Vitamin B12 Regulates the Transcriptional, Metabolic, and Epigenetic Programing in Human Ileal Epithelial Cells

Abstract

Vitamin B12 (VB12) is a micronutrient that is essential for DNA synthesis and cellular energy production. We recently demonstrated that VB12 oral supplementation coordinates ileal epithelial cells (iECs) and gut microbiota functions to resist pathogen colonization in mice, but it remains unclear whether VB12 directly modulates the cellular homeostasis of iECs derived from humans. Here, we integrated transcriptomic, metabolomic, and epigenomic analyses to identify VB12-dependent molecular and metabolic pathways in human iEC microtissue cultures. RNA sequencing (RNA-seq) revealed that VB12 notably activated genes involved in fatty acid metabolism and epithelial cell proliferation while suppressing inflammatory responses in human iECs. Untargeted metabolite profiling demonstrated that VB12 facilitated the biosynthesis of amino acids and methyl groups, particularly S-adenosylmethionine (SAM), and supported the function of the mitochondrial carnitine shuttle and TCA cycle. Further, genome-wide DNA methylation analysis illuminated a critical role of VB12 in sustaining cellular methylation programs, leading to differential CpG methylation of genes associated with intestinal barrier function and cell proliferation. Together, these findings suggest an essential involvement of VB12 in directing the fatty acid and mitochondrial metabolisms and reconfiguring the epigenome of human iECs to potentially support cellular oxygen utilization and cell proliferation.

Keywords: DNA methylation; cell proliferation; ileal epithelial cells; mitochondrial metabolism; vitamin B12.

Figure 1

VB12-dependent regulation of transcriptomic responses in human iECs. The organotypic 3D human small intestine microtissues were cultured in the presence of VB12 or PBS (n = 3 samples/group) using cells obtained from the ileal region of a 19-year-old female donor. Two weeks later, cells were collected for RNA-seq analysis. (A) PCA plot of transcriptomes of human iECs cultured in the presence of VB12 versus PBS. (B) Volcano plot of differentially expressed genes (DEGs; FDR < 0.05, fold change > 1.5) between VB12 and PBS groups. The number of upregulated (green) and downregulated (red) DEGs in the VB12 group is shown in parentheses. (C) Heat map showing top-50 DEGs. (D) STRING analysis depicting the networks of the top-50 DEGs. (E) qPCR validation of representative DEGs. (F) KEGG pathways of DEGs enriched in the VB12 versus PBS group. Bar graphs show mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001; two-tailed unpaired t-test (E).

Figure 2

Comparison of human and mouse iEC-enriched genes. Green dots indicate genes upregulated in both human iECs cultured with VB12 and iECs isolated from VB12-gavaged mice of different ages (1, 5, and 8 weeks old), compared to their PBS counterparts. Red dots indicate genes downregulated in the VB12 group of both human and mouse iECs. Dashed lines indicate fold changes of 1.5. log₂ FC, log₂ (fold change).

Figure 3

Metabolic programming of human iECs by VB12. (A) PCA plots of metabolite features identified by positive and negative ionization in human iECs cultured with VB12 or PBS for 2 weeks (n = 3 samples/group). (B) Significant metabolic pathway between VB12- and PBS-cultured iECs. (C) Heatmap showing differentially enriched metabolites. SAM, S-adenosylmethionine.

Figure 4

VB12-mediated alterations in genome-wide DNA methylation profiles of human iECs. (A) Global CpG methylation ratios of human iECs cultured with VB12 or PBS for 2 weeks (n = 3 samples/group). (B) Summary of the number of differential methylation regions (DMRs) between VB12- and PBS-cultured human iECs from WGBS analysis. (C) Donut chart representative of the DMR genomic distribution. UTR, untranslated region. (D) Comparison of DEGs and DMRs between VB12 and PBS groups. The number of DMRs within indicated locations of the DEGs is shown in parentheses. (E) Normalized plots of CpG methylation at sites surrounding or within DMRs of genes associated with barrier function (MUC13) and cell proliferation (PTGER2, FGFR1, GRK5 and SHH) obtained from WGBS analysis. The red and blue lines depict methylated and unmethylated CpG sites, respectively. * p < 0.05; two-tailed unpaired t-test (A).

Figure 5

Summary model based on transcriptomic, metabolomic, and epigenomic integration. As a crucial co-factor for MCM, VB12 supports the generation of succinyl-CoA, which can participate in the TCA cycle and serve as the precursor for heme biosynthesis, resulting in the activation of heme enzymes. VB12 also facilitates fatty acid metabolism, which potentially generates free fatty acids that can be transported into mitochondria to fuel the TCA cycle through the carnitine shuttle. Further, VB12 sustains the MS activity to enhance the biosynthesis of carnitine and SAM, which supports the carnitine shuttle and influences epigenetic programs of genes involved in cell proliferation. An elevated TCA cycle also contributes to the production of amino acids that are essential for cell proliferation.