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RESEARC H Open Access
Effect of the Cannabinoid Receptor-1 antagonist
SR141716A on human adipocyte inflammatory
profile and differentiation
Ravi Murumalla
1
, Karima Bencharif
1
, Lydie Gence
1
, Amritendu Bhattacharya
1
, Frank Tallet
2
, Marie-Paule Gonthier
1
,
Stefania Petrosino
3
, Vincenzo di Marzo
3
, Maya Cesari
1
, Laurence Hoareau
1*†
and Régis Roche
1†
Abstract
Background: Obesity is characterized by inflammation, caused by increase in proinflammatory cytokines, a key
factor for the development of insulin resistance. SR141716A, a cannabinoid receptor 1 (CB1) antagonist, shows
significant improvement in clinical status of obese/diabetic patients. Therefore, we studied the effect of SR141716A
on human adipocyte inflammatory profile and differentiation.
Methods: Adipocytes were obtained from liposuction. Stromal vascular cells were extracted and differentiated into
adipocytes. Media and cells were collected for secretory (ELISA) and expression analysis (qPCR). Triglyceride
accumulation was observed using oil red-O staining. Cholesterol was assayed by a fluorometric method. 2-AG and
anandamide were quantified using isotope dilution LC-MS. TLR-binding experiments have been conducted in HEK-
Blue cells.
Results: In LPS-treated mature adipocytes, SR141716A was able to decrease the expression and secretion of TNF-a.
This molecule has the same effect in LPS-induced IL-6 secretion, while IL-6 expression is not changed. Concerning
MCP-1, the basal level is down-regulated by SR141716A, but not the LPS-induced level. This effect is not caused by
a binding of the molecule to TLR4 (LPS receptor). Moreover, SR141716A restored adiponectin secretion to normal
levels after LPS treatment. Lastly, no effect of SR141716A was detected on human pre-adipocyte differentiation,
although the compound enhanced adiponectin gene expression, but not secretion, in differentiated pre-
adipocytes.
Conclusion: We show for the first time that some clinical effects of SR141716A are probably directly related to its
anti-inflammatory effect on mature adipocytes. This fact reinforces that adipose tissue is an important target in the
development of tools to treat the metabolic syndrome.
Keywords: human adipocyte, inflammation, SR141716A, TNF-a
Background
Obesity displays characteristics of a metabolic syndrome,
with hyperinsulinemia and resistance to insulin, leading
to type II diabetes, atherosclerosis, hypertension, hepatic
steatosis, and sometimes cancer [1]. The accumulation
of fat in organs and tissues leads to local inflammation,
characterized by an increase in pro-inflammatory
cytokines such as TNF-a [2]. This is probably one of the
decisive steps in the development of insulin-resistance
[2]. Obesity is also characterized by the existence of a
global inflammatory state, with raised levels of circulat-
ing pro-inflammatory cytokines such as TNF-a, C-reac-
tive protein, and IL-6 [3], as well as a reduction in anti-
inflammatory cytokines such as adiponectin [4]. Lastly,
major modifications of lipid metabolism are also asso-
ciated with raised circulating triglyceride and fatty acid
levels, and with reduction of HDL-C [5].
The development of pharmacological tools is of enor-
mous interest in the fight against obesity and its
* Correspondence: laurence.hoareau@adipsculpt.com
†Contributed equally
1
GEICO, Groupe d’Etude sur l’Inflammation et l’Obésité Chronique, Université
de La Réunion, plateforme CYROI, 15 avenue René Cassin, 97715 Saint-Denis
Messag Cedex, France
Full list of author information is available at the end of the article
Murumalla et al.Journal of Inflammation 2011, 8:33
http://www.journal-inflammation.com/content/8/1/33
© 2011 Murumalla et al; licensee BioMed Cent ral Ltd. This is an Open Access article distributed un der the terms of the Creative
Commons Attri bution License (http://creativecommons.org /licenses/by/2.0), which permits unrestricte d use, distribution, and
reproductio n in any medium, provided the original work is properly cited.
metabolic consequences. One new physiological pathway
of interest is the endocannabinoid system discovered in
the early 1990s and believed to influence body weight
regulation and cardiometabolic risk factors. This endo-
cannabinoid system consists of two G protein-coupled
receptors known as cannabinoid receptors CB1 and
CB2; their endogenous ligands, the endocannabinoids,
derived from lipid precursors; and the enzymes respon-
sible for ligand biosynthesis and degradation [6,7]. The
endocannabinoid system is said to be usually silent and
to become transiently activated in stressful conditions.
After ligand binding, signalling cascades of cannabinoid
receptors can occur through several mechanisms that
can act via G protein-dependent and independent path-
ways. Consequently, according to the signalling pathway
activated, multiple biological effects are attributed to the
endocannabinoid system which has been found to regu-
late appetite and energy expenditure, insulin sensitivity,
as well as glucose and lipid metabolism ([8] for review).
Moreover, it seems that the endocannabinoid system
exerts many anti-inflammatory actions ([9] for review).
