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• W2000864811 abstract "Background & Aims: Butyrate oxidation is impaired in intestinal mucosa of patients with inflammatory bowel diseases (IBD). Butyrate uptake by colonocytes involves the monocarboxylate transporter (MCT) 1. We aimed to investigate the role of MCT1 in butyrate oxidation deficiency during colonic inflammation. Methods: Colonic tissues were collected from patients with IBD or healthy controls and from rats with dextran sulfate sodium (DSS)-induced colitis. The intestinal epithelial cell line HT-29 was treated with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). MCT1 expression was analyzed by real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence. Butyrate uptake and oxidation in HT-29 cells was assessed using [14C]-butyrate. The mechanism of MCT1 gene regulation was analyzed by nuclear run-on and reporter gene assays. Results: MCT1 messenger RNA (mRNA) and protein levels were markedly decreased in inflamed colonic mucosa of IBD patients and rats. In HT-29 cells, down-regulation of MCT1 mRNA and protein abundance by IFN-γ and TNF-α correlated with a decrease in butyrate uptake and subsequent oxidation. IFN-γ and TNF-α did not affect MCT1 mRNA stability but rather down-regulated gene transcription. We demonstrate that the cytokine response element is located in the proximal −111/+213 core region of the MCT1 promoter. Conclusions: The data suggest that butyrate oxidation deficiency in intestinal inflammation is a consequence of reduction in MCT1-mediated butyrate uptake. This reinforces the proposition that butyrate oxidation deficiency in IBD is not a primary defect. Background & Aims: Butyrate oxidation is impaired in intestinal mucosa of patients with inflammatory bowel diseases (IBD). Butyrate uptake by colonocytes involves the monocarboxylate transporter (MCT) 1. We aimed to investigate the role of MCT1 in butyrate oxidation deficiency during colonic inflammation. Methods: Colonic tissues were collected from patients with IBD or healthy controls and from rats with dextran sulfate sodium (DSS)-induced colitis. The intestinal epithelial cell line HT-29 was treated with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). MCT1 expression was analyzed by real-time reverse-transcription polymerase chain reaction, Western blot, and immunofluorescence. Butyrate uptake and oxidation in HT-29 cells was assessed using [14C]-butyrate. The mechanism of MCT1 gene regulation was analyzed by nuclear run-on and reporter gene assays. Results: MCT1 messenger RNA (mRNA) and protein levels were markedly decreased in inflamed colonic mucosa of IBD patients and rats. In HT-29 cells, down-regulation of MCT1 mRNA and protein abundance by IFN-γ and TNF-α correlated with a decrease in butyrate uptake and subsequent oxidation. IFN-γ and TNF-α did not affect MCT1 mRNA stability but rather down-regulated gene transcription. We demonstrate that the cytokine response element is located in the proximal −111/+213 core region of the MCT1 promoter. Conclusions: The data suggest that butyrate oxidation deficiency in intestinal inflammation is a consequence of reduction in MCT1-mediated butyrate uptake. This reinforces the proposition that butyrate oxidation deficiency in IBD is not a primary defect. Butyrate is a short-chain fatty acid produced by colonic bacterial fermentation of dietary fibers. Butyrate plays an important part in maintaining the health and integrity of the colonic mucosa. It is the primary energy source for the colonic epithelium1Roediger W.E.W. Utilization of nutrients by isolated epithelial cells of the rat colon.Gastroenterology. 1982; 83: 424-429Abstract Full Text PDF PubMed Scopus (891) Google Scholar and regulates cell proliferation,2Siavoshian S. Segain J.P. Kornprobst M. et al.Butyrate and trichostatin A effects on the proliferation/differentiation of human intestinal epithelial cells: induction of cyclin D3 and p21 expression.Gut. 2000; 46: 507-514Crossref PubMed Scopus (243) Google Scholar differentiation, and apoptosis.3Heerdt B.G. Houston M.A. Augenlicht L.H. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines.Cancer Res. 1994; 54: 3288-3293PubMed Google Scholar In addition, butyrate has an important role in modulating mucosal inflammation.4Inan M.S. Rasoulpour R.J. Yin L. et al.The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line.Gastroenterology. 2000; 118: 724-734Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 5Segain J.P. Raingeard de la Blétière D. Bourreille A. et al.Butyrate inhibits inflammatory responses through NF-κB inhibition: implications for Crohn’s disease.Gut. 2000; 47: 397-403Crossref PubMed Scopus (952) Google Scholar Ulcerative colitis (UC) and Crohn’s disease (CD) are the 2 major inflammatory bowel diseases (IBD). Several studies have found that butyrate oxidation is decreased in the inflamed mucosa of patients suffering from UC6Roediger W.E.W. The colonic epithelium in ulcerative colitis: an energy deficiency disease.Lancet. 1980; 2: 712-715Abstract PubMed Scopus (563) Google Scholar, 7Den Hond E. Hiele M. Evenepoel P. et al.In vivo butyrate metabolism and colonic permeability in extensive ulcerative colitis.Gastroenterology. 1998; 115: 584-590Abstract Full Text Full Text PDF PubMed Google Scholar or CD8Duffy M.M. Regan M.C. Ravichandran P. et al.Mucosal metabolism in ulcerative colitis and Crohn’s disease.Dis Colon Rectum. 1998; 41: 1399-1405Crossref PubMed Scopus (28) Google Scholar and in animal models of experimental colitis.9Ahmad M.S. Krishnan S. Ramakrishna B.S. et al.Butyrate and glucose metabolism by colonocytes in experimental colitis in mice.Gut. 2000; 46: 493-499Crossref PubMed Scopus (135) Google Scholar However, these studies have suggested that butyrate oxidation deficiency is not a primary defect. Indeed, butyrate oxidation is impaired in patients with active UC but not with quiescent UC.7Den Hond E. Hiele M. Evenepoel P. et al.In vivo butyrate metabolism and colonic permeability in extensive ulcerative colitis.Gastroenterology. 1998; 115: 584-590Abstract Full Text Full Text PDF PubMed Google Scholar Also, in mice with dextran sulfate sodium (DSS)-induced colitis, butyrate oxidation was impaired only after 6 days of DSS treatment.9Ahmad M.S. Krishnan S. Ramakrishna B.S. et al.Butyrate and glucose metabolism by colonocytes in experimental colitis in mice.Gut. 2000; 46: 493-499Crossref PubMed Scopus (135) Google Scholar These studies did not consider a potential reduction in butyrate uptake by intestinal epithelial cells as a causative factor of this butyrate oxidation deficiency. The human monocarboxylate transporter 1 (MCT1) gene encodes for a plasma membrane protein of 45 kilodaltons containing 12 α-helical transmembrane domains with C- and N-termini located within the cytoplasm.10Halestrap A.P. Price N.T. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation.Biochem J. 1999; 15: 281-299Crossref Scopus (1093) Google Scholar We and others have demonstrated that MCT1 transports butyrate across the apical membrane of human colonocytes.11Ritzhaupt A. Wood I.S. Ellis A. et al.Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate.J Physiol. 1998; 513: 719-732Crossref PubMed Scopus (191) Google Scholar, 12Hadjiagapiou C. Schmidt L. Dudeja P.K. et al.Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1.Am J Physiol. 2000; 279: G775-G780PubMed Google Scholar Thus, a decrease in MCT1 expression, which reduces the intracellular availability of butyrate,13Cuff M. Dyer J. Jones M. et al.The human colonic monocarboxylate transporter isoform 1: its potential importance to colonic tissue homeostasis.Gastroenterology. 2005; 128: 676-686Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar could affect not only its oxidation but also its cell regulatory effects. Indeed, we have shown that silencing MCT1 expression by RNA interference in colonic epithelial cells decreases butyrate induction of cell-cycle arrest and differentiation.13Cuff M. Dyer J. Jones M. et al.The human colonic monocarboxylate transporter isoform 1: its potential importance to colonic tissue homeostasis.Gastroenterology. 