Inhibition of glutamine synthesis induces glutamate dehydrogenase-dependent ammonia fixation into alanine in co-cultures of astrocytes and neurons

It has been previously demonstrated that ammonia exposure of neurons and astrocytes in co-culture leads to net synthesis not only of glutamine but also of alanine. The latter process involves the concerted action of glutamate dehydrogenase (GDH) and alanine aminotransferase (ALAT). In the present study it was investigated if the glutamine synthetase (GS) inhibitor methionine sulfoximine (MSO) would enhance alanine synthesis by blocking the GS-dependent ammonia scavenging process. Hence, co-cultures of neurons and astrocytes were incubated for 2.5 h with [U-¹³C]glucose to monitor de novo synthesis of alanine and glutamine in the absence and presence of 5.0 mM NH₄Cl and 10 mM MSO. Ammonia exposure led to increased incorporation of label but not to a significant increase in the amount of these amino acids. However, in the presence of MSO, glutamine synthesis was blocked and synthesis of alanine increased leading to an elevated content intra- as well as extracellularly of this amino acid. Treatment with MSO led to a dramatic decrease in glutamine content and increased the intracellular contents of glutamate and aspartate. The large increase in alanine during exposure to MSO underlines the importance of the GDH and ALAT biosynthetic pathway for ammonia fixation, and it points to the use of a GS inhibitor to ameliorate the brain toxicity and edema induced by hyperammonemia, events likely related to glutamine synthesis.     

Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by (13)C and (31)P nuclear magnetic resonance

The relative contribution of glutamate dehydrogenase (GDH) and the aminotransferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum) tubers. Measurements of glutamate-dependent oxygen consumption by mitochondria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate. First, a new assay for aminotransferase activity, based on the exchange of deuterium between deuterated water and glutamate, provided a method for establishing the effectiveness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxidation to be assessed unambiguously. Secondly, the activity of GDH in the mitochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glutamate. Thirdly, the reversibility of the GDH reaction was investigated by monitoring the isotopic exchange between glutamate and [(15)N]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funneling carbon from glutamate into the TCA cycle.     

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.     

Effects of a GTP-insensitive mutation of glutamate dehydrogenase on insulin secretion in transgenic mice

Glutamate dehydrogenase (GDH) plays an important role in insulin secretion as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonemia syndrome (GDH-HI) and sensitize beta-cells to leucine stimulation. GDH transgenic mice were generated to express the human GDH-HI H454Y mutation and human wild-type GDH in islets driven by the rat insulin promoter. H454Y transgene expression was confirmed by increased GDH enzyme activity in islets and decreased sensitivity to GTP inhibition. The H454Y GDH transgenic mice had hypoglycemia with normal growth rates. H454Y GDH transgenic islets were more sensitive to leucine- and glutamine-stimulated insulin secretion but had decreased response to glucose stimulation. The fluxes via GDH and glutaminase were measured by tracing 15N flux from [2-15N]glutamine. The H454Y transgene in islets had higher insulin secretion in response to glutamine alone and had 2-fold greater GDH flux. High glucose inhibited both glutaminase and GDH flux, and leucine could not override this inhibition. 15NH4Cl tracing studies showed 15N was not incorporated into glutamate in either H454Y transgenic or normal islets. In conclusion, we generated a GDH-HI disease mouse model that has a hypoglycemia phenotype and confirmed that the mutation of H454Y is disease causing. Stimulation of insulin release by the H454Y GDH mutation or by leucine activation is associated with increased oxidative deamination of glutamate via GDH. This study suggests that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets and that this flux is tightly controlled by glucose.     

