Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

GLUTAMATE METABOLISM IN OCTOPUS BRAIN IN VIVO;ABSENCE OF A WAELSCH EFFECT

Abstract— The metabolism of [U-¹⁴C]glutamate was followed in vivo in the octopus Eledone cirrhosa following intracranial injection, and compared with that in the mammalian brain.
By contrast with the rat brain, the specific activity of glutamine recovered from Eledone optic and vertical lobes was lower than that of glutamate at short time intervals after injection. Thus the Waelsch effect was not apparent in this species. Again, in contrast with the rat brain, radioactivity could be found in alanine but not in GABA following [U-¹⁴C]glutamate injection. This was compatible with observations made previously in vitro.
The significance of these intraspecies differences in metabolism and compartmentation is discussed.     

[14C]GLUTAMATE UPTAKE AND COMPARTMENTATION IN GLIA OF RAT DORSAL SENSORY GANGLION

Abstract— The characteristics of the uptake of l -[U-¹⁴C] glutamate into rat dorsal sensory ganglia were investigated. The uptake was mediated by two distinct kinetic systems, with apparent K_m values of the order of 10⁻³ M (low affinity) and 10⁻⁵ m (high affinity). The high affinity uptake system was strongly dependent upon temperature and sodium ion concn, and was depressed by a number of metabolic inhibitors. Following uptake, [¹⁴C] glutamate was extensively metabolized, primarily to glutamine, although this was not so with cultured ganglia, where in addition to an increased uptake of [¹⁴C] glutamate, the specific radioactivity of glutamate was increased and that of glutamine decreased. The labelled substrates [U-¹⁴C]pyruvate and [U-¹⁴C] acetate were used to investigate this phenomenon and the results are discussed in relation to current knowledge of metabolic compartmentation in nervous tissue.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

METABOLISM OF d-[U-14C]RIBOSE IN RAT TISSUES

Abstract— d -[U-¹⁴C]Ribose injected subcutaneously into the rat enters the blood, liver and brain. At 30 min after injection 40-70 per cent of the radioactivity in the brain was found in amino acids and only 2-6 per cent in free sugars. In contrast, free sugars (mainly glucose) and carboxylic acids accounted for most of the radioactivity in liver and blood. Evidence for the entry of [U-¹⁴C]ribose into the brain was obtained by intracarotid or intravenous injection of [U-¹⁴C]ribose after interrupting the blood supply to the liver and kidney. Under these conditions the radioactivity in the brain was found in amino acids, carboxylic acids and ribose; no significant amount of [¹⁴C]glucose was detected in brain or heart. It is concluded that ribose is metabolized directly in vivo in the brain. d -[U-¹⁴C]Ribose was metabolized also by brain slices in vitro to form ¹⁴C-labelled amino acids and carboxylic acids; the rate was equivalent to the utilization of 0.65 μ mol of ribose/g/h. The specific radioactivity of glutamine and of γ -aminobutyrate was similar to or higher than that of glutamate in the brain. These results are discussed in the context of metabolic compartments.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

Glutamine--a major substrate for nerve endings

Abstract— Mammalian cortical synaptosomes incubated in the presence of glucose (2.5 MM) plus glutamine (0.5 mM) showed a 30% increase in transmitter amino acid content over controls with glucose alone and a doubling of glutamate release induced by Veratrine or high K⁺. Double-label experiments, i.e. [U-¹⁴C]glucose with [³H]glutamine, and single-label experiments, i.e. [U-¹⁴C]glucose or [U-¹⁴C]-glutamine showed that stimulus-released glutamate was derived principally (80%) from glutamine. Released glutamine-derived glutamate was of higher (x 2) specific radioactivity than its tissue equivalent. Glutamine alone (0.5–0.75 mM) was much less effective than equivalent amounts of glucose alone, in stimulating respiration and maintaining tissue K⁺ levels.     

