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An in vitro explant model for studies of intestinal amino acid metabolism
Clinical Nutrition Experimental xxx (xxxx) xxx
Contents lists available at ScienceDirect
Clinical Nutrition Experimental journal homepage: http:// www.clinicalnutritionexperimental.com
Methodology
An in vitro explant model for studies of intestinal amino acid metabolism Sandie Pestour b, 1, Morgane Couchet a, 1, Charlotte Breuillard a, de ric Lamarche a, Eric Fontaine a, b, Christelle Corne a, b, Nicolas Mathieu b, Fre c ,d a ,* €ffier , Christophe Moinard Moïse Coe Laboratoire de Bio energ etique Fondamentale et Appliqu ee, INSERM U 1055, Universit e Grenoble Alpes, France Centre Hospitalier Universitaire Grenoble Alpes, France c Centre Hospitalier Universitaire de Rouen, France d Institut de Recherche et d’Innovation Biom edicale INSERM U 1073, Universit e de Rouen, France a
b
a r t i c l e i n f o
s u m m a r y
Article history: Received 9 September 2019 Accepted 14 December 2019 Available online xxx
Background & aims: The main role of the small intestine to absorb the bulk of dietary nutrients, including proteins and amino acids (AA). Enterocytes have long been considered a major player in amino acid homeostasis, especially the glutamineecitrullineearginine crossroads. In vivo intestinal epithelium cell lines for studies of nitrogen metabolism have now reached their limitations. To push research forward, an interesting approach could be to use the explant incubation model of intestinal tissue, involving in vitro incubation of duodenal tissue biopsy specimens taken from human subjects. Methods: Eight duodenal biopsies were taken during endoscopy from patients without proven intestinal disease. Each biopsy was incubated in complete culture medium for 23 h then weighed, and cell viability was evaluated after 6 and 18 h. We then explored Lcitrulline (L-CIT) production (reflecting metabolic activity) and the presence of the main enzymes involved in AA metabolism in the intestine (arginase II, glutaminase I, ornithine aminotransferase, and ornithine carbamoyltransferase).
Keywords: Ornithine carbamoyltransferase Amino acid Enterocyte Enzyme
Abbreviations: AA, Amino acid; ARG, L-arginine; ARGase, Arginase; ASL, Argininosuccinate lyase; BMI, Body mass index; CIT, L-citrulline; DTT, Dithiothreitol; GLN, L-glutamine; GLNase, Glutaminase; GLU, L-glutamate; ICU, Intensive Care Unit; IFNg, Interferon-g; LDH, Lactate dehydrogenase; NO, Nitric oxide; OAT, Ornithine aminotransferase; OTC, Ornithine carbamoyltransferase; ORN, L-ornithine; SDS, Sodium dodecyl sulfate. nerge tique Fondamentale et Applique e, INSERM U 1055, Universite Grenoble * Corresponding author. Laboratoire de Bioe Alpes, CS 40700, 38058 Grenoble Cedex 9, France. E-mail address: [email protected] (C. Moinard). 1 These 2 authors contributed equally to the work. https://doi.org/10.1016/j.yclnex.2019.12.002 2352-9393/© 2019 Université Grenoble Alpes. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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Results: Mean weight of biopsies was 12.36 ± 1.00 mg. Mortality was around 20% after 6 h and 50% after 18 h of incubation. CIT was amply produced by the biopsies, and enzymes implicated in intestinal AA metabolism were expressed in this model. Conclusions: This in vitro explant model of intestinal tissue emerges as a reliable model for conducting ex vivo investigations on AA metabolism. © 2019 Université Grenoble Alpes. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction The small intestine is known to host the bulk of digestion and absorption of dietary nutrients, including proteins and amino acids (AA). However, its metabolic role has gained increasing attention, bringing important insights into the properties of AA metabolism and nitrogen homeostasis [1,2]. Enterocytes contain key enzymes for AA metabolism, such as arginase (ARGase) and glutaminase (GLNase). Intestinal mucosa thus plays a key role in the catabolism of several AA like L-arginine (ARG) and L-glutamine (GLN) (Fig. 1 [3]). Moreover, dietary AA is required precursors for intestinal synthesis of several molecules as nitric oxide (NO) and L-citrulline (Fig. 1 [3]). This makes dietary AA essential, both for maintaining intestinal mucosal mass and integrity and for intestinal motility and function [2e5]. The intestinal protein turnover represents about 15% of the total protein turnover [6]. Any effort to explore intestinal nitrogen metabolism requires a reliable in vitro model. Human intestinal Caco-2 cell lines, derived from a human colon carcinoma, are a popular model of the intestinal epithelial barrier, but the model has limitations [7]. First, immortalized cell lines have a specific metabolism that does not reflect physiological metabolism. Second, peptides and AA transporters express differently in Caco-2 cells compared to human gastrointestinal tract [8]. Caco-2 cells express a different pattern of enzymes involved in AA metabolism, and crucially, they do not express ornithine carbamoyltransferase (OTC) (which produces citrulline) but instead express argininosuccinate synthase and argininosuccinate lyase (which are not expressed in humans). For these reasons, Caco-2 cell lines are unsuitable for in vitro investigations of intestinal AA metabolism [5]. Primary cell cultures of human enterocytes could appear as an appropriate alternative model. However, isolating and culturing human enterocytes from the small intestine is a tricky technique that carries a number of challenges, including microbial infection risk and the short lifespan of enterocytes [7]. Moreover, primary cell culture fails to account for potential environmental interactions. The intestinal structure has a complex cell composition to ensure digestive, metabolic, immunologic and endocrine functions [9]. Here we propose an in vitro explant model of intestinal tissue, involving incubation of duodenal tissue biopsy specimens taken from human subjects. This model has been already developed to explore intestinal function [10e12] but without exploring the intestinal amino acid crossroads. The aim of this work was to perform viability experiments on the explants in order to define the best-adapted conditions of use for exploring AA metabolism and nitrogen homeostasis.
