|Burrin, Douglas - Doug|
Submitted to: American Journal of Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/23/2004
Publication Date: 3/25/2004
Citation: Fan, M.Z., Matthews, J.C., Etienne, N., Stoll, B., Lackeyram, D., Burrin, D.G. 2004. Expression of brush border L-glutamate transporters in neonatal porcine epithelial cells along the small intestinal crypt-villus axis. American Journal of Physiology. 287:G385-G398. Interpretive Summary: Glutamate is an acidic, non-essential amino acid that is abundant in many infant foods, especially milk. Previous studies have shown that glutamate is a major source of energy for the cells that line the intestinal tract, called epithelial cells. The function of the intestinal epithelial cells changes as they multiply and develop on the surface of the intestinal lining. The aim of our study was to determine the cellular and molecular basis that explains how glutamate is transported from the gut lumen into the epithelial cells. We measured the uptake of isotopically labeled glutamate in epithelial cells isolated from neonatal piglets. These transport measurements demonstrated that XAG is the major amino acid transport system for epithelial cell glutamate uptake and its activity was higher in crypt than in villus tip cells. We also measured the expression of the different acidic amino acid transporter genes and proteins in these cell fractions. Molecular analysis demonstrated that the excitatory amino acid carrier 1 (EAAC1) was the primary XAG transporter expressed in epithelial cells and its expression was higher in villus tip than crypt cells. The regulation of dietary glutamate transport into intestinal cells is altered depending on the stage of epithelial cell development.
Technical Abstract: Enteral L-glutamate is extensively utilized as an oxidative fuel by the gut mucosa in the neonate. To identify major uptake pathways and to understand uptake regulation, we examined transport kinetics and molecular identities of apical membrane L-glutamate transporters in epithelial cells sequentially isolated along the small intestinal crypt-villus axis from milk protein-fed, 16-day-old pigs. The distended intestinal sac method was used to isolate 12 sequential cell fractions from the tip villus to the bottom crypt. Initial rates and kinetics of L-glutamate uptake were measured with L-[G-3H]glutamate by fast filtration in apical membrane vesicles prepared by Mg2+ precipitation and differential centrifugation, with membrane potential clamped by SCN-. Initial L-glutamate uptake results suggested the presence of Bo and XAG- transport systems, but the XAG- system was predominant for uptake across the apical membrane. Kinetic data suggested that L-glutamate uptake through the XAG- system was associated with higher maximal transport activity but lower transporter affinity in crypt than in villus cells. Molecular identity of the XAG- glutamate transporter, based on immunoblot and RT-PCR analysis, was primarily the defined excitatory amino acid carrier EAAC-1. EAAC-1 expression was increased with cell differentiation and regulated at transcription and translation levels from crypt to upper villus cells. In conclusion, efficiency and capacity of luminal L-glutamate uptake across the apical membrane are regulated by changing expression of the XAG- system transporter gene EAAC-1 at transcription and translation levels as well as maximal uptake activity and transporter affinity along the intestinal crypt-villus axis in the neonate.