Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells.
We have shown recently that glucose-dependent insulinotropic polypeptide (GIP), but not glucagon-like peptide 1 (GLP-1) augments H(+) peptide cotransporter (PepT1)-mediated peptide absorption in murine jejunum. While we observed that inhibiting cAMP production decreased this augmentation of PepT1 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 [(3)H-glycyl-sarcosine (Gly-Sar), a relatively nonhydrolyzable dipeptide] uptake to measure PepT1 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 calphostin C (a potent PKC inhibitor) nor BAPTA (an intracellular Ca(2+) 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 PepT1 activity as well. Western blot analyses revealed that both GIP and Epac agonist pretreatment enhance the PepT1 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 PepT1 in intestinal absorptive epithelial cells.
Department of Medicine, Boston University School of Medicine, Boston, Massachusetts; Department of Medicine, Boston Veterans Affairs Healthcare System, Boston, Massachusetts; Department of Medicine, Boston University Clinical & Translational Science Institute, Boston, Massachusetts; and scoon@bu.edu.
Steven D
SD
Coon
Department of Biochemistry and Molecular Biology, West Virginia University School of Medicine, Morgantown, West Virginia.
Vazhaikkurichi M
VM
Rajendran
Department of Medicine, Boston University School of Medicine, Boston, Massachusetts;
John H
JH
Schwartz
Department of Medicine, Boston University School of Medicine, Boston, Massachusetts; Department of Medicine, Boston Veterans Affairs Healthcare System, Boston, Massachusetts;
Satish K
SK
Singh
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005749:Gastric Inhibitory Polypeptide / Q000494:pharmacology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D007408:Intestinal Absorption / Q000187:drug effects*; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000187:drug effects*; D000074011:Peptide Transporter 1; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D027981:Symporters / Q000378:metabolism*; D014162:Transfection; D015854:Up-Regulation
D004151:Dipeptides; D020662:Guanine Nucleotide Exchange Factors; D018398:Homeodomain Proteins; D000074011:Peptide Transporter 1; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; C526973:Rapgef3 protein, rat; D011964:Receptors, Gastrointestinal Hormone; C489940:Slc15a1 protein, rat; D027981:Symporters; C004194:glycylsarcosine; D005749:Gastric Inhibitory Polypeptide; C041086:gastric inhibitory polypeptide receptor; D000242:Cyclic AMP; D058539:Phosphatidylinositol 3-Kinase; D051057:Proto-Oncogene Proteins c-akt
CDX2 expression; IEC-6 cells; Intestinal absorptive epithelial cells; dipeptide uptake Gly-Sar uptake.