The effect of dehydroleucodine in adipocyte differentiation

Article abstract of DOI:10.1016/j.ejphar.2011.09.033

Dehydroleucodine (DhL) is a sesquiterpene lactone of the guaianolide group with gastric cytoprotective activity. Recent studies have also demonstrated that DhL inhibits the proliferation of vascular smooth muscle cells. In this study we examined the effect of DhL in the differentiation of 3T3-L1 preadipocytes. The addition of DhL significantly inhibited the differentiation of 3T3-L1 preadipocytes along with a significant decrease in the accumulation of lipid content by a dramatic downregulation of the expression of adipogenic-specific transcriptional factors PPARγ and C-EBPα. However, phosphorylation of AMPKα, Erk1/2 and Akt1 was not inhibited by DhL treatment. Interestingly, we also found that 11,13-dihydrodehydroleucodine, a derivative of DhL with inactivated α-methylene-γ-lactone function, also inhibited the differentiation of 3T3-L1 preadipocytes. Taken together, these data suggest that DhL has an important inhibitory effect in cellular pathways regulating adipocyte differentiation by modulating the PPARγ expression, which is known to play a pivotal role during adipogenesis.

Full text of DOI:10.1016/j.ejphar.2011.09.033

European Journal of Pharmacology 671 (2011) 18–25
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and Cellular Pharmacology
The effect of dehydroleucodine in adipocyte differentiation
Adriana Galvis a, Adriana Marcano a, Chad Stefancin a, Nicole Villaverde a, Horacio A. Priestap a,
Carlos E. Tonn b, Luis A. Lopez c, Manuel A. Barbieri a,
⁎
a
Department of Biological Sciences, Florida International University, Miami, FL 33199, USA
b
Cátedra de Química Orgánica, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Argentina
Laboratory of Cytoskeleton and Cell Cycle, Instituto de Histología y Embriología, Facultad de Ciencias Medicas, Universidad Nacional de Cuyo, 5500 Mendoza, Argentina
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Dehydroleucodine (DhL) is a sesquiterpene lactone of the guaianolide group with gastric cytoprotective activity.
Recent studies have also demonstrated that DhL inhibits the proliferation of vascular smooth muscle cells. In this
study we examined the effect of DhL in the differentiation of 3T3-L1 preadipocytes. The addition of DhL signifi-
cantly inhibited the differentiation of 3T3-L1 preadipocytes along with a significant decrease in the accumulation
of lipid content by a dramatic downregulation of the expression of adipogenic-specific transcriptional factors
PPARγ and C-EBPα. However, phosphorylation of AMPKα, Erk1/2 and Akt1 was not inhibited by DhL treatment.
Interestingly, we also found that 11,13-dihydrodehydroleucodine, a derivative of DhL with inactivated α-
methylene-γ-lactone function, also inhibited the differentiation of 3T3-L1 preadipocytes. Taken together, these
data suggest that DhL has an important inhibitory effect in cellular pathways regulating adipocyte differentiation
by modulating the PPARγ expression, which is known to play a pivotal role during adipogenesis.
© 2011 Elsevier B.V. All rights reserved.
Received 17 December 2010
Received in revised form 8 September 2011
Accepted 11 September 2011
Available online 19 September 2011
Keywords:
Dehydroleucodine
Adipocyte
α-Methylene-γ-lactone function
Transcriptional factor
1. Introduction
considered potentially promising treatments of obesity (Roberts et al.,
2010).
Obesity is a prevalent health risk in industrialized countries and is
associated with multiple pathological disorders, including diabetes
(Sartipy and Loskutoff, 2003), hypertension (Pi-Sunyer, 2002), cancer
(Lagra et al., 2004), gallbladder disease (Galal, 2003), and aterosclerosis
(Wofford et al., 1999). Obesity has also been associated with cancer and
cardiovascular disease, which have reached epidemic proportions
worldwide (Roberts et al., 2010; Yun, 2010). It has been reported that
weight loss reduces lipid levels, blood pressure, and the incidence of
type 2 diabetes mellitus (Sheard, 1993).
