NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

The influence of adenosine on intermediary metabolism of isolated hepatocytes.

The effect of adenosine was tested on the energetic metabolism of fed rat liver cells after isolation. The cells were incubated in a buffered saline medium with glucose (5 mM) and adenosine (1 mM) for 30 minutes at 37 degrees C. This increased the concentration of the adenylic nucleotides ATP (+57 per cent, ADP (+39 per cent). Cyclic AMP was increased (+50 per cent) and the intracellular inorganic phosphate decreased (-22 per cent). These changes were accompaned by a decrease of glycogenolysis, glucose consumption and lactate production. Measurement of glycolytic intermediates showed decreased concentrations of fructose 1,6-bis-phosphate and 3-phosphoglycerate proportional to the increase in ATP concentration. The near-equilibrium of the glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase system was not modified by adenosine. The decrease of the NAD+/NADH ratio along with the increase of the ATP/ADP X PO4 ratio explains the decrease of 3-phosphoglycerate. The decrease in glucose consumption can be explained by the cross over at the phosphofructokinase stage with the decrease of fructose 1,6-bisphosphate. The major part of adenosine was deaminated as indicated by an increase in the production of ammonia and urea. The effects of inosine, or adenosine along with an inhibitor of adenosine deaminase (pentostatin) suggest that adenosine acts on the glucose consumption through adenylic nucleotides. However the increase of the adenylic nucleotide level cannot totally explain the other metabolic changes: decrease of the NAD+/NADH cytoplasmic ratio, constancy of this ratio in mitochondria, decrease of gluconeogenesis from lactate. A direct action of adenosine can therefore be expected.     

Control of the shift from homolactic acid to mixed-acid fermentation in Lactococcus lactis: predominant role of the NADH/NAD+ ratio.

During batch growth of Lactococcus lactis subsp. lactis NCDO 2118 on various sugars, the shift from homolactic to mixed-acid metabolism was directly dependent on the sugar consumption rate. This orientation of pyruvate metabolism was related to the flux-controlling activity of glyceraldehyde-3-phosphate dehydrogenase under conditions of high glycolytic flux on glucose due to the NADH/NAD+ ratio. The flux limitation at the level of glyceraldehyde-3-phosphate dehydrogenase led to an increase in the pool concentrations of both glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate and inhibition of pyruvate formate lyase activity. Under such conditions, metabolism was homolactic. Lactose and to a lesser extent galactose supported less rapid growth, with a diminished flux through glycolysis, and a lower NADH/NAD+ ratio. Under such conditions, the major pathway bottleneck was most probably at the level of sugar transport rather than glyceraldehyde-3-phosphate dehydrogenase. Consequently, the pool concentrations of phosphorylated glycolytic intermediates upstream of glyceraldehyde-3-phosphate dehydrogenase decreased. However, the intracellular concentration of fructose-1,6-bisphosphate remained sufficiently high to ensure full activation of lactate dehydrogenase and had no in vivo role in controlling pyruvate metabolism, contrary to the generally accepted opinion. Regulation of pyruvate formate lyase activity by triose phosphates was relaxed, and mixed-acid fermentation occurred (no significant production of lactate on lactose) due mostly to the strong inhibition of lactate dehydrogenase by the in vivo NADH/NAD+ ratio.     

Synechococcus sp. strain PCC 7002 nifJ mutant lacking pyruvate:ferredoxin oxidoreductase

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry (F(v)/F(m)). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H(2) production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H(2) production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H(2) revealed two temporally resolved phases of H(2) production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD(+) ratio revealed that the reductant generated by PFOR contributing to the first phase of H(2) production was not in equilibrium with bulk NADH/NAD(+) and that the second phase corresponded to the equilibrium NADH-mediated process.     

The effect of NAPRTase overexpression on the total levels of NAD,the NADH/NAD+ ratio,and the distribution of metabolites in Escherichia coli

Escherichia coli (E. coli) maintains its total NADH/NAD+ intracellular pool by synthesizing NAD through the de novo pathway and the pyridine nucleotide salvage pathway. The salvage pathway recycles intracellular NAD breakdown products and preformed pyridine compounds from the environment, such as nicotinic acid (NA). The enzyme nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11), encoded by the pncB gene, catalyzes the formation of nicotinate mononucleotide (NAMN), a direct precursor of NAD, from NA. This reaction is believed to be the rate-limiting step in the NAD salvage pathway. The current study investigates the effect of overexpressing the pncB gene from Salmonella typhimurium on the total levels of NAD, the NADH/NAD+ ratio, and the production of different metabolites in E. coli under anaerobic chemostat conditions and anaerobic tube experiments. In addition, this paper studies the effect of combining the overexpression of the pncB gene with an NADH regeneration strategy that increases intracellular NADH availability, as we have previously shown. (The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures, Metabolic Eng. 4, 230-237; Metabolic engineering of Escherichia coli: Increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229.) Overexpression of the pncB gene in chemostat experiments increased the total NAD levels, decreased the NADH/NAD+ ratio, and did not significantly redistribute the metabolic fluxes. However, under anaerobic tube conditions, overexpression of the pncB gene led to a significant shift in the metabolic patterns as evidenced by a decrease in lactate production and an increase as high as two-fold in the ethanol-to-acetate (Et/Ac) ratio. These results suggest that under chemostat conditions the total level of NAD is not limiting and the metabolic rates are fixed by the system at steady state. On the other hand, under transient conditions (such as those in batch cultivation) the increase in the total level of NAD can increase the rate of NADH-dependent pathways (ethanol) and therefore change the final distribution of metabolites. The effect of combining overexpression of the pncB gene with the substitution of the native cofactor-independent formate dehydrogenase (FDH) with an NAD(+)-dependent FDH was also investigated under anaerobic tube conditions. This manipulation produced a metabolic pattern that combines a high Et/Ac ratio similar to that obtained with the new FDH with an intermediate lactate level similar to that obtained with the overexpression of the pncB gene. It was found that addition of the pncB gene to the FDH system does not increase further the production of reduced metabolites because the system for NADH regeneration already reached the maximum theoretical yield of approximately 4 mol NADH/mol of glucose.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig.

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase. The calculated cytosolic NAD+/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

Effect of Lactacidosis on Pyridine Nucleotide Stability During Ischemia in Mouse Brain

Brain levels of NADH and NAD+ were measured in three models of cerebral ischemia to determine whether degradation of the pyridine nucleotides is enhanced in models that generate high concentrations of lactic acid. Complete ischemia (decapitation), in which lactate increased to 14 mmol/kg, caused a gradual decrease in the NAD pool to 50% of control by 2 h. During focal ischemia (occlusion of the middle cerebral artery), the decrease in the NAD pool was less pronounced (82% of control at 2 h) despite the accentuated accumulation of lactate to 33 mmol/kg. In a third model (unilateral hypoxia-ischemia), pretreatment of animals with glucose augmented the ischemic elevation of lactate from 30 mmol/kg to 40 mmol/kg and greatly impaired restoration of energy metabolites during recirculation. However, glucose pretreatment had no effect on the size of the NAD pool during ischemia or early recovery. These results, therefore, demonstrate that the pyridine nucleotide pool is not rapidly degraded during ischemic insults that accumulate high concentrations of lactic acid. The stability of the NAD pool may have been enhanced by the limited increase in brain levels of NADH that occurred in these models of incomplete ischemia.     

Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H(2)O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H(2)O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15 ± 0.08 mM to 9.94 ± 0.07 mM, and the corresponding diacetyl production increased from 1.07 ± 0.03 mM to 4.16 ± 0.06 mM with the intracellular NADH/NAD(+) ratios varying from 0.711 ± 0.005 to 0.383 ± 0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD(+) ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H(2)O-forming NADH oxidase activity led to 76.95% lower H(2)O(2) concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H(2)O(2) accumulation and prolong cell survival.     

Increased production of succinic acid in Escherichia coli by overexpression of malate dehydrogenase

Escherichia coli NZN111 is a double mutant with inactivated lactate dehydrogenase and pyruvate formate-lyase. It cannot utilize glucose anaerobically because of its unusually high intracellular NADH/NAD(+) ratio. We have now constructed a recombinant strain, E. coli NZN111/pTrc99a-mdh, which, during anaerobic fermentation, produced 4.3 g succinic acid l(-1) from 13.5 g glucose l(-1). The NADH/NAD(+) ratio decreased from 0.64 to 0.26. Furthermore, dual-phase fermentation (aerobic growth followed by anaerobic phase) resulted in enhanced succinic acid production and reduced byproduct formation. The yield of succinic acid from glucose during the anaerobic phase was 0.72 g g(-1), and the productivity was 1.01 g l(-1) h(-1).     

Inhibition of Lactate Production in Rat Brain Extracts and Synaptosomes by 3-[4-(Reduced 3-Pyridine Aldehyde-Adenine Dinucleotide)]-Pyruvate

In basic solutions, pyruvate enolizes and reacts (through its 3-carbon) with the 4-carbon of the nicotinamide ring of NAD+, yielding an NAD-pyruvate adduct in which the nicotinamide ring is in the reduced form. This adduct is a strong inhibitor of lactate dehydrogenase, presumably because it binds simultaneously to the NADH and pyruvate sites. The potency of the inhibition, however, is muted by the adduct's tendency to cyclize to a lactam. We prepared solutions of the pyruvate adduct of NAD+ and of NAD+ analogues in which the -C(O)NH2 of NAD+ was replaced with -C(S)NH2, -C(O)CH3, and -C(O)H. Of the four, only the last analogue, 3-[4-(reduced 3-pyridine aldehyde-adenine dinucleotide)]-pyruvate (RAP) cannot cyclize and it was found to be the most potent inhibitor of beef heart and rat brain lactate dehydrogenases. The inhibitor binds very tightly to the NADH site (Ki approximately 1 nM for the A form). Even at high concentrations (20 microM), RAP had little or no effect on rat brain glyceraldehyde-3-phosphate, pyruvate, alpha-ketoglutarate, isocitrate, soluble and mitochondrial malate, and glutamate dehydrogenases. The glycolytic enzymes, hexokinase and phosphofructokinase, were similarly unaffected. RAP strongly inhibited lactate production from glucose in rat brain extracts but was less effective in inhibiting lactate production from glucose in synaptosomes.     

The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures

It is generally known that cofactors play a major role in the production of different fermentation products. This paper is part of a systematic study that investigates the potential of cofactor manipulations as a new tool for metabolic engineering. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ and producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. We have previously investigated a genetic means of increasing the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase and have demonstrated that this manipulation provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically (Berríos-Rivera et al., 2002, Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase, Metabolic Eng. 4, 217-229). The current work explores further the effect of substituting the native cofactor-independent formate dehydrogenase (FDH) by an NAD(+)-dependent FDH from Candida boidinii on the NAD(H/+) levels, NADH/NAD+ ratio, metabolic fluxes and carbon-mole yields in Escherichia coli under anaerobic chemostat conditions. Overexpression of the NAD(+)-dependent FDH provoked a significant redistribution of both metabolic fluxes and carbon-mole yields. Under anaerobic chemostat conditions, NADH availability increased from 2 to 3 mol NADH/mol glucose consumed and the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol to acetate ratio and a decrease in the flux to lactate. It was also found that the NADH/NAD+ ratio should not be used as a sole indicator of the oxidation state of the cell. Instead, the metabolic distribution, like the Et/Ac ratio, should also be considered because the turnover of NADH can be fast in an effort to achieve a redox balance.     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD(+) system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.     

Pyruvate metabolism in Lactococcus lactis is dependent upon glyceraldehyde-3-phosphate dehydrogenase activity

Modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity from Lactococcus lactis was undertaken during batch fermentation on lactose, by adding various concentrations of iodoacetate (IAA), a compound which specifically inhibits GAPDH at low concentrations, to the culture medium. As IAA concentration is increased, GAPDH activity diminishes, provoking a decrease of both the glycolytic flux and the specific growth rate. This control exerted at the level of GAPDH was due partially to IAA covalent fixation but also to the modified NADH/NAD+ ratio. The mechanism of inhibition by NADH/NAD+ was studied in detail with the purified enzyme and various kinetic parameters were determined. Moreover, when GAPDH activity became limiting, the triose phosphate pool increased resulting in the inhibition of pyruvate formate lyase activity, while the lactate dehydrogenase is activated by the high NADH/NAD+ ratio. Thus, modifying the GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.     

RESTITUTION OF CEREBRAL ENERGY STATE, AS WELL AS OF GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS AFTER 30 MINUTES OF COMPLETE ISCHEMIA IN RATS ANAESTHETIZED WITH NITROUS OXIDE OR PHENOBARBITAL

Restitution of cerebral cortex concentrations of organic phosphates, glycolytic metabolites, citric acid cycle intermediates, associated amino acids, and ammonia, following a 30 min period of complete ischemia, was studied in rats anaesthetized with either 70% N₂O or 150 mg·kg^-1 of phenobar-bital. Following a 90 min period of recirculation the pattern of restitution was similar in the two groups. Thus, all animals showed recovery of phosphocreatine concentrations, restitution of the adenylate energy charge to about 99% of control, and disappearance of lactate accumulated during the ischemia. Analyses of glycolytic metabolites indicated inhibition of glycolysis at the phosphofructokinase step, possibly caused by accumulation of citrate. Measured citric acid cycle intermediates indicated extensive normalization of mitochondrial metabolism. Changes in amino acid concentrations consisted of a fall in glutamate concentration, a rise in aspartate/glutamate ratio, a fall in GABA concentration, and a rise in alanine concentration. However, ammonia concentration was close to normal, and the size of the amino acid pool did not change. It is concluded that although the results do not exclude damage to a small part of the neuronal population, they demonstrate that, irrespective of the type of anaesthesia used, the majority of brain cells must have survived 30 min of complete ischemia without signs of irreversible metabolic damage.     

