CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.     

Renal tissue metabolism in the rat during chronic metabolic alkalosis: importance of glycolysis

Chronic metabolic alkalosis was induced in rats drinking 0.3 M NaHCO3 and receiving 1 mg furosemide/100 g body weight per day intraperitoneally. Another group of animals received a potassium supplement in the form of 0.3 M KHCO3. In this group, hypokalemia did not develop and muscle potassium fell by only 18% versus 50% in those not receiving potassium. In vitro renal production of ammonia and uptake of glutamine fell by 40% with a decrease in the activity of glutaminase I and glutamate dehydrogenase. Activity of phosphofructokinase, a major enzyme of glycolysis, rose only in the kidney of animals receiving a potassium supplement. Fructose-1,6-diphosphatase fell as well as phosphoenolpyruvate carboxykinase. Malate dehydrogenase also fell. The activity of phosphofructokinase also rose in the liver, heart, and leg muscle. The major biochemical changes in the renal cortex were the following: glutamate, alpha-ketoglutarate, malate, lactate, pyruvate, alanine, aspartate, and citrate rose as well as calculated oxaloacetate. The concentration of intermediates like 2-phosphoglycerate, 3-phosphoglycerate, and glucose-6-phosphate fell. The cytosolic redox potential (NAD+/NADH) decreased. In addition to the fall in ammoniagenesis, it could be demonstrated in vitro that the renal tubules incubated with glutamine showed decreased glucose production and increased production of lactate and pyruvate. The concentration of lactate was elevated in all tissues examined including liver, heart, and leg muscle. This study confirms in the rat that decreased renal ammoniagenesis takes place following decreased uptake of glutamine in metabolic alkalosis. All other changes are accounted for by the process of increased glycolysis, which appears to take place in all tissues in metabolic alkalosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diversity of Metabolic Patterns in Human Brain Tumors: Enzymes of Energy Metabolism and Related Metabolites and Cofactors

Biopsies from 15 human gliomas, five meningiomas, four Schwannomas, one medulloblastoma, and four normal brain areas were analyzed for 12 enzymes of energy metabolism and 12 related metabolites and cofactors. Samples, 0.01-0.25 microgram dry weight, were dissected from freeze-dried microtome sections to permit all the assays on a given specimen to be made, as far as possible, on nonnecrotic pure tumor tissue from the same region. Great diversity was found with regard to both enzyme activities and metabolite levels among individual tumors, but the following generalities can be made. Activities of hexokinase, phosphorylase, phosphofructokinase, glycerophosphate dehydrogenase, citrate synthase, and malate dehydrogenase levels were usually lower than in brain; glycogen synthase and glucose-6-phosphate dehydrogenase were usually higher; and the averages for pyruvate kinase, lactate dehydrogenase, 6-phosphogluconate dehydrogenase, and beta-hydroxyacyl coenzyme A dehydrogenase were not greatly different from brain. Levels of eight of the 12 enzymes were distinctly lower among the Schwannomas than in the other two groups. Average levels of glucose-6-phosphate, lactate, pyruvate, and uridine diphosphoglucose were more than twice those of brain; 6-phosphogluconate and citrate were about 70% higher than in brain; glucose, glycogen, glycerol-1-phosphate, and malate averages ranged from 104% to 127% of brain; and fructose-1,6-bisphosphate and glucose-1,6-bisphosphate levels were on the average 50% and 70% those of brain, respectively.     

EFFECT OF HYPOTHERMIA UPON ORGANIC PHOSPHATES, GLYCOLYTIC METABOLITES, CITRIC ACID CYCLE INTERMEDIATES AND ASSOCIATED AMINO ACIDS IN RAT CEREBRAL CORTEX

—The influence of hypothermia upon the metabolism of the brain was studied by reducing body temperature in N₂O-anaesthetized rats to 32, 27 or 22°C, with subsequent measurements of organic phosphates, glycolytic metabolites, citric acid cycle intermediates and associated amino acids. Hypothermia was maintained for either 1 or 2 h and the effect of anaesthesia was evaluated by maintaining unanaesthetized animals at 22°C. Hypothermia had no influence on the cerebral cortical concentrations of ATP, ADP or AMP and there was only a small increase in phosphocreatine. Since the tissue concentrations of glucose and glycogen were reduced, it is concluded that the well known resistance of the hypothermie brain to ischaemia is unrelated to increased energy stores. Hypothermia was accompanied by decreases in the tissue concentrations of fructose-1,6-diphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, pyruvate, lactate, α-ketoglutarate, succinate and malate, but not of glucose-6-phosphate or citrate. These results indicate that metabolic flux is retarded mainly at the phosphofructokinase and isocitrate dehydrogenase steps. The largest relative reduction was seen in α-ketoglutarate, which was possibly secondary to accumulation of ammonia. There was no change in GABA, but a decrease in glutamate and increases in aspartate and alanine. These, changes are compatible with shifts in the aspartate and alanine aminotransferase reactions, possibly induced by the fall in α-ketoglutarate.     

THE EFFECT OF HYPERCAPNIC ACIDOSIS UPON SOME GLYCOLYTIC AND KREBS CYCLE-ASSOCIATED INTERMEDIATES IN THE RAT BRAIN

Abstract— In order to study the influence of intracellular pH on the carbohydrate metabolism of brain tissue, the concentrations of glucose, glucose-6-phosphate, pyruvate, lactate, citrate, α-oxoglutarate, malate, glutamate, aspartate and ammonia were measured in rats exposed to 6–40% CO₂, for 45 min. Hypercapnia of increasing severity gave rise to progressive increases in the concentrations of glucose, glucose-6-phosphate and ammonium ion and to progressive decreases in the concentrations of all metabolic acids measured. The results fit with aH⁺ inhibition of a rate-limiting step between glucose-6-phosphate and pyruvate, and by inference from the results published by others it may be assumed that this step is the phosphofructokinase reaction. Since the proportionally largest decrease occurred in a α-oxoglutarate, the results might be compatible either with an inhibition of a second rate-limiting step such as isocitrate dehydrogenase, or with a loss of α-oxoglutarate through carboxylation to citrate.     

PATTERNS OF CHANGES IN BRAIN CARBOHYDRATE METABOLITES, AMINO ACIDS AND ORGANIC PHOSPHATES AT INCREASED CARBON DIOXIDE TENSIONS

—In order to study the time course of changes in cerebral metabolites in hypercapnia, anaesthetized and artificially ventilated rats were exposed to 11% CO₂ for 5, 15, 45, 90 and 180 min. In addition, the effect of anaesthetic levels of carbon dioxide was studied by exposing animals to 30 and 50% CO₂ for 45 min. In none of the groups were there significant changes in ATP, ADP or AMP, and a normal energy state was therefore obtained even in short-lasting hypercapnia, and at anaesthetic CO₂ concentrations (50% CO₂). In the group exposed to 11% CO₂ for 5 min there was a fall in glycogen but normalization occurred when the hypercapnia was prolonged. There were no changes in fructose 1,6-diphosphate, dihydroxyacetone phosphate or 3-phosphoglycerate but decreases in pyruvate, lactate, citrate, α-oxoglutarate, malate and glutamate at all exposure times. With 30 and 50% CO₂ glucose 6-phosphate accumulated. The results do not support the view that the depletion of pyruvate and of citric acid cycle intermediates is caused by H⁺-inhibition of rate-limiting enzymatic steps like the phosphofructokinase reaction. The glutamate concentration fell progressively during exposure to 11% CO₂. In the 5 and 15 min groups aspartate increased significantly indicating that the initial loss of glutamate was partly due to transamination to aspartate. With prolonged hypercapnia there was a secondary fall in aspartate to subnormal values. At 45 min and thereafter the glutamine concentration increased significantly. However, the sum of glutamate, aspartate and glutamine fell progressively after the initial 5 min period. Hypercapnia gave rise to similar increases in the lactate/pyruvate and malate/oxaloacetate ratios, and since the calculated NADH/NAD⁺ ratios remained close to normal in all groups, the results indicate that pH-dependent shifts occurred in the lactate and malate dehydrogenase equilibria.     

