Effect of ATP on purified L(+) lactate dehydrogenase from electric organ of Electrophorus electricus (L.)

L(+) lactate dehydrogenase (LDH) activity from the electric organ of Electrophorus electricus was measured in the presence of ATP in the forward (substrate lactate) and reverse (substrate pyruvate) enzymatic reactions. The I50 for ATP was first determined and then the kinetics of the reactions were investigated with either constant coenzyme (NAD or NADH) concentration and varying substrate (lactate or pyruvate) concentration, or, constant substrate and varying coenzyme concentration. The kinetic data showed that ATP inhibits LDH uncompetitively with respect to the reduced and the oxidized coenzyme. As for the substrates, ATP gives a mixed type inhibition for lactate and a noncompetitive inhibition for pyruvate.     

Impact of adding 5.5 mM glucose to SOF medium on the development,metabolism and quality of in vitro produced bovine embryos from the morula to the blastocyst stage

Although toxic for early stages of embryo development, glucose is a physiological metabolic substrate at the morula and blastocyst stages. We evaluated the effect of adding 5.5 mM glucose from the morula stage on bovine blastocyst development and quality. In vitro matured and fertilised bovine oocytes were cultured in modified Synthetic Oviduct Fluid medium containing 5% fetal calf serum, but without added glucose, up to day 5 post-insemination (pi). Morulae were selected and further cultured in the presence or absence of 5.5 mM glucose. Blastocyst and hatched blastocyst rates were recorded. Oxygen, glucose and pyruvate uptakes as well as lactate release were evaluated. The quality of the resulting blastocysts was evaluated by the cell allocation to the inner cell mass (ICM) and trophectoderm (TE) and by the apoptotic index. Adding glucose increased the blastocyst rate at day 8 pi (80% vs 65%) but had no impact on hatching rate (25% vs 28%). A 22% decrease in oxygen uptake was observed in the presence of glucose, concomitant with an increase in lactate release, although no change was observed in pyruvate uptake. A slight decrease in blastocyst cell number was observed at day 7 in the presence of glucose while neither the ICM/TE cell ratio nor the apoptotic index were affected. In conclusion, adding 5.5 mM glucose from the morula stage has a limited impact on blastocyst rate and quality although important modifications were observed in embryo metabolism. It remains to be determined whether those modifications could influence embryo viability after transfer.     

Inhibition kinetics of lactate dehydrogenase isoenzymes by gossypol acetic acid

The effect of gossypol acetic acid, a potent male sterilent was studied on LDH from goat liver (LDH-A4), heart (LDH-B4) and testis (LDH-C4) in vitro. All the preparations of LDH were inhibited by gossypol when the reaction was carried out in pyruvate-lactate (direct) or lactate to pyruvate (reverse) directions. The IC50 of gossypol for the pyruvate oxidation by LDH isozymes varied between 16 and 42 microM in presence of 0.27 mM pyruvate and 0.15 mM NADH at 25 degrees C and pH 7.4 whereas for the lactate oxidation, IC50 was 125 microM in a system containing 3.3 mM lactic acid and 1.8 mM NAD at 25 degrees C and pH 9.0. Reciprocal plots due to Lineweaver-Burk showed that these isozymes are inhibited in a non-competitive manner with respect to pyruvate and lactate, and in a competitive fashion when NAD and NADH were varied as substrates. Ki values of LDH-A4, -B4 and -C4 isozymes in presence of gossypol were 20, 34 and 29 microM against pyruvate; 33, 43 and 45 microM against NADH; 85, 85 and 125 microM against lactate and 94, 108 and 83 microM against NAD respectively.     

Requirement for glucose during in vitro culture of sheep preimplantation embryos.

Glucose utilization by sheep embryos was examined in 8-cell (N = 36) and blastocyst (N = 36) stages, by measuring conversion of [5-3H]glucose to 3H2O. Fifty percent glucose utilization occurred at 0.79 +/- 0.69 mM for 8-cell embryos and -0.06 +/- 0.15 mM for blastocysts. Development of 1- and 2-cell sheep embryos (N = 264) was examined under different glucose concentrations (0, 1.5, 3, or 6 mM) and in the presence or absence of 0.33 mM pyruvate and 3.3 mM lactate (PL). Overall, the presence of glucose was detrimental (P less than 0.001) to embryonic development. By contrast, the presence of pyruvate and lactate was beneficial (P less than 0.001) to development. An interaction was observed between the concentration of glucose and presence or absence of PL (P less than 0.05). An optimum level of glucose occurs at 0-3 mM in the presence of PL (P less than 0.1). Development to the blastocyst stage was observed in medium when supplemented with amino acids and albumin alone. Thus, glucose metabolism is not critical for embryonic development, but beneficial at low concentrations. High concentrations can inhibit development, possibly by inhibiting the tricarboxylic acid (TCA) cycle. Sheep embryos may also be using amino acids as an energy source for development.     

