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.     

CEREBRAL METABOLIC CHANGES IN PROFOUND, INSULIN-INDUCED HYPOGLYCEMIA, AND IN THE RECOVERY PERIOD FOLLOWING GLUCOSE ADMINISTRATION

Severe hypoglycemia was induced by insulin in lightly anaesthetized (70°_o N₂O) and artificially ventilated rats. Brain tissue was frozen in situ after spontaneous EEG potentials had disappeared for 5. 10. 15 or 30 min and cerebral cortex concentrations of labile organic phosphates, glycolytic metabolites, ammonia and amino acids were determined. In other experiments, recovery was induced by glucose injection at the end of the period of EEG silence. All animals with an isoelectric EEG showed extensive deterioration of the cerebral energy state. and gross perturbation of amino acid concentrations. The latter included a 4-fold rise in aspartate concentration and reductions in glutamate and glutamine concentrations to 20 and 5^o_o of control levels respectively. There was an associated rise in ammonia concentration to about 3μmol-g^-1. Administration of glucose brought about extensive recovery of cerebral energy metabolism. For example, after an isoelectric period of 30 min tissue concentrations of phosphocreatine returned to or above normal, the accumulation of ADP and AMP was reversed, there was extensive resynthesis of glycogen and glutamine and full normalisation of tissue concentrations of pyruvate. α-ketoglutarate. GABA and ammonia. However, even after 3 h of recovery there was a reduction in the ATP concentration and thereby in adenine nucleotide pool, moderate elevations of lactate content and the lactate pyruvate ratio, and less than complete restoration of the amino acid pool. It is concluded that some cells may have been irreversibly damaged by the hypoglycemia.     

LEVELS OF CEREBRAL CORTICAL GLYCOLYTIC AND CITRIC ACID CYCLE METABOLITES DURING HYPOGLYCEMIC STUPOR AND ITS REVERSAL

Abstract— Blood glucose, cerebral cortical glucose, and eight metabolites of the glycolytic pathway and citric acid cycle were measured during insulin hypoglycemic stupor and during the first 100s after glucose administration. In hypoglycemic mice that had lost righting ability, blood and brain glucose were decreased 89% and 96% respectively, but glucose-6-phosphate fell only 23%. Other glycolytic and citric acid cycle intermediates were decreased 31–77%. Fructose bisphosphate, 3-phosphoglycerate and phosphopyruvate fell more than glucose-6-phosphate, but less than pyruvate and lactate. Citrate fell less than a-ketoglutarate and malate. These results suggest that in severe hypoglycemia there is a decrease in brain glucose utilization, mediated by phosphofructokinase, but probably caused by decreased neuronal activity. An intravenous injection of glucose restored brain glucose to 75% of normal within 10s and caused return of righting ability within 60s. Glucose-6-phosphate, fructose bisphosphate, 3-phosphoglycerate, and phosphopyruvate rose to normal or near normal levels within 60s, whereas pyruvate, lactate, citrate, ã-ketoglutarate, and malate changed little in this period. This suggests that although glucose given to hypoglycemic animals rapidly enters the glycolytic pathway in brain (and behavior is almost normal), total neuronal activity, and hence overall glucose metabolism, remains subnormal for several minutes.     

Renal metabolism during four types of lactic acidosis in the dog including anoxia

The present study was undertaken to evaluate the metabolic response of the kidney to lactic acidosis. Four types of lactic acidosis were induced in the dog: infusion of lactic acid, infusion of lactic acid with phenformin, administration of phenformin alone, and hypoxia by breathing 95% nitrogen. In all groups of animals, the same degree of acidosis was observed with plasma bicarbonate ranging from 12.8 to 14.9 mM. Plasma lactate concentration ranged from 3.0 to 8.1 mumol/mL. Renal ammoniagenesis failed to be influenced by lactic acidosis. As a matter of fact, it fell during anoxia. The extraction of glutamine by the kidney rose except during anoxia where it fell. The renal production of alanine rose during the infusion of lactic acid with and without phenformin. This coincided with the extraction of glutamine. The renal extraction of lactate rose in all forms of acidosis as well as the production of pyruvate. In the renal cortical tissue, the concentration of malate, pyruvate, and lactate rose. Alanine also rose except during anoxia. An important fall in cytosolic redox potential (NAD+/NADH lactate dehydrogenase) was observed, as well as a fall in mitochondrial redox (NAD+/NADH beta-hydroxybutyrate dehydrogenase). Lactate also accumulated in the liver and in the muscle. We propose that the kidney is unable to respond to lactic acidosis in terms of ammonia production and that this phenomenon is explained by transamination of pyruvate and glutamate into alanine and also by the observed fall in cytosolic redox potential. It is likely that renal gluconeogenesis is also inhibited and this is reflected by the rise in the concentration of malate in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in blood metabolite concentrations in response to repeated capture,anaesthesia and blood sampling in the golden perch,Macquaria ambigua

• 1.1. The major metabolic changes associated with repeated capture, aquarium transfer, anaesthesia and blood sampling were investigated in an Australian freshwater fish, the golden perch (Macquaria ambigua),
• 2.2. A compounded stress response was seen after repetition of the procedure, in which the plasma glucose rose within 3 hr and amino acid concentrations rose and the serum free fatty acids concentration fell after 24 hr.
• 3.3. Alanine was identified as an important circulating energy store in the stress response of golden perch.
• 4.4. No change was noted in the serum protein, plasma lactate or β-hydroxybutyrate concentrations, indicating that tissue damage and hypoxia were absent, and that degradation of free fatty acids did not produce metabolites excess to the requirements of gluconeogenesis and the tricarboxylic acid cycle.

     

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.     

Stimulation of flux through hepatic pyruvate dehydrogenase by 3-mercaptopicolinate

Isolated hepatocytes from 24-h-starved rats were used to assess the possible effect of Ahe hypoglycaemic agent 3-mercaptopicolinate on flux through the hepatic pyruvate dehydrogenase complex. Increasing the extraceIIular pyruvate concentration from 1 mM to 2 mM or 5 mM resulted in an increase in flux through pyruvate dehydrogenase and the tricarboxylic acid cycle as measured by¹⁴CO₂ evolution from [1-¹⁴C]pyruvate and [3-¹⁴C]pyruvate. Gluconeogenesis was inhibited by 3-mercaptopicolinate from both 1 mM and 2 mM pyruvate, but significant increases in malate and citrate concentrations only occurred in cells incubated with 1 mM pyruvate. Flux through pyruvate dehydrogenase was stimulated by 3-mercaptopicolinate with 1 mM pyruvate but was unaltered with 2 mM pyruvate. Dichloroacetate stimulated flux through pyruvate dehydrogenase with no effect on gluconeogenesis in the presence of I mM pyruvate. There was no effect of 3-mercaptopicolinate, administered in vivo, to 24-h-starved rats on the activity of pyruvate dehydrogenase in freeze-clamped heart or liver tissue, although the drug did decrease blood glucose concentration and increase the blood concentrations of lactate and alanine. Dichloroacetate, administered in vivo to 24-h-starved rats, increased the activity of pyruvate dehydrogenase in freeze-clamped heart and liver, and caused decreases in the blood concentrations of glucose, lactate , and alanine. The results suggest that 3-mercaptopicolinate increases flux through hepatocyte pyruvate dehydrogenase by an indirect mechanism.     

