CHANGES WITHIN METABOLIC COMPARTMENTS IN THE BRAINS OF YOUNG RATS INGESTING LEAD

—[2-¹⁴C]Glucose and [³H]acetate were injected simultaneously into 19-day-old rats suckling from mothers fed either a normal diet or a diet containing 4·5% lead acetate. Changes in the rate of conversion of both precursors into amino acids associated with the tricarboxylic acid cycle were observed. [^I4C]Glucose. In the brain of young rats ingesting lead, the specific radioactivity of glutamate, aspartate, γ-aminobutyrate and glutamine were all significantly lowered relative to that of glucose. Glutamine labelling was the most affected. [³H]Acetate. In comparison with controls, the total amount of ³H in either water or acid-soluble constituents of the brain was the same, but the ³H content of the amino acids was significantly reduced in the lead-treated rats. In both groups, glutamine had the highest specific radioactivity but the time courses of the labelling of glutamine were different. In the control the peak incorporation was reached during the first 5 min, whereas in the experimental animals this occurred at about 10 min after the injection of the precursor, and the specific radioactivity even at that time was less than in controls. When compared with controls, the depression in the labelling of glutamine was accompanied at 5 min by an increase in the specific radioactivity of aspartate. In the lead-treated rats the labelling of GABA was also slowed and the time course seemed to follow that of glutamine rather than glutamate. In spite of the differences in the metabolism of [³H]acetate, metabolic compartmentation of glutamate, assessed by a glutamine : glutamate specific radioactivity ratio higher than 1, was evident even in the brain of the lead-treated animals, although the values of the ratio at 5 and 10 min were less than in controls. There was no evidence of a diminished supply of substrates to the brain in lead intoxication. The overall changes would be consistent with a retardation in the biochemical maturation of the brain in terms of development of glucose metabolism and metabolic compartmentation.     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

MANIFESTATION OF METABOLIC COMPARTMENTATION DURING THE MATURATION OF THE RAT BRAIN

—(1) The fate of [U-¹⁴C]leucine was studied in rat brain in vivo from birth to five weeks of age. The major route of leucine metabolism at all ages was conversion into protein. The rate of protein synthesis was low in the newborn; it reached a peak at about 15 days and slowed down moderately later. Incorporation into brain lipids was relatively low under the experimental conditions (less than 2 per cent of the total tissue ¹⁴C). (2) The conversion of leucine-carbon into amino acids associated with the tricarboxylic acid cycle was low in the first 9 days after birth (less than 4 per cent of the acid-soluble ¹⁴C at 10 min after injection) and increased rapidly until 15 days when the level characteristic of the adult was approached (about 20 per cent of the acid-soluble ¹⁴C). The results indicated that the oxidation of acetyl-CoA derived from leucine reached the adult level at an earlier age than that derived from glucose. (3) The glutamine/glutamate specific radioactivity ratio was 0·3 in the brain of newborn animals and increased progressively; it was 1·3 and 2·4 at 15 and 35 days of age respectively. The specific radioactivity of aspartate and of GABA relative to that of glutamate was less than 1 throughout the experimental period. (4) The factors involved in the development of metabolic compartmentation in brain were analysed. It is proposed that although the experimental results show that a 'small’compartment becomes functionally manifested with maturation the primary cause is the development of the‘large’metabolic compartment. (5) Morphological correlates of the metabolic compartments in brain tissue are suggested and it is concluded that the manifestation of metabolic compartmentation is related to maturational changes in glia-neuronal relations rather than to developmental processes affecting the individual components only.     

EFFECT OF UNDERNUTRITION ON AMINO ACID COMPARTMENTATION IN THE DEVELOPING RAT BRAIN

—Rat pups undernourished through 21 days of age show abnormal patterns of cerebral amino acid metabolism. The pattern of incorporation of radioactivity from l -[U-¹⁴C]leucine into amino acids derived from tricarboxylic acid cycle intermediates was altered, with significantly more ¹⁴C being incorporated into glutamate and aspartate in the underfed rats than in controls. Glutamate compartmentation, manifested in the ratio of specific radioactivities of glutamine to glutamate, developed more slowly in the. diet-restricted group. These results are similar to those seen in neonatally-thyroidectomized rats and suggest decreased growth of neuronal processes. This impairment of amino acid metabolism returns to normal after a 7-week period of adequate nutrition.     