Animal models as well as in vitro systems have been used extensive-
ly in diabetes research. 3T3-L1 preadipocytes have been a useful in vitro
model for the study of obesity, due to an observable accumulation of
triglycerides upon differentiating in culture (Rosen and Spiegelman,
2000). Adipocyte differentiation is induced by the expression and/or
phosphorylation of specific genes, such as AMPK, Akt1, Erk1/2, (Kemp
et al., 2003; Rosen and Spiegelman, 2000; Rosen et al., 2000; Spiegelman
et al., 1993), PPARγ and C-EBPα (Bost et al., 2005). It is anticipated that
compounds, which inhibit adipocyte differentiation could beneficially
prevent and/or treat obesity. Thus, natural products such as crude aque-
ous and chloroform plant extracts as well as other pure active com-
pounds that specifically target and inhibit adipogenesis have been
Sesquiterpene lactones are a large and structurally diverse group
of plant second metabolites (Heinrich et al., 1998) with distinctive
biological activities, including gastric cytoprotector effects (Penissi et
al., 1998), anti-migraine (Beekman et al., 1997), antiviral and antimicro-
bial activities (Hayashi et al., 1996; Perry and Foster, 1995), anti-tumor
(Robles et al., 1995) and neurotoxic effect (Cheng et al., 1992).
Sesquiterpene lactones are also blockers of smooth muscle con-
tractility (Hay et al., 1994) aromatase activity (Blanco et al., 1997)
and NF-kappa B activation (Hehner et al., 1998; Lyss et al., 1998).
Also, it has been found that sesquiterpene lactones inhibit the activation
of cyclooxygenase and proinflammatory cytokines in macrophages
(Hwang et al., 1996). Within the group of sesquiterpene lactones,
helenalin, which occurs in the aerial portion of Arnica montana L.,
was also found to block the hormonally induced Sky2 mRNA and
Akt phosphorylation during early stages of adipocyte differentiation
(Auld et al., 2006). Inhibitory activities have been principally linked
to the α-methylene-γ-lactone function (Heinrich et al., 1998). However,
the reduction of the α-methylene-γ-lactone limited its cytotoxicity ef-
fect without affecting the anti-proliferative and anti-aromatase activity
(Blanco et al., 1997).
Dehydroleucodine (DhL) is a sesquiterpene lactone of the guaianolide
group, which also contains an α-methylene-γ-lactone ring in its mole-
cule. It was first isolated from Lidbeckia pectinata (Bohlmann and Zdero,
1972). The aerial parts of Artemisia douglasiana Besser are also rich in
DhL (Giordano et al., 1990). Chloroform extracts of the air-dried aerial
parts of A. douglasiana showed significant gastric cytoprotective activity
⁎
Corresponding author at: Department of Biological Sciences, Florida International
University, 11220 SW 8th Street, Miami, FL 33199, USA. Tel.: +1 305 348 7536;
fax: +1 305 348 1986.
E-mail address: barbieri@fiu.edu (M.A. Barbieri).
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.09.033

A. Galvis et al. / European Journal of Pharmacology 671 (2011) 18–25
19
(Giordano et al., 1992). In addition, DhL inhibited cell proliferation (Polo
2.5. Triglyceride assay
et al., 2007) and growth of Trypanosoma cruzi in culture (Brengio et al.,
2000). In this study, we investigated the effects of both DhL and
11,13-dihydro-dehydroleucodine (DH-DhL) on the differentiation of
3T3-L1 preadipocytes, and its mechanism of action at the cellular and
molecular levels.
Nine day 3T3-L1 adipocytes were washed with PBS, scraped and
centrifuged. Pellets were resuspended in PBS and then homogenized
by sonication and the cell suspension was assayed for total triglyceride
(Triglyceride Assay Kit; Cayman Chemical Company, Ann Arbor, MI)
according to the method of Mendez et al. (1986). Results were
expressed as total triglyceride per cellular protein (DC Protein Assay;
Bio-Rad, Hercules, CA).
2. Material and methods
2.1. Materials
2.6. Western blot analysis
DhL was isolated from A. douglasiana as previously described
(Giordano et al., 1990) and DH-DhL was obtained for DhL reduction
as previously described (Giordano et al., 1992). 3T3-L1 cells were
purchased from American Type Culture Collection (ATCC, Manassas,
VA). Dulbecco's modified Eagle's medium high glucose (DMEM), pen-
icillin/streptomycin and L-glutamine were purchased from Media-
tech, Inc. (Manassas, VA). C-EBPα antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). PPARγ, total (T)-Erk, phospho
(P)-Erk, T-Akt, P-Akt, T-AMPKα and P-AMPKα antibodies were from
Cell Signaling Technology (Boston, MA). Tubulin antibodies were from
Sigma-Aldrich (St. Louis, MO). All secondary antibodies were purchased
from Jackson ImmunoResearch Laboratories (West Grove, PA). All other
chemicals were obtained from Sigma-Aldrich unless otherwise stated.
