FATE OF l-GLUTAMATE IN THE BRAIN

Abstract— It is shown, using aminooxyacetate as metabolic inhibitor, that the process of oxidation of endogenous glutamate in incubated rat brain cortex slices follows a different course from that of exogenous l -glutamate. Whereas endogenous glutamate is largely oxidized by an initial reaction with glutamate dehydrogenase with release of ammonia, exogenous l -glutamate undergoes initial transamination to aspartate and α-oxoglutarate before oxidation occurs. In the presence of 2·5 m m l -glutamate, it is found that, of the total exogenous glutamate utilized, 49 per cent is converted to aspartate, 37 per cent is converted to glutamine and the rest is f uily oxidized through glutamate dehydrogenase. It is suggested that endogenous glutamate is normally oxidized in the neurons, and that glutamate released from neurons during excitation, and acting therefore as exogenous glutamate, is taken up by the glia where, besides conversion to glutamine, it largely undergoes initial transamination before oxidation takes place.     

INTERACTION OF CATECHOLAMINE AND AMINO ACID METABOLISM IN BRAIN: EFFECT OF PARGYLINE AND l-DOPA

Abstract— The combination of l -DOPA and pargyline caused a decrease in level of aspartate and an increase in that of glutamine in vivo in cerebral cortex, cerebellum, brain stem, hypothalamus, neostriatum and cervical cord of rat. There was also a decreased incorporation of radioactivity from [1-¹⁴C]acetate into amino acids in vivo , most notably in cerebellum and brain stem. The labelling of glutamine was especially affected. In addition, cortical slices were prepared from guinea pigs which had been pretreated with pargyline. These slices were incubated with and without 1 m m l -DOPA in media containing [1-¹⁴C]acetate. Pargyline alone caused a stimulation of the labelling of glutamate and aspartate but not glutamine and GABA; the levels of aspartate and GABA were greater than in control slices. The addition of l -DOPA to slices from pargylinized animals caused a severe decrease in glutamine labelling but not in that of glutamate or aspartate; the level of glutamine was increased while that of glutamate was decreased. The results are discussed in terms of the known biochemical and morphological compartmentation of amino acids in brain. It is suggested that catecholamines, in the process of functioning as transmitters, may also function as metabolic regulators of other transmitters, e.g. amino acids, as well as of the energy required for balanced neuronal function.     

AMINO ACID METABOLISM AND AMMONIA FORMATION IN BRAIN SLICES

The formation of ammonia and changes in the contents of free amino acids have been investigated in slices of guinea pig cerebral cortex incubated under the following conditions: (1) aerobically in glucose-free saline; (2) aerobically in glucose-free saline containing 10 mM-bromofuroic acid, an inhibitor of glutamate dehydrogenase (EC 1.4.1.2); (3) aerobically in saline containing 11-1 mM-glucose and (4) anaerobically in glucose-free saline. Ammonia was formed at a steady rate aerobically in glucose-free medium. The formation of ammonia was largely suppressed in the absence of oxygen or in the presence of glucose whereas the inhibitor of glutamate dehydrogenase produced about 50 per cent inhibition. Other inhibitors of glutamate dehydrogenase exerted a similar effect. Ammonia formation was also inhibited by some inhibitors of aminotransferases but not by others. Inhibition was generally more pronounced during the second and third hour of incubation. With the exception of glutamine which decreased slightly, the contents of all amino acids increased markedly during the anaerobic incubation. During aerobic incubation in a glucose-free medium, there was an almost complete disappearance of glutamic acid and GABA. Glutamine also decreased, but to a relatively smaller extent. The content of all other amino acids increased during aerobic incubation in glucose-free medium, although to a lesser extent than under anaerobic conditions. The greater increase of amino acids appearing anaerobically in comparison to the increase or decrease occurring under aerobic conditions corresponded closely to the greater amount of ammonia formed aerobically over that formed anaerobically. This finding is interpreted as indicating a similar degree of proteolysis under anaerobic and aerobic conditions; aerobically, the amino acids are partly metabolized with the concomitant liberation of ammonia. In glucose-supplemented medium, the content of glutamine was markedly increased. The content of glutamate and aspartate remained unchanged, whereas that of some other amino acids increased but to a lesser extent than in the absence of glucose. Proteolysis in the presence of glucose was estimated at about 65 per cent of that in its absence. In the presence of bromofuroate the rate of disappearance of glutamate was unchanged, but there was a larger increase in the content of aspartate and a smaller decrease of GABA and glutamine. Other changes did not differ significantly from those observed in the absence of bromofuroate. We conclude that the metabolism of amino acids in general and of glutamic acid in particular differs according to whether they are already present within the brain slice or are added to the incubation medium. Only the endogenous amino acids appear to be able to serve as precursors of ammonia and as substrates for energy production.     

