Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway.
ABSTRACT Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood.
As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes.
The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein β (C/EBPβ), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT element binding protein α (C/EBPα) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished.
Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway. There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity.
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ABSTRACT: The nuclear receptors REV-ERB (consisting of REV-ERBα and REV-ERBβ) and retinoic acid receptor-related orphan receptors (RORs; consisting of RORα, RORβ and RORγ) are involved in many physiological processes, including regulation of metabolism, development and immunity as well as the circadian rhythm. The recent characterization of endogenous ligands for these former orphan nuclear receptors has stimulated the development of synthetic ligands and opened up the possibility of targeting these receptors to treat several diseases, including diabetes, atherosclerosis, autoimmunity and cancer. This Review focuses on the latest developments in ROR and REV-ERB pharmacology indicating that these nuclear receptors are druggable targets and that ligands targeting these receptors may be useful in the treatment of several disorders.dressNature Reviews Drug Discovery 02/2014; 13(3):197-216. · 33.08 Impact Factor
Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes
through LKB1/AMPK Pathway
Yonghan He1,2, Ying Li1, Tiantian Zhao2,3, Yanwen Wang2,4*, Changhao Sun1*
1Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, Harbin, People’s Republic of China, 2Aquatic and Crop Resource
Development, Life Sciences Branch, National Research Council Canada, Charlottetown, Prince Edward Island, Canada, 3Department of Psychology, University of Toronto,
Toronto, Ontario, Canada, 4Department of Biomedical Sciences, University of Prince Edward Island, Charlottetown, Prince Edward Island, Canada
Background: Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently
been reported to possess an anti-obesity effect; however, the mechanisms are less understood.
Objective: As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA
on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes.
Methods and Results: The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days.
The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular
targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at
concentrations ranging from 2.5 mM to 10 mM dose-dependently attenuated adipogenesis, accompanied by reduced
protein expression of CCAAT element binding protein b (C/EBPb), peroxisome proliferator-activated receptor c (PPARc),
CCAAT element binding protein a (C/EBPa) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic
acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase
1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid
increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of (silent mating type
information regulation 2, homolog) 1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was
reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of
AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK
activation was diminished.
Conclusions: Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway.
There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity.
Citation: He Y, Li Y, Zhao T, Wang Y, Sun C (2013) Ursolic Acid Inhibits Adipogenesis in 3T3-L1 Adipocytes through LKB1/AMPK Pathway. PLoS ONE 8(7): e70135.
Editor: Miguel Lo ´pez, University of Santiago de Compostela School of Medicine – CIMUS, Spain
Received March 5, 2013; Accepted June 15, 2013; Published July 26, 2013
Copyright: ? 2013 He et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was supported by funding from the Canadian Institutes of Health Research (CIHR)-China-Canada Joint Health Research Initiative (CCI-92219)
and the NRC-MOE Research and Post-doctoral Fellowship Program of Chinese Scholarship Council (CSC). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (YW); firstname.lastname@example.org (CS)
Obesity has become an epidemic in developed countries and
also many developing countries. The rates of obesity and
overweight have been continuing to grow in adults, and
unfortunately that the situation has been worsening by penetrating
into the child and adolescent population. In addition to morbidity,
obesity is associated with many metabolic complications, including
type-II diabetes, insulin resistance, hyperlipidemia, hypertension
and coronary heart disease [1,2]. These complications result in a
considerably higher rate of mortality in obese than lean subjects.
Although a number of drugs have been developed and used to
treat obese patients through regulating appetite, fat absorption and
fat oxidation [3,4], low efficacy and side effects are of great
concerns and result in the withdraw of many anti-obesity drugs
from market, leaving few drugs that can be prescribed [5,6].
Various programs including lifestyle change and intensive exercise
have been used to loose and help to control body weight, successful
rate is marginal. It is still of demand to develop more efficacious
and safer anti-obesity products/drugs. In recent years, numerous
bioactive compounds in food items and plants, such as resveratrol
, quercetin , and epigallocatechin gallate , have been
explored for their potential anti-obesity activities. Ursolic acid is a
natural pentacyclic triterpenoid, which is present in many different
plants, fruits and herbs. Evidence from in vitro and in vivo studies
suggests that UA possesses many nutritional and pharmacological
functions, including anti-inflammatory , anti-oxidative ,
anti-mutagenic , anti-carcinogenic , hepatoprotective
, anti-microbial , anti-atherosclerotic, and anti-hyperlip-
idemic effects . Recent studies demonstrated that UA inhibited
abdominal adiposity in mice fed a high-fat diet [17,18]. It is
reported that UA may reduce adiposity by enhancing lipolysis
PLOS ONE | www.plosone.org1 July 2013 | Volume 8 | Issue 7 | e70135
[19,20] and/or inhibiting protein tyrosine phosphatase 1B
(PTP1B) activity . On the other hand, it is well known that
adipogenesis plays a vital role in the development of obesity;
however, information is lacking regarding whether and how UA
Adipogenesis is determined by multi-processes, which include
preadipocyte proliferation, differentiation, and fatty acid oxidation
and synthesis, and controlled by a number of molecular factors.
