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ALIMENTARY |
Department of
1 Veterinary Physiology
2 Veterinary Biochemistry
3 Veterinary Pathology, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan
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Abstract |
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reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. Immunohistochemistry and immunofluorescence confocal analyses revealed widespread immunoreactive positivities for MCT1 in the caprine stomach and large intestine. Amongst the stratified squamous epithelial cells of the forestomach, MCT1 was predominantly expressed on the cell boundaries of the stratum basale and stratum spinosum. Double-immunofluorescence confocal laser-scanning microscopy confirmed the co-localization of MCT1 with its ancillary protein, CD147 in the caprine gastrointestinal tract. In vivo and in vitro functional studies, under the influence of the MCT1 inhibitors, p-chloromercuribenzoate (pCMB) and p-chloromercuribenzoic acid (pCMBA), demonstrated significant inhibitory effect on acetate and propionate transport in the rumen. This study provides evidence, for the first time in ruminants, that MCT1 has a direct role in the transepithelial transport and efflux of the SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side.
(Received 26 June 2006;
accepted after revision 3 August 2006;
first published online 10 August 2006)
Corresponding author S. Kato: Department of Veterinary Physiology, School of Veterinary Medicine, Rakuno Gakuen, University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan. Email: kato{at}rakuno.ac.jp
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Introduction |
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60–70% acetate, 15–20% propionate, and 10–15% butyrate (Titus & Ahearn, 1992). The net absorption of SCFA reaching the blood is dependent on its concentration in the rumen as well as the quantity used by the rumen wall. The rates of utilization by the rumen wall are butyrate > propionate > acetate (Stevens & Stettler, 1966); however, their respective concentrations in the blood were found to be in the reverse order (Masson & Phillipson, 1951). In ruminants, SCFA constitute the major source of energy, providing up to 80% of their maintenance energy requirements (Bergman, 1990). Propionate serves as the primary precursor of glucose, which must be synthesized de novo because little glucose is absorbed into the hepatic portal system (Baird et al. 1980; Huntington et al. 1981). In contrast, a high proportion of the acetate is not taken up by hepatocytes, but passes into the systemic circulation to be utilized for lipogenesis by peripheral tissues such as skeletal muscle, adipose tissue, and myocardium as well as mammary gland (Bergman, 1975). In addition to the energetic or nutritional contributions of SCFA to the body, the SCFA help in regulation of endocrine (Bassett, 1975) and exocrine (Harada & Kato, 1983) secretions of the pancreas. Moreover, SCFA production and absorption have a significant effect on epithelial cell growth, blood flow, and the normal secretory and absorptive functions of gastrointestinal tract (Bergman, 1990). Prior to use by the animal, these microbial products must be transferred and effectively absorbed across the gastrointestinal epithelium.
Despite the significance of SCFA in maintaining the ruminant physiology, the mechanism of SCFA absorption is still not fully studied. SCFA transfer across the apical membrane of ruminal epithelium is thought to occur by either passive permeation of undissociated acids, or exchange of dissociated SCFA for anions like bicarbonate (Kramer et al. 1996; Gäbel & Sehested, 1997). However, no mechanism for the transport of SCFA anions across the basolateral membrane into the bloodstream has so far been identified in the gastrointestinal tract of ruminants.
Monocarboxylate transporter 1 (MCT1) has been demonstrated to mediate the entry of SCFA in the small intestine of the rat (Tamai et al. 1999) and human colon (Ritzhaupt et al. 1998b; Cuff et al. 2002) as well as in Caco-2 cells (Hadjiagapiou et al. 2000). MCT1 is an isoform of the MCT family (also termed the SLC16 gene family), which has 14 members (Halestrap & Meredith, 2004), six of which have been functionally characterized but only MCT1–MCT4 have been shown to catalyse proton-coupled transport of monocarboxylates such as lactate, pyruvate and ketone bodies (Halestrap & Price, 1999; Halestrap & Meredith, 2004). Although studies carried out on MCT1 have focused exclusively on the human and monogastric animals (Halestrap & Meredith, 2004), we have recently studied the expression and regional distribution of MCT1 along the entire length of bovine and ovine gastrointestinal tract (Kirat et al. 2005; Kirat et al. 2006). However, whether MCT1 has a role in the transport of SCFA in ruminant gastrointestinal tract has not studied so far.
