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,**
* Department of Medicine,
Department of Biochemistry and the
** Center for Clinical and Molecular Nutrition, Emory University School of Medicine, Atlanta, GA 30322
3To whom correspondence should be addressed. E-mail: tzieg01{at}emory.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • apoptosis • cytokines • glutathione • intestine • TRAIL
Both availability of nutrient substrates and apoptosis play key roles in small bowel and colonic mucosal turnover. For example, fasting and other forms of protein-energy malnutrition are associated with gut mucosal atrophy and dysfunction (e.g., enhanced permeability to macromolecules, increased bacterial translocation from the lumen and reduced activity and expression of digestive enzymes), whereas refeeding results in rapid stimulation of mucosal cell growth, increased expression of digestive enzymes and nutrient transporters and normalization of gut barrier function (1). However, surprisingly little information is available on the regulation of intestinal epithelial cell apoptosis by nutrient substrates.
Apoptosis plays a critical role in mucosal restitution following injury and inflammation (2). Apoptosis prevents the release of cellular material (e.g., lysosomal proteases) that occurs with cellular necrosis, which may lead to mucosal injury (3). However, exaggerated apoptosis is observed in inflamed/ulcerated areas of colonic mucosa in inflammatory bowel disease (IBD)3 and this may impair mucosal restitution (4). Cytokines such as tumor necrosis factor-alpha (TNF-
), interferon-gamma (IFN-
) and Fas ligand mediate gut mucosal inflammation in IBD and induce apoptosis in human intestinal cell lines (5,6). The cytokine tumor necrosis factor-alpha–related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that induces apoptosis in the intestinal epithelial cell lines, Caco-2 and HT-29 (7). However, factors that control the rate of cytokine-induced intestinal epithelial cell apoptosis and the underlying mechanisms have been infrequently studied, particularly with regard to modulation by nutrients.
Glutamine (Gln) has been shown to have antiapoptotic effects in intestinal cells during both spontaneous and induced apoptosis. In rat intestinal epithelial (RIE-1) cells, Gln depletion alone induces apoptosis, whereas culture media containing Gln prevents apoptosis (8,9). In rat small intestine-derived cells (IEC-6 and IEC-18), Gln prevents cell death induced by oxidants and heat shock (10,11). As a substrate for glutamate (Glu), Gln serves as a precursor to glutathione (GSH), a potent antioxidant that detoxifies reactive oxygen species and has potent antiapoptotic effects in cultured cells (12). Although Gln prevents oxidant-induced apoptosis, the effect of Gln on cytokine-induced apoptosis and the role of GSH in intestinal cells have not previously been investigated.
The purpose of this study was to determine the ability of Gln to inhibit apoptosis induced by the cytokine, TRAIL, in HT-29 cells. HT-29 cells are a well-characterized human colon carcinoma cell line that retain many properties of the colonic epithelium and are commonly used to investigate modulation of intestinal cell growth. The cytokines TRAIL and TNF/IFN are effective inducers of apoptosis in HT-29 cells (7). TRAIL was chosen for this investigation because of its robust ability to induce apoptosis. Our results demonstrate that Gln protects against TRAIL-induced apoptosis in HT-29 cells through a mechanism independent of intracellular GSH redox status.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.
Cell culture.
HT-29 cells were purchased from American Type Culture Collection (Manassas, VA). Before treatment, cells were cultured in DMEM (Cellgro, Atlanta, GA) containing 4.5 g/L of glucose without Gln supplementation with 10% fetal calf serum, 2.0 mmol/L of Gln and 1% antibiotics (10000 U/L of penicillin and 100 g/L of streptomycin sulfate) at 37°C with 5% CO2/95% O2. Media were changed every 2 d and cultures were passaged every 5–7 d. For experimentation, cells (passages 145–175) were seeded at a density of 7.5 x 105 cells/cm2 in six-well plates (Fisher Scientific, Pittsburgh, PA), unless otherwise indicated, and allowed to adhere using the above medium for 24 h. All treatments outlined below were formulated in DMEM without serum or Gln with 1% antibiotics.
Cell treatments.