Energy metabolism in preimplantation bovine embryos derived in vitro or in vivo

This study was an investigation of metabolism during bovine preimplantation development from the oocyte up to the hatched blastocyst derived in vitro or in vivo. Metabolism was determined by estimating the consumption of radiolabeled glucose, pyruvate, or lactate during a 4-h incubation period in a closed noninvasive system with NaOH as trap for the continuous collection of CO(2). The postincubation medium was analyzed for the presence of lactate. Embryonic metabolism from the matured oocyte to the 12-cell stage was more or less constant, with pyruvate being the preferred substrate. The first marked increase in oxidation of glucose occurred between the 12- and 16-cell stage. Compaction of morula and blastocyst expansion was accompanied by significant increases in oxidation of all three energy substrates. The incorporation of glucose increased steadily 15-fold from the 1-cell to the blastocyst stage. In general, the pattern of metabolism was similar between the embryos derived in vitro and in vivo but with some distinct differences. The most apparent feature of glucose metabolism by in vitro-produced embryos was a 2-fold higher rate of aerobic glycolysis as compared to that in their in vivo counterparts. In vitro-matured oocytes produced measurable amounts of lactate, whereas in vivo-matured oocytes exhibited a significantly lower metabolic activity and did not produce any lactate. When in vivo-collected embryos were preexposed to culture conditions, lactate production increased significantly and at the hatched blastocyst stage matched that of their in vitro counterparts. In vitro-produced embryos up to the 8-cell stage oxidized significantly higher amounts of lactate and had a lower ratio of pyruvate-to-lactate oxidation than the in vivo-obtained embryos. The results of this study show that under our culture conditions, important differences exist at the biochemical level between bovine embryos produced in vitro and those generated in vivo that may well affect the developmental capacity.     

Glycollate inhibition of growth of Pseudomonas aeruginosa on lactate medium

Glycollate inhibited growth of Pseudomonas aeruginosa in media containing either pyruvate or lactate as carbon sources. Glycollamide, but not glyoxylate, showed similar effects. Spontaneous mutants (L/G strains) were isolated that were able to grow on lactate medium in the presence of glycollate: their growth in pyruvate medium was still inhibited by glycollate. Synthesis of membrane-bound NAD+-independent D(-)- and L(+)-lactate dehydrogenase (iLDHs) was inducible by D- or L-lactate in the parent strain but was constitutive in the L/G strains. Glycollate inhibited induction of the synthesis of iLDHs in the parent strain growing in succinate medium but had no effect under the same conditions on strain L/G1. Glycollate was a competitive inhibitor of L(+)-iLDH (Ki = 11 mM). No differences were found in the kinetic properties of L(+)-iLDH in cell-free extracts from strain L/G1 and the parent organism. Glycollate appears to inhibit growth on lactate medium predominantly through prevention of lactate induction of iLDH synthesis.     

Responsiveness to glucagon by isolated rat hepatocytes controlled by the redox state of the cytosolic nicotinamide--adenine dinucleotide couple acting on adenosine 3'':5''-cyclic monophosphate phosphodiesterase.

