Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activity in these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to the known formation of glutamine, the enzyme glutamine synthetase shows unknown activity in endothelial cell migration during pathological angiogenesis through RHOJ palmitoylation.

Ammonia induces changes in carbamoyl phosphate synthetase I and its regulation of glutamine synthesis and urea cycle in yellow catfish Pelteobagrus fulvidraco.

Fishes can adapt to certain levels of environmental ammonia in water, but the strategies utilized to defend against ammonia toxicity are not exactly the same. The carbamyl phosphate synthase I (CPS I) plays an important role in the regulation of glutamine synthesis and urea cycle, which are the most common strategies for ammonia detoxification. In this study, CPS I was cloned from the yellow catfish. The full-length cDNAs of the CPS I was 5 034 bp, with open reading frames of 4 461 bp. Primary amino acid sequence alignment of CPS I revealed conserved similarity between the functional domains of the yellow catfish CPS I protein with CPS I proteins of other animals. The mRNA expression of CPS I was significantly up-regulated in liver and kidney tissues after acute ammonia stress. The CPS I RNA interference (RNAi) down-regulated the mRNA expressions of CPS I and ornithine transcarbamylase (OTC), but up-regulated glutamine synthetase (GS) and glutamate dehydrogenase (GDH) expressions in primary culture of liver cell after acute ammonia stress. Similarly, the activity of enzymes related to urea cycle decreased significantly, while the activity of enzymes related to glutamine synthesis increased significantly. The results of RNAi in vitro suggested that when the urea cycle is disturbed, the glutamine synthesis will be activated to cope with ammonia toxicity.

Deficient astrocyte metabolism impairs glutamine synthesis and neurotransmitter homeostasis in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) leads to cerebral accumulation of insoluble amyloid-ß plaques causing synaptic dysfunction and neuronal death. Neurons rely on astrocyte-derived glutamine for replenishment of the amino acid neurotransmitter pools. Perturbations of astrocyte glutamine synthesis have been described in AD, but whether this functionally affects neuronal neurotransmitter synthesis is not known. Since the synthesis and recycling of neurotransmitter glutamate and GABA are intimately coupled to cellular metabolism, the aim of this study was to provide a functional investigation of neuronal and astrocytic energy and neurotransmitter metabolism in AD. To achieve this, we incubated acutely isolated cerebral cortical and hippocampal slices from 8-month-old female 5xFAD mice, in the presence of 13C isotopically enriched substrates, with subsequent gas chromatography-mass spectrometry (GC-MS) analysis. A prominent neuronal hypometabolism of [U-13C]glucose was observed in the hippocampal slices of the 5xFAD mice. Investigating astrocyte metabolism, using [1,2-13C]acetate, revealed a marked reduction in glutamine synthesis, which directly hampered neuronal synthesis of GABA. This was supported by an increased metabolism of exogenously supplied [U-13C]glutamine, suggesting a neuronal metabolic compensation of the reduced astrocytic glutamine supply. In contrast, astrocytic metabolism of [U-13C]GABA was reduced, whereas [U-13C]glutamate metabolism was unaffected. Finally, astrocyte de novo synthesis of glutamate and glutamine was hampered, whereas the enzymatic capacity of glutamine synthetase for ammonia fixation was maintained. Collectively, we demonstrate that deficient astrocyte metabolism leads to reduced glutamine synthesis, directly impairing neuronal GABA synthesis in the 5xFAD brain. These findings suggest that astrocyte metabolic dysfunction may be fundamental for the imbalances of synaptic excitation and inhibition in the AD brain.

Transcriptomic and genome-wide association study reveal long noncoding RNAs responding to nitrogen deficiency in maize.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in essential biological processes. However, our understanding of lncRNAs as competing endogenous RNAs (ceRNAs) and their responses to nitrogen stress is still limited. RESULTS: Here, we surveyed the lncRNAs and miRNAs in maize inbred line P178 leaves and roots at the seedling stage under high-nitrogen (HN) and low-nitrogen (LN) conditions using lncRNA-Seq and small RNA-Seq. A total of 894 differentially expressed lncRNAs and 38 different miRNAs were identified. Co-expression analysis found that two lncRNAs and four lncRNA-targets could competitively combine with ZmmiR159 and ZmmiR164, respectively. To dissect the genetic regulatory by which lncRNAs might enable adaptation to limited nitrogen availability, an association mapping panel containing a high-density single-nucleotide polymorphism (SNP) array (56,110 SNPs) combined with variable LN tolerant-related phenotypes obtained from hydroponics was used for a genome-wide association study (GWAS). By combining GWAS and RNA-Seq, 170 differently expressed lncRNAs within the range of significant markers were screened. Moreover, 40 consistently LN-responsive genes including those involved in glutamine biosynthesis and nitrogen acquisition in root were identified. Transient expression assays in Nicotiana benthamiana demonstrated that LNC_002923 could inhabit ZmmiR159-guided cleavage of Zm00001d015521. CONCLUSIONS: These lncRNAs containing trait-associated significant SNPs could consider to be related to root development and nutrient utilization. Taken together, the results of our study can provide new insights into the potential regulatory roles of lncRNAs in response to LN stress, and give valuable information for further screening of candidates as well as the improvement of maize resistance to LN stress.