2005; 128: 676-686Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar This finding suggests that a decrease in MCT1 expression could negatively affect colonic tissue homeostasis. In human colonic tissues, during transition from normality to malignancy, decreased MCT1 expression has been reported.14Lambert D.W. Wood I.S. Ellis A. et al.Molecular changes in the expression of human colonic nutrient transporters during the transition from normality to malignancy.Br J Cancer. 2002; 86: 1262-1269Crossref PubMed Scopus (116) Google Scholar However, very little is known about MCT1 expression in IBD. We hypothesize that the impairment in butyrate oxidation reported in active IBD could be related to a decrease in MCT1 expression in the inflamed colonic mucosa. We observed that inflammation caused down-regulation of MCT1 expression in the colonic tissue. Furthermore, treatment of intestinal epithelial cell lines with proinflammatory cytokines induced down-regulation of MCT1 expression that was associated with a reduction in butyrate uptake and subsequent oxidation. Therefore, butyrate oxidation deficiency in intestinal inflammation appears to be a consequence of reduction in MCT1-mediated butyrate uptake and sustains the idea that butyrate oxidation deficiency in IBD is not a primary defect. Colonic biopsy specimens were obtained from inflamed and noninflamed mucosa of 14 patients with CD (9 women, 5 men; mean age, 36 years; range, 21–78 years) and 9 patients with UC (4 women, 5 men; mean age, 46 years; range, 30–62 years). All patients underwent colonoscopy for an active disease. At the time of the study, 9 patients were receiving steroids; 2 patients, 5-aminosalicylic acid; 2 patients, azathioprine; 1 patient, 6-mercaptopurin plus infliximab; 1 patient, cyclosporin; and 8 patients, no medication. Colonic biopsy specimens were collected from healthy mucosa of 10 asymptomatic subjects undergoing routine colonoscopy. The study was approved by the “Fédération des Biothèques” of the University Hospital, Nantes. All patients gave informed consent to take part in the study. Principles of laboratory animal care and guidelines according to the Declaration of Helsinki were followed. Sprague-Dawley male rats were treated with 4% DSS in drinking water or with water alone (control) for 5 days. Rats were then killed and colonic segments removed for RNA or protein isolation. The parental intestinal epithelial cell line HT-29 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 μg/mL streptomycin, 100 IU/mL penicillin, and 2 mmol/L glutamine. Cells were treated with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) at the indicated time and doses. Cell viability was assessed using the [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) viability assay (Promega, Charbonnieres, France). To study the effect of IFN-γ and TNF-α on the stability of the MCT1 transcript, cells were stimulated in the presence of 1 μg/mL actinomycin D (Sigma) for 2, 4, 6, 8, and 24 hours. In other experiments, cells were preincubated for 1 hour with 10 μmol/L of the nuclear factor (NF)-κB inhibitor caffeic acid phenylethyl ester (CAPE) (Calbiochem, Nottingham, UK) before treatment with cytokines as described above. pGL3-basic luciferase reporter plasmids containing successive deletions of the 5′-end of the MCT1 promoter (−1525, −1319, −1106, −896, −703, −476, −307, −111) were used to assess promoter activity, as previously described.15Cuff M.A. Shirazi-Beechey S.P. The human monocarboxylate transporter, MCT1: genomic organization and promoter analysis.Biochem Biophys Res Comm. 2002; 292: 1048-1056Crossref PubMed Scopus (40) Google Scholar HT-29 cells were transfected in 96-well plates with 250 ng pGL3-MCT1 plasmid construct or pNFκB-Luc vector (Clontech, Saint-Germain-en-Laye, France) and 100 ng pIRES-EGFP vector (internal control; Clontech) using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, cells were stimulated with increasing doses of TNF-α and IFN-γ for 24 hours. Cell lysates were assayed for luciferase activity using the firefly luciferase 1-step assay kit (Fluoprobes, Montlucon Cedex, France). Luminescence and fluorescence were then measured with a luminometer/fluorimeter (VICTOR3Heerdt B.G. Houston M.A. Augenlicht L.H. Potentiation