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• W2034261640 abstract "In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na+-independent, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V max) without any modification in the apparent Michaelis-Menten constant (K m ). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers. In the intestine, butyrate constitutes the major energy fuel for colonocytes. However, little is known about the transport of butyrate and its regulation in the intestine. In this study we demonstrate that the monocarboxylate transporter (MCT-1) is apically polarized in model human intestinal epithelia and is involved in butyrate uptake by Caco2-BBE cell monolayers. The butyrate uptake by Caco2-BBE cell monolayers displayed conventional Michaelis-Menten kinetics and was found to be pH-dependent, Na+-independent, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid-insensitive, and inhibited by the monocarboxylate transporter inhibitor α-cyano-4-hydroxycinnamate and by an excess of unlabeled butyrate. We show that MCT-1 associates with CD147 at the apical plasma membrane in Caco2-BBE cell monolayers. Using antisense CD147, we demonstrate that the association of CD147 with MCT-1 is critical for the butyrate transport activity. Interestingly, we show for the first time hormonal regulation of CD147/MCT-1 mediated butyrate uptake. Specifically, luminal leptin significantly up-regulates MCT-1-mediated butyrate uptake by increasing its maximal velocity (V max) without any modification in the apparent Michaelis-Menten constant (K m ). Finally, we show that luminal leptin up-regulates butyrate uptake in Caco2-BBE monolayers by two distinct actions: (i) increase of the intracellular pool of MCT-1 protein without affecting CD147 expression and (ii) translocation of CD147/MCT-1 to the apical plasma membrane of Caco2-BBE cell monolayers. short chain fatty acid 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid monocarboxylate transporter α-cyano-3-hydroxycinnamate phosphate-buffered saline apolipoprotein Hanks' balanced salt solution 4-morpholineethanesulfonic acid bovine serum albumin Bacterial fermentation is high in the proximal large bowel, as is the production of short chain fatty acids (SCFA)1 that constitute the major end product of the microbial digestion of carbohydrates and dietary fibers (1Topping D.L. Clifton P.M. Physiol. Rev. 2001; 8: 11031-11064Google Scholar). At least 60% of SCFA uptake occurs by simple diffusion of the unionized form across the cell membrane; the remainder occurs by active cellular uptake of ionized SCFA involving an acid microclimate on the surface of the intestinal epithelium. SCFA are metabolized rapidly by colonocytes and are the major respiratory fuels in the intestine; indeed, oxidation of SCFA supplies 60–70% of the energy needs in isolated colonocytes (2Roediger W.E. Millard S. Gut. 1996; 38: 792-793Crossref PubMed Scopus (14) Google Scholar). Of the three major SCFA (acetate, propionate, and butyrate), butyrate is the major intestinal fuel even when competing substrates such as glucose and glutamine are available (3Fleming S.E. Fitch M., D. DeVries S. Liu M.L. Kight C. J. Nutr. 1991; 121: 869-878Crossref PubMed Scopus (135) Google Scholar, 4Clausen M.R. Mortensen P.B. Gastroenterology. 1994; 106: 423-432Abstract Full Text PDF PubMed Scopus (84) Google Scholar, 5Roediger W.E. Rae D.A. Br. J. Surg. 1982; 69: 23-25Crossref PubMed Scopus (68) Google Scholar). Apart from its function as the dominant energy source for the colonocytes, butyrate also affects cellular proliferation, differentiation, and apoptosis (6Scheppach W. Christl S., U. Bartram H.P. Richter F. Kasper H. Scand. J. Gastroenterol. Suppl. 1997; 222: 53-57Crossref PubMed Google Scholar, 7Augeron C. Laboisse C.L. Cancer Res. 1984; 44: 3961-3969PubMed Google Scholar, 8McIntyre A. Gibson P.R. Young G.P. Gut. 1993; 34: 386-391Crossref PubMed Scopus (490) Google Scholar, 9Gamet L. Daviaud D. Denis-Pouxviel C. Remesy C. Murat J.C. Int. J. Cancer. 1992; 52: 286-289Crossref PubMed Scopus (174) Google Scholar, 10Siavoshian S. Blottiere H.M. Bentouimou N. Cherbut C. Galmiche J.P. Eur J. Clin. Invest. 1996; 26: 803-810Crossref PubMed Scopus (26) Google Scholar). Recently, it has been suggested that the proton-linked monocarboxylate transporter 1 (MCT-1) may play a major role in the uptake of butyrate by the intestinal epithelial cells in vivo (11Ritzhaupt A. Ellis A. Hosie K.B. Shirazi-Beechey S.P. J. Physiol. 1998; 507: 819-830Crossref PubMed Scopus (104) Google Scholar, 12Ritzhaupt A. Wood I.S. Ellis A. Hosie K.B. Shirazi-Beechey S.P. Biochem. Soc. Trans. 1998; 26: S120Crossref PubMed Scopus (23) Google Scholar) as well as in vitro in Caco2 cells (13Hadjiagapiou C. Schmidt L. Dudeja P.K. Layden T.J. Ramaswamy K. Am. J. Physiol. 2000; 279: G775-G780Crossref PubMed Google Scholar, 14Stein J. Zores M. Schroder O. Eur. J. Nutr. 2000; 39: 121-125Crossref PubMed Scopus (72) Google Scholar). MCT-1 belongs to the monocarboxylate transporter family including nine MCT-related sequences that have been so far identified in mammals, each having a different tissue distribution (15Halestrap A.P. Price N.T. Biochem. J. 1999; 343: 281-299Crossref PubMed Scopus (1067) Google Scholar). Hydropathy plots predict the number of transmembrane domains to be 12 for MCT-1 with the N and C termini located within the cytoplasm (16Price N.T. Jackson V.N. Halestrap A.P. Biochem. J. 1998; 329: 321-328Crossref PubMed Scopus (304) Google Scholar). MCT-1 can transport a wide range of short chain monocarboxylates, the K m values (5–10 mm) decreasing as the chain length increases from two to four carbon atoms. Monocarboxylates with longer branched aliphatic or aromatic side chains also bind to the transporter, but are not released following translocation and may act as potent inhibitors. One of these is the classical inhibitor, α-cyano-3-hydroxycinnamate (CHC) (17Halestrap A.P. Brand M.D. Denton R.M. Biochim. Biophys. Acta. 1974; 367: 102-108Crossref PubMed Scopus (78) Google Scholar). MCT-1 (and MCT-4) has been shown to interact specially with CD147, a member of the immunoglobulin superfamily. This interaction appears to assist MCT expression at the cell surface in heart cells and transfected cells; thus, CD147 acts as a chaperone to increase MCT-1 translocation from the endoplasmic reticulum to the Golgi and plasma membrane (18Kirk P. Wilson M.C. Heddle C. Brown M. Barclay H. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar, 19Wilson M.C. Meredith D. Halestrap A.P. J. Biol. Chem. 2002; 277: 3666-3672Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 20Poole R.C. Halestrap A.P. J. Biol. Chem. 1997; 272: 14624-14628Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Although the regulation of MCT-1 expression has been extensively studied in skeletal muscle (21Hajduch E. Heyes R.R. Watt P.W. Hundal H.S. FEBS Lett. 2000; 479: 89-92Crossref PubMed Scopus (28) Google Scholar, 22Pilegaard H. Juel C. Wibrand F. Am. J. Physiol. 1993; 264: E156-E160PubMed Google Scholar, 23Pilegaard H. Domino K. Noland T. Juel C. Hellsten Y. Halestrap A.P. Bangsbo J. Am. J. Physiol. 