Cerebral carbohydrate metabolism during acute hypoxia and recovery

Abstract— The levels of ATP, ADP, AMP and phosphocreatine, of four amino acids, and of 11 intermediates of carbohydrate metabolism in mouse brain were determined after: (1) various degrees of hypoxia; (2) hypoxia combined with anaesthesia; and (3) recovery from severe hypoxia. Glycogen decreased and lactate rose markedly in hypoxia, but levels of ATP and phosphocreatine were normal or near normal even when convulsions and respiratory collapse appeared imminent. During 30 s of complete ischaemia (decapitation) the decline in cerebral ATP and phosphocreatine and the increase in AMP was less in mice previously rendered hypoxic than in control mice. From the changes we calculated that the metabolic rate had decreased by 15 per cent or more during 30 min of hypoxia. Hypoxia was also associated with decreases of cerebral 6-phosphogluconate and aspartate, and increases in alanine, γ-aminobutyrate, α-ketoglutarate, malate, pyruvate, and the lactate :pyruvate ratio. Following recovery in air (10 min), increases were observed in glucose (200 per cent), glucose-6-phosphate, phosphocreatine and citrate, and there was a fall in fructose-1, 6-diphosphale. Similar measurements were made in samples from cerebral cortex, cerebellum, midbrain and medulla. Severe hypoxia produced significant increases in lactate and decreases in glycogen in all areas; γ-aminobutyrate levels increased in cerebral cortex and brain stem, but not in cerebellum. No significant changes occurred in ATP and only in cerebral cortex was there a significant fall in phosphocreatine. Phosphocreatine, ATP and glycogen were determined by quantitative histochemical methods in four areas of medulla oblongata, including the physiological respiratory centre of the ventromedial portion. After hypoxia, ATP was unchanged throughout and the changes (decreases) in phosphocreatine and glycogen were principally confined to dorsal medulla, notably the lateral zone. Thus there is no evidence that respiratory failure is caused by a ‘power’ failure in the respiratory centre. It is suggested that in extremis a protective mechanism may cause neurons to cease firing before high-energy phosphate stores have been exhausted.     

METABOLIC CHANGES IN THE BRAINS OF MICE FROZEN IN LIQUID NITROGEN

Abstract— Autolytic changes in the mouse brain, occurring during immersion of the animal in liquid nitrogen, were evaluated by measuring the tissue concentrations of glucose, lactate, pyruvate, α-oxoglutarate, phosphocreatine, creatine, ATP, ADP and AMP. The values thus obtained were compared with those obtained in paralysed mice under nitrous oxide anaesthesia, the brains of which were frozen in such a way that arterial blood pressure and oxygénation were upheld during the freezing. Immersion of unanaesthetized mice in liquid nitrogen gave rise to significant alterations in phosphocreatine, creatine, lactate, lactate/pyruvate ratio, ADP and AMP. A comparison with values obtained in paralysed and anaesthetized mice that were frozen by immersion in liquid nitrogen showed that the metabolic changes observed in the unanaesthetized animals could not be caused by an anaesthetic effect on the metabolic pattern. It is concluded that autolysis in the mouse brain occurs during immersion of the animal in a coolant, mainly because arterial hypoxia develops before the tissue is frozen. A comparison with previous results on rat cerebral cortex indicates that mice offer no advantage for studies of cerebral metabolites in unanaesthetized animals. In both species, accurate analyses of labile cerebral metabolites require that the brain is frozen in a way that prevents arterial hypoxia during the fixation of the tissue.     

Effects of dichloroacetate on the metabolism of glucose, pyruvate, acetate, 3-hydroxybutyrate and palmitate in rat diaphragm and heart muscle in vitro and on extraction of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo

1. The extractions of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo were calculated from measurements of their arterial and coronary sinus blood concentration. Elevation of plasma free fatty acid concentrations by infusion of intralipid and heparin resulted in increased extraction of free fatty acids and diminished extractions of glucose, lactate and pyruvate by the heart. It is suggested that metabolism of free fatty acids by the heart in vivo, as in vitro, may impair utilization of these substrates. These effects of elevated plasma free fatty acid concentrations on extractions by the heart in vivo were reversed by injection of dichloroacetate, which also improved extraction of lactate and pyruvate by the heart in vivo in alloxan diabetes. 2. Sodium dichloroacetate increased glucose oxidation and pyruvate oxidation in hearts from fed normal or alloxan-diabetic rats perfused with glucose and insulin. Dichloroacetate inhibited oxidation of acetate and 3-hydroxybutyrate and partially reversed inhibitory effects of these substrates on the oxidation of glucose. In rat diaphragm muscle dichloroacetate inhibited oxidation of acetate, 3-hydroxybutyrate and palmitate and increased glucose oxidation and pyruvate oxidation in diaphragms from alloxan-diabetic rats. Dichloroacetate increased the rate of glycolysis in hearts perfused with glucose, insulin and acetate and evidence is given that this results from a lowering of the citrate concentration within the cell, with a consequent activation of phosphofructokinase. 3. In hearts from normal rats perfused with glucose and insulin, dichloroacetate increased cell concentrations of acetyl-CoA, acetylcarnitine and glutamate and lowered those of aspartate and malate. In perfusions with glucose, insulin and acetate, dichloroacetate lowered the cell citrate concentration without lowering the acetyl-CoA or acetylcarnitine concentrations. Measurements of specific radioactivities of acetyl-CoA, acetylcarnitine and citrate in perfusions with [1-(14)C]acetate indicated that dichloroacetate lowered the specific radio-activity of these substrates in the perfused heart. Evidence is given that dichloroacetate may not be metabolized by the heart to dichloroacetyl-CoA or dichloroacetylcarnitine or citrate or CO(2). 4. We suggest that dichloroacetate may activate pyruvate dehydrogenase, thus increasing the oxidation of pyruvate to acetyl-CoA and acetylcarnitine and the conversion of acetyl-CoA into glutamate, with consumption of aspartate and malate. Possible mechanisms for the changes in cell citrate concentration and for inhibitory effects of dichloroacetate on the oxidation of acetate, 3-hydroxybutyrate and palmitate are discussed.     

