PepT1-knockout mice harbor a protective metabolome beneficial for intestinal wound healing

Abstract

Genetic knockout (KO) of peptide transporter-1 (PepT1) protein is known to provide resistance to acute colitis and colitis-associated cancer (CAC) in mouse models. However, it was unclear which molecule(s) or pathway(s) formed the basis for these protective effects. Recently, we demonstrated that the PepT1^-/- microbiota is sufficient to protect against colitis and CAC. Given that PepT1 KO alters the gut microbiome and thereby changes the intestinal metabolites that are ultimately reflected in the feces, we investigated the fecal metabolites of our PepT1 KO mice. Using a liquid chromatography-mass spectrometry (LC-MS)-based untargeted-metabolomics technique, we found that the fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. Among the altered fecal metabolites, tuberonic acid (TA) was sevenfold higher in KO mouse feces than in WT mouse feces. Accordingly, we studied whether the increased TA could direct an anti-inflammatory effect. Using in vitro models, we discovered that TA not only prevented lipopolysaccharide (LPS)-induced inflammation in macrophages but also improved the epithelial cell healing processes. Our results suggest that TA, and possibly other fecal metabolites, play a crucial role in the pathway(s) associated with the anticolitis effects of PepT1 KO.NEW & NOTEWORTHY Fecal metabolites were significantly different between the KO and normal wild-type (WT) mice. One fecal metabolite, tuberonic acid (TA), was sevenfold higher in KO mouse feces than in WT mouse feces. TA prevented lipopolysaccharide (LPS)-induced inflammation in macrophages and improved the epithelial cell healing process.

Keywords: fecal metabolites; inflammatory bowel disease; knockout; peptide transporter 1 (PepT1); tuberonic acid.

Graphical abstract

Figure 1.

Flowchart of metabolite analysis of fecal samples from WT and PepT1-KO mice. KO, knockout; PepT1, peptide transporter-1; WT, wild type.

Figure 2.

Knockout PepT1 alters the metabolomics profiles in feces. A: upregulated and downregulated metabolites in feces (PepT1^−/−[KO] vs. PepT1^+/+[WT]). B: PCA analysis of KO versus WT fecal metabolites. C: altered metabolites with significant fold change (>7). D: heat map of metabolites with significant fold change (>7) (n = 5). dmodx, distance to model; KO, knockout; m/z, mass to charge ratio; PCA, principal component analysis; PC1, principal component 1; PC2, principal component 2; PepT1, peptide transporter-1; TA, tuberonic acid; WT, wild type.

Figure 3.

LC-MS/MS identification of tuberonic acid (TA). A: extracted ion chromatogram (EIC) shows the peak at retention time ∼3.3 min is much higher in KO samples compared with WT samples (n = 5). B: the MS/MS data show that this compound's high-resolution MS and MS fragmentation behavior is identical to tuberonic acid. KO, knockout; WT, wild type.

Figure 4.

In vitro anti-inflammatory assay. Gene expression fold changes of TNF-α (A), IL-1β (B), IL-6 (C), IL-10 (D), and IL-22 (E) in inflamed RAW 264.7 cells treated with control (medium), LPS, or LPS+TA (2 or 4 µg/mL). (*P < 0.05, **P < 0.01, n = 3). LPS, lipopolysaccharide; NS, not significant; RAW 264.7, mouse macrophage cells; TA, tuberonic acid.

Figure 5.

Representative cell growth curves (as indicated by electrical resistances) in an in vitro wound-healing assay of TA. Confluent Caco2-BBE cells seeded to ECIS plates were wounded using an elevated voltage pulse of 40 kHz frequency, 4.5 V amplitude, and 30-s duration. After the wounding, TA (4.0 µg/mL), 6-shogaol (1.0 µg/mL), culture medium (blue), or Trizol (purple, 1% in medium) were added to the wounded Caco2-BBE cell monolayers. The wounded Caco2-BBE cell monolayers were then allowed to heal (presumably from cells that did not undergo the elevated voltage pulse and surround the small active electrode). Resistance was measured before and after applying the elevated voltage pulse, as described in the materials and methods section (n = 3). Caco2-BBE, colonic epithelium cells; ECIS, electric cell-substrate impedance sensing; TA, tuberonic acid.