TMPA Ameliorates Lipid Accumulation By Disturbing The Combination Of LKB1 With Nur77 And Activating The AMPK Pathway In Hepatocytes

Background:Metabolic associated fatty liver disease(MAFLD)is one of the most common chronic disease in the world.It’s well-known as nonalcoholic fatty liver disease(NAFLD)before.Although it do not threaten the length of life,it will damage other organs and the balance of metabolism of patients and then increase the susceptibility of other disease.With the development of MAFLD,it will turn into end-stage liver disease,but there are no effective measures to treat it.Several studies has proved that AMP-activated protein kinase(AMPK)pathway has widely been considered a key factor in energy metabolism.Abnormal activity of AMPK is a key factor of MAFLD pathogenesis.The activity of AMPK is influenced by Nerve growth factor inducible B protein(Nur77)and Liver kinase B1(LKB1).AMPK is regarded as a target to prevent and treat MAFLD.Ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate(TMPA)is known as a novel AMPK agonist,having the ability of disturb the combination of Nur77 and LKB1,following activating AMPK.However,Whether TMPA has the function in the treatment of MAFLD is unclear.Objective:Investigating the expression of Nur77/LKB1/AMPK pathway related proteins in MAFLD and explore the molecular mechanism of TMPA in preventing MAFLD.Method:Establishing the model of MAFLD and detecting Nur77/LKB1/AMPK pathway proteins expression in MAFLD mice and Normal mice.Studying the hepatocytes proteins change in Nur77/LKB1/AMPK pathway under Free fatty acids(FFAs)stimulation in vitro.Further investigating the molecular mechanism of TMPA in preventing hepatocyte steatosis.Result:1.Comparing with normal fed mice,mice fed high fat forage for 8 weeks had significant lipid deposition in liver.The proteins expression of Nur77,3-hydroxy-3methylglutaryl coenzyme A reductase(HMGCR),Phosphorylated Acetyl-CoA carboxylase(p-ACC)and Carnitine palmitoyltransferase 1(CPT1A)in MAFLD mice liver were higher than normal diet mice liver,the level of AMPKa and LKB1 shown lower in MAFLD group besides the total ACC had no significant difference in both forms in vivo.2.Comparing with Control group,FFAs stimulation induced Nur77 decreased,p-AMPKα(Thr172)and CPT1A increased.LKB1,AMPK,HMGCR,Sterol regulatory element binding protein 1(SREBP1)and ACC were not changed in vitro.3.Hepatocytes had the significant lipid deposition and augmented the expression of Adipose differentiation-related protein(ADFP)compared with Control group.TMPA ameliorated the lipid deposition in hepatocytes under the influence of FFAs.Compared with FFAs group,TMPA group increased the expression of p-LKB1(Ser428),p-AMPKα(Thr172),p-ACC(S79)and CPT1A.Nur77,LKB1,AMPKα,ACC,HMGCR and SREBP1 do not had significant change.4.Separating cytoplasm and nucleus in hepatocytes and found that in TMPA group,the content of LKB1 in cytosol was higher than nucleus and p-AMPKα(Thr172)effectively increased in cytoplasm but not detected in nucleus compared with FFAs group.Immunofluorescent staining of LKB1 also indicated that the fluorescence intensity of LKB1 in cytoplasm was stronger than in nucleus.Conclusion:1.Nur77/LKB1/AMPK pathway proteins expression are significantly changed in MAFLD mice liver and FFAs induced hepatocytes.2.TMPA ameliorates lipid deposition in hepatocytes by activating Nur77/LKB1/AMPK pathway.3.TMPA inhibits hepatocyte steatosis by inhibiting Nur77 binding to LKB1 in the nucleus and inducing LKB1 shuttling from nucleus to cytoplasm and phosphorylation to activate AMPKα.