β-Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine

Abstract— Rat brain cortex slices were incubated with 10 mm -glucose and trace amounts of [6-³H]glucose and [3-¹⁴C]β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and ³H:¹⁴C ratios calculated. Incorporation of [¹⁴C]β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of ¹⁴C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [³H-,¹⁴C]-glucose. (–)-Hydroxycitrate (5.0 m^M) reduced incorporation of [³H]glucose and [¹⁴C]β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm ) showed a decrease in ¹⁴C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm ) also produced a decreased ¹⁴C incorporation into ACh and its precursors. At 10 mm , acetoacetate reduced ³H and ¹⁴C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.     

Metabolic effects of acetate in perfused rat liver studies on ketogenesis,glucose output,lactate uptake and lipogenesis

1. Livers from fed male rats were perfused in situ in a non-recirculating system with whole rat blood containing acetate at six concentrations, from 0.04 to 1.5 μmol/ml, to cover the physiological range encountered in the hapatic portal venous blood in vivo. 2. Below a concentration of 0.25 μmol/ml there was net production of acetate by the liver, while above it there was ner uptake with a fractional extraction of 40%. 3.No relationship was observed between blood [acetate] and hepatic ketogenesis, the ration [3-hydroxybutyrate]/[acetoacetate] or glucose output, either at low fatty acid concentration s or during oleate infusion. 4. Following the increase in serum fatty acid concentration, induced by oleate infusion, there were suquential incresase in ketogenesis and the ratio of [3-hydroxybutyrate]/[acetoacetate] while glucose output rose and lactate uptake fell significantly after in redox state. 5. There was a highly significant negative correlation between blood [acetate] and hepatic lactate uptake during oleate infusion. At the highest acetate concentration of 1.5 μmol/ml there was a small net hepatic lactate output. After oleate infusion ceased, lactate uptake increased, but the negative correlation between blood [acetate] and hepatic lactate uptake persisted. 6. Livers were also perfused with iether [1-¹⁴C]acetate or [U-¹⁴C]lactate at a concentration of acetate of either 0.3 or 1.3 μmol/ml of blood. With [1-¹⁴C]acetate, most of the radioactivity was recovered as fatty acids at the lower concentration of blood acetate. At the higher blood [acetate] a considerably smaller proportion of the radioactivity was recovered in lipids. With [U-¹⁴C]lactate the reverse pattern obtained i.e., recovery was greater at the high concentration of acetate and fell at the low concentration. Fatty acid biosynthesis, measured with ³H₂O, was stimulated from 2.4 to 6.6 μmol of fatty acid/g of liver per h by high blood [acetate] although the contribution of (acetate+lactate) to synthesis remained constant at 33–38% of the total. 7. These results emphasize the important role of the liver in regulating blood acetate concentrations and indicate that it can be major hepatic substrate. Acetate taken up by the liver appeared to compete directly with lactate, for lipogenesis and metabolism and acetate uptake was inhibited by raised bloodd [lactate].     