Emerging evidence suggests that AMP-activated protein kinase
(AMPK) functions as a sensor of cellular energy status. Once
activated, it switches on the catabolic pathways and simultaneously
switches off the ATP-consuming anabolic pathways . AMPK
provides an upstream signal of peroxisome proliferator-activated
receptor c (PPARc) and inhibits differentiation of preadipocytes
into adipocytes [23,24]. Furthermore, (silent mating type infor-
mation regulation 2, homolog) 1 (Sirt1) is an NAD-dependent
deacetylase that also serves as a master metabolic sensor, regulated
by NAD+concentration, and modulates cellular energy metabo-
lism . Sirt1 has been reported to inhibit adipogenesis in 3T3-
L1 cells by repressing PPARc and is involved in the regulation
of the number and function of adipocytes . Therefore, the
present study was conducted to determine the effect of UA on
adipogenesis and mechanism of action, with the primary focus on
the regulation of UA on the energy sensors AMPK and Sirt1 and
further their downstream lipogenic targets in 3T3-L1 adipocytes.
Materials and Methods
Chemicals and reagents
Ursolic acid, nicotinamide, radicicol, insulin, 3-isobutyl-1-
methylxanthine (IBMX), dexamethasone, propidium iodide,
ribonuclease (Rnase) and protease inhibitor were purchased from
Sigma (St. Louis, MO, USA). High glucose Dulbecco’s modified
Eagle’s medium (DMEM) was from Mediatech, Inc. (Cellgro
Mediatech, Inc. Manassas, VA). Fetal bovine serum (FBS) was
from PAA Laboratories (Etobicoke, ON, Canada). Bovine calf
serum (BCS) and adipogenesis assay kits were purchased from
Cayman Chemical Company (Ann Arbor, Michigan, USA).
Lipolysis assay kits were purchased from Zen-Bio, Inc. (Research
Triangle Park, NC, USA). The BCA protein assay kit was from
Thermo Scientific (San Jose, CA, USA). RIPA lysis buffer was
from Millpore (MA, USA). Protein loading buffer was from Bio-
Rad (Montreal, QC, Canada). Antibodies against sterol regulatory
element binding protein 1c (SREBP-1c), pAMPKa (Thr 172),
AMPKa, fatty acid-binding protein 4 (FABP4), b-actin, carnitine
palmitoyltransferase 1 (CPT1), acetyl-CoA carboxylase (ACC) and
pACC were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA). Antibodies against liver kinase B1 (LKB1),
pLKB1, PPARc, CCAAT element binding protein a (C/EBPa)
and C/EBPbwere from Cell Signaling Technology, Inc. (Beverly,
Massachusetts, USA). Fatty acid synthase (FAS) antibody was from
Novus Biologicals (Oakville, ON, Canada). BM chemilumines-
cence blotting substrate kit was from Roche Diagnosis (Laval, QC,
Canada). LKB1 and AMPK small interfering RNAs (siRNAs),
Opti-MEM, reduced serum medium and lipofectamine RNAi-
MAX were obtained from Life Technologies (Rockville, MD).
3T3-L1 mouse embryo fibroblasts were obtained from Amer-
ican Type Culture Collection (Rockville, MD) and cultured in
DMEM containing 10% BCS until confluent, and were then
maintained in the same medium for an additional 2 days.
Differentiation was induced 2 days post-confluence (day 0 of
differentiation) by adding 0.5 mM IBMX, 1 mM dexamethasone,
and 5 mg/mL insulin in DMEM with 10% FBS. After 2 days of
incubation, culture medium was changed to fresh DMEM
containing 10% FBS and 5 mg/mL insulin. Two days later, the
medium was replaced with DMEM supplemented with 10% FBS
and incubated for another two days. UA was added two days after
confluence (day 0) and maintained during cell differentiation until
the time when the cells were harvested for the following described
3T3-L1 preadipocytes were seeded onto 96-well plates at a
density of 104cells/cm2and differentiated with induction medium
in the presence of 2.5 to 20 mM UA for 6 days, as described above.
