Study On The Mechanism Of Hepatic SphK2 Regulating The Homeostasis Of Lipid Metabolism In NAFLD

Objective:Non-alcoholic fatty liver disease（NAFLD）is a metabolic stress-induced liver injury without alcohol and other definite liver injury factors,which is characterized by diffuse hepatic bullous steatosis and closely related to obesity and insulin resistance.NAFLD has a wide spectrum of diseases,including simple hepatic steatosis,nonalcoholic steatohepatitis（NASH）,progressive liver fibrosis,cirrhosis and even hepatocellular carcinoma.The pathogenesis of NAFLD is complex,and there is no specific drug for NAFLD clinical treatment.Sphingosine kinase（SphK）is a key rate limiting enzyme that catalyzes the phosphorylation of sphingosine to sphingosine 1-phosphate（S1P）.It plays a central role in the metabolic homeostasis of ceramide,sphingosine and S1P.There are two subtypes of SphK,namely SphK1 and SphK 2,which are isoenzymes.At present,the role of SphK2 in hepatic lipid metabolism has not been fully elucidated.Therefore,this study aims to reveal the regulatory role and the molecular mechanism of hepatic SphK2 in NAFLD induced by high-fat diet（HFD）in mice.This study will provide new insights and ideas for the targeted regulation of disorders of hepatic lipid metabolism and the exploration of new therapeutic target.Methods:In this study,wild-type（WT）,hepatocyte-specific SphK2conditional knockout(SphK2^Hep-/-),and SphK2 overexpression mediated by adeno-associated virus（AAV）mice were fed with high-fat diet（Research Diets,#D12492）for 20 weeks to induce NAFLD.Moreover,we used 0.5 mM palmitic acid（PA）to treat primary mouse hepatocytes for 24 hours to induce a cellular fatty liver model.Lipid metabonomics was used to analyze and screen the different metabolites significantly affected by hepatic SphK2.In addition,we used co IP,homology modeling,molecular docking and dynamics simulation techniques to construct the three-dimensional structure of proteins,and observed the interaction and binding sites between proteins.The overexpression tag plasmid,the SphK2 mutant plasmid and the ubiquitination assay in vivo were used to detect protein ubiquitination levels.Results:Compared with WT mice,conditional deletion of hepatocellular SphK2 aggravated hepatic lipid deposition and steatosis,hepatocellular injury and insulin resistance induced by HFD.Overexpression of SphK2 by adeno-associated virus markedly ameliorated hepatic steatosis and insulin resistance in mice fed by HFD.Furthermore,lipid metabolomics analysis indicated that hepatocellular SphK2 deficiency promoted hepatic triglyceride（TG）deposition in mice fed with HFD.Mechanistically,we observed that the protein expression of ATGL was significantly down-regulated in SphK2-deficient hepatocytes compared with that of WT.However,the mRNA levels of ATGL did not change in SphK2-deficient hepatocytes We first revealed that hepatic SphK2 via its H335 competes with COP1 to occupy the VP motif of ATGL to prevent COP1 from binding to ATGL,thereby inhibiting ubiquitination degradation of ATGL and up regulating the expression of ATGL protein,which in turn reverses disorders of hepatic lipid metabolism and reduces TG deposition in hepatocyte.Conclusion:Hepatic SphK2 can competitively bind to ATGL with COP1,and then inhibit the ubiquitination of ATGL,accelerate the hydrolysis of TG in hepatocytes,so as to maintain the homeostasis of hepatic lipid metabolism and defer the progression of NAFLD.Taken together,this study is expected to unravel a new molecular mechanism of maintaining the homeostasis of hepatic lipid metabolism,which provides a new understanding that SphK2 corrects disorders of hepatic lipid metabolism to improve NAFLD,and an important theoretical basis for the exploration of new therapeutic target for NAFLD...