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Hepatocyte-Secreted Autotaxin Exacerbates Nonalcoholic Fatty Liver Disease Through Autocrine Inhibition of the PPARα/FGF21 Axis

Background & Aims The prevalence of nonalcoholic fatty liver disease (NAFLD) has reached epidemic proportions globally as a result of the rapid increase in obesity. However, there is no Food and Drug Administration–approved pharmacotherapy available for NAFLD. This study investigated the role of autotaxin, a secreted enzyme that hydrolyzes lysophosphatidylcholine to produce lysophosphatidic acid (LPA), in the pathogenesis of NAFLD and to explore whether genetic or pharmacologic interventions targeting autotaxin ameliorate NAFLD. Methods The clinical association of autotaxin with the severity of NAFLD was analyzed in 125 liver biopsy-proven NAFLD patients. C57BL/6N mice or fibroblast growth factor 21 (FGF21)-null mice were fed a high-fat diet or a choline-deficient diet to investigate the role of the autotaxin–FGF21 axis in NAFLD development by hepatic knockdown and antibody neutralization. Huh7 cells were used to investigate the autocrine effects of autotaxin. Results Serum autotaxin levels were associated positively with histologic scores and NAFLD severity. Hepatocytes, but not adipocytes, were the major contributor to increased circulating autotaxin in both patients and mouse models with NAFLD. In mice, knocking-down hepatic autotaxin or treatment with a neutralizing antibody against autotaxin significantly reduced high-fat diet–induced NAFLD and high fat– and choline-deficient diet–induced nonalcoholic steatohepatitis and fibrosis, accompanied by a marked increase of serum FGF21. Mechanistically, autotaxin inhibited the transcriptional activity of peroxisome proliferator-activated receptor α through LPA-induced activation of extracellular signal-regulated kinas, thereby leading to suppression of hepatic FGF21 production. The therapeutic benefit of anti-autotaxin neutralizing antibody against NAFLD was abrogated in FGF21-null mice. Conclusions Liver-secreted autotaxin acts in an autocrine manner to exacerbate NAFLD through LPA-induced suppression of the peroxisome proliferator-activated receptor α–FGF21 axis and is a promising therapeutic target for NAFLD.

BACKGROUND & AIMS: The prevalence of nonalcoholic fatty liver disease (NAFLD) has reached epidemic proportions globally as a result of the rapid increase in obesity. However, there is no Food and Drug Administration-approved pharmacotherapy available for NAFLD. This study investigated the role of autotaxin, a secreted enzyme that hydrolyzes lysophosphatidylcholine to produce lysophosphatidic acid (LPA), in the pathogenesis of NAFLD and to explore whether genetic or pharmacologic interventions targeting autotaxin ameliorate NAFLD.

METHODS:
The clinical association of autotaxin with the severity of NAFLD was analyzed in 125 liver biopsy-proven NAFLD patients. C57BL/6N mice or fibroblast growth factor 21 (FGF21)-null mice were fed a high-fat diet or a cholinedeficient diet to investigate the role of the autotaxin-FGF21 axis in NAFLD development by hepatic knockdown and antibody neutralization. Huh7 cells were used to investigate the autocrine effects of autotaxin.
RESULTS: Serum autotaxin levels were associated positively with histologic scores and NAFLD severity. Hepatocytes, but not adipocytes, were the major contributor to increased circulating autotaxin in both patients and mouse models with NAFLD. In mice, knocking-down hepatic autotaxin or treatment with a neutralizing antibody against autotaxin significantly reduced high-fat diet-induced NAFLD and high fat-and cholinedeficient diet-induced nonalcoholic steatohepatitis and fibrosis, accompanied by a marked increase of serum FGF21.
Mechanistically, autotaxin inhibited the transcriptional activity of peroxisome proliferator-activated receptor a through LPAinduced activation of extracellular signal-regulated kinas, thereby leading to suppression of hepatic FGF21 production. The therapeutic benefit of anti-autotaxin neutralizing antibody against NAFLD was abrogated in FGF21-null mice.
diabetes. 1,2 The pathohistologic spectrum of NAFLD ranges from benign steatosis to nonalcoholic steatohepatitis (NASH), the latter of which is characterized by lobular inflammation and ballooning of hepatocytes, with or without perisinusoidal fibrosis. Patients with NAFLD are more susceptible to endstage liver diseases such as cirrhosis and hepatocellular carcinoma, and are at increased risk for type 2 diabetes, cardiovascular disease, cancer, and overall mortality. 3 NAFLD is expected to become a major indication for liver transplantation in the coming decade. 2,4,5 Despite the rapid increase in the health care burden of NAFLD, there are currently no approved pharmacotherapies for this chronic liver disease Liver-secreted hepatokines and adipose tissue-secreted adipokines play important roles in controlling metabolic homeostasis, insulin sensitivity, and immunometabolism in the liver. 6 Aberrations in the production and/or function of a large number of hepatokines and adipokines have been implicated in the pathogenesis of NAFLD and its related liver complications. 6,7 A number of hepatokines and adipokines, such as fibroblast growth factor 21 (FGF21) and adiponectin, have been developed as potential therapeutic agents or noninvasive diagnostic biomarkers for NAFLD. 6,8 Autotaxin (ATX), also known as ectonucleotide pyrophosphatase/phosphodiesterase 2, is an adipokine secreted abundantly from mature adipocytes, 9,10 although it also is expressed in many other tissues including the liver. ATX possesses lysophospholipase D (lysoPLD) activity, thereby converting lysophosphatidylcholine to lysophosphatidic acid (LPA), 11 the latter of which is a bioactive lipid that activates multiple cell signaling pathways involved in cell aggregation, proliferation, chemotaxis, and inflammation via binding to its G-protein-coupled receptors. 12,13 An increasing body of studies has suggested the involvement of the ATX-LPA axis in obesity and its related metabolic complications. [14][15][16][17] In adipose tissue, the ATX-LPA axis acts in an autocrine manner to impair insulin actions in white adipocytes, inhibit thermogenesis in brown adipocytes, and in a paracrine manner to promote adipogenesis of preadipocytes. 9 Adipose deletion of ATX has been shown recently to protect against high-fat diet (HFD)-induced obesity, insulin resistance, and glucose intolerance in mice. 18,19 However, another earlier study reported a more obese phenotype of adipose-specific ATX knockout mice. 20 In older human beings with obesity, serum autotaxin correlates positively with adiposity and is an independent predictor of insulin resistance. 21 Aberrant serum autotaxin levels have been observed in patients with severe obesity, viral hepatitis, and fibrosis, 16,[22][23][24][25][26][27] and inhibition of hepatic autotaxin or adipose-derived autotaxin has been reported to alleviate the development of hepatocellular carcinoma 16 or steatosis. 17 However, the pathophysiological role of the ATX-LPA axis in the onset and progression of NAFLD remains to be established.
In this study, we measured the dynamic changes in circulating levels of autotaxin and its hepatic expression in several different mouse models with NAFLD, and explored the relationship of serum autotaxin with histologic severity and progression of NAFLD in liver biopsy-proven patients. Furthermore, we investigated the role of liver-secreted autotaxin in NAFLD development by both knockdown of hepatic autotaxin and treatment with a neutralizing antibody against autotaxin, and delineated the pathogenic mechanism whereby the aberrant ATX-LPA axis exacerbates NAFLD via dysregulation of FGF21.

Serum Autotaxin Levels Are Closely Associated With Abnormal Liver Function and NAFLD Severity in Obese Individuals
To investigate the clinical association of autotaxin with liver function and the severity of NAFLD, we consecutively recruited 151 obese individuals undergoing bariatric surgery. The clinical, biochemical, and histologic characteristics of the 151 obese subjects included in this study are shown in Table 1. The mean ± SD age and body mass index (BMI) of the participants were 30.35 ± 1.35 y and 40.23 ± 1.24 kg/m 2 , respectively. Histologic examination of liver biopsy specimens showed that the prevalence of hepatic steatosis (at least 5% of fat within hepatocytes), ballooning, inflammation, and fibrosis occurred in 132 (87.4%), 80 (53.0%), 70 (46.3%), and 38 (25.2%) patients, respectively. A total of 14 (9.3%) patients showed normal liver histology, while 31 (20.5%), 62 (41.1%), and 44 (29.1%) patients were classified into simple steatosis, borderline NASH, and NASH, respectively, according to the fatty liver inhibition of progression algorithm. 28 Consistent with previous reports, 21 serum autotaxin levels in women (508.53 ± 21.58 ng/mL; n ¼ 85) were significantly higher than in men (417.33 ± 22.48 ng/mL; n ¼ 66; P ¼ .004, Mann-Whitney U test). Notably, serum autotaxin levels showed significant positive correlations with hepatic fat content (determined by magnetic resonance imaging); serum levels of the liver injury markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ( Figure 1A-C); severity scores of hepatic steatosis, ballooning, and inflammation grade; as well as histologic progression of NAFLD from normal liver, benign steatosis to NASH. These significances remained even after adjustment for sex, age, and BMI (Table 2). Realtime polymerase chain reaction (PCR) analysis showed a significantly higher protein level of autotaxin in the liver tissue than in subcutaneous and visceral adipose tissues of obese individuals ( Figure 1D). Furthermore, there was a strong positive association between serum autotaxin levels and the autotaxin messenger RNA (mRNA) expression in the liver, but not in adipose tissues ( Figure 1E). Taken together, these findings suggest that increased serum autotaxin is associated closely with the development of liver dysfunction and NAFLD progression in obese individuals, and the liver is perhaps an important contributor to increased circulating autotaxin.

Liver But Not Adipose Tissue Contributes to Increased Circulating Autotaxin in Mouse Models With NAFLD
To confirm our findings in the earlier-described clinical study, we monitored the dynamic changes of the autotaxin mRNA expression in major organs, serum autotaxin levels, as well as lysoPLD activity in HFD-induced obese mice with NAFLD and lean controls for a period of 20 weeks. Serum autotaxin levels and lysoPLD activity showed a progressive increase with the progression of diet-induced obesity (Figure 2A and B), but remained little changed in standard chow diet (STC)-fed lean mice. At 20 weeks after HFD start, serum autotaxin levels and lysoPLD activity in obese mice were 1.65-and 2-fold higher than in lean mice, respectively (Figure 2A and B). In line with previous reports showing autotaxin as an adipokine, 9,18,29 real-time PCR analysis detected highly enriched mRNA expression of autotaxin in several adipose depots, including epididymal white adipose tissue and interscapular brown adipose tissue ( Figure 2C). However, the autotaxin mRNA expression in these 2 adipose depots paradoxically were decreased in HFD-induced obese mice compared with that in lean mice, although there was a modest increase of autotaxin gene expression in subcutaneous white adipose tissue of obese mice. In contrast, the autotaxin mRNA expression level in the liver of HFDinduced obese mice was increased significantly by almost 2-fold, whereas its mRNA abundance in other tissues was comparable between obese and lean mice, except for lymph nodes, where autotaxin mRNA expression in obese mice was lower than in lean mice. Further cellular fractionation analysis showed that the autotaxin gene expression in hepatocytes was more than 5-fold higher than in nonparenchymal cells, while obesity-induced up-regulation of the autotaxin gene expression occurred predominantly in hepatocytes, but not in nonparenchymal cells ( Figure 2D).
To further dissect the contribution of adipose tissue and liver to circulating autotaxin in mice with NAFLD, we next used adipocyte-specific murine double-minute 2 (MDM2) knockout (KO) mice, which showed complete loss of adipose tissues, severe hyperglycemia, dyslipidemia, and massive fatty liver. 30,31 Surprisingly, the autotaxin mRNA expression in the liver of the lipodystrophic mice was more than 3-fold higher than in wild-type (WT) littermates, accompanied by a significant increase of serum autotaxin ( Figure 2E and F). On the other hand, the autotaxin gene expression level in other tissues was comparable between these 2 genotypes of mice ( Figure 2F). Taken together, these findings suggest that hepatocytes, but not adipose tissues, are the major production site for increased circulating autotaxin in both dietary obese mice and lipodystrophic mice with NAFLD.

