N-3 PUFA Regulate Mitochondrial Energy Metabolism By Alteration Of DNA Methylation In FAT-1 Transgenic Bovine Fibroblast Cells

Due to the lack of n-3 polyunsaturated fatty acid dehydrogenases,the mammalian animals can not synthesize n-3 polyunsaturated fatty acids(n-3PUFA)themselves.The transgenic technologies make it possible to transfer the exogenous fat-1 gene into mammals and obtain genetically modified animals with increased endogenous n-3 PUFA.In this experiment,both transcriptome and DNA methylation analyses were performed to investigate the differences of fat-1 transgene(FT group)and wild type(WT group)bovine fetal fibroblasts in mRNA and DNA methylation levels.The global mRNA expressions and DNA methylations in FT cells had significantly changed in comprison with the WT cells.The transcriptome differential genes are enriched to the pathways related to energy metabolisms and methylations,and the pathways associated with energy metabolism are also enriched in DNA methylation sequencing.The activities and expressions of the rate-limiting enzymes and/or products involved in energy metabolism were detected.Theresults showed that the increase of n-3 PUFA in the FT cells obviously inhibited fat synthesis,increased the rate of fatty acid synthesis and increased the rate of ?-oxidation.The high proportion of n-3 PUFA in the FT cells significantly reduced the activities and expressions of the glycolytic enzymes HK,PFK and PK,which resulted in the inhibition of the glycolysis process.Furthermore,the high level of n-3 PUFA negative affected the TCA cycle by the decreases of IDH,NADH-CoQ reductase,cytochrome C oxidase and ATP synthase,which significantly reduced the production of ATP,and inhibited the oxidative phosphorylation of mitochondria.The results also indicated that the methylations of total DNA in FT group were significantly higher than that in WT group,but the hydroxymethylation state were with similar between two groups.However,the methyltransferase DNMT3 B and demethyltransferase TET2 changed significantly at mRNA and protein levels in the fat-1 transgenic cells.Results from MeDIP-qPCR analysis showed that DNA methylation status in the upstream 2000 bp of the promoters of the genes associated with energy metabolism rate-limiting enzymes was negatively correlated with the enzymes' activities.Therefore,we infer that the increase of the n-3 PUFA in the FT cells resulted in significant changes of the DNA methylations of the metabolic related rate-limiting enzymes,which subsequently affected the enzymes' expressions and then changed the whole energy metabolism.