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,**,3
,**,**,4
* Department of Nutrition Science, University of Bonn, Germany;
Department of Medicine and
** Center for Clinical and Molecular Nutrition, Emory University School of Medicine, Atlanta, GA 30322; and
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912
4To 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: • oxidative stress • glutamine • intestine • redox status
In the human gastrointestinal tract, products of dietary protein digestion are absorbed in the form of free amino acids and oligopeptides. Transport of di- and tripeptides as well as peptidomimetic-like drugs across the gut epithelium is dependent upon the H+-dependent transporter PepT1 (1–4). Human PepT1 is found throughout the gastrointestinal tract, with the highest levels on the apical surface of human small bowel villous epithelial cells (5). Interestingly, PepT1 is also expressed at low levels in normal human colonocytes, indicating that the colon has a mechanism to accrue luminal small peptides (5,6). Furthermore, colonic mucosal PepT1 expression is upregulated in patients with inflammatory bowel disease (IBD)5 or short bowel syndrome (5,7). Small bowel PepT1 expression and/or activity is increased in rats fed high-protein or dipeptide-enriched diets (8,9) and by certain hormones, including thyroid hormone, insulin, and luminal leptin (10–12).
The amino acid glutamine (Gln) has trophic and cytoprotective effects in intestinal mucosal cells (13,14). For example, in rodent and pig models, enteral or parenteral Gln supplementation of nutrition support diminishes gut mucosal injury after abdominal irradiation, systemic chemotherapy, and experimental sepsis (15). Additionally, several studies demonstrated beneficial effects of Gln in animal models of experimental colitis, including reduction of endotoxin levels, inhibition of mucosal damage, and local production of inflammatory cytokines (16,17). In human duodenal mucosal cells cultured ex vivo, Gln decreased production of the proinflammatory cytokines interleukin (IL)-6 and IL-8 and enhanced production of the anti-inflammatory cytokine IL-10 (18).
An increasing number of clinical studies have demonstrated beneficial effects of Gln and Gln dipeptides as components of enteral and parenteral nutrition support in catabolic states (13,19). Given the abundance of PepT1 in human small bowel and its presence in colon, studies to define the effects of Gln dipeptide administration on the expression and function of this transporter are of interest. Anabolic hormones can also improve nutrient utilization during catabolic states, and some studies suggest that treatment with recombinant growth hormone (GH), without or with Gln supplementation, improves nitrogen absorption in patients with short bowel syndrome (20,21). GH treatment was also shown to decrease disease activity scores in patients with Crohn’s disease (22). In transgenic mice overexpressing GH, animal survival and gut mucosal repair were enhanced during experimental colitis compared with responses in wild-type mice (23).
Oxidative stress occurs when high levels of reactive oxygen species (ROS; H2O2, superoxide, hydroxyl radical) overwhelm the protection mediated by cellular antioxidants such as glutathione (GSH) and thioredoxin (24). Although ROS are normal products of cellular aerobic metabolism and appear to play an important role in cell signaling pathways, increased amounts of potentially damaging ROS may be generated from luminal sources such as toxins, bacteria, bile acids, and food components (24–26). In IBD and other catabolic conditions, ROS-mediated injury to gut epithelial cells may impair mucosal restitution, gut barrier function, and nutrient transport (27–30).
The effect of oxidative stress on PepT1-mediated functions and the underlying mechanisms by which Gln and GH may improve nitrogen absorption and enhance gut mucosal repair remain unclear. Therefore, the aims of this study were as follows: 1) to determine whether PepT1-mediated dipeptide uptake is compromised in oxidatively stressed human gut epithelial Caco-2 cells, and 2) to evaluate the effects of Ala-Gln dipeptide and GH, alone and in combination, on dipeptide transport during oxidative stress induced by H2O2.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Cell culture. Human colonic epithelial Caco-2 cells were obtained from ATCC (American Type Culture Collection). Caco-2 cells spontaneously differentiate and retain many properties of the small intestinal epithelium. All experiments were performed at cell passages 23–40. Cells were cultured in MEM (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (Invitrogen), 100 kU/L penicillin, 100 mg/L streptomycin (Invitrogen), and 2 mmol/L L-Gln (Mediatech) at 37°C and 5% CO2. Cells were passaged every 5–7 d upon reaching 80–90% confluency. Unless otherwise indicated, cells were subcultured in 6-well plates, seeded at a density of 5 x 105 cells/well. Cell media were changed every 2 d.
