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PubMed2003

Glutamine Analogues As Adjunctive Therapy for Infectious Diarrhea.

Glutamine is the major fuel for the gut as well as for many cells in the immune system that becomes conditionally essential during catabolic states. Glutamine supplementation improves intestinal mucosal repair and function. Glutamine, even at high doses, is without side effects and is well tolerated. Though unstable in solution, this is overcome by creating stable dipeptides such as alanyl-glutamine. In HIV-positive patients with wasting, glutamine enhances intestinal absorptive function and weight gain. Glutamine enhances sodium and water absorption in a rabbit model of cholera and Cryptosporidium-infected piglet intestine. Both glutamine and alanyl-glutamine have recently proven effective in a bovine model of Cryptosporidium as well. Finally, a rat model of cholera toxin-induced diarrhea also showed that alanyl-glutamine enhanced water and electrolyte intestinal absorption even better than the traditional glucose solutions. Clearly glutamine and its stabler derivatives hold promise for enhancing repair of mucosal injury by a wide range of infections or toxic agents, and hence have great potential as a new oral rehydration and nutrition therapy for patients with enteric infection, malnutrition, or chemotherapy- or radiation-induced enteritis.

Journal Article↗
PubMed1998

Regulation of the spatiotemporal pattern of expression of the glutamine synthetase gene.

Glutamine synthetase, the enzyme that catalyzes the ATP-dependent conversion of glutamate and ammonia into glutamine, is expressed in a tissue-specific and developmentally controlled manner. The first part of this review focuses on its spatiotemporal pattern of expression, the factors that regulate its levels under (patho)physiological conditions, and its role in glutamine, glutamate, and ammonia metabolism in mammals. Glutamine synthetase protein stability is more than 10-fold reduced by its product glutamine and by covalent modifications. During late fetal development, translational efficiency increases more than 10-fold. Glutamine synthetase mRNA stability is negatively affected by cAMP, whereas glucocorticoids, growth hormone, insulin (all positive), and cAMP (negative) regulate its rate of transcription. The signal transduction pathways by which these factors may regulate the expression of glutamine synthetase are briefly discussed. The second part of the review focuses on the evolution, structure, and transcriptional regulation of the glutamine synthetase gene in rat and chicken. Two enhancers (at -6.5 and -2.5 kb) were identified in the upstream region and two enhancers (between +156 and +857 bp) in the first intron of the rat glutamine synthetase gene. In addition, sequence analysis suggests a regulatory role for regions in the 3' untranslated region of the gene. The immediate-upstream region of the chicken glutamine synthetase gene is responsible for its cell-specific expression, whereas the glucocorticoid-induced developmental appearance in the neural retina is governed by its far-upstream region.

Aging↗
PubMed1999

Interdomain signaling in glutamine phosphoribosylpyrophosphate amidotransferase.

The glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase-catalyzed synthesis of phosphoribosylamine from PRPP and glutamine is the sum of two half-reactions at separated catalytic sites in different domains. Binding of PRPP to a C-terminal phosphoribosyltransferase domain is required to activate the reaction at the N-terminal glutaminase domain. Interdomain signaling was monitored by intrinsic tryptophan fluorescence and by measurements of glutamine binding and glutamine site catalysis. Enzymes were engineered to contain a single tryptophan fluorescence reporter in key positions in the glutaminase domain. Trp(83) in the glutamine loop (residues 73-84) and Trp(482) in the C-terminal helix (residues 471-492) reported fluorescence changes in the glutaminase domain upon binding of PRPP and glutamine. The fluorescence changes were perturbed by Ile(335) and Tyr(74) mutations that disrupt interdomain signaling. Fluoresence titrations of PRPP and glutamine binding indicated that signaling defects increased the K(d) for glutamine but had little or no effect on PRPP binding. It was concluded that the contact between Ile(335) in the phosphoribosyltransferase domain and Tyr(74) in the glutamine site is a primary molecular interaction for interdomain signaling. Analysis of enzymes with mutations in the glutaminase domain C-terminal helix and a 404-420 peptide point to additional signaling interactions that activate the glutamine site when PRPP binds.

