Carbenoxolone treatment attenuates symptoms of

Transcription

Am J Physiol Endocrinol Metab 293: E1517–E1528, 2007.
First published September 18, 2007; doi:10.1152/ajpendo.00522.2007.
Carbenoxolone treatment attenuates symptoms of metabolic syndrome
and atherogenesis in obese, hyperlipidemic mice
Alli M. Nuotio-Antar,1 David L. Hachey,2 and Alyssa H. Hasty1
Departments of 1Molecular Physiology and Biophysics and 2Pharmacology, Vanderbilt University Medical Center,
Nashville, Tennessee
Submitted 10 August 2007; accepted in final form 11 September 2007
is a world-wide health problem (5). Of
major concern to health care professionals is the accompanying
rise in other metabolic syndrome risk factors associated with
severe obesity: dyslipidemia, hypertension, and impaired glucose homeostasis, which further predispose patients to cardiovascular disease and diabetes (6). Cushing’s syndrome, characterized by excess circulating endogenously or exogenously
derived glucocorticoids, results in metabolic syndrome symptoms, such as central obesity, increased plasma triglycerides
(TGs), hypertension, and elevated fasting glucose (38, 55).
Glucocorticoids impact body fat distribution and stimulate
adipocyte differentiation and lipolysis (19, 21, 48). In hepato-
cytes, glucocorticoids modulate gluconeogenic and lipogenic
processes (14, 53). Because obesity is not frequently associated
with excess circulating glucocorticoids, there is speculation
that enhanced actions of glucocorticoids in key metabolic
tissues, such as adipose tissue and liver, may play a causative
role in the altered physiology observed in patients with metabolic syndrome (43).
11␤-Hydroxysteroid dehydrogenase (11␤-HSD) type 1
(11␤-HSD1) is a bidirectional NADP⫹/NADPH-dependent dehydrogenase/reductase that is highly expressed in key metabolic tissues such as liver and adipose tissue, where it acts
primarily to regenerate glucocorticoids from inactive, 11-keto
metabolites (33). A second isoform, 11␤-HSD2, the product of
a different gene, acts solely to metabolize glucocorticoids and
is highly expressed in tissues such as kidney, where it confers
protection against excess binding of glucocorticoids to the
mineralocorticoid receptor (32, 49). Thus, 11␤-HSD1 is the
only enzyme capable of regenerating corticosterone from 11dehydrocorticosterone (11-DHC) in rodents or cortisol from
cortisone in humans. As these 11-keto metabolites circulate at
high concentrations (24, 57), 11␤-HSD1 activity may serve to
potentiate local concentrations of active glucocorticoids in a
tissue-specific manner, preventing the need for the body to
produce excess circulating receptor-competent glucocorticoid
isoforms, which may have deleterious effects in other tissues.
Dysregulated 11␤-HSD1 activity can profoundly affect metabolic phenotype in mice. Adipose tissue-specific 11␤-HSD1
overexpression results in hypertension, increased serum free
fatty acid (FFA) and TG levels, insulin resistance, central
obesity, and hypertension (30, 31). Liver-specific 11␤-HSD1
amplification results in hypertension, a proatherogenic lipoprotein profile, and non-obesity-associated hyperinsulinemia (42).
Conversely, global 11␤-HSD1 deficiency in mice fed a highfat diet is associated with decreased weight gain and improved
serum HDL lipoprotein profiles, despite increased adrenocortex-derived circulating glucocorticoids (24, 34, 35). Therefore,
it appears that 11␤-HSD1 activity may potentially impact more
than one risk factor for metabolic syndrome. Inasmuch as
11␤-HSD1 expression has been reported to be increased in the
adipose tissue of obese patients (10, 22, 46), this enzyme may
prove to be a promising drug target for patients with the
metabolic syndrome.
No study has assessed metabolic outcomes of 11␤-HSD1
inhibition in the context of combined obesity, atherogenesis,
and hyperlipidemia, nor have the effects of 11␤-HSD1
inhibition on lipoprotein metabolism in such a context been
Address for reprint requests and other correspondence: A. H. Hasty, 702
Light Hall, 23rd Ave. South at Pierce, Nashville, TN 37027 (e-mail:
alyssa.hasty@vanderbilt.edu).
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.
11␤-hydroxysteroid dehydrogenase type 1; obesity; agouti; low-density lipoprotein receptor
THE OBESITY EPIDEMIC
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0193-1849/07 $8.00 Copyright © 2007 the American Physiological Society
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Downloaded from http://ajpendo.physiology.org/ by 10.220.33.2 on October 25, 2016
Nuotio-Antar AM, Hachey DL, Hasty AH. Carbenoxolone
treatment attenuates symptoms of metabolic syndrome and atherogenesis in obese, hyperlipidemic mice. Am J Physiol Endocrinol
Metab 293: E1517–E1528, 2007. First published September 18,
2007; doi:10.1152/ajpendo.00522.2007.—Glucocorticoids, which are
well established to regulate body fat mass distribution, adipocyte
lipolysis, hepatic gluconeogenesis, and hepatocyte VLDL secretion,
are speculated to play a role in the pathology of metabolic syndrome.
Recent focus has been on the activity of 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD1), which is capable of regenerating,
and thus amplifying, glucocorticoids in key metabolic tissues
such as liver and adipose tissue. To determine the effects of global
11␤-HSD1 inhibition on metabolic syndrome risk factors, we subcutaneously injected “Western”-type diet-fed hyperlipidemic mice displaying moderate or severe obesity [LDL receptor (LDLR)-deficient
(LDLR⫺/⫺) mice and mice derived from heterozygous agouti (Ay/a)
and homozygous LDLR⫺/⫺ breeding pairs (Ay/a;LDLR⫺/⫺ mice)]
with the nonselective 11␤-HSD inhibitor carbenoxolone for 4 wk.
Body composition throughout the study, end-point fasting plasma, and
extent of hepatic steatosis and atherosclerosis were assessed. This
route of treatment led to detection of high levels of carbenoxolone in
liver and fat and resulted in decreased weight gain due to reduced
body fat mass in both mouse models. However, only Ay/a;LDLR⫺/⫺
mice showed an effect of 11␤-HSD1 inhibition on fasting insulin and
plasma lipids, coincident with a reduction in VLDL due to mildly
increased VLDL clearance and dramatically decreased hepatic triglyceride production. Ay/a;LDLR⫺/⫺ mice also showed a greater effect of
the drug on reducing atherosclerotic lesion formation. These findings
indicate that subcutaneous injection of an 11␤-HSD1 inhibitor allows
for the targeting of the enzyme in not only liver, but also adipose
tissue, and attenuates many metabolic syndrome risk factors, with
more pronounced effects in cases of severe obesity and hyperlipidemia.
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CARBENOXOLONE TREATMENT OF MURINE METABOLIC SYNDROME
MATERIALS AND METHODS
Mice. All mouse procedures were approved by the Vanderbilt
University Institutional Animal Care and Use Committee. Mice were
allowed food and water ad libitum and were kept under 12:12-h
light-dark cycles. Mice were derived from heterozygous agouti (Ay/a)
and homozygous LDLR⫺/⫺ breeding pairs on a C57BL/6 background
(Jackson Laboratories). For generation of Ay/a;LDLR⫺/⫺ mice, heterozygous agouti (Ay/a) mice were bred with homozygous LDLR⫺/⫺
mice to yield heterozygous Ay/a;LDLR⫺/⫺ and non-agouti LDLR⫺/⫺
littermates.
Experimental design. At 2 mo of age, LDLR⫺/⫺ and Ay/a;
LDLR⫺/⫺ mice were placed on a 42% fat, 31% sucrose, 15% protein,
0.2% cholesterol Western-type diet (WD; TD.88137, Harlan Teklad)
for a total of 10 wk to accelerate atherogenesis and obesity. After they
were fed the WD diet for 6 wk, the mice were divided into body
weight-matched groups and subcutaneously injected once daily between 5 and 6 PM with 250 ␮l of PBS vehicle, 25 mg/kg CBX
solution (Sigma-Aldrich), or 50 mg/kg CBX solution for 4 wk while
Fig. 1. 11␤-Hydroxysteroid dehydrogenase (11␤HSD1) expression and activity in LDL receptordeficient (LDLR⫺/⫺) and heterozygous agouti
(Ay/a) mice bred with homozygous LDLR⫺/⫺
mice (Ay/a;LDLR⫺/⫺ mice). 11␤-HSD1 expression was quantitated in gonadal fat pads (A) and
livers (B) excised from chow diet-fed, 4-mo-old
and 2-yr-old female LDLR⫺/⫺ and Ay/a;
LDLR⫺/⫺ mice (n ⫽ 3). C and D: 11␤-HSD1
enzymatic activity in gonadal fat pads and livers,
respectively, dissected from female Western diet
(WD)-fed Ay/a;LDLR⫺/⫺ mice subcutaneously
injected with vehicle or 50 mg 䡠 kg⫺1 䡠 day⫺1 carbenoxolone (CBX) for 4 wk. E: ultraperformance
liquid chromatography-tandem mass spectrometry analysis of tissue 11-deoxycorticosterone (11DHC) and corticosterone levels in gonadal fat
pads dissected from female WD-fed LDLR⫺/⫺
and Ay/a;LDLR⫺/⫺ mice subcutaneously injected
with vehicle or 50 mg 䡠 kg⫺1 䡠 day⫺1 CBX for 4
wk. Values are means ⫾ SE for 3–5 LDLR⫺/⫺
and 9 Ay/a;LDLR⫺/⫺ mice. *P ⬍ 0.001 vs. 2-moold LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice and P ⬍
0.01 vs. age-matched, lean LDLR⫺/⫺ control.
#P ⬍ 0.05 vs. vehicle.
AJP-Endocrinol Metab • VOL
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characterized, which would be key to determining effects of
such drugs on dyslipidemia in patients with metabolic syndrome. In this study, we administered subcutaneous injections of the nonselective 11␤-HSD inhibitor carbenoxolone
(CBX) to hyperlipidemic, Western-type diet (WD)-fed moderately obese LDL receptor (LDLR)-deficient (LDLR⫺/⫺)
and severely obese mice derived from heterozygous agouti
(Ay/a) and homozygous LDLR⫺/⫺ breeding pairs (Ay/a;
LDLR⫺/⫺ mice) (11, 12) and examined outcomes on various
metabolic syndrome risk factors. We found that systemic
inhibition of 11␤-HSD1 led to dramatic improvements in
body composition, basal metabolic rate, insulin resistance,
lipoprotein metabolism, hepatic steatosis, and atherosclerosis in the more severely obese and insulin-resistant mice,
suggesting that targeting this enzyme may become a key
therapy for patients with multiple metabolic syndrome risk
factors.
by including in the food weight assessments ⬎0.1-g remnants of food
pellets found in cage bedding. Food intake efficiency over the 4-wk
treatment period was calculated using the following equation: efficiency ⫽ (total change in body weight/cumulative food intake) ⫻ 100.
Indirect calorimetry. At the end of the 1st wk of treatment, singly
housed, weight-matched female Ay/a;LDLR⫺/⫺ mice were placed in
metabolic cages for 24 h of acclimation followed by 24 h of measurement. Whole body O2 consumption (V̇O2), CO2 production
(V̇CO2), and activity were measured continuously for 1 min at 15-min
intervals using an indirect calorimetry system (Oxymax Deluxe,
Columbus Instruments) with an airflow rate of 0.6 l/min. Oxymax
software used the following equations to calculate respiratory exchange ratio (RER) and energy expenditure (heat): RER ⫽ V̇CO2/V̇O2
and heat ⫽ (3.815 ⫹ 1.232 ⫻ RER) ⫻ V̇O2 (40). Heat was then
normalized to lean body mass. Basal metabolic rate for each mouse
was calculated by averaging heat from three 45-min intervals of least
energy expenditure during the light cycle.
