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Adiponectin is required to mediate rimonabant-induced improvement of
insulin sensitivity but not body weight loss in diet-induced obese mice
Ste´phanie Migrenne,
1
Ame´lie Lacombe,
1
Anne-Laure Lefe`vre,
1
Marie-Pierre Pruniaux,
2
Etienne Guillot,
2
Anne-Marie Galzin,
2
and Christophe Magnan
1
1
University Paris-Diderot, Centre National de la Recherche Scientifique, Paris, France; and
2
Sanofi-Aventis Research
and Development, Rueil-Malmaison, France
Submitted 9 October 2008; accepted in final form 3 February 2009
Migrenne S, Lacombe A, Lefe` vre AL, Pruniaux MP, Guillot E,
Galzin AM, Magnan C. Adiponectin is required to mediate rimonabant-
induced improvement of insulin sensitivity but not body weight loss
in diet-induced obese mice. Am J Physiol Regul Integr Comp Physiol
296: R929 –R935, 2009. First published February 11, 2009;
doi:10.1152/ajpregu.90824.2008.—The increase in adiponectin levels
in obese patients with untreated dyslipidemia and its mRNA expres-
sion in adipose tissue of obese animals are one of the most interesting
consequences of rimonabant treatment. Thus, part of rimonabant’s
metabolic effects could be related to an enhancement of adiponectin
secretion and its consequence on the modulation of insulin action, as
well as energy homeostasis. The present study investigated the effects
of rimonabant in adiponectin knockout mice (Ad
⫺/⫺
) exposed to
diet-induced obesity conditions. Six-week-old Ad
⫺/⫺
male mice and
their wild-type littermate controls (Ad
⫹/⫹
) were fed a high-fat diet for
7 mo. During the last month, animals were administered daily either
with vehicle or rimonabant by mouth (10 mg/kg). High-fat feeding
induced weight gain by about 130% in both wild-type and Ad
⫺/⫺
mice. Obesity was associated with hyperinsulinemia and insulin
resistance. Treatment with rimonabant led to a significant and similar
decrease in body weight in both Ad
⫹/⫹
and Ad
⫺/⫺
mice compared
with vehicle-treated animals. In addition, rimonabant significantly
improved insulin sensitivity in Ad
⫹/⫹
mice compared with Ad
⫹/⫹
vehicle-treated mice by decreasing hepatic glucose production and
increasing glucose utilization index in both visceral and subcutaneous
adipose tissue. In contrast, rimonabant failed to improve insulin
sensitivity in Ad
⫺/⫺
mice, despite the loss in body weight. Rimon-
abant’s effect on body weight appeared independent of the adiponec-
tin pathway, whereas adiponectin seems required to mediate rimon-
abant-induced improvement of insulin sensitivity in rodents.
endocannabinoid system; cannabinoid receptor 1 antagonist; meta-
bolic syndrome
THE DISCOVERY OF THE ENDOCANNABINOID system has led to the
development of new treatments for patients with obesity and
associated cardiometabolic risk factors (33). Basic research has
demonstrated that such a system plays an integrative role in the
control of food intake, metabolism, and fat storage through
cannabinoid receptors 1 (CB1) located in both the brain and the
periphery, notably in adipose tissue (18, 21). Regarding their
central effects, endogenous cannabinoids anandamide and
2-arachidonoyl glycerol (2AG) stimulate appetite and food
intake in both rodents (8, 10) and humans (13), and CB1
receptor knockout (KO) mice are resistant to diet-induced
obesity (23). The role of the peripheral endocannabinoid sys-
tem in human obesity is also being extensively investigated.
