| BackgroundNonalcoholic fatty liver disease(NAFLD)is the most common chronic liver disease in the world.In recent years,with the improvement of living standards,changes in diet and lifestyle,the incidence of NAFLD has increased year by year,which has now affected 1/4 of the world's human health.Without treatment,NAFLD can develop into severe liver damage,with 25%of patients developing hepatic fibrosis,1.5-8%developing cirrhosis,and a few even developing liver cancer.Meanwhile,atherosclerosis,hypertension,diabetes and cardiovascular diseases induced by NAFLD are the risk factors of death.However,there are still no effective drugs approved by FDA for the treatment of NAFLD,so it is urgent to study the pathogenesis of NAFLD and find effective treatment.At present,it is generally accepted that NAFLD is a kind of chronic liver disease,which is caused by many factors,such as lipid metabolism disorder,neuroendocrine disorders,chronic inflammatory response,oxidative stress,insulin resistance and intestinal barrier dysfunction.Among them,the excessive accumulation of TG in liver caused by abnormal fatty acid metabolism is the driving factor for the occurrence and development of NAFLD.Studies have shown that factors,such as obesity and type ?diabetes,lead to the disorder of lipid synthesis,uptake and utilization and promote the occurrence and development of NAFLD:On one hand,obesity and type II diabetes cause insulin resistance(IR)can increase the level of free fatty acids(FFA)in the blood by enhancing the decomposition of peripheral fat,thus enhancing the fatty acid intake and TG synthesis in liver;On the other hand,the overload of mitochondrial oxidation in hepatocytes leads to the decrease of oxidation capacity of fatty acid,further increasing the lipid accumulation in liver.On the contrary,the increase of FFA in blood will further enhance IR,which further promotes the disorder of lipid metabolism.At present,it has been found that fatty acid sensors play an important role in regulation of lipid metabolic and have potential clinical application value.PPAR ?,a fatty acid sensor,is involved in the regulation of fatty acid uptake,transport,fatty acid-oxidation,de novo synthesis and other lipid metabolism processes,Fibrates,an agonist of PPAR?a,has good clinical application in primary and mixed hyperlipidemia.BMS309403,an A-FABP inhibitor,can significantly improve atherosclerosis and diabetes in mice.CPDA and PAHSA,the FFA4 agonists,can also significantly inhibit the progression of diabetes in mice,which are expected to go into clinical application.Therefore,the search for fatty acid sensors involved in the regulation of lipid metabolism in hepatocytes will provide new ideas for the treatment of NAFLD.TIPE(TNF?-induced protein 8)family is a protein family with potential lipid binding ability discovered in recent years,including TIPE,TIPE1,TIPE2 and TIPE3.They have high sequence homology and play important roles in cell proliferation,apoptosis,immune regulation and tumor development.With the profound research of the TIPE family,the crystal structures of human TIPE2,TIPE3 and mouse TIPE have been successfully resolved.They all have TH domains with 6 helixes,forming the central hydrophobic cavity,which are rich hydrophobic amino acids,providing binding sites for lipid.It was also found that TIPE2 and TIPE3 could recognize and transport PIP2,PIP3 and other phospholipid molecules to regulate phospholipid signaling pathway.Although the structure of TIPE1 has not been reported in literature,the PyMOL predicted that TIPE 1 also has a central hydrophobic cavity,and has the potential to bind with lipid molecules.TIPE1(TNFa-induced protein 8 like 1)is one member of the TIPE family.At present,it has been found that TIPE1 has an obvious anti-cancer effect.Previous studies in our research group found that TIPE1 can promote the apoptosis of hepatocellular carcinoma cells through negative regulation of Racl pathway,and it has also been reported that TIPE 1 can significantly inhibit the growth and metastasis of lung cancer and gastric cancer.In addition,TIPE1 can also participate in the occurrence of Parkinson's disease by inhibiting the FBXW5-dependent degradation of TSC2 protein.TIPE1 can also induce increased ROS production to accelerate the formation of atherosclerosis.Previous results in our laboratory showed that protein level of TIPE1 was decreased in HFD induced NAFLD model mice,suggesting that TIPE1 may be involved in the occurrence and development of NAFLD.Meanwhile,PyMOL analysis indicate that TIPE1 has potential ability to bind with fatty acid.Thus,we hypothesized that TIPE1 might sense fatty acids and participate in the occurrence and development of NAFLD.Objectives:1.To explore the effect of TIPE1 on hepatocyte steatosis and the development NAFLD.2.To clarify the molecular mechanism of TIPE1 regulating hepatic steatosis.