d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

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.     

Compartmentation of citric acid cycle metabolism in brain: labelling of glutamate, glutamine, aspartate and gaba by several radioactive tracer metabolites

—(1) Compartmentation of the metabolism of amino acids in brain has been studied in slices of cerebral cortex incubated with sodium [1-¹⁴C]acetate, sodium [1-¹⁴C]-bicarbonate, [1-¹⁴C]GABA or l-[1-¹⁴C]glutamate and in samples of brain after injection in vivo of [1-¹⁴C]- or [³H]acetate. (2) The method of treatment of the slices (a) maintained in ice-cold medium prior to incubation; (b) preincubation at 37°C and transfer to fresh medium affected the metabolism of the added, labelled substrate, particularly its labelling of glutamine. (3) The specific activity of glutamine labelled from the above metabolites was greater than that of glutamic acid in experiments of 10–30 minutes duration, whether or not subjected to pretreatment in the cold. (4) Incubation in medium containing 27 mm-K⁺ was associated with a decrease in the relative specific activity (RSA) of glutamine, except for the increase when l-[1-¹⁴C]glutamate was the precursor. (5) The data have been discussed in terms of metabolic compartmentation and their consistency with the concept of the presence in brain of more than one citric acid cycle, one containing the relatively smaller pools of intermediates and associated with synthetic processes; the other containing the relatively larger pools of intermediates and functioning as a homeostatic buffer for energy metabolism.     

METABOLISM OF MALONIC ACID IN RAT BRAIN AFTER INTRACEREBRAL INJECTION

Labeled malonic acid ([1-¹⁴C] and [2-¹⁴C]) was injected into the left cerebral hemisphere of anesthetized adult rats in order to determine the metabolic fate of this dicarboxylic acid in central nervous tissue. The animals were allowed to survive for 2, 5, 10. 15 or 30min. Blood was sampled from the torcular during the experimental period and labeled metabolites were extracted from the brain after intracardiac perfusion. There was a very rapid efflux of unreacted malonate in the cerebral venous blood. Labeled CO₂ was recovered from the venous blood and the respired air after the injection of [1-¹⁴C]malonate but not after [2-¹⁴C]malonate. The tissue extracts prepared from the brain showed only minimal labeling of fatty acids and sterols. Much higher radioactivity was present in glutamate, glutamine, aspartate, and GABA. The relative specific activities (RSA) of glutamine never rose above 1.00. Aspartate was labeled very rapidly and revealed evidence of ¹⁴CO₂ fixation in addition to labeling through the Krebs cycle. GABA revealed higher RSA after [1-¹⁴C]malonate than after [2-¹⁴C]malonate. Sequential degradations of glutamate and aspartate proved that labeling of these amino acids occurred from [1-¹⁴C] acetyl-CoA and [2-¹⁴C] acetyl-CoA, respectively, via the Krebs cycle. Malonate activation and malonyl-CoA decarboxylation in vivo were similar to experiments with isolated mitochondria. However, labeled malonate was not incorporated into the amino acids of free mitochondria. The results were compared to data obtained after intracerebral injection of [1-¹⁴C]acetate and [2-¹⁴C]acetate.     

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.     

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.     

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

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

Utilization in vivo of glucose and volatile fatty acids by sheep brain for the synthesis of acidic amino acids

1. Free glutamic acid, aspartic acid, glutamic acid from glutamine and, in some instances, the glutamic acid from glutathione and the aspartic acid from N-acetyl-aspartic acid were isolated from the brains of sheep and assayed for radioactivity after intravenous injection of [2-¹⁴C]glucose, [1-¹⁴C]acetate, [1-¹⁴C]butyrate or [2-¹⁴C]propionate. These brain components were also isolated and analysed from rats that had been given [2-¹⁴C]propionate. The results indicate that, as in rat brain, glucose is by far the best precursor of the free amino acids of sheep brain. 2. Degradation of the glutamate of brain yielded labelling patterns consistent with the proposal that the major route of pyruvate metabolism in brain is via acetyl-CoA, and that the short-chain fatty acids enter the brain without prior metabolism by other tissue and are metabolized in brain via the tricarboxylic acid cycle. 3. When labelled glucose was used as a precursor, glutamate always had a higher specific activity than glutamine; when labelled fatty acids were used, the reverse was true. These findings add support and complexity to the concept of the metabolic `compartmentation' of the free amino acids of brain. 4. The results from experiments with labelled propionate strongly suggest that brain metabolizes propionate via succinate and that this metabolic route may be a limited but important source of dicarboxylic acids in the brain.     