Therefore, the present study aimed to investigate the possible involvement of MCT1 in the SCFA transport across the caprine rumen epithelium using in vivo and in vitro functional studies, as well as to delineate the precise cellular localization and levels of MCT1 protein along the caprine gastrointestinal tract, utilizing different molecular biological techniques. Because of the importance of CD147 for targeting MCT1 to the plasma membrane, the study also intended to explore the co-localization of CD147 with MCT1 within the ruminant gastrointestinal epithelia.
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Adult goats of both sexes were used in all experiments. Prior to use, the goats were housed under standard conditions and fed hay (100 g) and Lucerne pellets (2.5% of the body weight) daily. Water was available ad libitum. The experimental protocol used in the present study was approved by the Ethics Committee for Animal Experiments in the School of Veterinary Medicine, Rakuno Gakuen University. This committee was established under the Laboratory Animal Control Guidelines, which are basically consistent with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health in the USA (NIH publication no. 86–23, revised in 1985).
Tissue samples
Eight adult goats were killed by bleeding from the carotid artery following intravenous injection with sodium pentobarbital (35 mg kg–1). Immediately after killing, the gastrointestinal tract was removed from the abdominal cavity. Epithelial preparations were prepared from the ventral ruminal sac and mounted into Ussing chambers. For the molecular biological studies, samples were collected from all of the gastrointestinal segments; including the rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, caecum, proximal colon, and distal colon. The tissues were washed in ice cold 0.9% (w/v) NaCl (pH 7.0). Ruminal and reticular epithelia were peeled from underlying and connective tissues, while individual omasal plies were removed. In the case of the abomasum and intestinal sections, the epithelium of each region was scraped off using glass slides on ice. All the collected samples were immediately frozen in liquid nitrogen, and subsequently stored at –80°C until use for Western blotting and RT-PCR analyses. For immunohistochemical and immunofluorescence confocal studies, tissue samples were immediately fixed in 4% paraformaldehyde for 24 h. After fixation, the tissues were dehydrated through a series of graded concentrations of ethanol and xylene, embedded in paraffin, sectioned serially at 4 µm, and mounted on poly-L-lysine-coated slides.
RT-PCR
Total RNA was extracted from the caprine gastrointestinal tissues using RNeasy Mini Kit (Qiagen Sciences, Maryland, USA) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed into cDNA in a 20 µl reaction volume using Superscript II and oligo-d(T)12–18 (Invitrogen, Carlsbad, CA, USA). PCR amplification was conducted on synthesized cDNAs using Taq DNA polymerase (Takara, Bio, Inc., Otsu, Japan). To date, the sequence of caprine MCT1 is not identified, therefore MCT1 primer pairs were derived from the predicted Bos taurus MCT1 (GenBank accession no. XM-614552). The primer sequences were; 5'-GTCCTATCAGCAGTGTCCTAGTG-3' and 5'-ACCTAAAACTGGTGGTCCCAGGAG-3' for sense and antisense, respectively. After an initial denaturation at 94°C for 2 min, 35 cycles of amplification with a thermocycler (iCycler, Bio-Rad) were performed under the following conditions: 94°C for 30 s, 55°C for 30 s and 72°C for 1 min followed by a final extension at 72°C for 10 min. To provide an appropriate internal PCR control as well as to assess the quality of the extracted RNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was amplified with primer sets designed against published rat sequences (sense: 5'-ATCACCATCTTCCAGGAG-3'; antisense: 5'-TCATCATACTTGGCAGGT-3'). As negative controls, PCR reactions were performed in the absence of cDNA. PCR products were analysed by electrophoresis in 1% agarose gels, and visualized by ethidium bromide staining.
