Glutamine and asparagine as nitrogen donors for reductant-dependent glutamate synthesis in pea roots.
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ABSTRACT:
SUBMITTER: Miflin BJ
PROVIDER: S-EPMC1165634 | biostudies-other | 1975 Aug
REPOSITORIES: biostudies-other
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Project description:Glutamine dependence is a prominent feature of cancer metabolism, and here we show that melanoma cells, irrespective of their oncogenic background, depend on glutamine for growth. A quantitative audit of how carbon from glutamine is used showed that TCA-cycle-derived glutamate is, in most melanoma cells, the major glutamine-derived cataplerotic output and product of glutaminolysis. In the absence of glutamine, TCA cycle metabolites were liable to depletion through aminotransferase-mediated α-ketoglutarate-to-glutamate conversion and glutamate secretion. Aspartate was an essential cataplerotic output, as melanoma cells demonstrated a limited capacity to salvage external aspartate. Also, the absence of asparagine increased the glutamine requirement, pointing to vulnerability in the aspartate-asparagine biosynthetic pathway within melanoma metabolism. In contrast to melanoma cells, melanocytes could grow in the absence of glutamine. Melanocytes use more glutamine for protein synthesis rather than secreting it as glutamate and are less prone to loss of glutamate and TCA cycle metabolites when starved of glutamine.
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Project description:Bacteria in nature often reside in differentiated communities termed biofilms, which are an active interphase between uni-cellular and multicellular life states for bacteria. Here we demonstrate that the development of B. subtilis biofilms is dependent on the use of glutamine or glutamate as a nitrogen source. We show a differential metabolic requirement within the biofilm; while glutamine is necessary for the dividing cells at the edges, the inner cell mass utilizes lactic acid. Our results indicate that biofilm cells preserve a short-term memory of glutamate metabolism. Finally, we establish that drugs that target glutamine and glutamate utilization restrict biofilm development. Overall, our work reveals a spatial regulation of nitrogen and carbon metabolism within the biofilm, which contributes to the fitness of bacterial complex communities. This acquired metabolic division of labor within biofilm can serve as a target for novel anti-biofilm chemotherapies.
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Project description:BackgroundThe cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis.ResultsWe observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain.ConclusionsThe physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.
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Project description:Although glutamine is considered the main precursor for citrulline synthesis, the current literature does not differentiate between the contribution of glutamine carbon skeleton vs. nonspecific nitrogen (i.e., ammonia) and carbon derived from glutamine oxidation. To elucidate the role of glutamine and nonspecific nitrogen in the synthesis of citrulline, l-[2-(15)N]- and l-[5-(15)N]glutamine and (15)N-ammonium acetate were infused intragastrically in mice. The amino group of glutamine labeled the three nitrogen groups of citrulline almost equally. The amido group and ammonium acetate labeled the ureido and amino groups of citrulline, but not the delta-nitrogen. D(5)-glutamine also infused in this arm of the study, which traces the carbon skeleton of glutamine, was utilized poorly, accounting for only 0.2-0.4% of the circulating citrulline. Dietary glutamine nitrogen (both N groups) incorporation was 25-fold higher than the incorporation of its carbon skeleton into citrulline. To investigate the relative contributions of the carbon skeleton and nonspecific carbon of glutamine, arginine, and proline to citrulline synthesis, U-(13)C(n) tracers of these amino acids were infused intragastrically. Dietary arginine was the main precursor for citrulline synthesis, accounting for approximately 40% of the circulating citrulline. Proline contribution was minor (3.4%), and glutamine was negligible (0.4%). However, the glutamine tracer resulted in a higher enrichment in the ureido group, indicating incorporation of nonspecific carbon from glutamine oxidation into carbamylphosphate used for citrulline synthesis. In conclusion, dietary glutamine is a poor carbon skeleton precursor for the synthesis of citrulline, although it contributes both nonspecific nitrogen and carbon to citrulline synthesis.
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Project description:Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development.IMPORTANCE Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.
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Project description:Prior evidence suggests panic disorder (PD) is characterized by neurometabolic abnormalities, including increased brain lactate responses to neural activation. Increased lactate responses could reflect a general upregulation of metabolic responses to neural activation. However, prior studies in PD have not measured activity-dependent changes in brain metabolites other than lactate. Here we examine activity-dependent changes in both lactate and glutamate plus glutamine (glx) in PD.Twenty-one PD patients (13 remitted, 8 symptomatic) and 12 healthy volunteers were studied. A single-voxel, J-difference, magnetic resonance spectroscopy editing sequence was used to measure lactate and glx changes in visual cortex induced by visual stimulation.The PD patients had significantly greater activity-dependent increases in brain lactate than healthy volunteers. The differences were significant for both remitted and symptomatic PD patients, who did not differ from each other. Activity-dependent changes in glx were significantly smaller in PD patients than in healthy volunteers. The temporal correlation between lactate and glx changes was significantly stronger in control subjects than in PD patients.The novel demonstration that glx responses are diminished and temporally decoupled from lactate responses in PD contradicts the model of a general upregulation of activity-dependent brain metabolic responses in PD. The increase in activity-dependent brain lactate accumulation appears to be a trait feature of PD. Given the close relationship between lactate and pH in the brain, the findings are consistent with a model of brain metabolic and pH dysregulation associated with altered function of acid-sensitive fear circuits contributing to trait vulnerability in PD.
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Project description:1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22mumol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7-0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37 degrees C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.
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Project description:Elucidating the amino acid (AA) metabolism patterns of Streptococcus thermophilus has important effects on the precise design of nitrogen sources for high-cell-density culture. Transcriptomics and metabolomics were combined to reveal the cysteine, methionine, glutamate, glutamine, arginine, aspartate, asparagine and alanine metabolic pathways in S. thermophilus MN-ZLW-002, including glutathione. The changes in the synthesis, consumption and concentration of AAs and their metabolites, as well as regulatory genes with time were revealed. The metabolism of L-cysteine, L-glutamate, L-aspartate and L-alanine generated some potential functional metabolites. The metabolism of methionine and glutamate generated potential harmful metabolites. S. thermophilus MN-ZLW-002 can synthesize glutathione. Some potential functional metabolites have similar biological functions, indicating that S. thermophilus can resist environmental stresses through multiple mechanisms. The expression of some key genes in synthesis pathway of AA indicated that cysteine, methionine, asparagine, aspartate, arginine and lysine were insufficient or imbalance between nutrient components. The accumulation of large amounts of AA metabolites might be the primary cause of the overconsumption of AAs and influence the growth of S. thermophilus. The present study revealed the metabolic profiles of abovementioned AAs as well as those of regulatory genes and metabolites. These results were beneficial to the precise design of nitrogen sources and regulation of functional metabolites for the high-cell-density culture of S. thermophilus.
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Project description:While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.IMPORTANCE We have previously found that Kaposi's sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.
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