Several recent data obtained from studies carried out on
animals or humans have demonstrated a close associa-
tion between obesity and the endocannabinoid system
dysregulation, illustrated either by an overproduction of
endocannabinoids or by an upregulation of CB1 expres-
sion in tissues involved in energy homeostasis ([8] for
review). Interest in blocking stimulation of this pathway
to aid weight loss and reduce cardiometabolic risk factor
development is an area of interest and research. One of
the first approaches proposed to reduce the hyperactiv-
ity of the endocannabinoid system related to obesity was
the development of selective CB1 receptor antagonists
such as SR141716A or rimonabant, which has already
demonstrated its capacity to improve the clinical picture
in obese patients with metabolic disorders. Results from
various clinical studies (RIOstudies,STRADIVARIUS,
SERENADE and ADAGIO) clearly show that treatment
with SR141716A leads to weight reduction, an increase
in HDL-C levels, a reduction in triglycerides and arterial
blood pressure, an improvement in insulin response and
glucose uptake, and an increase in adiponectin levels
[10-15]. In addition, studies in animal models show that
SR141716A is able to reduce the local, hepatic and
macrophage levels of pro-inflammatory cytokines
[16-18], as effectively as their circulating levels [17,19].
A certain number of clinical effects of SR141716A
have been attributed to its direct action on the adipose
tissue. This is due to the fact that this tissue is a major
player in the development of metabolic disturbances
associated with obesity [20], but also because adipocytes
express the CB1 receptor and are able to produce and
release endocannabinoids [21-23]. Interestingly, it has
been postulated that body weight reduction can be
linked to inhibition of the cellular proliferation of pre-
adipocytes [24] and that the increase in circulating adi-
ponectin is related to increased adipocyte expression of
cannabinoid receptors [24,25]. In addition, it has been
shown that the treatment of murine pre-adipocytes with
SR141716A leads to the inhibition of their differentia-
tion [26], which is in agreement with the finding that
CB1 activation instead stimulates pre-adipocyte differen-
tiation [21]. Another recent study demonstrates that a
CB1 agonist increases the sensitivity of adipocytes to
insulin, whereas SR141716A has the opposite effect [27],
which again would agree with the pro-lipogenic role
suggested for endocannabinoids acting at CB1 receptors
[21]. It is surprising, however, that no studies have been
conducted with SR141716A and human adipose cells,
which represent the best model to predict the in vivo
actions of this CB1 antagonist in human white adipose
tissue.
Here, we aimed at filling this gap by investigating the
effects of SR141716A in human pre-adipocytes and
mature adipocytes (exhibiting full fat accumulation) in
primary culture. In particular, we have investigated
whether the clinical effects of SR141716A have any cor-
relation with the action of this antagonist on human
adipose tissue.
Methods
Materials
Lipopolysaccharide (LPS from E. coli 0111:B4 strain,
batch #LPE-32-02) was purchased from Sigma (Saint
Quentin Fallavier, France). 2-Arachidonoyl glycerol and
R1-Methanandamide (2-AG and R1-Met, CB1 agonist,
Cayman) were obtained from SpiBio (Massy, France).
SR141716 (rimonabant, CB1 antagonist) was a generous
gift of SANOFI-SYNTHELABO (Montpellier, France).
Origin of human adipose tissue samples
Subcutaneous (abdominal, buttocks, hips and thighs) tis-
sue samples of human white fat were obtained from
normal weight or slightly overweight human subjects
(exclusively females, mean body mass index = 23.3)
undergoing liposuction, performed under general anaes-
thesia, for cosmetic reasons (aged between 25 and 60
years, mean 39 years). Apart from oral contraception,
the subjects were not receiving treatment with pre-
scribed medication at the time of liposuction. A total of
21sampleswereobtainedfrom24patients.Thestudy
was approved by the Ile de la Réunion ethics committee
for the protection of persons undergoing biomedical
research.
Primary culture of human adipocytes
Cultures were carried out as previously described [22].
Briefly, tissue samples obtained by liposuction were
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 2 of 15
digested for 30 min at 37°C in Ringer-Lactate buffer
containing 1.5 mg/mL collagenase (NB5, SERVA, Ger-
many, PZ activity 0.175 U/mg). The floating adipocytes
(mature adipocytes) were rinsed three times in Ringer-
Lactate. Cells were plated in 24-well (30 000 cells) or 6-
well (120 000 cells) tissue culture plates with 199 cul-
ture medium supplemented with: 1% Fetal Bovine
Serum (FBS) (PAN Biotech, France), amphotericin B, (5
mg/mL), streptomycin (0.2 mg/mL) & penicillin (200 U/
mL) (PAN Biotech, France), 66 nM insulin (Umuline
Rapide,Lilly,France),2g/Lglucose,8mg/mLbiotin
and 4 mg/mL pantothenate. Cells were then maintained
at 37°C in 5% CO
2
for a period of 24 hours prior to the
experiments.
Endocannabinoid quantification
Mature adipocytes isolated as described above, were
treated or not with 1 μg/ml LPS for 1 or 2 hours.
Extraction, purification and quantification of endocanna-
binoids, 2-AG and anandamide, was achieved as pre-
viously described [28]. Briefly, cells with their medium
were Dounce-homogenized and total lipids extracted
with chloroform/methanol/Tris-HCl 50 mM, pH 7.5
(2:1:1, v/v/v) containing internal deuterated standards
(200 pmol [
2
H
5
]-2-AG or [
2
H
8
]-anandamide). After
determination of the total lipid content (mg), lipid
separation was carried out by using open bed chromato-
graphy on silica mini-columns. The pre-purified lipid
extracts were then injected on to an HPLC-APCI-MS
system (LC2010, Shimadzu, Japan) and compounds
identified by single ion monitoring according to the
method previously described [28]. Quantification of
endocannabinoids was achieved by the isotopic dilution
method with amounts expressed as pmol per mg of
total lipid extract.