Unless otherwise indicated, all experiments were conducted with n = 3 and repeated twice. To determine the role of Gln during cytokine-induced apoptosis, cultures were pretreated in serum-free media for 24 h with increasing amounts of Gln (0, 5, 50 or 500 µmol/L). Media were then replaced with fresh serum-free media containing TRAIL (100 µg/L) and the above concentrations of Gln for 8 h. Gln doses were based on pilot data with 0, 0.2, 2.0 and 10.0 mmol/L, which showed that 2.0 and 10.0 mmol/L of Gln did not further inhibit apoptosis over that of 0.2 mmol/L (data not shown). Additional studies used 100 µg/L of TNF and 100 U/mL of IFN- to test the specificity of the antiapoptotic effects of Gln in response to other apoptosis inducers. The doses of TRAIL and TNF/IFN- were based on pilot studies demonstrating a dose-related increase in apoptosis (data not shown).
To test responses to exogenous Gln or provision of another amino acid, additional cultures were treated as above with 0 µmol/L of Gln, 500 µmol/L of Gln, 500 µmol/L of alanine (Ala) or 500 µmol/L of 6-diazo-5-norleucine [(DON) a Gln analogue and an inhibitor of glutaminase, the rate-limiting enzyme for Gln metabolism]. To assess amino acid specificity, additional cultures were treated as above with either medium alone, 500 µmol/L of Gln, amino acid mixture 1 [(AA-1) containing 500 µmol/L each of aspartate, tryptophan (Trp), asparagine, leucine, methionine, tyrosine, isoleucine and phenylalanine (Phe)], amino acid mixture 2 [(AA-2) containing 500 µmol/L each of alanine (Ala), arginine, histidine, proline, lysine, serine, threonine and valine], 500 µmol/L of Glu or 500 µmol/L each of glycine (Gly) and cysteine (Cys). In all experiments, controls consisted of Gln-free, nonTRAIL-treated cells cultured in serum-free DMEM.
To assess the effects of altering the intracellular GSH redox state on apoptosis, cells were pretreated with either 500 µmol/L of Gln, 500 µmol/L of Glu, 500 µmol/L of DON, 500 µmol/L of DON + 500 µmol/L of Gln, 100 µg/L of butathione sulfoximine [BSO, a specific inhibitor of -glutamyl cysteine ligase (GCL), the key regulatory enzyme in GSH biosynthesis], or 100 µg/L of BSO + 500 µmol/L of Gln. After 24 h, fresh media containing TRAIL and the above treatments were added for 8 h and apoptosis was assessed by flow cytometry.
Flow cytometry.
Cells were collected and analyzed using fluorescent-activated cell sorting (FACS) analysis to determine the percentage of cells in the sub-G1 peak as the primary index of apoptosis. Nonadherent cells were collected and combined with adherent cells obtained by trypsinization, pelleted, rinsed, fixed in 70% ethanol and stored at -20°C. On the day of analysis, cells were resuspended and treated as previously described (13). The percentage of cells in the sub-G1 peak was immediately determined using FACS analysis with a Becton-Dickinson FACSort flow cytometer (Becton-Dickinson, Franklin Lakes, NJ).
Nuclear condensation.
The nuclear condensation assay was used as a morphological index of apoptosis. Cells were collected and processed as described previously (13) by a researcher with knowledge of the research design (M.E.E.). Cultures were observed with an Olympus IX70 fluorescent microscope (Olympus America, Melville, NY) under UV light. Images were captured using Metamorph software (Universal Imaging, Downingtown, PA).
Caspase-8 activation.
Activation of caspase-8 was determined by the production of caspase-8 cleavage products as assessed by Western blotting. Cells were seeded in 10-cm dishes at a density of 10 x 105 cells/cm2 and treated as above. Cells were harvested in lysis buffer [1% (v/v) of Triton X-100, 0.01 g/L of sodium deoxycholate, 0.1% (v/v) of SDS, 0.15 mol/L of NaCl and 0.1 mol/L of sodium phosphate, pH 7.2) with Complete Mini protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), homogenized and the cytosolic fraction obtained by centrifugation. The supernatant was removed and stored at -80°C until analysis. Protein concentrations were determined using a bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) and gamma globulin as the standard. Total protein (100 µg) was resolved on 10–20% Tris-tricine gels (Bio-Rad, Hercules, CA) and transferred to Hybond ECL nitrocellulose membranes (Amersham, Arlington Heights, IL). Cleaved caspase-8 was detected using anticaspase-8 (Cell Signaling Technology, Beverly, MA) and antimouse horseradish peroxidase-conjugated secondary antibody (Santa Cruz, Santa Cruz, CA) with ECL chemiluminescence (Amersham). Protein bands were scanned on an HP ScanJet 5490c (Palo Alto, CA) and quantified using ImageJ software (NIH, Bethesda, MD). Caspase-8 antibodies were removed using stripping buffer [62.5 mmol/L of Tris-HCL, 2% (v/v) of SDS and 100 mmol/L of ß-mercaptoethanol] at 50°C for 45 min and membranes were reprobed with anti-ß–actin (Santa Cruz) to normalize for protein loading.