Cells were treated with MTT assay reagents (1 mg/mL) for
4 hours and the resulting formazan was solubilized in 150 mL
dimethyl sulfoxide (DMSO) and further diluted 10 times with
DMSO. The absorbance was measured at 570 nm on a Varioskan
Flash spectral scanning multimode plate reader (Thermo Fisher
Scientific, Waltham, MA).
density of 36103cells/cm2in 96-well plates. The cells were grown
in DMEM containing 10% BCS supplemented with increasing
doses of UA for 24, 48, and 72 hours. MTT solution (1 mg/mL)
was added to the medium and incubated for 4 hours. The purple
formazan crystals were dissolved in 150 mL DMSO and the
absorbance was read at 570 nm on the Varioskan Flash spectral
scanning multimode plate reader as described above.
3T3-L1 preadipocytes were seeded at a low
3T3-L1 preadipocytes were seeded onto 24-well plates at a low
density of 36103cells/cm2. The cells were treated with different
concentrations of UA for 24, 48, and 72 hours, respectively. The
number of adherent cells was determined by direct counting using
a hemocytometer and a mini-automated cell counter from Orflo
(http://www.orflo.com) after being trypsinized. Three indepen-
dent experiments were performed with triplicates in each
Flow cytometric analysis of cell cycle and apoptosis
Three days post-confluence, 3T3-L1 preadipocytes were
differentiated in the presence of 0, 2.5, 5, 10 mM ursolic acid for
24 hours. The cells were collected and fixed overnight with 70%
ethanol at 4uC. Following wash with PBS, the cells were stained
with propidium iodide solution containing 20 mg/ml of RNase for
30 minutes. Fluorescence-activated cell sorting (FACS) analysis
was performed on a Becton–Dickinson FACScan system and data
were analyzed using the Flowjo software (Version 7.6.1, Tree Star
Software; San Carlos, CA, USA).
Intracelluar lipid accumulation
3T3-L1 preadipocytes were seeded onto 96-well plates and were
induced to differentiate in the presence or absence of ursolic acid
as described above. Lipid accumulation was measured 6 days after
the induction using a commercial adipogeneis assay kit. Briefly, the
cells were fixed with lipid fixative solution, stained with Oil Red O
and washed with distilled water. Intracellular lipids were extracted
with the extraction solution included in the kit. The absorbance
was read at 520 nm on the same plate reader as described above.
Images were captured under a Nikon inverted microscope
(ECLIPSE TE200) after washing with distilled water.
Ursolic Acid Iinhibits Adipogenesis
PLOS ONE | www.plosone.org2July 2013 | Volume 8 | Issue 7 | e70135
3T3-L1 preadipocytes were seeded onto 96-well plates and
induced to differentiate 2 days after confluence. The differentiated
Cells were treated with different concentrations of ursolic acid for
24 and 48 hours to detect cell viability as described above.
Lipolysis was measured using a commercial 3T3-L1 lipolysis assay
kit. Briefly, cells were washed twice with 200 mL wash buffer and
then treated for 3 hours with 150 mL of 10 mM ursolic acid
suspended in the assay buffer in a humidified incubator at 37uC.
One hour prior to the assay, glycerol standards were prepared
following the kit’s instructions (Zen-Bio, Inc., Research Triangle
Park, NC, USA). At the end of the incubation, 100 mL of medium
was collected from each well and transferred to a new plate with
addition of 100 mL of the glycerol reagent. After 15 minutes at
room temperature, the absorbance was measured at the
wavelength of 540 nm on the Varioskan Flash spectral scanning
multimode plate reader
After 6-day differentiation in the presence of UA, 3T3-L1
adipocytes were collected and lysed in ice-cold RIPA lysis buffer
for 30 minutes. Protein content was determined using a BCA
protein assay kit. Equal amount of protein for each sample was
loaded and separated on a 10% SDS-PAGE. After electrophoretic
separation, the proteins were transferred to a nitrocellulose filter
using a semi-dry transfer cell (Bio-Rad) at 13 V for 1 hour,
blocked with 5% skim milk for 1 hour at room temperature, and
incubated with primary antibodies at 4uC overnight. The
nitrocellulose filters were then incubated with horseradish-
peroxidase conjugated secondary antibody at room temperature
for 3 hours. Immunoreactive proteins were detected using the
chemiluminescent ECL assay and quantified using the Molecular
Imager software (Bio-Rad). C/EBPbexpression was determined 2
days after the induction of cell differentiation in the presence or
absence of the indicated concentrations of UA. The expression of
each protein was present as fold of the loading control, b-actin.
