Compartmentation of citric acid cycle metabolism in brain: effect of aminooxyacetic acid, ouabain and Ca2+ on the labelling of glutamate, glutamine, aspartate and gaba by [1-14C]acetate, [U-14C]glutamate and [U-14C]asparate

—(1) The effects of aminooxyacetic acid, ouabain and Ca²⁺ on the compartmentation of amino acid metabolism have been studied in slices of brain incubated with sodium-[1-¹⁴C]acetate, l-[U-¹⁴C]glutamate and l-[U-¹⁴C]aspartate as tracer metabolites. (2) Aminooxyacetic acid (10^-3 m) inhibited the labelling of aspartate from [¹⁴C]acetate and [¹⁴C]glutamate, as well as the incorporation of label from [¹⁴C]aspartate into glutamate and glutamine. It also inhibited the labelling of GABA from all three radioactive precursors, as would be anticipated if there was inhibition of several transaminases as well as glutamate decarboxylase. The RSA of glutamine labelled from [1-¹⁴C]acetate was increased. This finding indicated that the glutamate pool which is utilized for glutamine formation is associated with glutamate dehydrogenase, and this enzyme appears to be related to the ‘synthetic tricarboxylic acid cycle’. AOAA exerted its major inhibitory effects on the citric acid‘energy cycle’with which transaminases are associated. (3) Ouabain (10^-5 m) inhibited the labelling of glutamine to a much greater extent than the labelling of glutamate from [1-¹⁴C]acetate. It also caused leakage of amino acids from the tissue into the medium. Its effect on the glutamate–glutamine system was interpreted to be a selective inhibition of the 'synthetic’citric acid cycle. (4) The omission of Ca²⁺ from the incubation medium was associated with formation of glutamine with RSA less than 1·0 when labelled from [U-¹⁴C]glutamate, [U-¹⁴C]aspartate and lower than normal when labelled from [1-¹⁴C]acetate.     

Tricarboxylic acid-cycle metabolism in brain. Effect of fluoroacetate and fluorocitrate on the labelling of glutamate aspartate, glutamine and γ-amino butyrate

1. The effect of fluoroacetate and fluorocitrate on the compartmentation of the glutamate-glutamine system was studied in brain slices with l-[U-(14)C]glutamate, l-[U-(14)C]aspartate, [1-(14)C]acetate and gamma-amino[1-(14)C]butyrate as precursors and in homogenates of brain tissue with [1-(14)C]acetate. The effect of fluoroacetate was also studied in vivo in mouse brain with [1-(14)C]acetate as precursor. 2. Fluoroacetate and fluorocitrate inhibit the labelling of glutamine from all precursors but affect the labelling of glutamate to a much lesser extent. This effect is not due to inhibition of glutamine synthetase. It is interpreted as being due to selective inhibition of the metabolism of a small pool of glutamate that preferentially labels glutamine.     

