Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition

Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition

Accepted Manuscript Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition Giang Nguyen, So...

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Accepted Manuscript Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition Giang Nguyen, So Young Park, Cong Thuc Le, Won Sun Park, Dae Hee Choi, EunHee Cho PII:

S0006-291X(17)32543-3

DOI:

10.1016/j.bbrc.2017.12.143

Reference:

YBBRC 39143

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 20 December 2017 Accepted Date: 23 December 2017

Please cite this article as: G. Nguyen, S.Y. Park, C.T. Le, W.S. Park, D.H. Choi, E.-H. Cho, Metformin ameliorates activation of hepatic stellate cells and hepatic fibrosis by succinate and GPR91 inhibition, Biochemical and Biophysical Research Communications (2018), doi: 10.1016/j.bbrc.2017.12.143. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

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Metformin ameliorates activation of hepatic stellate cells and hepatic

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fibrosis by succinate and GPR91 inhibition.

Giang Nguyen, 1So Young Park, 1Cong Thuc Le, 2 Won Sun Park, 1Dae Hee Choi*, 1Eun-

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Hee Cho*

Department of Internal Medicine, School of Medicine, Kangwon National University, Korea

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Department of Physiology, Kangwon National University School of Medicine, Chuncheon, Republic of Korea.

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*DHC and E-HC are joint corresponding authors Address correspondence to:

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Eun-Hee Cho

Department of Internal Medicine, School of Medicine, Kangwon National University, 26

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Kangwondaehak-gil, Chuncheon-si, Gangwon-do, 200-701, Korea Tel:+82-33-258-9167

Fax:+82-33-258-2455

E-mail:[email protected]

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ABSTRACT Background: Chronic liver disease is becoming a major cause of morbidity and mortality worldwide. During liver injury, hepatic stellate cells (HSCs) trans-

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differentiate into activated myofibroblasts, which produce extracellular matrix. Succinate and succinate receptor (G-protein coupled receptor91, GPR91) signaling pathway has now emerged as a regulator of metabolic signaling. A previous study

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showed that succinate and its specific receptor, GPR91, are involved in the activation

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of HSCs and the overexpression of α-smooth muscle actin (α-SMA).

Metformin, a well-known anti-diabetic drug, inhibits hepatic gluconeogenesis in the liver. Many studies have shown that metformin not only prevented, but also reversed, steatosis and inflammation in a nonalcoholic steatohepatitis (NASH) animal model.

has not been clarified. Methods:

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However, the role of metformin in HSC activation and succinate-GPR91 signaling

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The immortalized human HSCs, LX-2 cells, were used for the in vitro study. For the in vivo study, male C57BL/J6 mice were randomly divided into 3 groups and were

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fed with a methionine- choline-deficient diet (MCD diet group) as a nonalcoholic steatohepatitis (NASH) mouse model with or without 0.1% metformin for 12 weeks, or were fed a control methionine- choline-sufficient diet (MCS diet group). Results: In our study, metformin and 5-aminoimidazole-4-carboxamide 1-β-Dribofuranoside (AICAR), which is an analog of adenosine monophosphate, were shown to suppress α-SMA expression via enhanced phosphorylation of AMP2

ACCEPTED MANUSCRIPT activated protein kinase (AMPK) and inhibition of succinate-GPR91 signaling in activated LX-2 cells induced by palmitate- or succinate. Metformin and AICAR also reduced succinate concentration in the cell lysates when LX-2 cells were treated with

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palmitate. Moreover, metformin and AICAR reduced interleukin-6 and, transforming growth factor-β1 production in succinate-treated LX-2 cells. Both metformin and AICAR inhibited succinate-stimulated HSC proliferation and cell migration.

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Mice fed a MCD diet demonstrated increased steatohepatitis and liver fibrosis compared to that of mice fed control diet. Metformin ameliorated steatohepatitis,

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liver fibrosis, inflammatory cytokine production and decreased α -SMA and GPR91expression in the livers of the MCD diet- fed mice.

Conclusion: This study shows that metformin can attenuate activation of HSCs by activating the AMPK pathway and inhibiting the succinate-GPR91 pathway.

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Metformin has therapeutic potential for treating steatohepatitis and liver fibrosis.

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Keywords: Metformin; Liver fibrosis; Succinate; GPR91.

