Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, and Jesse Brown Veterans Affairs Medical Center, Chicago, Illinois
Submitted 11 March 2005 ; accepted in final form 9 May 2005
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ABSTRACT |
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monocarboxylate transporter; short-chain fatty acids; absorption; short-chain fatty acid transport; mammalian colon
Several studies have attempted to investigate the mechanism(s) of absorption of SCFAs across the plasma membrane of the intestinal epithelial cells. In this regard, SCFA absorption across the intestinal mucosa has been shown to occur through the nonionic diffusion of protonated SCFAs and/or a carrier-mediated anion exchange (7, 9). We have clearly demonstrated (16, 17) the presence of HCO3-dependent, carrier-mediated anion exchange for SCFA uptake in the apical membranes of the human ileum and colon. Recent studies from our laboratory (12) and others (28, 29) have demonstrated the involvement of monocarboxylate transporter (MCT)1 in the absorption of SCFAs across the apical membrane in the human intestine. It was shown previously that absorbed butyrate is not completely metabolized by colonocytes and thus could exit across the basolateral membrane into the bloodstream (30, 34). In this regard, previous functional studies from our laboratory and others have characterized a kinetically distinct basolateral SCFA/anion exchanger in the basolateral membrane of human (34) and rat colon (27). The basolateral membrane SCFA/anion exchangers exhibited Km for butyrate of 17.5 ± 4.5 mM compared with the luminal SCFA/anion exchangers (Km for butyrate 1.5 ± 0.2 mM) (34), indicating the involvement of possibly distinct molecular isoforms of the MCTs expressed in the colon. However, the molecular identity of the basolateral membrane SCFA transporter and whether it is encoded by a MCT isoform other than MCT1 are not known yet.
Fourteen MCT isoforms have been identified to date in mammals, each having a unique tissue distribution (13). Direct demonstration of the functional transport for mammalian MCT14 has been demonstrated (14). MCT1 is ubiquitously expressed but is especially prominent in heart and red muscle (24). MCT2 and MCT3 distribution has been shown to be more restricted (18, 21, 23, 35). MCT5 is primarily found in placenta (26), MCT6 in kidney and placenta (26), MCT7 in pancreas and brain, and MCT8 in liver, kidney, and myocardium (26). Our RT-PCR studies (26) showed that MCT1, -3, -4, -5, and -6 isoforms were expressed in the human colonic carcinoma cell line Caco-2. The possibility of multiple MCT isoforms expressed in the human intestine makes the establishment of the molecular identity of the apical and basolateral SCFA transporters complex. Therefore, detailed studies are needed to understand the membrane localization as well as the regional expression of MCT isoforms to clearly define their role in the transport of SCFAs, especially across the basolateral membrane of polarized human intestinal epithelial cells.
The current studies were designed to assess the expression and membrane localization of different MCT isoforms along the length of the human intestine with immunoblotting and immunohistochemical studies. Our findings demonstrate that the MCT1, -4, and -5 isoforms are the predominant isoforms expressed in the human colon, with MCT1 localized to the apical membrane and MCT4 and -5 isoforms localized to the basolateral membranes. MCT3 expression was found to be significantly low compared with other MCT isoforms, and it was found to be basolaterally localized.
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MATERIALS AND METHODS |
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Isolation of human small intestinal and colonic plasma membrane vesicles.
These investigations were approved by the Human Investigation Committee of the Jesse Brown VA Medical Center and the Institutional Review Board of the University of Illinois at Chicago. Small intestine and colon from healthy adult organ donors (primarily trauma victims) were obtained immediately after harvest of transplantation organs. The small intestine was divided into two equal parts after discarding one-third of the middle portion. The first half was considered to be jejunum and the second half to be ileum. After the cecum was discarded, the remaining large intestine was divided equally into proximal and distal colon. The mucosa was scraped from the seromuscular layer of these segments and stored at 80°C. The apical membranes from these four regions of the intestine were prepared as described previously (10, 15, 17). The purity of membrane vesicles and the degree of contamination with intracellular organelles were assessed using appropriate marker enzymes (10, 15, 17). The small intestinal membranes exhibited 15- to 20-fold purity over the crude homogenate as checked by the activities of alkaline phosphatase and sucrase-isomaltase. The colonic membranes showed 7- to 10-fold purity over the crude homogenate as checked by the activity of cysteine-sensitive alkaline phosphatase.
The human small intestinal and colonic basolateral plasma membranes were purified by using a Percoll density gradient technique recently established in our laboratory (33, 34, 36). The final membrane pellet was suspended in 1x PBS by passing it 10 times through a 25-gauge (1.5 in.) needle. The purity of the membrane vesicles and the degree of contamination with intracellular organelles were assessed by appropriate marker enzymes. Enrichment with the marker enzyme activity Na+-K+-ATPase was 8- to 11-fold in the basolateral membrane over that of homogenate. The membranes showed minimal contamination with markers of the apical or microsomal membranes. Membrane protein was assessed as described by Bradford (3), using BSA as the standard.