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.     

Cerebral energy metabolism during experimental status epilepticus.

—The concentration of ATP, ADP, AMP, phosphocreatine and of 5 intermediates of carbohydrate metabolism were determined in rodent brain after single and repeated seizures induced by either electroshock (ES), flurothyl or pentylenetetrazol (PTZ). In paralysed-ventilated rats, one ES produced a 4–5 fold increase in cortical glycolytic flux (estimated from changes in glucose and lactate), and associated increases in pyruvate and in the lactate/pyruvate ratio. Total high energy phosphates declined during the seizure; a decrease was also calculated in cortical tissue pH and in the cytoplasmic [NAD⁺]/[NADH] ratio. Similar changes in brain were observed in ventilated mice after ES, but in paralysed animals, no decrease in high energy phosphates occurred during the first seizure. More vigorous and prolonged chemically-induced seizures in both rats and mice elicited a decrease in the cerebral energy reserves with a rise in lactate and in the lactate/pyruvate ratio. At all times during the seizures the cerebral venous blood had a higher oxygen tension than that of control animals (rats) or was visibly reddened (mice), implying that oxygen availability to brain exceeded metabolic demands. It is proposed that the development of‘non-hypoxic’cerebral lactacidosis during seizures is part of the overall metabolic response of the brain to an abrupt increase in energy consumption. The response constitutes a homeostatic influence which promotes cerebral vasodilatation, thereby increasing blood flow and the delivery of substrates. With repeated seizures, delivered 2 min apart, glycogen declined progressively, but concentrations of the adenine nucleotides appeared to plateau, suggesting that a new energy balance had been established. However, after 20–25 seizures, the attacks became self-generating and there was a further reduction in the tissue high energy phosphate stores, a fall in brain glucose and in the brain/blood glucose ratio. It is concluded that the brain possesses a limited capacity to adjust its metabolism to meet the increased energy requirements of single or repeated seizures, but that this mechanism ultimately fails during status epilepticus unless the abnormal electrical discharges, themselves, are brought under control.     

Metabolic interactions of dichloroacetate and insulin in experimental diabetic ketoacidosis.

1. The infusion of sodium dichloroacetate into rats with severe diabetic ketoacidosis over 4h caused a 2mM decrease in blood glucose, and small falls in blood lactate and pyruvate concentrations. Similar findings had been reported in normal rats (Blackshear et al., 1974). In contrast there was a marked decrease in blood ketone-body concentration in the diabetic ketoacidotic rats after dichloroacetate treatment. 2. The infusion of insulin alone rapidly decreased blood glucose and ketone bodies, but caused an increase in blood lactate and pyruvate. 3. Dichloroacetate did not affect the response to insulin of blood glucose and ketone bodies, but abolished the increase of lactate and pyruvate seen after insulin infusion. 4. Neither insulin nor dichloroacetate stimulated glucose disappearance after functional hepatectomy, but both agents decreased the accumulation in blood of lactate, pyruvate and alanine. 5. Dichloroacetate inhibited 3-hydroxybutyrate uptake by the extra-splachnic tissues; insulin reversed this effect. Ketone-body production must have decreased, as hepatic ketone-body content was unchanged by dicholoracetate yet blood concentrations decreased. 6. It was concluded that: (a) dichloroacetate had qualitatively similar effects on glucose metabolism in severely ketotic rats to those observed in non-diabetic starved animals; (b) insulin and dichloroacetate both separately and together, decreased the net release of lactate, pyruvate and alanine from the extra-splachnic tissues, possibly through a similar mechanism; (c) insulin reversed the inhibition of 3-hydroxybutyrate uptake caused by dichloroacetate; (d) dichloroacetate inhibited ketone-body production in severe ketoacidosis.     

Anaerobic energy metabolism of goldfish,Carassius auratus (L.)