Plasma analyses. Plasma TG, total cholesterol (TC), and FFA were
measured using TG and cholesterol reagents (Raichem) and the NEFA
C kit (Wako), respectively. Blood glucose was measured using a
Lifescan One Touch basic glucometer kit (Johnson & Johnson).
Insulin and leptin were determined using modified insulin doubleantibody RIA kits (Linco Research). Lipoprotein fractionation was
achieved by fast protein LC using aliquots of pooled plasma samples
from each group of mice. Plasma samples from mice used for VLDL
turnover and hepatic TG production experiments were omitted from
end-point plasma lipid analysis.
VLDL turnover. Plasma from fasted age-matched, WD-fed, overnight-fasted Ay/a;LDLR⫺/⫺ mice was pooled, and VLDL (density
⬍1.019 g/l) was isolated by density gradient ultracentrifugation in a
centrifuge (Optima TLX, Beckman Coulter). IODO-GEN precoated
reaction tubes (Pierce Biotechnology) were used to incorporate 125I
Fig. 2. Body weight and composition were affected by CBX. Vehicle- and CBX-treated female (A–C) and male (D–F) mice were weighed and body composition
was analyzed throughout the 4-wk injection study. Values are means ⫾ SE of 16 –17 female LDLR⫺/⫺, 14 female Ay/a;LDLR⫺/⫺, 8 –9 male LDLR⫺/⫺, and 9 –11
Ay/a;LDLR⫺/⫺ mice. *P ⬍ 0.05; #P ⬍ 0.01 vs. vehicle.
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being maintained on WD. Mice were weighed and total fat and lean
mass were assessed weekly using an NMR analyzer (Minispec,
Bruker Optics) during the 4 wk of treatment. Unless otherwise noted,
at the end of the study, all mice were fasted for 5 h and injected 1 h
before they were killed, anesthetized, bled by puncture of the retroorbital venous plexus, euthanized with isoflurane inhalation followed
by cervical dislocation, perfused with PBS, and dissected for further
tissue analyses. Plasma was separated from whole blood by centrifugation at 4°C.
11␤-HSD1 enzyme assays. 11␤-HSD1 activity was analyzed as
previously described (39) using 5 ␮g of liver and 50 ␮g of gonadal fat
pad protein from tissue homogenates and a total concentration of 20
␮M cortisone substrate and 1.2 mM NADPH co-substrate per assay
after determination of optimal protein concentrations of liver and
adipose tissue homogenates.
Ultraperformance liquid chromatography-tandem mass spectrometry analysis of tissues. Sample preparation and liquid chromatography (LC)-tandem mass spectrometry (MS-MS) analysis were performed according to the method of Ronquist-Nii and Edlund (50) with
modifications (see supplemental information online at the American
Journal of Physiology-Endocrinology and Metabolism website).
Systolic blood pressure measurement. Systolic blood pressure was
measured in conscious, resting mice by tail cuff plethysmography as
previously described (59). During the last week of treatment, vehicle
and 50 mg 䡠 kg⫺1 䡠 day⫺1 CBX were administered to male and female
Ay/a;LDLR⫺/⫺ and LDLR⫺/⫺ mice, the animals were trained for 3
consecutive days, and baseline systolic blood pressure was measured
and averaged over the 2 succeeding days.
Food intake. For studies measuring food intake, mice were separated by genotype and housed in initially weight-matched groups of
two or three mice per cage. Weighing error due to food spillage was
minimized by using large, intact food pellets throughout the study and
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Table 1. Fasting plasma lipid and insulin levels were attenuated by CBX treatment in Ay/a;LDLR⫺/⫺, but not
LDLR⫺/⫺, mice
n
TC, mg/dl
TG, mg/dl
FFA, meq/l
Glucose, mg/dl
Insulin, ng/ml
Leptin, ng/ml
Females
⫺/⫺
LDLR
Vehicle
CBX (50 mg 䡠 kg⫺1 䡠 day⫺1)
Ay/a;LDLR⫺/⫺
Vehicle
17
16
754⫾35
757⫾24
296⫾27
274⫾19
1.08⫾0.12
1.20⫾0.11
121⫾6
125⫾6
0.50⫾0.08
0.33⫾0.07
16.2⫾2.1
10.2⫾1.1*
14
14
14
1,858⫾76
1,487⫾81†
1,464⫾82†
661⫾51
520⫾45*
498⫾29*
1.56⫾0.11
1.24⫾0.10
1.08⫾0.10†
109⫾4
120⫾5
116⫾5
2.66⫾0.33
1.74⫾0.26*
1.38⫾0.21†
110.4⫾8.6
84.7⫾6.9
65.2⫾9.1†
Males
9
8
9
9
11
1,117⫾95
1,025⫾88
538⫾64
487⫾67
1.04⫾0.17
0.97⫾0.14
131⫾8
122⫾11
0.78⫾0.10
0.71⫾0.08
14.2⫾3.7
6.1⫾1.9
1,723⫾83
1,528⫾43
1,257⫾114†
977⫾42
931⫾37
743⫾86*
1.37⫾0.13
1.19⫾0.11
0.88⫾0.06†
120⫾9
124⫾6
126⫾9
6.01⫾0.98
5.85⫾0.75
3.14⫾0.27*
99.6⫾9.2
71.3⫾7.1*
45.4⫾5.2†
Values are means ⫾ SE. Plasma lipids were measured in 5-h-fasted LDL receptor-deficient (LDLR⫺/⫺) mice and heterozygous agouti (Ay/a) mice bred with
homozygous LDLR⫺/⫺ mice (Ay/a;LDLR⫺/⫺ mice) at the end of the study. TG, triglyceride; TC, total cholesterol; FFA, free fatty acid; CBX, carbenoxolone.
