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PubMed2007

Glutamine signalling in bacteria.

Glutamine is a metabolite of central importance in bacterial physiology. In addition to its function as one of the 20 standard amino acids in protein synthesis, glutamine is required for the biosynthesis of a variety of nitrogen-containing compounds. Of particular importance is glutamine synthesis as primary reaction of ammonium assimilation. Because of this versatile role, glutamine metabolism is tightly controlled in response to the cellular nitrogen status in bacteria. Recent progress in elucidating the molecular basis of nitrogen signalling has shed light on the role of glutamine as a signalling molecule. Bacteria belonging to the phylogenetic domains of proteobacteria and low G+C gram-positives (firmicutes) have evolved different mechanisms to monitor glutamine as an indicator of the state of nitrogen metabolism, which then regulates nitrogen metabolism at the transcriptional and post-transcriptional levels. Using the conserved PII signal transduction system, major groups of prokaryotes, including the cyanobacteria, have evolved yet another strategy to monitor the cellular nitrogen status, which relies on 2-oxoglutarate instead of glutamine as the signalling molecule. In addition to monitoring the intracellular glutamine level, bacteria may respond to extracellular glutamine, which is used as a nutrient. This overview details our current knowledge of glutamine-regulated processes in bacteria.

Bacteria↗
PubMed1999

Examination of metabolism of viscera drained by the portal vein in neonatal calves, using short-term intravenous infusions of glutamine and other nutrients.

OBJECTIVE: To quantify glutamine use in viscera drained by the portal vein in neonatal calves and to assess the relative nutritional importance of glutamine, glucose, and acetate for enterocytes. ANIMALS: 5 healthy neonatal calves. PROCEDURE: A femoral artery, jugular vein, and the portal vein were surgically cannulated in each calf. Blood flow in the portal vein was measured by use of an ultrasonographic transit-time flow probe. A series of solutions was infused on 4 days for each calf. On the infusion days, acetate, glucose, glutamine, and saline (0.9% NaCl; control) solutions were administered IV during 1-hour periods via the jugular vein. Venous and arterial blood samples were collected during the last 15 minutes of each 1-hour infusion. RESULTS: Uptake of glutamine and glucose by viscera drained by the portal vein was 0.3+/-1.1 and 1.9+/-3.1 micromol/kg0.75/min, respectively, during saline infusion. During acetate, glucose, and saline infusions, glucose was a greater source of energy for the intestines than was glutamine. However, during glutamine infusion, uptake of glutamine by viscera drained by the portal vein increased significantly (29.9+/-11.2 micromol/kg0.75/min), which was associated with an increase in ammonia production (7.0+/-0.5 micromol/kg0.75/min). Toxicosis was not associated with IV administration of glutamine. CONCLUSION: Glutamine infusion resulted in an increase in glutamine uptake by viscera drained by the portal vein, which was associated with an increase in ammonia production and a slight increase in oxygen consumption. CLINICAL RELEVANCE: These solutions may be used to develop treatments that enhance healing of intestines of diarrheic calves.

Acetates↗
PubMed1999

Glutamine oxidation and utilization by rat and human oesophagus and duodenum.

The rates of utilization and oxidation of glutamine and glucose by oesophageal and duodenal tissues have been investigated in both rats and human subjects. In the rat, glutamine utilization by oesophageal tissue was 2-3-fold lower than that in the duodenum, and this substrate contributed less than 10% to the total oxidative metabolism of the tissue, even when glutamine was the only substrate provided. In contrast, rat duodenal tissue derived about 34% of the total CO2 production from glutamine-C, and this contribution was not suppressed by the addition of either glucose or a mixture of the other substrates. Rates of glucose utilization and oxidation by the duodenum were lower than those for glutamine, and were significantly (P < 0.001) suppressed by addition of glutamine. In both oesophageal and duodenal tissues, less than 10% of the glutamine-C utilized was fully oxidized, approximately 60-70% was converted to glutamate, and 30-40% to alanine. Results obtained using human biopsy tissue samples were similar to those observed in the rat. Glutamine oxidation contributed 34 (SD 4)% of the total CO2 production by the duodenal tissue, but only 8 (SD 4)% to oesophageal tissue oxidation. The findings suggest that glutamine is not an important or preferred fuel for oesophageal tissue, whereas it is for duodenal tissue. Thus, these tissues can be expected to respond differently to glutamine administration.

Analysis of Variance↗
PubMed2002

The role of L-alanyl-L-glutamine in the immune response in vitro.

INTRODUCTION: The amino acid glutamine plays an important role in the immune system by providing energy and precursors for biosynthetic processes. For lack of stability it could not so far be generally supplied in total parenteral nutrition. The development of dipeptides consisting of glutamine and a second amino acid offers a solution to this problem. METHODS: In vitro effects of the dipeptide L-alanyl-L-glutamine on different cells of the immune system are assessed and compared to those of glutamine on its own. RESULTS: T-lymphocyte proliferation stimulated with mitogens and alloantigens increased significantly and dose-dependently after addition of L-alanyl-L-glutamine or glutamine. Maximal effects were observed with a concentration of 2 mmol/l of either substance. The stimulatory effects were partly attributed to enhanced cytokine production following glutamine or L-alanyl-L-glutamine treatment. In contrast, the activity of natural killer and cytotoxic T-cells was not influenced by neither amino acid at concentrations of 0.2 and 2 mmol/l, and suppressed at 20 mmol/l. In all experiments, early addition of the amino acids to the cultures proved crucial. CONCLUSION: In this series of in vitro experiments the dipeptide L-alanyl-L-glutamine exerted almost identical immunostimulatory activities to glutamine alone. Its provision in parenteral nutrition appears commendable.

Cytokines↗
PubMed1992

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed2003

Improvement of pancreatic islet isolation outcomes using glutamine perfusion during isolation procedure.

During procurement, isolation, and transplantation, islets are exposed to high levels of oxidative stress triggering a variety of signaling pathways that can ultimately lead to cell death. Glutamine is an important cellular fuel and an essential precursor for the antioxidant glutathione. The aim of this study was to examine the role of intraductal glutamine administration in facilitating recovery of isolated rat islets from pancreases subjected to a clinically relevant period of warm ischemia. Islets were isolated in Sprague-Dawley (SD) rats (n = 18 per group). Pancreata in groups 1 and 2 were procured immediately while groups 3 and 4 were subjected to 30-min warm ischemia. Groups 2 and 4 were treated intraductally with 5 mM glutamine prior to pancreatectomy. Exposure to 30-min warm ischemia significantly reduced islet yield [groups 1 & 2 (nonischemia): 503 +/- 29 islets/rat vs. groups 3 & 4 (ischemia): 247 +/- 26 islets/rat; p < 0.05]. Intraductal glutamine treatment significantly improved islet yield when pancreata were subjected to 30-min warm ischemia [144 +/- 16 islets/rat without glutamine (group 3) vs. 343 +/- 36 islets/rat with glutamine (group 4), p < 0.05]. Glutamine also significantly improved islet viability (values were 50 +/- 4% in group 4 vs. 27 +/- 3% in group 3, p < 0.05). Similarly, glutathione (reduced) levels were significantly elevated in both glutamine-treated groups; however, this increase was greatest in tissues exposed to ischemia (2.76 +/- 0.04 nmol/mg protein in group 4 vs. 1.66 +/- 0.04 nmol/mg protein in group 3, p < 0.05). Intraductal glutamine administration considerably improves the islet yield, viability, and augments endogenous glutathione levels in pancreata procured after a clinically relevant period of ischemia. Intraductal administration of glutamine at the time of digestive enzyme delivery into the harvested pancreas may represent a simple yet effective tool to improve islet yields in clinical isolations.

Animals↗
PubMed2006

[The effect of glutamine on the growth, metabolism and endostatin production of microencapsulated rCHO cells].

