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1 Department of Veterinary Physiology, School of Veterinary Medicine, Rakuno Gakuen University, 582 Bunkyodai-Midorimachi, Ebetsu, Hokkaido 069-8501, Japan
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Abstract |
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(Received 2 March 2006;
accepted after revision 10 July 2006; first published online 20 July 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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Short-chain fatty acids (SCFA) are the products of the anaerobic microbial fermentation of complex carbohydrates in the forestomach and large intestine. Acetate, propionate and butyrate, the predominant SCFA, are readily absorbed and assimilated as a nutrient source by the ruminant animal (Bergman, 1990). Ruminants depend on SCFA for up to 80% of their maintenance energy requirements (Bergman, 1990). Caecal SCFA provides, on average, 8.6% of metabolizable energy intake in bovines (Siciliano-Jones & Murphy, 1989). Caecal and colonic fermentation accounts for 8.6–16.8% of total SCFA production in ruminants (Ulyatt et al. 1975). In addition to their involvement as the major source of energy, the SCFA also serve as building blocks for milk synthesis; acetate is a necessary component in the formation of milk fat, while propionate is used for glucose production, which is needed for the synthesis of milk sugar (lactose). In ruminants, propionate is also the major substrate of hepatic gluconeogenesis (Herdt, 1988). Thus, effective absorption of SCFA from the forestomach and large intestine is essential for these species. Much work has been conducted concerning the production and absorption of SCFA from the rumen (Rechkemmer et al. 1995; Remond et al. 1996; Kramer et al. 1996; Gäbel & Sehested, 1997), but there have been few reports concerning SCFA absorption from the lower digestive tract of ruminants.
The transport of short-chain monocarboxylates across the plasma membrane in most cells is largely dependent on a family of specific MCTs (monocarboxylate transporters). The family of MCTs includes up to 14 subtypes (MCT1–MCT14), although the monocarboxylate substrate specificity has been demonstrated for only MCT1–MCT4 (Halestrap & Meredith, 2004). The first MCT, designated MCT1, is a membrane-bound protein with 12 predicted transmembrane regions that facilitates the transport of lactic acid, pyruvic acid, short-chain fatty acids and ketone bodies across biological membranes (Halestrap & Price, 1999). MCT1 is an obligatory symporter that carries a dissociated proton–monocarboxylate pair with each transport cycle (Halestrap & Meredith, 2004).
We have previously shown that MCT1 is expressed along the gastrointestinal tract of preruminant calves (Kirat et al. 2005) and adult sheep (Kirat et al. 2006), and we suggested that it may possibly play a role in the transport of SCFA across the ruminant gastrointestinal tract. The present study was designed to determine, by in vitro studies, the functional role of MCT1 in the bovine large intestine.
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Methods |
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Nine lactating Holstein-Friesian cows (4–6 years old) weighing 450–644 kg were used in this study. 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 is 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).
Sample preparation
Animals were killed by exsanguination following intravenous injection of sodium pentobarbital (35 mg kg–1). Immediately, segments from the large intestine (caecum, proximal colon and distal colon) were excised, opened along the mesenteric border, and rinsed in a chilled cold saline (0.9% NaCl) solution. For reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot studies, the epithelia of each region were scraped off using glass slides on ice, immediately frozen in liquid N2, and subsequently stored at –80°C until use. For immunohistochemical studies, samples were immediately fixed in 4% paraformaldehyde for 24 h then the tissues were dehydrated through a series of graded concentrations of ethanol and xylene, embedded in paraffin and sectioned serially at 4 µm. For in vitro studies, pieces of caecum were carefully cleaned and prepared in ice-cold, carbogen (95% O2–5% CO2)-gassed Krebs–Ringer solution containing SCFA (Table 1). The serosa and outer muscle layer were removed by placing a section of the sheet of caecum, serosal side up, on a silicon rubber plate and pinning it in place with needles. A transverse incision was made through the muscle layers with a razor blade, and the layers were peeled off longitudinally with fine curved forceps. The stripped caecal mucosa was cut into 3-cm-long sections and mounted in an Ussing chamber (exposed surface area, 2 cm2). Edge damage was minimized by rings of silicon rubber on both sides of the tissue. Immediately before transport studies, the mounted tissues were washed twice (15 min each) on both mucosal and serosal sides with 10 ml of Krebs–Ringer buffer (KRB) solution (Table 1) to remove any remaining SCFA. Solutions were aerated by being bubbled with carbogen and were maintained at 37°C.