Circulating levels of anandamide and 2AG are increased in
obese compared with control subjects (7), whereas CB1 mRNA
expression is correlated with visceral fat mass (2), thus sug-
gesting an upregulated peripheral endocannabinoid system in
human obesity. Rimonabant, the first selective CB1 antagonist,
has been shown to reduce fat mass and improve multiple
cardiometabolic risk factors in overweight/obese patients, as
evidenced in the “Rimonabant In Obesity” (RIO) program (24,
30). One of the most interesting consequences of rimonabant
treatment was the increase in plasma adiponectin concentra-
tions in obese patients with untreated dyslipidemia (5), as well
as an increased adiponectin mRNA expression in adipose
tissue of obese fa/fa rats and diet-induced obese (DIO) mice
(11) and in cultured adipocytes (1). From these data, it may be
proposed that rimonabant metabolic effects could be related, at
least in part, to an enhancement of adiponectin secretion and its
insulin-sensitizing actions (19). Indeed, hyperinsulinemic/
euglycemic clamp studies evidenced that plasma adiponectin
levels correlated significantly with insulin sensitivity in obese
humans (4). The present study was aimed at investigating
whether the effect of rimonabant on both body weight loss and
increased insulin sensitivity required adiponectin. To that end,
adiponectin (Ad
⫺/⫺
) KO mice (15) and their control littermates
(Ad
⫹/⫹
) were fed a high-fat diet during 7 mo and treated with
rimonabant or vehicle during the last month.
MATERIALS AND METHODS
Animals. The study protocol was approved by the Institutional
Animal Care and Use Committee of Paris Diderot University. Adi-
ponectin-deficient mice (Ad
⫺/⫺
) were given to us by P. Froguel and
T. Kadowaki. To construct the targeting vector for disruption of the
adiponectin gene, a neomycin resistance gene (neoR) was substituted
for exon 2 and exon 3, the coding region of the adiponectin gene (15).
The adiponectin allele was screened by PCR from tail DNA. Duplex
PCR was run using specific primers (15). All experiments were
performed using littermate mice as controls (Ad
⫹/⫹
).
Diet and rimonabant treatment. Mice fed ad libitum were housed
under controlled temperature and lighting. Six-week-old male Ad
⫹/⫹
and Ad
⫺/⫺
mice were fed for 7 mo a 54% high-fat diet (HFD) (3) or
regular chow (RC). During the last month of the diet, half of the obese
mice received rimonabant daily (10 mg/kg, dilution in saline-Tween
80% by mouth, Ad
⫺/⫺R
and Ad
⫹/⫹R
), and half received vehicle
(saline-Tween 80%, Ad
⫺/⫺V
and Ad
⫹/⫹V
).
Food intake, body weight, plasma, and liver parameters. Food
intake was measured by weighing the HFD pellets every 2 days. Body
weight was measured before and after vehicle or rimonabant treat-
Address for reprint requests and other correspondence: S. Migrenne, Centre
National de la Recherche Scientifique-Univ. Paris Diderot-Baˆtiment Buffon, 4,
rue Marie Andre´e Lagroua Weill-Halle-75205 Paris Cedex 13, France (e-mail:
stephanie.migrenne@univ-paris-diderot.fr).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Regul Integr Comp Physiol 296: R929–R935, 2009.
First published February 11, 2009; doi:10.1152/ajpregu.90824.2008.
0363-6119/09 $8.00 Copyright ©2009 the American Physiological Societyhttp://www.ajpregu.org R929
ment. Blood was sampled from caudal vessels for determination of
glycemia, and plasma was collected to measure hormone using ELISA
kits (insulin, leptin, and adiponectin), triglyceride (TG) and free fatty
acid (FFA) concentrations. Liver TG was extracted as previously
described (34). At the end, animals were killed, and subcutaneous
adipose tissue (SCAT) and visceral adipose tissue (VAT) were re-
moved and weighed.
Oral glucose tolerance test. Oral glucose tolerance tests (3 g/kg
body wt) were performed in overnight fasted mice. Glycemia was
monitored from a blood drop collected from caudal vessels (glucose
analyzer; Roche Diagnostics, Meylan, France) at 0, 5, 10, 15, 20, 30,
40, 50, 60, 90, and 120 min, and plasma insulin was measured at 30
min. The insulinogenic index (⌬I/⌬G) was calculated by dividing the
incremental plasma insulin by the incremental plasma glucose con-
centrations at 30 min.
