Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepTl expression in intestinal epithelial cells

摘要

We have shown recently that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1) augments H+ peptide cotransporter (PepTl)-mediated peptide absorption in murine jejunum. While we observed that inhibiting cAMP production decreased this augmentation of PepTl activity by GIP, it was unclear whether PKA and/or other regulators of cAMP signaling pathway(s) were involved. This study utilized tritiated glycyl-sarcosine [3H-glycyl-sarcosine (Gly-Sar), a relatively nonhydrolyzable dipeptide] uptake to measure PepTl activity in CDX2-transfected IEC-6 (IEC-6/CDX2) cells, an absorptive intestinal epithelial cell model. Similar to our earlier observations with mouse jejunum, GIP but not GLP-1 augmented Gly-Sar uptake (control vs. +GIP: 154 ± 22 vs. 454 ± 39 pmol/mg protein; P < 0.001) in IEC-6/CDX2 cells. Rp-cAMP (a PKA inhibitor) and wortmannin [phosophoinositide-3-kinase (PI3K) inhibitor] pretreatment completely blocked, whereas neither calphos-tin C (a potent PKC inhibitor) nor BAPTA (an intracellular Ca2+ chelator) pretreatment affected the GIP-augmented Gly-Sar uptake in IEC-6/CDX2 cells. The downstream metabolites Epac (control vs. Epac agonist: 287 ± 22 vs. 711 ± 80 pmol/mg protein) and AKT (control vs. AKT inhibitor: 720 ± 50 vs. 75 ± 19 pmol/mg protein) were shown to be involved in GIP-augmented PepTl activity as well. Western blot analyses revealed that both GIP and Epac agonist pretreatment enhance the PepTl expression on the apical membranes, which is completely blocked by wortmannin in IEC-6/CDX2 cells. These observations demonstrate that both cAMP and PI3K signaling pathways augment GIP-induced peptide uptake through Epac and AKT-mediated pathways in intestinal epithelial cells, respectively. In addition, these observations also indicate that both Epac and AKT-mediated signaling pathways increase apical membrane expression of PepTl in intestinal absorptive epithelial cells.