1999; 276: E255-E261PubMed Google Scholar, 24Juel C. Halestrap A.P. J. Physiol. 1999; 517: 633-642Crossref PubMed Scopus (317) Google Scholar, 25McCullagh K.J. Poole R.C. Halestrap A.P. Tipton K.F. O'Brien M. Bonen A. Am. J. Physiol. 1997; 273: E239-E246Crossref PubMed Google Scholar), little is known about the regulation of MCT-1 or its association with CD147 in the intestine. Leptin, the ob gene cloned in 1994 by Zhang et al. (26Zhang Y. Proenca R. Maffei M. Barone M. Leopold L. Friedman J.M. Nature. 1994; 372: 425-432Crossref PubMed Scopus (11671) Google Scholar), is a hormone mainly secreted by adipocytes and is involved in central regulation of body weight homeostasis (27Halaas J.L. Gajiwala K.S. Maffei M. Cohen S.L. Chait B.T. Rabinowitz D. Lallone R.L. Burley S.K. Friedman J.M. Science. 1995; 269: 543-546Crossref PubMed Scopus (4208) Google Scholar, 28Campfield L.A. Smith F.J. Guisez Y. Devos R. Burn P. Science. 1995; 269: 546-549Crossref PubMed Scopus (3062) Google Scholar, 29Pelleymounter M.A. Cullen M.J. Baker M.B. Hecht R. Winters D. Boone T. Collins F. Science. 1995; 269: 540-543Crossref PubMed Scopus (3855) Google Scholar) via its specific receptors in the hypothalamus (30Couce M.E. Burguera B. Parisi J.E. Jensen M.D. Lloyd R.V. Neuroendocrinology. 1997; 66: 145-150Crossref PubMed Scopus (183) Google Scholar). Subsequent studies have established that nonadipose tissues, such skeletal muscle (31Wang X. Levi A.J. Halestrap A.P. Am. J. Physiol. 1996; 270: H476-H484PubMed Google Scholar), pituitary gland (32Jin L. Zhang S. Burguera B.G. Couce M.E. Osamura R.Y. Kulig E. Lloyd R.V. Endocrinology. 2000; 141: 333-339Crossref PubMed Scopus (204) Google Scholar), and stomach (33Bado A. Levasseur S. Attoub S. Kermorgant S. Laigneau J.P. Bortoluzzi M.N. Moizo L. Lehy T. Guerre-Millo M., Le Marchand-Brustel Y. Lewin M.J. Nature. 1998; 394: 790-793Crossref PubMed Scopus (1033) Google Scholar) also produce luminal leptin in the nanomolar range as concentration (33Bado A. Levasseur S. Attoub S. Kermorgant S. Laigneau J.P. Bortoluzzi M.N. Moizo L. Lehy T. Guerre-Millo M., Le Marchand-Brustel Y. Lewin M.J. Nature. 1998; 394: 790-793Crossref PubMed Scopus (1033) Google Scholar, 34Sobhani I. Bado A. Vissuzaine C. Buyse M. Kermorgant S. Laigneau J.P. Attoub S. Lehy T. Henin D. Mignon M. Lewin M.J. Gut. 2000; 47: 178-183Crossref PubMed Scopus (272) Google Scholar). Moreover under secretin, pentagastrin, or vagal stimulation, the gastric luminal leptin output increased by ∼50 times (34Sobhani I. Bado A. Vissuzaine C. Buyse M. Kermorgant S. Laigneau J.P. Attoub S. Lehy T. Henin D. Mignon M. Lewin M.J. Gut. 2000; 47: 178-183Crossref PubMed Scopus (272) Google Scholar, 35Sobhani I. Buyse M. Goiot H. Weber N. Laigneau J.P. Henin D. Soul J.C. Bado A. Gastroenterology. 2002; 122: 259-263Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In addition, it has been recently demonstrated that some of the stomach-derived leptin secreted in the gastric juice is not fully degraded by proteolysis, suggesting that it reaches the intestine in an active form, and thus can initiate biological processes involved in controlling functions of the intestinal tract, such as absorption and secretion (34Sobhani I. Bado A. Vissuzaine C. Buyse M. Kermorgant S. Laigneau J.P. Attoub S. Lehy T. Henin D. Mignon M. Lewin M.J. Gut. 2000; 47: 178-183Crossref PubMed Scopus (272) Google Scholar). Recently, we have found that the concentration of luminal leptin from normal colon is in the low nanomolar range. We suggest that this leptin is coming from the gastric gland because no leptin staining was detected from the epithelial cells along normal small and large intestine. 