Amidophosphoribosyltransferase↗
PubMed1990

ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro.

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [alpha-32P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [alpha-32P]ATP- and [alpha-32P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [alpha-32P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes (Moss, J., Watkins, P.A., Stanley, S.J., Purnell, M.R., and Kidwell, W.R. (1984) J. Biol. Chem. 259, 5100-5104). A 32P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H3 32PO4-grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification.

Adenosine Diphosphate Ribose↗
PubMed1979

Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties.

Glutamine 5-phosphoribosylamine:pyrophosphate phosphoribosyltransferase (amidophosphoribosyl-transferase) has been purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was 194,000 by sedimentation equilibrium centrifugation and 224,000 by gel filtration. A subunit Mr = 57,000 was estimated by gel electrophoresis in sodium dodecyl sulfate. Cross-linking experiments gave species of Mr = 57,000, 117,000, and 177,000. A trimer or tetramer of identical subunits is indicated for the native enzyme. Highly active E. coli amidophosphoribosyl-transferase lacks significant nonheme iron. Enzyme activity was not enhanced by addition of iron salts and sulfide. Amidophosphoribosyltransferase exhibited both NH3- and glutamine-dependent activities. Glutaminase activity was detected in the absence of other substrates. Both glutamine- and NH3-dependent activities were subject to end product inhibition by purine 5'-ribonucleotides. AMP and GMP, in combination, gave synergistic inhibition. AMP and GMP exhibited positive cooperativity. In addition, GMP promoted cooperativity for saturation by 5-phosphoribosyl-1-pyrophosphate. Glutamine utilization was inhibited by NH3, suggesting that the amide of glutamine is transferred to the NH3 site prior to amination of 5-phosphoribosyl-1-pyrophosphate. The glutamine-dependent activity was selectively inactivated by the glutamine analogs L-2-amino-4-oxo-5-chloropentanoic acid and 6-diazo-5-oxo L-norleucine (DON) and by iodoacetamide. Incorporation of 1 eq of DON/subunit (Mr = 57,000) caused complete inactivation of the glutamine-dependent activity, thus providing evidence for one glutamine site per monomer and for the functional identity of the subunits. Following alkylation with iodoacetamide, carboxymethylcysteine was the only modified amino acid isolated from an acid hydrolysate. The glutamine-dependent activity was sensitive to oxidation. Inactivation by exposure to air was reversed by incubation with high concentrations of dithiothreitol.

Amidophosphoribosyltransferase↗
PubMed1989

The effect of glutamine concentration on the activity of carbamoyl-phosphate synthase II and on the incorporation of [3H]thymidine into DNA in rat mesenteric lymphocytes stimulated by phytohaemagglutinin.

The maximum catalytic activities of carbamoyl-phosphate synthase II, a limiting enzyme for pyrimidine nucleotide synthesis, are very much less than those of glutaminase, a limiting enzyme for glutamine utilization, in lymphocytes and macrophages; and the flux through the pathway for pyrimidine formation de novo is only about 0.4% of the rate of glutamine utilization by lymphocytes. The Km of synthase II for glutamine is about 16 microM and the concentration of glutamine necessary to stimulate lymphocyte proliferation half-maximally is about 21 microM. This agreement suggests that the importance of glutamine for these cells is provision of nitrogen for biosynthesis of pyrimidine nucleotides (and probably purine nucleotides). However, the glutamine concentration necessary for half-maximal stimulation of glutamine utilization (glutaminolysis) by the lymphocytes is 2.5 mM. The fact that the rate of glutamine utilization by lymphocytes is markedly in excess of the rate of the pathway for pyrimidine nucleotide synthesis de novo and that the Km and 'half-maximal concentration' values are so different, suggests that the glutaminolytic pathway is independent of the use of glutamine nitrogen for pyrimidine synthesis.