Staphylococcus aureus grown in anaerobic conditions exhibits elevated glutamine biosynthesis and biofilm units.

The enormous spread of Staphylococcus aureus infections through biofilms is a major concern in hospital-acquired infections. Biofilm formation by S. aureus on any surface is facilitated by adjusting its redox status. This organism is a facultative anaerobe shift more towards reductive conditions by enhancing nitrogen metabolism where glutamine synthesis plays a key role. Glutamine is synthesized by glutamine synthetase (GS) encoded by the glnA gene. The gene was amplified by PCR from the chromosomal DNA of S. aureus, sequenced (HQ329146.1), and cloned. The pure recombinant GS exhibited Km of 11.06 ± 0.05 mmol·L-1 for glutamate and 2.4 ± 0.03 mmol·L-1 for ATP. The glnA gene sequence showed a high degree of variability with its human counterpart, while it was highly conserved in bacteria. Structural analysis revealed that the GS structure of S. aureus showed close homology with other Gram-positive bacteria and exhibited a high degree of variation with Escherichia coli GS. In the present study, we observed the increased presence of GS activity in multidrug-resistant strains of S. aureus with elevated biofilm units, grown in brain heart infusion broth; among them methicillin-resistant strains S. aureus LMV 3, 4, and 5 showed higher biofilm units. All these results explain the important role of glutamine biosynthesis with elevated biofilm units in the pathogenesis of S. aureus.

Role of metabolic spatiotemporal dynamics in regulating biofilm colony expansion.

Cell fate determination is typically regulated by biological networks, yet increasing evidences suggest that cell-cell communication and environmental stresses play crucial roles in the behavior of a cell population. A recent microfluidic experiment showed that the metabolic codependence of two cell populations generates a collective oscillatory dynamic during the expansion of a Bacillus subtilis biofilm. We develop a modeling framework for the spatiotemporal dynamics of the associated metabolic circuit for cells in a colony. We elucidate the role of metabolite diffusion and the need of two distinct cell populations to observe oscillations. Uniquely, this description captures the onset and thereafter stable oscillatory dynamics during expansion and predicts the existence of damping oscillations under various environmental conditions. This modeling scheme provides insights to understand how cells integrate the information from external signaling and cell-cell communication to determine the optimal survival strategy and/or maximize cell fitness in a multicellular system.

Loss of RBF1 changes glutamine catabolism.

Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo (13)C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H(2)O(2) preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.

Role of GlnR in Controlling Expression of Nitrogen Metabolism Genes in Listeria monocytogenes.

Listeria monocytogenes is a fastidious bacterial pathogen that can utilize only a limited number of nitrogen sources for growth. Both glutamine and ammonium are common nitrogen sources used in listerial defined growth media, but little is known about the regulation of their uptake or utilization. The functional role of L. monocytogenes GlnR, the transcriptional regulator of nitrogen metabolism genes in low-G+C Gram-positive bacteria, was determined using transcriptome sequencing and real-time reverse transcription-PCR experiments. The GlnR regulon included transcriptional units involved in ammonium transport (amtB glnK) and biosynthesis of glutamine (glnRA) and glutamate (gdhA) from ammonium. As in other bacteria, GlnR proved to be an autoregulatory repressor of the glnRA operon. Unexpectedly, GlnR was most active during growth with ammonium as the nitrogen source and less active in the glutamine medium, apparently because listerial cells perceive growth with glutamine as a nitrogen-limiting condition. Therefore, paradoxically, expression of the glnA gene, encoding glutamine synthetase, was highest in the glutamine medium. For the amtB glnK operon, GlnR served as both a negative regulator in the presence of ammonium and a positive regulator in the glutamine medium. The gdhA gene was subject to a third mode of regulation that apparently required an elevated level of GlnR for repression. Finally, activity of glutamate dehydrogenase encoded by the gdhA gene appeared to correlate inversely with expression of gltAB, the operon that encodes the other major glutamate-synthesizing enzyme, glutamate synthase. Both gdhA and amtB were also regulated, in a negative manner, by the global transcriptional regulator CodY.IMPORTANCEL. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

Targeting Glutamine Synthesis Inhibits Stem Cell Adipogenesis in Vitro.

