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.     

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.     

The effect of L-3,4-dihydroxyphenylalanine administration on glucose metabolism in brain

The effect of the administration of l -3,4-dihydroxyphenylalanine (l -DOPA) on the metabolism of glucose in brain was studied by administering [U-¹⁴C]glucose to three groups of rats: (1) those injected previously with l -DOPA, 100 mg/kg; (2) those fed 1 % (w/w) l -DOPA in their diet for several months and also injected 15 min before the administration of glucose with l -DOPA, 100 mg/kg; and (3) appropriate controls. Chronic treatment with l -DOPA caused a decrease in the flux of carbon from glucose in plasma to those amino acids in brain that are in equilibrium with the tricarboxylic acid cycle intermediates but not to lactate and alanine. Similar differences from controls, but of smaller magnitude, were observed in rats given a single injection of l -DOPA. Concentrations of glucose in plasma and in brain were increased after acute or chronic treatment with l -DOPA. A single injection of l -DOPA did not cause changes in the levels of the most abundant amino acids in brain, but after chronic treatment with l -DOPA modest changes were noted in the brain levels of some ninhydrin-reacting substances; the contents of taurine and aspartate were lower and those of threonine, serine, glutamine, and glycine were higher.     

三甲双酮对斑马鱼胚胎早期发育的影响

针对抗癫痫药物的临床神经性毒副作用及致畸性,以三甲双酮为探针药建立了抗癫痫药毒性的斑马鱼胚胎模型。结果显示,斑马鱼胚胎暴露于三甲双酮后出现浓度依赖性的畸形和死亡。畸形表型有生长迟缓,脑区、眼和听囊变小,半规管和耳石受损,以及心血管系统异常。这些表型与临床病例和文献报道很相似。毛细胞染色显示听囊ML2神经丘毛细胞数明显减少。原位杂交检测发现脑标志基因zic1和xb51、自噬基因atg5的表达图式发生了异常变化。RT-PCR检测显示听觉基因val和hmx2的表达水平也发生了异常变化。这些结果提示脑组织和控制身体平衡及听力的神经感受器是三甲双酮的主要毒性靶位。斑马鱼胚胎和幼体可以模拟三甲双酮对哺乳动物的致畸和神经毒性反应。     

The effect of excess K+ on two different tricarboxylate cycles in rat brain cortex slices

The authors compared, in rat brain cortex slices, the oxidation of labelled glucose and acetate and the conversion of these precursors into amino acids during incubation in control salt-glucose medium and in medium with 47 mM K+, with the aim of determining with which of the two determinable tricarboxylate cycles raised oxygen consumption is associated in the presence of excess K+. Under the experimental conditions it was found that from U-[14C]-glucose more than double the amount of [14C]-CO2 was formed and that the rate of [14C] incorportation into the amino acids was likewise roughly doubled. This is indicative of activation of processes in the tricarboxylate cycle associated with the large glutamate pool. Incorporation from 1-[14C]-acetate into the total amino acids was not affected. Specific activity in glutamate and asparate was more than doubled, while glutamine specific activity fell to less than half. [14C]-CO2 production fell to 65%. This shows that the tricarboxylate cycle associated with the small glutamate pool, which is probably localized in the glia cells, did not participate in raised oxygen consumption in the presence of excess K+.     

Supplement of TCA cycle intermediates protects against high glucose/palmitate-induced INS-1 beta cell death

The aim of this study is to investigate the effect of mitochondrial metabolism on high glucose/palmitate (HG/PA)-induced INS-1 beta cell death. Long-term treatment of INS-1 cells with HG/PA impaired energy-producing metabolism accompanying with depletion of TCA cycle intermediates. Whereas an inhibitor of carnitine palmitoyl transferase 1 augmented HG/PA-induced INS-1 cell death, stimulators of fatty acid oxidation protected the cells against the HG/PA-induced death. Furthermore, whereas mitochondrial pyruvate carboxylase inhibitor phenylacetic acid augmented HG/PA-induced INS-1 cell death, supplementation of TCA cycle metabolites including leucine/glutamine, methyl succinate/α-ketoisocaproic acid, dimethyl malate, and valeric acid or treatment with a glutamate dehydrogenase activator, aminobicyclo-heptane-2-carboxylic acid (BCH), significantly protected the cells against the HG/PA-induced death. In particular, the mitochondrial tricarboxylate carrier inhibitor, benzene tricarboxylate (BTA), also showed a strong protective effect on the HG/PA-induced INS-1 cell death. Knockdown of glutamate dehydrogenase or tricarboxylate carrier augmented or reduced the HG/PA-induced INS-1 cell death, respectively. Both BCH and BTA restored HG/PA-induced reduction of energy metabolism as well as depletion of TCA intermediates. These data suggest that depletion of the TCA cycle intermediate pool and impaired energy-producing metabolism may play a role in HG/PA-induced cytotoxicity to beta cells and thus, HG/PA-induced beta cell glucolipotoxicity can be protected by nutritional or pharmacological maneuver enhancing anaplerosis or reducing cataplerosis.     

