ITRAQ-based Liver Proteomic Study Of Obese Rats With Hypoxic Exercise And Mechanism Of Wnt/β-catenin Signaling Pathway

Objectives:Hypoxic exercise has an obvious effect on body weight loss and body fat reduction as it can improve lipid metabolism,reduce blood lipid level,inhibit the synthesis of fatty acids and promote the decomposition and oxidation of fatty acids.Based on the iTRAQ proteomics technology,a high throughput and high efficient approach for screening differentially expressed proteins in the liver of the obese rats with hypoxic exercise,the author has conducted a systematic study of the different proteins and signal pathways that affect the lipogenesis of obese rats under hypoxic exercise environment.The proteomics study suggests that Wnt/β-catenin signaling pathway may be involved in hypoxia lipogenesis;accordingly,the role of Wnt/β-catenin in hypoxia lipogenesis is validated in mRNA and protein level and the DNA methylation is studied as well,further improving the theoretical system how hypoxia exercise affects the adipocyte differentiation and lipid metabolism.Methods:Obesity rat model was established and divided into four groups: normoxic quiet group,hypoxic quiet group,normoxic exercise group and hypoxic exercise group;hypoxia concentration was 13.6%(equivalent to 3500 m above sea level),and horizontal running platform was used for endurance training of the exercise groups.The training intensity was 25 m/min for normoxic exercise group,and 20 m/min for hypoxic exercise group,and both groups were subject to training 1 hour per day,5days per week,lasting 4 weeks.The iTRAQ proteomics technology was used to screen the differentially expressed proteins of the four groups,and Western Blot(WB)assay was applied to detect the protein-expression changes of FABP4,CacyBP/SIP and ACAA1 associated with PPAR signaling pathway,Wnt signaling pathway,and fatty acid decomposition.Real time PCR and WB were used to detect the expression changes of Wnt/β-catenin correlated mRNA and proteins.The Reduced Representation Bisulfite Sequencing(RRBS)was used to analyze DNA motion high throughput sequencing expression in both hypoxic exercise group and normoxiagroup,and finally the CpG island and DMR of the Wnt/β-catenin signaling pathway correlated DNA in the two groups were analyzed and screened.Results:Result 1: 1)Hypoxia and exercise reduced the body weight,lipid ratio,Lee’S index,TC concentrations,TG concentrations and LDL-C concentrations of the obese rats and could improve blood lipid level and reduce body fat content.Of all the groups,hypoxic exercise had the most significant effect.2)There were 70 significantly different protein expressions in the two quiet groups,of which 33 proteins were up-regulated and the other 37 down-regulated.There were 110 significantly different protein expressions in the two exercise groups,of which 61 proteins were up-regulated and the other 49 down-regulated.3)KEGG analysis showed that 14 signal pathways had significant changes in normoxic quiet and hypoxic quiet groups,while 11 signal pathways in normoxic exercise and hypoxic exercise groups.4)The expressing quantity of FABP4 protein increased more significantly in hypoxic quiet group,normoxic exercise group and hypoxic exercise group than the normoxic quiet group.The expressing quantity of CacyBP/SIP decreased significantly in hypoxic exercise group,and the expression of ACAA1 increased significantly in hypoxic exercise group.FABP4,CacyBP/SIP,ACAA1 proteins were involved in lipogenesis of obese rats under hypoxic exercise environment.Result 2: 1)Compared with normoxia exercise group,the mRNA expressing quantity of Wnt/β-catenin signaling pathway correlated molecules c-myc,β-catenin,significantly increased(P<0.05)in hypoxic exercise group,while that of APC,GSK3βsignificantly decreased(P<0.05);yet,there were no significant difference in CCND1 in the two groups.2)Compared with normoxia exercise group,the protein expression content of Wnt/β-catenin signaling pathway correlated molecules c-myc,β-catenin significantly increased in hypoxic exercise group(P<0.05).Result 3: 1)Based on a comparative analysis of the genome-wide DNA methylation profiling of CpG islands in hypoxic exercise and normoxic exercise obese rats,there were 675 genes with significant differences;the methylation of the hypoxic exercise group was 1.1 % higher than that of the normoxia exercise group,and there were 213 DMR different regions.2)Wnt signaling pathway correlated genes,the CpG promoter methylation gene sites included Fzd2,Fzd9,Wnt16,Wnt2 b,Sfrp2,Sfrp1,Sox2,Sox1,etc.,and the different regions of DMR methylation genes included cdc,Fzd10,Gsk-3β,PI3 k,Wnt16 and Wnt6.The methylation of Wnt signaling pathway correlated genes CpG and DMR was significant.Conclusions:1.The results of iTRAQ proteomics analysis show that there are 70 significantly different protein expressions and 14 significantly different signal pathways in normoxic quiet and hypoxic quiet groups;there were 110 significantly different protein expressions and 11 significantly different signal pathways in hypoxic exercise and normoxia exercise groups.2.Wnt/β-catenin signaling pathway is activated in the lipogenesis of obese rats under hypoxic exercise group,which indicates that Wnt/β-catenin signaling pathway play roles in the lipogenesisn and lipid metabolism of of obese rats under hypoxic exercise environment.3.There were 675 genes with significant differences and 213 DMR different regions based on the comparative analysis of genome-wide DNA methylation profiling of CpG islands of the rats in both hypoxic exercise and normoxic exercise groups.The methylation of Wnt signaling pathway correlated genes CpG and DMR was significant.