Fig. 3: H⁺-coupled dipeptide absorption is impaired in EEC-deficient small intestine.

a EEC-deficient human and mouse small intestine did not respond to the dipeptide Gly-Sar after exposure to VIP (mouse, n = 9 wild-type, n = 6 mutant, ****P < 0.0001; human, n = 11 wild-type, n = 5 mutant, **P = 0.006). Pre-treatment of EEC-deficient tissue with exogenous PYY (mouse, n = 6, ns; human, n = 5, *P = 0.03), or of wild-type tissue with BIBO3304 (mouse, n = 9; human, n = 6) did not improve the I_sc response to Gly-Sar. Statistics calculated by one-way ANOVA with Tukey’s multiple comparisons test. b Expression and localization of peptide transporter PEPT1 is unchanged in EEC-deficient human small intestine. Representative images from eight independent organoids are shown. Scale bars = 50 μm. c The PYY-VIP axis regulates intracellular pH in human small intestinal cells. EEC-deficient enteroids differentiated with VIP for 5–7 days developed an H⁺ imbalance with an acidic cytoplasm whereas wild-type enteroids were able to maintain their intracellular pH (**P = 0.004). Concurrent treatment with 10 nM PYY normalized the pH in EEC-deficient enteroids and was dependent on NPY1R. pHrodo MFI was analyzed by flow cytometry and normalized to vehicle-treated wild-type. n = 3 independent experiments. Statistics calculated by mixed effects analysis using the Holm–Sidak method. d Small intestinal EECs regulate proton transport in a paracrine fashion. Using reporter animals with mosaic loss of EECs we found that regions of jejunal epithelium that escaped recombination had normal pH as measured by pHrodo MFI. Adjacent regions that expressed tdTomato exhibited increased pHrodo MFI, indicating elevated cytosolic H⁺ (n = 4 mice, ***P = 0.0002). There was no difference in pHrodo MFI between mosaic regions in wild-type reporter jejunum (n = 8 mice). Statistics calculated by two-way ANOVA with Sidak’s multiple comparisons test. All error bars are + SEM.