Knock-Down of Hepatic Autotaxin Expression Alleviates NAFLD by Increasing Fatty Acid Oxidation in Obese Mice
Conventional autotaxin KO mice are embryonic lethal owing to the pivotal role of autotaxin in angiogenesis and development. 32 To investigate the role of liver-secreted autotaxin in obesity-induced metabolic complications and fatty liver disease, we used the adeno-associated virus (AAV) 2/8 delivery system to knock-down (KD) hepatic autotaxin in adult mice by intravenous injection of AAV directly to the liver. Compared with the mice injected with AAV expressing scramble control, the mRNA expression of autotaxin in the liver of STC-fed lean mice and HFD-fed obese mice was decreased significantly by 40% and 45%, respectively, after injection of AAV bearing short hairpin RNA (shRNA) specific to the autotaxin (KD) gene for 15 weeks ( Figure 3A), whereas autotaxin gene expression in adipose tissues remained unchanged ( Figure 3B). Notably, HFD-induced increases of serum autotaxin levels and lysoPLD activity largely were abrogated by AAV-mediated KD of hepatic autotaxin ( Figure 3C and D), further confirming the liver as a major contributor to increased circulating autotaxin in obesity.
KD of hepatic autotaxin did not exert an obvious effect on body weight, total fat mass, and food intake ( Figure 3E-G), but significantly improved lipid profiles in  dietary obese mice, including reductions in serum triglyceride, cholesterol, and free fatty acids ( Figure 4A). Moreover, hepatic knockdown of autotaxin markedly alleviated HFD-induced increases of liver weight ( Figure 4B) and hepatic steatosis, as determined by H&E and Oil Red O staining ( Figure 4C), and biochemical analysis for hepatic triglyceride contents ( Figure 4D). Accordingly, increased serum levels of ALT and AST, the 2 well-established markers of liver injury, were decreased markedly in obese mice with knockdown of hepatic autotaxin ( Figure 4E). Excessive hepatic lipid accumulation often is caused by an imbalance between fatty acid oxidation (FAO) and de novo lipogenesis. 4 We next examined the impact of knockdown of hepatic autotaxin on expression of genes involved in hepatic lipid metabolism and found that several FAOrelated genes, including Cpt1a, Mcad/Lcad/Scad, and Acox1 were increased greatly in both lean and obese mice with AAV-mediated knockdown of hepatic autotaxin ( Figure 4F), whereas genes involved in fatty acid synthesis such as Fasn and Scd-1 were not affected. Furthermore, increased hepatic FAO gene expression in the autotaxin knockdown mice was accompanied by a significant increase in FAO activity in the liver, as determined by reduced nicotinamide adenine dinucleotide-dependent production of iodonitrotetrazolium (INT)-formazan ( Figure 4G). Taken together, these findings suggest that blocking augmented hepatic autotaxin expression is sufficient to counteract obesity-related NAFLD, possibly by increasing FAO.

Treatment With Anti-Autotaxin Neutralizing Antibody Potently Ameliorates Hepatic Steatosis and Increases Hepatic FGF21 in Obese Mice
Given that autotaxin is a secreted enzyme with lysoPLD activity, we generated a neutralizing antibody (IgG) against autotaxin (anti-ATX) and evaluated whether neutralization of secreted autotaxin has therapeutic benefits on obesityrelated NAFLD. Ex vivo incubation of serum from obese mice with anti-ATX IgG dose-dependently inhibited the endogenous lysoPLD activity with half maximal inhibitory concentration and maximum inhibitory effects at concentrations of 0.069 and 0.251 umol/L, respectively, whereas nonimmune IgG had no such inhibitory effect ( Figure 5A). Furthermore, treatment with anti-ATX but not nonimmune IgG blocked the lysoPLD activity of recombinant mouse autotaxin (rmATX) and suppressed the stimulatory effects of rmATX on phosphorylation of extracellular signalregulated kinase (ERK)1/2 in HepG2 hepatocytes in a concentration-dependent manner ( Figure 5B and C). The circulating half-life of anti-ATX IgG in mice was approximately 5 days ( Figure 5D). The relative mRNA abundance of (C) mouse autotaxin in different tissues (shown as fold change relative to brown adipose tissue [BAT] of the STC group), and in (D) different fractions of the liver (shown as fold change relative to hepatocytes of the STC group) determined by real time PCR analysis. *P < .05, **P < .01, and ***P < .001 vs STC group. Data are presented as the means ± SEM. Twelveweek-old male adipocyte-specific MDM2 KO mice and their WT littermates (adipo-MDM2-WT) were killed to collect (E) serum and various tissues for measurement of circulating autotaxin (n ¼ 8-12) and (F) relative mRNA abundance of autotaxin (n ¼ 5, shown as fold change relative to lymph node of adipo-MDM2-WT group) by enzyme-linked immunosorbent assay and realtime PCR, respectively. (A-F) Statistical significance was determined by a 2-tailed Student t test. *P < .05, and ***P < .001 vs the Adipo-MDM2-WT group. Data are presented as the means ± SEM. eWAT, epididymal adipose tissue; msWAT, mesenteric adipose tissue; NPC, nonparenchymal cell.
Treatment of mice with anti-ATX IgG for a period of 7 weeks decreased circulating lysoPLD activity ( Figure 6A) and significantly reduced HFD-induced increase of serum triglycerides, cholesterol, and free fatty acids (FFAs) ( Figure 6B), although it had no significant effect on body weight, body composition, and food intake ( Figure 5E-G). In line with the findings in mice with knockdown of hepatic autotaxin, obese mice treated with anti-ATX IgG showed obvious improvements in NAFLD, as evidenced by significant reductions in liver weight ( Figure 6C), hepatic steatosis ( Figure 6D), triglyceride accumulation ( Figure 6E), and serum levels of ALT and AST ( Figure 6F). These improvements in anti-ATX IgG-treated mice were accompanied by up-regulated expression of hepatic genes involved in FAO ( Figure 6G), and increased FAO activity in the liver ( Figure 6H).
Crosstalk between liver-secreted hepatokines and adipose-secreted adipokines play important roles in regulating hepatic lipid metabolism. 6 Therefore, we next examined the effects of anti-ATX IgG on serum levels of a panel of well-established hepatokines and adipokines involved in the pathogenesis of NAFLD. 6 Treatment of mice with anti-ATX IgG had no obvious effects on mRNA expression levels of lipocalin-2, Fgf15, Angptl3/4/6, Fetuin-a, Fgl-1, Dpp4, Fabp4, leptin, and Apelin ( Figure 7A and B), but markedly increased serum levels of FGF21 as well as the hepatic mRNA expression of FGF21 ( Figure 7C and D), a well-known hepatokine with protective effects against NAFLD. 8 Accordingly, serum level of adiponectin, a downstream effector of FGF21 with anti-steatosis activity, 33 also was increased by anti-ATX IgG ( Figure 7E). Likewise, significant increases in hepatic FGF21 expression and serum levels of FGF21 and adiponectin also were observed in mice with AAV-mediated knockdown of hepatic autotaxin expression ( Figure 7F-H).