2. Methods 2.1. Patients Patients followed by GrenobleeAlpes University Medical Center's Gastroenterology Department and requiring a gastroduodenal endoscopy were enrolled in the experimental protocol after giving informed written consent. Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
S. Pestour et al. / Clinical Nutrition Experimental xxx (xxxx) xxx
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
Fig. 1. AA metabolism in the intestine. (From Wu, 1998). The enzymes that catalyze the indicated reactions are as follows: 1) phosphate-dependent glutaminase; 2) D1-pyrroline-5-carboxylate synthetase; 3) ornithine aminotransferase; 4) ornithine carbamoyltransferase; 5) argininosuccinate synthetase; 6) argininosuccinate lyase; 7) arginase; 8) ornithine decarboxylase; 9) nitric oxide synthase; 10) carbamoyl phosphate synthetase; 11) proline oxidase; 12) pyrroline-5-carboxylate reductase; 13) BCAA transaminase; 14) BCKA dehydrogenase; 15) aspartate transaminase; 16) alanine transaminase; 17) a-KG dehydrogenase; 18) possibly via NADP-linked malic enzyme, phosphoenolpyruvate carboxykinase/pyruvate kinase and oxaloacetate decarboxylase; 19) pyruvate dehydrogenase; 20) Krebs cycle enzymes; 21) serine transaminase; 22) serine hydroxymethyltransferase; 23) glutathione-synthesizing enzymes; 24) serine dehydratase; 25) glucosesynthesizing enzymes; and 26) purine and pyrimidine-synthesizing enzymes. The symbol “?” denotes unknown reactions in intestinal mucosa. Abbreviations: Asp, aspartate; BCAA, branched-chain amino acids; BCKA, branched-chain a-ketoacid dehydrogenase; CP, carbamoyl phosphate; Glu, glutamate; a-KG, a-ketoglutarate; MTF, N5,N10-methylenetetrahydrofolate; OAA, oxaloacetate; Pyr, pyruvate; TF, tetrahydrofolate. 3
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This study was approved by the GrenobleeAlpes University Hospital institutional review board (‘Comit e de Protection des Personnes Sud-Est V’, 15-CHUG-44). All 16 subjects enrolled required a gastroduodenal endoscopy based on clinical digestive symptoms or biological data. Patients with known or suspected duodenal disease were ruled out. After examination, only patients without altered intestinal mucosa and without proven intestinal disease (based on biological and histological data) were selected for the study.
2.2. Endoscopy During the endoscopy, 8 biopsy specimens were taken from each patient, from the second duodenum using surgical pliers after testing several models to get the largest and well-shaped biopsies. Large pliers (Captura serrated large 2.4-mm forceps, spiked 230 cm, DBF-2.4SL-230SP-S, Cook Medical, Limerick, Ireland) were found to be adequate as they allowed to obtain larger less fragmented biopsies, and were used for the experiments (Fig. 2). During this exploration, no hemorrhage occurred. Postendoscopy bleeding is negligible and stops spontaneously. Moreover, the number of biopsy removed and the materials used are common for this clinical act. Biopsies were immediately transferred to separate vials containing complete cell culture medium composed of standard medium with glucose 4.5 g/L (Dulbecco's modified Eagle medium, Dutscher, Brumath, France) supplemented with 1% non-essential AA (Minimum Essential Mediume100X solution, Gibco, Invitrogen, Saint-Aubin, France), 2 mM L-GLN (PAN-Biotech, Aidenbach, Germany), penicillin (100 U/mL; PAN-Biotech), streptomycin (0.1 mg/mL; PAN-Biotech) and 10% fetal bovine serum (SigmaeAldrich, Saint-Louis, MO). Samples were kept at 4 C throughout transport within 1 h to the cell culture laboratory.
2.3. Biopsy culture Biopsy specimens were transferred into separated plastic culture dishes (24-well plates) containing 1 ml of the complete culture medium. Medium from tube transport was sampled for viability analysis and snap-frozen at 80 C. Biopsies were put in an incubator in a humidified atmosphere equilibrated with 21% O2/5% CO2 and incubated at 37 C for 23 h. In order to avoid damaging the tissue, biopsies were not handled with pliers but with a pipette (after cutting the plastic cone tip) or with a spoon.
Fig. 2. Endoscope and picking site in the intestine. A. Endoscope. B. The biopsy is removed from the second duodenum using large pliers.
