Kynurenine promotes porcine intestinal epithelial cell proliferation by activating the AHR-MST1-YAP1 axis

TL;DR

This study shows that kynurenine, a metabolite of tryptophan, promotes growth of pig intestinal cells through a specific signaling pathway involving AHR, MST1, and YAP1.

Contribution

The study identifies kynurenine as a key metabolite that activates the AHR-MST1-YAP1 axis to promote intestinal epithelial cell proliferation in pigs.

Findings

L-tryptophan supplementation increases villus height and decreases crypt depth in pig intestines.

Kynurenine promotes IPEC-J2 cell proliferation by activating the AHR-MST1-YAP1 signaling pathway.

Kyn-induced cell proliferation is confirmed to depend on the AHR-MST1-YAP1 axis using specific inhibitors.

Abstract

The objective of this study was to investigate the effect of L-tryptophan (L-Trp) and its metabolite kynurenine (Kyn) on the regulation of porcine intestinal epithelial cell proliferation.

Dietary supplementation of L-Trp significantly increased villus height and decreased crypt depth in the jejunum and ileum of weaned pigs. mRNA sequencing data and qPCR analysis found that L-Trp activated the expression of cell proliferative genes and the AHR (aryl hydrocarbon receptor)-MST1 (mammalian STE20-like kinase 1)-YAP1 (Yes-associated protein 1) axis in the ileum. Further in vitro analysis revealed that L-Trp treatment significantly enhanced cell proliferation of intestinal porcine epithelial cells-jejunum 2 (IPEC-J2) cells by activating the MST1-YAP1 signaling pathway. Further targeted metabolomics analysis identified Kyn as the core Trp metabolite involved in promoting IPEC-J2 cell proliferation. Mechanistically, Kyn interacted with AHR, which in turn bound to the upstream promote region of MST1 to initiate the transcription of downstream target gene YAP1 to activate intestinal epithelial cell proliferation. Furthermore, porcine intestinal organoid model also demonstrated that Kyn promoted intestinal organoid-budding efficiency and intestinal stem cell proliferation. Importantly, by using the AHR- or YAP1-specific inhibitors, the data confirmed that the Kyn-induced intestinal epithelial cell proliferation in IPEC-J2 cells and intestinal organoids was dependent on the activation of the AHR-MST1-YAP1 axis.

Together, this study has revealed a regulatory mechanism of Trp metabolism-derived Kyn in promoting porcine intestinal epithelial cell proliferation, offering insights into the connection between nutrient metabolism and intestinal epithelial homeostasis.

The online version contains supplementary material available at 10.1186/s40104-026-01368-0.

Figures

Effect of dietary L-Trp on the intestinal epithelial cell proliferation of weaned pigs. A Representative images of hematoxylin and eosin staining in the duodenum, jejunum and ileum of control (DLY_Ctrl) and L-Trp supplemented (DLY_Trp) weaned pigs (*n* = 6). DLY, Duroc × Landrace × Large White. Red lines indicated villus height and crypt depth. scale bar: 100 μm. B Volcano plot of differential expressed genes (DEGs) in the ileum of DLY_Ctrl and DLY_Trp weaned pigs. C GO biological process analyses of DEGs in control and L-Trp-supplemented pigs. D Heatmap analysis of cell proliferative gene exp

L-Trp promotes proliferation of IPEC-J2 cells. A The mRNA expression of proliferative genes *PCNA*, *SYR* and *MUC13* in IPEC-J2 cells treated with different concentrations of L-Trp (*n* = 6). ^a–c^Mean values with unlike letters were significantly different (*P* < 0.05). B and C EdU staining and quantification of the ratio of EdU-positive cells in IPEC-J2 cells treated with 4 mmol/L L-Trp or 5 mmol/L L-Glu. D The mRNA expression of cell proliferation associated genes *Cyclin E1, Cyclin D1, TGF-β, SMAD4, MST1, YAP1* in IPEC-J2 cells after 4 mmol/L L-Trp or 5 mmol/L L-Glu treatment (*n* = 6). A

L-Trp is metabolized into Kyn to activate cell proliferation in IPEC-J2 cells. A Schematic presentation of Trp metabolic pathways and qRT-PCR analyses of the expression of key enzymes related to the Trp-Kyn and Trp-serotonin pathways in IPEC-J2 cells treated with 4 mmol/L L-Trp (*n* = 6). B Targeted metabolomic analysis of Trp metabolism in IPEC-J2 cells upon 4 mmol/L L-Trp treatment. C qRT-PCR analysis of the expression of cell proliferation associated genes in IPEC-J2 cells in response to kynurenine (Kyn), kynurenic acid (KYNA), and 3-hydroxyanthranilic acid (3-HAA) treatment, respectively (

Molecular docking analysis between Kyn and AHR. A Prediction of the binding pocket of the porcine AHR with Kyn. Light blue structure indicated the AHR protein, pink sticks denoted Kyn, and the hydrogen bonds in Å units were depicted as yellow dashed lines. B 2D Visualization of the binding site in Kyn-AHR docking complex. C Surface electrostatic force visualization at the docking interface of Kyn with AHR. D Calculation of the binding energy between Kyn and AHR. Generally, the binding energy below −5.0 kcal/mol is considered to have good binding affinity

Kyn promotes the proliferation of IPEC-J2 cells via activating the AHR-MST1-YAP1 axis. A Quantification of the mRNA expression of *AHR* and *CYP1A1* in IPEC-J2 cells after Kyn or Kyn + Verteporfin treatment (*n* = 6). B Illustration of the AHR binding sites in the promote region of the *MST1* gene. C Quantification of the mRNA expression of *MST1*, *YAP1*, *PCNA* and *Cyclin E1* after Kyn or Kyn + Verteporfin treatment (*n* = 6). D, E EdU staining and scratch assay in IPEC-J2 cells treated with Kyn alone or Kyn + Verteporfin (Scale bar: 20 μm). F Quantification of the ratio of EdU-positive cel

Kyn enhances porcine intestinal organoids budding rate. A and B Representative images of porcine intestinal organoids and the quantification of the budding efficiency from the control, 25 mmol/L, 50 mmol/L and 250 mmol/L Kyn groups at d 1, d 2 and d 3 respectively. C Quantification of the mRNA expression cell proliferation-related genes in Kyn-treated intestinal organoids (*n* = 6). Asterisks denote statistically significant differences (^*^*P* < 0.05, ^^*P* < 0.01). Error bars denote SEM

Kyn-induced intestinal organoids proliferation is dependent on the activation of the MST1-YAP1 pathway. A–D Representative images of porcine intestinal organoids and the quantification of budding efficiency from the control, Kyn and Kyn + Verteporfin treated groups, respectively (scale bar: 100 μm). E–H Immunostaining and the fluorescence intensity of OLFM4 in the control, Kyn and Kyn + Verteporfin treated groups, respectively (scale bar: 20 μm). I and J The protein expression of PCNA, AHR, MST1 and Cyclin D1 in intestinal organoids treated with Kyn or Kyn + Verteporfin (*n* = 3). K Proposed m

Full text

Introduction

Intestinal epithelium undergoes constantly self-renewal to replenish damaged or dead cells during development. The maintenance of intestinal epithelium homeostasis is modulated by the activity of intestinal stem cells (ISCs) that are located at the base of intestinal crypt. Surrounded intestinal epithelial cells are able to secrete endocrine hormones and transmit metabolic intermediates, such as insulin, leptin, and lactate, to modulate ISC activity [1–3]. Regulation of ISC proliferation is governed by several conserved signaling pathways [4–7]. For instance, the Hippo signaling pathway plays an important role in modulating ISC proliferation. Under normal condition, mammalian STE20-like kinase 1 (MST1) activates the Hippo pathway to suppresses ISC proliferation by inhibiting the downstream transcriptional activator Yes-associated protein 1 (YAP1) [8–10]. While inactivation of Hippo pathway triggers YAP1 translocation into the nucleus to initiate the transcription of cell proliferation genes, thus promoting ISC proliferation [11, 12]. Therefore, the MST1-YAP1 axis is essential for controlling the proliferative activity of ISCs.