MCT isoform-specific antibodies. The polyclonal MCT isoform-specific antibodies were raised commercially (Alpha Diagnostics) by immunizing rabbits with synthetic peptides corresponding to the COOH terminus of MCT1 [amino acids (aa) 482500], MCT4 (aa 201219), MCT5 (aa 488505), MCT3 (aa 202221), and MCT6 (aa 510523). A search in the BLAST database revealed no membrane proteins with significant homologies to the peptide sequences used for immunization: MCT1, ESPDQKDTEGGPKEEESPV; MCT4, KSENNSGIKDKGSSLSAHG; MCT5, PKAVLQAKQTALGWNSPT; MCT3, VTAQPGSGPPRPSRRLLDL; and MCT6, FLEMDLAKNHERVHVQME.
Immunoblotting. For immunoblotting studies, briefly, 150 µg each of purified human small intestinal and colonic apical membranes were solubilized in Laemmli sample buffer (2% SDS, 100 mM dithiothreitol, 60 mM Tris, pH 6.8, 0.01% bromophenol blue) and separated on 12% Tris-glycine SDS-polyacrylamide gels. The separated proteins were electroblotted onto nitrocellulose membranes and stained with Ponceau S to ensure equal loading of the protein in each lane. The blot was then probed with MCT1, MCT4, or MCT5 (overnight, 4°C, diluted 1:2,0001:5,000) or MCT3 (overnight, 4°C, diluted 1:50) antibody. The bands were visualized using enhanced chemiluminescence according to the manufacturer's instructions (ECL; Amersham).
Immunohistochemical analysis. Formalin-fixed paraffin-embedded tissues of human colonic crypts were obtained from human pinch biopsy samples or tissues that had been surgically removed. Collection of these specimens was performed according to protocols approved by the Human Investigation Committee of the Jesse Brown VA Medical Center and the Institutional Review Board of the University of Illinois at Chicago. Immunohistochemical studies were carried out on 5- or 7-µm paraffin sections of colonic tissues mounted on (3-aminopropyl)triethoxysilane-treated slides. The slides were deparaffinized in xylene for 20 min to remove the embedding media and washed in absolute ethanol for 5 min. The slides were then gradually rehydrated gently in a series of alcohol washes, including 96%, 85%, and 50%, and placed in distilled water for 5 min each. The activity of endogenous peroxidase was blocked by incubating the slides for 1 h in 97% methanol solution containing 3% hydrogen peroxide and 0.01% sodium azide. After permeabilization with 0.1% Triton X-100, the slides were then incubated for 1 h at room temperature (RT) in 20% normal goat serum in PBS containing 1% BSA to block nonspecific antibody binding. This was followed by incubation of the slides with rabbit polyclonal antibodies to human MCT isoform-specific antibodies, diluted 1:125 (MCT1, 4°C, overnight) or 1:100 (MCT5, RT, 1 h). After the slides were washed three times for 5 min with PBS, liquid DAB+chromogen (3,3'-diaminobenzidine solution, Dako) was applied (until the brown chromogen stain was detected). The reaction was stopped by immersing the slides in distilled water. The slides were counterstained with hematoxylin for 30 s to counterstain the cell nuclei. For control experiments, the sections were incubated with preimmune rabbit serum and with primary antibody omitted.
Image acquisition. The immunostained slides were examined with a Nikon Eclipse E400 microscope. Digital images were captured with a spot insight digital camera.
Statistical analysis. All experiments were performed with at least three or four freshly isolated membrane preparations from different organ donors.
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RESULTS |
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DISCUSSION |
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MCT1 was the first isoform to be cloned from Chinese hamster ovary cells and has been shown to transport monocarboxylates such as lactate and pyruvate (14, 24). With respect to membrane localization studies of MCT1 in intestine, Garcia et al. (11), utilizing immunohistochemistry, previously demonstrated that MCT1 was localized to basolateral membranes in hamster cecal epithelial cells. In the rat jejunum, immunohistochemistry studies have shown the localization of MCT1 predominantly to the basolateral membranes of immature crypt cells, but in mature surface cells MCT1 was shown to shift to the luminal membranes (32). In contrast, studies conducted by Ritzhaupt et al. (28) using Western blotting have demonstrated localization of MCT1 to the pig and human colonic luminal membranes. Similarly, Buyse et al. (6), using immunofluorescence, have shown the apical localization of MCT1 in Caco-2 cells. MCT1 protein expression was shown to follow a gradient across the crypt-surface axis, with a peak in the apical membrane of the surface cells of human colon (20). Using two different but complementary techniques of immunoblotting and immunohistochemistry, we have now shown that MCT1 expression is restricted to the luminal membranes of human intestinal epithelial cells as well as in colonic crypts. Consistent with the protein expression, parallel studies to examine MCT1 mRNA relative abundance by RNase protection assay suggested higher MCT1 mRNA expression (relative to GAPDH as an internal control) in the human colon compared with duodenum. Our present study is the first to determine MCT1 expression (at both protein and mRNA levels), distribution, and localization in different regions of the human intestine. We have shown that the expression of MCT1 increased along the length of the human intestine, with maximum expression in the distal colon. This observation is not surprising, because the colon represents the major site for SCFA production and absorption (9). Our immunoblot studies showing a size of 39 kDa for the MCT1 band (which was competed by specific peptide) mainly in the apical membranes also detected faint bands in the basolateral membranes of the ileum and colon. This faint expression can be explained by a possible cross-contamination of basolateral membranes with apical membranes.
