title: The apical (hPepT1) and basolateral peptide transport systems of Caco-2 cells are regulated by AMP-activated protein kinase.
abstract: The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) activation of the AMP-activated protein kinase (AMPK) on the transport of the model radiolabeled dipeptide [(3)H]-D-Phe-L-Gln was investigated in the human epithelial colon cancer cell line Caco-2. Uptake and transepithelial fluxes of [(3)H]-D-Phe-L-Gln were carried out in differentiated Caco-2 cell monolayers, and hPepT1 and glucose transporter 2 (GLUT2) protein levels were quantified by immunogold electron microscopy. AICAR treatment of Caco-2 cells significantly inhibited apical [(3)H]-D-Phe-L-Gln uptake, matched by a decrease in brush-border membrane hPepT1 protein but with a concomitant increase in the facilitated glucose transporter GLUT2. A restructuring of the apical brush-border membrane was seen by electron microscopy. The hPepT1-mediated transepithelial (A-to-B) peptide flux across the Caco-2 monolayers showed no significant alteration in AICAR-treated cells. The electrical resistance in the AICAR-treated monolayers was significantly higher compared with control cells. Inhibition of the sodium/hydrogen exchanger 3 (NHE3) had an additive effect to AICAR, suggesting that the AMPK effect is not via NHE3. Fluorescence measurement of intracellular pH showed no reduction in the proton gradient driving PepT1-mediated apical uptake. The reduction in apical hPepT1 protein and dipeptide uptake after AICAR treatment in Caco-2 cells demonstrates a regulatory effect of AMPK on hPepT1, along with an influence on both the microvilli and tight junction structures. The absence of an associated reduction in transepithelial peptide movement implies an additional stimulatory effect of AICAR on the basolateral peptide transport system in these cells. These results provide a link between the hPepT1 transporter and the metabolic state of this model enterocyte.
authors.affiliation: School of Life Sciences, Oxford Brookes University, Headington, Oxford OX3 0BP, UK.
authors.forename: Myrtani
authors.initials: M
authors.lastname: Pieri
authors.forename: Helen C
authors.initials: HC
authors.lastname: Christian
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wilkins
authors.forename: C A R
authors.initials: CA
authors.lastname: Boyd
authors.forename: David
authors.initials: D
authors.lastname: Meredith
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D001692:Biological Transport; D018938:Caco-2 Cells; D016764:Cell Polarity; D048430:Cell Shape; D004151:Dipeptides / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D005470:Fluorometry; D051273:Glucose Transporter Type 2 / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008689:Methacrylates / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D000074011:Peptide Transporter 1; D012265:Ribonucleotides / Q000494:pharmacology; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism; D027981:Symporters / Q000378:metabolism*
chemical_list: C118956:3-(2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methylphenyl)-N-isopropylidene-2-methyl-acrylamide dihydrochloride; D004151:Dipeptides; D020536:Enzyme Activators; D051273:Glucose Transporter Type 2; D006146:Guanidines; D008689:Methacrylates; D000074011:Peptide Transporter 1; D012265:Ribonucleotides; C082186:SLC15A1 protein, human; C495680:SLC2A2 protein, human; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D027981:Symporters; D000620:Aminoimidazole Carboxamide; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide