Epigenetic Alterations In Non-alcoholic Fatty Liver Disease And The Mechanism Of Bariatric Surgery On Non-alcoholic Fatty Liver Disease

BACKGROUNDNon-alcoholic fatty liver disease(NAFLD)is a very common disease in clinical practice,and the incidence of this disease has increased significantly in recent years along with changes in people’s diet and exercise patterns.The prevalence of this disease is close to 30%in China.The clinical development of NAFLD is often accompanied by lipid accumulation in the liver,and as the disease progresses,it can progress to nonalcoholic steatohepatitis(NASH),cirrhosis and even hepatocellular carcinoma(HCC).However,the current treatment for this disease is still based on controlled diet and exercise instruction.There is no effective drug treatment for this disease.Because there are no obvious clinical symptoms in the early stages of the disease,patients often do not choose to receive treatment,which makes the research on the molecular mechanisms related to the early stages of the disease very lacking.Liver tissue biopsy from patients undergoing bariatric surgery is the gold standard for the diagnosis of NAFLD in patients.Combined with 2 generation sequencing technology,additional mechanisms in the process of liver lipid accumulation in the early stages of NAFLD have been revealed.Research in this process will greatly assist in the diagnosis,treatment and prevention of this disease.In recent years bariatric surgery has been rapidly developing in China as an important treatment modality for obesity and obesity-related diseases.Sleeve gastrectomy(SG)is currently the main surgical procedure for weight loss surgery in China.Mechanistically,SG can significantly alleviate NAFLD and T2DM by improving glucose homeostasis and enhancing insulin sensitivity through mechanical diet restriction,altering intestinal flora and bile acid metabolism,and regulating gastrointestinal hormone secretion.however,the underlying mechanisms remain elusive.METHOD AND RESULTIn this paper we investigated the molecular mechanisms of early lipid accumulation in NAFLD based on whole genome sequencing of liver tissue specimens from clinical patients.We also combined with existing studies to reveal the potential molecular mechanisms of insulin resistance in NAFLD patients treated with weight loss surgery.The specific results include the following two aspects.PART Ⅰ The role of RAB7B in the development of NAFLDMETHODWe performed whole genome sequencing and gene library creation on liver tissue specimens from 12 clinical patients.These included 6 liver tissue specimens receiving intraoperative liver tissue biopsy for bariatric surgery and 6 normal liver tissue specimens adjacent to liver tissue resection tumors for hepatic hemangioma.GO,KEGG and GSEA analyses were performed on the mRNA fraction of whole-genome sequencing results to observe macroscopic changes in epigenetics of early NAFLD patients and to screen for target genes.Further,mouse models of NAFLD and in vitro cellular models were established to verify the role of target genes in the disease and the specific molecular mechanisms.RESULTA total of 30,982 genes were detected in mRNA sequencing,of which 649 genes were upregulated and 178 genes were downregulated.RAB7B was found to be upregulated in NAFLD liver.RAB7B is a small GTPase that plays an important role in autophagy.The aim of this study was to determine the role of RAB7B in nonalcoholic fatty liver disease(NAFLD).RAB7B was found to be upregulated in NAFLD livers,and overexpression of RAB7B in HepG2 cells and HFD mouse models inhibited the LKB1-AMPKα signaling pathway,activated the mTOR-PS6K pathway,and induced excessive lipid accumulation.In addition,RAB7B overexpression exacerbated inflammation and fibrosis in the MCD diet mouse model.Mechanistically,we used Co-IP-based mass spectrometry(MS/MS)analysis and ubiquitination experiments to demonstrate the binding relationship between RAB7B and FBXO22.RAB7B promoted the recruitment of FBXO22 to LKB1 and increased the ubiquitination of LKB1.These results suggest that RAB7B is a novel regulator of the AMPK pathway that exacerbates hepatic lipid accumulation and inflammatory responses in NAFLD.PART Ⅱ Bariatric surgery alleviate insulin resistance in the liverMETHODWe constructed an animal model of obese fatty liver combined with insulin resistance in mice by high-fat diet.The mice were subjected to SG surgery to observe the therapeutic effect of SG on the disease.Further,protein immunoblotting,immunohistochemistry and real-time quantitative PCR were used to detect the altered expression of FBXO2,a key protein for insulin receptor ubiquitination.Further FBXO2 specific overexpression in mouse liver was performed using tail vein.Glucose metabolism in mice was assessed by tissue staining sections,oral glucose tolerance and insulin tolerance assays.To investigate the role of SG in promoting glucose metabolism by improving insulin resistance through FBXO2 and the molecular mechanism.RESULTLipid metabolism was assessed by histology and lipid analysis.Glucose metabolism was assessed by oral glucose tolerance(OGTT)and insulin tolerance test(ITT).The SG group showed reduced hepatic lipid accumulation and glucose tolerance compared to the sham-operated group,and western blot analysis showed activation of AMPK and PI3K-AKT pathways.fbxO2 is a member of the E3 ubiquitin ligase family and degrades insulin receptors in obese mice by ubiquitination,disrupting glucose homeostasis.Mice fed a high-fat diet(HFD)for 16 weeks were subjected to SG and sham surgery.transcriptional and translational levels of FBXO2 were reduced after SG.the improvement in glucose metabolism observed after SG was partially reversed after liver-specific overexpression of FBXO2;however,remission of fatty liver was not significantly affected by FBXO2 overexpression.This suggests that downregulation of FBXO2 by bariatric surgery is and one of the important molecular mechanisms of insulin resistance alleviation by bariatric surgery,and this result complements the molecular mechanisms of insulin resistance treatment by bariatric surgery.CONCLUSIONS:1.RAB7B was icnereased in liver in NAFLD patients.2.RAB7B can activate mTOR,NFκB through downregulation of LKB1-AMPK signaling pathway,thus causing increased lipid accumulation and activation of inflammatory response.3.RAB7B can promote the specific binding of FBXO22 and LKB1 to promote the ubiquitination of FBXO22 to LKB1.4.SG can cause hepatic lipid accumulation and insulin resistance remission in NAFLD.5.SG can cause FBXO2 downregulation and increased insulin receptor protein levels.6.FBXO2 liver-specific overexpression partially reversed the SG-induced remission of insulin resistance.