Study On The Molecular Mechanism Of GP73 Regulating Hepatic Lipid Metabolism Through Its GAP Activity

Objective: Non-alcoholic fatty liver disease(NAFLD)is a common cause of chronic liver disease.Due to defects in lipoprotein assembly or transport,NAFLD is usually associated with increased triglycerides in the liver and insulin resistance.VLDL are large particles of about 150-500 nm,mainly composed of triglycerides,phospholipids,and cholesterol.The lipid core of these particles is decorated with apolipoprotein B(Apo B).These lipid particles are synthesized and assembled in liver cells and secreted into the serum for transmission to other organs through circulation.Defects in the export of VLDL-Apo B impair lipid homeostasis.This article aims to investigate the effects of high expression of Golgi Protein GP73(GP73)on lipid metabolism in liver,and to elucidate the molecular mechanism that regulates lipid metabolism through the inhibition of Apo B secretion by GAP activity,in order to provid potential diagnostic and therapeutic targets for the prognosis of NAFLD.Potential diagnostic and therapeutic targets.Methods: 1.The mice were given a high-fat diet(HFD)in groups for 0,3,and 7 days.The intake of high-fat diet on the m RNA and protein levels of GP73 in mice liver was detected by fluorescent quantitative PCR and Western blotting,respectively.The impact of expression.2.Inject the empty vector(AAV-V)of hepatocyte-specific adeno-associated virus(AAV)and AAV(AAV-GP73)that highly expresses GP73 into the tail vein of mice,and give them a regular diet(Reg)or a high-fat diet(HFD);Evaluate glucose metabolism in mice by monitoring fasting blood glucose,fasting insulin levels,and glucose tolerance experiments;after successful model construction,use enzyme-linked immunosorbent assay to determine TG and CHO levels in mouse liver;use protein immunoimprinting Detection of the expression levels of GP73,Apo B,and Apo E in mouse liver;TG,CHO,low-density lipoprotein(LDL),glutamate aminotransferase(ALT),and aspartate in mouse serum were detected using a fully automatic biochemical analyzer Acid aminotransferase(AST)levels;hematoxylin eosin staining and oil red O staining were used to detect the morphology of liver cells and the degree of lipid accumulation in the liver.3.Using transcriptome expression chip sequencing technology(RNA-seq)to determine the molecular changes that cause dyslipidemia phenotypes to compare the transcriptome differences in livers of GP73 overexpressing mice and control mice.4.The effect of WT GP73 and GP73-RQ overexpression on apolipoprotein secretion was detected by Elisa method.5.Inject AAV-V,AAV-GP73 and AAV(AAV-GP73-RQ)with high expression of GP73-RQ into the tail vein of mice to build a GP73 GAP activity model;by measuring fasting blood glucose,fasting insulin levels,and glucose tolerance experiments To evaluate and monitor the glucose metabolism of mice;after the model was successfully constructed,TG and CHO levels in mouse liver were measured by enzyme-linked immunosorbent assay;TG,CHO,LDL,ALT,and AST in mouse serum were detected by a fully automatic biochemical analyzer s level.Results: 1.The m RNA level of GP73 in the liver of mice was detected by fluorescent quantitative PCR.The level of GP73 in the liver increased with the time of normal high-fat diet.Immunoblotting was used to detect the protein of GP73 in the liver on the seventh day of high-fat diet.The level started to increase.2.Throughout the observation process,AAV-GP73 mice showed an increase in fasting blood glucose,a decrease in fasting insulin,and an abnormal glucose tolerance test;high expression was confirmed by high expression of liver GP73 in mice tested for AAV-GP73 by immunoblotting.The expression of Apo B100 in the liver of AAV-GP73 mice was significantly increased.As determined by enzyme-linked immunosorbent assay,the contents of TG and CHO in the liver of AAV-GP73 mice were significantly higher than those of the control group.Weighing found that the specific gravity of the liver was also higher than that of the control group;oil red O staining revealed that in the conventional diet,the red lipid droplets in the liver of AAV-GP73 mice were significantly more than in the control;and in the high-fat diet,the liver of AAV-GP73 mice was significantly higher Significant yellowing and swelling,hematocyte staining and obvious balloon-like changes and damages were detected by hematoxylin staining.The serum TG,CHO in AAV-V and AAV-GP73 mice were detected by a fully automatic biochemical analyzer compared to the control group.Esters were significantly reduced,while ALT and AST were significantly increased.3.Use transcriptome sequencing technology to detect the molecular changes that cause the dyslipidemia phenotype to compare liver transcriptomes isolated from the control group or GP73 overexpressing mice.Overexpression of GP73 in liver cells(1.5-fold change in expression,adjusted p value <0.05)makes 20% of all genes differentially expressed;in GP73 high-expressing mice fed on a regular diet,it affects up-regulated genes The types of genes up-regulated in HFD-fed control mice are very similar;in the liver of AAV-GP73 high mice,genes related to fatty acid secretion,de novo fatty acid synthesis and NAFLD,and genes related to gluconeogenesis and insulin resistance are all significant Increased,genes related to abnormal glucose metabolism were differentially expressed in mice with high liver GP73;and the levels of tumor-associated genes in livers of mice with high GP73 were significantly higher than those in control mice.4.In the Huh7.5 cell model,we found that WT GP73 inhibited the secretion of Apo B and Apo B100,while GP73-RQ mutants were compared with controls The groups are not very different.The secretion of Apo A1 was not affected by WT GP73 or GP73-RQ,and WT GP73 promoted the secretion of Apo E.5.In the model of GP73 GAP activity,liver GP73-RQ mutant overexpressing mice did not show significant fasting blood glucose and fasting insulin reduction in WT GP73 mice during the entire observation process;by enzyme-linked immunosorbent assay It was determined that the contents of TG and CHO in the liver of AAV-GP73 mice were significantly higher than those in the control group,while AAV-GP73-RQ was not significantly different from the control AAV-V mice;the serum of AAV-GP73 mice was detected by a fully automatic biochemical analyzer TG and CHO were significantly reduced,and ALT and AST were significantly increased,while TG and CHO in serum of AAV-GP73-RQ mice were not significantly different from the control,but ALT and AST were still increased compared with the control.Conclusion: Based on the above results,we found that a high-fat diet can up-regulate the abnormal expression of GP73 in the liver,and high-expressed GP73 in the liver can affect lipid metabolism by inhibiting Apo B secretion,leading to severe lipid disorders in the body;further research found that GP73 mutants showed no such functional changes,proving that GP73 regulates lipid metabolism and part of glucose metabolism is achieved through its GAP activity.