A novel bile acid analog, A17, ameliorated non-alcoholic steatohepatitis in high-fat diet-fed hamsters

Article abstract of DOI:10.1016/j.taap.2020.115169

Being endocrine signaling molecules that regulate lipid metabolism and affect energy balance, bile acids are potential drug candidates for non-alcoholic steatohepatitis (NASH). Obeticholic acid (OCA) could improve NASH accompanied by significant side effects. Therefore, it is worthwhile to develop safer and more effective bile acid analogs. In this study, a new bile acid analog A17 was synthesized and its potential anti-NASH effects were assessed in vitro and in vivo. The impact of A17 on steatosis was investigated in the rat primary hepatocytes challenged with oleic acid. It was found that A17 alleviated lipid accumulation by reducing fatty acid (FA) uptake and promoting FA oxidation. The reduction of FA uptake came from inhibiting fatty acid translocase (Cd36) expression. The promotion of FA oxidation came from stimulating the phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha (AMPKα). In addition, A17 reduced lipopolysaccharide-induced inflammation in Raw264.7 cells by activating Takeda G protein-coupled receptor 5 (TGR5). In in vivo study, male Golden Syrian hamsters were fed with high fat (HF) diet and then treated with 50 mg/kg/d A17 for 6 weeks. A17 lowered the lipid profiles and liver enzyme levels in serum and improved liver pathological conditions with less side effects compared with OCA. Further studies confirmed that the molecular mechanisms of A17 in vivo were similar to those in vitro. In conclusion, a novel bile acid analog A17 was identified to ameliorate NASH in HF-fed hamsters. The potential mechanisms could be contributed to reducing FA uptake, stimulating FA oxidation and relieving inflammation.

Full text of DOI:10.1016/j.taap.2020.115169

Journal Pre-proof
A novel bile acid analog, A17, ameliorated non-alcoholic
steatohepatitis in high-fat diet-fed hamsters
Ying Wang, Yao Zhu, Junxing Niu, Qiangqiang Deng, Shimeng
Guo, Haowen Jiang, Zhaoliang Peng, Yaru Xue, Huige Peng,
Lijiang Xuan, Guoyu Pan
PII:
S0041-008X(20)30295-7
DOI:
https://doi.org/10.1016/j.taap.2020.115169
YTAAP 115169
Reference:
To appear in:
Toxicology and Applied Pharmacology
Received date:
Revised date:
Accepted date:
9 May 2020
12 July 2020
27 July 2020
Please cite this article as: Y. Wang, Y. Zhu, J. Niu, et al., A novel bile acid analog, A17,
ameliorated non-alcoholic steatohepatitis in high-fat diet-fed hamsters, Toxicology and
Applied Pharmacology (2020), https://doi.org/10.1016/j.taap.2020.115169
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© 2020 Published by Elsevier.

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A novel bile acid analog, A17, ameliorated non-alcoholic
steatohepatitis in high-fat diet-fed hamsters
Ying Wangab#, Yao Zhuab#, Junxing Niuab, Qiangqiang Dengab, Shimeng Guoabc, Haowen Jiangabc,
Zhaoliang Pengab, Yaru Xueab, Huige Pengab, Lijiang Xuanab*, Guoyu Panab*
aShanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
bUniversity of Chinese Academy of Sciences, Beijing 100049, China
cNational Center for Drug Screening, Shanghai 201203, China
#These authors contributed equally to this work.
*Corresponding authors.
Guoyu Pan, Ph.D., Professor
Shanghai Institute of Materia Medica
Chinese Academy of Sciences
501 Hai-ke Rd, Shanghai, 201203
Tel: +86-21-20231000 ext 1715
Email: gypan@simm.ac.cn
Lijiang Xuan, Ph.D., Professor,
Shanghai Institute of Materia Medica
Chinese Academy of Sciences
501 Hai-ke Rd, Shanghai, 201203
Tel: +86-21-20231968
Email: ljxuan@simm.ac.cn
Abstract
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Being endocrine signaling molecules that regulate lipid metabolism and affect energy
balance, bile acids are potential drug candidates for non-alcoholic steatohepatitis (NASH).
Obeticholic acid (OCA) could improve NASH accompanied by significant side effects.
Therefore, it is worthwhile to develop safer and more effective bile acid analogs. In this study,
a new bile acid analog A17 was synthesized and its potential anti-NASH effects were assessed
in vitro and in vivo. The impact of A17 on steatosis was investigated in the rat primary
hepatocytes challenged with oleic acid. It was found that A17 alleviated lipid accumulation by
reducing fatty acid (FA) uptake and promoting FA oxidation. The reduction of FA uptake
came from inhibiting fatty acid translocase (Cd36) expression. The promoting of FA oxidation
came from stimulating the phosphorylation of adenosine monophosphate (AMP)-activated
protein kinase alpha (AMPKα). In addition, A17 reduced lipopolysaccharide-induced
inflammation in Raw264.7 cells by activating Takeda G protein-coupled receptor 5 (TGR5).
In in vivo study, male Golden Syrian hamsters were fed with high fat (HF) diet and then
treated with 50 mg/kg A17 for 6 weeks. A17 lowered the lipid profile and liver enzyme levels
in serum and improved liver pathological conditions with less side effects compared with
OCA. Further studies confirmed that the molecular mechanisms of A17 in vivo were similar
to those in vitro. In conclusion, a novel bile acid analog A17 was identified to ameliorate
NASH in HF-fed hamsters. The potential mechanisms could be contributed to reducing FA
uptake, stimulating FA oxidation and relieving inflammation.
Keywords: bile acid analog; NASH; TGR5; Cd36; AMPKα.