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Total RNA was collected from the caecum, as well as proximal and distal colon, using RNeasy Mini Kit (Qiagen Sciences, Germantown, MD, USA) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed into cDNA using Superscript II and oligo-d(T)12–18 (Invitrogen, Carlsbad, CA, USA). Polymerase chain reaction amplification was conducted on synthesized cDNAs using Taq DNA polymerase (New England BioLabs Inc., Beverly, MA, USA). MCT1 primer pairs were derived from Bos taurus MCT1 (GenBank accession number: NM-001037319). The primer sequences were 5'-ACAATGCCACCAGCAGTTGGAGGTC-3' and 5'-TACAGGACAGCACTCCACAATGGTC-3' for sense and antisense, respectively. After an initial denaturation at 94°C for 2 min, 35 cycles of amplification with a thermocycler (iCycler, Biorad) were performed under the following conditions: 94°C for 30 s, 58°C for 30 s and 72°C for 3 min followed by a final extension for 10 min at 72°C. 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. Products of the PCR were analysed by electrophoresis in 1% agarose gels and visualized by ethidium bromide staining.
The amplified cDNA fragments were then extracted from the agarose gels using the Quantum Prep Freeze 'N Squeeze DNA gel extraction spin column (Biorad) and cloned into pSTBlue-1 AccepTorTM 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 (Biorad). Insertion of the PCR product into the plasmid was confirmed by restriction endonuclease digestion with EcoRI and subsequent gel electrophoresis. Sequencing of DNA 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).
Immunohistochemical analysis
Four-micrometer paraffin sections of caecum, proximal colon and distal colon were processed for immunohistochemical analysis using the avidin–biotin peroxidase (Vectastain Elite ABC Kit; Vector Laboratories, Inc.) as previously described (Kirat et al. 2005). For immunostaining, sections were incubated overnight at 4°C with a polyclonal affinity-purified antibody raised in chickens against rat MCT1 (chicken anti-MCT1; AB1286; Chemicon International Inc., Temecula, CA, USA) diluted at 1:200 in phosphate-buffered saline (PBS; g l–1: 8.0 NaCl, 1.15 Na2HPO4, 0.2 KCl, 0.2 KH2PO4; pH 7.4). Thereafter, they were treated with secondary antibody (biotinylated goat anti-chicken IgY; Santa Cruz Biotechnology) at a dilution of 1:200 for 30 min. To detect immunoreactivity, the sections were treated with 0.5% 3,3'-diaminobenzidine tetrachloride (Kanto Chemical Co., Inc., Tokyo, Japan) in PBS containing 0.01% H2O2. For negative control slides, the tissue sections were treated with the chicken anti-rat MCT1 antibody that had been pre-incubated with rat MCT1 peptide (10 µg ml–1; Alpha Diagnostic International, San Antonio, TX, USA) for 24 h at 4°C before use. The sections were then counterstained with Mayer's Haematoxylin.
Immunofluorescence confocal laser microscopy
After deparafinization, 4 µm tissue 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). Non-specific binding sites were then blocked for 30 min with normal goat serum (G 9023; Sigma-Aldrich, Inc.). Subsequently, sections were incubated overnight with the diluted (1:200 in PBS) primary antibody, chicken anti-rat MCT1 antibody, in a humidified chamber at 4°C. The sections were then washed (3 x 5 min) in PBS. For visualization, sections were labelled with goat anti-chicken IgG conjugated to fluorescein isothiocyanate (FITC; 60360; Alpha Diagnostic International, San Antonio, TX, USA) at a dilution of 1:100 for 30 min at room temperature. After washes (3 x 5 min) in PBS, sections were mounted using Vectashield mounting medium (H-1000; Vector Laboratories, Inc., Burlingame, CA, USA). The coverslipped sections were then examined under an Olympus Fluoview confocal laser-scanning microscope (Olympus, Tokyo, Japan). Negative control slides were included in each staining run. These controls involved the omission of the primary antibody as well as the use of the primary antibody preabsorbed with its peptide antigen.
