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PubMed2005

Cultures of rat astrocytes challenged with a steady supply of glutamate: new model to study flux distribution in the glutamate-glutamine cycle.

Glutamate metabolism in astrocytes was studied using an experimental setup that simulates the role of neurons (glutamate producers and glutamine consumers) by the addition of glutaminase to the culture medium. Thereby, a steady supply of glutamate was imposed at the expense of glutamine, and the stress intensity was manipulated by changing the glutaminase concentration. Glutamate supply rates in the range 8-23 nmol/min/mg protein were examined for periods of up to 48 h. When the glutamate supply rate exceeded the uptake rate of this amino acid, a transient increase in the extracellular concentration of glutamate was observed. In response to this stress, the fluxes through the glutamate transporter and glutamine synthetase were increased considerably, and the extracellular concentration of glutamate was eventually restored to a low level. The increased levels of glutamine synthetase were demonstrated by immunoblotting analysis. The effect on glutamate metabolism of the transaminase inhibitor, aminooxyacetic acid (AOAA), and of NH4Cl was also investigated. The supply of glutamate caused a concomitant reduction in the levels of phosphocreatine, phosphoethanolamine, and phosphocholine without affecting the ATP pool. Glutamine synthetase was shown to be is a key element in the control of glutamate metabolism in astrocytic cultures. The metabolic fate of glutamate depends greatly on the time of endurance to the challenge: in naive cells, glutamate was primarily metabolized through the transaminase pathway, while in well-adapted cells glutamate was converted almost exclusively through glutamine synthetase.

Animals↗

PubMed1978

Reciprocal regulation of glucose and glutamine utilization by cultured human diploid fibroblasts.

Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 micrometer, (2) the total glutamine utilization by fibroblasts increase at least 30% in medium with 25 micrometer to 70 micrometer glucose compared to medium with 5.5 mM glucose, while the rate of glutamine-1 or 5-14C oxidation to CO2 increased 5-fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose-6-14C oxidation by 88% and stimulated glucose-1-14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.

Cell Division↗

PubMed1984

The relationship between purines, pyrimidines, nucleosides, and glutamine for fibroblast cell proliferation.

Previous studies indicate that glutamine is a critical requirement for cell proliferation in vitro. We recently showed that depletion of glutamine from the culture medium supporting growing cells significantly reduced the proportion of cells undergoing DNA synthesis. Similarly glutamine depletion significantly reduced the stimulatory response of quiescent cells to 10% serum. This study shows that the inhibitory effects of depletion of glutamine--in either of these two situations--can be overcome by the addition of adenine or adenosine. Adenine was the only nitrogen base and adenosine was the only nucleoside for which this effect was observed. Such effects could, however, also be achieved by addition of the purine metabolites hypoxantine and inosine. Furthermore, it was found that glutamine (or adenine/adenosine) is only required during a limited interval coinciding with the late part of the G1-phase and the beginning of S-phase. These data suggest the possibility that glutamine exerts its main regulatory effects on cell proliferation by acting as a precursor for adenine and adenosine.

Adenine↗

PubMed1987

Production of tumor growth-inhibiting protein by the neonatal mouse brain: dependence on intracellular glutamine metabolism.

We previously reported that the mouse brain at the neonatal stage but not at the adult stage secreted a carcinostatic factor of 62,000 Da, termed NBCF, which inhibited clonal growth and DNA synthesis of malignant cells preferentially over those of normal cells. In the present study, NBCF production by the neonatal mouse brain in culture was investigated. Addition of L-glutamine to the culture medium markedly promoted NBCF production in a concentration-dependent manner. The production seemed to be specific to glutamine, since no promotive effect was exerted by L-glutamic acid, its analogue DL-alpha-aminoadipic acid, L-aspargine, or L-aspartic acid or by other amino acids or vitamins. NBCF production was markedly reduced in culture medium either devoid of L-glutamine or containing 6-diazo-5-oxo-L-norleucine, a glutamine antagonist, or L-methionine sulfoximine, an inhibitor of glutamine synthetase. Thus NBCF production was promoted by extracellular supply, intracellular synthesis, and utilization of L-glutamine but was not affected by its deamidated form or homologue amino acids. On the other hand, NBCF production was completely repressed by addition of cycloheximide to the culture medium. The repressive effect was also exerted by actinomycin D although not completely, whereas cytosine beta-D-arabinofuranoside did not repress NBCF production. These results indicated that NBCF production by cultivation was independent of DNA replication but dependent mostly on a transcription stage and its following stages and partly on a translation stage from the preexisting mRNA to the protein.

