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Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Monocarboxylate Transporter 1 Mediates DL-2-Hydroxy-(4-Methylthio)Butanoic Acid Transport across the Apical Membrane of Caco-2 Cell Monolayers¹

² Departament de Fisiologia, Facultat de Farmàcia, Universitat de Barcelona, 08028 Barcelona, Spain and ³ Adisseo France S.A.S., 92160 Antony, France

^* To whom correspondence should be addressed. E-mail: rutferrer{at}ub.edu.

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 (pH_i) 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 pH_i recovery, suggesting cooperation between H⁺-dependent DL-HMB transport and NHE3 activity. cis-Inhibition experiments with L-Ala, ß-Ala, D-Pro, betaine, or glycyl-sarcosine excluded the participation of systems proton amino acid transporter 1 and peptide transporter 1. In contrast, -cyano-4-hydroxycinnamate, phloretin, L-lactate, ß-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 (K_D, 1.9 nL/µg protein; K_m, 13.1 ± 0.04 mmol/L; and V_max, 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.

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