Diet-derived macronutrients like carbohydrates and fats generate a variety of bioactive compounds that interact with proliferative signaling pathways to modulate the fate of ISCs both under homeostatic and injury conditions [13–16]. In particular, dietary supplementation of amino acids such as glutamate, arginine and glutamine have been reported to enhance ISC proliferation in response to different stimuli [17–20], highlighting the physiological importance of amino acid metabolism in the maintenance of intestinal health.

Tryptophan (Trp) is one of essential amino acids that can be metabolized into various bioactive molecules through the kynurenine (Kyn), 5-HT and indole pathways. Dietary supplementation of Trp improves growth performance and strengthens intestinal barrier integrity in pigs [21–24]. Recent studies also highlighted the regulatory function of indole derivatives including indole-3-aldehyde and indole-3-carboxaldehyde in promoting porcine ISC proliferation by activating the aryl hydrocarbon receptor (AHR) pathway [25, 26]. But whether Trp metabolism coordinates with the Hippo signaling pathway to regulate porcine intestinal epithelial cell proliferation is not known.

In this study, by utilizing both in vivo weaned pigs, and in vitro IPEC-J2 cells and porcine intestinal organoids, we characterized the regulatory mechanism by which L-Trp and its metabolite Kyn promote porcine intestinal epithelial cell proliferation by activating the AHR-MST1-YAP1 axis. Strikingly, inhibition of YAP1 expression blocked Kyn treatment-induced intestinal epithelial cell proliferation in both IPEC-J2 cells and porcine intestinal organoids, highlighting the physiological relationship between Trp metabolism and the Hippo signaling pathway.

Materials and methods

Animal experiment

All animal experimental procedures used in this experiment were approved by Institutional Animal Care and Use Committee of College of Animal Science and Technology, Hunan Agricultural University (No. 43321809) (Changsha, China). A total of 16 male Duroc × Landrace × Large white (DLY) pigs weaned at 28 days of age were obtained from the Hunan New Wellful Co., Ltd. (Changsha, China) and used in this study. After 5 days of adaptation period, pigs with the initial body weight (BW) of 9.1 ± 0.9 kg were divided into two groups: the control group (DLY_Ctrl) and 0.2% of L-tryptophan-supplemented group (DLY_Trp). Each group had 8 replicates and pigs were individually kept in pens in a mechanically ventilated and temperature-controlled room at 22–24 °C, humidity of 60%−65%. All pigs had ad libitum access to drinking water and feed throughout the experimental period. Experimental diets were formulated to meet the recommended nutrient requirements suggested by the National Research Council (NRC) [27] (Table 1). All pigs were fed with experimental diets for 4 weeks and then euthanized by the penetration of a captive bolt followed by exsanguination. Approximately 10 cm of the ileum was dissected longitudinally and gently washed with PBS to remove luminal contents for intestinal mucosa collection. The ileal mucosa was gently scraped with a glass slide and immediately frozen in liquid nitrogen for further gene expression analysis. The middle of duodenal and jejunal, the distal of ileal tissues were collected in lengths of approximately 5 cm per segment after flushing with 0.9% saline solution, and fixed in 4% paraformaldehyde (BL539A, Biosharp, China) for 24 h for histological analysis. Table 1. Composition of experimental diets (air-dry basis)ItemTryptophan supplementation0%0.20%Ingredient, % Corn (yellow dent)6565 Soybean meal (ground)1212 Fermented Soybean meal1010 Fish meal44 Whey (dehydrated)44 Salt0.30.3 L-Lysine0.740.74 L-Threonine0.220.22 L-Tryptophan00.2 DL-Methionine0.110.11 Limestone powder0.590.59 DCP1.1041.104 Zeolite powder0.210.21 Premix^1^1.7261.726Calculated nutrient level DE, MJ/kg14.214.2 CP, %17.5817.58 Tryptophan, %0.1780.378^1^Premix: phytase, 0.02%; enzyme preparation, 0.05%; multivitamin and minerals 726, 0.05%; choline, 0.16%; flavouring agent, 0.04%; sweetening agent, 0.03%; feed acidifier, 0.3%; ZnO, 0.16%; CuSO_4_·5H_2_O, 0.07%; MnSO_4_·H_2_O, 0.03%; ZnSO_4_·H_2_O, 0.01%; FeSO_4_·H_2_O, 0.06%; 1% KI, 0.002%; NaSeO, 0.002%; CoCl·6H_2_O, 0.002%; glucose, 0.6%; antioxidant, 0.05%; mould inhibitor, 0.05%DCP CaHPO_4_·2H_2_O, DE Digestible energy, CP Crude protein

RNA sequencing

The total RNA from ileal mucosa (1 g) in control and Trp-supplemented groups were extracted using TRIzol (Invitrogen, Carlsbad, California, USA). To avoid the variations among each individual piglet, the RNA from two piglets from the same group were mixed as one biological sample. Based on this standard, each group contained four biological replicates for mRNAseq. The RNA concentration was measured by the Thermo Scientific NanoDrop 2000 Spectrophotometer (Thermo Scientific, Inc, Waltham, MA, USA). The RNA quality was evaluated by the Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). The cDNA library preparation and RNA sequencing were performed by BGI (Shenzhen, China), generating paired-end reads at 150 bp length on the Illumina HiSeq sequencing platform. The sequencing raw data was filtered with SOAPnuke [28] by (1) removing reads containing sequencing adapter; (2) removing reads whose low-quality base ratio (base quality less than or equal to 15) is more than 20%; (3) removing reads whose unknown base (‘N’ base) ratio is more than 5%. Afterwards the clean reads were obtained and stored in FASTQ format. The subsequent analysis and data mining were performed on Dr. Tom Multi-omics Data mining system (https://biosys.bgi.com). The clean reads were mapped to the reference genome (NCBI_GCF_000003025.6_Sscrofa11.1) using HISAT2 [29]. After that, Ericscript (v0.5.5) [30] and rMATS (v4.1.2) [31] were used to detect fusion genes and differential splicing genes (DSGs), respectively. Bowtie2 was applied to align the clean reads to the gene set in which known and novel, coding and noncoding transcripts were included. Expression level of gene was calculated by RSEM (v1.3.1) [32]. The heatmap was drawn by pheatmap (v1.0.12) according to the gene expression difference in different samples. Statistically significant differentially expressed genes were selected based on a P value threshold of ≤ 0.05. Essentially, differential expression analysis was performed using the DESeq2 (v1.34.0) [33] with P value ≤ 0.05 (or FDR ≤ 0.001). To gain insight into the change of phenotype, Gene Ontology (GO; http://www.geneontology.org/) enrichment analysis of the annotated differentially expressed genes (DEGs) was performed using Phyper, which is based on the hypergeometric test. The significant levels of terms and pathways were corrected by P value with a rigorous threshold (P value ≤ 0.05).