2S. V. Sitaraman and D. Merlin, unpublished observations. 2S. V. Sitaraman and D. Merlin, unpublished observations. Interestingly, under inflammatory states, we have detected a strong leptin staining from colonic epithelial cells and the luminal leptin concentration increased significantly (∼10 times greater compared with noninflamed tissues).2 During inflammation the luminal colonic leptin concentration is likely to be the addition of the leptin produced by the gastric gland and the leptin produced by the colonic epithelial cells. These results suggest that luminal leptin could have an important physiological and/or pathological role in the colon. Indeed, the different leptin receptor isoforms including the functional long isoform (Ob-Rb) have been detected in the rat intestine from duodenum to colon and in the model intestinal cell line Caco2 (36Buyse M. Berlioz F. Guilmeau S. Tsocas A. Voisin T. Peranzi G. Merlin D. Laburthe M. Lewin M., J. Roze C. Bado A. J. Clin. Invest. 2001; 108: 1483-1494Crossref PubMed Scopus (198) Google Scholar, 37Lostao M.P. Urdaneta E. Martinez-Anso E. Barber A. Martinez J.A. FEBS Lett. 1998; 423: 302-306Crossref PubMed Scopus (115) Google Scholar, 38Morton N.M. Emilsson V. Liu Y.L. Cawthorne M.A. J. Biol. Chem. 1998; 273: 26194-26201Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 39Doi T. Liu M. Seeley R.J. Woods S.C. Tso P. Am. J. Physiol. 2001; 281: R753-R759Crossref PubMed Google Scholar, 40Stan S. Levy E. Bendayan M. Zoltowska M. Lambert M. Michaud J. Asselin C. Delvin E.E. FEBS Lett. 2001; 508: 80-84Crossref PubMed Scopus (30) Google Scholar). The demonstration of leptin receptor in intestinal tract has initiated several investigations on the possible role of leptin in the digestive physiology as absorption and secretion. Evidence has been provided that leptin can regulate intestinal triglyceride transport by inhibiting apolipoprotein AIV expression via activation of a jejunal leptin receptor in mice (38Morton N.M. Emilsson V. Liu Y.L. Cawthorne M.A. J. Biol. Chem. 1998; 273: 26194-26201Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Similarly, in rat, intravenous leptin infusion attenuates the increase in synthesis and secretion of apoAIV induced by intraduodenal infusion of lipids (39Doi T. Liu M. Seeley R.J. Woods S.C. Tso P. Am. J. Physiol. 2001; 281: R753-R759Crossref PubMed Google Scholar). In addition, leptin administered to the basolateral side of Caco2 cells inhibits the triglyceride secretion, the biosynthesis of apoB-100 and apoB-48, as well as the output of chylomicron and low density lipoproteins (40Stan S. Levy E. Bendayan M. Zoltowska M. Lambert M. Michaud J. Asselin C. Delvin E.E. FEBS Lett. 2001; 508: 80-84Crossref PubMed Scopus (30) Google Scholar). More recently, we have reported that luminal leptin improves the transport of oligopeptides across the intestinal epithelium through the H+-dependent, di- and tripeptide transporter PepT-1 in vitro and in vivo (36Buyse M. Berlioz F. Guilmeau S. Tsocas A. Voisin T. Peranzi G. Merlin D. Laburthe M. Lewin M., J. Roze C. Bado A. J. Clin. Invest. 2001; 108: 1483-1494Crossref PubMed Scopus (198) Google Scholar). Together, the results clearly demonstrate that leptin is a key hormone of the intestinal tract. This study aims to investigate the regulation of butyrate uptake by luminal leptin using the model human intestinal epithelial cell line Caco2-BBE. Caco2-BBE (41Merlin D., Si- Tahar M. Sitaraman S.V. Eastburn K. Williams I. Liu X. Hediger M.A. Madara J.L. Gastroenterology. 