Animals↗
PubMed1998

Oxidation of glutamine in HeLa cells: role and control of truncated TCA cycles in tumour mitochondria.

The oxidative metabolism of glutamine in HeLa cells was investigated using intact cells and isolated mitochondria. The concentrations of the cytoplasmic amino acids were found to be aspartate, 8.0 mM; glutamate, 22.2 mM; glutamine, 11.3 mM; glycine, 9.8 mM; taurine, 2.3 mM; and alanine, < 1 mM. Incubation of the cells with [14C]glutamine gave steady-state recoveries of 14C-label (estimated as exogenous glutamine) in the glutamine, glutamate, and aspartate pools, of 103%, 80%, and 25%, respectively, indicating that glutamine synthetase activity was absent and that a significant proportion of glutamate oxidation proceeded through aspartate aminotransferase. No label was detected in the alanine pool, suggesting that alanine aminotransferase activity was low in these cells. The clearance rate of [14C]glutamine through the cellular compartment was 65 nmol/min per mg protein. There was a 28 s delay after [14C]glutamine was added to the cell before 14C-label was incorporated into the cytoplasm, while the formation of glutamate commenced 10 s later. Aspartate was the major metabolite formed when the mitochondria were incubated in a medium containing either glutamine, glutamate, or glutamate plus malate. The transaminase inhibitor AOA inhibited both aspartate efflux from the mitochondria and respiration. The addition of 2-oxoglutarate failed to relieve glutamate plus malate respiration, indicating that 2-oxoglutarate is part of a well-coupled truncated cycle, of which aspartate aminotransferase has been shown to be a component [Parlo and Coleman (1984): J Biol Chem 259:9997-10003]. This was confirmed by the observation that, although it inhibited respiration, AOA did not affect the efflux of citrate from the mitochondria. Thus citrate does not appear to be a cycle component and is directly transported to the medium. Therefore, it was concluded that the truncated TCA cycle in HeLa cells is the result of both a low rate of citrate synthesis and an active citrate transporter. DNP (10 microM) induced a state III-like respiration only in the presence of succinate, which supports the evidence that NAD-linked dehydrogenases were not coupled to respiration, and suggests that these mitochondria may have a defect in complex I of the electron transport chain. Arising from the present results with HeLa cells and results extant in the literature, it has been proposed that a major regulating mechanism for the flux of glutamate carbon in tumour cells is the competitive inhibition exerted by 2-oxoglutarate on aspartate and alanine aminotransferases. This has been discussed and applied to the data.

Amino Acids↗
PubMed1991

Selective uptake of glutamine in the gastrointestinal tract: confirmation in a human study.

Recent animal data suggest that the gut plays a far more important metabolic role than previously thought. During critical illness, disruption in bowel barrier function may result in a chronic hypermetabolic state and contribute to multiorgan failure. Animal studies have demonstrated that enterocytes of the gastrointestinal tract use glutamine as a respiratory fuel and during critical illness the consumption of glutamine by the gut significantly increases. The selective uptake of glutamine by the gut, to date, has not been confirmed in humans. Seven patients who sustained multisystem trauma necessitating laparotomy underwent portal venous catheterization. This was done by carefully reopening the obliterated umbilical vein and facilitating access to the left branch of the portal vein using a standard central venous catheter. Portal venous and systemic blood samples were recorded for 5 days after operation. Amino acid levels in both circulations were recorded at 48 h and 5 days. Using Student's t test for related samples, the differences between individual amino acids in portal and systemic circulations were compared. At 48 h, mean(s.d.) portal venous glutamine was 85(5) per cent of the systemic levels (253(80) compared with 296(90) mumol/ml, P less than 0.002). At 5 days, portal glutamine was 87(3) per cent of the systemic levels (255(69) compared with 292(83) mumol/ml, P less than 0.003). Levels of citrulline, a breakdown product of glutamine metabolism, were elevated in the portal venous circulation at 48 h (20(4) compared with 16(3) mumol/ml, P less than 0.005) and at 5 days (21(5) compared with 14(3) mumol/ml, P less than 0.002). No significant differences between any of the other amino acids analysed were identified. This study confirms, for the first time in humans, that selective uptake of glutamine occurs in the gut. In stressed states, glutamine deficiency is associated with gut mucosal atrophy. This has significant implications as glutamine is not provided in most commercially available parenteral and enteral nutrition formulations.

