The Role And Mechanism Of MPST In Nonalcoholic Fatty Liver Disease

Backgounds and Aims:Accumulation of fatty acids in hepatocytes leads to nonalcoholic fatty liver disease(NAFLD)via its direct lipotoxicity,in which the potential mediators and involved pathways at the molecular level remain unclear.The cystathionine-?-synthase(CBS)and cystathionine-y-lyase(CSE)system,which contributes to endogenous hydrogen sulfide(H2S)biosynthesis,may be regulated by fatty acids and has been actively investigated in the pathogenesis of NAFLD.This study aimed to investigate the role of 3-mercaptopyruvate sulfurtransferase(MPST),another H2S-generating enzyme,in the development of NAFLD.Methods and Materials:Hepatic MPST expression was evaluated in NAFLD cell and mouse models induced by free fatty acids(FFA)stimulation for 24h and high fat diet(HFD)-feeding for 8 weeks,respectively.We measured the levels of MPST protein expression in liver biopsies from NAFLD patients by using immunohistochemistry staining.To investigate the role of MPST in diet-induced fatty liver disease,we applied a recombinant adenovirus-mediated RNA interference approach to inhibit hepatic MPST expression in HFD-fed mice.Heterozygous MPST-deficient(MPST+/-)mice and their wild type controls(MPST+/+),which were generated through frameshift mutation by TAL-effector nuclease(TALEN)system on a C57BL/6 background,were fed with HFD for 8 weeks.To investigate the effect of MPST regulation in hepatocyte steatosis in vitro,MPST expression was knocked down by MPST siRNA and overexpressed by transfecting with pIRES2-GFP-MPST plasmid in the FFA-stimulated L02 cells.H&E and Oil Red O staining were used to evaluate hepatic steatosis,combined with the quantitative tests including triglyceride(TG),total cholesterol(TC)contents and FFA levels.A variety of molecular approaches,containing the administration of NaHS(H2S donor),determination of H2S production,MDA and SOD activity,in addition to measurement of SREBP-1 pathway as well as JNK phosphorylation and Co-Immunoprecipitation(CoIP)were used to investigate the the effects and underlying mechanism of MPST regulation on hepatic steatosis and oxidative stress in vivo and in vitro.Results:FFA treatment of human hepatocytes induced significant upregulation of MPST,which was partially dependent on NF-?B/p65.Hepatic protein expression of MPST was markedly increased in HFD-induced mouse model of NAFLD and in patients with NAFLD as well.Partial knockdown of MPST via the adenovirus delivery of MPST shRNA or heterozygous deletion of the MPST gene significantly ameliorated hepatic steatosis in HFD-fed mice.Consistently,siRNA-mediated partial knockdown of MPST in L02 cells reduced FFA-induced fat accumulation.Intriguingly,partial knockdown of MPST significantly enhanced rather than decreased H2S production;whereas MPST over-expression markedly inhibited H2S production.CoIP experiments showed that MPST directly interacted with and negatively regulated CSE,a major source of H2S production in the liver.Mechanistically,H2S mediated the regulatory effect of MPST on hepatic steatosis by suppressing the SREBP-1 pathway,JNK phosphorylation,and hepatic oxidative stress.Conclusions:Fatty acids upregulate hepatic expression of MPST and subsequently inhibit the CSE/H2S pathway,leading to NAFLD.MPST may be a potential therapeutic target for NAFLD.