LKB1 and AMPK gene silencing
3T3-L1 preadipocytes were transfected with LKB1 or AMPK
siRNA oligonucleotide duplexe 1 day post the confluence with
lipofectamine RNAiMax. Generally, 75 nM siRNA was transfect-
ed with 4 ml/well of lipofectamine in a 6-well plate. Lipofectamine
RNAiMax and siRNA were individually diluted in 50 mL Opti-
MEM medium, mixed, incubated for 20 minutes at room
temperature, and then added to each well. The medium was
removed and replaced with the induction medium in the presence
or absence of UA after 24 hours of transfection. The effectiveness
of siRNA knockdown was determined after 24 hours of transfec-
tion and on day 6 of cell differentiation, respectively, by measuring
the expression of LKB1 or AMPK using the Western blotting.
The statistical analyses were performed using SPSS 13.0
statistical program (version 13.01S; Beijing Stats Data Mining
Co. Ltd). The treatment effect was determined using one-way
ANOVA and followed by a post-hoc Dunnett’s or Bonferroni’s
multiple comparisons test, where a P value less than 0.05 was
considered significant. Data are presented as means 6 SD.
Effect of UA on fat cell viability
3T3-L1 preadipocytes were induced to differentiate in the
presence of 0, 2.5, 5, 10, 15, or 20 mM of UA for 6 days. The
MTT assay revealed that UA at concentrations of 2.5 to 10 mM
did not affect cell viability while 15 mM was toxic (Fig. 1A).
Therefore, the concentration range of 2.5–10 mM was chosen for
further experiments. In mature adipocytes, 2.5 to 30 mM of UA
did not affect cell viability after 24 hours of treatment whereas
after 48 hours 20 mM and higher concentrations of UA were toxic
(Fig. S1A, B).
Effect of UA on preadipocyte proliferation, cell cycle and
To investigate the effect of UA on preadipocyte proliferation,
3T3-L1 preadipocytes were grown in basal DMEM supplemented
with different concentrations of UA for 24, 48 and 72 hours,
respectively. As shown in Fig. 1B and Fig. 1C, neither
microscopic counting nor MTT assay revealed any effect of UA
on preadipocyte proliferation. The differentiation medium initiat-
ed cell cycle progression (Fig. 1D). The addition of different
concentrations of UA did not affect cell cycle (Fig. 1D) nor did
induce cell apoptosis (Fig. 1E).
UA inhibits lipid accumulation and increases lipolysis in
3T3-L1 preadipocytes underwent morphological changes from
the spindle-like features to round shape and accumulated
intracellular lipids after adding the induction reagents. Ursolic
acid at the doses of 2.5, 5, or 10 mM decreased intracellular fat
content compared to the control as revealed by microscopic
examination following Oil Red O staining in differentiated 3T3-
L1 adipocytes (Fig. 2A). Consistently, the intracellular fat content
was significantly reduced by UA at the concentrations ranging
from 2.5 to 10 mM (Fig. 2B). Ursolic acid at the concentrations of
2.5, 5, and 10 mM reduced the lipid content in 3T3-L1 adipocytes
by 10%, 19%, and 30%, respectively compared to the control.
These findings suggest that UA was involved in the process of
preadipocyte differentiation and adipogenesis. On the other hand,
10 mM UA significantly increased glycerol release compared to the
control group (Fig. S1C), indicating that UA stimulated lipolysis
in mature 3T3-L1 adipocytes.
UA decreases protein expression of adipogenic
Differentiation of preadipocyte into adipocyte is tightly regulat-
ed by a sequential activation of several transcriptional factors,
including C/EBPb, C/EBPa, PPARcand SREBP-1c. Normally,
C/EBPbfunctions quickly following the induction of preadipocyte
differentiation, followed by the expression of C/EBPaand PPARc.
As shown in Fig. 3A–3C, UA at 2.5 to 10 mM significantly
decreased the expression of C/EBPbon day 2 and subsequently
inhibited the expressions of PPARcand C/EBPaon day 6 after the
induction of differentiation. SREBP-1c expression was also
significantly down-regulated by UA at 10 mM after 6 days
UA modulates the expression of lipogenic and fatty acid
Since the adipogenic transcription factors were down-regulated
by UA, we further determined the expression and activation of
their downstream protein targets such as ACC, FAS and FABP4,
which are important adipogenic proteins involved in fatty acid and
triacylglycerol biosyntheses. Ursolic acid inactivated ACC by
increasing the phosphorylation of ACC (pACC) and reduced the
expression of FAS and FABP4 at the concentrations of 5 and
10 mM, respectively (Fig. 4A–C). By contrast, UA at the
Ursolic Acid Iinhibits Adipogenesis
PLOS ONE | www.plosone.org3July 2013 | Volume 8 | Issue 7 | e70135
concentrations of 2.5, 5 and 10 mM increased the protein
expression of CPT-1 (Fig. 4D).
