FXR agonist INT-747 upregulates DDAH expression and enhances insulin sensitivity in high-salt fed Dahl rats

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DOI: 10.1371/journal.pone.0060653 · Source: PubMed
Abstract
Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor, asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element, we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin sensitivity. In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747 protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls. Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or activity using small molecules.
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FXR Agonist INT-747 Upregulates DDAH Expression and
Enhances Insulin Sensitivity in High-Salt Fed Dahl Rats
Yohannes T. Ghebremariam
1
, Keisuke Yamada
1
, Jerry C. Lee
1
, Christine L. C. Johnson
2
, Dorothee Atzler
3
,
Maike Anderssohn
3
, Rani Agrawal
4
, John P. Higgins
5
, Andrew J. Patterson
3
, Rainer H. Bo
¨ger
3
,
John P Cooke
1
*
1Division of Cardiovascular Medicine, Stanford University, Stanford, California, United States of America, 2Department of Pediatrics, Stanford University, Stanford,
California, United States of America, 3Institute of Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4Division of
Anesthesia, Stanford University, Stanford, California, United States of America, 5Division of Pathology, Stanford University, Stanford, California, United States of America
Abstract
Aims:
Genetic and pharmacological studies have shown that impairment of the nitric oxide (NO) synthase (NOS) pathway is
associated with hypertension and insulin-resistance (IR). In addition, inhibition of NOS by the endogenous inhibitor,
asymmetric dimethylarginine (ADMA), may also result in hypertension and IR. On the other hand, overexpression of
dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes ADMA, in mice is associated with lower
ADMA, increased NO and enhanced insulin sensitivity. Since DDAH carries a farnesoid X receptor (FXR)-responsive element,
we aimed to upregulate its expression by an FXR-agonist, INT-747, and evaluate its effect on blood pressure and insulin
sensitivity.
Methods and Results:
In this study, we evaluated the in vivo effect of INT-747 on tissue DDAH expression and insulin
sensitivity in the Dahl rat model of salt-sensitive hypertension and IR (Dahl-SS). Our data indicates that high salt (HS) diet
significantly increased systemic blood pressure. In addition, HS diet downregulated tissue DDAH expression while INT-747
protected the loss in DDAH expression and enhanced insulin sensitivity compared to vehicle controls.
Conclusion:
Our study may provide the basis for a new therapeutic approach for IR by modulating DDAH expression and/or
activity using small molecules.
Citation: Ghebremariam YT, Yamada K, Lee JC, Johnson CLC, Atzler D, et al. (2013) FXR Agonist INT-747 Upregulates DDAH Expression and Enhances Insulin
Sensitivity in High-Salt Fed Dahl Rats. PLoS ONE 8(4): e60653. doi:10.1371/journal.pone.0060653
Editor: Yu Huang, The Chinese University of Hong Kong, Hong Kong
Received December 31, 2012; Accepted March 1, 2013; Published April 4, 2013
Copyright: ß2013 Ghebremariam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants to JPC from the National Institutes of Health (NIH) (grant numbers RC2HL103400, 1U01HL100397,
K12HL087746 and 1R01EY02060901A1); the American Heart Association (AHA) (grant number 11IRG5180026); Stanford SPARK Translational Research Program;
and by the Tobacco-Related Disease Research Program of the University of California (grant number 18XT-0098). YTG was a recipient of the Stanford School of
Medicine Dean’s fellowship (grant number 1049528-149- KAVFB); and is currently supported by Tobacco-Related Disease Research Program of the University of
California (grant number 20FT-0090). Other than the listed authors, no individual employed or contracted by the sponsors played any role in: study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The INT-747 used in this study was provided by Intercept Pharmaceuticals. JPC and YTG are inventors on patents, owned by Stanford
University, that protect the use of agents that therapeutically modulate the DDAH/NOS pathway. This does not alter the authors’ adherence to all the PLOS ONE
policies on sharing data and materials.
* E-mail: john.cooke@stanford.edu
Introduction
Salt-sensitivity (SS), dysregulated changes in blood pressure (BP)
in response to salt-intake, is principally influenced by genetics, diet,
age and socio-economic factors and is strongly associated with
increased cardiovascular (CV) risk and mortality [1,2]. One of the
outcomes of high-salt consumption by salt-sensitive individuals is
an increase in BP that may be refractory to multiple antihyper-
tensive therapies [3]. Elevated BP increases the risk of coronary
heart disease and stroke [2,4]. In addition, high salt intake is
associated with renal calculi and other kidney diseases [4,5].