TCA循环中间产物对酿酒酵母胞内代谢关键酶活性的影响

对酿酒酵母在添加苹果酸、柠檬酸和琥珀酸的混合培养基与其在YEPD培养基中胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活力差异进行了对比分析。结果表明:添加苹果酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.82%、57.23%、39.15%、12.10%;添加柠檬酸使胞内丙酮酸激酶、异柠檬酸脱氢酶、苹果酸脱氢酶的酶活分别下降50.17%、42.20%、48.40%;添加琥珀酸使胞内丙酮酸激酶、葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、苹果酸脱氢酶、乙醇脱氢酶的酶活分别下降34.16%、34.16%、50.87%、50.87%、12.37%。丙酮酸激酶、异柠檬酸脱氢酶和苹果酸脱氢酶对3种有机酸的耐受性较差,葡萄糖-6-磷酸脱氢酶、乙醇脱氢酶对3种有机酸的耐受具有选择性。     

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate

1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40-50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD(+)]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP(+)]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD(+)]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.     

The role of calcium in the stimulation of gluconeogenesis by catecholamines

In hepatocytes isolated from fasted normal rats and incubated without albumin or gelatin, norepinephrine stimulated gluconeogenesis from fructose or dihydroxyacetone only in the absence of added calcium and from sorbitol or glycerol only in the presence of added calcium. The effects of calcium, norepinephrine, or calcium in combination with norepinephrine on the concentration of intermediary metabolites were therefore studied in hepatocytes metabolizing fructose or sorbitol as the representative oxidized or reduced substrate, respectively. With fructose as the substrate, addition of calcium increased the concentrations of lactate, pyruvate, glyceraldehyde 3-phosphate, and β-hydroxybutyrate, but decreased the concentrations of phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate, glucose 6-phosphate, malate, citrate, and α-oxoglutarate. With sorbitol as the substrate, calcium increased the concentrations of pyruvate, malate, β-hydroxybutyrate, and glucose. With either substrate, calcium caused a decrease in the lactate/ pyruvate ratio and an increase in the β-hydroxybutyrate/acetoacetate ratio, indicating the stimulation of transfer of reducing equivalents from cytosol to mitochondria. With sorbitol as the substrate, and with calcium present, norepinephrine promoted further electron transfer from cytosolic to mitochondrial NAD. Enhanced cytosolic calcium concentrations, when cells are exposed to catecholamines in the presence of medium calcium, stimulate the mitochondrial α-glycerophosphate dehydrogenase and thus the transfer of electrons between cell compartments.     

Enzymes of Energy Metabolism in the Mudpuppy Retina

Abstract: The distributions of glycogen phosphorylase, hexokinase, phosphofructokinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, citrate synthase, malate dehydrogenase, β-hydroxyacyl CoA dehydrogenase, and adenylokinase were determined in the mudpuppy retina. Distinct differences were found in regard to the glycolytic and oxidative capacities of the various layers. In the outer retina, citric acid cycle enzymes were high while glycolytic enzymes were low. Synaptic zones were distinctly enriched in all energy-producing enzymes. Mudpuppy photoreceptors were found to be rich in phosphorylase but poor in glucose-6-phosphate dehydrogenase, suggestive of some evolutionary divergence from mammals in the metabolic machinery which is used to support the visual process.     

THE EFFECTS OF INTOXICATING DOSES OF ETHANOL UPON INTERMEDIARY METABOLISM IN RAT BRAIN

Abstract— The effect of acute (8-min) and prolonged (13-h) exposures to high doses of ethanol upon the intermediary metabolites of rat brain has been studied, with the use of a new freezing technique which minimizes post-mortem changes. Injection of ethanol (80 mmol/kg body wt) produced general anaesthesia within 8 min after administration. At this time there were increases in the brain contents of glucose, glucose-6-phosphate and citrate; there was no change in arterial pCO₂. Rats under ethanol anaesthesia for 13 h showed increases in brain contents of glycogen, glucose and glucose 6-phosphate; and decreases in lactate, pyruvate, α-oxoglutarate and malate. Under similar experimental conditions, arterial pCO₂, increased from 37 to 51 Torr. The changes in levels of metabolites after injection of ethanol were similar to those after administration of many volatile anaesthetic agents or elevation of brain CO₂ by other means. Although brain levels of malate and α-oxoglutarate decreased after prolonged exposure to ethanol, the mitochondrial redox state was maintained. Accordingly, the levels of glutamate and aspartate fell in accordance with the law of mass action. The maintenance of the cytoplasmic and mitochondrial redox states in the brain during ethanol intoxication was in marked contrast to the effects on the liver. We suggest that the different effects observed in brain and liver result from the action of ethanol upon the nerve cell membrane in brain, whereas the primary target in liver is alcohol dehydrogenase.     

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [(3)H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40-50% of values in parietal cortex. In contrast, formation of aspartate from [U-(14)C]glucose in awake rats (which reflects the passage of (14)C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, alpha-ketoglutarate dehydrogenase, and fumarase in white structures were 10-23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex ( approximately 3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20-40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.     

The effect of audiogenic seizures on regional CNS energy reserves, glycolysis and citric acid cycle flux

Selected energy reserves, glycolytic intermediates and citric acid cycle intermediates were measured in the cerebral cortex, thalamus, brain stem, cerebellum and spinal cord of susceptible mice during audiogenic seizures. Changes in energy reserves (ATP, phosphocreatine and glucose) differed strikingly in extent and temporal pattern from region to region. The audiogenic seizure produced a transient, large decrease in thalamic energy reserves during the early, pretonic phase of the seizure. Less extensive decreases were observed in brain stem and spinal cord; but in these latter regions the changes persisted throughout the pretonic and tonic phases of the seizures. In cerebellum there was a biphasic decrease in energy reserves; a small decrease was observed immediately after the sound stimulus and a second much greater decrease was observed during the tonic phase of the seizure. No change in energy reserves was observed in cerebral cortex. Changes in glycolytic intermediates (glucose 6-phosphate, fructose diphosphate, pyruvate and lactate) also varied from region to region in response to the decreases in energy reserves. In contrast, changes in the two citric acid cycle intermediates, α-oxoglutarate and malate, were essentially the same in all regions studied. α-Oxoglutarate decreased during the tonic phase of the seizure and rose during recovery. Malate remained at control levels throughout the seizure and then slowly increased. These findings are interpreted as indicating regional variations in nueronal activity during audiogenic seizures. During the period when clinical seizure activity is apparent neuronal activity increases in the subcortical regions. This is reflected by an increase in energy utilization and an increase in glycolytic flux in these areas. However, a concomitant increase in citric acid cycle flux does not seem to occur during this period. Citric acid cycle flux does appear to increase after the seizure is over.     

Capacities of pea chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis

The aim of this work was to measure the capacities of pea (Pisum sativum) shoot chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis. Of the total activities in the unfractionated homogenates, appreciable proportions of those of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase, and smaller but significant proportions of those of phosphopyruvate hydratase and pyruvate kinase were recovered in crude preparations of chloroplasts, and co-purified with intact chloroplasts on sucrose gradients. The activities in the chloroplasts showed considerable latency that was closely correlated with chloroplast integrity. Phosphoglyceromutase activity in the above preparations of chloroplasts did not exceed that expected from cytoplasmic contamination. The mass-action ratio for phosphoglyceromutase in illuminated isolated chloroplasts differed markedly from the enzyme's equilibrium constant. Isolated chloroplasts converted 2-phosphoglycerate to pyruvate. The enzyme activities of the chloroplasts were compared with the rates of respiration and starch breakdown in pea leaves in the dark. It is concluded that in the dark chloroplasts could metabolize all the products of starch breakdown and catalyse much of the respiration of pea shoots via the oxidative pentose phosphate pathway and/or glycolysis as far as 3-phosphoglycerate. It is suggested that pea shoot chloroplasts lack phosphoglyceromutase but contain some phosphopyruvate hydratase and pyruvate kinase.     

Carbohydrate metabolism of malarial parasites--I. Metabolism of lactate in Plasmodium knowlesi infected monkey erythrocytes

Lactate metabolism by Plasmodium knowlesi infected erythrocytes was examined after careful removal of leucocytes from cell preparations. Infected cells were able to metabolize glucose, pyruvate and lactate. Respiration of infected erythrocytes was maximally stimulated by lactate and to a lesser degree by pyruvate and glucose. Respiration of infected cells was insensitive to stimulation by succinate or glutamate or inhibition by malonate. Mepacrine was found to be a potent respiratory inhibitor. Chromatographic analysis of end products of lactate metabolism showed incorporation of carbon from [2-C14]lactate into phosphoenol pyruvate, 3-phosphoglycerate and malate. Experimental data failed to provide evidence for the presence of a functional citric acid cycle activity in infected cells.     

Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Induction of rat kidney gluconeogenesis during acute liver intoxication by carbon tetrachloride.

1. Glucose production from L-lactate was completely inhibited 24h after carbon tetrachloride treatment in liver from 48h-starved rats. The activities of phosphoenolpyruvate carboxykinase, fructose diphosphatase and glucose 6-phosphatase were decreased by this treatment in fed and starved rats, whereas lactate dehydrogenase activity was only decreased in fed animals. 2. The production of glucose by renal cortical slices from fed rats previously treated with carbon tetrachloride was enhanced when L-lactate, pyruvate and glutamine but not fructose were used as glucose precursors. Renal phosphoenolpyruvate carboxykinase activity was increased in this condition. 3. This increase was counteracted by cycloheximide or actinomycin D, suggesting that the effect was due to the synthesis de novo of the enzyme. 4. The pattern of hepatic gluconeogenic metabolites in treated animals was characterized by an increase in lactate, pyruvate, malate and citrate as well as a decrease in glucose 6-phosphate, suggesting an impairment of liver gluconeogenesis in vivo. 5. In contrast, the profile of renal metabolites suggested that gluconeogenesis was operative in the treated rats, as indicated by the marked increase in the content of phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate and glucose 6-phosphate. 6. It is postulated that renal gluconeogenesis could contribute to the maintenance of glycaemia in carbon tetrachloride-treated rats.     

CHANGES IN CARBOHYDRATE SUBSTRATES, AMINO ACIDS AND AMMONIA IN THE BRAIN DURING INSULIN-INDUCED HYPOGLYCEMIA

—The influence of insulin-induced hypoglycemia upon carbohydrate substrates, amino acids and ammonia in the brain was studied in lightly anaesthetized rats, and the changes observed were related to the blood glucose concentration and to the EEG. Calculations from glucose concentrations in tissue, CSF and blood indicated the presence of appreciable amounts of free intracellular glucose at blood glucose concentrations above 3 μmol/g. When the blood glucose concentration fell below 3 μmol/g, there was no calculated intracellular glucose and decreases in the concentrations of glycogen, G-6-P, pyruvate, lactate and of citric acid cycle intermediates were observed. At blood glucose levels of below 1 μmol/g the tissue was virtually depleted of glycogen, G-6-P, pyruvate and lactate. When the blood glucose concentration was reduced below about 2·5 μmol/g there were progressive increases in aspartate and progressive decreases in alanine, GABA, glutamine and glutamate, and at blood glucose concentrations below 2 μmol/g the ammonia concentration increased. It is suggested that most of the changes observed can be explained as a result of a decreased availability of pyruvate and of NADH. The decrease in the concentration of free NADH was reflected in reductions of the lactate/pyruvate and malate/oxaloacetate ratios at an unchanged intracellular pH. Slow wave activity appeared in the EEG when the hypoglycemia gave rise to reduction of the intracellular glucose concentration to zero. Convulsive activity continued until carbohydrate stores in the form of glycogen and G-6-P were depleted. When this occurred the EEG became isoelectric. In all convulsive animals the concentration of the nervous system activity inhibitor, GABA, was decreased and stimulant, aspartate, was increased.     

Effect of copper intrauterine contraceptive device on carbohydrate metabolism in rat endometrium

Activities of Phosphorylase, glyceraldehyde-3 -phosphate dehydrogenase, lactate dehydrogenase, malate dehydrogenase and succinate dehydrogenase in the rat endometrial tissue are significantly inhibited by an intrauterine copper device, while it stimulated glucose-6-phosphate dehydrogenase activity. The copper device decreased the lactate/pyruvate ratio in the tissue; pyruvate utilizationin vitro by the rat endometrium is also blocked by copper. These findings suggested that the normal carbohydrate metabolism of the tissue may be affected in presence of copper, thus resulting in a change of the endometrial function, which may be one of the factors responsible for the contraceptive and pharmacological action of an intrauterine copper device.