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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To measure cell viability, samples of culture medium were removed at different incubation times (1, 3, 6, 9 and 23 h) and immediately frozen at 80 C. After a 23 h incubation period, each biopsy (n ¼ 16) was removed from its medium and frozen at 80 C. To measure CIT concentration and protein expression of AA metabolism, a second batch of biopsies we cultured for 6 (n ¼ 18) or 18 h (n ¼ 12). At the end of the incubation period, culture media and biopsy specimens were removed and immediately frozen at 80 C. Before each further experiment, biopsies were defrosted, any excess medium was removed with Whatman paper, and the tissue was weighed (precision: 0.0001, Mettler-Toledo, Viroflay, France). Given the tiny size of the biopsy specimens, it was impossible to perform all the biological measurements on the same one sample, and so the number of samples per experiment varies from 5 to 18 depending on the analysis. 2.4. Cell viability For each biopsy, viability was checked based on LDH activity levels. LDH activity was measured in each culture medium removed after transport and then at different incubation times (1, 3, 6, 9 and 23 h) and in the biopsy lysate using an enzymatic activity assay kit (SigmaeAldrich). Each biopsy (n ¼ 16) was weighed, lysed in 1 mL of a 1% Triton solution, then centrifuged (15000 g for 10 min at 4 C) to collect the supernatants. For each biopsy specimen, at each measurement timepoint, the ratio of LDH in the medium to total LDH (LDH released in culture medium at 23 h plus the LDH contained in the tissue) was calculated and expressed as percent cell death. 2.5. CIT measurement CIT concentration in the culture media was measured by ion exchange chromatography followed by post-column derivatization with ninhydrin (Hitachi L8800 analyzer, Sciencetec, Villebon-sur-Yvette, France) as previously described [13] or by UPLC-MS (Acquity UPLC H-Class QDa, Waters, Guyancourt, France) using a CORTECS UPLC C18 column after a derivatization step (AccQTag Ultra®kit, Waters). CIT concentration in the culture media was evaluated at 6 h (n ¼ 18; n ¼ 9 with the Hitachi L8800; n ¼ 9 by UPLC) and at 18 h (n ¼ 12; n ¼ 5 with the Hitachi L8800; n ¼ 7 by UPLC) and was ratioed to tissue weight for each biopsy specimen. 2.6. Protein expression Western Blot analysis was performed to verify ARGase II (36 KDa), GLNase II (73 KDa), ornithine aminotransferase (OAT) (49 KDa) and OTC (40 KDa) enzyme expression. Tissues were lysed in a commercial RIPA buffer (50 mM TriseHCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate added with protease inhibitor cocktail (both from SigmaeAldrich) and phosphatase inhibitor cocktail (Fischer Scientific, Illkirch-Graffenstaden, France). Proteins were quantified by the Pierce method using a BCA (bicinchoninic acid assay) kit (Interchim, Montluçon, France). Proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Protein immunodetection was performed using anti-OTC antibodies (1:1000 dilution; Sigma Aldrich) with anti-ARGase II (1:250 dilution, Sigma Aldrich), anti-GLNase I (1:1000, Abcam, Cambridge, United Kingdom) and anti-OAT (1:200, Sigma Aldrich) as primary antibodies. Membranes were incubated overnight with primary antibodies at 4 C. Primary antibodies were detected with secondary anti-rabbit antibodies conjugated to horseradish peroxidase (1:1000 dilution; Jackson ImmunoResearch, Ely, United Kingdom). Signals were revealed using a chemiluminescence system (Amersham ECL Prime kit, GE Healthcare, France) and quantified using Image J software (version 1.48, National institutes of Health, MD).
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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3. Results 3.1. Clinical patient data A histological analysis of the duodenal mucosa was performed for all patients. No duodenal pathology was found. Clinical patient data was checked and recorded (Table 1). No hemorrhage complication has occurred during the clinical endoscopy. 3.2. Biopsy weight Tissue weight was around 12.36 mg [7.3e27.0]. 3.3. Cell viability Mortality was around 20% at 6 h incubation and increased progressively throughout the incubation period to reach around 50% at 18 h and 70% at 23 h incubation (Fig. 3). 3.4. CIT concentrations CIT release was around 0.215 mM/mg biopsy [0e9] over the 6 h period and 0.300 ± 0.040 mM/mg biopsy [0e7] at 18 h. Table 1 Clinical patient data. Clinical data Age (years) Gender Body Mass Index (BMI) (KG/M2)
Average Range Female Male Average Range
40 19e73 47% 53% 26 18.0e31.1
Fig. 3. Cell viability over 23 h. Biopsy specimens (n ¼ 16) were incubated for 23 h. LDH activity was measured in culture media at several timepoints (at 1 h, 3 h, 6 h, 9 h and 23 h of incubation) and in each biopsy after tissue lysis. Cell mortality percentages were calculated. Individual cell mortality values for each biopsy are given for each incubation time. (- - -) corresponds to the percentage of cell mortality at 6 h (approx. 20%) and 18 h (approx. 50%).
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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3.5. Protein expression ARGase II, GLNase I, OAT, and OTC enzymes were found in the biopsy specimens after 6 h (Fig. 4) and 18 h of incubation. 4. Discussion Here we repurposed a preexisting model of duodenal tissue previously used to study the production of cytokines or other metabolic processes in the gut [10e12] in order to explore AA metabolism in the intestine. The main strength of this model is that it includes not only the epithelial enterocyte barrier but also the lamina propria that contains other tissue elements [14]. It is consequently more representative of real conditions in terms of functionality and metabolism. However, this may make experimental results more difficult to interpret. For example, if the culture medium shows altered release of an AA, then further experiments will be needed to determine the metabolic pathways involved and whether these pathways take place inside or outside the enterocyte. The production of CIT by the biopsy is proof of enterocyte functionality [15]. Indeed, a very small difference in CIT production is observed between 6 and 18 h of incubation, whereas mortality doubled over these two same timepoints, suggesting CIT release is not due to cell mortality but reflects enterocyte functionality. Moreover, the presence of the main enzymes of the CIT metabolism, such as OTC, ARGase II, GLNase I and OAT, confirms enterocyte functionality. Quantifying enzyme activity would bring sharper results but was impossible to do here due to the restrictive size of the biopsies. Another set of experiments measured OTC enzyme activity and found that the enzyme was active and stable in human enterocytes after 6 or 18 h of incubation (personal data). Based on the literature, duodenal biopsies can be cultured over 24e48 h with well-maintained differentiation and metabolic activities [10]. Here, viability was evaluated in order to define the appropriate incubation time for further experimental studies on AA metabolism. Environmental modifications to improve explants viability were not envisaged to stay as close as possible to physiological conditions in order not to influence intestinal amino acid metabolism. Cell mortality was still around 20% up to 6 h incubation and then increased to reach 50% after 18 h and 60%e80% after 23 h. These figures are in the same range as those described for other cell types in primary culture, such as Beta cells [16], and our data is consistent with the rapid turnover of intestinal cell lines (i.e. between 24 and 48 h) [17]. Here we studied two incubation periods: short term (6 h) and long-term (18 h). Neither of these two incubation periods makes it possible to reach the half-life of OTC, which is estimated at between 6 and 9 days [18,19]. Given the speed of intestinal turnover, it is practically impossible to capture the half-specific activity of the enzyme. Indeed, a long half-life time masks the cell's adaptation to a changing environment as stress-induced damage. The advantage of 6 h incubation is mostly the low percent mortality compared to 18 h incubation. However, the longer 18 h incubation time could better serve to find out certain cell adaptations because the period is closer to OTC half-life time found in light hepatocytes. Finally, incubation time should obviously be dictated by the objectives of each study.
