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PubMed2003

Glutamine attenuates post-traumatic glutathione depletion in human muscle.

Glutathione is quantitatively the most important endogenous scavenger system. Glutathione depletion in skeletal muscle is pronounced following major trauma and sepsis in intensive care unit patients. Also, following elective surgery, glutathione depletion occurs in parallel with a progressive decline in muscle glutamine concentration. The present study was designed to test the hypothesis that glutamine supplementation may counteract glutathione depletion in a human trauma model. A homogeneous group of patients (n = 17) undergoing a standardized surgical procedure were prospectively randomly allocated to receive glutamine (0.56 g x day(-1) x kg(-1)) or placebo as part of isonitrogenous and isocaloric nutrition. Percutaneous muscle biopsies and blood samples were taken pre-operatively and at 24 and 72 h after surgery. The concentrations of muscle glutathione and related amino acids were determined in muscle tissue and plasma. In the control (unsupplemented) subjects, total muscle glutathione had decreased by 47+/-8% and 37+/-11% and reduced glutathione had decreased by 53+/-10% and 45+/-16% respectively at 24 and 72 h after surgery (P < 0.05). In contrast, in the glutamine-supplemented group, no significant post-operative decreases in total or reduced glutathione were seen following surgery. Muscle free glutamine had decreased at 72 h after surgery in both groups, by 41.4+/-14.8% (P < 0.05) in the glutamine-supplemented group and by 46.0+/-14.3% (P < 0.05) in the control group. In conclusion, the present study demonstrates that intravenous glutamine supplementation attenuates glutathione depletion in skeletal muscle in humans following standardized surgical trauma.

Aged↗
PubMed1999

Effect of glutamine on the intestinal permeability changes induced by indomethacin in humans.

BACKGROUND: Long-term non-steroidal anti-inflammatory drug (NSAID) intake may induce increased intestinal permeability, eventually resulting in enteropathy. Because increased permeability might be related to cell damage resulting from energy depletion, it was hypothesized that glutamine--the major energy source of the intestinal mucosal cell--might prevent permeability changes. METHODS: The 6-h urinary excretion of 51Cr-EDTA after an oral load of 51Cr-EDTA was used in this study as a measure for intestinal permeability. Healthy volunteers underwent a series of permeability tests: (i) basal test; (ii) test following NSAID (indomethacin); (iii) test following NSAID in combination with glutamine and/or misoprostol. RESULTS: The NSAID induced increased permeability in all volunteers. Pre-treatment with glutamine (3x7 g daily, 1 week before NSAID-dosing) did not prevent the NSAID-induced increase in permeability. Multiple doses of glutamine close in time to NSAID-dosing resulted in significantly lower permeability compared to the NSAID without glutamine. Co-administration of misoprostol with the multiple-dose scheme of glutamine resulted in a further reduction in the NSAID-induced increase in permeability. CONCLUSIONS: Glutamine decreases the permeability changes caused by NSAID-dosing when it is administered close in time, and misoprostol has a synergistic effect.

Adenosine Triphosphate↗
PubMed2002

Steady-state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis. The role of the allosteric activator GTP incoupling between glutamine hydrolysis and CTP synthesis.

CTP synthase catalyzes the reaction glutamine + UTP + ATP --> glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half-reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to the active site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP-activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP-binding analog adenosine 5'-[gamma-thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.

Allosteric Regulation↗
PubMed1995

Excess extracellular and low intracellular glutamate in poorly differentiating wobbler astrocytes and astrocyte recovery in glutamine-depleted culture medium.

