Mechanism(s) of butyrate transport in Caco-2 cells: role of monocarboxylate transporter 1

Christos Hadjiagapiou, Larry Schmidt, Pradeep K. Dudeja, Thomas J. Layden, and Krishnamurthy Ramaswamy

Section of Digestive and Liver Diseases, Department of Medicine, University of Illinois at Chicago and the West Side Veterans Affairs Medical Center, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The short-chain fatty acid butyrate was readily taken up by Caco-2 cells. Transport exhibited saturation kinetics, was enhanced by low extracellular pH, and was Na+ independent. Butyrate uptake was unaffected by DIDS; however, alpha -cyano-4-hydroxycinnamate and the butyrate analogs propionate and L-lactate significantly inhibited uptake. These results suggest that butyrate transport by Caco-2 cells is mediated by a transporter belonging to the monocarboxylate transporter family. We identified five isoforms of this transporter, MCT1, MCT3, MCT4, MCT5, and MCT6, in Caco-2 cells by PCR, and MCT1 was found to be the most abundant isoform by RNase protection assay. Transient transfection of MCT1, in the antisense orientation, resulted in significant inhibition of butyrate uptake. The cells fully recovered from this inhibition by 5 days after transfection. In conclusion, our data showed that the MCT1 transporter may play a major role in the transport of butyrate into Caco-2 cells.

short-chain fatty acids; intestinal absorption; antisense cDNA


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SHORT-CHAIN FATTY ACIDS (SCFA) are the most abundant anions in the colonic lumen of many mammals, including humans (4, 11). They are the products of bacterial fermentation of undigested carbohydrates and proteins that escape absorption in the small intestine. Acetate, propionate, and butyrate, the predominant SCFAs, are readily absorbed by colonocytes, causing rapid cellular acidification followed by Na+-independent pH recovery and used as the preferred source of fuel by these cells (4, 7, 8, 11, 22, 30). Previous experiments showed a preferential utilization of SCFA in the order of butyrate > propionate > acetate (10). SCFA are metabolized by colonocytes into glucose, ketone bodies, and amino acids (28, 29). Their importance to the normal function of human colon has been well established by their use to treat colonic diseases like diversion colitis and ulcerative colitis (18, 31). Butyrate is oxidized more readily to CO2 than propionate and acetate by colonocytes, and its oxidation is independent of the presence of other SCFA (10). It is implicated in the regulation of growth of colonic mucosa and in vitro cell proliferation and differentiation, involving the activation of several differentiation-specific genes (19, 23, 35). Butyrate has also been shown to reduce in vivo and in vitro growth rates in colorectal cancer cells (3, 12, 21, 32, 38).

A number of recent studies have focused on elucidation of the mechanism of SCFA transport into colonic cells. Studies in our laboratory indicated that SCFA are transported via a SCFA/HCO3- antiporter that is independent of the Cl-/HCO3- exchanger and of the presence of Na+ (16, 17). Although several studies came to similar conclusions (20, 33, 37), others suggested different mechanisms, such as nonionic diffusion (5, 37), a combination of nonionic diffusion and transporter activity (6, 8, 9), or an anion-exchange process involving the AE2 exchanger (39). It is also possible that a monocarboxylate transporter (MCT), such as MCT1 (13), can be involved, as recently shown (27). Therefore, our studies were carried out to elucidate the mechanism of SCFA transport in the human intestine by characterizing the uptake of butyrate in Caco-2 cells and by investigating the possible role of MCT1 in butyrate transport. In the present study, we demonstrate that the transport of butyrate in Caco-2 cells exhibits typical Michaelis-Menten kinetics and that the process is pH dependent, Na+ independent, and DIDS insensitive. Furthermore, inhibition of butyrate transport in Caco-2 cells with the antisense MCT1 cDNA demonstrates the possible involvement of the MCT1 transporter in the uptake process.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Caco-2 cells were obtained from Dr. Gail Hecht's laboratory (Univ. of Illinois at Chicago, Chicago, IL), and [1-14C]butyrate (specific activity 15 mCi/mmol) was purchased from NEN (Boston, MA). Butyrate, DIDS, and alpha -cyano-4-hydroxycinnamate were obtained from Sigma (St. Louis, MO), and the Bradford reagent was obtained from Bio-Rad (Hercules, CA). LipofectAMINE PLUS reagent, Opti-MEM medium, Superscript II RT, and oligonucleotides were purchased from GIBCO BRL (Grand Island, NY). Radioactivity was measured in a liquid scintillation counter made by Packard (Downers Grove, IL).

Cell culture. Caco-2 cells were grown routinely in T-75 cm2 plastic flasks at 37°C in a 5% CO2-95% air environment. The culture medium consisted of high-glucose DMEM (GIBCO BRL), 10% FBS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cells reached confluence after 5-7 days in culture. They were used for this study between passages 26 and 55 and plated in 12-well plates. Cells were used for experiments 2 days after confluence and fed with fresh incubation medium the day before an experiment was carried out.

Transport experiments. Cells were washed twice with 1 ml Hanks' balanced salt solution (HBSS; in mM: 1.3 CaCl2, 5.4 KCl, 0.44 K2HPO4, 0.4 MgSO4, 0.4 Na2HPO4, 4 NaHCO3, 0.5 MgCl2, 135 choline Cl, and 10 HEPES, pH 7.5) and incubated for 30 min at room temperature in the same buffer. To initiate the transport experiments, 0.5 ml HBSS-PIPES (pH 6.5) containing 0.2 mM [1-14C]butyrate was added to each well and incubated for 5 min at room temperature. The supernatant was removed, and cells were washed twice with 1 ml ice-cold HBSS-PIPES (pH 6.5). One milliliter of ethanol was added to each well to extract radioactivity and incubated for 30 min at room temperature, and the extract was counted in a liquid scintillation counter. Cells were dissolved in 0.1 N NaOH/PBS, and the amount of protein in each well was assayed using the Bradford method.

Cloning of MCT. Total RNA from Caco-2 cells was obtained using STAT-60 (Tel-Test). An oligo(dT) primer was used to reverse transcribe 10 µg of total RNA at 42°C for 60 min using SuperScript II RT. The reaction was terminated by heating at 70°C for 15 min, followed by the addition of RNase H and incubation at 37°C for 25 min. Two microliters of RT reaction were used to amplify MCT1 and fragments of MCT isoforms using PCR. The reaction was carried out using primers designed from the published human MCT cDNA sequences. The amplification conditions were one cycle at 94°C for 2 min, followed by 35 cycles at 94°C for 45 s, annealing at 68°C for 1 min, and extension at 72°C for 2 min for MCT1 cDNA or 1 min for cDNA fragments of MCT isoforms. Final extension was carried out at 72°C for 10 min. The primers used were GI 236 and GI 154 for the MCT1 cDNA and GI 120 and GI122, GI 347 and GI 348, GI 350 and GI 351, GI 353 and GI 352, and GI 354 and GI 355 for MCT1, MCT3, MCT4, MCT5, and MCT6 fragments, respectively. The resulting PCR products were cloned into pGEM-T vector (Promega, Madison, WI) and sequenced (Sequenase v. 2, Amersham). A list of the oligonucleotide sequences is provided in Table 1.

                              
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Table 1.   Oligonucleotides

Transient transfection experiments. The pRc/CMV vector was cut with Hind III restriction endonuclease, and the resulting 3' protruding end was made blunt with Klenow enzyme. The MCT1 cDNA was ligated into the blunt-ended pRc/CMV, and its antisense orientation was used for transfection. Caco-2 cells were plated at 50% confluency the day before transfection in 12-well plates. Antisense MCT1 construct (0.8 µg) or vector alone was complexed with 5 µl LipofectAMINE PLUS reagent in 50 µl Opti-MEM medium and incubated for 15 min. In other tubes, 2 µl of LipofectAMINE were diluted in 50 µl Opti-MEM medium and combined with precomplexed DNA, mixed, and incubated for 15 min at room temperature. The cells were washed once with Opti-MEM medium, and the DNA-LipofectAMINE PLUS complexes were added into each well along with 400 µl Opti-MEM medium. Incubations were carried out for 3-5 h at 37°C. Media were removed, and 1 ml DMEM/10% FBS was added to each well. Butyrate transport experiments were done 48 h after transfection.

Ribonuclease protection assay. [32P]CTP-labeled antisense MCT transcript was synthesized from cDNA template using SP6 or T7 RNA polymerase. Radioactive probe (400,000 cpm) was hybridized with 20 µg of total RNA, extracted from Caco-2 cells, and incubated at 42°C for 20 h. After hybridization, the samples were digested with RNase T for 45 min at 37°C and ethanol precipitated. They were resuspended in sequencing buffer, run on a 6% polyacrylamide/urea gel, and visualized by autoradiography, exposed on an X-ray film at -80°C for 20 h.