To prepare cell lysates, cell monolayers were washed with ice-cold
lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM Na2 EDTA,
10% NP40 and 10% Na Deoxycholate) containing protease and phospha-
tase inhibitors. Lysates were collected by centrifugation and protein
concentrations were estimated by using BCA protein assay (Thermo
Fisher Scientific, Inc., Pittsburgh, PA) following the manufacturer's
instructions. Proteins were resolved by SDS-PAGE and transferred to
nitrocellulose membranes, blocked, probed with the specific antibodies
A
0.6
0.5
2.2. Cell culture and differentiation
0.4
*
3T3-L1 cells were grown to confluence in Dulbecco's modified
Eagle's medium (DMEM) high glucose supplemented with 10% fetal
bovine serum (Invitrogen, Carlsbad, CA), 1% penicillin/streptomycin
and 1% L-glutamine (growth media) in a humidified atmosphere of
5% CO2 at 37 °C. Culture was fed every 48 h, both for cell growth
and differentiation. To trigger differentiation cells were exposed to
induction medium (IM) (growth media supplemented with 670 nM in-
sulin, 65 nM dexamethasone and 0.5 mM 3-isobutyl-1-methylxanthine
[IBMX]) for the first two days, then fed with post-differentiation media
(DMEM high glucose supplemented with 5% fetal bovine serum, 1%
penicillin/streptomycin and 1% L-glutamine) for the next seven days.
Cells were exposed to DhL throughout the entire differentiation process,
unless otherwise indicated.
*
0.3
*
*
0.2
*
*
0.1
*
0
0
0.65 1.2
3
4
5
6
7
8
9
10
B
Day 1
Day 9
0.6
0.5
0.4
0.3
0.2
0.1
0
2.3. Cell viability assay
Cells were cultured on 6-well plates and treated with DhL as indi-
cated for each experiment. Cell viability was measured using the
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)
colorimetric assay following the manufacturer's instructions (ATCC).
In brief, MTT reagent was added to cells at a final concentration of
10 μl/ml. Then cells were incubated as indicated in Fig. 1 at 37 °C and
5% CO2. After incubation, the medium was removed, and formazan
crystals were dissolved in detergent reagent. Optical density for each
condition was determined at 570 nm.
2.4. Oil Red O staining and microscopy
On the ninth day cells were fixed with 10% formalin (Thermo Fisher
Scientific, Inc., Pittsburgh, PA) in phosphate buffered saline (PBS) for 1 h
at 4 °C. Oil Red O (Allied Chemical, Morristown, NJ) stock solution (0.6 g
in 100 ml of isopropanol) was diluted with 0.6 parts of water, filtered
and added to the fixed cells for 15 min at room temperature. Cells
were then washed with water and analyzed in a Leica DM IRB inverted
microscope. Photos of lipid droplets were taken using digital Leica DC
500 camera. To quantify the lipid droplets, Oil Red O was eluted with
100% isopropanol for 10 min at 37 °C, collected, and its optical density
was measured at 540 nm.
Fig. 1. Dehydroleucodine inhibited adipogenesis of 3T3-L1 preadipocytes without
reducing cell viability. (A) 3T3-L1 preadipocytes were differentiated into adipocytes in
the absence or in the presence of various amounts of DhL (0.65 to 10 μM) as described
in Material and methods. Results were represented as relative lipid contents. Data repre-
sent the mean S.E.M. of three independent experiments. *Pb0.05 by Student's t-test
compared to DMSO and only induction media-treated cells. (B) Cells were treated with
8 μM DhL for either 24 h (day 1) or for 9 days (day 9). Cell viability was measured using
the MTT assay as described in Material and methods. Data represent the mean S.E.M.
of three independent experiments.

20
A. Galvis et al. / European Journal of Pharmacology 671 (2011) 18–25
and then visualized by Western analysis. Relative levels of Erk1/2 and
Akt1 proteins were determined by densitometry using the ratio of
P-Erk1/2 to T-Erk1/2 and P-Akt1 to T-Akt1, respectively. Relative levels
of PPARγ and C-EBPα were determined using the ratio of PPARγ to
tubulin and C-EBPα to tubulin, respectively.
the mean (S.E.M.) of triplicates and the statistical significance was
analyzed by one-way ANOVA or Student's test. Results with *Pb0.05
and **Pb0.01 were considered as statistically significant.
