Ca2+ exchange in isolated hepatocytes at different Ca2+ concentrations, at 20° C and in the presence of inhibitors of mitochondrial Ca2+ transport

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