THE METABOLISM OF [14C]GLUCOSE BY THE BRAINS OF SUCKLING RATS INTOXICATED WITH INORGANIC LEAD

—The development with age of the pattern of distribution of glucose carbon characteristic of the adult brain was studied in 7-, 13-, 19- and 24-day-old rats suckling from mothers maintained on a diet containing 4·5% lead acetate or on a normal diet. In normal rats the rapid and extensive conversion of glucose carbon into amino acids associated with the tricarboxylic acid cycle has been shown to develop in the cerebellum and the cerebral cortex during the same period as previously observed for the whole forebrain. A significant retardation was observed in both brain parts of rats suckling from mothers ingesting lead. In comparison with glutamate, aspartate and γ-aminobutyrate, the labelling of glutamine was particularly low. The concentration of glutamine was not affected in the forebrain, but it was elevated in the cerebellum. The age-dependent rise in the amounts of glutamate and aspartate was also retarded, but it would appear that the contribution of this effect to the depressed labelling of amino acids was small. There was no evidence of impaired entry of glucose into the brain from the blood. Although in comparison with undernourished animals, the growth retardation of the lead-treated rats is similar, several of the effects observed on the developing brain seem to be distinct.     

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.     

Changes in Amino Acid Content and the Metabolism of γ-Aminobutyrate in Cucurbita moschata Seedlings

Ungerminated pumpkin (Cucurbita moschata Poir.) cotyledons contained 30 % of their dry weight as lipid and 26 % as protein, of which 93 % was globulin. There was a rapid degradation of these reserves 4 to 8 days after planting when the cotyledons had their maximum metabolic activity. About half of the mole percent of amino acids found in the globulin reserve was in arginine, glutamate, aspartate, and their amides. The cotyledons had a large soluble pool of arginine, glutamine, glutamate, and leucine. Most amino acids increased steadily in amount in the cotyledons during germination, except glutamine, ornithine, alanine, serine, glycine, and γ-aminobutyrate and these appeared in large amounts in the translocation stream to the axis tissue. Little arginine or proline was translocated. By 10 days, when translocation had decreased, amino acids accumulated. Ornithine, γ-aminobutyrate, and aspartate were rapidly utilized in the hypocotyl, while glutamine, glycine, and alanine accumulated there. Cysteine and methionine levels were low in the reserve, trans-location stream and soluble fractions. γ-Aminobutyrate-U^?14C injected into cotyledons or incubated with hypocotyls was utilized in a similar fashion. The label appeared in citric acid cycle acids and in the amino acids closely related to this cycle, but the bulk of the label appeared in CO₂. The labeling pattern suggests that γ-aminobutyrate was utilized via succinate, and thus entered the citric acid cycle. A close relationship between arginine, ornithine, glutamate, and γ-aminobutyrate exists in the cotyledon with all but arginine being translocated rapidly to the axis tissue where these amino acids are rapidly metabolized.     

Metabolic changes associated with adaptation of plant cells to water stress

Suspension cultured cells of tomato (Lycopersicon esculentum Mill. cv VFNT Cherry) adapted to water stress induced with polyethylene glycol 6000 (PEG), exhibit marked alterations in free amino acid pools (Handa et al. 1983 Plant Physiol 73: 834-843). Using computer simulation models the in vivo rates of synthesis and utilization and compartmentation of free amino acid pools were determined from ¹⁵N labeling kinetics after substituting [¹⁵N]ammonium and [¹⁵N]nitrate for the ¹⁴N salts in the culture medium of cell lines adapted to 0% and 25% PEG. The 300-fold elevated proline pool in 25% PEG adapted cells is primarily the consequence of a 10-fold elevated rate of proline synthesis via the glutamate pathway. Ornithine was insufficiently labeled to serve as a major precursor for proline. Our calculations suggest that the rate of proline synthesis only slightly exceeds the rate required to sustain both protein synthesis and proline pool maintenance with growth. Mechanisms must operate to restrict proline oxidation in adapted cells. The kinetics of labeling of proline in 25% PEG adapted cells are consistent with a single, greatly enlarged metabolic pool of proline. The depletion of glutamine in adapted cells appears to be a consequence of a selective depletion of a large, metabolically inactive storage pool present in unadapted cultures. The labeling kinetics of the amino nitrogen groups of glutamine and glutamate are consistent with the operation of the glutamine synthetase-glutamate synthase cycle in both cell lines. However, we could not conclusively discriminate between the exclusive operation of the glutamine synthetase-glutamate synthase cycle and a 10 to 20% contribution of the glutamate dehydrogenase pathway of ammonia assimilation. Adaptation to water stress leads to increased nitrogen flux from glutamate into alanine and γ-aminobutyrate, suggesting increased pyruvate availability and increased rates of glutamate decarboxylation. Both alanine and γ-aminobutyrate are synthesized at rates greatly in excess of those simply required to maintain the free pools with growth, indicating that these amino acids are rapidly turned over. Thus, both synthesis and utilization rates for alanine and γ-aminobutyrate are increased in adapted cells. Adaptation to stress leads to increased rates of synthesis of valine and leucine apparently at the expense of isoleucine. Remarkably low ¹⁵N flux via the aspartate family amino acids was observed in these experiments. The rate of synthesis of threonine appeared too low to account for threonine utilization in protein synthesis, pool maintenance, and isoleucine biosynthesis. It is possible that isoleucine may be deriving carbon skeletons from sources other than threonine. Tentative models of the nitrogen flux of these two contrasting cell lines are discussed in relation to carbon metabolism, osmoregulation, and nitrogenous solute compartmentation.     