Cell transplantation is a promising technology in cancer therapy, however, immunological rejection is the major problem of cell transplantation. Based on the permselective property of microcapsule membrane, encapsulated cells can be immuno-protected. The normal physiological state and function expression of cells can be maintained so as to realize allo- or xenotransplantation. The microencapsulated cells grow in three dimensions, giving a more biologically representative in vivo model, which hints difference in characters of growth and metabolism compared to the monolayer cells. Therefore, characterization of growth and metabolism of microencapsulated recombinant CHO cells is essential for further large-scale culture. In present study, the effect of concentration of glutamine on the growth, metabolism and endostatin production of microencapsulated cells was investigated. In the experimental range of initial glutamine concentrations from 2.69mmol/L to 9.05mmol/L in the culture of microencapsulated recombinant CHO cells, the maximum density of active cells and multiplication ratios almost kept constant. The specific consumption rate of glucose increased with lower initial glutamine concentration (2.69mmol/L). When initial glutamine concentration was much higher (7.91mmol/L to approximately 9.05mmol/L), the specific consumption rates of both glucose and glutamine increased while the efficiencies of glucose and glutamine decreased. The highest efficiencies of glucose and glutamine utilization were observed with initial glutamine concentration of 4.97mmol/L. It was also demonstrated that glutamine had significant effect on the accumulation of endostatin. The accumulative concentration of endostain reached its peak of 546.36 ng/mL with the initial glutamine concentration of 4.97mmol/L.

Animals↗
PubMed2006

[Effect of glutamine on apoptosis of the small intestine in young rats with endotoxemia and its mechanism].

OBJECTIVE: To study the effect of glutamine on intestinal epithelial apoptosis by examining changes regarding Bcl-2 and Bax mRNA expressions in the small intestine of young rats with endotoxemia and to explore the protective mechanism that glutamine may have. METHODS: A total of 120 18-day-old rats were randomly assigned into Endotoxemia, Glutamine-treated and Control groups (n = 40 each). The endotoxemia model was established by intraperitoneal injection of endotoxin (4 mg/kg of O55B5 Escherichia coli lipopolysaccharide). Rats in the Glutamine-treated group were intraperitoneally injected with N (2)-L-alanyl-L-glutamine (2 g/kg) along with endotoxin. Rats in the Control group were intraperitoneally injected with an equal volume of normal saline. The entire ileum was collected at 2, 4, 6, 24, and 72 hrs after injection. Bcl-2 and Bax mRNA expressions were detected by semi-quantities reverse transcriptase chain reaction. RESULTS: Bcl-2 mRNA was not expressed in the Control and the Endotoxemia groups but increased in the Glutamine-treated group at each time point. Bax mRNA expression was weak in the Control group, and significantly increased in the Endotoxemia group at each time point. The Glutamine-treated group showed noticeably reduced Bax mRNA expression at 2 hrs post-injection while other time points were similar to the Control group. The ratio of Bax and Bcl-2 mRNA expression at each time point in the Endotoxemia group was significantly higher than that in the Control group while the Glutamine-treated group demonstrated significantly lower ratio of Bax and Bcl-2 mRNA expression than both. CONCLUSIONS: Glutamine treatment increased Bcl-2 mRNA expression and decreased Bax mRNA expression, as a result, the ratio of Bax and Bcl-2 mRNA expression decreased. The effects of glutamine resulted in a suppression of intestinal epithelial apoptosis and maintained the integrity of the gut barrier structure.

Animals↗
PubMed1992

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.

Animals↗
PubMed1991

Maximal activity of phosphate-dependent glutaminase and glutamine metabolism in septic rats.