Hematoxylin and eosin staining

The middle of the duodenum, jejunum and the distal of ileum from control and Trp-supplemented piglets were collected and immediately fixed in 4% paraformaldehyde for 24 h. Then, samples were embedded in paraffin, sectioned (5 μm), and stained with haematoxylin and eosin (H&E) for histological examination. Images were photographed by the Moticam 3000 microscope (US) under 30 × magnification. Measurements of villus height and crypt depth from duodenal, jejunal and ileal samples were conducted by using image processing and analysis software (Image J, NIH). The average 10 measurements of villi and their associated crypts in each sample were selected and calculated.

Cell culture and treatment

The porcine intestinal epithelial cells (IPEC-J2) cell line was a gift from Prof. Bi’e Tan (College of Animal Science and Technology, Hunan Agricultural University, Hunan, China). IPEC-J2 cells (5 × 10^5^ cells/mL) were seeded in 6-well Costar plates (Corning, New York, USA) and maintained in Dulbecco’s Modified Eagle Medium (D2650, Sigma), containing 10% fetal calf serum and 10% fetal bovine serum (FBS; #A3160901, GIBCO) and 1% antibiotic cocktail (10 KU/mL penicillin and 10 mg/mL streptomycin, #15070063; 25 μg/mL amphotericin B, #15290026; GIBCO, USA). IPEC-J2 cells were cultured in a 37 °C incubator supplemented with 5% CO_2_. When the cells reached approximately 70% confluence, they were treated as follows: 4 mmol/L L-tryptophan (T0254-5G, Sigma) or 5 mmol/L L-glutamate (G0355000, Sigma, USA) or 4 mmol/L L-kynurenine (HY-104026, MedChemExpress, USA) dissolved in complete medium were added and incubated for 24 h, respectively. The control group was treated with complete medium or PBS. For inhibiting YAP1 and AHR expression, 0.5 µmol/L Verteporfin ( HY-B0146, MedChemExpress; dissolved in 0.1% DMSO) and 0.2 µmol/L AHR inhibitor CH-223191 (HY-12684, MedChemExpress, USA; dissolved in complete medium) were co-incubated with 4 mmol/L L-kynurenine for 24 h, respectively. The 0.1% DMSO did not affect IPEC-J2 cell survival (Fig. S1). Then the total RNA and protein samples from each group were collected for further analysis.

5-Ethynyl-2'-deoxyuridine (EdU) staining

IPEC-J2 cells were plated at a density of 1–5 × 10^5^ cells/mL into a 24-well plate, when the cells reached approximately 70% confluence. Then IPEC-J2 cells were incubated with 4 mmol/L L-tryptophan or 5 mmol/L L-glutamate, 4 mmol/L L-kynurenine or 4 mmol/L L-kynurenine + 0.5 µmol/L Verteporfin for 24 h, respectively. Then the EdU assay was performed using BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (C0071S, Beyotime, China), following the manufacturer’s protocol. Fluorescent images were captured with an inverted fluorescence microscope and the ratio of EdU-positive cells and the mean gray value were analyzed by Image J.

Immunofluorescence (IF) analysis

IPEC-J2 cells were seeded on glass coverslips in 24-well plates. After reaching 70% confluence, the cells were treated 4 mmol/L L-kynurenine, or 4 mmol/L L-kynurenine + 0.2 µmol/L AHR inhibitor CH-223191 (HY-12684, MedChemExpress, USA) for 24 h. Subsequently, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. The cells were then incubated overnight at 4 °C with primary antibodies against PCNA (60097-1-Ig, Proteintech, China) and AHR (1:300, GTX22770, GeneTex, USA), followed by incubation with Alexa Fluor 488-conjugated or Alexa Fluor 594-conjugated goat anti-mouse secondary antibody for 2 h at room temperature. Nuclei were counterstained with DAPI. Fluorescence images were captured via a confocal microscope (LSM 710, Zeiss, Oberkochen, Germany). The fluorescence intensity was quantified with ImageJ software. All experiments were performed at least three independent replicates.

Wound healing assay

IPEC-J2 cells were plated at a density of 1–5 × 10^5^ cells/mL into a 6-well plate until the cell density reached 90%–100% confluence at the bottom of the culture dish. Then the monolayer was scratched with a sterilized micropipette tip to generate wounds. IPEC-J2 cells were incubated with 4 mmol/L L-kynurenine or 4 mmol/L L-kynurenine + 0.5 µmol/L Verteporfin, or 4 mmol/L L-kynurenine + 0.2 µmol/L AHR inhibitor CH-223191 (HY-12684, MedChemExpress, USA) for 24 h, the PBS group was used as a control. The migration of IPEC-J2 cells into the gap of each group was imaged using a transmitted-light microscope and the wound width in each group were measured using Image J.

Metabolomic analysis

50 μL of sample from IPEC-J2 cells were mixed with 10 μL of sulfosalicylic acid solution, and diluted to 1 mL with 0.1% formic acid aqueous solution. After ultrasonic extraction and high-speed centrifugation, the supernatant was collected for analysis. Analysis was performed using a Thermo Fisher Vanquish/TSQ Altis LC–MS/MS system with a Kinetex F5 column (4.6 mm × 250 mm, 5 μm) at 40 °C. The mobile phase consisted of 10 mmol/L ammonium formate aqueous solution with 0.1% formic acid and methanol–water (9:1) solution with 0.1% formic acid for gradient elution. Mass spectrometry employed electrospray ionization (ESI) in positive ion mode with multiple reaction monitoring (MRM). The ion source temperature was set at 350 °C with a spray voltage of 4,000 V. Quantification was performed using the external standard method by establishing calibration curves, with quality control samples interspersed throughout the analysis for quality assurance.

Culturing of porcine intestinal organoids

The isolation and culture procedures for porcine jejunal organoids were obtained from a published protocol by Yin et al. [34]. Seven-day-old piglets were euthanized and a 3-cm segment of the proximal jejunum best for organoid growth was removed and immediately irrigated with ice-cold PBS (HyClone) to remove the intestinal contents. Fat and mesentery attached to the jejunum were removed with forceps and scissors. The villi were gently scraped off by using an ice-cold cover slip as complete as possible. Then, 3 cm segment of the proximal jejunum was cut into small pieces (0.2 to 0.3 cm) and incubated with 2 mmol/L ethylenediaminetetraacetic acid (Sigma-Aldrich) in a 50-mL tube at 4 °C for 1 h for epithelial isolation. Next, the tube was swayed for 5 min to obtain a high purity of crypts, which were further filtered through a 70-μm cell strainer. The isolated crypts were washed with 10% (vol/vol) fetal calf serum (FCS) and resuspended in 15-mL advanced DMEM-GF (Gibco, Grand Island, NY, USA) with 0.5 μmol/L A83-01 (TGF-β inhibitor, Cat. No. 2939, Tocris) and 2.5 μmol/L CHIR99021 (GSK-3β inhibitor, Cat. No. SML1046, Sigma). After a short centrifugation and resuspension, the crypts were embedded in a growth factor-reduced phenol-red Matrigel (Cat. No. 356231, Corning, NY, USA). Subsequently, a 50-µL of Matrigel-Crypts mix in each well was incubated at 37 °C with 5% CO_2_ for 15 min in a 24-well plate. Next, 500 µL of culture medium containing 50% (vol/vol) of Wnt3A (conditioned medium), 20% (vol/vol) of R-Spondin 1 (conditioned medium), 10% (vol/vol) of Noggin (conditioned medium), B27 supplement (Invitrogen), N2 supplement (Invitrogen, Carlsbad, CA, USA), glutamine (Sigma-Aldrich), N-acetyl cysteine (Sigma-Aldrich), recombinant murine epidermal growth factor (PeproTech, Rocky Hill, NJ, USA), nicotinamide (Sigma-Aldrich), and SB202190 (Sigma-Aldrich), was added per well after Matrigel solidification. The growth media were refreshed every 2 to 3 d.