Metabolic effects of D-glyceraldehyde in isolated hepatocytes.

The effects of D-glyceraldehyde on the hepatocyte contents of various metabolites were examined and compared with the effects of fructose, glycerol and dihydroxyacetone, which all enter the glycolytic/gluconeogenic pathways at the triose phosphate level. D-Glyceraldehyde (10 MM) caused a substantial depletion of hepatocyte ATP, as did equimolar concentrations of fructose and glycerol. D-Glyceraldehyde and fructose each caused a 2-fold increase in fructose 1,6-bisphosphate and the accumulation of millimolar quantities of fructose 1-phosphate in the cells. D-Glyceraldehyde caused an increase in the glycerol 3-phosphate content and a decrease in the dihydroxyacetone phosphate content, whereas dihydroxyacetone increased the content of both metabolites. The increase in the [glycerol 3-phosphate]/[dihydroxyacetone phosphate] ratio caused by D-glyceraldehyde was not accompanied by a change in the cytoplasmic [NAD+]/[NADH] ratio, as indicated by the unchanged [lactate]/[pyruvate] ratio. The accumulation of fructose 1-phosphate from D-glyceraldehyde and dihydroxyacetone phosphate in the hepatocyte can account for the depletion of the intracellular content of the latter. Presumably ATP is depleted as the result of the accumulation of millimolar amounts of a phosphorylated intermediate, as is the case with fructose and glycerol. It is suggested that the accumulation of fructose 1-phosphate during hepatic fructose metabolism is the result of a temporary increase in the D-glyceraldehyde concentration because of the high rate of fructose phosphorylation compared with triokinase activity. The equilibrium constant of aldolase favours the formation and thus the accumulation of fructose 1-phosphate.     

Is the glycolytic flux in Lactococcus lactis primarily controlled by the redox charge? Kinetics of NAD(+) and NADH pools determined in vivo by 13C NMR

The involvement of nicotinamide adenine nucleotides (NAD(+), NADH) in the regulation of glycolysis in Lactococcus lactis was investigated by using (13)C and (31)P NMR to monitor in vivo the kinetics of the pools of NAD(+), NADH, ATP, inorganic phosphate (P(i)), glycolytic intermediates, and end products derived from a pulse of glucose. Nicotinic acid specifically labeled on carbon 5 was synthesized and used in the growth medium as a precursor of pyridine nucleotides to allow for in vivo detection of (13)C-labeled NAD(+) and NADH. The capacity of L. lactis MG1363 to regenerate NAD(+) was manipulated either by turning on NADH oxidase activity or by knocking out the gene encoding lactate dehydrogenase (LDH). An LDH(-) deficient strain was constructed by double crossover. Upon supply of glucose, NAD(+) was constant and maximal (approximately 5 mm) in the parent strain (MG1363) but decreased abruptly in the LDH(-) strain both under aerobic and anaerobic conditions. NADH in MG1363 was always below the detection limit as long as glucose was available. The rate of glucose consumption under anaerobic conditions was 7-fold lower in the LDH(-) strain and NADH reached high levels (2.5 mm), reflecting severe limitation in regenerating NAD(+). However, under aerobic conditions the glycolytic flux was nearly as high as in MG1363 despite the accumulation of NADH up to 1.5 mm. Glyceraldehyde-3-phosphate dehydrogenase was able to support a high flux even in the presence of NADH concentrations much higher than those of the parent strain. We interpret the data as showing that the glycolytic flux in wild type L. lactis is not primarily controlled at the level of glyceraldehyde-3-phosphate dehydrogenase by NADH. The ATP/ADP/P(i) content could play an important role.     

The behaviour of NAD+ and NADH inAcinetobacter calcoaceticus duringn-alkane assimilation

The behaviour of the nicotinamide adenine dinucleotides NAD+ and NADH in Acinetobacter calcoaceticus during n-alkane assimilation was studied, acetate and succinate being used as reference carbon sources. The intracellular concentration of the two nucleotides was found to increase during the exponential growth phase, reaching its maximum in the phase of decreasing growth rates. In the exponential phase, the NAD+/NADH quotients were less than 1 and showed only unimportant variations. In the phase of decreasing growth rates, the concentration of NADH showed a distinct decrease, reaching its minimum in the stationary phase. Parallel to this, the concentration of NAD+ showed a continuous increase until the stationary phase was reached. This resulted in an increase, during the phase of decreasing growth rates, of the NAD+/NADH quotients to values greater than 1, similarly as recorded in the stationary phase. There were no fundamental differences in this behaviour between the individual carbon sources.     

The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.