Autotaxin Inhibits Hepatic FGF21 Production Through LPA-Induced ERK Activation and Peroxisome Proliferator-Activated Receptor a Inhibition
The earlier-described findings suggest that autotaxin may exacerbate NAFLD via inhibition of hepatic FGF21 production. Therefore, we next investigated the direct effects of autotaxin and its enzyme product LPA on FGF21 expression in Huh7 hepatocytes. The Fgf21 mRNA abundance and FGF21 secretion into conditioned medium were decreased significantly by treatment with recombinant human autotaxin (rhATX), but were obviously increased by incubation with PF8380 (a chemical compound that selectively inhibits the lysoPLD activity of autotaxin to produce LPA 34 ) and Ki16425 (a pharmacologic LPA-receptor antagonist 35 ) ( Figure 7I and J), suggesting the ATX-LPA axis acts in an autocrine manner to suppress endogenous FGF21 production in hepatocytes. Likewise, treatment of Huh7 hepatocytes with rhATX and LPA dose-dependently inhibited the peroxisome proliferator-activated receptor a (PPARa) agonist fenofibrate-induced Fgf21 mRNA were performed by 1-way analysis of variance followed by Tukey multiple comparisons using Prism 6. *P < .05, **P < .01, and ***P < .001. Data are presented as the means ± SEM. eWAT, epididymal adipose tissue; scWAT, subcutaneous adipose tissue. expression and its protein secretion, whereas such inhibitory effects of endogenous autotaxin were reversed by preincubation with PF8380 and Kil6465 ( Figure 7K and L). Autotaxin-derived LPA exerts its pleiotropic effects by activating several intracellular signaling cascades, especially phosphoinositide 3-kinases/protein kinase B (PI3K/Akt) and mitogen-activated protein kinase/extracellular signalregulated kinases (MAPK/ERK) pathways. 36 Our results showed that treatment with the selective inhibitor of mitogen-activated protein kinase kinase/extracellular signal-regulated kinases (MEK/ERK, U0126) markedly reversed the inhibitory effect of LPA on fenofibrate-induced FGF21 production, whereas the PI3K inhibitor (wortmannin) had no obvious effect ( Figure 7O). Given that PPARa is a key regulator for FGF21 gene expression in hepatocytes, 37,38 we next investigated whether LPA suppresses FGF21 production through ERK-mediated inhibition of PPARa. In Huh7 cells transfected with the PPAR response element X3-TK-luc promotor plasmid, 39 LPA treatment significantly decreased fenofibrate-induced transcriptional activity of PPARa as determined by the luciferase reporter analysis, whereas such a suppressive effect of LPA was largely reversed by U0126, but not wortmannin ( Figure 7P).
Likewise, LPA significantly reduced the expression of another well-known PPARa target gene, Vlcad, through ERK activation ( Figure 7O). In contrast, PPARa mRNA abundance was not affected by either LPA or U0126 ( Figure 7O).
PPARa activity can be modulated post-translationally by ERK-mediated phosphorylation, 40-42 and 2 ERK phosphorylation sites (S12 and S21) have been identified in mouse PPARa, which are highly conserved between species. 41,42 We next explored the effect of LPA on PPARa phosphorylation at these 2 sites with Western blot. As expected, LPA induced ERK phosphorylation at Ser 21, which was blocked by U0126 ( Figure 7M and N), whereas neither LPA nor U0126 had any effect on phosphorylation at Ser 12. Taken together, these findings suggest that autotaxin inhibits hepatic FGF21 expression via LPA-induced ERK activation and subsequent PPARa suppression.   were performed by 1-way analysis of variance followed by Tukey multiple comparisons using Prism 6. *P < .05, **P < .01. Data are presented as the means ± SEM. BAT, brown adipose tissue; eWAT, epididymal adipose tissue; scWAT, subcutaneous adipose tissue. models and patients. 8,43 We next investigated whether the therapeutic effect of autotaxin neutralization on fatty liver requires hepatic up-regulation of FGF21 using FGF21 knockout mice. 44,45 Treatment with anti-ATX IgG led to a comparable degree of suppression on lysoPLD activity in HFD-fed FGF21 knockout mice and WT littermates ( Figure 8A). However, the effects of anti-ATX IgG on improvement of serum lipid profiles, reduction of liver weight, and alleviation of hepatic steatosis and liver injury were observed only in WT mice, but not in FGF21 knockout mice ( Figure 8B-F). Similarly, anti-ATX IgG induced upregulation of FAO-related genes, increases in hepatic FAO activity, and in serum adiponectin levels were blunted markedly in FGF21 knockout mice ( Figure 8G-I), supporting the role of FGF21 as an indispensable downstream effector conferring hepatoprotective effects of anti-ATX IgG in obese mice.
In light of the fact that recent clinical trials have shown promising therapeutic potential of FGF21 analogs for patients with NASH and fibrosis, 8,43,46 we next explored whether neutralization of autotaxin with anti-ATX IgG ameliorates advanced stages of NAFLD in choline-deficient, L-amino acid-defined, 60% HFD (CDAHF60)-fed mice, which develop all of the histologic features of NAFLD, including severe steatosis, inflammation, and fibrosis. 47,48 Although CDAHF60 feeding did not increase body weight and fat mass, it caused a time-dependent increase in the serum level of autotaxin and its lysoPLD activity, as well as the autotaxin mRNA expression in the liver but not in adipose tissue, as compared with age-matched STC-fed mice ( Figure 9A-C and E-G). The typical histopathology of NAFLD and liver injury were readily detected at 4 weeks after feeding CDAHF60 ( Figure 9D). Treatment with anti-ATX IgG led to a comparable reduction in circulating lysoPLD activity between FGF21 knockout mice and WT littermates ( Figure 10A), and markedly increased serum FGF21 levels in WT mice, but not in FGF21 knockout mice ( Figure 10B). Notably, the significant effects of anti-ATX antibody on amelioration of serum triglycerides, cholesterol, and FFAs, and increase of serum adiponectin were observed only in WT mice, but not in FGF21 knockout mice ( Figure 10C and  D). Furthermore, anti-ATX IgG significantly decreased liver weights, attenuated steatosis inflammation, fibrosis, and NASH scores (NAFLD Activity Score, NAS) as determined by histologic examination ( Figure 10E-G), obviously reduced triglyceride accumulation in the liver and serum levels of ALT and AST ( Figure 10H and I), improved FAO rate ( Figure 10J), and inhibited the expression of several fibrosis-related genes including Acta2, Col1a1, Timp-1, and Fn1 ( Figure 10K). However, all of these therapeutic benefits of anti-ATX IgG on NASH and fibrosis was largely abolished in FGF21 knockout mice, indicating that anti-ATX IgGmediated neutralization of autotaxin is effective in treating CDAHF60-induced NASH and fibrosis by induction of FGF21.

Discussion
In the present study, we provide both clinical and experimental evidence that increased autotaxin is an important contributor to the pathogenesis of NAFLD through LPA-induced inhibition of PPARa. Furthermore, small interfering RNA-mediated blockage of hepatic autotaxin production or treatment with an antibody that neutralizes the lysoPLD activity of autotaxin potently ameliorates fatty liver, NASH, and hepatic fibrosis in 2 different mouse models with NAFLD by promoting FGF21 production ( Figure 11). These findings identify the ATX-LPA axis as an endogenous inhibitor of hepatic FGF21 expression, and a promising therapeutic target for the treatment of NAFLD.
The ATX-LPA axis regulates a plethora of cellular functions including proliferation and growth, development, chemotaxis, vasoregulation, calcium dynamics, and energy homeostasis by binding LPA to its G-protein-coupled receptors (LPA1-6). 36 Augmented autotaxin expression has been implicated in multiple types of cancers, chronic inflammatory diseases, and cardiomyopathy. [49][50][51] Although autotaxin previously was thought to be an adipokine expressed abundantly in adipose tissue in rodents, our present study shows that liver, but not adipose tissue, is a major contributor to increased circulating autotaxin in both human beings and several types of mouse models with NAFLD. In obese subjects, autotaxin expression in liver is much higher than in visceral and subcutaneous adipose tissues. Furthermore, autotaxin expression in liver, but not in adipose tissues, correlates strongly with serum autotaxin, which is closely associated with several histopathologic features as well as staging of biopsy-proven NAFLD. In a lipodystrophic mouse model without adipose tissues, circulating autotaxin remains increased owing to its markedly increased hepatic expression. Furthermore, in NAFLD mouse models induced by both dietary obesity and CDAHF60 diet, autotaxin expression is increased markedly in liver but remains little changed in other tissues, while selective knockdown of hepatic autotaxin expression alone is sufficient to counteract dietary obesity and a CDAHF60induced increase of circulating autotaxin, accompanied with obvious amelioration of NAFLD and liver injury. Taken together, these findings support the causal role of liversecreted autotaxin in the development of NAFLD in both animals and human beings. In line with our finding, a previous study found that hepatocyte-secreted autotaxin contributes to lipid accumulation and carcinogenesis in the liver in a N-nitrosodiethylamine/carbon tetrachloride (DEN/CCL4) induced hepatocellular carcinoma mouse model. 16 FGF21, a hormone secreted predominantly from hepatocytes, possesses pleiotropic protective activities against obesity and its related metabolic complications including NAFLD. 8,46,52,53 In mice, FGF21 knockout exacerbates methionine and choline-deficient diet-induced hepatic steatosis, inflammation, and fibrosis, 44,54 while treatment with recombinant FGF21 or its analogs markedly ameliorates these pathologic changes in the liver of both mice and rodents. 44,54 FGF21 has been shown to exert its hepatoprotective effects through its direct actions in the liver by promoting fatty acid oxidation, enhancing hepatic insulin sensitivity, attenuating endoplasmic reticulum and oxidative stresses, 8,52,53 limiting endotoxin-induced liver inflammation and injury, blocking nuclear factor-kB, 55 and mitigating fibrogenic gene expression in hepatic stellate cells. 56 Furthermore, administration of FGF21 or its analogs in both mice and human beings induces the production of adiponectin, an adipokine with insulin-sensitizing, anti-inflammatory, and hepatoprotective properties. 33 Notably, several recent clinical trials have shown highly promising therapeutic benefits of FGF21 analogs (such as pegylated long-acting forms of FGF21) and FGF21-receptor agonists in the treatment of biopsy-confirmed NASH patients. 8,43,46,57,58 In another clinical study with biopsy-confirmed F1-F3 NASH patients, treatment with efruxifermin (a long-acting FGF21 fusion protein with fragment crystallizable (Fc) fragment) lead to a more than 70% reduction in liver fat contents, accompanied by resolution of histologic features of NASH and fibrosis, reduction of liver injury, and increase of adiponectin (NCT05039450). 59 A remarkable reduction in liver fat content and markers of liver fibrosis and injury also have been observed in obese adults treated with another genetically engineered FGF21 analogue: LLF580. 60 In the present study, we observed a marked increase in both circulating levels of FGF21 and its hepatic expression in both dietary obese mice and CDAHF60-fed mice with autotaxin knockdown or antibody neutralization, while the effects of knocking down or neutralizing autotaxin in ameliorating steatosis, NASH, and fibrosis, and in promoting hepatic fatty acid oxidation, largely were abrogated in FGF21 knockout mice, suggesting that increase of endogenous FGF21 production by pharmacologic or genetic inhibition of hepatic autotaxin may represent another therapeutic approach for treatment of NAFLD by targeting FGF21.
Another notable finding of our study was that the ATX-LPA axis acts in an autocrine manner to suppress PPARa activity, which is a master regulator of hepatic lipid metabolism and an obligatory transcription factor for hepatic FGF21 production. 37,38,61 In Huh7 hepatocytes, both autotaxin and LPA dose-dependently inhibit fenofibrate-induced PPARa activation and FGF21 production, possibly through ERK1/2-induced Ser21 phosphorylation of PPARa located within the A/B domain of PPARa, and such effects are reversed by pharmacologic inhibition of autotaxin or ERK. In line with our findings, ERK-induced phosphorylation and deactivation of PPARa has been implicated in cardiac hypertrophic growth and myocadiac lipid accumulation. 42 However, another study reported that PPARa can be activated by ERK1/2 in response to insulin or Pseudomonas aeruginosa stimulation in lung epithelial cells, 40,41 suggesting that ERK regulates PPARa activity in a cellular contextdependent manner. PPARa decreases triglycerides by Data are presented as the means ± SEM. (K-L) Huh7 hepatocytes were pretreated with rhATX, LPA, PF8380 (inhibitor of autotaxin), Ki16425 (LPA-receptor antagonist) for 30 minutes, followed by treatment with or without fenofibrate for 24 hours. (K) FGF21 protein was secreted into conditioned medium and (L) hepatic FGF21 mRNA were determined by ELISA and realtime PCR, respectively. Huh7 hepatocytes were pretreated with U0126 (ERK inhibitor), wortmannin (PI3K), or LPA for 30 minutes, (M and N) followed by treatment with or without fenofibrate for 30 minutes to collect cell lysates for Western blot analysis, or (O) 24 hours to measure the relative mRNA abundance of FGF21, PPARA, and VLCAD (very-long-chain acylcoenzyme-A dehydrogenase, a downstream target of PPARa). (P) Relative luciferase activity in Huh7 hepatocytes transfected with a luciferase reporter vector carrying PPAR response element and treated with LPA, U0126, and wortmannin in the presence or absence of fenofibrate for 48 hours. Data are normalized against human b-ACTIN and expressed as the fold change relative to dimethyl sulfoxide-treated controls. (A-L and N-P) Statistical calculations were performed by 1-way analysis of variance followed by Tukey multiple comparisons using Prism 6. *P < .05, **P < .01, and ***P < .001. n ¼ 5. Data are presented as the means ± SEM. HSP90, heat shock protein 90; p-ERK, phosphorylated_extracellular signal-related kinase; p-PPARa, phosphorylated peroxisome proliferator-activated receptor a; t-ERK, total extracellular signal-related kinase; t-PPARa, total peroxisome proliferator-activated receptor a. transactivation of a cluster of genes involved in fatty acid metabolism and ketogenesis, in which FGF21 acts as an obligatory downstream effector. 37,[61][62][63][64] Mice with global and hepatocyte-specific PPARa are more susceptible to HFDinduced hepatic steatosis and methionine/choline-deficient diet-induced NASH, 65 while treatment with PPARa agonists ameliorates hepatic steatosis and inflammation in several different types of NAFLD mouse models. 66,67 However, clinical trials failed to show any therapeutic benefits of either PPARa agonists or a PPARa/d agonist on liver histology in NASH patients despite significant improvements in circulating lipid profiles (NCT02704403), 68 suggesting that PPARa activation alone is insufficient for the treatment of NAFLD. In this connection, an augmented ATX-LPA axis has been linked to several pathophysiological pathways involved in NAFLD development, including exacerbation of proinflammatory responses, 69 induction of insulin resistance, 18,49 apoptosis, 70 and fibrosis 15,16,71 in different disease models. Therefore, pharmacologic interventions targeting autotaxin are expected to produce additional therapeutic benefits beyond PPARa activation and FGF21 production and may represent a more efficacious strategy for treatment of NAFLD.
A large number of chemical inhibitors of autotaxin and LPA-receptor antagonists have been developed recently for the treatment of different chronic diseases, such as idiopathic pulmonary fibrosis, systemic sclerosis, and neurodegenerative diseases. 36 However, several clinical trials on the inhibitors of autotaxin (such as GLPG1690) and lysophosphatidic acid receptor antagonist (such as BMS-986020) were terminated because of severe adverse effects of these chemical compounds (https://clinicaltrials. gov). 72 Therapeutic antibodies have several advantages over small-molecule drugs with respect to target specificity, safety, and stability. In this connection, our study provides proof-of-concept evidence that antibody-mediated neutralization of autotaxin activity is highly efficacious in ameliorating fatty liver and NASH in 2 different mouse models, suggesting that the neutralizing antibodies against autotaxin may represent a promising strategy for drug development.

Study Approval
The human study was approved by the Institutional Review Board of Jinan University (2016-017) and the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 20-700), and conducted according to Declaration of Helsinki principles. Written informed consent was received from participants before their inclusion in the study. Animal studies were approved by the Committee on the Use of Live Animals in Teaching and Research at the University of Hong Kong.

Clinical Studies
A total of 151 obese patients were recruited consecutively from the Bariatric Surgery Clinic at the First Affiliated Hospital of Jinan University. Overweight and obesity class were classified by BMI, according to the World Health Organization Asian Standard. Overweight, class I obesity, and class II obesity were defined as a BMI of 23.0-24.9 kg/m 2 , BMI of 25-29.9 kg/m 2 , and BMI !30 kg/m 2 , respectively. Twentysix individuals were excluded because of the following reasons: (1) age younger than age 18 years; (2) had syphilis or human immunodeficiency virus infection; (3) heavy alcohol drinking history (!40 g/d for up to 2 weeks); and (4) lack of sufficient information. Demographic, clinical, and laboratory data (after an overnight fast of at least 10 hours) for each patient were collected within 1 week before their surgery and liver biopsy specimens were obtained during the bariatric surgery. Liver biopsy specimens were taken from the middle of the right lobe during bariatric surgery. Thin sections of liver biopsy specimens were stained with H&E or Masson trichrome, and were evaluated by 3 independent experienced liver pathologists (Dr Subrata Chakrabarti, Western University, London, Ontario, Canada; Dr Hema Mahajan, Western Sydney University, Greater Western Sydney, New South Wales, Australia; and Dr Sen Yan, Dr Everett Chalmers Hospital, New Brunswick, Canada). The scores of steatosis (0-3), hepatocellular ballooning (0-2), lobular inflammation (0-3), and fibrosis (0-4) were Statistical calculations were performed by the Student t test using Prism 6. (C and E-G) Statistical calculations were performed by 1-way analysis of variance followed by Tukey multiple comparisons using Prism 6. *P < .05, **P < .01, and ***P < .001. Data are presented as the means ± SEM. SR, Sirius Red. evaluated according to the NASH Clinical Research Network scoring system. 73 For measurement of autotaxin mRNA and protein levels, biopsy specimens of intra-abdominal (visceral) and subcutaneous adipose tissues and liver obtained during bariatric surgery were immediately frozen in liquid nitrogen and stored at À80 C. Serum was isolated by centrifugation, and immediately stored at À80 C for further analysis.

Animal Experiments
C57BL/6N mice were obtained from the Animal Unit of the University of Hong Kong. Mice were housed at a controlled temperature (23 C ± 1 C) with a 12-hour light/12-hour dark cycle and free access to drinking water and different types of diet: standard chow (catalog #5053, LabDiet, St. Louis, MO), rodent high-fat diet with 45 kcal% fat (D12451, Research Diets), or L-amino acid rodent diets with 60 kcal% fat with low methionine and no added choline diet (CDAHF60, Dyets). Fgf21 KO mice and their WT littermates in C57BL/6N were generated as previously described. 33,74 Genotyping was conducted as previously described. 45 Adipocyte-specific MDM2 KO mice were generated by crossing MDM2 floxed/floxed mice with Adipoq-Cre mice (expressing Cre recombinase under the control of the adiponectin promoter; The Jackson Laboratory, Bar Harbor, ME) as previously described. 31 Mice were subjected to AAV infection or antibody treatment as indicated in each figure legend. Body composition was determined by the Bruker Minispec LF90 II Body Composition Analyzer (Bruker Biospin, Billerica, MA). Food intake was measured every week and shown as the calculated average daily calorie intake per mouse.

Histologic Analysis and Oil Red O Staining
A liver specimen (approximately 5 mm Â 5 mm) taken from the right lobe near the portal vein was fixed in 4% (w/ v) paraformaldehyde, dehydrated, and embedded in paraffin as previously described. 75 The paraffin-embedded liver sections (5 mm) were deparaffinized, rehydrated, and stained with H&E, Sirius Red (Picro Sirius Red Staining Kit, ab150681; Abcam, Cambridge, UK), or Trichrome Stain (Masson's) (SDHT15; Sigma-Aldrich, Burlington, MA) according to the manufacturer's instructions. For Oil Red O staining, liver embedded in optimal cutting temperature compound (OCT compound) was immediately frozen on dry ice. Frozen sections (7 mm) were obtained using a Leica CM 1900 Cryostat (Leica Biosystems, Nussloch, Germany). Rehydrated sections were rinsed with 60% isopropanol and stained for 20 minutes with 0.3% Oil Red O solution, followed by rinsing 3 times in 60% isopropanol and twice in double-distilled water. Slides were further counterstained with hematoxylin and mounted.
visualized by enhanced chemiluminescence reagents (GE Healthcare) and quantified using ImageJ software (National Institutes of Health, Bethesda, MD) based on the intensities of bands. For the determination of serum levels of anti-ATX neutralizing IgG, a standard curve was generated by densitometry using a series of rabbit nonimmune IgG standards with known quantity, and concentration of anti-ATX neutralizing IgG was calculated based on the equation generated from the standard curve.
Total RNA was extracted from cells or various mouse tissues using TRIzol (Invitrogen, Waltham, MA) following the manufacturer's instructions and subsequently reversetranscribed into complementary DNA using a reversetranscription kit (PrimeScript RT reagent kit, RR037A; Takara, Kusatsu, Shiga, Japan). Real-time PCR was performed using SYBR Green master mix (RR420D; Takara) on the Applied Biosystems Prism 7000 sequence detection system (Thermo Fisher Scientific, Waltham, MA), using the primers listed in Table 3. All mouse genes were normalized against the mouse b-actin gene and human genes were normalized against the human b-actin gene.

Liver Fractionation
Liver fractionation was performed as previously described. 76 In brief, liver was perfused with ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid solution (calcium-free Hank's balanced salt solution with 25 mmol/L HEPES and 0.2 g/L ethylene glycol-bis(b-aminoethyl ether)-N,N,N 0 ,N 0 -tetraacetic acid) and sequentially perfused with digestion solution (Iscove's modified Dulbecco's medium with GlutaMAX (35050061; Thermo Fisher Scientific), 500 mg/L collagenase type 4) under a warming infrared light. The liver then carefully was removed from the abdominal cavity and placed in a Petri dish filled with 5 mL postdigestion solution (Dulbecco's modified Eagle medium [DMEM] with GlutaMAX, 25 mmol/L HEPES, and 10% fetal bovine serum [FBS]) and was mechanically disrupted by a scalpel blade. Minced liver was collected and diluted with 45 mL postdigestion solution into a 50-mL conical tube on ice. The suspension was passed through a 70-mm cell strainer and centrifuged at 68 Â g for 5 minutes. The pellet then was washed with phosphate-buffered saline 2 times at 68 Â g for 10 minutes to obtain parenchymal cells. The top 40 mL was transferred into a new tube and centrifuged at 460 Â g for 10 minutes. The pellet was resuspended in 37.5% Percoll (P1644; Sigma) containing 5 mmol/L EDTA, followed by centrifugation at 850 Â g for 30 minutes. The pellet was resuspended in red cell lysis buffer (00-4333-57; Invitrogen), followed by centrifugation at 460 Â g for 10 minutes to collect nonparenchymal cells in the pellets.

Biochemical and Immunologic Analyses
Mouse autotaxin, FGF21, and adiponectin were measured with enzyme-linked immunosorbent assay kits (32770, 32180, and 32010, respectively; Immunodiagnostics, Ltd, Hong Kong, China). Serum levels of human FGF21 and human autotaxin were quantified with enzyme-linked immunosorbent assay kits (31180 and 31770, respectively; Immunodiagnostics, Ltd). Serum FFAs were determined by a FFA Half Micro Test kit (Roche). Serum triglyceride, cholesterol, ALT, and AST levels were measured using commercial kits from STANBIO Laboratory (Kolkata, West Bengal, India). To measure liver triglyceride content, liver tissues (w50 mg) were homogenized in chloroform/ methanol (2:1 v/v) using a tissue homogenizer. Lipid extracts were prepared by the Folch method, as previously described, 77 and then were dried under nitrogen flow and further dissolved in ethanol. The FAO rate was measured using a commercial kit (E-141; Biomedical Research Service, University at Buffalo, Buffalo, NY) following the manufacturer's instructions. 78,79 The FAO activity assay is based on the oxidation of octanoyl-CoA (an intermediate during boxidation). Generation of reduced nicotinamide adenine dinucleotide in this reaction is coupled to the reduction of the tetrazolium salt INT to formazan. The intensity of the redcolored formazan is proportional to FAO activity. Briefly, 10 mg liver tissue was lysed in 200 uL lysis buffer (provided in the kit) and incubated on ice for 5 minutes, followed by centrifugation at 11,000 Â g for 5 minutes at 4 C. The supernatant was transferred to a new tube, and lysis buffer was added to adjust the protein concentration of the supernatant to 5 mg/mL. A total of 10 uL supernatant then was mixed with 50 uL blank solution or reaction solution (provided in the kit) in a 96-well plate and incubated at 37 C for 1 hour. A total of 50 mL of 3% fresh-made acetic acid solution subsequently was added to each well to stop the reaction, and the absorbance was measured at 492 nm for the calculation of formazan production (mol). FAO activity was presented as INT-formazan (mol) per gram of protein.
Production, Purification, and Validation of Anti-Autotaxin Neutralizing Antibody rmATX (42771; Immunodiagnostics, Ltd) was used as antigen for 4 consecutive immunizations of New Zealand white rabbits for 2 months. After the antibody titer in the serum reached more than 10 -6 , blood samples were taken from marginal ear veins of sedated rabbits, followed by sequential affinity purification of anti-autotaxin IgG with protein G-agarose and rmATX-conjugated sepharose 4b resin. Nonimmune rabbit IgG purified from preimmune rabbit serum was used as control. The neutralizing activity of anti-autotaxin IgG was validated by its ability to block the activity of rmATX in vitro and to inhibit rmATX-induced Erk activation in HepG2 cells (ATCC, Manassas, VA) maintained in DMEM (Gibco, Carlsbad, CA).

Generation and Titration of AAVs
The shRNA targeting autotaxin (target sequence: CCTGTACCAAATCTGACATAT) and control shRNA with scrambled sequence (CCTAAGGTTAAGTCGCCCTCG) in pAAV-EGFP-U6 vectors were purchased from VectorBuilder (Santa Clara, CA). The sequence targeting the 3' untranslated region of autotaxin mRNA was chosen from the database provided by VectorBuilder, based on the predicted high knockdown efficiency. In vitro validation for the knockdown efficiency of shRNA was conducted by transfection of shRNA targeting autotaxin or control shRNA into Hepa1-6 cells (ATCC, Manassas, VA) cultured in DMEM (supplemented with 10% FBS, 100 IU/mL penicillin, and 100 ug/mL streptomycin) and examination of the mRNA level of autotaxin at 4 days after transfection.
To generate AAV, 500 mL DMEM was mixed with 120 mL of 1 mg/mL PEI to generate solution A, while another 500 mL DMEM was mixed with 12 mg of shRNA plasmid, 10 mg pXX6, and 10 mg p5E18-VD2/8 to make solution B. After mixing solutions A and B vigorously, the PEI-DNA complex was incubated at room temperature for 20 minutes and added into the 150-mm culture dish of HEK293T cells (ATCC). Six hours after transfection, the medium was replaced with fresh complete DMEM medium. The medium of HEK293T cells was harvested 3 days after transfection. The concentration and purification of AAV were conducted following the standard protocol of AAVanced Concentration Reagent (AAV110A-1; System Biosciences, Inc, Palo Alto, CA). For the titration of AAV, 5 mL concentrated AAV was added with buffer AL (19075; QIAGEN, Inc, Santa Clarita, CA) and incubated at 55 C for 30 minutes. The mix then was proceeded to Zymoclean Gel DNA Recovery Kit (Zymo Research Corporation, Irvine, CA ). Eluted DNA was used as the amplification template for the titration of AAV by realtime PCR, and serially diluted shRNA plasmids were used to generate a standard curve showing the correlation between the cycle threshold (CT) value and the copy number of vector genome.

Statistics
For animal studies, data statistical analyses were performed using GraphPad Prism 6 (GraphPad Software, Inc La Jolla, CA). The results are expressed as means ± SEM. Animal sample size (N number) for each experiment was chosen according to previous reports of similar types of experiments. For comparison between 2 groups, statistical significance was determined by the 2-tailed Student t test. For multiple comparisons within more than 2 groups, statistical calculations were performed by 1-way analysis of variance followed by Tukey multiple comparisons using