UA regulates the expression or activation of energy
sensors AMPK and Sirt1
AMPK and Sirt1 are two important regulators of preadipocyte
differentiation and adipogenesis. In this study, UA at concentra-
tions of 5 mM or 10 mM increased AMPK phosphorylation
(pAMPK) and thus activation, while showing no effect on its total
protein expression (Fig. 4E). Similarly, UA at 5 mM and 10 mM,
respectively stimulated the protein expression of Sirt1 (Fig. 4F).
Effect of AMPK and Sirt1 on adipogenic differentiation
To further investigate the role of AMPK and Sirt1 in
preadipocyte differentiation, both proteins were inhibited by
AMPK siRNA and Sirt1 inhibitor nicotinamide, respectively. As
shown in Fig. S2A, the AMPK siRNA significantly reduced
AMPK protein expression, indicative of the effectiveness of
AMPK siRNA transfection. The transfection of AMPK siRNA
significantly increased lipid storage in differentiated 3T3-L1 cells
as compared with the control (Fig. 5B, D). Ursolic acid at 10 mM
significantly reduced fat content and strikingly, this effect was
completely reversed by AMPK siRNA (Fig. 5B, D). Sirt1
inhibitor did not show a significant effect on intracellular fat
accumulation as compared to the control nor did reverse the anti-
adipogenic effect of UA (Fig. 5C, D).
AMPK siRNA reversed the effect of UA on the AMPK
downstream adipogenic targets
To confirm whether UA modulates adipogenesis through
AMPK, we detected the expression of adipogenic transcription
factors, C/EBPa and PPARc, and the key lipogenic gene FAS and
Figure 1. Effects of UA on the viability, proliferation, cell cycle and apotosis of 3T3-L1 preadipocytes. (A) 3T3-L1 preadipocytes were
incubated in differentiation medium with or without UA. After 6 days, MTT reagent was added to the medium. After 4 hours of incubation, the
medium was aspirated and 150 mL DMSO was added to each well. The absorbance was read at 570 nm. (B) 3T3-L1 preadipocytes were seeded onto
96-well plates at a density of 36103cell/cm2and treated with indicated concentrations of UA for 24, 48 and 72 hours, respectively. MTT assay was
performed as described above to reflect the proliferation. (C) 3T3-L1 preadipocytes were seeded onto 24-well plates at a density of 36103cell/cm2
and treated with different concentrations of UA for 24, 48 and 72 hours, respectively. The number of adherent cells was determined using an
automatic cell counter. (D, E) Three days post-confluence, 3T3-L1 preadipocytes were differentiated in the presence or absence of 2.5, 5, 10 mM ursolic
acid for 24 hours. The cells were collected and fixed overnight with 70% ethanol at 4uC, and stained with propidium iodide solution. Cell cycle and
apoptosis rate were analyzed on a flow cytometry. Data are expressed as means 6 SD (n=3). * P,0.05 and ** P,0.001 vs. the control.
Ursolic Acid Iinhibits Adipogenesis
PLOS ONE | www.plosone.org4 July 2013 | Volume 8 | Issue 7 | e70135
ACC after cells were treated by AMPK siRNA. The application of
AMPK siRNA diminished the effect of UA on PPARc and C/
EBPa (Fig. 6A, B). Consistently, the decreased expression of FAS
by UA was reversed by the AMPK siRNA (Fig. 6C). The
phosphorylation of ACC was inhibited by AMPK siRNA without
affecting the expression of total ACC (Fig. 6D). It was
demonstrated that UA regulated adipogenic process in 3T3-L1
preadipocytes through the AMPK pathway.
LKB1 destabilizer or small interfering RNA diminishes the
effect of UA on AMPK activation
To understand whether UA modulates AMPK activity in 3T3-
L1 preadipocytes through its upstream regulator LKB1, we further
investigated the effect of UA on the expression and activation of
LKB1. It was observed that UA at 10 mM increased the LKB1
activity by increasing its phosphorylation (Fig. 7 A). To further
explore the role of LKB1 in the upregulation of UA on
adipogenesis, LKB1 siRNA and its destabilizer radicicol were
used, respectively. As shown in Fig. S2B and 2D, total LKB1
expression was inhibited by either radicicol or LKB1 siRNA.
Consequently, the increased phosphorylation of AMPK by UA
was diminished when LKB1 siRNA or inhibitor was applied.
(Fig. 7B and Fig. S2C), suggesting that UA activated AMPK via
LKB1. To further evaluate the effect of LKB1 knockdown on
adipogenesis in the presence of UA, we detected two key
adipogenic proteins, FAS and FABP4. As shown in Fig. 7C–
7D, LKB1 siRNA itself promoted the expression of FAS and
FABP4 compared to the control group, which is similar to the
effect of AMPK siRNA as described above. Furthermore, LKB1
siRNA abolished the inhibitory effect of 10 mM UA on FAS and
Ursolic acid is present in many fruits and plants and the major
component of several traditional medicine herbs. It is well known
to that UA possess a wide range of biological functions .