Purification and differentiation of Stromal Vascular
Fraction
Tissue samples obtained by liposuction were digested
for 30 min at 37°C in Ringer-Lactate buffer containing
1.5 mg/ml collagenase (NB5, SERVA, Germany, PZ
activity 0.175 U/mg). Digested tissue was centrifuged
at 900 g for 3 min. The cell pellet (SVF, Stromal Vas-
cular Fraction) harvested after centrifugation was
resuspended and incubated twice for 10 min in BLB
(blood lysis buffer pH 7, NH4Cl 155 mM, KHCO3 10
mM, Na
2
EDTA 1 mM) to eliminate red blood cells.
Cells were then centrifuged at 900 g for 3 min and the
pellet was resuspended in ringer lactate and filtered
through Steriflip 100 μm (Millipore, France). After
centrifugation at 900 g for 3 min, cells were resus-
pended in 199 medium (PAN Biotech, France). Cell
number and viability were assessed by trypan blue dye
exclusion.
Around 1 million cells were plated in 60 mm culture
flask with Media-1 [M199 + Amphotericin B, (5 mg/
mL), streptomycin (0.2 mg/mL) and penicillin (200 U/
mL) (PAN Biotech, France), 66 nM insulin (Umuline
Rapide,Lilly,France),2g/Lglucose)]with20%Fetal
Bovine Serum (FBS) (PAN Biotech, France). Cells were
then maintained at 37°C in 5% CO
2
for a period of 24
hours prior to the experiments.
Cells were cultured for proliferation in Media-1 with
10% FBS. After 3 days, cells were treated with differen-
tiating Media-2 [M199 + T3 (1 nM), Cortisol (0.2 μM),
Ciglitazone (5 μg/mL), Transferrin (0.1 μg/mL)], without
FBS, for 3 days.
Cells were then treated with appropriate concentra-
tions of drugs along with Media-3 [M199 + T3 (1 nM),
Cortisol (0.2 μM), biotin (8 μg/L) and pantothenate (4
μg/mL)] for 10 days. Media were changed every 3 days.
After 6 days of differentiation and 10 days of treat-
ment, media samples were collected, and the differen-
tiated adipocytes were scraped from the culture plates
using TRIzol reagent for RNA extraction, or wells were
assayed for lipid accumulation by oil-red-O staining.
ELISA assays for TNF-a, IL-6 and MCP-1
Following LPS stimulation for 6 hours, with or without
SR141716A, media were assayed for TNF-a, IL-6 con-
tent with Ready-SET-Go human ELISA kits
(eBioscience, Cliniscience, Montrouge, France), and for
MCP-1 content with RayBio human MCP-1 ELISA kit
(RayBioTech, Clinisciences, France), according to the
manufacturer’s instructions. ELISA sensitivity: 4 pg/mL
for TNF-a, 2 pg/mL for IL-6 and MCP-1.
ELISA assay for adiponectin
Mature adipocytes cultured in 24 well culture plates
were stimulated with LPS with or without SR141716A
for 12 and 24 h. Media were assayed for adiponectin
levels by using a commercial Human Adiponectin
ELISA kit (RayBiotech, Cliniscience, Montrouge,
France). ELISA sensitivity: 10 pg/mL.
TLR2- and TLR4-binding experiments
HEK-Blue™LPS Detection Kit and PlasmoTest™were
purchased from Invivogen,France.HEK-Blue-2and
HEK-Blue-4cellsarestablytransfected with multiple
genes from the TLR2 and TLR4 pathways respectively,
and with a reporter gene (secreted alkaline phosphatase)
which monitors the TLR binding through NFkappaB
activation.
Cells were maintained and plated according to the
manufacturers instructions. HEK-Blue-4 cells were then
treated with 100 nM and 200 nM SR141716A, with or
without 10 ng/mL LPS. Similarly, HEK-Blue-2 cells were
treated with 100 nM and 200 nM SR141716A, with or
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 3 of 15
without 1× Positive Control (stock 1000×, provided
along with the kit). HEK-Blue-2 and HEK-Blue-4 cells
wereincubatedfor16and20hours respectively, fol-
lowed by collection of OD values at 640 nm.
RNA extraction, reverse transcription and real-time
quantitative PCR
Cells from 6 well plates (3 × 10
5
cells) for mature adipo-
cytes and 60 mm culture plates for Pre -adipocytes were
extracted with 500 μLofTRIzol™reagent (Invitrogen,
France). Total RNA was isolated and precipitated
according to the manufacturer’s instructions. 2 μgof
total RNA was reverse-transcribed using random hepta-
mer primers (Eurogentec, Belgium) with MMLV (Invi-
trogen,France).1μl of reverse-transcribed RNA was
amplified by PCR on an ABI PRISM 7000 thermal cycler
(Applied Biosystems, France) using the Taqman™Mas-
ter Mix Kit (Eurogentec, Belgium). The 18S ribosomal
RNA (rRNA) gene was used as a reference. Primers and
probes sequences of TNF-a, IL-6, A-FABP, Adiponectin
and 18S are in Table 1. Quantification of target mRNA
was carried out by comparison of the number of cycles
required in order to reach the reference and target
threshold values (DDCT method). Each analysis reaction
was performed in duplicate, with 6 samples per
condition.