Fig. 4. Protein expression after 6 h and 18 h of incubation in tissue. Biopsy specimens (n ¼ 2) were incubated for 6 or 18 h. Arginase II (ARG II), glutaminase I (GLNase I), ornithine aminotransferase (OAT) and ornithine carbamoyltransferase (OTC) protein expression were all verified.
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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Here, a 6 h incubation period looks the most appropriate time window to avoid broad cell death, but other incubation times can be employed and designed to meet other study purposes. Stable CIT production over 6 and 18 h of incubation further supports the use of a 6 h incubation period for studies on AA metabolism. This in vitro explant model of intestinal tissue emerges as a reliable model for conducting further experiments on intestinal AA metabolism. It seems well adapted both for in vitro explorations (i.e. modification of the biopsies environment) and for ex vivo explorations (i.e. patients with a specific treatment or nutritional intervention). Conflict of Interest None of the authors have any conflict of interest to report. Acknowledgments Grenoble University Hospital was promoter of the study. The authors thank the Endoscopy Unit staff at the Hepato-Gastroenterology Department headed by Pr Leroy for their valuable assistance, and in particular Dr Patrick Tuvignon, Dr Pierre-Yves Eyraud, Dr Nicolas Mathieu and Dr Audrey Picot for their participation as investigators in the clinical strand of the study. Statement of authorship S.P., M.Ct., C.B., E.F., and C.M contributed to the Conceptualization. S.P., M.Ct., C.B., F.L., E.F., M.Cr., and C.M., contributed to the methodology. S.P., M.Ct., C.B., C.C., N.M., and F.L., contributed to the investigation. S.P., M.Ct., C.B., and C.M., wrote the original draft. S.P., M.Ct., C.B., C.C., N.M., F.L., E.F., M.Cr., and C.M., contributed to reviewing and editing. S.P., M.Ct., C.B., and C.M., contributed to the supervision. Funding sources This work was supported in part by a grant from the nonprofit association ‘Agir pour les Maladies Chroniques’. References [1] Brosnan JT. Interorgan amino acid transport and its regulation. J Nutr 2003;133(6):2068Se72S. https://doi.org/10.1093/jn/ 133.6.2068S. [2] Breuillard C, Cynober L, Moinard C. Citrulline and nitrogen homeostasis: an overview. Amino Acids 2015;47(4):685e91. https://doi.org/10.1007/s00726-015-1932-2. [3] Wu G. Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs. Am J Physiol Gastrointest Liver Physiol 1997;272(6):G1382e90. [4] Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis. J Nutr 2007;137(6):1621Se5S. https://doi.org/10.1093/jn/137.6.1621S. re V, Lasserre V, Cynober L, et al. Evidence for a role of the ileum in the control of [5] Ventura G, Moinard C, Sinico F, Carrie nitrogen homeostasis via the regulation of arginine metabolism. Br J Nutr 2011;106(2):227e36. https://doi.org/10.1017/ S0007114511000079. re B. Me tabolisme prote ique. Cahiers de Nutrition et de Die t e tique 2005;40(1):53e64. https://doi.org/10. [6] Boirie Y, Beaufre 1016/S0007-9960(05)80467-5. [7] Chougule P, Herlenius G, Hernandez NM, Patil PB, Xu B, Sumitran-Holgersson S. Isolation and characterization of human primary enterocytes from small intestine using a novel method. Scand J Gastroenterol 2012;47(11):1334e43. https://doi. org/10.3109/00365521.2012.708940. [8] Jochems P, Garssen J, van Keulen A, Masereeuw R, Jeurink P. Evaluating human intestinal cell lines for studying dietary protein absorption. Nutrients 2018;10(3):322. https://doi.org/10.3390/nu10030322. [9] Kong S, Zhang YH, Zhang W. Regulation of intestinal epithelial cells properties and functions by amino acids. BioMed Res Int 2018;2018:1e10. https://doi.org/10.1155/2018/2819154. €ffier M, Miralles-Barrachina O, Le Pessot F, Lalaude O, Daveau M, Lavoinne A, et al. Influence of glutamine on cytokine [10] Coe production by human gut in vitro. Cytokine 2001;13(3):148e54. https://doi.org/10.1006/cyto.2000.0813. €ffier M. Modulating effect of glutamine on IL-1b-induced cytokine production by human gut. Clin Nutr 2003;22(4): [11] Coe 407e13. https://doi.org/10.1016/S0261-5614(03)00040-2. [12] Lecleire S, Coeffier M, Leblond J, Hubert A, Lemoulan S, Petit A, et al. Modulation of nitric oxide and cytokines production by l-arginine in human gut mucosa. Clin Nutr 2005;24(3):353e9. https://doi.org/10.1016/j.clnu.2004.11.011.
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[13] Le Boucher J, Charret C, Coudray-Lucas C, Giboudeau J, Cynober L. Amino acid determination in biological fluids by automated ion-exchange chromatography: performance of Hitachi L-8500A. Clin Chem 1997;43(8):1421e8. [14] Achkar E, Carey WD, Petras R, Sivak MV, Rn RR. Comparison of suction capsule and endoscopic biopsy of small bowel mucosa. Gastrointest Endosc 1986;32(4):278e81. https://doi.org/10.1016/S0016-5107(86)71846-4. [15] Crenn P, Vahedi K, Lavergne-Slove A, Cynober L, Matuchansky C, Messing B. Plasma citrulline: a marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterology 2003;124(5):1210e9. https://doi.org/10.1016/S00165085(03)00170-7. [16] Lablanche S, Cottet-Rousselle C, Argaud L, Laporte C, Lamarche F, Richard M-J, et al. Respective effects of oxygen and energy substrate deprivation on beta cell viability. Biochim Biophys Acta 2015;1847(6e7):629e39. https://doi.org/10.1016/ j.bbabio.2015.04.002. [17] Darwich AS, Aslam U, Ashcroft DM, Rostami-Hodjegan A. Meta-analysis of the turnover of intestinal epithelia in preclinical animal species and humans. Drug Metab Dispos 2014;42(12):2016e22. https://doi.org/10.1124/dmd.114.058404. [18] Wallace R, Knecht E, Grisolía S. Turnover of rat liver ornithine transcarbamylase. FEBS Lett 1986;208(2):427e30. https:// doi.org/10.1016/0014-5793(86)81062-6. [19] Vargas JL, Roche E, Knecht E, Grisolía S. Differences in the half-lives of some mitochondrial rat liver enzymes may derive partially from hepatocyte heterogeneity. FEBS Lett 1987;224(1):182e6. https://doi.org/10.1016/0014-5793(87)80444-1.
