Effects Of PPAR?Ligand On The Differentiation Of Preadipocytes Of Yanbian Cattle And Mechanism Of Lipid Formation

In recent years,with the continuous improvement of people's living standards,consumers have increasingly higher requirements for the quality of meat products Meat quality indexes include color,texture,hardness,tenderness,marbling,etc.,among which intramuscular fat content is an important index to evaluate the quality of beef.Muscular adipocyte deposition in beef cattle involves the accumulation and consumption of lipid droplets in adipocytes and tissues and the dynamic balance of fatty acid metabolism.It is closely related to adipocyte differentiation and is regulated by a series of transcription factors related to adipocyte differentiation.PPARy,as an important factor promoting adipocyte differentiation and transcriptional regulation of various genes,plays an extremely important role in the induction and differentiation of bovine adipocytes and fatty acid metabolism.Yanbian cattle is one of the five best local beef cattle in China.It has the breeding potential comparable with Korean cattle and Japanese wagyu cattle.For an in-depth understanding of yanbian cattle fat tissue first fat cells of biological characteristics and related regulatory mechanism,for the future of yanbian cattle molecular breeding work and provide a valuable reference for fat metabolism regulation,this study with oleic acid and ciglitazone as excited PPAR?ligands regulation gene expression,through induction and mutual induction of yanbian cattle alone before the fat cell differentiation,aiming to clarify the influence on yanbian cattle fat cell differentiation and the related mechanism,clear differentially expressed genes,further clarify the key signaling pathways in the main and the interaction relationship between the change rule of differentially expressed genes.The specific research results are as follows:1.Study on the effect of oleic acid on the differentiation of preadipocytes from Yanbian cattle and related mechanismIn vitro culture of 18 month old Yanbian bovine adipocytes was successfully isolated by collagenase digestion.The cells were induced to differentiate by 100?Mol/L oleic acid.The effects of oleic acid on adipocyte differentiation were studied and the transcriptomics of 48 h and 96 h differentiated cells were studied,and the effects of PPAR inhibitor GW9662 at different concentrations(0,0.25,0.5,1,2 and 4 ?Mol/L)on the differentiation of preadipocytes were further verified.The results showed that:l)The formation of lipid droplets was observed 24 h after oleic acid induction,and the differentiation was almost complete at 120 h;2)With the increase of treatment time,the triglyceride concentration increased,reached the highest at 96 h(P<0.05),and slightly decreased at 120 h,but remained higher than the control group(P<0.05).3)after oleic acid induced into genes PPAR?,C/EBP?,LPL,FABP4,SCD,CPT1?,SREBP1 and PLIN2 expression with different processing time show different trends,PPAR?,C/EBP?,and SREBP1,CPT1?,FABP4 differentiation and PLIN2 gene expression in different time is always higher than that of control group(P<0.05),LPL gene expression after falling short gradually increased(P<0.05),only the SCD gene expression is always lower than the control group(P<0.05);4)According to the results of transcriptome sequencing,at 48 h of differentiation,there were 3,926 differentially expressed genes(2,263 up-regulated genes and 1,663 down-regulated genes),and at 96 h of differentiation,there were 7,445 differentially expressed genes(7,088 up-regulated genes and 357 down-regulated genes).5)The molecular functions,biological processes and cell composition of genes with different differentiation time were changed,and the main biochemical metabolic pathways and signal transduction pathways of genes with different differentiation time were significantly different;6)According to the regulation results of oleic acid on PPAR signaling pathway in adipocytes,PPAR expression was up-regulated,CPT1,FABP4,PLIN2 and SREBP1 were up-regulated,and SCD expression was down-regulated at 48 and 96 h,compared with the control group at 0 h.Compared with 48 h of differentiation,PPARy and SCD expression in 96 h of differentiation were significantly down-regulated,CPT1,PLIN2 and SREBP1 were significantly up-regulated,and FABP4 expression was not significantly changed;7)With the increase of inhibitor concentration,the expression of PPAR decreased continuously,the concentration of triglyceride and the secretion of adiponectin decreased continuously(P<0.05),and the relative expression of FABP4,CD36,PLIN2 and ADP decreased continuously(P<0.05).2.The effect of ciglitazone on the differentiation of proadipocytes in Yanbian cattle and the mechanism of actionWith 10 ?g/mL ciglitazone of yanbian cattle fat cell differentiation,before the ciglitazone before on the role of adipocyte differentiation,48 h and 96 h and the differentiation of cells in the transcriptome studies,the results show that:1)can be observed in lipid droplets,divide 72 h triglyceride concentrations rising,144 h reached the highest(P<0.05);2)after ring Glenn methadone induced into fat genes PPARy,C/EBPa,LPL,FABP4,SCD,CPT1?,SREBP1 and PLIN2 represents different changing trends,among them,PPAR?,FABP4,CPT1? and SREBP1 expression quantity is always higher than the control group(P<0.05),C/EBP?,LPL,SCD,and PLIN2 gene expression to fall after rise,in addition to the SCD gene expression is always lower than the control group,The expression of C/EBP?,LPL and PLIN2 genes increased gradually after reaching the minimum at 48 h,and was significantly higher than that of the control group at 144 h(P<0.05).3)According to the results of transcriptome sequencing,there were 1,282 differentially expressed genes(687 up-regulated genes and 595 down-regulated genes)at 48 h of differentiation,and 6,172 differentially expressed genes(5,708 up-regulated genes and 464 down-regulated genes)at 