TIPE1 Is Involved In NAFLD By Sensing Fatty Acid And Regulat-ing MTOR Signalling Pathway

BackgroundIn recent years,the incidence of nonalcoholic fatty liver(NAFLD)has increased year by year with the increased number of obese people and patients with metabolic syndrome.The incidence of NAFLD is about 20%-30%in West-em countries,while in Asia it is about 5-18%.The incidence of NAFLD in our country is 15%?40%.NAFLD is the most common chronic liver disease.In the past ten years,the incidence of NAFLD related hepatocellular carcinoma(HCC)has increased by 3?10 times,which brings huge social and medical burdens.It is predict-ed that by 2030,the incidence of NAFLD will increase to 33.5%and becomes the main cause of end-stage liver disease and liver transplantation.Therefore,to probe into the molecular mechanisms involved in NAFLD pathogenesis and to find out novel diagnostic and therapeutic targets for NAFLD,and to achieve dynamic monitoring and effective intervention in the early stage of patients is an important clinical and basic scientific problem.According to the course of disease,NAFLD can be divided into non-alcoholic fatty liver disease(NAFL),nonalcoholic steatohepatitis(NASH),cirrhosis and hepato cellular carcinoma(HCC).Although the pathogenesis of NAFLD is extremely com-plicated,the accumulation of triglyceride(TG)in hepatocytes is theinitial step of the whole process.Accumulated lipids are peroxided and generate toxic products,and lead to endoplasmic reticulum oxidative stress,which further activates inflammatory signals and aggravates the progress of the disease.Previous studies have shown that the imbalance of Fatty Acid(FA)metabolic pathways,namely the imbalance of fatty acid uptake,synthesis and utilization,is the intrinsic mechanism of TG accumulation in hepatocytes.Many metabolic enzymes and related transcriptional factors and regu-latory molecules are known to play an important role in maintaining the homeostasis of liver cell lipid metabolism.Among them,the proteins which can bind with and sense different fatty acids(fatty acid binding protein FABPs,peroxisome proliferator activated receptor family PPARs,e.g.)is highly concerned and deeply studied.The abnormal expression of these molecules may lead to the disorder of fatty acid metabo-lism in the liver cells,which is one of the important mechanisms to promote the de-velopment of NAFLD.The corresponding agonists or inhibitors based on fatty acid binding proteins have been carried out a clinical trial for the treatment of NAFLD[4].Therefore,to search the new fatty acid sensors and to study their regulation on fatty acid metabolism would shed new light on elucidating the mechanism of NAFLD and finding new therapeutic targets.Tumor necrosis factor alpha-induced protein 8(TNF alpha-induced protein 8,TIPE)family is a newly identified protein family with the potential lipid binding ac-tivity.The TIPE family includes TIPE,TIPE1,TIPE2,and TIPE3 proteins,which share high homology in the amino acid sequence and protein structure.The crystal structure analysis shows that TIPE,TIPE2 and TIPE3 proteins contain a central hy-drophobic cavity composed of multiple alpha helix,which can accommodate lipid molecules.Studies also showed that TIPE,TIPE2 and TIPE3 could regulate the downstream signaling pathway by mediating the translocation of lipid molecules(phosphatidylinositoltwo phosphate PIP2,phosphatidylinositol three phosphate PIP3)or forming protein lipid complex with phosphatidylethanolamine PE,which then af-fect cell growth,death chemotaxis,and other important biological processes.These studies strongly suggest that TIPE family can bind to different lipid molecules by the unique protein structure,which is important for their biological functions.TIPE1,a member of the TIPE family,was first discovered in 2008 as a regulator for apoptosis and necrosis.Under the condition of oxidative stress,TIPE1 can bind to FBXW5 to inhibit proteasome degradation of TSC2 protein and to enhance the au-tophagy of dopamine neurons.In 2015,our research group found that TIPE1 showed high abundance expression in adult liver,but significantly downregulated in liver cancer tissues,and it can promote the death of hepatocellular carcinoma cells by in-hibiting the activity of Rac1.The cDNA microarray data showed that the expression of TIPE1 in the heart tissues of db/db mice was significantly lower than that of db/+mice.It is suggested that TIPE1 may be involved in the development of metabolic diseases.According to the research progress from ours and other groups,we speculate that TIPE1 may be involved in the development of metabolic diseases of the liver.In this study,We explore the roles of TIPE1 in the development of NAFLD and its mo-lecular mechanism.ObjectivesIn this project,we explore the role of TIPE1 in the development of NAF LD and its molecular mechanism.The specific purpose is described as followi-ngs:1.To detect the changes in TIPE1 expression in hepatocyte steatosis;2.To clarify the roles of TIPE1 in hepatocyte steatosis;3.To investigate the molecular mechanism of TIPE1 regulating hepatocyte lipid metabolism and participating in hepatic steatosis.Methods and results1.TIPE1 expression is downregulated in hepatic steatosis1.1 Hepatocyte TIPE1 expression is significantly downregulated in NAFLD mouse model.We first established a mouse NAFLD model with high fat diet(High-fat diet,HFD).Western blot and qPCR showed that the expression of TIPE1 protein and mRNA was significantly lower than that of the normal diet fed mice with.The im-munofluorescence staining further confirmed the decreased expression of TIPE1 in the hepatocytes of the HFD fed mice.Methionine-Choline-Deficient(MCD)diet inducing NAFLD mouse model further verified the above results,All these findings suggest that the expression of TIPE1 is downregulated in the NAFLD model.1.2 Decreased TIPE1 expression in hepatocyte steatosis modelHuman liver cell line Huh7 cells were stimulated with high concentration of un-saturated fatty acid-oleic acid(OA)and saturated fatty acid-palmitic acid(PA)to es-tablish hepatocyte steatosis model in vitro.TIPE1 mRNA expression was detected at different time points.qPCR results showed that the expression of TIPE1 decreased gradually with the time of stimulation,and PA had a stronger inhibitory effect.The results further clarified that the expression of TIPE1 is down-regulated in hepatocyte steatosis,which suggested that TIPE1 may be involved in the development of NAFLD.2.TIPE1 alleviates hepatocyte steatosis2.1 TIPE1 attenuate hepatic steatosis in vitro.2.1.1 TIPE1 mitigates hepatocyte lipid deposition in vitro modelTo investigate the role of TIPE1 in hepatic steatosis,Huh7 cells transfected with TIPE1 siRNA or NC siRNA was stimulated with OA/PA hepatoma cell model.Oil red O staining was performed to detect the lipid droplet deposition and ELISA was used to detect intracellular triglyceride(TG)content.Results showed that TIPE1 knock-down aggravated lipid deposition and TG accumulation.On the contrary,HepG2 cells transfected with pcDNA3.0-TIPE1-HA alleviate the lipid droplet deposition and TG accumulation stimulated with OA.2.1.2 TIPE1 regulates the expression of lipid metabolism related genesTG excessive accumulation in the liver is the main reason for the formation of hepatocyte steatosis.And,the increase of de novo lipid synthesis of fatty acids,the inhibition of beta oxidation and the blocked TG transport are the main mechanisms leading to excessive accumulation of TG in the liver.In order to further verify the role of TIPE1 in hepatic steatosis,Huh7 cells were transfected with NC-siRNA or TIPE1-siRNA and the expression of lipid metabolism related genes were detected by qPCR and WB.Results showed that TIPE1 knockdown upregulated the expression of PPARy,SREBP1,FASN and CD36.With the stimulation of OA/PA,the upregulation of the above genes was more significant.On the contrary,pcDNA3.0-hTIPE1-HA transfection into HepG2 cellsdownregulated the expression of PPAR?,SREBP1 and CD36.These results further confirm that TIPE1 can regulate the expression of lipid metabolism related genes and participate in modulating hepatocyte steatosis.2.2 TIPE1 attenuates hepatic steatosis in vivoIn order to further clarify the role of TIPE1 in hepatic steatosis,liver-specific TIPE1 overexpression or TIPE1 knockout mice was used to establish MCD diet and HFD diet-induced NAFLD models.2.2.1 TIPE1 overexpression mitigates MCD induced hepatic steatosisC57BL/6 mice,hydrodynamically injected with TIPE1 overexpression plasmid pcTIPE1 and control plasmid pcDNA3 at day-1 and day-6,were fed with MCD diet for 2 weeks.Body weight,lipid content in serum and liver,lipid droplet deposition in liver and the expression of proinflammatory genes was detected.Results showed that TIPE1 overexpression alleviated the body weight decrease,liver lipid accumulation,and serum lipid depletion.Coincidently,TIPE1 overexpressed mouse liver tissues weakened the expression of TNF-a and IL-1?2.2.2 Hepatocyte specific TIPE1 overexpression reduces the lipid metabolism disorder induced by HFDWe construct a hepatocyte specific AAV vectors containing TIPE1 gene and fire-fly luciferase AAV-TBG-mTIPE1-Luc.The C57BL/6 mice were hydrodynamically injected with AAV-TBG-mTIPE1-Luc virus or control virus by the tail vein and then fed with HFD diet.Optical in vivo imaging showed that AAV-TBG-TIPE1-Luc virus and control virus was specifically expressed in the liver.Importantly,TIPE1 overex-pression weakened the weight gain of mice and downregulates the serum lipid level(the experiment is still in progress).2.2.3 Hepatocyte specific TIPE1 knockout aggravates lipid metabolic disorders induced by HFDWe used the Cre-loxp system to construct hepatocyte specific TIPE1 knockout mice(Alb-cre+;TIPE1flox/flox)and control mice(Alb-cre-;TIPE1flox/flox).RT-PCR and Western blot showed a significant decrease of TIPE1 expression in the liver tissues of mice.Liver TIPE1 KO mice or wild-type mice were fed with HFD diet.ELISA showed that liver TIPE1 knockout downregulated serum TG and ALT level(the ex-periment was still in progress).3.The molecular mechanism of TIPE1 involvement in hepatic steato-sisThe crystal structure of TIPE,TIPE2 and TIPE3 proteins showed a central hy-drophobic cavity structure surrounded by multiple alpha helices.It is suitable for,which could accommodate lipid molecules.Further experiments have proved that TIPE2 and TIPE3 can be combined with lipid second messenger molecules(PIP2 and PIP3),then mediate the transfer of PIP2/PIP3 to the cell membrane and activate the downstream signaling pathway PI3K-AKT and MEK-ERK pathway,which then reg-ulate cytoskeleton remolding,cell proliferation and cell migration.Similarly,TIPE molecules formed a protein-lipid complex with phosphatidyl ethanolamine PE,acti-vated the mTOR pathway,and then inhibited autophagy.These findings suggested that TIPE1 might have the lipid-binding potential.3.1 TIPE1 binds with fatty acidsThe analysis of amino acid sequence showed that TIPE 1 was highly homologous to other family members,and the protein structure share high similarity.The software prediction showed that the central cavity structure of TIPE 1 protein could hold the OA molecule.Surface plasmon resonance(SPR)experiments suggest that TIPE1 mole-cules have high affinity with OA.These results suggest that TIPE1 has the potential of lipid binding,which can bind fatty acid molecules with high affinity.3.2 TIPE1 affects lipid metabolism by regulating the mTORC2 complexThe mTOR complex,including mTORC1 and mTORC2,plays an important roles in lipid metabolism by receiving signals from external amino acids and growth factors.3.2.1 TIPE1 interacts with the mTORC2 complexIn order to study the molecular mechanism of TIPE1 regulating lipid metabolism,we transfected pcDNA3.0-TIPEl-HA plasmid and control pcDNA3.0 into HEK293 cells,and HA antibody was used to carry out CO-IP test.It was found that TIPE1 bound with mTOR,Rictor and G?L protein,but had no interaction with Raptor.Ac-cordingly,mTOR and Rictor antibody were used for reverse immunoprecipitation,which further confirmed the interaction between TIPE1 and mTORC2 complex.In addition,SPR experiments further clarified the direct interaction between TIPE1 and Rictor protein.3.2.2 OA enhances the interaction between TIPE1 and RictorTo further study the effect of OA on the relationship between TIPE1 and mTORC2 complex,we carry out the sequential combination experiment by SPR assay.We found that the pre-exposure of Chip-bound TIPE1 protein to OA solution en-hanced the binding affinity of TIPE1 protein with Rictor.3.2.3 TIPE1 regulates the expression and activation of mTORC2 complex and the downstream signaling moleculesIn order to further explain the molecular mechanism of TIPE1 involved inregu-lating lipid metabolism,we detected the effect of TIPE1 on the expression and acti-vation of mTORC2 and downstream signaling molecules.TIPE1 knockdown upregu-lated the expression of mTOR and Rictor in unstimulated or OA/PA stimulated Huh7 cells.In addition,TIPE1 knockdown enhanced the phosphorylation of SGK1 and AKT,which are important downstream molecules.But therewas no significant differ-ence in the expression of raptor protein.3.2.4 TIPE1 involves in hepatic steatosis by regulating mTORC2 pathwayIn order to further explore the role of mTORC2 pathway in the regulation of he-patic steatosis by TIPE1,we used SGK1 inhibitor(GSK650394)to pretreat hepato-cytes for rescue experiments.Huh,cells,transfected with TIPE1 siRNA or NC siR-NA and pretreated with GSK650394,were stimulated with OA for another 24h.Neu-tral lipid was stained with fluorescent dye BODIPY and then observed under the flu-orescence microscope and detected by flow cytometry.The results showed that TIPE1 interference significantly aggravated lipid deposition of hepatocyte.GSK650394 pre-treatment almost abrogated the enhancement of TIPE1 knockdown on lipid droplet deposition of Huh7 cells.Western Blot also disclosed that GSK650394 neutralized the upregulation of PPAR-r and FASN.These results preliminarily suggested that mTORC2 pathway involved in TIPE1 regulation on hepatic steatosis.ConclusionIn the present project,we preliminarily clarify the molecular mechanism of TIPE1 regulation in the development of non-alcoholic fatty liver disea'se and make the following conclusions:1.The expression of TIPE1 in the hepatic steatosis model is significantly downregulated.2.TIPE1 alleviates hepatocyte steatosis.3.TIPE1 can bind with fatty acids,regulate the expression and activation mTORC2 complex and downstreatm signaling molecules.Innovations and significancesIn this study,we firstly detected the changes in the expression of TIPE1 in hepatic steatosis,preliminarily studied the role of TIPE1 in hepatic steatosis,and proved that mTORC2 signaling pathway involve in TIPE1 regulation on he patic steatosis.It might provide a new potential target for the clinical treatment of nonalcoholic fatty liver diseases.