Statistical analysis: Statistical analysis was performed
using Microsoft Excel software. Differences were tested
for significance by the unpaired Student’s t-test. *P <
0,05; **P < 0,005; ***P = ###P < 0.001.
Results
Effect of SR141716A on basal- and LPS-induced TNF-a, IL-
6, and MCP-1 secretion and gene expression in mature
adipocytes
It has already been demonstrated that adipocytes
express innate immune receptors, such as TLR-4 and
TLR-2 and are capable of secreting TNF-a when stimu-
lated with bacterial LPS. When mature adipocytes were
treated with LPS (1 μg/mL), the addition of SR141716A
at a concentration still selective for CB1 versus CB2
receptors (50 to 400 nM) for 6 hours led to a significant
decrease in the secretion of TNF-a (around 30%, Figure
1A) and IL6 after 12 hours (around 25%, Figure 2A).
Comparable results were obtained when the expres-
sion of TNF-a mRNA was investigated. Co-treatment of
adipocytes with LPS (1 μg/mL) + SR141716A (200 nM)
brought about a 30% reduction in LPS-induced TNF-a
mRNA (Figure 1B). However, we found no significant
change in IL-6 gene expression after the co-treatment
(Figure 2B).
Concerning MCP-1, SR141716A seems to have an
effect on basal secretion, whereas secretion induced by
LPS was not significantly affected (Figure 3).
Thus, SR141716A seems to have a broad anti-inflam-
matory effect on mature human adipocytes, but the
mode of action is specific to each cytokine.
Anti-inflammatory effect of SR141716A is not TLR4-, nor
TLR2-dependant
The secretion of TNF-a is mediated by the activation of
the NFkappaB pathway, following the binding of LPS to
TLR4, with CD14 mediating this effect. In order to find
out if the anti-inflammatory effect of SR141716A is due
to a TLR4-blocking effect, we treated the HEK4-Blue
cells (and the HEK2-Blue cells) with 100 nM and 200
nM of SR141716A, with or not a positive control (10
ng/mL LPS for HEK4-Blue cells and 1X Positive Control
for HEK2-Blue cells), for 20 and 16 hours respectively.
The Figure 4 shows the reporter protein activity normal-
ized to control cells, which represents NFkappaB activa-
tion, and thus TLR-binding. It is quite evident that
there is no binding between SR141716A and TLR4 (Fig-
ure 4A), nor with TLR2 (Figure 4B).
LPS induces secretion of the endocannabinoid 2-AG in
mature adipocytes
In order to assess whether the effect of SR141716A on
TNF-a secretion was due to inverse agonism or to
antagonism of tonically active endocannabinoids, we
analysed whether or not LPS induces the formation of 2
endocannabinoids, 2-arachidonoyl glycerol (2-AG) and
Table 1 Primers and probes sequences
Gene Primers Probes
TNF-a 5’-AACATCCAACCTTCCCAAACG-3’
3’-CTCTTAACCCCCGAATCCCAG-5’
5’-FAM-CCCCCTCCTTCAGACACCCTCAACC-TAMRA-3’
IL-6 5’-TCACCTCTTCAGAACGAATTGACA-3’
3’-AGTGCCTCTTTGCTGCTTTCAC-5’
5’-FAM-TACATCCTCGACGGCATCTCAGCCC-TAMRA-3’
18S 5’-CGCCGCTAGAGGTGAAATTCT-3’
3’-CTTTCGTAAACGGTTCTTAC-5’
5’-FAM-ACCGGCGCAAGACGGACCAGA-TAMRA-3’
A-FABP 5’-TGAAAGAAGTAGGAGTGGGCTTTG-3’
3’-ACTAGTAGTCACACTTACCCCT-5’
5’-FAM-AGGAAAGTGGCTGGCATGGCCAA-TAMRA-3’
Adiponectin 5’-TCAATGGCCCCTGCACTACT-3’
3’-CAGGTGGCCTTGAGGAACAG-5’
5’-FAM-CCAACTCCATCTCTAAGTGCCGAACTCATC-TAMRA-3
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 4 of 15
**
gene expression
(arbitrary units)
A
B
0
100
200
300
400
500
600
*** ***
**
0
5
10
15
20
25
30
Control
LPS
LPS+SR1-50
LPS+SR1-100
LPS+SR1-200
LPS+SR1-400
SR1-50
SR1-100
SR1-200
SR1-400
Control
LPS
L
PS+SR1-200
SR1-200
TNF-a secretion (pg/mL)
TNF-a
Figure 1 SR141716A decreases TNF-a secretion and expression in LPS-stimulated mature adipocytes. Panel A: The concentrations of TNF-
a in the medium of mature adipocyte cultures, treated or not with LPS 1 μg/mL alone or in combination with SR141716A were measured at 6
hours by ELISA. SR141716A was used at 50, 100, 200 and 400 nM. Results are expressed in pg/mL. The graph shows the mean ± SD of the
results from 3 patients (n = 6 for each condition, for each patient). ***P < 0.001 and *P < 0.05, versus LPS-treated cells. Panel B: TNF-a gene
expression was determined at 4 hours of treatment in mature adipocyte cultures, treated or not with LPS 1 μg/mL alone or in combination with
200 nM SR141716A. The graph shows the mean ± SD of the results from 2 patients (n = 6 for each condition, for each patient). **P < 0.005,
versus LPS-treated cells.