3. Results
2.7. High-performance liquid chromatography analysis
3.1. DhL inhibits differentiation of preadipocytes
The high-performance liquid chromatography (HPLC) equipment
consisted of a SpectraSystem SMC1000 solvent delivery system, vacuum
membrane degasser, P4000 gradient pumps and AS3000 autosampler
(Thermo Electro Corporation, San Jose, CA). Column effluent was moni-
tored at 254 nm with Spectra System UV6000LP variable wavelength
PDA detector and ChromQuest 4.1 software. DhL, (11S)DH-DhL and
(11R)DH-DhL were separated using a C18 YMC column (A-302, 150×
4.3 mm i.d., S-5 μm, 12 nm; Waters) and the following solvents: A.
acetonitrile; and B. 0.1% TFA in water. System 1: linear gradient 10% to
100% A in 120 min; flow rate 1 ml/min. Preparative HPLC was per-
formed in the above equipment with a XTerra Prep MS C18 OBD column,
15 μm, 19×50 mm (Waters) and the solvent system 25% acetonitrile–
75% 0.1% TFA in water (isocratic); flow rate 3 ml/min.
To examine the potential role of DhL on the differentiation of preadi-
pocytes, cells were incubated with induction media in the presence of
various concentrations of DhL. In Fig. 1A, we show that the addition of
DhL inhibited the lipid content in a dose-dependent manner with an
IC50 of 6 μM. Furthermore, our experimental conditions demonstrate
that the addition of DMSO (0.2%) exclusively does not hinder 3T3-L1
preadipocyte differentiation (Fig. 1A).
We were further interested in examining the effect of DhL on the
viability of 3T3-L1 preadipocytes. Cells were incubated with induction
media in the presence of 8 μM DhL for 24 h (Day 1) or throughout the
entire 9 days of differentiation (Day 9). Cell viability was measured
A
B
2.8. Gas chromatography (GC)/flame ionization detector (FID) and GC/
mass spectrometry (MS) analysis
GC/FID analyses were performed on a Trace GC Ultra apparatus
(Thermo Electro Corporation, San Jose, CA) equipped with a flame ioni-
zation detector. The output was recorded using a ChromQuest version
4.1 data system. A DB-5MS capillary column (0.25 mm i.d.×30 m; film
thickness 0.25 μm; J & W Scientific, Folsom, CA) was employed. The tem-
perature was programmed 105 to 240 °C at 3 °C/min (linear increase)
and then the temperature was held at 240 °C for 10 min. The injector
temperature was 250 °C with a split ratio of 1/20. The detector temper-
ature was 270 °C. Helium was used as gas carrier at 1 ml/min. DHL,
(11S)DH-DHL and (11R)DH-DhL were dissolved in ethyl acetate and
2 μl of the solution was injected. GC/MS determinations were carried
out in a Hewlett Packard model 6890 instrument coupled to a Q-Mass
910 quadrupole selective detector at 70 eV and equipped with a DB-
5MS capillary column. Temperature program and other conditions
were as indicated above.
0.8
0.6
0.4
0.2
C
D
2.9. Compound identification
Dehydroleucodine {(1S,6S,2R)-9,13-dimethyl-5-methylene-3 oxatri-
cyclo[8.3.0.0b2,6N]trideca-9,12-diene-4,11-dione, IUPAC nomenclature}.
DhL (compound 1): HPLC (System 1), Rt 27.74 min; GC/MS, Rt
*
22.72 min; UV/PDA λmax 256 nm; MS m/z (rel. int.), 244 (100) M+
,
173 (18.2), 145 (19.8), 129 (18.9), 115 (18.5), 105 (18.3), 91 (62.9),
79 (17.7), 77 (28.4), 65 (21.6), 53 (30.8).
0
_
(1S,2S,5S,6S)-5,9,13-trimethyl-3-oxatricyclo[8.3.0.0b2,6N]trideca-9,
12-diene-4,11-dione;11,13-dihydro-dehydroleucodine (IUPAC nomen-
clature), (11S)DH-DhL (compound 2): HPLC (System 1), Rt 28.16 min;
GC/MS, Rt 21.87; UV/PDA λmax 257 nm; MS, m/z (rel. int.): 246 (100)
M+, 217 (26.7), 173 (37.6), 172 (31.2), 145 (26.2), 105 (23.2), 91
(60.7), 77 (26.2), 55 (24.3).