INFLUENCE OF ANAESTHETICS ON THE BALANCE BETWEEN PRODUCTION AND UTILIZATION OF ENERGY IN THE BRAIN

Abstract— The influence of general anaesthesia upon the metabolic state of the brain was evaluated from the tissue concentrations of ATP, ADP and AMP, and from the concentrations of glycolytic and citric acid cycle intermediates, in immobilized and artificially ventilated rats anaesthetized either with 70% N₂O, 1% halothane or 60 mg/kg of pentobarbitone. The results were compared to the results obtained on awake animals in fentanyl-analgesia. The adenylate energy charge was identical in all groups studied and there were no H⁺-independent changes in the phosphocreatine/creatine ratios. In pentobarbitone anaesthesia there was an accumulation of glucose 6-phosphate and a fall in fructose 1,6-diphosphate, indicating inhibition of phosphofructokinase. No significant changes in these metabolites were observed with halothane or nitrous oxide anaesthesia and the substrate patterns differed from that obtained with pentobarbitone.
The blood glucose concentrations were higher in the unanaesthetized, immobilized rats given fentanyl than in those anaesthetized. There was a direct relationship between the glucose concentrations in blood and in tissue. The glucose concentration ratios intracellular water to blood were higher in the anaesthetized than in the unanaesthetized animals, increasing with increasing depth of anaesthesia. The intracellular lactate concentrations were lowest in the groups given pentobarbitone and fentanyl citrate, and there was thus no direct relationship between lactate concentration and depth of anaesthesia.     

Hexose and pentose phosphates in brain during convulsions

The Metabolism of glucose via glycolysis and the citric acid cycle represents the chief source of energy supplies in the brain. However, enzymes for the metabolism of glucose via the hexosemonophosphate (O'Neill, Simon and Shreeve, 1965) and pentose phosphate (Dreyfus and Moniz, 1962) pathway are also present in brain. A major difficulty in studying intermediates of the hexose and pentose phosphate pathway in brain is their extremely low absolute levels. Recently, fluorimetric, enzymic assays for these substrates have been developed (Kauffman, Brown, Passonneau and Lowry, 1969), and these assays, combined with rapid freezing techniques (Lowry, Passonneau, Hasselberger and Schulz, 1964) have made it possible to study these intermediates in brains of mice. Electrically-induced tonic-clonic convulsions are associated with a striking increase in metabolic rate in brain, but phenobarbitone minimizes changes in metabolic rate during convulsions (King, Lowry, Passonneau and Venson, 1967). In order to determine whether or not levels of hexose and pentose phosphates are altered under these conditions, assays for these substrates have been carried out in brains of convulsing mice with or without administration of phénobarbital prior to stimulus.     

UV-Photoconversion of Ethosuximide from a Longevity-Promoting Compound to a Potent Toxin

The anticonvulsant ethosuximide has been previously shown to increase life span and promote healthspan in the nematode Caenorhabditis elegans at millimolar concentrations. Here we report that following exposure to ultraviolet irradiation at 254 nm, ethosuximide is converted into a compound that displays toxicity toward C. elegans. This effect is specific for ethosuximide, as the structurally related compounds trimethadione and succinimide do not show similar toxicities following UV exposure. Killing by UV-irradiated ethosuximide is not attenuated in chemosensory mutants that are resistant to toxicity associated with high doses of non-irradiated ethosuximide. Non-irradiated ethosuximide extends life span at 15°C or 20°C, but not at 25°C, while irradiated ethosuximide shows similar toxicity at all three temperatures. Dietary restriction by bacterial deprivation does not protect against toxicity from irradiated ethosuximide, while non-irradiated ethosuximide further extends the long life spans of restricted animals. These data support the model that ethosuximide extends life span by a mechanism that is, at least partially, distinct from dietary restriction by bacterial deprivation and demonstrates an unexpected photochemical conversion of ethosuximide into a toxic compound by UV light.     