1. The effects of changes in the cytoplasmic [NADH]/[NAD+] ratio on the efficacy of glucagon to alter rates of metabolism in isolated rat hepatocytes were examined. 2. Under reduced conditions (with 10mM-lactate), 10nM-glucagon stimulated both gluconeogenesis and urea synthesis in isolated hepatocytes from 48h-starved rats; under oxidized conditions (with 10mM-pyruvate), 10nM-glucagon had no effect on either of these rates. 3. The ability of glucagon to alter the concentration of 3':5'-cyclic AMP and the rates of glucose output, glycogen breakdown and glycolysis in cells from fed rats were each affected by a change in the extracellular [lactate]/[pyruvate] ratio; minimal effects of glucagon occurred at low [lactate]/[pyruvate] ratios. 4. Dose-response curves for glucagon-mediated changes in cyclic AMP concentration and glucose output indicated that under oxidized conditions the ability of glucagon to alter each parameter was decreased without affecting the concentration of hormone at which half-maximal effects occurred. 5. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.05 mM) significantly reversed the inhibitory effects of pyruvate on glucagon-stimulated glucose output. 6. For exogenously added cyclic [3H]AMP(0.1 mM), oxidized conditions decreased the stimulatory effect on glucose output as well as the intracellular concentration of cyclic AMP attained, but did not alter the amount of cyclic [3H]AMP taken up. 7. The effects of lactate, pyruvate, NAD+ and NADH on cyclic AMP phosphodiesterase activities of rat hepatocytes were examined. 8. NADH (0.01--1 MM) inhibited the low-Km enzyme, particularly that which was associated with the plasma membrane. 9. The inhibition of membrane-bound cyclic AMP phosphodiesterase by NADH was specific, reversible and resulted in a decrease in the maximal velocity of the enzyme. 10. It is proposed that regulation of the membrane-bound low-Km cyclic AMP phosphodiesterase by nicotinamide nucleotides provides the molecular basis for the effect of redox state on the hormonal control of hepatocyte metabolism by glucagon.     

Effect of exogenous carbohydrates in a serum-free culture medium on the development of in vitro matured and fertilized porcine embryos

This study was conducted to examine the effect of energy substrates in a serum-free culture medium on in vitro development of porcine embryos. Presumptive zygotes derived from in vitro fertilization were cultured in glucose-free North Carolina State University (NCSU)-23 medium with glucose, pyruvate, fructose and lactate added to the culture medium singly or in various combinations. In experiment 1, a higher percentage of embryos cleaved (53-63% vs 10-13%) and developed to the blastocyst stage (18-27% vs 0) after the single addition of glucose (5.6 mM), pyruvate (0.5 mM) or lactate (10 mM) than with no energy substrate addition or the addition only of fructose (5.6 mM). In experiment 2, the addition of pyruvate and lactate resulted in higher blastocyst formation (25%) than other combinations (6-22%), while the addition of glucose and pyruvate significantly inhibited blastocyst formation. Increasing lactate concentration, as a single energy supplement, from 5 to 20 mM significantly improved blastocyst formation (7% vs 14-18%), while no benefit was achieved from increasing pyruvate concentration up to 2 mM (experiment 3). Glucose-free NCSU-23 medium supplemented with 0.5 mM pyruvate and 5 mM lactate significantly improved blastocyst formation (28% vs 17%) compared with NCSU-23 medium supplemented with 5.6 mM glucose (experiment 4). In conclusion, pyruvate and lactate are preferable energy substrates to support in vitro development of porcine embryos cultured in a serum-free NCSU-23 medium.     

Mitochondrial malate-aspartate shuttle regulates mouse embryo nutrient consumption

Pyruvate has been considered the sole substrate that can support development of the mouse zygote to the two-cell stage, with lactate able to support development from the two-cell stage. This study has determined for the first time that mitochondrial reducing equivalent shuttles regulate metabolism in the early embryo. Activity of the malate-aspartate shuttle was found to be essential for the metabolism of lactate in the two-cell embryo. Furthermore, the inability of the mouse zygote to use lactate as an energy source was a result of a lack of malate-aspartate shuttle activity. The mRNA for the four enzymes for shuttle activity were detected at all stages of development. It was determined that aspartate was a rate-limiting factor in the activity of the malate-aspartate shuttle in mouse zygotes probably due to the high K(m) of the cytoplasmic aspartate aminotransferase. Addition of high concentrations of exogenous aspartate to the culture medium enabled mouse zygotes to utilize lactate in the absence of pyruvate and develop normally to the blastocyst stage as well as produce normal viable offspring. This study determined that the malate-aspartate shuttle is a key regulator of embryo metabolism and therefore viability and is the first report that mouse zygotes can develop normally to term in the absence of pyruvate.     

Metabolism, protein content, and in vitro embryonic development of goat cumulus-oocyte complexes matured with physiological concentrations of glucose and L-lactate

No information is available concerning how the maturation environment controls the metabolism of goat oocytes. The objectives of this experiment were to: (1) Determine the concentrations of glucose, lactate, and pyruvate in caprine follicular fluid; and (2) Investigate the effects of physiological concentrations of glucose and lactate in the in vitro maturation (IVM) medium on the metabolism (glycolysis and pyruvate oxidation), protein content, and developmental competence of caprine oocytes and cumulus-oocyte complexes (COCs). Abattoir-derived COCs were matured for 18-20 hr in a defined, SOF-based medium containing 0.75, 1.5 (follicular fluid = 1.4 mM), or 3.0 mM glucose, and 3.0, 6.0 (follicular fluid = 7.1 mM), or 12.0 mM L-lactate. The protein content of oocytes and COCs was not affected (P > 0.05) by the concentration of glucose and lactate in the maturation medium. Increasing glucose and lactate decreased (P < or = 0.05) glycolytic activity of oocytes, without affecting (P > 0.05) pyruvate oxidation. In COCs, increasing glucose concentrations tended (P = 0.07) to decrease glycolysis. When metabolic activity was corrected for protein content (pmol/microg protein/3 hr), increasing glucose or lactate concentrations in the medium decreased (P < or = 0.05) pyruvate oxidation in oocytes, but increased (P < or = 0.05) pyruvate oxidation in COCs. Embryonic development (cleavage and blastocyst development, hatching, and cell number) was not affected (P > 0.05) by the glucose and lactate concentrations tested. These results indicate that concentrations of glucose and lactate in the medium have cell type-specific effects on metabolism of oocytes and COCs, but do not affect developmental competence within the range of concentrations tested.     

Oxygen consumption and energy metabolism of the early mouse embryo

Oxygen consumption of preimplantation and early postimplantation mouse embryos has been measured using a novel noninvasive ultramicrofluorescence technique, based on an oil-soluble, nontoxic quaternary benzoid compound pyrene, whose fluorescence is quenched in the presence of oxygen. Pyruvate and glucose consumption, lactate production, and glycogen formation from glucose were also measured. Preimplantation mouse embryos of the strain CBA/Ca × C57BL/6 were cultured in groups of 10–30 in 2 μl of modified M2 medium containing 1 mmol l⁻¹ glucose, 0 mmol l⁻¹ lactate, and 0.33 mmol l⁻¹ pyruvate, for between 4–6 hr. Day 6.5 and 7.5 embryos were cultured singly in 40 μl M2 medium for between 2–3 hr. Oxygen consumption was detected at all stages of development, including, for the first time, in the early postimplantation embryo. Consumption remained relatively constant from zygote to morula stages before increasing in the blastocyst and day 6.5–7.5 stages. When expressed as QO₂ (μl/mg dry weight/hr), oxygen consumption was relatively constant from the one-cell to morula stages before increasing sharply at the blastocyst stage and declining to preblastocyst levels on days 6.5 and 7.5. Pyruvate was consumed during preimplantation stages, with glucose uptake undetectable until the blastocyst stage. Glucose was the main substrate consumed by the 6.5 and 7.5 day embryo. The proportions of glucose accounted for by lactate appearance were 81%, 86%, and 119% at blastocyst, day 6.5, and day 7.5 stages, respectively. The equivalent figures for glucose incorporated into glycogen were 10.36%, 0.21%, and 0.19%, respectively. The data are consistent with a switch from a metabolism dependent on aerobic respiration during early preimplantation stages to one dependent on both oxidative phosphorylation and aerobic glycolysis at the blastocyst stage, a pattern which is maintained on days 6.5 and 7.5. Our technique for measuring oxygen consumption may have diagnostic potential for selecting viable embryos for transfer following assisted conception techniques in man and domestic animals. © 1996 Wiley-Liss, Inc.     

Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semi-defined medium

Three experiments were conducted in which 2-cell bovine embryos were prepared from oocytes, obtained from abattoir ovaries, by in-vitro maturation for 22 to 24 hours, followed by exposure to spermatozoa for 8 hours and culture for 40 hours within the cumulus. The cumulus cells were then removed, and the cleaved embryos were cultured for a further 120 hours or longer, in the presence or absence of glucose, pyruvate and lactate. Very few embryos developed in the complete absence of energy substrates. Lactate and pyruvate, alone or combined, supported development to the 8-cell stage, but pyruvate was required to support development to the morula stage (Experiment 1). When present throughout culture or when added at 48 or 96 hours postinsemination, 5.56 mM glucose was detrimental to development (Experiments 1 and 2). However, when added at 120 hours postinsemination, 5.56 mM glucose improved development to the blastocyst and expanded blastocyst stages, compared with no glucose or 11.12 mM glucose (Experiment 3).     

