Glycolytic and oxidative metabolism in relation to retinal function

Measurements of lactate production and ATP concentration in superfused rat retinas were compared with extracellular photoreceptor potentials (Fast PIII). The effect of glucose concentration, oxygen tension, metabolic inhibition, and light were studied. Optimal conditions were achieved with 5-20 mM glucose and oxygen. The isolated retina had a high rate of lactate production and maintained the ATP content of a freshly excised retina, and Fast PIII potentials were similar to in vivo recordings. Small (less than 10%) decreases in aerobic and anaerobic lactate production were observed after illumination of dark-adapted retinas. There were no significant differences in ATP content in dark- and light-adapted retinas. In glucose-free medium, lactate production ceased, and the amplitude of Fast PIII and the level of ATP declined, but the rates of decline were slower in oxygen than in nitrogen. ATP levels were reduced and the amplitude of Fast PIII decreased when respiration was inhibited, and these changes were dependent on glucose concentration. Neither glycolysis alone nor Krebs cycle activity alone maintained the superfused rat retina at an optimal level. Retinal lactate production and utilization of ATP were inhibited by ouabain. Mannose but not galactose or fructose produced lactate and maintained ATP content and Fast PIII. Iodoacetate blocked lactate production and Fast PIII and depleted the retina of ATP. Pyruvate, lactate, and glutamine maintained ATP content and Fast PIII reasonably well (greater than 50%) in the absence of glucose, even in the presence of iodoacetate. addition of glucose, mannose, or 2-deoxyglucose to medium containing pyruvate and iodoacetate abolished Fast PIII and depleted the retina of its ATP. It is suggested that the deleterious effects of these three sugars depend upon their cellular uptake and phosphorylation during the blockade of glycolysis by iodoacetate.     

Isoproterenol fails to produce myocardial necrosis in streptozotocin-induced diabetic rats.

Biochemical alterations in the hearts of non-diabetic and 5 weeks of streptozotocin-induced diabetic rats following isoproterenol (ISO) administration were compared. Serum lactate dehydrogenase (LDH) and myocardial adenosine triphosphate (ATP), creatine phosphate (CP), lactate and glycogen were used as indices of myocardial injury. Hearts from diabetic rats (blood glucose greater than 350 mg/dl), before ISO administration, had normal lactate levels but significantly low high-energy phosphate (HEP) levels and high glycogen levels in comparison to non-diabetic rats. No difference was observed in serum LDH levels between these two groups. ISO administration to non-diabetic rats caused myocardial necrosis as evidenced by a significant depletion of myocardial glycogen and HEPs along with significant myocardial lactate accumulation and an increase in serum LDH. However, the hearts from diabetic rats failed to show any significant HEP depletion, accumulation of lactate and leakage of LDH into serum following ISO-administration, though myocardial glycogen level was significantly lowered. These observations, in conjunction with earlier reports, point to the hypothesis that, in diabetes, there are certain alterations in the sarcolemma which hamper the process by which ISO causes myocardial necrosis.     

Lactate prevents the alterations in tissue amino acids, decline in ATP, and cell damage due to aglycemia in retina

Under conditions of energy impairment, CNS tissue can utilize substrates other than glucose to maintain energy metabolism. Retinas produce large amounts of lactate, although it has not been shown that lactate can be utilized by retina to prevent the cell damage associated with hypoglycemia. To investigate this, intact, isolated retinas were subjected to aglycemic conditions in the presence or absence of 20 mM lactate. Retinas incubated in the absence of glucose for 60 min showed a threefold elevation in tissue aspartate and 60% decreases in tissue glutamate and glutamine, demonstrating a mobilization of carbon from glutamine and glutamate to the tricarboxylic acid cycle. Lactate prevented these changes in tissue amino acids, indicating metabolism of lactate with sparing of tissue glutamate and glutamine. Tissue ATP was 20 and 66% of control values with zero glucose or zero glucose plus lactate, respectively. Consistent with previous findings, incubation of retinas in the absence of glucose caused acute swelling of retinal neurons and release of GABA into the medium at 60 min. These acute toxic affects caused by the absence of glucose were completely prevented by the presence of lactate. At 24 h of recovery following 60 min of zero glucose, many pyknotic profiles were observed and lactate dehydrogenase (LDH) release into the medium was elevated sevenfold, indicating the extent of cell death. In contrast, no elevation in LDH was found and histology appeared normal in retinas exposed to zero glucose in the presence of lactate. alpha-Cyano-4-hydroxy cinnamate (4-CIN; 0.5 mM), an inhibitor of the monocarboxylic acid transporter and mitochondrial pyruvate carrier, blocked the ability of lactate to maintain ATP and protect retinas from aglycemia but had no effect on ATP or toxicity per se. Derangements in tissue aspartate, glutamate, and glutamine, which were prevented by lactate during zero glucose incubation, were again observed with lactate plus zero glucose in the presence of 4-CIN. However, 0.5 mM 4-CIN alone in the presence of glucose produced similar increases in aspartate and decreases in glutamate and glutamine as observed with zero glucose while having only modest inhibitory effects on [U-(14)C]lactate uptake, suggesting the mitochondrial pyruvate carrier as the main site of action. The above findings show that lactate is readily utilized by the chick retina during glucose deprivation to prevent derangements in tissue amino acids and ATP and retinal neuronal cell death.     

Energy dependence of enzyme release from hypoxic isolated perfused rat heart tissue

There is a sudden release of intracellular constituents upon reoxygenation of isolated perfused hypoxic heart tissue (O2 paradox) or on perfusion with calcium-free medium after a period of hypoxia. Rat hearts were perfused by the method of Langendorff (Pfluegers Arch. 61: 291-332, 1895) with Krebs-Henseleit medium containing 10 mM glucose. Hearts were equilibrated for 30 min, followed by 90 min of hypoxia or 60 min of hypoxia and 30 min of reoxygenation. The massive enzyme release observed upon reoxygenation after 60 min of hypoxia was prevented by infusing 0.5 or 5 mM cyanide 5 min before reoxygenation. Lactate dehydrogenase (LDH) release commenced immediately upon withdrawal of cyanide. Hearts perfused with calcium-free medium throughout hypoxia did not release increased amounts of LDH at reoxygenation. Perfusing heart tissue with medium containing 0 or 25 microM calcium, but not 0.25 or 2.5 mM, after 50 min of hypoxia initiated a release of cardiac LDH, which was not further enhanced by reoxygenation. Enzyme release was significantly inhibited when the calcium-free perfusion medium included 10 mM 2-deoxyglucose (replacing glucose), 0.5 mM dinitrophenol, or 2.5 mM cyanide. Histologically, hearts perfused with calcium-free medium after 50 min of hypoxia showed areas of severe necrosis and contracture without any evidence of the contraction bands that were seen in hearts reoxygenated in the presence of calcium. Cardiac ATP and creatine phosphate (PCr) levels were significantly decreased after 50-60 min of hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between energy reserves and function in rat superior cervical ganglion

—Major components of the energy reserves of the isolated superior cervical ganglion (ATP, phosphocreatine, glucose, glycogen and lactate) were measured under aerobic and anaerobic conditions. Complete anaerobiosis was maintained by incubation in mineral oil through which N₂ had been bubbled. From the initial rate of change in the energy reserves, a metabolic rate was calculated which would be equivalent to the consumption of 93 m-moles of O₂ per kg per hour. Under aerobic conditions (oxygenated moist chamber) a similar metabolic rate was calculated. In contrast to the anaerobic state, initial energy expenditure was almost exclusively at the expense of glucose. Continuous supramaximal stimulation in O₂ increased energy expenditure by a factor of three; both glucose and glycogen were utilized from the outset, and lactate accumulated in the initial periods. Ganglionic transmission failed in both resting and stimulated states in spite of the continued presence of very substantial levels of ATP and phosphocreatine. Failure seemed to be associated not with ATP depletion but rather with the complete disappearance of glucose and glycogen.     

Thyroid hormones and muscle metabolism in dogs

Muscle contents of ATP, ADP, AMP, creatine phosphate and creatine as well as glycogen, some glycolytic intermediates, pyruvate and lactate were compared in the intact, thyroidectomized and triiodothyronine (T3) treated dogs under resting conditions. After thyroidectomy muscle glycogen, glucose 1-phosphate and glucose 6-phosphate contents were significantly elevated while in T3-treated animals these variables were decreased in comparison with control dogs. Muscle free glucose was not altered by thyroidectomy but T3 treatment significantly increased its content. Muscle lactate content was elevated both in hypo- and hyperthyroid animals. Muscle ATP and total adenine nucleotide contents were significantly increased in hyperthyroid dogs while no differences were found between the three groups in the muscle creatine phosphate content. It is assumed that in T3-treated animals carbohydrate catabolism is enhanced in the resting skeletal muscle in spite of high tissue ATP content. Muscle metabolite alterations in hypothyroid dogs seem to reflect the hypometabolism accompanied by a diminished rate of glycogenolysis with inhibited rate of pyruvate oxidation or decreased rate of lactate removal from the cells.     