The wobbler mouse develops an inherited motoneuronal degeneration of unknown origin in the spinal cord. Primary cultures of adult wobbler spinal cord astrocytes display abnormal morphological characteristics with fewer processes and paucity of cell-cell contacts. We have searched for a possible involvement of glutamate and glutamine intra- and extracellular accumulations in vitro in the abnormal differentiation of mutant astrocytes. We have found significantly higher glutamate and glutamine concentrations in the culture media of mutant astrocytes over a 3-day period compared with normal control astrocytes. Moreover, intracellular glutamate concentrations decreased substantially in mutant astrocytes, but intracellular glutamine concentrations remained unchanged. Furthermore, decreasing initial glutamine concentrations in the culture medium (glutamine-depleted medium) led to the recovery of normal extra- and intracellular concentrations of glutamate and recovery of quasi-normal morphological differentiation and increased cell-cell contacts, leading to an essentially normal looking astrocyte network after 3 days of culture. Under these conditions, which lead to recovery, the only remaining abnormality was the higher glutamine extracellular concentration attained in the originally depleted glutamine media. These findings suggest that mechanisms regulating glutamate/glutamine synthesis and/or influx/efflux are defective in wobbler astrocytes, leading to metabolic imbalance and possible cytotoxic effects characterized by disturbed intercellular networks and poor differentiation.

Animals↗
PubMed1997

Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of alpha-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of alpha-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of alpha-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.

Ammonia↗
PubMed1998

Glutamine uptake at the blood-brain barrier is mediated by N-system transport.

The mechanism of unidirectional transport of glutamine from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Amino acid uptake into brain across the blood-brain barrier (BBB) is classically thought to be via the Na-independent large neutral (L-system), acidic and basic amino acid transporters. In the presence of physiological concentrations of amino acids in the perfusate, which should saturate the known amino acid transporters at the BBB, the cortical transfer constant (Ki) for L-[14C]glutamine was 11.6 +/- 1.1 microl/g/min. The addition of either 10 mM 2-amino-2-norbornanecarboxylic acid or 10 mM 2-amino-2-norbornanecarboxylic acid and 5 mM cysteine had no effect on the cortical Ki for L-[14C]glutamine, indicating that glutamine transport under these conditions does not occur by the L-, A-, or ASC-systems. Decreasing perfusate Na from 140 to 2.4 mM by Tris substitution reduced the cortical Ki for L-[14C]glutamine by 62% (p < or = 0.001). The Na-dependent uptake has the characteristics of N-system transport. It was inhibited by L-histidine and L-glutamine, both N-system substrates, and it was pH sensitive and moderately tolerant of Li substitution for Na. This putative N-system transporter at the luminal membrane of the BBB plays an important role in mediating brain glutamine uptake.

Animals↗
PubMed2002

Glutamate-glutamine cycling in the epileptic human hippocampus.

PURPOSE: Several findings suggest that energy metabolism and the glutamate-glutamine cycle may be impaired in epilepsy. Positron emission tomography often shows interictal hypometabolism of the epileptogenic hippocampus. In vivo microdialysis studies show that seizure-associated glutamate release is doubled, and clearance is slowed. We hypothesized that the glutamate-glutamine cycle between neurons and glia may be decreased in the epileptic human hippocampus. METHODS: A 20% solution of 2-13C-glucose was infused before resection of the epileptogenic hippocampus. Blood glucose isotopic fractions were measured every 30 min. Blood and brain specimens were frozen quickly; perchloric acid extracts of the small metabolites were prepared and analyzed by proton and carbon magnetic resonance spectroscopy (MRS) at 11.75 Tesla. RESULTS: Standard histology showed 12 with hippocampal sclerosis and five with minimal neuron loss. The relative rates of glutamate-glutamine cycling with respect to glutamate synthesis were decreased in biopsies affected by hippocampal sclerosis (mean, 0.08; 95% confidence interval, 0.04-0.12) compared with those with minimal neuron loss (0.52; 95% CI, 0.30-0.75). Mean cellular glutamate concentrations were higher in minimal neuron loss (8.9 mM; 95% CI, 7.4-10.4) than hippocampal sclerosis (7.3 mM; 95% CI, 5.9-8.7). Cellular glutamine concentrations (mean, 2.8 mM; 95% CI, 2.4-3.2; n = 17) were the same in all groups. CONCLUSIONS: The epileptogenic, gliotic human hippocampus appears to be characterized metabolically by slow rates of glutamate-glutamine cycling, decreased glutamine content, and a relative increase in glutamate content. We hypothesize that the low rate of glutamate-glutamine cycling that results from a failure of glial glutamate detoxification could account for slow glutamate clearance from synapses and continuing low-grade excitotoxicity.