Effect of osmolytes on protein dynamics in the lactate dehydrogenase-catalyzed reaction

Laser-induced temperature jump relaxation spectroscopy was used to probe the effect of osmolytes on the microscopic rate constants of the lactate dehydrogenase-catalyzed reaction. NADH fluorescence and absorption relaxation kinetics were measured for the lactate dehydrogenase (LDH) reaction system in the presence of varying amounts of trimethylamine N-oxide (TMAO), a protein-stabilizing osmolyte, or urea, a protein-destabilizing osmolyte. Trimethylamine N-oxide (TMAO) at a concentration of 1 M strongly increases the rate of hydride transfer, nearly nullifies its activation energy, and also slightly increases the enthalpy of hydride transfer. In 1 M urea, the hydride transfer enthalpy is almost nullified, but the activation energy of the step is not affected significantly. TMAO increases the preference of the closed conformation of the active site loop in the LDH·NAD(+)·lactate complex; urea decreases it. The loop opening rate in the LDH·NADH·pyruvate complex changes its temperature dependence to inverse Arrhenius with TMAO. In this complex, urea accelerates the loop motion, without changing the loop opening enthalpy. A strong, non-Arrhenius decrease in the pyruvate binding rate in the presence of TMAO offers a decrease in the fraction of the open loop, pyruvate binding competent form at higher temperatures. The pyruvate off rate is not affected by urea but decreases with TMAO. Thus, the osmolytes strongly affect the rates and thermodynamics of specific events along the LDH-catalyzed reaction: binding of substrates, loop closure, and the chemical event. Qualitatively, these results can be understood as an osmolyte-induced change in the energy landscape of the protein complexes, shifting the conformational nature of functional substates within the protein ensemble.     

Presence of bound substrate in lactate dehydrogenase from carp liver

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45 degrees C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.     

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.     

Glucose inhibits development of hamster 8-cell embryos in vitro

Relative preferences of energy substrates (glucose, pyruvate, and lactate) for in vitro development of hamster 8-cell embryos were investigated. Using protein-free modified Tyrode's medium (TLP-PVA) containing 10 mM lactate (L), 0.1 mM pyruvate (P), and amino acids (Phe, Ile, Met and Gln), we found that development of hamster 8-cell embryos to blastocysts was supported better in the absence of glucose than in medium containing (standard) 5 mM glucose (88.1% and 50%, respectively). Addition of even 0.25 mM glucose to the medium significantly inhibited blastocyst formation (54.1%). Medium T-PVA, containing 5 mM glucose as sole energy substrate (without pyruvate, lactate, and amino acids), very poorly supported embryo development (less than or equal to 7.9% blastocysts), but addition of 0.1 mM pyruvate enhanced blastocyst formation (52%). Elimination of pyruvate in TL-PVA medium containing 5 mM glucose and amino acids markedly reduced blastocyst formation by 4-fold (13.5%); the optimal pyruvate concentration was 0.2 mM. However, if the same medium was devoid of glucose, blastocyst formation was high both in the absence (71.1%) and presence (83.3%) of 0.1 mM pyruvate. Similarly, in glucose-free T-PVA medium, addition of either 10 mM lactate or amino acids supported 8-cell embryo development to blastocysts (61.7% and 60.5%, respectively) as opposed to 18.8% and 30.6%, respectively, in the presence of 5 mM glucose. This augmented development in the absence of glucose is suggested to the due to the efficient conversion of lactate to pyruvate and of amino acids to amphibolic intermediates and hence their utilization via the Krebs cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactate dehydrogenase isoenzymes in the eye, cardiac and skeletal muscles of several decapods]