Metabolism of [U-13C5]Glutamine in Cultured Astrocytes Studied by NMR Spectroscopy: First Evidence of Astrocytic Pyruvate Recycling

Abstract: Metabolism of [U-¹³C₅]glutamine was studied in primary cultures of cerebral cortical astrocytes in the presence or absence of extracellular glutamate. Perchloric acid extracts of the cells as well as redissolved lyophilized media were subjected to nuclear magnetic resonance and mass spectrometry to identify ¹³C-labeled metabolites. Label from glutamine was found in glutamate and to a lesser extent in lactate and alanine. In the presence of unlabeled glutamate, label was also observed in aspartate. It could be clearly demonstrated that some [U-¹³C₅]glutamine is metabolized through the tricarboxylic acid cycle, although to a much smaller extent than previously shown for [U-¹³C₅]glutamate. Lactate formation from tricarboxylic acid cycle intermediates has previously been demonstrated. It has, however, not been demonstrated that pyruvate, formed from glutamate or glutamine, may reenter the tricarboxylic acid cycle after conversion to acetyl-CoA. The present work demonstrates that this pathway is active, because [4,5-¹³C₂]glutamate was observed in astrocytes incubated with [U-¹³C₅]glutamine in the additional presence of unlabeled glutamate. Furthermore, using mass spectrometry, mono-labeled alanine, glutamate, and glutamine were detected. This isotopomer could be derived via the action of pyruvate carboxylase using ¹³CO₂ produced within the mitochondria or from labeled intermediates that had stayed in the tricarboxylic acid cycle for more than one turn.     

PYRUVATE METABOLISM IN THE LOBSTER NERVE AS AFFECTED BY THE PARTIAL PRESSURE OF CARBON DIOXIDE: OBSERVATIONS ON THE SYNTHESIS OF ACETYLCHOLINE AND ON METABOLIC COMPARTMENTATION

Abstract— In the lobster nerve the fixation of CO, at various levels of _pCO2 was studied by the incorporation of [l-¹⁴C]pyruvate. Incorporation of ¹⁴C was solely dependent on CO₂ fixation since the C-1 was decarboxylated in the formation of acetyl-CoA. Paired-nerve studies with [2-¹⁴C]pyruvate afforded a study of pyruvate metabolism in the lobster nerve. [I¹⁴C]Pyruvate was incorporated to nearly the same extent at all levels of pCO₂ including zero pCO₂, a finding that suggested metabolic recycling of CO₂. The magnitude of the metabolic recycling of C-1 of pyruvate or pyruvate dismutation was estimated to be nearly 20 per cent of total CO₂ fixation. Re-evaluation of the relative contributions of the CO₂ fixation. and acetyl-CoA pathways on the basis of more extensive data gave a ratio of 2:3.
The pCO₂ affected synthesis of ACh and the level of citrate. With increasing pCO₂, the specific radioactivity of ACh decreased much more than the content of ACh. The decrease in the specific radioactivity of ACh but not that of citrate further suggested metabolic compartmentation. The implication of these findings is discussed.
Alanine functioned as a metabolic sink for the incorporated pyruvate. Pyruvate levels were estimated to be approximately 0.1 nmol/mg of protein.     

Altered cerebral glucose and acetate metabolism in succinic semialdehyde dehydrogenase-deficient mice: evidence for glial dysfunction and reduced glutamate/glutamine cycling

Succinic semialdehyde dehydrogenase (SSADH) catalyzes the NADP-dependent oxidation of succinic semialdehyde to succinate, the final step of the GABA shunt pathway. SSADH deficiency in humans is associated with excessive elevation of GABA and γ-hydroxybutyrate (GHB). Recent studies of SSADH-null mice show that elevated GABA and GHB are accompanied by reduced glutamine, a known precursor of the neurotransmitters glutamate and GABA. In this study, cerebral metabolism was investigated in urethane-anesthetized SSADH-null and wild-type 17-day-old mice by intraperitoneal infusion of [1,6-¹³C₂]glucose or [2-¹³C]acetate for different periods. Cortical extracts were prepared and measured using high-resolution ¹H-[¹³C] NMR spectroscopy. Compared with wild-type, levels of GABA, GHB, aspartate, and alanine were significantly higher in SSADH-null cortex, whereas glutamate, glutamine, and taurine were lower. ¹³C Labeling from [1,6-¹³C₂]glucose, which is metabolized in neurons and glia, was significantly lower (expressed as μmol of ¹³C incorporated per gram of brain tissue) for glutamate-(C4,C3), glutamine-C4, succinate-(C3/2), and aspartate-C3 in SSADH-null cortex, whereas Ala-C3 was higher and GABA-C2 unchanged. ¹³C Labeling from [2-¹³C]acetate, a glial substrate, was lower mainly in glutamine-C4 and glutamate-(C4,C3). GHB was labeled by both substrates in SSADH-null mice consistent with GABA as precursor. Our findings indicate that SSADH deficiency is associated with major alterations in glutamate and glutamine metabolism in glia and neurons with surprisingly lesser effects on GABA synthesis.     