BACKGROUND/AIMS: Glutamine is the most abundant amino acid in the body and has a metabolic role as a precursor for protein, amino sugar and nucleotide synthesis. After glucose, glutamine is the main source of energy in cells and has recently been shown to be an important carbon source for de novo lipogenesis. Glutamine is synthesized by the enzyme glutamine synthetase, a mitochondrial enzyme that is active during adipocyte differentiation suggesting a regulatory role in this process. The aim of our study was therefore to investigate whether glutamine status impacts on the differentiation of adipocytes and lipid droplet accumulation. METHODS: Mouse mesenchymal stem cells (MSCs) were submitted to glutamine deprivation (i.e. glutamine-free adipogenic medium in conjunction with irreversible glutamine synthetase inhibitor, methionine sulfoximine - MSO) during differentiation and their response was compared with MSCs differentiated in glutamine-supplemented medium (5, 10 and 20 mM). Differentiated MSCs were assessed for lipid content using Oil Red O (ORO) staining and gene expression was analysed by qPCR. Intracellular glutamine levels were determined using a colorimetric assay, while extracellular glutamine was measured using liquid chromatography-mass spectrometry (LC-MS). RESULTS: Glutamine deprivation largely abolished adipogenic differentiation and lipid droplet formation. This was accompanied with a reduction in intracellular glutamine concentration, and downregulation of gene expression for classical adipogenic markers including PPARγ. Furthermore, glutamine restriction suppressed isocitrate dehydrogenase 1 (IDH1) gene expression, an enzyme which produces citrate for lipid synthesis. In contrast, glutamine supplementation promoted adipogenic differentiation in a dose-dependent manner. CONCLUSION: These results suggest that the glutamine pathway may have a previously over-looked role in adipogenesis. The underlying mechanism involved the glutamine-IDH1 pathway and could represent a potential therapeutic strategy to treat excessive lipid accumulation and thus obesity.

Effects of ammonia-N exposure on the growth, metabolizing enzymes, and metabolome of Macrobrachium rosenbergii.

Ammonia nitrogen elevated is one of the commonest problem in the aquatic system, which caused a great threat to the survival and growth of prawn. However, little is know about the ammonia metabolism and detoxification strategy of prawn. In this study, the effects of ammonia-N (0, 0.108, 0.216, 0.324, or 0.54 mg L-1) on growth and metabolizing enzymes in hepatopancreas of Macrobrachium rosenbergii, including glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamate dehydrogenase (GDH), were investigated. The metabolome of its muscle was also analyzed after exposure to ammonia-N (0, 0.108, 0.324, or 0.54 mg L-1) for 20 days. The survival rate of M. rosenbergii decreased significantly after treatment with 0.54 mg L-1 ammonia-N compared with that in the other groups. However, ammonia-N had no significant effect on the growth of the river prawn after exposure for 20 days. GS activity increased significantly after exposure to 0.108 mg L-1 ammonia-N compared with the control and other ammonia-N-treated groups. Hepatopancreatic GDH activity was lower in the prawns treated with 0.216, 0.324, or 0.54 mg L-1 ammonia-N than in the control by 34.70%, 38.80%, or 41.94%, respectively. Ammonia-N had no significant effect on hepatopancreatic AST or ALT activity. Urea nitrogen was higher in the prawns treated with 0.216 mg L-1 ammonia-N than in the control or those treated with 0.54 mg L-1 ammonia-N. Ammonia-N had significant effects on the lipid, carbohydrate. and protein metabolism of M. rosenbergii, including purine metabolism, amino sugar and nucleotide sugar metabolism, α-linolenic acid metabolism, arginine and proline metabolism, glutathione metabolism, and phosphonate and phosphate metabolism, and on the terpenoid biosynthesis, lysine degradation, and lysine biosynthesis pathways. High concentrations of ammonia-N stress increased the content of glutamate and arginine, which may participate in the urea cycle, which synthesizes glutamine or urea to eliminate ammonia toxicity.

Astrocytic glycogen metabolism in the healthy and diseased brain.