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 5 of 15
B
A
IL-6 secretion (%)
Control
LPS
LPS+SR1-50
0
20
40
60
80
100
120
140
LPS+SR1-100
LPS+SR1-200
LPS+SR1-400
SR1-50
SR1-100
SR1-200
SR1-400
***
***
0
5
10
15
20
25
30
35
IL-6 gene expression (arbitrary units)
Control
SR1-200
L
PS+SR1-200
LPS
Figure 2 SR141716A decreases LPS-induced IL-6 secretion but not gene expression in mature adipocytes. Panel A: Adipocytes were
treated with 1 μg/mL LPS and/or not with SR141716A from 50 to 400 nM. IL-6 secretion was measured in media after 12 hours treatment by
ELISA. Results are expressed in percentage, normalised to LPS (100% represents from 1 to 10 ng/mL IL6, depending on the patients). The graph
shows the mean ± SD of the results from 3 patients (n = 6 for each condition, for each patient). ***P < 0.001, **P < 0.005 and *P < 0.05, versus
LPS-treated cells. Panel B: IL6 gene expression was determined at 4 hours of treatment in mature adipocyte cultures, treated or not with LPS 1
μg/mL alone or in combination with 200 nM SR141716A. The graph shows the mean ± SD of the results from 2 patients (n = 6 for each
condition, for each patient).
Murumalla et al.Journal of Inflammation 2011, 8:33
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arachidonoyl ethanolamine (AEA or anandamide) in
human adipocytes. Over short incubation time intervals
(1 and 2 hours), LPS (1 μg/ml) induced the secretion of
the classical CB1 agonist, 2-AG, in mature adipocytes
(Figure 5A). The maximal effect of LPS was observed at
2 hours post treatment. No effect on the other endocan-
nabinoid, anandamide, was observed (Figure 5B).
SR141716A restores secretion of adiponectin in LPS-
treated mature adipocytes
Adiponectin is one of the most important adipokines
secreted by adipocytes. It has been previously shown in a
murine cell line that SR141716A stimulates adiponectin
protein as well as gene expression [25]. We thus decided
to study the effect of 200 nM, SR141716A, for 12 and 24
hours, on mature human adipocytes. We did not, how-
ever, find any significant change in adiponectin protein
secretion in SR141716A-treated adipocytes (Figure 6A).
In order to measure adiponectin secretion in LPS-sti-
mulated mature adipocytes, as well as the effect of
SR141716A on these cells, we treated adipocyte cells
with 1 μg/mL LPS alone or with SR141716A (200 nM).
As shown in Figure 6B, LPS caused a decrease in
mature adipocyte adiponectin secretion (approximately
30%), at 24 hours of treatment. In this case, co-treat-
ment with SR141716A and LPS reversed this effect, and
restored the adiponectin levels to those of the control
cells.
Effect of SR141716A on pre-adipocyte differentiation and
gene expression
In order to understand the effect of the CB1 receptor
antagonist SR141716A on the differentiation process
and its particular effect on fat accumulation, we differ-
entiated human stromal vascular cells (SVF) into adipo-
cytes and observed the level of differentiation using oil-
red-O staining, as well as by measuring the expression
of well known differentiation gene markers. Pre-adipo-
cytes were treated with SR141716A at 200 nM and 500
nM for 10 days.
SR141716A neither increased nor decreased fat accu-
mulation in these differentiated cells (Figure 7), nor
0
50
100
150
200
250
300
M
C
P-1 secret
i
on
(%)
Control
LPS
L
PS+SR1-50
LPS+SR1-100
LPS+SR1-200
LPS+SR1-400
SR1-50
SR1-100
SR1-200
SR1-400
***
**
Figure 3 SR141716A decreases the basal MCP-1 secretion, and slightly the LPS-induced MCP-1 secretion in mature adipocytes.
Adipocytes were treated with 1 μg/mL LPS and/or not with SR141716A from 50 to 400 nM. MCP-1 secretion was measured in media after 6
hours treatment by ELISA. Results are expressed in percentage, normalised to LPS (100% represents from 2 to 5 ng/mL MCP-1, depending on
the patients). The graph shows the mean ± SD of the results from 3 patients (n = 6 for each condition, for each patient). **P < 0.005 and *P <
0.05, versus control cells.
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 7 of 15
A
B
HEK-4 Cells Reporter protein activity
(arbitrary units)
Control
LPS
LPS+SR1-100
SR1-100
SR1-200
LPS+SR1-200
0
0.5
1
1.5
2
0
0.5
1
1.5
2
2.5
3
Control
PC-1/100
SR1-100
SR1-200
P
C-1/100+SR1-100
P
C-1/100+SR1-200
HEK-Blue-4 Cells Reporter protein activity
(arbitrary units)
Figure 4 SR141716A does not bind to TLR4, nor to TLR2. HEK-4-Blue (panel A) and HEK-2-Blue cells (panel B) were treated with SR141716A
(100 nM and 200 nM), with or without their respective positive control: 10 ng/mL LPS and 1/100X Positive Control (PC), for 20 and 16 hours
respectively. The graphs show the mean ± SD of the results of 2 experiments (n = 12 for each conditions).