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
Contents lists available at ScienceDirect
Clinical Nutrition Experimental journal homepage: http:// www.clinicalnutritionexperimental.com
Methodology
An in vitro explant model for studies of intestinal amino acid metabolism Sandie Pestour b, 1, Morgane Couchet a, 1, Charlotte Breuillard a, de ric Lamarche a, Eric Fontaine a, b, Christelle Corne a, b, Nicolas Mathieu b, Fre c ,d a ,* €ffier , Christophe Moinard Moïse Coe Laboratoire de Bio energ etique Fondamentale et Appliqu ee, INSERM U 1055, Universit e Grenoble Alpes, France Centre Hospitalier Universitaire Grenoble Alpes, France c Centre Hospitalier Universitaire de Rouen, France d Institut de Recherche et d’Innovation Biom edicale INSERM U 1073, Universit e de Rouen, France a
b
a r t i c l e i n f o
s u m m a r y
Article history: Received 9 September 2019 Accepted 14 December 2019 Available online xxx
Background & aims: The main role of the small intestine to absorb the bulk of dietary nutrients, including proteins and amino acids (AA). Enterocytes have long been considered a major player in amino acid homeostasis, especially the glutamineecitrullineearginine crossroads. In vivo intestinal epithelium cell lines for studies of nitrogen metabolism have now reached their limitations. To push research forward, an interesting approach could be to use the explant incubation model of intestinal tissue, involving in vitro incubation of duodenal tissue biopsy specimens taken from human subjects. Methods: Eight duodenal biopsies were taken during endoscopy from patients without proven intestinal disease. Each biopsy was incubated in complete culture medium for 23 h then weighed, and cell viability was evaluated after 6 and 18 h. We then explored Lcitrulline (L-CIT) production (reflecting metabolic activity) and the presence of the main enzymes involved in AA metabolism in the intestine (arginase II, glutaminase I, ornithine aminotransferase, and ornithine carbamoyltransferase).
Keywords: Ornithine carbamoyltransferase Amino acid Enterocyte Enzyme
Abbreviations: AA, Amino acid; ARG, L-arginine; ARGase, Arginase; ASL, Argininosuccinate lyase; BMI, Body mass index; CIT, L-citrulline; DTT, Dithiothreitol; GLN, L-glutamine; GLNase, Glutaminase; GLU, L-glutamate; ICU, Intensive Care Unit; IFNg, Interferon-g; LDH, Lactate dehydrogenase; NO, Nitric oxide; OAT, Ornithine aminotransferase; OTC, Ornithine carbamoyltransferase; ORN, L-ornithine; SDS, Sodium dodecyl sulfate. nerge tique Fondamentale et Applique e, INSERM U 1055, Universite Grenoble * Corresponding author. Laboratoire de Bioe Alpes, CS 40700, 38058 Grenoble Cedex 9, France. E-mail address: [email protected] (C. Moinard). 1 These 2 authors contributed equally to the work. https://doi.org/10.1016/j.yclnex.2019.12.002 2352-9393/© 2019 Université Grenoble Alpes. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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Results: Mean weight of biopsies was 12.36 ± 1.00 mg. Mortality was around 20% after 6 h and 50% after 18 h of incubation. CIT was amply produced by the biopsies, and enzymes implicated in intestinal AA metabolism were expressed in this model. Conclusions: This in vitro explant model of intestinal tissue emerges as a reliable model for conducting ex vivo investigations on AA metabolism. © 2019 Université Grenoble Alpes. Published by Elsevier Ltd on behalf of European Society for Clinical Nutrition and Metabolism. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction The small intestine is known to host the bulk of digestion and absorption of dietary nutrients, including proteins and amino acids (AA). However, its metabolic role has gained increasing attention, bringing important insights into the properties of AA metabolism and nitrogen homeostasis [1,2]. Enterocytes contain key enzymes for AA metabolism, such as arginase (ARGase) and glutaminase (GLNase). Intestinal mucosa thus plays a key role in the catabolism of several AA like L-arginine (ARG) and L-glutamine (GLN) (Fig. 1 [3]). Moreover, dietary AA is required precursors for intestinal synthesis of several molecules as nitric oxide (NO) and L-citrulline (Fig. 1 [3]). This makes dietary AA essential, both for maintaining intestinal mucosal mass and integrity and for intestinal motility and function [2e5]. The intestinal protein turnover represents about 15% of the total protein turnover [6]. Any effort to explore intestinal nitrogen metabolism requires a reliable in vitro model. Human intestinal Caco-2 cell lines, derived from a human colon carcinoma, are a popular model of the intestinal epithelial barrier, but the model has limitations [7]. First, immortalized cell lines have a specific metabolism that does not reflect physiological metabolism. Second, peptides and AA transporters express differently in Caco-2 cells compared to human gastrointestinal tract [8]. Caco-2 cells express a different pattern of enzymes involved in AA metabolism, and crucially, they do not express ornithine carbamoyltransferase (OTC) (which produces citrulline) but instead express argininosuccinate synthase and argininosuccinate lyase (which are not expressed in humans). For these reasons, Caco-2 cell lines are unsuitable for in vitro investigations of intestinal AA metabolism [5]. Primary cell cultures of human enterocytes could appear as an appropriate alternative model. However, isolating and culturing human enterocytes from the small intestine is a tricky technique that carries a number of challenges, including microbial infection risk and the short lifespan of enterocytes [7]. Moreover, primary cell culture fails to account for potential environmental interactions. The intestinal structure has a complex cell composition to ensure digestive, metabolic, immunologic and endocrine functions [9]. Here we propose an in vitro explant model of intestinal tissue, involving incubation of duodenal tissue biopsy specimens taken from human subjects. This model has been already developed to explore intestinal function [10e12] but without exploring the intestinal amino acid crossroads. The aim of this work was to perform viability experiments on the explants in order to define the best-adapted conditions of use for exploring AA metabolism and nitrogen homeostasis.