by specific short-chain fatty acids of differentiation and apoptosis in human colonic carcinoma cell lines.Cancer Res. 1994; 54: 3288-3293PubMed Google Scholar, PerkinElmer, Courtaboeuf Cedex, France). Luciferase activity was normalized to fluorescence intensity and expressed as relative light units (RLU). Total RNA was isolated with TRIzol reagent (Invitrogen, Cergy Pontoise Cedex, France) and treated for 45 minutes at 37°C with 2 U RQ1 DNAse (Promega). One microgram RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen). One microliter of the complementary DNA (cDNA) solution was subjected to real-time quantitative polymerase chain reaction (PCR) in a Bio-Rad iCycler iQ system using the QuantiTect SYBR Green PCR kit (Qiagen, Courtaboeuf Cedex, France). Quantitative PCR consisted of 45 cycles, each PCR cycle consisting of 30 seconds at 95°C and 30 seconds at 60°C. The sequences of human and rat primers for MCT1, interleukin (IL)-1β, β2-microglobulin, and β-actin are included in Supplementary Table 1 (see Supplementary Table 1 online at www.gastrojournal.org). The expression level of β-actin was used as a reference value to normalize MCT1 and IL-1β gene expression. Relative quantitative gene expression was calculated by the 2−ΔΔCt method,16Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (124688) Google Scholar using normal tissues from healthy subjects, control rats, or untreated control cells as the calibrator samples. Membrane protein samples were either from HT-29 postnuclear membranes, prepared as previously described,17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar or from patient biopsy specimens and rat colonic tissues. The latter were coextracted with RNA using TRIzol reagent. Samples were analyzed by Western blot using anti-MCT1 antibody (1:1000; Santa Cruz, CA) and a mouse monoclonal anti-β-actin antibody (1:10,000; Sigma, Dorset, UK). Immunoreactive bands were visualized with horseradish peroxidase-conjugated secondary antibody (Dako, Cambridgeshire, UK) and subsequent ECL detection (Amersham International, Buckinghamshire, UK). Band intensities were quantified by scanning densitometry (Phoretix ID quantifier; Non-linear Dynamics, Newcastle, UK). HT-29 cell monolayers were fixed in −20°C methanol, permeabilized, and incubated with the anti-MCT1 antibody or goat IgG control (sc-2028; Santa Cruz) for 1 hour at room temperature, as previously described.5Segain J.P. Raingeard de la Blétière D. Bourreille A. et al.Butyrate inhibits inflammatory responses through NF-κB inhibition: implications for Crohn’s disease.Gut. 2000; 47: 397-403Crossref PubMed Scopus (952) Google Scholar This was followed by incubation with FITC-conjugated anti-goat antibody (1:500; Jackson ImmunoResearch, Newmarket, UK). Nuclei were counterstained with Hoechst. Serial x-y and x-z sections were collected every 0.25 μm using a laser scanning confocal microscope (Leica, Rueil-Malmaison Cedex, France). Paraffin-embedded human colonic tissue sections were obtained from the Department of Pathology (Nantes). After microwave treatment in Antigen Unmasking Solution (Vector Laboratories Inc, Peterborough, UK), sections were incubated with the anti-MCT1 antibody (1:100; Santa Cruz) overnight at 4°C, followed by incubation with FITC-conjugated anti-goat antibody. Images were acquired using a digital camera (DXM1200F; Nikon, Champigny sur Marne, France) coupled to a fluorescence microscope (Nikon). Fluorescence intensity was quantified using the LUCIA software (Laboratory Imaging, Champigny sur Marne, France) in 4 regions of interest within the colonic epithelium. Two different tissue sections were analyzed for each patient. Mean fluorescence intensity was calculated from these 8 (4 × 2) values. Image acquisition and analysis were identical between controls (n = 5) and patients (n = 5), allowing a comparative semiquantitative estimation of MCT1 protein expression. Run-on assays were performed as described previously.17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar Nuclei were isolated from untreated or cytokine-treated HT-29 cells and incubated in transcription solution (5 mmol/L Tris-HCl [pH 8.0]; 2.5 mmol/L MgCl2; 150 mmol/L KCl; 0.25 mmol/L each of ATP, GTP, and CTP; and 250 μCi [α-32P]UTP [3000 Ci/mmol, 10 μCi/μL]) at 30°C for 20 minutes. The labelled RNA was hybridized to slot-blotted cDNA probes on Hybond-XL nylon membrane and exposed to Biomax-MS film (−80°C for 5 days). Band intensities were assessed densitometrically, and the values were normalized to the β-actin signals. Measurement of cellular uptake and oxidation was performed as previously described,12Hadjiagapiou C. Schmidt L. Dudeja P.K. et al.Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1.Am J Physiol. 2000; 279: G775-G780PubMed Google Scholar, 17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar with slight modifications. Briefly, HT-29 cells were grown in 24-well plates and treated with IFN-γ and TNF-α as described above. Cells were incubated in Hank’s balanced salt solution (HBSS; Invitrogen) containing 10 mmol/L Hepes, pH 7.5, for 30 minutes at 37°C and then in HBSS-10 mmol/L Mes, pH 6.4, containing [14C]-butyrate (16 mCi/mmol) for various incubation times (1–30 minutes). Preliminary kinetic experiments using increasing concentrations of butyrate showed that butyrate uptake was linear between 1 and 10 minutes. Thus, a 5-minute incubation time was used to measure the initial rate of butyrate uptake using butyrate concentrations ranging from 0.1 to 10 mmol/L. Km and Vmax values were determined by linear regression using Eadie-Hofstee plots.17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar To assess the effect of cytokines on the steady state rates of butyrate uptake and oxidation, cells were incubated with 40 μmol/L [14C]-butyrate for 5 minutes (uptake) or 90 minutes (oxidation) at 37°C. For uptake measurements, cells were placed in 100% ethanol for 30 minutes, and the radioactivity in the supernatants was counted by liquid scintillation. For measurement of butyrate oxidation,18Leschelle X. Delpal S. Goubern M. et al.Butyrate metabolism upstream and downstream acetyl-CoA synthesis and growth control of human colon carcinoma cells.Eur J Biochem. 2000; 267: 6435-6442Crossref PubMed Scopus (67) Google Scholar the reaction was stopped by adding perchloric acid to the medium (final concentration 4%), and 14CO2 was recovered hermetically in 3 mol/L NaOH for counting of radioactivity. Butyrate uptake and oxidation results were normalized to cell protein content. The normality of data distribution was analyzed by the Smirnoff-Kolmogorov test. The significance of differences was determined using the Mann–Whitney U test for nonnormally distributed data (patients and rats) and 1-way ANOVA for normally distributed data (cell line experiments). Nonnormally distributed data (patients and rats) are presented as median ± interquartiles (IQ). Normally distributed data (cell line experiments) are presented as mean ± SEM. Simple regressions were used for statistical correlations. We assessed MCT1 messenger RNA (mRNA) and protein expression in segments of rat colon with DSS-induced colitis and control rats. Western blot analysis of protein lysates showed that MCT1 protein levels were significantly lower in the cecum and proximal and distal colon of rats with DSS-induced colitis in comparison with controls (P < .01) (Figure 1A, i and ii). Similarly, reverse-transcription (RT) PCR analysis showed a significant decrease in MCT1 mRNA expression in these DSS-inflamed colonic segments as compared with normal tissues (Figure 1B). Expression of MCT1 protein and mRNA was also determined in colonic biopsy specimens of patients with IBD. MCT1 immunofluorescence staining of colonic tissue sections showed that MCT1 protein expression was dramatically reduced in inflamed mucosa of patients with UC and CD in comparison with healthy mucosa of controls (Figure 1C, i). In healthy subjects, MCT1 expression was almost exclusively confined to the epithelium. MCT1 staining was also found in cells of the lamina propria—probably immune cells—as recently reported.19Merezhinskaya N. Ogunwuyi S.A. Mullick F.G. et al.Presence and localization of three lactic acid transporters (MCT1, -2 and -4) in separated human granulocytes, lymphocytes and monocytes.J Histochem Cytochem. 