Actions of quercetin on gluconeogenesis and glycolysis in rat liver

1. The action of quercetin on glucose catabolism and production was investigated in the perfused rat liver. 2. Quercetin inhibited lactate production from glucose: 80% inhibition was found at a quercetin concentration of 100 µM, and at higher concentrations inhibition was complete. 3. Pyruvate production from glucose presented a complex pattern, but stimulation was evident at 100 and 300 µM quercetin. Oxygen uptake tended to be increased. 4. Glucose synthesis from lactate and pyruvate was inhibited. Inhibition was already evident at 50 µM quercetin and almost complete at 300 µM. Concomitantly, the increment in oxygen uptake caused by lactate plus pyruvate was stimulated by 50 µM quercetin, but clearly inhibited by higher concentrations (100-500 µM). 5. Glucose phosphorylation in the high-speed supernatant fractions of liver homogenates was inhibited by quercetin, but only at concentrations above 150 µM. 6. It is concluded that quercetin can inhibit both glucose degradation and production and increase the cytosolic NAD + /NADH ratio. 7. These effects are likely to arise from many causes. Reduction of oxidative phosphorylation, inhibition of Na + -K + -ATPase, inhibition of glucokinase and inhibition of glucose 6-phosphatase could all contribute to the overall action of quercetin.     

Action of quercetin on glycogen catabolism in the rat liver

1. The influence of quercetin on glycogen catabolism and related parameters was investigated in the isolated perfused rat liver and subcellular systems. 2. Quercetin stimulated glycogenolysis (glucose release). This effect was already evident at a concentration of 50 microM maximal at 300 microM and declined at higher concentrations. Quercetin also stimulated oxygen consumption, with a similar concentration dependence. 3. Lactate production from endogenous glycogen (glycolysis) was diminished by quercetin without significant changes in pyruvate production. 4. Quercetin did not inhibit glucose transport into cells but decreased intracellular sequestration of [5-(3)H]glucose under conditions of net glucose release. 5. In isolated mitochondria, quercetin diminished the energy transduction efficiency. It also inhibited several enzymatic activities, e.g. the K(+)-ATPase/Na(+)-ATPase of plasma membrane vesicles and the glucose 6-phosphatase of isolated microsomes. 6. No significant changes of the cellular contents of AMP, ADP and ATP were found. The cellular content of glucose 6-phosphate, however, was increased (3.12-fold). 7. Some of the effects of quercetin (glycogenolysis stimulation) can be attributed to its action on mitochondrial energy metabolism, as, for example, uncoupling of oxidative phosphorylation. However, the multiplicity of the effects on several enzymatic systems certainly produces an intricate interplay that also generates complex and apparently contradictory effects.     

The action of quercetin on the mitochondrial NADH to NAD(+) ratio in the isolated perfused rat liver

It has been suggested that active forms of quercetin ( o-semiquinones) are able to oxidize NADH in mammalian cells. The purpose of this study was to investigate this proposition by measuring the beta-hydroxybutyrate to acetoacetate ratio as an indicator of the mitochondrial NADH/NAD (+) redox ratio in the isolated perfused rat liver. The NADH to NAD (+) ratio was reduced by quercetin; half-maximal reduction occurred at a concentration of 32.6 microM. Additionally, quercetin (25 to 300 microM) stimulated the Krebs cycle ( (14)CO (2) production) and inhibited oxygen uptake (50 to 300 microM). Low quercetin concentrations (25 microM) stimulated oxygen uptake. The results of the present work confirm the hypothesis that quercetin is able to participate in the oxidation of NADH in mammalian cells, shifting the cellular conditions to a more oxidized state (prooxidant activity). Stimulation of the Krebs cycle was probably caused by the increased NAD (+) availability whereas the decreased NADH availability and the inhibition of mitochondrial energy transduction could be the main causes for oxygen uptake inhibition.     

