Role Of VEGF-B Signaling Pathway In Ectopic Deposition Of Fatty Acid In Skeletal Muscle During Catch-up Growth

Part 1 Changes of VEGF-B signaling pathway in skeletal muscle of catchup growth mice and its role in mediating lipid transport and storageObjective: 1.To explore whether VEGF-B signaling pathway changes in skeletal muscle of cath-up growth mice.2.To explore the role of VEGF-B signaling pathway in mediating lipid transport and storage in skeletal muscle of cath-up growth mice.Methods: 1.A total of 72 C57BL/6J aged 6 weeks were divided into three groups:(1)Normal control(NC)which were fed with normal diets for 12 weeks;(2)Catch-up growth group(RN)which were refed with normal diets for 8 weeks after caloric restricted to 50% for 4 weeks;(3)Catch-up growth group accompanying AAV9 injected into TA(RN+AAV9)which were refed with normal diets for 8 weeks after caloric restricted to 50% for 4 weeks.2.After caloric restriction for 4 weeks or refeeding for 8 weeks,measurements were carried out as follows:(1)Evalution of insulin sensitivity of mice by IPGTT and IIPITT;(2)Evalution of the lipid transport ability of skeletal muscle in different groups by given a bolus of 3H-OA through oral gavage;(3)Detecting the changes of lipid in serum of different groups;(4)Detecting the lipid storage in TA of different groups;(5)Detecting the changes of VEGF-B signaling pathway in skeletal muscle of different groups.Results: 1.After caloric restricted for 4 weeks,the VEGF-B signaling pathway in the skeletal muscle of the CUG mice muscle tissue was activated,and the sensitivity of insulin was increased,as well as the lipid transport.2.After 8 weeks of refeeding,the lipid transport and deposition in the skeletal muscle of the CUG mice was increased compared with the control group.In addtion,the VEGF-B signaling pathway is continuously activated,and local insulin resistance is observed in skeletal muscle.3.After down-regulating the expression of VEGF-B,the lipid transport and the ectopic lipid deposition in skeletal muscle of CUG mice was alleviated after refeeding for 8 weeks.The insulin sensitivity of skeletal muscle was improved.Conclusion: 1.VEGF-B signaling pathway mediates lipid deposition in skeletal muscle of CUG mice.2.Interfering with VEGF-B signaling pathway can alleviate lipid transport and ectopic lipid storage,and improve insulin sensitivity in skeletal muscle of CUG mice.Part 2 Role of epigenetic modification in regulating VEGF-B expression in skeletal muscle during catch-up growthObjective: To explore role of DNA methylation modification and histone methylation modification played in mediating VEGF-B changes in the skeletal muscle during catch-up growth.Methods: 1.Using the bisulfite treatment sequencing(BSP)method to detect changes of DNA methylation in the promoter region of Vegfb in the tibialis anterior(TA)muscle of catch-up rats(RN)and normal diet rats(NC).The rat model of catch-up growth has been successful constructed in our previous group.2.Real-time quantitative PCR(q PCR)was used to screen related histone methylases that may regulate histone methylation modification,and then verified by Western Blotting and chromatin immunoprecipitation(CHIP-q PCR)were conduced to verify whether the altered methylase or demethylase is involved in the regulation of Vegfb changes.3.L6 myoblasts were cultured in vitro,and the regulation of VEGF-B expression by methylase was further confirmed by interference or overexpression of related methylase expression.Results: 1.The DNA methylation level of the Vegfb promoter region was not significantly abnormal between the normal group and the catch-up group.2.As a member of demethylases,PHF2 showed a significant decrease at the end of food restriction and refeeding for 8 weeks,and the expression of H3K9me1 at the promoter of Vegfb was also increased.3.In vitro experiments further confirmed that PHF2 can regulate the expression of VEGF-B through regulating H3K9me1 at the promoter of Vegfb.Conclusions: During catch-up growth,demethylation transferase PHF2 regulates VEGF-B expression by regulating H3K9me1.