Murumalla et al.Journal of Inflammation 2011, 8:33
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0.000
0.010
0.020
0.030
0.040
0.050
0.060
0.000
0.010
0.020
0.030
0.040
0.050
0.060
*
Control
LPS
Control
LPS
2-AG (pmol/mg of total lipid extract)
AEA (pmol/mg of total lipid extract)
1 hour 2 hours
1 hour 2 hours
A
B
Figure 5 2-AG, but not anandamide (AEA), is induced by LPS in human mature adipocytes. Adipocytes were incubated with LPS 1 μg/mL
for 1 and 2 hours. Medium and cells were collected and total lipids were extracted. The CB1 agonists 2-AG (panel A) and anandamide (AEA)
(panel B) were identified and quantified by HPLC-APCI-MS analysis. The graphs shows the mean ± SD of the results from 3 patients (n = 6 for
each condition, for each patient), *P < 0.05, versus control cells.
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0
100
200
300
400
500
600
Control
Adiponectin secretion (pg/mL)
***
###
LPS
L
PS+SR1
0
100
200
300
400
500
Control
SR1
12 hours 24 hours
A
B
Adiponectin secretion (pg/mL)
Figure 6 SR141716A has no effect on basal adiponectin secretion, but restores basal level in LPS-treated mature adipocytes . Panel A:
Mature adipocytes were treated or not with 200 nM SR141716A for 12 and 24 hours. The medium was assayed for adiponectin using ELISA. The
graph shows the mean ± SD of the results from 3 patients (n = 6 for each condition, for each patient). Panel B: Mature adipocytes were treated
or not with LPS 1 μg/mL, alone or in combination with 200 nM SR141716A for 24 hours. The medium was assayed for adiponectin using ELISA.
The graph shows the mean ± SD of the results from 5 patients (n = 6 for each condition, for each patient). ***P < 0.001, versus control cells.
###P < 0.001, versus LPS-treated cells.
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 10 of 15
changed the expression of the Adipocyte-Fatty Acid
Binding Protein (A-FABP) gene (Figure 8A).
Lastly, we found that treatment with SR141716A led
to an increase in adiponectin gene expression (Figure
8B). This effect was significant at 200 nM and 500 nM
concentrations. The increase in adiponectin gene
expression was not accompanied by an increase in pro-
tein secretion as measured by ELISA (Figure 8C).
Discussion
The adipose tissue is now recognized as being an endo-
crine tissue capable of secreting a large number of various
types of molecules, the adipokines, which are more or less
specific to this tissue. Although not an exhaustive list, the
following are the main adipokines: leptin, TNF-a, IL-6,
adiponectin, MCP1 and IL-10. It has largely been demon-
strated that these mediators are implicated in pathologies
associated with obesity, in particular those associated with
local and global inflammation [4,20,29,30].
Furthermore, the adipose tissue should no longer be
considered as a passive, fatty acid storage tissue, since
numerous studies now demonstrate that it acts in fact
like a transitory reservoir also for circulating cholesterol
[31-33]. Reverse cholesterol transport occurs following
mobilization of cholesterol and adipocyte apoE by devel-
oping HDLs (Bencharif et al., 2010, under review).
Moreover, the interactions between pro- or anti-inflam-
matory molecules and cholesterol efflux are currently
being investigated [33,34].
Lastly, the adipose tissue is also able to produce endo-
cannabinoids, i.e. mediators acting at cannabinoid CB1
and CB2 receptors (anandamide, 2-AG) and endocanna-
binoid-like molecules, such as PEA and OEA, which act
at PPAR-alpha receptors [22,35]. These mediators dis-
play an important paracrine or autocrine pro- or anti-
inflammatory actions [29,36], since their receptors are
expressed on the surface of adipocytes, and in particular
in fully differentiated mature adipocytes [23].
Some of the beneficial clinical effects of the CB1
antagonist, SR141716A, have until recently been attribu-
ted both to the peripheral action of the molecule on adi-
pose tissue [24,25,37,38], particularly with regard to
weight loss and the increase in circulating adiponectin
levels [17,24,25] and to the anti-inflammatory action of
the molecule against hepatic steatosis and pro-athero-
sclerotic processes [16-19]. At least some of these per-
ipheral effects of SR141716A can be explained by an
overactivity of CB1 receptors caused by permanently
elevated levels of endocannabinoids, anandamide and 2-
AG, in the visceral adipose tissue, liver and atherosclero-
tic plaques, as assessed, in vitro, in murine adipocytes
and, in vivo, in animal models of obesity and athero-
sclerosis [21,36,39]; see [40] for review.
In this study, we show that SR141716A possesses an
anti-inflammatory activity also upon mature human adi-
pocytes in primary culture, consisting of a partial but
significant inhibition of LPS-induced expression and
secretion of TNF-a (Figure 1A and 1B). This result is in
agreement with those of Miranville et al., who showed
that SR141716A could decrease the macrophage TNF-a
production, resulting in a rescue of insulin signaling in
adipocyte (Miranville et al., Obesity, 2010). So, the per-
ipheral anti-inflammatory effect of SR141716A on adi-
pose tissue is first due to the direct action of this
molecule on adipocytes, but also to an indirect action
on infiltrated macrophages.
