GPR180 Knockdown Ameliorates Non-Alcoholic Fatty Liver Diseases And Its Underlying Mechanism

Background:Nonalcoholic fatty liver disease has a high prevalence,great harm and heavy medical burden in the world.It is difficult for existing drugs to have significant curative effect.Therefore,the development of new drug targets will have positive clinical significance.GPR180,a member of G protein coupled receptor family,is also known as intimal thickness related receptor(ITR).It has been originally reported to be critical for the growth of smooth muscle cells.Current understanding on GPR180 is limited.In addition to vascular remodeling,GPR180 may contribute to the pathogenesis of tumors.Recent genome wide association studies(GWAS)also found that single nucleotide polymorphism(SNP)of GPR180 may be associated with hypertriglyceridemia.These GWAS analyses suggest that GPR180 may contribute to the regulation of lipid metabolism.The physiological function of GPR180 in lipid metabolism,its underlying mechanism and endogenous ligand remain largely unknown.Method:Mice fed normal chow diet and high-fat diet,which can induce nonalcoholic fatty liver disease were randomly divided into control and treatment groups.Control group was injected via tail vein with AAV virus containing scramble shRNA,while the treatment group received shRNA-GPR180 AAV to knockdown hepatic GPR180.After 12.5 weeks,the effects of GPR180 deficiency on body metabolism was assessed.In vitro,the effect of GPR180 on triglyceride and cholesterol synthesis was verified by overexpression or knockdown of GPR180 using cultured hepa1-6 cells.The molecular signal pathway underlying the action of GPR180 was explored.Potential endogenous ligands for GPR180 were explored using mass spectrometric analysis of proteins pull-downed by immunocoprecipitation and bioinformatic prediction.Result:1.Effect of GPR180 knockdown on lipid metabolism in normal diet miceThrough the detection of GPR180 expression level,the liver GPR180 knockdown efficiency of AAV shRNA-GPR180 treatment group in normal diet mice was about 50%compared with the control group.Knockdown of hepatic GPR180 demonstrated no significant effect on hepatic lipid metabolism.Body weight,plasma and liver lipid content were largely unaltered.However,increasing energy metabolism rate,reducing fat mass and enhancing insulin sensitivity were observed.2.Effect of GPR180 knockdown on lipid metabolism in diet-induced obese miceKnockdown of hepatic GPR180 by 45%in animals fed high-fat diet significantly improved the lipid metabolism relevant to the control group.Body weight,subcutaneous and epididymal fat mass,hepatic lipid contents,and lipid synthesis transcription factors Srebpl and Srebp2 were significantly reduced.These alterations were associated with substantial increase in energy metabolic rate and insulin sensitivity.3.Molecular mechanism underlying the metabolic effect of GPR180In Hepa1-6 cells,overexpression of GPR180 significantly increased levels of Srebpl and Srebp2 and their target genes,leading to the increment of triglycerides and cholesterol.On the other hand,knockdown of GPR180 substantially reduced hepatic levels of triglyceride and cholesterol.Double luciferase reporter gene experiments using pcDNA-GPR180 and pGL3-CREB in 293T cells showed a significant reduction in the fluorescence signal,indicating a suppression of cAMP signaling by GPR180.In addition,the phosphorylation level of PKA was timedependently decreased by overexpression of GPR180 in Hepa1-6 cells.These observations indicate that GPR180 is a Gi protein coupled receptor,which inhibits the activity of PKA.4.Identification of potential endogenous ligands for GPR180Mass spectrometric analysis of proteins pull-down by immunoprecipitation and bioinformatic prediction identified Asprosin and Clusterin as the potential endogenous ligands for GPR180.Conclusion:GPR180 knockdown ameliorates hepatic steatosis in high-fat induced obese mice.GPR180 promotes the synthesis of triglycerides and cholesterol by up-regulating the expression of transcription factors Srebpl and Srebp2.GPR180,may function through the Gi protein to inhibit the cAMP-PKA signaling.Asprosin and Clusterin may be the endogenous ligands for GPR180.