Different concentrations of L-kynurenine (25 µmol/L, 50 µmol/L, and 250 µmol/L) were added to the culture medium after intestinal organoids were passaged by mechanically breaking them into single crypts without buds. All groups in the current experiment were treated at the same time using the same batch of organoids, and different concentrations of L-kynurenine were compared to the control. The number of budding organoids as well as the number of buds on each budding organoid were counted at d 1 to d 3 after L-kynurenine treatment. The organoid budding efficiency was measured by the ratio of the number of budding organoids to the total number of organoids.

Quantitative reverse transcription polymerase chain reaction

The total RNA from ileum samples (1 g) or IPEC-J2 cells or porcine intestinal organoids were extracted using TRIzol reagent (Takara Biomedical Technology Co., Ltd., Beijing, China), and the genomic DNA was removed by using the gDNA Eraser Kit (Takara Biomedical Technology, Beijing, China). The RNA purity (absorbance ratios at 260 and 280 nm) of each sample was measured by the NanoDrop 2000 Spectrophotometer (Thermo Scientific, Inc, Waltham, MA, USA). Then 1 µg of the total RNA of each sample was used to synthesize cDNA in a 20-µL of reaction volume using the PrimeScript™ RT reagent Kit following the manufacturer’s protocol. Next, the mRNA expression of target genes were measured by RT-qPCR in a 10 µL reaction volume using SYBR Green mix kit (Takara Biomedical Technology, Beijing, China) on the Light Cycler R480II Machine (Roche, USA). Primer sequences were designed according to published literatures and all primers underwent thorough verification and optimization before performing the RT-qPCR analysis. The detailed primer sequence was listed in Table S1. All samples were analyzed as least in biological triplicate and the relative amounts of each target were quantified relative to a set standard curve pooled from all samples in the analysis and finally normalized relative to those of GAPDH or β-actin. Comparative cycle threshold (2^−ΔΔ^^CT^) was conducted during data analysis. Statistical significance was calculated using unpaired paired t-test and P-values of < 0.05, < 0.01 and < 0.001 were considered significant.

Molecular docking between kynurenine and aryl hydrocarbon receptor

Molecular docking was performed to investigate the binding interactions between endogenous tryptophan metabolites and the AHR of Sus scrofa (UniProt ID: I3LF82). The crystal structure of AHR was obtained from the UniProt database and prepared for docking using AutoDockTools (Version 1.5.7), including hydrogen addition, charge assignment, and removal of non-essential molecules. The docking grid was defined to encompass the entire ligand-binding domain of the receptor, with grid center coordinates set to center_x = 1.338, center_y = 4.402, and center_z = 1.265, and grid box dimensions set to size_x = 126.0 Å, size_y = 124.0 Å, and size_z = 126.0 Å. These parameters ensured that all potential binding pockets within the AHR cavity were fully covered. Docking simulations were conducted using AutoDock Vina (Version 1.2.3), which applies an iterated local search optimization algorithm to identify the lowest-energy binding conformations. The pose with the minimum binding free energy was selected as the final predicted binding mode. Visualization and interaction analysis, including hydrogen bond identification and pocket mapping, were performed using PyMOL (Version 2.3.0) and LigPlot + (Version 2.2.8).

Western blotting analysis

The protein from ileum (1 g) or IPEC-J2 cells or porcine intestinal organoids were isolated by homogenizing samples using the lysis buffer (50 mmol/L Tris-HCl, 5 mmol/L EDTA, 150 mmol/L NaCl, 0.5% Nonidet-40, 1 mmol/L PMSF, pH 8.0). After centrifugation at 12,000 × g for 20 min at 4 °C, the supernatants were collected and the concentration were analyzed using Bradford assay. Then protein samples were mixed with loading buffer and denatured at 90 °C, 10 min before electrophoresis. Forty micrograms of proteins from each sample were loaded and run on the sodium dodecyl sulfate–polyacrylamide gel, and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA), followed by 1 h incubation with blocking solution (Tris-buffered saline containing 5% BSA). Then, membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies used in this study were as follows: AHR (1:300, GTX22770, GeneTex, USA), MST1 (1:500, T58571, Abmart, USA), PCNA (1:2,000, 60097-1-Ig, Proteintech, China), Cyclin D1 (1:500, WL01435a, Wanlei, China), LATS1 (1:1,000, PA3032, Abmart, USA), Cyclin E1 (1:700, WL01072, Wanlei, China). The β-Actin (1:5,000, 66009-1-Ig, Proteintech, China) or GAPDH (1:5,000, 60004-1-Ig, Proteintech, China) were used as internal controls. The next day, membranes were washed and incubated for 1 h at room temperature with HRP-conjugated secondary antibodies (goat anti-mouse, RGAM001; goat anti-rabbit, RGAR001; Proteintech, China). The protein bands were visualized using an enhanced chemiluminescence (ECL) kit (LI-COR ink, USA), and the density of the specific protein bands were analyzed using Image J.

Statistical analysis

For comparison between the control and tryptophan-supplemented weaned pigs, the unpaired Student’s t-test was performed. For statistical analyses in IPEC-J2 cells or porcine intestinal organoids, the unpaired Student’s t-test were performed. For multiple comparisons in IPEC-J2 cells treated with different concentrations of L-Trp, one-way ANOVA was carried out combined with Tukey’s HSD post-hoc test. Statistical significances were calculated by SPSS version 29.0 (SPSS, Inc., Chicago, USA). Data are presented as mean ± SEM (standard error of the mean). P-values < 0.05 were considered statistically significant. All figures were graphed by GraphPad Prism 9.0 (GraphPad Software, La Jolla, CA, USA).