Properties of lactate dehydrogenase (LDH) in the eye, heart and muscles of Hemigrapsus sanguineus, Paralithodes camtschatica, Erimacrus isenbeckii, Pandalus latirastrus, Pagurus brachiomastus have been studied with acrylamide gel electrophoresis and kinetics analysis. LDH in all the tissues of all the representatives studied was found to be specific for L-pyruvate and lactate; it migrated in electrophoresis as a single band revealing low mobility towards anode. The isoenzyme from P. camtschatica and P. latirastrus differed from the isoenzymes of other animals studied by higher mobility towards anode that reflected higher negative value of its total charge. The LDH isoenzymes in all the animals studied resembled the A4 (LDH5) of the vertebrates being unstable to the denaturing action of high temperature and being unaffected by high concentrations of pyruvate up to 1.0.10-3M. On the other hand, in conrast to the A4 of mammals, the LDH in question displayed enhancement of the reaction rate and decrease of the Km values upon increase in the NAD+ and NAD.H concentrations both in the presence of high or low lactate and pyruvate concentrations. The isoenzymes displayed catalytic activity also in the presence of NADP, the Km values for pyruvate in the presence of equimolar (2.25 mM) concentrations of NAD.H or NADP.H were practically identical and were found to be within the limits of 14-26.10-5 M. Molecular weight of the LDH studied assessed by the gel filtration method was found to be 130-140,000. It is suggested that the LDH isoenzyme from the representatives of the decapod crayfish studied is homologous in its certain properties to the homotetrameric A4 form of the vertebrates.     

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.     

Utilization of Lactate Isomers by Propionibacterium freudenreichii subsp. shermanii: Regulatory Role for Intracellular Pyruvate

Five strains of Propionibacterium freudenreichii subsp. shermanii utilized the l-(+) isomer of lactate at a faster rate than they did the d-(-) isomer when grown with a mixture of lactate isomers under a variety of conditions. ATCC 9614, grown anaerobically in defined medium containing 160 mM dl-lactate, utilized only 4 and 15% of the d-(-)-lactate by the time 50 and 90%, respectively, of the l-(+)-lactate was used. The intracellular pyruvate concentration was high (>100 mM) in the initial stages of lactate utilization, when either dl-lactate or the l-(+) isomer was the starting substrate. The concentration of this intermediate dropped during dl-lactate fermentation such that when only d-(-)-lactate remained, the concentration was <20 mM. When only the d-(-) isomer was initially present, a similar relatively low concentration of intracellular pyruvate was present, even at the start of lactate utilization. The NAD-independent lactate dehydrogenase activities in extracts showed different kinetic properties with regard to pyruvate inhibition, depending upon the lactate isomer present. Pyruvate gave a competitive inhibitor pattern with l-(+)-lactate and a mixed-type inhibitor pattern with d-(-)-lactate. It is suggested that these properties of the lactate dehydrogenases and the intracellular pyruvate concentrations explain the preferential use of the l-(+) isomer.     

An increase in the redox state during reperfusion contributes to the cardioprotective effect of GIK solution

This study aimed at determining whether glucose-insulin-potassium (GIK) solutions modify the NADH/NAD(+) ratio during postischemic reperfusion and whether their cardioprotective effect can be attributed to this change in part through reduction of the mitochondrial reactive oxygen species (ROS) production. The hearts of 72 rats were perfused with a buffer containing glucose (5.5 mM) and hexanoate (0.5 mM). They were maintained in normoxia for 30 min and then subjected to low-flow ischemia (0.5% of the preischemic coronary flow for 20 min) followed by reperfusion (45 min). From the beginning of ischemia, the perfusate was subjected to various changes: enrichment with GIK solution, enrichment with lactate (2 mM), enrichment with pyruvate (2 mM), enrichment with pyruvate (2 mM) plus ethanol (2 mM), or no change for the control group. Left ventricular developed pressure, heart rate, coronary flow, and oxygen consumption were monitored throughout. The lactate/pyruvate ratio of the coronary effluent, known to reflect the cytosolic NADH/NAD(+) ratio and the fructose-6-phosphate/dihydroxyacetone-phosphate (F6P/DHAP) ratio of the reperfused myocardium, were evaluated. Mitochondrial ROS production was also estimated. The GIK solution improved the recovery of mechanical function during reperfusion. This was associated with an enhanced cytosolic NADH/NAD(+) ratio and reduced mitochondrial ROS production. The cardioprotection was also observed when the hearts were perfused with fluids known to increase the cytosolic NADH/NAD(+) ratio (lactate, pyruvate plus ethanol) compared with the other fluids (control and pyruvate groups). The hearts with a high mechanical recovery also displayed a low F6P/DHAP ratio, suggesting that an accelerated glycolysis rate may be responsible for increased cytosolic NADH production. In conclusion, the cardioprotection induced by GIK solutions could occur through an increase in the cytosolic NADH/NAD(+) ratio, leading to a decrease in mitochondrial ROS production.