Animals↗

PubMed1994

Glutathione stimulates A549 cell proliferation in glutamine-deficient culture: the effect of glutamate supplementation.

Extracellular glutathione (GSH) is degraded by an external cell-surface enzyme, gamma-glutamyltranspeptidase (gamma-GT). The products are transported into cells to participate in important cellular processes. In the present study, we tested the hypothesis that extracellular GSH is a source of glutamic acid for cells that express gamma-GT. Under a glutamine-deficient culture condition, the extracellular GSH-supplemented glutamic acid would enhance intracellular glutamine synthesis, thereby stimulating cell proliferation. Human lung carcinoma A549 cells were cultured in glutamine-deficient Dulbecco's modified Eagle medium, and they did not proliferate unless glutamine was supplemented. Extracellular GSH, however, provoked a partial proliferation. The GSH effect correlated with a high level of gamma-GT activity and an increased intracellular level of glutamic acid. A constituent amino acid of GSH, glutamic acid but not cysteine, produced the same growth-stimulatory effect as GSH. Furthermore, neither oxothiazolidine-4-carboxylate (OTC), a cellular cysteine-delivery compound, nor cysteinylglycine, a dipeptide released from the gamma-GT reaction, stimulated cell proliferation. Moreover, buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, enhanced the GSH growth stimulatory effect, suggesting that increased cellular GSH synthesis does not correlate with cell growth stimulation. The results obtained demonstrated that glutamine is required for A549 cell proliferation and exogenous GSH partially substitutes for the growth stimulatory action of glutamine. It also suggests that the glutamic acid rather than the cysteine released from the GSH is responsible for the cell proliferation.

Buthionine Sulfoximine↗

PubMed2005

Arginine and glutamine availability and macrophage functions in the obese insulin-resistant Zucker rat.

Increased susceptibility to infections in obese patients may be related to decreased availability of arginine and glutamine, which may affect immune cell functions. Our aim was to evaluate the in vitro effects of these amino acids on the function of macrophages from obese insulin-resistant Zucker rats. Macrophages, isolated from male Zucker obese or lean rats by peritoneal lavage, were incubated in Dulbecco's modified Eagle medium (DMEM) without arginine or glutamine. Arginine or glutamine was added to the medium at increasing final concentrations (0, 0.25, 0.5, 1 or 2 mM). After stimulation by lipopolysaccharide (LPS) from E. coli (40 microg/ml), productions of tumour necrosis factor alpha (TNFalpha) and of nitric oxide (NO) were measured after 3 or 48 h incubation, respectively. NO production, lower in macrophages from obese rats, decreased in macrophages from lean rats (0 mM: 2,423 +/- 1,174 vs. 2 mM: 198 +/- 31 microM/mg protein/24 h; P < 0.05), but not in those from obese rats, when glutamine was added. TNFalpha production, lower in macrophages from obese rats, was inversely correlated with glutamine concentration. In the presence of arginine, NO production was constantly higher in macrophages from obese rats. It peaked at 0.5 mM arginine and decreased thereafter in both groups. TNFalpha production in macrophages from lean rats was unaffected by arginine, but decreased in macrophages from obese rats (0 mM: 1920 +/- 450 vs. 2 mM: 810 +/- 90 microM/mg protein/3 h; P < 0.05). These results suggest that abnormalities in cell signalling or in arginine and glutamine metabolism in macrophages of obese rats, resulting in decreased TNFalpha production and increased NO release, may contribute to increased susceptibility to infection in insulin-resistant states.