Introduction
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Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver
disease around the world [1], which comprises non-alcoholic fatty liver (NAFL) and
non-alcoholic steatohepatitis (NASH) [2]. NAFL is the first stage of NAFLD and considered as
a benign condition [3], which is featured by simple lipid accumulation (i.e. lipid accumulation
in ≥ 5% hepatocytes) in clinic [4]. Though NAFL can be reversed via changing lifestyle and
treating with lipid-lowering medicine [5], 10-25% patients progress to NASH [6], which is
regarded as a more serious disease with intralobular inflammation, hepatocyte ballooning and
damage [7]. Moreover, compared to NAFL patients, the patients with NASH carry a higher
risk of adverse hepatic outcomes including fibrosis, cirrhosis and hepatocellular carcinoma
(HCC) [8]. Increasing data demonstrates that the recent elevation in HCC incidence is driven
by NASH in developed countries [9]. Hence, NASH is of the major health concern worldwide.
The pathogenesis of NASH is complex and multifactorial [10]. So far, the most popular
theory is ‘two hits hypothesis’. The first hit is intrahepatic lipid accumulation, which results
from four mechanisms, namely (1) the increase in hepatic fatty acids (FA) uptake, (2) the
increase in liver lipolysis, (3) the reduction of mitochondrial FA oxidation and (4) the
reduction of triglycerides (TG) export from the liver [11]. These lipid metabolism
abnormalities sensitize the liver to a ‘second hit’, such as the induction of oxidation stress and
the excess production of inflammatory cytokines, resulting in the progression to NASH [12]
.
Bile acids (BAs) are endocrine signaling molecules that regulate lipid and glucose
metabolism and affect energy balance [13]. The function of BAs in regulating lipid metabolism
is mediated by activating the nuclear farnesoid X receptor (FXR) [14]. FXR activation reduces
hepatic lipogenesis and promotes FA oxidation, which results in the decreases of hepatic lipid
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accumulation [15]. Another main receptor of BAs is Takeda G protein-coupled receptor 5
(TGR5) whose activation increases energy expenditure, improves glucose tolerance and
inhibits inflammation [16]. Interestingly, total BAs concentrations and the primary to
secondary BAs ratio are increased in NASH patients [17]. Moreover, the increase in circulating
BAs level is relevant to the metabolic benefits after bariatric surgery [18]. These appearances
indicate that BAs may take an important part in the development, progression, and regression
of NASH.
Considering the effects of BAs signaling on lipid homeostasis and energy expenditure,
modulating BAs receptor activities is a promising therapeutic strategy to treat NASH.
Obeticholic acid (OCA), a chenodeoxycholic acid derivative, is a potent and selective FXR
agonist [19]. Clinical results indicated that OCA improved the liver histologic score in NASH
patients and may become the first approved agent to treat NASH. However, OCA resulted in a
concentration-dependent pruritus and elevated total cholesterol (TC) and LDL-cholesterol
(LDL-C) levels in serum, which increased cardiovascular risk [20]. INT-777, a cholic acid
derivative, is a potent and selective TGR5 agonist [21]. Administration of INT-777 in high fat
(HF) diet-fed C57BL/6J mice ameliorated hepatic steatosis and decreased liver enzyme levels
[22]. In addition, dual agonist of FXR and TGR5, INT-767, which is also a bile acid analog,
reduced hepatic lipid accumulation and proinflammatory cytokine expression in db/db obese
mice [23]. Notably, norursodesoxycholic acid, a side-chain shortened derivative of
[24]
ursodesoxycholic acid (UDCA), was found to ameliorate NASH in mouse models
and
[25]
clinic
without activating FXR or TGR5, suggesting that bile acid analogs may have the
potential to treat NASH with different mechanisms.
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Given that preclinical and clinical studies of bile acid analogs in treating NASH are
promising, other bile acid analogs deserve intensively explored to develop safer and more
effective therapies, which may have different pharmacological mechanisms from exist. In this
report, a new bile acid analog A17 was synthesized by protecting the sensitive hydroxyl
groups and using condensation agent to connect amino acids. A17 was found to reduced lipid
accumulation in oleic acid (OA)-induced rat primary hepatocytes (RPHs) steatosis model and
inhibit lipopolysaccharide (LPS)-induced inflammation in Raw264.7. It also ameliorated
NASH in HF-induced hamsters by reducing lipid uptake, promoting FA oxidation and
inhibiting inflammation in liver. The reduction of lipid uptake came from inhibiting the
expression of fatty acid translocase (Cd36). The promoting of FA oxidation came from
stimulating adenosine monophosphate (AMP)-activated protein kinase alpha
(AMPKα)/acetyl-CoA carboxylase (ACC)/carnitine palmitoyltransferase-1α (Cpt-1α)
pathway. The anti-inflammation effect came from activating TGR5/nuclear factor of kappa
light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) pathway.
Materials and Methods
The synthetic routes of A17
UDCA (1.0 mmol) was dissolved in anhydrous tetrahydrofuran (THF, 10 mL), under N2
protection, 5 mL 98% HCOOH and 20 μL 70% HClO4 were added at the same time under
stirring at 55°C. And then, the mixture was stirred at 55°C for 2-4 h and subjected to
evaporation of the solvent to generate compound 1. Under N2 protection, compound 1 (1.0
mmol), HATU (1.2 mmol) and L-(+)-VALINOL (1.3 mmol) was dissolved in anhydrous
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dichloromethane (DCM, 15 mL). Under stirring, triethylamine (TEA, 170 μL, 1.5 mmol) was
added to the mixture, which was then stirred at room temperature for 3-4 h, generating the
target compound A17. Silica gel column chromatography (petroleum ether: acetone = 8:1 ~
3:1, v:v, contains 0.1% formic acid); Yield 82%; White Powder; 1H NMR (400 MHz, CDCl3 )
δ 8.05 (s, 1H), 8.01 (s, 1H), 4.80 – 4.70 (m, 2H), 3.66 (td, J = 6.5, 4.6 Hz, 1H), 3.53 (qd, J =
11.2, 5.4 Hz, 2H), 3.29 (q, J = 1.7 Hz, 1H), 2.28 (dd, J = 8.9, 4.6 Hz, 1H), 2.18 – 2.08 (m, 1H),
2.04 (dd, J = 12.5, 3.4 Hz, 1H), 1.91 – 1.80 (m, 4H), 1.79 – 1.69 (m, 4H), 1.68 – 1.55 (m, 4H),
1.54 – 1.36 (m, 6H), 1.36 – 1.28 (m, 3H), 1.24 – 1.15 (m, 1H), 1.15 – 1.03 (m, 2H), 1.00 (s,
3H), 0.96(d, J = 6.3 Hz, 3H), 0.92 (d, J = 6.8 Hz, 3H), 0.88 (d, J = 6.8 Hz, 3H), 0.70 (s, 3H).
