Glutamine (Gln), a preferred fuel source for enterocytes, is critical for intestinal epithelial cell integrity and barrier function. Chronic enteritis inhibits apical Na+–Gln cotransport. It is not known whether inflammatory cytokines that are secreted during inflammation
inhibit Na+–Gln cotransport. Thus, this study aimed to examine whether TNF-α would affect apical Na+–Gln cotransport in intestinal epithelial cells. In this study, the presence of Na+–Gln cotransport was established by measuring Gln
uptake in 10 days postconfluent IEC-6 cells grown on transwell plates. Cation, amino acid specificity, and siRNA transfection studies established that Na+–Gln cotransport is mediated via B0AT1. Immunoblotting and immunofluorescence studies established the apical
membrane localization of B0AT1 in IEC-6 cells. Tumour necrosis factor α (TNF-α) inhibited Na+–Gln cotransport in a concentration- and time-dependent manner with an inhibitory concentration of 1.53 nmol·L−1. Quantitative real-time
PCR and Western blot analyses indicated that TNF-α did not alter B0AT1-specific transcripts or protein expression level. Kinetic studies revealed that TNF-α inhibited Na+–Gln cotransport by reducing the affinity of the cotransporters for Gln, and this
effect was antagonized by genistein. Thus, we conclude that the TNF-α inhibition of Na+–Gln cotransport occurs at the post-translational level, and that the IEC-6 cell line is an excellent system to study the role of cytokines in Na+–Gln cotransport.
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B0AT1 protein expression;
B0AT1 transcript abundance;
abondance des transcrits de B0AT1;
captation de glutamine;
expression de B0AT1;
Document Type: Research Article
Department of Biology, LeMoyne-Owen College, 807 Walker Avenue, Memphis, TN 38126, USA.
Department of Biochemistry and Molecular Biology, West Virginia University, School of Medicine, Morgantown, WV 26506, USA.
Publication date: January 1, 2013
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