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.