ARRB1 inhibits non-alcoholic steatohepatitis progression by promoting GDF15 maturation

Journal Pre-proof ARRB1 inhibits non-alcoholic steatohepatitis progression by promoting GDF15 maturation Zechuan Zhang, Xiaoliang Xu, Wenfang Tian, Runqiu Jiang, Yijun Lu, Qikai Sun, Rao Fu, Qifeng He, Jincheng Wang, Yang Liu, Hailong Yu, Beicheng Sun PII:

S0168-8278(19)30716-0

DOI:

https://doi.org/10.1016/j.jhep.2019.12.004

Reference:

JHEPAT 7559

To appear in:

Journal of Hepatology

Received Date: 11 September 2019 Revised Date:

20 November 2019

Accepted Date: 3 December 2019

Please cite this article as: Zhang Z, Xu X, Tian W, Jiang R, Lu Y, Sun Q, Fu R, He Q, Wang J, Liu Y, Yu H, Sun B, ARRB1 inhibits non-alcoholic steatohepatitis progression by promoting GDF15 maturation, Journal of Hepatology (2020), doi: https://doi.org/10.1016/j.jhep.2019.12.004. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

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ARRB1 inhibits non-alcoholic steatohepatitis progression by promoting GDF15

2

maturation.

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Zechuan Zhang1,2,*, Xiaoliang Xu1,3,*, Wenfang Tian1,*, Runqiu Jiang1,*, Yijun Lu1,2,

4

Qikai Sun1, Rao Fu1, Qifeng He1, Jincheng Wang1, Yang Liu1, Hailong Yu1 and

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Beicheng Sun1,2,4,§ 1

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Nanjing University Medical School, Nanjing, China; 2

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Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical

University, Nanjing, China; 3

School of Medicine, Southeast University, Nanjing, China;

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State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,

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11

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Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of

Nanjing. *

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Authors share co-first authorship.

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Correspondence: Beicheng Sun, M.D., Ph.D.

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Department of Hepatobiliary Surgery, The Affiliated Drum Tower Hospital of Nanjing

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University Medical School, 321 Zhongshan Road, Nanjing 210008, Jiangsu Province.

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TEL: 86-25-83105892; FAX: 86-25-86560946. Email: [email protected].

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Abstract word count:

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Word count

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Tables

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Figures

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Supplementary Tables

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251 7145 (manuscript, references, figure legends)

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Supplementary Figures

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Abbreviations:

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NASH, non-alcoholic steatohepatitis; ARRB1, β-arrestin1; HFD, high-fat diet; MCD,

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methionine- and choline-deficient diet; LFD, low-fat diet; CD, chow diet; GDF15,

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growth differentiation factor 15; pro-GDF15, GDF15 precursor; NAFLD,

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non-alcoholic fatty liver disease; PPAR, Peroxisome Proliferator-Activated Receptor;

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NAS, non-alcoholic fatty liver disease activity score; IP, immunoprecipitation; Chx,

30

cycloheximide; Baf, bafilomycin; PCSK, proprotein convertase subtilisin/kexin;

31

VEGF-C,

32

receptor-α-like;

33

TEM, transmission electron microscopy; PA, palmitic acid; β-HB, β-hydroxybutyrate;

34

PACE4, Paired basic amino acid cleaving enzyme 4.

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Key words: ARRB1; GDF15; non-alcoholic steatohepatitis; GFRAL.

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Conflict of interest: The authors declared no conflict of interest.

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Financial support: This work was supported by grants from the National Key

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Research and Development Program of China (Grant Number 2016YFC0905900 to

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B.S.), State Key Program of the National Natural Science Foundation (Grant Number

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81430062, 81930086 to B.S.), National Natural Science Youth Foundation (Grant

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Number 81600487 to W.T.) and Innovative Research Groups of the National Natural

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Science Foundation (Grant Number 81521004 to B.S.). B.S. is Yangtze River Scholar

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of Distinguished Professor.

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Author contributions: Z. Zhang, X. Xu, W. Tian and R. Jiang drafted the manuscript;

vascular

6

endothelial

growth

factor

C;

GFRAL,

GDNF-family

45

Z. Zhang, X. Xu, R. Jiang, Y. Lu, Q. Sun, R. Fu, Q. He, J. Wang, Y. Liu and H. Yu

46

conducted experiments; Z. Zhang, X. Xu, R. Jiang, W. Tian and B. Sun participated in

47

research design; W. Tian and B. Sun contributed to the writing of the manuscript

48

discussing data and supervised the study; and all authors performed data analysis and

49

interpretation and read and approved the final manuscript.

50

51

Abstract

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Background & Aims: Non-alcoholic steatohepatitis (NASH) is associated with

53

dysregulation of lipid metabolism and hepatic inflammation. The causal mechanism

54

underlying NASH is not fully elucidated. We aim to investigate the role of β-arrestin1

55

(ARRB1) in the progression of NASH.

56

Methods: Human liver tissues from patients with NASH and control subjects were

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obtained to evaluate ARRB1 expression. NASH models were established in ARRB1

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knockout and wild type mice fed high-fat diet (HFD) for 26 weeks or

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methionine/choline deficient (MCD) diet for 6 weeks.

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Results: ARRB1 expression was diminished in NASH patient liver samples.

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Moreover, diminished ARRB1 levels were detected in mice NASH models. ARRB1

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deficiency accelerated steatohepatitis development in HFD-/MCD diet-fed mice

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accompanied by upregulation of lipogenic genes and downregulation of β-oxidative

64

genes. Intriguingly, ARRB1 was found to interact with GDF15 and facilitated the

65

transportation of GDF15 precursor (pro-GDF15) to Golgi apparatus for cleavage and

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maturation. Treatment with recombinant GDF15 ablated the lipid accumulation in the

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presence of ARRB1 deletion in vitro and in vivo. Re-expression of ARRB1 in the

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NASH models ameliorated the liver disease, and the effect was greater in the presence

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of pro-GDF15 overexpression. In contrast, the effect of pro-GDF15 overexpression

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alone was impaired in ARRB1-deficient mice. In addition, the severity of liver disease

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in patients with NASH was negatively correlated with ARRB1 expression.

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Conclusion: ARRB1 acts as a vital regulator in the development of NASH via

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facilitating GDF15’s translocation to the Golgi apparatus and subsequent maturation.

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ARRB1 thus is a potential therapeutic target for the treatment of NASH.

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Key words: ARRB1; GDF15; non-alcoholic steatohepatitis; GFRAL.

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Lay summary

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Non-alcoholic steatohepatitis is associated with progressing dysfunction of lipid

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metabolism and a consequent inflammatory response. Decreased ARRB1 is observed

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in patients with NASH and mouse NASH models. Deletion of ARRB1 aggravates

81

NASH in mice fed HFD and MCD diet. Furthermore, ARRB1 is responsible for the

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maturation and secretion of GDF15 by facilitating the transport of pro-GDF15 to the

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Golgi apparatus. Re-expression of ARRB1 in NASH model ameliorated the liver

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disease, and the effect was more pronounced in the presence of pro-GDF15

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overexpression, highlighting a promising strategy for NASH therapy.

86 87

Introduction

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With the dramatic changes in people’s dietary choices and life styles, metabolic

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disorders including obesity, insulin resistance, and nonalcoholic fatty liver disease

90

(NAFLD) have become a public health issue worldwide [1, 2]. Excessive nutritional

91

intake and decreased energy expenditure appear to be crucial in the pathogenesis of

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NAFLD. NAFLD comprises a spectrum of liver diseases ranging from simple fatty

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liver to non-alcoholic steatohepatitis (NASH), which can potentially progress to

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cirrhosis and liver cancer [3, 4]. NASH is associated with reprogrammed hepatic

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metabolic profiles that lead to excessive lipid accumulation in the liver and

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imbalances in lipid metabolism and lipid catabolism [5, 6]. More advanced NASH is

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associated with impaired lipid metabolism, thus leading to the accumulation of

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triglycerides and other lipids in hepatocytes [7]. Lipotoxicity in the liver is the

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primary insult that initiates and propagates damage leading to hepatocyte injury and

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resultant inflammation [8]. Hepatic lipid homeostasis is fine-tuned by a complex

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machinery comprising hormones, signaling/transcriptional pathways, and downstream

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genes associated with lipogenesis and lipolysis [9]. Although many molecular

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regulatory networks have been described, the underlying mechanisms initiating the

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metabolic rearrangement and inflammatory response underlying NASH remain

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incompletely elucidated.

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β-arrestin1 (ARRB1), originally identified as a negative regulator of G

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protein-coupled receptor signaling, has been demonstrated to function as molecular

108

scaffold that regulates cellular function by interacting with other partner proteins, and

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to be involve in multiple physiological process including immune response,

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tumorigenesis and inflammation [10-13]. ARRB1 has been found to regulate the

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NF-κB pathway in multiple inflammatory disease models [14, 15]. Our previous study

112

has shown that ARRB1 participates in the regulating hepatocellular carcinoma

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aggressiveness through mediating the desensitization and internalization of CD97 [16].

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Moreover, ARRB1 partially represses diet-induced obesity and improves glucose

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tolerance through interaction with PPARγ in preadipocytes [17]. However, the

116

regulatory roles of ARRB1 in hepatic inflammation and lipid metabolism disorder

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during the progression of NASH remain unknown.

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The regulation of energy balance in the liver and other peripheral tissues is

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influenced by humoral factors that influence various metabolic activities such as

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lipolysis and lipogenesis. Dysregulation of hormones or cytokines including leptin,

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adiponectin, and insulin are well documented to contribute to metabolic disorders and

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hepatic lipid accumulation. Thus, more comprehensive elucidation of the causal

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mechanism underlying abnormal expression of these hormones or cytokines may

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enable the development of new therapeutic approaches for NASH. GDF15 (also

125

known as macrophage inhibitor 1), is predominantly expressed in the liver and is a

126

member of the TGF-beta superfamily [18]. GDF15 is initially translated to

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pro-GDF15 in dimeric form and is subsequently cleaved and secreted as mature

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GFD15 dimers [19, 20]. Recent studies have shown that GDF15 activates AKT,

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ERK1/2, and PLCγ through binding GFRAL and through a GFRAL–RET complex

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present in cells, thus, reducing food intake, driving weight loss and enhancing glucose

131

homeostasis [18, 21-23]. In addition, GDF15 alleviates fatty acid metabolic

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dysfunction in the liver, thus indicating that the liver is the direct target organ of

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GDF15 [24]. However, the post-translational regulation of GDF15, such as the

134

maturation of pro-GDF15, and the downstream molecular mechanisms of GDF15 in

135

hepatocytes, remain to be investigated.

136

Here, our results demonstrate that ARRB1 expression is diminished in NASH

137

patient liver samples and in mouse NASH models. ARRB1 deficiency accelerates the

138

development of steatohepatitis in HFD-/MCD diet-fed mice and upregulates of

139

lipogenic genes and downregulates β-oxidative genes. Functionally, ARRB1 interacts

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with GDF15 and facilitates the transportation of pro-GDF15 to the Golgi apparatus

141

for cleavage and maturation, thereby promoting fatty acid β-oxidation and inhibiting

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de novo lipogenesis. Thus, our results collectively indicate that ARRB1 is a critical

143

regulator linking GDF15 maturation to the development of NASH.

144

145

Materials and Methods

146

Human Liver Samples

147

Human liver samples with NASH were obtained from patients with NAFLD who

148

were undergoing bariatric surgery (n=40). All liver specimens were evaluated

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independently by three experienced pathologists, who are blinded to clinical data,

150

according to the NAFLD activity score (NAS), defined as the sum of steatosis,

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inflammation and hepatocyte ballooning. Patients with a NAS score ≥ 5 were

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considered likely to have NASH. The exclusion criteria were the presence of other

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causes of liver disease, including alcohol ingestion greater than 20 g/day, chronic

154

infection with hepatitis B and/or C virus, and other liver diseases. Normal human liver

155

samples were collected from 40 patients without NASH (patients with hepatic

156

hemangioma) and were used as normal controls. The study was approved by the

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Institutional Ethics Committee of Nanjing Drum Tower Hospital. Informed consent

158

for tissue analysis was obtained before surgery. Detailed characteristics of patients

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with NASH are listed in Supplementary Table 1. All research was performed in

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compliance with government policies and the Helsinki declaration. All experiments

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were undertaken with the understanding and written consent of each subject.

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Animal Studies

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C57BL/6 mice were obtained from the Animal Research Center of Nanjing Medical

164

University. ARRB1 knockout (C57BL/6J background, Arrb1-/-) mice were a kind gift

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from Dr. Bin Wei (Shanghai University, Shanghai, China). Male wild type (WT) and

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Arrb1-/- mice 8 weeks of age were fed either low-fat diet (LFD) or HFD (Bio Serv) for

167

26 weeks. In another NASH model, male 8 weeks of age WT and Arrb1-/- mice were

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fed with MCD diet (Research Diets) or chow diet for 6 weeks. All mice were housed

169

under specific pathogen-free and controlled temperature conditions with a 12-h

170

light-dark cycle at 22°C to 24°C, and 6–10-week-old male mice were used for the

171

experiments. All animal studies were approved by the Animal Care and Use

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Committee of Nanjing Drum Tower Hospital and were carried out in accordance with

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the Guide for the Care and Use of Laboratory Animals.

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Statistical Analyses

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All data were analyzed with two-tailed Student’s t test or one-way ANOVA followed

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by post hoc t tests. Data are presented as mean ± SD unless stated. Significance was

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established at p ≤ 0.05. Statistical analysis was performed in GraphPad Prism Version

178

7.0.

179

Details on other materials and methods are provided in the Supplementary

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Materials.

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182

Results

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Patients with NASH and mouse models of NASH have low hepatic levels of

184

ARRB1.

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To investigate the involvement of ARRB1 in NASH, we first analyzed the expression

186

of ARRB1 in liver samples from 40 normal patients without NASH and 40

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pathologically diagnosed patients with NASH. Both hepatic ARRB1 mRNA and

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protein levels were significantly lower in patients with NASH than those without

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NASH (Fig. 1A-1C). Moreover, ARRB1expression was lower at both mRNA and

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protein levels in the liver of WT mice fed with HFD than LFD for 26 weeks, or MCD

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diet than CD for 6 weeks (Fig. 1D-1F). To explore the potential role of ARRB1-in

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NASH progression, we used Arrb1-/- and WT mice fed with HFD for 26 weeks or

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MCD diet for 6 weeks to construct NASH models. H&E, Oil Red O, Sirius Red and

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IHC staining revealed that the degree of steatosis, ballooning fat droplet and fibrosis

195

accumulation were aggravated in the livers of mice fed with HFD or MCD diet, and

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these effects were further exacerbated by ARRB1 deficiency (Fig. 1G). Although WT

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mice on the HFD showed nearly no liver fibrosis, the Arrb1-/- mice showed liver

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fibrosis to some extent (Fig. 1G). Significantly higher liver injury markers (ALT and

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AST) and levels of liver triglycerides (TG) were observed in Arrb1-/- mice than in WT

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mice under HFD or MCD diet (Fig. 1H). To obtain further insight into steatohepatitis,

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we evaluated the transcriptional levels of genes implicated in hepatic inflammation,

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fibrogenesis, lipogenesis and β-oxidation. ARRB1 deficiency significantly increased

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the mRNA levels of inflammatory, fibrosis and lipogenesis associated genes, but

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decreased the mRNA levels of β-oxidation associated genes (Supplementary Fig. 1A

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and 1B). Furthermore, F4/80 immunofluorescence staining showed that there were

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more macrophages infiltrated in the liver of Arrb1-/- mice than WT mice fed with HFD,

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as expected in the MCD diet induced NASH model (Supplementary Fig. 1C). In

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addition, infiltrated macrophages and neutrophils were detected by flow cytometry,

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and significantly higher levels were observed in the CD11b+F4/80+ and

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CD11b+Ly6G+ cell infiltrating fractions in the liver in mice receiving HFD or MCD

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diet treatment than in WT mice (Supplementary Fig. 1D). Together, these findings

212

indicate that ARRB1 is downregulated in the liver tissues in NASH and ARRB1

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deficiency promotes the development of steatohepatitis.

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Loss of ARRB1 induces lipid metabolism disorder and the activation of NF-κB in

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vitro.

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Studies have revealed that both hepatocytes and Kupffer cells participate in the

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regulation of steatohepatitis. To further investigate the function of ARRB1 in the

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development of steatohepatitis, we isolated primary hepatocytes and Kupffer cells for

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further study. Followed qPCR, western blot, and immunofluorescence staining

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analyses revealed that ARRB1 was not expressed in Kupffer cells (Fig. 2A–2C). Next,

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we focused on the function of ARRB1 in hepatocytes. Similarly, both the mRNA and

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protein levels of ARRB1 were downregulated after treatment with palmitic acid (PA)

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(Fig. 2D and 2E). Oil Red O staining and transmission electron microscopy (TEM)

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revealed that loss of ARRB1 resulted in more accumulation of larger lipid droplets in

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hepatocytes (Fig. 2F). Similar results were also obtained in HepG2 cells (in which

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ARRB1 was knocked out by CRISPR Cas9; Fig. 2G). The NF-κB signaling pathway

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plays a crucial role in the pathogenesis of steatohepatitis, as demonstrated by a

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previous study, in which treatment with PA promoted the phosphorylation of P65 and

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elicited recruitment of Brg1 and Brm to the promoter regions of Il1b, Il6, and Mcp1 in

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cultured hepatocyte [25]. In addition, ARRB1 has been reported to be involved in

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regulation of the NF-κB signaling pathway [26]. Therefore, we hypothesized that

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ARRB1 might participate in the activation of P65 during PA treatment. As shown in

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Supplementary Fig. 2A–2D, loss of ARRB1 augmented the protein level of p-P65,

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nuclear translocation of P65, and the transcriptional levels of Il1b, Il6, Tnfa and Mcp1

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after PA treatment. To further examine the function of ARRB1 on steatohepatitis, we

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overexpressed ARRB1 in primary hepatocytes and HepG2 cells, and found that

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overexpression of ARRB1 attenuated the accumulation of lipid droplets and inhibited

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the phosphorylation of P65 induced by ARRB1 deficiency (Fig. 2G and 2H and

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Supplementary Fig. 2E, 2F). ARRB1 overexpression also decreased the mRNA levels

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of lipogenesis associated genes and increased the mRNA levels of β-oxidation

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associated genes (Fig. 2I). Collectively, these data illustrate that loss of ARRB1

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promotes the accumulation of lipid droplets and the activation of NF-κB in

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hepatocytes.

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ARRB1 interacts with pro-GDF15 and decreases the expression of pro-GDF15 in

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cytoplasm.

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Work over the past several decades has shown that ARRB1 regulates specific cellular

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functions by interacting with specific partner proteins [27]. To explore the molecular

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mechanisms through which ARRB1 exerts its function in NASH, we performed

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co-immunoprecipitation (Co-IP) experiments and liquid chromatography tandem

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mass spectrometry (LC-MS/MS) to qualitatively analyze ARRB1 binding protein.

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LC-MS/MS analysis identified that ARRB1 interacts with GDF15 (Fig. 3A), a distant

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member of the TGF-beta superfamily [20] that is expressed predominantly in the liver

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[28] and plays a prominent role in obesity and energy metabolism [29]. Then, Co-IP

254

experiments and double immunofluorescence staining demonstrated a direct

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interaction between ARRB1 and GDF15 (Fig. 3B and 3C). To further explore which

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domain of GDF15 is essential for the interaction with ARRB1, we constructed

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His-GDF15 truncations and co-transfected them with Flag-ARRB1 into HepG2 cells.

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Immunoprecipitation results revealed that residues 1 – 196 of GDF15 were

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indispensable for binding to ARRB1 (Fig. 3D, left panel), and that this domain

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belonged to the GDF15 propeptide. Furthermore, the interaction between ARRB1 and

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GDF15 was completely abolished by mutation of residues 1–196 of Flag-GDF15 (Fig.

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3D, right panel). Interestingly, western blot analysis and immunofluorescence

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revealed that ARRB1 knockout increased the accumulation of pro-GDF15 in the

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cytoplasm, whereas ARRB1 overexpression decreased the accumulation of

265

pro-GDF15 (Fig. 3E and 3F). Similar observations were found in HepG2 cells with

266

ARRB1 knockdown via ARRB1-shRNA, in case there might have been off-target

267

effects of CRISPR/Cas9 (Fig. 3E and Supplementary Fig. 3A). However, ELISA

268

revealed that knockout or knockdown of ARRB1 decreased the secretion of mature

269

GDF15, whereas ARRB1 overexpression increased the production of mature GDF15

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in HepG2 cells (Fig. 3G and 3H). The interaction between ARRB1 and the propeptide

271

of GDF15 may promote the conversion from pro-GDF15 to mature GDF15.

272

ARRB1 enhances the transportation of pro-GDF15 to the Golgi apparatus and

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promotes the secretion of mature GDF15.

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To further clarify that ARRB1 influences the maturation of GDF15 by interacting with

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pro-GDF15 propeptide, we confirmed that neither knockout nor overexpression of

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ARRB1 altered the mRNA levels of GDF15 in HepG2 cells (Supplementary Fig. 3B

277

and 3C). Then, HepG2 cells were transfected with either pro-GDF15-His or

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pro-GDF15-His/ARRB1-Flag. Cycloheximide (Chx) or bafilomycin (Baf) was added

279

to decrease possible interference from overexpression of proteins and lysosomal

280

processing. In the presence of ARRB1, increased amounts of mature GDF15 peptide

281

was detected in cell lysates. Simultaneously, more mature GDF15 was also detected in

282

the culture medium of the co-transfected cells (Fig. 4A). Moreover, previous studies

283

have shown that pro-GDF15 is cleaved by PCSK3, PCSK5, PCSK6 and MMP26 at

284

the proconvertase cleavage site, thus generating mature GDF15 [30-32]. We found

285

that ARRB1 did not change the expression of PCSK3, PCSK5, PCSK6, and MMP26

286

(Fig. 4B and Supplementary Fig. 3D), nor did it influence the activity of PCSK3, -5

287

and -6 , on the basis of detection of concentrations of VEGF-C, the product of

288

PCSK3,-5and -6 [33](Supplementary Fig. 3E). Because studies have validated that the

289

maturation process of GDF15 occurs in the Golgi apparatus [34], we next sought to

290

detect the subcellular localization of pro-GDF15 by immunostaining. We found that

291

pro-GDF15 was abundantly distributed throughout the cytoplasm of the ARRB1

292

knockout HepG2 cells. In contrast, most of the pro-GDF15 localized in the Golgi

293

apparatus in the presence of ARRB1 overexpression (Fig. 4C). In addition, the

294

concentration of serum GDF15 was diminished in ARRB1 knockout mice, whereas

295

the concentration of serum GDF15 was elevated in mice with hepatocytes specific

296

overexpression of Arrb1 by injection of AAV-Arrb1 (Fig. 4D). Because exposure to

297

chronic high-fat or acute lysine-deficient diets increases GDF15 levels [35], we

298

detected the alterations in serum GDF15 in HFD-fed or MCD diet-fed mice. The

299

concentration of serum GDF15 was significantly decreased in Arrb1 knockout mice

300

and was clearly increased in AAV-Arrb1 injected mice (Fig. 4E and 4F). Growing

301

evidence indicates that the anorectic effects of GDF15 are mediated through GFRAL,

302

and RET is a known coreceptor for the GFRAL [21-23]. Because ARRB1 promotes

303

the maturation and secretion of GDF15 in hepatocytes, we sought to examine whether

304

ARRB1 might affect the downstream pathway of GDF15. We first validated the

305

expression of GFRAL and RET in the human liver with or without NASH by

306

immunofluorescence and western blot analysis and made similar observations in

307

primary mouse hepatocytes and HepG2 cells (Fig. 4G, 4H and Supplementary Fig.

308

3F). Next, we investigated the activation status of the downstream signaling pathways

309

of GDF15 after ARRB1 alteration. As shown in Fig. 4I, the expression of p-RET,

310

p-AKT, and p-ERK was decreased by ARRB1 knockout and was increased by

311

ARRB1 overexpression; furthermore, the phosphorylation status of RET, AKT and

312

ERK was positively correlated with GDF15 stimulation, regardless of ARRB1

313

expression. Collectively, these data supported our hypothesis that ARRB1 promotes

314

the maturation of GDF15 in vitro and in vivo and activates the downstream pathway

315

of GDF15 in the liver.

316

ARRB1 regulates fatty acid de novo lipogenesis and β-oxidation in a

317

GDF15-dependent manner.

318

Hepatocytes harvested from Arrb1-/- and WT mice fed with HFD for 26 weeks were

319

subjected to RNA sequencing analysis (n=3) (Supplementary Fig. 3G). The fatty acid

320

degradation and PPAR signaling pathway were markedly inhibited in ARRB1

321

knockout hepatocytes according to KEGG pathway enrichment analysis (Fig. 5A).

322

Additionally, Gene Ontology (GO) enrichment analysis showed that ARRB1

323

deficiency downregulated the category of lipid metabolic process and upregulated the

324

category of inflammatory response (Supplementary Fig. 3H). Previous studies have

325

indicated

326

adipogenesis related genes [36], and the GDF15 downstream molecule, p-ERK,

327

represses PPARγ transcriptional activity by inhibiting ligand binding and altering

328

cofactor recruitment [37]. Moreover, GDF15 has been reported to promote fatty acid

329

β-oxidation in the liver by upregulating PPARα [24]. Increased hepatic p-AKT levels

330

protect the liver against steatosis in diabetes [38]. Therefore, we addressed the

that

ARRB1

represses

diet-induced

obesity by down-regulating

331

question of whether ARRB1 might regulate metabolic pathways by interacting with

332

GDF15. As expected, our results revealed that ARRB1 inhibited expression of fatty

333

acid lipogenesis related proteins and promote β-oxidation associated proteins by

334

regulating GDF15 in primary hepatocytes (Fig. 5B). Similar results were obtained in

335

HepG2 cells (Supplementary Fig. 4A and 4B). Then, we used a PPARα antagonist

336

(GW6471) to inhibit PPARα activity and used the ligands of PPARα (Fenofibrate) and

337

PPARα agonist (GW7647) to stimulate PPARα activity. As shown in Fig. 5C, both

338

basal and ARRB1 overexpression or recombinant GDF15 induced expression of

339

lipogenesis associated proteins was inhibited by GW6471. In contrast, basal ARRB1

340

knockout or si-GDF15 induced downregulation of lipogenesis related proteins was

341

increased by Fenofibrate or GW7647 (Fig. 5D). Similar observations were obtained

342

from the levels of β-hydroxybutyrate (β-HB), a product of FA oxidation (Fig. 5E, 5F

343

and Supplementary Fig. 4C). Furthermore, we observed that overexpression of

344

ARRB1 or the use of recombinant GDF15 result in significantly lower TG levels and

345

liver lipid accumulation in ARRB1 knockout hepatocytes and HepG2 cells than in

346

controls (Fig. 5G and Supplementary Fig. 4D, 4E). Similar observations were found

347

regarding lipogenesis inhibition with lipogenesis inhibitors (C75 and cerulenin) (Fig.

348

5H). Together, these data suggest that ARRB1 inhibits lipogenesis and promotes

349

β-oxidation in a GDF15 dependent manner.

350

Re-expression of ARRB1 in mice alleviates HFD or MCD diet induced

351

steatohepatitis.

352

To explore whether re-expression of ARRB1 might attenuate NASH in vivo, we used

353

AAV to overexpress ARRB1 (Fig. 6A and Fig. 7A). Interestingly, various analysis of

354

HFD- or MCD diet-induced NASH livers indicated that mice with ARRB1

355

re-expression had less steatosis, inflammation, fibrosis, liver weight, serum liver

356

enzymes, hepatic TG contents, and inflammatory cytokines, but more β-HB (Fig. 6B–

357

6D and Fig. 7B, C). In addition, WT mice injected with AAV-pro-Gdf15 showed

358

improvement in steatohepatitis to some degree, whereas, no amelioration was

359

observed in ARRB1 knockout mice (Fig. 6B–6D and Fig. 7B, 7C). Similarly, lower

360

levels of serum GDF15 were observed in ARRB1 knockout mice fed with HFD or

361

MCD diet after injection with AAV-pro-Gdf15 than in WT mice (Supplementary Fig.

362

5A and 5B). Conversely, mice injected with AAV-Arrb1 and AAV-pro-Gdf15 showed

363

dramatically decreased degrees of steatohepatitis (Fig. 6B–6D and Fig. 7B, 7C).

364

Furthermore, the GDF15 Fc fusion protein markedly reversed steatohepatitis induced

365

by ARRB1 deficiency (Fig. 6B-6D and Fig. 7B, 7C). A previous study has shown

366

GDF15

367

immunofluorescence showed fewer infiltrated macrophages in livers of HFD or MCD

368

diet fed mice after injection with AAV-Arrb1 or AAV-Arrb1+AAV-pro-Gdf15

369

(Supplementary Fig. 5C and 5D). Overall, these data further imply the essential

370

function of ARRB1 in promoting the maturation of GDF15, and that ARRB1

371

overexpression in the liver protects liver against NASH pathogenesis in a GDF15

372

dependent manner.

373

The severity of liver disease in patients with NASH is negatively correlated with

374

ARRB1 expression.

protects

cell

against

macrophage-mediated

killing

[39].

F4/80

375

To explore the clinical relevance of the above animal-based observation, we divided

376

patients with NASH into two groups, an ARRB1 high expression group (20/40,

377

ARRB1high) and an ARRB1 low expression group (20/40, ARRB1low), according to

378

the median value of ARRB1 detected by qPCR. There were statistical differences

379

observed between the two groups. The degree of steatosis, fibrosis, inflammation and

380

liver lipogenesis was significantly lower, whereas β-oxidation was higher in the

381

ARRB1high group than the ARRB1low group (Fig. 8A-8C). Furthermore, Pearson

382

correlation analysis revealed that NAS, serum ALT, AST, and TG were negatively

383

correlated with ARRB1 mRNA level (Fig. 8D), Moreover, the level of mature GDF15

384

was significantly higher in the liver in the ARRB1high group than the ARRB1low group

385

(Fig. 8E). Similarly, the serum level of circulating GDF15 was higher in the

386

ARRB1high group than the ARRB1low group (Fig. 8F). Together, these data indicate

387

that the severity of liver disease in patients with NASH was negatively correlated with

388

ARRB1 expression.

389

Discussion

390

This study demonstrates that ARRB1 plays an important role in the pathogenesis of

391

NASH. The expression of ARRB1 was diminished in livers from patients or mice

392

with NASH. Moreover, deletion of ARRB1 significantly exacerbated hepatic steatosis,

393

fibrosis, and inflammation in both HFD- and MCD diet-fed mouse models.

394

Mechanistically, ARRB1 interacts with pro-GDF15 and promotes its localization in

395

the Golgi apparatus for the process of cleavage/maturation.

396

ARRB1 was originally identified and characterized on the basis of its function of

397

desensitizing activated G protein-coupled receptors (GPCRs)[10]. In addition,

398

ARRB1 is sought to function as a molecular scaffold that controls the spatiotemporal

399

distribution of partner proteins [11]. However, the mechanisms underlying the

400

regulation of downstream pathways by ARRB1 and its regulatory role in NASH

401

development remain unclear. Here, we observed diminished expression of ARRB1 in

402

NASH samples from patients and mice. Indeed, ARRB1 expression was negatively

403

correlated with the severity of liver damage in patients with NASH. Moreover,

404

genetic deletion of ARRB1 promoted NASH development in mice fed HFD or MCD

405

diets. These results indicated that ARRB1 is a potential protective factor against

406

NASH and its downregulation might be a key event during NASH development.

407

ARRB1 has been reported to be inhibited by microRNAs in glioma and breast cancer

408

[40, 41]. We did not identify the regulatory mechanism of ARRB1 downregulation in

409

this article and our further studies will aim to elucidate its expression regulation. A

410

previous study showed that both ARRB1 and ARRB2 interact with IκBα, and only

411

ARRB1, but not ARRB2, regulates the NF-κB response to TNFα [26]. We found that

412

ARRB1 deletion increased the activation of p65 in primary hepatocytes and its

413

downstream gene expression levels. Given the vital role of macrophages in

414

steatohepatitis [42], we sought to investigate whether ARRB1 deficiency might alter

415

the function of Kupffer cells. In liver with steatohepatitis, ARRB1 knockout increased

416

the infiltration of macrophages. However, our results indicated that ARRB1 is not

417

expressed in Kupffer cells. Thus, we presumed that the reasons why ARRB1

418

deficiency in the liver increases macrophage infiltration are mainly due to the damage

419

to hepatocytes and the hepatic microenvironment.

420

As a distant member of the TGF-beta superfamily, human GDF15 is a product of

421

a two exon gene, which is translated into a 308 amino acid protein including a

422

propetide of 167 amino acids and a mature domain of 112 amino acids, separated by

423

proconvertase cleavage [32, 34]. The propeptide can facilitate correct folding of

424

GDF15 [34]. We found that ARRB1 binds the propeptide of GDF15. Furthermore, the

425

metabolic effect of GDF15 is conveyed by GFRAL, whose expression is restricted to

426

the central nervous system. However, GFRAL is expressed in other organs and cell

427

lines, many of which have been reported to exhibit a biological response after

428

treatment with GDF15 [22]. GDF15 is clearly a protective factor against metabolic

429

disorders including obesity, insulin resistance and liver steatohepatitis [43-45].

430

Emerging evidence has indicated that treatment or overexpression of GDF15

431

decreases the body weight and improves metabolic profiles in mice and monkeys [29,

432

46]. Moreover, increasing studies reveal that hepatic GDF15 promotes fatty acid

433

β-oxidation and ketogenesis of the liver during fasting and is highly effective in

434

decreasing adiposity and correcting the metabolic dysfunction in mice fed with HFD

435

[24, 47]. Although HFD exposure increases GDF15 levels, the increase in GDF15

436

does not reverse obesity [35]. Nevertheless, deletion of GDF15 increases body weight,

437

levels of serum TNF-α and IL-6, and inflammatory changes within liver tissue [46,

438

48]. Therefore, we speculated that the increase in GDF15 might not be sufficient to

439

decrease body weight by reducing food intake, but this aspect of GDF15 may play an

440

indispensable role in balancing energy metabolism. Most studies in animal models of

441

inflammation strongly suggest that GDF15 has an overall beneficial effect on disease

442

outcomes. In our work, we firstly demonstrated that ARRB1 interacts with

443

pro-GDF15 in the cytoplasm and promotes the secretion of mature GDF15.

444

Furthermore, RNA sequencing results revealed that loss of ARRB1 upregulates

445

inflammatory genes and downregulates lipid metabolic processes. We propose that

446

ARRB1 participates in hepatic lipid metabolism by regulating GDF15.

447

Similar to many other proteins, GDF15 is regulated at the levels of transcription,

448

translation, and maturation. Inhibition of PACE4 decreases the serum level of mature

449

GDF15 but increases the intracellular levels of pro-GDF15 [32], mainly because

450

PACE4 and PCSK isoforms are involved in GDF15 cleavage to produce mature

451

GDF15 [31]. However, whether other mechanisms might function in the

452

post-transcriptional regulation of GDF15 remains unclear. Here, we observed that

453

ARRB1 increased the transport of pro-GDF15 to the Golgi apparatus and our data

454

indicated that ARRB1 deficiency impeded GDF15 secretion via inhibiting the

455

maturation of pro-GDF15 both in vitro and in vivo. As p-ERK is activated by

456

GFRAL-RET complex, loss of ARRB1 downregulates the level of p-ERK and p-AKT.

457

Therefore, the ARRB1-GDF15-GFRAL-RET axis can significantly alter the fatty acid

458

de novo lipogenesis and β-oxidation in hepatocytes. Importantly, we observed that

459

simultaneous overexpression of ARRB1 and GDF15 improved hepatic steatosis,

460

fibrosis, and inflammation, however, the promoting effect of GDF15 was not

461

observed in ARRB1 knockout mice. We speculated that the main reason for these

462

findings might be that pro-GDF15 was not transported to the Golgi apparatus for

463

cleavage and production of mature GDF15 in the absence of ARRB1. This function

464

may help to determine the precise regulation of GDF15 in post-translational level and

465

may explain why overexpression of ARRB1 alleviates diet-induced steatohepatitis.

466

In summary, our findings summarized in the graphical abstract indicate that

467

ARRB1 plays an essential role in the maturation of GDF15 and the pathogenesis of

468

steatohepatitis. Thus, we propose ARRB1 as a potential therapeutic target for

469

counteracting the development of NASH by using an ARRB1 agonist or manipulating

470

the expression of ARRB1 combined with GDF15.

471

472

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473

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609

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612

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614

615

Figure legends

616

Fig. 1. ARRB1 is downregulated in the liver in patients with NASH or mouse

617

NASH models. (A) ARRB1 mRNA levels in the liver tissues of human NASH (n =

618

40) compared with normal controls (n = 40), as determined by qPCR (*P < 0.05 as

619

indicated). (B) ARRB1 protein in liver extracts from people with NASH compared

620

with normal control as indicated by Western blot. The quantification of bands

621

represented in a bar graph (n = 6/group, *P < 0.05 as indicated). (C) ARRB1

622

immunostaining in liver sections of normal individuals and patients with NASH.

623

Scale bars:100 µm. (D-F) The mRNA and protein levels of ARRB1 were detected in

624

the liver in WT mice fed with HFD compared with LFD, or MCD diet compared with

625

CD (n = 6/group, *P < 0.05 as indicated). (G) Paraffin-embedded liver sections were

626

stained with H&E, Oil Red O, Sirius Red, and α-SMA immunostaining. Scale bars:

627

200 µm. (H) Graphs showed the liver weight and serum ALT, AST, and hepatic TG (n

628

= 6/group, *P < 0.05 as indicated). Data are expressed as mean ± SD. For A, B, D, and

629

H, significance determined by Student’s two-tailed t test.

630

631

Fig. 2. Loss of ARRB1 exacerbates lipid metabolism dysfunction in vitro. (A and

632

B) Expression levels of ARRB1 in mouse liver hepatocytes and Kupffer cells were

633

measured by qPCR and western blot (*P < 0.05 as indicated). (C) Representative

634

immunofluorescence staining showing the expression of ARRB1 (green) and F4/80

635

(red) in the mouse liver. Scale bars: 25 µm. (D and E) Expression of ARRB1 mRNA

636

and protein levels in primary hepatocyte treated with 0.2 mM palmitic acid

637

(Representative of three independent experiments, *P < 0.05 as indicated). (F)

638

Representative Oil Red O staining and TEM in primary hepatocytes after addition of

639

palmitic acid for 48 hours. (G) Representative Oil Red O staining and TEM in HepG2

640

cells by treated with 0.4 mM palmitic acid for 48 hours. (H) Representative Oil Red O

641

staining and TEM images in primary hepatocytes. (LD, lipid droplets; N, nuclei).

642

Scale bars: 50 µm in Oil Red O staining and 2 µm in TEM images. (I) Expression

643

levels of genes involved in de novo lipogenesis and β-oxidation were measured by

644

qPCR (Representative of three independent experiments, *P < 0.05 versus WT +

645

Vector group; #P < 0.05 versus Arrb1-/- + Vector group). All data are presented as the

646

mean ± SD; significance determined by Student’s two-tailed t test (A, D and E) and

647

one-way ANOVA (I).

648

649

Fig. 3. ARRB1 interacts with GDF15 precursor. (A) Mass spectrum showing the

650

structural diagram of the GDF15 protein. (B) Co-IP assays showed that ARRB1

651

interacted with GDF15 precursor. Immunoprecipitations were performed by using

652

anti-ARRB1 (left panel) and anti-GDF15 (right panel). (C) Representative double

653

immunofluorescence staining images showing the co-localization of ARRB1 (green)

654

and GDF15 (red) proteins in HepG2. Scale bars: 25 µm. (D) HepG2 cells were

655

co-transfected

656

Immunoprecipitations were performed with using anti-Flag antibodies to identify the

657

individual binding sites of GDF15 (left panel). HepG2 cells were co-transfected with

658

Flag-ARRB1, His-GDF15, or lentiviral vector carrying mutant GDF15 (His-GDF15

659

197-308 aa mut) and/or the mutant GDF15 (His-GDF15 1-196 aa mut) (right panel).

660

(E) Protein levels of ARRB1 and GDF15 were measured in HepG2 cells. (F)

with

Flag-ARRB1

and

different

His-GDF15

truncations.

661

Representative immunofluorescence staining showing the relationship between the

662

expression of ARRB1(green) and GDF15 (red) in the cytoplasm of HepG2 cells.

663

Scale bars: 50 µm. (G) The concentration of GDF15 in HepG2 culture supernatant

664

was measured by ELISA (n = 6/group, *P < 0.05 versus CRISPR-CON group; #P <

665

0.05 versus Vector group). (H) The concentration of GDF15 in HepG2 culture

666

supernatant was measured by ELISA (n = 6/group, *P < 0.05 versus shNC group). All

667

data are presented as the mean ± SD; significance determined by Student’s two-tailed

668

t test (G and H).

669

670

Fig. 4. ARRB1 promotes transportation of pro-GDF15 to the Golgi apparatus for

671

cleavage and maturation. (A) Representative images showed that ARRB1 promoted

672

the intracellular maturation of pro-GDF15. The scheme of transfections and chemical

673

treatments of HepG2 cells is shown at the top. Baf, bafilomycin; Chx, cycloheximide.

674

(B) The protein levels of PCSK3, PCSK5, PCSK6, and MMP26 were measured in

675

HepG2 cells (Representative of three independent experiments). (C) Confocal

676

microscopy images of Giantin (green) and ARRB1(red) in HepG2 cells. Scale bars:

677

25 µm. (D–F) The concentration of serum GDF15 in mice treated with SCD, HFD or

678

MCD. WT mice were injected with AAV-Arrb1 to improve the expression of ARRB1

679

(n = 6/group; *P < 0.05 versus WT group; #P < 0.05 versus AAV-CON group). (G)

680

Representative immunofluorescence staining showing the GFRAL expressed in

681

mouse hepatocytes and HepG2 cells. Scale bars: 25 µm. (H) Expression of GFRAL

682

was measured by western blot. (I) Representative images showing the expression

683

levels of downstream pathway of GDF15. All data are presented as the mean ± SD;

684

significance determined by Student’s two-tailed t test (B–F).

685

686

Fig. 5. ARRB1 regulated fatty acid de novo lipogenesis and β-oxidation in a

687

GDF15-dependent manner. (A) KEGG analysis showed the top 20 pathway

688

enrichment in the Arrb1-/- group versus WT group. (B) Representative images

689

showing the expression levels of fatty acid de novo lipogenesis and β-oxidation

690

markers after ARRB1 knockout or overexpression in primary hepatocytes.

691

Recombinant mouse GDF15 (10 nM) or Fc fusion GDF15 (100 ng/ml) was used to

692

stimulate cells. (C) Expression of de novo lipogenesis markers in HepG2 cells either

693

treated with PPARα antagonist GW6471 (1 µM) or untreated. (D) Expression of de

694

novo lipogenesis markers in the indicated cells treated with the PPARα agonist

695

Fenofibrate (5 µM) or GW7647 (100 nM), or not treated. (E) Measurement of β-HB

696

level in HepG2 cells. (*P < 0.05 as indicated). (F) Measurement of β-HB levels in

697

HepG2 cells. (*P < 0.05 as indicated). (G) TG levels were measured in hepatocytes of

698

WT and Arrb1-/- mice transfected with lentiviral ARRB1 or recombinant mouse

699

GDF15 (*P < 0.05 as indicated). (H) TG levels were measured in hepatocytes of WT

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and Arrb1-/- mice treated with FASN inhibitors, C75 (10 µg/ml) and cerulenin (10

701

µg/ml), or not treated. (*P < 0.05 versus WT-DMSO group; #P < 0.05 versus Arrb1-/-

702

-DMSO group). n = 3 independent experiments for the in vitro study. All data are

703

presented as the mean ± SD; significance determined by one-way ANOVA (E–H).

704

705

Fig. 6. Re-expression of ARRB1 alleviated steatohepatitis in mice fed with HFD.

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(A) Schematic showing the administration protocol for AAV-CON, AAV-ARRB1,

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AAV-pro-GDF15, AAV-ARRB1 + AAV-pro-GDF15, and Fc-GDF15 in HFD-fed mice

708

for experiments shown in (B–D). (B) Paraffin-embedded liver sections were stained

709

with H&E, Oil Red O, Sirius Red, and α-SMA immunostaining. Scale bars: 200 µm.

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(C) Graphs show levels of NAS, liver weight, serum ALT, AST, and hepatic IL-1β,

711

TNF-α and MCP-1 secretion. (D) TG levels were measured in liver extracts (left

712

panel), and β-HB was measured in the serum (right panel). For C and D, n = 6/group;

713

*P < 0.05 as indicated; #P < 0.05 versus WT mice fed with HFD; $P < 0.05 versus

714

Arrb1-/- mice fed with HFD. All data are presented as the mean ± SD; significance

715

determined by one-way ANOVA (C and D).

716

717

Fig 7. Re-expression of ARRB1 in mice on MCD diet alleviated steatohepatitis.

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(A) Schematic showing the administration protocol for AAV-CON, AAV-Arrb1,

719

AAV-pro-Gdf15, AAV-Arrb1 + AAV-pro-Gdf15 and Fc-GDF15 in MCD diet-fed mice

720

for experiments shown in (B and C). (B) Paraffin-embedded liver sections stained

721

with H&E, Oil Red O, Sirius Red, and α-SMA immunostaining. Scale bars: 200 µm.

722

(C) Graphs showed levels of NAS, serum ALT, AST, hepatic IL-1β, TNF-α and

723

MCP-1 secretion (n = 6/group; *P < 0.05 as indicated; #P < 0.05 versus WT mice fed

724

with MCD diet; $P < 0.05 versus Arrb1-/- mice fed with MCD diet). All data are

725

presented as the mean ± SD; significance determined by one-way ANOVA (C).

726

727

Fig 8. The severity of liver disease in patients with NASH was negatively

728

correlated with ARRB1 expression. (A) ARRB1 immunostaining, H&E and

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Masson-trichrome staining in liver sections of normal individual and patients with

730

NASH. Scale bars: 200 µm. (B) Representative graphs showing FASN and CPT1A

731

(green and red) in liver sections of normal and NASH livers. Scale bars: 50 µm. (C)

732

Representative immunofluorescence staining showing CD11b and CD68 (green and

733

red; left panel) and CD3 and α-SMA (green and red; right panel). (D) Graphs show

734

the correlation between ARRB1 mRNA levels and NAS, levels of serum ALT, AST,

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and TG in patients with NASH. (E) GDF15 precursor and mature form were detected

736

in human liver extracts by western blot. (F) GDF15 circulating levels in 40 healthy

737

volunteers, 20 patients with NASH (ARRB1low) and 20 patients with NASH

738

(ARRB1high) (*P < 0.05 versus Normal group; #P < 0.05 versus NASH ARRB1low

739

group). For D, data are presented by Pearson correlation coefficient. For F, data are

740

presented as the mean ± SD; significance determined by one-way ANOVA.

Highlights


ARRB1 is downregulated in NASH samples from both patients and mouse models.




ARRB1 deficiency accelerates NASH development by increasing lipogenesis and decreasing β-oxidation.




ARRB1 protects against NASH by facilitating GDF15 precursor maturation and secretion.




ARRB1 may serve as a novel potential target for NASH treatment.