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• W2040201181 abstract "Glucagon-like peptide (GLP)-2, secreted by L cells in the small intestine, has anti-inflammatory effects in the gastrointestinal tract. A GLP-2 analogue has been an effective treatment for Crohn disease (CD). G-protein–coupled receptor (GPR) 40 and GPR120 are probably involved in GLP-2 production, the mechanisms of which remain unclear. In our experiments, normal ileal mucosa expressed GPR40, but rarely expressed GPR120. However, both GPRs were overexpressed in the L cells of the inflamed ileal mucosa of CD patients. Mucosal inflammation induced the overexpression of GPR40, GPR120, and several inflammatory cytokines, with correlations between ileal concentrations of tumor necrosis factor (TNF)-α and GPR expression levels; however, inflammation did not induce the expression of proglucagon, a precursor of GLP-2 in CD patients. In rat L cells and GLUTag cells, TNF-α treatment increased GPR120 mRNA expression without affecting GPR40 mRNA expression. Dual agonists of GPR40 and GPR120, GW9508 and linoleic acid, respectively, increased GLP-2 production from L cells, but these agonists decreased it in the presence of TNF-α. The GPR40 antagonist, GW1100, inhibited the GW9508-induced increase in GLP-2 production, and silencing GPR120 resulted in further elevation of GLP-2 production. Thus, GPR120-dependent signaling inhibited the stimulatory effects of GPR40 on GLP-2 expression, and TNF-α treatment decreased GLP-2 expression by up-regulating GPR120 expression in L cells. Glucagon-like peptide (GLP)-2, secreted by L cells in the small intestine, has anti-inflammatory effects in the gastrointestinal tract. A GLP-2 analogue has been an effective treatment for Crohn disease (CD). G-protein–coupled receptor (GPR) 40 and GPR120 are probably involved in GLP-2 production, the mechanisms of which remain unclear. In our experiments, normal ileal mucosa expressed GPR40, but rarely expressed GPR120. However, both GPRs were overexpressed in the L cells of the inflamed ileal mucosa of CD patients. Mucosal inflammation induced the overexpression of GPR40, GPR120, and several inflammatory cytokines, with correlations between ileal concentrations of tumor necrosis factor (TNF)-α and GPR expression levels; however, inflammation did not induce the expression of proglucagon, a precursor of GLP-2 in CD patients. In rat L cells and GLUTag cells, TNF-α treatment increased GPR120 mRNA expression without affecting GPR40 mRNA expression. Dual agonists of GPR40 and GPR120, GW9508 and linoleic acid, respectively, increased GLP-2 production from L cells, but these agonists decreased it in the presence of TNF-α. The GPR40 antagonist, GW1100, inhibited the GW9508-induced increase in GLP-2 production, and silencing GPR120 resulted in further elevation of GLP-2 production. Thus, GPR120-dependent signaling inhibited the stimulatory effects of GPR40 on GLP-2 expression, and TNF-α treatment decreased GLP-2 expression by up-regulating GPR120 expression in L cells. Crohn disease (CD) involves an abnormal immune status, including the enhanced production of inflammatory cytokines, such as ILs and tumor necrosis factor (TNF)-α. Among these cytokines, TNF-α plays a key role in the pathogenesis of CD, and this cytokine is a main target in CD treatment.1Targan S.R. Hanauer S.B. van Deventer S.J.H. Mayer L. Present D.H. Braakman T. DeWoody K.L. Schaible T.F. Rutgeerts P.J. A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn's disease.N Engl J Med. 1997; 337: 1029-1035Crossref PubMed Scopus (3067) Google Scholar, 2Hanauer S.B. Feagan B.G. Lichtenstein G.R. Mayer L.F. Schreiber S. Colombel J.F. Rachmilewitz D. Wolf D.C. Olson A. Bao W.H. Rutgeerts P. ACCENT I Study GroupMaintenance infliximab for Crohn's disease: the ACCENT I randomised trial.Lancet. 