Adolescent↗
PubMed1997

The effect of hypotonicity, glutamine, and glycine on red cell preservation.

BACKGROUND: Red cells (RBCs) stored in hypo-osmolar additive solutions with the same concentrations of adenine, dextrose, mannitol, and sodium chloride and varied amounts of ammonium, phosphate, glycerol, and glutamine were better preserved than RBCs in the standard additive solution (Adsol). Cell swelling occurred in all the experimental additives. This observation prompted the evaluation of glutamine and glycine alone, as well as a combination of glutamine and glycine, all of which have been described as producing swelling of rat liver cells. STUDY DESIGN AND METHODS: Aliquots of RBCs were stored at 4 degrees C in Adsol or experimental additive solutions (EASs) all containing adenine, 2 mM; dextrose, 110 mM; mannitol, 55 mM; and sodium chloride, 50 mM. EAS 42 had, in addition, glutamine, 10 mM; glycine 5 mM, and phosphate, 20 mM. EAS 43 had glutamine, 10 mM; glycine, 10 mM; and phosphate 20 mM. EAS 44 had glutamine, 10 mM; EAS 45 had glutamine, 10 mM, and phosphate, 20 mM, and EAS 46 had only glycine, 10 mM. At intervals, measurements were made of mean corpuscular volume, mean corpuscular hemoglobin concentration, morphology, ATP, hemolysis, supernatant potassium, ammonia, pH, and microvesicles shed. RESULTS: The initial mean corpuscular volumes were larger in all EASs than in Adsol, but the greatest difference was between EASs 44 and 46 (108 fL) and Adsol (86 fL) (p < 0.001). The morphology scores were significantly better in all the EASs (p < 0.04). The ATPs were significantly greater in all the EASs (p < 0.001), and highest in those with phosphate. potassium leakage and hemolysis were less in the EASs (p < 0.001). The ammonia levels higher in all the EASs than in Adsol, with the exception of EAS 46. During storage, the extracorpuscular and intracorpuscular pH levels were essentially identical. The shedding of microvesicles was greatly reduced in all the EASs. CONCLUSION: Cell swelling induced in RBCs after collection appears to improve preservation. Ammonia and phosphate enhance RBC ATP maintenance. Glycine decrease the formation of ammonia by RBCs stored in a hypotonic medium.

Adenine↗
PubMed2000

Cost containment through L-alanyl-L-glutamine supplemented total parenteral nutrition after major abdominal surgery: a prospective randomized double-blind controlled study.

BACKGROUND & AIMS: Glutamine is recognized as a conditionally essential amino acid. Recent studies indicate that glutamine-containing total parenteral nutrition improves nitrogen economy, enhances gastrointestinal and immune functions and shortens hospital stay. METHODS: Thirty-seven patients (19 w and 18 m; age 61. 4+/-10.4 years; BMI 23.7+/-2.8 kg/m(2)) following major abdominal surgery receiving an isonitrogenous isoenergetic TPN with or without alanyl-glutamine supplementation (0.5 g/kg BW/day), were evaluated in a double-blind, randomized, controlled trial over a five-day period by measuring nitrogen balance, selected biochemical parameters and length of hospital stay. RESULTS: Supplemental alanyl-glutamine improved the overall mean (-3.5+/-1.6 vs. -5.5+/-1. 4 g N;P<0.05) and cumulative nitrogen balance (-14.1+/-9.1 vs. -21.7+/-11.4 g N;P<0.05) compared with the isonitrogenous, isoenergetic standard regimen. Alanyl-glutamine normalized plasma glutamine concentration and reduced the length of hospital stay (12.8+/-2.6 vs. 17.5+/-6.4 days;P<0.05). CONCLUSIONS: The results of the study confirm that supplementation with synthetic alanyl-glutamine dipeptide is associated with cost containment due to shortened hospitalization and improved nitrogen economy.

Abdomen↗
PubMed2002

Effect of glutamine-enriched total parenteral nutrition in patients with acute pancreatitis.

