title: Inflammatory cytokine TNF-α inhibits Na(+)-glutamine cotransport in intestinal epithelial cells.
abstract: 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 B(0)AT1. Immunoblotting and immunofluorescence studies established the apical membrane localization of B(0)AT1 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 B(0)AT1-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.
authors.affiliation: Department of Biology, LeMoyne-Owen College, Memphis, TN 38126, USA. Jamil_Talukder@loc.edu
authors.forename: Jamilur R
authors.initials: JR
authors.lastname: Talukder
authors.forename: Brittney
authors.initials: B
authors.lastname: Boyd
authors.forename: Ashley
authors.initials: A
authors.lastname: Griffin
authors.forename: Antara
authors.initials: A
authors.lastname: Jaima
authors.forename: Vazhaikkurichi M
authors.initials: VM
authors.lastname: Rajendran
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D016207:Cytokines / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005973:Glutamine / Q000235:genetics / Q000378:metabolism*; D007249:Inflammation / Q000235:genetics / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D007422:Intestines / Q000166:cytology / Q000187:drug effects; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D016207:Cytokines; D008565:Membrane Proteins; D047428:Protein Kinase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D005973:Glutamine; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase