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.     

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

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

Release-Regulating Serotonin 5-HT1D Autoreceptors in Human Cerebral Cortex

Abstract: Primary cultures of cerebral cortical astrocytes were incubated with [U^-13C]glutamate (0.5 m M ) in modified Dulbecco's medium for 2 h. Perchloric acid (PCA) extracts of the cells as well as redissolved lyophilized media were subjected to NMR spectroscopy to identify ¹³C-labeled metabolites. NMR spectra of the PCA extracts exhibited distinct multiplets for glutamate, aspartate, glutamine, and malate. The culture medium showed peaks for a multitude of compounds released from the astrocytes, among which lactate, glutamine, alanine, and citrate were readily identifiable. For the first time incorporation of label into lactate from glutamate was clearly demonstrated by doublet formation in the C-3 position and two doublets in the C-2 position of lactate. This labeling pattern can only occur by incorporation from glutamate, because natural abundance will only produce singlets in proton-decoupled ¹³C spectra. Glutamine, released into the medium, was labeled uniformly to a large extent, but the C-3 position not only showed the expected apparent triplet but also a doublet due to ¹³C incorporation into the C-4 position of glutamine. The doublet accounted for 11% of the total label in the glutamine synthesized and released within the incubation period. The corresponding labeling pattern of [¹³C]glutamate in the PCA extracts showed that 19% of the glutamate contained ¹²C. Labeling of lactate, citrate, malate, and aspartate as well as incorporation of ¹²C into uniformly labeled glutamate and glutamine could only arise via the tricarboxylic acid cycle. The relative amount of glutamate metabolized via this route is at least 70% as calculated from the areas of the C-3 resonances of these compounds. Only a maximum of 30% was converted to glutamine directly.     

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.     

In vivo neurochemical profiling of rat brain by 1H-[13C] NMR spectroscopy: cerebral energetics and glutamatergic/GABAergic neurotransmission

The quantification of excitatory and inhibitory neurotransmission and the associated energy metabolism is crucial for a proper understanding of brain function. Although the detection of glutamatergic neurotransmission in vivo by ¹³C NMR spectroscopy is now relatively routine, the detection of GABAergic neurotransmission in vivo has remained elusive because of the low GABA concentration and spectral overlap. Using ¹H-[¹³C] NMR spectroscopy at high magnetic field in combination with robust spectral modeling and the use of different substrates, [U-¹³C₆]-glucose and [2-¹³C]-acetate, it is shown that GABAergic, as well as glutamatergic neurotransmitter fluxes can be detected non-invasively in rat brain in vivo .     

ALANINE METABOLISM IN RAT CORTEX IN VITRO

Abstract— (1) The metabolism of [U-¹⁴C]alanine was followed in slices of rat cerebral cortex and its interaction with glucose, pyruvate and the metabolic inhibitors fluoracetate and malonate was studied.
(2) Alanine did not stimulate respiration above endogenous levels or affect the rate of oxygen uptake with glucose or pyruvate as cosubstrate. Radioactivity found in CO₂ from labelled alanine was only 6 per cent of that from labelled pyruvate. Lactate was not formed from alanine.
(3) After 2 h incubation with [U-¹⁴C]alanine the specific activities of glutamate, glutamine and GABA were 20–30 per cent that of alanine. All these specific activities except glutamate were lowered by addition of glucose, but with pyruvate as cosubstrate the specific activity of glutamate was increased by 87 per cent above the level with alanine alone.
(4) The effect of alanine as cosubstrate with [U-¹⁴C]pyruvate was to reduce the specific activity of GABA and of glutamine, but not glutamate or lactate; thus there was not an equal dilution of all the metabolites of pyruvate.
(5) Fluoracetate diminished respiration and the production of CO₂ from [U-¹⁴C]-alanine only slightly; the addition of malonate as well practically abolished both. Fluoracetate lowered incorporation from alanine into all the amino acids, and radioactivity could not be detected in glutamine at all; addition of malonate lowered the specific activity of glutamate to 25 per cent but increased that into aspartate, GABA and glutamine.
(6) The interpretation of these data in terms of known pathways of alanine metabolism is discussed.     

Carbon metabolism of Listeria monocytogenes growing inside macrophages

The intracellular metabolism of Listeria monocytogenes was studied by ¹³C-isotopologue profiling using murine J774A.1 macrophages as host cells. Six hours after infection, bacteria were separated from the macrophages and hydrolyzed. Amino acids were converted into tert-butyl-dimethylsilyl derivatives and subjected to gas chromatography/mass spectrometry. When the macrophages were supplied with [U-¹³C₆]glucose prior to infection, but not during infection, label was detected only in Ala, Asp and Glu of the macrophage and bacterial protein with equal isotope distribution. When [U-¹³C₆]glucose was provided during the infection period, ¹³C label was found again in Ala, Asp and Glu from host and bacterial protein, but also in Ser, Gly, Thr and Val from the bacterial fraction. Mutants of L. monocytogenes defective in the uptake and catabolism of the C₃-metabolites, glycerol and/or dihydroxyacetone, showed reduced incorporation of [U-¹³C₆]glucose into bacterial amino acids under the same experimental settings. The ¹³C pattern suggests that (i) significant fractions (50–100%) of bacterial amino acids were provided by the host cell, (ii) a C₃-metabolite can serve as carbon source for L. monocytogenes under intracellular conditions and (iii) bacterial biosynthesis of Asp, Thr and Glu proceeds via oxaloacetate by carboxylation of pyruvate.     