A possible role of alanine for ammonia transfer between astrocytes and glutamatergic neurons

The metabolism of [U-(13)C]lactate (1 mM) in the presence of unlabeled glucose (2.5 mM) was investigated in glutamatergic cerebellar granule cells, cerebellar astrocytes, and corresponding co-cultures. It was evident that lactate is primarily a neuronal substrate and that lactate produced glycolytically from glucose in astrocytes serves as a substrate in neurons. Alanine was highly enriched with (13)C in the neurons, whereas this was not the case in the astrocytes. Moreover, the cellular content and the amount of alanine released into the medium were higher in neurons than astrocytes. On incubation of the different cell types in medium containing alanine (1 mM), the astrocytes exhibited the highest level of accumulation. Altogether, these results indicate a preferential synthesis and release of alanine in glutamatergic neurons and uptake in cerebellar astrocytes. A new functional role of alanine may be suggested as a carrier of nitrogen from glutamatergic neurons to astrocytes, a transport that may operate to provide ammonia for glutamine synthesis in astrocytes and dispose of ammonia generated by the glutaminase reaction in glutamatergic neurons. Hence, a model of a glutamate-glutamine/lactate-alanine shuttle is presented. To elucidate if this hypothesis is compatible with the pattern of alanine metabolism observed in the astrocytes and neurons from cerebellum, the cells were incubated in a medium containing [(15)N]alanine (1 mM) and [5-(15)N]glutamine (0.5 mM), respectively. Additionally, neurons were incubated with [U-(13)C]glutamine to estimate the magnitude of glutamine conversion to glutamate. Alanine was labeled from [5-(15)N]glutamine to 3.3% and [U-(13)C]glutamate generated from [U-(13)C]glutamine was labeled to 16%. In spite of the modest labeling in alanine, it is clear that nitrogen from ammonia is transferred to alanine via transamination with glutamate formed by reductive amination of alpha-ketoglutarate. With regard to the astrocytic part of the shuttle, glutamine was labeled to 22% in one nitrogen atom whereas 3.2% was labeled in two when astrocytes were incubated in [(15)N]alanine. Moreover, in co-cultures, [U-(13)C]alanine labeled glutamate and glutamine equally, whereas [U-(13)C]lactate preferentially labeled glutamate. Altogether, these results support the role proposed above of alanine as a possible ammonia nitrogen carrier between glutamatergic neurons and surrounding astrocytes and they show that lactate is preferentially metabolized in neurons and alanine in astrocytes.     

Glutathione Turnover in Cultured Astrocytes: Studies with [15N]Glutamate

The incorporation of [15N]glutamic acid into glutathione was studied in primary cultures of astrocytes. Turnover of the intracellular glutathione pool was rapid, attaining a steady state value of 30.0 atom% excess in 180 min. The intracellular glutathione concentration was high (20-40 nmol/mg protein) and the tripeptide was released rapidly into the incubation medium. Although labeling of glutathione (atom% excess) with [15N]glutamate occurred rapidly, little accumulation of 15N in glutathione was noted during the incubation compared with 15N in aspartate, glutamine, and alanine. Glutathione turnover was stimulated by incubating the astrocytes with diethylmaleate, an electrophile that caused a partial depletion of the glutathione pool(s). Diethylmaleate treatment also was associated with significant reductions of intraastrocytic glutamate, glycine, and cysteine, i.e., the constituents of glutathione. Glutathione synthesis could be stimulated by supplementing the steady-state incubation medium with 0.05 mM L-cysteine, such treatment again partially depleting intraastrocytic glutamate and causing significant reductions of 15N labeling of both alanine and glutamine, suggesting that glutamate had been diverted from the synthesis of these amino acids and toward the formation of glutathione. The current study underscores both the intensity of glutathione turnover in astrocytes and the relationship of this turnover to the metabolism of glutamate and other amino acids.     

