Mechanisms Of ACAT2 In Muscle Tissue Fluid Affects Intramuscular Fat Deposition Through Regulation Of SREBP2 And LDLR

The intramuscular fat(IMF)is considered as the fat deposited between muscle fibers.The extra-cellular matrix microenvironment of adipose tissue is of critical importance for the differentiation,remodeling and function of adipocytes.Therefore,in this study we extracted the muscle tissue centrifugal fluid(MTF)of the longissimus dorsi of Erhualian pigs to mimic the microenvironment of intramuscular preadipocytes.MTF of pigs with low intramuscular fat level can inhibit pig intramuscular pre-adipocytes differentiation.Then proteomics technology(iTRAQ)was used to analyze the MTF with different IMF content,and found that individuals with high IMF had low ACAT2(Acyl-Co A: cholesterol acyltransferases 2)levels,while individuals with low IMF had high ACAT2 levels.Significant changes have taken place in the pathways involved in coenzyme A,which is closely related to fat and cholesterol metabolism.Therefore,we speculate that ACAT2,as an important role involved in cholesterol metabolism,may become a potential molecular marker for the mechanism of pig intramuscular preadipocytes differentiation.Overexpression of ACAT2 in pig intramuscular pre-adipocytes can inhibit their differentiation,while adding ACAT2 inhibitor avasimibe can rescue the process.Knockdown of srebp2 or ldlr,which are two key genes closely related to ACAT2 and cholesterol metabolism,can inhibit pig intramuscular pre-adipocytes differentiation.At the end we verified the effect of ACAT2 on intramuscular fat deposition by in vivo experiments.Overall,our results suggest that ACAT2 is a novel negative regulator of intramuscular adipocyte differentiation through regulation of srebp2/ldlr signaling involved in cholesterol metabolism.1 MTF of low IMF individuals inhibits the differentiation of pig intramuscular preadipocytesTo examine the exact role of MTF in intramuscular pre-adipocytes differentiation,we treated pre-adipocytes with MTF.The triglyceride content and cholesterol content in the culture medium of our treatment system were tested and we found no significant differences in the cholesterol and triglyceride content between the culture medium with and without muscle tissue fluid.Then we detected the m RNA and protein expression of pparγ and cebpα and Oil Red O staining to confirm the function of MTF on intramuscular pre-adipocytes differentiation.The muscle tissue fluid of individuals with very high intramuscular fat content had no effect on the differentiation of intramuscular pre-adipocytes(p>0.05).The muscle tissue fluid of individuals with very low intra-muscular fat content can reduce the expression levels of pparγ and cebpα(p<0.01).Oil Red O staining,triglyceride content determination and cholesterol content determination results showed that the differentiation of intramuscular pre-adipocytes was inhibited(p<0.01).2 iTRAQ Analysis of Muscle Tissue FluidIn order to explore which factors in MTF have an important inhibitory effect on pig intramuscular fat differentiation,proteomic(iTRAQ)analysis on MTF of individuals with very high and very low IMF was conducted,respectively.A total of1671 proteins were identified,of which 10 were significantly up-regulated and 16 were significantly down-regulated.GO analysis and KEGG analysis of the proteomic data showed that the individual’s cholesterol metabolism pathways changed significantly,and the synthesis pathways of pantothenic acid and coenzyme A changed significantly.Among them,ACAT2 is an extremely significant downregulated protein in individuals with high IMF.The expression level of ACAT2 in the MTF of individuals with high IMF is extremely low.ACAT2,acetyltransferase2,is an important factor involved in cholesterol metabolism and coenzyme A synthesis pathway.Combined with the inhibitory effect of MTF on the differentiation of pig intramuscular preadipocytes,we speculate that ACAT2 may become a novel negative molecular marker affecting pig intramuscular fat deposition.3 Validation of MTF proteomic analysisIn order to verify the results of iTRAQ in the second part of this study,we detected the ACAT2 protein level in the MTF of low IMF individuals and found that the ACAT2 protein level in the MTF of low IMF individuals was significantly higher than that of high IMF individuals(p<0.01).At the same time,we carried out immunofluorescence detection and analysis of MTF-treated pig intramuscular preadipocytes from individuals with low IMF,and found that ACAT2 in MTF entered the cells and played an important role.Since a large amount of ACAT2 is produced in the liver,in order to find out the source of the variation of ACAT2 levels in the MTF of individuals with different IMF,we detected the expression levels of ACAT2 in the livers of individuals with very high and very low IMF,and