Comparison of in vivo energy metabolism in the brain of rainbow trout, Salmo gairdneri and bullhead catfish, Ictalurus nebulosus during anoxia

1. Rainbow trout and bullhead catfish (Ictalurus nebulosus) were exposed to anoxic water inside a plastic tube until death (12 min for trout; 62 min for catfish). Immediately upon death, the brain was removed and analyzed for metabolites, high energy phosphate compounds, and metabolic fuel while the blood was analyzed for metabolites. 2. Control bullhead brains had higher concentrations of glycogen, ATP, creatine phosphate (CrP), and glucose than control trout. 3. After anoxia bullheads showed a significant decrease in ATP, CrP, and glycogen while lactate more than doubled in concentration. 4. After anoxia trout showed a doubling in brain lactate and a decline in glycogen, but no decline in ATP or CrP. There were no changes in brain glucose, ketone bodies, or alternative anaerobic end-products in either species although an elevation in blood isobutyrate was noted. 5. Brain death in the catfish may be due to depletion of fuel for anaerobiosis and a subsequent loss of ATP. In the trout there may be other causes such as a greater permeability of its neuronal membranes and alterations in intracellular free calcium stores.     

Relating Cerebral Ischemia and Hypoxia to Insult Intensity

The contributions of five variables believed to influence the brain's metabolism of O2 during hypoxia [duration, PaO2, delta CMRO2 (the difference between normal and experimental oxygen uptake), O2 availability (blood O2 content.CBF), and O2 deficit (delta CMRO2.duration)] were assessed by stepwise and multiple linear regression. Levels of brain tissue carbohydrates (lactate, glucose, and glycogen) and energy metabolites [ATP, AMP, and creatine phosphate (CrP)] were significantly influenced by O2 deficit during hypoxia, as was final CMRO2. After 60 min of reoxygenation, levels of tissue lactate, glucose, ATP, and AMP were related statistically to the O2 deficit during hypoxia; however, CMRO2 changes were always associated more significantly with O2 availability during hypoxia. Creatine (Cr) and CrP levels in the brain following reoxygenation were correlated more to delta CMRO2 during hypoxia. Changes in some brain carbohydrate (lactate and glucose), energy metabolite (ATP and AMP) levels, and [H+]i induced by complete ischemia were also influenced by O2 deficit. After 60 min of postischemic reoxygenation, brain carbohydrate (lactate, glucose, and glycogen) and energy metabolite (ATP, AMP, CrP, and Cr) correlated with O2 deficit during ischemia. We conclude that "O2 deficit" is an excellent gauge of insult intensity which is related to observed changes in nearly two-thirds of the brain metabolites we studied during and following hypoxia and ischemia.     

Effect of glucose on ATP dephosphorylation in rat spermatids

Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.     

The relation between the action potential duration,the increase in resting tension,and ATP content during metabolic inhibition in guinea pig ventricular muscles

To investigate whether the action potential duration (APD) or resting tension was dependent on global ATP content, and whether they were preferentially dependent on glycolytic ATP, APD and resting tension were measured under various metabolic inhibition with corresponding measurement of ATP content in guinea pig ventricular muscles. Oxidative phosphorylation was inhibited by either hypoxic perfusion, the perfusion of sodium cyanide, or 2,4-dinitrophenol. Glycolysis was blocked by the perfusion of iodoacetic acid, and hypoxia with variable glycolytic activities was achieved by hypoxic perfusion in the presence of glucose (5, 10, and 50 mM). APD began to decrease when ATP content decreased to less than 3 mM/kg w.w. from the control level of 4.35 mM/kg w.w. APD shortened significantly and resting tension increased steeply, when ATP content decreased below 1 mM/kg w.w. The dependence of APD and the increase in resting tension on ATP content was not affected by the mode of metabolic block, that is, the inhibition of glycolysis and/or oxidative phosphorylation. Though other factors can affect APD and resting tension, we found no evidence of functional ATP compartmentation, with respect to APD and the increase in resting tension during metabolic inhibition.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

Intracellular ATP in a glucosephosphate isomerase mutant ofSaccharomyces cerevisiae

The degree of ATP depletion caused by glucose in a glucosephosphate isomerase-deficient strain of Saccharomyces cerevisiae was determined. Even in the presence of a sugar normally fermentable by the mutant, the addition of glucose can decrease the intracellular ATP, depending on the competition of the sugars for transport and subsequent phosphorylation. For both parent and mutant cells, a correlation exists between the calculated velocity of ATP formation or ATP consumption during the utilization of different concentrations of sugars and the experimental intracellular ATP level. For initially resting yeast cells, a rate increase of 35 mumol per min per g ATP was calculated to increase the intracellular level of this nucleotide by 1 mumol per g cell mass.     

