Mechanism Of Hepatic CGI-58 And PAT Proteins Regulating Lipid Droplet Homeostasis And Lipid Metabolism

BackgroundAbnormal lipid metabolism is the main manifestation of metabolic syndrome,inducing diseases including hyperlipidemia,fatty liver and atherosclerosis.Abnormal hepatic lipid metabolism is closely related to the occurrence and development of cardiovascular diseases in addition to the liver disease itself,and the degree of abnormal liver lipid metabolism is positively correlated with the risk of cardiovascular diseases.Lipid droplet is the main form of lipid accumulation in hepatocytes which is generally believed that its formation can help to reduce the free fatty acids in hepatocytes,thus alleviating the lipid toxicity.However,long-term high-fat diet or gene mutation may lead to the disorder of liver triglyceride and other lipid catabolism,and the large accumulation of lipid droplets in liver cells will induce liver injury,promote immune cell infiltration,and further develop liver inflammation and fibrosis.CGI-58（Comparative Gene Indentification-58）,as the fifth member of theα/βhydrolase folding protein family,which can’t be able to decompose lipids directly.However,it can as an activator of Adipose Triglyceride Lipase（ATGL）.Human CGI-58 mutation can cause Chanarin-Dorfman Syndrome（CDS）,which is manifested by the accumulation of lipid droplets in the cytoplasm of most types of cells in the body.CDS is a clinically heterogeneous group of inherited disorders characterized by the cytoplasmic accumulation of lipid droplets（LDs）in most tissues.Clinical phenotypes include hepatomegaly（hepatic steatosis and steatohepatitis）,ichthyosis,myopathy,hearing loss,ataxia,mental retardation,etc,for which there is no effective treatment.PAT protein family（Perilipin-Adr P-TIP47 protein family）is an important part of the lipid coated protein,including five members:PLIN1（Perilipin）,PLIN2（Perilipin-2;ADRP）,PLIN3（Perilipin-3;TIP47）,PLIN4（Perilipin-4;S3-12）,PLIN5（Perilipin-5;OXPAT）.The function of PAT protein family molecules in lipid metabolism is different.Previous studies have confirmed that liver Plin2,Plin3 and Plin4 expressions are up-regulated in mouse models with liver CGI-58 specific knockout.Therefore,screening the effects of Plin2,Plin3 and Plin4 on fatty liver degeneration in mice with liver CGI-58specific knockout and exploring its specific mechanism will provide the most effective treatment for CDS,and find the possible regulatory molecules and their pathways regulating liver lipid metabolism and even systemic lipid metabolism.ObjectiveTo explore the role of Plin2,Plin3 and Plin4 in lipid metabolism disorders caused by specific knockout of liver CGI-58,and find possible treatment plans for CDS through screening and PAT protein.And evaluate which is the most effective in regulating lipid metabolism in liver to provide new target molecules and pathways for systemic lipid metabolism abnormalities.Methods1.AML-12 hepatocytes as the cell model,and the expression of Plin2 or Plin3 in PAT protein family was targeted by Si-RNA.Detect the expression of PAT protein family in cells with or without oleic acid induction,and observe lipid droplet formation and quantitatively detect triglycerides.2.Using CGI-58^Flox/Flox mice as animal model,the AAV virus targeting mouse liver was constructed,and CGI-58 knockout and Plin2 knockdown were achieved separately by co-expressing Cre protein and Micro-RNA targeting Plin2.Liver-specific knockout CGI-58mice were used as control.Investigate the effect of hepatic PLIN2 on fatty liver induced by CGI-58 specific deletion and explore the possible mechanism.3.Using CGI-58^Flox/Flox mice as animal model,the AAV virus targeting mouse liver was constructed,and CGI-58 knockout and Plin3 knockdown were achieved separately by co-expressing Cre protein and Micro-RNA targeting Plin3.Liver-specific knockout CGI-58mice were used as control.Investigate the effect of liver PLIN3 on fatty liver induced by CGI-58 specific deletion and explore the possible mechanism.4.Using CGI-58^Flox/Flox mice as animal model,the AAV virus targeting mouse liver was constructed to achieve co-expression of Cre protein and Micro-RNA targeting Plin4.Liver-specific knockout CGI-58 mice were used as control.Investigate the effect of liver PLIN4 on fatty liver induced by CGI-58 specific deletion and explore the possible mechanism.5.Comparing the results of hepatic Plin2,Plin3 and Plin4 knockdown on liver gross,serological and liver pathology of liver CGI-58 knockout mouse model to find the most effective target molecule for intervention.Results1.Plin2/Plin3 knockdown could reduce the lipid droplet formation and lipid accumulation in AML-12 cells induced by OA,and the effect of Plin3 knockdown on lipid accumulation was more significant than that of Plin2.2.Liver Plin2 knockdown can improve the glucose tolerance of liver CGI-58 knockout mice,significantly reduce liver enlargement and liver triglyceride content,and alleviate liver microvesicular fatty degeneration.3.Hepatocyte Plin3 knockdown can significantly reduce liver enlargement,reduce liver triglyceride content caused by liver CGI-58 knockout mice,relieve liver inflammation and reduce the activation of liver necroptosis pathway.4.Micro-RNA targeting mouse liver Plin4 can significantly reduce liver CGI-58knockout induced hepatomegaly,reduce serum ALT/AST content and activate the liver PPAR pathway.5.Comparing targeting Plin4/Plin3/Plin2 on hepatic CGI-58 specific knockout mice,Plin4 was stronger than Plin3 in improving liver weight and serum ALT/AST efficacy than Plin2.Conclusion1.Cell experiments confirm that PLIN2/PLIN3 are involved in the formation of lipid droplets in AML-12 cells,and the effect of PLIN3 on lipid droplet formation and lipid accumulation is stronger than PLIN2.2.The knockdown of Plin2 gene in mouse liver can alleviate the hepatic microvesicular steatosis caused by CGI-58 gene knockout.3.The knockdown of Plin3 gene in mouse liver can relieve liver enlargement and steatohepatitis caused by CGI-58 gene knockout,and reduce its necrosis and apoptosis pathway.4.The expression of Micro-RNA targeting mouse liver Plin4 can activate the PPAR signaling pathway and alleviate hepatomegaly and steatohepatitis caused by CGI-58 gene knockout.5.The effect of PAT protein family members on the regulation of lipid metabolism is ranked as PLIN4>PLIN3>PLIN2.