It is to be noted that the anti-inflammatory effect, in
our adipocyte cellular model, includes IL-6 secretion,
but not its gene expression (Figure 2A and 2B). These
results are in accordance to those obtained by Sugamura
et al. [18] who demonstrated, in human macrophages
treated with LPS, that SR141716A is able to decrease
both TNF-a and IL-6 secretion levels. However, Dol-
Gleizes et al. showed a decrease in LPS-induced IL6
gene expression [16]. However, to obtain significant
results, the authors have used a concentration of
C
ontrol SR1
Figure 7 SR141716A has no effect on oil accumulation in differentiated pre-adipocyte. Cultures of SVF cells were stained with Oil-red-O
after 10 days of SR141716A (200 nM) treatments. Photographs are representative of 3 different experiments on 3 different tissue samples.
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 11 of 15
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
0
100
200
300
400
500
600
700
800
900
1000
0
0.5
1
1.5
2
2.5
3
**
**
Control
SR1-200
SR1-500
A-FABP gene expression
(arbitrary units)
Adiponectin gene expression
(arbitrary units)
Adiponectin secretion (pg/mL)
A
B
C
Control
Control
SR1-200
SR1-200
S
R1-500
SR1-500
Figure 8 SR141716A increases adiponectin gene expression, but not secretion, and has no effect on A-FABP gene expression in
differentiated pre-adipocytes. Panel A: A-FABP gene expression was determined at 10 days in differentiated pre-adipocytes cultures, treated or
not with SR141716A (200 and 500 nM). The graph shows the mean ± SD of the results from 2 patients (n = 6 for each condition, for each
patient). Panel B: Adiponectin gene expression was determined at 10 days in differentiated pre-adipocytes cultures, treated or not with
SR141716A (200 and 500 nM). The graph shows the mean ± SD of the results from 3 patients (n = 6 for each condition, for each patient). **P <
0.005, versus control cells. Panel C: Differentiated SVF cells were treated or not with SR141716A (100 nM and 200 nM) for 48 hours. The medium
was assayed for adiponectin using ELISA. The graph shows the mean ± SD of the results from 2 patients (n = 6 for each condition, for each
patient).
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 12 of 15
SR141716A of 1 μM, which can be considered as nota-
bly high, or unselective for this kind of molecule. It is
probable that the concentration of 200 nM, which was
used in the present study, is more selective for CB1, and
this, together with the different cell type used here,
could explain the difference between the two sets of
results. Moreover, this concentration is in accordance
with several previous studies on adipocytes [24].
In order to confirm the broad anti-inflammatory
action of SR141716A, we have also checked the LPS-
induced MCP-1 section. Although the results are not
significant, SR141716A seems to decrease this secretion
(Figure 3). The same result was obtained by Dol-Greizes
et al. on MCP-1 gene expression [16]. Moreover, it
should be noted that SR141716A is able to reduce the
basal MCP-1 secretion, while it’snotthecaseforthe
other cytokines secreted. This result is crucial, because
it shows that SR141716A could act long before the
establishment of chronic inflammation, especially as the
deleterious effect of MCP-1 has been demonstrated,
notably in the macrophages infiltration process [41].
In low-grade inflammatory status, and in our cellular
model, activation of “Toll-like receptor 4”(TLR4) with
LPS (linked to the LPS-binding protein) is the primor-
dial step leading to cytokines secretion, via the NFkap-
paB pathway. So, the anti-inflammatory effect of
SR141716A could be explained by its binding to TLR4,
which could block the receptor and then limit the LPS
binding. We verified this hypothesis by using HEK4-
Blue cells, which have high expression of TLR4 and all
genes downstream, and we have proved here that there
is no binding between SR141716A and TLR4, nor with
TLR2 (another PAMPs receptor) (Figure 4A and 4B).
This anti-inflammatory effect of SR141716A seems to be
specific to CB1.
We also report here that LPS induces an increase in
the secretion of 2-AG by adipocytes (Figure 5A), but
not in anandamide synthesis (Figure 5B). This is in
accordance with the fact that 2-AG are expressed at a
permanently elevated level in inflammated adipose tis-
sue, whereas it’snotthecaseforanandamide[21].
Moreover, we’ve tested the effect of 2-AG on LPS-
induced TNF-a secretion and we are not able to find
any pro-inflammatory effect of 2-AG (data not shown).
According to that, we can conclude that the anti-inflam-
matory effect of SR141716A is not due to the blockade
of 2-AG binding. SR141716A has thus its own effect.
Contrary to the results obtained by Bensaid et al. [25]
and Matias et al. [21], in mouse 3T3 adipocytes, we
were unable to show that treatment of mature human
adipocytes with SR141716A alone results in an increase
in the expression or secretion of adiponectin (Figure
6A). It is likely that the choice of the cellular model,
and in particular of a different species, is the cause of
this discordance. Alternatively, it is possible that the sti-
mulatory effect of SR141716A on adiponectin expression
and release from adipocytes is only observed in the visc-
eral adipose tissue, which is characterised by the stron-
gest pro-inflammatory profile during obesity. Indeed,
SR141716A, in clinical use, does restore adiponectin
levels in abdominally obese patients (ADAGIO-lipids
study, [15]), whose levels are low compared to non-
obese patients. This effect could potentially be related
also to a reduction in the levels of circulating TNF-a,
since there exists a well established inverse regulation
between these two molecules [42]. We thus verified this
hypothesis by measuring the levels of adiponectin
secreted when the cells were treated with LPS (with sub-
sequent increase in TNF-a secretion), or with LPS +
SR141716A. Indeed, LPS reduced the release of adipo-
nectin from cells, and co-treatment with SR141716A
effectively counteracted this effect in this case (Figure
6B). These results support some of the claims made
about the peripheral effects of SR141716A, and in parti-
cular the effect upon the adipose tissue. However, it is
necessary to stress that SR141716A exhibited here no
effect upon adiponectin when the cells were in a non-
inflammatory state. Moreover, it is also possible that, in
a clinical setting, the effect on adiponectinemia is par-
tially related to weight loss [43], or to a reduction in the
visceral vs. subcutaneous white adipose mass as a result
of the lipolytic effect of the molecule [15,44], or also,
that the peripheral effect of SR141716A on the adipose
tissue concerns cells other than the adipocytes. This last
point is supported by evidence showing that the secre-
tion of adiponectin is not specific to adipose cells [45].