+ + +
IM
_ _ _
_
+
DhL
_
_
DMSO
+
(1S,2S,6S,5R)-5,9,13-trimethyl-3-oxatricyclo[8.3.0.0b2,6N]trideca-9,
12-diene-4,11-dione, 11,13-dihydro-dehydroleucodine (IUPAC nomen-
clature), (11R)DH-DhL (compound 3): HPLC (System 1), Rt 26.23 min;
GC/MS, Rt 22.84; UV/PDA λmax 257 nm; MS, m/z (rel. int.): 246 (100)
M+, 217 (31.5), 173 (35.5), 172 (33.1), 145 (27.5), 105 (26.1), 91
(67.1), 77 (29.0), 55 (25.8).
Fig. 2. Dehydroleucodine blocked the formation of lipid droplet by induction media in
3T3-L1 cells. Adipocyte differentiation was induced by treating confluent 3T3-L1 prea-
dipocytes with induction media in the absence or presence of 8 μM DhL. Morphological
changes of 3T3-L1 preadipocytes were monitored by a microscope and photographed
after 9 days from the onset of differentiation. (A) Vehicle only, (B) cells treated with in-
duction media in the presence of DMSO, or (C) in the presence of DhL. (D) Nine days
after induction of differentiation, cells were lysed for triglyceride and protein assays
as described in Material and methods. Vehicle only (line 1), cells treated with induction
media alone (line 2), cells treated with induction media in the presence of DMSO (line
3), and cells treated with induction media in the presence of 8 μM DhL (line 4).
Bars=10 μm. Data represent the mean S.E.M. of three independent experiments.
*Pb0.05 by Student's t-test compared to DMSO-treated cells and only induction
media-treated cells.
2.10. Statistical analysis
All experiments were done in duplicates and they were repeated
at least three times. Values are represented as the standard error of

A. Galvis et al. / European Journal of Pharmacology 671 (2011) 18–25
21
using the MTT colorimetric assay as described in Material and methods.
In Fig. 1A, we show that the addition of DhL blocked the differentiation
of 3T3-L1 preadipocytes whereas cell viability was not affected as com-
pared with DMSO or untreated cells (Fig. 1B). However, at higher DhL
concentrations (N10 μM), 3T3-L1 preadipocytes detached from the
plate, which was coupled with a significant reduction in the MTT
assay (data not shown). These results suggest that, up to a maximum
concentration of 10 μM, DhL has a strong inhibitory activity of 3T3-L1
preadipocyte differentiation without significant effect on cell viability.
Adipocyte differentiation can be also monitored by the formation of
intracellular lipid droplets (Rosen and Spiegelman, 2000). As described
above, 3T3-L1 preadipocytes were cultivated and induced to differenti-
ate into adipocytes with induction media in the absence or presence of
8 μM DhL. At day 9, Oil Red O staining showed an abundant number of
lipid droplets suggesting a significant lipid accumulation in untreated
differentiated cells. However, lipid droplets were not observed in
untreated non-differentiated cells (compare Fig. 2A and B). More im-
portantly, formation of lipid droplets was inhibited by 8 μM DhL
treatment (Fig. 2C). These observations were further supported with
the quantitative measurement of lipid content by determining the ab-
sorbance at 540 nm (Fig. 1A). In addition, DhL treatment significantly
inhibited adipogenic morphology (i.e., transition from a fibroblast-like
shape to an increasingly rounded-up appearance with an accumulation
of cytoplasmic lipid droplets; compare Fig. 2, B and C).
Given that DhL inhibited differentiation of 3T3-L1 preadipocytes,
we next considered whether DhL would inhibit triglyceride accumu-
lation. Cells treated with induction medium in the presence of 8 μM of
DhL accumulated roughly 31 5% of the intracellular triglyceride
contained in controls (Fig. 2D). As expected, DMSO-treated cells did
not affect the formation of triglyceride accumulation as compared
with cells incubated with induction media alone (Fig. 2D).
3.2. DhL inhibits key adipogenic transcriptional factors
Adipogenesis is a highly regulated process requiring coordinated
expression and activation of key transcriptional factors and signaling
A
B
PPAR
C-EBP
Erk1/2
1.2
1
1.2
1
1.2
1
P-Erk1/2
T-Erk1/2
PPAR
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
CEBP
Tubulin
*
*
C
D
AMPK
1.2
1
Akt1
2.0
1.0
0
*
P-Akt1
T-Akt1
P-AMPK
T-AMPK
0.8
0.6
0.4
0.2
0
Fig. 3. Dehydroleucodine attenuated the expression of PPARγ during 3T3-L1 preadipocyte differentiation. 3T3-L1 preadipocytes were induced to differentiate by induction media
into adipocytes in the absence (inset: −DhL, control) or in the presence (inset: +DhL) of 8 μM DhL. Total protein extracts were prepared at day 9 from each sample. The proteins
were subset to 12% SDS-PAGE electrophoresis, blotted to a nitrocellulose membrane, and probed with anti-bodies specific to (A) PPARγ, C-EBPα and tubulin, (B) P-Erk1/2, T-Erk1/2,
(C) P-Akt1, T-Akt1, and (D) P-AMPKα and T-AMPKα respectively. Relative levels of proteins were determined by densitometry as described in Material and methods. Data repre-
sent the mean S.E.M. of three independent experiments. *Pb0.05 by Student's t-test compared to DMSO induction media-treated cells (control).

22
A. Galvis et al. / European Journal of Pharmacology 671 (2011) 18–25
molecules (Rosen and Spiegelman, 2000). To investigate whether DhL
affects the expression of PPARγ and C-EBPα, 3T3-L1 preadipocytes
were incubated with induction media in the absence or presence of
8 μM DhL as described in Fig. 1, and then harvested at day 9 for Western
blot analysis using anti-PPARγ and C-EBPα antibodies. In Fig. 3A, we
show that the addition of DhL clearly attenuated the expression of
PPARγ and C-EBPα. We also examined the effect of DhL on the phos-
phorylation of Akt1 and Erk1/2 proteins. To our surprise we found
that the addition of DhL did not attenuate phosphorylation of
Erk1/2 and Akt1 (Fig. 3B and C). Furthermore, we tested whether
the addition of DhL affected the phosphorylation of AMPKα during
3T3-L1 preadipocyte differentiation. This result shows that phosphory-
lation of AMPKα (P-AMPKα) was not inhibited by the addition of DhL
(Fig. 3D). In contrast, DhL further enhanced phosphorylation of
AMPKα (Fig. 3D). Furthermore, we also found that the level of total
AMPKα appeared relatively constant in the presence or absence of
DhL. Therefore, these results indicated that the level of phosphorylated
AMPKα increased in the presence of DhL. Interestingly, both PPARγ and
C-EBPα are selectively expressed during the differentiation of 3T3-L1
preadipocytes. The levels of expression of these transcription factors
are undetectable in preadipocytes; however, expression increases
2 days after induction and they are expressed 5 days after the induction
of differentiation (Rosen and Spiegelman, 2000). Consistent with these
observations, we also observed that the expression of PPARγ and
C-EBPα was significantly increased with the progression of the differ-
entiation of 3T3-L1 preadipocyte (Supplementary Fig. 3A and B). How-
ever, DhL treatment at the concentration of 8 μM significantly blocked
the expression of PPARγ and C-EBPα (Supplementary Fig. 3A and B).
In addition, the differentiation of 3T3-L1 preadipocyte progressed
with an increase of the phosphorylation status of AMPKα (Supplemen-
tary Fig. 3C). Treatment with DhL further significantly increased the
phospho-status of AMPKα, suggesting that DhL also induced the activa-
tion of AMPKα (Supplementary Fig. 3C). Taken together, these data sug-
gest that DhL selectively blocks the expression of PPARγ and C-EBPα
and also increases the phosphorylation of AMPKα during the differenti-
ation of 3T3-L1 preadipocytes.
A
DMSO
DhL
1
2
3
4
5
6
DhL
DhL
DhL
DhL
1
3
5
7
9
days
B
0.60
0.45
0.30
0.15
0
**
*
1
2
3
4
5
6
Treatments
Fig. 4. Dehydroleucodine blocked adipocyte differentiation in a time-dependent man-
ner. (A) 3T3-L1 preadipocyte cells were treated with 8 μM DhL for the time indicated
in the schematic representation of the experiment. (B) In each treatment (1–6), the
accumulation of lipid droplets was measured by the incorporation of Oil Red O as de-
scribed in Material and methods. Data represent the mean S.E.M. of three indepen-
dent experiments. *Pb0.05 and **Pb0.01 by Student's t-test compared to DMSO
induction media-treated cells (control).