Action of quercetin on glycogen catabolism in the rat liver

1. The influence of quercetin on glycogen catabolism and related parameters was investigated in the isolated perfused rat liver and subcellular systems. 2. Quercetin stimulated glycogenolysis (glucose release). This effect was already evident at a concentration of 50 µ M maximal at 300 µ M and declined at higher concentrations. Quercetin also stimulated oxygen consumption, with a similar concentration dependence. 3. Lactate production from endogenous glycogen (glycolysis) was diminished by quercetin without significant changes in pyruvate production. 4. Quercetin did not inhibit glucose transport into cells but decreased intracellular sequestration of [5- 3 H]glucose under conditions of net glucose release. 5. In isolated mitochondria, quercetin diminished the energy transduction efficiency. It also inhibited several enzymatic activities, e.g. the K + -ATPase/Na + -ATPase of plasma membrane vesicles and the glucose 6-phosphatase of isolated microsomes. 6. No significant changes of the cellular contents of AMP, ADP and ATP were found. The cellular content of glucose 6-phosphate, however, was increased (3.12-fold). 7. Some of the effects of quercetin (glycogenolysis stimulation) can be attributed to its action on mitochondrial energy metabolism, as, for example, uncoupling of oxidative phosphorylation. However, the multiplicity of the effects on several enzymatic systems certainly produces an intricate interplay that also generates complex and apparently contradictory effects.     

The action of oxybutynin on haemodynamics and metabolism in the perfused rat liver

The present study was planned to investigate the possible action of oxybutynin on liver haemodynamics and its influence on metabolic variables. The isolated liver perfused either bivascularly or monovascularly in the non-recirculating system was used for the experiments and Krebs/Henseleit-bicarbonate buffer (pH 7.4) as a perfusion fluid. Oxybutynin (25-200 microM) was infused, the infusion time for each concentration being 14 min. Portally infused oxybutynin increased the perfusion pressure starting at 100 microM. Oxygen uptake was diminished, also starting at 100 microM. Arterially infused oxybutynin also increased the perfusion pressure in the hepatic artery. Lactate and pyruvate releases were considerably diminished by oxybutynin. Glucose release showed a small initial stimulation, then returned to values slightly below the basal ones. Cessation of oxybutynin infusion resulted in progressive stimulation of glucose release. When Ca2+ was omitted all effects of oxybutynin vanished. The hepatic contents of glucose, glucose 6-phosphate and lactate in the presence of 200 microM oxybutynin increased 7.8-, 4.6- and 5.1 times, respectively. The pyruvate content was not changed. The ATP content was diminished by 26.6% in the presence of 200 microM oxybutynin, but the AMP content was increased by 64.3%. The ADP content was not changed. Apparently, upon administration of oxybutynin, a considerable fraction of the liver parenchyma ceased to be irrigated or almost so, which is apparent from the concomitant inhibition of oxygen uptake, pressure increase and inhibition of glucose, lactate and pyruvate release together with the simultaneous intracellular accumulation of glucose, lactate and glucose 6-phosphate.     

Metabolic effects of propofol in the isolated perfused rat liver

Inhibitory effects of the intravenous anaesthetic propofol on mitochondrial energy metabolism have been reported by several authors. Impairment of energy metabolism is usually coupled to reduction in ATP production, which in turn is expected to lead to several alterations in cell metabolism such as stimulation of glycolysis and inhibition of gluconeogenesis. The present work aimed at finding an answer to the question of how propofol affects energy metabolism-linked parameters in the isolated perfused rat liver. In the fed state, propofol increased glycogenolysis (glucose release), glycolysis (lactate and pyruvate production) and oxygen uptake in the range between 10 and 500 microM. In the liver of fasted rats, propofol up to 100 microM increased oxygen uptake but decreased gluconeogenesis from three different substrates: lactate, alanine and glycerol. When lactate was the substrate 50% inhibition occurred at a propofol concentration of 50 microM. Propofol (100 microM) decreased the ATP content of the liver (-33.3%), increased the AMP content (+25%) and decreased the ATP/ADP and ATP/AMP ratios (49 and 45%, respectively). Most effects of propofol are probably due to impairment of oxidative phosphorylation. Particularly, the combined differential action on oxygen uptake (stimulation) and gluconeogenesis (inhibition) is strongly suggestive of an uncoupling action also under the conditions of the intact cell. This effect, in turn, is consistent with the reported high affinity of the cellular hepatic structure, especially membranes, for propofol.     

