Font Size: a A A

Folate Inhibits Lipid Deposition Via The Autophagy Pathway In Chicken Hepatocytes

Posted on:2024-06-14Degree:MasterType:Thesis
Country:ChinaCandidate:Y Q PangFull Text:PDF
GTID:2543307094467684Subject:Aquaculture
Abstract/Summary:PDF Full Text Request
The production efficiency and product quality of broiler industry will be affected by excessive fat deposition.Liver fat synthesis plays an important role in deposition of broilers.Other studies have shown that folic acid is also involved in the regulation of lipid metabolism,which can reduce lipid deposition in abdominal fat and liver,thereby improving the body health of broilers.At present,the specific molecular mechanism of how folic acid regulates liver lipid deposition in broilers remains unclear.Therefore,in this study,key genes and signaling pathways related to fat deposition are identified by liver transcriptome sequencing of high and low fat broiler strains.Then,taking primary liver cells of broilers as the research object,folic acid and palmitic acid are added to explore the role of folic acid in regulating lipid metabolism for liver cells,which provide a new perspective and reference for the study of folic acid in regulating liver fat deposition of broilers.The main results obtained in this study are as follows:(1)Liver tissue transcriptome sequencing.Northeast agricultural university chicken belly fat two-way choice are high in fat and low fat department,each group take 5 liver transcriptome sequencing samples(including m RNA and lnc RNA),differential expression analysis founds that 722 significant differences in expression of m RNA(cut)rise with 444 and 278(| log2 Fold change | 1 or more,Padj.< 0.05).GO enrichment analysis shows that these differential genes are significantly(Padj.< 0.05)involved in multiple biological processes such as lipid,sterol,cofactor and coenzyme metabolism(Padj.< 0.05).KEGG analysis reveales that differential genes(Padj.< 0.05)are significantly involved in pathways such as fatty acid metabolism and extension,folic acid biosynthesis and metabolism.In addition,102 new lncrnas are identified as having no coding potential,being relatively short,with a small number of exons and a typical pattern of genomic distribution,among which 18 lncrnas are significantly differentially expressed(10 with up-regulated,8 with down-regulated).Lnc RNA and m RNA network analysis also identifie key node genes and signaling pathways related to fatty acid biosynthesis,elongation and metabolism,metabolic pathways,and folic acid biosynthesis).The expression levels of folic acid and lipid metabolism related genes(FPKM)are significantly different between high and low lipid lines.Therefore,folic acid signaling may be involved in the regulation of liver lipid metabolism in broilers.(2)Primary hepatocytes were treated with folic acid and palmitic acidTo explore how folic acid affects lipid deposition in the liver,primary hepatocytes from single broiler embryos are further isolated and cultured,and the effects of folic acid(FA)and/or palmitic acid(PA)treatments on hepatocytes are examined.The hepatocytes are sexed(examining the gonads of each embryo)and examined for morphology and characteristics(PAS staining and LDH activity assay).Hepatocytes are treated with FA concentration gradient(0,5,10,15,20μM)and LDH activity was determined.The optimal FA concentration is 15μM.The concentration gradients of PA(0,50,75,100,125μM)are preliminarily set.It is found that the activity of LDH was the highest with 50 μM concentration.In the range of 0 ~ 50μM,we set the concentration of 10,20,30,40,50 μM,and determine 40 μM as the optimal treatment concentration.In addition,the oil red O method also shows that 40μM PA is the best concentration.The optimal combination of FA and PA is 15 μM FA and 40 μM PA.(3)Folic acid treatment and hepatocyte gene expression profile.FA and/or PA treatments have sex-specific effects on the expression of most of the genes examined.First,we detecte the expression levels of folate transporter(PCFT),key metabolic enzymes(DHFR),DNA methylation-related genes(MTHFD2,MTHFR,and DNMT1),and glucose and energy metabolism-related genes(GCK and PGC-1α).FA treatment significantly increase the level of PCFT,while the addition of PA only slightly change the expression trend.However,DHFR expression levels in female hepatocytes do not increase significantly until day 4.FA treatment significantly increased the expression level of DNMT1,MTHFD2 in male and MTHFR in female,respectively.However,further supplementation of PA inhibited their expression levels.In addition,the levels of GCK and PGC-1α are significantly decreased by FA treatment.PA treatment further significantly improve its expression level.For genes related to lipid synthesis,FA treatment has a relatively greater effect on males.FA treatment could increase FAS and significantly inhibit ACC expression.Male SCD and female ELOVL6 levels are significantly inhibited.The simultaneous treatment of FA+PA only affecte the expression of FAS and ACC,significantly inhibite the expression of SCD,and has little effect on ELOVL6.For lipolysis related genes,FA treatment increased the expression levels of FABP1,CREB,PPARα and ACOX1,and significantly increase the expression level of CREB.Further processing of PA do not change their expression patterns.(4)Folate and m TOR-SREBP1-PI3 K and autophagy signaling pathways in hepatocytes.Firstly,we evaluate the Mtor-Srebp-1-PI3 K pathway involved in the regulation of nutrient metabolism.Gene expression detection showes that FA treatment significantly increased the levels of m TOR,SREBP-1c and PI3 K in male and female hepatocytes.Further combine treatment with PA significantly inhibited m TOR and PI3 K,but enhance the expression level of SREBP-1c.Western blot confirmes a significant increase in the abundance of m TOR and SREBP-1c proteins in female hepatocytes.Analysis of ATG5 and LAMP2 transcription levels in the autophagy pathway shows that FA treatment significantly increased the expression of ATG5 in females,as well as LAMP2 expression in both males and females.After further treatment with PA,the expression level of ATG5 increases significantly,while the expression level of LAMP2 has no significant change.Western blot confirmes that the levels of ATG5 and LAMP2 in female hepatocytes are significantly increased.In conclusion.The integrated sequencing of m RNA and lnc RNA from liver tissues of broilers with different abdominal fat content reveale key genes and signaling pathways,metabolic pathways and folic acid biosynthesis related to fatty acid biosynthesis.Detailed expression profiles of folic acid and/or palmitic acid-treated primary hepatocytes(sexdetermined)shows that folic acid inhibits lipid deposition in a sex-dependent manner,by regulating transcriptional gene expression and protein levels associated with DNA methylation,lipid metabolism(m TOR/SREBP-1c/PI3K)and autophagy(LAMP2/ATG5)pathways.Through our scientific research,we hope to provide reliable and effective solutions for poultry breeding.At the same time,The results of this study provide theoretical support and basis for further research on the apparent regulation mechanism of folic acid biosynthesis involved in liver fat deposition.
Keywords/Search Tags:broiler, liver, folate, hepatocyte, lipid deposition, autophagy
PDF Full Text Request
Related items