Transport and metabolism of acetate in rat brain cortex in vitro

1. [1-(14)C]Acetate undergoes metabolism when incubated aerobically at 37 degrees in the presence of rat brain-cortex slices, forming (14)CO(2) and (14)C-labelled amino acids (glutamate, glutamine, aspartate and relatively small quantities of gamma-aminobutyrate). In the absence of glucose the yield of (14)C-labelled aspartate exceeds that of (14)C-labelled glutamate and glutamine. The addition of glucose brings about a doubling of the rate of formation of (14)CO(2) and a greatly increased yield of (14)C-labelled glutamate or glutamine, whereas that of (14)C-labelled aspartate is diminished. 2. The addition of potassium chloride (100mm) to the incubation medium causes an increased rate of (14)CO(2) formation in the presence or absence of glucose and an increased rate of utilization of acetate. 3. The addition of 2,4-dinitrophenol (0.1mm) suppresses the rate of utilization of [1-(14)C]acetate. 4. The presence of ouabain (10mum) suppresses the rate of formation of (14)CO(2) from [1-(14)C]acetate and the rate of acetate utilization. Acetate conversion into carbon dioxide in the rat brain cortex is both Na(+)- and K(+)-dependent and controlled by operation of the active sodium-transport process. Only the Na(+)-stimulated rate is suppressed by ouabain. 5. Sodium fluoroacetate (1mm) decreases the rate of (14)CO(2) evolution from [1-(14)C]acetate in the presence of rat brain cortex without affecting the respiratory rate. The results are consistent with the conclusion that fluoroacetate competes with, or blocks, a transport carrier for acetate, so that in its presence only the passive diffusion rate of acetate takes place. 6. The presence of sodium propionate or sodium butyrate suppresses the utilization of [1-(14)C]acetate in rat brain cortex and leads to a concentration ratio (tissue/medium) of [1-(14)C]-acetate greater than unity. 7. The presence of NH(4) (+) diminishes acetate utilization, this being attributed to a diminished ATP concentration. Glycine is also inhibitory. It is concluded that acetate transport into the brain is carrier-mediated and dependent on the operation of the sodium pump.     

Response of brain amino acid metabolism to ketosis

Our objective was to study brain amino acid metabolism in response to ketosis. The underlying hypothesis is that ketosis is associated with a fundamental change of brain amino acid handling and that this alteration is a factor in the anti-epileptic effect of the ketogenic diet. Specifically, we hypothesize that brain converts ketone bodies to acetyl-CoA and that this results in increased flux through the citrate synthetase reaction. As a result, oxaloacetate is consumed and is less available to the aspartate aminotransferase reaction; therefore, less glutamate is converted to aspartate and relatively more glutamate becomes available to the glutamine synthetase and glutamate decarboxylase reactions. We found in a mouse model of ketosis that the concentration of forebrain aspartate was diminished but the concentration of acetyl-CoA was increased. Studies of the incorporation of 13C into glutamate and glutamine with either [1-(13)C]glucose or [2-(13)C]acetate as precursor showed that ketotic brain metabolized relatively less glucose and relatively more acetate. When the ketotic mice were administered both acetate and a nitrogen donor, such as alanine or leucine, they manifested an increased forebrain concentration of glutamine and GABA. These findings supported the hypothesis that in ketosis there is greater production of acetyl-CoA and a consequent alteration in the equilibrium of the aspartate aminotransferase reaction that results in diminished aspartate production and potentially enhanced synthesis of glutamine and GABA.     

Selective inhibition of glucose oxidation by triethyltin in rat brain in vivo

Results are reported of a comparative study in vivo of the metabolism of [2-(14)C]-glucose and [1-(14)C]acetate in brains of rats intoxicated with triethyltin sulphate. The incorporation of (14)C from glucose into glutamate, glutamine, gamma-aminobutyrate and aspartate was greatly decreased. The incorporation of (14)C from acetate into these amino acids was unaffected. The experimental data indicated that the main action of triethyltin was to decrease the rate at which pyruvate formed from glucose is oxidized. Glycolysis was not inhibited. Changes in glucose metabolism in the brain are shown not to be directly due to hypothermia. Some of the advantages of measuring the labelling of intermediates at very short time intervals after the injection of the labelled glucose are demonstrated.     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

Quantification of carbon fluxes through the tricarboxylic acid cycle in early germinating lettuce embryos