ESI-MS: m/z 534.4 [M + H] +; HRMS (ESI): m/z calcd. For C31H52NO6 + [M + H] +534.3795,
found 534.3767.
Fatty acid preparation
50 mM sodium oleate (Sigma-Aldrich, Saint Louis, MO, USA) stock was prepared in 50%
ethyl alcohol by heating at 70°C for 2 min. Meanwhile, 2% fatty acids-free bovine serum
albumin (BSA, Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in William’s E medium
(GIBCO, GrandIsland, NY, USA) and preheated at 37°C. After completely vortexed, the
sodium oleate stock was added into 2% BSA dropwise to make 2.5 mM OA-BSA conjugate
and mixed completely at 37°C on a shaker for 1 h. The conjugate was later filtered with 0.25
μM pore sized polyvinylidene fluoride hydrophilic membrane filter and stored at -20°C.
Hepatocytes culture and steatosis induction
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Isolated RPHs were suspended in plating medium and plated onto collagen-coated
96-well (3 × 104 cells/well) plate. After 4 h incubation in a humidified atmosphere containing
95% air and 5% CO2 at 37°C, RPHs adhered to the collagen completely and spread out. Then,
the medium was replaced with feeding medium containing 100, 250, 500, 750, 1000 μM OA.
Corresponding concentrations of BSA were used as negative controls. Post 24 h treatment,
cells were assayed for their viability and intracellular TG levels.
Hepatocytes intracellular A17 measurement
RPHs were seeded in collagen-coated 6-well (1 × 106 cells/well) plates and treated with
10, 25 or 50 μM A17 for 24 h. After washed with phosphate buffer solution (PBS) for 3 times,
the cells were collected and intracellular A17 concentrations were detected using
HPLC/MS-8030 triple quadrupole system (Shimadzu Corp, Kyoto, Japan). The positive
electrospray ionization interface (ESI) mode was chosen. A17 and the internal standard
(tolbutamide) were separated by Sunfire C18 columns (2.1 ×100 mm, i.d. 3.5 μm). The
precursor and product ion values were m/z 534.2 (Q1) > m/z 442.3 (Q3) for A17 and m/z
271.0 (Q1) > m/z 172.0 (Q3) for tolbutamide, with collision energy (CE) of 23 eV and 20 eV
respectively. The mobile phase (MP) consisted of acetonitrile (A) and 0.1% formic acid (C)
with a gradient elution of 60% MPC at 0-0.1 min, 5% MPC at 1-2 min, and 60% MPC at 3-7
min, at a flow rate of 0.25 mL/min. The oven temperature was 40 °C, and the injection
volume was 20 μL.
Hepatocytes chemical treatment
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As shown in the results section, 500 μM OA was chosen to induce RPHs steatosis model.
RPHs were seeded in collagen-coated 96-well (3 × 104 cells/well) and 6-well (1 × 106
cells/well) plates. After attached to the collagen, the cells were treated with 500 μM OA and
10 μM or 25 μM A17 for 24 h. OCA (purity > 98%, Dalian Meilun Biotechnology Co., LTD,
Dalian, China) at the same concentrations were used as positive controls and 0.1% dimethyl
sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO, USA) was used as a negative control.
Cells in the 96-well plate was assayed for viability, intracellular TG levels and fatty acid
uptake, while those in the 6-well plate were harvested for intracellular malondialdehyde
(MDA) concentration measurement, qPCR and western blot assay.
In another experiment, RPHs were seeded in collagen-coated 96-well (3 × 104 cells/well)
and pretreated with or without 10 μM Etomoxir (Selleck Chemicals, Houston, TX, USA),
which is a Cpt-1α irreversible inhibitor, for 24 h. Then, RPHs were treated with or without
500 μM OA, 25 μM A17 and 10 μM Etomoxir for another 24 h. Intracellular TG levels and
cell viability were measured.
FXR binding assay
The binding potency of A17 to FXR was determined by AlphaScreen-GST detection kit
(PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. OCA was
used as positive control. After different concentrations of A17 reacted with GST-FXR system
for 2 h at room temperature, the ligand-induced interaction between the steroid receptor
coactivator 1 (SRC1) and FXR were detected by an EnVision multiplate reader (PerkinElmer,
Waltham, MA, USA).
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HEK293 transient transfection and TGR5 activation assay
Human embryonic kidney (HEK) 293 cell line was purchased from the American Type
Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS, GIBCO, GrandIsland,
NY, USA) and 1% streptomycin/penicillin (GIBCO, GrandIsland, NY, USA). Human TGR5
plasmid was obtained from the UMR cDNA Resource Center (Rolla, MO, USA). Nearly 2 ×
106 HEK293 cells were suspended in 200 μL transfection buffer and were transiently
transfected with human TGR5 plasmid by electroporation, which was performed with a
Scientz-2C electroporation apparatus (Scientz Biotech, Ningbo, China). Intracellular
cyclic-AMP (cAMP) of TGR5-transfected HEK293 treated with different concentrations of
A17 was measured as described previously [26]. INT-777 (MedChemExpress, Monmouth
Junction, NJ, USA) at the same concentrations were used as positive controls and 0.1%
DMSO was used as a negative control.