Locations of amino acids in brain slices from the rat. Tetrodotoxin-sensitive release of amino acids

1. Amino acids, particularly glutamate, gamma-aminobutyrate, aspartate and glycine, were released from rat brain slices on incubation with protoveratrine (especially in a Ca(2+)-deficient medium) or with ouabain or in the absence of glucose. Release was partially or wholly suppressed by tetrodotoxin. 2. Tetrodotoxin did not affect the release of glutamine under various incubation conditions, nor did protoveratrine accelerate it. 3. Protoveratrine caused an increased rate of formation of glutamine in incubated brain slices. 4. Increased K(+) in the incubation medium caused release of gamma-aminobutyrate, the process being partly suppressed by tetrodotoxin. 5. Incubation of brain slices in a glucose-free medium led to increased production of aspartate and to diminished tissue contents of glutamates, glutamine and glycine. 6. Use of tetrodotoxin to suppress the release of amino acids from neurons in slices caused by the joint action of protoveratrine and ouabain (the latter being added to diminish reuptake of amino acids), it was shown that the major pools of glutamate, aspartate, glycine, serine and probably gamma-aminobutyrate are in the neurons. 7. The major pool of glutamine lies not in the neurons but in the glia. 8. The tricarboxylic cycle inhibitors, fluoroacetate and malonate, exerted different effects on amino acid contents in, and on amino acid release from, brain slices incubated in the presence of protoveratrine. Fluoroacetate (3mm) diminished the content of glutamine, increased that of glutamate and gamma-aminobutyrate and did not affect respiration. Malonate (2mm) diminished aspartate and gamma-aminobutyrate content, suppressed respiration and did not affect glutamine content. It is suggested that malonate acts mainly on the neurons, and that fluoroacetate acts mainly on the glia, at the concentrations quoted. 9. Glutamine was more effective than glutamate as a precursor of gamma-aminobutyrate. 10. It is suggested that glutamate released from neurons is partly taken up by glia and converted there into glutamine. This is returned to the neurons where it is hydrolysed and converted into glutamate and gamma-aminobutyrate.     

The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

FLUCTUATIONS IN FREE AMINO ACID POOLS OF GYMNODINIUM SIMPLEX (DINOPHYCEAE) IN RESPONSE TO AMMONIA PERTURBATION: EVIDENCE FOR GLUTAMINE SYNTHETASE PATHWAY1,2

An ammonia limited chemostat culture of Gymnodinium simplex (Lohm.) Kofoid & Swezy was perturbed with ammonia and fluctuations in the free intracellular amino acid pools were followed 80 min. The steady-state value of glutamate was 2.07 ± 10^-15 mol cell^-1 and of glutamine was 0.31 ± 10^-15 mol cell^-1. Five minutes after the perturbation, a substantial rise in glutamine was observed with a corresponding decrease in glutamate. This is considered a result of glutamine synthetase acting as the primary ammonia assimilating enzyme. The level of ammonia and the major free amino acids reached a maximum 10 min after the perturbation and then slowly decreased.     