*P ⬍ 0.05; †P ⬍ 0.01 vs. vehicle.
label (Na125I, Amersham Biosciences) into the protein component of
the VLDL fraction. Specific activity of the 125I-VLDL preparation
was ⬃350 cpm/ng protein. The 125I-VLDL preparation was diluted
with PBS, 200,000 cpm were injected in a final volume of 200 ␮l into
the retroorbital space of one eye of each mouse, and plasma was
collected 1.5, 10, 30, and 90 min later. GraphPad Prism 4 software
was used for curve fitting to a biexponential decay curve.
Hepatic TG production. Blood was collected from overnight-fasted
male and female mice for baseline TG analysis. Mice were injected
with tyloxapol (Sigma-Aldrich; 500 mg/kg body wt) to inhibit TG
clearance. Mice were bled 1 and 2 h after injection, and plasma was
analyzed for TG content. Hepatic TG production rate was linear over
the time course and was calculated as the slope of the plasma TG vs.
time curve from baseline to 2 h.
Aortic root lesion area quantification. Sections (10 ␮m) of aortic
root from frozen, OCT-embedded hearts were cut according to the
method of Paigen et al. (41). Cryosections were stained with oil red O
(Sigma-Aldrich) for detection of neutral lipids and counterstained
with Mayer’s hematoxylin. A Q-Imaging Micropublisher camera
attached to an Olympus light microscope and Histometrix 6 software
(Kinetic Imaging) were used to capture images and quantify aortic
root lesion areas.
Liver neutral lipid staining. Cryosections (10 ␮m) from livers of
representative female mice from each treatment group were cut,
mounted, and stained, and images were captured as described previously for aortic root lesions.
Liver lipid quantification. Liver lipids were extracted and quantified using a gas chromatography-flame ionization detector system as
described previously (52).
Gene expression quantification. Total RNA was extracted from
livers, and gene expression was quantified using an ABI Prism 7700
sequence detection system (Applied Biosystems) and validated Assays-on-Demand primers with 6-carboxyfluorescein dye-MGB-labeled TaqMan probes as previously described (39).
Table 2. Total body weight and end weights of gonadal fat pad, liver, and kidney
Tissue Wt, g
n
Total Body Wt, g
Gonadal fat pad
Kidney
Liver
Females
LDLR⫺/⫺
Vehicle
Ay/a;LDLR⫺/⫺
Vehicle
17
16
23.6⫾0.5
22.0⫾0.5*
0.46⫾0.04
0.35⫾0.04*
0.126⫾0.004
0.124⫾0.003
1.17⫾0.04
1.22⫾0.06
14
14
14
38.0⫾0.7
35.9⫾0.7
33.5⫾1.5†
2.34⫾0.13
1.91⫾0.12
1.74⫾0.17†
0.139⫾0.004
0.134⫾0.007
0.137⫾0.006
2.11⫾0.08
1.84⫾0.07*
1.66⫾0.10†
Males
LDLR⫺/⫺
Vehicle
y
A /a;LDLR⫺/⫺
Vehicle
9
8
29.1⫾0.7
26.5⫾0.6*
0.89⫾0.14
0.59⫾0.09
0.171⫾0.007
0.172⫾0.011
1.39⫾0.12
1.47⫾0.05
9
9
11
42.5⫾1.0
39.9⫾1.1
38.5⫾0.8*
2.29⫾0.10
2.21⫾0.08
1.86⫾0.11*
0.157⫾0.009
0.160⫾0.005
0.173⫾0.004
2.71⫾0.19
2.25⫾0.10*
2.01⫾0.06†
Values are means ⫾ SE. Total body weight and tissue weights were measured on the last day of the study. *P ⬍ 0.05; †P ⬍ 0.01 vs. vehicle.
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LDLR⫺/⫺
Vehicle
Ay/a;LDLR⫺/⫺
Vehicle
Statistical analyses. Values are means ⫾ SE. GraphPad Prism 4
software was used to assess significance for all data sets, and P ⬍ 0.05
was considered statistically significant. For studies comparing chow
diet-fed LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice, one-way analysis of
variance with Bonferroni’s post hoc test was used to determine
significance. For comparisons between vehicle-treated LDLR⫺/⫺ and
Ay/a;LDLR⫺/⫺ mice, LDLR⫺/⫺ mice treated with vehicle and those
treated with 50 mg 䡠 kg⫺1 䡠 day⫺1 CBX and Ay/a;LDLR⫺/⫺ mice
treated with vehicle and those treated with 50 mg 䡠 kg⫺1 䡠 day⫺1,
unpaired, two-way Student’s t-test was used to evaluate significance.
For studies comparing Ay/a;LDLR⫺/⫺ mice treated with vehicle, 25
mg 䡠 kg⫺1 䡠 day⫺1 CBX, and 50 mg 䡠 kg⫺1 䡠 day⫺1 CBX, one-way analysis of variance with Dunnett’s post hoc test was used to determine
significance.
Measurement of CBX and its effects in liver and adipose
tissue. To study effects of 11␤-HSD1 inhibition in a mouse
model of hyperlipidemia and obesity, we chose to use hyperlipidemic hyperphagic Ay/a;LDLR⫺/⫺ mice, which are a
mouse model of maturity-onset obesity when fed a chow diet
(11, 12). A preliminary analysis of 11␤-HSD1 expression in
chow diet-fed LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice indicated
that a significant increase in enzyme expression in a representative fat pad was associated with obesity in aged Ay/a;
LDLR⫺/⫺ mice (Fig. 1A). We observed no such change for
liver 11␤-HSD1 expression levels (Fig. 1B).