Raw264.7 culture and treatment
Mouse macrophage cell line Raw264.7 was obtained from ATCC and maintained in
DMEM-High Glucose medium containing 10% FBS. When the confluent reached 90%, the
cells were seeded on 6-well (6 × 105 cells/well) plate. After 12 h culture, the cells were
pretreated with 25 μM A17 for 24 h. 10 μM dexamethasone (Dex, Sigma-Aldrich, Saint Louis,
MO, USA) was used as a positive control and 0.1% DMSO was used as a negative control.
Subsequently, the cells were simulated with 1 μg/mL LPS (Sigma-Aldrich, Saint Louis, MO,
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USA) supplemented with 25 μM A17 or 10 μM Dex for another 8 h. the supernatants were
collected for nitric oxide (NO), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6)
measurement.
In another experiment, the cells were seeded on 6-well and pretreated with 25 μM A17
for 24 h. 25 μM INT-777 was used as a positive control and 0.1% DMSO was used as a
negative control. Afterwards, the cells were simulated with 1 μg/mL LPS supplemented with
25 μM A17 or 25 μM INT-777 for another 1 h, and then harvested for western blot assay.
Cell viability assay
Cell viability was evaluated by Cell Counting Kit 8 (CCK8, Yeasen Biotech Co. Ltd,
Shanghai, China) according to the manufacturer’s instructions. Briefly, after the cells in
96-well plates underwent different treatments for 24 h, 10 μL CCK8 was added to each well
and incubated for another 1 h. The absorbance of 450 nm was measured by the automatic
microplate reader (Biotek, Winooski, VT, USA).
Cellular TG measurements
The content of TG in cells was measured to determine the extent of steatosis. At the end
of the treatment, the cells in 96-well plates were washed with PBS and lysed by 10% Triton
(Beyotime Biotechnology, Shanghai, China) for 30 min. the lysates of each well were
collected and measured by commercial kits (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China) following the protocol of manufacturer. The absorbance at 510 nm was
assayed and the results were normalized to protein concentration measured by Pierce BCA
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Protein Assay Kit (Thermo Fisher Scientific, Madison, WI, USA).
Cellular malondialdehyde (MDA) measurements
Intracellular MDA concentration was measured to examine the effect of A17 on lipid
peroxidation. After treatment of OA and A17 or OCA, the cells in 6-well plates were washed
with PBS and lysed by RIPA buffer (Beyotime Biotechnology, Shanghai, China), the lysates
of each well were collected and measured by commercial kits (Beyotime Biotechnology,
Shanghai, China) following the instructions of manufacturer. The absorbance at 532 nm was
assayed and the results were normalized to protein concentration.
Fatty acid uptake assay
Free fatty acid uptake assays (Molecular Devices, San Jose, USA) were performed after
the cells in 96-well plates were incubated with compounds for 24 h. 10 μM Triacsin C
(Sigma-Aldrich, Saint Louis, MO, USA) served as positive control and incubated with cells
for 2 h before the assay. Assay buffer containing BODIPY®-dodecanoic acid fluorescent fatty
acid analog (BODIPY® 500/512 C1, C121) coupled with quench was prepared and added 100
μL to each well. Then, the excitation/emission 485/515 nm was read immediately using a
kinetic mode (every 1 min for 30 min). For blank wells, the compound diluent was added, and
the fluorescence was subtracted from the values for those wells with cells treated with
compounds. The results were normalized to cell viability measured by CCK-8.
Fatty acid oxidation (FAO) assay
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The oxygen consumption rate (OCR) due to oxidation of fatty acids was measured using
a Seahorse XFe96 extracellular flux analyzer (Agilent Technologies, Santa Clara, California).
RPHs were seeded into collagen coated 96-well Seahorse microplates at 1 × 104 cells/well.
Four hours later, media was replaced with feeding medium containing 500 μM OA and 25 μM
A17 or OCA, 50 μM amiodarone was used as positive control. After 24 h treatment, OCR was
measured as previously described in the protocol [27]. The median antimycin/rotenone (A/R)
measurements at time point 10, 11 and 12 was subtracted from the median of FCCP
measurements at time point 7, 8 and 9 to identify chemical effects on maximal respiration.
Measurement of NO and inflammatory cytokines
NO assay was conducted using commercial kit (Beyotime Biotechnology, Shanghai,
China). TNF-α and IL-6 levels were quantified with mouse TNF-α and IL-6 ELISA kits
(Multi Sciences Biotech. Co. Ltd, Hangzhou, China). All the operations were strictly
performed according to the operating manual.
Animal experiments
The animal experiments were performed according to protocols approved by the
Institutional Animal Care and Use Committee (IACUC) of Shanghai Institute of Materia
Medica, People’s Republic of China Academic Science. Male Sprague-Dawley rats (180 ± 10
g, IACUC permission number: 2018-05-PGY-24) and Male Golden Syrian hamsters (100 ±
10 g, IACUC permission number: 2019-05-PGY-32) were purchased from Shanghai SLAC
Laboratory Animal Co. (Shanghai, People’s Republic of China) and housed under controlled
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temperature (23 ± 2°C), relative humidity (50 ± 10%) and lighting (12 h light/dark cycle).
Experiments were performed after animals were acclimated for 7 days.
RPHs were isolated from male Sprague-Dawley rats by two-step collagenase perfusion
following the previous protocols [28]
.
Hamsters were fed a standard diet (n = 10) or a HF diet (n = 15) containing 60% fat
(#12492, Research Diets, Inc., New Brunswick, NJ, USA), and provided with normal
drinking water. The weights of animals were measured once a week until the end of the
experiment.
After 16 weeks of diet, the hamsters fed the standard diet were randomly divided into
two groups (n = 5 per group) and were given a daily dose of A17 at 50 mg/kg (A17 group) or
vehicle (vehicle group) by intraperitoneal injection. Hamsters under HF diet were kept on the
same diet and allocated into the following 3 treatment groups (n = 5 per group): (1) HF group,
the HF diet group treated with vehicle daily by intraperitoneal injection; (2) HF + OCA group,
the HF diet group treated with OCA at 20 mg/kg/d by oral gavage and (3) HF + A17 group,
the HF diet group treated with A17 at 50 mg/kg/d by intraperitoneal injection. The drug
treatment lasted for 6 weeks. OCA was suspended in 0.5% carboxyl-methyl cellulose
(Sigma-Aldrich, Saint Louis, MO, USA) and sonicated for 1 h to form homogenous liquid.
A17 was distributed in 20% hydroxypropyl-beta-cyclodextrin (purity > 98%, Dalian Meilun
Biotechnology Co., LTD, Dalian, China) and sonicated overnight to get clear liquid.
At the end of the study, serum samples were collected after an overnight fast for
determination of serum TG, TC, LDL-C, alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) levels. Then, hamsters were euthanized and livers were immediately
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removed, washed and cut into two parts. One part of the livers was fixed in 4% formaldehyde
solution to perform histology analysis, the other was stored at -80℃ for lipid measurement,
qPCR and western blot assay.
Biochemical analysis
The serum TG, TC, LDL-C, ALT and AST concentrations were assayed using
commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to
the manufacturer’s protocols.
For the measurements of the TG and TC levels in liver, liver samples were homogenized
in 9-fold diluted absolute ethyl alcohol and centrifuged at 2500 rpm for 10 min at 4°C. The
supernatants were collected for analysis using commercial available kits (Nanjing Jiancheng
Bioengineering Institute, Nanjing, China).
Histology analysis and scoring
The liver tissue samples fixed in 4% formaldehyde solution were embedded in paraffin
and subsequently stained with Hematoxylin-Eosin (H & E) and Oil Red. After staining,
steatosis and inflammation variables of each liver slices were assessed based on a previous
reported scoring system [29] in a blinded way by Zuocheng Biotechnology Co., LTD, Shanghai,
China.
RNA extraction and real-time PCR analysis
Total RNA was extracted from cells and liver samples by TRIzol reagent (Life
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Technology, CA, USA) and purified with the EZ-10 Spin column & Collection Tube (Sangon
Biotech, Shanghai, China). Then, isolated RNA was dissolved in water-DEPC treated water
(Sangon Biotech, Shanghai, China) and quantitated using the ScanDrop 100
spectrophotometer (Analytik Jena, Jena, German). 1000 ng of total RNA was reversely
transcribed into cDNA using PrimeScriptTM RT Master Mix (Takara, Shiga, Japan) by T100TM
thermal cycler (Bio-Rad, Hercules, California). Later, 2 μL cDNA was used as template in a
20 μL PCR mix containing 7.2 μL water-DEPC treated water, 0.4 μL each forward and
reverse primers (Sangon Biotech, Shanghai, China) and 10 μL Hieff® qPCR SYBR Green
Master Mix (Low Rox Plus, Yeasen Biotech, Shanghai, China) for PCR setup. PCR reactions
were performed using the Applied BiosystemsTM 7500 Fast real-time PCR system (Thermo
Fisher Scientific, Madison, WI, USA) following the manufacture’s protocol. Primers for
genes were list in Table 1. The detective mRNA levels were normalized against Gapdh.
Western blot analysis
Total protein was extracted with RIPA buffer supplemented with 1% protease inhibitor
cocktail (Sigma-Aldrich, Saint Louis, MO, USA) and quantitated using Pierce BCA Protein
Assay Kit (Thermo Fisher Scientific, Madison, WI, USA). After diluted to the same
concentration and conjugated to the sodium dodecyl sulfate (SDS) by being heated at 95°C
for 10 min, the proteins were separated on a 10% denaturing SDS gel and then transferred to
polyvinylindene difluoride membrane (Merck Millipore, Darmstadt, Germany). Then, the
blots were blocked using 5% skim milk in TBS-T (25 mM Tris-HCl pH 7.6, 150 mM NaCl,
2.5 mM KCl and 0.3% Tween-20) for 1 h at room temperature and incubated with the
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following primary antibodies overnight at 4°C. Gapdh (1:2000), p-AMPKα (1:1000), AMPKα
(1:1000), p-ACC (1:1000), ACC (1:1000), p-IκBα (1:1000) and IκBα (1:1000) were bought
from Cell Signaling Technology (Danvers, MA, USA); β-actin (1:1000),Cpt-1α (1:1000),
fatty acid binding protein (plasma membrane) (Fabp[pm], 1:1000), fatty acid transport protein
2 (Fatp2, 1:1000) and IL-6 (1:1000) were purchased from Proteintech Group (Wuhan, China);
fatty acid transport protein 5 (Fatp5, 1:1000) and Cd36 (1:1000) were from Abcam
(Cambridge, MA, USA). After washing with TBS-T, the blots were incubated with respective
secondary antibodies (1:10000, Yeasen Biotech, Shanghai, China) at room temperature for 1 h.
Later, the protein bands were soaked with the electrochemiluminescence (ECL) detection
reagent (Thermo Fisher Scientific, Madison, WI, USA) and captured by the ChemiScope
3300 mini system (CLINX, Shanghai, China). The relative blot intensity of each group was
quantified by Image J software (National Institute of Health, MD, USA).
Statistical analysis
All the data were analyzed using GraphPad Prism 7.0 software (GraphPad Software Inc.,
La Jolla, CA) and represent as mean ± SD. The Student’s t test was used to precede the
comparison between two groups. One-way analysis of variance (ANOVA) was used in case of
multiple testing. *P < 0.05, **P < 0.01, ***P < 0.001 were shown as statistical difference,
and the significance increases progressively.