2002; 359: 1541-1549Abstract Full Text Full Text PDF PubMed Scopus (3617) Google Scholar Currently, anti–TNF-α agents are widely used in CD patients and have been effective in inducing remission and mucosal healing. However, some CD patients exhibit an insufficient response or tolerance to these agents. Glucagon-like peptides (GLPs), designated GLP-1 and GLP-2, have been recently shown to be involved in metabolic disorders, such as obesity and type 2 diabetes.3van Bloemendaal L. Ten Kulve J.S. la Fleur S.E. Ijzerman R.G. Diamant M. Effects of glucagon-like peptide 1 on appetite and body weight: focus on the CNS.J Endocrinol. 2014; 221: T1-T16Crossref PubMed Scopus (166) Google Scholar GLP-1 is a 30–amino acid peptide that regulates glycemia by stimulating glucose-dependent insulin secretion and inhibiting glucagon release and gastric emptying.4Drucker D.J. The biology of incretin hormones.Cell Metab. 2006; 3: 153-165Abstract Full Text Full Text PDF PubMed Scopus (1690) Google Scholar, 5Plummer M.P. Jones K.L. Annink C.E. Cousins C.E. Meier J.J. Chapman M.J. Horowitz M. Deane A.M. Glucagon-like peptide 1 attenuates the acceleration of gastric emptying induced by hypoglycemia in healthy subjects.Diabetes Care. 2014; 37: 1509-1515Crossref PubMed Scopus (28) Google Scholar, 6Meloni A.R. DeYoung M.B. Lowe C. Parkes D.G. GLP-1 receptor activated insulin secretion from pancreatic β-cells: mechanism and glucose dependence.Diabetes Obes Metab. 2013; 15: 15-27Crossref PubMed Scopus (214) Google Scholar In contrast, GLP-2 is a highly conserved 33–amino acid peptide that regulates gastric motility, gastric acid secretion, and intestinal hexose transport.7Drucker D.J. Glucagon-like peptide 2.J Clin Endocrinol Metab. 2001; 86: 1759-1764Crossref PubMed Scopus (93) Google Scholar, 8Guan X. Shi X. Li X. Chang B. Wang Y. Li D. Chan L. GLP-2 receptor in POMC neurons suppresses feeding behavior and gastric motility.Am J Physiol Endocrinol Metab. 2012; 303: E853-E864Crossref PubMed Scopus (46) Google Scholar, 9Meier J.J. Nauck M.A. Pott A. Heinze K. Goetze O. Bulut K. Schmidt W.E. Gallwitz B. Holst J.J. Glucagon-like peptide 2 stimulates glucagon secretion, enhances lipid absorption, and inhibits gastric acid secretion in humans.Gastroenterology. 2006; 130: 44-54Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar In addition to these effects, GLP-2 has anti-inflammatory effects in the gastrointestinal tract. Experimental studies have demonstrated that GLP-2 reduces mucosal injury and cytokine expression in several models of colitis.10Boushey R.P. Yusta B. Drucker D.J. Glucagon-like peptide 2 decreases mortality and reduces the severity of indomethacin-induced murine enteritis.Am J Physiol Endocrinol Metab. 1999; 277: E937-E947Google Scholar, 11Drucker D.J. Yusta B. Boushey R.P. DeForest L. Brubaker P.L. Human Gly(2) GLP-2 reduces the severity of colonic injury in a murine model of experimental colitis.Am J Physiol Gastrointest Liver Physiol. 1999; 276: G79-G91Google Scholar, 12L'Heureux M.C. Brubaker P.L. Glucagon-like peptide-2 and common therapeutics in a murine model of ulcerative colitis.J Pharmacol Exp Ther. 2003; 306: 347-354Crossref PubMed Scopus (67) Google Scholar, 13Alavi K. Schwartz M.Z. Palazzo J.P. Prasad R. Treatment of inflammatory bowel disease in a rodent model with the intestinal growth factor glucagon-like peptide-2.J Pediatr. 2000; 35: 847-851Scopus (92) Google Scholar, 14Arthur G.L. Schwartz M.Z. Kuenzler K.A. Birbe R. Glucagonlike peptide-2 analogue: a possible new approach in the management of inflammatory bowel disease.J Pediatr Surg. 2004; 39: 448-452Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Furthermore, the effects of GLP-2 on the gastrointestinal tract have been confirmed by several clinical trials. Teduglutide, a GLP-2 analogue, increases mucosal healing and remission rates in patients with active moderate to severe CD.15Buchman A.L. Katz S. Fang J.C. Bernstein C.N. Abou-Assi S.G. Teduglutide Study GroupTeduglutide, a novel mucosally active analog of glucagon-like peptide-2 (GLP-2) for the treatment of moderate to severe Crohn's disease.Inflamm Bowel Dis. 2010; 16: 962-973Crossref PubMed Scopus (103) Google Scholar This drug also improves intestinal functions by increasing villus height in patients with short bowel syndrome.16Jeppesen P.B. Pertkiewicz M. Messing B. Iyer K. Seidner D.L. O'Keefe S.J.D. Forbes A. Heinze H. Joelsson B. Teduglutide reduces need for parenteral support among patients with short bowel syndrome with intestinal failure.Gastroenterology. 2012; 143: 1473-1481Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 17Jeppesen P.B. Gilroy R. Pertkiewicz M. Allard J.P. Messing B. O'Keefe S.J. Randomised placebo-controlled trial of teduglutide in reducing parenteral nutrition and/or intravenous fluid requirements in patients with short bowel syndrome.Gut. 2011; 60: 902-914Crossref PubMed Scopus (294) Google Scholar Thus, drugs affecting GLP-2 expression and GLP-2 itself and its analogue could be effective novel therapies against CD. GLP-2 and GLP-1 are produced by the unique enteroendocrine L cells in the distal small intestine and colon. Both GLP-1 and GLP-2 are simultaneously produced via post-transcriptional processing of proglucagon by prohormone convertase 1/3 (PC 1/3) in L cells.18Dhanvantari S. Seidah N.G. Brubaker P.L. Role of prohormone convertases in the tissue-specific processing of proglucagon.Mol Endocrinol. 1996; 10: 342-355Crossref PubMed Scopus (197) Google Scholar, 19Rouille Y. Martin S. Steiner D.F. Differential processing of proglucagon by the subtilisin-like prohormone convertases PC2 and PC3 to generate either glucagon or glucagon-like peptide.J Biol Chem. 1995; 270: 26488-26496Crossref PubMed Scopus (173) Google Scholar, 20Drozdowski L. Thomson A.B.R. Intestinal hormones and growth factors: effects on the small intestine.World J Gastroenterol. 2009; 15: 385-406Crossref PubMed Scopus (45) Google Scholar Although mechanisms of GLP-1 secretion are well established, those of GLP-2 remain unclear, and recent studies have suggested that different mechanisms may exist for GLP-1 and GLP-2 secretion.21Izumi H. Ishizuka S. Inafune A. Hira T. Ozawa K. Shimizu T. Takase M. Hara H. alpha-Lactalbumin hydrolysate stimulates glucagon-like peptide-2 secretion and small intestinal growth in suckling rats.J Nutr. 2009; 139: 1322-1327Crossref PubMed Scopus (21) Google Scholar, 22Ipharraguerre I.R. Tedo G. Menoyo D. Cabero N.D. Holst J.J. Nofrarias M. Mereu A. Burrin D.G. Bile acids induce glucagon-like peptide 2 secretion with limited effects on intestinal adaptation in early weaned pigs.J Nutr. 2013; 143: 1899-1905Crossref PubMed Scopus (19) Google Scholar G-protein–coupled receptors (GPRs) are important signaling molecules for many aspects of cellular function and are members of a large receptor family that share common structural motifs, such as seven transmembrane helices, and the ability to activate heterotrimeric G proteins, such as Gs, Gi, and Gq. Ligands bind specifically to GPRs to stimulate a variety of cellular responses via second messengers (eg, cAMP, Ca2+, inositol 1,4,5-triphosphate, and diacyl glycerol).23Ulloa-Aguirre A. Stanislaus D. Janovick J.A. Conn P.M. Structure-activity relationships of G protein-coupled receptors.Arch Med Res. 1999; 30: 420-435Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Gether U. Uncovering molecular mechanisms involved in activation of G protein-coupled receptors.Endocr Rev. 2000; 21: 90-113Crossref PubMed Scopus (1002) Google Scholar The activation of Gs proteins results in an increase in the cellular concentration of cAMP, leading to phosphorylation of protein kinase (PK) A, whereas the activation of Gi proteins results in a decrease in the cellular concentration of cAMP. GPRs that are coupled to Gq proteins use diacyl glycerol as a second messenger. Several GPRs, such as GPR40, GPR41, GPR43, and GPR120, have been reported to function as receptors for free fatty acids (FFAs).25Nilsson N.E. Kotarsky K. Owman C. Olde B. Identification of a free fatty acid receptor, FFA(2)R, expressed on leukocytes and activated by short-chain fatty acids.Biochem Biophys Res Commun. 2003; 303: 1047-1052Crossref PubMed Scopus (411) Google Scholar, 26Brown A.J. Goldsworthy S.M. Barnes A.A. Eilert M.M. Tcheang L. Daniels D. Muir A.I. Wigglesworth M.J. Kinghorn I. Fraser N.J. Pike N.B. Strum J.C. Steplewski K.M. Murdock P.R. Holder J.C. Marshall F.H. Szekeres P.G. Wilson S. Ignar D.M. Foord S.M. Wise A. Dowell S.J. The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids.J Biol Chem. 2003; 278: 11312-11319Crossref PubMed Scopus (1580) Google Scholar, 27Le Poul E. Loison C. Struyf S. Springael J.Y. Lannoy V. Decobecq M.E. Brezillon S. Dupriez V. Vassart G. Van Damme J. Parmentier M. Detheux M. Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation.J Biol Chem. 2003; 278: 25481-25489Crossref PubMed Scopus (1101) Google Scholar, 28Edfalk S. Steneberg P. Edlund H. Gpr40 is expressed in enteroendocrine cells and mediates free fatty acid stimulation of incretin secretion.Diabetes. 2008; 57: 2280-2287Crossref PubMed Scopus (469) Google Scholar, 29Habib A.M. Richards P. Rogers G.J. Reimann F. Gribble F.M. Co-localisation and secretion of glucagon-like peptide 1 and peptide YY from primary cultured human L cells.Diabetologia. 2013; 56: 1413-1416Crossref PubMed Scopus (137) Google Scholar, 30Hirasawa A. Tsumaya K. Awaji T. Katsuma S. Adachi T. Yamada M. Sugimoto Y. Miyazaki S. Tsujimoto G. Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120.Nat Med. 2005; 11: 90-94Crossref PubMed Scopus (1148) Google Scholar Some studies have demonstrated that L cells express both GPR40 and GPR120 and secrete GLPs when activated by medium- and long-chain FFAs, including linoleic acid (LA), suggesting that FFAs are ligands for GPR40 and GPR120 on L cells.28Edfalk S. Steneberg P. Edlund H. Gpr40 is expressed in enteroendocrine cells and mediates free fatty acid stimulation of incretin secretion.Diabetes. 2008; 57: 2280-2287Crossref PubMed Scopus (469) Google Scholar, 29Habib A.M. Richards P. Rogers G.J. Reimann F. Gribble F.M. Co-localisation and secretion of glucagon-like peptide 1 and peptide YY from primary cultured human L cells.Diabetologia. 2013; 56: 1413-1416Crossref PubMed Scopus (137) Google Scholar, 30Hirasawa A. Tsumaya K. Awaji T. Katsuma S. Adachi T. Yamada M. Sugimoto Y. Miyazaki S. Tsujimoto G. Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120.Nat Med. 2005; 11: 90-94Crossref PubMed Scopus (1148) Google Scholar However, the mechanisms of regulation of GLP-2 production by these ligands and the interactions between GPR40 and GPR120 are currently unclear. Therefore, we evaluated the expression of GPR40 and GPR120 in the ileal mucosa of patients with CD and correlated the expression of these GPRs with several inflammatory parameters. We also studied the mechanisms underlying GLP-2 production using rat primary L cells and mouse GLUTag L cells. During the 6-year period between 2004 and 2010 at Osaka City University Hospital (Osaka, Japan), we enrolled the 16 consecutive CD patients who underwent an ileocecal resection and had small-bowel pathological conditions, such as ulceration, edema, and redness in the distal ileum. The patient cohort consisted of 13 men and 3 women, with a median age of 36 years (range, 22 to 47 years). Some patients had been previously treated with TNF-α antibody therapy2Hanauer S.B. Feagan B.G. Lichtenstein G.R. Mayer L.F. Schreiber S. Colombel J.F. Rachmilewitz D. Wolf D.C. Olson A. Bao W.H. Rutgeerts P. ACCENT I Study GroupMaintenance infliximab for Crohn's disease: the ACCENT I randomised trial.Lancet. 2002; 359: 1541-1549Abstract Full Text Full Text PDF PubMed Scopus (3617) Google Scholar (4 of 16 patients) or an immune modulator (2 of 16 patients), but no patients were previously treated with corticosteroids. Normal ileal mucosa samples were obtained from patients with colon cancer who had undergone a colectomy (n = 15). The normal patient cohort consisted of seven men and eight women with a median age of 64 years (range, 34 to 83 years). Western blot analysis, real-time RT-PCR, and enzyme immunoassay (EIA) were performed on inflamed and normal mucosa samples. Our investigation was conducted according to the Declaration of Helsinki principles. All of the patients provided informed consent for the use of their ileum as a human sample. The ethics committee of Osaka City University Hospital approved our study (protocol number 2286). We used the rat primary L cells and the mouse GLUTag L cells.31Hira T. Mochida T. Miyashita K. Hara H. GLP-1 secretion is enhanced directly in the ileum but indirectly in the duodenum by a newly identified potent stimulator, zein hydrolysate, in rats.Am J Physiol Gastrointest Liver Physiol. 2009; 297: G663-G671Crossref PubMed Scopus (84) Google Scholar Rat primary L cells were purified from the ileum of 8-week-old male Sprague-Dawley rats for in vitro studies with a medium kit (Reprocell Inc., Yokohama, Japan). As a preliminary study, we characterized these cells by flow cytometric analysis. These cells expressed GPR40, GPR120, G-protein–coupled bile acid receptor Gpbar1 (TGR5), and proglucagon, confirming that these GLP-producing cells were L cells (Supplemental Figure S1).28Edfalk S. Steneberg P. Edlund H. Gpr40 is expressed in enteroendocrine cells and mediates free fatty acid stimulation of incretin secretion.Diabetes. 2008; 57: 2280-2287Crossref PubMed Scopus (469) Google Scholar, 30Hirasawa A. Tsumaya K. Awaji T. Katsuma S. Adachi T. Yamada M. Sugimoto Y. Miyazaki S. Tsujimoto G. Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120.Nat Med. 2005; 11: 90-94Crossref PubMed Scopus (1148) Google Scholar, 32Katsuma S. Hirasawa A. Tsujimoto G. Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1.Biochem Biophys Res Commun. 2005; 329: 386-390Crossref PubMed Scopus (568) Google Scholar Mouse GLUTag L cells were kindly provided by Tohru Hira (University of Hokkaido, Hokkaido, Japan) and Noriyasu Hirasawa (University of Tohoku, Tohoku, Japan) and used with the permission of Daniel J. Drucker (University of Toronto, Toronto, ON, Canada). GLUTag cells were cultured in Dulbecco's modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) (Hyclone; Thermo Fisher Scientific Inc., Waltham, MA) and 1% penicillin/streptomycin (Gibco by Life Technologies, Carlsbad, CA). Recombinant rat IL-1β and IL-6 (both from R&D Systems Inc., Minneapolis, MN) and recombinant rat and mouse TNF-α (Shenandoah Biotechnology Inc., Warwick, PA, and Life Technologies, respectively) were used as proinflammatory cytokines. GW9508 (Cayman Chemical Company, Ann Arbor, MI) and LA (MP Biomedicals, Santa Ana, CA), which are agonists for both GPR40 and GPR120, and GW1100 (Haoyuan Chemexpress Co, Ltd, Shanghai, China), a specific antagonist for GPR40,33Briscoe C.P. Peat A.J. McKeown S.C. Corbett D.F. Goetz A.S. Littleton T.R. McCoy D.C. Kenakin T.P. Andrews J.L. Ammala C. Fornwald J.A. Ignar D.M. Jenkinson S. Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules.Br J Pharmacol. 2006; 148: 619-628Crossref PubMed Scopus (350) Google Scholar were used to evaluate the involvement of GPRs in GLP-2 production. U73122 and H89, which are inhibitors of phospholipase C (PLC) and protein kinase A (PKA), respectively, were purchased from Millipore (Billerica, MA). GF109203X, an inhibitor of PKC, and cholera toxin (CTX) were supplied by Wako Pure Chemical Industries, Ltd (Osaka, Japan). Go6983, an inhibitor of PKC, and forskolin were obtained from Sigma Chemical (St. Louis, MO). All other reagents were of the highest purity commercially available. Rat L cells (1 × 106 cells per well) and GLUTag L cells (5 × 105 cells per well) were cultured for 24 hours with or without proinflammatory cytokines in HEPES buffer (140 mmol/L NaCl, 4.5 mmol/L KCl, 20 mmol/L HEPES, 1.2 mmol/L CaCl2, 1.2 mmol/L MgCl2, and 0.1% bovine serum albumin, pH 7.4). After washing, the cells were incubated for 1.5 hours to assess GLP-2 production in the absence or presence of agonists and antagonists of GPR40 and GPR120 and then centrifuged (200 × g for 4 minutes at room temperature). In some experiments, the L cells were treated with agonists of both GPR40 and GPR120 and incubated with several agents that interact with G-protein–dependent signaling pathways with or without TNF-α. The supernatant and pellet were collected for GLP-2 EIA and extraction of mRNA, respectively. Western blot analysis was performed using antibodies that recognize GPR40 (Abnova Corp., Taipei, Taiwan) and GPR120 (Abcam Plc, Cambridge, UK), GLP-2 and proglucagon (both from Santa Cruz Biotechnology Inc., Dallas, TX), and phospho-PKA (Cell Signaling Technology, Danvers, MA). Briefly, 20 μg of protein per well was loaded onto a gel for Western blot analysis. SDS-PAGE was performed using SuperSep Ace agarose gels (Wako Pure Chemical Industries, Ltd), according to molecular weight of each protein. After SDS-PAGE, proteins were transferred to a polyvinylidene difluoride membrane, which was then blocked using 5% skim milk and incubated with a primary antibody recognizing GPR40, GPR120, GLP-2, proglucagon, or phospho-PKA. Finally, membranes were incubated with horseradish peroxidase–conjugated anti-IgG (Amersham ECL Plus Western blotting reagent pack; GE Healthcare, Buckinghamshire, UK). Proteins were detected using ECL Plus detection reagent (GE Healthcare) and imaged using a LAS-3000 analyzer (Fujifilm, Tokyo, Japan). Levels of TNF-α, IL-1β, and IL-6 in the ileal mucosa and levels of GLP-2 in cell culture supernatants were determined using an EIA kit [Human TNF-α ELISA Kit (Thermo Fisher Scientific Inc.), Human IL-1β and IL-6 ELISA kits (R&D Systems Inc.), and rat and mouse GLP-2 EIA (Yanaihara Institute Inc., Shizuoka, Japan)]. Cytokine concentrations were displayed as the amount per 100 μg of protein. GLP-2 levels were displayed as percentages of the control. Total mRNA from human samples and L cells was extracted using the RNAqueous and RNAqueous micro kits (Life Technologies), respectively. Real-time RT-PCR (TaqMan; Life Technologies) products were examined using the Fast7500 System (Applied Biosystems by Life Technologies, Carlsbad, CA). A One Step PrimeScript RT-PCR kit (TAKARA Bio Inc., Shiga, Japan), along