Anaerobic Lactic Acid Production Research Articles

Degradation of phosphate polymer polyP enhances lactic fermentation in mice.

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Effects of l‐glutamate/d‐aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.

Modulation of the Pasteur effect in retinal cells: implications for understanding compensatory metabolic mechanisms

The purpose of the present experiments was to enhance understanding of the factors that are critical for the survival of retinal cells exposed to mitochondrial inhibition. Confluent cultures of Müller cells (rMC-1) and human retinal pigment epithelial cells (hRPE) were incubated in Dulbecco's minimal essential medium in the presence and absence of 1×10 −5 m Antimycin A, an inhibitor of mitochondrial electron transport. To modulate the rates of aerobic and anaerobic glycolysis, cells were incubated in media containing varying concentrations of glucose and 1–100 μ m of iodoacetic acid (IAA), an inhibitor of glyceraldehdye-3-phosphate dehydrogenase (G3PDH). Measurements were made of G3PDH, lactic acid production, and cellular ATP levels, along with an examination of cellular morphology, the latter providing an index of cellular viability. Control rMC-1 and hRPE produced lactate aerobically, respectively, at 0·48 and 1·50 μmol hr −1/10 6 cells. Anaerobically, lactate production increased 2-fold in rMC-1 and 3-fold in hRPE. Anaerobic ATP levels in both types of cells were maintained at control levels over 8 hr. Experimental conditions were sought that would modulate only the capacity of rMC-1 and hRPE to increase glycolysis following mitochondrial inhibition, i.e. alter their Pasteur effect. We used low concentrations of IAA to partially inhibit G3PDH. Incubation of rMC-1 with IAA for 6 hr caused a graded inhibition of G3PDH: 70% inhibition with 1 μ m, 90% with 5 μ m, 97% with 10 μ m, and 100% with 100 μ m. While the aerobic and anaerobic rates of lactic acid production were not altered by 1 μ m IAA, both were suppressed completely by 100 μ m IAA. However, incubation of rMC-1 with 5 μ m IAA caused a decrease of 30% in the rate of anaerobic lactic acid production but no change in the rate of aerobic glycolysis. Moreover, with 5 μ m IAA, rMC-1 incubated aerobically maintained ATP levels, but anaerobic ATP content decreased to a low level and cell morphology and viability were compromised. Essentially similar results were observed with hRPE. Both rMC-1 and hRPE are remarkably resistant to mitochondrial inhibition. This resistance is linked directly to the magnitude of the increase in the Pasteur effect. When the capacity of rMC-1 and hRPE to generate a Pasteur effect is selectively curtailed, these cells no longer are resistant to mitochondrial inhibition. It is suggested that in an intact tissue the ability of a cell to withstand a metabolic challenge will depend very much on the adequacy of the supply of glucose. Even a small limitation in the availability of this utilizable substrate and in the rate of the compensatory increase in the rate of anaerobic glycolysis could put the cell at greater risk during the challenge.

Aerobic and anaerobic metabolism in smooth muscle cells of taenia coli in relation to active ion transport.

1. The O2 consumption and lactic acid production of the guinea-pig's taenia coli have been studied in relation to the active Na-K transport, in order to estimate the ratio: active Na extrusion/active K uptake/ATP hydrolysis. 2. By applying different procedures of partial metabolic ingibition, it was found that a reactivation of the active Na-K transport in K-depleted tissues could occur in an anaerobic medium, provided glucose was present and in an aerobic medium free of added metabolizable substrate. The active Na-K transport was rapidly blocked in an anaerobic-substrate free medium. 3. Readmission of K to K-depleted tissues under aerobic conditions stimulates both O2 consumption and lactic acid production. While the O2 consumption creeps up slowly and requires 50 min to reach control values, the aerobic lactic acid production increases to a maximum within 10 min and decreases again during the next 50 min to its steady-state value. 4. A reactivation of the Na-pump in K-depleted cells in a N2-glucose medium causes an immediate increase of the lactic acid production, which decreases to its control value after 60 min. The maximal increase in anaerobic lactic acid production during reactivation of the Na-K pump is a function of [K]O. The system can be cescribed with first order kinetics having a Vmax = 0-72 mumole.g-1 f. wt. min-1 and a Km = 1-1 mM. 5. By varying the glucose concentration of [K]O during reactivation of the Na-K pump, different Na-K pumping rates can be obtained. The ratios net Na extrusion/ATP or net K accumulation/ATP amount to -1-32 +/- 0-19 (36) and 1-02 +/- 0-11 (36), in the experiments with different glucose concentrations. Taking into account the interference by net passive fluxes, one can estimate a ratio:active Na transport/active K transport/ATP, of 1-7/0-8/1. This ratio is not very different from the values observed in other tissues.