A method involving labeling to isotopic steady state and modeling of the tricarboxylic acid cycle has been used to identify the respiratory substrates in lettuce embryos during the early steps of germination. We have compared the specific radioactivities of aspartate and glutamate and of glutamate C-1 and C-5 after labeling with different substrates. Labeling with [U-14C]acetate and 14CO2 was used to verify the validity of the model for this study; the relative labeling of aspartate and glutamate was that expected from the normal operation of the tricarboxylic acid cycle. After labeling with 14CO2, the label distribution in the glutamate molecule (95% of the label at glutamate C-1) was consistent with an input of carbon via the phosphoenolpyruvate carboxylase reaction, and the relative specific radioactivities of aspartate and glutamate permitted the quantification of the apparent rate of the fumarase reaction. CO2 and intermediates related to the tricarboxylic acid cycle were labeled with [U-14C]acetate, [1-14C] hexanoate, or [U-14C]palmitic acid. The ratios of specific radioactivities of asparate to glutamate and of glutamate C-1 to C-5 indicated that the fatty acids were degraded to acetyl units, suggesting the operation of beta-oxidation, and that the acety-CoA was incorporated directly into citrate. Short-term labeling with [1-14C]hexanoate showed that citrate and glutamate were labeled earlier than malate and aspartate, showing that this fatty acid was metabolized through the tricarboxylic acid cycle rather than the glyoxylate cycle. This was in agreement with the flux into gluconeogenesis compared to efflux as respiratory CO2. The fraction of labeled substrate incorporated into carbohydrates was only about 5% of that converted to CO2; the carbon flux into gluconeogenesis was determined after labeling with 14CO2 and [1-14C]hexanoate from the specific radioactivity of aspartate C-1 and the amount of label incorporated into the carbohydrate fraction. It was only 7.4% of the efflux of respiratory CO2. The labeling of alanine indicates a low activity of either a malic enzyme or the sequence phosphoenolpyruvate carboxykinase/pyruvate kinase. After labeling with [U-14C]glucose, the ratios of specific radioactivities indicated that the labeled carbohydrates contributed less than 10% to the flux of acetyl-CoA. The model indicated that the glycolytic flux is partitioned one-third to pyruvate and two-thirds to oxalacetate and is therefore mainly anaplerotic. The possible role of fatty acids as the main source of acetyl-CoA for respiration is discussed.     

Alteration in Neuronal-Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid

The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.     

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+.     

Metabolism of [1-(13)C)glucose and [2-(13)C]acetate in the hypoxic rat brain.

The effects of hypoxia on the metabolism of the central nervous system were investigated in rats submitted to a low oxygen atmosphere (8% O(2); 92% N(2)). [1-(13)C]glucose and [2-(13)C]acetate were used as substrates, this latter being preferentially metabolized by glial cells. After 1-h substrate infusion, the incorporation of 13C in brain metabolites was determined by NMR spectroscopy. Under hypoxia, an important hyperglycemia was noted. As a consequence, when using labeled glucose, the specific enrichment of brain glucose C1 was lower (48.2+/-5.1%) than under normoxia (66.9+/-2.5%). However, relative to this specific enrichment, the (13)C incorporation in amino acids was increased under hypoxia. This suggested primarily a decreased exchange between blood and brain lactate. The glutamate C2/C4 enrichment ratio was higher under hypoxia (0.62+/-0.01) than normoxia (0.51+/-0.06), indicating a lower glutamate turnover relative to the neuronal TCA cycle activity. The glutamine C2/C4 enrichment ratio was also higher under hypoxia (0.87+/-0.07 instead of 0.65+/-0.11), indicating a new balance in the contributions of different carbon sources at the acetyl-CoA level. When using [2-(13)C]acetate as substrate, no difference in glutamine enrichment appeared under hypoxia, whereas a significant decrease in glutamate, aspartate, alanine and lactate enrichments was noted. This indicated a lower trafficking between astrocytes and neurons and a reduced tricarboxylic acid cycle intermediate recycling of pyruvate.     

COMPARTMENTATION OF AMINO ACID METABOLISM IN THE RAT POSTERIOR PITUITARY

—Isolated rat posterior pituitary glands were incubated with [¹⁴C]glucose or [¹⁴C]acetate and the incorporation of radioactivity into several amino acids was followed. The results indicated that radioactivity was incorporated from [¹⁴C]glucose into a large pool of glutamate which appeared to be responsible for a large proportion of GABA synthesis in the gland. The specific activity of glutamine was always less than that of glutamate when [¹⁴C]glucose was the precursor employed, whereas [¹⁴C]acetate labelled a glutamate pool which had approximately the same specific activity as that of glutamine. The results are discussed with reference to the compartmentation of amino acid metabolism in the nervous system.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