Summary The concentrations of pyruvate, lactate, oxalo-acetate, aceto-acetate -hydroxybutyrate, -ketoglutarate, glutamate, NH ₄ ⁺ , NAD⁺ and NADH were measured in goldfish tissues after previous conditioning to normal and anoxic (12h) conditions. For 11 different metabolites efficiency of different extraction methods was tested by means of internal standards. The recoveries were generally over 80%. The substrate/product couples of the reactions catalysed by lactate dehydrogenase, malate dehydrogenase, -hydroxybutyrate dehydrogenase and glutamate dehydrogenase were used as redox parameters. In the lateral red muscle the redox state did not change during 12 h of anoxia. In the dorsal white muscle only the cytoplasmic redox state underwent a change, as indicated by the increase of the lactate/pyruvate ratio from 20 to 110. In liver both cytoplasm and mitochondria were reduced during anoxia. From the measured values the NAD⁺/NADH ratio was found to change only in white muscle, while the calculated free NAD⁺/NADH ratios were reduced in anoxic white muscle cytoplasm, anoxic liver mitochondria, and anoxic liver cytoplasm. Oxalo-acetate concentrations calculated from the equilibrium constants of lactate dehydrogenase and malate dehydrogenase were at least one order of magnitude smaller than the measured values. The data obtained from anoxic goldfish are in contrast to available data on other animals and support earlier reports which indicate that this animal has a special anaerobic metabolism. The results are discussed especially with respect to the role of ethanol as a sink for reducing equivalents.Abbreviations LDH lactate dehydrogenase - MDH malate dehydrogenase - HBDH -hydroxybutyrate dehydrogenase - GIDH glutamate dehydrogenase     

MECHANISMS ACTIVATING GLYCOLYSIS IN THE BRAIN IN ARTERIAL HYPOXIA

Abstract— In order to study regulatory steps responsible for the activation of anaerobic glycolysis in the brain during hypoxia, cerebral concentrations of carbohydrate substrates and organic phosphates were measured in rats after reduction of the arterial P_O2 to 23-25 mm Hg for 2, 5 and 15 min. The results demonstrated a progressive accumulation of lactate as well as of pyruvate and malate in the absence of changes in ATP, A DP, AMP, citrate and ammonia. The pattern of substrate changes obtained indicate that hypoxia is accompanied by activation of pyruvate kinase and of hexokinase, but not of phosphofructokinase. There was a progressive fall in intracellular pH and a moderate increase in the calculated cytoplasmic NADH/NAD⁺ ratio. The changes in pyruvate and in the NADH/NAD⁺ ratio may be responsible for the observed increase in the malate concentration.     

CEREBRAL METABOLIC CHANGES DURING PROLONGED EPILEPTIC SEIZURES IN RATS

Abstract— Sustained epileptic seizures were induced in paralysed, artificially ventilated and anaesthetized (70% N₂O) rats by means of intravenous bicuculline (1.2mgkg^?1), and cerebral cortical tissue was frozen in situ after periods varying between 10 s and 2 h for analyses of labile phosphates, glycolytic metabolites, citric acid cycle intermediates, and associated amino acids and ammonia, using enzymic fluorometric techniques. Body temperature was kept at 37°C, and arterial hypotension, arterial hypoxaemia and hypoglycaemia were prevented. Cortical glycogen concentrations fell progressively (to 23% of control levels) between 1 and 20 min after seizure onset but returned to control concentrations after 120 min of seizure activity. Cortical glucose concentration fell to 30% of control after 1 min of seizure activity, remained close to 50% of control for 1 h, and fell again to 30% after 2 h of seizure activity. Cortical lactate concentration was doubled in brains frozen 10 s after bicuculline injection. It rose over the following 20 min, reaching a steady concentration of about 10μmolg^?1 wet wt. The changes in lactate and glucose concentration indicated a 34-fold increase in the rate of glycolysis during the first minute of seizure. Phosphocreatine concentration was reduced by nearly 50% after 10 and 30 s of seizure activity, and subsequently stabilized at a concentration 2/3 of normal. ATP concentration was maximally reduced (by 7%) after 30 s and remained close to normal thereafter. Larger, initial reductions occurred in ATP/ADP and ATP/ AMP ratios, as well as in the adenylate energy charge. All these parameters remained significantly reduced for the rest of the 2 h seizure period. However, the changes were moderate since the energy charge was maintained within 2% of control. Changes in citric acid cycle intermediates included initial reductions in α-ketoglutarate and oxaloacetate (calculated) and progressive increases in fumarate, malate and citrate. After long periods of seizures all citric acid cycle intermediates except oxaloacetate were increased in concentration. Ammonia increased during the first min to reach steady state values of 200% of control. Alanine increased progressively during the first 20 min, to stabilize at 200% of control thereafter. GABA increased at 5 min and subsequently rose to almost twice the control value (120 min). At 20 min and onwards there were progressive decreases in glutamate and aspartate, and a progressive increase in glutamine. The sum of amino acids measured increased significantly and the sum of ammonia equivalents rose substantially. Intracellular pH calculated from the creatine kinase equilibrium decreased by 0.25 units during the first minute. However, since the pH calculated from P_co, and cellular buffer base changes remained close to normal during this period, it is concluded that the components of the creatine kinase reaction were not in equilibrium, and the pH values calculated from this equilibrium were incorrect. Tentative calculations of NADH/NAD⁺ ratios indicated that redox changes of opposite direction occurred in cytoplasm (reduction) and mitochondria (oxidation).     

