A novel combined glucocorticoid-mineralocorticoid receptor selective modulator markedly prevents weight gain and fat mass expansion in mice fed a high-fat diet

Abstract

Background:
We have previously shown that antagonism of the mineralocorticoid receptor (MR) results in a potent antiadipogenic activity, in vitro and in vivo. Excessive glucocorticoid exposure is associated with obesity and related disorders in humans and mice.

Methods:
In this study responses to a novel combined glucocorticoid receptor (GR)/MR antagonist were investigated in a model of diet-induced obesity. Female 10-week-old C57BL/6J mice were fed with normal chow or a high fat diet (HFD) for 9 weeks. Mice fed a HFD were concomitantly treated for 9 weeks with the GR antagonist mifepristone (80 mg/kg/day) or the novel combined GR/MR antagonist CORT118335 (80 mg/kg/day). Male, juvenile 6-week-old C57BL/6J mice fed HFD were treated with CORT118335 for 4 weeks.

Results:
Mice fed a HFD showed a significant increase in total body weight and white fat mass, with impaired glucose tolerance and increased fat infiltration in livers. Interestingly, only CORT118335 completely prevented the HFD-induced weight gain and white fat deposition, whereas mifepristone showed no effect on body weight and modestly increased subcutaneous fat mass. Importantly, food intake was not affected by either treatment, and CORT118335 dramatically increased PGC-1α protein expression in adipose tissue, without any effect on UCP1. Both CORT118335 and mifepristone produced metabolic benefit, improving glucose tolerance, increasing adiponectin plasma levels, decreasing leptin and reducing mean adipocyte size. When tested in vitro, CORT118335 markedly reduced 3T3-L1 differentiation and reversed MR-mediated pro-adipogenic effects of aldosterone; differently, GR-mediated effects of dexamethasone were not antagonised by CORT118335, suggesting that it mostly acts as an antagonist of MR in cultured preadipocytes.

Conclusions:
Combined GR/MR pharmacological antagonism markedly reduced HFD-driven weight gain and fat mass expansion in mice through the increase in adipose PGC-1α, suggesting that both receptors represent strategic therapeutic targets to fight obesity. The effects of CORT118335 in adipocytes seem predominantly mediated by MR antagonism.International Journal of Obesity accepted article preview online, 02 February 2016. doi:10.1038/ijo.2016.13.