Seasonal and Individual Metabolic Vibrations of Hairless Mouse Skin

Oxygen consumption with and without glucose and aerobic and anaerobic lactic acid production have been measured in vitro in skin pieces from different groups of hairless mice, totaling 1051 animals of both sexes. The oxygen consumption showed significant and identical seasonal variations during two consecutive years. Highest values were found in spring and early autumn, while the lowest values were seen in winter and mid-summer. The anaerobic lactic acid production was inverse to the oxygen consumption with lowest values in spring and autumn, indicating that the metabolic variations are actively regulated in the cells. Possible causes of this biphasic pattern are discussed. No diurnal variations were found and region of the body, age or weight did not influence the metabolic parameters. Females had higher respiration and glycolysis than males but lower average weight.

The Effect of Cooling and Freezing on the Aerobic and Anaerobic Lactic Acid Production of Human Semen

The Effect of Cooling and Freezing on the Aerobic and Anaerobic Lactic Acid Production of Human Semen

Tricarboxylic acid cycle in wound healing by granulation.

SummaryThe existence of all the enzymes of Tricarboxylic Acid Cycle in granulation tissue was demonstrated by the increase in oxygen consumption in the presence of appropriate intermediates. No effect was observed with added glucose and values for aerobic and anaerobic lactic acid production by 10-day-old granulation tissue were identical. These results suggest the absence of Pasteur effect at this stage of wound healing.

ANAEROBIC GLYCOLYSIS IN AMPHIBIAN DEVELOPMENT. HOMOGENATES

1. Freshly prepared homogenates of R. pipiens embryos contain considerable amounts of lactic acid, aerobically produced from an unknown source. The amount present is an increasing function of the age of the embryos from which the homogenates are made.2. In the presence of suitable exogenous substrates, anaerobic homogenates of R. pipiens embryos, and of hybrid R. pipiens ♀ x R. sylvatica ♂ embryos, produce lactic acid at constant rates for at least two hours. Anaerobic lactic acid production from endogenous substrates occurs at very low rates.3. The relation between homogenate concentration and the rate of lactic acid production under anaerobiosis is linear, but graphs of the relation do not pass through the origin.4. Roughly speaking, the rates at which homogenized embryos glycolyze exogenous glycogen, glucose-1-phosphate, glucose-6-phosphate and fructose-1,6-diphosphate are at least as great for hybrid embryos as for normal R. pipiens controls.5. Exogenous glucose is not glycolyzed by homogenized R. pipiens embryos, even in the presence of yeast hexokinase. Provided that exogenous glucose is absent, the presence of exogenous hexokinase is a powerful stimulant to the glycolysis of some unidentified endogenous substrate.6. The energetics of hybrid development is reviewed briefly. It is concluded that the system of state-transformations is blocked in some manner independent of energy production, transfer or storage.

Metabolic properties of quinidine; effects of quinidine sulfate on anaerobic carbohydrate metabolism of rat diaphragm.

Summary1. Quinidine sulfate at a concentration of 1 × 10-3 M strongly inhibits the uptake of glucose and fructose by intact rat diaphragm under anaerobic conditions. 2. Quinidine is without effect on endogenous anaerobic lactic acid production by rat diaphragm. 3. Glucose at a concentration of 1 mg/ml or 4 mg/ml markedly stimulates anaerobic glycolysis while fructose at a concentration of 5 mg/ml produces only a slight stimulation. 4. Stimulation of glycolysis by glucose or fructose is abolished by 1 × 10-3 M quinidine and partially inhibited by 5 × 10-4 M quinidine. 5. It is suggested that quinidine inhibits glucose or fructose uptake prior to the step at which these sugars and glycogen share a common metabolic pathway.