The activity of phosphate-dependent glutaminase and glutamine metabolism by tissues known markedly to utilize or synthesize glutamine (or both) were studied in rats made septic by cecal ligation and puncture technique and compared with the same measures in rats that underwent sham operation (laparotomy). Blood glucose level was not markedly different in septic rats, but lactate, pyruvate, alanine, and glutamine levels were markedly increased. Conversely, blood ketone body concentrations were significantly decreased in septic rats. Both plasma insulin and glucagon levels were markedly elevated in response to sepsis. The maximal activity of phosphate-dependent glutaminase was decreased in the small intestine, increased in the kidney and mesenteric lymph nodes, and unchanged in the liver of septic rats. Arteriovenous concentration difference measurements across the gut showed a decrease in the net glutamine removed from the circulation in septic rats. Arteriovenous concentration difference measurements for glutamine showed that both renal uptake and skeletal muscle release of the amino acid were increased in response to sepsis, whereas measurements across the hepatic bed showed a net uptake of glutamine in septic rats. Enterocytes isolated from septic rats exhibited a decreased rate of utilization of glutamine and production of glutamate, alanine, and ammonia, whereas lymphocytes isolated from septic rats showed an enhanced rate of utilization of glutamine and production of glutamate, aspartate, and ammonia. It is concluded that, during sepsis, glutamine uptake and metabolism are enhanced in renal and lymphoid tissue but decreased in that of the small intestine, with increased rates of release by skeletal muscle; however, the liver appears to utilize glutamine in septic rats.

Alanine↗
PubMed1984

Glutamine-dependent synthesis of citrulline by isolated hepatic mitochondria from Squalus acanthias.

High levels of both glutamine synthetase and a unique L-glutamine- and N-acetyl-L-glutamate-dependent carbamoyl phosphate synthetase are present in the mitochondria in livers of marine urea-retaining elasmobranchs (Casey, C. A., and Anderson, P. M. (1982) J. Biol. Chem. 257, 8449-8453). On the basis of these observations it has been suggested that in these species carbamoyl phosphate and, consequently, one of the nitrogen atoms of citrulline and, ultimately, urea, are derived directly from glutamine rather than from ammonia as occurs in mammalian ureotelic species. The purpose of this study was to obtain evidence for this role of glutamine. Isolated hepatic mitochondria from Squalus acanthias incubated with ammonia plus glutamate, ornithine, bicarbonate, inorganic phosphate, and succinate as an energy source were found to synthesize citrulline at a rate comparable to the rate of urea synthesis observed in vivo. Citrulline synthesis proceeds at maximal rates even when the ammonia concentration is as low as 0.05 mM and is stoichiometric with the amount of ammonia initially present. Synthesis from ammonia does proceed in the absence of glutamate, but a much higher concentration of ammonia (congruent to 4 mM) is required to achieve a half-maximal rate. Glutamine can substitute for ammonia plus glutamate as the nitrogen-donating substrate for citrulline synthesis. Selective inhibition of the glutamine-dependent activity of the carbamoyl phosphate synthetase in the isolated mitochondria completely inhibits the ability of the mitochondria to synthesize citrulline from glutamine or from ammonia plus glutamate, whereas selective inhibition of glutamine synthetase inhibits citrulline synthesis from ammonia plus glutamate, but not from glutamine. These observations provide direct evidence that ammonia assimilation for citrulline synthesis (and, therefore, urea synthesis) in these species involves intermediate formation of glutamine.

Ammonia↗
PubMed1994

Glutamine-dependent nitrogen transfer in Escherichia coli asparagine synthetase B. Searching for the catalytic triad.

