Glucose-dependent insulinotropic polypeptide-mediated signaling pathways enhance apical PepT1 expression in intestinal epithelial cells

Abstract

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.

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…The augmentative effect of GIP can be blocked by Rp-cAMP, wortmannin, or an Akt inhibitor, but not by calphostin C, a PKC inhibitor, or BAPTA, an intracellular Ca 2ϩ chelator. Importantly, activation of EPAC by 007 mimics GIP stimulation, dose-dependently enhances glycylsarcosine uptake, and increases the expression of PepT1 on apical membranes (200). These data suggest that EPAC and PI3K/Akt signaling pathways are involved in GIP-induced peptide uptake of the small intestine.…”

“…Earlier studies have established that glucose-dependent insulinotropic polypeptide (GIP), but not GLP-1, enhances PepT1-mediated peptide absorption in murine jejunum by a cAMP-dependent signaling pathway (201). Coon et al (200) show that PepT1 and GIP receptor (GIPR) proteins are expressed in villus epithelial cells of jejunum where these membrane-bound proteins localize on the apical and basolateral membranes, respectively. The use of a nonhydrolyzable dipeptide, glycylsarcosine, permitted Coon's group to further demonstrate that GIP activates PepT1 enhancing glycylsarcosine uptake in CDX2-transfected IEC-6 cells, an absorptive intestinal epithelial cell model.…”

Functional intercalated nanocomposites with chitosan-glutathione-glycylsarcosine and layered double hydroxides for topical ocular drug delivery

“…HCEpiC were seeded on 96-well plates at a density of 2×10 4 cells per well. When 90% confluence was reached, the cells were treated with medium containing different nanocomposites with various concentrations of LDH carriers (25,50,75, 100 and 200 μg/mL). After incubation for 12 h, the medium was removed and the cells were washed twice with Dulbecco's phosphatebuffered saline (DPBS; pH 7.4), and then 180 μL of culture medium and 20 μL of MTT reagent (5 mg/mL in DPBS) were added.…”

“…[21][22][23] Glycylsarcosine (GS) is one of the best known reference substrates of PepT-1 and commonly used as an inhibitor in the study of transport mechanism of PepT-1. [24][25][26] GS, as an active target PepT-1 ligand, was conjugated with thiolated CTS hybrid LDH to form a multifunctional nanocomposite. We proposed that specific recognition between GS and PepT-1 might significantly enhance corneal permeation for topical ocular drug delivery.…”

Taste and move: glucose and peptide transporters in the gastrointestinal tract

“…Interestingly, PEPT1 activity was shown to be affected by GIP (Coon et al . ), and also leptin was shown to regulate PEPT1 and GLUT5 function but not SGLT1 and GLUT2, suggesting obesity‐mediated adaptation of intestinal transport processes (Tavernier et al . ).…”