2001; 120: 1666-1679Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) cells (between passage 30 and 50) were grown in high glucose Dulbecco's Vogt modified Eagle's medium (DMEM, Invitrogen) supplemented with 14 mmol/liter NaHCO3 and 10% newborn calf serum. Cells were kept at 37 °C in 5% CO2 and 90% humidity, and medium was changed every day. Monolayers were subcultured every 7 days by trypsinization with 0.1% trypsin and 0.9 mmol/liter EDTA in Ca2+/Mg2+-free phosphate-buffered saline (PBS). Uptake, confocal immunofluorescence were performed with confluent monolayers plated on collagen-coated permeable supports (area, 1 cm2; pore size, 0.4 μm; Transwell-Clear polyester membranes from Costar) and examined 15 days after plating. For protein, membrane or RNA extractions, cells were plated on six-well cluster trays at a density of 104 cells/cm2 and examined 15 days after plating. For treatments, leptin was added to the luminal compartment for 1–24 h in medium without serum. Cells were washed twice with Hanks' balanced salt solution (Sigma Aldrich) complemented with 4 mm NaHCO3 (HBSS) and 10 mm HEPES, pH 7.5, and stabilized 30 min in the same buffer. Caco2-BBE cells were then incubated in HBSS plus 10 mm MES, pH 6.4, containing 20 μm [14C]butyrate (specific activity, 16 mCi/mmol; Sigma) in apical compartment for 1 h at 37 °C. We choose 1-h butyrate uptake to visualize the steady state butyrate uptake. However to investigate the pH-dependent activity of MCT-1, 5-min incubation was used to avoid the equilibration of H+ concentration across the monolayer. The supernatant was then removed, and cells were washed twice with ice-cold HBSS-HEPES, pH 7.5. Cell-associated radioactivity was determined by liquid scintillation counting in a β-counter. Caco2-BBE pellets was resuspended and homogenized in HEPES (5 mm) containing protease inhibitors. The pellet was then incubated for 30 min at 4 °C and centrifuged at 13,000 × g at 4 °C for 30 min. The resulting pellet was suspended in PBS by repeated passage through an 18-gauge needle. The protein solution was then boiled 5 min at 100 °C in Laemmli buffer supplemented with 0.5% β-mercaptoethanol. Cross-linking was carried out using the bifunctional stilbene disulfonate (DIDS; Sigma), as described previously (20Poole R.C. Halestrap A.P. J. Biol. Chem. 1997; 272: 14624-14628Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Cells seeded on six-well plates were washed twice with PBS and incubated with 100 μm DIDS during 1 h at 37 °C. The cells were then washed in ice-cold PBS and lysed with the lysis buffer (1% Triton X-100 in 20 mmTris, pH 5.0, 50 mm NaCl, 5 mm EDTA, 0.2% bovine serum albumin (BSA), and protease inhibitors). The resulting supernatants were immunoprecipitated with the appropriate amount of specific antibody (0.05 μg/ml mouse anti-human CD147 (Cymbus Biotechnology Ltd, Chandlers Ford, UK); 0.05 μg/ml of the polyclonal rabbit anti-human MCT-1 (Alpha Diagnostic, San Antonio, TX) was added and gently rocked overnight at 4 °C. Subsequently, 50 μl of protein G suspension was added to the mixture and incubated overnight at 4 °C. The complexes were collected by centrifugation at 12,000 × g for 1 min by microcentrifuge. The beads were washed one time with buffer 1 (1% Triton X-100 in 20 mm Tris, pH 5.0, 50 mm NaCl, 5 mmEDTA, and 2% BSA) and two times with buffer 2 (20 mmTris-HCl, pH 8.0). The protein solution was then boiled 5 min at 100 °C in Laemmli buffer and subjected to SDS-PAGE and transferred at 4 °C to nitrocellulose membranes. The blots were blocked 1 h with 5% nonfat dry milk in blocking buffer. After washing with blocking buffer, the blots were incubated for 1 h at room temperature with 1:2000 dilution of a mouse anti-human CD147 or rabbit anti-human MCT-1. They were further incubated for 30 min at room temperature with anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibody diluted 1:1000 and probed using chemiluminescence system (ECL, Amersham Biosciences). The expression of CD147 in Caco2-BBE cells was determined using a reverse transcription-polymerase chain reaction (PCR) method with oligonucleotide primers specific for CD147. Poly(A)T RNA was isolated from Caco2-BBE cells with a Micro Fast Track 2.0 kit (Invitrogen). The yield of RNA was determined by ultraviolet spectrophotometry. One microgram of Poly(A)T RNA was primed with oligo(dT) and reverse transcribed with avian myeloblastosis virus-reverse transcriptase (cDNA Cycle Kit; Invitrogen). A dilution of the reverse transcription reaction was used as a template for amplification by PCR. PCR conditions are determined according the primer characteristics. CD147 coding sequence was amplified using the following primers from Invitrogen: 5′-GGAATAGGAATCATGGCG-3′ and 5′-CCACCTGCCTCAGGAAGA-3′. The product was visualized and purified from a 1% agarose gel using a DNA extraction kit (Qiagen, Valencia, CA) in the antisense orientation into the mammalian expression vector, pTarget (Promega). The constructed plasmids were verified by DNA sequencing. Plasmids were purified using the Qiagen Maxiplasmid kit. Subconfluent Caco2-BBE cells were plated on permeable filters 48 h prior to transfection with vector alone or antisense CD147 into vector using Lipofectin (Invitrogen) in serum-free medium for 48 h, and uptake experiments were performed. Uptake experiments were performed as described above with the exception that 20 μm[14C]butyrate was added to the apical plasma membrane of Caco2-BBE for only 5 min. Proteins were separated by SDS-polyacrylamide gel electrophoresis on a 4–20% gradient gel (Bio-Rad) and then transferred to nitrocellulose membranes. The blots were blocked for 1 h with 5% nonfat milk in blocking buffer, and then the blots were incubated 1 h with 0.05 μg/ml CD147 human monoclonal antibody (Cymbus Biotechnology) or with 0.05 μg/ml MCT-1 antibody (Alpha Diagnostic). After washing three times for 15 min in blocking buffer, they were further incubated for 1 h with the corresponding secondary horseradish peroxidase-conjugated (anti-rabbit or anti-mouse) antibody diluted 1:2000. The nitrocellulose was washed three times for 20 min in blocking buffer and then probed using a chemiluminescence system (ECL, Amersham Biosciences). Caco2-BBE cells grown on filters were washed twice in HBSS, pH 7.4, and then fixed with 3.7% paraformaldehyde in Hanks' balanced salt solution with calcium, pH 7.4 (HBSS+). Caco2-BBE cells were then permeabilized with 0.5% Triton for 30 min at 25 °C. The cells were rinsed and incubated with rhodamine-phalloidin (Molecular Probes Inc., Eugene, OR) diluted 1:60 for 40 min. Caco2-BBE cells were then blocked 1 h in a blocking solution containing 0.2% gelatin and 0.08% saponin in HBSS+. These monolayers were incubated 1 h with 0.05 μg/ml mouse anti-human CD147 human antibody (Cymbus Biotechnology) or with 0.05 μg/ml rabbit anti-human MCT-1. These monolayers were then stained with appropriate fluorescein isothiocyanate (anti-goat or anti-mouse) antibody diluted at 1:1000. Microscopy was performed using a Zeiss epifluorescence microscope equipped with a Bio-Rad MRC600 confocal unit, computer, and laser scanning microscope image analysis software (Carl Zeiss, Jena, Germany). MCT-1 cDNA was obtained