To test whether 11␤-HSD1 inhibition affected metabolic
syndrome risk factors in the context of obesity and hyperlipidemia, 2 mo-old LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice
(11, 12) were fed WD for 6 wk and subsequently subcutaneously injected with vehicle, 25 mg䡠kg⫺1 䡠day⫺1 CBX, or 50
mg䡠kg⫺1 䡠day⫺1 CBX for another 4 wk while maintained on
WD. Leshchenko et al. (26) established that intraperitoneal
injection of rats with 50 mg/kg CBX resulted in maximal
circulating levels of CBX 40 –70 min after injection. At 1 h
after subcutaneous injection, ultraperformance LC (UPLC)MS-MS analysis of tissue homogenates from LDLR⫺/⫺ and
Ay/a;LDLR⫺/⫺ mice revealed that ⬃11–12% and 3– 4%, respectively, of the 50 mg/kg dose was detectable in liver and
gonadal fat, respectively: 5.9 ⫾ 0.8 and 5.6 ⫾ 1.2 ␮g/g for
8 –10 LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ livers, respectively, and
1.7 ⫾ 0.8 and 2.0 ⫾ 0.7 ␮g/g for 4 –7 LDLR⫺/⫺ and Ay/a;
LDLR⫺/⫺ gonadal fat pads, respectively.
To directly assess the impact of CBX on 11␤-HSD1 activity,
we next measured enzymatic activity in homogenates of gonadal fat pads and livers dissected from Ay/a;LDLR⫺/⫺ mice at
the end of the study, 1 h after injection. Enzyme activity assays
revealed a trend toward a decrease in 11␤-HSD1 activity in
Fig. 3. CBX treatment resulted in negative energy balance. A: cumulative food intake was
measured for 25 days throughout the drug treatment period in male and female LDLR⫺/⫺ and
Ay/a;LDLR⫺/⫺ mice treated with vehicle or 50
mg 䡠 kg⫺1 䡠 day⫺1 CBX. Values are means ⫾ SE
of 2–3 mice per cage in 5–7 cages. B: food
intake efficiency throughout the drug treatment
period in male and female LDLR⫺/⫺ and Ay/a;
LDLR⫺/⫺ mice treated with vehicle or 50
mg 䡠 kg⫺1 䡠 day⫺1 CBX. Values are means ⫾ SE
of 2–3 mice per cage in 5–7 cages. C: energy
expenditure in weight-matched female Ay/a;
LDLR⫺/⫺ mice treated with vehicle or 50
mg 䡠 kg⫺1 䡠 day⫺1 CBX (n ⫽ 5) at the end of the
1st wk of the drug study. Values are means ⫾
SE. *P ⬍ 0.01 vs. vehicle. #P ⬍ 0.001. ^P ⫽
0.0002.
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RESULTS
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LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ controls: 59 ⫾ 4 and 75 ⫾ 2 g,
respectively (P ⫽ 0.01; Fig. 3A). However, subcutaneous
injection of CBX at the highest dose did not significantly
impact cumulative food intake in LDLR⫺/⫺ or Ay/a;LDLR⫺/⫺
mice after 25 days of treatment. Food intake efficiency was
decreased in CBX- compared with vehicle-treated LDLR⫺/⫺
and Ay/a;LDLR⫺/⫺ mice (Fig. 3B).
Indirect calorimetry revealed a significant increase in energy
expenditure in Ay/a;LDLR⫺/⫺ mice treated with 50 mg䡠kg⫺1 䡠day⫺1
CBX compared with vehicle-treated controls that was attributable to a significant increase in light cycle energy expenditure
(Fig. 3C). In addition, basal metabolic rate was significantly
greater in 50 mg䡠kg⫺1 䡠day⫺1 CBX- than in vehicle-treated in
Ay/a;LDLR⫺/⫺ mice: 1,187 ⫾ 24 vs. 1,328 ⫾ 34 kcal䡠kg body
mass⫺1 䡠min⫺1 (P ⫽ 0.0022).
CBX significantly decreased hyperinsulinemia in Ay/a;
LDLR⫺/⫺ mice. Because previous studies reported an insulinsensitizing effect of 11␤-HSD1 inhibition in mice and humans
(1, 2, 20), we measured end-point glucose and insulin levels in
all mice. Although subcutaneous CBX administration had no
effect on plasma glucose and insulin in LDLR⫺/⫺ mice, a
significant reduction in fasting insulin, but not glucose, levels
was observed in the more severely obese Ay/a;LDLR⫺/⫺ mice
(P ⬍ 0.05; Table 1).
Hyperlipidemia in Ay/a;LDLR⫺/⫺ mice is attenuated by CBX
treatment. Previous reports indicated that 11␤-HSD1 may play
a role in regulating plasma TG and HDL levels in rodents (9,
20, 28, 29, 34, 42). In our study, neither male nor female
LDLR⫺/⫺ mice showed an effect of CBX treatment on circulating TG, TC, and FFA levels (Table 1). However, CBXtreated male and female Ay/a;LDLR⫺/⫺ mice showed dosedependent and significant reductions in all measured lipids
(P ⬍ 0.01 for TC and FFA, P ⬍ 0.05 for TG). Analysis of
lipoprotein fractions revealed a selective reduction in the
VLDL fraction (Fig. 4A).
Fig. 4. Fasting VLDL, postprandial VLDL
turnover, and triglyceride (TG) production rate
were significantly affected by CBX treatment
in Ay/a;LDLR⫺/⫺, but not LDLR⫺/⫺, mice.
A: lipoprotein distribution shown as fast protein liquid chromatography-fractionated lipoproteins from pooled plasma samples from
5-h-fasted female mice collected at the end of
the study. B: VLDL clearance in plasma collected from nonfasted male mice injected with
125
I-VLDL (n ⫽ 3– 4). C: hepatic TG production determined by injection of overnightfasted male and female mice with tyloxapol
(n ⫽ 5– 6). Values are means ⫾ SE. *P ⬍ 0.05.
#P ⫽ 0.0008, Ay/a;LDLR⫺/⫺ CBX vs. Ay/a;
LDLR⫺/⫺ vehicle.