The mechanism of nitrogen transfer in glutamine-dependent amidotransferases remains to be unambiguously established. We now report the overexpression, purification, and kinetic characterization of both the glutamine- and ammonia-dependent activities of Escherichia coli asparagine synthetase B (AS-B) and a series of mutants. In common with other members of the purF family of amidotransferases, the recombinant enzyme possesses an NH2-terminal cysteine residue. Replacement of Cys-1 by either alanine or serine results in a loss of glutaminase and glutamine-dependent activity, without out any significant effect upon ammonia-dependent asparagine synthesis. As previously observed for human AS (Sheng, S., Moraga-Amador, D., Van Heeke, G., Allison, R. D., Richards, N. G. J., and Schuster, S. M. (1993) J. Biol. Chem. 268, 16771-16780), glutamine is an inhibitor of the ammonia-dependent reaction catalyzed by both the Cys-1-->Ala (C1A) and Cys-1-->Ser (C1S) mutants of AS-B. In the case of C1A, the inhibition pattern suggests that an abortive complex is formed. This is consistent with a recent proposal implicating the formation of an imide intermediate in the nitrogen transfer reaction (Richards, N. G. J., and Schuster, S. M. (1992) FEBS Lett. 313, 98-102). In contrast, glutamine appears to be only a competitive inhibitor of the ammonia-dependent activity of C1S. Cys-1 does not appear to be required for glutamine binding. Replacement of Asp-33 by either asparagine or glutamic acid has little effect on the kinetic properties of the mutant enzymes when compared to wild-type AS-B. Cys-1 and Asp-33 are cognate to residues Cys-1 and Asp-29 in glutamine phosphoribosylpyrophosphate amidotransferase which have been proposed to be members of a catalytic triad responsible for mediating nitrogen transfer in this enzyme (Mei, B., and Zalkin, H. (1989) J. Biol. Chem. 264, 16613-16619). In the case of AS-B, although Cys-1 is essential for glutamine-dependent activity, Asp-33 does not appear to participate in mediating nitrogen transfer. In an effort to locate other residues which might form part of a "catalytic triad" in the glutamine amidotransferase domain of AS-B, we have expressed and characterized mutant proteins in which His-29 and His-80, which are conserved within the glutamine amidotransferase domain of purF amidotransferases, are replaced by alanine (H29A and H80A).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1994

Glutamine modulates phenotype and stimulates proliferation in human colon cancer cell lines.

Glutamine supplementation has been advocated for patients requiring parenteral nutritional support. However, the direct effect of glutamine on neoplastic cells is poorly understood. We therefore investigated the effects of glutamine on the proliferation, differentiation, and cell-matrix interactions of two human colon carcinoma cell lines (Caco-2 and SW620) adapted to glutamine-free media. Doubling times were calculated by logarithmic transformation of serial cell counts. Alkaline phosphatase, cathepsin C (dipeptidyl peptidase), lactase, and isomaltase expression (markers of differentiation) were assayed by digestion of synthetic substrates. Adhesion to matrix proteins was assessed by colorimetric quantitation of toluidine blue staining of adherent cells. Surface expression of Caco-2 receptors for matrix proteins (integrins) was studied by biotinylation and immunoprecipitation with specific antibodies. Glutamine (1-10 mM) dose-dependently stimulated Caco-2 proliferation on all matrices studied with maximal effect at 7 mM. For instance, Caco-2 doubling time on collagen IV decreased by 57 +/- 0.2% (SE) (P < 0.001). Glutamine inhibited the expression of all four digestive enzymes with maximal inhibition ranging from 10 to 40% (P < 0.05 for all). Adhesion to matrix proteins was markedly diminished (51 +/- 1%, P < 0.01) by glutamine (5 mM) treatment, correlating with decreased alpha 2 and beta 1 integrin subunit surface expression. Glutamine had similar effects on SW620 cells, stimulating proliferation, inhibiting digestive enzyme expression, and diminishing both adhesion and integrin surface expression. Glutamine supplementation modulates the phenotype of at least two human colon carcinoma cell lines, increasing proliferation, decreasing differentiation, and decreasing adhesion to matrix proteins in association with decreased integrin expression. Although the mechanisms of these effects await elucidation, such characteristics would appear to predict more aggressive tumor behavior and raise the possibility that nutritional supplementation with glutamine may be deleterious in patients with cancer.

Alkaline Phosphatase↗
PubMed1997

L-glutamine inhibits nitric oxide synthesis in bovine venular endothelial cells.