Adolescent↗
PubMed1995

Experimental study to show that growth hormone treatment before trauma increases glutamine uptake in the intestinal tract.

This study examined whether growth hormone treatment deprived the intestinal tract of glutamine after trauma. Piglets were treated with growth hormone 24 units daily 3 days before and at the start of the trauma (GH-3, n = 8) or at the start of the trauma only (GH-1, n = 8). Eight piglets acted as non-treated controls. The trauma consisted of a standardized abdominal surgical procedure. Primed constant infusions of U-14C-glutamine were given. Intestinal, hepatic, renal and hindleg glutamine fluxes were measured. Growth hormone treatment increased mean(s.e.m.) net intestinal glutamine uptake: GH-3, 39.7(9.4) and 48.7(12.7) mumol/min; GH-1, 33.2(5.5) and 25.7(12.3) mumol/min; controls, 19.5(10.3) and 2.0(15.3) mumol/min at 1 h and 5 h after trauma, respectively, (P = 0.02). The treatment increased glutamine oxidation (P = 0.025), and decreased hindleg glutamine net (P = 0.0052) and absolute release (P = 0.0063), glutamine rate of appearance (P = 0.01), and percentage of glucose coming from glutamine (P = 0.05). Growth hormone treatment before trauma increased intestinal glutamine uptake.

Abdomen↗
PubMed2006

Glutamine: a Trojan horse in ammonia neurotoxicity.

Mechanisms involved in hepatic encephalopathy still remain to be defined. Nonetheless, it is well recognized that ammonia is a major factor in its pathogenesis, and that the astrocyte represents a major target of its CNS toxicity. In vivo and in vitro studies have shown that ammonia evokes oxidative/nitrosative stress, mitochondrial abnormalities (the mitochondrial permeability transition, MPT) and astrocyte swelling, a major component of the brain edema associated with fulminant hepatic failure. How ammonia brings about these changes in astrocytes is not well understood. It has long been accepted that the conversion of glutamate to glutamine, catalyzed by glutamine synthetase, a cytoplasmic enzyme largely localized to astrocytes in brain, represented the principal means of cerebral ammonia detoxification. Yet, the "benign" aspect of glutamine synthesis has been questioned. This article highlights evidence that, at elevated levels, glutamine is indeed a noxious agent. We also propose a mechanism by which glutamine executes its toxic effects in astrocytes, the "Trojan horse" hypothesis. Much of the newly synthesized glutamine is subsequently metabolized in mitochondria by phosphate-activated glutaminase, yielding glutamate and ammonia. In this manner, glutamine (the Trojan horse) is transported in excess from the cytoplasm to mitochondria serving as a carrier of ammonia. We propose that it is the glutamine-derived ammonia within mitochondria that interferes with mitochondrial function giving rise to excessive production of free radicals and induction of the MPT, two phenomena known to bring about astrocyte dysfunction, including cell swelling. Future therapeutic approaches might include controlling excessive transport of newly synthesized glutamine to mitochondria and its subsequent hydrolysis.

Ammonia↗
PubMed1991

Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells.

