Adiponectin Treatment Regulats The Expression Of The Mitochondrial-derived Peptide MOTS-c Via APPL1/SIRT1/PGC-1α And The Signal Mechanism Of Exercise Improving Insulin Resistance

Objective:Adiponectin is a hormone derived from adipocytes and skeletal muscle that regulates energy homeostasis through increasing insulin sensitivity.APPL1（adaptor protein,phosphotyrosine interacting with PH domain and leucine zipper 1）is the first identified signaling molecule that mediates adiponectin function.In mammalian cells,APPL1 interacts with adiponectin receptors and mediates many pathways,among which the interaction between APPL1 and SIRT1 has been confirmed in the study of primary mouse hepatocytes.SIRT1 can deacetylate oxidase proliferator activated receptor coactivator-1α（PGC-1α）in skeletal muscle,and PGC-1αis the main regulator of mitochondrial biogenesis.MOTS-c is a new bioactive mitochondrial open reading frame of the 12S rRNA-c,which is expressed in various tissues of human body and animals.With the blood circulation,MOTS-c mainly acts on skeletal muscle,increasing glucose uptake and utilization,thus improving insulin resistance and regulating metabolic balance.These observations suggest that adiponectin regulates the production and/or secretion of MOTS-c in skeletal muscle cells by increasing the levels of APPL1,SIRT1,and PGC-1α,and whether exercise can stimulate MOTS-c production by skeletal muscle and result in the parallel increase in the systemic level of MOTS-c.Whether exercise can stimulate the production of MOTS-c in skeletal muscle and increase the level of MOTS-c in the body.Methods:In order to solve these problems,we employed Adipoq-KO(Adipoq^–/–)and their wild type littermates male mice（C57BL/6 background）were injected Acrp30intraperitoneally for one week;C57BL/6 wild-type male mice were selected for high-fat diet intervention,and after successful modeling,eight weeks of continuous exercise and one week of intraperitoneal injection of adiponectin or eight weeks of intraperitoneal injection of MOTS-c were applied respectively;On the other hand the mouse C2C12myotube cells were treated with SIRT1 stimulants or SIRT1 inhibitors or PGC-1αinhibitors respectively,the day following differentiation,cells were transfected with pcDNA-SIRT1,pcDNA-PGC-1α,pLDNA-APPL1,and/or siRNAs and sgRNAs using Lipofectamine3000.After the intervention,the mice serum and gastrocnemius muscle were collected,and C2C12 cells were collected,western bolt and qRT-PCR were used to detect the protein and mRNA levels of APPL1,SIRT1,PGC-1αexpression in the skeletal muscle and cells of mice after pharmacological or gene intervention.ELISA was used to detect the levels of insulin,adiponectin and MOTS-c in the serum of mice and MOTS-c in the skeletal muscle to explore the mechanism of the pathway.Results:We found that Adipoq^–/–mice decreased the levels of MOTS-c in serum and skeletal muscle,and adiponectin treatment could restore the expression of MOTS-c in serum and skeletal muscle.C2C12 myotube experiment confirmed that adiponectin increased the expression of MOTS-c mRNA in skeletal muscle;the level of APPL1 protein treated by adiponectin was consistent with the expression trend of MOTS-c protein and mRNA in skeletal muscle;After the cells were transfected withpLDNA-APPL1orsiRNAcould increase or decrease the expression of SIRT1,PGC-1αand MOTS-c,and overexpression of APPL1 could further improve SIRT1 induced by adiponectin,The expression of PGC-1α,MOTS-c protein and mRNA increased,while adiponectin treatment could reverse SIRT1 induced by siRNA-APPL1,PGC-1α,MOTS-c protein and mRNA expression were down regulated;overexpression of SIRT1 or SIRT1 activator treatment could increase the expression of MOTS-c,SIRT1 and PGC-1αprotein and mRNA induced by adiponectin;siRNA-SIRT1 or SIRT1 inhibitor treatment could reduce the expression of MOTS-c,SIRT1 and PGC-1αprotein and mRNA,which could be reversed after adiponectin treatment;pharmacology and gene inhibition of PGC-1αcould reduce the overexpression of SIRT1 induced MOTS-c protein and mRNA levels.MOTS-c and adiponectin in serum and skeletal muscle decreased significantly in the state of insulin resistance.Exercise,adiponectin or MOTS-c treatment restore MOTS-c or adiponectin in serum and skeletal muscle,to improve insulin resistance.Exercise can increase the expression of MOTS-c protein and mRNA in serum and skeletal muscle of mice.Conclusion:This is the first study to uncover that adiponectin is a regulator of MOTS-c,and that it regulates the production and/or secretion of skeletal muscle MOTS-c through the APPL1/SIRT1/PGC-1αpathway in Adipoq^-/-mice andC2C12myotubes.Furthermore,this study preliminarily demonstrated that MOTS-c responds to exercise and un raveled the signaling mechanism by which exercise increases MOTS-c expression and improves insulin resistance in C57BL/6 mice.That shows promise in yielding a better understanding of the cellular and molecular pathways underlying the pathogenesis of diabetes.