from ATCC MGC-1187 (ATCC, Manassas, VA) and used as probe for Northern blot analysis. Total RNA was isolated from Caco2-BBE cells with Tri-Reagent (Molecular Research Center, Cincinnati, OH). Total RNA (20 μg) was denatured by heating at 65 °C in 20 mm HEPES pH 7.2, 1 mm EDTA, 50% formamide, and 6% formaldehyde for 15 min and subjected to electrophoresis on a 1% agarose gel containing 2% formaldehyde. Resolved RNA was transferred to a nylon membrane (PerkinElmer Life Sciences) and covalently cross-linked by exposure to UV light. Hybridization was performed in a solution that contains 7% SDS, 1% BSA, 10% polyethylene glycol 8000, 250 mm NaCl, 1.25 mm EDTA, 125 mmNaPO4 and 1 mg of salmon sperm DNA (Ambion, Austin, TX). MCT-1 cDNA probe was labeled with [α-32P]CTP using the Rediprime II random prime labeling system (Amersham Biosciences). A mouse glyceraldehyde-3-phosphate dehydrogenase probe was used as control (Ambion, Austin, TX). Results are expressed as means ± S.E. Statistical significance was determined using the paired ttest. The existence of a specific transporter system mediating butyrate flux across the apical membranes of Caco2-BBE cell monolayers was investigated. The butyrate transport across apical plasma membranes of Caco2-BBE monolayers was saturable, as was evident from uptake measurements done at varying concentrations of butyrate in the range of 0.5–10 mm (Fig.1 A). Kinetic parameters were calculated from the Michaelis-Menten equation. The apparentK m and V max values for uptake across the apical membrane of Caco2-BBE were 2.6 ± 0.2 mm and 8.25 ± 1.75 μmol/cm2/h, respectively. The Eadie-Hofstee transformation of the data for the carrier-mediated uptake yielded a linear plot (r = 0.82) (Fig. 1 A, inset). The substituted aromatic monocarboxylates such as CHC, known as one of the now classical inhibitors of MCT, decreased the butyrate uptake by 40% in Caco2-BBE monolayer (without CHC: 210 ± 18 pmol/cm2/h; with CHC: 126 ± 18 pmol/cm2/h) (Fig. 1 B). The uptake of 20 μm radiolabeled [14C]butyrate in Caco2-BBE monolayers was reduced by 95% in the presence of 50 mm unlabeled butyrate (402 ± 32 versus12 ± 1 pmol/cm2/h), indicating that the butyrate uptake was almost completely carrier-mediated (Fig. 1 B). However, these results suggest that other transporter(s) in addition to MCT are involved in the butyrate transport. When Na+ was replaced by choline in the uptake solution, butyrate uptake by Caco2-BBE monolayers was not affected (with NaCl: 717 ± 31 pmol/cm2/h; with choline chloride: 716 ± 86 pmol/cm2/h), demonstrating that the butyrate uptake was Na+-independent (Fig. 1 B). In addition, butyrate uptake was not affected in the presence of 100 μm DIDS (without DIDS: 583 ± 35 pmol/cm2/h; with DIDS: 529 ± 27 pmol/cm2/h), excluding the involvement of an anion-exchange mechanism in the butyrate uptake by Caco2-BBE monolayers (Fig. 1 B). Furthermore, uptake of butyrate by Caco2-BBE monolayers increased with H+ concentration (pH 7.5: 121 ± 10; pH 6.5: 169 ± 23; pH 5.6: 257 ± 34 pmol/cm2/h) (Fig. 1 C). These observations are consistent with those reported by others, showing that the transfer of butyrate across apical plasma membranes of Caco2-BBE involves co-transport of butyrate and a proton. Because MCT-1 is the most abundant MCT isoform expressed in Caco2 cells (13Hadjiagapiou C. Schmidt L. Dudeja P.K. Layden T.J. Ramaswamy K. Am. J. Physiol. 2000; 279: G775-G780Crossref PubMed Google Scholar) and because our kinetic parameters are similar to those reported in a previous study using Caco2 cells (13Hadjiagapiou C. Schmidt L. Dudeja P.K. Layden T.J. Ramaswamy K. Am. J. Physiol. 