The brain contains a fairly low amount of glycogen, mostly located in astrocytes, a fact that has prompted the suggestion that glycogen does not have a significant physiological role in the brain. However, glycogen metabolism in astrocytes is essential for several key physiological processes and is adversely affected in disease. For instance, diminished ability to break down glycogen impinges on learning, and epilepsy, Alzheimer's disease, and type 2 diabetes are all associated with abnormal astrocyte glycogen metabolism. Glycogen metabolism supports astrocytic K+ and neurotransmitter glutamate uptake and subsequent glutamine synthesis-three fundamental steps in excitatory signaling at most brain synapses. Thus, there is abundant evidence for a key role of glycogen in brain function. Here, we summarize the physiological brain functions that depend on glycogen, discuss glycogen metabolism in disease, and investigate how glycogen breakdown is regulated at the cellular and molecular levels.

Sucrose breakdown within guard cells provides substrates for glycolysis and glutamine biosynthesis during light-induced stomatal opening.

Sucrose has long been thought to play an osmolytic role in stomatal opening. However, recent evidence supports the idea that the role of sucrose in this process is primarily energetic. Here we used a combination of stomatal aperture assays and kinetic [U-13 C]-sucrose isotope labelling experiments to confirm that sucrose is degraded during light-induced stomatal opening and to define the fate of the C released from sucrose breakdown. We additionally show that addition of sucrose to the medium did not enhance light-induced stomatal opening. The isotope experiment showed a consistent 13 C enrichment in fructose and glucose, indicating that during light-induced stomatal opening sucrose is indeed degraded. We also observed a clear 13 C enrichment in glutamate and glutamine (Gln), suggesting a concerted activation of sucrose degradation, glycolysis and the tricarboxylic acid cycle. This is in contrast to the situation for Gln biosynthesis in leaves under light, which has been demonstrated to rely on previously stored C. Our results thus collectively allow us to redraw current models concerning the influence of sucrose during light-induced stomatal opening, in which, instead of being accumulated, sucrose is degraded providing C skeletons for Gln biosynthesis.

Yeast two-hybrid screening reveals a dual function for the histone acetyltransferase GcnE by controlling glutamine synthesis and development in Aspergillus fumigatus.

The acetyltransferase GcnE is part of the SAGA complex which regulates fungal gene expression through acetylation of chromatin. Target genes of the histone acetyltransferase GcnE include those involved in secondary metabolism and asexual development. Here, we show that the absence of GcnE not only abrogated conidiation, but also strongly impeded vegetative growth of hyphae in the human pathogenic fungus Aspergillus fumigatus. A yeast two-hybrid screen using a Saccharomyces cerevisiae strain whose tRNA molecules were specifically adapted to express A. fumigatus proteins identified two unprecedented proteins that directly interact with GcnE. Glutamine synthetase GlnA as well as a hypothetical protein located on chromosome 8 (GbpA) were identified as binding partners of GcnE and their interaction was confirmed in vivo via bimolecular fluorescence complementation. Phenotypic characterization of gbpA and glnA deletion mutants revealed a role for GbpA during conidiogenesis and confirmed the central role of GlnA in glutamine biosynthesis. The increase of glutamine synthetase activity in the absence of GcnE indicated that GcnE silences GlnA through binding. This finding suggests an expansion of the regulatory role of GcnE in A. fumigatus.

Farnesoid X Receptor Activation Promotes Hepatic Amino Acid Catabolism and Ammonium Clearance in Mice.

BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice to evaluate these functions and investigate whether FXR regulates amino acid metabolism. METHODS: To study the role of FXR in mouse liver, we used mice with a disruption of Nr1h4 (FXR-knockout mice) and compared them with floxed control mice. Mice were gavaged with the FXR agonist obeticholic acid or vehicle for 11 days. Proteome analyses, as well as targeted metabolomics and chromatin immunoprecipitation, were performed on the livers of these mice. Primary rat hepatocytes were used to validate the role of FXR in amino acid catabolism by gene expression and metabolomics studies. Finally, control mice and mice with liver-specific disruption of Nr1h4 (liver FXR-knockout mice) were re-fed with a high-protein diet after 6 hours fasting and gavaged a 15NH4Cl tracer. Gene expression and the metabolome were studied in the livers and plasma from these mice. RESULTS: In livers of control mice and primary rat hepatocytes, activation of FXR with obeticholic acid increased expression of proteins that regulate amino acid degradation, ureagenesis, and glutamine synthesis. We found FXR to bind to regulatory sites of genes encoding these proteins in control livers. Liver tissues from FXR-knockout mice had reduced expression of urea cycle proteins, and accumulated precursors of ureagenesis, compared with control mice. In liver FXR-knockout mice on a high-protein diet, the plasma concentration of newly formed urea was significantly decreased compared with controls. In addition, liver FXR-knockout mice had reduced hepatic expression of enzymes that regulate ammonium detoxification compared with controls. In contrast, obeticholic acid increased expression of genes encoding enzymes involved in ureagenesis compared with vehicle in C57Bl/6 mice. CONCLUSIONS: In livers of mice, FXR regulates amino acid catabolism and detoxification of ammonium via ureagenesis and glutamine synthesis. Failure of the urea cycle and hyperammonemia are common in patients with acute and chronic liver diseases; compounds that activate FXR might promote ammonium clearance in these patients.