This study was conducted to test the hypothesis that L-glutamine has differential effects on nitric oxide (NO) synthesis from L-arginine in bovine venular endothelial cells (EC) stimulated by A23187 (a Ca++ ionophore) and receptor-mediated vasodilators (bradykinin and substance P). EC were cultured at 37 degrees C for 24 h in the presence of 0.4 mM L-arginine and 0.0 to 2.0 mM L-glutamine with or without 1 microM A23187, 1 microM bradykinin or 10 microM substance P. The release of nitrite and nitrate by EC was used as an indicator of NO synthesis. A23187, bradykinin or substance P increased NO synthesis from L-arginine by EC in the presence or absence of L-glutamine. The addition of L-glutamine (0.5 and 2 mM) markedly increased intracellular concentrations of L-glutamine, L-glutamate and L-aspartate and decreased NO synthesis by EC in a concentration-dependent manner in the presence or absence of A23187, bradykinin or substance P. L-Glutamine had no effect on L-arginine uptake by EC or on intracellular L-arginine concentration. Neither L-glutamine nor its glutaminase metabolites (ammonia, L-glutamate and L-aspartate) had any effect on endothelial NO synthase activity. Taken together, these results suggest that the inhibition by L-glutamine of NO synthesis from L-arginine is unlikely to result from an effect of L-glutamine on L-arginine transport or NO synthase activity. Although the mechanism involved remains unknown, regulation of the arginine-NO pathway by L-glutamine may have pharmacologic and therapeutic implications in such conditions as inflammation and septic shock by inhibiting NO generation from L-arginine in endothelial cells.

Animals↗
PubMed1998

Glutamine deprivation induces apoptosis in intestinal epithelial cells.

BACKGROUND: Glutamine is the most abundant amino acid in the blood, and its deprivation leads to gut mucosal atrophy. The small intestinal mucosa is maintained by a balance between cell proliferation and cell death by apoptosis. We reported that glutamine is required for nitrogen-stimulated proliferation in intestinal epithelial cells. We do not know whether glutamine regulates apoptosis in the gut. The purpose of this study is to determine whether glutamine deprivation induces apoptosis in rat intestinal epithelial (RIE-1) cells and to compare the effect of glutamine starvation with that of methionine and cysteine (Met/Cys) starvation. METHODS: RIE-1 cells were deprived of either glutamine or Met/Cys for 24 hours. Cell numbers were determined by cell counting and tetrazolium enzymatic assay. Apoptosis was quantified by Annexin V assay and confirmed by DNA gel electrophoresis and Hoecsht nuclear staining. RESULTS: Deprivation of glutamine or Met/Cys resulted in decreased cell numbers. However, only the glutamine-deprived group showed significant induction of apoptosis with increased Annexin V staining, DNA laddering, and nuclear condensation. CONCLUSIONS: This study provides biochemical and morphologic evidence that glutamine deprivation induces apoptosis in rat intestinal epithelial cells. In contrast, Met/Cys starvation suppresses cell number without induction of apoptosis. These results suggest that glutamine serves as a specific survival factor in enterocytes.

Animals↗
PubMed1991

Exceptional codon recognition by the glutamine tRNAs in Saccharomyces cerevisiae.

Recently, it was shown that wild-type glutamine tRNAs in yeast cause low-level nonsense suppression that can be enhanced by increasing glutamine tRNA gene copy number. In order to investigate glutamine tRNA behavior further, anticodon mutations that confer nonsense suppression were identified in yeast sup70 gene, which codes for glutamine tRNA(CAG). In this study we show that suppressors derived by mutation severely limit growth such that suppressor-bearing spores germinate but arrest cell division at approximately the 50 cell stage. Analysis of a sup70 deletion was used to establish that growth limitation results from loss of wild-type glutamine tRNA(CAG) function. By exploiting the growth inhibition of sup70 alleles, some exceptional codon recognition properties of glutamine tRNAs were revealed. Our results indicate that amber suppressor glutamine tRNA(UAG) can translate 5'-CAG-3' glutamine codons with low efficiency in the presence of an A/C mismatch at the first position of the codon, suggesting that reading may occur at a low level by a two-out-of-three reading mechanism. In addition, when glutamine tRNA(CAA) is over-expressed in vivo, it translates 5'-CAG-3' codons using a mechanism that resembles prokaryotic-like U/G wobble, which normally does not occur in yeast. Our studies also suggest that the yeast glutamine tRNA suppressors could potentially be exploited to express ciliated protozoan genes that normally contain internal 5'-UAG-3' and 5'-UAA-3' codons.