Effect of Anticonvulsant Drugs on 7-Hydroxybutyrate Release from Hippocampal Slices: Inhibition by Valproate and Ethosuximide

The effects of some anticonvulsant drugs have been investigated on gamma-hydroxybutyrate release from rat hippocampal and striatal slices. Sodium valproate and ethosuximide inhibited the depolarization-evoked release of gamma-hydroxybutyrate induced by 40 mM K+. The IC50 values for these two drugs are in the concentration range of valproate and ethosuximide that exists in rat brain after administration of anticonvulsant doses to the animals. Trimethadione and pentobarbital are without significant effects. It can be concluded that the inhibition of gamma-hydroxybutyrate release, particularly that observed for hippocampus, might explain the protective effect of valproate and ethosuximide on gamma-hydroxybutyrate-induced seizures and perhaps on other kinds of epileptoid phenomenon.     

The temporal dimensions of anticonvulsant action of some newer benzodiazepines against metrazol induced seizures in mice and rats

In a time-distribution study, the anticonvulsant effects of four benzodiazepine compounds were compared with those of three standard antiepileptics against metrazol-induced seizures in mice and rats. Ethosuximide and trimethadione had the shortest duration of action in mice, but protected the rats up to 6 hr. Phenobarbitone, diazepam, flurazepam and nitrazepam protected the mice up to 12 hr, but the rats were effectively protected only up to 3-4 hr. Clonazepam, the most potent and effective agent, protected the mice from clonic-tonic seizures up to 18-20 hr and the rats up to 6-7 hr. Comparison of the PD50 from clonic seizure at the peak-effect hours revealed that the benzodiazepines were 16 to 96 times more potent than phenobarbitone on a molar basis, while phenobarbitone itself was 12 to 26 times more potent than ethosuximide and trimethadione. Tonic seizures and mortality were largely suppressed by all drugs until 18-20 hr in mice and 6-7 hr in rats. Seizure latency and mortality patterns varied from drug to drug but not in a dose-dependent manner.     

Evidence for involvement of the astrocytic benzodiazepine receptor in the mechanism of action of convulsant and anticonvulsant drugs

The anticonvulsant drugs carbamazepine, phenobarbital, trimethadione, valproic acid and ethosuximide at pharmacologically relevant concentrations inhibit [3H]diazepam binding to astrocytes in primary cultures but have much less effect on a corresponding preparation of neurons. Phenytoin as well as pentobarbital (which is not used chronically as an anticonvulsant) are equipotent in the two cell types. The convulsants picrotoxinin and pentylenetetrazol, the convulsant benzodiazepine RO 5-3663 and the two convulsant barbiturates DMBB and CHEB similarly inhibit diazepam binding to astrocytes but have little effect on neurons. On the basis of these findings it is suggested that these convulsants and anticonvulsants owe at least part of their effect to an interaction with the astrocytic benzodiazepine receptor, perhaps by interference with a calcium channel.     

Effect of 2-deoxy-D-glucose on lipolytic processes in blood and adipose tissue of rat

The effect of 2-deoxy-D-glucose on lipolytic processes in the blood and adipose tissue was studied. Rats treated with this antimetabolite showed a significant increase in serum glucose, FFA and glycerol level, as well as in the lipid mobilizing activity. On the other hand, the lipolytic activity of rat serum decreased when compared to control group. From these results it may be concluded that during hypothermia induced by administration of 2-deoxy-D-glucose intracellular, but not intravascular, lipolysis is enhanced.     

Effects of antiepileptic drugs on brain energy reserves during convulsions

Cerebral cortical ATP, P-creatine, glucose, and lactate were measured 6 sec after 1 sec of 150/sec rectangular pulses, at 0,20 v, 40 v, 60 v, 80 v, or 100 v, applied to the heads of intact mice which had been given either no drug, phenobarbitone (25 mg/kg), trimethadione (600 mg/kg), or diphenylhydantoin (40 mg/kg), intraperitoneally. In general, regardless of stimulus strength or drug used, animals which exhibited maximal (tonic-clonic) convulsions showed similar striking decreases in brain P-creatine, decreases in ATP and glucose, and increases in lactate. On the other hand, in animals which exhibited less than maximal clinical response, there was little or no change in these metabolites. An exception was the case of diphenylhydantoin. Tonic-clonic seizures did not occur after diphenylhydantoin administration, even with 100 v stimuli, but substrate changes at this voltage were, nevertheless, similar to those observed in brains of other mice undergoing maximal convulsions.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na+-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na+-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na+-independent pathways, possibly mediated by an organic anion transporter, and Na+-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na+-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na+-dependent transport of succinate by NaDC1.     

