| Goat milk is rich in short to medium-chain fatty acids and a variety of unsaturated fatty acids.On goat milk,the short to medium-chain fatty acids(C8-C14)and parts of long-chain fatty acids(C16)come from de novo synthesis in mammary epithelial cells,and long-chain fatty acids(C16 and above)are mainly obtained through exogenous uptake.Trans10,cis12 conjugated linoleic acid(t10c12-CLA)is a kind of biohydrogenation intermediate in rumen,which forms conjugated double bonds in the 10 th and 12 th carbon chain.t10c12-CLA reduces milk fat synthesis,inhibiting short to medium-chain fatty acid synthesis in goat mammary gland.However,the regulation mechanism of t10c12-CLA on fatty acid metabolism is still unclear.The investigation of the molecular mechanism of t10c12-CLA on lipid metabolism in goat mammary epithelial cells has important theoretical significance and practical value,including regulating milk fatty acid composition and content,improving the milk flavor and milk quality.The present study investigated the molecular functions of t10c12-CLA in regulation of goat milk lipid metabolism,by dietary feeding t10c12-CLA in lactating Xinong Saanen goats,establishment of the in vitro model of goat mammary epithelial cells in the treatment of t10c12-CLA,as well as combing with the transcriptome sequencing technology,cell function and morphology studies,gene and protein expression analysis,transcriptional regulation mothods,and cellular signal transduction research methods.The main results are as follows: 1.Effects of t10c12-CLA on lactation performance on dairy goats.A total of 30 multiparity Xinong Saanen goats were divided into three groups of 10 according to weight and milk yield.Goats were supplied with 0,8,and 16 g/day of lipid-encapsulated CLA(LE-CLA)referred to as control,CLA-1,and CLA-2 treatments,respectively.The results showed that compared with control group,no significant effects were observed on milk yield,milk lactose and milk protein in CLA groups(P > 0.05).Milk fat was significantly decreased in CLA groups(P < 0.05).The content of short to medium-chain fatty acids(< C16:0)in the two experimental groups was decreased significantly,and the difference between CLA-1 and CLA-2 group was significant(P < 0.05).The content of C16:0 & C16:1 was decreased significantly in CLA-2 group(P < 0.05),while the content of long-chain fatty acids(> C16)was not significantly different between comparision groups(P > 0.05).The content of saturated fatty acid(SFA)or unsaturated fatty acid(UFA)was significantly reduced or increased(P < 0.01)with the increasing concentrations of dietary CLA.Besides that,the desaturation indexes of C16 and C18 in CLA-2 group were significantly higher than the control group(P < 0.01).2.Effects of t10c12-CLA on the growth of goat mammary epithelial cells(GMECs).Different concentrations of t10c12-CLA were added on in vitro cultured GMECs.Using cell vitality assay,hoechst 33342 staining,real-time quantitative PCR(RT-qPCR),western blot and wound healing assay,it is found that cell proliferation was not affected,and no obvious cell apoptosis was observed in the treatment of 100 μM t10c12-CLA;however,cell migration was inhibited.The cell proliferation was not affected in the treatment of 200 μM t10c12-CLA within 12 h,but inhibited more than 12 h.3.RNA sequencing(RNA-seq)and bioinformatics analysis on effects of t10c12-CLA in GMECs.Concentrations of 0,100 and 200 μM t10c12-CLA were added on GMECs referred to as CTR,TR1 and TR2,respectively.Total RNA was extracted and the RNA-seq analysis was performed.Using goat genome(Capra hircus genome,ID: 10731)as the reference genome,25153 annotated transcripts were obtained.Considering RPKM > 0.01 as “expressed”,17559,17455,and 17982 expressed genes were identified in CTR,TR1 and TR2,respectively.Using the corrected P value < 0.05 as threshold,there were 49,854 and 623 differential expressed genes in TR1 versus CTR,TR2 versus CTR,and TR1 versus TR2,respectively.Accoring to GO and KEGG cluster analysis of the 41 differential expressed genes shared by TR1 versus CTR,TR2 versus CTR,and TR1 versus TR2,it is found that most significantly-enriched cell signaling pathways were related to lipid metabolism.The reliability of RNA-seq was verified through RT-qPCR.Meanwhile,it is also found that gene expression levels of ACSL4,FDPS,HMGCS1,PLIN2,SCD1 and SLC2A1 showed a significant dose-response effect with the increasing concentrations of t10c12-CLA treatment(P < 0.05).4.Effects of t10c12-CLA on lipid metabolism related genes and transcriptional regulation in GMECs.Concertrations of 0 and 100 μM t10c12-CLA were added on in vitro cultured GMECs.Using oil red O staining,determination of fatty acids composition,RT-qPCR,western blot and immunofluorescence staining,it is found that t10c12-CLA inhibited de novo fatty acid synthesis and desaturation processes;however,long-chain fatty acid(LCFA)intake,lipid droplets formation and accumulation processes were activated.Using luciferase reporter assay,it is found that t10c12-CLA inhibited the promoter activities of SREBP1 and SCD1 genes.Furthermore,the activity of SRE site located in the core region of SCD1 gene promoter was inhibited by t10c12-CLA as well.5.The molecular pathway of t10c12-CLA on regulating lipid metabolism through AMPK and mTOR in GMECs.Concertrations of 0,100 and 200 μM t10c12-CLA were added on in vitro cultured GMECs.It is found that t10c12-CLA significantly reduced the phosphorylation level of mTOR,and increased the phosphorylation level of AMPK.Both the Akt/mTOR pathway and AMPK pathway were affected after treatment of Akt/mTOR pathway specific inhibitors MK-2206 and Rapamycin,and AMPK pathway inhibitor Dorsomorphin,respectively.After co-treatment with inhibitors and t10c12-CLA,it is found that t10c12-CLA negatively regulated the up-regulation of ACACA and SCD1 expression levels caused by Dorsomorphin;Rapamycin and t10c12-CLA showed synergistic effect on the inhibition of SREBF1 gene expression.In summary,t10c12-CLA has important regulatory effects on goat milk lipid metabolism.It not only reduces the milk fat content and short to medium-chain fatty acids,but also inhibits de novo fatty acid synthesis and desaturation processes;and regulates the transcription of SCD1 gene through SREBP1.At the same time,t10c12-CLA regulates goat mammary lipid metabolism network by affecting the kinase activities of AMPK and mTOR. |
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