A novel AMPK activator, WS070117, improves lipid metabolism discords in hamsters and HepG2 cells.

Abstract

WS070117 is a novel small molecule compound that significantly improves lipid metabolism disorders in high-fat-diet (HFD) induced hyperlipidemia in hamsters.
We evaluated liver/body weight ratio, liver histology, serum and hepatic lipid content in HFD-fed hamsters treated with WS070117 for 8 weeks. Comparing with HFD fed hamsters, WS070117 (2 mg/kg per day and above) reduced serum triglyceride (TAG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and hepatic cholesterol and triglyceride contents. Oil Red O staining of liver tissue also showed that WS070117 improved lipid accumulation. We then carried out an experiment in the oleic acid (OLA)-induced steatosis model in HepG2 cell to investigate the lipid-lowering effect of WS070117. Oleic acid (0.25 mM) markedly induced lipid accumulation in HepG2 cells, but WS070117 (10 μM) inhibited cellular lipid accumulation. In OLA-treated HepG2 cells, WS070117 (above 1 μM) treatment reduced lipid contents which synthesized from [1-(14)C] labeled acetic acid. Because WS070117 is an analog of adenosine, we evaluated the effect of WS070117 on AMP-activated protein kinase (AMPK) signaling. The results showed that the activation of AMPK in OLA-induced steatosis in HepG2 cells was up-regulated by treatment with 0.1, 1 and 10 μM WS070117. The hepatic cellular AMPK phosphorylation is also up regulated by WS070117 (6 and 18 mg/kg) treatment in HFD fed hamsters.
These new findings identify WS070117 as a novel molecule that regulates lipid metabolism in the hyperlipidemia hamster model. In vitro and in vivo studies suggested that WS070117 may regulate lipid metabolism through stimulating the activation of AMPK and its downstream pathways.

Full text

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RESEARCHOpen Access
A novel AMPK activator, WS070117, improves
lipid metabolism discords in hamsters and HepG2
cells
Zeqin Lian, Yan Li, Jian Gao, Kai Qu, Jin Li, Linghua Hao, Song Wu*and Haibo Zhu*
Abstract
Background: WS070117 is a novel small molecule compound that significantly improves lipid metabolism
disorders in high-fat-diet (HFD) induced hyperlipidemia in hamsters.
Methods and Results: We evaluated liver/body weight ratio, liver histology, serum and hepatic lipid content in
HFD-fed hamsters treated with WS070117 for 8 weeks. Comparing with HFD fed hamsters, WS070117 (2 mg/kg per
day and above) reduced serum triglyceride (TAG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C)
and hepatic cholesterol and triglyceride contents. Oil Red O staining of liver tissue also showed that WS070117
improved lipid accumulation. We then carried out an experiment in the oleic acid (OLA)-induced steatosis model
in HepG2 cell to investigate the lipid-lowering effect of WS070117. Oleic acid (0.25 mM) markedly induced lipid
accumulation in HepG2 cells, but WS070117 (10 μM) inhibited cellular lipid accumulation. In OLA-treated HepG2
cells, WS070117 (above 1 μM) treatment reduced lipid contents which synthesized from [1-14C] labeled acetic acid.
Because WS070117 is an analog of adenosine, we evaluated the effect of WS070117 on AMP-activated protein
kinase (AMPK) signaling. The results showed that the activation of AMPK in OLA-induced steatosis in HepG2 cells
was up-regulated by treatment with 0.1, 1 and 10 μM WS070117. The hepatic cellular AMPK phosphorylation is also
up regulated by WS070117 (6 and 18 mg/kg) treatment in HFD fed hamsters.
Conclusion: These new findings identify WS070117 as a novel molecule that regulates lipid metabolism in the
hyperlipidemia hamster model. In vitro and in vivo studies suggested that WS070117 may regulate lipid
metabolism through stimulating the activation of AMPK and its downstream pathways.
Background
Metabolism syndromes are often linked to the macronu-
trient content of the diet and there is evidence that
excessive consumption of macronutrients such as carbo-
hydrates, fats, and even protein may eventually lead to
the development of metabolic syndromes [1]. Diets high
in fats and cholesterol have been demonstrated to
induce weight gain, insulin resistance and hyperlipide-
mia in humans and animal models [2]. Control of
serum lipids is important to prevent cardiovascular
events. For many patients, however, achievement of
tight serum lipid control by diet is difficult, requiring
therapy with drugs [3]. Statins are one of the most
widely used classes of drugs in clinical anti-
hyperlipidemia therapy. However, availability of drugs
that alleviate dyslipidemia syndromes is somewhat lim-
ited, warranting the discovery and characterization of
novel molecules targeting various pathways involved in
the pathogenesis of hyperlipidemia.