We have investigated the effects of extracellular anions on the regulation of expression of the heat shock response in Drosophila Kc cells incubated in defined balanced salt solutions. Widely varying chloride concentrations had no effect on normal or heat shock protein (hsp) expression. Increasing glutamate concentrations from zero to 15 mM increased hsp expression more than 100-fold while affecting expression of non-heat-shock proteins minimally. Glutamine was 20-100-fold more potent than glutamate in supporting hsp expression, while other amino acids were less effective or supported no detectable hsp synthesis in heat shock. Inhibition of glutamine synthetase with methionine-sulfoximine resulted in very low hsp expression with glutamate and normal high level expression with glutamine, confirming the importance of glutamine. The absence of glucose and treatment with 2-deoxyglucose did not change the requirement for adequate glutamine for hsp expression. Cells heat shocked under conditions which gave very low hsp expression resumed growth when returned to normal medium as well as cells which expressed normal levels of hsps. Measurements of free amino acid levels in cells heat shocked in the presence and absence of glutamine showed a correlation between glutamine levels and amount of hsp expression. We conclude that a physiological process regulated by glutamine or a glutamine metabolite is important for normal hsp expression in heat shock conditions in Drosophila.

Amino Acids↗
PubMed1989

Development of a noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos.

A noninvasive ultramicrofluorometric method for measuring net uptake of glutamine by single preimplantation mouse embryos is described. A linear relationship was found between fluorescence intensity of NADH produced and glutamine concentration (R2 = 0.985). Single embryos were placed in 20 nl drops of medium containing 0.5 mM glutamine, and the medium was sampled after 2 hr incubation at 37 degrees C. Changes in net glutamine uptake were determined in one-cell, two-cell, and eight-cell embryos and blastocysts incubated in medium containing no energy substrates (glucose, pyruvate, and lactate). The median glutamine uptake increased significantly from 0.480 and 0.270 pmoles/embryo/2 hr at the one-cell and two-cell stages, respectively, to 1.610 pmoles/embryo/2 hr at the blastocyst stage. Mean glutamine uptake was compared in the presence or absence of energy substrates at several developmental stages. A highly significant reduction of glutamine uptake in the presence of substrates was observed at the one-cell and two-cell stages of development. At the eight-cell stage, glutamine uptake was only marginally reduced in the presence of substrates, and no effect was found at the blastocyst stage. These data may partially explain the beneficial effect of glutamine on the culture of early mouse embryos through the two-cell block of development.

Animals↗
PubMed2001

Role of glutamine in cerebral nitrogen metabolism and ammonia neurotoxicity.

Ammonia enters the brain by diffusion from the blood or cerebrospinal fluid, or is formed in situ from the metabolism of endogenous nitrogen-containing substances. Despite its central importance in nitrogen homeostasis, excess ammonia is toxic to the central nervous system and its concentration in the brain must be kept low. This is accomplished by the high activity of glutamine synthetase, which is localized in astrocytes and which permits efficient detoxification of incoming or endogenously generated ammonia. The location also permits the operation of an intercellular glutamine cycle. In this cycle, glutamate released from nerve terminals is taken up by astrocytes where it is converted to glutamine. Glutamine is released to the extracellular fluid to be taken up into the nerve cells, where it is converted back to glutamate by the action of glutaminase. Most extrahepatic organs lack a complete urea cycle, and for many organs, including the brain, glutamine represents a temporary storage form of waste nitrogen. As such, glutamine was long thought to be harmless to the brain. However, recent evidence suggests that excess glutamine is neurotoxic. Hyperammonemic syndromes (e.g., liver disease, inborn errors of the urea cycle, Reye's disease) consistently cause astrocyte pathology. Evidence has been presented that hyperammonemia results in increased formation of glutamine directly in astrocytes, thereby generating an osmotic stress to these cells. This osmotic stress results in impaired astrocyte function, which in turn leads to neuronal dysfunction. In this review a brief overview is presented of the role of glutamine in normal brain metabolism and in the pathogenesis of hyperammonemic syndromes.

Ammonia↗
PubMed1998

Cytokine production by human peripheral blood mononuclear cells: differential senstivity to glutamine availability.