Full text

ORIGINAL ARTICLE
A novel combined glucocorticoid-mineralocorticoid
receptor selective modulator markedly prevents weight
gain and fat mass expansion in mice fed a high-fat diet
C Mammi
1
, V Marzolla
1
, A Armani
1
, A Feraco
1
, A Antelmi
1
, E Maslak
2
, S Chlopicki
2,3
, F Cinti
1,4
, H Hunt
5
, A Fabbri
6
and M Caprio
1,7
BACKGROUND: We have previously shown that antagonism of the mineralocorticoid receptor (MR) results in a potent
antiadipogenic activity, in vitro and in vivo. Excessive glucocorticoid exposure is associated with obesity and related disorders in
humans and mice.
METHODS: In this study, responses to a novel combined glucocorticoid receptor (GR)/MR antagonist were investigated in a model
of diet-induced obesity. Female 10-week-old C57BL/6J mice were fed with normal chow or a high-fat diet (HFD) for 9 weeks.
Mice fed a HFD were concomitantly treated for 9 weeks with the GR antagonist mifepristone (80 mg kg
−1
per day) or the novel
combined GR/MR antagonist CORT118335 (80 mg kg
−1
per day). Male, juvenile 6-week-old C57BL/6J mice fed HFD were treated
with CORT118335 for 4 weeks.
RESULTS: Mice fed a HFD showed a significant increase in total body weight and white fat mass, with impaired glucose tolerance
and increased fat infiltration in livers. Interestingly, only CORT118335 completely prevented the HFD-induced weight gain and
white fat deposition, whereas mifepristone showed no effect on body weight and modestly increased subcutaneous fat mass.
Importantly, food intake was not affected by either treatment, and CORT118335 dramatically increased PGC-1αprotein expression
in adipose tissue, without any effect on UCP1. Both CORT118335 and mifepristone produced metabolic benefit, improving glucose
tolerance, increasing adiponectin plasma levels, decreasing leptin and reducing mean adipocyte size. When tested in vitro,
CORT118335 markedly reduced 3T3-L1 differentiation and reversed MR-mediated pro-adipogenic effects of aldosterone; differently,
GR-mediated effects of dexamethasone were not antagonized by CORT118335, suggesting that it mostly acts as an antagonist
of MR in cultured preadipocytes.
CONCLUSIONS: Combined GR/MR pharmacological antagonism markedly reduced HFD-driven weight gain and fat mass expansion
in mice through the increase in adipose PGC-1α, suggesting that both receptors represent strategic therapeutic targets to fight
obesity. The effects of CORT118335 in adipocytes seem predominantly mediated by MR antagonism.
International Journal of Obesity advance online publication, 22 March 2016; doi:10.1038/ijo.2016.13
INTRODUCTION
The incidence of obesity and type 2 diabetes has markedly
increased over recent decades and novel therapeutic strategies
seem to be necessary to fight such an epidemic. Unbalanced
corticosteroid metabolism in adipose tissue has a pivotal role in the
development of obesity and related cardiovascular and metabolic
disorders.
1,2
In fact, patients with glucocorticoid (GC) excess, such as
in Cushing’s Syndrome, develop insulin resistance and central
obesity, a phenotype characteristic of the metabolic syndrome.
3,4
Interestingly, treatment of obese Zucker rats, carrying a dysregula-
tion of 11 beta-hydroxysteroid dehydrogenase type 1, with the GC
receptor (GR) antagonist mifepristone or adrenalectomy reverses
the obese phenotype in these animals.
5,6
Notably, GCs regulate almost every aspect of adipose tissue
biology, from the early regulation of adipocyte differentiation
7
to
the fine control of lipolysis, glucose uptake and gluconeogenesis.
8
Hence, integrity of the HPA axis is of fundamental importance for
healthy metabolic homeostasis.
9
Interestingly, the effects of GCs in
adipose tissue are not exclusively mediated by the GR, but also by
the mineralocorticoid receptor (MR).
10
Recently, it has been found
that the MR is expressed in white adipocytes as well as in brown
adipocytes,
11,12
and has a key role in corticosteroid-induced
adipogenesis.
13,14
The MR binds not only to aldosterone but also
to GCs with equivalent affinity.
15
Moreover, the affinity of GCs
(cortisol in humans, corticosterone in rodents) for MR is 10-fold
higher than that for GR.
16
Importantly, GCs represent the major
MR ligand in adipocytes, and MR knockdown in primary murine
adipocytes as well as in 3T3-L1 cells blocks corticosteroid-induced
adipose differentiation.
13,17
In contrast to these observations,
recent data provide evidence that the GR has a more important
role than the MR in promoting the early steps of adipogenesis in
human preadipocytes.
18
However, it is clear that both receptors
have an important role in adipogenesis, and their reciprocal
interplay is crucial for adipocyte differentiation.
17
Beneficial effects of MR antagonists have been demonstrated
in several animal models of genetic and diet-induced obesity. Guo
1
Laboratory of Cardiovascular Endocrinology, IRCCS San Raffaele Pisana, Rome, Italy;
2
Jagiellonian Centre for Experimental Therapeutics, Jagiellonian University, Krakow, Poland;
3
Chair of Pharmacology, Jagiellonian University, Medical College, Krakow, Poland;
4
Department of Experimental and Clinical Medicine, Center for Obesity, Università Politecnica
delle Marche, Ancona, Italy;
5
Corcept Therapeutics, Menlo Park, CA, USA;
6
Department of Systems Medicine, Endocrinology Unit, S. Eugenio & CTO A. Alesini Hospitals, University
Tor Vergata, Rome, Italy and
7
Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy. Correspondence: Professor M
Caprio, Laboratory of Cardiovascular Endocrinology, IRCCS San Raffaele Pisana, Via di Val Cannuta 247, Rome 00166, Italy.
E-mail: massimiliano.caprio@sanraffaele.it
Received 5 June 2015; revised 30 November 2015; accepted 27 December 2015; accepted article preview online 2 February 2016
International Journal of Obesity (2016), 1–9
© 2016 Macmillan Publishers Limited All rights reserved 0307-0565/16
www.nature.com/ijo

et al.
19
reported that systemic MR antagonism with eplerenone
reversed the adverse metabolic consequences of genetic obesity
in db/db mice, improving glucose tolerance, insulin resistance and
adipokine expression in adipose tissue. Similar effects were obtained
in a model of diet-induced obesity, in which MR blockade induced
a marked reduction of hypertrophic adipocytes and macrophage
infiltration with a concomitant decrease of pro-inflammatory
adipokine levels and excess of reactive oxygen species in adipose
tissue.
20
Importantly, our group recently showed that pharmaco-
logical MR antagonism with spironolactone was able to counter
metabolic dysfunctions in a model of HFD-induced obesity in
female mice, and that beneficial effects on body fat composition
and glucose tolerance were associated with ‘browning’of adipose
tissue.
21
To date, the exact role, interaction and relative importance
of GR vs MR in murine adipogenesis as well as in adipose tissue
expansion during a high-fat challenge is still controversial.
For these reasons, we explored adipose tissue responses to the
novel combined GR/MR antagonist CORT118335 in a model of
diet-induced obesity in mice. CORT118335 displays eightfold
selectivity for GR over MR, as assessed in reporter gene assays.
22
MATERIALS AND METHODS
Animal model
Animal procedures were approved by the Italian National Institute of
Health Care and Use Committees. Female 10-week-old (study 1) and male
6-week-old (study 2) C57BL/6J mice were purchased from Charles River
Laboratories, Calco (LC), Italy. Mice were divided into four groups (n= 10)
as follows: (i) ND: mice fed a normal diet (10% kcal as fat; D12450B;
Research Diets, New Brunswick, NJ, USA) plus vehicle by oral gavage;
(ii) HFD: mice fed a high-fat diet (HFD; 45% kcal as fat; D12451; Research
Diets) plus vehicle by oral gavage; (iii) mifepristone: mice fed a HFD plus
mifepristone (80 mg kg
−1
per day) by oral gavage; (iv) CORT118335: mice
fed a HFD, plus CORT 118335 (80 mg kg
−1
per day) by oral gavage.
Glucose tolerance assessment
After 8 weeks of treatment, animals from study 1 (n= 10) were fasted for
6 h and a tail vein blood sample was collected to measure basal blood
glucose levels.
The animals were then injected with a glucose solution into the peritoneum
(0.09 g ml
−1
glucose, 112 μl solution per 10 g body weight) and blood
glucose concentrations measured using a commercial glucometer (Contour
XT, BAYER Healthcare, Leverkusen, Germany) over the next 2 h at 20-min
intervals for the first hour and at 30-min intervals in the second hour.
Body weight, food intake, caloric efficiency and adipokine
secretion assessment
Total body weight was measured weekly and when the mice were killed
(day 63 for study 1 or day 28 for study 2). We assessed the wet weight
of dissected inguinal subcutaneous and visceral abdominopelvic fat
depots, as well as tibia and femur length. Food intake was measured
each week throughout the study in mice (n= 6) housed in individual cages,
by providing a fixed amount of food and weighing uneaten food for five
consecutive days. Quantification of food intake and change in body weight
over the 63-day treatment period were used to evaluate caloric efficiency,
which is expressed as body weight gain per unit of energy intake.
23
At the
time the mice were killed, peripheral blood samples were collected via
retro-orbital bleeding. Leptin and adiponectin serum levels were measured
by a commercially available ELISA kit (RD System, Minneapolis, MN, USA),
sodium and potassium were measured by Hitachi Cobas C 311 analyzer
(Roche Diagnostic Ltd, Rotkreuz, Switzerland).
Gene expression analysis
Total RNA from fat depots was isolated using RNeasy lipid tissue mini kit
(Qiagen, Milan, Italy) following the manufacturer’s instructions. Total
RNA from skeletal muscles (tibialis anterior) and adipocyte cultures was
isolated in Trizol reagent (Life Technologies, Milan, Italy) according to the
manufacturer’s recommendations. Preparation of cDNA and real-time
qPCR were performed as previously described.
21
See Supplementary Table
1 for primer sequences.
Micro-CT studies
The femur samples from juvenile male mice (n= 4, study 2) were analyzed,
after careful dissection, with a microcomputed cone-beam X-ray system
(Skyscan 1072 μ-CT, Kartuizersweg 3B, 2550 Kontich, Belgium) without
addition of contrasting agent. Image reconstruction and analysis were
conducted using the software package provided by Skyscan, as already
described.
24
For each sample, an internal region of interest was selected
and histomorphometric parameters for the corresponding volume of
interest were calculated from the most proximal point of the trochanter
and were extended proximally for 4 mm; then, the volume of the cortical
bone of femur neck was analyzed.
Cell culture studies and Oil Red O staining
Murine 3T3-L1 preadipocytes were grown until confluence at 37 °C
in Dulbecco's modified Eagle's medium (Invitrogen, Milan, Italy) containing
4.5 g l
−1
D-glucose, 10% fetal calf serum (Invitrogen), 100 U ml
−1
penicillin
and 100 μgml
−1
streptomycin. To achieve adipogenic differentiation
in the absence of serum steroids, confluent cells were transferred in
Dulbecco's modified Eagle's medium containing 10% dextran-coated
charcoal-stripped fetal calf serum, and treated as previously reported.
13
Aldosterone (10
−8
M) or dexamethasone (10
−7
M) were added from day 2
of differentiation, with or without CORT118335 (10
−6
M).
Primary cultures of murine preadipocytes derived from the vascular
stromal fraction of inguinal fat depots of 10-week-old female C57/BL6J
were prepared as previously described.
25
Cells were allowed to differ-
entiate until day 6. Differentiating cells were treated from confluence until
day 6 with spironolactone (10
−5
M), mifepristone (10
−5
M) or CORT118335
(10
−6
M). Unless otherwise indicated, all chemicals were obtained from
Sigma-Aldrich (Milan, Italy). For determination of lipid content, cells were
processed as previously published.
26
To quantify the lipid content, the
stained cells were permeabilized with 10% sodium dodecyl sulfate and the
eluate was measured using a spectrophotometer at 520 nm. For every cell
culture study, two independent experiments were performed in triplicate.
Western blot analysis
Specimens of inguinal subcutaneous fat (n= 6) were lysed in HNTG lysis
buffer containing 1% Triton X-100, 50 mMHepes, 10% glycerol, 150 mM
NaCl, 1 mMNaVO4 and 75 U of aprotinin and allowed to stand for 30 min.
After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
blotting,
21
membranes were probed with rabbit polyclonal anti-uncoupling
protein 1 (UCP1, Abcam 23841, Cambridge, MA, USA), rabbit polyclonal
anti-peroxisome proliferator-activated receptor gamma coactivator 1-alpha
(PGC-1α, Santa Cruz Biotechnology sc-13067, Heidelberg, Germany) or goat
polyclonal anti-actin (Santa Cruz Biotechnology sc-1615, Heidelberg,
Germany). Bound antibodies were visualized with horseradish peroxidase-
conjugated anti-rabbit IgG (Sigma-Aldrich) and immunoreactivity assessed
by chemiluminescence reaction, using the ECL Western detection system
(General Electric Healthcare, Milan, Italy). Densitometric scanning analysis
was performed by Mac OS X (Apple Computer International, Milan, Italy)
using NIH Image 1.62 software.
Histological analysis of fat depots and livers
Dissected adipose tissue depots (n= 6) were fixed overnight in 10%
formaldehyde at 4 °C and rinsed with 0.1M phosphate buffer before
embedding in paraffin. Each paraffin-embedded depot was cut (3-μm
thick) and the cutting plane corresponding to the largest surface was used
for histological examination, morphometry (hematoxylin-eosin). Ten fields
at × 40 final magnification for each slide were analyzed to determine mean
adipocyte area using a digital image system (Lucia imaging, version 4.82,
Praha, Czech Republic).
Liver samples (n=6)after fixation in formalin solution were cryoprotected
by immersion in 30% sucrose solution overnight and afterwards embedded
in Tissue-Tek OCT (optimum cutting temperature) gel, frozen at −80 °C and
cut into 7-μm slides. Prepared slides were stained using the Oil Red O
method for fat deposition. The Oil Red O sections were photographed under
× 100 magnification. At least six images of each section were randomly
obtained. The images were subsequently analyzed by using Columbus
Image Data Storage and Analysis System (Perkin Elmer, Boston, MA, USA),
with an algorithm adapted for Oil Red O-stained sections.
GR/MR antagonism in adipose tissue
C Mammi et al
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International Journal of Obesity (2016), 1 –9 © 2016 Macmillan Publishers Limited

Statistical analysis
Ten mice per group were required to allow the assessment for primary
outcome (total body weight) of a standardized difference among any
pair of means of at least 0.65 s.d. at the significance level alpha = 0.05
(two-tailed test), with power (1–beta) = 0.80.
Data are reported as the mean +/ −s.e.m. Data points greater or less than
two standard deviations from the mean were considered statistical outliers
and were excluded from all analyses. Statistical comparisons were made
by one- or two-way analysis of variance, followed by Bonferroni multiple
comparison post hoc analysis, using Prism 6.0 (GraphPad, San Diego, CA,
USA). Po0.05 was considered significant.
RESULTS
Treatment with CORT118335 protects from HFD-induced weight
gain and fat storage, whereas mifepristone shows no effect on
body weight and fat mass
In study 1, female mice were fed a diet with 45% calories from fat,
which is similar in macronutrient composition to a typical western
diet,
27
and treated daily with vehicle, CORT118335 or mifepristone
for 9 weeks. Mice fed a HFD showed an increase in total body
weight of approximately 4 g, as compared with the ND group
(Figure 1a), with a parallel increase in the weight of inguinal and
abdominopelvic fat pads. Treatment with CORT118335 dramati-
cally attenuated the HFD-induced body weight gain (Figure 1a,
Po0.001 vs HFD), preventing the increase in fat pad weight
(Figure 1b); this effect was already significant after 2 weeks
of treatment with CORT118335. In contrast, mifepristone had no
effect on total body weight (Figure 1a) and abdominopelvic fat
pad weight (Figure 1b, lower panel), and modestly increased
subcutaneous fat (Figure 1b, upper panel). Average kilocalorie
intake was not modified by mifepristone or CORT118335 at any
time point of the study (Figure 1c); interestingly, caloric efficiency
was dramatically reduced only in the CORT118335 group, when
compared with the HFD group (Figure 1d), suggesting a different
metabolic profile in mice treated with the combined GR/MR
antagonist. To confirm these observations in the male gender, and
to exclude any potential adverse effects of CORT118335 on
growth, skeletal muscle and bone quality, we performed a shorter
study (4 weeks, study 2) on juvenile male mice. CORT118335
treatment did not affect body growth, tibia and femur length
(data not shown), but markedly blunted HFD-induced weight
gain; this effect was significant after only 1 week of treatment
(Figure 2a). The marked difference in total body weight observed
at the end of the study was due to a dramatic reduction in visceral
and subcutaneous fat depots in treated mice, compared with
controls (Figure 2b). Importantly, transcript expression of Myosin
Heavy Chain and MyoD, two markers of skeletal muscle
differentiation, and atrogin-1 (a marker of muscular atrophy) in
Figure 1. CORT118335 counters body weight gain in female mice, prevents the increase in fat pad weight from inguinal and abdominopelvic
depots, and reduces caloric efficiency in mice fed a HFD. (a) Body weight gain of female 10-week-old C57BL/6J mice during a 63-day period of ND or
HFD, treated or not with mifepristone (MIFE) or CORT118335 (Cort335). (b) Wet weight percentage increase vs ND of subcutaneous (inguinal, upper
panel) and visceral (abdominopelvic, lower panel) depots from mifepristone and CORT118335-treated mice, as compared with the HFD group
(n=10). (c) Average kilocaloric intake was measured in mice housed in individual cages (n=6pergroup).(d)Caloricefficiency calculated over the
entire study period (n=6). Values are expressed as means ±s.e.m.
#
Po0.05,
##
Po0.01 vs ND group; *Po0.05, **Po0.01, ***Po0.001 vs HFD group.
GR/MR antagonism in adipose tissue
C Mammi et al
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© 2016 Macmillan Publishers Limited International Journal of Obesity (2016), 1 –9

tibialis anterior were not modified by treatment with CORT118335.
Moreover, trabecular thickness and cortical bone volume were not
affected by CORT118335, as shown by micro-CT analysis of femurs
(Figure 2d). Interestingly, porosity of trabecular bone was even
slightly decreased in CORT118335-treated animals, thus excluding
a potential adverse effect of the compound on bone develop-
ment. CORT118335 did not alter Na and K plasma levels (data not
shown) and treated mice never displayed any sign or symptom
suggestive of adrenal insufficiency, throughout the study.
Treatment with CORT118335 improves glucose tolerance and
adipokine secretion profile in HFD-fed mice
After 8 weeks of treatment (study 1), an intraperitoneal glucose
tolerance test was performed. Before injection, CORT118335- and
mifepristone-treated groups showed lower plasma glucose levels
compared with untreated HFD mice. Interestingly, 20 min after the
intraperitoneal injection of glucose, CORT118335-treated mice
showed a marked reduction in plasma glucose levels as compared
with the HFD group (Figure 3a), which led to a significant decrease
in the area under the curve of plasma glucose (Figure 3b).
In accordance with previous studies,
28
mifepristone-treated mice
also showed a significant improvement in glucose tolerance,
which was slightly delayed compared with CORT118335 (Figure 3a),
but demonstrated a similar reduction in plasma glucose area under
the curve (Figure 3b). Accordingly, both treatments significantly
increased plasma adiponectin levels (Po0.001, Figure 3c), and
concomitantly decreased leptin levels (Po0.001, Figure 3d), as
compared with the HFD group.
Treatment with CORT118335 prevents liver steatosis and
adipocyte hypertrophy in HFD-fed mice, but does not increase
brown adipocyte content in WAT
To gain more insights in the favorable metabolic effects of
CORT118335, we studied its effects on HFD-induced liver steatosis.
As expected, histological analysis of Oil Red O stained liver lipid
droplets showed moderate, microvesicular steatosis within the
liver of HFD-treated mice. Treatment with CORT118335 signifi-
cantly reduced liver steatosis (Po0.05; Figures 4a and b), similarly
to results observed for mifepristone, as previously reported.
28,29
As expected, the average adipocyte cross-sectional area of the
inguinal and abdominopelvic depots was significantly larger in the
HFD group compared with the ND group (data not shown). Cell
Figure 2. CORT118335 counters body weight gain in male juvenile mice, prevents the increase in abdominopelvic fat depots, without any
adverse effect on growth, skeletal muscle and bone quality. (a) Body weight gain of male 6-week-old C57BL/6J mice during a 28-day period
of ND or HFD, treated or not with CORT118335 (Cort335). (b) Exposed abdominopelvic and inguinal fat of two representative mice at the end
of the study. (c) qRT-PCR analysis of genes involved in skeletal muscle differentiation (MyHC and MyoD) and atrophy pathway (Atrogin-1)
performed in tibialis anterior of male 6-week-old mice fed a HFD, treated or not with CORT118335. (d) micro-CT analysis of femurs dissected
after killing the mice in study 2. The panel represents the following structural parameters: Tissue Volume (total volume of the volume-of-
interest), Trabecular thickness, Porosity and Cortical Bone Volume.
GR/MR antagonism in adipose tissue
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International Journal of Obesity (2016), 1 –9 © 2016 Macmillan Publishers Limited

size distribution analysis, performed in both depots, showed
a significant reduction of cross-sectional area of adipocytes of
mice treated with either CORT118335 or mifepristone, as
compared with the HFD group (Figure 4c, upper panels).
In mice, browning of subcutaneous WAT has been observed to
improve metabolic profile, protecting from diet-induced obesity.
30
To determine whether alterations in brown fat content con-
tributed to the protective effect of both treatments on glucose
tolerance and body composition, we investigated the occurrence
of browning in hematoxylin-eosin-stained sections of inguinal
subcutaneous fat depots; we did not, however, observe any increase
in the number of brown adipocytes in either of the treated groups
(Figure4c,lowerpanel).Thesedatawereconfirmed by western blot
analysis for UCP1 in subcutaneous fat, which did not show any
increase in UCP1 protein expression, by either CORT118335 or
mifepristone treatment (Figures 5a and c). These data indicate that
CORT118335 does not induce browning of white adipose tissue, in
contrast to observations with selective MR antagonists, as recently
published.
21
Treatment with CORT118335 markedly increases adipose PGC-1α
expression
Importantly, treatment with CORT118335 induced a dramatic
increase in PGC-1αprotein expression in subcutaneous fat, which
was also observed, although at a lower extent, in mice treated
with mifepristone (Figures 5a and b). Accordingly, both com-
pounds were able to upregulate transcript expression of PGC-1αin
primary cultured murine preadipocytes (Figure 5g).
We recently demonstrated that selective antagonism of adipocyte
MR determines browning of white adipose tissue, in vivo and
in vitro.
21
To explore whether MR antagonistic properties displayed
by CORT118335 were able to affect browning, murine preadipocytes
isolated from the stroma-vascular fraction of the inguinal sub-
cutaneous fat depot were differentiated in the absence or in the
presence of spironolactone (10
−5
M), mifepristone (10
−5
M) or
CORT118335 (10
−6
M) for 6 days. As previously shown,
21
specificMR
antagonism by spironolactone significantly increased UCP1,
PRDM16, CIDEA and PGC-1-αtranscript levels (Figures 5d–g).
Interestingly, CORT118335 only modestly increased UCP1 gene
expression (Po0.05), whereas it did not modify the expression of
PRDM16 and CIDEA mRNA. None of the brown adipocyte-specific
transcripts were affected by GR antagonism with mifepristone.
Taken together, these data suggest that the modest MR antagonism
displayed by CORT118335 was not able to fully trigger the brown
adipogenic gene program. This is confirmed by the lack of increase
in UCP1 protein, observed in vivo, in the adipose tissue of mice
treated with CORT118335 (Figure 5a). We can therefore hypothesize
that the metabolically beneficial effects of CORT118335 can be
explained by the increase in adipose tissue protein expression of
PGC-1α, a master regulator of mitochondrial biogenesis, rather than
browning of white adipose tissue.
CORT118335 inhibits lipids accumulation of 3T3-L1 preadipocytes
in vitro, mainly through MR antagonism
To further explore a potential direct role of adipocyte MR and GR in
the metabolic effects of the compound in vivo, we investigated the
effects of CORT118335 (10
−6
M) in 3T3-L1 preadipocyte differentia-
tion. Treatment began when cells reached confluence and was
maintained for the entire duration of adipocyte differentiation
(8 days).
26
CORT118335 markedly reduced lipid accumulation as
determined by Oil Red O staining (Po0.001, Figure 6a). The
inhibitory effects of CORT118335 were observed also at concentra-
tions of 10
−7
M, were maximal at 10
−6
M and did not increase at
10
−5
M (data not shown). As expected, mifepristone also showed a
marked inhibition of adipocyte differentiation. We next dissected
the mixed GR/MR antagonist activity of CORT118335 in this model.
3T3-L1 cells were maintained in a steroid-depleted medium from
confluence, omitting dexamethasone from the adipogenic cocktail
during induction of differentiation (day 0–2), and treated from day 2
with aldosterone (10
−8
M) or dexamethasone (10
−7
M), in the
presence or in the absence of CORT118335 (10
−6
M). Under these
culture conditions, aldosterone and dexamethasone are known to
Figure 3. CORT118335 treatment improves glucose tolerance, increases plasma adiponectin levels and markedly reduces plasma leptin, as
compared with the HFD group. (a) Plasma glucose levels during an intraperitoneal glucose tolerance test following food withdrawal for 6 h
(n=10) in female 10-week-old C57BL/6J mice fed a HFD in the presence or absence of mifepristone or CORT118335 for 63 days. (b) Area under
the curve (AUC) glucose was calculated using the trapezoidal rule (n=10). (c) Measurement of plasma adiponectin and (d) leptin levels in all
groups of mice before killing after fasting for 6 h (n=10). Values are expressed as means ±s.e.m. ***Po0.001 vs HFD group.
GR/MR antagonism in adipose tissue
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© 2016 Macmillan Publishers Limited International Journal of Obesity (2016), 1 –9

display opposite effects on adipocyte differentiation.
13
We then
measured Oil Red O accumulation at day 8, and mRNA levels for
leptin and resistin, which are selectively activated by aldosterone
13
and angiotensinogen, which is specifically induced by dexametha-
sone through GR. As expected, aldosterone significantly increased
adipose differentiation (Figure 6b), and its effects were selectively
countered by CORT118335, indicating functional MR antagonism. As
previously shown,
13
dexamethasone displayed a GR-mediated
inhibitory effect on adipose differentiation, which was not reversed
by CORT118335 (Figure 6b), whereas coincubation with mifepris-
tone was able to counter the inhibitory action of dexamethasone,
thereby favoring adipose differentiation (data not shown). More-
over, we found that treatment with aldosterone increased gene
expression of leptin and resistin and co-treatment with CORT118335
significantly inhibited this response (Po0.001 and Po0.05,
respectively, Figures 6c and d). Treatment with dexamethasone
decreased leptin and resistin mRNA levels, and co-treatment with
CORT118335 did not prevent this effect (Figures 6c and d). To
further investigate whether CORT118335 exhibited antagonistic
effects on adipocyte GR, we analyzed transcript levels of
angiotensinogen, a well-known GC target gene.
13
Importantly, co-
treatment with CORT118335 did not prevent the upregulation of
angiotensinogen mRNA by dexamethasone (Figure 6e), suggesting
that, in vitro, CORT118335 affects adipocyte function mainly through
MR antagonism.
DISCUSSION
In the present study, we investigated for the first time the effects
of a novel combined MR/GR antagonist in a validated model of
HFD-induced obesity in mice, and showed a powerful inhibition of
fat mass expansion that was associated with beneficial metabolic
effects on glucose tolerance, serum adipokines and liver steatosis.
Importantly, the compound did not cause any sign or symptom
suggestive of adrenal insufficiency, nor altered growth, skeletal
muscle and bone quality, when administered to juvenile growing
male mice. GR antagonism with mifepristone had no effect on
body weight and fat deposition, even though mifepristone still
exhibited favorable metabolic effects. Our data confirm previous
results, showing that mifepristone improved insulin sensitivity
but did not affect body weight and fat mass in a model of HFD-
induced obesity in mice.
28
Importantly, the concomitant antagon-
ism of the MR, provided by the novel compound, demonstrated a
dramatic inhibition of adipose mass expansion, driven by a HFD.
We have recently shown that pharmacological antagonism
of the MR, obtained with spironolactone and drospirenone,
countered weight gain, as well as the increase in WAT mass
expansion and impaired glucose tolerance induced by a HFD in
mice.
21
Therefore, we hypothesize that the pharmacological MR
antagonistic properties of CORT118335 have an important role in
the marked inhibition of HFD-driven weight gain and white fat
mass expansion observed in mice treated with this compound.
Interestingly, the combined antagonism of GR and MR completely
Figure 4. Treatment with CORT118335 and mifepristone prevents liver steatosis and adipocyte hypertrophy in HFD-fed mice.(a)
Representative histological sections of livers obtained from six animals in each treatment condition, stained by Oil Red O; scale
bars =50 μm. (b) Percentage of Oil Red O-stained area, as compared with the HFD group (n=6). (c) Representative sections of inguinal fat
depots of all groups (n=6), stained with hematoxylin-eosin. Scale bars =40 μm(upperpanels);scalebar=200 μm(lowerpanels).(d)Mean
cross-sectional area of adipocytes in inguinal fat depots from all groups. Values are expressed as means ±s.e.m. *Po0.05, ***Po0.001 vs
HFD group.
GR/MR antagonism in adipose tissue
C Mammi et al
6
International Journal of Obesity (2016), 1 –9 © 2016 Macmillan Publishers Limited