Results

Dietary L-Trp activates intestinal epithelial cell proliferation in weaned pigs

To investigate the effect of L-Trp on the regulation of the small intestinal epithelial development, the histological characteristics of duodenum, jejunum and ileum in weaned pigs-fed with 0.2% of L-Trp were examined. Dietary L-Trp supplementation increased the villus height in all three segments of the small intestine (P < 0.05; Fig. 1A). While the crypt depth was selectively decreased in the jejunum and ileum of L-Trp supplemented pigs when compared to control (P < 0.05; Fig. 1A). These data indicate that dietary L-Trp supplementation promotes the development of the small intestinal epithelium. Next, since the ileum exhibited the most dominant changes in the villus height after L-Trp addition, the mRNA sequencing was performed in the ileum to profile the global mRNA expression. As shown in Fig. 1B, the mRNA sequencing analysis identified 181 DEGs, with 69 upregulated and 112 downregulated genes, in the ileum of L-Trp supplemented weaned pigs when compared to control. Gene Ontology (GO) analysis further revealed that the biological functions of DEGs were largely enriched in epithelial cell proliferation, lipid hydroxylation, hydrogen metabolic process, all of which have been implicated in gut development (Fig. 1C). Furthermore, a variety of cell proliferative genes, such as leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), Wnt family member 3 A (WNT3A), SMAD family member 4 (SMAD4), and NOTCH1 were drastically increased in the ileum of L-Trp supplemented weaned pigs when compared to control (P < 0.05; Fig. 1D), implying the potential function of L-Trp in promoting intestinal epithelial cell proliferation. As expected, RT-qPCR data confirmed the upregulation of cell proliferative genes including olfactomedin-4 (OLFM4), LGR5, proliferating cell nuclear antigen (PCNA), NOTCH1,* SYR* (YES1 pseudogene 1; P < 0.05), but not WNT3A and β-Catenin, in the ileum of L-Trp supplemented weaned pigs when compared to control (Fig. 1E). Notably, dietary L-Trp activated the AHR pathway as evident by the upregulation of AHR and its target gene cytochrome P450 family 1 subfamily A member 1 (CYP1A1) (P < 0.05; Fig. 1F and G). Moreover, dietary L-Trp also promoted the expression of MST1 and its target gene YAP1, and cell cycle regulatory gene Cyclin D1 (P < 0.05; Fig. 1F and G). Collectively, these data suggest that dietary L-Trp stimulates intestinal epithelial cell proliferation in weaned pigs.Fig. 1. Effect of dietary L-Trp on the intestinal epithelial cell proliferation of weaned pigs. A Representative images of hematoxylin and eosin staining in the duodenum, jejunum and ileum of control (DLY_Ctrl) and L-Trp supplemented (DLY_Trp) weaned pigs (n = 6). DLY, Duroc × Landrace × Large White. Red lines indicated villus height and crypt depth. scale bar: 100 μm. B Volcano plot of differential expressed genes (DEGs) in the ileum of DLY_Ctrl and DLY_Trp weaned pigs. C GO biological process analyses of DEGs in control and L-Trp-supplemented pigs. D Heatmap analysis of cell proliferative gene expression in the ileum of control and L-Trp-supplemented pigs (n = 4). E RT-qPCR validation of the expression of proliferation-associated genes (PCNA, OLFM4, LGR5, β-Catenin, WNT3A, NOTCH1, SYR) in the ileum of control and L-Trp-supplemented pigs (n = 6). F The mRNA expression of effective genes related to the AHR and MST1-YAP1signaling pathways in the ileum of control and Trp-supplemented pigs (n = 6). G The protein expression of AHR, MST1, PCNA and Cyclin D1 in the ileum of control and Trp-supplemented pigs (n = 3). Asterisks denote statistically significant differences (^*^P < 0.05, ^**^P < 0.01). Error bars denote SEM

L-Trp activates intestinal porcine epithelial cells-jejunum 2 cells proliferation

Next, the expression of cell proliferative genes in IPEC-J2 cells were measured to further examine the in vitro activity of L-Trp in regulating intestinal epithelial cell proliferation. Increasing L-Trp concentration from 4 mmol/L to 8 mmol/L increased the expression of PCNA in IPEC-J2 cells (P < 0.05; Fig. 2A). Similarly, 4 mmol/L L-Trp also activated Mucin 13 (MUC13) and SYR expression in IPEC-J2 cells (P < 0.05), suggesting that 4 mmol/L L-Trp is sufficient to enhance IPEC-J2 cell proliferation. Next, EdU incorporation assay was conducted to visualize the proliferative activity of IPEC-J2 cells upon L-Trp treatment. Similar to the pro-proliferative effect as L-glutamate (L-Glu) [19], L-Trp also increased the fluorescence intensity of EdU signal and the ratio of EdU-positive cells in IPEC-J2 cells (P < 0.05; Fig. 2B and C). Furthermore, the expression of cell proliferative gene Cyclin E1, and effective genes related to the TGF-β/SMAD4 and MST1/YAP1 signaling pathways were upregulated in IPEC-J2 cells after L-Trp treatment (P < 0.05; Fig. 2D). These data indicate that L-Trp promotes the proliferation of IPEC-J2 cells.Fig. 2L-Trp promotes proliferation of IPEC-J2 cells. A The mRNA expression of proliferative genes PCNA, SYR and MUC13 in IPEC-J2 cells treated with different concentrations of L-Trp (n = 6). ^a–c^Mean values with unlike letters were significantly different (P < 0.05). B and C EdU staining and quantification of the ratio of EdU-positive cells in IPEC-J2 cells treated with 4 mmol/L L-Trp or 5 mmol/L L-Glu. D The mRNA expression of cell proliferation associated genes Cyclin E1, Cyclin D1, TGF-β*, SMAD4, MST1, YAP1 in IPEC-J2 cells after 4 mmol/L L-Trp or 5 mmol/L L-Glu treatment (n = 6). Asterisks denote statistically significant differences (^^P < 0.05, ^**^P < 0.01). Error bars denote SEM

L-Trp is metabolized into kynurenine to activate cell proliferative gene expression in IPEC-J2 cells

To further delineate the metabolic outcomes of L-Trp, we analyzed the expression of core enzymes governing the kynurenine (Kyn) and serotonin (5-HT) pathways in IPEC-J2 cells (Fig. 3A). The data showed that L-Trp treatment increased indoleamine 2,3-dioxygenase 1 (IDO1) expression (P < 0.05), one enzyme that essential for initiating the Kyn pathway, while the mRNA levels of its downstream enzymes including kynureninase (KYNU), kynurenine aminotransferase (KAT), 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), and quinolinate phosphoribosyltransferase (QPRT) remained unchanged (Fig. 3A). Additionally, the expression of tryptophan hydroxylase 1(TPH1), one initial enzyme in the biosynthesis of 5-HT pathway, was decreased in IPEC-J2 cells after L-Trp treatment (P < 0.05; Fig. 3A), indicating the inactivation of 5-HT pathway in IPEC-J2 cells. Next, Trp-targeted metabolomics analysis identified the increased level of Kyn, tryptamine, xanthurenic acid, indole-3-lactic acid, indole-3-glyoxylic acid in IPEC-J2 cells treated with L-Trp (P < 0.05; Fig. 3B). Conversely, the level of nicotinic acid, 5-hydroxyindole-3-acetic acid, 5-hydroxyl-l-tryptophan and 5-hydroxytryptophol were reduced in L-Trp-treated IPEC-J2 cells when compared to control (P < 0.05; Fig. 3B). These data suggest the activation of the Trp-Kyn metabolic pathway in IPEC-J2 cells upon L-Trp treatment.Fig. 3L-Trp is metabolized into Kyn to activate cell proliferation in IPEC-J2 cells. A Schematic presentation of Trp metabolic pathways and qRT-PCR analyses of the expression of key enzymes related to the Trp-Kyn and Trp-serotonin pathways in IPEC-J2 cells treated with 4 mmol/L L-Trp (n = 6). B Targeted metabolomic analysis of Trp metabolism in IPEC-J2 cells upon 4 mmol/L L-Trp treatment. C qRT-PCR analysis of the expression of cell proliferation associated genes in IPEC-J2 cells in response to kynurenine (Kyn), kynurenic acid (KYNA), and 3-hydroxyanthranilic acid (3-HAA) treatment, respectively (n = 6). D The protein expression of LAST1, MST1 and Cyclin E1 in IPEC-J2 cells treated with 4 mmol/L Kyn (n = 3). E Targeted metabolomic analysis of Trp metabolism in IPEC-J2 cells after 4 mmol/L Kyn treatment. Asterisks denote statistically significant differences (^*^P < 0.05, ^**^P < 0.01). Error bars denote SEM