Results
Synthesis of A17
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Previous researches showed that modifications of carboxyl in the C17 side chain could
improve the activities of BAs in activating FXR/TGR5 [30-32]. In contrast, bile acid synthetic
intermediates which lack carboxyl or hydroxyl in the C17 side chain showed no effects on
FXR/TGR5 activation [33]. These evidences suggest that the C17 side chain of BAs has a large
pocket with hydrogen bond donor/acceptor groups [34]. Considering the reactivity of carboxyl
groups, amide was formed to connect different hydrogen bond donors and receptor groups. In
addition, to prevent the hydroxyl reaction, formyl was used to protect and replace the
hydroxyl to form new hydrogen bond receptor groups. Thus, A17 was synthesized by
connecting amino acids to the C17 side chain of UDCA and protecting the sensitive hydroxyl
groups using formyl.
The synthesis of A17 started from commercially available UDCA; formylation of UDCA
in HCOOH gave compound 1, which then reacted with L-(+)-VALINOL to form A17 in the
presence of HATU and TEA (Scheme 1).
A17 ameliorated OA-induced steatosis in rat primary hepatocytes
Given that A17 could be transported into RPHs (Fig. S1), RPHs induced by OA was
chosen to be the cell model to assess the effect of A17 on steatosis in vitro. OA (≥ 500 μM)
caused a significant decrease in cell viability and increase in TG accumulation (Fig. 1A-B).
Therefore, 500 μM OA was used to induce lipid accumulation in RPHs in the later
experiments. OCA could decrease intracellular TG concentrations dose-dependently in
OA-induced RPHs (Fig. S2), so it was used as the positive control. A17 treatment
dramatically reduced intracellular TG level and increased viability in a dose-dependent
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manner in RPHs induced by OA (Fig. 1C-D). In addition, excessive lipid peroxidation results
in oxidation stress, which is a hallmark of NASH progression [35]. Thus, Intracellular MDA
concentrations were measured and found to be lowered to normal value in OA-induced RPHs
after A17 treatment (p < .05, Fig. 1E). Notably. The inhibitory effect of A17 on steatosis was
greater than that of OCA. These results indicated that A17 alleviated steatosis in OA-induced
RPHs model.
A17 treatment inhibited FA uptake via reducing Cd36 protein expression
Lipid accumulation in hepatocytes partly attributes to the increase in lipid influx. Hence,
the effect of A17 on hepatocellular FA uptake was measured. Triacsin C, a long-chain
acyl-CoA synthetases inhibitor was used as positive control. 25 μM A17 significantly
inhibited the uptake of FA in RPHs induced by OA (p < .001), while 25 μM OCA made no
differences (Fig. 2A). Western blot analysis demonstrated that A17 treatment significantly
decreased the expression of Cd36 (p < .01), but not other FA uptake transporters like
Fabp(pm) , Fatp2 and Fatp5 (Fig. 2B). These results suggested that A17 decreased the protein
level of Cd36, which resulted in the reduction of FA entered into hepatocytes.
A17 treatment promoted FA oxidation via AMPKα/ACC/Cpt-1α pathway
Hepatocellular lipid accumulation also depends on energy balance, especially fatty acid
oxidation. In hepatocytes, fatty acid are broken down in mitochondria to provide energy by
beta-oxidative pathways which require oxygen consumption [36]. Thus, the OCR was
measured to assess FA oxidation. RPHs induced by OA showed significant lower OCR
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compared to the vehicle group (p < .001, Fig. 3A), which could be one of the causes of lipid
accumulation. 25 μM A17 markedly increased FA oxidation in RPHs induced by OA (p
< .001), while 25 μM OCA made no differences in OCR (Fig. 3A). Analysis of the proteins
involved in fatty acid oxidation revealed that 25 μM A17 increased the expression of Cpt-1α
(p < .01) and the phosphorylation levels of AMPKα and ACC (p < .05, p < .05, Fig. 3B).
Additionally, the mRNA expression of Cpt-1α was significantly elevated by A17 treatment (p
< .001, Fig. 3C). Further inhibitor experiment showed that Cpt-1α inhibitor Etomoxir (10 μM)
significantly reversed the effects of A17 on reducing intracellular TG levels (p < .05, Fig. 3D)
and increasing cell viability (p < .001, Fig. 3E). These results suggested that A17 increased
the expression of Cpt-1α by AMPKα/ACC pathway, promoting FA oxidation and clearing
intracellular fatty acids.
A17 activated TGR5 and inhibited inflammatory response in Raw264.7
Being a bile acid analog, A17 may be the ligand of FXR and/or TGR5. To verify if the
potential anti-steatosis function of A17 was relevant to FXR activation, the binding potency
of A17 to FXR was measured. Compared to OCA whose EC50 was 201.55 nM, A17 was
unable to bind FXR (Fig. 4A) and thus could not activate FXR directly. Furthermore, the
mRNA levels of FXR-targeted genes such as small heterodimer partner (Shp), bile salt export
pump (Bsep) and cytochrome P450 7a1 (Cyp7a1) were not affected by A17 in OA-induced
RPHs (Fig. S3A) or in vivo (Fig. S3B), implying that A17 was not an agonist of FXR.
The effect of A17 on TGR5 was further investigated. A17 was an activator of TGR5 with
an EC50 of 117.5 nM, reaching 90% response of INT-777 (Fig. 4B). Activating TGR5 in
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macrophages has been reported to suppress multiple inflammatory diseases by blunting the
phosphorylation of IκBα [37]. To further verify the effect of A17 on TGR5 activation, the
potential anti-inflammation function of A17 in LPS-induced Raw264.7 cell model was
evaluated. Pretreating with 25 μM A17 for 24 h significantly inhibited NO, TNF-α and IL-6
levels in the supernatant of LPS-induced Raw264.7 (p < .001, p < .01, p < .05, Fig. 4C). A17
also decreased phosphorylation of IκBα (p < .05, Fig. 4D), implying A17 attenuated
LPS-induced cytokine expression via TGR5/IκBα pathway.
