Effects And Mechanisms Of Liraglutide Ameliorated Non-Alcohol Fatty Liver Disease

[Background]Nonalcoholic fatty liver disease(NAFLD)has become the most common chronic liver disease throughout the world,with the increasing prevalence in mainland China which has reached to 15%in 2012.Although many drugs have been used to improve NAFLD,there is no specific preferable treatment for NAFLD.Hence,finding a widely effective drug has become an urgent problem to be solved.Liraglutide,a prospective recommended anti-diabetes drugs,has recently suggested by several clinical trials to be effective in improving NAFLD in Type 2 Diabetes patients.However,no mice model has indicated that Liraglutide could relieve NAFLD,and the potential mechanism of how Liraglutide relieved NAFLD still remains unclear[Objectives]To explore the effects and specific mechanisms of how Liraglutiderelieved NAFLD[Methods]1.20 weight matched C57BL/6J mice were selected and randomly divided into normal diet group(n=8)and high-fat diet group(n=12),respectively for 16 weeks.2.The normal diet group and high-fat diet group mice were further randomly divided into normal control group(normal diet + normal saline injection),control group(normal diet +Liraglutide injection),high fat diet group(high fat diet+normal saline injection)and Liraglutide group(high fat diet+Liraglutide inj ection).Liraglutide was subcutaneous inj ected at the dose of 150ug per gram of body weight of mice for 4 weeks.3.Body weight and body fat content were assessed to determine the growth and obesity of mice.Intraperitoneal glucose tolerance test(ipGTT)and imtraperitoneal insulin tolerance test(ipITT)were administrated to evaluate the glucose tolerance and insulin sensitivity in mice.Mouse micro-CT imaging was used to determine nonalcoholic fatty liver disease remission in mice in vitro,while the mouse liver tissue sections with HE staining and lipid deposition assessment by TG kit were administrated to quantitatively determine the level of nonalcoholic fatty liver disease remission.4.To determine the liver oxidative stress,that is,the oxidation-antioxidant balance in mice.MDA(malondialdehyde)kit was used to detect the membrane lipid peroxidation,while the expression of antioxidant enzymes in the liver indicating the antioxidative process was detected by Western blot analysis.5.QPCR and Western Blot was used to assess the mRNA and protein levels of markers of mitochondrial fusion and fission process in order to explore the role of Liraglutide in reducing the ingeneration of Reactive Oxygen Species(ROS).6,QPCR and Western Blot was used to assess the expression of autophagy pathway in liver tissue which may elucidate the role of Liraglutide in eliminating ROS.[Results]1.After high fat inducement for 16 weeks,the body weight as well as body fat content of mice was remarkably increased,while micro-CT showed a significant decrease in liver CT value,suggesting the successful establishment of NAFLD.2.Liraglutide subcutaneous injection for 4 weeks significantly ameliorated non-alcoholic fatty liver and also improved systematically metabolism:(1)mice body weight and body fat content decreased significantly,suggesting that Liraglutide could reduce the degree of obesity in mice.(2)the amount of area under the curve of glucose tolerance test as well as insulin resistant test were both significantly reduced,suggesting that Liraglutide could improve glucose tolerance and insulin sensitivity in mice(3)the reservation of decreased CT value induced by high fat liver assessed by micro-CT provided the radiographic evidence of Liraglutide's role in relieving nonalcoholic fatty liver disease(4)the HE staining of liver sections showed that Liraglutide can significantly improve fatty liver fat deposition and inflammatory reaction,while the liver tissue TG assessment indicated that Liraglutide 4 weeks after injection can significantly reduce the overwhelmed content of TG in liver caused by high-fat diet3.Liraglutide can reduce oxidative stress causing by the breaking of oxidative and anti-oxidative balance(1)MDA content assessment showed that Liraglutide injection can significantly reverse the high fat diet induced overwhelmed MDA level in liver tissue,suggesting that Liraglutide can alleviate the high fat diet caused membrane lipid peroxidation(2)qPCR and Western blot proved that Liraglutide can significantly increase the mRNA and protein levels of antioxidant enzymes CATALASE,MnSOD,which indicates an increase in antioxidant levels in the liver4.Liraglutide can strengthen the mitochondrial structure stability through dynamic fusion and fission process,thereby reducing the generation of reactive oxygen species:(1)qPCR and Western blot experiment results show that the dynamic process of mRNA and liver mitochondrial fission protein DRP1 protein and mitochondrial fusion related MFN2 expression level was significantly increased,suggesting that Liraglutide could strengthen the dynamic fusion and fission process to maintain mitochondrial stability.(2)qPCR and Western blot proved that Liraglutide could significantly reverse the high fat diet caused inhibition of mitochondrial respiratory chain structure protein Complex ? and UCP2,indicating that Liraglutide can enhance the mitochondrial structure.5.Liraglutide can strengthen autophagy by up-regulating the eliminating of ROS:(1)the qPCR and Western blot experiment showed that autophagosome structural protein LC3 and Beclinl exert significantly increases in both mRNA and protein levels while the mRNA and protein level of p62 was significantly inhibited,suggesting that Liraglutide could significantly enhance hepatic autophagy.(2)the autophagy pathway,SIRT3-FOXO3a-LC3,was assessed by Western blot procedure.Interestingly,it is suggested that the protein levels of SIRT3 and p-FOXO3a were significantly increased while the protein level of FOXO3a had no obvious change,indicating SIRT3-FOXO3a-LC3 pathway may be involved in Liraglutide enhanced liver autophagy.[Conclusions]1.Liraglutide can significantly improve nonalcoholic fatty liver disease.2.Liraglutide can remarkably reduce oxidative stress in liver by enhancing the oxidative and antioxidative balance.3.Liraglutide could inhibit the liver oxidative stress through strengthening of mitochondrial fusion fission process and enhancing mitochondrial structure management to reduce generation of reactive oxygen species.4.Liraglutide can strengthen autophagy by scavenging reactive oxygen species in order to improve the oxidative stress of liver.The SIRT3-FOXO3a-LC3 pathway may be involved in the process of Liraglutide promoted the autophagy.