Effects of undigested protein-rich ingredients on polarised small intestinal organoid monolayers

Here, we describe the use of monolayers of intestinal epithelial cells derived from intestinal organoids and transcriptomics to investigate the direct effects of dietary protein sources on epithelial function. Mechanically dissociated 3D organoids of mouse duodenum were used to generate a polarized epithelium containing all cell types found in the tissue of origin. The organoid-derived cell monolayers were exposed to 4% (w/v) of 'undigested (non-hydrolysed)-soluble' fraction of protein sources used as feed ingredients [soybean meal (SBM) and casein], or alternative protein sources (spray dried plasma protein, and yellow meal worm), or controls for 6 h prior to RNA isolation and transcriptomics. All protein sources altered expression of unique biological processes in the epithelial cells. Exposure of intestinal organoids to SBM downregulated expression of retinol and retinoid metabolic processes as well as cholesterol and lipid biosynthetic pathways, consistent with the reported hypotriglyceridaemic effect of soy protein in vivo. These findings support the use of intestinal organoids as models to evaluate complex interactions between dietary ingredients and the intestinal epithelium and highlights some unique host effects of alternative protein sources in animal feed and potentially human food. Graphical abstract: Schematic representation of the study. 3-dimensional organoids were generated from mouse duodenum (1). The organoids were subsequently dissociated into single cells (2) and grown as 2-dimensional polarised monolayers (3). Polarized monolayers of organoid cells were exposed to different protein sources [CAS, SBM, SDPP, YMW, or medium control (MC)] for 6 h (4) and further processed for imaging (5) gene expression (6), and biochemical assays (7), to investigate the effects of undigested protein sources on the duodenal epithelium. [Figure not available: See fulltext.]</p

Dietary protein sources differentially affect microbiota, mTOR activity and transcription of mTOR signaling pathways in the small intestine

Dietary protein sources can have profound effects on host-microbe interactions in the gut that are critically important for immune resilience. However more knowledge is needed to assess the impact of different protein sources on gut and animal health. Thirty-six wildtype male C57BL/6J mice of 35 d age (n = 6/group; mean ± SEM body weight 21.9 ± 0.25 g) were randomly assigned to groups fed for four weeks with semi synthetic diets prepared with one of the following protein sources containing (300 g/kg as fed basis): soybean meal (SBM), casein, partially delactosed whey powder, spray dried plasma protein, wheat gluten meal and yellow meal worm. At the end of the experiment, mice were sacrificed to collect ileal tissue to acquire gene expression data, and mammalian (mechanistic) target of rapamycin (mTOR) activity, ileal digesta to study changes in microbiota and serum to measure cytokines and chemokines. By genome-wide transcriptome analysis, we identified fourteen high level regulatory genes that are strongly affected in SBM-fed mice compared to the other experimental groups. They mostly related to the mTOR pathway. In addition, an increased (P < 0.05) concentration of granulocyte colony-stimulating factor was observed in serum of SBM-fed mice compared to other dietary groups. Moreover, by 16S rRNA sequencing, we observed that SBM-fed mice had higher (P < 0.05) abundances of Bacteroidales family S24-7, compared to the other dietary groups. We showed that measurements of genome-wide expression and microbiota composition in the mouse ileum reveal divergent responses to diets containing different protein sources, in particular for a diet based on SBM

Immunohistochemistry of PFA-fixed paraffin-embedded tissue sections with anti-mTOR antibody in ileal tissue of mice fed with different experimental diets.

<p><b>(</b>A) Brown color indicates positive reaction in the image of tissue type control (testis) and a positive ileal tissue section (CAS). No positivity was observed in the same tissue by withholding the primary antibody (i.e. anti-mTOR antibody) during the staining procedure. (B) Immunohistochemistry of PFA-fixed paraffin-embedded ileum sections with anti-mTOR antibody of mice fed with different experimental diets. Brown color in the tissue section indicates positive reaction. SBM, soybean meal; CAS, casein; SDPP, spray dried porcine plasma; WGM, wheat gluten meal and YMW, yellow meal worm. Scale bar: 50 μm.</p

Endogenous levels of phosphorylated mTOR in cells derived from ileal tissue of mice fed with different experimental diets.

<p>Bars and whiskers represents means values ± SEM (n = 12, six biological replicates and two technical replicates); *P < 0.05, **P < 0.005, ***P < 0.0005 compared to SBM-fed mice fed. Here, SBM, soybean meal; CAS, casein; SDPP, spray dried porcine plasma; WGM, wheat gluten meal and YMW, yellow meal worm.</p

The ileal microbiome composition profiles based on the relative abundance at the family level.

<p>Bacterial families are represented with different colors (shown in key). Hierarchical clustering of the microbial family composition is indicated above the composition profile. * Represents significant difference (<i>P</i> < 0.05) of microbiota at family level compared to SBM-fed mice. Members of microbial family belonging to ‘others’ are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188282#pone.0188282.s007" target="_blank">S3 Table</a>. SBM, soybean meal; CAS, casein; DWP, partially delactosed whey powder; SDPP, spray dried porcine plasma; WGM, wheat gluten meal and YMW, yellow meal worm.</p

Concentrations of serum granulocyte-colony stimulating factor (G-CSF) of mice fed diets containing different dietary proteins.

<p>Bars and whiskers represents means values ± SEM (n = 6), recorded at the end of experiment. *<i>P</i> < 0.05 compared to SBM-fed mice fed. SBM, soybean meal; CAS, casein; DWP, partially delactosed whey powder; SDPP, spray dried porcine plasma; WGM, wheat gluten meal and YMW, yellow meal worm.</p

Key or hub genes of the functional network downregulated in mice fed soybean meal (SBM)-based diet.

<p>Key or hub genes of the functional network downregulated in mice fed soybean meal (SBM)-based diet.</p

Venn diagram featuring common and unique enriched gene-set expression patterns.

<p>(A) Colored spheres represent different experimental diets. The white number at the core of the Venn diagram represents the number of common overlapping gene-sets and black numbers towards the periphery of each sphere represent the number of unique gene-sets. (B) Functional interaction network for the common overlapping gene-sets. The colored nodes denote the GO term significantly (FDR < 0.001) overrepresented in the enriched gene-sets. The edges represent interactions between gene-set as determined by BiNGO. Sky blue color nodes relate to immune processes and light pink color nodes relate to metabolic processes. Arrows represent directed interactions. The diameter of the nodes represents the number of genes associated with that particular GO term. SBM, soybean meal; CAS, casein; DWP, partially delactosed whey powder; SDPP, spray dried porcine plasma; WGM, wheat gluten meal and YMW, yellow meal worm.</p

Differential gene expression and number of enriched gene-sets in ileal mucosa of mice fed experimental diets with indicated protein sources relative to a diet with SBM.

<p>Differential gene expression and number of enriched gene-sets in ileal mucosa of mice fed experimental diets with indicated protein sources relative to a diet with SBM.</p