The metabolic role of isoleucine in detoxification of ammonia in cultured mouse neurons and astrocytes

Cerebral hyperammonemia is a hallmark of hepatic encephalopathy, a debilitating condition arising secondary to liver disease. Pyruvate oxidation including tricarboxylic acid (TCA) cycle metabolism has been suggested to be inhibited by hyperammonemia at the pyruvate and -ketoglutarate dehydrogenase steps. Catabolism of the branched-chain amino acid isoleucine provides both acetyl-CoA and succinyl-CoA, thus by-passing both the pyruvate dehydrogenase and the -ketoglutarate dehydrogenase steps. Potentially, this will enable the TCA cycle to work in the face of ammonium-induced inhibition. In addition, this will provide the -ketoglutarate carbon skeleton for glutamate and glutamine synthesis by glutamate dehydrogenase and glutamine synthetase (astrocytes only), respectively, both reactions fixing ammonium. Cultured cerebellar neurons (primarily glutamatergic) or astrocytes were incubated in the presence of either [U-¹³C]glucose (2.5 mM) and isoleucine (1 mM) or [U-¹³C]isoleucine and glucose. Cell cultures were treated with an acute ammonium chloride load of 2 (astrocytes) or 5 mM (neurons and astrocytes) and incorporation of ¹³C-label into glutamate, aspartate, glutamine and alanine was determined employing mass spectrometry. Labeling from [U-¹³C]glucose in glutamate and aspartate increased as a result of ammonium-treatment in both neurons and astrocytes, suggesting that the TCA cycle was not inhibited. Labeling in alanine increased in neurons but not in astrocytes, indicating elevated glycolysis in neurons. For both neurons and astrocytes, labeling from [U-¹³C]isoleucine entered glutamate and aspartate albeit to a lower extent than from [U-¹³C]glucose. Labeling in glutamate and aspartate from [U-¹³C]isoleucine was decreased by ammonium treatment in neurons but not in astrocytes, the former probably reflecting increased metabolism of unlabeled glucose. In astrocytes, ammonia treatment resulted in glutamine production and release to the medium, partially supported by catabolism of [U-¹³C]isoleucine. In conclusion, i) neuronal and astrocytic TCA cycle metabolism was not inhibited by ammonium and ii) isoleucine may provide the carbon skeleton for synthesis of glutamate/glutamine in the detoxification of ammonium.     

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

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

Metabolic 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.     

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.     

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.     

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

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

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

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

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

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

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.     

Glutamine--a major substrate for nerve endings

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

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.     

[14C]AMINO ACID FORMATION FROM LABELLED GLUCOSE AND/OR ACETATE IN BRAIN CORTEX SLICES WITH EXPERIMENTALLY ELICITED PROLIFERATION OF ASTROGLIA. CORRELATION OF BIOCHEMICAL AND MORPHOLOGICAL CHANGES

Abstract— Changes in morphology and in transformations of [U-¹⁴C]glucose and [1-¹⁴C]acetate into amino acids of the brain cortex were followed on the Sth, 10th and 21st days after production of mechanical lesions and compared with control tissue. In the experimental tissue, proliferation of astroglia and reduction of the number of neurons had taken place. On the 10th day, accumulation of mitochondria and of some gliofilaments in the cytoplasm of astroglia was observed. On the 21st day, the gliofilaments occupied a substantial portion of the astroglial cytoplasm and the mitochondria were reduced in number and compressed to the cell membrane. Incorporation of ¹⁴C from acetate into amino acids was substantially increased on the 10th day (up to 240% with respect to controls) and normalized again on the 21st day. Incorporation of [¹⁴C]glucose into amino acids decreased somewhat during the experimental period. It has been proposed that the proliferation of astrocytes and their ultrastructural changes may account for the increased transformation of [¹⁴C]acetate into amino acids, in particular into glutamine which is formed from the small glutamate pool.     

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [¹³C]-α-toluene or [¹³C₇]-toluene as growth substrates. Cells incubated with [¹³C]-α-toluene or [¹³C₇]-toluene incorporated 8–15 at.%¹³C and 51–57 at.%¹³C into total lipid fatty acids, respectively, indicating a lower specific incorporation of ¹³C from [¹³C₇]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [¹³C]-α-toluene and [¹³C₇]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.