Glutamate Catabolism Research Articles

NAD(+)-specific glutamate dehydrogenase (EC.1.4.1.2) in Streptomyces coelicolor; in vivo characterization and the implication for nutrient-dependent secondary metabolism.

While glutamate and glutamate-rich compounds are widely used for culturing Streptomyces sp., little is known regarding glutamate catabolism at molecular level. Noting the presence of two distinct putative glutamate dehydrogenases (GDH), we constructed knockout mutants of each gene with Streptomyces coelicolor M145 and examined the functionality related to antibiotic production. Out of the two, the sco2999 knockout (ΔgdhB, NAD(+)-specific) showed outstanding effects; it decreased the growth sevenfold but initiated the undecylprodigiosin (RED) production in complex Difco nutrient media which otherwise does not support the production from M145. With glucose supplementation, the growth difference by ΔgdhB disappeared but we could obtain significantly increased actinorhodin (ACT) and RED biosynthesis with the mutant by limiting the glucose content (0.5∼1.0%, w/v). Complementing the gene to the knockout mutant inhibited the production, confirming its gene specificity. Along with the extended impacts on overall nitrogen metabolism based on the intracellular metabolite analysis and enzyme assays, GdhB and glutamate utilization were shown to interfere with N-acetylglucosamine metabolism and the activity of its associated global transcriptional regulator (DasR). Taken together, GdhB-subjected to the nutritional context-dependent regulation-is proposed as a key member of central nitrogen metabolism to control the secondary metabolism initiation in exploiting the organic nitrogen sources.

Glutamate Dehydrogenase Is Required by Mycobacterium bovis BCG for Resistance to Cellular Stress.

We recently reported on our success to generate deletion mutants of the genes encoding glutamate dehydrogenase (GDH) and glutamine oxoglutarate aminotransferase (GOGAT) in M. bovis BCG, despite their in vitro essentiality in M. tuberculosis. We could use these mutants to delineate the roles of GDH and GOGAT in mycobacterial nitrogen metabolism by using M. bovis BCG as a model for M. tuberculosis specifically. Here, we extended our investigation towards the involvement of GDH and GOGAT in other aspects of M. bovis BCG physiology, including the use of glutamate as a carbon source and resistance to known phagosomal stresses, as well as in survival inside macrophages. We find that gdh is indispensable for the utilization of glutamate as a major carbon source, in low pH environments and when challenged with nitric oxide. On the other hand, the gltBD mutant had increased viability under low pH conditions and was unaffected by a challenge with nitric oxide. Strikingly, GDH was required to sustain M. bovis BCG during infection of both murine RAW 264.7 and bone-marrow derived and macrophages, while GOGAT was not. We conclude that the catabolism of glutamate in slow growing mycobacteria may be a crucial function during infection of macrophage cells and demonstrate a novel requirement for M. bovis BCG GDH in the protection against acidic and nitrosative stress. These results provide strong clues on the role of GDH in intracellular survival of M. tuberculosis, in which the essentiality of the gdh gene complicates knock out studies making the study of the role of this enzyme in pathogenesis difficult.

Comparative responses to water deficit stress and subsequent recovery in the cultivated beet Beta vulgaris and its wild relative B. macrocarpa

The effects of water deficit stress and recovery on growth, photosynthesis, physiological and biochemical parameters were investigated in the cultivated Beta vulgaris and in two Tunisian provenances (Soliman and Enfidha) of its wild relative B. macrocarpa. Seedlings were cultivated for 4 weeks under optimal or limiting water supply (respectively, 100% and 25% of field capacity, FC). After 2 weeks of treatment, a lot of stressed plants were rehydrated to 100% FC. In the Control, B. vulgaris was more productive than B. macrocarpa, whereas Enfidha provenance showed the highest biomass production (1.6- and 3-fold compared with B. vulgaris and Soliman, respectively), under water deficit stress. A partial re-establishment of growth occurred in both species upon recovery at 100% FC. The sensitivity of B. vulgaris and Soliman provenance to drought was associated with the disturbance of leaf water status and the sharp decrease in net CO2 assimilation (–66% and –82% as compared with the Control, respectively). On the contrary, the better behaviour of Enfidha provenance was related to its better photosynthetic capacity and leaf relative water content, along with a higher accumulation of amino acids (proline, glycine, and glutamine) implied in the osmotic adjustment. Leaf hexose concentration increased significantly under drought stress in both species whereas leaf sucrose concentration declined only in drought-stressed B. vulgaris and Soliman provenance. Leaf glutamate dehydrogenase activity increased under water deficit in both species despite to a higher extent in B. vulgaris. As glutamate dehydrogenase is implied in catabolism of glutamate to oxoglutarate, it might contribute to provide stressed plants with carbon skeletons. Enfidha provenance of the spontaneous species B. macrocarpa could be used in the marginal arid ecosystems in order to limit the deficit in fodder and to improve the pastoral value of these regions. In addition, this species could serve as a source of genes for genetic improvement to water deficit stress.

Astrocytic Control of Biosynthesis and Turnover of the Neurotransmitters Glutamate and GABA

Glutamate and GABA are the quantitatively major neurotransmitters in the brain mediating excitatory and inhibitory signaling, respectively. These amino acids are metabolically interrelated and at the same time they are tightly coupled to the intermediary metabolism including energy homeostasis. Astrocytes play a pivotal role in the maintenance of the neurotransmitter pools of glutamate and GABA since only these cells express pyruvate carboxylase, the enzyme required for de novo synthesis of the two amino acids. Such de novo synthesis is obligatory to compensate for catabolism of glutamate and GABA related to oxidative metabolism when the amino acids are used as energy substrates. This, in turn, is influenced by the extent to which the cycling of the amino acids between neurons and astrocytes may occur. This cycling is brought about by the glutamate/GABA – glutamine cycle the operation of which involves the enzymes glutamine synthetase (GS) and phosphate-activated glutaminase together with the plasma membrane transporters for glutamate, GABA, and glutamine. The distribution of these proteins between neurons and astrocytes determines the efficacy of the cycle and it is of particular importance that GS is exclusively expressed in astrocytes. It should be kept in mind that the operation of the cycle is associated with movement of ammonia nitrogen between the two cell types and different mechanisms which can mediate this have been proposed. This review is intended to delineate the above mentioned processes and to discuss quantitatively their relative importance in the homeostatic mechanisms responsible for the maintenance of optimal conditions for the respective neurotransmission processes to operate.

Deletion of glutamate dehydrogenase 1 (Glud1) in the central nervous system affects glutamate handling without altering synaptic transmission

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS-specific GDH-null mice (CnsGlud1(-/-)); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns-Glud1(-/-) brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns-Glud1(-/-) mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns-Glud1(-/-) mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up-regulation of astrocyte-type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.

Strategies and tools for studying the metabolism and function of γ-aminobutyrate in plants. II. Integrated analysis

γ-Aminobutyrate (GABA) is a ubiquitous nonprotein amino acid that accumulates in plants in response to abiotic and biotic stresses. In a companion paper, we discussed the origin of GABA from glutamate and subsequent catabolism to succinic semialdehyde and either succinate or γ-hydroxybutyrate (GHB), and the characteristics of genes and proteins responsible for GABA permease, glutamate decarboxylase, GABA transaminase, succinic semialdehyde dehydrogenase, and succinic semialdehyde reductase activities. In this paper, we explore gene expression and transcript–metabolite relationships during the response to abiotic stress, and describe phenotypes of genetic mutants and relationships of GABA metabolism to other plant functions. Evidence indicates that both gene-dependent and -independent processes are involved in the response of the GABA pathway to abiotic stresses. Study of stress-specific responses and their interplay with the C/N network and various signalling pathways would be more informative if circadian rhythms and light–dark transitions upon imposition of the stress were always taken into account, and relevant genes and metabolites simultaneously profiled in wild-type plants or genetic mutants.

Differential Proteomic Analysis of a Polymicrobial Biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H₂¹⁶O/H₂¹⁸O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.

13C-flux Analysis Reveals NADPH-balancing Transhydrogenation Cycles in Stationary Phase of Nitrogen-starving Bacillus subtilis

In their natural habitat, microorganisms are typically confronted with nutritional limitations that restrict growth and force them to persevere in a stationary phase. Despite the importance of this phase, little is known about the metabolic state(s) that sustains it. Here, we investigate metabolically active but non-growing Bacillus subtilis during nitrogen starvation. In the absence of biomass formation as the major NADPH sink, the intracellular flux distribution in these resting B. subtilis reveals a large apparent catabolic NADPH overproduction of 5.0 ± 0.6 mmol g(-1)h(-1) that was partly caused by high pentose phosphate pathway fluxes. Combining transcriptome analysis, stationary (13)C-flux analysis in metabolic deletion mutants, (2)H-labeling experiments, and kinetic flux profiling, we demonstrate that about half of the catabolic excess NADPH is oxidized by two transhydrogenation cycles, i.e. isoenzyme pairs of dehydrogenases with different cofactor specificities that operate in reverse directions. These transhydrogenation cycles were constituted by the combined activities of the glyceraldehyde 3-phosphate dehydrogenases GapA/GapB and the malic enzymes MalS/YtsJ. At least an additional 6% of the overproduced NADPH is reoxidized by continuous cycling between ana- and catabolism of glutamate. Furthermore, in vitro enzyme data show that a not yet identified transhydrogenase could potentially reoxidize ∼20% of the overproduced NADPH. Overall, we demonstrate the interplay between several metabolic mechanisms that concertedly enable network-wide NADPH homeostasis under conditions of high catabolic NADPH production in the absence of cell growth in B. subtilis.

Architecture of the hypothalamo-posthypophyseal complex is controlled by monoamines

The hypothalamo-neurohypophyseal system displays significant plasticity when subjected to physiological stimuli, such as dehydration, parturition, or lactation. This plasticity arises at the neurochemical and electrophysiological levels but also at a structural level. Several studies have demonstrated the role of monoaminergic afferents in controlling neurochemical and electrophysiological plasticity of the supraoptic nucleus (SON) and of the neurohypophysis (NH), but little is known about how the changes in structural plasticity are triggered. We used Tg8 mice, disrupted for the monoamine oxidase A gene, to study monamine involvement in the architecture of the SON and of the NH. SON astrocytes in Tg8 mice displayed an active status, characterized by an increase in S100β expression and a significant decrease in vimentin expression, with no modification in glial fibrillary acidic protein (GFAP) levels. Astrocytes showed a decrease in glutamate dehydrogenase (GDH) levels, whereas glutamine synthetase (GS) levels remained constant, suggesting a reduction in astrocyte glutamate catabolism. Tenascin C and polysialic acid-neural cell adhesion molecule (PSA-NCAM) expressions were also elevated in the SON of Tg8 mice, suggesting an increased capacity for structural remodelling in the SON. In the NH, similar date were obtained with a stability in GFAP expression and an increase in PSA-NCAM immunostaining. These results establish monoamine (serotonin and noradrenaline) involvement in SON and NH structural arrangement. Monoamines therefore appear to be crucial for the coordination of the neurochemical and structural aspects of neuroendocrine plasticity, allowing the hypothalamo-neurohypopyseal system to respond appropriately when stimulated.

Glutamate racemization and catabolism inFusobacterium varium

The pathways of glutamate catabolism in the anaerobic bacterium Fusobacterium varium, grown on complex, undefined medium and chemically defined, minimal medium, were investigated using specifically labelled (13)C-glutamate. The metabolic end-products acetate and butyrate were isolated from culture fluids and derivatized for analysis by nuclear magnetic resonance and mass spectrometry. On complex medium, labels from L-[1-(13)C]glutamate and L-[4-(13)C]glutamate were incorporated into C1 of acetate and equally into C1/C3 of butyrate, while label derived from L-[5-(13)C]glutamate was not incorporated. The isotopic incorporation results and the detection of glutamate mutase and 3-methylaspartate ammonia lyase in cell extracts are most consistent with the methylaspartate pathway, the best known route of glutamate catabolism in Clostridium species. When F. varium was grown on defined medium, label from L-[4-(13)C]glutamate was incorporated mainly into C4 of butyrate, demonstrating a major role for the hydroxyglutarate pathway. Upon addition of coenzyme B(12) or cobalt ion to the defined medium in replicate experiments, isotope was located equally at C1/C3 of butyrate in accord with the methylaspartate pathway. Racemization of D-glutamate and subsequent degradation of L-glutamate via the methylaspartate pathway are supported by incorporation of label into C2 of acetate and equally into C2/C4 of butyrate from D-[3-(13)C]glutamate and the detection of a cofactor-independent glutamate racemase in cell extracts. Together the results demonstrate a major role for the methylaspartate pathway of glutamate catabolism in F. varium and substantial participation of the hydroxyglutarate pathway when coenzyme B(12) is not available.

RNAi-Mediated Knock-Down of Arylamine N-acetyltransferase-1 Expression Induces E-cadherin Up-Regulation and Cell-Cell Contact Growth Inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.

Smoking-mediated up-regulation of GAD67 expression in the human airway epithelium

BackgroundThe production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium.MethodsWe assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis.ResultsThe data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking.ConclusionsIn the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking.Trial registrationNCT00224198; NCT00224185

Glutamate dehydrogenase and glutamine synthetase are regulated in response to nitrogen availability in Myocbacterium smegmatis

BackgroundThe assimilation of nitrogen is an essential process in all prokaryotes, yet a relatively limited amount of information is available on nitrogen metabolism in the mycobacteria. The physiological role and pathogenic properties of glutamine synthetase (GS) have been extensively investigated in Mycobacterium tuberculosis. However, little is known about this enzyme in other mycobacterial species, or the role of an additional nitrogen assimilatory pathway via glutamate dehydrogenase (GDH), in the mycobacteria as a whole. We investigated specific enzyme activity and transcription of GS and as well as both possible isoforms of GDH (NAD+- and NADP+-specific GDH) under varying conditions of nitrogen availability in Mycobacterium smegmatis as a model for the mycobacteria.ResultsIt was found that the specific activity of the aminating NADP+-GDH reaction and the deaminating NAD+-GDH reaction did not change appreciably in response to nitrogen availability. However, GS activity as well as the deaminating NADP+-GDH and aminating NAD+-GDH reactions were indeed significantly altered in response to exogenous nitrogen concentrations. Transcription of genes encoding for GS and the GDH isoforms were also found to be regulated under our experimental conditions.ConclusionsThe physiological role and regulation of GS in M. smegmatis was similar to that which has been described for other mycobacteria, however, in our study the regulation of both NADP+- and NAD+-GDH specific activity in M. smegmatis appeared to be different to that of other Actinomycetales. It was found that NAD+-GDH played an important role in nitrogen assimilation rather than glutamate catabolism as was previously thought, and is it's activity appeared to be regulated in response to nitrogen availability. Transcription of the genes encoding for NAD+-GDH enzymes seem to be regulated in M. smegmatis under the conditions tested and may contribute to the changes in enzyme activity observed, however, our results indicate that an additional regulatory mechanism may be involved. NADP+-GDH seemed to be involved in nitrogen assimilation due to a constitutive aminating activity. The deaminating reaction, however was observed to change in response to varying ammonium concentrations which suggests that NADP+-GDH is also regulated in response to nitrogen availability. The regulation of NADP+-GDH activity was not reflected at the level of gene transcription thereby implicating post-transcriptional modification as a regulatory mechanism in response to nitrogen availability.

Glutamate Is the Major Anaplerotic Substrate in the Tricarboxylic Acid Cycle of Isolated Rumen Epithelial and Duodenal Mucosal Cells from Beef Cattle

In this study, we aimed to determine the contribution of substrates to tricarboxylic acid (TCA) cycle fluxes in rumen epithelial cells (REC) and duodenal mucosal cells (DMC) isolated from Angus bulls (n = 6) fed either a 75% forage (HF) or 75% concentrate (HC) diet. In separate incubations, [13C6]glucose, [13C5]glutamate, [13C5]glutamine, [13C6]leucine, or [13C5]valine were added in increasing concentrations to basal media containing SCFA and a complete mixture of amino acids. Lactate, pyruvate, and TCA cycle intermediates were analyzed by GC-MS followed by 13C-mass isotopomer distribution analysis. Glucose metabolism accounted for 10–19% of lactate flux in REC from HF-fed bulls compared with 27–39% in REC from HC and in DMC from bulls fed both diets (P < 0.05). For both cell types, as concentration increased, an increasing proportion (3–63%) of α-ketoglutarate flux derived from glutamate, whereas glutamine contributed <3% (P < 0.05). Although leucine and valine were catabolized to their respective keto-acids, these were not further metabolized to TCA cycle intermediates. Glucose, glutamine, leucine, and valine catabolism by ruminant gastrointestinal tract cells has been previously demonstrated, but in this study, their catabolism via the TCA cycle was limited. Further, although glutamate's contribution to TCA cycle fluxes was considerable, it was apparent that other substrates available in the media also contributed to the maintenance of TCA fluxes. Lastly, the results suggest that diet composition alters glucose, glutamate, and leucine catabolism by the TCA cycle of REC and DMC.