Western blot analysis
Western blot analysis was performed as previously described (Kirat et al. 2005). Briefly, plasma membrane proteins (60 µg) prepared from caecum, proximal and distal colon of cows were separated by electrophoresis on a 10% SDS-polyacrylamide gel and electroblotted into nitrocellulose membranes (Amersham Pharmacia Biotech). The blotted membrane was blocked in PBS containing 5% skimmed milk, incubated for 60 min with the primary antibody (chicken anti-rat MCT1) diluted in the same buffer (1:500), washed with 0.1% Tween 20 in PBS, and then incubated for 30 min with horseradish peroxidase-conjugated rabbit anti-chicken IgY diluted at 1:2000 (Upstate Biotechnology, Lake Placid, NY, USA). After washing, signals were detected using the Enhanced Chemiluminescense System (ECL; Amersham International, Buckinghamshire, UK). To verify the specificity of immunoreactions, the membranes were probed with the primary antibody that had been preabsorbed overnight at 4°C with its peptide antigen (rat MCT1, 10 µg ml–1). Quantification of band intensities was performed by scanning the immunostaining band and analysing the image with Scion Image analysis software (Scion Corporation, Frederick, MD, USA).
Transport studies
The effect of pH. The effect of pH on acetate transport across the cow caecal epithelium was determined. Caecal epithelia were incubated for 30 min in KRB solution containing 50 mM sodium acetate at mucosal side pH values of 5.5, 6.0, 6.5, 7.0 or 7.4, whereas the respective serosal buffer, which contained an equimolar amount of sodium gluconate instead of the acetate, was set at pH 7.4. The sodium concentration was kept constant by reducing the amount of sodium chloride in the KRB solution as appropriate (see Table 1). The solutions were continuously gassed with carbogen and maintained at 37°C.
The effect of MCT1 inhibitor. We examined whether the MCT1 inhibitor ‘p-chloromercuribenzoic acid (pCMB; Sigma-Aldrich, Inc.)’ can affect the acetate transport across the bovine caecal epithelia. In the control chamber, the buffer solution in the mucosal compartment was replaced by 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; detailed composition is described in Table 1). In the experiment chambers, pCMB was added at a final concentration of 0.1, 0.5 or 1.0 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 6.5 or 7.4, respectively. All solutions were continuously gassed with carbogen and maintained at 37°C.
The effect of substrate analogue. The possible competitive effect of the substrate analogue ‘propionate’ on transport of acetate was also investigated. The amount of acetate transported across the cow caecal epithelium was measured for 60 min in the absence (control chamber) or presence of sodium propionate. Acetate was applied at the same concentration and in the same conditions used for the MCT1 inhibitor experiment, while propionate was added on the mucosal side at a final concentration of 100 mM.
Samples were drawn from the serosal bathing solution every 10 min over the experimental period and stored at –30°C until analysis.
Analyses
Acetate concentrations in the serosal buffer samples were analysed using a gas chromatograph (GC-9A, Shimadzu Corporation, Kyoto, Japan). Two microlitres of the sample were injected into a FAL-M glass column (2.1 m long x 3.2 mm i.d.). The column and injector temperature were 140 and 200°C, respectively. Nitrogen, 52 ml min–1, was used as carrier gas.
Statistical analysis
All measurements were carried out in triplicate, and values are expressed as means ± S.E.M. Values obtained in the analogue experiment were subjected to Student's unpaired t test, while data from all other experiments 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 < 0.01. All the data analysis was performed using Statistica program (StatSoft Inc.Tokyo, Japan).