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.     

ALTERATION OF TRICARBOXYLIC ACID CYCLE METABOLISM IN RAT BRAIN SLICES BY HALOTHANE

Abstract—

• 1 Metabolism of [2-¹⁴C]pyruvate, [1-¹⁴C]acetate and [5-¹⁴C]citrate in the rat cerebral cortex slices was studied in the presence of halothane. Metabolites assayed include acetylcholine (ACh), citrate, glutamate, glutamine, γ-aminobutyrate (GABA) and aspartate. The trichloroacetic acid soluble extract, the trichloroacetic acid insoluble precipitate and its lipid extract were also studied.
• 2 In control experiments, pyruvate preferentially labelled ACh, citrate, glutamate, GABA and aspartate. Acetate labeled ACh, but to a lesser extent than pyruvate. Acetate also labeled lipids and glutamine. Citrate labeled lipids but not ACh and served as a preferential precursor for glutamine. These data support a three-compartment model for cerebral tricarboxylic acid cycle metabolism.
• 3 Halothane caused increases in GABA and aspartate contents and a decrease in ACh content. It has no effect on the contents of citrate, glutamate and glutamine.
• 4 Halothane preferentially inhibited the metabolic transfer of radioactivity from pyruvate into almost all metabolites, an effect probably not related to pyruvate permeability. This is interpreted as halothane depression of the‘large metabolic compartment’ which includes the nerve endings.
• 5 Halothane increased the metabolic transfer of radioactivity from acetate into lipids but did not alter such a transfer into the trichloracetic acid extract.
• 6 Halothane increased the metabolic transfer of radioactivity from citrate into the trichloroacetic acid precipitate, lipids and especially glutamine. Transfer of citrate radioactivity into GABA was somewhat decreased.
• 7 The differential effects of halothane on acetate and citrate utilization suggest that the ‘small metabolic compartment’ should be subdivided. Therefore, at least three metabolic compartments are demonstrated.
• 8 Halothane did not interfere with the dicarboxylic acid portion of the tricarboxylic acid cycle.

     

Metabolic fate of a high concentration of glutamine and glutamate in rat brain slices: a ¹³C NMR study

This study was performed to analyze the metabolic fate of a high concentration (5 mM) of glutamine and glutamate in rat brain slices and the participation of these amino acids in the glutamine-glutamate cycle. For this, brain slices were incubated for 60 min with [3-13C]glutamine or [3-13C]glutamate. Tissue plus medium extracts were analyzed by enzymatic and 13C NMR measurements and fluxes through pathways of glutamine and glutamate metabolism were calculated. We demonstrate that both substrates were utilized and oxidized at high rates by rat brain slices and served as precursors of neurotransmitters, tricarboxylic acid (TCA) cycle intermediates and alanine. In order to determine the participation of glutamine synthetase in the appearance of new glutamine molecules with glutamine as substrate, brain slices were incubated with [3-13C]glutamine in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase. Our results indicate that 36.5% of the new glutamine appeared was glutamine synthetase-dependent and 63.5% was formed from endogenous substrates. Flux through glutamic acid decarboxylase was higher with glutamine than with glutamate as substrate whereas fluxes from α-ketoglutarate to glutamate and through glutamine synthetase, malic enzyme, pyruvate dehydrogenase, pyruvate carboxylase and citrate synthase were in the same range with both substrates.     