Caspase-3 activity.
Adherent and nonadherent cells were treated as above, collected via trypsinization, pelleted by centrifugation and caspase-3 activity was determined using a caspase-3 activity kit (Calbiochem, San Diego, CA) following the manufacturer’s protocols on a Becton-Dickinson FACSort flow cytometer.
GSH redox.
GSH redox was assessed as previously described (12) to determine whether intracellular GSH redox status was mechanistically related to the antiapoptotic effects of Gln. In the first experiment, cells were pretreated with DMEM alone or 500 µmol/L of Gln, Ala or DON for 24 h, followed by 4 h of TRAIL and the above preparations. In the second experiment, cells were treated with Gln or Glu alone during the entire experiment or pretreated with Gln, Glu, Gln + DON, BSO or Gln + BSO for 24 h and then treated with TRAIL and the previous treatments for 4 h. The 4-h time point for GSH redox analysis was chosen because our methods required adherent cells and a significant proportion of cells were nonadherent after 6 or more hours of TRAIL treatment. Pilot data also showed that Gln was unable to prevent TRAIL-induced apoptosis when added >2 h after TRAIL treatment (data not shown) indicating that cells commited to apoptosis well before the 4-h time point chosen for assessment of GSH redox. Cellular thiol levels [GSH and glutathione disulfide (GSSG)] were measured as previously described (14). Quantification of thiols was calculated based on integration relative to the -glutamyl-glutamate internal standard and expressed as nmol/mg of protein. Eh (mV) was calculated using the Nernst equation {Eh = Eo + RT/2F ln [GSSG/(GSH)2]} and used as an index of the redox state of the GSH/GSSG pool (12). An Eo value of -258.0 mV was used to estimate Eh from GSH and GSSG values (15).
Statistical analysis.
Differences between endpoints were compared across groups by one-way ANOVA. Specific treatment doses were compared post hoc using the Fisher’s least significant difference test (SPSS for Windows; SPSS, Chicago, IL). Values were considered significant at P < 0.05. Data are expressed as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Treatment with 100 µg/L of TRAIL for 8 h increased apoptosis (16.2% of cells in the sub-G1 peak), whereas control cells cultured without Gln demonstrated only a small percentage (0.8%) of apoptotic cells (Fig. 1A). The induction of apoptosis was also observed following treatment with TNF + IFN- for 8 h (Fig. 1B), however, the induction was not as great (3.5 versus 0.9% for controls) as with TRAIL-induction. After 4 h of TRAIL treatment, nuclei were also condensed and highly fluorescent (Fig. 2C) compared with controls (Fig. 2A) indicating induction of apoptosis. In addition, TRAIL treatment induced a 64-fold activation of caspase-8 compared with controls (Fig. 3) and a 186% increase in caspase-3 activity compared with controls (Fig. 4). Thus, TRAIL is an effective inducer of apoptosis in HT-29 cells, and this model is therefore useful for the purpose of determining whether Gln can protect against cytokine-mediated apoptosis.
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Addition of Gln inhibited TRAIL-induced apoptosis in a dose-dependent manner (Fig. 1A). Supplementation with 500 µmol/L of Gln reduced the percentage of apoptotic cells to a level similar to nonTRAIL-treated control cells (1.6%). Similar antiapoptotic effects of Gln were observed when apoptosis was induced with TNF + IFN- (Fig. 1B). Treatment with 500 µmol/L of Gln completely prevented TNF + IFN--induced apoptosis (Gln 0.8 versus 0.9% for untreated cells). In contrast to the Gln response, both Ala (an isomolar nitrogen source) and DON inhibited, but did not prevent, TRAIL-induced apoptosis.
Gln also prevented the nuclear condensation observed with TRAIL treatment (Fig. 2). Gln alone (Fig. 2B) did not alter nuclear condensation compared with control treatment. In contrast, the nuclei of TRAIL-treated cells were condensed and highly fluorescent (Fig. 2C), whereas TRAIL + Gln-treated cells (Fig. 2D) exhibited noncondensed diffuse nuclei, indicating that apoptosis was prevented. Neither Ala (Fig. 2E) nor DON (Fig. 2F) treatment prevented induction of nuclear condensation by TRAIL.