Glucose turnover rate. Glucose turnover rate was assessed at the
end of HFD-feeding period, using the euglycemic-hyperglycemic
clamp technique. Ten days before the experiment, an indwelling
catheter (Becton Dickinson, Rabalot, France) was inserted into the
right jugular vein in anesthetized mice (100 mg/kg body wt ketamine/
xylazine; Sigma-Aldrich, La Courneuve, France). Hyperinsulinemic-
euglycemic clamp was conducted in 5-h fasted conscious mice, as
previously described (29). Briefly, a 5-Ci bolus of [3-
3
H]glucose and
a priming dose of insulin (100 mU/kg; Actrapid, Novo, Copenhagen,
Denmark) dissolved in isotonic saline were injected through the
jugular vein, followed by a continuous infusion of [3-
3
H]glucose (15
Ci) and insulin (6.66 mU䡠kg
⫺1
䡠min
⫺1
) at a constant rate of 1
l/min to maintain blood glucose levels at 100 mg/dl. During the
clamp, blood was sampled from the cut tail every 10 min to determine
glucose levels and to adjust the rate of unlabeled glucose infusion to
maintain euglycemia. The euglycemic conditions were reached within
30 – 40 min and then maintained for 60 min thereafter. Steady-state
specific glucose radioactivity, plasma glucose, and insulin concentra-
tions were determined during the last 20 min of the clamp. During the
glucose clamp (50 –70 min after the onset of insulin infusion), the
glucose disposal rate (GDR) that reflects glucose utilization is equal to
the rate of glucose appearance, resulting from hepatic glucose pro-
duction (HGP), added to the amount of glucose infused necessary to
maintain euglycemia (glucose infusion rate, GIR, expressed in
mg䡠min
⫺1
䡠kg
⫺1
). For the assay of [3-
3
H]glucose radioactivity, blood
samples were deproteinized with Ba(OH)
2
and ZnSO
4
, and the super-
natant was evaporated to dryness at 50°C to remove tritiated water.
The dry residue was dissolved in 0.5 ml water to which 10 ml
scintillation solution was added (Aqualuma plus, Lumac, The Neth-
erlands), and radioactivity was determined in a Packard Tri-Carb
460C liquid scintillation system.
Glucose utilization index (GUI) during hyperinsulinemic-euglyce-
mic clamp. When the steady state was reached, a bolus (3 Ci) of
2-deoxy-D-[1-
14
C] glucose (2DG; Amersham Pharmacia Biotechnol-
ogy, Orsay, France) was injected by the jugular vein. Blood was
sampled at time 1, 5, 10, 20, 30 40, and 60 min to measure [
14
C]2DG
specific activity. Mice were then euthanized, and tissues were col-
lected for analysis: soleus, extensor digitorum longus, SCAT, and
VAT. GUI was calculated in individual tissues from the plasma
[
14
C]2DG profile, which was fitted with a double exponential curve
using Origin (OriginLab Corp, Northampton, MA) and tissue
[
14
C]2DG-6-phosphate content, as previously described (17).
Statistical analysis. Statistical analyses were performed using a
two-factor repeated-measures ANOVA. Comparisons between groups
were carried out using a nonpaired Student’s t-test, except when
mentioned. A Pvalue of less than 0.05 was considered statistically
significant.