It is, therefore, possible that the peripheral effects of
SR141716A on adiponectinemia in human obesity are,
in the end, a summation of all of these effects. Interest-
ingly, in rodents, the amelioration of glucose intolerance
and insulin resistance observed following treatment of
mice with high fat diet- or leptin deficiency-induced
obesity with SR141716A was, to a large extent, depen-
dent on the presence of adiponectin [46,47].
Lastly, certain authors put forth the hypothesis that
the reduction in body weight observed with SR141716A
and other CB1 antagonists/inverse agonists could be
related to inhibition of pre-adipocyte cellular prolifera-
tion [24]. With this in mind we decided to study the
effect of SR141716A on adipocyte differentiation. The
evaluation of proliferation in cells of the human SVF is
complicated by the fact that is difficult to determine the
actual proportion of pre-adipocytes in this fraction. In
fact, it is difficult to prove conclusively that there exists
a specific anti-proliferation effect in pre-adipocytes or
on any other cellular type. On the other hand, adipocyte
differentiation can easily be observed via lipid
accumulation.
Murumalla et al.Journal of Inflammation 2011, 8:33
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Page 13 of 15
Our results show that SR141716A has no effect upon
human adipocyte differentiation of SVF cells since we
did not observe any change in lipid accumulation (Fig-
ure 7), nor any variation in the expression of a key gene
of adipocyte differentiation: A-FABP (Figure 8A). The
expression of CD36, PPARalpha and PPARgamma genes
was also investigated but again no effect was detected
(data not shown).
Lastly, unlike what we observed before using differen-
tiated adipocytes, SR141716A increased the expression
of the adiponectin gene when administered to pre-adi-
pocytes (Figure 8B). Our results agree with the findings
of Bensaid et al. [25] obtained in a murine cell line.
However, the secretion of adiponectin by differentiated
adipocytes was not modified in our set-up (Figure 8C).
Conclusion
In conclusion, the present results lead us to suggest that
some of the effects observed during clinical treatment
with SR141716A are due, at least in part, to an effect of
the molecule on the white adipose tissue, which is now
considered as an important target in the development of
molecules to treat the metabolic syndrome.
On the other hand, we could not confirm all the
observations obtained previously with CB1 antagonists
in murine adipocytes [21,25,48], thus indicating that
there might be species-differences in the actions of
these compounds in the adipose tissues, and emphasiz-
ing again the importance of using human cells to predict
possible effects in vivo of pharmacological and therapeu-
tic tools.
The clinical development of SR141716A was discon-
tinued because of psychiatric side effects, which are
inherent to the central action of CB1 antagonists [49].
The present data confirm that it might be worthwhile,
in order to limit these side effects, to develop molecules
which exert only peripheral effects, such as CB1 antago-
nists that do not cross the hemato-encephalic barrier.
These compounds, by acting directly on the white adi-
pose tissue of obese individuals, might reduce systemic
inflammation and hence contribute to counteract ather-
osclerosis and insulin resistance.
Acknowledgements
We are grateful to the plastic surgeons: Hulard O., Delarue P. and Gonçalves
J. who took part in this study and allowed the collection of subcutaneous
adipose tissue samples, to the entire team of the Biochemistry Department
of the Félix Guyon Hospital, Reunion island, and to the Regional Council for
the financial support. Finally, we would like to thank all patients who
consented to the collection of tissue samples, and thus made this study
possible.
Author details
1
GEICO, Groupe d’Etude sur l’Inflammation et l’Obésité Chronique, Université
de La Réunion, plateforme CYROI, 15 avenue René Cassin, 97715 Saint-Denis
Messag Cedex, France.
2
Service de biochimie, Centre Hospitalier Félix Guyon,
97400 Saint-Denis, La Réunion, France.
3
Endocannabinoid Research Group at
the Institute of Biomolecular Chemistry of the National Research Council,
Pozzuoli (NA), Italy.
Authors’contributions
RR and MC conceived of the study and LH and FT participated in its design.
MP, SP and VdM carried out the endocannabinoid quantification. RM and LG
carried out the primary culture and the ELISA, with the help of AB and KB.
RM and LH carried out the gene expression. RR, RM and LH participated in
drafting the manuscript. All authors read and approved the final manuscript.
Competing interests
No potential conflict of interest relevant to this article was reported.
Received: 9 August 2011 Accepted: 16 November 2011
Published: 16 November 2011
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doi:10.1186/1476-9255-8-33
Cite this article as: Murumalla et al.: Effect of the Cannabinoid Receptor-
1 antagonist SR141716A on human adipocyte inflammatory profile and
differentiation. Journal of Inflammation 2011 8:33.
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