The Function And Mechanisms Of Apolipoprotein J In The Progression Of Nonalcoholic Fatty Liver Disease

With the changes in dietary habits and lifestyles,non-alcoholic fatty liver disease(NAFLD)has become an emerging global health problem.Although the underlying mechanisms of NAFLD progression are complicated,the "Second hit hypothesis" regarding its pathogenesis is generally accepted.Lipid deposition is considered as the "first hit",leading to increased susceptibility of the liver to a subsequent "second hit",thereby promoting liver injury.Lipophagy is a process that autophagically digests lipid droplets(LDs)and plays a crucial role in maintaining hepatic lipid homeostasis.The lysosomal biogenesis and autophagy are restricted by the mammalian target of rapamycin(mTOR)in response to free fatty acid and glucose stress.Therefore,an impaired lipophagy might cause hepatic LD deposition and contribute to the development of NAFLD.Apolipoprotein J(ApoJ)belongs to the small heat shock protein family,which is also known as clusterin.Our group previously demonstrated that the chaperone activity ApoJ is important for both hepatitis C virus(HCV)particles assembly or sterol O-acyltransferase 2(SOAT2)-induced LD accumulation.Supportively,other groups have reported that ApoJ maintains the stability of the LC3-Atg3 complex and involves in autophagosome biogenesis.Thus,we hypothesize that ApoJ may participate in hepatic lipid deposition by chaperoning proteins involved in lipophagy.The research is conducted as followed:1.Lipid stress up-regulated ApoJ expression.To investigate the role of ApoJ in NAFLD,the public single-cell databases,GEO databases,and serum samples from clinical NAFLD patients were analyzed.The result showed that ApoJ is mainly expressed in hepatocytes and up-regulated under lipid stress.By using gain-and loss-experiments,we found that ApoJ silencing alleviated the accumulation of intracellular lipid droplets.Next,the role of non-secreted ApoJ in lipid deposition was evaluated using an ApoJ R227 Q maturation deficit mutant.The result showed that the ApoJ R227 Q exerted comparable efficacy with wild-type ApoJ in restoring oleic acid(OA)-induced lipid accumulation in ApoJ knockdown cells.2.Silencing ApoJ activated mTOR-supprssed lipophagic flux.We explored the mechanism of ApoJ in regulation of intracellular lipid metabolism.The proteomic analysis showed that treatment of OA activates the mTORC1 and inhibited the pathways involved in lipid metabolism,leading to the accumulation of LD in control cells.In contrast,knockdown of ApoJ blocked the mTORC1 signaling pathway and restored lipid metabolism,resulting in alleviation of lipid accumulation.Functional analysis further demonstrated that ApoJ silencing reduces mTOR activity and restores autophagy.Furthermore,hepatic ApoJ level exerted association with the protein markers of autophagy and hepatic lipid contents in mouse model of NAFLD,3.Hepatocyte-specific knockout of ApoJ alleviated high fat diet-induced NAFLD progression.a liver-specific ApoJ knockout(HKO)mouse model was estabolished to verify the function of heaptic ApoJ on NAFLD.The proteomic approaches were applied to explore the underlying pathways in ApoJ HKO mice under high fat diet(HFD).The levels of fasting serum lipid,glucose,and insulin were detected by biochemical method and the insulin sensitivity was examined by GTT(Glucose Tolerance Test)and ITT(Insulin Tolerance Test).The results showed that ApoJ HKO improves liver steatosis,restores lipid and glucose homeostasis,and increases insulin sensitivity in mice fed with HFD.4.Antagonized ApoJ chaperone promoted mTOR/FBW7 interaction and restored mTOR-suppressed lipophagy.We demonstrated that ApoJ bound majorly to the kinase domain of mTOR and OA enhanced ApoJ-mTOR interaction in a dose-dependent manner.Functionally,ApoJ,as a molecular chaperone,prolonged the half-life of mTOR by preventing proteasomal degradation of mTOR through interfering mTOR and FBW7(F-box and WD repeat domain-containing 7)interaction.5.The ApoJ antagonist peptide improved lipid and glucose metabolism in mice with NAFLD.The therapeutic effect of ApoJ antagonist peptide on hepatic lipid deposition was examined using mouse model of NAFLD.The ApoJ antagonist peptide bound specifically to ApoJ could be internalized by cell and exerted minimal cell cytotoxicity.In vivo,the peptide was accumulated in the liver with a half-life of 3.5 hours.Functional experiments showed that antagonist peptide reduced serum lipid levels,improved liver steatosis,and increasing insulin sensitivity through inhibiting mTOR activation and restoration of mTOR-suppressed lipophagy in HFD-fed mice.6.The ApoJ antagonist peptide exhibited comparable efficacy with liraglutide in improving lipid and glycemic homeostasis in diabetic mice.The therapeutic effect of ApoJ antagonist peptides was tested on mouse model of T2DM(Type 2 diabetes mellitus).The findings showed that ApoJ antagonist peptide reduces serum lipid levels and insulin resistance,and prevents hepatic lipid deposition in db/db mice.In addition,ApoJ antagonist peptide restored mTOR-inhibited lipophagy in db/db mice.Taken together,our study demonstrated that lipid stress-induced ApoJ competes with the FBW7 ubiquitin ligase to prevent the ubiquitin-proteasomal degradation of mTOR.Accumulation of mTOR subsequently inhibits lipophagy and impairs lysosomal function,resulting in hepatic lipid deposition.On the other hand,ApoJ antagonist peptide binds to stress-induced ApoJ and promotes mTOR degradation,therefore restores hepatic lipid homeostasis and prevents the diseases progression of NAFLD and T2 DM.This study clarifies the regulation mechanism of ApoJ on NAFLD,and provides a new perspective and target for the treatment of NAFLD.