Gut Online First, published on September 6, 2017 as 10.1136/gutjnl-2017-313778 Hepatology ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2017-313778 on 6 September 2017. Downloaded from Fatty acids promote fatty liver disease via the dysregulation of 3-mercaptopyruvate sulfurtransferase/hydrogen sulfide pathway Meng Li,1 Chengfu Xu,1 Junping Shi,2 Jiexia Ding,1 Xingyong Wan,1 Dahua Chen,1 Jianguo Gao,1 Chunxiao Li,1 Jie Zhang,1 Yiming Lin,1 Zhenhua Tu,3 Xiaoni Kong,4 Youming Li,1 Chaohui Yu1 ► Additional material is ABSTRACT steatohepatitis (NASH); the latter may further published online only. To view Objective Accumulation of free fatty acids (FFAs) progress to liver cirrhosis and hepatocellular please visit the journal online 3 4 (http:// dx. doi. org/ 10. 1136/ in hepatocytes induces lipotoxicity, leading to non- carcinoma. The number of patients with gutjnl- 2017- 313778). alcoholic fatty liver disease (NAFLD). This study aimed NAFLD is growing at alarming rates world- to investigate the underlying mechanisms by which wide. However, its pathogenesis remains poorly 1Department of Gastroenterology, the First FFA contributes to the pathogenesis of NAFLD via the understood but is of great interest, since current Affiliated Hospital, College of regulation of 3-mercaptopyruvate sulfurtransferase therapeutic options are limited. Medicine, Zhejiang University, (MPST), a key enzyme that regulates endogenous Emerging data indicate that the flux of fatty Hangzhou, Zhejiang, China hydrogen sulfide (H S) biosynthesis. acids through the liver is increased in patients with 2Division of Hepatology, 2 Design Hepatic MPST expression was evaluated in NASH.5 Free fatty acids (FFAs) and their metabo- Hangzhou Normal University Affiliated Hospital, Hangzhou, mice and patients with NAFLD. A variety of molecular lites are important mediators of lipotoxicity via the Zhejiang, China approaches were used to study the effects of MPST induction of lipid overaccumulation, which causes 3 Division of Hepatobiliary and regulation on hepatic steatosis in vivo and in vitro. lipotoxic hepatocellular injury and the progression of Pancreatic Surgery, Department Results In vitro treatment of hepatocytes with NAFLD.6 However, the exact mechanisms by which of Surgery, the First Affiliated Hospital, College of Medicine, FFAs upregulated MPST expression, which was FFAs cause lipotoxicity remain unclear. partially dependent on NF-κB/p65. Hepatic MPST Zhejiang University, Hangzhou, Hydrogen sulfide (H2S), which has been Zhejiang, China expression was markedly increased in high fat diet known for centuries as a toxic gas with the smell 4 Department of Surgery, Renji (HFD)-fed mice and patients with NAFLD. Partial of rotten eggs,7 has been established as the third Hospital, School of Medicine, knockdown of MPST via adenovirus delivery of gaseous signalling molecule following nitric Shanghai Jiao Tong University, http://gut.bmj.com/ Shanghai, China MPST short hairpin RNA or heterozygous deletion oxide and carbon monoxide, participating in a of the Mpst gene significantly ameliorated hepatic wide range of physiological processes, including Correspondence to steatosis in HFD-fed mice. Consistently, inhibition inflammation, apoptosis, vasorelaxation and Youming Li and Professor of MPST also reduced FFA-induced fat accumulation 8–10 neuromodulation. The role of H2S in NAFLD Chaohui Yu, Department of in L02 cells. Intriguingly, inhibition of MPST has been received great attention over the last Gastroenterology, the First significantly enhanced rather than decreased H S Affiliated Hospital, College of 2 two decades. The liver is an important organ in production, whereas MPST overexpression markedly 11 Medicine, Zhejiang University, the regulation of H2S metabolism. Conversely, on September 25, 2021 by guest. Protected copyright. Hangzhou 310003, Zhejiang, inhibited H S production. Co-immunoprecipitation 2 H2S plays critical roles in pathophysiology of China; zlym@ zju. edu. cn, experiments showed that MPST directly interacted liver diseases.12 H S protected against liver zyyyych@ zju. edu. cn 2 with and negatively regulated cystathionine γ-lyase injury induced by ischemia reperfusion and (CSE), a major source of H S production in the 13–15 ML, CX and JS contributed 2 carbon tetrachloride in rodents. Recent equally. liver. Mechanistically, MPST promoted steatosis via studies revealed that the endogenous formation inhibition of CSE/H2S and subsequent upregulation of H S was impaired in high fat diet (HFD)-fed Received 16 January 2017 2 of the sterol regulatory element-binding protein 1c mice and rats with NASH.16 17 Treatment with Revised 14 July 2017 pathway, C-Jun N-terminal kinase phosphorylation Accepted 21 August 2017 sodium hydrosulfide (NaHS), a