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

Neuronal Glutamine Utilization: Pathways of Nitrogen Transfer tudied with [15N]Glutamine

Gas chromatography-mass spectrometry was used to evaluate the metabolism of [15N]glutamine in isolated rat brain synaptosomes. In the presence of 0.5 mM glutamine, synaptosomes accumulated this amino acid to a level of 25-35 nmol/mg protein at an initial rate greater than 9 nmol/min/mg of protein. The metabolism of [15N]glutamine generated 15N-labelled glutamate, aspartate, and gamma-aminobutyric acid (GABA). An efflux of both [15N]glutamate and [15N]aspartate from synaptosomes to the medium was observed. Enrichment of 15N in alanine could not be detected because of a limited pool size. Elimination of glucose from the incubation medium substantially increased the rate and amount of [15N]aspartate formed. It is concluded that: (1) With 0.5 mM external glutamine, the glutaminase reaction, and not glutamine transport, determines the rate of metabolism of this amino acid. (2) The primary route of glutamine catabolism involves aspartate aminotransferase which generates 2-oxoglutarate, a substrate for the tricarboxylic acid cycle. This reaction is greatly accelerated by the omission of glucose. (3) Glutamine has preferred access to a population of synaptosomes or to a synaptosomal compartment that generates GABA. (4) Synaptosomes maintain a constant internal level of glutamate plus aspartate of about 70-80 nmol/mg protein. As these amino acids are produced from glutamine in excess of this value, they are released into the medium. Hence synaptosomal glutamine and glutamate metabolism are tightly regulated in an interrelated manner.     

Nitrogen metabolism of asparagine and glutamate in Vero cells studied by <Superscript>1</Superscript>H/<Superscript>15</Superscript>N NMR spectroscopy

Glutamine-free culture of Vero cells has previously been shown to cause higher cell yield and lower ammonia accumulation than that in glutamine-containing culture. Nitrogen metabolism of asparagine and glutamate as glutamine replacer was studied here using nuclear magnetic resonance (NMR) spectroscopy. ¹⁵N-labelled glutamate or asparagine was added and their incorporation into nitrogenous metabolites was monitored by heteronuclear multiple bond coherence (HMBC) NMR spectroscopy. In cells incubated with l-[¹⁵N]glutamate, the ¹⁵N label was subsequently found in a number of metabolites including alanine, aspartate, proline, and an unidentified compound. No detectable signal occurred, indicating that glutamate was utilized by transamination rather than by oxidative deamination. In cells incubated with l-[2-¹⁵N]asparagine, the ¹⁵N label was subsequently found in aspartate, the amine group of glutamate/glutamine, and in two unidentified compounds. Incubation of cells with l-[4-¹⁵N]asparagine showed that the amide nitrogen of asparagine was predominantly transferred to glutamine amide. There was no detectable production of , showing that most of the asparagine amide was transaminated by asparagine synthetase rather than deaminated by asparaginase. Comparing with a glutamine-containing culture, the activities of phosphate-activated glutaminase (PAG), glutamate dehydrogenase (GDH) and alanine aminotransferase (ALT) decreased significantly and the activity of aspartate aminotransferase (AST) decreased slightly.     

Metabolism of 15NH3 in Organotypic Cerebellar Explants and Cultured Astrocytes: Studies with Gas Chromatography-Mass Spectrometry

Gas chromatography-mass spectrometry was used to study the metabolism of 15NH3 in organotypic cerebellar explants and cultured astrocyte monolayers. A steady-state level of 15NH3 was present by 1 min in both systems. Steady-state labeling in L-[amide-15N] glutamine, L-[15N]alanine, L-[15N]glutamate, and L-[15N]aspartate was attained by 1 min after 15NH3 addition in the organotypic cerebellar explants and by approximately 5 min in the cultured astrocytes. No measurable 15N labeling was noted in either glycine or serine in either system.     

Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation

Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to alpha-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native beta-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to alpha-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.     