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adipose tissue homogenates (P ⫽ 0.076), with no observable
effect of CBX administration on liver 11␤-HSD1 activity (Fig.
1, C and D, respectively). For LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺
mice, UPLC-MS-MS of 11-DHC-to-corticosterone ratios revealed a statistically significant accumulation of 11-DHC substrate over corticosterone product in gonadal fat pads of CBXvs. vehicle-treated mice, further confirming an inhibitory
effect of CBX on adipose tissue 11␤-HSD1 reductase activity (Fig. 1E).
CBX did not affect circulating systolic blood pressure. To
exclude the possibility that CBX impacted blood pressure and,
thus, possibly confounded an end point assessing the extent of
atherosclerosis, systolic blood pressure was measured. Systemic administration of CBX had no effect on systolic blood
pressure in LDLR⫺/⫺ or Ay/a;LDLR⫺/⫺ mice (data not
shown).
Effects of subcutaneously administered CBX on body weight
and composition. Total body weight of LDLR⫺/⫺ mice was
significantly lower than that of Ay/a;LDLR⫺/⫺ mice after 6 wk
of WD before CBX treatment (P ⬍ 0.0001; Fig. 2, A and D).
Male and female WD-fed LDLR⫺/⫺ mice treated with 50
mg䡠kg⫺1 䡠day⫺1 CBX showed a steady decline in body weight
that achieved significance by the end of the study. Male and
female WD-fed Ay/a;LDLR⫺/⫺ mice treated with 25 or 50
mg䡠kg⫺1 䡠day⫺1 CBX also showed a similar, dose-dependent
reduction in body weight. Body weight changes were reflected
in differences in total fat mass (Fig. 2, B and E). CBX
treatment did not impact end-point muscle mass (Fig. 2, C and
F). Consistent with the observed reductions in total fat mass,
circulating leptin levels and gonadal fat pad weight were also
dose dependently decreased with CBX treatment by the end of
the study (Tables 1 and 2).
CBX treatment results in negative energy balance. Confirming a hyperphagic effect of hypothalamic agouti protein overexpression in agouti mice (17), cumulative food intake data
revealed a significant difference between vehicle-treated
mice treated with 50 mg䡠kg⫺1 䡠day⫺1 CBX showed a slight,
but nonsignificant, decrease (15% in females and 18% in
males) in aortic root lesion area (Fig. 5). However, Ay/a;
LDLR⫺/⫺ mice showed a dose-dependent and significant decrease in atherosclerotic lesion area with treatment: 12% and
26% in 25 and 50 mg䡠kg⫺1 䡠day⫺1 CBX-treated females and
12% and 28% in 25 and 50 mg䡠kg⫺1 䡠day⫺1 CBX-treated
males (P ⬍ 0.05, vehicle vs. 50 mg䡠kg⫺1 䡠day⫺1 CBX).
Decreased hepatic steatosis due to CBX treatment. Because
end-point liver weights (Table 2) and hepatic TG production
rates were dramatically decreased by CBX in Ay/a;LDLR⫺/⫺
mice, liver lipid content was assessed in mice treated with
vehicle and 50 mg䡠kg⫺1 䡠day⫺1 CBX. Oil red O staining for
neutral lipids indicated an increase in lipid levels in liver
between LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ vehicle-treated controls
(Fig. 6A). Hepatic neutral lipid content was decreased by CBX
treatment in LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice. Variations in
Fig. 5. Atherosclerotic lesion area was significantly decreased in CBX-treated Ay/a;LDLR⫺/⫺,
but not LDLR⫺/⫺, mice. A: representative cross
sections of oil red O-stained aortic root lesions
from female mice. Magnification ⫻40. B and C:
quantification of aortic root lesion areas for female
LDLR⫺/⫺ (n ⫽ 16) and Ay/a;LDLR⫺/⫺ (n ⫽
13–14) mice (B) and male LDLR⫺/⫺ (n ⫽ 14–15)
and Ay/a;LDLR⫺/⫺ mice treated with vehicle (n ⫽
16) and Ay/a;LDLR⫺/⫺ mice treated with 25 (n ⫽
9) and 50 (n ⫽ 19) mg䡠kg⫺1 䡠day⫺1 CBX (C).
Values are means ⫾ SE. *P ⬍ 0.05.
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Because changes in circulating VLDL levels can be a reflection of altered clearance or hepatic secretion, we determined whether these processes were impacted by CBX treatment. Using 125I-VLDL tracer, we observed no effect on
VLDL clearance due to CBX treatment in LDLR⫺/⫺ mice (Fig.
4B). In Ay/a;LDLR⫺/⫺ mice, CBX treatment resulted in a
significant increase in VLDL clearance rate at an early time
point, 10 min after injection of the VLDL tracer (P ⫽ 0.0008).
Hepatic TG production rate was not impacted by CBX in
LDLR⫺/⫺ mice but was significantly reduced in CBX-treated
Ay/a;LDLR⫺/⫺ mice compared with vehicle-treated controls
(P ⬍ 0.05; Fig. 4C).
CBX reduces atherosclerosis in Ay/a;LDLR⫺/⫺ mice. Elevated plasma cholesterol levels play a well-established,
proatherogenic role in humans and mice. Therefore, we sought
to determine the effect of CBX treatment on lesion formation
in WD-fed LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice. LDLR⫺/⫺
E1523
E1524
neutral lipid content were attributable to significant differences
in liver TG and unesterified cholesterol levels, as well as a
trend toward decreased esterified cholesterol content; CBX
treatment had no effect on hepatic phospholipid or FFA composition (Fig. 6B and data not shown).