METABOLIC COMPARTMENTATION OF GLUTAMATE ASSOCIATED WITH THE FORMATION OF γ-AMINOBUTYRATE

Abstract— The distibution of ¹⁴C in the brains of rats that had been given [U-¹⁴C]glucose (10μCi/100g body wt.) at 10 min before death was followed for 20 min post mortem. The results indicated that the input of glucose-carbon into the tricarboxylic acid cycle stopped instantaneously after death. Although the proportion (more than 40 per cent) of tissue-¹⁴C combined in the amino acids associated with the cycle did not change significantly, there was a characteristic redistribution of ¹⁴C within the amino acid fraction after death: significantly, the ¹⁴C content of glutamate decreased andthat of GABA increased. The GABA/glutamate specific radioactivity ratio which in vivo was 0-58, increased progressively in the first 5 min after death, reaching a value of 0-93. However, by 5 min the rise in the ratio stopped abruptly, although GABA accumulation continued at about half the initial rate beyond that time. These results indicated that GA BA formation is compartmented in the brain andpermitted the evaluation of certain kinetic parameters of the two compartments which could be distinguished under the experimental conditions. One of the compartments was evidently a summation of a number of subcompartments which had certain features in common, such as a low GABA flux relative to the amount of glutamate. The properties of the other compartment were compatible with those of nerve terminals functioning with GABA as the transmitter. This compartment contained about 2 per cent of the total glutamate, but the glutamate pool was labelled about three times more than the average. Further, this compartment accounted for about 50 per cent of the total GABA formation flux andcontained GABA in high concentrations (the probable values were about seven times the mean).     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

Lactate Is Released and Taken Up by Isolated Rabbit Vagus Nerve During Aerobic Metabolism

Abstract: To determine if lactate is produced during aerobic metabolism in peripheral nerve, we incubated pieces of rabbit vagus nerve in oxygenated solution containing d -[U-¹⁴C]glucose while stimulating electrically. After 30 min, nearly all the radioactivity in metabolites in the nerve was in lactate, glucose 6-phosphate, glutamate, and aspartate. Much lactate was released to the bath: 8.2 pmol (µg dry wt)⁻¹ from the exogenous glucose and 14.2 pmol (µg dry wt)⁻¹ from endogenous substrates. Lactate release was not increased when bath P o ₂ was decreased, indicating that it did not come from anoxic tissue. When the bath contained [U-¹⁴C]lactate at a total concentration of 2.13 m M and 1 m M glucose, ¹⁴C was incorporated in CO₂ and glutamate. The initial rate of formation of CO₂ from bath lactate was more rapid than its formation from bath glucose. The results are most readily explained by the hypothesis that has been proposed for brain tissue in which glial cells supply lactate to neurons.     

GAMMA-HYDROXYBUTYRATE DEGRADATION IN THE BRAIN IN VIVO: NEGLIGIBLE DIRECT CONVERSION TO GABA

Abstract— The metabolism of γ-hydroxybutyrate (GHB) was studied by following the fate of [1-¹⁴C]GHB in mouse brain after an intravenous injection. Cerebral uptake of GHB was rapid and this substance disappeared from brain tissue with a half-life of approx 5 min. Degradation of [1-¹⁴C]GHB took place in the brain since ¹⁴C was incorporated in amino acids associated with the tricarboxylic acid cycle: the labelling pattern was consistent with the oxidation of GHB via succinate through the cycle, rather than with β-oxidation of GHB. Conversion of [¹⁴C]GHB into [¹⁴C]GABA prior to oxidation was negligible, thus it is unlikely that the pharmacological action of GHB would be mediated through GABA formation. [¹⁴C]GHB oxidation also elicited the signs of metabolic compartmentation of the tricarboxylic acid cycle in the brain (glutamine/glutamate specific radioactivity ratio was about 4).     