In this regard, the Dietary Approaches to Stop Hypertension
(DASH) study demonstrated a significant reduction in BP by
reducing salt-intake and/or by consuming the ‘‘DASH diet’’ [6]; a
good source of dietary nitrates which can be converted to nitric
oxide (NO) in vessel walls [7]. Further evidence that endogenous
NO regulates BP in humans comes from studies of the T-786C
polymorphism of the eNOS gene promoter, which reduces eNOS
activity, impairs endothelial function and increases the risk for
essential [8] and salt-induced hypertension (HTN) [9,10]. Endo-
thelium-derived NO is thought to play a crucial role in vascular
and metabolic homeostasis [11,12]. In pre-clinical models, genetic
disruption or pharmacological inhibition of NOS causes HTN and
insulin resistance (IR) [13–15].
The IR syndrome is manifested by compensatory hyperinsulin-
emia and is associated with increased CV risk [16,17]. The
association of IR with CV risk may be mediated by its adverse
effect on a variety of endothelial functions including those related
to vascular reactivity, structure, inflammation and thrombosis
[18]. In addition to its contribution to these endothelial
dysfunctions, a reduction in NO may also play a role in the
development of IR [15]. NO modulates insulin-mediated glucose
PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e60653
disposal as well as reactivity of vessels in insulin-sensitive tissues
[19]. Furthermore, mice with gene disruption of either endothelial
or neuronal NOS exhibit IR [13,20], possibly due to the
consequent reduction in microvascular recruitment and/or muscle
glucose uptake in response to insulin. Similarly, excessive
inhibition of NOS by its endogenous inhibitor, asymmetric
dimethylarginine (ADMA), is associated with IR [21]. In addition,
pre-clinical and human data suggest that salt sensitive HTN
(which is also associated with IR) may be mediated in part by
ADMA [22,23].
Plasma levels of ADMA are highly dependent upon the activity
of the enzyme dimethylarginine dimethylaminohydrolase (DDAH)
[24–26]. DDAH is present in all nucleated cells in one of two
isoforms [25]. DDAH is highly sensitive to oxidative stress [27],
and a number of CV risk factors impair its activity, leading to
elevated ADMA levels and impairment of the NOS pathway [25].
The DDAH1 transgenic mouse expresses greater DDAH activity
and has reduced plasma and tissue levels of ADMA [28]. As a
result, NOS activity is upregulated, plasma and urinary nitrogen
oxides are increased, and vascular resistance and mean arterial
pressure (MAP) are reduced [29]. Intriguingly, we have found that
the DDAH transgenic mouse has greater insulin sensitivity [28].
Accordingly, we hypothesized that pharmacological upregulation
of DDAH expression might enhance insulin sensitivity. Therefore,
we studied the effect of a farnesoid x receptor (FXR) agonist,
Obeticholic Acid (INT-747), on tissue DDAH expression and
insulin sensitivity in an animal model of salt-sensitive hypertension
[30,31]. The rationale for the use of an FXR agonist in this study
is based on the presence of putative FXR response element in the
DDAH1 promoter [32] and on previous studies that demonstrated
upregulation of DDAH expression using this approach [32–34].
FXR belongs to the family of nuclear receptors essential in the
regulation of lipids, glucose and bile acid. In this study, we found
that INT-747 upregulated liver DDAH1 expression and enhanced
insulin sensitivity in Dahl rats.
Materials and Methods
Animals and Experimental Design
Dahl salt-sensitive (SS/JrHsd) male 6-weeks old rats and low
(0.49% sodium chloride (NaCl)) and high-salt (8% NaCl) Teklad
Custom Research diets were all purchased from Harlan Labora-
tories (Indianapolis, IN). INT-747 was kindly provided by
Intercept Pharmaceuticals (Perugia, Italy). ADMA and nitrogen
oxides (NOx) were measured using kits from DLD Diagnostika
(Hamburg, Germany) and Assay Designs (Ann Arbor, MI)
respectively. Urinary albumin and creatinine measurement kits
were purchased from Exocell (Philadelphia, PA). Methylcellulose
and glucose were purchased from Sigma (St Louis, MO). Plasma
insulin was measured using the ultra-sensitive rat insulin ELISA kit
(Crystal Chem, Inc; Downers Grove, IL). Tail-cuff blood pressure
measurements were using the BP-2000 blood pressure analysis
system (Visitech Systems Inc; Apex, NC). Western blot antibodies
were purchased from suppliers described in the text. Histological
sectioning as well as standard H&E and Trichrome staining was
performed at Stanford University (Department of Comparative
Medicine).