96 h of differentiation.4)The molecular functions,biological processes and cell composition of differentially expressed genes at different times of differentiation are changed,and the main biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes are also different;5)According to the regulation result of cyglitazone on PPAR signaling pathway in adipocytes,the expression of ACSL6,NR1H3,MMP1,PLIN1,CPT1B,FABP4,ANGPTL4,GK and CD36 genes in the pathway can be up-regulated and the expression of SCD can be down-regulated after 48 h treatment.At 96 h differentiation,11 genes including SLC27A1,SLC27A4,ACSL1,SCD5,CPTIA,CPT1B,ANGPTL4,PLIN2,MMP1,PCK1 and CD36 were up-regulated,but no down-regulated genes were detected3.A comparative study on the effects of oleic acid and ciglitazone on the differentiation of preadipocytes of Yanbian cattleThe effects of different treatments(100 ?M/L oleic acid,10 ?g/mL cigiitazone,100 ?M/L oleic acid+10 ?g/mL ciglitazone)on the differentiation of preadipocytes at different differentiation times and transcriptome studies showed that:1)At 48 h of differentiation,oleic acid alone induction and oleic acid and ciglitazone together induction could promote the formation of lipid droplets.The effect on triglyceride concentration was oleic acid>oleic acid+ciglitazone>ciglitazone.Number of differentially expressed genes oleic acid(3926)>oleic acid+ciglitazone(2957)>ciglitazone(1282);The expression of ACSL6,FABP4,ANGPTL4,GK and CD36 genes was up-regulated and the expression of SCD genes was down-regulated in each treatment group.2)At 96 h differentiation,lipid droplets were generated in different treatment groups;The effect on triglyceride concentration was oleic acid+ ciglitazone>oleic acid>ciglitazone,which was higher than the control group.The number of differentially expressed genes was oleic acid and ciglitazone(7499)>oleic acid(7445)>(6172).The molecular functions of the differentially expressed genes,the biological processes involved,the cell composition,the main biochemical metabolic pathways and signal transduction pathways were all different,and the expressions of CPT1A,ANGPTL4,MMP1,PLIN2,PCK1 and CD36 genes were up-regulated in each treatment group.4.Effects of oleic acid and ciglitazone on the differentiation of preadipocytes of Yanbian Cattle10 ?g/mL ciglitazone+100 ?M/L oleic acid was used to induce the differentiation of yanbian bovine preadipocytes.The effects of oleic acid and ciglitazone on the differentiation of preadipocytes were studied.The results showed that:1)The formation of lipid droplets was observed at 48 h after induction.2)The triglyceride concentration increased with the treatment time,which was significantly higher than that of the control group(P<0.05).3)into a fat genes PPARy,C/EBPa,LPL,FABP4,SCD,CPT1?,SREBP1 and PL/IN2 expression with different processing time show different trends,PPARy,C/EBP?,FABP4,the expression of CPT1?,SREBP1 and PLIN2 quantity in different differentiation time always higher than that of control group,SCD gene expression began to decline after the differentiation,120 h to reach the lowest(P<0.05),LPL gene expression decreased in the early differentiation,48 h meet minimum,after rising,The expression was highest at 120 h(P<0.05).4)According to the results of transcriptome sequencing,there were 2957 differentially expressed genes(1060 up-regulated genes and 1897 down-regulated genes)at 48 h of differentiation,and 7499 differentially expressed genes(6912 up-regulated genes and 587 down-regulated genes)at 96 h of differentiation.5)The molecular functions,biological processes and cell composition of genes differentially expressed at different differentiation times,as well as the main biochemical metabolic pathways and signal transduction pathways involved in enrichment are significantly different;6)According to the research results of PPAR signaling pathway,PPAR?,ACSL6,CPTIA,CPT1B,MMP1 PLIN2,FABP4,ANGPTL4,GK and CD36 were up-regulated and SCD was down-regulated at 48 h differentiation under the joint induction of oleic acid and ciglitazone.At 96 h differentiation,NR1H3,ACSL6,SLC27A4,CPT1A,CPT1B,ANGPTL4,FABP4,PLIN2,MMP1,GK,PCK1,PCK2,and CD36 expression were up-regulated,while LPL expression was down-regulated.The regulation of differentially expressed genes in PPAR signaling pathway induced by oleic acid and ciglitazone is time-dependent or time-specific.5.Effect of FABP4 on the differentiation of proadipocytes in Yanbian cattleUsing oleic acid promote Yanbian cattle before join at the same time the adipocyte differentiation of different concentration(0,2.5,5,10,20 and 40 ?Mol/L)of BMS-309403,before the activation of PPAR? promote adipocyte differentiation at the same time,to suppress FABP4 research FABP4 in yanbian cattle before the influence of adipocyte differentiation,the results show that 1)adding different concentration of FABP4 inhibitors had no significant effect on the relative expression of FABP4;2)After adding inhibitors of different concentrations,lipid droplets could be generated in each treatment group,and the number and density of lipid droplets in cells in the high-concentration treatment group were significantly higher than those in the low-concentration treatment group;3)The concentration of triglyceride and the secretion of adiponectin in different treatment groups decreased with the increase of the added concentration(P<0.05),and the relative expression levels of PPARy,PLIN2,CD36 and ADP genes also decreased(P<0.05).To sum up,through the study of PPARy agonist-oleic acid and ciglitazone of Yanbian cattle and transcriptome study adipocyte differentiation process,cleared the oleic acid of adipocyte differentiation and PPAR signal pathway before concrete mechanism,gene regulation and molecular breeding in the future work to provide theoretical evidence for the selection of PPARy agonist,as well as the main function in the process of adipocyte differentiation gene research provides a theoretical basis.