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Results |
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The result of RT-PCR indicated that MCT1 mRNA is highly expressed in bovine large intestinal mucosa. Agarose gel electrophoresis revealed a PCR product of the predicted size, 1200 bp (Fig. 1). The product was absent in the negative control. The nucleotide and amino acid sequences of the amplified fragments of MCT1 have been deposited to the GenBank under the accession number AB250265 [GenBank] . The homology searches of the identified 1200 bp fragment against the previously published nucleotide sequences of MCT1 showed 99, 99, 98, 90, 88, 86 and 84% identities with the equivalent regions of bovine, ovine, caprine, equine, human, Chinese hamster and rat MCT1, respectively (GenBank accession numbers: BC104598 [GenBank] , AJ315929 [GenBank] , AB231662 [GenBank] , AY457175 [GenBank] , BC045664 [GenBank] , L25842 [GenBank] and NM-012716, respectively).
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Immunohistochemical analysis of bovine large intestine demonstrated that the MCT1 protein is present in the epithelial cells lining the caecum as well as the proximal and distal colon of cows (Fig. 2A–C). The transporter protein was abundant in the surface epithelium, and the amount decreased from the opening of the crypt to its base. This finding has been further substantiated by confocal laser-scanning microscopy, which revealed that MCT1 immunofluorescence was localized predominantly on the basolateral membranes of the epithelium lining the caecum and the proximal and distal colon (Fig. 3A–E). No immunostaining was seen in the in the negative control experiments, confirming the specificity of the immunoreactions (see Figs 2D and 3F).
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A single band of 43 kDa was recognized by Western blot analysis in the plasma membrane protein prepared from large intestinal epithelial scrapings, collected from adult cows (Fig. 4A). The band was absent when the MCT1 antibody was preabsorbed with its peptide, confirming the specificity of immunoreactions (data not shown). The densitometric analyses of the immunoblots confirmed that, among the large intestinal segments, MCT1 protein levels were higher (P < 0.01) in the caecum and proximal colon than in distal colon (Fig. 4B).
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Time course and pH dependence of acetate transport. The time-dependent transport of 50 mM acetate by caecal epithelium was examined. As shown in Fig. 5, increasing amounts of acetate were transported to the serosal side as the time increased. Moreover, acetate absorption across the caecal epithelium correlated positively with the concentration of protons. We found that the transport of acetate to the serosal side was significantly increased when the pH on the mucosal side was lowered from 7.4 to 5.5 (Fig. 5).
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Discussion |
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Reverse transcriptase-polymerase chain reaction studies and sequence analysis of the PCR product confirmed the expression of MCT1 in bovine large intestine (Fig. 1). The particular band of 43 kDa protein recognized by Western blot analysis is consistent with the predicted molecular mass of 40–50 kDa for the MCT1 protein (Poole et al. 1996). The presence of MCT1 at both mRNA and protein levels in bovine caecum and colon was consistent with our earlier studies on calf and sheep large intestine (Kirat et al. 2005, 2006).
Our immunofluorescence confocal laser-microscopy findings (Fig. 3) confirmed the results obtained by the immunohistochemistry (Fig. 2), and both indicated that MCT1 is predominantly localized on the basolateral membranes of epithelial cells lining the bovine caecum and colon. Studies of the intestinal distribution of MCT1 isoform have shown the localization of MCT1 to be either on the luminal or the basolateral plasma membrane. Regarding the membrane localization of MCT1 in 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 luminal membranes of human (Ritzhaupt et al. 1998b; Gill et al. 2005) and pig colon (Ritzhaupt et al. 1998b), as well as in Caco-2 cells (Buyse et al. 2002).
It is well known that the transport of monocarboxylates via MCT1 is proton linked (Juel & Halestrap, 1999; Halestrap & Meredith, 2004). Our in vitro studies showed that the transport of acetate by caecal cells was pH dependent (Fig. 5), indicating that acetate transport across the caecal epithelium involves cotransport of protons. This finding is in accordance with those of Ritzhaupt et al. (1998a) and Stein et al. (2000), who declared that the uptake of butyrate across human and pig colonic luminal membranes, as well as Caco-2 cells, was stimulated significantly with the reduction of extracellular pH.