prevented HFD-induced increase in body weight and fat mass, at a
greater extent to those observed with specific MR antagonism by
spironolactone and drospirenone.
21
In fact, CORT118335-treated
mice showed a marked difference in total body weight, as
compared with vehicle-treated, after only 2 weeks of treatment,
whereas at least 9 weeks of treatment with MR antagonists were
required to obtain a significant effect on body weight gain.
21
Moreover, CORT118335-treated mice never showed any signifi-
cant increase in total body weight induced by HFD, throughout
the study. The marked difference in total body weight was mostly
due to fat mass, as showed by the dramatic effect of the
compound on both visceral and subcutaneous fat pads weight.
Importantly, such effects were not due to reduced food intake,
which was unchanged among treatment groups throughout the
study. Caloric efficiency, estimated as body weight gain per unit of
energy intake, which represents a rough estimation of energy
expenditure,
23
was dramatically lower in mice treated with
CORT118335, as compared with the HFD and mifepristone groups.
This suggests a marked increase in energy expenditure in mice
treated with the combined GR/MR antagonist. The lack of
metabolic studies including energy expenditure through calori-
metry is a limitation of the present study. However, our results
suggest that combined GR/MR antagonism improves the meta-
bolic activity of mice and has a profound impact on adiposity,
glucose tolerance and resistance to HFD.
White fat mass reduction in CORT118335-treated mice was
associated with a significant improvement in glucose tolerance after
an intraperitoneal glucose tolerance test, higher levels of plasma
adiponectin and reduced concentrations of leptin. Several mechan-
isms may contribute to the improvement in glucose tolerance
induced by CORT118335. Our study showed that CORT118335 was
also able to counteract the dysfunctional enlargement of adipocyte
size and the subsequent altered adipokine expression induced by
HFD. Therefore, CORT118335 may indirectly affect blood glucose
disposal through the increase in adiponectin and the decrease in
leptin secretion by adipose tissue.
19,20,31,32
The metabolic benefits observed in mice treated with both
CORT118335 and mifepristone were confirmed by the protection
from liver steatosis induced by the HFD. Our data confirm previous
results showing that mifepristone improved liver injury,
28
and
indicate that the MR antagonistic activity of CORT118335 does
not add benefit to the protection from liver steatosis obtained
with GR antagonism, in contrast to the effects observed for white
fat mass expansion.
Figure 5. CORT118335 increases adipose PGC-1αprotein expression in vivo, without any major effects on UCP1 protein and brown adipose
tissue (BAT)-specific transcripts in vitro. CORT118335 treatment in vivo induces a dramatic increase of adipose tissue PGC-1αprotein
expression, without any major effect on UCP1. (a) Western blot analysis performed in subcutaneous adipose depots of mice fed a HFD, treated
with mifepristone or CORT118335, by using polyclonal antibodies to PGC-1αor UCP1. A representative experiment among three is shown.
(b) Densitometric analysis for PGC-1αand (c) UCP1 (using actin as loading control). (d–g) qRT-PCR analysis of genes involved in brown
adipogenesis in primary murine preadipocytes from inguinal fat depot differentiated for 6 days with or without spironolactone (SPIRO,
10
–5
M), mifepristone (10
–5
M) or CORT118335 (10
–6
M). Values are expressed as % of control untreated cells and represent the mean ±s.e.m. of
two independent experiments performed in triplicate. CORT118335 weakly upregulates UCP1 transcript levels (d), without any effect on
PRDM16 (e) and CIDEA (f) mRNA. (g) CORT118335 and mifepristone markedly increase PGC-1αmRNA expression. Values are expressed as
means ±s.e.m. *Po0.05, **Po0.01, ***Po0.001 vs UT.
GR/MR antagonism in adipose tissue
C Mammi et al
7
© 2016 Macmillan Publishers Limited International Journal of Obesity (2016), 1 –9