Activation of AMP-activated protein kinase (AMPK), a
heterotrimeric energy-sensing protein, acts to restore
cellular energy balance by promoting ATP-generating
pathways such as fatty acid oxidation while simulta-
neously inhibiting ATP-utilizing pathways such as fatty
acid synthesis [4]. Historically, the primary function of
AMPK was to generate ATP when cellular levels are
low. The AMPK system plays a major role in the regula-
tion of hepatic glucose and lipid metabolism through its
acute effects on energy metabolism and long-term
effects on gene expression patterns in the liver [5]. Simi-
larly, pharmacological activation of AMPK by metformin
* Correspondence: ws@imm.ac.cn; zhuhaibo@imm.ac.cn
Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking
Union Medical College, Beijing, 100050, China
Lian et al. Lipids in Health and Disease 2011, 10:67
http://www.lipidworld.com/content/10/1/67
© 2011 Lian et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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prevents intracellular accumulation of lipids, and
reverses fatty liver in obese humans and mice [6,7].
Therefore, it is clearly an attractive therapeutic target in
cardio-metabolic diseases.
During our screening for AMPK activators, we seren-
dipitously identified WS070117 as a lead compound
with potent lipid-regulating properties. The objective of
this study was to evaluate the impact of WS070117 on
an animal and cellular model of hyperlipidemia. Studies
in the hamster hyperlipidemia model revealed that
WS070117 markedly improved lipid abnormalities asso-
ciated with conditions characteristic of the high fat diet
(HFD)-induced lipid metabolism disorder. Investigations
into cellular signaling mechanisms suggested that
WS070117 may produce the lipid-regulating effects by
the phosphorylation of the AMPK signaling pathway.
Methods
Synthesis of WS070117
The synthesis of O2’,O3’,O5’-tri-acetyl-N6-(3-hydroxylani-
line)adenosine, named as WS070117 (Figure 1A), began
with acetylation of inosine using commercially available
acetic anhydride, chlorination with SOCl2, and substitution
with 3-aminophenol. Briefly, acetic anhydride was added
to a suspension of inosine in dry pyridine at 0°C and stir-
red at room temperature for 6 h to yield the pure product
O2’,O3’,O5’-tri-acetylinosine (Compound 1). Compound 1
was then dissolved in dry CH2Cl2:DMF (50:1) and a solu-
tion of SOCl2in CH2Cl2was added in a drop-wise manner.
After dilution with CH2Cl2, the organic layer of the reac-
tion mixture was washed with saturated NaHCO3solution
and brine and dried with anhydrous sodium sulfate. To a
solution of O2’,O3’,O5’-triacetyl-6-chloroadenosine in abso-
lute ethanol was added 3-aminophenol and dry triethyla-
mine. The mixture was then refluxed for 8 h at 60°C. The
solution was concentrated in vacuum and the residue was
chromatographed (ethyl acetate:petroleum ether, 2:1) to
give WS070117 as a white solid.
Animal
Golden hamsters (12-month-old) were purchased from
Vital River Laboratory Animal Technology Co. Ltd. (Beij-
ing, China). All animal treatment procedures described
in this study were approved by the Animal Care and Use
Committee at the Institute of Materia Medica, Chinese
Academy of Medical Sciences and Peking Union Medical
College (Beijing, China). Animals were housed under
well-controlled conditions of temperature (22 ± 2°C),
humidity (55 ± 5%) and a 12 h light-dark cycle with
access to food and water ad libitum.
Experiment design and drug administration
Except the control hamsters (n = 5), other hamsters (n
= 40) were fed high fat diet (HFD) accustomedly for 2
weeks, blood was collected from orbit vein for assaying
the content of serum total cholesterol and triglyceride
for grouping. Then, The HFD fed hamsters were divided
into the following groups with eight animals per group
respectively, accounted into their body weight and the
levels of serum total cholesterol and triglyceride, HFD
A
N
N
N
N
O
O O
O
O
HN
OH
O
O
B
C

Figure 1 A novel structure compound, WS070117, activated
AMPK in HepG2 cells. A: Structure of WS070117; B: Effect of
WS070117 on AMPK activity in HepG2 cells detected by AMPK
activity assays with SAMS peptide and [g-32P]ATP used as
substrates. HepG2 cells were treated with WS070117 (10 μM) for 1h,
6h and 12h. C: Effect of 12h treatment of WS070117 (1,10,100 μM)
or AICAR (1 mM) on AMPK activity of HepG2 cells. AMPK activities
are expressed relative to activity detected in HepG2 cells lysate.