2000; 279: G775-G780Crossref PubMed Google Scholar), MCT-1 seems to be one of the transporter involved in the butyrate uptake by Caco2-BBE. Previous studies from Kirk et al. (18Kirk P. Wilson M.C. Heddle C. Brown M. Barclay H. Halestrap A.P. EMBO J. 2000; 19: 3896-3904Crossref PubMed Scopus (517) Google Scholar) demonstrated that CD147 is associated to MCT-1 in the plasma membrane, and this association was shown to be critical for the MCT-1 activity in heart cell line and in transfected cells. We next investigated whether there was an interaction between CD147 and MCT-1 in Caco2-BBE cells by using DIDS to cross-link the two proteins. After DIDS treatment of cells for 1 h, Western blot of the MCT-1 immunoprecipitated with mouse anti-CD147 antibody revealed the presence of 100-kDa immunoreactive band corresponding to the expected size of MCT-1/CD147 complex (Fig. 2 A,lanes 7 and 8). Similarly, Western blotting of CD147 immunoprecipitated with a rabbit anti-MCT-1 revealed an identical 100-kDa immune complex (Fig. 2 A,lanes 3 and 4). When cell lysates were mot immunoprecipitated (lanes 1, 2,5, and 6), the presence of ∼40–45 kDa (lanes 1 and 2) and ∼50–55 kDa (lanes 5 and 6) corresponding to MCT-1 and CD147, respectively, were detected. These results demonstrate that CD147 and MCT-1 are associated to the Caco2-BBE cell membranes. To study the functional role of CD147 in the butyrate uptake, Caco2-BBE monolayers were transfected with vector alone, antisense CD147 cDNA (full-length) inserted into pTarget/CMC vector. The uptake of butyrate was determined 48 h after transfection. As shown in Fig.2 B, the antisense CD147 inhibited butyrate uptake by ∼25% when compared with the vector alone or nontransfected cells. This inhibition is significant, given that the transfection efficiency in Caco2-BBE is not 100%. These results confirm the functional role of CD147 in the butyrate uptake by Caco2-BBE cell monolayers. To investigate the effect of luminal leptin on butyrate uptake, Caco2-BBE cells were pre-incubated with 10 nm leptin for 1–24 h. Although 10 nm leptin added to the apical compartment of the Caco2-BBE monolayers did not increase butyrate uptake after 1–6 h, it did significantly increase butyrate uptake after 12 and 24 h (p < 0.01): 20 and 25% increase, respectively (without luminal leptin for 12 h: 439 ± 18 versus 525.5 ± 14 with 10 nm luminal leptin for 12 h; without luminal leptin for 24 h: 513 ± 33 versus 640 ± 25 pmol/cm2/h with 10 nm luminal leptin) (Fig.3 A). The effect of luminal leptin on butyrate uptake is specific to leptin, because an unrelated peptide, interleukin-8, did not induce any change in butyrate uptake in Caco2-BBE monolayers (data not shown). In addition, the effect of luminal leptin on butyrate uptake was concentration-dependent and reached a plateau at 10 nm (without luminal leptin: 231 ± 87; 10 nm luminal leptin: 367 ± 21; 100 nmluminal leptin: 370 ± 11 pmol/cm2/h) (Fig.3 B). Moreover, the MCT inhibitor, 1 mm CHC, reversed luminal leptin induced increase in butyrate uptake (507 ± 22 versus 203 ± 18 pmol/cm2/h) (Fig.3 C). Furthermore, the addition of an excess of butyrate (50 mm) to the apical compartment completely suppressed (98%) the leptin-induced increase in [14C]butyrate uptake (507 ± 22 versus 10 ± 1 pmol/cm2/h) (Fig. 3 C). In addi" @default.
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• W2034261640 title "Luminal Leptin Enhances CD147/MCT-1-mediated Uptake of Butyrate in the Human Intestinal Cell Line Caco2-BBE" @default.
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