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

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

Localized 13C NMR Spectroscopy in the Human Brain of Amino Acid Labeling from d-[1-13C]Glucose

Abstract: Cerebral metabolism of d [1-¹³C]glucose was studied with localized ¹³C NMR spectroscopy during intravenous infusion of enriched [1-¹³C]glucose in four healthy subjects. The use of three-dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time-resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4-¹³C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3-¹³C]glutamate clearly lagged behind that of [4-¹³C]glutamate and peaked at t = 110–140 min. Multiplets due to homonuclear ¹³C-¹³C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a ¹³C isotopic fraction at C4 glutamate of 27 ± 2% (n = 4), which was slightly less than one-half the enrichment of the C1 position of plasma glucose (63 ± 1%), p < 0.05. By comparison with an external standard the total amount of [4-¹³C]glutamate was directly quantified to be 2.4 ± 0.1 µmol/ml-brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 ± 0.7 µmol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in healthy human brain.     

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.     

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

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

Metabolism of Acetate to Amino Acids in Brains of Rats After Complete Hepatectomy

Abstract: The concentration of glutamine increases in the brain after hepatectomy. In the present studies the conversion of intravenously given [¹⁴C]acetate to [¹⁴C]glutamate and [¹⁴C]glutamine was studied in control rats and in rats at 6 h after complete hepatectomy. The incorporation of label into glutamate was only slightly inhibited, but the further incorporation into glutamine was greatly inhibited, after hepatectomy. These data, and previous data using [¹⁴C]glucose as precursor, indicate that synthesis of glutamine in brain is inhibited after hepatectomy, and suggest that its concentration must increase because degradation is inhibited to an even greater extent.     

Portacaval Anastomosis Results in Altered Neuron-Astrocytic Metabolic Trafficking of Amino Acids: Evidence from 13C-NMR Studies

Abstract: ¹³C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-¹³C]glucose, whereas [2-¹³C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-¹³C]glucose as precursor. Brain [1-¹³C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The ¹³C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-¹³C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

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

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

Selective Inhibition of the Tricarboxylic Acid Cycle of GABAergic Neurons with 3-Nitropropionic Acid In Vivo

Abstract: The effects of 3-nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase, on cerebral metabolism were investigated in mice by NMR spectroscopy. 3-NPA, 180 mg/kg, caused a dramatic buildup of succinate. Succinate was labeled 5.5 times better from [1-¹³C]glucose than from [2-¹³C]acetate, showing a predominantly neuronal accumulation. [1-¹³C]Glucose labeled GABA in the C-2 position only, compatible with inhibition of the tricarboxylic acid (TCA) cycle associated with GABA formation, at the level of succinate dehydrogenase. Aspartate was not labeled by [1-¹³C]glucose in 3-NPA-intoxicated animals. In contrast, [1-¹³C]glucose labeled glutamate in the C-2, C-3, and C-4 positions showing uninhibited cycling of label in the TCA cycle associated with the large, neuronal pool of glutamate. The labeling of glutamine, and hence GABA, from [2-¹³C]acetate showed that the TCA cycle of glial cells was unaffected by 3-NPA and that transfer of glutamine from glia to neurons took place during 3-NPA intoxication. The high ¹³C enrichment of the C-2 position of glutamine from [1-¹³C]glucose showed that pyruvate carboxylation was active in glia during 3-NPA intoxication. These findings suggest that 3-NPA in the initial phase of intoxication fairly selectively inhibited the TCA cycle of GABAergic neurons; whereas the TCA cycle of glia remained uninhibited as did the TCA cycle associated with the large neuronal pool of glutamate, which includes glutamatergic neurons. This may help explain why the caudoputamen, which is especially rich in GABAergic neurons, selectively undergoes degeneration both in humans and animals intoxicated with 3-NPA. Further, the present results may be of relevance for the study of basal ganglia disorders such as Huntington's disease.     

Exogenous Glutamate Concentration Regulates the Metabolic Fate of Glutamate in Astrocytes

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

Brain Carbohydrate Metabolism in Developing Rats During Hypercapnia

Abstract: Brain glucose metabolism was studied in developing rats at ages 10 and 20 days postnatal under normal and hypercapnic conditions. Brains were removed and frozen within 1 s with a freeze-blowing apparatus. Glucose utilization was measured with [2-¹⁴C]glucose and [³H]deoxyglucose as tracers. Metabolites were determined by standard enzymatic techniques. Data from [³H]deoxyglucose phosphorylation indicated that normal brain glucose utilization increased almost threefold between the 10th and 20th postnatal days. From the relative rates of utilization of the two isotopes in the 20-day-old control group, it appeared that about 25% of ¹⁴C label derived from metabolism of [2-¹⁴C]glucose was lost from brain (probably as lactate) rather than entering the Krebs cycle. Under hypercapnic conditions (20% CO₂-21% O₂-59% N₂), rates of glucose utilization by brain were decreased by one-half at both ages and there were progressive decreases in the concentrations of many intermediary metabolites. The bases for concluding that these metabolites were used to supplement glucose as a fuel for respiration, rather than being lost by leakage into blood, are discussed. Despite the differences in brain glucose metabolism between 10-day-old and 20-day-old rats, their responses to hypercapnia are remarkably similar: Rates of glucose utilization are reduced to approximately the same proportion of the original rate by 20% CO₂, and endogenous metabolites (particularly glutamate and lactate) appear to be oxidized as replacement fuels.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

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

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

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).     