Each point is the mean (± SEM) of 3 separate experiments. **P <
0.01, *P < 0.05 as compared with control.
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model group, HFD+Simvastatin (2 mg/kg) as a positive
group, HFD+WS070117 low dose group (2 mg/kg),
HFD+WS070117 middle dose group (6 mg/kg) and
HFD+WS070117 high dose group (18 mg/kg). The ani-
mals in normal control group were fed ordinary diet,
and the other groups were still fed HFD ad libitum for
8 weeks with the indicated drugs oral administration.
WS070117 was dissolved in distilled water prior to
administration. All drugs were given once daily by oral
gavage for 8 weeks. The animals in the normal control
and in the HFD model control group received the same
volum of distilled water as that of the drug-treated
groups.
Cell culture and treatment
The hepatic cell line HepG2 was purchased from the
Cell Resource Center(IBMS, CAMS/PUMC, Beijing
China) was grown in Dulbecco’s modified Eagle’s med-
ium (DMEM, GIBCO, NY, USA) supplemented with
10% fetal bovine serum (FBS, MDgenics, St. Louis, MO,
USA) and 1% penicillin-streptomycin under a humidi-
fied atmosphere of 5% CO2in air. After growth to 70%
to 80% confluence, cells were rendered quiescent by
incubating with serum-free medium for 12 h before
fatty acid treatment. For OLA-induced lipid deposition,
the medium for cells at 75% confluence was exchanged
for new medium with or without 0.25 mmol/L OLA
(Sigma, St. Louis, MO, USA) [8]. Meanwhile, different
concentration of WS070117 was added into medium
and treated with OLA for 12h.
Serum isolation and cholesterol determination
Blood samples were collected from the retro-orbital
plexus 1 h after drug treatments. Serum was isolated at
room temperature. Standard enzymatic methods were
used to determine TC, TAG, LDL-c and high density
lipoprotein cholesterol (HDL-C) levels with commer-
cially available kits purchased from Bio Sino Bio-tech-
nology and Science Inc. (Beijing, China). Each sample
was assayed in duplicate.
Measurement of hepatic TAG and TC contents in hamster
livers
100 mg of hamster liver tissue was taken for lipid
extraction. Briefly, the frozen tissue was thawed and
homogenized with a motor driven homogenizer in 2 ml
chloroform/methanol (2:1). After homogenization, lipids
were further extracted by rocking samples for 1 h at
room temperature, followed by centrifugation at 5,000
rpm for 10 min. The liquid phase was washed with 0.2
volume of 0.9% saline. The mixture was centrifuged
again at 2,000 rpm for 5 min to separate the two phases.
The lower phase containing lipids was evaporated and
lipids were dissolved in 0.5 ml isopropanol containing
10% Triton X-100 for TAG and TC measurements.
Oil red O stain
For histological examination, pieces of liver tissues were
fixed in 4% paraformaldehyde and dehydrated in 30%
sucrose solution at room temperature. Tissues were
then immersed in Optimal Cutting Temperature (OCT)
solution on dry ice for Oil Red O staining. Three ham-
ster livers were randomly picked from each group and
three different tissue sections from each liver were pro-
cessed and stained using routine laboratory procedures.
For fat accumulation examination in HepG2 cells, cul-
ture dishes were washed with cold phosphate-buffered
saline and fixed in 4% paraformaldehyde. After 2
changes of 60% isopropanol, Oil-Red-O was added with
agitation for 10 minutes, followed by washing in 50%
isopropanol. For each dish, 3 images were photo-
graphed, and a representative image is shown.
