Heterozygote Deletion Of Gfer Accelerates Nash In Mice By Inhibiting The Mitochondrial Fusion

Background & Aims:Hepatic stimulator substance(HSS), also named hepatocyte growth-promoting factor, or growth factor erv1(GFER), was initially reported in 1970 s by LaBrecque and his colleagues. In the following investigation, we found that HSS was able to induce the hepatocyte growth which might be related HSS regulation on EGF receptors. Consistent with many other studies,our results also demonstrated that HSS could protect hepatocytes from various injuries.Nonalcoholic fatty liver disease(NAFLD) is a metabolic syndrome which occurs in the patients without history of heavy drinking, but the liver histopathology is identical to what was found in in alcoholic lesion, such as hepatic fatty degeneration and inflammation. NAFLD spectrum includes simple hepatic steatosis, nonalcoholic steatohepatitis and develops into fibrosis and cirrhosis. At present, alternation in life style and rational diet are proved to be effective to hepatic steatosis. However, a few effective medicines are available for curing the disease once it develops into the NASH stage. The pathogenesis of NASH can’t summarize by one single factor, it is generally believed it has related with genetic, environmental and metabolic.It is previously found in our laboratory that development of NASH could be be effectively obstructed if HSS gene was highly expressed in the hepatocytes. HSS protection liver from steatosis was believed to reduce preserve mitochondrial function and reduce ER stress in vivo and in vitro. In this study, mice with partial deletion of HSS gene(presented by Gfer+/-) were generated with genetic manipulation. In Gfer+/-mice, HSS mRNA and protein were singnificantly reduced as compared to the wild-type animals. In attempt to preparation of NASH model, both Gfer+/- and Gfer+/+ mice were fed with choline-difficient diet(CD-diet) for 4 and 8 weeks, respectively. The lipotoxicity and lipid accumulation as well were accessed by determination of glycemia and histopatholgical staining. Firstly build a NASH model in low expression of HSS mice to observe the relationship of HSS and the development of NASH. Then research the mechanism of low expression of HSS lead to mitochondrial dysfunction and the accelerated development of NASH. Methods:1. Two groups of mice, genotype Gfer+/+ and Gfer+/- mice, weighting 20-25 g, were placed on choline-deficient diet(60 kcal% fat) and drinking water containing 0.165% DL-ethionine at the first week.2. Blood was collected from each group mouse by removalling eyeballs. Serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG) and total cholesterol(TC) levels were measured by biochemical analyzer.3. Tissue was sectioned for histopathology. Liver samples were respectively HE staining, Oil Red staining, Masson staining and Sirius Red staining.4. To extract the total DNA liver tissue, using the method of Real-time PCR analysis of the relative content of mt DNA.5. Total mRNA were extracted from liver tissue. Detect the HSS,PGC-1α,TFAM,ATPs,COXIV,Drp1,Fis1,Mfn1,Mfn2,Atg7 gene expression in two groups mice by Real-time PCR. And then evaluate the relationship between the HSS and NASH.6. Total protein were extracted from liver tissue. Detect the various indicators of two groups in the liver of mice by Western blot.7. Using the kit detected the content of MDA and ATP.8. Analysis of respiratory chain complex V and cytochrome C oxidase activity, and detect the change of mitochondrial membrane potential Results:1. The expression of HSS in Gfer+/- group is obviously lower than Gfer+/+ group both in protein and mRNA level. Our heterozygous mouse can be used for further experiment.2. The serum levels of ALT, AST and TG have no obvious difference, but Gfer+/- mice serum TC levels significantly lower than the Gfer+/+ mice, prompt the liver function damage, lipid metabolism disorders.3. Compare two model groups of serology and histological indexes. The Gfer+/-mice has a faster NASH process than Gfer+/+ mice, prompting the liver tissue lesion progress to fibrosis stage.4. The number of Gfer+/- mice mitochondria in the liver cell is more obviously, and the area of the mitochondria is bigger, the shape is more round.5. According to the results of Real-time PCR and Western blot, the expression of Mfn1 and Mfn2 associated with mitochondrial fusion in Gfer+/- mice liver tissue is decreased obviously prompting the mitochondria dynamic change as well as the fusion function obstacle.6. Real-time PCR results showed that Gfer+/- mice mt DNA content decreased obviously. Associated with mt DNA replication and transcription, the expression of PGC-1α and TFAM also significantly reduced. The expression of ATP and COXIV also reduced. With the corresponding, Gfer+/- mice liver tissue respiratory chain complex V and cytochrome C oxidase activity decreased, mitochondrial membrane potential also decreased.7. The ATP amount of Gfer+/- mice liver tissue has decreased significantly, the MDA amount of Gfer+/- mice liver tissue has increased significantly. Conclusions:The expression of Gfer in heterozygous mouse bred in our lab has an obviously lower level. The construction of mouse model can be applied to disease model.When the expression of HSS decreased, reducing the mitochondrial biogenesis, resulting in impaired the function of mitochondrial respiratory chain and restrained the fatty acid oxidation. Then eventually accelerate the development of NASH.