(1)To explore the fatty acid binding capacity of TIPE1 and its regulation on lipid metabolism in hepatocytes.(2)To elucidate the pathway involved in TIPE1 regulation on hepatic steatosis.Methods and results1.TIPE1 regulates lipid metabolism of hepatocyte and inhibits hepatocyte steatosis1.1 TIPE1 reduces the lipid level in hepatocytesTo test the potential ability of TIPE1 in regulating lipid metabolism of hepatocytes,we performed a lipidomics analysis with HUH7 cells transfected with siTIPEl and siNC.Results showed that the levels of TG,PC and ceramide was significantly increased in HUH7 cells with TIPE1 knockdown,which preliminarily indicate TIPE1 might regulate lipid metabolism of hepatocytes.1.2 TIPE1 inhibits hepatocyte steatosis in vitroIn order to clarify the role of TIPE1 in hepatote steatosis,HUH7 cells were transfected with siTIPE1 and siNC,then stimulated with OA or PA for 24h respectively.ORO and BODIPY staining showed that compared with the control group,TIPE1 knockdown significantly increased intracellular lipid deposition in HUH7 cells.In accordance,TIPE1 overexpression significantly reduced the lipid deposition of HUH7 cells stimulated by OA and PA.The results suggested that TIPE1 can improve the hepatocyte steatosis in vitro.1.3 TIPE1 regulates the expression of lipid metabolism related genes in hepatocytesNAFLD is a chronic liver disease caused by the disorder of lipid metabolism.Abnormal fatty acid metabolism can lead to excessive accumulation of TG in liver,which induce the occurance and development of NAFLD.We then detected four important lipid metabolism related genes,FASN,SREBP1,PPAR y and CD36.RT-PCR and western blot analysis showed that HUH7 cells,transfected with siTIPE1,had increased expression of FASN,SREBP1,PPAR? and CD36.Consistently,overexpression of TIPE1 in HUH7 cells significantly reduced the RNA and protein levels of FASN,SREBP1,PPAR? and CD36.It is further suggested that TIPE1 may affect the steatosis of hepatocytes by regulating the expression of lipid metabolism related genes.2.TIPE1 attenuates the occurrence and development of NAFLDTo further explore the effect of TIPE1 on the occurance and development of NAFLD,we constructed mouse model of NAFLD induced by high-fat diet,which was verified in both hepatocyte specific overexpression and knockout mice,and it was also verified in clinical fatty liver samples.2.1 Hepatocyte specific TIPE1 overexpression significantly inhibits the progression of NAFLD6-week-old C57BL/6 mice were hydro dynamically injected with adeno-associated virus with TBG-mTIPE1-Luciferase or TBG-Luciferase and fed with high-fat diet for 3 months.In vivo imaging results showed that the virus was specifically expressed in the liver.HE and ORO staining showed significant reduction of lipid deposition in AAV-mTIPE1 injected group,accompanied by decreased serum ALT level and downregulated TG,CHO level in both serum and liver homogenate.In accordance,the expression of pro inflammatory cytokines(IL-6,IL-1?,TNF-?)and the important lipid metabolism related genes(FASN,SREBP1 and CD36)were also decreased in AAV-mTIPE1 injected group.All these results further confirmed that TIPE1 could attenuate the development of NAFLD.2.2 Liver specific TIPE1 knockout significantly aggravates NAFLDThe liver specific TIPE1 knockout C57 mice,Alb-cre+;TIPE1 flox/flox and TIPE1 flox/flox,were constructed by the CRE-LOXP system,NAFLD model was constructed by high-fat feeding for 5 months.The results showed that,compared to WT mice,TIPE1-LKO mice had higher serum TG,CHO,FFA level and bigger livers,the expression of lipid metabolism related genes SREBP1,PPAR? and CD36 were also significantly increased.It further suggests that TIPE1 can improve NAFLD in mice 2.3 The expression of TIPE1 is significantly decreased in liver tissues of NAFLD patients We collected the paraffin sections of clinical NAFLD liver tissues and normal liver tissues,and performed immunohistochemical staining of TIPE1.The results showed that the expression of TIPE1 in the liver of patients with NAFLD was significantly lower than that of normal liver.This result further supported the hypothesis about the protective effect of TIPE1 on hepatocyte steatosis 3.TIPE1 regulates lipid metabolism through binding with fatty acidFatty acid sensors play an important role in the regulation of lipid metabolism.The crystal structures analysis showed that TIPE,TIPE2 and TIPE3 all contain central hydrophobic cavities formed by the al-a6 helix,which confer them the ability to bind with phospholipid such as PIP2 and PIP3 and then to regulate phospholipid signals.The PyMOL predicted that TIPE1 also has hydrophobic cavities,which are enough to contain fatty acids such as OA and PA.Therefore,it was speculated that TIPE1 is a potential fatty acid sensor that can sense fatty acids and regulate lipid metabolism.3.1 TIPE1 selectively binds with fatty acidsTo determine whether TIPE1 can bind with fatty acids,SPR experiment was performed.The purified TIPE1 protein was coated on a Biocore specific CM5 chip,and the fatty acids,including oleic acid(OA),linolenic acid(LA),arachidonic acid(AA),docosahexaenoic acid(DHA)and stearic acid(SA),were diluted to gradient concentration for mobile phase to detect their binding with TIPE1.The results showed that TIPE1 specifically bound with OA,LA,AA and DHA,but not SA,which confirmed that TIPE1 could selectively bind with fatty acids.3.2 Regulation of lipid metabolism of TIPE1 depends on its fatty acid binding ability3.2.1 Fatty acids can enhance the regulation on lipid metabolism of TIPE1In order to further clarify the role of fatty acid binding capacity in the regulation of hepatocyte lipid metabolism by TIPE1,we used fatty acid-free culture medium prepared with carbon adsorption serum,as the control group,and the experimental group was further stimulated with sodium oleate.HUH7 cells,transfected with siTIPE1 and siNC,were cultured with fatty acid-free culture medium or further stimulated with sodium oleate respectively.Western blot results showed that the protein levels of FASN,SREBP1,CD36 and PPAR ? increased more significantly in siTIPE1 group with sodium oleate stimulation.Consistantly,sodium oleate stimulation can also enhance the inhibitory effect of TIPE1 overexpression on the level of lipid metabolism related proteins.The results indicated that fatty acids could affect the regulation of hepatocyte lipid metabolism by TIPE1 to a certain extent3.2.2 Mutations in the candidate fatty acid binding region can weaken the regulatory effect of TIPE1 on lipid metabolism in hepatocytesTo further confirm whether the regulation of hepatocyte lipid metabolism by TIPE1 depends on its fatty acid binding ability,we constructed TIPE1 mutant plasmids based on the predicted protein structure of TIPE1 by PyMOL software,which either change the steric resistance(L42W)of hydrophobic cavity or the conserved positively charged amino acids(R75/91Q,K15/16/20/24Q,K58/61/65Q)in the hydrophobic cavity respectively to alter the binding ability of TIPE1 with fatty acids.HUH7 cells were transfected with wild-type TIPE1,four mutants of TIPE1 or pcDNA3.0 control plasmids respectively,and stimulated with sodium oleate.ORO staining showed that compared with the control group,lipid deposition was reduced in wild type TIPE1 group,while no significant changes were observed in cells transfected with four TIPE1 mutants.Western blot results showed that compared with the control group,the protein levels of FASN,SREBP1 and PPAR ? were decreased in wild type TIPE1 transfected cells,while no significant changes were observed in those cells transfected with four TIPE1mutants.These results indicats that the regulation on lipid metabolism by TIPE1 at least partially depends on fatty acids sensing4.mTOR pathway is involved in TIPE1 regulation on lipid metabolism in hepatocytesIt has been reported that mTOR pathway plays an important role in regulation of cellular metabolism.mTOR forms two distinct protein complexes with different regulatory mechanisms and functions,mTORCl and mTORC2.Among them,mTORC1 can sense growth factor,amino acid,energy level,oxygen and so on to regulate lipid synthesis,protein synthesis,autophagy,lysosomal and mitochondrial biogenesis.TIPE family has also been reported to regulate mTOR pathway.Does TIPE1 also participate in the regulation of lipid metabolism through mTORC1 pathway?4.1 TIPE1 can negatively regulate the expression and activation of mTOR complexFirst,we examined the effect of TIPE1 on the expression and activation of mTOR complex in hepatocytes both in vitro and in vivo.The protein level of mTOR complex was detected after transfection of siTIPE1 or siNC in HUH7 cells.The results showed that the levels of both phosphorylated proteins(p-mTOR,p-RAPTOR,p-RICTOR),total proteins(mTOR,G?L,RAPTOR,RICTOR)and downstream p-AKT and p-S6K in siTIPE1 group were significantly increased.In NAFLD mouse model,the protein levels of p-mTOR,p-RAPTOR,mTOR and RAPTOR were significantly decreased in the mTIPE1 group.These results suggest that TIPE 1 can negatively regulate the expression and activation of mTOR complex.4.2 Regulation of mTOR complex by TIPE1 depends on its fatty acid binding activityThe previous results showed that TIPE1 regulation on lipid metabolism of hepatocytes depends on its fatty acid binding activity.Thus,we further verify whether the fatty acid-binding activity also be critical for TIPE1 regulation on mTOR pathway HUH7 cells,transfected with wild-type TIPE1,four mutants of TIPE1 or pcDNA3.0 plasmids respectively,were stimulated by sodium oleate.Results of western blot showed that wild-type TIPE1 transfection decreased protein levels of mTOR and G?L,the four mutants lost this effect.These results indicate that the regulation of mTOR complex by TIPE1 also depends on its fatty acid binding activity.4.3 mTOR pathway participates in the regulation of hepatocyte lipid metabolism by TIPE1To further clarify whether mTOR complex is involved in the regulation on hepatocyte lipid metabolism by TIPE1,HUH7 cells transfected with siTIPE1 or siNC were treated with rapamycin and then stimulated with sodium oleate.ORO staining showed that lipid deposition was significantly increased in siTIPE1 group,while after rapamycin treatment,lipid deposition was weakened and the difference between siTIPEl and siNC groups disappeared.Western blot analysis also showed that rapamycin treatment almost abrogated the effect of TIPE1 on expression of lipid metabolism related proteins in hepatocytes.Similar results were obtained in HUH7 transfected with pcDNA3.0-TIPE1-HA and pcDNA3.0.Thus,these preliminarily suggest that mTOR pathway is involved in TIPE1 regulation on hepatic lipid metabolism4.4 mTORCl pathway is critical for TIPE1 regulation on hepatocyte lipid metabolismTo determine whether mTORC1 or mTORC2 are involved in the regulation of hepatocyte lipid metabolism by TIPE1,we used siRAPTOR or siRICTOR to destroy mTORC1 or mTORC2 complexes respectively.First,siRAPTOR or siRICTOR were transfected into HUH7 cells,western blot results showed that protein levels of FASN,SREBP1,PPAR ? and CD36 were decreased in siRAPTOR group,while there was no significant change in siRICTOR transfected group,suggesting that mTORC1 plays an important role in hepatocyte lipid metabolism.Then,siRAPTOR and siTIPE1 were co-transferred into HUH7 cells.ORO staining showed that lipid deposition in siTIPE1 group was significantly increased,while the co-transfection of siRAPTOR alleviated the effect of siTIPE1.Western blot results showed that protein levels of FASN,SREBP1 and PPAR ? increased in siTIPE1 group,while co-transfection of siRAPTOR abrogated this effect.These results indicate that mTORC1 pathway is involved in the TIPE1 regulation on hepatocyte lipid metabolism5.TIPE1 can bind with mTORCl complex and promote its degradation through proteasome pathwayIt was found that TIPE1 could regulate the expression and activation of mTORC1 complex,and then inhibit the steatosis of hepatocytes.Then,we further explore the molecular mechanism of the regulation of mTORC1 by TIPE1.5.1 TIPE1 does not affect the transcription of mTORCl complexThe mRNA level of mTORCl complex was detected after transfection of siTIPE1 or siNC into HUH7 cells.RT-qPCR results showed that there was no significant change of mRNA expression of mTOR,G?L,and RAPTOR in siTIPE1 group.It is suggested that TIPE1 does not affect the transcription of mTORC1 complex5.2 TIPE1 promotes the degradation of mTORC1 