RESULTS
Body weight and food intake. Before starting HFD feeding,
Ad
⫺/⫺
mice exhibited a body weight similar to that of the
wild-type Ad
⫹/⫹
mice (23.3 ⫾0.5 g vs. 22.7 ⫾0.5 g, ns). The
HFD induced an obesity in both Ad
⫹/⫹
and Ad
⫺/⫺
mice
compared with RC (Fig. 1A). Body weight in both Ad
⫹/⫹
and
Ad
⫺/⫺
mice treated with rimonabant was significantly de-
creased to the same extent compared with vehicle-treated
animals (Fig. 1A). Rimonabant had no effect on body weight in
both Ad
⫹/⫹
and Ad
⫺/⫺
fed a RC compared with vehicle-
treated animals (Fig. 1A). During the first 2 mo of HFD, the
food intake in Ad
⫹/⫹
and Ad
⫺/⫺
mice increased significantly
in a similar way (34 ⫾3to58⫾6 kcal/100 g body wt/24 h,
P⬍0.001 in Ad
⫹/⫹
mice and 37 ⫾4to52⫾5 kcal/100 g
body wt/24 h, P⬍0.001 in Ad
⫺/⫺
mice). Then, it stabilized
during the following months of the study. Food intake was
transiently decreased by rimonabant during the first week in
both Ad
⫹/⫹
and Ad
⫺/⫺
mice (48.4 ⫾1.4 and 34.3 ⫾3.8
kcal/100 g body wt/24 h, P⬍0.01 in vehicle and rimonabant-
treated Ad
⫹/⫹
mice, respectively, and 46.1 ⫾2.3 and 32 ⫾2.5
kcal/100 g body wt/24 h, P⬍0.01 in vehicle and rimonabant-
treated Ad
⫺/⫺
mice, respectively). There was no difference in
the weight of adipose tissue between vehicle-treated Ad
⫹/⫹
and Ad
⫺/⫺
mice fed a RC, and rimonabant treatment had no
effect under these conditions (Fig. 1B). HFD induced an
increase in fat mass in both VAT (Fig. 1C) and SCAT depots
in Ad
⫹/⫹
. In contrast, in Ad
⫺/⫺
mice, this increase was only
observed in VAT. SCAT and VAT mass was decreased by
rimonabant in Ad
⫹/⫹
, and only VAT mass was decreased in
Ad
⫺/⫺
mice compared with vehicle-treated mice (Fig. 1C).
Plasma and liver parameters. In RC-fed mice, all parame-
ters were similar in Ad
⫹/⫹
and Ad
⫺/⫺
mice treated either with
vehicle or rimonabant, except adiponectin, which was unde-
tectable in Ad
⫺/⫺
mice. In HFD-fed mice, rimonabant induced
a decrease in plasma FFA and TG concentrations in both
Ad
⫹/⫹
and Ad
⫺/⫺
mice (Table 1). Neither the HFD nor the
rimonabant treatment had an effect on TG liver content in both
Ad
⫹/⫹
and Ad
⫺/⫺
mice (5.6 ⫾0.9 mg TG/g liver in Ad
⫹/⫹
mice fed RC, 6.5 ⫾1.9 and 7.7 ⫾1.7 mg TG/g liver, ns, in
vehicle and rimonabant-treated Ad
⫹/⫹
mice, respectively, and
5.8 ⫾0.6 and 5.6 ⫾1.7 mg TG/g liver, ns, in vehicle and
rimonabant-treated Ad
⫺/⫺
mice, respectively). Both plasma
leptin and insulin concentrations increased in the HFD-fed
vehicle-treated Ad
⫹/⫹
and Ad
⫺/⫺
mice and decreased in Ad
⫹/⫹
and
Ad
⫺/⫺
mice treated with rimonabant. Plasma adiponectin con-
centration decreased in the HFD-fed control Ad
⫹/⫹
compared
with RC-fed Ad
⫹/⫹
mice, and rimonabant treatment increased
it (Table 1).
Insulin response to oral glucose overload. Both HFD-fed
Ad
⫹/⫹
and Ad
⫺/⫺
mice exhibited glucose intolerance com-
pared with Ad
⫹/⫹
mice fed RC, and rimonabant did not
improve glucose tolerance (Fig. 2, Aand B). However, plasma
insulin at 30 min after glucose load was markedly increased in
both HFD-fed Ad
⫹/⫹
and Ad
⫺/⫺
mice compared with Ad
⫹/⫹
mice fed RC (Fig. 2C). Rimonabant treatment normalized
insulin response in Ad
⫹/⫹
mice to the levels observed in mice
fed RC but not in Ad
⫺/⫺
mice fed a HFD (Fig. 2C). ⌬I/⌬G was
significantly increased in both vehicle-treated Ad
⫹/⫹
and
Ad
⫺/⫺
mice fed a HFD (Fig. 2D). In Ad
⫹/⫹
mice, ⌬I/⌬G was
significantly reduced by rimonabant, whereas it remained
markedly increased in Ad
⫺/⫺
mice, both fed a HFD (Fig. 2D).