Effects of Branched-Chain L-Amino Acids, L-Phenylalanine, and L-Methionine on the Transport of L-Glutamine in Rat Brain Cortex In Vitro. Influence of Cations

Abstract: Uptake of L-glutamine (2 mM) by rat brain cortex slices against a concentration gradient is markedly inhibited (40%) by branched-chain Lamino acids (1 mM), L-phenylalanine (1 mM), or L-methionine (1 mM); that of L-asparagine (2 mM) is much less affected by these amino acids. Other amino acids investigated have little or no effect on cerebral L-glutamine uptake. The suppressions of L-glutamine uptake by the inhibitory amino acids are apparently blocked by high [K⁺], which itself has little or no effect on glutamine uptake. This abolition of suppression is partly explained by high [K⁺] retention of endogenous glutamine; in the absence of Ca²⁺ such retention disappears. The inhibitory amino acids (1 mM) also enhance the release of endogenous glutamine, exogenous glutamine with which slices have been loaded, or glutamine synthesized in the slices from exogenous glutamate. The enhanced release of endogenous glutamine is diminished by high [K⁺]. The suppression of glutamine uptake by the branched-chain amino acids is independent of the concentration of glutamine at low concentrations (0.25–0.5 mM), indicating non-competition, but is reduced with high concentration of glutamine. The inhibition by L-phenylalanine is noncompetitive. L-Glutamine (2 mM) exerts no inhibition of the cerebral uptakes of the branched-chain L-amino acids or Lphenylalanine (0.25–2 mM). The inhibitory amino acids are as active in suppressing L-glutamine uptake with immature rat brain slices as with adult, although the uptake, against a gradient, of L-glutamine in the infant rat brain is about one-half that in the adult. They are also just as inhibitory on the concentrative uptake of L-glutamine by a crude synaptosomal preparation derived from rat brain cortex. Such a nerve ending preparation takes up L-glutamine (0.25 mM), against a gradient, at about ninefold the rate at which it is taken up by cortex slices (for equal amounts of protein), and the uptake process is markedly suppressed by high [K⁺] in contrast to the effects of high [K⁺] with slices. The possible physiological and pathological consequences of the suppression of glutamine uptake are discussed.     

HIGH AFFINITY UPTAKE OF l-GLUTAMINE IN RAT BRAIN SLICES

—l -Glutamine is taken up into rat brain slices by a specific‘high affinity’uptake system (K_m 52 μm ) which is not influenced by high concentrations of l -glutamate and l -asparagine. The uptake system appears to be associated with cellular structures that do not survive homogenization under conditions which yield synaptosomes. The‘high affinity’uptake of glutamine is dependent on the external sodium ion concentration and can be inhibited by p-chloromercuriphenylsulphonate, amino-oxyacetic acid, ouabain, dibenamine and allylglycine. The effects of several inhibitors indicate that l -asparagine uptake is mediated by a system different from the‘high affinity’system mediating l -glutamine uptake.     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

The Role of Glutamine Synthetase and Glutamate Dehydrogenase in Cerebral Ammonia Homeostasis

In the brain, glutamine synthetase (GS), which is located predominantly in astrocytes, is largely responsible for the removal of both blood-derived and metabolically generated ammonia. Thus, studies with [¹³N]ammonia have shown that about 25?% of blood-derived ammonia is removed in a single pass through the rat brain and that this ammonia is incorporated primarily into glutamine (amide) in astrocytes. Major pathways for cerebral ammonia generation include the glutaminase reaction and the glutamate dehydrogenase (GDH) reaction. The equilibrium position of the GDH-catalyzed reaction in vitro favors reductive amination of α-ketoglutarate at pH 7.4. Nevertheless, only a small amount of label derived from [¹³N]ammonia in rat brain is incorporated into glutamate and the α-amine of glutamine in vivo. Most likely the cerebral GDH reaction is drawn normally in the direction of glutamate oxidation (ammonia production) by rapid removal of ammonia as glutamine. Linkage of glutamate/α-ketoglutarate-utilizing aminotransferases with the GDH reaction channels excess amino acid nitrogen toward ammonia for glutamine synthesis. At high ammonia levels and/or when GS is inhibited the GDH reaction coupled with glutamate/α-ketoglutarate-linked aminotransferases may, however, promote the flow of ammonia nitrogen toward synthesis of amino acids. Preliminary evidence suggests an important role for the purine nucleotide cycle (PNC) as an additional source of ammonia in neurons (Net reaction: l-Aspartate?+?GTP?+?H₂O?→?Fumarate?+?GDP?+?P_i?+?NH₃) and in the beat cycle of ependyma cilia. The link of the PNC to aminotransferases and GDH/GS and its role in cerebral nitrogen metabolism under both normal and pathological (e.g. hyperammonemic encephalopathy) conditions should be a productive area for future research.     