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2-¹³C]acetate and [1-¹³C]glucose as shown by ¹³C NMR spectroscopy. In fasted mice that had received [2-¹³C]acetate intravenously, brain lactate C-2 and C-3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1-¹³C]glucose strongly labeled lactate C-3, reflecting glycolysis, whereas lactate C-2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate-metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2-¹³C]acetate reached ∼1% in both the C-2 and the C-3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C-2 and C-3 from [2-¹³C]acetate, whereas in fed mice, lactate C-3 was more highly labeled than the C-2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.     

Metabolic Precursors and Compartmentation of Cerebral GABA in Vigabatrin-Treated Rats

Abstract: The metabolic precursors and cerebral compartmentation of the augmented GABA pool induced by vigabatrin, an irreversible inhibitor of GABA transaminase, have been investigated by ¹³C NMR. Adult rats receiving rat chow ad libitum were given either drinking water only or drinking water containing 2.5 g/L vigabatrin for 7 days. Both groups of animals were infused either with [1,2-¹³C₂]acetate (15 µmol/min/100 g body weight), an exclusive precursor of GABA formation through the glial glutamine pathway, or with [1,2-¹³C₂]glucose (15 µmol/min/100 g body weight), a substrate that can produce GABA through the glial glutamine pathway or by direct metabolism in the neurons. The brains were frozen in situ, extracted with perchloric acid, and analyzed by ¹³C NMR. In vigabatrin-treated animals [¹³C]glutamine, a common intermediate for [¹³C]GABA synthesis from glucose or acetate, was accumulated to similar amounts during infusions with [1,2-¹³C₂]glucose or [1,2-¹³C₂]acetate. However, [¹³C]GABA accumulation was sevenfold higher during [1,2-¹³C₂]glucose infusions or twofold higher during [1,2-¹³C₂]acetate infusions. These results show that the direct pathway of GABA formation by neuronal metabolism of glucose predominates over the alternative pathway through glial glutamine. Near-equilibrium relationships of the aminotransferases of GABA and aspartate imply that the observed [¹³C]GABA accumulation occurs initially in the neuronal compartment.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

Incorporation of label from root-applied N6[8-14C]furfiuryIadenine into the guanine nucleotide fraction of pea bud ribonucleic acid

The pattern of incorporation of label into the nucleotides of axillary bud ribonucleic acid was investigated in Pisum sativum L. cv. Meteor following the application of N ⁶[8-^I4C]furfuryladenine or of [8-¹⁴C]adenine to the root system of decapitated plants and to cultured excised buds. When N ⁶[8-¹⁴C]furifaryladenine was applied to the root system label was confined to the guanine nucleotide moiety of the axillary bud ribonucleic acid; label from [8-¹⁴C]adenine was incorporated preferentially into adenine nucleotide in the molar ratio adenine nucleotide/guanine nucleotide = 3.23. When isolated buds were incubated in media containing [8-¹⁴C]adenine or N ⁶[8-¹⁴C]furfuryladenine, label was incorporated into both purine moieties of the ribonucleic acid. However, the relative incorporation into the guanine nucleotide fraction was considerably greater for N ⁶[8-^I4C]furfuryladenine (adenine nucleotide/guanine nucleotide = 2.23) than for [8-¹⁴C]adenine (ratio = 4.67).
It was concluded that the pattern of metabolism of adenine to guanine and its incorporation into the guanine nucleotide moiety of pea axillary bud ribonucleic acid, is influenced by the presence of a substitution in the N ⁶ position of the adenine base.     

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

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

Mammalian cerebral metabolism and amino acid neurotransmission during hibernation

This report demonstrates that during the torpor phase of hibernation, hamsters utilize ¹⁴C and ¹³C glucose in torpor-specific brain metabolic pathways. Microdialysis of ¹⁴C glucose into the striatum rapidly induced a steady state labeling of extracellular fluid (ECF) lactate and labeling of tissue GABA, glutamate, glutamine, and alanine in ipsilateral and contralateral striata. The same tissue metabolites were labeled in cortex, hypothalamus, and brainstem after microdialysis of ¹⁴C lactate into the lateral ventricle. Serine, aspartate, glycine, taurine, tyrosine, and methionine were not synthesized from glucose or lactate during torpor. ECF levels of amino and organic acids were low and unchanging during torpor and increased late during arousal to cenothermia. Labeled intracellular ¹⁴C GABA and glutamate were not communicated to the striatal ECF or ventricular space during torpor. ¹³C NMR demonstrated rapid formation of lactate and functional tricarboxylic acid cycles in GABAergic and glutamatergic neurons, and enrichment of glutamine and alanine after i.v. ¹³C glucose. Large changes in tissue levels of amino acids occur prior to or during entrance into torpor but not during torpor. It is proposed that cerebral intracellular dehydration, the enlargement of ECF and the biochemistries associated with brain water homeostasis may have a role in regulating hibernation.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.