2. Methods 2.1. Patients Patients followed by GrenobleeAlpes University Medical Center's Gastroenterology Department and requiring a gastroduodenal endoscopy were enrolled in the experimental protocol after giving informed written consent. Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
Fig. 1. AA metabolism in the intestine. (From Wu, 1998). The enzymes that catalyze the indicated reactions are as follows: 1) phosphate-dependent glutaminase; 2) D1-pyrroline-5-carboxylate synthetase; 3) ornithine aminotransferase; 4) ornithine carbamoyltransferase; 5) argininosuccinate synthetase; 6) argininosuccinate lyase; 7) arginase; 8) ornithine decarboxylase; 9) nitric oxide synthase; 10) carbamoyl phosphate synthetase; 11) proline oxidase; 12) pyrroline-5-carboxylate reductase; 13) BCAA transaminase; 14) BCKA dehydrogenase; 15) aspartate transaminase; 16) alanine transaminase; 17) a-KG dehydrogenase; 18) possibly via NADP-linked malic enzyme, phosphoenolpyruvate carboxykinase/pyruvate kinase and oxaloacetate decarboxylase; 19) pyruvate dehydrogenase; 20) Krebs cycle enzymes; 21) serine transaminase; 22) serine hydroxymethyltransferase; 23) glutathione-synthesizing enzymes; 24) serine dehydratase; 25) glucosesynthesizing enzymes; and 26) purine and pyrimidine-synthesizing enzymes. The symbol “?” denotes unknown reactions in intestinal mucosa. Abbreviations: Asp, aspartate; BCAA, branched-chain amino acids; BCKA, branched-chain a-ketoacid dehydrogenase; CP, carbamoyl phosphate; Glu, glutamate; a-KG, a-ketoglutarate; MTF, N5,N10-methylenetetrahydrofolate; OAA, oxaloacetate; Pyr, pyruvate; TF, tetrahydrofolate. 3
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This study was approved by the GrenobleeAlpes University Hospital institutional review board (‘Comit e de Protection des Personnes Sud-Est V’, 15-CHUG-44). All 16 subjects enrolled required a gastroduodenal endoscopy based on clinical digestive symptoms or biological data. Patients with known or suspected duodenal disease were ruled out. After examination, only patients without altered intestinal mucosa and without proven intestinal disease (based on biological and histological data) were selected for the study.
2.2. Endoscopy During the endoscopy, 8 biopsy specimens were taken from each patient, from the second duodenum using surgical pliers after testing several models to get the largest and well-shaped biopsies. Large pliers (Captura serrated large 2.4-mm forceps, spiked 230 cm, DBF-2.4SL-230SP-S, Cook Medical, Limerick, Ireland) were found to be adequate as they allowed to obtain larger less fragmented biopsies, and were used for the experiments (Fig. 2). During this exploration, no hemorrhage occurred. Postendoscopy bleeding is negligible and stops spontaneously. Moreover, the number of biopsy removed and the materials used are common for this clinical act. Biopsies were immediately transferred to separate vials containing complete cell culture medium composed of standard medium with glucose 4.5 g/L (Dulbecco's modified Eagle medium, Dutscher, Brumath, France) supplemented with 1% non-essential AA (Minimum Essential Mediume100X solution, Gibco, Invitrogen, Saint-Aubin, France), 2 mM L-GLN (PAN-Biotech, Aidenbach, Germany), penicillin (100 U/mL; PAN-Biotech), streptomycin (0.1 mg/mL; PAN-Biotech) and 10% fetal bovine serum (SigmaeAldrich, Saint-Louis, MO). Samples were kept at 4 C throughout transport within 1 h to the cell culture laboratory.
2.3. Biopsy culture Biopsy specimens were transferred into separated plastic culture dishes (24-well plates) containing 1 ml of the complete culture medium. Medium from tube transport was sampled for viability analysis and snap-frozen at 80 C. Biopsies were put in an incubator in a humidified atmosphere equilibrated with 21% O2/5% CO2 and incubated at 37 C for 23 h. In order to avoid damaging the tissue, biopsies were not handled with pliers but with a pipette (after cutting the plastic cone tip) or with a spoon.
Fig. 2. Endoscope and picking site in the intestine. A. Endoscope. B. The biopsy is removed from the second duodenum using large pliers.