DECARBOXYLATION STUDIES OF GLUTAMATE, GLUTAMINE, AND ASPARTATE FROM BRAIN LABELLED WITH [1-14C] ACETATE, l-[U-14C]-ASPARTATE, AND l-[U-14C]GLUTAMATE

Studies in vivo and in vitro of the distribution of label in C-1 of glutamate and glutamine and C-4 of aspartate in the free amino acids of brain were carried out. [1-¹⁴C]-Acetate was used both in vivo and in vitro and l -[U-¹⁴C]aspartate and l -[U-¹⁴C]glutamate were used in vitro.

• 1 The results obtained with labelled acetate and aspartate suggest that CO₂ and a 3-carbon acid may exchange at different rates on a CO_a-fixing enzyme.
• 2 The apparent cycling times of both glutamate and glutamine show fast components measured in minutes and slow components measured in hours.
• 3 With [1-¹⁴C]acetate in vitro glutamine is more rapidly labelled in C-1 than is glutamate at early time points; the curves cross over at about 7 min.
• 4 The results support and extend the concept of metabolic compartmentation of amino acid metabolism in brain.

     

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.     

METABOLIC COMPARTMENTATION IN THE BRAIN: EFFECTS OF A CENTRAL NERVOUS SYSTEM DEPRESSANT, 1-HYDROXY-3-AMINO-PYRROLIDONE-2

—The effect of 1-hydroxy-3-aminopyrrolidone-2(HA-966), a CNS depressant, was studied on the metabolism of [¹⁴C]glucose and [³H]acetate in the brain in mice. HA-966 had a marked effect on glucose metabolism. The conversion of glucose carbon into amino acids associated with the tricarboxylic acid cycle (‘cycle’) was severely reduced, while the concentration of brain glucose was approximately doubled. Relative to the specific radioactivity of glucose in the brain, the specific radioactivity of alanine was 60–70 per cent of the control, indicating a reduction in the rate of glycolysis, and those of the‘cycle’amino acids were also lowered. A reduction in‘cycle’flux of 30–35 per cent was estimated. It was established that the depressed glucose utilization flux was not due to either impaired uptake of glucose from blood to brain or to hypothermia. In contrast to [¹⁴C]glucose, there was no change in the labelling of the amino acid fraction from [³H]acetate, which is preferentially metabolized in the 'small’compartment believed to be associated with glia. Thus it seems that CNS depression caused by HA-966 resulted in a selective decrease in energy production in the‘large’metabolic compartment where glucose is oxidized preferentially and which is believed to be associated with neuronal structures. The results also suggested that communication between the metabolic compartments mediated via glutamine and GABA was reduced, since the labelling from [³H]acetate of glutamine was increased and that of GABA decreased by HA-966.     

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.

     

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.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

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.     

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.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

Intermediary metabolism of carbon compounds by nitrifying bacteria

Summary The assimilation of¹⁴CO₂ and [2-¹⁴C] acetate, [3-¹⁴C] pyruvate, [5-¹⁴C] -ketoglutarate, [2,3-¹⁴C] succinate, [U-¹⁴C] glutamate and [U-¹⁴C] aspartate was followed in cell suspensions ofNitrosomonas europaea andNitrobacter agilis respectively. There was appreciable incorporation of these substrates even without adding the inorganic nitrogen compounds that are oxidized by these bacteria yielding ATP. In the soluble amino acid fraction most of¹⁴C label was recovered in glutamate while in the protein amino acids a more uniform distribution was found. Acetate was rapidly incorporated to a high level in both nitrifying bacteria while inNitrobacter there was a relatively lower uptake of the other substrates especially succinate. High levels of the NAD malate dehydrogenase and NADP isocitrate dehydrogenase were measured but no significant amounts of the other tricarboxylic acid cycle enzymes or NADH oxidase were found. Glutamate decarboxylase was detected in both organisms and the transferase assay for glutamine synthetase indicated a 30-fold higher activity for this enzyme inNitrobacter. The amino acid composition of the water soluble fraction was determined in both bacteria.     

Metabolism of [1,6-13C]Glucose and [U-13C]Glutamine and Depolarization Induced GABA Release in Superfused Mouse Cerebral Cortical Mini-slices

Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K⁺) release of preloaded [2,3-³H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K⁺) conditions from either [1,6-¹³C]glucose or [U-¹³C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 μM) or by replacing Ca²⁺ with Co²⁺. In the presence of both tiagabine and Co²⁺ (1 mM), release was abolished completely. The release observed in the presence of 25 μM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-¹³C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-¹³C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity. Special issue article in honor of Dr. Ricardo Tapia.