Glucose infusion rate and glucose turnover rate during
hyperinsulinemic-euglycemic clamps. The HFD induced a de-
crease in GIR during hyperinsulinemic-euglycemic clamp (Fig. 3A).
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AJP-Regul Integr Comp Physiol •VOL 296 •APRIL 2009 •www.ajpregu.org
GIR was significantly increased by rimonabant in Ad
⫹/⫹
mice
(Fig. 3A) but remained unchanged in Ad
⫺/⫺
compared with
vehicle-treated Ad
⫺/⫺
mice fed a HFD (Fig. 3A). GDR was
decreased in all groups fed the HFD compared with RC-fed
mice (Fig. 3B), and rimonabant treatment significantly in-
creased GDR only in HFD-fed Ad⫹/⫹mice (Fig. 3B). HGP
was significantly raised in all groups fed the HFD compared
with RC-fed mice (Fig. 3C) and rimonabant treatment signif-
icantly reduced HGP only in HFD-fed Ad⫹/⫹mice (Fig. 3C).
GUI was similar in skeletal muscles from all groups, and
rimonabant treatment had no effect (Fig. 4A). In contrast, GUI
was increased in both SCAT and VAT of rimonabant-treated
Ad
⫹/⫹
but remained unchanged in rimonabant-treated Ad
⫺/⫺
mice (Fig. 4B).
DISCUSSION
Our data demonstrate that rimonabant’s effect on body
weight is adiponectin independent since it was similarly re-
duced in both wild-type and adiponectin KO obese mice. In
contrast, unlike wild-type animals, the decrease in body weight
in adiponectin KO mice was not associated with an improve-
Fig. 1. A: body weight of wild-type (Ad
⫹/⫹
) and adiponectin knockout (KO)
mice (Ad
⫺/⫺
) fed a regular chow (RC) or high-fat diet (HFD) and treated with
rimonabant (Ad
R⫹/⫹
and Ad
R⫺/⫺
) or vehicle (Ad
V⫹/⫹
and Ad
V⫺/⫺
).
§§§
P⬍
0.001 vs. same group under RC diet; **P⬍0.01 vs. vehicle treatment. B: Fat
mass of subcutaneous (SCAT) and visceral (VAT) adipose tissue in RC fed
mice.
§§§
P⬍0.001 vs. RC diet; ***P⬍0.001; **P⬍0.01 vs. vehicle. Values
are expressed as means ⫾SE of 9 to 12 animals in each group. C: Fat mass
of subcutaneous (SCAT) and visceral (VAT) adipose tissue in HFD fed mice.
§§§
P⬍0.001 vs. RC diet; ***P⬍0.001; **P⬍0.01 vs. vehicle. Values are
expressed as means ⫾SE of 9 to 12 animals in each group.
Table 1. Plasma substrate and hormone concentration in
regular chow and high-fat diet fed mice treated with vehicle
or rimonabant
RC HFD
Plasma triglyceride, g/l
Ad
V⫹/⫹
0.65⫾0.08 1.23⫾0.13‡
Ad
R⫹/⫹
0.56⫾0.07 0.75⫾0.11†
Ad
V⫺/⫺
0.62⫾0.09 1.34⫾0.10‡°
Ad
R⫺/⫺
0.72⫾0.12 1.17⫾0.09*
Plasma fatty acids, M
Ad
V⫹/⫹
220⫾38 330⫾52‡
Ad
R⫹/⫹
234⫾27 250⫾48*
Ad
V⫺/⫺
247⫾33 400⫾65‡
Ad
R⫺/⫺
256⫾23 290⫾28*
Plasma glucose, mM
Ad
V⫹/⫹
7.2⫾0.8 9.7⫾0.7
Ad
R⫹/⫹
8.0⫾0.7 8.4⫾0.6
Ad
V⫺/⫺
8.2⫾0.5 8.4⫾0.3
Ad
R⫺/⫺
7.8⫾0.7 8.2⫾0.3
Plasma insulin, pM
Ad
V⫹/⫹
234⫾56 364⫾50
Ad
R⫹/⫹
254⫾43 221⫾37†
Ad
V⫺/⫺
272⫾88 365⫾22
Ad
R⫺/⫺
212⫾37 195⫾31†
Plasma leptin, nM
Ad
V⫹/⫹
0.45⫾0.03 1.30⫾0.22‡
Ad
R⫹/⫹
0.52⫾0.06 0.72⫾0.20*
Ad
V⫺/⫺
0.56⫾0.05 1.39⫾0.19‡
Ad
R⫺/⫺
0.50⫾0.07 0.84⫾0.25*
Plasma adiponectin, g/ml
Ad
V⫹/⫹
7.35⫾1.5 5.90⫾0.80‡
Ad
R⫹/⫹
7.65⫾⫾1.2 8.20⫾0.67*
Ad
V⫺/⫺
undetectable undetectable
Ad
R⫺/⫺
undetectable undetectable
Ad
R⫹/⫹
and Ad
R⫺/⫺
, wild-type and adiponectin knockout (KO) mice treated
with rimonabant. Ad
V⫹/⫹
and Ad
V⫺/⫺
, wild-type and adiponectin KO mice
treated with vehicle. *P⬍0.01, †P⬍0.001 vs. mice treated with vehicle.