Complete sequence of the tumor-inducing plasmid pTiChry5 from the hypervirulent Agrobacterium tumefaciens strain Chry5.

Agrobacterium tumefaciens strain Chry5 is hypervirulent on many plants including soybean that are poorly transformed by other A. tumefaciens strains. Therefore, it is considered as a preferred vector for genetic transformation of plants. Here we report the complete nucleotide sequence of its chrysopine-type Ti-plasmid pTiChry5. It is comprised of 197,268 bp with an overall GC content of 54.5%. Two T-DNA regions are present and 219 putative protein-coding sequences could be identified in pTiChry5. Roughly one half of the plasmid is highly similar to the agropine-type Ti plasmid pTiBo542, including the virulence genes with an identical virG gene, which is responsible for the supervirulence caused by pTiBo542. The remaining part of pTiChry5 is less related to that of pTiBo542 and embraces the trb operon of conjugation genes, genes involved in the catabolism of Amadori opines and the gene for chrysopine synthase, which replaces the gene for agropine synthase in pTiBo542. With the exception of an insertion of IS869, these Ti plasmids differ completely in the set of transposable elements present, reflecting a different evolutionary history from a common ancestor.

Hepatic glutamate transport and glutamine synthesis capacities are decreased in finished vs. growing beef steers, concomitant with increased GTRAP3-18 content.

Hepatic glutamate uptake and conversion to glutamine is critical for whole-body N metabolism, but how this process is regulated during growth is poorly described. The hepatic glutamate uptake activities, protein content of system [Formula: see text] transporters (EAAC1, GLT-1) and regulatory proteins (GTRAP3-18, ARL6IP1), glutamine synthetase (GS) activity and content, and glutathione (GSH) content, were compared in liver tissue of weaned Angus steers randomly assigned (n = 8) to predominantly lean (growing) or predominantly lipid (finished) growth regimens. Steers were fed a cotton seed hull-based diet to achieve final body weights of 301 or 576 kg, respectively, at a constant rate of growth. Liver tissue was collected at slaughter and hepatic membranes fractionated. Total (75%), Na+-dependent (90%), system [Formula: see text]-dependent (abolished) glutamate uptake activity, and EAAC1 content (36%) in canalicular membrane-enriched vesicles decreased as steers developed from growing (n = 6) to finished (n = 4) stages, whereas Na+-independent uptake did not change. In basolateral membrane-enriched vesicles, total (60%), Na+-dependent (60%), and Na+-independent (56%) activities decreased, whereas neither system [Formula: see text]-dependent uptake nor protein content changed. EAAC1 protein content in liver homogenates (n = 8) decreased in finished vs. growing steers, whereas GTRAP3-18 and ARL6IP1 content increased and GLT-1 content did not change. Concomitantly, hepatic GS activity decreased (32%) as steers fattened, whereas GS and GSH contents did not differ. We conclude that hepatic glutamate uptake and GS synthesis capacities are reduced in livers of finished versus growing beef steers, and that hepatic system [Formula: see text] transporter activity/EAAC1 content is inversely proportional to GTRAP3-18 content.

Restricting Glutamine or Glutamine-Dependent Purine and Pyrimidine Syntheses Promotes Human T Cells with High FOXP3 Expression and Regulatory Properties.