Then, we evaluated the effect of the Kyn pathway-derived metabolites including Kyn, kynurenic acid (KYNA), and 3-hydroxyanthranilic acid (3-HAA) on the regulation of IPEC-J2 cell proliferation. As shown in Fig. 3C, only Kyn, but not KYNA or 3-HAA, activated the expression of cell proliferative genes Cyclin D1, Cyclin E1,* SMAD4,* and the MST1-YAP1 pathway responsive genes MST1 and YAP1 in IPEC-J2 cells when compared to control (P < 0.05). Interestingly, the classic Wnt and Notch pathway responsive genes β-Catenin and NOTCH1 were not activated in IPEC-J2 cells upon Kyn, KYNA or 3-HAA treatment (Fig. S2). Furthermore, the protein expression of large tumor suppressor 1 (LAST1) and MST1, two effector proteins of the MST1/YAP1 pathway, and Cyclin E1 were upregulated in IPEC-J2 cells upon Kyn treatment (P < 0.05; Fig. 3D). Trp-targeted metabolomics analysis in Kyn-treated IPEC-J2 cells revealed the increased levels of Kyn, 5-hydroxytryptophol and indole-3-carboxaldehyde, but the decreased levels of indole-3-propionic acid, indole-3-lactic acid and 5-methoxytryptamine (P < 0.05; Fig. 3E). Taken together, these data imply that the Kyn may act as the major Trp-Kyn pathway-derived metabolite to activate IPEC-J2 cell proliferation.

Kyn exhibits a high binding affinity with AHR

Trp metabolites have been reported to act as endogenous ligands of AHR [35]. Thus, molecular docking analysis was conducted to examine the binding affinity between Kyn and AHR. The data demonstrated that Kyn exhibited a favorable binding affinity toward the AHR ligand-binding pocket, with a predicted binding energy of −5.5 kcal/mol (Fig. 4A–D). The ligand fit well within the receptor cavity and formed four hydrogen bonds with residues aspartate-65 (ASP65), serine-68 (SER68), and aspartate-151 (ASP151; Fig. 4A), indicating a stable interaction. Additional nonbonded contacts, including hydrophobic interactions and electrostatic attractions, further contributed to complex stabilization (Fig. 4B–D). These results suggest that Kyn possesses a suitable structural conformation for binding to AHR under physiological conditions.Fig. 4. Molecular docking analysis between Kyn and AHR. A Prediction of the binding pocket of the porcine AHR with Kyn. Light blue structure indicated the AHR protein, pink sticks denoted Kyn, and the hydrogen bonds in Å units were depicted as yellow dashed lines. B 2D Visualization of the binding site in Kyn-AHR docking complex. C Surface electrostatic force visualization at the docking interface of Kyn with AHR. D Calculation of the binding energy between Kyn and AHR. Generally, the binding energy below −5.0 kcal/mol is considered to have good binding affinity

The AHR-MST1-YAP1 axis is required for Kyn-induced cell proliferation in IPEC-J2 cells

Next, the expression of AHR and its target gene CYP1A1 was measured to evaluate whether the AHR pathway is activated in IPEC-J2 cells upon Kyn treatment. The data showed that the expression of both AHR and CYP1A1 were upregulated (P < 0.05; Fig. 5A and Fig. S3), indicating the activation of the AHR pathway in IPEC-J2 cells. By searching and analyzing the JASPAR database, we identified two conserved AHR binding motifs (5′-GCGTG-3′) located in the upstream promoter region of MST1 gene (Fig. 5B), indicating that AHR may directly regulate MST1 transcription. Then, we co-treated IPEC-J2 cells with Kyn and Verteporfin, a specific pharmacological inhibitor of YAP1 (Fig. S4) [36], to further examine whether the MST1/YAP1 pathway is required for Kyn-induced cell proliferation. As shown in Fig. 5C, Verteporfin co-treatment alleviated Kyn-induced expression of MST1 and YAP1, and cell proliferative genes PCNA and Cycin E1 (P < 0.05). Moreover, the EdU staining showed that Verteporfin co-treatment drastically decreased Kyn-induced EdU fluorescence intensity (P < 0.05; Fig. 5D and F). Similarly, the scratch assay, a method to measure two-dimensional cell migration, also found that Verteporfin co-treatment blocked Kyn-induced cell migration (P < 0.05; Fig. 5E and F). These results imply that Kyn-induced cell proliferation in IPEC-J2 cells is dependent on the activation of YAP1 expression.Fig. 5. Kyn promotes the proliferation of IPEC-J2 cells via activating the AHR-MST1-YAP1 axis. A Quantification of the mRNA expression of AHR and CYP1A1 in IPEC-J2 cells after Kyn or Kyn + Verteporfin treatment (n = 6). B Illustration of the AHR binding sites in the promote region of the MST1 gene. C Quantification of the mRNA expression of MST1, YAP1, PCNA and Cyclin E1 after Kyn or Kyn + Verteporfin treatment (n = 6). D, E EdU staining and scratch assay in IPEC-J2 cells treated with Kyn alone or Kyn + Verteporfin (Scale bar: 20 μm). F Quantification of the ratio of EdU-positive cells and the width of cell migration in IPEC-J2 cells treated with Kyn alone or Kyn + Verteporfin. G and H Quantification of the mRNA expression (n = 6) of AHR and CYP1A1 and the protein expression (n = 3) of PCNA in IPEC-J2 cells treated with Kyn alone or Kyn + AHR inhibitor. I Quantification of the mRNA expression of MST1, YAP1, PCNA and Cyclin E1 after Kyn or Kyn + AHR inhibitor treatment (n = 6). J and K Immunostaining and scratch assay in IPEC-J2 cells treated with Kyn alone or Kyn + AHR inhibitor (Scale bar: 20 μm). The yellow arrowheads indicated the nuclear localization of AHR, while the red arrowheads indicated the cytoplasmic localization of AHR, respectively. L Quantification of the fluorescence intensity of PCNA and the width of cell migration in IPEC-J2 cells after Kyn or Kyn + AHR inhibitor treatment. Asterisks denote statistically significant differences (^*^P < 0.05, ^**^P < 0.01). Error bars denote SEM

Subsequently, the AHR inhibitor was also co-cultured with Kyn to investigate whether the AHR pathway is required for Kyn-induced cell proliferation in IPEC-J2 cells. As shown in Fig. 5G and H, AHR inhibitor co-treatment reduced the Kyn-induced the mRNA expression of AHR and CYP1A1, and the protein level of PCNA in IPEC-J2 cells (P < 0.05). Moreover, AHR inhibitor co-treatment also decreased the Kyn-induced expression of the MST1-YAP1 pathway responsive genes MST1 and YAP1, and cell proliferative genes PCNA and Cyclin D1 (P < 0.05; Fig. 5I). The immunostaining data showed that AHR inhibitor co-treatment greatly reduced the Kyn-induced nuclear translocation of AHR and the fluorescence intensity of PCNA (Fig. 5J–L). Similarly, the scratch assay also confirmed that AHR inhibitor co-treatment suppressed Kyn-induced cell migration in IPEC-J2 cells (P < 0.05; Fig. 5K and L). Collectively, these data suggest that the AHR-MST1-YAP1axis is required for Kyn-induced cell proliferation in IPEC-J2 cells.

Kyn promotes porcine intestinal organoid budding rate

Next, a porcine intestinal organoid model was utilized to investigate the role of Kyn in regulating intestinal stem cell (ISC) proliferation. As shown in Fig. 6A and B, 250 µmol/L Kyn incubation increased the organoid budding rate over d 1 to d 3 (P < 0.05). In contrast, 25 µmol/L or 50 µmol/L Kyn incubation only transiently increased the organoid budding rate on d 1 (P < 0.05; Fig. 6A and B), suggesting that Kyn modulates porcine intestinal organoid budding rate both in a dynamic and dosage-dependent manner. Moreover, 250 µmol/L Kyn incubation activated the AHR-MST1-YAP1 axis, as presented by the increased expression of AHR, MST1 and YAP1 in intestinal organoids (P < 0.05; Fig. 6C). Concurrently, the expression of cell proliferative genes PCNA, TGF-β, SMAD4, OLFM4, Cyclin E1, and Cyclin D1 were also increased in intestinal organoids upon 250 µmol/L Kyn incubation (P < 0.05; Fig. 6C). Together, these data imply that Kyn promotes porcine intestinal organoids budding rate by activating the expression of cell proliferative genes.Fig. 6. Kyn enhances porcine intestinal organoids budding rate. A and B Representative images of porcine intestinal organoids and the quantification of the budding efficiency from the control, 25 mmol/L, 50 mmol/L and 250 mmol/L Kyn groups at d 1, d 2 and d 3 respectively. C Quantification of the mRNA expression cell proliferation-related genes in Kyn-treated intestinal organoids (n = 6). Asterisks denote statistically significant differences (^*^P < 0.05, ^**^P < 0.01). Error bars denote SEM

Kyn-induced intestinal stem cell proliferation is dependent on the activation of the MST1-YAP1 pathway

Finally, the YAP1-specific inhibitor Verteporfin was also incubated in porcine intestinal organoid to examine whether the MST1-YAP1 pathway is required for Kyn-induced intestinal organoid budding. Intriguingly, Verteporfin incubation decreased Kyn-induced porcine intestinal organoid budding rate (P < 0.05; Fig. 7A–D). Furthermore, Kyn treatment increased the fluorescence intensity of OLFM4, a protein widely used as a maker for ISC, in porcine intestinal organoid, when compared to control (P < 0.05; Fig. 7E–H), indicating that Kyn promotes ISC proliferation in intestinal organoids. Similarly, Verteporfin incubation also greatly decreased the Kyn-induced fluorescence intensity of OLFM4 in intestinal organoids (P < 0.05; Fig. 7E–H). In addition, Kyn incubation increased the protein expression of AHR, MST1, and cell proliferation effectors PCNA and Cyclin D1 in intestinal organoid (P < 0.05; Fig. 7I and J). While Verteporfin incubation greatly suppressed Kyn-induced activation of these proteins in intestinal organoids (P < 0.05; Fig. 7I and J). Taken together, these data suggest that the Kyn-induced intestinal stem cell proliferation in porcine intestinal organoids is dependent on the activation of the MST1-YAP1 pathway (Fig. 7K).Fig. 7. Kyn-induced intestinal organoids proliferation is dependent on the activation of the MST1-YAP1 pathway. A–D Representative images of porcine intestinal organoids and the quantification of budding efficiency from the control, Kyn and Kyn + Verteporfin treated groups, respectively (scale bar: 100 μm). E–H Immunostaining and the fluorescence intensity of OLFM4 in the control, Kyn and Kyn + Verteporfin treated groups, respectively (scale bar: 20 μm). I and J The protein expression of PCNA, AHR, MST1 and Cyclin D1 in intestinal organoids treated with Kyn or Kyn + Verteporfin (n = 3). K Proposed model of Trp metabolism-derived Kyn in regulating intestinal epithelial cell proliferation via activation of the AHR-MST1-YAP axis. Trp, tryptophan; Kyn, kynurenine; AHR, aryl hydrocarbon receptor; ARNT, aryl hydrocarbon receptor nuclear translocator; ISC, intestinal stem cell; IEC, intestinal epithelial cell. Asterisks denote statistically significant differences (^*^P < 0.05, ^**^P < 0.01). Error bars denote SEM

Discussion

Intestinal epithelium renewal driven by intestinal stem cells occurs throughout the whole developmental stages of pigs and contributes to the maintenance of gut health. Trp acts as the critical amino acid that is essential for intestinal epithelial development and growth. Interestingly, dietary Trp levels exhibit both positive and negative effects on the regulation of intestinal morphology and barrier function in weaned pigs. Dietary supplementation of low levels of Trp (e.g., 0.20%, 0.21%, 0.28%, 0.35%, 0.40%) increases the villus height and the ratio of villus height to crypt depth, and enhances the expression of tight junction proteins in the jejunum and ileum of piglets [37–39]. While a high level of Trp (e.g., 0.75%) decreases the ratio of villus height to crypt depth and reduces intestinal integrity in the jejunum of weaned pigs [40]. It is likely that the effect of dietary Trp levels on intestinal epithelial renewal and barrier function in piglets is dosage-dependent. Low dietary Trp level generally has beneficial effect on intestinal development, while high dietary Trp level may generate negative effect on intestinal structure and function. Therefore, in this study, we selected 0.2% Trp in the experimental group to examine the positive regulatory function of Trp in intestinal epithelial development. Our data found that dietary supplementation of 0.2% Trp significantly increases the villus height and the ratio of villus height to crypt depth, which is consistence with above findings reported by Rao et al. [37] and Liang et al. [39]. Notably, these studies only measured intestinal morphological characteristics upon dietary Trp supplementation, without measuring gene expression that are linked to intestinal epithelial cell proliferation. By conducting mRNAseq, we profiled the global mRNA gene expression in the ilium of pigs fed with Trp, and found that the function of differentially expressed genes is enriched in epithelial cell proliferation. Moreover, the expression of a subset of cell proliferative genes such as PCNA, OLFM4, LGR5, and Cyclin E1 were also significantly activated. These data imply that the improved intestinal morphology in pigs fed with Trp is likely, but not exclusively, due to enhanced intestinal epithelial cell proliferation.

