Intermittent Fasting Regulates Adipocyte Mitochondrial Fusion Through Sirt3 Deacetylation Of Mdh2 In Mice

Increasing meat production and improving meat quality are important goals of animal genetic improvement,and fat deposition and muscle development are the core scientific issues in the formation of meat yield and meat quality traits.However,as people’s demand for lean pig meat increases,reducing pig fat deposition and increasing lean meat rate have become new research points.Porcine fat deposition and lipid metabolism are closely related to the morphology,structure and function of mitochondria.The morphology of mitochondria is regulated by the balance between fusion and fission processes,depends on the coordination of many proteins and is mainly regulated by post-translational modification(post-translational modification,PTM).Studies have shown that Sirt3 is highly expressed and mitochondrial fusion is increased in the liver of high-fat mice treated with intermittent fasting.Proteomics studies have found that more than 20% of the protein in mitochondria can be acetylated,which is partly involved in mitochondrial fusion and mitochondrial fusion.Fission regulation.However,the mechanism by which mitochondrial protein acetylation regulates the dynamics of mitochondrial fusion and fission in the adipose tissue of high-fat mice treated with intermittent fasting is not clear,and it is unclear whether this phenomenon is affected by the mitochondrial NAD+-dependent deacetylase silent mating type information regulation2 homolog-3(Sirt3)mediate.This paper uses non-standard quantitative analysis of acetylation modification,q PCR,immunoblotting,IP purification,fluorescent staining and other methods to explore the role and mechanism of Sirt3 deacetylation of mitochondrial protein to promote mitochondrial fusion during intermittent fasting.This experiment provides a theoretical basis for analyzing the regulatory role and mechanism of intermittent fasting protein deacetylation in mitochondrial fusion and functional improvement,and provides a control model and feasible approach for reducing pig fat deposition and improving its meat quality.The results of this study are as follows:1.Intermittent fasting promotes iWAT mitochondrial fusion and improves mitochondrial function in HFD mice.After intermittent fasting,the iWAT of HFD mice changed significantly(p<0.05).After the mitochondria were separated,the number of mitochondria was reduced and the morphology lengthened(p<0.05).The expression of mitochondrial fusion protein Opa1 was extremely significantly increased(p<0.01),the expression of fission protein Fis1 and Mff was extremely significantly reduced(p <0.01),the mRNA levels of fusion genes Opa1 and Mfn2 were extremely significantly increased(p<0.01),the split gene Fis1 and Drp1 mRNA levels were significantly reduced(p<0.05),further verifying that intermittent fasting promotes iWAT mitochondrial fusion in HFD mice.The same method detected that intermittent fasting made WT mice and ob mice iWAT mitochondria tend to fuse.Intermittent fasting promoted a very significant increase in the membrane potential of HFD mice(p<0.01),significantly increased the ATP content and mt DNA copy number of mouse iWAT(p<0.01),and promoted the mRNA level of mitochondrial function-related genes Uqcrc and Sdhb increased significantly(p<0.01),which significantly increased the mRNA expression levels of Tfam,ATP5a1,Cox4i1(p<0.05).It showed that intermittent fasting significantly improved iWAT mitochondrial function in HFD mice(p<0.05).The same method measured that intermittent fasting partially improved iWAT mitochondrial function in WT and ob mice.2.Intermittent fasting promotes protein deacetylation,mitochondrial fusion and mitochondrial function through Sirt3.Intermittent fasting can increase the level of protein deacetylation and intermittent fasting significantly promoted the deacetylation of iWAT protein in HFD mice(p<0.05).Intermittent fasting made NAD+ molecules and TCA metabolic pathways active,and the NAD+ content of mouse iWAT increased significantly(p<0.01).Intermittent fasting significantly increased the mRNA and protein levels of Sirt3(p<0.05).Constructing Sirt3 overexpression and interference vectors and processing adipocytes,measured that pc-Sirt3 significantly reduced the number of adipocyte mitochondria(p<0.05).pc-Sirt3 significantly increased the mRNA levels of the mitochondrial fusion genes Opa1 and Mfn2(p<0.05),and significantly decreased the mRNA levels of the split genes Drp1 and Fis1(p<0.05).pc-Sirt3 significantly promoted the expression of the fusion protein Opa1(p<0.01),and significantly inhibited the expression of the fission protein Fis1 and Mff(p<0.05).Then it was measured that pc-Sirt3 can significantly increase fat cell ATP content and mt DNA copy number(p<0.05),significantly increase the mRNA level of mitochondrial function-related genes ATP5a1,Sdhb,Tfam,Ndufb8(p<0.05),and significantly reduce fat Cellular ROS generation(p<0.05).It is determined that Sirt3 can significantly enhance mitochondrial function.The above studies have proved that intermittent fasting promotes Sirt3 expression and protein deacetylation in iWAT of HFD mice,and Sirt3 promotes adipocyte mitochondrial fusion and mitochondrial function.3.Sirt3 regulates the JNK-FIS1 pathway by deacetylating Mdh2 to promote mitochondrial fusion.After treating adipocytes with pc-Sirt3 and si-Sirt3,Sirt3 can significantly increase the NAD+ content of adipocytes(p<0.05),and significantly inhibit the level of total protein acetylation in adipocytes(p<0.01).Sirt3 significantly inhibited the acetylation level of purified Mdh2(p<0.01).Sirt3 can significantly inhibit the protein levels of p-JNK/JNK and Fis1(p<0.05).The inhibitor SP600125 and agonist Ani treatment of Sirt3 can enhance or weaken the inhibitory effects of p-JNK/JNK and Fis1,respectively,indicating that Sirt3 can pass JNK-FIS1 pathway regulates adipocyte mitochondrial fusion.After knocking out Mdh2 in adipocytes with CAS9-Mdh2 vector,Sirt3’s inhibitory effect on the JNK-FIS1 pathway was significantly reduced(p<0.05),and its promotion of Opa1 and Mfn2 was significantly weakened,confirming that Sirt3 is regulated by deacetylation of Mdh2 the JNK-FIS1 pathway thus promotes mitochondrial fusion.This study confirmed that intermittent fasting can promote iWAT mitochondrial fusion in HFD mice,improve mitochondrial function,promote high Sirt3 expression and promote protein deacetylation.Sirt3 inhibits the JNK-FIS1 pathway to promote mitochondrial fusion by deacetylating Mdh2.This study can provide research ideas and foundations for reducing pig feeding costs and fat deposition through intermittent fasting treatment,and promoting pig energy metabolism by adjusting mitochondrial phenotype,and provide a feasible way and powerful target for improving pig meat quality.