Human peripheral blood mononuclear cells (PBMCs) were cultured in the presence of different glutamine concentrations (0, 0.1, 0.4, 0.6, 2 mM) and stimulated with concanavalin A (Con A) or bacterial lipopolysaccharide (LPS). The concentrations of T lymphocyte- and monocyte-derived cytokines were measured in the culture medium 24 h later. The availability of glutamine significantly increased the production of interleukin (IL)-2 (2-fold), IL-10 (4-fold) and interferon (IFN)-gamma (4.5-fold) by Con A-stimulated PBMCs. Maximal production of these cytokines occurred at 0.1 mM glutamine and increasing the concentration of glutamine above that did not lead to a further increase in cytokine production. Glutamine availability resulted in small increases (24 to 35%) in the production of IL-1alpha, IL-1beta, IL-6 and tumour necrosis factor (TNF)-alpha by Con A-stimulated PBMCs; again maximal production occurred at a glutamine concentration of 0.1 mM. Glutamine availability did not influence the production of IL-1beta or TNF-alpha by LPS-stimulated PBMCs, while there was a small increase (17 to 32%) in the production of IL-1alpha, IL-6 and IL-10 by these cells at a glutamine concentration of 0.1 mM. It is concluded that glutamine enhances the production of T lymphocyte-derived cytokines with only minimal effects on production of cytokines by monocytes.

Adult↗
PubMed1993

Endotoxin increases hepatic glutamine transport activity.

Glutamine uptake by the liver is accelerated during endotoxemia, but little is known regarding the influence of sepsis on the plasma membrane transport systems catalyzing hepatic glutamine uptake. We hypothesized that this augmented uptake was due to an increase in hepatocyte plasma membrane transport activity. We investigated the activities of the Na(+)-dependent transport System N (transports glutamine into the hepatocyte) and the Na(+)-independent System n (transports glutamine out of the cell) in hepatocyte plasma membrane vesicles (HPMVs) prepared from livers of rats treated with Escherichia coli endotoxin (LPS) in vivo. HPMVs were prepared by differential centrifugation and the transport of [3H]glutamine was assayed by a rapid mixing/filtration technique in the presence and absence of sodium. Vesicle integrity and functionality were confirmed by enzyme marker enrichments and classic "overshoots" in the presence of sodium. Carrier-mediated Na(+)-independent glutamine transport activity was not altered by LPS administration. In contrast, endotoxemia resulted in a time- and dose-dependent two- to threefold increase in Na(+)-dependent glutamine transport activity in HPMVs secondary to an increase in the transport Vmax, consistent with the appearance of increased numbers of corresponding transporter proteins in the hepatocyte plasma membrane. The Km (affinity for glutamine) of the System N transporter was not affected by LPS treatment. Maximal increases in transport were observed 4 hr after exposure to endotoxin. System N transport activity returned to basal levels by 12 hr. This increase in transport activity represents an important mechanism regulating the accelerated hepatic glutamine uptake that occurs during severe infection.

Animals↗
PubMed1993

Intracellular glutamine concentration does not decrease in all muscles during sepsis.

The concentrations of glutamine and other amino acids were measured in plasma and intracellular fluid of soleus and extensor digitorum longus (EDL) muscles of rats 4, 8, and 16 hr after induction of sepsis by cecal ligation and puncture or after sham operation. Previous studies have shown that muscle protein breakdown is greatly increased in EDL, but not in soleus muscle, in this sepsis model. Corresponding to previous observations of protein breakdown in sepsis, muscle glutamine was markedly depleted (< 50%) in EDL by sepsis, while no significant fall in glutamine concentration in soleus was observed. Changes in muscle glutamine concentration in sepsis could not be attributed to changes in the precursor of glutamine, glutamic acid. Data were examined for changes consistent with hypothesized alterations in glutamine transport. Correlations among glutamine and other amino acids in muscle, histidine in particular, were consistent with a sepsis-induced alteration in activity of the sarcolemmal glutamine transporter, system Nm. These results thus strengthen the proposed connection between muscle glutamine content and muscle protein metabolism under catabolic conditions.