BACKGROUND: The management of acute pancreatitis (AP) frequently includes parenteral nutrition, but conditionally essential amino acids such as glutamine are not included in conventional total parenteral nutrition (TPN). AIM: This study was conducted to determine whether the inclusion of glutamine has a beneficial effect in patients with AP receiving TPN. METHODS: In a randomized, controlled study 28 patients with AP received either a standard TPN with 1.5 g/kg body weight protein or an isonitrogen, isocaloric TPN which contains 0.3 g/kg L -alanine- L -glutamine. Patients were assessed for nutritional and inflammatory parameters, infectious complications, length of TPN, length of hospital stay (LOS) and cost of TPN. RESULTS: There were no side-effects related to glutamine substitution observed. Glutamine was associated with a significant increase of cholinesterase, albumin and lymphocyte count in AP as well a decrease of C-reactive protein compared to standard TPN at day 14. There was a reduced length of TPN (10 [6-16] vs 16 [10-18] days, P<0.05) and a trend of reduced LOS (21 [14-32] vs 25 [19-40] days) in AP patients receiving glutamine. The overall cost per patient for TPN did not differ (gln+: 929+/-586 vs gln-: 981+/-507 euro/patient). CONCLUSION: Our results suggest that glutamine substitution is beneficial and does not increase the overall cost of parenteral feeding in patients with acute pancreatitis.

Acute Disease↗
PubMed2006

Monitoring 6 weeks of progressive endurance training with plasma glutamine.

The distinction between positive and negative training adaptation is an important prerequisite in the identification of any marker for monitoring training in athletes. To investigate the glutamine responses to progressive endurance training, twenty healthy males were randomly assigned to a training group or a non-exercising control group. The training group performed a progressive (3 to 6 x 90 minute sessions per week at 70 % V.O (2max)) six-week endurance training programme on a cycle ergometer, while the control group did not participate in any exercise during this period. Performance assessments (V.O (2max) and time to exhaustion) and resting blood samples (for haemoglobin concentration, haematocrit, cortisol, ferritin, creatine kinase, glutamine, uric acid and urea analysis) were obtained prior to the commencement of training (Pre) and at the end of week 2, week 4 and week 6. The training group showed significant improvements in time to exhaustion (p < 0.01), and V.O (2max) (p < 0.05) at all time points (except week 2 for V.O (2max)), while the control group performance measures did not change. In the training group, haemoglobin concentration and haematocrit were significantly lower (p < 0.01) than pretraining values at week 2 and 4, as percentage changes in plasma volume indicated a significant (p < 0.01) haemodilution (+ 6 - 9 %) was present at week 2, 4 and 6. No changes were seen in the control group. In the training group, plasma glutamine (week 2, 4 and 6), creatine kinase (week 2 and 4), uric acid (week 2 and 4) and urea (week 2 and 4) all increased significantly from pretraining levels. No changes in cortisol or ferritin were found in the training group and no changes in any blood variables were present in the control group. Plasma glutamine was the only blood variable to remain significantly above pretraining (966 +/- 32 micromol . 1 (-1)) levels at week 6 (1176 +/- 24 micromol . 1 (-1); p < 0.05) The elevation seen here in glutamine levels, after 6 weeks of progressive endurance training, is in contrast to previous reports of decreased glutamine concentrations in overtrained athletes. In conclusion, 6 weeks of progressive endurance training steadily increased plasma glutamine levels, which may prove useful in the monitoring of training responses.

Adult↗
PubMed2003

Enteral glutamine supplementation and morbidity in low birth weight infants.

OBJECTIVE: To determine if glutamine-supplemented enteral nutrition decreased the incidence of nosocomial sepsis in neonates. METHODS: In a multicenter (n = 20) clinical trial, we randomly allocated infants (n = 649) with birth weight between 500 and 1250 g, who were <7 days of age, and had no major anomalies to receive enteral glutamine supplementation (0.3 g/kg/day) or sterile water (placebo) for the first 28 days. The primary outcome variable was the number of infants who had blood culture-proven nosocomial sepsis between 7 days' and 36 weeks' postmenstrual age. RESULTS: Infants were assigned to placebo (n = 335) or to glutamine supplementation (n = 314). Neonates assigned to glutamine were similar to those assigned placebo for demographic characteristics and nutritional support during the first week. There was no difference in the occurrence of culture-proven nosocomial sepsis (33.7% vs 30.9%) or suspected sepsis (51.6% vs 47.1%) between the placebo and glutamine groups; however, neonates treated with glutamine less often had gastrointestinal dysfunction (7.5% vs 2.5%, P <.01) and severe neurologic sequelae (15.1% vs 10.4%, P =.08). CONCLUSIONS: At a dose of 0.3 g/kg/day, enteral glutamine does not appear to reduce nosocomial sepsis in premature neonates.

Adolescent↗
PubMed1993

Effect of culture from the zygote stage on the metabolism of glucose and glutamine by 2-cell embryos and blastocysts recovered from outbred or F1 hybrid female mice.

The oxidation and incorporation of glucose and glutamine by embryos derived from cultured zygotes was compared with the utilization of these substrates by embryos recovered directly from the reproductive tract of pregnant females. The oxidation of glutamine was greater at the blastocyst stage than at the 2-cell stage. Embryos derived from outbred females (Qs) were less active in the oxidation of glutamine than those from hybrid (B10D2F1) females and development in culture was detrimental to this oxidation, especially in blastocysts from the outbred stock. The oxidation of glutamine was stimulated by the presence of glucose at the 2-cell stage but reduced by its presence at the blastocyst stage. Maternal genotype had no effect on the oxidation of glucose at either the 2-cell or blastocyst stage, and only at the blastocyst stage was there evidence of a detrimental effect of culture. The oxidation of glucose was stimulated by the presence of glutamine at the 2-cell stage but depressed by its addition at the blastocyst stage. Incorporation of glutamine increased with development, but this was reduced at the blastocyst stage by development in culture, especially if the blastocysts were derived from outbred females. Incorporation of glucose also increased with development. At the 2-cell stage, culture reduced incorporation of this substrate, especially into the acid-soluble fraction of embryos from outbred females. In blastocysts, incorporation of glucose into the acid-insoluble fraction was depressed by culture and in embryos from outbred females. In contrast to glucose oxidation, incorporation of glucose into the acid-soluble fraction was reduced by the addition of glutamine at the 2-cell stage but increased by its addition at the blastocyst stage.

Animals↗
PubMed1998

Coagulation factor XIIIa undergoes a conformational change evoked by glutamine substrate. Studies on kinetics of inhibition and binding of XIIIA by a cross-reacting antifibrinogen antibody.

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

Amino Acid Sequence↗
PubMed2003

Glutamine stimulates argininosuccinate synthetase gene expression through cytosolic O-glycosylation of Sp1 in Caco-2 cells.

Glutamine stimulates the expression of the argininosuccinate synthetase (ASS) gene at both the level of enzyme activity and mRNA in Caco-2 cells. Searching to identify the pathway involved, we observed that (i) the stimulating effect of glutamine was totally mimicked by glucosamine addition, and (ii) its effect but not that of glucosamine was totally blocked by 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of amidotransferases, suggesting that the metabolism of glutamine to glucosamine 6-phosphate was required. Moreover, run-on assays revealed that glucosamine was acting at a transcriptional level. Because three functional GC boxes were identified on the ASS gene promoter (Anderson, G. M., and Freytag, S. O. (1991) Mol. Cell Biol. 11, 1935-1943), the potential involvement of Sp1 family members was studied. Electrophoretic mobility shift assays using either the Sp1 consensus sequence or an appropriate fragment of the ASS promoter sequence as a probe demonstrated that both glutamine and glucosamine increased Sp1 DNA binding. Immunoprecipitation-Western blot experiments demonstrated that both compounds increased O-glycosylation of Sp1 leading to its translocation into nucleus. Again, the effect of glutamine on Sp1 was inhibited by the addition of DON but not of glucosamine. Taken together, the results clearly demonstrate that the metabolism of glutamine through the hexosamine pathway leads to the cytosolic O-glycosylation of Sp1, which, in turn, translocates into nucleus and stimulates the ASS gene transcription. Collectively, the results constitute the first demonstration of a functional relationship between a regulating signal (glutamine), a transcription factor (Sp1), and the transcription of the ASS gene.