On the mechanism of sodium 2-5-4 chlorophenylpentyloxirane-2-carboxylate (POCA) inhibition of hepatic gluconeogenesis

Inhibition of hepatic long chain fatty acid oxidation by 2-5-4 chlorophenylpentyloxirane-2-carboxylate (POCA) leads to decreased gluconeogenic rates from lactate or from low concentrations of pyruvate. The inhibitory effect is fully overcome by concentrations of pyruvate above 0.8 mM or by the simultaneous administration of a medium chain fatty acid. At low pyruvate availability the energy cost of gluconeogenesis is mainly supported by fatty acid oxidation and POCA-induced inhibition of glucose production is secondary to a decreased energy availability. This is supported by the following observations: (i) POCA decreases hepatic respiration and phosphorylation potential: (ii) the rate of pyruvate-induced respiration was the same regardless of whether gluconeogenesis was inhibited or not by POCA: and (iii) concentrations of pyruvate above 0.8 mM, at which gluconeogenesis is not inhibited, prevented the POCA-induced decrease in the phosphorylation potential. It is concluded that inhibition of long chain fatty acid oxidation by POCA leads to a switch of energy fuel, and results in the oxidation of more pyruvate to meet the cellular energy demands. When pyruvate availability is low and thus, presumably, its mitochondrial transport restricted, pyruvate carboxylation most probably becomes limiting as a result of the increased flux through pyruvate dehydrogenase, in the presence of POCA.     

Contribution of Na+ -Ca2+ exchanger to pinacidil-induced relaxation in the rat mesenteric artery

1 Pinacidil relaxes blood vessels through opening the K(ATP) channels with a resultant membrane hyperpolarization and inhibition of Ca(2+) influx. The aim of this study was to examine the mechanisms thereby pinacidil induces K(+) channel-independent relaxation in isolated endothelium-denuded rat mesenteric artery. 2 Pinacidil-induced relaxation was inhibited by glibenclamide (1-10 micro M) in phenylephrine-preconstricted rings, but was unaffected by glibenclamide after inhibition of K(+) channels and VGCCs. Pinacidil-induced K(+) channel-independent relaxation remained unchanged after treatment with cyclopiazonic acid (10 micro M), thapsigargin (1 micro M), ouabain (100 micro M), propranolol (10 micro M), Rp-cAMPS triethylamine (30 micro M), L-NNA (100 micro M), or ODQ (10 micro M). 3 Pinacidil induced more relaxant effect in the presence of nifedipine than in the presence of 60 mM K(+) plus nifedipine. Pretreatment with Na(+)-Ca(2+) exchanger inhibitors, nickel (30-300 micro M) or benzamil (20 micro M) attenuated pinacidil-induced relaxation in normal or in nifedipine-containing solution. Pinacidil (1 micro M) produced less relaxant effect with decreasing extracellular Na(+) concentration. Na(+)-free condition abolished the inhibitory effect of benzamil. Both nickel and benzamil inhibited pinacidil-induced relaxation in the presence of glibenclamide (10 micro M). Nickel (300 micro M) did not affect the relaxant response to sodium nitroprusside. 4 Pinacidil relaxed the rings preconstricted by active phorbol and U46619 with similar potency. 5 The present results indicate that stimulation of the forward mode Na(+)-Ca(2+) exchange pathway is in part responsible for pinacidil-induced K(+) channel-independent vasorelaxation. Pinacidil also induces K(+) channel-dependent but VGCCs-independent relaxation. The PKC-mediated cellular pathway may be a target site for pinacidil only in higher concentrations.     

