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  • Title: Epidermal growth factor and insulin short-term increase hPepT1-mediated glycylsarcosine uptake in Caco-2 cells.
    Author: Nielsen CU, Amstrup J, Nielsen R, Steffansen B, Frokjaer S, Brodin B.
    Journal: Acta Physiol Scand; 2003 Jun; 178(2):139-48. PubMed ID: 12780388.
    Abstract:
    AIMS: 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.
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