Confirmation of dipeptide transport. We first examined the effects of the dipeptides alanylglutamine (Ala-Gln), glycylglutamine (Gly-Gln), and cysteinylglycine (Cys-Gly) on PepT1-mediated transport of [14C]glycylsarcosine (Gly-Sar). These experiments examined the ability of unlabeled dipeptide in the cell culture media to compete with radiolabeled [14C]Gly-Sar, a protease-resistant dipeptide, for uptake in Caco-2 cells at an extracellular pH of 6.0. Under these experimental conditions, there exists an inwardly directed H+ gradient across the Caco-2 cell apical membrane necessary to energize PepT1 (3). Carrier-mediated uptake of [14C]Gly-Sar (5 µmol/L) was specifically calculated by subtracting [14C]Gly-Sar uptake measured in the presence of an excess of unlabeled Ala-Gln, Gly-Gln, or Cys-Gly (10 mmol/L) from the total uptake. Dipeptide transport was confirmed via the method of Mackenzie et al. (31). Human PepT1 (hPepT1) RNA was functionally expressed in Xenopus laevis oocytes, and the transport of Ala-Gln, Gly-Gln, or Cys-Gly by hPepT1 was measured electrophysiologically as previously described (3,31). Briefly, hPepT1-expressing oocytes were maintained in ND96 transport buffer at pH 5.5 and perfused with 2 mmol/L Ala-Gln, Gly-Gln, or Cys-Gly (31). The transport function of hPepTl in X. laevis oocytes was determined from inward currents induced by hPepT1 substrates using a 2-microelectrode voltage-clamp technique (2,3). In this system, only transportable substrates induced inward current, whereas nontransportable peptides do not induce any detectable current.
Analysis of cellular apoptosis and viability. Three days after reaching confluency, cells were serum-starved for 24 h in Gln-free MEM. Oxidative stress was induced with H2O2 (0.1–5 mmol/L) for 24 h in cysteine/cystine-free, serum-free DMEM (Invitrogen); 93 µmol/L cystine and 14 µmol/L cysteine were added to obtain a physiologic extracellular thiol redox potential of –80 mV (32). For flow cytometric analysis of apoptosis, cells were harvested as previously described (14). Flow cytometric analyses were performed on a FACScan flow cytometer (FL2) (Becton-Dickinson) in linear mode and singlet gate, and analyzed using CellQuest software (Becton-Dickinson). The percentage of cells in the sub-G1 fraction was used as the index of apoptosis (14). Cell viability was determined by trypan blue exclusion.
Caco-2 cell treatment and dipeptide transport studies. Cells were seeded, serum-starved, and oxidative stress was induced with H2O2 (0.1–5 mmol/L) in serum-free DMEM as described above with or without concurrent Ala-Gln (10 mmol/L), GH (100 µg/L), or a combination of GH and Ala-Gln. Dipeptide transport was measured after 1 h or 24 h of H2O2-treatment as outlined below.
Dipeptide transport. All transport experiments were performed 3 d postconfluency—a stage that we previously showed was optimal for transport experiments in Caco-2 cells (33). Briefly, transport was initiated by aspiration of treatment medium and washing with transport buffer for 1 min. The transport medium consisted of 25 mmol/L Mes-Tris (pH 6.0) containing 140 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl2, 0.8 mmol/L MgSO4 and 5 mmol/L glucose. After washing, 1 mL of transport medium containing 20 µmol/L [14C]Gly-Sar (Cambridge Research Biochemicals; specific activity 56.7 mCi/mmol)] was added. For kinetic studies, the concentrations of Gly-Sar ranged from 20 µmol/L to 12.5 mmol/L. To confirm H+-dependent PepT1-mediated Gly-Sar transport, some experiments were carried out in transport media at neutral pH (7.5).
After incubation with [14C]Gly-Sar for 10 min at 37°C, the transport medium was removed, and the cells were quickly washed 4 times with ice-cold transport buffer. Cells were then dissolved in 1 mL of 0.2 mol/L NaOH containing 1% SDS. An aliquot was taken to assess protein concentration as described below. The remaining cells were transferred into vials containing Scinti Verse scintillation cocktail (Fisher Scientific) for quantification of radioactivity by liquid scintillation spectrometry in a Packard Tri Carb 4640 scintillation counter (Perkin Elmer).
Calculation of transport parameters. Non-PepT1-mediated dipeptide transport was determined by measuring the transport of [14C]-labeled Gly-Sar in the presence of an excess amount of unlabeled Gly-Sar (50 mmol/L). Carrier-mediated (saturable) transport was determined by subtracting noncarrier-mediated transport from total transport. Gly-Sar transport data were corrected for the nonsaturable component and expressed as nmol/(g protein · 10 min). Carrier-mediated transport values were used for construction of Eadie-Hofstee plots and calculation of the Michaelis constant, Km, and maximum velocity, Vmax [expressed in nmol/(g protein · 10 min)] (31).
Western blot analyses of PepT1. Caco-2 cells were cultured at a density of 4 x 103 cells/cm2 in 10-cm dishes as above. Cells were serum-starved for 24 h, then treated with or without H2O2 (1 mmol/L) ± Ala-Gln (10 mmol/L), GH (100 µg/L), or a combination of GH and Ala-Gln and harvested after 24 h. Cytosolic protein was isolated as previously described (5). Total protein (30 µg) was separated on Ready gel Tris-HCl 4–20% polyacrylamide gels (BioRad) and transferred to Hybond ECl nitrocellulose membrane (Amersham Pharmacia). Anti-human PepT1 (1:500) (custom-made by Zymed Laboratories) was used to detect PepT1 as previously described (5). ß-Actin was detected with anti-ß-actin (Sigma Chemical) to control for protein loading. Bound antibody was detected with anti-rabbit IgG (Santa Cruz Biotechnology). The amount of PepT1 was determined by chemiluminescence using ECL reagent (Amersham Pharmacia Biotech), quantified using a Molecular Dynamics Computing Densitometer, and expressed as a percentage of control cells normalized for ß-actin expression.
Intracellular GSH/glutathione disulfide (GSSG) redox. Cells were treated as above for transport studies and harvested for measurement of intracellular GSH and GSSG concentrations after 24 h as previously described (32,34). GSH and GSSG concentrations were determined using reversed-phase HPLC after derivatization with iodoacetic acid and dansyl-chloride (34). Thiols were quantified by integration relative to the internal standard and expressed as nmol/mg protein. GSH/GSSG redox (Eh) was calculated using the Nernst equation.
Protein assay. Protein concentrations for transport studies and Western blotting were measured using a BioRad DC-protein assay kit according to the manufacturer’s protocols.
Statistics. One-factor ANOVA and the post-hoc Fisher’s least significant difference test were used to detect significant differences between treatment groups. Differences 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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100 nA) (Fig. 1A). The dipeptide-induced currents were dependent on the testing membrane potential for all 3 dipeptides (Fig. 1B), demonstrating that the membrane potential also provides the driving force for hPepT1, in addition to the H+ gradient, as previously shown (31). Thus, Gly-Gln, Ala-Gln, and Cys-Gly are excellent substrates for the human intestinal peptide transporter PepT1.
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1% under control conditions to 2% at 3.0 and 5.0 mmol/L, respectively (Fig. 2). Cell viability assessed by trypan blue exclusion was not altered after treatment with H2O2 at 1.0–5.0 mmol/L for 24 h (95 ± 1.2% living cells in controls vs. 97 ± 0.3% at 5 mmol/L H2O2). Under these conditions, H2O2 treatment (1–5 mmol/L) decreased intracellular GSH after 1 h. Eh wasslightly but not significantly oxidized compared with controls (Table 2). At 24 h after H2O2 treatment, intracellular GSH concentrations were modestly increased at the higher H2O2 doses (3–5 mmol/L); however, Eh, an index of the reducing potential of the GSH/GSSG redox system, was not significantly altered (Table 2). Thus, these conditions are appropriate to study the effect of oxidative stress on dipeptide transport.
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1 mmol/L (Fig. 3B). Ala-Gln and GH maintain PepT1-mediated dipeptide transport after oxidative stress. Supplementation of Caco-2 cells with Ala-Gln alone significantly attenuated the decrease in dipeptide transport observed with 24 h of 1.0 mmol/L H2O2 treatment (Fig. 4). GH treatment completely prevented this decrease in dipeptide transport. Ala-Gln + GH in combination did not have additive or synergistic effects (P = 0.40) compared with Ala-Gln alone. Ala-Gln, GH or the combination of Ala-Gln + GH did not significantly alter dipeptide transport in nonoxidatively stressed cells (data not shown).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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PepT1 is responsible for the transport of oligopeptides from the lumen of the intestine into enterocytes in humans (1). Liang et al. (2) cloned and expressed the human PepT1 transporter and determined that it was capable of transporting di- and tripeptides. We confirmed that this transporter transports several dipeptides that are in clinical use, including Ala-Gln and Gly-Gln as documented by both transport into X. laevis oocytes transfected with the hPepT1 transporter RNA and competition studies in Caco-2 cells. Because PepT1 is the only known mechanism for oligopeptide transport in the gut, improvement of impaired PepT1 transporter function may be useful for the treatment of malabsorption associated with IBD and other conditions associated with increased local ROS production (30,35).
Several studies investigated the role of hormones and dipeptides on PepT1-mediated transport. For example, in Caco-2 cells, insulin was shown to stimulate dipeptide uptake through an increase in the translocation of PepT1 from the cytoplasmic pool to the plasma membrane (11). Apical, but not basolateral leptin treatment of Caco-2 cells also increased maximal velocity of Gly-Sar transport, an observation associated with an increase in the concentration of membrane-associated PepT1 expression (12). Transient transfection studies conducted with rat PepT1 indicated that dipeptides increased dipeptide (Gly-Sar, Gly-Phe, Lys-Phe, and Asp-Lys) transport through increased transcriptional activation (9). Walker et al. (36) found that PepT1-mediated dipeptide uptake was upregulated in Caco-2 cells in the presence of dipeptides through both an increase in transcription and mRNA stability. Consistent with our findings using GH in H2O2-treated cells, Sun et al. (37) recently determined that GH prevented the observed decrease in PepT1-mediated uptake of cephalexin (a ß-lactam antibiotic with a structure similar to dipeptides) in Caco-2 cells subjected to anoxia/reoxygenation injury, an effect associated with an increase in PepT1 mRNA. Therefore, substantial evidence shows that dipeptides regulate PepT1 expression and activity in human gut epithelial cells, and specific hormones may potentiate this effect.
Dipeptides such as Ala-Gln may also protect against oxidative stress. In our model, Ala-Gln increased cellular GSH because these values were significantly higher than either control or H2O2-treated cells at 1 h after H2O2 treatment (Table 3). Furthermore, Ala-Gln treatment maintained PepT1 protein expression after 24 h of oxidative stress. Increased cellular concentrations of GSSG and decreased GSH concentrations were documented in inflamed gut mucosa of patients with IBD (35,38,39). Moreover, replenishment of GSH ameliorated the gut mucosal injury associated with experimentally induced colitis in rats (40). Oral Gln supplementation also prevented the oxidative stress-associated impairment of brush border membrane activity in rats subjected to intestinal surgery (41). Thus, Ala-Gln may potentially protect dipeptide transport through an increase in GSH production which maintains the GSH/GSSG Eh redox potential.
Several gut-trophic agents were used successfully in models of gastrointestinal diseases associated with oxidative stress. Among these substances are hormones such as epidermal growth factor (EGF) and keratinocyte growth factor (13,32,42,43). For example, EGF had time-dependent effects on Gly-Sar transport in nonstressed Caco-2 cells. In cells treated for 1 h, EGF increased Gly-Sar transport, an observation accompanied by an increase in Vmax and a concomitant increase in PepT1 protein expression, as we show here (44). However, Gly-Sar transport and PepT1 Vmax, protein and mRNA expression were reduced in Caco-2 cells treated with EGF for 28 d (45). In the current study, we showed that administration of GH maintains PepT1-mediated dipeptide transport under conditions of oxidative stress. However, unlike Ala-Gln, GH had no effect on GSH and therefore appears likely to protect transport activity through a different mechanism.
Finally, in this model of oxidative stress, both GH and Ala-Gln treatment increased PepT1 dipeptide transport, whereas only Ala-Gln treatment increased PepT1 protein expression. One possible explanation for this discrepancy is that GH may alter PepT1 membrane expression or trafficking without altering total PepT1 expression, as observed with leptin treatment in Caco-2 cells (12). Thus, additional studies are warranted to further define the underlying mechanisms for improved PepT1 dipeptide transport with Ala-Gln and GH treatment in oxidatively stressed cells. This study was also limited by our use of H2O2 as the only oxidative insult. It would be interesting to address whether the PepT1 transport system responds to other redox perturbations, such as reactive nitrogen species donors, or whether the PepT1 transporter gene possesses redox stress sensors in its promoter region. Finally, this study did not examine potential trophic effects of Ala-Gln or GH in these oxidatively stressed cells. It is possible that such effects may be responsible in part for the protective effects of these agents on dipeptide transport.
In summary, PepT1-mediated dipeptide transport in Caco-2 cells was impaired by H2O2-induced oxidative stress in a time- and concentration-dependent manner. Both Ala-Gln dipeptide and GH treatment improved dipeptide transport through apparently different mechanisms. Therefore, these agents should be investigated further as potential therapies to improve dipeptide absorption in disorders associated with oxidative injury to the gut mucosal epithelia.
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FOOTNOTES |
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2 Supported in part by National Institutes of Health Grants R01 DK55850 (T.R.Z.), R01 ES011195 and R01 ES009047 (D.P.J.), F32 DK65345 (M.E.E.) and the Emory Digestive Diseases Research Development Core grant R24 DK064399.
3 These authors contributed equally to the work presented in this article.
5 Abbreviations used: Ala-Gln, alanylglutamine; Cys-Gly, cysteinylglycine; EGF, epidermal growth factor; Eh, GSH/GSSG redox potential; GH, growth hormone; Gln, glutamine; Gly-Gln, glycylglutamine; Gly-Sar, glycylsarcosine; GSH, glutathione; GSSG, glutathione disulfide; hPepT1, human PepT1; IBD, inflammatory bowel disease; IL, interleukin; ROS, reactive oxygen species.
Manuscript received 16 July 2004. Initial review completed 26 August 2004. Revision accepted 20 October 2004.
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