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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To measure cell viability, samples of culture medium were removed at different incubation times (1, 3, 6, 9 and 23 h) and immediately frozen at 80 C. After a 23 h incubation period, each biopsy (n ¼ 16) was removed from its medium and frozen at 80 C. To measure CIT concentration and protein expression of AA metabolism, a second batch of biopsies we cultured for 6 (n ¼ 18) or 18 h (n ¼ 12). At the end of the incubation period, culture media and biopsy specimens were removed and immediately frozen at 80 C. Before each further experiment, biopsies were defrosted, any excess medium was removed with Whatman paper, and the tissue was weighed (precision: 0.0001, Mettler-Toledo, Viroflay, France). Given the tiny size of the biopsy specimens, it was impossible to perform all the biological measurements on the same one sample, and so the number of samples per experiment varies from 5 to 18 depending on the analysis. 2.4. Cell viability For each biopsy, viability was checked based on LDH activity levels. LDH activity was measured in each culture medium removed after transport and then at different incubation times (1, 3, 6, 9 and 23 h) and in the biopsy lysate using an enzymatic activity assay kit (SigmaeAldrich). Each biopsy (n ¼ 16) was weighed, lysed in 1 mL of a 1% Triton solution, then centrifuged (15000 g for 10 min at 4 C) to collect the supernatants. For each biopsy specimen, at each measurement timepoint, the ratio of LDH in the medium to total LDH (LDH released in culture medium at 23 h plus the LDH contained in the tissue) was calculated and expressed as percent cell death. 2.5. CIT measurement CIT concentration in the culture media was measured by ion exchange chromatography followed by post-column derivatization with ninhydrin (Hitachi L8800 analyzer, Sciencetec, Villebon-sur-Yvette, France) as previously described [13] or by UPLC-MS (Acquity UPLC H-Class QDa, Waters, Guyancourt, France) using a CORTECS UPLC C18 column after a derivatization step (AccQTag Ultra®kit, Waters). CIT concentration in the culture media was evaluated at 6 h (n ¼ 18; n ¼ 9 with the Hitachi L8800; n ¼ 9 by UPLC) and at 18 h (n ¼ 12; n ¼ 5 with the Hitachi L8800; n ¼ 7 by UPLC) and was ratioed to tissue weight for each biopsy specimen. 2.6. Protein expression Western Blot analysis was performed to verify ARGase II (36 KDa), GLNase II (73 KDa), ornithine aminotransferase (OAT) (49 KDa) and OTC (40 KDa) enzyme expression. Tissues were lysed in a commercial RIPA buffer (50 mM TriseHCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate added with protease inhibitor cocktail (both from SigmaeAldrich) and phosphatase inhibitor cocktail (Fischer Scientific, Illkirch-Graffenstaden, France). Proteins were quantified by the Pierce method using a BCA (bicinchoninic acid assay) kit (Interchim, Montluçon, France). Proteins were then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Protein immunodetection was performed using anti-OTC antibodies (1:1000 dilution; Sigma Aldrich) with anti-ARGase II (1:250 dilution, Sigma Aldrich), anti-GLNase I (1:1000, Abcam, Cambridge, United Kingdom) and anti-OAT (1:200, Sigma Aldrich) as primary antibodies. Membranes were incubated overnight with primary antibodies at 4 C. Primary antibodies were detected with secondary anti-rabbit antibodies conjugated to horseradish peroxidase (1:1000 dilution; Jackson ImmunoResearch, Ely, United Kingdom). Signals were revealed using a chemiluminescence system (Amersham ECL Prime kit, GE Healthcare, France) and quantified using Image J software (version 1.48, National institutes of Health, MD).
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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3. Results 3.1. Clinical patient data A histological analysis of the duodenal mucosa was performed for all patients. No duodenal pathology was found. Clinical patient data was checked and recorded (Table 1). No hemorrhage complication has occurred during the clinical endoscopy. 3.2. Biopsy weight Tissue weight was around 12.36 mg [7.3e27.0]. 3.3. Cell viability Mortality was around 20% at 6 h incubation and increased progressively throughout the incubation period to reach around 50% at 18 h and 70% at 23 h incubation (Fig. 3). 3.4. CIT concentrations CIT release was around 0.215 mM/mg biopsy [0e9] over the 6 h period and 0.300 ± 0.040 mM/mg biopsy [0e7] at 18 h. Table 1 Clinical patient data. Clinical data Age (years) Gender Body Mass Index (BMI) (KG/M2)
Average Range Female Male Average Range
40 19e73 47% 53% 26 18.0e31.1
Fig. 3. Cell viability over 23 h. Biopsy specimens (n ¼ 16) were incubated for 23 h. LDH activity was measured in culture media at several timepoints (at 1 h, 3 h, 6 h, 9 h and 23 h of incubation) and in each biopsy after tissue lysis. Cell mortality percentages were calculated. Individual cell mortality values for each biopsy are given for each incubation time. (- - -) corresponds to the percentage of cell mortality at 6 h (approx. 20%) and 18 h (approx. 50%).
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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3.5. Protein expression ARGase II, GLNase I, OAT, and OTC enzymes were found in the biopsy specimens after 6 h (Fig. 4) and 18 h of incubation. 4. Discussion Here we repurposed a preexisting model of duodenal tissue previously used to study the production of cytokines or other metabolic processes in the gut [10e12] in order to explore AA metabolism in the intestine. The main strength of this model is that it includes not only the epithelial enterocyte barrier but also the lamina propria that contains other tissue elements [14]. It is consequently more representative of real conditions in terms of functionality and metabolism. However, this may make experimental results more difficult to interpret. For example, if the culture medium shows altered release of an AA, then further experiments will be needed to determine the metabolic pathways involved and whether these pathways take place inside or outside the enterocyte. The production of CIT by the biopsy is proof of enterocyte functionality [15]. Indeed, a very small difference in CIT production is observed between 6 and 18 h of incubation, whereas mortality doubled over these two same timepoints, suggesting CIT release is not due to cell mortality but reflects enterocyte functionality. Moreover, the presence of the main enzymes of the CIT metabolism, such as OTC, ARGase II, GLNase I and OAT, confirms enterocyte functionality. Quantifying enzyme activity would bring sharper results but was impossible to do here due to the restrictive size of the biopsies. Another set of experiments measured OTC enzyme activity and found that the enzyme was active and stable in human enterocytes after 6 or 18 h of incubation (personal data). Based on the literature, duodenal biopsies can be cultured over 24e48 h with well-maintained differentiation and metabolic activities [10]. Here, viability was evaluated in order to define the appropriate incubation time for further experimental studies on AA metabolism. Environmental modifications to improve explants viability were not envisaged to stay as close as possible to physiological conditions in order not to influence intestinal amino acid metabolism. Cell mortality was still around 20% up to 6 h incubation and then increased to reach 50% after 18 h and 60%e80% after 23 h. These figures are in the same range as those described for other cell types in primary culture, such as Beta cells [16], and our data is consistent with the rapid turnover of intestinal cell lines (i.e. between 24 and 48 h) [17]. Here we studied two incubation periods: short term (6 h) and long-term (18 h). Neither of these two incubation periods makes it possible to reach the half-life of OTC, which is estimated at between 6 and 9 days [18,19]. Given the speed of intestinal turnover, it is practically impossible to capture the half-specific activity of the enzyme. Indeed, a long half-life time masks the cell's adaptation to a changing environment as stress-induced damage. The advantage of 6 h incubation is mostly the low percent mortality compared to 18 h incubation. However, the longer 18 h incubation time could better serve to find out certain cell adaptations because the period is closer to OTC half-life time found in light hepatocytes. Finally, incubation time should obviously be dictated by the objectives of each study.
Fig. 4. Protein expression after 6 h and 18 h of incubation in tissue. Biopsy specimens (n ¼ 2) were incubated for 6 or 18 h. Arginase II (ARG II), glutaminase I (GLNase I), ornithine aminotransferase (OAT) and ornithine carbamoyltransferase (OTC) protein expression were all verified.
Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002
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Here, a 6 h incubation period looks the most appropriate time window to avoid broad cell death, but other incubation times can be employed and designed to meet other study purposes. Stable CIT production over 6 and 18 h of incubation further supports the use of a 6 h incubation period for studies on AA metabolism. This in vitro explant model of intestinal tissue emerges as a reliable model for conducting further experiments on intestinal AA metabolism. It seems well adapted both for in vitro explorations (i.e. modification of the biopsies environment) and for ex vivo explorations (i.e. patients with a specific treatment or nutritional intervention). Conflict of Interest None of the authors have any conflict of interest to report. Acknowledgments Grenoble University Hospital was promoter of the study. The authors thank the Endoscopy Unit staff at the Hepato-Gastroenterology Department headed by Pr Leroy for their valuable assistance, and in particular Dr Patrick Tuvignon, Dr Pierre-Yves Eyraud, Dr Nicolas Mathieu and Dr Audrey Picot for their participation as investigators in the clinical strand of the study. Statement of authorship S.P., M.Ct., C.B., E.F., and C.M contributed to the Conceptualization. S.P., M.Ct., C.B., F.L., E.F., M.Cr., and C.M., contributed to the methodology. S.P., M.Ct., C.B., C.C., N.M., and F.L., contributed to the investigation. S.P., M.Ct., C.B., and C.M., wrote the original draft. S.P., M.Ct., C.B., C.C., N.M., F.L., E.F., M.Cr., and C.M., contributed to reviewing and editing. S.P., M.Ct., C.B., and C.M., contributed to the supervision. Funding sources This work was supported in part by a grant from the nonprofit association ‘Agir pour les Maladies Chroniques’. References [1] Brosnan JT. Interorgan amino acid transport and its regulation. J Nutr 2003;133(6):2068Se72S. https://doi.org/10.1093/jn/ 133.6.2068S. [2] Breuillard C, Cynober L, Moinard C. Citrulline and nitrogen homeostasis: an overview. Amino Acids 2015;47(4):685e91. https://doi.org/10.1007/s00726-015-1932-2. [3] Wu G. Synthesis of citrulline and arginine from proline in enterocytes of postnatal pigs. Am J Physiol Gastrointest Liver Physiol 1997;272(6):G1382e90. [4] Moinard C, Cynober L. Citrulline: a new player in the control of nitrogen homeostasis. J Nutr 2007;137(6):1621Se5S. https://doi.org/10.1093/jn/137.6.1621S. re V, Lasserre V, Cynober L, et al. Evidence for a role of the ileum in the control of [5] Ventura G, Moinard C, Sinico F, Carrie nitrogen homeostasis via the regulation of arginine metabolism. Br J Nutr 2011;106(2):227e36. https://doi.org/10.1017/ S0007114511000079. re B. Me tabolisme prote ique. Cahiers de Nutrition et de Die t e tique 2005;40(1):53e64. https://doi.org/10. [6] Boirie Y, Beaufre 1016/S0007-9960(05)80467-5. [7] Chougule P, Herlenius G, Hernandez NM, Patil PB, Xu B, Sumitran-Holgersson S. Isolation and characterization of human primary enterocytes from small intestine using a novel method. Scand J Gastroenterol 2012;47(11):1334e43. https://doi. org/10.3109/00365521.2012.708940. [8] Jochems P, Garssen J, van Keulen A, Masereeuw R, Jeurink P. Evaluating human intestinal cell lines for studying dietary protein absorption. Nutrients 2018;10(3):322. https://doi.org/10.3390/nu10030322. [9] Kong S, Zhang YH, Zhang W. Regulation of intestinal epithelial cells properties and functions by amino acids. BioMed Res Int 2018;2018:1e10. https://doi.org/10.1155/2018/2819154. €ffier M, Miralles-Barrachina O, Le Pessot F, Lalaude O, Daveau M, Lavoinne A, et al. Influence of glutamine on cytokine [10] Coe production by human gut in vitro. Cytokine 2001;13(3):148e54. https://doi.org/10.1006/cyto.2000.0813. €ffier M. Modulating effect of glutamine on IL-1b-induced cytokine production by human gut. Clin Nutr 2003;22(4): [11] Coe 407e13. https://doi.org/10.1016/S0261-5614(03)00040-2. [12] Lecleire S, Coeffier M, Leblond J, Hubert A, Lemoulan S, Petit A, et al. Modulation of nitric oxide and cytokines production by l-arginine in human gut mucosa. Clin Nutr 2005;24(3):353e9. https://doi.org/10.1016/j.clnu.2004.11.011.
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Please cite this article as: Pestour S et al., An in vitro explant model for studies of intestinal amino acid metabolism, Clinical Nutrition Experimental, https://doi.org/10.1016/j.yclnex.2019.12.002