2004; 52: 1483-1493Crossref PubMed Scopus (41) Google Scholar Fluorescence quantification of MCT1 protein within the epithelium of patients with UC (median fluorescence intensity ± interquartiles, 27.0 ± 8.0) and CD (28.5 ± 17.5) showed lower MCT1 levels compared with normal controls (70.5 ± 36.5, P < .001) (Figure 1C, ii). This finding suggests that MCT1 expression is reduced in the epithelial layer of the inflamed mucosa of IBD patients. Similarly, MCT1 mRNA levels were significantly lower in colonic biopsy specimens of patients with UC or CD than in normal colonic mucosa of controls (Figure 1D). No differences in these parameters were noted between UC and CD patients. We next sought to verify whether the down-regulation in MCT1 expression was proportional to the degree of mucosal inflammation. We first confirmed the endoscopic estimation of the inflammatory status of colonic biopsy specimens by RT-PCR analysis of IL-1β mRNA levels. As expected, IL-1β mRNA levels were significantly higher in inflamed biopsy specimens than in noninflamed or control biopsy specimens, being lowest in controls (P < .01) (Figure 2A, i). In contrast, MCT1 mRNA levels were significantly decreased in inflamed biopsy tissues as compared with noninflamed (P < .05) and control tissues (P < .001) (Figure 2A, ii). MCT1 mRNA levels were also significantly decreased in noninflamed tissues compared with controls (P < .01) (Figure 2A, ii). The reduction in MCT1 mRNA abundance, induced by inflammation, was similar irrespective of the regional origin of the biopsy specimen (data not shown). Analysis of paired samples from inflamed and noninflamed biopsy specimens from both UC (n = 8) and CD (n = 10) patients showed that MCT1 mRNA levels were significantly reduced in inflamed mucosa (P < .05) (Figure 2B). Furthermore, Western blot analysis of protein extracts of the same biopsy specimens also showed decreased levels of MCT1 protein in inflamed colonic mucosa of both UC (P < .01) and CD (P < .05) patients in comparison with paired noninflamed mucosa (Figure 2C, i and ii). Collectively, these results provide evidence that, in IBD (both UC and CD), the decrease in MCT1 expression is closely related to the degree of mucosal inflammation. Because we observed a markedly decreased expression of MCT1 in the inflamed colonic epithelium, we aimed to determine the potential of proinflammatory cytokines to down-regulate MCT1 expression in vitro. Therefore, we tested the effect of IFN-γ and TNF-α, alone or in combination, on the intestinal epithelial cell line HT-29. Quantitative RT-PCR analysis of cells treated with increasing concentrations of IFN-γ (10–1000 U/mL) or TNF-α (1–100 ng/mL) showed a dose-dependent decrease of MCT1 mRNA expression (Figure 3A). The half maximal effect was obtained for 300 U/mL IFN-γ (P < .05) and 30 ng/mL TNF-α (P < .05) (Figure 3A). The combination of either IFN-γ or TNF-α at these doses with increasing concentrations of each synergistically down-regulated MCT1 mRNA expression (Figure 3B, i and ii). The down-regulation induced by the cytokine mixture (300 U/mL IFN-γ plus 30 ng/mL TNF-α) was time-dependent with a maximal effect at 24 hours (P < .05) (Figure 3C). This effect was also observed with the intestinal epithelial cell lines Caco-2 and SW-1116 (data not shown). Furthermore, RT-PCR and Western blot analysis showed that treatment of HT-29 cells for 24 hours with increasing concentrations of cytokines (at 10:1 ratio) induced a dose-dependent decrease in MCT1 mRNA (Figure 3D) and protein expression (Figure 3E). For the maximal concentration of the cytokine mixture (1000 U/mL IFN-γ plus 100 ng/mL TNF-α), the decreases in MCT1 mRNA and protein levels were 75% and 87%, respectively (P < .01) (Figure 3D and E). These data suggest that proinflammatory cytokines down-regulate MCT1 predominantly at the level of mRNA abundance. We have shown previously that modulation of the steady-state level of MCT1 mRNA can result from the regulation of both its transcription and stability.17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar Accordingly, we