Epigenetic modifier supplementation improves mitochondrial respiration growth rates and alters DNA methylation of bovine embryonic fibroblast cells cultured in divergent energy supply

Authors: Crouse, Matthew; CATON, JOEL - North Dakota State University; Larson, Kate; DINIZ, WELLISON - North Dakota State University; Lindholm-Perry, Amanda; REYNOLDS, LARRY - North Dakota State University; DAHLEN, CARL - North Dakota State University; BOROWICZ, PAWEL - North Dakota State University; WARD, ALISON - North Dakota State University

Submitted to: Frontiers in Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/2022
Publication Date: 2/24/2022
Citation: Crouse, M.S., Caton, J.S., Claycombe-Larson, K.J., Diniz, W.J., Lindholm-Perry, A.K., Reynolds, L.P., Dahlen, C.R., Borowicz, P.P., Ward, A.K. 2022. Epigenetic modifier supplementation improves mitochondrial respiration growth rates and alters DNA methylation of bovine embryonic fibroblast cells cultured in divergent energy supply. Frontiers in Genetics. 13. Article 812764. https://doi.org/10.3389/fgene.2022.812764.
DOI: https://doi.org/10.3389/fgene.2022.812764

Interpretive Summary: Prenatal vitamins are a common supplement for women to take prior to and during pregnancy, but their requirements and roles in beef cattle are not well understood. Prenatal vitamins consist of one-carbon metabolites (methionine, choline, folate, and vitamin B12) which are important for early embryonic development in mammals. It has been thought that one-carbon metabolites were provided in sufficient concentrations, but recent data has suggested that these may be limiting to beef cattle. Thus, we investigated the roles of supplementing increasing concentrations of one-carbon metabolites to embryonic cells to determine the effects on cell growth rate, mitochondrial function, and DNA methylation. We determined that increasing one-carbon metabolite supplementation to 2.5 and 5 times the basal levels improved measured outcomes of cell growth rate and mitochondrial function but increasing to 10 times the basal levels resulted in no improvement and even decreased growth compared with no supplementation. These data are impactful in that they demonstrate that current understanding of one-carbon metabolite requirements in beef cattle may be underestimated, and that cattle who may be limiting in one-carbon metabolites can improve embryonic growth by supplementing one-carbon metabolites during early gestation. Furthermore, these data show that finding the optimal supplementation level in cattle is necessary to increase embryonic growth and to identify the level at which growth rate is reduced or embryonic viability is decreased due to high levels of one-carbon metabolites.

Technical Abstract: Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/ L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.