T cell subsets differ in their metabolic requirements, and further insight into such differences might be harnessed to selectively promote regulatory T cells (Tregs) for therapies in autoimmunity and transplantation. We found that Gln restriction during human T cell activation favored CD4 T cells with high expression of the Treg transcription factor FOXP3. This resulted from shrinking numbers and reduced proliferation of activated FOXP3(lo/-)CD4 T cells while FOXP3(hi)CD4 T cell numbers increased. This gain was abolished by blocking Gln synthetase, an enzyme that responds to Gln and purine/pyrimidine deficiencies. The shift toward FOXP3(hi)CD4 T cells under Gln restriction was recapitulated with inhibitors of Gln-dependent pyrimidine and purine syntheses that together closely mimicked declining cell numbers and cell cycles, and by small interfering RNA knockdown of the respective rate-limiting Gln-consuming enzymes CAD and PPAT. FOXP3(hi)-enriched CD25(hi)CD4 T cells from these cultures inhibited proliferation, but they also produced effector cytokines, including IL-17A. The latter was largely confined to CTLA-4(hi)-expressing FOXP3(hi)-enriched CD25(hi)CD4 T cells that suppressed proliferation more weakly than did CTLA-4(lo/-)CD25(hi)FOXP3(hi)-enriched T cells. A causal link between high IL-17A production and impaired suppression of proliferation could not be demonstrated, however. Collectively, these results reveal a Gln synthetase-dependent increase and resilience of FOXP3(hi) cells under Gln restriction, and they demonstrate that impaired Gln-dependent nucleotide synthesis promotes FOXP3(hi) cells with regulator properties. It remains to be investigated to what extent the concomitant retention of IL-17A-producing CD4 T cells may limit the therapeutic potential of Tregs enriched through targeting these pathways in vivo.

Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis.

Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous 13 C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of 13 C-labeled acetate as an astrocyte specific metabolic substrate. Recent studies have, however, challenged the arguments used to anchor this astrocyte specificity of acetate and glutamate. The aim of the current study was to evaluate the specificity of acetate and glutamate as astrocyte substrates in brain slices. Acutely isolated hippocampal and cerebral cortical slices from female NMRI mice were incubated in media containing [1,2-13 C]acetate or [U-13 C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority of glutamine 13 C-labeling from [1,2-13 C]acetate as intended. However, 13 C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-13 C]acetate in the presence of MSO exceeded the level probable from exclusive labeling of the astrocytic pool, which likewise suggests neuronal acetate metabolism. Approximately 50% of glutamate was uniformly labeled in slices incubated with [U-13 C]glutamate in the presence of MSO, suggesting that neurons exhibit substantial uptake of exogenously provided glutamate. © 2017 Wiley Periodicals, Inc.

Multifactorial Effects on Different Types of Brain Cells Contribute to Ammonia Toxicity.

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate-aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH4+ and K+ in their effects on the Na+,K+-ATPase and the Na+,K+, 2 Cl- and water transporter NKCC1. Stimulation of the Na+,K+-ATPase driven NKCC1 in both astrocytes and endothelial cells is essential for the development of brain edema. Na+,K+-ATPase stimulation also activates production of endogenous ouabains. This leads to oxidative and nitrosative damage and sensitizes NKCC1. Administration of ouabain antagonists may accordingly have therapeutic potential in hyperammonemic diseases.

Glutamine: a major player in nitrogen catabolite repression in the yeast Dekkera bruxellensis.

In the present work we studied the expression of genes from nitrogen central metabolism in the yeast Dekkera bruxellensis and under regulation by the Nitrogen Catabolite Repression mechanism (NCR). These analyses could shed some light on the biological mechanisms involved in the adaptation and survival of this yeast in the sugarcane fermentation process for ethanol production. Nitrogen sources (N-sources) in the form of ammonium, nitrate, glutamate or glutamine were investigated with or without the addition of methionine sulfoximine, which inhibits the activity of the enzyme glutamine synthetase and releases cells from NCR. The results showed that glutamine might act as an intracellular sensor for nitrogen availability in D. bruxellensis, by activating NCR. Gene expression analyses indicated the existence of two different GATA-dependent NCR pathways, identified as glutamine-dependent and glutamine-independent mechanisms. Moreover, nitrate is sensed as a non-preferential N-source and releases NCR to its higher level. After grouping genes according to their regulation pattern, we showed that genes for ammonium assimilation represent a regulon with almost constitutive expression, while permease encoding genes are mostly affected by the nitrogen sensor mechanism. On the other hand, nitrate assimilation genes constitute a regulon that is primarily subjected to induction by nitrate and, to a lesser extent, to a repressive mechanism by preferential N-sources. This observation explains our previous reports showing that nitrate is co-consumed with ammonium, a trait that enables D. bruxellensis cells to scavenge limiting N-sources in the industrial substrate and, therefore, to compete with Saccharomyces cerevisiae in this environment.