L-GLUTAMIC ACID DECARBOXYLASE IN NON-NEURAL TISSUES OF THE MOUSE

Abstract— Low levels of γ-aminobutyric acid (GABA) and of glutamic acid decarboxylase (GAD) activity have been detected in mouse kidney, liver, spleen and pancreas. Quantitation of both ¹⁴CO₂ and [¹⁴C]GABA produced in radiometric assays from [U-¹⁴CJglutamic acid has shown that measurement of ¹⁴CO₂ evolution alone is not, in all cases, a valid estimate of true GAD activity. As evidenced by increased ^,14CO₂ production upon addition of NAD and CoA to assay mixtures, radiometric assay of GAD activity in crude homogenates may yield ¹⁴CO₂ via the coupled reactions of glutamic acid dehydrogenase and a-ketoglutarate dehydrogenase. The addition of 1 mM aminooxyacetic acid (AOAA) to assays of kidney homogenates inhibited [^,14C]GABA production 92 per cent while ¹⁴CO₂ production was inhibited only 53 per cent. No evidence was found to confirm the reported existence of a second form of the enzyme, GAD II. previously described by Haber el al. (H aber B., K uriyama K. & R oberts E. (1970) Biochem. Pharmac. 19, 1119-1136). Based on sensitivity-to AOAA and chloride inhibition, the GAD activity in mouse kidney is. apparently, indistinguishable from that of neural origin.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U-14C]GLUCOSE AND [U-14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN

Abstract— The incorporation of ¹⁴C into amino acids of the brain was determined at different times after injection of [U-¹⁴C]glucose and [U-¹⁴C]ribose to rats maintained on thiamine-supplemented and thiamine-deficient diets for 22 days.
The ¹⁴C-content of amino acids in the brain of thiamine-deficient rats decreased at times 2–10 min after injection of [U-¹⁴C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U-¹⁴C]ribose.
The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a-oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.
Thiamine deficiency in rats showed a 5% decrease in glutamate ( P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine ( P < 0–01) content of the brain.     

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

Abstract: The metabolic fate of glutamate in astrocytes has been controversial since several studies reported >80% of glutamate was metabolized to glutamine; however, other studies have shown that half of the glutamate was metabolized via the tricarboxylic acid (TCA) cycle and half converted to glutamine. Studies were initiated to determine the metabolic fate of increasing concentrations of [U-¹³C]glutamate in primary cultures of cerebral cortical astrocytes from rat brain. When astrocytes from rat brain were incubated with 0.1 m M [U-¹³C]glutamate 85% of the ¹³C metabolized was converted to glutamine. The formation of [1,2,3-¹³C₃]glutamate demonstrated metabolism of the labeled glutamate via the TCA cycle. When astrocytes were incubated with 0.2–0.5 m M glutamate, ¹³C from glutamate was also incorporated into intracellular aspartate and into lactate that was released into the media. The amount of [¹³C]lactate was essentially unchanged within the range of 0.2–0.5 m M glutamate, whereas the amount of [¹³C]aspartate continued to increase in parallel with the increase in glutamate concentration. The amount of glutamate metabolized via the TCA cycle progressively increased from 15.3 to 42.7% as the extracellular glutamate concentration increased from 0.1 to 0.5 m M , suggesting that the concentration of glutamate is a major factor determining the metabolic fate of glutamate in astrocytes. Previous studies using glutamate concentrations from 0.01 to 0.5 m M and astrocytes from both rat and mouse brain are consistent with these findings.     

THE FORMATION OF GLUTAMATE, ASPARTATE AND GABA IN THE RAT RETINA; GLUCOSE AND GLUTAMINE AS PRECURSORS

Abstract— The distribution of the neuroactive amino acids taurine, GABA, glycine, glutamate and aspartate, together with glutamine, have been studied in the rat retina. Peak levels of taurine were found in photoreceptor cells and of GABA and glycine in a retinal fraction enriched in amacrine cells and, synaptic terminals. In vitro , GABA formation from [³H]glutamine and [¹⁴C]glucose was also most prominent in this fraction; at 500 μ m [³H]glutamine was the better precursor.
Observations on metabolism in the photoreceptor cell layer of the tissue suggest an active turnover of glutamate, aspartate and GABA, and show that glutamine may serve as an alternative substrate to glucose here, perhaps via the GABA bypath.