Ethics Statement
The in vivo study adhered with the Guide for the Care and Use
of Laboratory Animals published by the US National Institutes of
Health (NIH Publication, 8th Edition, 2011). Isoflurane inhalation
(2%) was used in all the procedures involving anesthesia, prior to
performing these procedures, and was frequented as necessary to
assure adequate depth of anesthesia during procedures as
described below. The adequacy of the depth of anesthesia was
assured prior to procedures and maintained during procedures by
monitoring responsiveness to stimuli (such as footpad-pinch),
observing color changes to the ears and foot pads and monitoring
vital signs such as heart rate and breathing pattern. At the end of
the study period, the animals were sacrificed by cervical
dislocation under anesthesia following the American Veterinary
Medical Association (AVMA) guidelines on euthanasia. The study
was approved by Stanford’s Institutional Animal Care and Use
Committee (IACUC; permit #24045).
High salt-induced Hypertension
Initially, all animals (at 6-weeks age) were placed on a standard
rodent diet for a week. Baseline blood and urine samples were
collected and basal blood pressure (BP) was measured prior to
grouping the animals. Subsequently, the animals were randomized
into low (LS; n = 9) or high salt (HS) diet groups. Hypertension was
induced in the HS group by daily high-salt diet feeding and the
group was subdivided to receive one of two doses of INT-747: low
dose (10 mg/kg/day; n = 15) or high dose (30 mg/kg/day; n = 15)
in 1% methylcellulose; or vehicle (1% methylcellulose in distilled
water; n = 15) orally everyday for 6 weeks. In parallel, the LS
group also received 1% methylcellulose. BP was measured weekly
for the duration of the study as described below.
Hemodynamic measurements
Heart rate (HR) and BP were measured weekly in conscious
animals using a multi-channel noninvasive tail-cuff system as
illustrated in the schematic in Figure-1. Briefly, each animal was
placed in a restrainer attached to temperature-regulated BP-2000
platform. Subsequently, pre-calibrated manometer was attached
to the tail of each animal to record the BP and HR. Average values
of 10 consecutive recordings per animal were taken at each time
point for comparison.
Measurement of Pulmonary Arterial Pressure
Pulmonary arterial (PA) pressure was measured using an
invasive technique by passing a catheter through the jugular vein
into the PA. In brief, the animals were sedated by isoflurane
inhalation, ventilated using the TOPO volume/pressure small
animal ventilator (Kent Scientific Corp; Torrington, CT) and the
jugular vein was exposed using surgical scalpel in order to insert a
guiding catheter into the vein and then through the right atrium
and ventricle into the PA for pressure measurements. The values
were recorded in replicates and the average values were analyzed.
Glucose Challenge Test
At 5-weeks following the initiation of LS/HS feeding, the
animals were fasted overnight and baseline blood samples were
collected from tail vein prior to challenging them with glucose
solution (at 2 mg/g body weight) intraperitoneally. Blood samples
were collected using heparinized catheters every 30 minutes until
150 minutes post-challenge and blood glucose concentration was
measured using glucometer. Plasma was separated by centrifuging
the samples at 3,000 rpm for 15 minutes and was stored at 280uC
for insulin measurements.
Measurement of Insulin Concentration
Plasma insulin concentration was measured using an ELISA
colorimetric assay as per the supplier’s recommendations. Back-
ground absorbance (read at 630 nm) was subtracted from the
respective absorbencies (measured at 450 nm) and the sample
Upregulation of DDAH and Insulin Sensitivity
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plasma insulin concentration was calculated from a standard
curve.
Assessment of Insulin Sensitivity
The IR index, an indicator of insulin sensitivity, was calculated
as described before [35]. In brief, the glucose values during the
glucose challenge test above were averaged and expressed in mM.