Statistics. Results are expressed as means ± SE. Statistical significance was determined using the paired t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Time- and concentration-dependent uptake of butyrate. The time-dependent uptake of 0.2 mM [1-14C]butyrate by Caco-2 cells was examined. As shown in Fig. 1, increasing amounts of butyrate were transported into the cells as the time increased up to 10 min. This increase was found to be linear between 3 min and 10 min, and a 5-min incubation time was used for determining the initial rates of transport on the basis of these studies. For kinetic studies, the concentration-dependent uptake of butyrate by Caco-2 cells was examined by incubating them for 5 min with various concentrations of butyrate up to 10 mM (Fig. 2). The uptake was found to exhibit typical Michaelis-Menten kinetics with a calculated Michaelis-Menten constant (Km) of 2.6 mM and a maximal velocity (Vmax) of 96 nmol · 5min-1 · mg protein-1. Since Caco-2 cells differentiate fully up to 21 days after confluence, we decided to examine the uptake of butyrate by Caco-2 cells 22 days after confluence. The results (not shown) showed a calculated Km of 1.98 mM and Vmax of 82 nmol/5 min-1 · mg protein-1. These results are similar to those results obtained from 2 days postconfluent Caco-2 cells.


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Fig. 1.   Time-dependent uptake of butyrate. Caco-2 cells were preincubated in Hanks' balanced salt solution (HBSS)-HEPES (pH 7.5) for 30 min and incubated with 200 µM [1-14C]butyrate in HBSS-PIPES (pH 6.5) at room temperature. Values are means ± SE of 3 different cultures.



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Fig. 2.   Concentration-dependent uptake of butyrate. Cells were preincubated in HBSS-HEPES (pH 7.5) for 30 min and incubated with increasing concentrations of [1-14C]butyrate in HBSS-PIPES (pH 6.5) for 5 min. Values are means ± SE of 3 different cultures.

Effect of Na+ and pH on butyrate uptake. Considering that the electroneutral transport of anions may be Na+ or H+ linked, we decided to examine whether the uptake of butyrate was Na+ dependent. Cells were incubated with 200 µM [1-14C]butyrate for 5 min with or without 138 mM Na+. This inward Na+ gradient had no effect on butyrate uptake (results not shown), and therefore the transport of this SCFA was regarded as a Na+-independent process. Next, the effect of extracellular pH on butyrate transport into the cells was determined. Cells were incubated with butyrate in HBSS-PIPES at pH 7.5, 6.5, or 5.5 for 5 min at room temperature (Table 2). When the extracellular pH was reduced from 7.5 to 6.5, a twofold increase of butyrate uptake was observed, whereas further reduction of pH to 5.5 increased the uptake by sevenfold, demonstrating an acidic pH-stimulated uptake.

                              
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Table 2.   Effect of extracellular pH on [1-14C]butyrate uptake

Effect of inhibitors. To determine whether butyrate uptake could be associated with an anion-exchange mechanism, the effect of DIDS at concentrations of 0.05, 0.1, and 1 mM on butyrate uptake was determined. Cells were preincubated with or without DIDS for 15 min and incubated with butyrate in the presence or absence of DIDS for 5 min. As can be seen from Table 3, DIDS had no effect on butyrate transport. We also examined whether alpha -cyano-4-hydroxycinnamate, at concentrations of 0.2, 0.4, and 1 mM, can affect the butyrate uptake by Caco-2 cells. Cells were preincubated with or without the inhibitor for 15 min and incubated with butyrate in the presence or absence of inhibitor for 5 min. Our results (Fig. 3) showed that alpha -cyano-4-hydroxycinnamate, a monocarboxylate inhibitor, significantly inhibited the transport of butyrate into cells at all concentrations examined (P < 0.05). The highest inhibition (45%) was observed at 1 mM concentration.

                              
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Table 3.   Effect of DIDS on [1-14C]butyrate uptake



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Fig. 3.   Effect of alpha -cyano-4-hydroxycinnamate on butyrate uptake by Caco-2 cells. Cells were preincubated in HBSS-HEPES (pH 7.5) for 30 min and preincubated with or without alpha -cyano-4-hydroxycinnamate in the same buffer for 15 min. They were incubated with 200 µM [1-14C]butyrate in the presence or absence of inhibitor in HBSS-PIPES (pH 6.5) for 5 min. Values are means ± SE of 3 different cultures. * P < 0.05.