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 cultured human colonic epithelial cells, AA/C1. In the present study, the rate of acetate absorption under the influence of the potent MCT1 inhibitor, pCMB (Poole & Halestrap, 1993) is shown in Fig. 6A and B. The significant inhibitory effect of pCMB on the acetate transport that was achieved when it was applied into the serosal side versus the non-significant inhibitory effect when it was applied into the mucosal side (Fig. 6B) is in agreement with the results of the immunohistochemistry (Fig. 2) and the immunofluorescence confocal microscopy (Fig. 3), which revealed that MCT1 was mostly expressed in the basolateral membranes of the caecal epithelial cells.
It has been reported that MCT1 has the ability to transport a number of monocarboxylates in addition to butyrate; these include acetate and propionate (Tamai et al. 1999; Juel & Halestrap, 1999; Stein et al. 2000). Our results indicated that the structural analogue ‘propionate’ induced a significant inhibition of the acetate efflux (Fig. 6C), indicating a possible competition for the carrier by the alternative SCFA substrates (i.e. acetate and propionate compete for binding to the same transporter).
Equilibrium is necessarily established within and between the different compartments of the epithelium, i.e. luminal, intracellular and basolateral. Slight changes in one compartment will be reflected in changes in other compartments. Minor changes in pH across membranes may have a profound effect on the relative distribution of SCFA. This presents a challenge for physiologists studying transport to clarify the mechanisms of SCFA movement across epithelia. The pKa of SCFA is close to 4.8. As calculated from the Henderson–Hasselbalch relationship, 95% or more of the SCFA are present in the dissociated form (anions) at the physiological pH (6–7) of the intestine (Bergman, 1990; Titus & Ahearn, 1992). In contrast to the undissociated form, which is lipid soluble and therefore readily permeates the lipid bilayers of epithelial cell membranes by non-ionic diffusion, the dissociated form requires specific transport proteins to cross into or out of a cell.
In addition to the non-ionic diffusion of SCFA (Myers et al. 1967), a considerable amount of SCFA has also been anticipated to be absorbed in the large intestine via SCFA––HCO3– exchange. Such a SCFA––HCO3– mechanism was concluded to be present in the rat distal colon (Mascolo et al. 1991) and guinea-pig caecum and proximal colon (von Engelhardt et al. 1994), as well as in the human colon (Harig et al. 1996). Further evidence of the presence of such an antiport mechanism includes HCO3– secretion in the presence of SCFA in pig proximal colon (Argenzio & Whipp, 1979), pony ventral colon (Argenzio et al. 1977) and sheep colon (Rübsamen & von Engelhardt, 1981).
During transfer through the epithelial layers, SCFA function as direct or indirect carriers of protons (Gäbel et al. 2002). Since such a large amount of protons is taken up, there is a risk of lethal intracellular acidification. Therefore, protons have to be rapidly and effectively extruded by the intestinal epithelial cells to maintain a constant intracellular pH. Thus, the mechanisms of SCFA absorption must be complemented by epithelial mechanisms for stabilizing the intracellular pH. The MCT1 on the basolateral membrane provides a major mechanism that helps the intracellular pH to recover from SCFA-induced acidification. The increase in intracellular protons will lead to enhanced transport of the ionic form of SCFA across the basolateral membranes via MCT1.
The proposed model in Fig. 7 illustrates the role of MCT1 in the transport of SCFA and intracellular pH regulation. Short-chain fatty acids are known to be taken up either as the protonated form (HSCFA) by passive diffusion or as the ionic form (SCFA–) in exchange for bicarbonate. Both of these mechanisms will acidify the cytosol, since the entry of the protonated SCFA into cells results in the release of protons and ionized SCFA with a subsequent decrease in the intracellular pH. In addition, the intracellular carbonic anhydrase-catalysed hydration of CO2 results in the generation of protons and bicarbonate; the latter enters the lumen in exchange for ionized SCFA and leads to a reduction in the intracellular buffer capacity. The resultant protons, either from the intracellular dissociation of the protonated SCFA or from the hydration reaction, are suggested by this study to exit along with the ionized SCFA across the basolateral membrane by MCT1.
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References |
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500 bp) was used as an internal standard in all tissues (bottom gel).