complex through proteasome pathway5.2.1 TIPE1 promotes the degradation of mTORC1 complexHUH7 cells,transfected with pcDNA3.0-TIPE1-HA or pcDNA3.0 plasmids,were treated with actinomycin(CHX),a protein synthesis inhibitor,for 0h,12h,24h and 36h and the expression of mTORC1 was detected.Western blot results showed that the half-life period of mTOR,G?L and RAPTOR in TIPE1 group was significantly accelerated,suggesting that TIPE1 might promote the degradation of mTORC1 complex.Autophagy system and proteasome system are two main pathways of protein degradation in eukaryotic cells.Thus,we detected their potential roles in TIPE1 promotion on the degradation of mTORC1 complex5.2.2 TIPE1 does not regulate mTORC1 complex expression through autophagyThe expression of autophagy related proteins were detected after transfection of siTIPE1 or siNC in HUH7 cells.The results showed that there were no significant changes in P62 and LC3B between two groups,suggesting that TIPE1 might not affect the autophagy pathway.Then,HUH7 cells,transfected with siTIPE1 or siNC,were treated with actinomycin(CHX)and chloroquine(CQ).Results of western blot showed that protein levels of mTOR,G?L and RAPTOR were not accumulated after CQ treatment and CQ treatment did not affect the up-regulation of of mTORC1 complex expression by TIPE1 interference,suggesting that autophagy pathway is not involved in the accelerated degradation of mTORC1 complex by TIPE1.5.2.3 TIPE1 promotes the degradation of mTOR complex through proteasome pathwayHUH7 cells,transfected with siTIPE1 and siNC,were treated with actinomycetin(CHX),MG132 or DMSO.Results of western blot showed that protein levels of mTOR,G?L,RAPTOR were significantly accumulated after MG132 treatment,and the difference between the two groups disappeared,suggesting that TIPE1 might promote the degradation of mTORC1 complex by regulating proteasome pathway.Meanwhile,HEK-293 cells,transfected with Ub-HA,and pcDNA3.0-TIPE1-HA or pcDNA3.0 plasmids,were treated with CHX and MG132.CO-IP assay was used to detect the ubiquitination level of RAPTOR.Results of western blot showed that TIPE1 significantly increased the ubiquitination of RAPTOR.These results suggest that TIPE1 may promote the degradation of RAPTOR by the proteasome pathway5.3 TIPE1 interactes with mTORCl complexIt has been reported that TIPE1 can interact with a variety of molecules to regulate signal transduction and affect cellular biological functions.Therefore,we performed immunocoprecipitation assay to detect the interaction between TIPE1 and mTORCl complex.HEK-293 cells were transfected with pcDNA3.0-TIPE1-HA and pcDNA3.0 plasmids.CO-IP result showed that TIPE1 interacted with mTORCl complex,including mTOR,G?L and RAPTOR.5.4 Mutations in the potential fatty acid binding region weaken the interaction of TIPE1 with mTORCl complexPrevious results showed that the down-regulation of mTORC1 complex by TIPE1 depends on its fatty acid binding ability.Thus we further detected the interaction of four mutant TIPE1 with mTORCl complex.HEK-293 cells were transfected with wild-type TIPE1 or mutants of TIPE1 plasmids and CO-IP assay was perfomed.The results showed that compared to wild-type TIPE1 group,these mutants showed the weakened interaction with mTOR,G?L and RAPTOR,except for R75/91Q.It is suggested that the interaction of TIPE1 with mTORC1 complex also partially depends on its fatty acid binding activityConclusions and significances:1.Based on the previous work in our laboratory,it was further confirmed that TIPE1 could negatively regulate lipid metabolism of hepatocytes and improve NAFLD2.It was firstly demonstrated that TIPE1 has potential fatty acid binding capacity,and regulate lipid metabolism of hepatocytes by sensing fatty acids3.It was firstly revealed that TIPE1 could promote the degradation of mTORC1 complex through proteasome pathway,and then negatively regulating lipid metabolism of hepatocytes.Innovations and significances:In our study,we first revealed that TIPE1 is a fatty-acid sensor.By sensing fatty acids,TIPE1 regulates the expression and activation of mTORC1,then inhibits the lipid deposition of hepatocytes and improves the course of NAFLD.It would be helpful to elucidate the pathogenesis of NAFLD and provide a new way for clinical treatment. |
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