Effect of butyrate analogs. The effect of substrate analogs lactate and propionate on butyrate uptake was determined. Lactate is a monocarboxylic acid, known to be transported into cells via the MCT, and propionate, a SCFA structurally similar to butyrate, is found in the colonic lumen. Cells were incubated with [1-14C]butyrate in the presence or absence of lactate or propionate for 5 min, and its uptake was estimated. The results (Table 4) showed that the transport of butyrate into cells was inhibited by the presence of either analog. In the presence of lactate, the decrease was 70 and 80% at 10 and 20 mM, respectively, whereas in the presence of propionate the inhibition was 77 and 92% at 10 and 20 mM, respectively.

                              
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Table 4.   Effect of substrate analogs on [1-14C]butyrate uptake

MCT isoforms in Caco-2 cells. These results suggested that a MCT isoform plays a role in the transport of butyrate into Caco-2 cells. We therefore examined the presence of MCT isoforms in these cells by using RT-PCR. Our results showed that MCT1, MCT3, MCT4, MCT5, and MCT6 isoforms are expressed in Caco-2 cells (Fig. 4). To determine their relative abundance of expression, we employed the RNase protection assay using glyceraldehyde 3-phosphate dehydrogenase as an internal standard. The results (Fig. 5) showed MCT1 to be the most abundant MCT isoform expressed in Caco-2 cells. The MCT1 cDNA was amplified from Caco-2 total RNA with the use of PCR. Sequence analysis showed 100% homology between the Caco-2 MCT1 cDNA and the reported MCT 1 sequence from the human heart.


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Fig. 4.   Monocarboxylate transporter (MCT) isoforms expressed in Caco-2 cells using the PCR method. Total RNA (10 µg) from Caco-2 cells were reverse transcribed and amplified using the PCR method and specific primers for each MCT isoform. The conditions used were 94°C for 2 min for 1 cycle, 94°C for 45 s, annealing at 68°C for 1 min, and extension at 72°C for 1 min for 35 cycles. Ten microliters of the PCR reaction were run on 1% agarose gel. Numbers on left are numbers of base pairs.



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Fig. 5.   Relative abundance of mRNA of MCT isoforms in Caco-2 cells by RNase protection assay. MCT antisense probe (400,000 cpm) was hybridized with 10 µg total RNA from Caco-2 cells. The hybridization was carried out at 42°C for 20 h. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Left: protected fragments (P-); right: radioactive probes used.

Effect of antisense MCT1 transfection on butyrate transport. Studies were carried out to insert the MCT1 cDNA into pRc/CMV vector in the antisense orientation and to transfect Caco-2 cells with this construct or the vector alone as control. The uptake of butyrate was determined 48 h after transfection. Our results showed that the antisense MCT1 inhibited butyrate uptake by ~50%, whereas the vector alone showed no inhibition (Fig. 6). Furthermore, to determine cell recovery from the effects of antisense MCT1 inhibition, we determined butyrate uptake 3, 4, and 5 days after transfection (Fig. 7). Compared with a 50% reduction at day 2, at 3 days after transfection the uptake was inhibited by 27% and after 4 days by 11%, with no significant difference in the uptake between control and antisense MCT1. The cells fully recovered from the inhibition 5 days after transfection.


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Fig. 6.   Butyrate uptake in Caco-2 cells transfected with antisense MCT1 construct. Cells were transfected with 0.8 µg of antisense MCT1 construct or the vector alone (pRc/CMV) for 3 h using the LipofectAMINE PLUS reagent. Butyrate uptake was determined 2 days after transfection. Cells were preincubated in HBSS-HEPES (pH 7.5) and incubated with 200 µM [1-14C]butyrate in HBSS-PIPES (pH 6.5) for 5 min. Values are means ± SE of 3 separate cultures.