Purification and characterization of cytosolic aldolase from carrot storage root.

The time courses of incorporation of 13C from 13C-labelled glucose or acetate into cerebral amino acids (glutamate, glutamine and 4-aminobutyrate) and lactate were monitored by using 13C-n.m.r. spectroscopy. When [1-13C]glucose was used as precursor the C-2 of 4-aminobutyrate was more highly labelled than the analogous C-4 of glutamate, whereas no label was observed in glutamine. A similar pattern was observed with [2-13C]glucose: the C-1 of 4-aminobutyrate was more highly labelled than the analogous C-5 of glutamate. Again, no labelling of glutamine was detected. In contrast, [2-13C]acetate labelled the C-4 of glutamine and the C-2 of 4-aminobutyrate more highly than the C-4 of glutamate; [1-13C]acetate also labelled the C-1 and C-5 positions of glutamine more than the analogous positions of glutamate. These results are consistent with earlier patterns reported from the use of 14C-labelled precursors that led to the concept of compartmentation of neuronal and glial metabolism and now provide the possibility of distinguishing differential effects of metabolic perturbations on the two pools simultaneously. An unexpected observation was that citrate is more highly labelled from acetate than from glucose.     

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.     

EFFECT OF THYROID HORMONE ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: CONVERSION OF GLUCOSE-CARBON INTO AMINO ACIDS

• 1 The rapid and extensive conversion of glucose-carbon into amino acids is an index of the final coordination of the mechanisms underlying energy metabolism in the adult brain. This phenomenon develops in the rat during a short period extending from 10 to about 19 days after birth. The underlying factors have been analysed.

• 2 The development of the pattern of distribution of glucose-carbon characteristic of the adult brain was markedly influenced by the thyroid state of the animals. The age-curve for the conversion of glucose-carbon into brain amino acids was displaced to the left after treatment with thyroid hormone (T₃) in infancy thus indicating an accelerated maturation. Conversely, neonatal thyroidectomy resulted in a significant retardation in the conversion of glucose-carbon into amino acids.

• 3 The specific radioactivity of glutamate increased five-fold in the brain of normal rats from the 10th to the 19th day of age. The values (as a percentage of those for littermate controls) were 220 in the case of the 10 day-old thyroid treated rats and about 30 for the 19 day-old thyroid deficient animals. At the age of 10 days neither treatment affected the concentration of glutamate which was also only slightly less than the control values in the brain of 19 day-old thyroid deficient animals (–17 per cent).

• 4 Specific pool(s) of glutamate associated with the formation of GABA can be demonstrated in the brain of 19 day-old rats after administration of [U-¹⁴C]glucose as a result of anoxia post mortem. These pools did not develop in the brain of 10 day-old animals. Neonatal thyroidectomy retarded the development of these glutamate pools.

• 5 Evidence is summarized which indicates that the development of the rapid conversion of glucose-carbon into amino acids reflects the enlargement, during maturation, of the metabolic compartments which are associated with neuronal processes.

     

METABOLISM OF GLUCOSE AND FREE AMINO ACIDS IN BRAIN, STUDIED WITH 14C-LABELLED GLUCOSE AND BUTYRATE IN RATS INTOXICATED WITH CARBON DISULPHIDE

Abstract— The effect of 15 h continuous exposure to CS₂ on the metaboliam of glucose and free amino acids in the brain of rats was studied. CS₂ caused a moderate hypoglycaemia. There were also changes in the amounts of some amino acids in the brain. Glutamate and γ-aminobutyrate were lower whereas glutamine was markedly increased. Comparative studies in vivo of the metabolism of [2-¹⁴C]glucose and [1-¹⁴C]butyrate indicated that CS₂ did not affect glycolysis or the incorporation of ¹⁴C from glucose into amino acids except into γ-aminobutyrate which was reduced. Contrary to the findings with [¹⁴C]glucose, CS₂ provoked distinct changes in the labelling of amino acids when [¹⁴C]butyrate was the precursor. The most notable change was a markedly increased incorporation of ¹⁴C into glutamine. Based on the two-compartment model of brain glutamate the experimental findings indicated that CS₂ affected metabolism associated with the 'small' pool of glutamate but had a minimal effect on metabolism associated with the 'large' glutamate pool. The possibility is suggested that the changes observed involved an increased rate of ammonia removal. The low incorporation of ¹⁴C into γ-aminobutyrate from either precursor is consistent with other evidence showing that CS₂ interferes with pyridoxal phosphate-dependent enzymes.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.     