the results showed that the levels of ACAT2 in the livers of individuals with very high IMF is very low,and the level of ACAT2 in the liver of individuals with very low IMF is very high.Then we detected the m RNA and protein levels of ACAT2 in pig liver,subcutaneous fat,and muscle respectively.The results showed that the level of ACAT2 in muscle was significantly lower than that in liver.Thus,we speculate that ACAT2 levels in the MTF of our different IMF individuals were derived from the liver.We then performed transcription factor prediction on the promoter region of acat2 gene and obtained that the transcription factor HNF1 A could bind to two sites in the promoter region of the pig acat2 gene.Luciferase activity analysis and chromatin immunoprecipitation analysis showed that HNF1 A affects the activity of porcine acat2 gene by binding to its promoter.In addition,we detected the m RNA and protein levels of two key cholesterol pathway factors,srebp2 and ldlr,in pig intramuscular preadipocytes treated with MTF of low IMF individuals,and found that they were significantly inhibited(p<0.01).It was shown that the cholesterol pathway was significantly inhibited by MTF treatment in low IMF individuals.The above experimental results are consistent with our proteomic analysis results.4 ACAT2 inhibits the intramuscular fat deposition in pigs through the cholesterol pathwayIn order to explore the mechanism by which high levels of ACAT2 in MTF inhibit the differentiation of pig intramuscular preadipocytes,we transfected acat2-overexpressing plasmids into pig intramuscular pre-adipocytes.The m RNA and protein levels of cebpα and pparγ were significantly decreased(p<0.01),combined with the significant difference results of Oil Red O staining,triglyceride and cholesterol detection(p<0.01),indicating that the differentiation of pig intramuscular pre-adipocytes were inhibited.After adding the ACAT inhibitor avasimibe,the expression levels of pparγ and cebpα recovered.Therefore,we found that excessively high level of ACAT2 inhibits the pig intramuscular pre-adipocytes differentiation.Combined with the experimental results of the second and third parts,we speculate that srebp2/ldlr signaling is an important way that ACAT2 in MTF affects the differentiation of pig intramuscular preadipocytes.Therefore,we synthesized si RNAs for srebp2 and ldlr.After knocking down these two factors,the m RNA and protein levels of pparγ and cebpα were significantly inhibited(p<0.01),and the levels of lipid droplet aggregation,triglyceride and cholesterol were significantly inhibited(p<0.01).However,in the case of srebp2 and ldlr knockdown,neither overexpression of acat2 nor avasimibe had any effect(p>0.05).The above results indicated that ACAT2 could affect the differentiation of pig intramuscular pre-adipocytes through the cholesterol pathway involving srebp2 and ldlr,and the effect of ACAT2 disappeared after blocking the cholesterol pathway.5 Verification of the mechanism of ACAT2 affecting intramuscular fat deposition in vivoTo verify the role of ACAT2 in intramuscular fat deposition,we divided 40 8-week-old male C57BL/6 mice of similar body weight into 4 groups: control(control),high cholesterol fed(hcd),the intraperitoneal injection of avasimibe group(control+avasimibe)and the high cholesterol feeding group with injection of avasimibe group(hcd+avasimibe group).The hcd group and hcd+avasimibe were fed with high-cholesterol feed for 8 weeks,and the control was fed with the special control feed for high-cholesterol feed.At the 8th week,the hcd+avasimibe group and the control+avasimibe group were given intraperitoneal injection of avasimibe for 4weeks.After 12 weeks,we weighed the body weight,inguinal fat,epididymal fat,and quadriceps muscle of the mice,and detected the blood lipid indexes of the mice.After avasimibe injection,the triglyceride content,cholesterol content and low density lipoprotein content returned to normal levels(p<0.01),indicating after the injection of avasimibe to the high-cholesterol-fed model mice,the body weight,inguinal fat,and epididymal fat all decreased significantly(p<0.01),but there was no significant changes in the quadriceps(p>0.05),so we speculate that avasimibe may promote the accumulation of intramuscular fat deposition by inhibiting the high level of ACAT2.To verify this process,we detected the protein levels of LDLR,SREBP2 and ACAT2,and found that the level of ACAT2 decreased after the injection of the avasimibe,whereas the levels of LDLR and SREBP2 rebounded.Combined with the measurement results of quadriceps triglyceride content tests and the quantitative analysis of the slice results,we found that the high ACAT2 level activated by highcholesterol feeding was inhibited by avasimibe and promoted deposition of intramuscular fat through the cholesterol pathway involving LDLR and SREBP2.