Proinflammatory Cytokine TNF‐α Inhibits Na+‐glutamine Cotransport in Intestinal Epithelial Cell line (IEC‐6)

Abstract

Mucosal Na+-glutamine (Gln) cotransport is critical in absorbing exogenous Gln that the body needs during extreme stress (e.g. infections and trauma). Although stress has been shown to inhibit Gln synthesis, it is not known whether stress also inhibits Gln absorption. Thus, the aim of this study was to determine whether TNF-α, the major proinflammatory cytokine is produced during infection would inhibit Gln cotransport in rat intestinal epithelial (IEC-6) cells. [3H]-Gln uptake was measured in 10-day post-confluent normal and TNF-α (25 ng/ml) treated IEC-6 cells, grown in 6-well plates. Na+/K+-ATPase activity, and B0AT1 specific mRNA abundance and protein expression were measured by qRT-PCR and western blot analyses, respectively. Na+-Gln cotransport is present in IEC-6 cells. TNF-α exposure (24 hrs) inhibited Na+-Gln cotransport by 75% (1.9 ± 0.1 vs 0.5 ± 0.01 nmol/mg protein • 2 min). Kinetic studies revealed that TNF-α inhibited Na+-Gln cotransport by reducing the affinity for Gln (Km: normal vs TNF-α = 3.5 ± 0.3 vs 8.5 ± 1.0 mM). TNF-α did not alter either B0AT1 specific mRNA abundance and protein expression or Na+/K+-ATPase activity. We concluded from these results that, in addition to inhibiting Gln synthesis, extreme stress also inhibits exogenous Gln absorption. We speculate that TNF-α might regulate Na+-Gln cotransport mediated via B0AT1 by second messenger mediated signal transduction process(es).