AMP-activated protein kinase (AMPK) activity assay
AMPK activity assays were performed on lysates of
HepG2 cells cultured in 6-well plates with 10% FBS/
DMEM supplemented with 1,6,12 hours time course
(10 μM) and 1,10,100 μM dosage (12 hours) of
WS070117 by measurement of [g-32P]ATP (Perkin-
Elmer, Boston, MA, USA) radioactivity phosphorylated
by a synthetic peptide substrate, SAMS [9]. AMPK
activity was expressed as nmol of phosphate incorpo-
rated into the peptide substrate per min per mg of
lysate (nmol/min/mg).
Lipid synthesis assay
OLA HepG2 cells was co-incubated with 0.1,1 and 10
μM WS070117 for 12h. Incorporation of [1-14C] acetic
acid (Perkin-Elmer, Boston, MA, USA) into lipid was
performed as described previously [10]. After replace-
ment with 1 ml of fresh medium, the cellular lipid was
labeled by incubating for 2.5 h with [1-14C] acetic acid
(0.2 μCi/ml). Lipid was extracted with n-hexane:2-pro-
panol (3:2, v/v) and dried under a nitrogen stream. Ali-
quots of lipid samples were loaded onto thin-layer
chromatography plates and chromatographed in a sol-
vent mixture containing hexane-diethylether-acetic acid
(85:30:1, v/v/v). Bands corresponding to triglyceride and
cholesteryl ester were scraped off, immersed in non-
aqueous scintillation fluid, and counted for radioactivity.
Western blot analysis
The expression and phosphorylation of each protein
were analyzed by Western blot analysis. Briefly, protein
lysates were subjected to 10% SDS-polyacrylamide gel
electrophoresis. Proteins were transferred to a
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nitrocellulose membrane and the membranes were
blocked for 1 h at room temperature with 5% non-fat
dry milk in Tris-buffered saline (TBS) containing 0.1%
Tween 20. Immunostaining to detect each protein was
achieved after over-night incubation (4°C) with a 1:1000
dilution of anti-phospho (thr-172) AMPK (1:2000),
AMPK (1:2000) and anti-beta-actin (1:5000). Proteins
were visualized after subsequent incubation with a
1:5000 dilution of anti-mouse or rabbit IgG conjugated
to horseradish peroxidase and use of a Super Signal
Chemical luminescence detection procedure. All antibo-
dies used in this study were from Cell Signaling Tech-
nology Inc. (Beverly, MA, USA). Protein concentrations
were determined using a bicinchoninic acid assay
(Applygen Inc., Beijing China). Three independent
experiments were performed for each condition and the
intensity of immune-reactive bands was analyzed with a
Bio-Rad calibrated densitometer.
Statistical analyses
One-way ANOVA was used to determine significant dif-
ferences among groups, after which the modified Stu-
dent’s t-test with the Bonferroni correction was used for
comparison between individual groups. P < 0.05 was
considered statistically significant.
Results
WS070117 increased AMPK activity
AMPK has been proposed to act as a fuel gauge in
mammalian cells and to play a crucial role in regulating
fat metabolism in the liver. The major pathways that
regulate intracellular lipid metabolism are evidently pre-
sent in HepG2 cells. The change of AMPK activity in
HepG2 cells is strongly associated with intracellular
lipid metabolism. Because WS070117 is an adenosine
analogue, we studied the possibility that WS070117
could activate AMPK. Incubation of HepG2 cells with 1
~ 100 μM WS070117 for 12h significantly activated
AMPK, as measured in cell lysates. WS070117 also acti-
vated AMPK in a time dependently manner within 12
hours treatment. AICAR, one of the potent AMPK acti-
vators, also increased AMPK activity (Figure 1B, C).
In vivo reductions of plasma and hepatic lipid contents
by WS070117
HFD with obese hamsters fed a diet containing 20% fat
was used for producing the lipid metabolism dyslipide-
mia model. After 8 weeks, hamster body weights
increased about 20% with no difference among the HFD
treated groups. The liver/body weight ratio was mark-
edly increased at the end of the experiment. The
increases in the groups treated with WS070117 were
significantly decreased the liver hypertrophy as com-
pared with the HFD group (Table 1). WS070117 (2, 6 or
18 mg/kg/day) treatment resulted in significant
decreases in serum TC, TAG and LDL-c as well as
hepatic TAG and TC (Figure 2B).
Oil Red O staining of livers showed that the hepato-
cytes of animals in the HFD group were compressed
and separated by bulks of fat, demonstrating the abnor-
mally high levels of fat accumulation in liver (Figure
2A). 8 weeks treatment with 18 mg/kg WS070117 sub-
stantially repressed these changes in HFD hamsters.