Postprandial nitrogen metabolism and excretion in juvenile marble goby, Oxyeleotris marmorata (Bleeker, 1852)

The objective of this study was to examine postprandial nitrogen metabolism and excretion in the juvenile marble goby, Oxyeleotris marmorata (30–80 g), fed a high protein diet (cod fish fillet). Feeding led to only a slight increase in plasma ammonia concentration and had no significant effect on the ammonia content in the brain of juvenile O. marmorata during the subsequent 24 h. Unlike other fishes, juvenile O. marmorata could apparently avoid postprandial ammonia toxicity, and as a result there was no increase in the brain glutamine content, which decreased significantly instead at certain post-feeding time point. Since no prominent increases in tissue glutamine and urea contents were observed after feeding despite its ability to detoxify ammonia to glutamine during emersion and its possession of a full complement of hepatic ornithine-urea cycle enzymes, it can be concluded that only a moderate amount of ammonia was produced after feeding. Traditionally, it has been accepted that excess amino acid would be degraded in the liver through transdeamination which involves the deamination of glutamate catalyzed by glutamate dehydrogenase (GDH). However, we report for the first time that glutamate content in liver and intestine of juvenile O. marmorata increased to 10.8 µmol g − 1 and 3.3 µmol g − 1 , respectively, 6 h post-feeding. These results indicate that the rate of glutamate catabolism in the liver was lower than the total rate of glutamate released directly from protein degradation and produced indirectly from other amino acids through transamination. Indeed, feeding led to significant increases in hepatic GDH amination activity and intestinal GDH activity, in both amination and deamination directions, in juvenile O. marmorata. Thus, there could be an increase in the capacity of the liver, and perhaps the intestine, to produce and retain glutamate after feeding. As a result, only 33% of the ingested nitrogen was excreted during the 24-h post-feeding period, with 67% being retained for somatic growth. Results obtained from this study can be useful for designing re-circulating aquaculture systems for juvenile marble goby, and they offer insights into mechanisms that can be induced by feeding to retain ingested nitrogen.

Amino acid‐dependent growth of Campylobacter jejuni: key roles for aspartase (AspA) under microaerobic and oxygen‐limited conditions and identification of AspB (Cj0762), essential for growth on glutamate

Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking serine dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA-dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.

Elimination of KATP Channels in Mouse Islets Results in Elevated [U-13C]Glucose Metabolism, Glutaminolysis, and Pyruvate Cycling but a Decreased γ-Aminobutyric Acid Shunt

Pancreatic beta cells are hyper-responsive to amino acids but have decreased glucose sensitivity after deletion of the sulfonylurea receptor 1 (SUR1) both in man and mouse. It was hypothesized that these defects are the consequence of impaired integration of amino acid, glucose, and energy metabolism in beta cells. We used gas chromatography-mass spectrometry methodology to study intermediary metabolism of SUR1 knock-out (SUR1(-/-)) and control mouse islets with d-[U-(13)C]glucose as substrate and related the results to insulin secretion. The levels and isotope labeling of alanine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid (GABA) served as indicators of intermediary metabolism. We found that the GABA shunt of SUR1(-/-) islets is blocked by about 75% and showed that this defect is due to decreased glutamate decarboxylase synthesis, probably caused by elevated free intracellular calcium. Glutaminolysis stimulated by the leucine analogue d,l-beta-2-amino-2-norbornane-carboxylic acid was, however, enhanced in SUR1(-/-) and glyburide-treated SUR1(+/+) islets. Glucose oxidation and pyruvate cycling was increased in SUR1(-/-) islets at low glucose but was the same as in controls at high glucose. Malic enzyme isoforms 1, 2, and 3, involved in pyruvate cycling, were all expressed in islets. High glucose lowered aspartate and stimulated glutamine synthesis similarly in controls and SUR1(-/-) islets. The data suggest that the interruption of the GABA shunt and the lack of glucose regulation of pyruvate cycling may cause the glucose insensitivity of the SUR1(-/-) islets but that enhanced basal pyruvate cycling, lowered GABA shunt flux, and enhanced glutaminolytic capacity may sensitize the beta cells to amino acid stimulation.