The activation of caspase-8 observed in TRAIL-treated cells was also completely inhibited in the presence of Gln (Fig. 3). Isomolar amounts of Ala did not affect the caspase-8 activation induced by TRAIL. Treatment with DON decreased caspase-8 activation, but values were still 
30-fold higher than in the cells treated with Gln. The increase in caspase-3 activity was also completely prevented in the presence of Gln (0 versus 186% for TRAIL-treated cultures), whereas neither Ala nor DON altered the TRAIL-induced increase in caspase-3 activity (Fig. 4). These data indicate that Gln supplementation prevents cytokine-induced (TRAIL and TNF + IFN-) apoptosis in HT-29 cells.
Gln prevention of apoptosis is specific to Gln.
Gln specificity was demonstrated by evaluating the antiapoptotic effects of two mixtures of essential and nonessential amino acids, Glu alone or Gly + Cys (Fig. 1C). Glu was tested to evaluate whether this downstream metabolite of Gln could mimic Gln effects. As in the previous experiments, Gln treatment alone prevented TRAIL-induced apoptosis. In contrast, the AA-1 combination increased apoptosis (50%), in keeping with previous studies showing that Phe and Trp induces apoptosis in human leukemia MOLT-4 cells (15). The AA-2 combination decreased apoptosis (25%), whereas treatment with Glu or Cys + Gly had no effect. These results demonstrate that the antiapoptotic effects of Gln are highly specific and cannot be substituted with Glu or other amino acid combinations.
Gln metabolism is required for the prevention of TRAIL-induced apoptosis.
The addition of DON to TRAIL-treated cells had no effect on apoptosis (Fig. 5). Furthermore, treatment with Gln + DON prevented Gln antiapoptotic effects indicating that DON, acting either as a Gln analogue or antimetabolite, prevents the antiapoptotic effects observed with Gln supplementation.
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TRAIL treatment did not alter intracellular GSH concentrations compared with control cells (Fig. 6A). In contrast, Gln supplementation of TRAIL-treated cultures resulted in an increase in GSH concentration, whereas Ala and DON had no effect. TRAIL-treated cells demonstrated a 9-mV oxidation of intracellular GSH/GSSG Eh compared with control cells (Fig. 6B). In contrast, cells treated with Gln + TRAIL demonstrated more reducing Eh (by -32 mV) compared with controls, indicating increased reducing potential of the GSH/GSSG redox pool. Ala reduced GSH/GSSG Eh, but this response was less than with Gln. In contrast to the Gln response, inhibition of endogenous Gln metabolism by DON did not alter TRAIL-induced oxidation of the GSH/GSSG redox pool.
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To further determine the potential role of GSH in Gln antiapoptotic effects, cells were treated with Glu (as an alternative precursor to GSH) or BSO (to deplete intracellular GSH) (Figs. 5and 7). The addition of either Gln or isomolar Glu, with or without TRAIL, increased GSH concentrations three- to fourfold compared with controls (Fig. 7A). However, addition of DON to Gln-supplemented TRAIL-treated cells prevented the Gln-induced upregulation of intracellular GSH. The addition of BSO to TRAIL-treated cells decreased GSH to nearly undetectable levels; this response was significantly attenuated by the addition of Gln. However, GSH concentrations were still substantially lower than TRAIL + Gln-treated cultures (0.33 ± 0.04 versus 5.29 ± 0.58 nmol/mg protein).
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GCL inhibition with BSO. As in the previous experiments, TRAIL increased the percentage of HT-29 cells undergoing apoptosis (sub-G1 peak) compared with controls, whereas Gln prevented this response (Fig. 5). The percentage of apoptotic HT-29 cells was similar in TRAIL + BSO-treated cells to that of TRAIL treatment alone (Fig. 5), despite the depletion of GSH and oxidation of the GSH redox pool with BSO (Fig. 7). DON also inhibited the ability of Gln to upregulate GSH levels and reduce GSH/GSSG redox potential but did not prevent antiapoptotic effects from occurring (Figs. 5and 7). In addition, inhibition of endogenous GSH synthesis with BSO did not inhibit the ability of exogenous Gln supplementation to prevent TRAIL-induced apoptosis. These data support the concept that Gln effects on apoptosis and GSH redox are not linked.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Apoptosis and its dysregulation play an important role in pathophysiologic states involving the gastrointestinal mucosa including IBD, carcinogenesis, vascular insults, infectious diarrhea and drug-induced mucosal injury (19). The rate of apoptosis of gut mucosal cells is critical for normal mucosal turnover and is also important during illness because apoptotic cells can be recognized and scavenged by phagocytic cells (or are released into the lumen) without releasing cellular materials that may cause inflammation and loss of tissue integrity (3).