THE INCORPORATION OF DOUBLE-LABELLED ACETATE INTO GLUTAMATE AND RELATED AMINO ACIDS FROM ADULT MOUSE BRAIN: COMPARTMENTATION OF AMINO ACID METABOLISM IN BRAIN

Abstract– We have determined the incorporation of [³H]-, [1-¹⁴C]- and [2-¹⁴C]acetate into glutamate, glutamine and aspartate of the adult mouse brain. All these three acetates were incorporated more extensively into glutamine than into glutamate. This has been reported by several authors for each of these labelled acetates in separate experiments. It was shown that [³H, 2-¹⁴C]acetate can be used to obtain an acetate labelling ratio analogous to the previously used [2-¹⁴C]acetate/[1-¹⁴C]acetate labelling ratio. From these acetate labelling ratios of glutamine and glutamate conclusions can be deduced about the dynamic relationship of these amino acids with each other and with the tricarboxylic acid cycle.
A fairly large isotope effect between acetate and glutamate was observed. As this isotope effect is very likely caused by the citrate synthase reaction, it can be argued that citrate synthase involved in the conversion of labelled acetate into glutamate is far out of equilibrium in vivo. Comparing our data with literature data, the possibility can be suggested that citrate synthase in the acetate metabolizing compartment is in situ kinetically distinct from citrate synthase in other compartments of the brain.     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

Pentylenetetrazole decreases metabolic glutamate turnover in rat brain

Seizures were induced in rats by intraperitoneal injection of pentylenetetrazole (PTZ, 70 mg/kg), followed, 30 min later, by injection of [1-13C]glucose and [1,2-13C]acetate. Analyses of extracts from cortex, subcortex and cerebellum were performed using 13C magnetic resonance spectroscopy and HPLC. It could be shown that PTZ affected different brain regions differently. The total amounts of glutamate, glutamine, GABA, aspartate and taurine were decreased in the cerebellum and unchanged in the other brain regions. GABAergic neurones in the cortex and subcortex were not affected, whereas those in the cerebellum showed a pronounced decrease of GABA synthesis. However, glutamatergic neurones in all brain regions showed a decrease in glutamate labelling and in addition a decreased turnover in cerebellum. It could be shown that this decrease was in the metabolic pool of glutamate whereas release of glutamate was unaffected since glutamine labelling from glutamate was unchanged. Aspartate turnover was also decreased in all brain regions. Changes in astrocyte metabolism were not detected, indicating that PTZ had no effect on astrocyte metabolism in the early postictal stage.     

Activation of astrocytes in brain of conscious rats during acoustic stimulation: acetate utilization in working brain

To evaluate the response of astrocytes in the auditory pathway to increased neuronal signaling elicited by acoustic stimulation, conscious rats were presented with a unilateral broadband click stimulus and functional activation was assessed by quantitative autoradiography using three tracers to pulse label different metabolic pools in brain: [2-14C]acetate labels the 'small' (astrocytic) glutamate pool, [1-14C]hydroxybutyrate labels the 'large' glutamate pool, and [14C]deoxyglucose, reflects overall glucose utilization (CMR(glc)) in all brain cells. CMR(glc) rose during brain activation, and increased activity of the oxidative pathway in working astrocytes during acoustic stimulation was registered with [2-14C]acetate. In contrast, the stimulation-induced increase in metabolic activity was not reflected by greater trapping of products of [1-14C]hydroxybutyrate. The [2-14C]acetate uptake coefficient in the inferior colliculus and lateral lemniscus during acoustic stimulation was 15% and 18% (p < 0.01) higher in the activated compared to contralateral hemisphere, whereas CMR(glc) in these structures rose by 66% (p < 0.01) and 42% (p < 0.05), respectively. Calculated rates of brain utilization of blood-borne acetate (CMR(acetate)) are about 15-25% of total CMR(glc) in non-stimulated tissue and 10-20% of CMR(glc) in acoustically activated structures; they range from 28 to 115% of estimated rates of glucose oxidation in astrocytes. The rise in acetate utilization during acoustic stimulation is modest compared to total CMR(glc), but astrocytic oxidative metabolism of 'minor' substrates present in blood can make a significant contribution to the overall energetics of astrocytes and astrocyte-neuron interactions in working brain.     