Animals↗

PubMed2005

Rapid induction of the intrinsic apoptotic pathway by L-glutamine starvation.

While the amino acid L-glutamine is known to play a role in the survival of several cell types, the underlying molecular mechanisms are still poorly defined. We show in this report that L-glutamine starvation rapidly triggered apoptosis in Sp2/0-Ag14 hybridoma cells. This process involved the activation of both caspases-9 and -3, suggesting that L-glutamine deprivation initiated an intrinsic apoptotic pathway in Sp2/0-Ag14 cells. Supporting this idea, the cytosolic release of the mitochondrial proteins SMAC/DIABLO and cytochrome c (Cyt c) was observed, with an initial limited leakage occurring during the first 30 min of L-glutamine deprivation, followed by a greater release after 60 min. The latter occurred simultaneously with the translocation of the pro-apoptotic protein Bax to the mitochondria. Finally, a decline in XIAP levels and the activation of caspases-3 and -9 were observed. Thus, L-glutamine deprivation of Sp2/0-Ag14 cells rapidly triggers intracellular events, which target the mitochondria, leading to the cytosolic release of apoptogenic factors, the activation of caspases-9 and -3, and the commitment to the death program. This work introduces the Sp2/0Ag14 hybridoma as a unique model for the study of the molecular events underlying the pro-survival function of L-glutamine.

Animals↗

PubMed1992

Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media.

A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCl, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage. Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium. Even in the presence of 5 mM glucose, 70% of the embryos develop to at least four cells, and 60% reach the blastocyst stage. Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development. Moreover, the response to glutamine depends on the concentration of NaCl in the medium. When the NaCl concentration is low, glutamine inhibits development. In contrast, when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt. We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte.

Animals↗

PubMed1994

Glutamine transport in normal and acidotic rat kidney mitochondria.

Glutamine transport in both normal and acidotic rat kidney mitochondria was investigated using both isotopic techniques and by spectroscopic measurements in which glutamine metabolism was allowed to occur. Widely used criteria for demonstrating the occurrence of carrier-mediated transport were successfully applied in both cases. Three transport mechanisms were found to occur, namely glutamine uniport, active only during acidosis and glutamine/glutamate and glutamine/malate antiports, active in both normal and acidotic mitochondria. Efflux of glutamate, via a glutamate/OH- translocator, following glutamine uptake by mitochondria was experimentally ruled out. Glutamine uniport in acidotic mitochondria and glutamine/glutamate and glutamine/malate antiports in both normal and acidotic mitochondria were investigated in detail: differences found in Km and Vmax values, in pH and temperature dependence, and in the pattern of inhibitor sensitivity of glutamine transport demonstrated the existence of five different translocators whose activities were found to fit with the physiological requirements of renal ammoniogenesis.

Acidosis↗

PubMed1996

An important role for pentose cycle in the synthesis of citrulline and proline from glutamine in porcine enterocytes.

This study was designed to determine a role of pentose cycle in the provision of NADPH for the synthesis of citrulline and proline from glutamine in porcine enterocytes. Enterocytes from 4-day-old pigs were incubated at 37 degrees C for 0 to 30 min in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 2 mM glutamine and 5 mM glucose in the presence of 0, 0.1, or 0.25 mM dehydroepiandrosterone (DHEA), a potent inhibitor of glucose-6-phosphate dehydrogenase which is the key regulatory enzyme of pentose cycle. The activity of this cycle was estimated with the use of [1-14C]glucose and [6-14C]glucose. In some experiments, the medium included 2 mM ornithine and 2 mM NH4Cl (no glutamine). About 14% of glucose taken up by enterocytes was metabolized via pentose cycle. The flux from glucose into this cycle was decreased by 70 and 86%, respectively, in the presence of 0.1 and 0.25 mM DHEA compared with its absence. DHEA inhibited the synthesis of ornithine, citrulline, arginine, and proline from glutamine in a concentration-dependent manner, but had no effect on the formation of citrulline and arginine from ornithine. However, DHEA decreased the synthesis of proline from ornithine by 79%. DHEA had no effect on cellular ATP concentrations. These results provide the first line of evidence suggesting that glucose metabolism via pentose cycle plays an important role in providing NADPH for the conversion of glutamine into pyrroline-5-carboxylate in porcine enterocytes, which may explain the glucose-dependent synthesis of citrulline and proline from glutamine in these cells.