Base Sequence↗
PubMed2000

Glutamine synthetase and marker enzymes of the blood-retina barrier in fetal bovine retinal pigment epithelial cells.

BACKGROUND: Glutamine synthetase is involved in the recycling of synaptically released glutamate and GABA and in the detoxification of ammonia. It is present in the Müller cells of the neural retina but not in the retinal pigment epithelial (RPE) cells of adult mammals. In human retinal pathological conditions glutamine synthetase has also been detected in RPE cells. In this case glutamine synthetase may provide the cells with glutamine needed for proliferation. Proliferation is also intense during retinal development. METHODS: We studied the distribution of glutamine synthetase immunoreactivity in fetal bovine retinae, especially in the RPE. The maturity of the RPE was demonstrated by histochemical detection of gamma-glutamyltranspeptidase, alkaline phosphatase and Na/K-ATPase as marker enzymes for the blood-retina barrier and by electron microscopy. RESULTS: We found that in the first 3 months of gestation glutamine synthetase immunoreactivity is located exclusively in the RPE. During the 3rd month the marker enzymes of the blood-retina barrier begin to appear. From the 4th month on, RPE cells are glutamine synthetase immunonegative. CONCLUSION: Glutamine in RPE cells in early development may supply glutamine for the intensely proliferating cells in the retina. Glutamine synthetase immunoreactivity in human retinal pathological conditions may indicate reinduction of an enzyme used in earlier development.

Animals↗
PubMed2003

Glutamine synthetase in tilapia gastrointestinal tract: zonation, cDNA and induction by cortisol.

Glutamine synthetase, an enzyme generally associated with ammonia detoxication in the vertebrate brain and with hepatic nitrogen turnover in mammals, shows substantial activities in the gastrointestinal tract of teleostean fishes. Enzyme activity is highest in the central area of the stomach and reveals a distinct distribution pattern in stomach and along the intestine of tilapia (Oreochromis niloticus), rainbow trout (Oncorhynchus mykiss) and copper rockfish (Sebastes caurinus). In all three species, intestinal activity peaks in the distal region of the intestine. The brain contains the highest titre of the enzyme (46 U g(-1) in tilapia brain versus 15 U g(-1) in tilapia stomach), but because of the relative mass of the stomach, the largest glutamine synthetase pool in tilapia body appears to be localized in the stomach. Activities in white and red muscle are very modest at 0.1% of the brain. Independent of distribution, peak activities of glutamine synthetase in selected areas of tilapia stomach and intestine are significantly (two- to fourfold) increased after a 5-day treatment with an intraperitoneal cortisol deposit. Cortisol also increases glutamine synthetase activity in tilapia liver, white and red muscle, while activities in brain remain unaffected. We cloned and sequenced the predominant transcript of tilapia stomach glutamine synthetase (about 1.9 kb), encoding a 371-amino acid peptide. The open reading frame shows considerable identity with glutamine synthetase in toadfish (92% at peptide level, 87% at nucleotide level), but possesses a longer 3'-untranslated region than the toadfish. The tilapia glutamine synthetase mRNA contains a remnant of a putative mitochondrial leader sequence, but without a conserved second site for initiation of translation. We also find evidence for additional transcripts of glutamine synthetase in tilapia, suggesting multiple genes. Finally, we present evidence for similar abundance of glutamine synthetase transcripts in all regions of rockfish intestine. The physiological significance of the presence of glutamine synthetase in teleostean intestine is discussed.

Amino Acid Sequence↗