Regulation of mitochondrial glutamine/glutamate metabolism by glutamate transport: studies with (15)N

We focused on the role of plasma membrane glutamate uptake in modulating the intracellular glutaminase (GA) and glutamate dehydrogenase (GDH) flux and in determining the fate of the intracellular glutamate in the proximal tubule-like LLC-PK(1)-F(+) cell line. We used high-affinity glutamate transport inhibitors D-aspartate (D-Asp) and DL-threo-beta-hydroxyaspartate (THA) to block extracellular uptake and then used [(15)N]glutamate or [2-(15)N]glutamine to follow the metabolic fate and distribution of glutamine and glutamate. In monolayers incubated with [2-(15)N]glutamine (99 atom %excess), glutamine and glutamate equilibrated throughout the intra- and extracellular compartments. In the presence of 5 mM D-Asp and 0.5 mM THA, glutamine distribution remained unchanged, but the intracellular glutamate enrichment decreased by 33% (P < 0.05) as the extracellular enrichment increased by 39% (P < 0.005). With glutamate uptake blocked, intracellular glutamate concentration decreased by 37% (P < 0.0001), in contrast to intracellular glutamine concentration, which remained unchanged. Both glutamine disappearance from the media and the estimated intracellular GA flux increased with the fall in the intracellular glutamate concentration. The labeled glutamate and NH formed from [2-(15)N]glutamine and recovered in the media increased 12- and 3-fold, respectively, consistent with accelerated GA and GDH flux. However, labeled alanine formation was reduced by 37%, indicating inhibition of transamination. Although both D-Asp and THA alone accelerated the GA and GDH flux, only THA inhibited transamination. These results are consistent with glutamate transport both regulating and being regulated by glutamine and glutamate metabolism in epithelial cells.     

Glutamine metabolism regulation in Chlorella pyrenoidosa. Regulation of Chlorella glutamine synthetase activity by amino acids]

Effect of glutamine and its metabolites (amino acids) on Chlorella glutamine synthetase (GS) (E.C.6.3.1.2) in the presence of Mg or Mn was studied. Purified GS preparation was used, isolated from Chlorella grown in the presence of NH as a sole nitrogen source. Glutamate, aspartate, alanine and glycine inhibit GS activity in the presence of both Mg and Mn. Tryptophane and valine (up to 15 mM) activate GS in the presence of Mn. Tryptophane inhibits GS in the system with Mg. Sinergistic inhibition was observed under the combined effect of amino acids on GS in the presence of Mn and aspartate or alanine. The change of GS activity observed is supposed to be due to the inhibitory effect of glutamine and amino acids studied, since the glutamine content is increased (in 2.5 times for 5 min) and that of alanine and dicarbonic amino acids (for the following 15 min) under NH assimilation in Chlorella cells.     

Concerted modulation of alanine and glutamate metabolism in young Medicago truncatula seedlings under hypoxic stress

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. (15)NH(4) labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized (15)N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while (15)N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.     

Activation of Glutamate Dehydrogenase by Leucine and Its Nonmetabolizable Analogue in Rat Brain Synaptosomes

Leucine and beta-(+/-)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) stimulated, in a dose-dependent manner, reductive amination of 2-oxoglutarate in rat brain synaptosomes treated with Triton X-100. The concentration dependence curves were sigmoid, with 10-15-fold stimulations at 15 mM leucine (or BCH); oxidative deamination of glutamate also was enhanced, albeit less. In intact synaptosomes, leucine and BCH elevated oxygen uptake and increased ammonia formation, consistent with stimulation of glutamate dehydrogenase (GDH). Enhancement of oxidative deamination was seen with endogenous as well as exogenous glutamate and with glutamate generated inside synaptosomes from added glutamine. With endogenous glutamate, the stimulation of oxidative deamination was accompanied by a decrease in aspartate formation, which suggests a concomitant reduction in flux through aspartate aminotransferase. Activation of reductive amination of 2-oxoglutarate by BCH or leucine could not be demonstrated even in synaptosomes depleted of internal glutamate. It is suggested that GDH in synaptosomes functions in the direction of glutamate oxidation, and that leucine may act as an endogenous activator of GDH in brain in vivo.     

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.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

The Role of Ammonium Assimilating Enzymes in Lentil Roots and Nodules

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant. This revised version was published online in July 2006 with corrections to the Cover Date.     

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.