Glutamine Regulates Gene Expression Profiles to Increase the Proliferation of Porcine Intestinal Epithelial Cells and the Expansion of Intestinal Stem Cells

Abstract

The intestinal epithelium is known for its rapid self-renewal, and glutamine is crucial in providing carbon and nitrogen for biosynthesis. However, understanding how glutamine affects gene expression in the intestinal epithelium is limited, and identifying the essential genes and signals involved in regulating intestinal epithelial cell growth is particularly challenging. In this study, glutamine supplementation exhibited a robust acceleration of intestinal epithelial cell proliferation and stem cell expansion. RNA sequencing indicated diverse transcriptome changes between the control and glutamine supplementation groups, identifying 925 up-regulated and 1152 down-regulated genes. The up-regulated DEGs were enriched in the KEGG pathway of cell cycle and GO terms of DNA replication initiation, regulation of phosphatidylinositol 3-kinase activity, DNA replication, minichromosome maintenance protein (MCM) complex, and ATP binding, whereas the down-regulated DEGs were enriched in the KEGG pathway of p53 signaling pathway, TNF signaling pathway, and JAK-STAT signaling pathway and GO terms of inflammatory response and intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress. Furthermore, GSEA analysis revealed a significant up-regulation of the cell cycle, DNA replication initiation, ATP-dependent RNA helicase activity, and down-regulation of the TNF signaling pathway. The protein-protein association network of the intersecting genes highlighted the significance of DNA replication licensing factors (MCM3, MCM6, and MCM10) in promoting intestinal epithelial growth in response to glutamine. Based on these findings, we propose that glutamine may upregulate DNA replication licensing factors, leading to increased PI3K/Akt signaling and the suppression of TNF, JAK-STAT, and p53 pathways. Consequently, this mechanism results in the proliferation of porcine intestinal epithelial cells and the expansion of intestinal stem cells.

Keywords: gene expression profile; glutamine; intestinal epithelium; intestinal stem cells; minichromosome maintenance protein; transcriptomics.

Figure 1

Ex vivo glutamine administration increases the activity of intestinal organoids. (A) MTT assay was used to detect the effect of different concentrations of glutamine on IPEC proliferation. The results are expressed as mean ± SEM (n = 8 wells per group). Statistical significance was determined by one-way ANOVA. Different lowercase letters indicate significant differences (p < 0.05). (B,C) Organoid-forming efficiency and budding efficiency were measured on day 4. The results are expressed as mean ± SEM (n = 5 wells per group, * p < 0.05). Statistical significance was determined by Student’s t-test. (D) Representative images of intestinal organoids cultured from the additive-free culture medium group and the 2 mM glutamine treatment group on days 1, 2, 3, and 4 (40× magnification). (E,F) Ex vivo administration of glutamine enhanced the mitosis of intestinal stem cells, as indicated by the increasing fluorescence intensity (FI) of EdU (calculated as FIs normalized to the mean FI of the control group). Representative images of intestinal organoids stained with the EdU reagent are shown in the control and 2 mM glutamine groups. The results are expressed as mean ± SEM (n = 5 organoids per group, ** p < 0.01). Statistical significance was determined by the Student’s t-test.

Figure 2

Gene expression profiles between the control and 2 mM glutamine groups. (A) Principal component analysis of the groups based on log2(FMPK + 1) corrected expression data. (B) Distances within each sample. (C) Unbiased hierarchical clustering heat map based on Pearson’s correlation coefficient for 16,201 identified genes. The ordinate and coordinates represent sample names. Color intensity indicates the correlation between samples, with dark blue indicating a high correlation.

Figure 3

Identification of DEGs between the control (CON) and 2 mM glutamine (GLN) groups. (A) The volcano plot displays DEGs from GLN vs. CON. The x-axis represents the log2 (fold change) value, and the y-axis represents the log10 (p-value). Blue nodes represent down-regulated genes, while red nodes represent up-regulated genes. (B) Number of DEGs from GLN vs. CON. (C) Top 30 DEGs in the CON and GLN groups.

Figure 4

Identification of functional enrichment from DEGs. (A,B) KEGG functional enrichment for up-regulated DEGs and down-regulated DEGs. (C,D) GO pathways enriched for up-regulated DEGs and down-regulated DEGs.

Figure 5

Identifying the crucial pathways and genes involved in cell proliferation and anti-inflammation. (A–F) Performing gene set enrichment analysis (GSEA) on IPEC-J2 transcriptomics data ranked from the glutamine treatment group (red) to the control group (blue) reveals significant enrichment in various pathways, including cell cycle, focal adhesion, TNF signaling pathway, DNA replication initiation, DNA replication, and ATP-dependent RNA helicase activity. (G) Venn plot illustrates the intersecting genes among the six crucial pathways. (H) The protein–protein association network of the intersection genes displays nodes representing the genes, with the size of each node indicating the number of connections (degree). Larger node sizes indicate a higher number of connections.