Study On The Mechanism Of OPG Regulating Liver Triglyceride Metabolism

PART ? EXPRESSION OF OPG IN NAFLD ANIMAL MODELS AND PATIENTSObjective: To explore the changes of OPG in NAFLD animal models and NAFLD patients,and to understand the relationship between OPG and NAFLD.Methods:The expression of OPG m RNA in the liver of c57 male mice with normal food diet(20 weeks old)and high fat diet for 12 weeks at the age of 8 weeks,adiponectin deficient mice,ob / ob mice(20 weeks old),db / db mice(20 weeks old)and NAFLD patients were detected by q PCR.The expression of OPG protein in liver of c57 male mice fed with normal diet and HFD mice,ob / ob mice,db / db mice and NAFLD patients were detected by western blot.Immunohistochemistry was used to analyze the protein levels of OPG in livers of NAFLD patients and healthy people.The changes of OPG m RNA in stimulating L02 cells with different concentrations of glucose and FFAs were measured.The expression of OPG m RNA in the liver of c57 mice in different feeding states were measured by q PCR.Results: In the NAFLD animal model and NAFLD patients,the levels of OPG m RNA and protein were significantly reduced,glucose can be dose-dependent stimulation to OPG m RNA expression,and FFAs stimulation can inhibit the m RNA expression of OPG.Conclusion: OPG may be involved in the energy metabolism of hepatocytes.PART ? THE EFFECTS OF OPG EXPRESSION IN VITRO ON THE LEVELS OF TRIGLYCERIDES IN HEPATOCYTESObjective: To demonstrate the involvement of OPG in the regulation of triglyceride metabolism in vitroMethods: Hepatocyte steatosis was induced by FFAs after being infected by OPG overexpressing or inhibiting adenovirus.The changes of fat were observed by oil red O staining.The levels of Triglycerides in liver cells were measured by Triglycerides Kits.The changes of m RNA expression in triglyceride metabolism related genes FATP2,FATP4,FATP5,CD36,SREBP1 c.FASn,ACC,SCD-1,Ch REBP,Cpt-1a,Mcad,PPAR,MTTP,LXR,PXR,RXR,and PPAR? were detected by q PCR.the changes of protein levels in triglyceride metabolism related gene SREBP1 c,ACC,FAS,CD36,PPAR? were detected by Western blot.Western blot was used to screen the changes of PKA,Akt,JNK,P38 and ERK signaling pathways.Results: We constructed OPG overexpressed and supressed adnovirus.Overexpression of OPG increased the number of lipid droplets and levels of triglycerides in liver cells significantly.Further more,the expression of PPAR?,nuclear receptor gene,and CD36,fatty acid uptake related gene,were significantly increased by overexpressing OPG.And the activity of p-ERK decreased.While the fatty acid synthesis related genes 1C,FASn,ACC,SCD-1,fatty acid ? oxidation related gene PPAR ?,Cpt-1a,Mcad,VLDL secretion related genes MTTP did not show significant changes.Inhibition of OPG was resistant to hepatocyte fat accumulation induced by FFAs,decreased the levels of TG in liver cells.CD36,Fatty acid uptake related gene,and PPAR?,nuclear receptor gene were suppressed by inhibition of OPG.And the activity of p-ERK increased.While fatty acid synthesis related genes SREBP1 c,FASn,ACC,SCD-1,fatty acid ? oxidation related gene PPAR ?,Cpt-1a,Mcad,VLDL secretion related genes MTTP did not show significant changes.Conclusion: In vitro,OPG regulated triglycerides levels in hepatocytes by regulating the expression of fatty acid uptake related gene,CD36,and nuclear receptor gene,PPAR?.16 PART ? OPG KNOCKOUT MICE IS RESISTANT TO HEPATIC CELL STEATOSIS INDUCED BY HIGH FAT DIETObjective: To study the effect of OPG on hepatic triglyceride metabolism by studying OPG knockout mice.Methods: A large number of OPG gene knockout mice were harvested by heterozygous and heterozygous combinations.After enough male wild type and homozygous mice were obtained,they were divided into WT-SD,OPG-/--SD group and WT-HFD,OPG-/--HFD group,the