Lactate formation in Caldicellulosiruptor saccharolyticus is regulated by the energy carriers pyrophosphate and ATP

Caldicellulosiruptor saccharolyticus displays superior H₂ yields on a wide range of carbon sources provided that lactate formation is avoided. Nevertheless, a low lactate flux is initiated as the growth rate declined in the transition to the stationary phase, which coincides with a drastic decrease in the glucose consumption and acetate production fluxes. In addition, the decrease in growth rate was accompanied by a sudden increase and then decrease in NADH levels. The V′_MAX of the lactate dehydrogenase (LDH) doubled when the cells entered the stationary phase. Kinetic analysis revealed that at the metabolic level LDH activity is regulated through (i) competitive inhibition by pyrophosphate (PPi, k_i=1.7 mM) and NAD (k_i=0.43 mM) and (ii) allosteric activation by FBP (300%), ATP (160%) and ADP (140%). From these data a MWC-based model was derived. Simulations with this model could explain the observed lactate shift by displaying how the sensitivity of LDH activity to NADH/NAD ratio varied with different PP_i concentrations. Moreover, the activation of LDH by ATP indicates that C. saccharolyticus uses LDH as a means to adjusts its flux of ATP and NADH production. To our knowledge, this is the first time PPi is observed as an effector of LDH.     

Futile substrate cycles in the glycolytic pathway of boar and rat spermatozoa and the effect of alpha-chlorohydrin

In boar spermatozoa incubated with 0.1 mM-glucose about 20 nmol glucose were converted to lactate and CO2 and the rate of futile substrate cycling between glucose and glucose 6-phosphate was about 6 nmol/10(8) spermatozoa/30 min. Futile cycling was increased in the presence of 0.05 or 1 mM-alpha-chlorohydrin but not to an extent sufficient to account for the rapid decline in ATP concentration observed under these conditions. These estimates include a substantial rate of fructose formation from fructose phosphates. The addition of 10 mM-L-lactate plus 1 mM-pyruvate protected the spermatozoa against the effect of alpha-chlorohydrin and glucose on the ATP concentration but increased futile substrate cycling. Substrate cycling between fructose 6-phosphate and fructose 1,6-bisphosphate could not be measured in boar spermatozoa but in rat spermatozoa its rate (nmol/10(8) spermatozoa/30 min) was about 10 under control condition and about 25 in the presence of 1 mM-alpha-chlorohydrin. This increase was insufficient to account for the decline in ATP concentration. In both species futile substrate cycling consumed a significant proportion of the ATP synthesis during lactate production but only about 5% of that produced in the oxidation of glucose to acetyl carnitine and CO2.     

Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro

Sertoli cells actively metabolize glucose that is converted into lactate, which is used by developing germ cells for their energy metabolism. Androgens and oestrogens have general metabolic roles that reach far beyond reproductive processes. Hence, the main purpose of this study was to examine the effect of sex hormones on metabolite secretion/consumption in primary cultures of rat Sertoli cells. Sertoli cell-enriched cultures were maintained in a defined medium for 50?h. Glucose and pyruvate consumption, and lactate and alanine secretion were determined, by 1H-NMR (proton NMR) spectra analysis, in the presence or absence of 100?nM E2 (17β-oestradiol) or 100?nM 5α-DHT (dihydrotestosterone). Cells cultured in the absence (control) or presence of E2 consumed the same amount of glucose (29±2?pmol/cell) at similar rates during the 50?h. After 25?h of treatment with DHT, glucose consumption and glucose consumption rate significantly increased. Control and E2-treated cells secreted similar amounts of lactate during the 50?h, while the amount of lactate secreted by DHT-treated cells was significantly lower. Such a decrease was concomitant with a significant decrease in LDH A [LDH (lactate dehydrogenase) chain A] and MCT4 [MCT (monocarboxylate transporter) isoform 4] mRNA levels after 50?h treatment in hormonally treated groups, being more pronounced in DHT-treated groups. Finally, alanine production was significantly increased in E2-treated cells after 25?h treatment, which indicated a lower redox/higher oxidative state for the cells in those conditions. Together, these results support the existence of a relation between sex hormones action and energy metabolism, providing an important assessment of androgens and oestrogens as metabolic modulators in rat Sertoli cells.     

