Gene ablation for PEPT1 in mice abolishes the effects of dipeptides on small intestinal fluid absorption, short-circuit current, and intracellular pH

Abstract

PEPT1 function in mouse intestine has not been assessed by means of electrophysiology and methods to assess its role in intracellular pH and fluid homeostasis. Therefore, the effects of the dipeptide glycilsarcosin (Gly-Sar) on jejunal fluid absorption and villous enterocyte intracellular pH (pH(i)) in vivo, as well as on enterocyte[(14)C]Gly-Sar uptake, short-circuit current (I(sc)) response, and enterocyte pH(i) in vitro were determined in wild-type and PEPT1-deficient mice and in mice lacking PEPT1. Immunohistochemistry for PEPT1 failed to detect any protein in enterocyte apical membranes in Slc15a1(-/-) animals. Saturable Gly-Sar uptake in Slc15a1(-/-) everted sac preparations was no longer detectable. Similarly, Gly-Sar-induced jejunal I(sc) response in vitro was abolished. The dipeptide-induced increase in fluid absorption in vivo was also abolished in animals lacking PEPT1. Since PEPT1 acts as an acid loader in enterocytes, enterocyte pH(i) was measured in vivo by two-photon microscopy in SNARF-4-loaded villous enterocytes of exteriorized jejuni in anesthetized mice, as well as in BCECF-loaded enterocytes of microdissected jejunal villi. Gly-Sar-induced pH(i) decrease was no longer observed in the absence of PEPT1. A reversal of the proton gradient across the luminal membrane did not significantly diminish Gly-Sar-induced I(sc) response, whereas a depolarization of the apical membrane potential by high K(+) or via Na(+)-K(+)-ATPase inhibition strongly diminished Gly-Sar-induced I(sc) responses. This study demonstrates for the first time that proton-coupled electrogenic dipeptide uptake in the native small intestine, mediated by PEPT1, relies on the negative apical membrane potential as the major driving force and contributes significantly to intestinal fluid transport.

The peptide transporter PEPT1 is expressed in distal colon in rodents and humans and contributes to water absorption

“…However, the intestinal peptide uptake with subsequent intracellular hydrolysis has been demonstrated to be a driving force for water uptake (1). And the PEPT1-mediated di-and tripeptide absorption has been shown in this context to be accompanied by a substantial concomitant salt and fluid absorption (6). Evidence for this role was also demonstrated here based on increased water content on feces of Pept1 Ϫ/Ϫ mice.…”

Mice lacking the intestinal peptide transporter display reduced energy intake and a subtle maldigestion/malabsorption that protects them from diet-induced obesity

“…NHE3 is essential for PEPT1 activity by exporting protons back to the lumen, leading to the recovery of pH in from the acid load. As reported previously, PEPT1-deficient mice have impaired intestinal fluid absorption that is mediated via NHE3 in concert with a chloride-bicarbonate exchange system (5). This impairment in water absorption could also contribute to the observed dietindiced obesity (DIO) resistance phenotype.…”

“…PEPT1 is mainly expressed in the small intestine with higher levels in the proximal parts than in distal regions. Studies employing Pept1 Ϫ/Ϫ mice have demonstrated a lack of intestinal transport of model dipeptides such as glycylsarcosine (5,15), but animals otherwise do not show any obvious phenotypic alterations (21,22). We here provide on the analysis of changes in the gastrointestinal tract and of other phenotypic measures in mice deficient of PEPT1 when the animals are fed a diet containing 48 energy% from fat (HFD) compared with a high-carbohydrate/low-fat diet (13 energy% fat).…”

Effect of Dose Escalation on the In Vivo Oral Absorption and Disposition of Glycylsarcosine in Wild-Type andPept1Knockout Mice

“…Thus, it is obvious that in situ intestinal perfusions, although mechanistically valid, do not necessarily reflect expected outcomes under physiological in vivo conditions in which luminal drug concentrations and gastrointestinal residence times are operative. Other mechanisms (e.g., passive permeability and paracellular permeability), as suggested by Chen et al (2010) using everted jejunal sacs, may play a bigger role in the in vivo absorption of GlySar than previously believed, especially in the absence of PEPT1. GlySar is sufficiently small (mol.…”