‡P⬍0.001 vs. RC fed mice. HFD, high-fat diet; RC, regular chow. Values are
expressed as means ⫾SD.
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ment of insulin sensitivity, suggesting that the capacity of
rimonabant to decrease insulin resistance requires adiponectin.
Even if rimonabant decreased body weight in both pheno-
types, white adipose tissue distribution evolution pattern was
different in the two groups. Indeed, HFD induced an increase
in both SCAT and VAT mass in wild-type, whereas only VAT
mass was increased in adiponectin KO mice. In addition,
rimonabant-promoted body weight loss was associated with a
decrease in both SCAT and VAT in Ad
⫹/⫹
mice, whereas
VAT only was affected in Ad
⫺/⫺
mice (probably, since there is
no change in SCAT mass compared with RC-fed mice). These
data suggest that rimonabant action on body weight is effective
only in situations with increased fat mass depots and that its
effect is independent of adiponectin. This point was recently
reinforced by the data of Watanabe and colleagues (32), who
demonstrated that rimonabant treatment significantly de-
creased WAT mass and reduced the average adipocyte size to
similar degrees in the ob/ob and adipo(-/-)ob/ob mice (32).
These findings indicate that rimonabant treatment can induce a
reduction of adipocyte size and thus a decrease in fat mass in
the absence of adiponectin or leptin or both.
Our and other data converge to the idea of a transient
decrease of food intake induced by rimonabant, whereas its
effect on body weight gain and fat is persistent. This suggests
Fig. 2. A: time course of plasma glucose after oral
glucose overload (3 g/kg body wt). Vehicle-treated
Ad
⫹/⫹
mice (open square, dotted line) fed RC; Vehi-
cle-treated Ad
⫹/⫹
(solid square), and Ad
⫺/⫺
mice
(open square) fed a HFD; rimonabant-treated Ad
⫹/⫹
(solid triangle) and Ad
⫺/⫺
mice (open triangle) fed a
HFD; ***P⬍0.001 vs. Ad
⫹/⫹
mice fed a RC. B: area
under the curve of glycemia during an oral glucose
tolerance test (OGTT) in RC- or HFD-fed mice;
***P⬍0.001 vs. Ad
⫹/⫹
mice fed a RC. C: plasma
insulin at 30 min in RC- or HFD-fed mice;
§§§
P⬍
0.001 vs. Ad
⫹/⫹
mice fed RC, ***P⬍0.001 vs.
Ad
V⫹/⫹
fed a HFD. D: insulinogenic index (⌬I/⌬G) in
RC- or HFD-fed mice;
§§§
P⬍0.001 vs. Ad
⫹/⫹
mice
fed RC, ***P⬍0.001 vs. Ad
V⫹/⫹
fed a HFD. Values
are expressed as means ⫾SE of 8 to 12 cases per
group.