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1. INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is a wide spectrum of liver diseases ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and

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ultimately hepatocellular carcinoma. The prevalence of NAFLD has increased quickly in parallel with the marked increase in obesity and diabetes [1-2]. About 3% of patients with NAFLD progress to NASH [3]. Persistent NASH can lead to fibrosis

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and subsequently progress to cirrhosis, liver failure, and hepatocellular carcinoma. Unfortunately, the molecular mechanisms leading to NASH -fibrosis remain unclear,

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and there is currently no effective anti-fibrosis treatment [4-5].

Hepatic stellate cells (HSCs) are non-parenchymal cells that localize in the space of Disse. They were first described by Carl-von Kupffer in 1876 [6] and are the main collagen-producing cells in the liver [7]. In normal liver, HSCs constitute

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approximately 8% of total hepatic cells and are the primary site for vitamin A storage in the body [8-9]. Following liver injury, quiescent HSCs can undergo a phenotypic

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transformation from retinoid storage cells into highly proliferative and contractile myofibroblasts, with an increased expression of α -smooth muscle actin (α-SMA)

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and a loss of cytoplasmic vitamin A storage [9]. Metformin has been widely used as a first-line hypoglycemic agent in type 2 diabetes for more than 60 years [10-11]. In addition, there is increasing evidence suggesting that metformin also has strong anti-inflammatory, anti-oxidant and anti-tumor activities [12-14]. Metformin has been shown to ameliorate hepatic steatosis by reducing hepatocyte fat deposition and inflammation in a mouse model of diet1

ACCEPTED MANUSCRIPT induced obesity [15]. In another study, treatment with metformin significantly attenuated CCl4-induced liver fibrosis with suppression of transforming growth

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factor-β1 (TGF-β1) expression and inhibition of Smad3 phosphorylation [16].

Succinate, an intermediate of the citric acid cycle, is converted to fumarate by succinate dehydrogenase (SDH) [17, 18]. Besides the pivotal role of succinate in

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energy metabolism, it also plays a role in molecular signaling by binding to and

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activating its specifics G-protein coupled receptor, known as GPR91 [19- 20]. Concerning the role of succinate in the liver, succinate accumulation and GPR91 overexpression are important pathological hallmarks of further hepatic fibrosis [21]. Additionally, adeno- associated virus-mediated RNA knockdown of GPR91 gene

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expression in methionine- choline- deficient (MCD) diet- fed mice ameliorated steatohepatitis and fibrosis significantly [22]. However, there was no data on the relationship between metformin and succinate -

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GPR91 signaling in HSCs. The goals of this study are to investigate whether metformin or AICAR mitigates the activation of HSCs by regulating succinate -

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GPR91 signaling using LX-2 cells and an MCD diet - induced mouse model of NAFLD.

2. MATERIALS AND METHODS

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Materials

Palmitate,

succinate,

metformin,

and

5-aminoimidazole-4-carboxamide-1-4-

2.2.

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ribofuranoside (AICAR) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture

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LX-2 cells, an immortalized human HSC line, were kindly provided by Prof. Ja June Jang, Seoul National University, Korea. LX-2 cells were maintained in Dulbecco's

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modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin) at 37°C and, 5% CO2.

Animal experiments

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2.3.

The animal studies were approved by The National Kangwon University Animal Care and Use Committee. Male C57BL/J6 mice, 10-week-old and weighing 24–26 g,

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were purchased from Doo Yeol Biotech (Seoul, Korea). All of the mice were housed

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at ambient temperature (22 ± 1°C) with a 12/12-h light/dark cycle and with free access to water and food in The National Kangwon University animal care facility. They were randomly divided into 3 groups: 1) the Control group (n = 9) that was fed control methionine- choline-sufficient (MCS) diet, 2) the NASH model group (n = 12) that was fed MCD diet, and 3) the treatment group (n = 11) that was fed MCD diet mixed with 0.1% metformin. The study duration was 12 weeks.

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Cell migration assessment by wound healing assay

The cell migration capability was evaluated using a scratch test. First, LX-2 cells were seeded (5×105 cells) into 2 mL media per well of a 6-well plate. The cells were

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grown to confluence, and 1 µg/mL mitomycin C was added to prevent cell division. After incubation for 30 min, a scratch was made in each well using a pipette tip. Subsequently, the cells were washed gently with phosphate-buffered saline (PBS),

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and 1.6 mM succinate, 1 mM AICAR, or 1 mM metformin was added to the respective wells. An image was captured at time point 0. The cells were then

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incubated at 37 ̊°C in 5% CO2 and images were acquired after 16 h. Olympus cellSens standard imaging was used to quantify the results.

Proliferation assay

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2.5.

LX-2 cells were seeded (2.5×103 cells/well) in a 96-well plate and incubated at 37°C

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and, 5% CO2 for 24 h. The media was changed to DMEM containing 1% FBS. The cells were incubated at 37°C and 5% CO2 for 2 h, 1.6 mM succinate, 1 mM AICAR,

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or 1 mM metformin was added to the respective wells, and the incubation was continued for 24 and 48 h. Ten microliters of EZ-Cytox reagent (DoGen, Korea) was added to each well and the cells were incubated for 1 h. The plates were shaken gently for 1 min and the absorbance at 450 nm was measured using a plate reader.

2.6.

Western Blot Analysis 4

ACCEPTED MANUSCRIPT Mouse liver tissues and human LX-2 cell extracts were prepared on ice with RIPA lysis buffer (Cell Signaling Technology) containing protease inhibitors (Thermo Scientific, USA) on the ice. The total protein amount was measured by bicinchoninic

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acid (BCA) protein assay. An equal amount of protein from each lysate was electrophoresed on a 10% polyacrylamide gel by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-/PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes. After that, the membranes were blocked

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with 5% skim milk in Tris-buffered saline with 0.1% Tween® 20 (TBST) for 1 h at

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room temperature, washed and then incubated with primary antibodies at appropriate dilutions at 4°0C overnight. Primary antibodies included those specific to α-SMA (ab5694, Abcam), GPR91 (NBP1-00861, Novus Biologicals, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, (GTX627408, GeneTex), AMP-activated

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protein kinase alpha (AMPKα, 2532, Cell Signaling Technology), pPhosphoAMPKα (Thr172) (2535, Cell Signaling Technology), interleukin-6 (IL-6, (sc-32296, Santa Cruz Biotechnology), and TGF-β1 (GTX110630, GeneTex). The membranes

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were washed, incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature, and then washed again. The bands were visualized with Westsave

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Star Detection Reagent solution (AbFrontier, Seoul, Korea) and the bio-rad Chemidoc XRS system (Bio-Rad Laboratories, Inc., Hercules, California, U.S.A).

2.7.

Succinate Assay

Succinate levels were measured with a succinate colorimetric assay kit (BioVision, 5

ACCEPTED MANUSCRIPT California, USA) according to the manufacturer’s instructions. The samples were added into a 96-well plate and mixed with the provided reaction solution. The resultant mixtures were incubated at 37°C for 30 min. The absorbance at 450 nm was

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measured. The assay was performed in quadruplicate with the LX-2 cells and the murine samples.

Hematoxylin and -eosin (H&E) stain and Masson’s trichrome stain

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2.8.

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Samples of mouse liver were fixed in 4% (w/v) paraformaldehyde. After dehydration through a graded series of ethanol solutions, the tissues were embedded in paraffin wax. Serial frontal sections were cut at 5-µm intervals and stained with (H&E) and Masson’s trichrome for histopathology and ECM content, respectively, and then

Statistical Analyses

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2.9.

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visualized by light microscopy.

In vitro experiments were repeated at least three times. All results are expressed as

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the means ± S.D. for cell studies, and as the means ± S.E.M. for in vivo studies. Statistical comparisons between two groups were performed using a 2–tailed unpaired Student’s t test. Differences were considered statistically significant when P < 0.05, P < 0. 01, or P < 0.001.

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3. RESULTS

Metformin ameliorates HSCs activation through the AMPK and GPR91

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3.1.

pathway.

To investigate whether metformin and AICAR could impede the activation of HSCs

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by reducing succinate levels, LX-2 cells were activated for 24 h with 300 µM palmitate either with or without 1 mM AICAR or 1 mM metformin; the intracellular

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concentrations of succinate were then measured. Succinate levels were increased in palmitate-treated cells compared to that in control cells, and metformin and AICAR significantly reduced this palmitate–induced increase (Figure 1A). To directly investigate the influence of metformin and AICAR on the inhibition of palmitate-

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and succinate-induced HSCs activation, LX-2 cells were treated with 1.6 mM succinate or 300 µM palmitate and co-treated with 1 mM metformin or 1 mM AICAR for 24 h. Succinate and palmitate treatment significantly increased α-SMA

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and GPR91 expression in LX-2 cells compared to that of the control. However, αSMA and GPR91 protein expression were attenuated in LX-2 cells treated with

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metformin or AICAR in the presence of palmitate and or succinate. Moreover, metformin and AICAR treatments increased AMPK phosphorylation (Figure 1B and 1C). Taken together, succinate and palmitate leads to HSC activation, by direct activation of GPR91, and phosphorylation of AMPK by metformin or AICAR may block HSCs activation by inhibiting the succinate-GPR91 pathway.

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Metformin attenuates activated HSC proliferation, migration, and

inflammatory cytokine expression. During liver fibrosis, the activation, proliferation, and migration of HSCs occur

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rapidly in response to various stimuli present in the extracellular environment, and inflammatory cytokines are released during liver injury. To determine whether metformin attenuates succinate-induced HSC activation, we exposed LX-2 cells to

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1.6 mM succinate, with or without 1 mM metformin or 1 mM AICAR, and tested cells migration, proliferation, and inflammatory cytokines expression. Succinate

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significantly increased proliferation (Figure 2A) and migratory distance (Figure 2B) of LX-2 cells compared with that of untreated control cells. Particularly, metformin and AICAR ameliorated HSC migration and proliferation that was induced by succinate treatment (Figure 2A and 2B). Additionally, metformin or AICAR

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treatment increased p-AMPK and lowered the expression of IL-6 and TGF-β1 in succinate-induced LX-2 cells (Figure 2C). These results indicate that metformin and AICAR significantly ameliorate succinate-enhanced cell migratory capacity,

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proliferation, and inflammatory cytokine expression.

3.3.

Metformin decreases α-SMA and GPR91 overexpression in the liver and

improves steatohepatitis and liver fibrosis in MCD diet- fed mice. To examine the protective role of metformin in liver fibrosis in vivo, metformin was administered for 12 weeks to mice that were fed MCD diet as a model of NAFLD. Body weight change from baseline to final had no significant different between MCD 8

ACCEPTED MANUSCRIPT groups and MCD diet and metformin treated groups (-3.32±0.23g vs -3.05±0.59g, P = 0.096) after 12 weeks of metformin treatment. Amount of food consumption was also unchanged between MCD groups and MCD+ Met groups (2.19±0.51 g/day vs

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2.36±0.56, P=0.083). Metformin treatment prevented hepatic steatosis and fibrosis in the MCD diet – induced mouse model, as assessed by H&E and Masson’s- trichrome stain (Figure

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3A). Additionally, metformin administration also dramatically decreased succinate concentrations in the liver lysates and plasma compared with that of the MCD diet

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group, which showed significantly higher succinate levels than the control MCS diet group did (Figure 3B). The western blotting results show that MCD diet enhanced the expression of inflammatory cytokines, such as IL-6 and TGF-β1, and metformin administration decreased inflammatory cytokine production in the MCD diet-induced

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NAFLD mouse model (Figure 4A). Particularly, the MCD diet enhanced the expression of α-SMA and GPR91; however, metformin treatment increased AMPK phosphorylation and suppressed α-SMA protein expression by inhibiting GPR91

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expression in the liver (Figure 4B). Therefore, our results suggest that metformin activates the AMPK pathway and reduces liver steatosis and fibrosis by inhibiting the

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succinate-GPR91 pathway.

DISCUSSION

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ACCEPTED MANUSCRIPT In this study, we showed that metformin, which is a well-known AMPK activator, can be a potential therapeutic agent for steatohepatitis and liver fibrosis by inhibiting HSCs activation via suppressing the succinate-GPR91 signaling pathway in the

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MCD diet-induced mouse model.

Succinate, a crucial metabolic intermediate in several metabolic pathways,

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accumulates in extracellular spaces under pathological conditions, such as

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hyperglycemia or hypoxia [23]. High concentrations of succinate have been detected in the urine, plasma, and cerebral white matter of patients with metabolic diseases [19]. Dysfunction of SDH, an enzyme complex participating in both the citric acid cycle and electron transport chain, leads to succinate accumulation in the mitochondria and secretion outside the cell. In a prior study, increasing succinate

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levels were observed in the plasma, isolated hepatocytes, and isolated HSCs of mice fed with the MCD diet, compared to that of control mice, suggesting both systemic and local influence of succinate in HSC activation [21]. In another study, MCD diet-

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fed mice had elevated succinate levels and GPR91 overexpression, and knockout of

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GPR91 in the MCD diet- fed mice led to attenuation of steatosis and fibrosis [22]. Therefore, increasing levels of succinate can lead to hepatic stellate cell activation and can contribute to liver damage through the succinate-GPR91 pathway. To protect the liver and inhibit HSCs activation, reducing succinate concentrations and inhibiting GPR91 expression are our goals. In our current study, we obtained similar results to those of previous studies, in which palmitate treatment increased intracellular succinate concentrations in LX-2 cells [21]. Moreover, our study 10

ACCEPTED MANUSCRIPT showed that metformin or AICAR attenuated the palmitate-induced increased succinate levels in the lysates of LX-2 cells. These results suggest that metformin ameliorates activation of HSCs when they are activated by palmitate under stress

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conditions. Additionally, when we activated AMPK by metformin or AICAR treatment of LX-2 cells, both agents attenuated the upregulation of α-SMA and GPR91 induced by palmitate or succinate. Taken together, this suggests that

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succinate triggers α-SMA production through GPR91 activation in HSCs, and that AMPK activation by AICAR or metformin treatment decreases succinate levels,

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resulting in reduced α-SMA and GPR91 upregulation.

In normal liver, HSCs maintain a non-proliferative, quiescent phenotype. Following liver injury or culture in vitro, HSCs become activated, trans-differentiating from

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vitamin-A-storing cells to myofibroblasts, which are proliferative, contractile, chemotactic, and secrete inflammatory cytokines [24]. It has been shown that stimulation of HSCs with platelet-derived growth factor (PDGF), increases their

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mobility and proliferation, but reagents that activate AMPK, such as adiponectin,

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AICAR, and metformin, reduce the activity of HSCs [25]. TGF-β1 plays a crucial role in fibrosis, mediating a cross-talk between parenchymal, inflammatory, and collagen expressing cells. TGF-β1, derived from activated Kupffer cells and sinusoidal endothelial cells, causes apoptosis of hepatocytes, stimulates activation and recruitment of inflammatory cells into injured liver, and induces differentiation of liver- resident cells (e.g., fibroblasts, HSC, and epithelial cells) into myofibroblasts. In turn, activated HSCs themselves can secrete TGF-β1, increasing 11

ACCEPTED MANUSCRIPT hepatocyte damage and lymphocytes infiltration [26]. Compelling evidence indicates that the interactions between endotoxin and HSCs can play pivotal roles in liver pathogenesis. Endotoxin-induced release of multifunctional mediators and pro-

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inflammatory cytokines, such as IL-6, by HSCs could be a significant mechanism of liver pathology [27]. We hypothesize that succinate leads to HSC activation by increasing cells proliferation, and migration, and by releasing inflammatory

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cytokines. In our present study, we found that, stimulation of LX-2 cells with succinate led to increased migratory capacity and proliferation. In addition, succinate

compared to that of controls.

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enhanced inflammatory cytokines production of HSCs, such as IL-6 and TGF-β1,

In the in vivo experiment, we also confirmed that the administration of metformin

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ameliorated liver steatohepatitis and fibrosis; moreover, metformin decreased succinate concentrations in both the plasma and liver lysates. Considering this, we have demonstrated that the administration of metformin activates AMPK and

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decreases the expression of α-SMA and GPR91 in the liver of MCD diet-induced

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NASH by reducing the production of succinate.

In the future, additional studies are needed to elucidate the exact mechanism connecting the AMPK and succinate-GPR91 signaling pathways. Downstream pathways of succinate and GPR91 signaling, such as hypoxia-inducible factors- -1α (HIF-1α) and, extracellular signal-regulated kinases (ERK) pathways, in metformin’s 12

ACCEPTED MANUSCRIPT protective effect on HSC proliferation, fibrosis, and inflammatory responses should be clarified.

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In conclusion, the present study provides evidence to support the beneficial effect of metformin on reducing steatohepatitis and hepatic fibrosis of NAFLD via succinateGPR91 inhibition. This research demonstrates that AMPK activators, such as

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metformin or AICAR, ameliorate HSC activation by inhibiting the succinate –

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GPR91 signaling pathway, thereby decreasing migration, proliferation, and inflammatory responses of HSCs. This suggests a therapeutic role for the treatment and/or prevention of NAFLD.

ACKNOWLEDGMENTS

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5.

This work was supported by NRF-2016R1C1B2011968 and 2016 Kangwon National

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University Hospital Grant.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

7.

AUTHOR CONTRIBUTIONS

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Giang Nguyen performed experiments and wrote the manuscript, Cong Thuc Le, So Young Park , and Won Sun Park provided comments. Dae-Hee Choi and Eun-Hee Cho designed

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[1].

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ACCEPTED MANUSCRIPT Figure captions

Fig. 1. Metformin ameliorates HSCs activation by activating AMPK and GPR91

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pathway. A, LX-2 cells were treated with 1.6 mM succinate and co-treated with 1 mM AICAR or 1 mM metformin for 24 h, and succinate concentration was measured in whole cell lysate. *, P < 0.05, versus control, #, P < 0.05, versus succinate only

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treatment (mean ± SD, n=4). B, C, Western blotting analysis of p-AMPK, AMPK,

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α-SMA, and GPR91 expression in LX-2 cells, which were treated with 300 µM palmitate (B) or 1.6 mM succinate (C) and co-treated with 1 mM AICAR or 1 mM metformin for 24 h. Band intensities were quantified using ImageJ software and are plotted to the right of the representative blot images. The protein levels were

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normalized to the expression of GAPDH. *, P < 0.05, **, P < 0.01, ***, P < 0.001, versus control, #, P < 0.05, ##, P < 0.01, ###, P < 0.001, versus succinate or palmitate only treatment (mean ± SD, n=3). HSC, hepatic stellate cell; AICAR, 5-

AC C

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Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside.

Fig. 2. Metformin attenuates activated hepatic stellate cell proliferation, migration, and inflammatory cytokine expression in LX-2 cells. A, The EZ-Cytox assay kit was used to determine the proliferation of LX-2 cells, which were treated with 1.6 mM succinate and co-treated with either 1 mM AICAR or 1 mM metformin for 24 and 48 h. **, P < 0.01, ***, P < 0.001, versus control, #, P <0.05, versus succinate only treatment, ###, P <0.001 versus succinate only treatment (n=3). B, 18

ACCEPTED MANUSCRIPT Wound-healing migration assay was used to observe the effect of 1 mM AICAR or 1 mM metformin 1mM on LX-2 cells which that were treated with 1.6 mM succinate for 16 h. Black vertical lines delineate the area that was scratched with a pipette tip

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(magnification, ×100). C, LX-2 cells were treated with 1.6 mM succinate and cotreated with 1 mM AICAR or 1 mM metformin 1mM for 24 h. Whole cell lysates were subjected to western blotting for indicated proteins. Band intensities were

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calculated using ImageJ software and are plotted to the right of the representative blot images. Protein levels were normalized to the expression of GAPDH. ***, P

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<0.05, versus control, #, P <0.05, versus succinate or palmitate only treatment, ###, P <0.01 versus succinate or palmitate only treatment (mean ± SD, n=3).

AICAR, 5-

Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside.

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Fig. 3. Metformin improves steatohepatitis and liver fibrosis in MCD diet- fed mice model. A, H&E stain (left panels) and Masson’s trichrome stain (right panels) were performed to illustrate hepatitis and liver fibrosis. Image magnification is

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shown. B, All of the mouse liver samples were homogenized and succinate

AC C

concentrations were measured in the liver lysates (left plot) and mouse plasma (right plot) samples. ***, P < 0.001, versus control group, ###, P < 0.001, versus MCD diet group. MCD, methionine-choline-deficient.

Fig. 4. Metformin decreases α-SMA and GPR91 upregulation in the liver via AMPK signaling. A,B, Western blot analysis of p-AMPK, IL-6, TGF-β1, α-SMA 19

ACCEPTED MANUSCRIPT and GPR91 expression levels in liver from control, MCD diet- fed and MCD dietfed metformin mice. Representative blots are shown to the left of the plots of the corresponding band intensities. **, P < 0.01, versus control group, #, P < 0.05,

AC C

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versus MCD diet group (mean ± SD, n=3). MCD, methionine-choline-deficient.

20

ACCEPTED MANUSCRIPT Figures 1

A

Control Palmitate 300µM Palmitate +Aicar 1mM Palmitate +Metformin 1mM

1.6

*

1.4

Succinate concentrtion (fold)

1.2

#

RI PT

1 0.8 0.6

SC

0.4 0.2 0

3.5

Palmitate +Metformin 62kDa

AMPK

62kDa

α-SMA

42kDa

GPR91

#

#

##

α-SMA

GPR91

1

*

0 p-AMPK

62kDa

Protein level (fold)

AMPK

2.5

AMPK

GAPDH

CONTROL

3

62kDa

PALMITATE + AICAR 1mM PALMITATE + METFORMIN 1mM

3.5

P-AMPK

PALMITATE 300uM

0.5

EP Succinate + Metformin

Succinate + Aicar

Succinate

AC C

***

1.5

36kDa

Control

C

CONTROL

*

2

38kDa

GAPDH

##

2.5

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P-AMPK

###

3

Protein level (fold)

Palmitate +Aicar

Control

Palmitate

B

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LX-21cells

#

*

#

SUCCINATE 1.6mM SUCCINATE + AICAR 1mM SUCCINATE + METFORMIN 1mM

2 1.5

* # #

1

α-SMA

42kDa

GPR91

38kDa

0.5

GAPDH

36kDa

0

**

## ##

α-SMA

GPR91 p-AMPK AMPK

GAPDH

ACCEPTED MANUSCRIPT Figures 2 0.9 0.8

CONTROL 0 mmole/L succinate

1.4 ***

succinate 1.6 mmole/L SUCCINATE 1.6 Mm succinate 1.6 mmole/L + Aicar 1 mmole/L SUCCINATE + AICAR 1 mM

1.2

O. D. at 450 nm

0.7 0.6 0.5 0.4

###

0.3

O. D. at 450 nm

A

0.6

0.2

0 µmole/L succinate

B

Succinate 1.6mM

EP AC C

C

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0h

16h

48 h

0

SC

24 h

0h

M AN U

0h

#

0.8

0.4

0

***

1

0.2 0.1

CONTROL 0 mmole/L succinate succinate 1.6 mmole/L SUCCINATE 1.6 mM succinate 1.6 mmole/L + Metformin 2 mmole/L SUCCINATE 1.6 mM + METFORMIN 1 mM

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1

Succinate + Aicar 1mM

24 h

48 h

Succinate + Metformin 1mM

ACCEPTED MANUSCRIPT Figure 3: Masson’s trichrome staining

H&E staining

A

400×

100×

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MCD diet 5 4 3 2 1 0

###

1 Liver lysate

Succinate concentration (nmol/µl)

6

***

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7

Control MCD diet MCD diet + Metformin

AC C

8

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MCD diet +Metformin

B Succinate concentration nmol/µg

400×

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Normal

100×

1.2 1

0.8

Control MCD diet MCD diet + Metformin *** # # #

###

0.6 0.4 0.2 0

Mouse 1Plasma

ACCEPTED MANUSCRIPT Figure 4:

A Normal

MCD diet +Metformin

MCD diet

6

**

IL-6

AMPK GAPDH

#

4 3

#

2

#

1 0

TGFβ-1 p-AMPK AMPK GAPDH

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IL-6

B

**

MCD+Metformin

SC

p-AMPK

MCDdiet

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Protein level (fold)

TGF-β1

Normal

5

12

α SMA

GPR91

AC C

GAPDH

Protein levels (fold)

AMPK

10

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P-AMPK

MCD diet

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Normal

MCD diet +Metformin

Normal

**

MCD diet MCD+Metformin

8 6

#

4

# 2

**

#

0 α-SMA

GPR91

p-AMPK

AMPK

GAPDH