Adenosine Triphosphate↗

PubMed1996

Improvement of motility and fertilization potential of postthaw human sperm using glutamine.

The effectiveness of three amino acids, glutamine, proline, and histidine, and one amino acid-related compound, betaine, in preserving human sperm diluted v/v in a basal medium (BM) containing 14% glycerol during the freeze-thaw (FT) process was studied. At 80 mM in BM, only glutamine improved the 5- to 60-min postthaw total and progressive motilities and velocity. The presence of glutamine at 80 mM is not sufficient to achieve lower concentrations of the toxic agent glycerol in FT medium. Glutamine may therefore have a mechanism of protection on human spermatozoa that is independent from that of glycerol. The zona-free hamster egg penetration test showed that the percentage of eggs penetrated was significantly greater when spermatozoa were frozen-thawed with 80 mM glutamine in BM. Consequently, the presence of glutamine at 80 mM in a glycerol-FT medium maintains human sperm motility and fertilizing ability during the FT process.

Animals↗

PubMed1996

Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation.

Assimilation of nitrogen requires the synthesis of only two central intermediates, glutamate and glutamine, from which other compounds derive nitrogen by secondary transfers. We measured the internal pool sizes of glutamate and glutamine in Salmonella typhimurium under conditions of external nitrogen limitation or sufficiency. When growth was slowed by nitrogen limitation, the glutamine pool was lower by a factor of up to 10, whereas the glutamate pool remained high. The decrease in the glutamine pool was general in nature, being seen with various limiting nitrogen sources in batch culture and with ammonia, the optimal nitrogen source, as the limiting nutrient in continuous culture. The only nitrogen source that gave discordant results was alanine, and we present evidence that alanine has inhibitory effects which extend beyond simple nitrogen limitation. Studies with mutant strains having altered nitrogen assimilation indicated that the decreases in the glutamine pool observed in the wild-type strain under nitrogen-limiting conditions were probably sufficient to account for slow growth and were likely to be responsible for slow growth. Hence we postulate that external nitrogen limitation is first perceived by Salmonella as a drop in its internal glutamine pool.

Alanine↗

PubMed1990

Glutamine synthetase inhibition prevents cerebral oedema during hyperammonemia.

The relationship between cerebral oedema and cerebral glutamine accumulation was investigated during acute hyperammonemia in anesthetized rats. Six hours of ammonium acetate infusion resulted in an increase in cortical glutamine concentration and a decrease in specific gravity. Pretreatment with methionine sulfoximine inhibited glutamine synthetase, prevented the increase in glutamine during hyperammonemia, and prevented the decrease in specific gravity. We conclude that the increase in brain water content is linked to the glutamine accumulation derived from the detoxification of ammonia by glutamine synthetase.

Acetates↗

PubMed1984

Methionine and glutamine transport systems in D-methionine utilising revertants of Salmonella typhimurium.

In Salmonella typhimurium, methionine auxotrophs such as metB can use D-methionine as a methionine source. MetP mutations prevent this growth since D-methionine can enter only via the metP high-affinity methionine transport system. D-methionine utilising revertants ( Dmu +) were selected from metB23 metP760 ( HU76 ) following nitrosoguanidine mutagenesis. The properties of two such revertants, HU206 and HU415 , indicated that reversion was not due to backmutation of the metP760 mutation. Genetic analysis indicated that each strain possessed two mutations, designated dmu and gln, in addition to the original metB23 and metP760 mutations. The dmu mutation restores ability to grow on D-methionine, partly restores D- and L-methionine transport activity, and makes the cells particularly sensitive to inhibition by L-glutamine while growing on D but not L-methionine. The growth inhibition by L-glutamine was shown to be caused by competition by L-glutamine for D-methionine transport by the high-affinity methionine system. The gln mutation greatly reduces activity of the high-affinity glutamine transport system. The Dmu + strains are also partly defective in the glutamine low-affinity transport system, possibly because the partially-restored methionine high-affinity system, or a component of tis system, functions in the transport of glutamine by its low-affinity system.