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that, although reducing food intake may be the main initial
cause for reducing body weight, it is probably not the only
mechanism for the long-lasting antiobesity effect of rimon-
abant (22). Indeed, Liu and collaborators (16) showed that
rimonabant has a direct effect on energy expenditure, suggest-
ing that the antiobesity effect of SR141716 is due to activation
of thermogenesis, in addition to the initial hypophagia. Jbilo
et al. (11) showed that rimonabant activates several genes of
brown adipose tissue (BAT) involved in the regulation of
mitochondrial activity, and Doyon et al. (6) showed that
rimonabant increased BAT UCP1 mRNA levels in obese rats.
In the same way, Herling et al. (9) recently showed that the
weight-reducing effect of rimonabant was due to continuously
elevated energy expenditure based on increased fat oxidation
driven by lipolysis from fat tissue, as long as fat stores were
elevated. Finally, Tedesco et al. (28) demonstrated that CB1
receptor blockade increased mitochondrial biogenesis in white
adipocytes by inducing the expression of endothelial nitric
oxide synthase. This was linked to the prevention of high-fat
diet-induced fat accumulation, without concomitant changes in
food intake. This is of particular interest since Koh et al. (14)
demonstrated that induction of increased mitochondrial bio-
genesis in cultured adipocytes enhanced adiponectin synthesis,
suggesting a chain of cause-and-effect between rimonabant
treatment and stimulation of adiponectin expression.
Glucose homeostasis was first assessed by oral glucose load
in HFD-fed mice. A significant glucose intolerance associated
with a compensatory insulin hypersecretion was evidenced in
both Ad
⫹/⫹
and Ad
⫺/⫺
mice compared with RC-fed mice, thus
suggesting a trend for an acquired peripheral insulin resistance.
This phenomenon was confirmed during hyperinsulinemic-
euglycemic clamp and specifically by a significantly decreased
GIR in HFD-fed mice compared with their RC-fed littermates.
Such insulin resistance was related to sustained hepatic glucose
production and decreased glucose uptake. Rimonabant treat-
ment had no effect on the glycemic response to glucose
overload in both phenotypes. However, it normalized insulin
secretion in Ad
⫹/⫹
mice, thus leading to a normalization of
insulinogenic index and suggesting an improved insulin sensi-
tivity. This was also evidenced by the increase in GIR during
Fig. 4. A: glucose utilization index (GUI) in muscles of HFD-fed mice treated
with rimonabant (Ad
R⫹/⫹
and Ad
R⫺/⫺
) or vehicle (Ad
V⫹/⫹
and Ad
V⫺/⫺
).
B: GUI in adipose tissue (subcutaneous: SCAT or visceral VAT) of HFD-fed
mice treated with rimonabant (Ad
R⫹/⫹
and Ad
R⫺/⫺
) or vehicle (Ad
V⫹/⫹
and
Ad
V⫺/⫺
). ***P⬍0.001 vs. vehicle-treated mice.
Fig. 3. A: glucose infusion rate (GIR). B: glucose disposal rate (GDR).
C: hepatic glucose production (HGP). All three parameters were measured
during hyperinsulinemic-euglycemic clamp in mice fed a regular chow (RC) or
a high-fat diet (HFD) treated with rimonabant (Ad
R⫹/⫹
and Ad
R⫺/⫺
) or vehicle
(Ad
V⫹/⫹
and Ad
V⫺/⫺
).
§§§
P⬍0.001 vs. Ad
⫹/⫹
mice fed RC, **P⬍0.01 vs.
Ad
⫹/⫹
mice fed a HFD.
R933ADIPONECTIN INVOLVEMENT IN RIMONABANT’S METABOLIC EFFECTS
AJP-Regul Integr Comp Physiol •VOL 296 •APRIL 2009 •www.ajpregu.org
hyperinsulinemic-euglycemic clamp. Such an improvement of
insulin sensitivity by rimonabant in Ad
⫹/⫹
mice was associ-
ated with a decreased hepatic glucose production, as well as an
increase in glucose uptake mainly due to insulin-induced glu-
cose utilization in SCAT and VAT. Contrary to Liu and
collaborators (16) who showed that 7 days of rimonabant
treatment (10 mg/kg ip) induced an increase in glucose uptake
in isolated soleus muscle from ob/ob female mice, rimonabant
did not increase glucose uptake in skeletal muscles of our
Ad
⫹/⫹
mice. However, it is difficult to compare since it was an
ex vivo study performed in female mice.
Finally, increased insulin-dependent glucose uptake evi-
denced by our experiments strongly suggests increased insulin
signaling pathway, as also reported by Watanabe et al. (32),
who showed that insulin-stimulated Akt phosphorylation was
significantly increased in rimonabant-treated ob/ob mice com-
pared with untreated ob/ob mice.
In contrast to Ad
⫹/⫹
mice, rimonabant treatment in Ad
⫺/⫺
mice did not improve insulin sensitivity, since insulinemia
remained dramatically high during glucose tolerance test com-
pared with wild-type mice. GIR was also lower in Ad
⫺/⫺
mice
than in Ad
⫹/⫹
mice, demonstrating a persistent insulin resis-
tance in spite of rimonabant treatment. However, it must be
pointed out that there was a tendency to a decrease in hepatic
glucose production in Ad
⫺/⫺
mice, which could be related to a
reduction of both plasma TG and FFA concentrations. Indeed,
in spite of adiponectin deficiency, the body weight loss induced
by rimonabant was, at least in part, responsible for the decrease
in plasma TG and FFA, leading to improvement of hepatic
insulin sensitivity. Finally, there was no potentiation by rimon-
abant of insulin-mediated glucose disposal rate. This was
confirmed by the absence of improvement in glucose uptake in
either adipose tissue (both in SCAT and VAT) or in skeletal
muscles, thus in agreement with the maintained insulin-resis-
tant status observed in Ad
⫺/⫺
mice. In the same way, Watanabe
et al. (32) demonstrated that insulin-stimulated Akt phosphorylation
was impaired in the rimonabant-treated Adipo
⫺/⫺
ob/ob mice.
Thus, our data demonstrate that rimonabant-induced insulin-
sensitizing effect is mediated, at least in part, by the increase of
adiponectin expression, since improvement of insulin sensitiv-
ity has been only observed in Ad
⫹/⫹
-treated mice. In addition,
adiponectin has been widely reported to reduce hyperinsulin-
emia and insulin resistance (26). Interestingly, Watanabe et al.
(32) recently suggested that rimonabant treatment preferen-
tially upregulated the expression of high-molecular-weight
adiponectin, which is considered as the biologically active
isoform of adiponectin, according to in vitro and in vivo
data (12).
The increase in adiponectin concentration observed in Ad
⫹/⫹
mice treated with rimonabant could have also blocked the
inflammatory effect induced by HFD diet. Indeed, it has been
shown that adiponectin could counteract tumor necrosis fac-
tor-␣(TNF-␣)-mediated FFA-induced insulin resistance in
3T3-L1 adipocytes (20). It is also established that CB1 receptor
is coupled to the generation of the lipid second messenger
ceramide (31) involved in the induction of insulin resistance
(27). In primary cultures of rat astrocytes, D9-tetrahydrocan-
nabinol (THC) produced a rapid stimulation of sphingomyelin
hydrolysis that was concomitant to an elevation of intracellular
ceramide levels. These effects of THC were prevented by
rimonabant (25) and interestingly, adiponectin has been evi-
denced to inhibit de novo ceramide synthesis through the
activation of AMP kinase.
Perspectives and Significance
The present study directly demonstrates for the first time the
key role of adiponectin as a mediator of rimonabant to improve
insulin sensitivity, thus highlighting the importance of inter-
play between the cannabinoid pathway and adiponectin to
regulate glucose homeostasis. This study also emphasizes the
importance of a peripheral effect of rimonabant to normalize
glucose homeostasis, mainly through the remodeling of adi-
pose tissue mass.
GRANTS
This work was supported by the French Ministry of Industry and Ministry
of Research through the collaborative project GenObeCB1 “Integrated
genomic for obesity therapy by studying cannabinoid receptor (CB1R) and
effects of its modulators”.
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