Our data show for the first time that Gln prevents cytokine-induced apoptosis and associated activation of caspase-8 and -3 in a human colon-derived epithelial cell line. Furthermore, substitution of Gln with other amino acids demonstrated that Gln-induced antiapoptotic effects were highly specific for this amino acid. The antiapoptotic effects of Gln did not appear to be the result of nitrogen concentration in the media. Supplementation with cysteine and glycine (total: 1 mmol/L of amino acids) or combinations of multiple amino acids at 500 umol/L each (total: 4 mmol/L amino acids) did not prevent TRAIL-induced apoptosis despite providing nitrogen doses equal to or higher than Gln. In studies with RIE-1 cells, rat neutrophils, and human leukemia-derived CEM and HL-60 cells, Gln deprivation induced spontaneous apoptosis (8,9,20,21). Likewise, Jurkat T cells deprived of Gln became more sensitive to the apoptosis inducers, phorbol myristate acetate (PMA) and ionomycin (22) while Gln protected small intestine-derived rat IEC-18 and IEC-6 cells against heat shock and oxidant-induced cell death (10,11). Gln treatment also up-regulated Bcl-2 and decreased caspase-3 and -8 activities in PMA plus ionomycin-activated human T-cells (22). Our data extend previous studies by showing that Gln prevents cytokine-induced apoptosis in intestinal cells. Furthermore, our data show that Gln modestly inhibited TRAIL-induced apoptosis at doses as low as 5 and 50 µmol/L but completely prevented apoptosis at 500 µmol/L, a concentration within the normal plasma range (400–650 µmol/L) in healthy adults (23).
Previous research suggests that both Glu and Gln are equally effective for protein synthesis and maintenance of small intestinal cellularity (24,25). In addition, enteral administration of Gln and especially Glu stimulate small intestine mucosal GSH synthesis in neonatal piglets (26). Our data are the first to show that both Gln and Glu increase GSH/GSSG reducing potential (Eh) in human intestinal cells, with or without cytokine treatment.
We did not observe an association between intracellular GSH redox and apoptosis as observed in studies of nonintestinal cells (22,27–30). In our study, the addition of BSO oxidized GSH/GSSG Eh and decreased GSH concentrations but did not inhibit Gln antiapoptotic properties. Moreover, treatment with Glu resulted in similar GSH redox indices as did Gln treatment but did not inhibit TRAIL-induced apoptosis. However, it remains possible that GSH-dependent regulation of apoptosis is dependent upon the stimulus used (i.e., cytokine or Gln depletion), is cell-type specific, is altered as a function of the proliferation-differentiation stage of the cell or depends on GSH redox in specific subcellular (e.g., mitochondrial) fractions.
Although our results indicate that Gln prevents apoptosis through metabolic pathways independent of GSH production, the specific regulatory mechanism(s) remains unclear. Gln is a preferred fuel source for enterocytes and therefore may prevent apoptosis through maintenance of ATP concentrations (17). In vivo studies suggest that Glu is equally effective as a fuel source for enterocytes (25,26), although in the current study, Glu was unable to prevent apoptosis. Furthermore, in lymphoma-leukemia cells, Gln does not prevent apoptosis via an oxidative fuel pathway (31). It is possible that another downstream metabolite of Gln acts as the mediator of Gln’s antiapoptotic effects. Gln has also been shown to upregulate nuclear factor kappa B activation, an event that may inhibit apoptosis via induction of Bcl-xL and caspase-8/FLICE inhibiting protein (32) or by preventing the formation of apoptosomes (33). Additional studies to explore the role of these or other pathways in the regulation of apoptosis by Gln or its metabolites would be of interest. Studies to evaluate the effects of Gln on apoptosis in differentiated intestinal cells as well as nontransformed cells are also warranted.
In conclusion, we demonstrated that a physiological concentration of Gln completely prevents apoptosis induced by cytokines in HT-29 cells. The antiapoptotic effects were highly specific to Gln, required Gln metabolism and could not be exchanged with Glu. The protective effect of Gln was not attributable to alterations in intracellular GSH/GSSG redox. Given that cytokine-induced cellular injury and apoptosis are important in the pathobiology of a number of intestinal disorders, additional in vitro and in vivo studies to determine the mechanisms of Gln-cytokine interactions in intestinal cells are warranted.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 This work was supported by NIH Grants R01 DK-55850 (T.R.Z.) and R01 ES-011195 and R01 ES-009047 (D.P.J.).