Area of the glucose curve was then calculated as described
(60 min = 1 cm). The corresponding plasma insulin concentration
was also averaged and expressed in mU/mL to calculate the area
of the insulin curve. The IR index was then calculated by
multiplying the two areas [35].
Assessment of Renal Function
Renal function was assessed by measuring the volume of urine
output; urinary creatinine and albumin levels. Metabolic cages
were used to separate the feces from the urine and 24 hours urine
volume was measured in each animal. Urinary albumin concen-
tration was quantified using the Nephrat competitive ELISA assay
following the supplier’s recommendations. The sample albumin
concentration was estimated from a standard curve and was
expressed in mg/day after correlating with the 24 hours urine
output. In addition, the urinary creatinine was quantified using the
Creatinine Companion ELISA assay as per the instruction in the
kit. Sample creatinine concentration was obtained from a standard
curve and was expressed in mg/day. The urinary albumin-to-
creatinine ratio (UACR) was calculated from the above measure-
ments. Furthermore, renal fibrosis was assessed by the standard
Masson’s Trichrome stain.
Measurement of serum ADMA and NO
The ADMA concentration in serum samples at baseline and 6-
weeks post LS/HS-diet was measured using an ELISA assay as
described [36]. In parallel, the NO levels of the baseline and 6-
weeks samples were measured by ELISA as described by the
manufacturer. ADMA and total NO (NOx) levels in the samples
were calculated from the respective standard curves.
Measurement of Tissue DDAH Activity
DDAH activity in liver tissue homogenates was measured using
a stable isotope-based dilution assay as described [37]. In brief,
about 50 mg of the harvested liver tissues were homogenized in
Figure 1. Schematic showing the time frame and experimental protocol followed to induce hypertension by high-salt feeding.
Measurements period of BP, glucose challenge, urinary collection and blood draw are also shown. BP = blood pressure.
doi:10.1371/journal.pone.0060653.g001
Figure 2. High-salt diet progressively increases systemic blood pressure. Weekly measurement of: a) systolic blood pressure (SBP) and b)
heart rate (HR) by tail-cuff in Dahl salt-sensitive rats fed diet containing low salt (n= 9); high-salt and treated with vehicle (n = 15); high-salt and
treated with 10 mg/kg/day INT-747 (n = 15) or high-salt and treated with high dose of INT-747 at 30 mg/kg/day (n = 15). Data is expressed as
Mean6SEM. (*p,0.05 versus high-salt diet data. ANOVA followed by Bonferroni post-test).
doi:10.1371/journal.pone.0060653.g002
Upregulation of DDAH and Insulin Sensitivity
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PBS. Equal amounts of lysate were incubated with deuterium-
labeled substrate (10 mMof[
2
H
7
]-ADMA) in a 96-well microplate
for 1 hour at 37uC. Next, the reaction was stopped for
determination of [
2
H
7
]-ADMA by LC-tandem MS in the presence
of a double-isotope labeled ([
13
C
5
-
2
H
6
]-ADMA) internal standard.
DDAH activity was calculated by subtracting the remaining [
2
H
7
]-
ADMA from the amount added to the reaction and was expressed
in nmol/g protein/min as described [37].
Immunoblot Assay
After sacrificing the animals, tissues were harvested, snap frozen
and homogenized for the preparation of total lysate. Protein
concentration was measured using the Coomassie Plus BioRad
assay. Liver lysates from the different groups were SDS-PAGE
resolved and immunoblotted with anti- DDAH1 (Abcam) and JNK
(Sigma) antibodies. Protein content was normalized to b-actin
(ACTB; Sigma). Band intensities were compared using NIH’s
Image J analysis software (http://rsb.info.nih.gov/ij/docs/).
Histological Examination and Fibrosis
For the histology, paraformaldehyde-fixed kidney sections were
paraffin embedded and stained with H&E to assess tissue
morphology. In addition, the kidney sections were also stained
for fibrosis using Masson’s Trichrome stain. Multiple fields were
scanned microscopically by an expert renal pathologist who was
unaware of the treatment groups. The degree of fibrosis was
recorded as an estimate of the % of the renal cortex that was
affected by interstitial fibrosis.