Antidiabetic effects of Mukia maderaspatana and its phenolics: An in vitro study on gluconeogenesis and glucose uptake in rat tissues

Context: Traditional medicine is used by over 60% of the world’s population for health care. Mukia maderaspatana (L.) M. Roem. (Cucurbitaceae) (Mukia) is extensively used in folklore medicine as an antidiabetic plant. It is rich in phenolics that contribute to its medicinal properties.

Objective: Mukia extract and phenolics such as quercetin and phloroglucinol are investigated for their in vitro antidiabetic activity.

Materials and methods: Quercetin, phloroglucinol, and methanol extract of the dried whole plant (0.25 and 0.5?mg/ml) were studied for the inhibition of gluconeogenesis in rat liver slices and glucose uptake in isolated rat hemi-diaphragm (50 and 100?µg/ml). Phenolics of Mukia were analyzed by HPLC.

Results and discussion: Glucose (1.2?mg/g/h) was synthesized from pyruvate and the synthesis was completely inhibited by insulin (1?U/ml). Quercetin at 0.25 and 0.5?mg/ml caused 65% and 89% inhibition (0.42?mg/g/h and 0.13?mg/g/h glucose). Addition of insulin did not increase inhibition. Phloroglucinol inhibited 100% glucose production with or without insulin. Mukia (0.25?mg/ml) inhibited gluconeogenesis (0.65?mg/g/h) by 45%, and with insulin, inhibition increased to 50% (0.59?mg/g/h). At 0.5?mg/ml, glucose production was stimulated by1.2-fold, but with insulin it was inhibited by 89% (0.13?mg/g/h glucose). Mukia had no effect on glucose uptake, but potentiated the action of insulin mediated glucose uptake (152.82?±?13.30?mg/dl/g/30?min) compared with insulin control (112.41?±?9.14?mg/dl/g/30?min) (p?0.05). HPLC analysis revealed the presence of phenolics.

Conclusion: Results provide scientific rationale for the use of Mukia in folk medicine as an antidiabetic nutraceutical.     

The effect of 5-thioglucose on the energy metabolism of Schistosoma mansoni in vitro

5-Thioglucose (5-TG) had a marked effect on the energy metabolism of Schistosoma mansoni in vitro: the conversion of external glucose into lactate by intact worms was severely inhibited. This inhibition of glycolysis was instantaneous, independent of the oxygen concentration and competitive with respect to glucose. Degradation of 0.5 mM external (14C-labelled) glucose was inhibited for 80% in the presence of 20 mM 5-TG. On the other hand the degradation of endogeneous glycogen to lactate was uninhibited. This shows that the inhibition of glucose breakdown occurred at the entrance of glucose into the cell and/or at the hexokinase reaction. It was demonstrated that 5-TG inhibited both the uptake of glucose and the activity of hexokinase. However, it was concluded that in the intact worm 5-TG blocked glycolysis by its competitive inhibition of hexokinase. In intact S. mansoni worms hexokinase is probably the rate-limiting enzyme of glycolysis. Krebs-cycle activity and lactate production do not occur at a fixed ratio: at lower rates of pyruvate formation Krebs-cycle activity was favoured.     