STUDIES ON THE METABOLISM OF THE FILARIAL WORM, LITOMOSOIDES CARINII

The filarial worm, Litomosoides carinii, has a high rate of aerobic and anaerobic glucose metabolism. Aerobically 30 to 45 per cent of the glucose utilized was converted to lactic acid, 25 to 35 per cent to acetic acid, and 10 to 20 per cent to a polysaccharide. Anaerobically over 80 per cent of the total carbohydrate removed by the filariae was metabolized to lactic acid, the remainder was accounted for by the production of acetic acid. The high rates of aerobic and anaerobic lactic acid production and of aerobic polysaccharide synthesis, as well as the absence of a postanaerobic increase of the oxygen uptake, differentiate the filarial worm, L. carinii, from the known metabolic characteristics of all other helminths and of most other invertebrates. The rate of aerobic lactate and pyruvate utilization by the filariae appears to be much slower than that of glucose. Anaerobically, dismutation of two moles of pyruvate to one mole of lactate, one mole of acetate, and one mole of CO(2), occurred. Aerobically, acetate production from pyruvate exceeded that of lactate. A significant proportion of the pyruvate metabolized aerobically by the filariae was not oxidized to acetate. In the presence of fluoroacetate, aerobic incubation of the filariae in a glucose-containing medium produced a marked decrease in the respiration of the organisms, an accumulation of pyruvate, a decreased formation of acetate, and an increase in aerobic glycolysis. Low concentrations of fluoroacetate (1 x 10(-3)M) inhibited the oxidative metabolism of pyruvate which did not result in the conversion of pyruvate to acetate; higher concentrations of this inhibitor produced also a decreased oxidation of pyruvate to acetate. No evidence has been obtained that fluoroacetate inhibits the respiration of the filariae because of a competitive inhibition of acetate oxidation. Respiration and glycolysis of filariae were markedly decreased by low concentrations of p-chloromercuric benzoate. This inhibition could not be reversed by a large excess of thioglycollate, cystein, glutathione, or H(2)S. Respiration of the filariae was completely inhibited by cyanide (2 x 10(-4)M). The cyanine dyes, a group of compounds possessing high chemotherapeutic activity in filariasis of the cotton rat, inhibited in low concentrations (6.5 x 10(-8)M) the oxygen uptake of the filarial worms. This decrease in oxidative metabolism was associated with a compensatory increase in aerobic glycolysis of the worms and with decreased rates of acetate production and of polysaccharide synthesis. The same metabolic changes were observed in filariae removed from cotton rats to which subcurative doses of a cyanine dye had been administered. Concentrations of cyanine dyes which produced an almost complete inhibition of filarial respiration had no effect on the rate of anaerobic glycolysis of the worms nor on the activity of cytochrome C or of cytochrome oxidase. It is concluded that, in contrast to many other parasitic invertebrates, oxidative metabolism is essential for the survival of the filarial worm, L. carinii, and that the chemotherapeutic activity of the cyanine dyes in filariasis of the cotton rat is due to the inhibitory effect of this group of compounds on the respiratory metabolism of the parasite.

Glyceraldehyde and Embryonic Glucolysis

IT has already been reported1,2 that anaerobic lactic acid production from glucose in the chick embryo is a true case of non-phosphorylating glucolysis, strongly inhibited by dl-glyceraldehyde. As possible intermediates, glycerol, glyceric acid, dihydroxyacetone and methylglyoxal were excluded. We are now able to add to the list pyruvic acid and glyceraldehyde.* Of the two stereoisomers, only l-glyceraldehyde inhibits glucolysis ; this corresponds in its configuration to l-lactic acid (sarcolactic acid) which derives from glucose in the body. The inhibitory effect is complete at a concentration of about 2·5 × 10-3 M. The fact that dl-glyceraldehyde does not apparently inhibit glucolysis more than about 90 per cent is due to a slow enzymic lactic acid formation from glyceraldehyde itself. This process needs glutathione as co-enzyme and is not based upon a primary condensation of trioses to hexose, as it is not inhibited by amounts of fluoride or iodoacetate which would be enough to poison a secondary glucose breakdown. It is due rather to the non-enzymic formation of methylglyoxal under the experimental conditions, which is then converted to lactic acid by the glyoxalase present.