Statistics
Statistical analysis to calculate the number of animals per study
group was performed using an advanced power and sample size
calculation (PS v3.0.14; Vanderbilt University). The study was
designed to detect a difference in means (d) of 0.2 with an
estimated standard deviation (s) of 0.18 at a significance level (a)
of 0.05 with 85% power (b). All other statistical tests, unless stated
otherwise, described in the study were performed using GraphPad
Prism (La Jolla, CA). Comparison between two samples was
performed using an unpaired student’s t-test and multiple samples
were compared using one-way ANOVA followed by Bonferroni
posttest correction. Data was considered statistically significant at
p value,0.05.
Results
The effect of INT-747 on high-salt diet induced
hypertension
A high-salt diet is known to increase mean arterial pressure
(MAP) in salt-sensitive animals [38] and humans [22,23].
Similarly, we observed that a HS-diet elevated systolic blood
pressure (SBP) (Figure-2a). The SBP progressively increased over
time reaching over 200 mm Hg after 4-weeks of HS-diet;
significantly higher than that of LS animals (p,0.05). The
treatment with INT-747 did not improve the BP. The HR was
not statistically different between the groups (Figure-2b).
The effect of high-salt diet on organ weight
The Dahl-SS animals are known to develop cardiac hypertro-
phy in response to HS-diet (http://www.harlan.com/). In this
study, high-salt diet induced cardiac and renal hypertrophy, an
effect that was not attenuated by INT-747 treatment (Figure-S1).
In addition, pulmonary congestion was increased in the animals
receiving HS and the lower dose of INT-747 (10 mg/kg) by
comparison to the LS group (Figure-S1). By contrast, at the
higher dose of INT-747 this effect was not observed, and there was
also a trend towards lower pulmonary arterial (PA) pressure in this
group (Figure-S2).
The effect of INT-747 on renal function
In the salt-sensitive Dahl rat, a high-salt diet induces nephropathy
as manifested by albuminuria. In our study, assessment of renal
function showed that HS-diet profoundly impaired renal function as
shown by significant increases in urinary albumin and creatinine
values. In addition, the UACR analysis confirmed renal impairment
in all the HS groups (Figure-S3). In addition, HS-diet increased
kidney weight, an effect that was not regulated by INT-747 (Figure-
S1). Furthermore, INT-747 did not improve renal pathology in the
HS-fed animals. In all HS animals, severe thrombotic microangi-
opathy (TMA) was observed as well as fibrinoid necrosis of afferent
arterioles with an onion-skin pattern of periarterial fibrosis and
extravasation of erythrocytes (Figure-3a).
Evaluation of the corresponding Trichrome stained kidney
sections showed tubular atrophy and interstitial fibrosis affecting
20–30% of the cortical area in the HS-diet fed animals compared
to minimal (below 5%) tubular atrophy and fibrosis seen in the LS-
diet group (Figure-3b).
Figure 3. Assessing renal morphology and fibrosis. a: H&E staining to assess the renal morphology of Dahl rats following: LS-diet with nearly
normal morphology (A) or HS-diet and administration of vehicle, showing fibrinoid arteriolar necrosis with extravasation of erythrocytes and
thrombosis of glomerular capillaries (B); INT-747 at 10 mg/kg/day shows similar findings (C) as does INT-747 at 30 mg/kg/day (D) for 6 weeks.
Representative images are shown. (400X mag). b: Masson’s Trichrome staining to evaluate renal fibrosis (seen as blue-colored expansion of the
interstitium between the tubules) in Dahl rat kidneys following: LS-diet (A) or HS-diet and administration of vehicle (B); INT-747 at 10 mg/kg/day (C)
or INT-747 at 30 mg/kg/day (D) for 6 weeks. Representative images are shown. (100X mag).
doi:10.1371/journal.pone.0060653.g003
Upregulation of DDAH and Insulin Sensitivity
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The effect of high-salt diet on DDAH protein expression
and activity
Our western blot analysis indicated that high-salt diet down-
regulated DDAH expression by almost 50% in the liver of HS-fed
group receiving vehicle. By contrast, treatment with INT-747
reversed the salt-induced downregulation of DDAH expression
(Figure-4). Despite the increase in liver DDAH protein expres-
sion following INT-747 treatment, liver DDAH activity of INT-
747 group remained similar to the enzymatic activity in the tissues
derived from the LS and vehicle groups (Figure-S4). It is possible
that the oxidative stress that accompanies high-salt diet [39] could
reduce DDAH expression and activity, as DDAH is highly
sensitive to oxidative stress [27]. Such an effect on DDAH might
offset the effect of INT-747 to increase DDAH expression.