Inhibition of gluconeogenesis in isolated perfused rat liver by clanobutin

The effect of clanobutin {4-[p-chloro-N-(p-methoxyphenyl-)benzamido]butyric acid} on gluconeogenesis from lactate + pyruvate (1.6 + 0.2 mmoles/1) as precursors in isolated perfused liver of fasted rats was investigated. Glucose production was dose dependent inhibited up to a maximum of 81 ± 3 per cent; the half-maximum concentration of clanobutin was 0.33 ± 0.04 mmoles/1. Buformin and phenformin, respectively, used as references showed no effect on gluconeogenesis under the given experimental conditions. Oxygen uptake was not inhibited by clanobutin at concentrations up to 0.14 mmoles/1. At higher concentrations, the inhibitory effect was smaller than observed for comparable buformin doses. According to our results, clanobutin appears to be a more potent and probably more specific inhibitor of gluconeogenesis than the therapeutically used biguanides—at least in the isolated perfused rat liver.     

Influence of sulphite on the release of glycolytic metabolites in the perfused rat liver

Sulphite interacts reversibly and irreversibly in vitro with many compounds of biological relevance. A possible inhibitory action of sulphite on hepatic lactate dehydrogenase activity was studied using the perfused rat liver to determine glycolytic metabolites released by the liver during sulphite infusion. A sulphite-dependent reversible inhibition of lactate release but no increase in pyruvate production indicated that lactate dehydrogenase was inhibited by sulphite concentrations in the millimolar range. This effect was diminished by addition of pyruvate to the perfusion medium. It appears very unlikely that, under physiological conditions, sulphite levels in the portal blood or the liver of the rat would reach concentrations that inhibit lactate dehydrogenase activity.     

Effects of derivatives of indole carboxylic acids on carbohydrate metabolism of the rat

Several indole derivatives were characterized on their effects on carbohydrate metabolism in the rat and compared in their insulin-like effects on hepatic gluconeogenesis and glucose consumption in muscle with 5-O-methoxyindole-2-carboxylic acid (MICA) and with indole-3-butyric acid. The substances depressed blood glucose in the fasted adrenalectomized rat at an oral dose of 50–250 mg/kg. Glucose consumption in an isolated muscle preparation was altered only a small amount. In some cases glycogen content of muscle was reduced at a millimolar concentration. All compounds revealed strong inhibition of glucose production from pyruvate in isolated liver slices at a concentration of 10^?4 M. By chemical modification of the indole structure strong inhibitors of gluconeogenesis are obtained. It was not possible to find substances which show a high stimulation of glucose uptake and low activity in inhibiting gluconeogenesis.     

Effect of loperamide on Na+/D-glucose cotransporter activity in mouse small intestine

The mu-opioid agonist loperamide is an antidiarrhoeal drug which inhibits intestinal motility and secretion. Its anti-absorptive effects are less well investigated, but may be mediated through calmodulin. We have investigated further the effect of loperamide on the intestinal Na+-dependent D-glucose transporter (SGLT1). Brush-border membrane vesicles were prepared from mouse small intestine, and uptake of [3H]glucose was measured. Na+-dependent glucose uptake displayed the typical overshoot at 34 s; the peak value was 1.6 nmol mg(-1). The overshoot disappeared in the presence of phlorizin or when Na+ was replaced by K+. Extravesicular loperamide dose-dependently inhibited SGLT1 activity with an IC50 value of 450 micromol L(-1). Loperamide displayed a mixed inhibition type: the apparent Vmax decreased from 0.9 to 0.5 nmol mg(-1)/15 s, the apparent Km increased from 0.23 to 1.13 mmol L(-1) glucose. Na+ kinetics were more complex, but loperamide inhibited net glucose uptake by 90% at 100 mmol L(-1) Na+. Glucose uptake was unchanged by agents affecting calmodulin activity. Loperamide inhibited intestinal Na+, K+-ATPase activity, whilst sucrase activity was unaffected. SGLT1 activity was inhibited by loperamide, but this effect was not mediated through calmodulin. As this action is only evident at high concentrations of loperamide a nonspecific mechanism may be involved.     