Therefore, WS070117 helped to keep normal fat accu-
mulation and deposition under HFD feeding conditions.
WS070117 decreases lipid accumulation and de novo
fatty acid synthesis in vitro
Hepatic steatosis in humans is associated with accumu-
lation of excess OLA and the end-product of de novo
fatty acid synthesis [4]. AMPK-mediated phosphoryla-
tion of protein targets involved in cholesterol and fatty
acids synthesis, however, lead to inhibition of cholesterol
and TAG synthesis. In present study, Oil Red O staining
showed that 10 μM WS070117 decreased the lipid accu-
mulation in HepG2 cells induced by 250 μM OLA (Fig-
ure 3A). We then evaluated the impact of WS070117 on
TAG and cholesterol synthesis by incubating HepG2
cells for 2 h with [14C]acetate in the presence of increas-
ing concentrations of WS070117 (Figure 3B). Both
Table 1 Hamsters were fed with normal chow or high fat diet (HFD) for 10 weeks
Control(n = 5) HFD(n = 8)HDF+WS070117 HFD+Simvastatin 2 mg/kg(n = 8)
2 mg/kg(n = 8)6 mg/kg(n = 8)18 mg/kg(n = 8)
TAG (mmol/L)
TC (mmol/L)
HDL (mmol/L)
LDL (mmol/L)
2.05 ± 0.52
3.95 ± 0.54
1.48 ± 0.25
2.40 ± 0.36
10.98 ± 5.84##
9.22 ± 3.87##
3.18 ± 1.46#
5.30 ± 2.01##
168.8 ± 14.7##
0.049 ± 0.0046##
6.52 ± 2.75
7.33 ± 1.84
2.56 ± 0.89
4.43 ± 0.58
5.91 ± 1.98*
6.81 ± 1.49
2.45 ± 0.90
3.97 ± 0.75*
4.51 ± 1.88**
6.11 ± 0.65*
2.14 ± 0.26
3.16 ± 0.43**
6.18 ± 1.28*
7.73 ± 1.31*
2.51 ± 0.60
2.51 ± 0.45*
Body weight
Liver/Body weight
136 ± 16.5
0.039 ± 0.0054
166.5 ± 9.3
0.044 ± 0.0056*
165.2 ± 15.7
0.044 ± 0.0034*
164.5 ± 13.6
0.045 ± 0.0023*
166.7 ± 12.6
0.045 ± 0.0018*
Serum was separated after 8 weeks of WS070117 and Simvastatin treatment at the indicated doses from hamsters fed a high fat diet (HFD). The actual values of
mean ± SD from 8 animals (control group, n = 5) are presented.##P < 0.01,#P < 0.05, as compared to vehicle control; ** P < 0.01, *P< 0.05 as compared to HFD
model.
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cholesterol and TAG synthesis were dose-dependently
inhibited by WS070117 (0.1 ~ 10 μM).
Effect of WS070117 on phosphorylation and activation of
AMPK in HepG2 cells and liver tissue
AMPK activation is thought to be a key proximal event in
the cellular energy balance response, and AMPK
phosphorylation levels are currently accepted as a marker
of AMPK activity. Therefore, we determined the phos-
phorylation of AMPK in OLA overload HepG2 cells.
After 12h treatment of WS070117, AMPK phosphoryla-
tion is activated at 0.1 10 and 1 μM. In HFD-fed hamster
livers, AMPK phosphorylation was also increased by
treatment with 6 or 18 mg/kg WS070117 (Figure 4).
A

B
HFD
HFD+WS070117(18mg/kg)
Control
HFD+ Simvastatin (2mg/kg)
Figure 2 Effects of WS070117 on hepatic lipids accumulation in HFD fed hamsters. A: Representative frozen tissue sections of liver taken
from hamsters fed chow diet, HDF diet, HFD+ simvastatin (2 mg/kg) and HFD+WS070117 (18 mg/kg) were stained with Oil Red-O to
demonstrate the reduction in lipid droplets. B: Hepatic TC and TAG were measured in liver samples of control (n = 5), HFD, 8-weeks WS070117
(2, 6, 18 mg/kg) or simvastatin (positive control; 2 mg/kg) treated HFD fed hamsters (n = 8). **P < 0.01, *P < 0.05 as compared with HFD model.
##P < 0.01 as compared with control.
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