It is also possible that the extent of inhibition is dependent on the
timing of DhL addition. For this purpose, 8 μM DhL was added in dis-
crete periods during differentiation (Fig. 4A). We observed a significant
reduction in the differentiation of 3T3-L1 preadipocytes when com-
pared with DMSO-control cells upon early DhL addition, corresponding
to day 1, or 3 of treatment (Fig. 4A and B). However, this inhibitory
effect was not apparent when DhL was added on day 5, 7 or at day
8 post-induction. These results indicate that DhL may affect early adipo-
cyte gene expression during in vitro differentiation.
these epimers are denoted (11S)DH-DhL and (11R)DH-DhL, respective-
ly, for simplicity. After separation by preparative HPLC, the epimers
were obtained in pure form and confirmed by GC analysis (Fig. 5, C
and D). They were studied, in parallel with DhL, for their effect on the
differentiation of 3T3-L1 preadipocytes.
In Fig. 6A, we show that the addition of DH-DhL inhibited the differ-
entiation of 3T3-L1 preadipocytes. This inhibition was dose-dependent
and required a higher concentration of DH-DhL to produce a similar
inhibitory effect as DhL. Thus, it required 10 times the DH-DhL concen-
tration to achieve the same effect as DhL (Fig. 6A). Furthermore, we also
found that 100 μM of DH-DhL did not affect cell viability (control cells:
98 2% of viability vs. 80 μM DH-DhL treated cells: 97 2% of viability).
These results suggest that the reduction of the α-methylene-γ-lactone
group of the DhL was not required to block the differentiation of 3T3-
L1 preadipocytes.
3.3. Effect of 11,13-dihydro-dehydroleucodine on the differentiation of
3T3-L1 preadipocytes
Our results clearly show that the addition of DhL inhibited differen-
tiation of 3T3-L1 preadipocytes in a dose-dependent manner without a
significant effect of cell toxicity (Fig. 1). However, it has been postulated
that the non-saturated α-methylene-γ-lactone function of sesquiter-
pene lactones produces an unspecific toxic effect leading to cell death
(Polo et al., 2007). Therefore, we then investigated the effect of
DH-DhL, which is a derivative of DhL lacking alkylating function, on
the differentiation of 3T3-L1 preadipocytes.
DhL (Fig. 5A, compound 1) can be gently reduced with sodium
borohydride to give the corresponding 11,13-dihydro derivative (clas-
sical nomenclature; Giordano et al., 1990). Analysis of the reaction
product (DH-DhL) by GC shows that two epimers (Fig. 5B, compounds
2 and 3) are formed in different amounts (Fig. 5B) due to the generation
of a chiral center at C-11 of the molecule during reduction. The 11S-
epimer (compound 2) is the major reaction product (Giordano et al.,
1992). It is accompanied by the minor 11R-epimer (compound 3), and
by traces of unreacted DhL (compound 1) (Fig. 5B). In the following
To further investigate the role of these epimers of DH-DhL on the dif-
ferentiation of 3T3-L1 preadipocytes, the two methyl epimers at C-11
produced during the in vitro reduction of DhL were separated by pre-
parative HPLC, concentrated, confirmed by GC analysis and then their
effect on adipogenesis was examined. For this, 3T3-L1 pre-adipocytes
were incubated with 80 μM of either (11S)DH-DhL or (11R)DH-DhL
during the entire differentiation process. We found that the addition
of (11R)DH-DhL epimer, but not (11S)DH-DhL epimer, inhibited the
differentiation of 3T3-L1 preadipocytes (Fig. 6B). These results suggest

A. Galvis et al. / European Journal of Pharmacology 671 (2011) 18–25
23
1
A
B
C
D
Dehydroleucodine
2
3 #1
2
(11S)DH-DhL
(11R)DH-DhL
3
0
10
20
30
40
O
O
O
10
1
3
5
7
CH3
H
H
11
12
H
H
O
H
13
CH3
O
O
O
O
O
1
3
2
Fig. 5. GC analysis of dehydroleucodine and 11,13-dihydrodehydroleucodine epimers. (A) DhL isolated from Artemisia douglasiana; (B) mixture DH-DhL epimers as obtained by
reduction of DhL, (#) denotes small amount of DhL after the reduction reaction; (C) DH-DhL epimer S, and, (D) DH-DhL epimer R after separation from the mixture. Chemical struc-
tures of DhL (compound 1), DH-DhL epimer 11S (compound 2) and DH-DhL epimer 11R (compound 3) (numbering according to classical nomenclature).
that (11R)DH-DhL epimer may be responsible in inhibiting adipocyte
differentiation.
Based on our observations, it is clear that in the adipocyte differenti-
ation model, the α-methylene-γ-lactone moiety causes a significant
decrease of 3T3-L1 preadipocyte differentiation at concentrations
≤10 μM without altering cell viability as demonstrated by the MTT
assay. However, the addition of N10 μM DhL (i.e., 12 μM) caused cell
toxicity as evidenced either by the incorporation of trypan blue dye or
by the induction of detachment of cells from the tissue culture plate.