Cloning and sequencing of amplified cDNA fragments
Amplified cDNA fragments were extracted from the agarose gels using the Quantum Prep Freeze 'N squeeze DNA gel extraction spin column (Bio-Rad laboratories, Hercules, CA, USA) and cloned into pSTBlue-1 AccepTor. Vector (Novagen, Darmstadt, Germany) by using DNA ligase (DNA ligation kit, Takara Bio, Inc., Otsu, Japan). Recombinant plasmids were isolated from the colonies using Quantum Prep plasmid miniprep kit (Bio-Rad laboratories, Hercules, CA, USA). Insertion of the PCR product into the plasmid was confirmed by restriction endonuclease digestion with EcoRI and subsequent gel electrophoresis. DNA sequencing was performed with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems, Foster City, CA, USA), according to the manufacturer's instructions, on an ABI Prism 3100 automated sequencer (Applied Biosystems Inc.). Nucleotide sequence data were then analysed by the GENETYX-MAC software, Version 12 (Genetyx Corp., Tokyo, Japan). Homology searches of the cDNA sequences were carried out against the previously identified genes using the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST/) of the GenBank database (National Center for Biotechnology Information, Washington, DC, USA).
Polyacrylamide gel electrophoresis (PAGE) and Western blot analysis
Membrane proteins for Western blot were prepared from mucosal samples of stomach and intestines. Samples were homogenized in a hypotonic buffer (20 mM Tris-HCl at pH 7.4, 5 mM MgCl2, 1 mM sodium EDTA, 1 mM dithiothreitol) containing a protease inhibitor cocktail (Nacalai Tesque Inc., Kyoto, Japan) and centrifuged at 200 g for 10 min at 4°C. The resulting supernatant was then centrifuged at 200 000 g for 30 min at 4°C, and the membrane pellet was resuspended in a buffer containing 62.5 mM Tris-HCl at pH 6.8, 15% (w/v) SDS, 8 M urea, 10% (w/v) sucrose, 100 mM dithiothreitol, and 10 mM sodium EDTA. Protein content was determined by the Lowry method (Lowry et al. 1951) following precipitation of samples with 20% (w/v) trichloroacetic acid in 0.015% (w/v) sodium deoxycholate. For immunoblotting, 25 µg of membrane protein from the indicated tissues were separated by 10% sodium dodecyl sulphate (SDS)-polyacrylamide gels according to Laemmli (1970), and transferred to nitrocellulose membrane (Toyo Roshi Kaisha, Ltd, Japan). The membranes were then blocked with 5% (w/v) non-fat dry milk in PBS-T (0.1% Tween 20 in phosphate-buffered saline) overnight at 4°C. Subsequently, the membranes were incubated for 1 h at room temperature with a polyclonal affinity-purified antibody raised in chickens against rat MCT1 (chicken anti-MCT1; AB1286; Chemicon International Inc., Temecula, CA, USA) at a dilution of 1 : 1000 in PBS. After being washed with PBS-T (3 x 5 min), the membranes were probed for 30 min with horseradish peroxidase-conjugated rabbit antichicken IgY (Upstate Biotechnology, NY, USA) diluted 1 : 2000 in PBS. The immunoreactive protein was visualized by the chemiluminescence protocol (ECL, Amersham International, Buckinghamshire, UK). Negative control blots were probed with the MCT1 antibody that had been preabsorbed overnight at 4°C with MCT1 peptide (10 µg ml–1) (Alpha Diagnostic International, Inc., San Antonio, USA). Densitometric analysis for the MCT1 bands was determined using Scion Image analysis software (Scion Corporation, Frederick, MD, USA).
Immunohistochemistry
The immunohistochemical localization of MCT1 protein was performed using Vectastain Elite ABC Kit according to the manufacturer's protocols. After deparafinization, sections were subjected to antigen retrieval by heating for 15 min in a microwave oven in the presence of sodium citrate buffer (0.01 M, pH 6.0). Sections were incubated in 3% (v/v) H2O2–methanol at room temperature for 10 min to quench endogenous peroxidase activity, and then washed (3 x 5 min) in PBS, followed by incubation with Block Ace (Dainippon Pharmaceutical Co., Osaka, Japan) at 37°C for 30 min, to prevent non-specific reactions. Subsequently, sections were incubated overnight with the diluted (1 : 200 in PBS) chicken anti-rat MCT1 antibody in a humidified chamber at 4°C. After washing with PBS, sections were further incubated with biotinylated bovine anti-chicken IgY (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1 : 200 for 30 min. The sections were then washed (3 x 5 min) with PBS, and then treated with 2% avidin–biotin–peroxidase complex (ABC) reagent for 30 min. Afterwards, sections were reacted with 0.5% (w/v) 3,3'-diaminobenzidine tetrachloride (Kanto Chemical Co., Inc., Tokyo, Japan) in PBS containing 0.01% H2O2, to visualize the bound antibody, and counterstained with Mayer's haematoxylin. Negative immunohistochemical controls were included in each staining run. These controls involved the omission of the primary antibody, as well as the use of MCT1 antibody that had been preabsorbed with 10 µg ml–1 of its peptide antigen.