Like other amino acids, Trp is primarily digested and absorbed in the small intestine, where it can be further metabolized by the gut microbiota. The small intestine is divided into duodenum, jejunum and ileum, among which jejunum and ileum are capable of absorbing and metabolizing Trp for gut development [41, 42]. Absorption is largely complete at the end of the small intestine. Proteins are degraded into peptides in jejunum and approximately 90% of amino acids are absorbed in ileum [43, 44]. In this study, we found that dietary supplementation of Trp increases the villus height and the ratio of villus height to crypt depth in all three segments of the small intestine in weaned pigs. In particular, the ileum exhibited the most dominant phenotypes in villus height when compared to duodenum and jejunum. Thus, we selected the ileum as the major site for profiling gene expression related to epithelial cell proliferation. However, because the porcine ileum-derived epithelial cell lines are not available, we chose the IPEC-J2 cells, a well-characterized intestinal epithelial cell line derived from the jejunum of a piglet, and porcine jejunum-derived organoids as in vitro models to investigate the mechanism of how Trp activates intestinal epithelial cell proliferation. Akin to the proliferative effects observed in the ileum of piglets, Trp or Kyn treatment also activates intestinal epithelial cell proliferation in two in vitro models, suggesting that the effect of Trp and its metabolites on regulating the proliferation of intestinal epithelial cells may be similar, but not identical, between jejunum and ileum. This is consistent with a report shown that dietary Trp has no obvious effect on morphology and development between jejunum and ileum of finishing pigs [45].

The growth and development of porcine intestine is driven by different types of luminal nutrients including amino acids that affect the morphology and function of intestinal epithelial cells. The proliferative activity of intestinal epithelial cells is linked to intestinal barrier and immune function, thus maintaining intestinal homeostasis. For instance, glutamate activates ISC expansion to promote intestinal epithelial growth by integrating the canonical FZD7/Wnt signaling pathway and mTORC1 activity [19, 20]. Glutamine accelerates porcine ISC proliferation via activating the Wnt signaling to alleviate weaning stress-induced intestinal epithelial atrophy [17, 46]. Additionally, arginine increases porcine intestinal epithelial cell proliferation and prevents cell apoptosis [47]. In this study, we showed that oral Trp administration increases the villus height and the ratio of villus height to crypt depth, as well as the expression of cell proliferative genes. Further in vitro analyses revealed that Trp is metabolized into Kyn to promote intestinal epithelial cell proliferation in an AHR-dependent manner. These findings imply that nutrients-derived functional amino acids including Trp play pivotal roles in regulating the activity of intestinal epithelial cells to maintain gut health in pigs. In agreement with this hypothesis, dietary supplementation of Trp has been shown to improve intestinal epithelial function and growth performance of pigs both under physiological and pathological conditions [37, 48].

Interestingly, we found that Trp promotes ISC proliferation by activating the Hippo signaling pathway, not the classic Wnt or Notch signaling pathways. MST1 and YAP1 are two core mediators of the Hippo signaling pathway that involves epithelial cell repair in multiple tissues, including the intestine [49–51]. YAP1 reprograms the activity of LGR5^+^ ISCs by inhibiting the Wnt activity, and sustains transient amplifying cells proliferation to modulate the self-renewal capacity of intestinal epithelial cells [11, 12, 52]. Recently, the Trp-indole pathway-derived indole-3-lactic acid (ILA) has been reported to mitigate intestinal damage and accelerate ISC proliferation by upregulating YAP1 expression in mice upon ischemia/reperfusion injury [53]. This suggest that the YAP1 may act as a downstream target gene of Trp metabolic pathways in modulating ISC proliferation. To support this hypothesis, we found that YAP1 inhibition drastically blocks Kyn treatment-induced intestinal epithelial cell proliferation in IPEC-J2 cells and porcine intestinal organoids. Moreover, we also identified several AHR binding sites on the promoter region of MST1, an upstream effector of YAP1. Collectively, these data imply that the Trp metabolism-derived Kyn promotes intestinal epithelial cell proliferation via the Kyn-AHR-MST1-YAP1 axis.

One limitation in this study is that both IPEC-J2 cells and porcine intestinal organoids cannot fully mimic the physiological environment in the gut of piglets, due to lack of some complex structures such as the absence of microbiome. By performing Trp-targeted metabolomic analysis in IPEC-J2 cells, we found the decreased amount of a few indole derivatives including indole-3-propionic acid (IPA), indo-3-acetamide and ILA in Kyn-treated IPEC-J2 cells, suggesting the inactivation of the indole pathway in IPEC-J2 cells. As expected, the most increased Trp metabolites identified in IPEC-J2 cells was the Kyn pathway-derived metabolites, including Kyn and its downstream products KYNA and 3-HAA. However, only Kyn treatment significantly increased the expression of Cyclin D1 and Cyclin E1, thus activating intestinal epithelial cell proliferation in IPEC-J2 cells. These data imply that Kyn may act as the primary cue from the Trp-Kyn pathway to promote intestinal epithelial cell proliferation. However, the effect of dietary supplementation of Kyn on the activation of intestinal stem cell proliferation in piglets awaits further investigation.

In addition, indole and its derivatives have also been shown to activate the AHR pathway to regulate intestinal epithelial cell proliferation. The IPA and indole-3-acetic acid participate in regulating intestinal barrier function and intestinal stem cell proliferation [54–56]. Indole-3-carbaldehyde activates the AHR/IL-10/Wnt signaling pathway to promote intestinal stem cell proliferation in mice [57]. Indole-3-aldehyde mitigates weaning stress-induced intestinal epithelial injury via promoting ISC expansion in piglets [26]. Thus, we cannot exclude the possibility that the Trp-indole pathway is also involved in regulating intestinal epithelial cell proliferation in piglets fed with Trp. Since both indole derivatives and Kyn serve as intracellular AHR ligands, these two pathways may regulate each other through the AHR pathway. Interestingly, the interaction between the Kyn pathway and indole pathway has been recently reported. Indole-3-carboxaldehyde competes with Kyn to bind to AHR to inhibit T-cell function [58]. Establishment of the AHR-IDO1-Kyn-AHR feedback loop in immune regulation can be enhanced by indoles [59]. Therefore, it is also interesting to further examine whether and how indole metabolites act together or compete with Kyn to activate the AHR pathway to modulate intestinal epithelial cell proliferation in the gut.

Conclusion

In summary, this study has demonstrated that the Kyn acts as a key metabolite of the Trp-Kyn metabolic pathway to drive intestinal epithelial cell proliferation in pigs. Mechanistically, Kyn serves as a ligand binding to AHR to activate the downstream MST1-YAP1 signaling pathways, which in turn stimulates the expression of cell proliferative genes to promote intestinal epithelial cell proliferation. The Kyn-induced intestinal epithelial cell proliferation is dependent on the activation of the AHR-MST1-YAP1 axis.

Supplementary Information

Additional file 1: Table S1. The primer sequences of RT-qPCR used in this study. Fig. S1. Survival assay of IPEC-J2 cells after treatment with different concentrations of DMSO. Fig. S2. RT-qPCR analysis of the expression of effective genes in Wnt and Notch pathways after L-Kyn, KYNA and 3-HAA treatment in IPEC-J2 cells. Fig. S3. RT-qPCR analysis of the efficiency of different concentration of Kyn in promoting AHR expression in IPEC-J2 cells. Fig. S4. RT-qPCR analysis of the efficiency of different concentrations of Verteporfin in suppressing YAP1 expression in IPEC-J2 cells.Additional file 2: The original gel and blot images.