Recent studies have demonstrated anti-obesity effect of UA;
however, the mechanism of action is not well known. Adipogenesis
and lipolysis determine, to a large degree, the adipose tissue mass.
We previously demonstrated that UA stimulated lipolysis through
the cAMP-dependent PKA pathway in primary-cultured rat
adipocytes . In this study, we also found that UA stimulated
lipolysis in mature 3T3-L1 adipocytes. Most importantly, we
report here, for the first time, that UA suppresses preadipocyte
differentiation and adipogenesis through LKB1/AMPK pathway.
Adipose tissue is determined by the number and size of
adipocytes. The increase in the number of adipocytes involves the
proliferation of preadipocytes. Therefore, the growth and prolif-
eration of preadipocytes has a profound implication in the
development of obesity therapeutics or health products. Our
results suggest UA did not affect preadipocyte number, cell cycle
or apoptosis. However, UA significantly inhibited the differenti-
ation of preadipocytes. After treatment with 10 mM of UA for 6
days, adipogenesis in differentiated 3T3-L1 cells was reduced by
approximately 30%. Further studies demonstrated that several
transcription factors were involved in the regulation of UA on
adipogenic differentiation, including PPARc, C/EBP members
and SREBP-1c [29,30].
PPARc functions through coordinating the expression of genes
responsible for the establishment of mature adipocyte phenotype.
The expression of PPARc is sufficient to initiate and control
adipogenesis in growing fibroblast cells by binding to PPAR
response elements located in the promoter of target genes . In
addition, C/EBP proteins play critical roles in preadipocyte
differentiation . Among the C/EBP isoforms, C/EBPb is
responsible for the initial transcriptional activation of C/EBPa
gene , which is essential and sufficient to induce differentiation
of 3T3-L1 preadipocytes . Besides, SREBP-1c is a transcrip-
tional factor that is involved in lipid metabolism , controls fatty
acid synthase and is an additional regulator of adipogenesis.
SREBP-1c promotes preadipocyte differentiation and gene
expression associated with fatty acid metabolism . In the
current study, UA decreased C/EBPb expression in 3T3-L1
preadipocytes during the first 48 hours after the induction of
differentiation, and subsequently PPARc, C/EBPaand SREBP-1c
expressions on day 6, suggesting that UA inhibited preadipocyte
differentiation through altering the expression of transcriptional
factors involved at different differentiation stages. C/EBPa,
PPARc and SREBP-1c coordinately drive the expression of
adipocyte-specific genes, such as FAS, ACC and FABP4, which
determine the later stages of adipocyte differentiation and
associated biosynthesis of fatty acids and triacylglycerols .
FABP4 is responsible for facilitating the influx of fatty acids across
the plasma membrane and modulates the activity of enzymes
involved in fatty acid metabolism . ACC and FAS are the key
enzymes controlling fatty acids synthesis [38,39]. Our results
revealed that these two proteins were inhibited by UA. Besides
Figure 2. Effect of UA on lipid accumulation in 3T3-L1
adipocytes. (A) Post-confluent 3T3-L1 preadipocytes were induced
to differentiate in the absence or presence of UA (added on day 0 of
differentiation) for 6 days. The morphological changes associated with
cell differentiation were photographed after Oil Red O staining. (B)
Stained lipids were extracted and quantified by measuring absorbance
at 520 nm. Data are expressed as means 6 SD (n=3). * P,0.05 and **
P,0.001 vs. the control.
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PLOS ONE | www.plosone.org5July 2013 | Volume 8 | Issue 7 | e70135
fatty acids synthesis, fatty acid oxidation is also critically important
in the regulation of fat accumulation. CPT1 transports long chain
fatty acids into mitochondria for beta oxidation and thus acts as a
key regulatory enzyme in modulating fatty acid oxidation. The
dose-dependent increase of CPT1 expression by UA suggests an
increase of fatty acid oxidation. The findings of the current study
suggest that UA can inhibit preadipocyte differentiation and
adipogenesis through inhibiting the expression and/or activity of
the adipogenic transcriptional factors and their target genes.
Meanwhile, UA induced fatty acid oxidation could have also
contributed to the inhibition of adipogenesis and reduction of fat
accumulation by UA in differentiated 3T3-L1 preadipocytes.