Thus, the inhibition of 3T3-L1 differentiation by DhL and (11R)
DH-DhL epimers may be induced by a process distinct from cell death
induced by toxicity of these compounds.
Adipocyte differentiation involves a complex progression of changes
in morphology, hormone sensitivity, and gene expression (Rosen and
Spiegelman, 2000). 3T3-L1 preadipocytes transform into adipocytes
through growth arrest, clonal selection and expression of adipocyte
selective markers following hormonal induction (Rosen et al., 2000).
The addition of induction media containing IBMX, dexamethasone,
and insulin activated several transcriptional factors, including PPARγ,
C-EBPα, C-EBPβ, and C-EBPδ, which in turn regulate a number of
genes required during adipocyte differentiation (Spiegelman et al.,
4. Discussion
In this study we investigated the effect of DhL in 3T3-L1 preadipo-
cyte differentiation. The addition of DhL to the medium inhibited
significantly the accumulation of lipid droplets in a dose-dependent
manner. DhL attenuated dramatically the production of adipogenic
transcriptional factors PPARγ and C-EBPα during adipogenesis
without altering the activation of Erk1/2 and Akt1. In contrast, DhL
increased the phosphorylation of AMPKα proteins. In addition, we
also found that DH-DhL, a derivative of DhL with inactivated α-
methylene-γ-lactone function, also inhibited the formation of adipo-
cytes. However, it required ten times the DH-DhL concentration to
achieve the same effect as DhL. More importantly, we have identified
a specific DH-DhL epimer [i.e., (11R)DH-DhL], which may be responsi-
ble for such inhibitory activity.

24
A. Galvis et al. / European Journal of Pharmacology 671 (2011) 18–25
A
Therefore, these results indicated that the addition of DhL significantly
0.6
increased the phosphorylation of AMPKα during 3T3-L1 differentiation,
which was detrimental to 3T3-L1 differentiation. In addition, our obser-
vations showed that the effect of DhL on adipocyte differentiation may
be selective since both Akt1 and Erk1/2 activities, which are thought to
be sensitive to insulin signal, were not affected by the addition of DhL.
Thus, the inhibitory effect of DhL does not seem to be related to these
two signaling molecules.
DH-DhL
0.3
In summary, this report suggests that DhL inhibits the adipogenic
differentiation process. The mechanisms by which DhL regulates
adipogenesis include the inhibition of expression of the adipogenic
transcription factors, PPARγ and C-EBPα. In addition, we also found
that a DH-DhL epimer, lacking a highly reactive and nonspecific α-
methylene-γ-lactone moiety, may improve its use without a cytotoxic
effect. Therefore, it is anticipated that the inhibition of differentiation
into adipocytes by DhL and its derivates may be beneficial for the
prevention of obesity. For this reason, natural products that specifically
inhibited adipogenesis might be considered with regard to their poten-
tial in treatment of obesity. However, it remains to be determined
whether manipulating adipogenesis can be beneficial and/or healthy
without leading to other metabolic diseases.
DhL
0
0
20
40
60
80
100
Compound (μM)
B
Relative Lipid Content
0
0.3
0.6
Supplementary materials related to this article can be found online
at doi:10.1016/j.ejphar.2011.09.033.
DMSO
(R)DH-DhL
(S)DH-DhL
Acknowledgment
∗
This work was partially supported by the National Institutes of
Health grant SC1DK084343 (to MAB) and by SECyTP, UNCuyo 06J
213 grant and ANPCYT PICT-R 2005 32850 grant (to LAL).
Fig. 6. 11,13-Dihydro-dehydroleucodine inhibited 3T3-L1 preadipocyte differentiation.
(A) 3T3-L1 preadipocytes were differentiated into adipocytes in the absence or in the pres-
ence of either DHL or DH-DhL. Results were represented as relative lipid content. Data rep-
resent the mean S.E.M. of three independent experiments. (B) 3T3-L1 preadipocyte cells
were incubated with induction media supplemented with either DMSO, 80 μM DH-DhL
epimer S or 80 μM DH-DhL epimer R and the incorporation of Oil Red O was measured
by as described in Material and methods. Results were represented as relative lipid
content. Data represent the mean S.E.M. of three independent experiments. **Pb0.01
by Student's t-test compared to DMSO-treated cells.
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