| Goat milk has gradually become recognized by consumers as the high nutritional value,which are rich in short,medium and polyunsaturated fatty acids(PUFA).Plenty of studies have shown that docosahexaenoic acid(DHA),as an important of PUFA,plays an important role in human health,such as resisting obesity,slowing down inflammation,maintaining normal fertility of the body,inhibiting the occurrence and development of tumors,and promoting the development of fetal brain and retina.The low activity of fatty acid desaturase2(FADS2)and very long chain fatty acid elongation enzyme 2(ELOVL2)in the process of mammalian biosynthesis of DHA restricts the synthesis in vivo.Therefore,exogenous DHA supplementation is an important approach to increase the content of PUFA in dairy goat milk.However,the effect on lipid metabolism and regulation molecular mechanism of exogenous DHA supplementation in dairy goat mammary epithelium cells(GMEC)remain unclear.In this study,the effects of DHA on lipid contents,fatty acid profiles and transcription profiles of GMEC were investigated.The role of histone H3 lysine 9 acetylation(H3K9ac)epigenetic modification in the process was explored by Ch IP-seq.The mechanism of epigenetic modification of H3K9 ac on lipid metabolism was explored through multi-omics analysis(lipidomics,RNA-seq and H3K9 ac Ch IP-seq).The function of differential expression gene-PDK4 in GMEC was investigated by overexpression and interference techniques.The mechanism of CD36-PDK4-AMPK signaling in DHA induced lipid remodeling was explored.The main results were as follows:1.DHA induces lipid remodeling in GMEC.The most appropriate amount of DHA treatment for GMEC was 100 μM.Ultrastructural analysis by TEM showed that DHA supplementation in GMEC promoted lipid droplet accumulation and the lipid droplets were uniformly distributed in the cytoplasm.Lipidomics analysis showed that,compared with the control group(CTR),490 different lipids were obtained in GMEC supplemented with DHA,among which 294 were up-regulated and 196 were down-regulated.The analysis of TOP 40 differential lipids showed that the supplementation of DHA in GMEC significantly increased the DHA enriched in TG(18:2＿20:5＿22:6),TG(14:0＿20:5＿22:6),PC(20:2＿22:6),PS(18:1＿22:6)and PE(14:0＿22:6);The contents of PG(16:1＿20:4),PI(18:1＿20:1),PC(14:1＿16:1)and other lipid were significantly reduced with 16:1,14:1,18:1,and 20:1monounsaturated fatty acid acyl chains.In addition,DHA changed the fatty acid profile in GMEC and significantly up-regulated the contents of DHA,EPA,DPA,TPA and THA.2.DHA regulates transcription of lipid metabolism related genes in GMEC.RNA-seq analysis showed that DHA significantly induced changes in GMEC transcriptional profiles and 520 differentially expressed genes(DEG)were screened.GO enrichment analysis showed that DEG were enriched in cellular lipid metabolism,lipid biosynthesis,unsaturated fatty acid synthesis and other biological processes.KEGG enrichment analysis showed that DEG were enriched in AMPK signaling pathway,unsaturated fatty acid biosynthesis and fatty acid metabolism.DHA significantly inhibited the transcription of fatty acid metabolism genes(SREBP1,FASN,FADS2,SCD1).DHA activated AMPK signaling pathway,and AMPK inhibitors weakened the inhibitory effect of DHA on lipid metabolism genes.3.DHA induces genome-wide changes of H3K9 ac epigenetic profile in GMEC.DHA induced H3K9 ac epigenetic profile changes in GMEC,and most of the signals were concentrated near the TSS.Compared with CTR,1,013 different peaks were identified,and the genes related to these different peaks were enriched in homeostasis metabolic pathways of lipid metabolism such as cholesterol metabolism and fatty acid synthesis.DHA significantly inhibited the m RNA and protein levels of histone acetylase PCAF.Interference of PCAF significantly inhibited the expression of fatty acid synthesis-related genes and PUFA biosynthesis genes.Interfering PCAF significantly reduced the protein levels of fatty acid metabolism genes(SREBP1,FASN and FADS2),suggesting that PCAF could be used as a regulatory target of lipid metabolism in mammary gland.4.DHA regulates lipid metabolism through epigenetic modification of H3K9 ac in GMEC.Multi-omics(RNA-Seq,H3K9 ac Ch IP-Seq,and lipidomics)analysis showed that DHA induced the expression of lipid metabolism-related genes(e.g.PDK4,FASN,SCD1,FADS1,FADS2,LPIN1,DGAT1,MBOAT2,HMGCR,FDFT1,DHCR7)was altered,which were regulated by H3K9 ac epigenetic modification.The supplementation of DHA in GMEC significantly increased the enrichment of H3K9 ac in the PDK4 promoter region and promoted its transcription.The supplementation of DHA significantly inhibited the enrichment of H3K9 ac in FADS2,FASN and SCD1 genes in their promoter regions,and inhibited their transcription.DHA treatment significantly reduced the expression of upstream transcription factor SREBP1 in GMEC nucleus,inhibited the enrichment of H3K9 ac in SREBP1 promoter region,and inhibited its transcription.5.CD36-PDK4-AMPK signaling mediates the regulation of lipid metabolism by DHA in GMEC.The screened DEG-PDK4 inhibited lipid metabolism in GMEC using overexpression and interference techniques,suggesting that PDK4 gene may be an important target to regulate lipid metabolism in goat.Mechanically,PDK4 activated AMPK signaling pathway,and overexpression of PDK4 weakened the activation of AMPK inhibitors on downstream genes(FASN,FADS2,SCD1,SREBP1),revealing the role of PDK4-AMPK axis in lipid metabolism of goat mammary gland.Fatty acid transporter CD36 mediated DHA transport into GMEC,and knockdown of CD36 inhibited the activation of PDK4 and the activation of AMPK signaling pathway by DHA.What’s more,mutated CD36 fatty acid binding domain resisted the inhibitory effect of DHA on AMPK downstream lipid metabolism genes(FASN,FADS2,SCD1,SREBP1).In conclusion,DHA regulates lipid metabolism and lipid remodeling through epigenetic modification of H3K9 ac and CD36-PDK4-AMPK signaling axis in GMEC,laying a theoretical foundation for the development of functional goat milk and breed of high quality dairy goats. |
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