Accumulating evidence suggests that AMPK and Sirt1 act as
intracellular energy sensors and regulate energy metabolism
through a concert process, and thus are of great interest in recent
years as molecular targets in obesity research. AMPK directly
modulates fatty acid synthesis and oxidation by altering the
expression and activation of enzymes and proteins involved in fat
metabolism. AMPK also regulates preadipocyte differentiation
and adipogenesis . It is reported that AMPK pathway is
responsible for the inhibition of adipocyte differentiation by several
natural compounds, such as apigenin , dioxinodehydroeckol
, chitin , ginsenoside Rg3 , and epigallocatechin
gallate . Sirt1 inhibits adipogenesis by repressing PPARc
expression in cultured adipocytes  and decreases adipocyte
formation during osteoblast differentiation of mesenchymal stem
cells . In this study, UA increased the phosphorylation and
activation of AMPK and the protein expression of Sirt1 in a dose-
dependent manner, indicating that UA might have inhibited
preadipocyte proliferation and adipogenesis through AMPK and/
or Sirt1. Further experiments showed that anti-adipogenesis of UA
was abolished by AMPK siRNA but not by Sirt1 inhibitor.
Consistently, the UA-induced reductions in the protein expression
of transcription factors C/EBPa, PPARcand their downstream
protein FAS and phosphorylation of ACC, were interestingly
reversed by blocking AMPK expression using AMPK siRNA.
LKB1 and calmodulin kinase kinase b are two main upstream
kinases of AMPK . LKB1 activates AMPK protein in adipose
tissue , while the role of calmodulin kinase kinase b in AMPK
activation is unclear. Therefore, we have moved a step further to
determine the effect of UA on LKB1 expression. The observed
increase in the phosphorylation and activition of LKB1 by UA
suggests that UA might upregulate AMPK activity via LKB1. To
verify this finding, LKB1 gene silencing and its destabilizer
Figure 3. Effect of UA on the protein expression of differentiation-related transcriptional factors. (A)–(B) 3T3-L1 preadipocytes were
incubated in differentiation medium without or with different concentrations of UA (added on day 0 of differentiation) for 6 days. The expression of
PPARcand C/EBPawere assessed by Western blotting as described in the Materials and Methods. (C) The C/EBPbexpression was determined after
preadipocytes were incubated in differentiation medium in the presence or absence of different concentrations of UA for 2 days. (D) The SREBP-1c
expression was determined after preadipocytes were incubated in differentiation medium in the presence or absence of 10 mM UA for 6 days. Data
are expressed as means 6 SD (n=3). *P,0.05 and **P,0.001 vs. the corresponding controls.
Ursolic Acid Iinhibits Adipogenesis
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radicicol were employed to determine the effect of UA on AMPK
activation in 3T3-L1 preadipocytes following the induction of
differentiation. Surprisingly, the UA-induced AMPK phosphory-
lation and activation were abolished by either LKB1 gene
silencing or LKB1 inhibition. Moreover, the UA induced
reductions in the protein expression of FAS and FABP4 was
reversed by blocking LKB1 expression using LKB1 siRNA. These
results indicate the importance of LKB1 in the regulation of
adipogenesis by UA. These results collectively demonstrated that
UA stimulated LKB1 activity, resulting in the increase of AMPK
Figure 4. Effect of UA on the protein expression of lipogenic proteins, Cpt1, AMPK and Sirt1. 3T3-L1 preadipocytes were incubated in
differentiation medium without or with different concentrations of UA (added on day 0 of differentiation) for 6 days. The expression of pACC, ACC,
FAS, FABP4, Cpt1, pAMPK, AMPK and Sirt1 were assessed by Western blotting as described in the Materials and Methods. Data are expressed as
means 6 SD (n=3). *P,0.05 and **P,0.001 vs. the corresponding controls.
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phosphorylation and activation, and consequently the inhibition of
AMPK downstream preadipocyte differentiation regulatory fac-
tors and their target lipogenic enzymes and proteins, and
ultimately the decrease of adipogenesis.
Although we identified AMPK as the regulator through which
UA exerted its anti-adipogenic effect, it is not possible to rule out
other regulators involved in adipocyte differentiation and adipo-
genesis, such as Wnt signaling, GATA factors, and KLFs .
Figure 5. Effect of AMPK siRNA or Sirt1 inhibitor on lipid accumulation in 3T3-L1 adipocytes. (A–C) Post-confluent 3T3-L1 cells were
induced for differentiation and treated with 10 mM UA (added on day 0 of differentiation) in the absence or presence of AMPK siRNA or 10 mM
nicotinamide for 6 days. Images were captured using an inverted microscope after Oil Red O staining. (D) Stained lipids were extracted and quantified
by measuring absorbance at 520 nm. Data are expressed as means 6 SD (n=3). *P,0.05 and **P,0.001 vs. the corresponding controls;#P,0.05
and##P,0.001 vs. nicotinamide treated cells.
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LKB1 is not the only upstream regulator of the AMPK signaling.