Immunofluorescence confocal laser-scanning microscopy
Four-micrometre tissue sections were subjected to deparafinization followed by antigen retrieval by heating in sodium citrate buffer (0.01 M, pH 6.0) for 15 min in a microwave oven. Non-specific binding sites were then blocked for 30 min at room temperature with normal donkey serum (D 9663; Sigma-Aldrich, Inc., MO, USA). The sections were then washed in PBS prior to incubation with a mixture of chicken anti-rat MCT1 antibody (diluted 1 : 200 in PBS) and rabbit anti-human CD147 antibody (diluted 1 : 10 in PBS; Abcam, Cambridge, UK) in a humidified chamber at 4°C overnight. Subsequently, the sections were washed (3 x 5 min) in PBS. For detection of MCT1, the sections were incubated with the diluted (1 : 50 in PBS) donkey anti-chicken IgG conjugated to fluorescein isothiocyanate (FITC) (AP194F; Chemicon International Inc., CA, USA) for 30 min at room temperature; while for detection of CD147, the sections were incubated with the diluted (1 : 50) Alexa Fluor-594-labelled donkey anti-rabbit IgG (A21207; Molecular probes Inc., Invitrogen) for 30 min at room temperature. After washing (3 x 5 min) in PBS, sections were mounted using Vectashield mounting medium (Vector Laboratories Inc., CA, USA). The cover-slipped sections were then examined under an Olympus Fluoview confocal laser-scanning microscope (Olympus, Tokyo, Japan). For negative control studies, sections were incubated without the primary antibodies and with the primary antibodies preincubated with blocking peptides specific for the anti-human CD147 antibody (Santa Cruz Biotechnology Inc.) and anti-rat MCT1 antibody.
In vivo studies
p-chloromercuribenzoate (pCMB; Sigma-Aldrich, Inc., MO, USA), a potent inhibitor for MCT1, was used to examine the possible role of MCT1 in the transport of SCFA in goat rumen. Polyethylene catheters (Catheter kit, Terumo, Tokyo, Japan) for injection and sampling were inserted into the right ruminal artery and vein of the goats (n = 4) under general anaesthesia with pentobarbital sodium (35 mg kg–1). The catheters were kept patent by flushing with a sterile isotonic solution of trisodium citrate (3.8%). A 10 ml solution of pCMB (1, 10, 100 mM) was injected over 1 min via the catheter that inserted into the right ruminal artery (i.e. each animal received an amount of 0.01, 0.1, and 1.0 mmole pCMB in three consecutive steps). For the control, injections were carried out using the same volume of saline. Blood samples were collected from the right ruminal vein at 5 min intervals (15 min for each concentration) using syringes containing EDTA, as an anticoagulant, then immediately transferred into polyethylene test tubes, cooled on ice, and centrifuged at 4°C. The plasma supernatant was transferred to microcentrifuge tubes and stored at –30°C until analysis.