Effect of increased cardiac output on liver blood flow,oxygen exchange and metabolic rate during longterm endotoxin-induced shock in pigs

1. We investigated hepatic blood flow, O₂ exchange and metabolism in porcine endotoxic shock (Control, n=8; Endotoxin, n=10) with administration of hydroxyethylstarch to maintain arterial pressure (MAP)>60 mmHg.
2. Before and 12, 18 and 24 h after starting continuous i.v. endotoxin we measured portal venous and hepatic arterial blood flow, intracapillary haemoglobin O₂ saturation (Hb-O₂%) of the liver surface and arterial, portal and hepatic venous lactate, pyruvate, glyercol and alanine concentrations. Glucose production rate was derived from the plasma isotope enrichment during infusion of [6,6-²H₂]-glucose.
3. Despite a sustained 50% increase in cardiac output endotoxin caused a progressive, significant fall in MAP. Liver blood flow significantly increased, but endotoxin affected neither hepatic O₂ delivery and uptake nor mean intracapillary Hb-O₂% and Hb-O₂% frequency distributions.
4. Endotoxin nearly doubled endogenous glucose production rate while hepatic lactate, alanine and glycerol uptake rates progressively decreased significantly. The lactate uptake rate even became negative (P<0.05 vs Control). Endotoxin caused portal and hepatic venous pH to fall significantly concomitant with significantly increased arterial, portal and hepatic venous lactate/pyruvate ratios.
5. During endotoxic shock increased cardiac output achieved by colloid infusion maintained elevated liver blood flow and thereby macro- and microcirculatory O₂ supply. Glucose production rate nearly doubled with complete dissociation of hepatic uptake of glucogenic precursors and glucose release. Despite well-preserved capillary oxygenation increased lactate/pyruvate ratios reflecting impaired cytosolic redox state suggested deranged liver energy balance, possibly due to the O₂ requirements of gluconeogenesis.

     

The effects of adenosine-5′-ethylcarboxamide on liver blood flow and hepatic glucose,lactate and pyruvate balances in dogs

Summary The actions of adenosine-5-ethylcarboxamide (744–96), a long-acting adenosine analoge, on liver, portal and intestinal balances of glucose, lactate and pyruvate and on hepatic and portal blood flow were investigated in 6 chloralose-anaesthetized mongrel dogs. 744–96 led to an increase in portal and hepatic blood flow. Glucose release by the liver and glucose uptake by the non-hepatic splanchnic area (portal balance) were markedly increased by 744–96. Hepatic lactate and pyruvate balances were reversed from uptake to release by the adenosine analogue. The changes in glucose balances compare closely to the actions of glucagon, which is known to be released by 744–96. Apart from these possible glucagon-mediated actions, a direct action of the adenosine analogue must be assumed from the changes in lactate metabolism. The results of this study are indicative of a substratemobilising action of adenosine in addition to its wellknown vasodilatory action.     

Inhibitory effect of valproate on weak organic acid uptake in rat renal proximal tubules.

The effects of an antiepileptic drug, valproic acid (VPA), on transport mechanisms involved in renal excretion of anionic xenobiotics were investigated on rat renal proximal tubules in vitro. It was found that VPA (0.1-1 mM) dose dependently inhibited the baseline uptake of a marker organic anion, fluorescein, in the tubules. The inhibition could not be exclusively accounted for by competition between VPA and fluorescein. Taking into account a proposed relationship between the weak organic anion uptake and ammoniagenesis, the influence of VPA (0.5 mM) on the effects of glutamine and glutamate (both at 5 mM) on fluorescein uptake and ammonia production were examined. Glutamine stimulated ammonia production by the tubules, with the glutamine-induced ammoniagenesis being further augmented by VPA, while glutamate failed to affect the basal ammoniagenesis. Both glutamine (5 mM) and glutamate (5 mM) slightly inhibited fluorescein uptake, with the inhibitory effects not modified by VPA. Thus, there was no coincidence in the effects of VPA on organic anion uptake and renal ammoniagenesis. At the same time, the inhibitory effect of VPA (0.5 mM) on fluorescein uptake was largely overcome by addition of pyruvate (5 mM) to the incubation medium. In addition, VPA strongly inhibited glucose production from pyruvate. A known modulator of pyruvate metabolism, dichloroacetic acid (DCA, 1 mM), also inhibited fluorescein uptake, although its inhibitory effect was less pronounced than that of VPA. Both inhibitors failed to alter the tissue content of alpha-ketoglutarate or lactate but did slightly augment the pyruvate level. The inhibitory effects of VPA and DCA on the baseline fluorescein uptake were not additive, suggesting their similar intracellular targeting. It is assumed that the inhibitory effect of VPA on baseline fluorescein uptake in rat renal proximal tubules in vitro may be associated with its action on pyruvate metabolism.     