A17 administration ameliorated steatosis and inhibited inflammatory response in
hamsters
After feeding HF diets for 16 weeks, the body weights of the hamsters in HF group were
markedly increased compared to that of control group (p < .001, Fig. 5A). Administration of
A17 for 6 weeks slightly decreased the weights of hamsters compared to HF group, while no
weight loss were found in HF + OCA group (Fig. 5A).
The serum biochemistry results (Table 2) revealed that HF diets dramatically increased
the levels of lipid parameters such as TG, TC, LDL-C, ALT and AST (p < .001, p < .01, p
< .01, p < .001, p < .001). Compared to those in HF group, hamsters in HF + A17 group
showed a down-regulated profile in serum lipid parameters, with lower TG by 50% (p < .001),
decreased TC and LDL-C levels by 26% and 32% (p < .05, p < .05), respectively. It is
worthwhile to note that OCA treatment did not change serum TC and LDL-C levels. Besides,
hamsters in HF + A17 group had significant lower serum ALT and AST levels by 75% and 85%
(p < .001, p < .001), respectively, which were even lower than those of HF + OCA group.
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Consistent with the results of serum biochemistry, liver Oil Red and H & E staining
indicated that hamsters in HF group showed severe fatty accumulation and inflammation with
the score of 5.4 and 4.8 (p < .001, p < .001), respectively. Administration of A17 resulted in
reversal of hepatic steatosis and inflammation and lowered the score to 2.8 and 1.8 (p < .001,
p < .001, Fig. 5B-C), respectively. Measurement of the TG levels in liver further confirmed
that A17 significantly alleviated steatosis compared to the HF-fed hamsters (p < .001).
However, the TC level in liver was neither reduced by OCA nor A17 (Table 2). Altogether,
these findings indicated that A17 prevented liver injury in HF-induced NASH model.
A17 alleviated NASH via inhibiting Cd36, stimulating AMPKα/ACC/Cpt-1α pathway
and TGR5/IκBα pathway
Western blot results showed that HF induced a significant increase in the protein level of
Cd36 (p < .01), which was dramatically reversed by A17 treatment (p < .001), but neither HF
nor A17 changed the expression of other FA uptake transporters such as Fabp(pm), Fatp2 or
Fatp5 (Fig. 6A). In regard to FA oxidation, HF inhibited the AMPK signaling pathway by
markedly decreasing the ratios of p-AMPKα/AMPKα and p-ACC/ACC (p < .001, p < .001),
while A17 treatment elevated the phosphorylation of AMPKα and ACC (p < .05, p < .05),
which resulted in the increase of Cpt-1α expression (p < .01, Fig. 6B). Moreover, A17
significantly increased the mRNA expression of Cpt-1α in hamster livers (Fig. 6C). These
results showed that the anti-steatosis mechanism of A17 in hamsters was in accordance to that
in RPHs. In addition, A17 treatment substantially decreased the production of p-IκBα and
IL-6 (p < .001, p < .001) in liver induced by HF (p < .001, p < .001, Fig. 6D), suggesting that
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A17 suppressed cytokine production by activating the TGR5 in liver.
Discussion
Although NASH is a complex and multisystem disease, one of its most important end
points is lipid accumulation [38]. Therefore, OA-induced RPH steatosis model was applied to
assess the anti-steatosis effect of A17 in vitro. It was found that A17: (1) dramatically reduced
intracellular TG and MDA levels and increased hepatocyte viability (Fig. 1); (2) inhibited FA
uptake via reducing Cd36 protein expression (Fig. 2); and (3) promoted FA oxidation via
AMPKα/ACC/Cpt-1α pathway (Fig. 3). Then, A17 was found to be an agonist of TGR5 and
inhibited LPS-induced inflammation in Raw264.7 via TGR5/IκBα pathway (Fig. 4). Further
animal studies confirmed that A17 alleviated NASH in HF-induced hamsters (Fig. 5), and the
mechanism was the same as that in vitro (Fig. 6).
In this report, A17 was found to be a potent TGR5 agonist (Fig. 4B). Previous studies
have reported that the specific TGR agonist INT-777 inhibited LPS-induced inflammation in
[39]
[40]
Raw264.7 model
and hepatic inflammatory response in mice via antagonizing NF-κB
.
Consistent with these results, A17 treatment in Raw264.7 significantly reduced the
phosphorylation of IκBα and resulted in decreased NO, IL-6 and TNF-α concentrations in
supernatant, confirming that the anti-inflammatory effect of A17 was link with TGR5
activation (Fig. 4C-D). In the liver, TGR5 is expressed in Kupffer cells, which are the resident
macrophages [37]. The TGR5 expressed in Kupffer cells may enable A17 to attenuate hepatic
inflammation in hamsters developed NASH. Decreasing the lipid content in liver and
reducing inflammation have been two therapeutic directions to treat NASH. Notably, besides
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diminishing inflammation, A17 ameliorated steatosis in both OA-induced RPHs and hamster
model of NASH by inhibiting FA uptake and stimulating FA oxidation in hepatocytes,
implying parenchymal hepatic cells was another action site of A17. In contrast, INT-777 had
no effects on steatosis in OA-induced RPHs (Fig. S4). Though INT-777 was reported to
reduce hepatic steatosis and obesity in high-fat feeding C57BL/6J mice, the underlying
mechanism was the activation of TGR5 in brown adipose/muscle tissue and intestinal L cells
[22]. Therefore, not only the activation of TGR5 in Kupffer cells, but also the direct action on
hepatocytes contributed to the anti-NASH effect of A17.
Compared to OCA, which directly acts on hepatocytes by activating FXR, A17 had no
agonistic effect on FXR (Fig. 4A), suggesting their mechanisms are quite different. FXR
activation reduces hepatic lipogenesis via inhibiting the expression of hepatic sterol
responsive element binding protein 1c [41] and promotes FA oxidation by inducing peroxisome
proliferator-activated receptor alpha [42]. However, our results indicated the elevation effect of
OCA on FA oxidation was not as great as that of A17 in OA-induced RPHs (Fig. 3A).