Biological Transport, Active↗

PubMed1981

Nitrogen control in Pseudomonas aeruginosa: a role for glutamine in the regulations of the synthesis of nadp-dependent glutamate dehydrogenase, urease and histidase.

In Pseudomonas aeruginosa the formation of urease, histidase and some other enzymes involved in nitrogen assimilation is repressed by ammonia in the growth medium. The key metabolite in this process appears to be glutamine or a product derived from it, since ammonia and glutamate did not repress urease and histidase synthesis in a mutant lacking glutamine synthetase activity when growth was limited for glutamine. The synthesis of these enzymes was repressed in cells growing in the presence of excess glutamine. High levels of glutamine were also required for the derepression of NADP-dependent glutamate dehydrogenase formation in the glutamine synthetase-negative mutant.

Ammonia↗

PubMed1981

The stimulus-secretion coupling of amino acid-induced insulin release. IV. Ionic response to L-Leucine and L-Glutamine.

L-Glutamine enhances insulin release evoked by L-leucine in isolated rat pancreatic islets. The enhancing action of L-glutamine, which is a rapid but steadily increasing and not rapidly reversible phenomenon is not attributable to any major change in either K+ or Ca2+ outflow from the islet cells. It coincides with an apparent increase in Ca2+ inflow rate and, hence, with Ca accumulation in the islets. The initial ionic response to L-leucine is not qualitatively altered by the presence of L-glutamine. In their combined capacity to stimulate 45Ca net uptake in the islets, L-glutamine can be replaced by L-asparagine but not by L-glutamate, whereas L-leucine can be replaced by L-norvaline or L-isoleucine, but not by L-valine, glycine or L-lysine. Such a specificity is identical to that characterizing the effect of these various amino acids upon insulin release. It is postulated that the release of insulin evoked by the combination of L-leucine and L-glutamine involves essentially the same remodelling of ionic fluxes as that evoked by other nutrient secretagogues with, however, an unusual time course for the functional response to L-glutamine.

Amino Acids↗

PubMed1989

Glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA: correlation of regional uptake by rat brain synaptosomes.

To more clearly define the roles of glutamine and 2-oxoglutarate as metabolic precursors of the transmitter pools of glutamate and GABA we have determined the relative rates at which these four substances, and adenosine and serotonin are accumulated by synaptosomes derived from twelve regions of the rat brain. Initial transport conditions and low substrate concentrations were used to maximize uptake by high-affinity systems, except the uptake of glutamine was determined at both low and high concentrations. Because the uptake of 2-oxoglutarate is markedly enhanced by glutamine, 2-oxoglutarate uptake was determined with and without glutamine (0.2 mM) added to the incubation medium. For each substrate, regional differences in uptake ranged from approximately two- to fourteen-fold. An anaylsis of uptake kinetics revealed that the regional differences were due primarily to differences in transport capacity rather than substrate affinities, at least for glutamate, GABA, and 2-oxoglutarate. Thirty-four correlation analyses of relative uptake values were performed. Strong correlations were found between 2-oxoglutarate and glutamate, and between glutamine and glutamate, whereas no strong correlations occurred between these substrates and GABA. Our results support the view that both glutamine and 2-oxoglutarate are major precursors of the transmitter pool of glutamate throughout the rat brain, but their relative contributions toward replenishing the transmitter pool of GABA are less certain.

Animals↗

PubMed1978

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles.

Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes with Km values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8--7.1. Glutamic acid uptake also occurred by a two-component system with Km values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The low Km system for glutamic acid had a pH optimum of 7.2--7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20--30% by 3 mM phenylalanine, valine, alpha-aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.

Amino Acids↗