Metabolism of glucose into glutamate via the hexose monophosphate shunt and its inhibition by 6-aminonicotinamide in rat brain in vivo

The treatment of rats for 4 h with 6-aminonicotinamide (60 mg kg-1) resulted in an 180-fold increase in the concentration of 6-phosphogluconate in their brains; glucose increased 2.6-fold and glucose 6-phosphate, 1.7-fold. Moreover, lactate decreased by 20%, glutamate by 8% and gamma-aminobutyrate by 12%, and aspartate increased by 10%. No significant changes were found in glutamine and citrate. In blood, 6-phosphogluconate increased 5-fold; glucose, 1.4-fold and glucose 6-phosphate, 1.8-fold. The metabolism of glucose in the rat brain, via both the Embden-Meyerhof pathway and the hexose monophosphate shunt, was investigated by injecting [U-14C]glucose or [2-14C]glucose, and that via the hexose monophosphate shunt alone by injecting [3,4-14C]glucose. The total radioactive yield of amino acids in the rat brain was 5.63 mumol at 20 min after injection of [U-14C]glucose, or 5.82 mumol after injection of [2-14C]glucose; by contrast, it was 0.62 mumol after injection of [3,4-14C]glucose. The treatment of rats with 6-aminonicotinamide showed significant decreases in these values, owing to decreases in the radioactive yields of glutamate, glutamine, aspartate, gamma-aminobutyrate, and alanine+glycine+serine. Glutamate isolated from the brain contained approximately 43% of its radioactivity in carbon 1 after injection of [3,4-14C]glucose, in contrast to 13% and 18% after injection of [U-14C]glucose and [2-14C]glucose, respectively, in both the control and treated rats. The calculations based on these findings showed that approximately 69% of the 14C-labelled glutamate was formed from [14C]acetyl coenzyme A (acetyl CoA) and the residual 31% by 14CO2 fixation of pyruvate after injection of [3,4-14C]glucose in both control and treated rats. The results gave direct evidence that glutamate and gamma-aminobutyrate in the brain were formed by metabolism of glucose via the hexose monophosphate shunt as well as via the Embden-Meyerhof pathway. From the radioactive yields of glutamate formed via [14C]acetyl CoA it was estimated that approximately 7.8% of the total glucose utilized was channelled via the hexose monophosphate shunt. Assuming that [14C]glutamate formed by carbon-dioxide fixation of pyruvate was also dependent on the metabolism of glucose through the hexose monophosphate shunt, the estimated value was approximately 9.5% of the total glucose converted into glutamate. The results of the present investigation, taken in conjunction with other findings, suggest that the utilization of glucose via the hexose monophosphate shunt is functionally important in the rat brain.     

The operation of the γ-aminobutyrate bypath of the tricarboxylic acid cycle in brain tissue in vitro

1. Cerebral-cortex slices prelabelled with gamma-amino[1-(14)C]butyrate (GABA) were incubated in a glucose-saline medium. After the initial rapid uptake there was no appreciable re-entry of (14)C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03mumol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of (14)C into amino acids associated with the tricarboxylic acid cycle was followed with [1-(14)C]GABA and [U-(14)C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of (14)C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-(14)C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by alpha-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [(14)C]GABA into gamma-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-(14)C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of (14)C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-(14)C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-(14)C]GABA were different from those labelled by the metabolism of [(14)C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-(14)C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ;transmitter pool' was estimated to be 25-30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.     

Inhibition of glutamine transport depletes glutamate and GABA neurotransmitter pools: further evidence for metabolic compartmentation

The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.