MCT4 has been shown to be the predominant isoform of skeletal muscles, where it is primarily responsible for lactate extrusion out of the muscle (14). With immunoblot studies, we have shown that MCT4 (54 kDa, which was competed by its specific peptide) is expressed in ileum and colon, with more predominant expression in the distal colon. MCT4 expression was restricted to the basolateral membranes in the human intestine. Studies of MCT1 and MCT4 expressed in Xenopus oocytes have shown that kinetic characteristics of these two isoforms are substantially different (Km for lactate: MCT1 3.5 mM, MCT4 1734 mM) (2). The affinity of MCT1 expressed in Xenopus oocytes for various monocarboxylates ranges from Km of 0.7 mM for pyruvate to 46 mM for acetate and 1012 mM for
-hydroxybutyrate (13). On the other hand, MCT4 expressed in Xenopus oocytes exhibits much lower affinity for substrates compared with MCT1, with Km values
5- to 10-fold higher (13). Our previous studies (34) in human proximal colonic basolateral membranes demonstrated the presence of a distinct SCFA/HCO3 transporter with a Km for butyrate of 17.5 mM. Although these studies compared the affinity of the basolateral transporter for butyrate, but not lactate, it is likely that MCT4 represents the basolateral SCFA transporter in the human colon. Similar to MCT4, MCT5 expression was highest in distal colon, followed by proximal colon and ileum. Both immunoblot and immunohistochemical studies confirmed that MCT5 was also localized to the basolateral membranes. MCT5 has not yet been functionally characterized (14). Future studies on MCT5 isoform function are needed to determine whether it could also represent one of the potential candidates mediating SCFA transport in the basolateral membranes of human colonic epithelial cells.
MCT3 was found to be expressed at relatively low levels compared with other isoforms, because it was detected only at a primary antibody concentration 20 times higher than that used for the detection of other isoforms. Studies have shown that MCT3 is exclusively localized to basolateral membranes of retinal pigment epithelium (RPE), where it plays an important role in lactate efflux from RPE into the choroidal blood supply (23, 35). Our studies showed that MCT3 was present in both the small intestine and colonic regions, localized to the basolateral membranes. However, unlike other basolaterally expressed isoforms (MCT4 and MCT5), MCT3 expression was in the order ileum >> proximal colon > distal colon > jejunum. Therefore, it could be speculated that MCT3 might not play an important role in SCFA transport in the human colon. Although our previous RT-PCR studies showed the expression of MCT1, -3, -4, -5, and -6 isoforms in the human colonic carcinoma cell line Caco-2 (26), the present studies showed that MCT6 protein expression was absent from the human intestine.
Our findings showing that human colonic epithelial cells express three different MCT isoforms on the basolateral membrane appear intriguing, but not unusual. Such a phenomenon is also very common for Na+- and Cl-absorbing isoforms in the human intestine. For example, Na+/H+ exchanger (NHE) isoforms NHE2 and NHE3 are both expressed on the apical surface of intestinal epithelial cells but are regulated differentially and even reciprocally under some conditions (8). NHE1, in contrast, is present on the basolateral surface (31). Similarly, anion exchanger (AE) isoforms AE2 and AE3 are both localized to the basolateral surface of human intestinal epithelial cells (1).
Therefore, the apparent question that remains to be answered is, What could be the possible reason for the coexpression of different MCT isoforms on the basolateral surface? It could be speculated that these different isoforms exhibit distinct kinetic properties for different SCFAs or, on the other hand, may be important in maintaining housekeeping functions. Also, it is possible that different MCT isoforms are differentially regulated under acute or chronic conditions and might play distinct roles in the pathophysiology of different inflammatory conditions where SCFAs are implicated.
In summary, a speculative model demonstrating the expression of potential transporters involved in SCFA absorption in the human intestinal epithelial cell is shown in Fig. 7. Our immunoblotting studies using purified apical and basolateral membrane fractions from organ donor intestines demonstrated that MCT4 and MCT5 proteins were expressed on the basolateral membrane domains, whereas MCT1 expression was predominantly on the apical membrane domains. It could be speculated that both MCT4 and MCT5, or either one of them, might represent the key basolateral SCFA transporter. The present studies have formed an important background for future investigations into further understanding of SCFA influx and efflux across the polarized membranes of human intestinal epithelial cells and establishing the function and identity of the basolateral SCFA transporter(s).
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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