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Fig. 7.   Butyrate uptake recovery in cells transfected with antisense MCT1 construct. Cells were transfected with 0.8 µg of antisense MCT1 in pRc/CMV vector or vector alone for 3-5 h using the LipofectAMINE PLUS reagent. Butyrate uptake was determined 3, 4, and 5 days after transfection. Cells were preincubated in HBSS-HEPES (pH 7.5) for 30 min and incubated with 200 µM [1-14C]butyrate for 5 min in HBSS-PIPES (pH 6.5). Values are means ± SE of 3 separate cultures.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrates that the transport of butyrate across the brush-border membrane of Caco-2 cells involves the MCT1 transporter. In this study, we have shown that butyrate was taken up by Caco-2 cells in a concentration- and time-dependent manner. This uptake was pH dependent, Na+ independent, and DIDS insensitive. It was also blocked by the monocarboxylate transport inhibitor alpha -cyano-4-hydroxycinnamate and the substrate analogs L-lactate and propionate. Furthermore, we have shown that the MCT isoforms MCT1, MCT3, MCT4, MCT5, and MCT6 (13, 25) were expressed in Caco-2 cells, and of these isoforms MCT1 was determined to play a role in the transport of butyrate in these cells.

Earlier studies from our laboratory showed that butyrate and propionate were readily taken up by colonic and ileal luminal membrane vesicles via a Na+ independent SCFA/HCO3- transporter (16, 17). Similar results were reported in other studies using various animals (20, 33, 37). In agreement with these reports, our data showed that butyrate was Na+ independent but readily taken up in a saturable manner, which is a characteristic of a carrier-mediated process. Also, the uptake was stimulated significantly with the reduction of extracellular pH, in agreement with those results obtained from pig and human colonic luminal membranes (16, 26). This stimulated uptake was shown to predominantly acidify the apical site of cells, as opposed to the basal site, which is alkaline and resistant to pH perturbations (14). It is well known that the transport via the MCT is H+ linked, with the exception of kidney (24, 34). In correlation with an earlier report showing that the absorption of SCFA in the colon increased the luminal pH (36), the uptake of SCFA via the MCT could account for the reduction of luminal H+ concentration, leading to an increased pH.

A recent study reported an increased transport of monocarboxylic acids, including butyrate and propionate, in HEK-293 cells transfected with mouse AE2 cDNA, and the transport was inhibited by DIDS (39). In addition, high concentrations of L-lactate and propionate were transported in human and rat erythrocytes by the band 3 protein, a Cl-/HCO3- exchanger (2, 15). Our data clearly showed that the butyrate transporter activity was not inhibited by DIDS, excluding the possibility that butyrate uptake occurred via a Cl-/HCO3- exchanger. Also, a recent study by Alrefai et al. (1) from our laboratory demonstrated that AE2 was found to be localized on the human colonic basolateral membranes, as demonstrated through Western blots of membranes with antipeptide antibodies. This is in agreement with previous SCFA transport studies from our laboratory and others that used human and other intestinal membrane vesicles (16, 17, 26, 33). In addition, we have shown that the monocarboxylate inhibitor alpha -cyano-4-hydroxycinnamate and the butyrate analogs L-lactate and propionate significantly inhibited the uptake of butyrate by Caco-2 cells. Similar results were obtained by other laboratories by using pig and human colonic luminal membranes (26) and human ileal and colonic luminal membranes and rat distal colon (16, 17, 20, 33). Furthermore, in Xenopus oocytes transfected with MCT1 cDNA, butyrate uptake was inhibited by pyruvate and L-lactate (27).

To further define the butyrate transporter in Caco-2 cells, we employed antisense technology to suppress the expression of MCT1 transporter. An antisense MCT1 construct transiently transfected into Caco-2 cells inhibited the butyrate uptake by ~50%, supporting the involvement of MCT1 in butyrate transport. This rate of inhibition appeared to be due to a binding between the antisense MCT1 mRNA and its target mRNA. The cells were found to start recovering from this inhibition by day 3 after transfection, and by day 5 the recovery was complete.

In summary, our current results demonstrate that the MCT1 isoform plays a major role in the transport of butyrate in Caco-2 cells. The extent of SCFA transport via the MCT1 pathway under physiological conditions is not known at present. Our studies also demonstrate that MCT1 is not the only MCT isoform expressed in these cells, and more studies are needed to evaluate the contribution of other isoforms to the transport of butyrate.


    ACKNOWLEDGEMENTS

These studies were supported by the Department of Veterans Affairs and National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-33349 (K. Ramaswamy) and DK-54016 (P. K. Dudeja).


    FOOTNOTES

Address for reprint requests and other correspondence: K. Ramaswamy, Dept. of Medicine, Univ. of Illinois at Chicago, M/C 787, Section of Digestive and Liver Diseases, 840 S. Wood St., Chicago, IL 60612 (E-mail: kramaswa{at}uic.edu).

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.

Received 9 March 2000; accepted in final form 10 May 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 279(4):G775-G780