Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport L-lactate as well as butyrate

Abstract

1. Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence of the RT-PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT-PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well-characterized human and pig colonic lumenal membrane vesicles (LMV). 2. Properties of the colonic lumenal membrane L-lactate transporter were studied by the uptake of L-[U-14C]lactate into human and pig colonic LMV. L-Lactate uptake was stimulated in the presence of an outward-directed anion gradient at an extravesicular pH of 5.5. Transport of L-lactate into anion-loaded colonic LMV appeared to be via a proton-activated, anion exchange mechanism. 3. L-Lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl- and SO4(2-). The uptake of L-lactate was inhibited by phloretin, mercurials and alpha-cyano-4-hydroxycinnamic acid (4-CHC), but not by the stilbene anion exchange inhibitors, 4,4'-diisothiocyanostilbene-2, 2'-disulphonic acid (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2, 2'-disulphonic acid (SITS). 4. The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of L-lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.

Figure 1. Detection of MCT1 transcript in pig and human colon by Northern blot analysis

Human and pig colonic RNA (5 μg lane⁻¹, each) was fractionated on a 1% (w/v) denaturing agarose gel containing 0.66 m formaldehyde, transferred to nylon membrane and probed with the radiolabelled human colonic RT-PCR product. Lane a, pig ileal RNA; lane b, pig colonic RNA; lane c, human ileal RNA; lane d, human colonic RNA.

Figure 2. Immunodetection of MCT1 protein in pig and human colonic LMV

LMV were dissolved in sample buffer containing SDS. Samples (20 μg (lane of protein)⁻¹, each) were separated on an 8% polyacrylamide gel and electrotransferred to nitrocellulose membrane. Immunoblotting was carried out as described in Methods. The specificity of the immunoreaction was determined by comparing blots carried out under standard conditions (A) with those in which the MCT1 antibody had been pre-incubated with the immunizing peptide antigen (B). Lane a, pig colonic homogenate; lane b, pig colonic LMV; lane c, human colonic homogenate; lane d, human colonic LMV; lane e, rat erythrocyte ghosts.

Figure 3. Time course of l-lactate uptake into pig colonic LMV

LMV were preloaded with standard loading buffer (100 mm mannitol, 100 mm sodium l-lactate, 20 mm Hepes-Tris pH 7.5 and 0.1 mm MgSO₄). LMV (100 μg of protein per assay) were incubated in the standard uptake media containing 100 mm mannitol, 100 mm sodium gluconate, 20 mm Mes-Tris pH 5.5, 0.1 mm MgSO₄, and 0.1 mm L-[U-¹⁴C]lactate. Uptake was measured at 37 °C as described. Values are presented as means ±s.e.m. for three experiments. The inset shows the linear phase of l-lactate uptake.

Figure 4. Saturability of l-lactate transport into pig colonic LMV

Initial rates of l-lactate transport with increasing lactate concentrations over the range of 1–40 mm were determined. LMV were loaded with 100 mm mannitol, 100 mm NaHCO₃, 20 mm Hepes-Tris pH 7.5, and 0.1 mm MgSO₄. LMV (100 μg protein per assay) were incubated in media containing 100 mm mannitol, 0.1 mm MgSO₄, 20 mm Mes-Tris pH 5.5 and varying concentrations of sodium gluconate, sodium l-lactate (to maintain a constant media osmolarity and sodium concentration) and tracer amounts of L-[U-¹⁴C]lactate. Uptake was measured at 37 °C for 5 s. The data are presented as a Michaelis-Menten curve and (inset) as Wolf-Hanes plot of substrate concentration (S) against S/V, where V is the velocity, (r² = 0.96). Values are presented as means ±s.e.m. for three experiments.

Figure 5. Inhibition of l-lactate transport by structural analogues

LMV isolated from pig () and human () colon were preloaded with 100 mm mannitol, 100 mm sodium l-lactate, 20 mm Hepes-Tris pH 7.5 and 0.1 mm MgSO₄. LMV (100 μg of protein per assay) were incubated in medium containing 100 mm mannitol, 80 mm sodium gluconate (60 mm sodium gluconate when Na₂SO₄ and sodium succinate were present in the media and 95 mm sodium gluconate when 4-CHC was present in the media), 20 mm Mes-Tris pH 5.5, 0.1 mm MgSO₄, 0.1 mm L-[U-¹⁴C]lactate and 20 mm of the following sodium salts: acetate, propionate, butyrate, pyruvate, succinate, Cl⁻, SO₄²⁻. 4-CHC was added at a concentration of 5 mm. Uptake was measured at 37 °C for 5 s. Values are presented as means ±s.e.m. for three experiments. * Statistically significant (P < 0.05) from control.

Figure 6. Immunodetection of MCT1 protein in healthy and diseased human colonic biopsies

Lumenal plasma membrane fractions were isolated from biopsy-size samples of several healthy, pre-cancerous and cancerous human colonic tissues. Protein fractions were dissolved in sample buffer containing SDS. Samples (20 μg (lane of protein)⁻¹, each) were separated on an 8% polyacrylamide gel and electrotransferred to nitrocellulose membrane. Immunoblotting was carried out as described in Methods. Lane a, colon adenoma; lane b, colon adenoma; lane c, colon adenoma; lane d, rectal carcinoma; lane e, colon adenoma; lane f, colon carcinoma; lane g, healthy colon; lane h, healthy colon.