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

A peculiar phenomenon, differing from the response of mammalian cells, occurred when Chinook salmon embryo (CHSE) cells were passaged in the medium lacking of both glucose and glutamine. To elucidate metabolic mechanism of CHSE cells, the metabolism parameters, key metabolic enzymes, and ATP levels were measured at different glucose and glutamine concentrations. In the glutamine-free culture, hexokinase activity kept constant, and lactate dehydrogenase (LDH) activity decreased. This indicated that lack of glutamine did not expedite glucose consumption but made it shift to lower lactate production and more efficient energy metabolism. The results coincided with the experimental results of unaltered specific glucose consumption rate and decreased yield coefficients of lactate to glucose. In the glucose-free culture, simultaneous increase of glutaminase activity and of specific ammonia production rate suggested an increased flux into the glutaminolysis pathway, and increases of both glutamate dehydrogenase activity and yield coefficient of ammonia to glutamine showed an increased flux into deamination pathway. However, when glucose and glutamine were both lacking, the specific consumption rates of most of amino acids increased markedly, together with decrease of LDH activity, indicating that pyruvate derived from amino acids, away from lactate production, remedied energy deficiency. When both glucose and glutamine were absent, intracellular ATP contents and the energy charge remained virtually unaltered.Revisions requested 16 December 2004; Revisions received 24 January 2005     

Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Cultured cerebellar granule cells were co-loaded with Ca²⁺-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (????_m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca²⁺]ⁱ. This sMD was insensitive to a removal of external Ca²⁺ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca²⁺ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6?C8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]_c) and ????_m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]_c and ????_m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ????_m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca²⁺ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.     

Lactate regulates pyruvate uptake and metabolism in the preimplantation mouse embryo

This study was an investigation of the interaction of lactate on pyruvate and glucose metabolism in the early mouse embryo. Pyruvate uptake and metabolism by mouse embryos were significantly affected by increasing the lactate concentration in the culture medium. In contrast, glucose uptake was not affected by lactate in the culture medium. At the zygote stage, the percentage of pyruvate taken up and oxidized was significantly reduced in the presence of increasing lactate, while at the blastocyst stage, increasing the lactate concentration increased the percentage of pyruvate oxidized. Lactate oxidation was determined to be 3-fold higher (when lactate was present at 20 mM) at the blastocyst stage compared to the zygote. Analysis of the kinetics of lactate dehydrogenase (LDH) determined that while the V(max) of LDH was higher at the zygote stage, the K(m) of LDH was identical for both stages of development, confirming that the LDH isozyme was the same. Furthermore, the activity of LDH isolated from both stages was reduced by 40% in the presence of 20 mM lactate. The observed differences in lactate metabolism between the zygote and blastocyst must therefore be attributed to in situ regulation of LDH. Activity of isolated LDH was found to be affected by nicotinamide adenine dinucleotide(+) (NAD(+)) concentration. In the presence of increasing concentrations of lactate, zygotes exhibited an increase in autofluorescence consistent with a depletion of NAD(+) in the cytosol. No increase was observed for later-stage embryos. Therefore it is proposed that the differences in pyruvate and lactate metabolism at the different stages of development are due to differences in the in situ regulation of LDH by cytosolic redox potential.     

Metabolic imaging in multicellular spheroids of oncogene-transfected fibroblasts.

Four rat embryo fibroblast (REF) cell lines with defined oncogenic transformation were used to study the relationship between tumorigenic conversion, metabolism, and development of cell death in a 3D spheroid system. Rat1 (spontaneously immortalized) and M1 (myc-transfected) fibroblasts represent early nontumorigenic transformation stages, whereas Rat1-T1 (T24Ha-ras-transfected Rat1) and MR1 (myc/T24Ha-ras-co-transfected REF) cells express a highly tumorigenic phenotype. Localized ATP, glucose, and lactate concentrations in spheroid median sections were determined by imaging bioluminescence. ATP concentrations were low in the nonproliferating Rat1 aggregates despite sufficient oxygen and glucose availability and lack of lactate accumulation. In MR1 spheroids, a 50% decrease in central ATP preceded the development of central necrosis at a spheroid diameter of around 800 micrometer. In contrast, the histomorphological emergence of cell death at a diameter of around 500 micrometer in Rat1-T1 spheroids coincided with an initial steep drop in ATP. Concomitantly, reduction in central glucose and increase in lactate before cell death were recorded in MR1 but not in Rat1-T1 spheroids. As shown earlier, myc transfection confers a considerable resistance to hypoxia of MR1 cells in the center of spheroids, which is reflected by their capability to maintain cell integrity and ATP content in a hypoxic environment. The data obtained suggest that small alterations in the genotype of tumor cell lines, such as differences in the immortalization process, lead to substantial differences in morphological structure, metabolism, occurrence of cell death, and tolerance to hypoxia in spheroid culture.