THE INFLUENCE OF HYPOCAPNIA UPON INTRACELLULAR pH AND UPON SOME CARBOHYDRATE SUBSTRATES, AMINO ACIDS AND ORGANIC PHOSPHATES IN THE BRAIN

Abstract— In order to evaluate the influence of hypocapnia upon the energy metabolism of the brain, lightly anaesthetized rats were hyperventilated to arterial CO₂ tensions of 26, 15 and 10 mm Hg respectively, with subsequent measurements of intracellular pH and of tissue concentrations of carbohydrate substrates, amino acids and organic phosphates. At P_co1= 26 there was a moderate increase in the intracellular pH but when the P_co2 was reduced further to 10 mm Hg the intracellular pH returned to normal, or slightly subnormal, values. The reduction in P_Co2 was accompanied by increased cerebral cortical concentrations of lactate, pyruvate, citrate, α-ketoglutarate, malate and glutamate and by decreased aspartate concentrations. It is concluded that the accumulation of metabolic acids explains the normal value for intracellular pH at very low CO₂ tensions. Previous results obtained in man indicate that there is an increased anaerobic production of lactic acid in the brain in extreme hypocapnia. At comparable CO₂ tensions the present results showed a small fall in phosphocreatine and a small rise in ADP. However, since the ammonia concentrations were normal or decreased and since there was an increase in citrate, the results give no direct support to the hypothesis of an activation of phosphofructokinase. Since the cerebral venous P_o2 was reduced to 20 mm Hg at an arterial CO₂ tension of 10 mm Hg the accumulation of acids was probably secondary to tissue hypoxia. However, since there was no, or only a very small, increase in the calculated cytoplasmic NADH/NAD⁺ ratio, it appears less likely that acids accumulated due to lack of NAD⁺.     

Alanine metabolism in skeletal muscle in tissue culture

Alanine production by skeletal muscle in tissue culture was studied using an established myogenic line (L₆) of rat skeletal muscle cells. Correlation analyses were performed on rates of metabolism of alanine, glucose, lactate and pyruvate over incubation periods up to 96 h. Alanine production did not correlate significantly with glucose utilization (r = 0.24, P < 0.20). Alanine production, however, did correlate with lactate production (r = 0.72, P < 0.0005) as well as medium (r = 0.50, P < 0.025) and intracellular (r = 0.85, P < 0.0005) pyruvate concentrations. The intercepts of the latter two correlation analyses indicated that when medium or cell pyruvate fell below 0.28 mM or 1 nmol/mg protein, respectively, net alanine consumption occurred. Alanine synthesis also correlated (r = 0.71, P < 0.0005) with the percent change in the cell mass action ratio for the sum of the alanine and aspartate aminotransferase reactions, i.e., [alanine] [malate]/[aspartate] [lactate]. These results suggest that alanine production is not necessarily linked to the rate of glucose utilization but rather to pyruvate overflow above a critical intracellular level; under conditions of pyruvate overflow, alanine synthesis is driven by the tendency to establish equilibrium between metabolites of the linked amino acid transaminases in skeletal muscle.