Epidermal growth factor and insulin short-term increase hPepT1-mediated glycylsarcosine uptake in Caco-2 cells.
OBJECTIVE
Little is known about the physiological regulation of the human intestinal di/tri-peptide transporter, hPepT1. In the present study we evaluated the effects of epidermal growth factor (EGF) and insulin on hPepT1-mediated dipeptide uptake in the intestinal cell line Caco-2.


METHODS
Caco-2 cells were grown on filters for 23-27 days. Apical dipeptide uptake was measured using [14C]glycylsarcosine([14C]Gly-Sar). HPepT1 mRNA levels were investigated using RT-PCR, cytosolic pH was determined using the pH-sensitive fluorescent probe BCECF.


RESULTS
Basolateral application of EGF increased [14C]Gly-Sar uptake with an ED50 value of 0.77 +/- 0.25 ng mL-1 (n = 3-6) and a maximal stimulation of 33 +/- 2% (n = 3-6). Insulin stimulated [14C]Gly-Sar uptake with an ED50 value of 3.5 +/- 2.0 ng mL-1 (n = 3-6) and a maximal stimulation of approximately 18% (n = 3-6). Gly-Sar uptake followed simple Michaelis-Menten kinetics. Km in control cells was 0.98 +/- 0.11 mM (n = 8) and Vmax was 1.86 +/- 0.07 nmol cm-2 min-1 (n = 8). In monolayers treated with 200 ng mL-1 of EGF, Km was 1.11 +/- 0.05 mM (n = 5) and Vmax was 2.79 +/- 0.05 nmol cm-2 min-1 (n = 5). In monolayers treated with 50 ng mL-1 insulin, Km was 1.03 +/- 0.08 mM and Vmax was 2.19 +/- 0.06 nmol cm-2 min-1 (n = 5). Kinetic data thus indicates an increase in the number of active transporters, following stimulation. The incrased Gly-Sar uptake was not accompanied by changes in hPepT1 mRNA, nor by measurable changes in cytosolic pH.


CONCLUSIONS
Short-term stimulation with EGF and insulin caused an increase in hPepT1-mediated uptake of Gly-Sar in Caco-2 cell monolayers, which could not be accounted for by changes in hPepT1 mRNA or proton-motive driving force.
Nielsen
C U
CU
Department of Pharmaceutics, Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark.
Amstrup
J
J
Nielsen
R
R
Steffansen
B
B
Frokjaer
S
S
Brodin
B
B
D020126:Brefeldin A / Q000494:pharmacology; D018938:Caco-2 Cells / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D004151:Dipeptides / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D005947:Glucose / Q000493:pharmacokinetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology; D007930:Leucine / Q000493:pharmacokinetics; D000074011:Peptide Transporter 1; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D027981:Symporters
D002352:Carrier Proteins; D004151:Dipeptides; D007328:Insulin; D000074011:Peptide Transporter 1; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C082186:SLC15A1 protein, human; D027981:Symporters; D020126:Brefeldin A; C004194:glycylsarcosine; D004815:Epidermal Growth Factor; D007930:Leucine; D005947:Glucose; D003078:Colchicine