In vitro studies
The Ussing chamber technique was used to examine the involvement of MCT1 in SCFA transport across isolated ruminal epithelia collected from eight goats. Approximately 150 cm2 of rumen wall was quickly taken from the ventral sac. The rumen wall was then cleaned, transferred and prepared in ice-cold Krebs–Ringer buffer (KRB) solution containing SCFA (mM: 67 NaCl, 5 KCl, 21 NaHCO3, 0.4 NaH2PO4, 2.4 Na2HPO4, 1.2 MgCl2, 1.2 CaCl2, 10 glucose, 35 sodium acetate, 10 sodium propionate, and 5 sodium butyrate; adjusted to pH 7.4). After removing serosa and muscle layers, the isolated mucosa was cut into squares and mounted between two halves of an Ussing chamber with an exposed area of 2 cm2. Rings of silicon rubber on both sides of the tissue were used to minimize edge damage. The ruminal epithelia were then washed twice (15 min each) on both the mucosal and serosal sides with 10 ml of standard KRB solution (mM: 117 NaCl, 5 KCl, 21 NaHCO3, 0.4 NaH2PO4, 2.4 Na2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose; pH 7.4) to remove any remaining SCFA. In the control chamber, the buffer solution in the mucosal compartment was replaced by standard KRB solution (pH 6.5) containing 50 mM sodium acetate, while an equimolar amount of sodium gluconate instead of the acetate was used for the respective serosal buffer (pH 7.4). In the experiment chambers, p-chloromercuribenzoic acid (pCMBA; Sigma-Aldrich, Inc., MO, USA) was added at a final concentration of 1 mM either to the mucosal or the serosal buffer. When MCT1 inhibitor was added to either the mucosal or serosal solutions the pH was retitrated to pH 6.5 or 7.4, respectively. The temperature of the bathing buffers was kept at 37°C. All solutions were continuously gassed with carbogen (5% CO2–95% O2). Throughout the 80 min of the experiment, samples were drawn from the serosal bathing solution every 10 min and stored at –30°C until analysis.
Analyses
SCFA (acetate and propionate) concentrations in plasma and buffer samples were analysed using a gas chromatograph (GC-9A, Shimadzu Corporation, Kyoto, Japan). A portion of plasma was mixed with one-half volume of a 25% metaphosphoric acid solution containing crotonic acid (25 mM) as internal standard. Two microlitres of the sample were injected into a FAL-M glass column (2.1 m length x 3.2 mm i.d.). The column and injector temperature were 140°C and 200°C, respectively. Nitrogen, 52 ml min–1, was used as carrier gas. Plasma 3-hydroxybutyrate was measured enzymatically with a Ketone test B kit (Sanwa Kagaku Kenkyusho Co., Ltd, Nagoya, Japan) according to the manufacturer's instructions.
Statistical analysis
All measurements were carried out in triplicate, and values are expressed as means ± S.E.M. Data were subjected to analysis of variance (ANOVA), and differences among the mean values were determined by the least significant difference (LSD) test. Differences were considered significant at P < 0.05, and highly significant at P < 0.01. All the data analysis was performed using Statistica program (StatSoft Inc.).
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RT-PCR analysis was performed with primers for the MCT1 on total RNA collected from various regions of the caprine gastrointestinal tract, and revealed a PCR product of the expected size of 966 bp in all of the tissues examined (Fig. 1A). The product was absent in the negative control. The nucleotide sequence of the amplified fragments of MCT1 (Fig. 1B) has been deposited to the GenBank under the accession number AB231662. The homology searches of the identified 966 bp fragment against the previously published nucleotide sequences of MCT1, showed 99, 97, 89, 87, and 83% identities with the equivalent regions of ovine, bovine, equine, human, and rat MCT1, respectively (GenBank accession numbers: AJ315929, XM-614552, AY457175, NM-003051, and NM-012716, respectively).
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Cellular localization of MCT1 protein along the caprine gastrointestinal tract was further studied by immunohistochemical analysis using the MCT1 antibody. Widespread immunoreactive positivity for MCT1 was clearly detected within the caprine stomach and large intestine. Among the stratified squamous epithelial cells of the forestomach (rumen, reticulum, omasum), MCT1 density was mainly expressed in the cell boundaries of the stratum basale and stratum spinosum (Fig. 3A–C). In the abomasum, however, MCT1 was mainly localized at the basolateral membranes of the cells lining the surface epithelium and the gastric pit (Fig. 3D).
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The role of MCT1 in the transport of SCFA across the ruminal epithelia
To determine the contribution of MCT1 in the transport of SCFA across the rumen epithelium, we performed in vivo and in vitro functional studies using the well-documented MCT1 inhibitors, pCMB and pCMBA. The rumen was selected as a representative tissue for the gastrointestinal tract to carry out the functional studies, since this organ contains the great majority of SCFA, and it largely expressed the MCT1.