H2S donor, and hepatic oxidative stress. prevented NASH in rodents by abating oxidative Conclusions FFAs upregulate hepatic expression of stress and suppressing inflammation.17–19 These MPST and subsequently inhibit the CSE/H2S pathway, findings suggest that H2S homeostasis plays an leading to NAFLD. MPST may be a potential therapeutic important role in hepatic lipotoxicity. However, target for NAFLD. how H2S biosynthesis is regulated in NAFLD remains unclear. In mammalian tissues, H2S can be produced INTRODUCTION from cysteine by pyridoxal-5′-phosphate Non-alcoholic fatty liver disease (NAFLD) is (PLP)-dependent enzymes including cysta- the most common liver disorder in Western thionine β-synthase (CBS) and cystathionine To cite: Li M, Xu C, Shi J, et al. Gut Published Online countries, with a prevalence of 20%–30% in γ-lyase (CSE); the former is mainly expressed 1 2 First: [please include Day the general population. The disease encom- in the brain and the latter is expressed in the Month Year]. doi:10.1136/ passes a broad spectrum of clinicopathology, vasculature and liver.20 3-Mercaptopyruvate gutjnl-2017-313778 ranging from simple steatosis to non-alcoholic sulfurtransferase (MPST) is another enzyme Li M, et al. Gut 2017;0:1–12. doi:10.1136/gutjnl-2017-313778 1 Copyright Article author (or their employer) 2017. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Hepatology MATERIALS AND METHODS Significance of this study Human samples Gut: first published as 10.1136/gutjnl-2017-313778 on 6 September 2017. Downloaded from Liver biopsies from liver transplant donors who were healthy What is already known about this subject? adults (n=19) and from patients with NAFLD (n=37) who ► Free fatty acids (FFAs) and their metabolites are important underwent liver biopsies for suspected NAFLD or NASH or mediators of lipotoxicity, leading to the progression of during liver surgeries were randomly selected from the liver non-alcoholic fatty liver disease (NAFLD). biopsy samples recruited at the First Affiliated Hospital, College ► The liver is an important organ in the regulation of hydrogen of Medicine, Zhejiang University; The Affiliated Hospital of sulfide (H2S) metabolism, and H2S homeostasis plays an Hangzhou Normal University; Renji Hospital, School of Medi- important role in hepatic lipotoxicity. cine, Shanghai Jiao Tong University; Hangzhou Sixth People’s ► Reported evidence suggests that the Hospital; and Ningbo Medical Treatment Center Lihuili cystathionine β-synthase (CBS) and cystathionine γ-lyase Hospital. The inclusion criteria and clinical characteristics of (CSE) system, which contributes to endogenous H2S patients were provided in the online supplementary data. All liver biosynthesis, may be regulated by fatty acids. The role of tissue samples from patients used in this study were approved by the CBS/CSE system in the pathogenesis of NAFLD has been the Ethics Committee of the First Affiliated Hospital, College of actively investigated, and the system is proposed to serve as Medicine, Zhejiang University, and all patients provided their a potential therapeutic target for NAFLD. written informed consent. ► 3-Mercaptopyruvate sulfurtransferase (MPST) is also an important enzyme that contributes to the generation of H2S by using 3-mercaptopyruvate as substrate in a pyridoxal-5′- Animal experiments phosphate (PLP)-independent way, which is different from An intravenous hydrodynamic injection of adenovirus plasmids the CBS/CSE system by way of using cysteine as substrate that contained green fluorescent protein (AD-GFP) coding that is dependent on PLP. sequence and MPST short hairpin RNA (AD-shMPST) was administered to male C57BL/6 mice to inhibit hepatic MPST What are the new findings? expression. Heterozygous MPST-deficient (MPST+/-) mice on FFAs induced upregulation of hepatic MPST protein expression ► a C57BL/6 background were generated through frameshift in high-fat diet (HFD)-fed mice and patients with NAFLD. mutation by TAL-effector nuclease system by Beijing View- ► Hepatic H S synthesis is impaired in NAFLD models in 2 Solid Biotechnology (Beijing, China). Detailed explanations of vivo and in vitro. Partial knockdown of MPST significantly generation strategy and all animal treatments are provided in elevated H S levels by directly interacting with and 2 the online supplementary data and online supplementary figure negatively regulating CSE. 1. All of the animal experiments were performed according to Partial knockdown of MPST via adenovirus delivery of ► the guidelines approved by the Animal Care and Use Committee MPST short hairpin RNA or heterozygous deletion of the of the First Affiliated Hospital, College of Medicine, Zhejiang Mpst gene significantly ameliorated hepatic steatosis in University.