CBX treatment impacts hepatic gene expression. Hepatic
TG and cholesterol content may be modulated by lipogenic
processes, cholesterol synthesis, and cholesterol metabolism
within the liver. Real-time PCR gene expression analysis
revealed striking differences between LDLR⫺/⫺ and Ay/a;
LDLR⫺/⫺ vehicle-treated controls. Triacylglycerol hydrolase/
carboxylesterase, liver X receptor-␣, and cytochrome P-450
(CYP27A1) expression levels were increased 1.5-, 6.2-, and
2.2-fold in Ay/a;LDLR⫺/⫺ vehicle-treated relative to LDLR⫺/⫺
vehicle-treated mice (P ⬍ 0.01, P ⫽ 0.0002, and P ⬍ 0.0001,
respectively). Consistent with the reductions in hepatic lipid
content, key genes regulating hepatic TG and cholesterol
accumulation were downregulated in CBX-treated LDLR⫺/⫺
and Ay/a;LDLR⫺/⫺ mice (Table 3).
DISCUSSION
We have shown that subcutaneous administration enables
CBX to target liver and adipose tissue, both key metabolic
tissues, ultimately resulting in profound metabolic changes in
WD-fed LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ hyperlipidemic mouse
models of obesity. In Ay/a;LDLR⫺/⫺ mice, CBX treatment
reduced plasma VLDL levels due to a small increase in the rate
of VLDL uptake and a more dramatic decrease in the rate of
hepatic TG production. The extent of atherogenesis, as assessed at the aortic root, was also significantly reduced in
CBX-treated Ay/a;LDLR⫺/⫺ mice despite the short (4-wk)
treatment period. In LDLR⫺/⫺ and Ay/a;LDLR⫺/⫺ mice, CBX
treatment ameliorated hepatic steatosis due to reductions in
liver TG and unesterified cholesterol content. This was associated with decreased expression of genes involved in hepatic
lipogenesis, cholesterol synthesis, and cholesterol metabolism.
These results highlight a role of 11␤-HSD1 activity in the
regulation of VLDL metabolism and secretion, as well as
adiposity, insulin sensitivity, hepatic steatosis, and atherosclerosis, all of which are associated with metabolic syndrome in
humans.
We chose to use the nonselective 11␤-HSD inhibitor CBX at
the onset of our study, because it was the only commercial
11␤-HSD inhibitor available at the time. Since the initiation of
our study, several specific 11␤-HSD1 inhibitors have been
characterized and utilized in rodent studies (1, 8, 20). However,
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Fig. 6. CBX treatment decreased liver TG and
free cholesterol levels in LDLR⫺/⫺ and Ay/a;
LDLR⫺/⫺ mice. A: oil red O neutral lipid staining
of representative livers from 5-h-fasted female
mice. Magnification ⫻200. B: gas chromatography quantification of lipid content of livers dissected from 5-h-fasted female mice (n ⫽ 6).
Values are means ⫾ SE. *P ⬍ 0.05. #P ⬍ 0.005.
E1525
Table 3. CBX treatment impacted expression levels of genes involved in lipogenesis, cholesterol metabolism,
and cholesterol synthesis
LDLR⫺/⫺
Gene
Hepatic lipase
TGH/CES3
Ay/a;LDLR⫺/⫺
Vehicle
CBX
Vehicle
CBX
1.00⫾0.15
1.00⫾0.08
0.84⫾0.11
0.51⫾0.07‡
1.00⫾0.09
1.00⫾0.06
1.45⫾0.18*
0.60⫾0.07‡
1.00⫾0.17
1.00⫾0.10
1.00⫾0.26
1.00⫾0.16
1.39⫾0.21
0.87⫾0.10
0.88⫾0.11
0.83⫾0.03
1.00⫾0.13
1.00⫾0.07
1.00⫾0.14
1.00⫾0.15
0.49⫾0.07†
0.55⫾0.06§
0.90⫾0.06
0.36⫾0.07‡
1.00⫾0.27
1.00⫾0.33
0.69⫾0.14
0.47⫾0.05
Lipogenesis
DGAT1
DGAT2
GPAT
SREBP1c
1.00⫾0.11
1.00⫾0.05
1.00⫾0.20
1.00⫾0.08
0.74⫾0.08
0.62⫾0.07‡
0.49⫾0.08*
0.83⫾0.17
Cholesterol metabolism
1.00⫾0.20
1.00⫾0.07
1.00⫾0.09
1.00⫾0.08
HMG-CoA reductase
HMG-CoA synthase
1.00⫾0.16
1.00⫾0.17
0.47⫾0.11*
1.09⫾0.05
0.74⫾0.10
0.99⫾0.02
Cholesterol synthesis
0.53⫾0.09*
0.42⫾0.06*
Values are means ⫾ SE. Gene expression was measured in livers of representative, 5-h-fasted female mice (n ⫽ 6) at the end of the study. Expression levels
for mice treated with 50 mg 䡠 kg⫺1 䡠 day⫺1 CBX are normalized to those for vehicle-treated LDLR⫺/⫺ or Ay/a;LDLR controls. *P ⬍ 0.05; †P ⬍ 0.01; ‡P ⬍ 0.005;
§P ⬍ 0.001 vs. vehicle. DGAT, diacylglycerol acyltransferase; GPAT, glycerol-3-phosphate acyltransferase; SREBP1c, sterol regulatory binding protein 1c;
TGH/CES3, triacylglycerol hydrolase/carboxylesterase 3; CYP, cytochrome P-450; FXR, farnesoid X-activated receptor; LXR-␣, liver X receptor-␣; HMG-CoA,
3-hydroxy-3-methylglutaryl coenzyme A.