Amino Acids↗
PubMed2001

Glutamine utilization in activated lymphocytes from rats receiving endotoxin.

BACKGROUND: A beneficial effect of supplemental glutamine for lymphocyte function in patients under metabolic stress has been suggested. Nevertheless, it is not clear how glutamine is used by lymphocytes when under stress. This time course study investigated the effect of endotoxin-induced stress on in vitro glutamine utilization and glutamine-dependent proliferation of activated lymphocytes. METHODS: Metabolic stress was modeled by intraperitoneal (ip) administration of endotoxin (5 mg/kg body wt) to rats. Control animals were injected with sterile saline. Cervical lymph node lymphocytes collected from animals 6, 12, 24, and 48 h following injection were activated with concanavalin A. Proliferation of these activated lymphocytes in the presence of 0.1-2 mM glutamine was determined. The glutamine utilization rate and glutaminase activity in the activated lymphocytes were also determined. RESULTS: The proliferation rate of lymphocytes was not affected by ip administration of endotoxin 6 h following the insult, however, 12, 24, and 48 h following the insult, the maximal response was suppressed (P < 0.05). In addition, at 12, 24, and 48 h, the concentration of glutamine for the maximal response of lymphocytes was lower than that for the control group (P < 0.05). Throughout the investigation period, both the glutamine utilization rate and glutaminase activity in the activated lymphocytes were decreased time-dependently. CONCLUSION: The present study demonstrates that glutamine utilization by lymphocytes under a mitogenic challenge in vitro is significantly decreased in the late period after endotoxin injection. This is at least partly due to decreased glutaminase activity and is associated with decreased proliferation rate of mitogen-activated lymphocytes.

Animals↗
PubMed2002

Protein kinase C activation of intestinal glutamine transport is mediated by mitogen-activated protein kinases.

BACKGROUND: Glutamine is essential for the preservation of intestinal structure and function and its uptake by the bowel is augmented during catabolic states. However, the signal transduction pathways implicated in brush border glutamine transport have not been examined. The aim of this study was to investigate the intracellular signaling pathways involved in the regulation of accelerated intestinal glutamine transport. Our hypothesis was that the activation of intestinal glutamine transport involves protein kinase C (PKC) and is mediated by mitogen-activated protein kinases (MAPKs). METHODS: [3H]L-Glutamine (50 microM) transport activity and mRNA levels for the intestinal glutamine transporter ATB(0) were measured in intestinal epithelial Caco-2 cells. Confluent cells were treated with phorbol ester (PMA, 0-10 microM), the MAPK MEK inhibitor PD 98059 (0-100 microM), actinomycin (0-0.1 microM), MAPK p38 inhibitor SB 203580 (0-10 microM), protein kinase C inhibitor chelerythrine chloride (0-6.6 microM), or cycloheximide (0-10 microM) for 24 h. Data were analyzed by ANOVA with significance set at P < 0.05. RESULTS: Phorbol ester treatment increased intestinal System B glutamine transport activity by 75%, an increase that was blocked individually by PD 98059, chelerythrine chloride, actinomycin, and cycloheximide, but not SB 203580, an effect first noted at 6 h. The resulting activity increase was consistent with de novo synthesis of transporter units and enhanced expression of transporter gene ATB(0) as indicated by a threefold increase of ATB(0) mRNA levels in PMA-treated cells. CONCLUSIONS: Activation of glutamine transport in Caco-2 cells by phorbol ester occurs via signaling pathways that lead to transcription of the glutamine transporter gene. PKC and mitogen-activate protein kinase MEK are key intracellular mediators involved in this signal transduction cascade.

Amino Acid Transport System ASC↗