In vivo study was carried out to elucidate the effects of pCMB, in consecutive amounts, on the absorption of acetate and propionate in the rumen. The results indicated that the concentrations of acetate and propionate in the ruminal vein were decreased as the amount of pCMB increased (Fig. 4A and B). pCMB in an amount of 0.1 and 1.0 mmole resulted in a significant (P < 0.05) decrease of 24% and 44% (pooled data over 15 min for each of the inhibitor amount used), respectively, for acetate concentration in comparison with the control (inset to Fig. 4A). The equivalent amounts of pCMB also resulted in significant (P < 0.05) decrease of 41% and 53% (pooled data over 15 min for each of the inhibitor amounts used), respectively, for propionate concentration when compared with the control (inset to Fig. 4B). Nonetheless, 0.01 mmole pCMB does not show any inhibitory effect (P > 0.05) on acetate or propionate concentrations in ruminal vein when compared with the control. This result indicates that the highest inhibition for acetate and propionate transport is achieved when 1.0 mmole pCMB is used. Furthermore, 1.0 mmole pCMB revealed a significant reduction of 48% (P < 0.05) in 3-hydroxybutyrate concentration in the ruminal vein (data not shown). To ensure that the amounts of pCMB used were not toxic to the tissue in general rather than only inhibiting MCT1, we analysed the concentrations of some selected ions using the atomic absorption spectrometry (for zinc and ferrous iron) and molybdenum blue method (for phosphorous). The mean values of the measurements indicated no significant differences for zinc (P = 0.521), ferrous iron (P = 0.244), and phosphorus (P = 0.112) in the goat plasma before and after the injection of pCMB.
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To study the parallelism between the expression of MCT1 and its chaperone, CD147, we next examined the co-localization of the CD147 with MCT1 in caprine gastrointestinal sections using double immunofluorescence confocal laser-scanning microscopy. Consistent with the immunohistochemical observations, the fluorescence labelling of MCT1 was mostly visualized in the stratum spinosum and stratum basale of the forestomach epithelia (Fig. 6A), and on the basolateral domains of the epithelium lining the abomasum (Fig. 7A) and large intestine (Fig. 8A and D) of goats. The immunofluorescence of the ancillary protein, CD147, displayed a close spatial co-localization, with MCT1 immunoreactivity being present in the epithelial cells of the strata spinosum and basale of the forestomach (Fig. 6) and on the basolateral membranes of cells lining the surface epithelium and gastric pit of the abomasum (Fig. 7). In the large intestine, CD147 and MCT1 were mostly co-localized on the basolateral membranes of the cells lining the surface epithelium and the upper third of the crypts (Fig. 8).
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Distribution and precise localization of MCT1 along the caprine gastrointestinal tract
The existence of MCT1 in the caprine gastrointestinal tract was verified by RT-PCR (Fig. 1A). The sequence analysis as well as the homology searches of the amplified MCT1 cDNA against the analogous region of various animal species showed that mRNA encoding for MCT1 is present in all regions of the caprine gastrointestinal tract. The MCT1 has previously been shown to be a ubiquitous isoform in the great majority of tissues of all species studied (Halestrap & Meredith, 2004). Our current findings of MCT1 mRNA being present in all segments of the caprine gastrointestinal tract are consistent with the ubiquitous nature of this MCT isoform.
MCT1 is a 55 kDa protein containing 12 membrane-spanning regions (Poole et al. 1996), that runs at 45 kDa on SDS-PAGE. In Western blot study, a band with the expected molecular mass (45 kDa) of MCT1 was detected in the plasma membrane protein prepared from each of the tissues examined (Fig. 2A). This band matches that of the 43 kDa protein seen in blots of calf forestomach and large intestine (Kirat et al. 2005), sheep gastrointestinal tract (Kirat et al. 2006), and hamster tissues (Garcia et al. 1995), also it matches the 48 kDa band detected in the colonic luminal membrane of human and pig (Ritzhaupt et al. 1998b). Densitometric analysis revealed that the MCT1 protein was found most abundantly in the forestomach, at intermediate levels in the large intestine and abomasum, and at a very low level in the small intestine of goats (Fig. 2B). The order of MCT1 intensity was; rumen reticulum > omasum > caecum > proximal colon > distal colon > abomasum > small intestine. This finding is consistent with the expression levels previously reported for bovine and ovine MCT1 (Kirat et al. 2005; Kirat et al. 2006). The considerable regional differences observed in the protein level of MCT1 in the forestomach as well as along the length of the caprine intestine are in harmony with the production and absorption site of the SCFA in ruminants. Elsden et al. (1946) compared total SCFA concentrations at different sites in the gastrointestinal tract of many mammalian species, and found that ruminants had the highest SCFA concentrations in the rumen, a fall to less than one-fifth of these values in the small intestine, and a second peak in the caecum and colon.