Importantly, histological examination of inguinal fat samples from
CORT118335-treated mice did not reveal any increase in brown
adipocyte content, as we recently observed in mice treated with
spironolactone.
21
In line with this, western blot analysis failed to
show any increase of UCP1 expression in inguinal fat of mice treated
with CORT118335. We have recently shown that pharmacological
MR blockade determines browning of the adipose organ.
21
These
effects were not observed with the GR antagonist mifepristone, nor
with CORT118335, probably because of the moderate antagonistic
activity of the compound at MR, as compared with spironolactone.
22
This could explain a reduced ability of CORT118335 in inducing
UCP1 protein expression in WAT. This is also confirmed by the lack
of major effects exerted by CORT118335 on brown adipose tissue-
specific transcripts in primary preadipocytes in vitro,suchas
PRDM16 and CIDEA, while UCP1 mRNA was only modestly
increased by CORT118335.
Conversely, we observed a dramatic increase of PGC-1αprotein
expression in inguinal fat from mice treated with CORT118335.
These data show that combined MR/GR blockade causes a marked
increase in adipose PGC-1αprotein, enhancing the effects obtained
with specific GR antagonism by mifepristone. PGC-1αis a
transcriptional coactivator and master regulator of mitochondrial
biogenesis.
33
We hypothesize that the marked upregulation of
adipose PGC-1αrepresents one of the leading mechanisms
explaining the effects of the novel compound on body fat. In fact,
recent findings suggest that adipose tissue PGC-1αhas an important
role in glucose homeostasis and fatty acid oxidation,
34
and that the
adipose organ in several murine models of obesity shows a reduced
mitochondrial function.
35
PGC-1αis a master gene linking mito-
chondrial number and function to external environmental and
hormonal stimuli,
33
and directly controls oxygen consumption,
oxidative phosphorylation, uncoupling and mitochondrial prolifera-
tion. In our model, we did not observe any increase in UCP1 protein
in any of the studied adipose depots, suggesting that the marked
increase in adipocyte PGC-1αregulates the biogenesis and electron
transport systems of mitochondria in WAT, without stimulating
uncoupling, hence without induction of browning. A limitation of
the present work stems from the systemic effects of the
pharmacological receptor blockade, which affects the activity of
GR and MR expressed not only in adipocytes but also in other cell
types (pancreatic beta cells, skeletal muscle cells, neurons). There-
fore, we cannot exclude the possibility that receptor blockade in
tissues other than fat may generate signals to control the metabolic
response to a HFD. For this reason, we treated in vitro primary
murine preadipocytes and 3T3-L1 cells with CORT118335, confirm-
ing its direct effects on adipose tissue biology, and suggesting
that adipose tissue may represent a direct target for CORT118335
in vivo. CORT118335, in fact, markedly reduced 3T3-L1 lipid
accumulation as measured by Oil Red O staining. To dissect
the mixed GR/MR antagonist activity of CORT118335 in this
model, we tested its effects under steroid-deprived conditions in
3T3-L1 cells.
13
Interestingly, only the MR-mediated pro-adipogenic
effects of aldosterone were antagonized by CORT118335, whereas
GR-mediated effects of dexamethasone were not affected. This
suggests that CORT118335 mostly acts as an antagonist of MR,
rather than GR, in murine cultured preadipocytes, and highlights the
relative importance of MR signaling as a strategic target to modulate
adipocyte differentiation. On the basis of our data, we can infer that
reduction in white fat mass and induction of browning may not
necessarily occur together, in response to pharmacological MR
antagonism.
Figure 6. CORT118335 inhibits adipose differentiation of 3T3-L1 preadipocytes in vitro, mainly through MR antagonism. (a) Both CORT118335
and mifepristone markedly reduce 3T3-L1 differentiation, as measured by Oil Red O staining. (b) Under steroid-deprived conditions,
CORT118335 counters adipose differentiation induced by aldosterone but does not reverse dexamethasone (DEXA)-driven inhibitory action
on adipose conversion. CORT118335 counters the increase in transcript levels of leptin (c) and resistin (d) stimulated by aldosterone, but does
not prevent the upregulation of angiotensinogen mRNA (e) induced by dexamethasone. Values are expressed as means ±s.e.m. **Po0.01,
***Po0.001 vs UT;
#
Po0.05;
###
Po0.001 vs ALDO.
GR/MR antagonism in adipose tissue
C Mammi et al
8
International Journal of Obesity (2016), 1 –9 © 2016 Macmillan Publishers Limited

In conclusion, this is the first study showing that combined
GR/MR pharmacological blockade markedly prevents fat mass
expansion in a model of HFD-induced obesity both in female and
male mice, confirming that both MR and GR have a relevant role in
adipose tissue function, and that such combined pharmacological
approach could represent a novel strategy to prevent HFD-induced
obesity and its metabolic complications.
CONFLICT OF INTEREST
Dr Hazel Hunt is employed by CORCEPT Therapeutics, which developed and provided
CORT118335 and mifepristone for the experiments. All the other authors declare no
conflict of interest.
ACKNOWLEDGEMENTS
We thank Dr Raffaella Pecci and Dr Rossella Bedini for precious technical assistance,
Prof Silvia Migliaccio and Prof Paolo Bianco for scientific support. This work
was supported by a grant from Ministero della Salute (BANDO 2011-2012 Progetti
Collaborazione Ricercatori Italiani all’Estero; project grant PE-2011-02347070 to MC),
and from Corcept Therapeutics. This work was presented, in part, at the
COST-ADMIRE Annual Meeting, Padua, Italy, 16th–17th October 2014. We would
like to acknowledge networking support by the COST Action ADMIRE BM1301. This
work is dedicated to the memory of Prof Paolo Bianco, outstanding scientist, shining
example of dedication to work and freedom of thought.
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Supplementary Information accompanies this paper on International Journal of Obesity website (http://www.nature.com/ijo)
GR/MR antagonism in adipose tissue
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© 2016 Macmillan Publishers Limited International Journal of Obesity (2016), 1 –9