Argininosuccinate Synthase↗
PubMed2005

Expression of four glutamine synthetase genes in the early stages of development of rainbow trout (Oncorhynchus mykiss) in relationship to nitrogen excretion.

The incorporation of ammonia into glutamine, catalyzed by glutamine synthetase, is thought to be important in the detoxification of ammonia in animals. During early fish development, ammonia is continuously formed as yolk proteins and amino acids are catabolized. We followed the changes in ammonia and urea-nitrogen content, ammonia and urea-nitrogen excretion, glutamine synthetase activity, and mRNA expression of four genes coding for glutamine synthetase (Onmy-GS01-GS04) over 3-80 days post fertilization and in adult liver and skeletal muscle of the rainbow trout (Oncorhynchus mykiss). Both ammonia and urea-nitrogen accumulate before hatching, although the rate of ammonia excretion is considerably higher relative to urea-nitrogen excretion. All four genes were expressed during early development, but only Onmy-GS01 and -GS02 were expressed at appreciable levels in adult liver, and expression was very low in muscle tissue. The high level of expression of Onmy-GS01 and -GS03 prior to hatching corresponded to a linear increase in glutamine synthetase activity. We propose that the induction of glutamine synthetase genes early in development and the subsequent formation of the active protein are preparatory for the increased capacity of the embryo to convert the toxic nitrogen end product, ammonia, into glutamine, which may then be utilized in the ornithine-urea cycle or other pathways.

Ammonia↗
PubMed2005

A feedback-resistant mutant of Bacillus subtilis glutamine synthetase with pleiotropic defects in nitrogen-regulated gene expression.

The Bacillus subtilis TnrA transcription factor regulates gene expression during nitrogen-limited growth. When cells are grown with excess nitrogen, feedback-inhibited glutamine synthetase forms a protein-protein complex with TnrA and prevents TnrA from binding to DNA. A mutation in glutamine synthetase with a phenylalanine replacement at the Ser-186 residue (S186F) was isolated by screening for B. subtilis mutants with constitutive TnrA activity. Although S186F glutamine synthetase has kinetic properties that are similar to the wild-type protein, the S186F enzyme is resistant to feedback inhibition by glutamine and AMP. Ligand binding experiments revealed that the S186F protein had a lower affinity for glutamine and AMP than the wild-type enzyme. S186F glutamine synthetase was defective in its ability to block DNA binding by TnrA in vitro. The properties of the feedback-resistant S186F mutant support the model in which the feedback-inhibited form of glutamine synthetase regulates TnrA activity in vivo.

Adenosine Monophosphate↗
PubMed2006

Heterologous expression of the glutamine transporter SNAT3 in Xenopus oocytes is associated with four modes of uncoupled transport.

The glutamine transporter SNAT3 (SLC38A3, former SN1) plays a major role in glutamine release from brain astrocytes and in glutamine uptake into hepatocytes and kidney epithelial cells. Here we expressed rat SNAT3 in oocytes of Xenopus laevis and reinvestigated its transport modes using two-electrode voltage clamp and pH-sensitive microelectrodes. In addition to the established coupled Na+-glutamine-cotransport/H+ antiport, we found that there are three conductances associated with SNAT3, two dependent and one independent of the amino acid substrate. The glutamine-dependent conductance is carried by cations at pH 7.4, whereas at pH 8.4 the inward currents are still dependent on the presence of external Na+ but are carried by H+. Mutation of threonine 380 to alanine abolishes the cation conductance but leaves the proton conductance intact. Under Na+-free conditions, where the substrate-dependent conductance is suppressed, a substrate-independent, outwardly rectifying current becomes apparent at pH 8.4 that is carried by K+ and H+. In addition, we identified a glutamine-dependent uncoupled Na+/H+ exchange activity that becomes apparent upon removal of Na+ in the presence of glutamine. In conclusion, our results suggest that, in addition to coupled transport, SNAT3 mediates four modes of uncoupled ion movement across the membrane.

Amino Acid Sequence↗