Regulation Of TIPE2 Expression In Hepatocytes And Its Role In Reducing Fasting-induced Hepatic Steatosis

Research purposeTumor necrosis factor alpha-induced protein 8-like 2(TIPE2),a negative regulator of immunity,is preferentially expressed in immune cells.It has been found that TIPE2 is downregulated in hematopoietic cells of patients with autoimmune diseases or infectious diseases.In addition,TIPE2 is also downregulated in some malignant tumors such as primary hepatocellular carcinoma.At present,the regulation mechanisms of TIPE2 expression mainly focus on mRNA degradation or protein ubiquitination degradation.The transcriptional regulation for TIPE2 expression is largely unclear.Liver,as the main metabolic organ,regulates the metabolism of glucose,lipids,proteins and vitamins in different nutritional states.Feeding and fasting are two important nutritional conditions that affect liver metabolism.In the fasted state,triglycerides in adipose tissue are broken down into fatty acids,which are released into the bloodstream to provide nutrients for the liver.However,prolonged fasting will cause lipid metabolism disorders in the liver.Fatty acid accumulation in the liver may increase the synthesis of triglycerides.Meanwhile,the inhibition of lipid autophagy and the dysfunction of lipid transport may increase the risk of hepatic steatosis.At present,studies on TIPE2 mainly focus on its immunosuppressive or anti-tumor functions.It remains unclear whether and how TIPE2 is expressed in hepatocytes under different nutritional conditions.It is unknown how TIPE2 functions in hepatic lipid metabolism.Our research mainly focuses on two points:1)TIPE2 expression in hepatocytes under starvation condition and its regulatory mechanism;2)The effects of TIPE2 in fasting-induced hepatic steatosis and its regulatory mechanism.Research method1)TIPE2 expression were detected in AML 12 cells or HepG2 cells that were starved by serum-free M199 medium.2)The transcription factor of TIPE2 was predicted by PROMO and JARSPAR.3)AML 12 cells were treated by siPPAR-a.HepG2 cells and 293T cells were overexpressed PPAR-a.HepG2 cells overexpressing PPAR-a were treated with MK886.The changes of TIPE2 expression were detected in the above cells.4)PPAR-a was overexpressed in HepG2 cells and 293T cells.And the effect of PPAR-a on the transcriptional activity of TIPE2 promoter was examined.5)CUT&RUN experiment was used to analyze the putative PPAR-α binding sites in human or mouse TIPE2 promoter region.6)MK886 was administrated in wild-type mice to block PPAR-α activation,the mRNA and protein levels were detected in liver tissue.7)TIPE2-LKO and control mice were fasted for 24 hours.The effect of TIPE2 on hepatic lipid metabolism was explored.The gene expression related to fatty acid oxidation,de novo fatty acid synthesis and triglyceride synthesis and related signaling pathways were analyzed in liver tissue by western blot and quantitative PCR.8)Primary hepatocytes from TIPE2-KO and wild-type mice were starved to explore the regulatory pathways of TIPE2 in hepatic lipid metabolism.Research results1)The mRNA and protein levels of TIPE2 were up-regulated in starved AML 12 cells and HepG2 cells.2)PPAR-α was predicted as a transcription factor of TIPE2 by PROMO and JARSPAR.3)PPAR-α overexpression induced the upregulation of TIPE2 expression in HepG2 cells and 293T cells,whereas PPAR-α knockdown decreased TIPE2 expression in AML12 cells.MK886 inhibited TIPE2 upregulation induced by PPAR-α overexpression in HepG2 cells.4)PPAR-α overexpression increased the luciferase activity of TIPE2 reporter gene in HepG2 cells and 293T cells.5)CUT&RUN assay showed four PPREs in human TIPE2 promoter and three PPREs in mouse TIPE2 promoter.6)MK886 that blocked PPAR-α activation inhibited the mRNA and protein expression of TIPE2 in liver tissue eof the wild-type mice.7)After fasting,TIPE2-LKO mice showed more obvious hepatic steatosis than control mice.And the content of hepatic triglyceride was also higher in TIPE2-LKO mice than control mice.TIPE2-LKO mice expressed higher levels of nSREBPlc,FASN,SCD1 proteins and Fasn,Scd1,Dgat1,Dgat2 mRNA than control mice.8)Primary hepatocytes from TIPE2-KO mice express higher levels of p-mTOR and p-S6 than those from wild-type mice.ERK inhibitor U0126 blocked the increase of p-mTOR and p-S6 in primary hepatocytes from TIPE2-KO mice,suggesting that TIPE2 may inhibit mTORC1 pathway to reduce hepatic steatosis via suppressing ERK.Conclusion and innovation1)In starved hepatocytes TIPE2 is upregulated by transcription factor PPAR-α.2)TIPE2 plays an important role in reducing fasting-induced hepatic steatosis through inhibiting the mTORC1 pathway.Limitation1)This study demonstrates TIPE2 inhibits mTORC1 to reduce fasting-induced hepatic steatosis,while in vivo validation in animal models remains to be studied;and other pathways possibly involved in this process remain to be determined.2)The potential clinical application of this study remains to be further verified through clinical data and experiments.