Actions of juglone on energy metabolism in the rat liver

Juglone is a phenolic compound used in popular medicine as a phytotherapic to treat inflammatory and infectious diseases. However, it also acts as an uncoupler of oxidative phosphorylation in isolated liver mitochondria and, thus, may interfere with the hepatic energy metabolism. The purpose of this work was to evaluate the effect of juglone on several metabolic parameters in the isolated perfused rat liver. Juglone, in the concentration range of 5 to 50 μM, stimulated glycogenolysis, glycolysis and oxygen uptake. Gluconeogenesis from both lactate and alanine was inhibited with half-maximal effects at the concentrations of 14.9 and 15.7 μM, respectively. The overall alanine transformation was increased by juglone, as indicated by the stimulated release of ammonia, urea, l-glutamate, lactate and pyruvate. A great increase (9-fold) in the tissue content of α-ketoglutarate was found, without a similar change in the l-glutamate content. The tissue contents of ATP were decreased, but those of ADP and AMP were increased. Experiments with isolated mitochondria fully confirmed previous notions about the uncoupling action of juglone. It can be concluded that juglone is active on metabolism at relatively low concentrations. In this particular it resembles more closely the classical uncoupler 2,4-dinitrophenol. Ingestion of high doses of juglone, thus, presents the same risks as the ingestion of 2,4-dinitrophenol which comprise excessive compromising of ATP production, hyperthermia and even death. Low doses, i.e., moderate consumption of natural products containing juglone, however, could be beneficial to health if one considers recent reports about the consequences of chronic mild uncoupling.     

Effects of physiologic concentrations of lactate, pyruvate and ascorbate on glucose metabolism in unstressed and oxidatively stressed human red blood cells

Glucose metabolism was studied in human red blood cells incubated in the presence of physiologic concentrations of ascorbate (0.1 mM) and/or lactate (2 mM) plus pyruvate (0.1 mM). The total flux through glycolysis, as measured by 14C-labeling of glycolytic intermediates, was increased about 15% by ascorbate, 30% by lactate plus pyruvate, and 40% by ascorbate plus lactate plus pyruvate. We found, however, that physiologic concentrations of ascorbate and/or lactate plus pyruvate had no effect on flux of glucose or recycling of pentoses through the hexose monophosphate shunt. Increased formation of lactate accounted for most of the observed increase in glycolysis with little change in pyruvate formation, indicating that the increased flux of reducing equivalents from glucose was stored as lactate rather than being consumed by red cell metabolism. In all experiments, there was a net increase with time in the absolute amount of both lactate and pyruvate in red cell suspensions, indicating that lactate or pyruvate present at zero time did not function as a stoichiometric source or sink for reducing equivalents. There was little effect on steady-state levels of ATP or 2,3-diphosphoglycerate. Equilibration of ascorbate between red cells and the medium was complete before the addition of 14C-labeled glucose to the medium. Glucose metabolism prevented net oxidation of ascorbate in the incubation medium. Physiologic concentrations of ascorbate, lactate and pyruvate appear to increase flux through glycolysis by increasing the turnover of ATP and/or 2,3-diphosphoglycerate. Red cells were exposed to mild oxidative stress by incubation with 0.27 mM 6-hydroxydopamine, 0.27 mM 6-aminodopamine, 0.13 mM 1,4-naphthoquinone-2-sulfonic acid or 0.27 mM phenylhydrazine. The metabolic response to oxidative stress was determined by measuring the formation of methemoglobin, pyruvate, lactate and CO2 in the presence and absence of physiologic concentrations of lactate, pyruvate and ascorbate. Lactate, pyruvate and ascorbate had no effect on the net methemoglobin accumulation but rather on the distribution of the metabolic sources of reducing equivalents and on the flux of reducing equivalents to oxygen. Physiologic lactate and pyruvate allowed increased flow of reducing equivalents from glycolysis to methemoglobin and ultimately oxygen without the necessity of increased flux through glycolysis. This was accomplished by a decrease in the ratio of newly formed lactate to newly formed pyruvate with no increase in total lactate plus pyruvate.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of cyanamide on the metabolism of ethanol and acetaldehyde and on gluconeogenesis by isolated rat hepatocytes

Previous experiments demonstrated that acetaldehyde stimulated glucose production from pyruvate, whereas gluconeogenesis from glycerol, xylitol and sorbitol was inhibited [A.I. Cederbaum and E. Dicker, Archs Biochem. Biophys. 197, 415 (1979)]. To determine the mechanism whereby acetaldehyde affects glucose production from these precursors, and to evaluate the role of acetaldehyde in the actions of ethanol, experiments with cyanamide were carried out. The oxidation of acetaldehyde by isolated rat liver cells was inhibited by cyanamide after a brief incubation period. Associated with this inhibition of acetaldehyde oxidation was an inhibition of ethanol oxidation by cyanamide and an increase in the amount of acetaldehyde which arose during the oxidation of ethanol. Ethanol oxidation was decreased because of the ineffective removal of acetaldehyde in the presence of cyanamide. Cyanamide had no effect on hepatic oxygen uptake. The increase in the β-hydroxybutyrate/acetoacetate ratio produced by acetaldehyde was completely prevented by cyanamide, whereas the slight increase in the lactate/pyruvate ratio was not prevented by cyanamide. Cyanamide partially reversed the ethanol-induced increase in the lactate/pyruvate ratio, but it completely prevented the ethanol-induced increase in the β-hydroxybutyrate/acetoacetate ratio. The ethanol-induced change in the mitochondrial redox state may, therefore, be due primarily to the mitochondrial oxidation of the acetaldehyde which arises during the oxidation of ethanol. The inhibitory effects of acetaldehyde on gluconeogenesis from glycerol, xylitol and sorbitol, as well as the stimulation of acetaldehyde of glucose production from pyruvate, were completely prevented by cyanamide. These results indicate that the effects of acetaldehyde on gluconeogenesis represent metabolic effects, rather than direct effects of acetaldehyde. Changes in the cellular NADH/NAD^? ratio as a consequence of acetaldehyde metabolism are postulated to be responsible for these actions of acetaldehyde. Ethanol stimulated glucose production from pyruvate, while inhibiting gluconeogenesis from glycerol, xylitol and sorbitol. Cyanamide, which prevented the effects of acetaldehyde on gluconeogenesis, also prevented the effects of ethanol on gluconeogenesis. This prevention by cyanamide may be suggestive for a role for acetaldehyde in the actions of ethanol on gluconeogenesis. The possibility cannot be ruled out, however, that the prevention of the effects of ethanol by cyanamide may be due to the partial inhibition of ethanol oxidation by cyanamide. These results indicate that cyanamide is an effective inhibitor of acetaldehyde oxidation by isolated liver cells and therefore can be used to determine the mechanism whereby acetaldehyde affects metabolic function. Depending on the reaction under investigation, acetaldehyde can have direct or indirect effects on cellular metabolism.