with primers and probes (Applied Biosystems by Life Technologies), was used to examine GPR40, GPR120, proglucagon, and PC 1/3 mRNA expression. Ileal specimens were fixed in periodate-lysine-paraformaldehyde, embedded in optimal cutting temperature compound (Tissue-Tek; Sakura Fine Technical Co, Tokyo, Japan), cut into sections (4 μm thick), and stained using an immunostaining method. Briefly, ileal sections were blocked with 5% normal donkey serum (Sigma-Aldrich, St. Louis, MO) in phosphate-buffered saline. After washing, those sections were incubated with goat anti-human GPR40, rabbit anti-human GPR120, or goat anti-human proglucagon primary antibodies (Santa Cruz Biotechnology Inc.), which were diluted in Antibody Diluent with Background Reducing Components (Dako, Glostrup, Denmark). The sections were incubated with Alexa Fluor 488 donkey anti-rabbit IgG or Alexa Fluor 594 donkey anti-goat IgG (Molecular Probes, Life Technologies) after washing. Finally, DAPI Fluoromount-G (Southern Biotech, Birmingham, AL) was added to the sections. We then evaluated the sections using a fluorescent microscope. Silencemag (Oz Bioscience, Marseille, France) as well as negative control and GPR120 siRNAs (both from Ambion, Silencer Select Pre-designed siRNA; Life Technologies) were used as transfection reagents. L cells were cultured for 3 days in DMEM containing 10% FBS and 1% penicillin/streptomycin before transfection. Silencemag reagent was mixed with each siRNA and incubated for 20 minutes at 37°C. The complexes were added to primary L cells in only DMEM for 15 minutes on a magnetic plate. Finally, DMEM containing 10% FBS and 1% penicillin/streptomycin was added to the cells, and the cells were cultured for 3 days. The final concentration of siRNA was 20 to 50 nmol/L. The results are expressed as means ± SEM. Significant differences between means were analyzed using Student's t-test and one- or two-way analysis of variance, followed by Tukey's post hoc test using StatView 5.0 (Hulinks Inc., Tokyo, Japan). Significant correlations were also analyzed using StatView. Statistical significance was set at P < 0.05. GPR40 and GPR120 mRNA and protein expression levels were detected in the normal ileal mucosa from control patients (Figure 1, A and B). The expression levels of both GPR40 and GPR120 were significantly increased in the inflamed mucosa from CD patients, compared with those from control patients, at both the protein and mRNA levels, and inflammation had a much stronger effect on GPR120 expression than GPR40 expression (Figure 1, A and B). We also observed a significant correlation between GPR40 and GPR120 expression (r = 0.699, P = 0.0018) (Figure 1C). By IHC, intestinal epithelial cells expressed GPR40, but they rarely expressed GPR120, in the normal ileal mucosa. Both GPR40 and GPR120 proteins were overexpressed in the inflamed ileal mucosa from CD patients. Double staining with GPR40 and GPR120 antibodies showed that these two GPRs were co-expressed on spindle-shaped cells in the intestinal glands (Figure 1D), which were L cells.34Bohorquez D.V. Chandra R. Samsa L.A. Vigna S.R. Liddle R.A. Characterization of basal pseudopod-like processes in ileal and colonic PYY cells.J Mol Histol. 2011; 42: 3-13Crossref PubMed Scopus (58) Google Scholar Furthermore, we determined the correlation between GPR expression and clinical CD activity, known as the Harvey-Bradshaw Index (HBI). HBI values significantly correlated with GPR120 expression (r = 0.57, P = 0.02), but not that of GPR40 (r = 0.432, P = 0.096) (Figure 1E). Next, we determined the levels of proinflammatory cytokines in the ileal mucosa and their correlations with GPR expressions. TNF-α levels in CD patients were significantly greater than those in control patients (Figure 2A). The levels of IL-1β and IL-6 in CD patients tended to be higher than those in control patients, but the differences were not statistically significant. We analyzed the correlations between GPR40 or GPR120 expression and proinflammatory cytokine production in the ileal mucosa of CD patients (Figure 2, B and C). Both GPR40 and GPR120 expression levels significantly correlated with levels of TNF-α [r = 0.832 (P < 0.0001) and r = 0.718 (P = 0.001), respectively], but not those of IL-1β (r = −0.123 and r = −0.09, respectively) or IL-6 (r = −0.144 and r = −0.259, respectively). We tried to detect GLP-2 protein in the ileal mucosa by Western blot analysis. However, because GLP-2 was rapidly degraded by dipeptidyl peptidase IV, which was in the epithelial layer during intestinal tissue sampling and protein preparation, this could not be done. Therefore, we examined the expression of proglucagon, a precursor of GLP-2, in the ileal mucosa. Proglucagon protein expression in the ileal mucosa of CD patients was similar to that of control patients (Figure 3A). The expression levels of proglucagon and PC 1/3 mRNAs in the ileal mucosa of CD patients also did not differ from those of the control patients (Figure 3B). IHC staining showed that proglucagon was expressed on spindle-like cells in the epithelial glands, suggesting that L cells produced proglucagon (Figure 3C). TNF-α at a dose of 30 ng/mL significantly increased the expression of GPR120 mRNA in L cells, but it did not affect that of GPR40 mRNA, even at a 300 ng/mL dose (Figure 4, A and B). Neither IL-1β nor IL-6 affected GPR40 and GPR120 mRNA expression levels. GW9508, at a dose of 1 μmol/L, significantly induced GLP-2 production from L cells. However, when combined with TNF-α treatment, GW9508 inhibited GLP-2 production in a dose-dependent manner (Figure 5A). Similarly, LA, another GPR40 and GPR120 agonist,33Briscoe C.P. Peat A.J. McKeown S.C. Corbett D.F. Goetz A.S. Littleton T.R. McCoy D.C. Kenakin T.P. Andrews J.L. Ammala C. Fornwald J.A. Ignar D.M. Jenkinson S. Pharmacological regulation of insulin secretion in MIN6 cells through the fatty acid receptor GPR40: identification of agonist and antagonist small molecules.Br J Pharmacol. 2006; 148: 619-628Crossref PubMed Scopus (350) Google Scholar increased GLP-2 production in L cells when used alone, but inhibited GLP-2 production in the presence of TNF-α (Figure 5B). Both GW9508 and LA increased proglucagon mRNA expression in L cells alone, whereas these agonists decreased that expression in the presence of TNF-α (Figure 5, C and D). By using GLUTag cells, we assessed the reproducibility of TNF-α treatment on GPR expression and GLP-2 production. TNF-α at doses of 30 and 100 ng/mL significantly increased GPR120 mRNA expression in GLUTag cells, whereas TNF-α did not affect GPR40 mRNA expression (Figure 6A). GW9508 induced GLP-2 production from GLUTag cells in the absence of TNF-α in a dose-dependent manner. However, in the presence of TNF-α, GW9508 inhibited GLP-2 production in a dose-dependent manner (Figure 6B). GW1100, a specific antagonist of GPR40, significantly inhibited the GW9508-induced increase in GLP-2 production in a dose-dependent manner (Figure 7A). This inhibitory effect of GW1100 was stronger when combined with TNF-α treatment, with inhibitions of GW9508-induced GLP-2 production of 51% and 45% with and without TNF-α, respectively (Figure 7B). GPR120 silencing did not affect GLP-2 production in rat L cells treated with GW9508, whereas it markedly increased GLP-2 produ" @default.
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