EFFECTS OF SODIUM FLUOROACETATE ON THE METABOLISM OF N-ACETYLASPARTATE AND ASPARTATE IN MOUSE BRAIN

A subconvulsant dose of sodium fluoroacetate inhibited the metabolic utilization of intracerebrally-administered N-acetyl-l -[U-¹⁴C]asparticacid and the labelling of glutamine from this precursor in mouse brain, but not the labelling of glutamate or aspartate. A convulsant dose also inhibited the utilization of l -[U-¹⁴C]aspartic acid. When intraperitoneal injection of a convulsant dose of sodium fluoroacetate was followed by intracerebral injection of N-acetyl-l -[U-¹⁴C]asparticacid, the levels of N-acetylaspartate, aspartate and glutamate in brain were lowered, while the glutamine content was increased. The specific radioactivity of glutamine relative to that of glutamate was much lower when these compounds were labelled from l -[U-¹⁴C]aspartic acid than when N-acetyl-l -[U-¹⁴C]aspartic acid was used as the precursor. Intracerebral injection of tracer amounts of l -[U-¹⁴C]aspartic acid reduced the content of N-acetylaspartate in brain and raised the glutamine content. Sodium fluoroacetate had no additional effect on the relative specific radioactivity of glutamine or the content of N-acetylaspartate, aspartate, glutamate or glutamine when l -[U-¹⁴C]aspartic acid was the precursor. We consider the results to be consistent with a selective inhibition both by sodium fluoroacetate and by exogenous aspartic acid of the tricarboxylic acid cycle in brain associated with the biosynthesis of glutamine. We suggest that the activity of this pathway may regulate the metabolism of N-acetylaspartate and aspartate.     

Control of aerobic glycolysis in the brain in vitro

Protoveratrine-(5 M) stimulated aerobic glycolysis of incubated rat brain cortex slices that accompanies the enhanced neuronal influx of Na⁺ is blocked by tetrodotoxin (3 M) and the local anesthetics, cocaine (0.1 mM) and lidocaine (0.5 mM). On the other hand, high [K⁺]-stimulated aerobic glycolysis that accompanies the acetylcholine-sensitive enhanced glial uptakes of Na⁺ and water is unaffected by acetylcholine (2 mM). Experiments done under a variety of metabolic conditions show that there exists a better correlation between diminished ATP content of the tissue and enhanced aerobic glycolysis than between tissue ATP and the ATP-dependent synthesis of glutamine. Whereas malonate (2 mM) and amino oxyacetate (5 mM) suppress ATP content and O₂ uptake, stimulate lactate formation, but have little effect on glutamine levels, fluoroacetate (3 mM) suppresses glutamine synthesis in glia, presumably by suppressing the operation of the citric acid cycle, with little effect on ATP content, O₂ uptake, and lactate formation. Exogenous citrate (5 mM), which may be transported and metabolized in glia but not in neurons, inhibits lactate formation by cell free acetone-dried powder extracts of brain cortex but not by brain cortex slices. These results suggest that the neuron is the major site of stimulated aerobic glycolysis in the brain, and that under our experimental conditions glycolysis in glia is under lesser stringent metabolic control than that in the neuron. Stimulation of aerobic glycolysis by protoveratrine occurs due to diminution of the energy charge of the neuron as a result of stimulation of the sodium pump following tetrodotoxin-sensitive influx of Na⁺; stimulation by high [K⁺, NH₄ ⁺, or Ca²⁺ deprivation occurs partly by direct stimulation of key enzymes of glycolysis and partly by a fall in the tissue ATP concentration.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.     