latter of which were fed with high fat diet for 12 weeks at the age of 8 weeks.The body weight,feeding were measured every week.GTT,ITT and other experiments were conducted at the age of 20 weeks.Serological indicators were analyzed from the blood of the eyeballs.The liver slices were taken from the liver.The lipid droplets of the liver were observed by oil red O staining.The fatty degeneration of the hepatocyte was observed by HE staining.The contents of triglycerides were measured by commercial kits.The expression of FATP2,FATP4,FATP5,CD36,SREBP1 c,FASn,ACC,SCD-1,Ch REBP,PPAR?,Cpt-1a,Mcad,MTTP,LXR,RXR,PXR,FXR,PPAR? were detected by PCR.The changes of proteins including PPAR?,CD36,p-ERK and others related to triglyceride metabolism were detected by western blot.Results: The body weight of OPG knockout mice was significantly lower than that of WT mice,but no significant changes in feeding were observed.GTT,ITT experiments showed that OPG knockout mice were resistant to insulin res istance induced by high fat diet.The serum levels of AST,ALT,TG,TC,FFA and GLU were significantly increased After high fat diet,which were significantly lower in OPG knockout mice than those in the control group(P <0.05).The results of HE staining showed that the degree of Steatosis and the vacuoles in liver were significantly increased after high fat diet.Whereas compared with WT-HFD group,OPG-/--HFD was significantly better than that in WT-HFD group.In normal diet groups,triglycerides levels in liver were lower in OPG-/-group than that in control group.OPG-/-mice reduced the expression of CD36 in hepatocyte fatty acid uptake,while the levels of the fatty acid synthesis-related genes SREBP1 c,FAS,ACC,SCD-1,fatty acid ?-oxidation related genes PPAR?,Cpt-1a,Mcad,VLDL and secretion-related gene MTTP did not show obvious changes.The expression of PPAR? decreased in the liver of OPG-/-group mice,compared with control group.While the activity of p-ERK increased.Conclusion: In vivo,OPG regulates the expression of triglycerides in,,,,hepatocytes by modulating the expression of fatty acid uptake related genes.PART ? THE MECHANISM OF TRIGLYCERIDE METABOLISM REGULATED BY OPGObjective: To explore the molecular mechanisms of triglyceride metabolism in liver regulated by OPG.Method: To verify the influence of OPG overexpression on TG,and fatty acid intake related gene CD36,nuclear receptor gene PPAR gamma,p-ERK,in c57 original generation liver cells,we first used primary liver cell separation.Then by cultivating primary liver cell,we analys is its influence on TG content in liver cells and the fatty acid intake related genes CD36,nuclear receptor gene PPAR gamma,p-ERK in OPG-/-mice,compare to the WT control group.Furthermore,by the culture of CD36-/-liver cells in primary generation,we proved that OPG manages content of TG in liver cells through CD36.Finally by use of PPAR? inhibitor GW9662,ERK inhibitors SCH772984 to verify whether OPG regulate the expression of CD36 by p-ERK,to PPAR?.Results: After treated by OPG-Fc,in the original generation of liver cells in c57 mice,intracellular TG content,CD36 fatty acid intake and PPAR? nuclear receptor gene expression had increased,whereas p-ERK activity declined.Contrast to the WT control group,in OPG-/-primary hepatocytes,TG concets reduced intracellular,the expression of fatty acid intake gene CD36 reduced,the expression of nuclear receptor genes PPAR? decreased,and p-ERK activity increased.In the CD36-/-original generation,OPG-Fc can not increase the content of intracellular TG.After the application of PPAR ? inhibitor GW9662,OPG-Fc can not increase the expression of CD36.After pretreated by ERK inhibitors SCH772984,OPG-Fc can't increase the PPAR?,CD36 expression.Conclusion : OPG regulates the content of triglyceride in liver by axle of ERK-PPAR?-CD36.