Supporting data for “Autotaxin as a New Mediator for Non-alcoholic Fatty Liver Disease: Mechanism and Therapeutic Interventions”

Non-alcoholic fatty liver disease (NAFLD) refers to a spectrum of conditions caused by excessive accumulation of fat in the liver. NAFLD has emerged as the leading cause of chronic liver disease, and it is a major indication for liver transplantation globally. Therefore, the identification of new therapeutic targets for the development of clinical interventions for NAFLD is of great importance. Autotaxin, also known as ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted glycoprotein originally discovered in melanoma cells. Autotaxin is an enzyme responsible for producing lysophosphatidic acid (LPA)which binds with LPA receptors and exerts various pathophysiological effects. Previous studies have reported that serum autotaxin levels increase in NAFLD, but the exact metabolic role of autotaxin in NAFLD is unclear. Therefore, the aim of this study is to investigate 1) whether autotaxin plays a pathological role in the development of NAFLD, as well as 2) the mechanism underlying the action of autotaxin.In this study, a novel role of autotaxin as a mediator to exacerbate NAFLD was discovered for the first time. Our clinical and animal studies demonstrated that liver-derived circulating autotaxin is positively associated with NAFLD. Knockdown of hepatic autotaxin markedly attenuated liver steatosis in mice subjected to high-fat diet (HFD), accompanied by significantly elevated hepatic fatty acid oxidation (FAO) rate. Moreover, anti-autotaxin neutralizing antibody mediated inhibition of circulating autotaxin activity significantly alleviated HFD-induced fatty liver. Notably, the expression levels of fibroblast growth factor 21 (FGF21), a well-known beneficial regulator in NAFLD, was also markedly increased after the inhibition of autotaxin activity. These results demonstrated autotaxin is a new mediator and a potential therapeutic target for NAFLD.The beneficial effects of anti-autotaxin neutralizing antibody on NAFLD disappeared in mice with global FGF21 deficiency, indicating that the detrimental effect of autotaxin on NAFLD is mediated through altering FGF21 levels. Mechanistically, autotaxin inhibits FGF21 expression via LPA-Erk pathway induced phosphorylation of peroxisome proliferator-activated receptor alpha (PPARα) on its site of Serine (Ser) 21. Anti-autotaxin neutralizing antibody treatment blocks serum autotaxin activity, alleviates the inhibitory effect of autotaxin-LPA axis on FGF21 and promotes hepatic FGF21 expression. FGF21 further exerts direct or indirect beneficial metabolic effects on liver and alleviates the progression of NAFLD. Collectively, our data suggest that liver-derived circulating autotaxin is closely associated with NAFLD, and autotaxin-LPA axis exacerbates NAFLD through inhibition of hepatic PPARα-regulated FGF21 expression. Thus, autotaxin mediates the development of NAFLD and it is a potential therapeutic target for the treatment of NAFLD.

非酒精性脂肪性肝病（NAFLD）是指由肝脏脂肪过度积累引起的一系列疾病。NAFLD已成为慢性肝病的主要病因，并且是全球肝脏移植的主要适应症。因此，为NAFLD的临床干预开发新的治疗靶点具有重要的意义。自分泌酶，亦称为外核苷酸焦磷酸酶/磷酸二酯酶2（ENPP2），是一种最初在黑色素瘤细胞中发现的分泌型糖蛋白。自分泌酶是一种负责产生溶血磷脂酸（LPA）的酶，LPA与LPA受体结合并发挥多种病理生理作用。先前的研究报告指出，在NAFLD患者中血清自分泌酶水平升高，但自分泌酶在NAFLD中的确切代谢作用尚不明确。因此，本研究旨在探讨1）自分泌酶是否在NAFLD的发展中发挥病理作用，以及2）自分泌酶作用的潜在机制。本研究首次发现了自分泌酶作为加剧NAFLD的介导者的新角色。我们的临床和动物研究证明了肝脏来源的循环自分泌酶与NAFLD呈正相关。敲低肝细胞自分泌酶显著减轻了高脂饮食（HFD）小鼠的肝脂肪变性，并伴随着肝脏脂肪酸氧化（FAO）率的显著提高。此外，抗自分泌酶中和抗体介导的循环自分泌酶活性抑制显著缓解了HFD引起的脂肪肝。值得注意的是，在抑制自分泌酶活性后，已知在NAFLD中具有益调节作用的成纤维细胞生长因子21（FGF21）的表达水平也显著升高。这些结果表明，自分泌酶是NAFLD的新介导者和潜在治疗靶点。在全局FGF21缺陷小鼠中，抗自分泌酶中和抗体对NAFLD的有益作用消失，表明自分泌酶对NAFLD的损害作用是通过改变FGF21水平介导的。从机制上讲，自分泌酶通过LPA-Erk途径诱导的过氧化物酶体增殖物激活受体α（PPARα）Ser21位点的磷酸化抑制FGF21的表达。抗自分泌酶中和抗体治疗通过阻断血清自分泌酶活性，缓解自分泌酶-LPA轴对FGF21的抑制作用，并促进肝脏FGF21的表达。FGF21进一步在肝脏上发挥直接或间接的有益代谢作用，缓解NAFLD的进展。总体而言，我们的数据表明，肝脏来源的循环自分泌酶与NAFLD密切相关，自分泌酶-LPA轴通过抑制肝脏PPARα调控的FGF21表达加剧NAFLD。因此，自分泌酶介导NAFLD的发展，并且是NAFLD治疗的潜在靶点。