DECREASED METABOLISM IN VIVO OF GLUCOSE INTO AMINO ACIDS OF THE BRAIN OF THIAMINE-DEFICIENT RATS AFTER TREATMENT WITH PYRITHIAMINE

Abstract— Thiamine deficiency produced by administration of pyrithiamine to rats maintained on a thiamine-deficient diet resulted in a marked disturbance in amino acid and glucose levels of the brain. In the two pyrithiamine-treated groups of rats (Expt. A and Expt. B) there was a significant decrease in the levels of glutamate (23%, 9%) and aspartate (42%, 57%), and an increase in the levels of glycine (26%, 27%) in the brain, irrespective of whether the animals showed signs of paralysis (Expt. A) or not (Expt. B). as a result of thiamine deficiency. A significant decrease in the levels of γ-aminobutyrate (22%) and serine (28%) in the brain was also observed in those pyrithiamine-treated rats which showed signs of paralysis (Expt. A). Threonine content increased by 57% in Expt. A and 40% in Expt. B in the brain of pyrithiamine-treated rats, but these changes were not statistically significant. The utilization of [U-¹⁴C]glucose into amino acids decreased and accumulation of glucose and [U-¹⁴C]glucose increased significantly in the brain after injection of [U-¹⁴C]glucose to pyrithiamine-treated rats which showed abnormal neurological symptoms (Expt. A). The decrease in ¹⁴C-content of amino acids was due to decreased conversion of [U-¹⁴C]glucose into alanine, glutamate, glutamine, aspartate and γ-aminobutyrate. The flux of [¹⁴C]glutamate into glutamine and γ-aminobutyrate also decreased significantly only in the brain of animals paralysed on treatment with pyrithiamine. The decrease in the labelling of, amino acids was attributed to a decrease in the activities of pyruvate dehydrogenase and α-oxoglutarate dehydrogenase in the brain of pyrithiamine-treated rats. The measurement of specific radioactivity of glucose, glucose-6-phosphate and lactate also indicated a decrease in the activities of glycolytic enzymes in the brain of pyrithiamine-treated animals in Expt. A only. It was suggested that an alteration in the rate of oxidation in vivo of pyruvate in the brain of thiamine-deficient rats is controlled by the glycolytic enzymes, probably at the hexokinase level. The lack of neurotoxic effect and absence of significant decrease in the metabolism of [U-¹⁴C]glucose in the brain of pyrithiamine-treated animals in Expt. B were probably due to the fact that animals in Expt. B were older and weighed more than those in Expt. A, both at the start and the termination of the experiments.     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

INTRACELLULAR AMINO ACID CONTENT OF NEURONAL, GLIAL, AND NON-NEURAL CELL CULTURES: THE RELATIONSHIP TO GLUTAMIC ACID COMPARTMENTATION

Abstract— A protocol for the accurate determination of intracellular levels of amino acids in tissue cultured cells has been developed and used in the measurement of intracellular amino acids levels in neuronal, glial, and non-neural cell lines, with the objective of establishing morphological correlates for large and small glutamic acid compartments and of examining hypotheses for the morphological basis of glutamic acid compartmentation. This survey of intracellular amino acid levels has revealed striking differences among the cell lines tested, but these differences did not correlate with cell type, i.e. neuronal vs glial, in contrast to earlier results (R ose , 1968) based on bulk separated neuronal and pial fractions from rat brain. Amino acid levels were found to be dependent upon tissue culture conditions, yet reproducible differences could be observed when growth and experimental conditions were carefully controlled. Glutamic acid levels for various cell lines ranged from 50.8 ± 14.3 to 158 ± 8.5 nmol/mg protein. Intracellular glutamine levels demonstrated even greater difference, with values ranging from 0.8 ± 0.2 to 107 ± 42.4 nmol/mg protein. Statistically significant differences in intracellular amino acid levels between cell lines were also observed for aspartic acid, praline, glycine, alanine, valine, cystathionine, isoleucine, and leucine. A number of cell lines demonstrating highly elevated elevated levels of γ-aminobutyrate and β-alanine were identified. The significance of neuronal and glial levels of glutamic acid, glutamine and γ-aminobutyrate to models for glutamic acid compartmentation is discussed.     

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).     

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.