TAK1  and KSR2 [51,52] have been shown to affect AMPK
functions and, therefore, should be taken into considerations in
future studies. Moreover, other proteins involved in adipocyte
differentiation and adipogenesis await investigation. For example,
Fsp27, an adipocyte-specific lipid droplet-associated protein, has
been shown to play a role in the lipid droplet clustering and
triglyceride accumulation . The intracellular localization of C/
EBPband the activity of PPARc affect adipogenesis through a
different way than their expressions. In addition, we used only
3T3-L1 adipocyte cell line to study the effect of UA on
differentiation and adipogenesis. Our results and the above-
Figure 6. Effect of AMPK siRNA on the expression of differentiation transcriptional factors and lipogenic protein. Post-confluent 3T3-
L1 cells were differentiated and treated with 10 mM UA for 6 days after the silencing of AMPK. PPARc, C/EBPaand FAS, pACC and ACC expressions
were assessed by Western blotting as described in the Materials and Methods. Data are expressed as means 6 SD (n=3). *P,0.05 and **P,0.001 vs.
the corresponding controls.
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mentioned items could be confirmed in other origins of adipocytes,
such as human preadipocytes and pluripotent mesenchymal stem
Encouragingly, we have conducted an in vivo study to evaluate
the effects of UA on anti-obesity and improving the fatty liver in
rats with high fat diet–induced obesity. The results revealed that
UA at doses of 0.25% and 0.50% significantly reduced body
weight and fat content, increased the fatty acid oxidation in
skeletal muscle but without affecting the food intake (unpublished
data). Thus, both in vitro and in vivo studies suggest the potential of
UA to be an anti-obesity agent. Of course, the side effect of UA
should be evaluated prior to the clinical application, especially in
obese patients who are also diabetic as described in our studies
Taken together, our results have demonstrated that UA inhibits
3T3-L1 preadipocyte differentiation and lipid accumulation by
regulating the transcriptional factors and their downstream
lipogenic targets via the activation of LKB1/AMPK pathway.
UA is a promising naturally-occurring therapeutic agent for the
prevention and treatment of obesity.
Figure 7. Effect of UA on the expression of LKB1, and the effect of LKB1 gene silencing on the expression of pAMPK, AMPK, FAS
and FABP4. (A) 3T3-L1 preadipocytes were incubated in differentiation medium with or without 10 mM UA for 6 days. (B–D) Preadipocytes were
transfected with LKB1 siRNA 1 day post confluence. Medium was removed and replaced with the differentiation-induction medium in the presence or
absence of UA 24 hours after the transfection and maintained for differentiation for 6 days. pAMPK, AMPK, FAS and FABP4 expressions were assayed
by Western blotting as described in the Materials and Methods. Data are expressed as means 6 SD (n=3). *P,0.05 and **P,0.001 vs. their
corresponding controls;#P,0.05 vs. UA treated cells.
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mature 3T3-L1. (A–B) Differentiated 3T3-L1 adipocytes were
incubated in different concentrations of UA for 24, 48 hours,
respectively. MTT reagent was added to the medium. After
4 hours of incubation, the medium was aspirated and 150 mL
DMSO was added to each well. The absorbance was read at
570 nm. (C) Mature adipocytes were treated with 10 mM ursolic
acid for 3 hours. Lipolysis was quantified by measuring absor-
bance at 520 nm. Data are expressed as means 6 SD (n=3). *
P,0.05 and ** P,0.001 vs. the control.
Effect of UA on the viability and lipolysis in
the effect of radicicol on the expression of LKB1, pAMPK
and AMPK. (A) and (D) 3T3-L1 preadipocytes were transfected
with AMPK or LKB1 siRNA oligonucleotide duplexes 1 day post
the confluence with lipofectamine RNAiMax. The effectiveness of
siRNA knockdown after 24 hours of transfection and on day 6 of
cell differentiation was determined by measuring the expression of
Effectiveness of AMPK and LKB1 siRNA, and
AMPK and LKB1 using the Western blotting as described in the
Materials and Methods. (B–C) Post-confluent 3T3-L1 cells were
differentiated in the absence or presence of 5 mM radicicol for 6
days. The expression of LKB1, pAMPK, AMPK was measured
using the Western blotting as described in the Materials and
Methods. The bands of LKB1 and AMPK expression on day 6 of
cell differentiation were shown. *P,0.05 and **P,0.001 vs. the
Thanks to Ben Perry for proofreading this manuscript and to Shuocheng
Zhang for his technical support during the experiments.
Conceived and designed the experiments: YWW CHS. Performed the
experiments: YHH YL TTZ. Analyzed the data: YHH TTZ. Contributed
reagents/materials/analysis tools: YL TTZ. Wrote the paper: YHH
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