4 Abbreviations used: Ala, alanine; BSO, buthionine sulfoximine; Cys, cysteine; DON, 6-diazo-5-norleucine; FACS, fluorescent-activated cell sorting; GCL, gamma-glutamyl cysteine ligase; Gln, glutamine; Glu, glutamate; Gly, glycine; GSH, glutathione; GSSG, glutathione disulfide; IBD, inflammatory bowel disease; IFN, interferon-gamma; Phe, phenylalanine; PMA, phorbol myristate acetate; RIE-1, rat intestinal epithelial; TNF, tumor necrosis factor-alpha; TRAIL, tumor necrosis factor-alpha related apoptosis inducing ligand; Trp, tryptopham; Tx, treatments.
Manuscript received 9 June 2003. Initial review completed 17 July 2003. Revision accepted 30 July 2003.
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C. F. Estivariz, D. P. Griffith, M. Luo, E. E. Szeszycki, N. Bazargan, N. Dave, N. M. Daignault, G. F. Bergman, T. McNally, C. H. Battey, et al. Efficacy of Parenteral Nutrition Supplemented With Glutamine Dipeptide to Decrease Hospital Infections in Critically Ill Surgical Patients JPEN J Parenter Enteral Nutr, July 1, 2008; 32(4): 389 - 402. [Abstract] [Full Text] [PDF]
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 H.-Y. Zhang, H.-Q. Wang, H.-M. Liu, Y. Guan, and Z.-X. Du Regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by DJ-1 in thyroid cancer cells Endocr. Relat. Cancer, June 1, 2008; 15(2): 535 - 544. [Abstract] [Full Text] [PDF]
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 J. Tian, N. Washizawa, L. H. Gu, M. S. Levin, L. Wang, D. C. Rubin, S. Mwangi, S. Srinivasan, D. P. Jones, and T. R. Ziegler Local Glutathione Redox Status Does Not Regulate Ileal Mucosal Growth after Massive Small Bowel Resection in Rats J. Nutr., February 1, 2007; 137(2): 320 - 325. [Abstract] [Full Text] [PDF]
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A. Hubert-Buron, J. Leblond, A. Jacquot, P. Ducrotte, P. Dechelotte, and M. Coeffier Glutamine Pretreatment Reduces IL-8 Production in Human Intestinal Epithelial Cells by Limiting I{kappa}B{alpha} Ubiquitination J. Nutr., June 1, 2006; 136(6): 1461 - 1465. [Abstract] [Full Text] [PDF]
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 B. A. Carneiro, J. Fujii, G. A. C. Brito, C. Alcantara, R. B. Oria, A. A. M. Lima, T. Obrig, and R. L. Guerrant Caspase and Bid Involvement in Clostridium difficile Toxin A-Induced Apoptosis and Modulation of Toxin A Effects by Glutamine and Alanyl-Glutamine In Vivo and In Vitro Infect. Immun., January 1, 2006; 74(1): 81 - 87. [Abstract] [Full Text] [PDF]
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 M. E. Evans, D. P. Jones, and T. R. Ziegler Glutamine inhibits cytokine-induced apoptosis in human colonic epithelial cells via the pyrimidine pathway Am J Physiol Gastrointest Liver Physiol, September 1, 2005; 289(3): G388 - G396. [Abstract] [Full Text] [PDF]
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P. Rittler, B. Schiefer, H. Demmelmair, B. Koletzko, A. A. Roscher, R. Jacobs, M. Krick, K.-W. Jauch, and W. H. Hartl Effect of Amino Acid Infusion on Human Postoperative Colon Protein Synthesis in Situ JPEN J Parenter Enteral Nutr, July 1, 2005; 29(4): 255 - 261. [Abstract] [Full Text] [PDF]
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B. Alteheld, M. E. Evans, L. H. Gu, V. Ganapathy, F. H. Leibach, D. P. Jones, and T. R. Ziegler Alanylglutamine Dipeptide and Growth Hormone Maintain PepT1-Mediated Transport in Oxidatively Stressed Caco-2 Cells J. Nutr., January 1, 2005; 135(1): 19 - 26. [Abstract] [Full Text] [PDF]
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