Self-enhanced PepT1-mediated transport of egg white-derived tripeptide FGR in apical-out mouse jejunal organoids

Apical-out organoids enable direct FGR-PepT1 contact, overcoming model limitations. • FGR activates three pathways remodeling apical niche for PepT1 self-enhancement. • Proteomics reveals self-enhanced absorption mechanism of egg-white tripeptide FGR. Our previous study has verified efficient PepT1-mediated transport of the egg white-derived tripeptide FGR (Phe-Gly-Arg), but its transport mechanism under physiological intestinal conditions remains elusive and is difficult to fully recapitulate using conventional culture models. This study established an apical-out mouse jejunal organoid model combined with Astral-DIA quantitative proteomics to investigate the molecular mechanisms and regulatory factors underlying egg white-derived tripeptide FGR transport via PepT1. Immunofluorescence confirmed that the model achieved successful polarity reversal, exposed the apical brush border, maintained the integrity of the F-actin cytoskeleton, tight junctions, and adherens junctions, and preserved normal intestinal epithelial cell lineages including absorptive cells and Paneth cells. After 4 h of apical-side FGR exposure, 682 differentially expressed proteins were identified, which were mainly enriched in the apical plasma membrane, microvillar structures, cytoskeleton remodeling, and nutrient-sensing pathways. KEGG analysis showed that glucagon signaling, PPAR signaling, and tight junction pathways were significantly activated, with key proteins such as GCG, ACOX2, EZR, and VASP markedly upregulated. These results demonstrate that FGR acts as a nutrient signal to trigger multi-dimensional epithelial adaptation and optimize the PepT1 transport microenvironment by regulating nutrient sensing, lipid-energy metabolism, and apical cytoskeleton remodeling, thereby modulating the molecular process of FGR transport via PepT1. This study provides proteomic evidence for the transport mechanism of food-derived peptides targeting PepT1. Download: Download high-res image (196KB) Download: Download full-size image