The immunohistochemical results were confirmed using confocal laser-scanning microscopy for MCT1 in the caprine gastrointestinal tract, and both corroborated the results of Western blot analysis and provided insight into the possible role of MCT1 in SCFA transport across the rumen epithelium. Morphologically, the ruminal epithelium is a stratified squamous epithelium consisting of, from the blood side, four cell layers: the stratum basale, stratum spinosum, stratum granulosum and stratum corneum. In the present study, the immunoreactive staining for MCT1 was mostly present in the stratum basale and stratum spinosum (Figs 3A—C and 6A). In contrast to these findings, studies conducted by Müller et al. (2002) on sheep rumen have demonstrated that the MCT1 immunoreactivity was present only in stratum basale cells of the intact ruminal epithelium, although they could detect MCT1 protein in the vast majority of cultured ruminal epithelial cells.
The overall distribution of MCT1 in the plasma membranes of the stratum basale and stratum spinosum suggested that it might play a role in the flux of SCFA across the functional thickness of the rumen epithelium into the bloodstream. Recently, a functional model of the rumen epithelium has been suggested on the basis of the expression of junctional markers, gap junctions and the Na+–K+ ATPase; that is the cells of the stratum granulosum, spinosum and basale form a functional syncitium interconnected by gap junctions, and the Na+–K+ ATPase is concentrated in the stratum basale (Graham & Simmons, 2005). The pattern of MCT1 distribution in the ruminal epithelium in our study is consistent with intercellular communication within and between the cells of the strata granulosum, spinosum, and basale, forming a syncytium (Saez et al. 2003; Graham & Simmons, 2005). In addition, the cells of the strata basale and spinosum have significant numbers of mitochondria, while the stratum granulosum contains progressively fewer numbers of mitochondria (Baldwin, 1998), and consequently stratum basale and spinosum cells are the most important cells of the rumen with regard to whole-animal energy metabolism (Baldwin, 1998).
Furthermore, the present work also studied the precise cellular localization of MCT1 protein in the caprine large intestine (Figs 3E–G and 8A and D), and provides evidence that MCT1 protein expression was mostly in the basolateral membrane of the epithelial cells lining the caprine caecum and colon. With respect to membrane localization studies of MCT1 in the large intestine, Garcia et al. (1995) demonstrated that MCT1 was localized to the basolateral membranes of hamster caecal epithelial cells. In contrast, other studies have demonstrated the localization of MCT1 to the colonic luminal membranes of human and pig (Ritzhaupt et al. 1998b; Gill et al. 2005), as well as in Caco-2 cells (Buyse et al. 2002).
MCT1 has a direct role in the transport of SCFA across the ruminal epithelia
The molecular data in the present study are strongly suggestive of a role of MCT1 in the transport of SCFA. This proposal was confirmed directly by in vivo and in vitro functional studies based on the utilization of powerful inhibitors for MCT1. The organomercurial thiol reagents that modify cysteine residues, such as pCMBA and pCMB, have been reported as potent inhibitors for MCT1 (Poole & Halestrap, 1993; Garcia et al. 1994, 1995). Studies by Ritzhaupt et al. (1998a) and Cuff et al. (2002) showed that pCMB induced an inhibitory effect on butyrate uptake across the human colonic luminal membrane vesicles as well as in the cultured human colonic epithelial cell, AA/C1.
Our in vivo studies revealed that the injection of pCMB (0.1 or 1 mmole) into the ruminal artery resulted in a significant reduction in the venous acetate and propionate concentrations, and this indicates the direct role of MCT1 in the absorption of SCFA in rumen. Furthermore, under the same conditions, pCMB was found to provoke a significant reduction in 3-hydroxybutyrate, which substantiates what had been reported by Müller et al. (2002) who claimed that MCT1 can mediate the extrusion of ketone bodies and lactate from the sheep rumen epithelium.
In vitro studies by Ussing chamber technique, indicated that the flux of acetate from rumen to blood was significantly (55%; P < 0.01) reduced, when pCMBA (1 mM) was applied to the blood side of the isolated ruminal epithelial sheets; however, a low percentage inhibition (34%; P < 0.05) in acetate flux from rumen to blood was achieved when the inhibitor was applied on the rumen side, which may be indicative of the existence of MCT1 toward the lumen of the rumen, but at a low level. The significant difference (P < 0.05) in acetate transport between the mucosal and serosal application of the MCT1 inhibitor (34% versus 55%) was in harmony with the results of immunohistochemistry and immunofluorescence, which revealed that MCT1 was mostly expressed on the cell membranes of stratum basale and stratum spinosum. In ruminants, however, the identity of the SCFA transporter on the basolateral side of the gastrointestinal epithelia is not known. Our data provided strong molecular and functional evidence for the presence of MCT1 that is able to transport the SCFA across the ruminal epithelium to the blood.
Interestingly, MCT1 appears to play a major role in rumen physiology. We recognized that MCT1 can act primarily to transport SCFA to the circulation, where they are subsequently used by liver and peripheral tissues for the synthesis of glucose and fatty acids, respectively. In addition, MCT1 is involved in the transport of ketone bodies. Since the transport via MCT1 is proton linked (Halestrap & Meredith, 2004), it is also likely that it can maintain the intracellular pH (pHi) of the ruminal epithelium. The ruminal epithelial cells are permanently exposed to high concentrations of SCFA in vivo, and although SCFA are beneficial for the animal, they are implicated in the intraruminal release of large amounts of protons. Therefore, the mechanisms of SCFA absorption must be complemented by epithelial mechanisms for stabilizing the intracellular pH. In this case, MCT1 is probably one of the major mechanisms that help in the transepithelial transfer of the dissociated form of SCFA into the blood, and thus would contribute directly to maintaining the intracellular pH homeostasis as well as stabilization of the intraruminal pH, since the protons are eliminated together with the substrate via MCT1.
Co-localization of MCT1 with CD147 in the caprine gastrointestinal tract
MCT1 requires an ancillary protein known as CD147. CD147 is a glycoprotein containing a single transmembrane span that facilitates targeting of MCT1 to the plasma membrane, where they remain tightly bound to each other, and this association appears to be important in determining its activity and location (Kirk et al. 2000). Herein we demonstrated, for the first time, the precise cellular localization of CD147, as well as its co-localization with MCT1 in the gastrointestinal tract in ruminants. Double-immunofluorescence confocal laser-scanning microscopy revealed the co-localization of MCT1 with CD147 in the stratum spinosum and stratum basale cells of the forestomach, and on the basolateral membranes of the cells lining the surface epithelium and the upper third of the crypts. The remarkably similar distribution of these two proteins is demonstrated by the overlay images of Figs 6–8. These observations substantiate the fact that CD147 is required for the proper localization of MCT1 in a wide range of cells of ruminant gastrointestinal epithelium.
The extended localization of CD147 on the plasma membranes throughout the stratified squamous epithelia of the forestomach and the simple epithelial cells of the abomasum and large intestine highlights the possible presence of other MCT isoforms such as MCT3 and/or MCT4 in the cell layers toward the lumen of the forestomach, as well as toward the base of the crypts in the large intestine. It has been demonstrated that CD147 is necessary for delivery of MCT3 and MCT4 to the plasma membrane (Deora et al. 2005). These findings could explain the uneven effect of pCMBA that resulted from our in vitro study.
Based on the results of in vivo and in vitro functional studies, in cooperation with the immunohistochemical and immunofluorescence confocal analyses, we can provide evidence, for the first time, that MCT1 has a direct role in the transepithelial transport and efflux of SCFA across the stratum spinosum and stratum basale of the forestomach toward the blood side. Furthermore, the study illustrates the coexpression of MCT1 with CD147, and substantiates the fact that CD147 is required for the proper localization of MCT1 in a wide range of cells in the ruminant gastrointestinal epithelium.
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): control; (
): 0.01 mmole pCMB; (•): 0.1 mmole pCMB; (
): 1 mmole pCMB. Reduction percentage (relative to control) in acetate (A, inset) and propionate (B, inset) concentrations induced by injection of different amounts of pCMB (pooled data over 15 min for each amount). All data points are means ±
S.E.M., represented by vertical bars, from four goats. Significant difference was identified at P < 0.05 (*).
