Extracellular glucose concentration alters functional activity of the intestinal oligopeptide transporter (PepT-1) in Caco-2 cells.
The objective of this study was to determine the effect of different cell culture media glucose concentrations on the functional activity of PepT-1 in Caco-2 cells. Uptake kinetics of Gly-Sar into Caco-2 cells that were maintained in iso-osmotic media containing 25 or 5.5 mM glucose were determined in the presence and absence of amino acid-selective chemical modifiers and dithiothreitol. Inhibition of Gly-Sar uptake into Caco-2 cells was measured in the presence of dipeptides and xenobiotics exhibiting various binding affinities for the PepT-1. The effect of extracellular glucose on PepT-1 gene expression was assessed using comparative RT-PCR. Long-term exposure of Caco-2 cells to 25 mM glucose reduced maximum transport capacity for Gly-Sar uptake without altering PepT-1 gene expression. In contrast, binding affinity of Gly-Sar and other dipeptides or xenobiotics was not significantly changed. Chemical modification of Lys and Tyr residues decreased V(max), while Cys modification increased the maximum transport capacity of the carrier. Preincubation of Caco-2 cells with dithiothreitol restored PepT-1 activity in cells maintained at 25 mM glucose. In conclusion, cell culture media containing 25 mM glucose decreases maximum transport capacity of PepT-1 in Caco-2 cells without affecting substrate recognition, at least in part, mediated via an oxidative pathway.
D'Souza
Vanessa M
VM
College of Pharmacy, University of Cincinnati Medical Center, 3223 Eden Avenue, Cincinnati, OH 45267, USA.
Buckley
Donna J
DJ
Buckley
Arthur R
AR
Pauletti
Giovanni M
GM
D018938:Caco-2 Cells / Q000187:drug effects* / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D000074011:Peptide Transporter 1; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D027981:Symporters
D002352:Carrier Proteins; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; C082186:SLC15A1 protein, human; D027981:Symporters; D005947:Glucose