Moreover, OCA had no effects on FA uptake, which was markedly inhibited by A17 (Fig. 2A).
Regarding safety issues, increased TC and LDL-C levels in NASH patients, which increases
the risk of cardiovascular diseases, is a significant side effect of OCA in clinical trials. In
contrast, hamster experiment results indicated that A17 markedly decreased the serum TC and
LDL-C contents (Table 2), implying the lower risk of developing related adverse event.
Furthermore, treating HF-fed hamsters with 30 mg/kg/d OCA for 2 weeks dramatically
lowered the weight of animals and increased ALT, AST and bilirubin (TBIL) levels in serum
(p < .01, p < .01, p < .001, Fig. S5). While administrating HF-fed hamsters with 50 mg/kg/d
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A17 for 6 weeks did not show any liver injuries (Fig. 5B-C). Therefore the safety window of
A17 was much wider than that of OCA.
Hepatic FA uptake is mainly mediated by the scavenger receptor Cd36 and the Fatp
family (Fatp2 and Fatp5) [43]. The expression of Cd36 is low in hepatocytes under normal
conditions, but could be drastically enhanced in livers of obese and diabetic murine models
[44]. Moreover, in human NAFLD, Cd36 expression correlates with liver TG content and
insulin resistance [45], implying that Cd36 is important in regulating hepatic lipid content
under high FA flux conditions. Disruption of hepatic Cd36 in HF-fed mice protected them
against inflammation and insulin resistance [46], which highlights the possibility of treating
NAFLD via modulating FA uptake in liver. A17 was found to decrease the protein expression
of Cd36 (Fig. 2B), which resulted in the reduction of FA uptake. However, the mechanism
remains to be determined. Cd36 was reported to be a shared target of liver X receptor,
pregnane X receptor and peroxisome proliferator-activated receptor gamma, and the former
two regulation were liver-specific [47]. Therefore, the regulation of Cd36 may be mediated by
nuclear receptors. In addition, it was reported that specific bile acids can inhibit the function
of hepatic fatty acid uptake transporters. For example, UDCA and deoxycholic acid are the
potent inhibitors of Fatp5 [48]. As a bile acid analog, A17 has the potential to be the inhibitor
of hepatic fatty acid uptake transporters as well. Considering the high expression of Cd36 on
macrophages, adipocytes, cardiomyocytes and muscle cells [49], the potential side effect of
A17 will be addressed in a future study.
AMPK serves as an energy sensor in cellular metabolism, harmonizing metabolic
pathways and maintaining the energy balance [50]. Liver-specific activation of AMPK has been
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reported to prevent mice from high-fructose-induced steatosis [51]. In this report, A17
upregulated the phosphorylation level of AMPKα, and phosphate-AMPKα past
phosphorylation to ACC. Phosphate-ACC decreased the level of malonyl-CoA, which has
inhibitory effect on Cpt-1α. Thereby, phosphorylating AMPKα indirectly activated Cpt-1α to
promote fatty acid β-oxidation. Previous studied have proved that Cd36 overexpression
elevated the src kinase Fyn mediated liver kinase B1 phosphorylation, which hindered the
activation of AMPK [52]. While inhibiting the palmitoylation of Cd36 enhanced the
[53]
phosphorylation of AMPK, promoting FA oxidation and inhibiting lipid accumulation
.
Whether the stimulation effect of A17 on AMPKα phosphorylation is relevant to Cd36
deserves further research.
In summary, our study suggested A17, a novel bile acid analog, ameliorated NASH in
HF-fed hamsters in three ways, reducing FA uptake via inhibiting the expression of Cd36,
stimulating FA oxidation via AMPKα/ACC/Cpt-1α pathway, and relieving inflammation via
TGR5/IκBα pathway (Fig. 7).
Author contributions
Ying Wang, Guoyu Pan, Junxing Niu and Lijiang Xuan designed the research; Ying
Wang, Yao Zhu, Qiangqiang Deng, Shimeng Guo, Haowen Jiang, Zhaoliang Peng, Yaru Xue
conducted the experiments; Ying Wang, Shimeng Guo, Haowen Jiang and Zhaoliang Peng
were responsible for the data analysis; Ying Wang and Guoyu Pan wrote the manuscript; Yao
Zhu, Shimeng Guo, Huige Peng and Lijiang Xuan helped revise the manuscript. All the
authors reviewed and agreed on the final version.
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Declaration of competing interest
The authors declare no competing financial interests.
Acknowledgements
This study was supported by the ‘Organ Reconstruction and Manufacturing’ Strategic
Priority Research Program of the Chinese Academy of Sciences [grant number
XDA16020205], the National Science Foundation of China [grant number 81872927], the
International Partnership Program of Chinese Academy of Sciences [grant number
153631KYSB20160004], the Independent Deployment Program of the Institute of
Pharmaceutical Innovation of the Chinese Academy of Sciences [grant number
CASIMM0120184005], and the China Postdoctoral Science Foundation [grant number
2019M651623].
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Figure legends
Scheme 1. Synthesis of A17. Cat. catalyst, DCM dichloromethane, HATU
2-(7-Azabenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate, r.t. room
temperature, TEA triethylamine, THF tetrahydrofuran, UDCA ursodesoxycholic acid.
Fig. 1. Intracellular TG concentrations were up-regulated by OA in RPHs, while A17
ameliorated the elevation, improved cell viability and decreased lipid peroxidation. Cells were
treated with various concentrations of OA for 24 h. Corresponding concentrations of BSA
were used as negative controls. (A) Cell viability was measured by CCK-8 assay and the cells
were harvested for intracellular TG measurement (B). Then, 500 μM OA was used to induce
64