CBX is commonly used in studies assessing the outcomes of
modulation of 11␤-HSD1 enzyme activity in humans (4, 47,
51, 56, 58). Although use of CBX as a long-term treatment for
patients with the metabolic syndrome is limited because of its
well-established hypertensive and hypokalemic side effects,
resulting from inhibition of renal 11␤-HSD2 (45), the results of
the present study suggest that specific inhibition of 11␤-HSD1
may be particularly beneficial in humans diagnosed with more
than one metabolic syndrome risk factor. For the purpose of
our study, the lack of effect of CBX on systolic blood pressure
eliminated the need for concern regarding possible confounding effects of hypertension on atherogenesis. Moreover, we
sought to address metabolic outcomes in liver and adipose
tissue, both of which highly express 11␤-HSD1 and only
minimally, if at all, express 11␤-HSD2. Similar to its naturally
occurring analog glycyrrhetinic acid, CBX is also a potent gap
junction blocker (13), and this activity itself may impact
adipogenesis (60), hormonal regulation of hepatocyte function
(16, 36, 37), and macrophage response at sites of inflammation
(15). Therefore, results reporting CBX effects on metabolism
may be subject to the confounding effects of CBX nonspecificity and, thus, must be interpreted with caution.
Route of administration may impact the ability of CBX to
reach various adipose tissue depots. Previously published results in rats and humans utilizing orally administered CBX to
inhibit 11␤-HSD1 indicated that this regimen impacts predominantly liver, with no detectable effect on adipose 11␤-HSD1
activity (28, 51). More recently, however, effective targeting of
11␤-HSD1 activity in adipose tissue of healthy human subjects
has been shown using orally administered CBX (56). For our
study, subcutaneous injection was chosen as a route of treatment, as subcutaneous injection was hypothesized to allow
more of the CBX to bypass first-pass hepatic metabolism that
may occur with oral ingestion, thus potentially enabling more
of the drug to target adipose tissue. Indeed, tissue levels of CBX
1 h after injection were ⬃175- and 60-fold higher than published
IC50 values in liver and adipose tissue, respectively (7).
In striking contrast to studies utilizing orally administered
CBX to inhibit liver 11␤-HSD1, subcutaneous CBX injection
resulted in additional metabolic improvements, such as blunted
weight gain and reduced fasting insulin, in our unique mouse
models (28, 51). The effect of CBX on weight gain was due to
selectively and dose dependently decreased total body fat
mass, with no detectable change in lean body mass, revealing
that the drug treatment was not causing wasting in the mice. In
CBX-treated Ay/a;LDLR⫺/⫺ mice, decreased body fat mass
was attributable to negative energy balance due to increased
basal metabolic rate. Transgenic modulation of adipose tissue
glucocorticoid regeneration in mice has been shown to impact
energy expenditure (23, 30), and it is possible that inhibition of
adipose tissue 11␤-HSD1 activity was key to the effects on
energy expenditure, and thus obesity, in our mice. However,
effects of CBX-mediated 11␤-HSD1 inhibition on other metabolic tissues involved in energy expenditure, such as skeletal
muscle and brown adipose tissue, cannot be excluded. For
instance, Berthiaume et al. (9) recently reported that, in a rat
model of diet-induced obesity, 3 wk of treatment with a
specific 11␤-HSD1 inhibitor resulted in elevated lipid oxidation product accumulation and/or increased expression of
genes involved in fatty acid oxidation in brown adipose tissue,
heart, and skeletal muscle.
Hermanowski-Vosatka et al. (20) utilized a specific 11␤HSD1 inhibitor to determine effects on plasma lipids in lean,
hyperlipidemic apolipoprotein E⫺/⫺ mice. During the preparation of our manuscript, two more reports emerged regarding
the effects of 11␤-HSD1-specific inhibition on white adipose
tissue lipolysis and plasma TGs (9, 56). The results from the
present study not only confirm that 11␤-HSD1 inhibition
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CYP7A1
CYP27A1
FXR
LXR-␣
E1526
Only Ay/a;LDLR⫺/⫺ mice showed significant decreases in
aortic root lesion area with CBX treatment, and this effect is
most likely due to effects of the drug on plasma lipid levels.
However, because VLDL induces lipid loading as well as
proinflammatory cytokine expression in macrophages (52, 54),
reductions in circulating VLDL may have attenuated atherogenesis not only by impacting lipid accumulation, but also by
decreasing inflammation at the site of the lesions. In addition,
as speculated by Hermanowski-Vosatka et al. (20), local inhibition of 11␤-HSD1 in the arterial wall may also impact
atherogenesis. This hypothesis is supported by our data showing a trend in reduction of lesion area, despite the absence of
effect on plasma lipids, in CBX-treated LDLR⫺/⫺ mice. Alternatively, as increased adiposity is associated with elevated
circulating proinflammatory cytokines and reduced secretion of
the atheroprotective adipokine adiponectin, the effect of differences in adiposity due to CBX treatment on these factors,
and thus atherogenesis, cannot be excluded.
Taken together, our results support the hypothesis that 11␤HSD1 activity influences obesity, dyslipidemia, and atherosclerosis, which are risk factors or outcomes of the metabolic
syndrome. Our findings hold key implications for future therapies aiming to inhibit 11␤-HSD1 in patients with metabolic
syndrome, highlighting the importance of inhibitors created to
selectively target adipose tissue enzyme activity.
ACKNOWLEDGMENTS
We thank Drs. Lawrence Chan, Owen McGuinness, and David Wasserman
for thoughtful reading and critique of our manuscript.
GRANTS
UPLC-MS-MS experiments were conducted in the Vanderbilt Mass Spectrometry Core of Vanderbilt Digestive Disease Research Center [supported by
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Grant DK-058404]. Body composition analysis and indirect calorimetry experiments were performed in the Metabolic Pathophysiology Core of the
Vanderbilt University Mouse Metabolic Phenotyping Center (MMPC; supported by NIDDK Grant DK-59637). Plasma insulin and leptin levels were
measured at the Vanderbilt Diabetes Research and Training Center (supported
by NIDDK Grant DK-020593)/MMPC Hormone Assay and Analytical Services Core. Tissue lipid content was determined in the Lipid Core Laboratory
of the Vanderbilt MMPC. A. M. Nuotio-Antar is a postdoctoral fellow in the
Department of Medicine, Baylor College of Medicine. A. H. Hasty is supported by American Diabetes Association Career Development Award 1-07CD-10.
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