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.     

Blood-Brain Glucose Transfer in Spreading Depression

Abstract Spreading depression in rat brain cortex is associated with a twofold increase of cerebral blood flow. It is not known whether this increase is coupled to increases of cerebral metabolic rate and glucose transport from blood to brain. During the passage of a single spreading depression, we measured blood-brain glucose transport and glucose metabolism in rat cerebral cortex by single intravenous injection of tracer glucose. Blood flow and tissue content of glucose were measured as well. Reduction of tissue glucose and the consequent increase of net transfer of glucose from blood to brain were consistent with a threefold increase of the consumption of glucose before the increase of blood flow. There was no increase of unidirectional blood-brain transfer.     

Effect of Reducing Brain Glutamine Synthesis on Metabolic Symptoms of Hepatic Encephalopathy

Abstract: Liver failure, or shunting of intestinal blood around the liver, results in hyperammonemia and cerebral dysfunction. Recently it was shown that ammonia caused some of the metabolic signs of hepatic encephalopathy only after it was metabolized by glutamine synthetase in the brain. In the present study, small doses of methionine sulfoximine, an inhibitor of cerebral glutamine synthetase, were given to rats either at the time of portacaval shunting or 3–4 weeks later. The effects on several characteristic cerebral metabolic abnormalities produced by portacaval shunting were measured 1–3 days after injection of the inhibitor. All untreated portacaval-shunted rats had elevated plasma and brain ammonia concentrations, increased brain glutamine and tryptophan content, decreased brain glucose consumption, and increased permeability of the blood–brain barrier to tryptophan. All treated rats had high ammonia concentrations, but the brain glutamine content was normal, indicating inhibition of glutamine synthesis. One day after shunting and methionine sulfoximine administration, glucose consumption, tryptophan transport, and tryptophan brain content remained near control values. In the 3–4-week-shunted rats, which were studied 1–3 days after methionine sulfoximine administration, the effect was less pronounced. Brain glucose consumption and tryptophan content were partially normalized, but tryptophan transport was unaffected. The results agree with our earlier conclusion that glutamine synthesis is an essential step in the development of cerebral metabolic abnormalities in hyperammonemic states.     

Identification of transport pathways for citric acid cycle intermediates in the human colon carcinoma cell line, Caco-2

Citric acid cycle intermediates are absorbed from the gastrointestinal tract through carrier-mediated mechanisms, although the transport pathways have not been clearly identified. This study examines the transport of citric acid cycle intermediates in the Caco-2 human colon carcinoma cell line, often used as a model of small intestine. Inulin was used as an extracellular volume marker instead of mannitol since the apparent volume measured with mannitol changed with time. The results show that Caco-2 cells contain at least three distinct transporters, including the Na⁺-dependent di- and tricarboxylate transporters, NaDC1 and NaCT, and one or more sodium-independent pathways, possibly involving organic anion transporters. Succinate transport is mediated mostly by Na⁺-dependent pathways, predominantly by NaDC1, but with some contribution by NaCT. RT-PCR and functional characteristics verified the expression of these transporters in Caco-2 cells. In contrast, citrate transport in Caco-2 cells occurs by a combination of Na⁺-independent pathways, possibly mediated by an organic anion transporter, and Na⁺-dependent mechanisms. The non-metabolizable dicarboxylate, methylsuccinate, is also transported by a combination of Na⁺-dependent and -independent pathways. In conclusion, we find that multiple pathways are involved in the transport of di- and tricarboxylates by Caco-2 cells. Since many of these pathways are not found in human intestine, this model may be best suited for studying Na⁺-dependent transport of succinate by NaDC1.     

INCREASE OF GLUCOSE AND HIGH ENERGY PHOSPHATE RESERVE IN THE BRAIN AFTER HYDROCORTISONE1

Abstract— Young mice treated with hydrocortisone (50 mg/kg) subcutaneously for 10 days showed a doubling of brain glucose. Brain phospho-creatine, glucose-6-phosphate, and ATP increased slightly. Brain glycogen and lactate were unchanged. Total energy reserve of the brain was 23 per cent higher than the control value. Liver glycogen was increased 47 per cent; liver and blood glucose levels were 11 per cent lower than in control animals. Since the animals showed no evidence of sedation, these findings suggest a facilitated transport of glucose from blood into the brain under the influence of hydrocortisone. Other possible explanations include an inhibition of the hexose monophosphate shunt and a proportionate decrease in both the oxidative and glycolytic pathways of the brain, but it was concluded that these explanations are less likely.