first investigated the effect of proinflammatory cytokines on the stability of the MCT1 transcript in HT-29 cells stimulated in the presence of the transcriptional inhibitor actinomycin D. The decay in MCT1 mRNA level, after inhibition of RNA synthesis, was similar in cytokine-treated and control cells (respective relative half-lives of MCT1, 6.1 vs 7.2 hours) (P = .95), indicating no effect of cytokines on the stability of the MCT1 transcript (Figure 4A). We next analyzed the effect of cytokines specifically on MCT1 gene transcription. Nuclear run-on reactions were performed using nuclei isolated from untreated or cytokine-treated HT-29 cells. In comparison with untreated controls, cytokine treatment strongly reduced the abundance of MCT1 nascent transcripts (P < .01) (Figure 4B). For the maximal cytokine concentrations, the magnitude of decrease in MCT1 mRNA (75%), as estimated by real-time RT-PCR (Figure 3B), is accounted for by the specific decrease (67%) in MCT1 transcription as determined by run-on assay (Figure 4B). Together, these data suggest that the cytokine mixture has a repressive effect on MCT1 transcription. To confirm this proposition, HT-29 cells were transfected with a luciferase reporter plasmid containing the −1525/+213 sequence of the human MCT1 promoter region15Cuff M.A. Shirazi-Beechey S.P. The human monocarboxylate transporter, MCT1: genomic organization and promoter analysis.Biochem Biophys Res Comm. 2002; 292: 1048-1056Crossref PubMed Scopus (40) Google Scholar or an NF-κB-Luciferase reporter vector to be used as a positive control for the action of proinflammatory cytokines. Treatment of cells with increasing concentrations of IFN-γ and TNF-α (10:1 ratio) drastically reduced MCT1 promoter activity (P < .01) (Figure 4C, i). In contrast, treatment with IFN-γ (300 U/mL) plus TNF-α (30 ng/mL) stimulated NF-κB-reporter gene activity (Figure 4C, ii). Furthermore, cells were also stimulated by butyrate as a positive control for transcriptional stimulation of MCT1.17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar Contrary to the inhibitory effect observed with cytokines, treatment of cells with butyrate stimulated MCT1 promoter activity (Figure 4C, iii). Therefore, run-on and reporter gene assays clearly support the proposition that cytokines repress MCT1 gene transcription. To eliminate further any potential involvement of cell death in cytokine-induced down-regulation of MCT1, we performed MTT viability cell assays in cells treated with increasing concentrations of IFN-γ and TNF-α for 24 hours. Whatever the dose of cytokines, no loss in cell viability was observed (see Supplementary Figure 1A online at www.gastrojournal.org). As controls, we showed that treatment of cells for 24 hours with 10 mmol/L butyrate, a well-known proapoptotic agent, induced approximately 38% loss in cell viability (P < .05) (see Supplementary Figure 1B online at www.gastrojournal.org). However, butyrate treatment also stimulated MCT1 expression (P < .05) (see Supplementary Figure 1C online at www.gastrojournal.org), consistent with previous observations.17Cuff M.A. Lambert D.W. Shirazi-Beechey S.P. Substrate-induced regulation of the human colonic monocarboxylate transporter, MCT1.J Physiol. 2002; 539: 361-371Crossref PubMed Scopus (161) Google Scholar Moreover, staining of HT29 cell nuclei with Hoechst showed that treatment of cells with 1000 U/mL IFN-γ plus 100 ng/mL TNF-α did not induce chromatin condensation, a typical sign of apoptosis. On the contrary, 10 mmol/L butyrate treatment of HT29 cells did induce chromatin condensation (see Supplementary Figure 2 online at www.gastrojour" @default.
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• W2000864811 date "2007-12-01" @default.
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• W2000864811 title "Down-Regulation of the Monocarboxylate Transporter 1 Is Involved in Butyrate Deficiency During Intestinal Inflammation" @default.
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• W2000864811 doi "https://doi.org/10.1053/j.gastro.2007.08.041" @default.
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