Monocarboxylate transporter 1 mediates DL-2-hydroxy-(4-methylthio)butanoic acid transport across the apical membrane of Caco-2 cell monolayers

Abstract

The methionine hydroxy analogue DL-2-hydroxy-(4-methylthio)butanoic acid (DL-HMB) is a supplementary source of methionine commonly added to commercial animal diets to satisfy the total sulfur amino acid requirement. In this study, we characterized DL-HMB transport across the apical membrane of Caco-2 cells to identify the transport mechanism involved in the intestinal absorption of this methionine source. DL-HMB transport induced a significant decrease in intracellular pH (pHi) and was inhibited in the presence of the protonophore carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone. Moreover, both Na+ removal and 5-(N-ethyl-N-isopropyl)amiloride, an inhibitor of apical Na+/H+ exchanger (NHE3), significantly reduced substrate uptake and pHi recovery, suggesting cooperation between H+-dependent DL-HMB transport and NHE3 activity. cis-Inhibition experiments with L-Ala, β-Ala, D-Pro, betaine, or glycylsarcosine excluded the participation of systems proton amino acid transporter 1 and peptide transporter 1. In contrast, α-cyano-4-hydroxycinnamate, phloretin, L-lactate, β-hydroxybutyrate, butyrate, and pyruvate, inhibitors and substrates of monocarboxylate transporter 1 (MCT1), significantly reduced DL-HMB uptake. Dixon plot analysis of L-lactate transport in the presence of DL-HMB revealed a competitive interaction (inhibition constant, 17.5 ± 0.11 mmol/L), confirming the participation of system MCT1. The kinetics of DL-HMB uptake was described by a model involving passive diffusion and a single low-affinity, high-capacity transport mechanism (KD, 1.9 nL/μg protein; Km, 13.1 ± 0.04 mmol/L; and Vmax, 43.6 ± 0.14 pmol/μg protein) compatible with MCT1 kinetic characteristics. In conclusion, the methionine hydroxy analogue is transported in Caco-2 cell apical membrane by a transport mechanism with functional characteristics similar to those of MCT1. © 2007 American Society for Nutrition.