Expression of glutamine synthetase and glutamate dehydrogenase in the latent phase and chronic phase in the kainate model of temporal lobe epilepsy

It has been suggested that astrocytic glutamate release or perturbed glutamate metabolism contributes to the proneness to epileptic seizures. Here we investigated whether astrocytic contents of the major glutamate degrading enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) decreases on moving from the latent phase (prior to seizures) to the chronic phase (after onset of seizures) in the kainate (KA) model of temporal lobe epilepsy. Western blotting and immunogold analysis of hippocampal formation indicated similar levels of GDH in the latent and chronic phases of KA injected rats and in corresponding controls. In contrast, the level of GS was increased in the latent phase compared with controls, as assessed by Western blots of whole hippocampal formation and subregions. The increase in GS paralleled that of glial fibrillary acidic protein (GFAP). Compared with the latent phase, the chronic phase revealed a lower level of GS (approaching control levels) but an unchanged GFAP content. The decrease in GS from latent to chronic phase was significant in whole hippocampal formation, dentate gyrus and CA3. It is concluded that kainate treated rats show an initial increase in GS, pari passu with the increase in GFAP, and a secondary decrease in GS that is not accompanied by a similar loss of GFAP. In a situation where glutamate catabolism is in high demand the secondary reduction in GS level may be sufficient to contribute to the seizure proneness that develops between the latent and chronic phases.

NAD(H)-dependent glutamate dehydrogenase is essential for the survival ofArabidopsis thalianaduring dark-induced carbon starvation

Interconversion between glutamate and 2-oxoglutarate, which can be catalysed by glutamate dehydrogenase (GDH), is a key reaction in plant carbon (C) and nitrogen (N) metabolism. However, the physiological role of plant GDH has been a controversial issue for several decades. To elucidate the function of GDH, the expression of GDH in various tissues of Arabidopsis thaliana was studied. Results suggested that the expression of two Arabidopsis GDH genes was differently regulated depending on the organ/tissue types and cellular C availability. Moreover, Arabidopsis mutants defective in GDH genes were identified and characterized. The two isolated mutants, gdh1-2 and gdh2-1, were crossed to make a double knockout mutant, gdh1-2/gdh2-1, which contained negligible levels of NAD(H)-dependent GDH activity. Phenotypic analysis on these mutants revealed an increased susceptibility of gdh1-2/gdh2-1 plants to C-deficient conditions. This conditional phenotype of the double knockout mutant supports the catabolic role of GDH and its role in fuelling the TCA cycle during C starvation. The reduced rate of glutamate catabolism in the gdh2-1 and gdh1-2/gdh2-1 plants was also evident by the growth retardation of these mutants when glutamate was supplied as the alternative N source. Furthermore, amino acid profiles during prolonged dark conditions were significantly different between WT and the gdh mutant plants. For instance, glutamate levels increased in WT plants but decreased in gdh1-2/gdh2-1 plants, and aberrant accumulation of several amino acids was detected in the gdh1-2/gdh2-1 plants. These results suggest that GDH plays a central role in amino acid breakdown under C-deficient conditions.

Fasting induces a biphasic adaptive metabolic response in murine small intestine.

BackgroundThe gut is a major energy consumer, but a comprehensive overview of the adaptive response to fasting is lacking. Gene-expression profiling, pathway analysis, and immunohistochemistry were therefore carried out on mouse small intestine after 0, 12, 24, and 72 hours of fasting.ResultsIntestinal weight declined to 50% of control, but this loss of tissue mass was distributed proportionally among the gut's structural components, so that the microarrays' tissue base remained unaffected. Unsupervised hierarchical clustering of the microarrays revealed that the successive time points separated into distinct branches. Pathway analysis depicted a pronounced, but transient early response that peaked at 12 hours, and a late response that became progressively more pronounced with continued fasting. Early changes in gene expression were compatible with a cellular deficiency in glutamine, and metabolic adaptations directed at glutamine conservation, inhibition of pyruvate oxidation, stimulation of glutamate catabolism via aspartate and phosphoenolpyruvate to lactate, and enhanced fatty-acid oxidation and ketone-body synthesis. In addition, the expression of key genes involved in cell cycling and apoptosis was suppressed. At 24 hours of fasting, many of the early adaptive changes abated. Major changes upon continued fasting implied the production of glucose rather than lactate from carbohydrate backbones, a downregulation of fatty-acid oxidation and a very strong downregulation of the electron-transport chain. Cell cycling and apoptosis remained suppressed.ConclusionThe changes in gene expression indicate that the small intestine rapidly looses mass during fasting to generate lactate or glucose and ketone bodies. Meanwhile, intestinal architecture is maintained by downregulation of cell turnover.