THE DISTRIBUTION OF AMINO ACIDS, Na+ AND K+ FROM SURFACE TO CENTRE IN INCUBATED SLICES OF MOUSE BRAIN

—The effect of tissue damage on the uptake of amino acids by brain slices was investigated by measuring uptake in slices of different thickness and measuring the distribution of [¹⁴C]-labelled amino acid on the surface and in the centre of incubated slices. The uptake of glutamate, aspartate, and GABA was greater in 0.1 mm-thick slices than in 0.42 mm-thick slices in short and in long (up to 120 min) incubations; the uptake of other amino acids was equal or greater in the 0.42 mm-thick slices. The water content of incubated slices did not change greatly from surface to centre; inulin space was greater at the surface, and in slices from cortex, especially higher at the cut surface. Na⁺ and K⁺ concentrations were also higher at the surface. In the rest of the slice space, inulin, Na⁺ and K⁺ distribution was quite uniform. The distribution of ATP was inhomogeneous: in thinner slices the centre concentration was higher; in thicker slices the centre concentration was lower. Amino acid uptake initially (at 5 min) was higher at the surface, especially in the thicker slices; after longer time (30 min) incubation, the distribution of lysine and leucine was uniform, and glutamate uptake was greater at the surface. The inhomogeneity of distribution increased with increasing thickness of the slices. We concluded that the uptake of some amino acids (perhaps those for which, beside a low affinity transport, also a higher affinity transport system exists) is greater in thinner slices and greater on the surface of slices, and there is an initially inhomogeneous distribution during amino acid uptake. The uptake on the surface constitutes only a small portion of the total uptake, and tissue damage does not explain the greater uptake of amino acids by slices in comparison to the brain in vivo. This shows the higher transport capacity of cells in the brain and emphasizes the importance of mechanisms controlling the metabolite composition of the extracellular fluid in finally influencing the metabolite composition of the brain itself.     

Cortical metabolism in pyruvate dehydrogenase deficiency revealed by ex vivo multiplet C NMR of the adult mouse brain

The pyruvate dehydrogenase complex (PDC), required for complete glucose oxidation, is essential for brain development. Although PDC deficiency is associated with a severe clinical syndrome, little is known about its effects on either substrate oxidation or synthesis of key metabolites such as glutamate and glutamine. Computational simulations of brain metabolism indicated that a 25% reduction in flux through PDC and a corresponding increase in flux from an alternative source of acetyl-CoA would substantially alter the ¹³C NMR spectrum obtained from brain tissue. Therefore, we evaluated metabolism of [1,6-¹³C₂]glucose (oxidized by both neurons and glia) and [1,2-¹³C₂]acetate (an energy source that bypasses PDC) in the cerebral cortex of adult mice mildly and selectively deficient in brain PDC activity, a viable model that recapitulates the human disorder. Intravenous infusions were performed in conscious mice and extracts of brain tissue were studied by ¹³C NMR. We hypothesized that mice deficient in PDC must increase the proportion of energy derived from acetate metabolism in the brain. Unexpectedly, the distribution of ¹³C in glutamate and glutamine, a measure of the relative flux of acetate and glucose into the citric acid cycle, was not altered. The ¹³C labeling pattern in glutamate differed significantly from glutamine, indicating preferential oxidation of [1,2-¹³C]acetate relative to [1,6-¹³C]glucose by a readily discernible metabolic domain of the brain of both normal and mutant mice, presumably glia. These findings illustrate that metabolic compartmentation is preserved in the PDC-deficient cerebral cortex, probably reflecting intact neuron–glia metabolic interactions, and that a reduction in brain PDC activity sufficient to induce cerebral dysgenesis during development does not appreciably disrupt energy metabolism in the mature brain.