In addition, INT-747 reduced expression of liver JNK-1 and
JNK-2 (Figure-S5); proinflammatory proteins that may be
upregulated by high-salt diet to interfere with normal insulin
signaling [39].
The effect of INT-747 on ADMA and NO concentration
Serum ADMA and NO concentration remained unchanged
between the baseline and 6-weeks study period in the HS-fed
Figure 4. The effect of INT-747 treatment on DDAH1 expression in Liver. Animals were fed low (control)- or high-salt diet and treated with
vehicle or INT-747 at 10 or 30 mg/kg/day for 6 weeks. Liver lysates were compared for DDAH1 expression by Western blot. rDDAH1: purified
recombinant human DDAH1 described previously [53]. The DDAH1 expression was normalized to b-Actin (ACTB). DDAH = dimethylarginine
dimethylaminohydrolase. Data is expressed as Mean6SEM. (*p,0.05 versus control value. ANOVA followed by Bonferroni post-test).
doi:10.1371/journal.pone.0060653.g004
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animals despite the treatment group. Both ADMA and NO (NOx)
levels were not favorably affected by treatment with INT-747 in
this model (Figure-5).
The effect of high-salt diet on insulin sensitivity
In addition to being hypertensive, Dahl rats are known to be
insulin resistant with an exacerbation in their IR after HS-feeding
for about 4 weeks [39,40]. In the current study, a glucose challenge
increased blood glucose to a greater degree in HS animals, an
effect which was blunted by INT-747 (Figure-6a). Similarly, after
glucose challenge, there was a greater initial increase in insulin
levels in the HS animals, which did not persist at later time points
in the INT-747 group (Figure-6b).
The IR index was increased by HS-diet, (p,0.05), indicating
deterioration in insulin sensitivity. This effect of HS-diet was
reduced by INT-747; specifically the IR index was respectively
reduced by 22.6% and 23.6% in the 10 mg/kg and 30 mg/kg
INT-747 treated HS animals compared to the vehicle-treated HS
animals, and was not statistically different compared to the LS-
group (p.0.05). These results indicated that INT-747 improved
insulin sensitivity (Figure-6c).
Discussion
Novelty and Significance:
The simultaneous effect of INT-747, a small molecule FXR
agonist, on blood pressure and insulin sensitivity has not been
studied before in Dahl Rats; an animal model that displays both
systemic hypertension and IR. Our study addressed the scientific
curiosity of whether it is possible to concurrently modulate both
BP and insulin sensitivity using INT-747. We have also addressed
the effect of high-salt diet on DDAH expression. Our key findings
are: 1) INT-747 does not reduce systemic or pulmonary vascular
pressure in high-salt fed Dahl rats. 2) INT-747 induces hepatic
DDAH expression and enhances insulin sensitivity in this animal
model as described below.
Role of DDAH/ADMA/NO in salt-sensitive hypertension
and insulin resistance. There is mounting pharmacological and
genetic evidence that implicate the NOS/ADMA/DDAH pathway
in salt-sensitive HTN and IR. For example, studies have shown that
high salt intake or increased salt retention impairs NO production
[41,42] and as a result endothelial function is blunted [43], causing
an increase in mean arterial pressure (MAP) in pre-clinical models
[38,44] and in patients [45]. On the other hand, NO regulates salt-
sensitivity [46] by blocking the entry of Na
+
/Cl
2
into the thick
ascending limb of the loop of Henle; a segment responsible for up to
30% of salt reabsorption [47], and by inhibiting the reabsorption of
Na
+
in cortical collecting ducts [48]. Furthermore, inhibition of NO
using a NOS inhibitor reduces salt excretion, glomerular filtration
rate (GFR) and diuresis [46], which is reversed by co-administration
of the NO precursor L-arginine [49,50], indicating the role of this
pathway in vascular and renal pathophysiology. In addition, ADMA
is elevated in patients with HTN and IR and is implicated in the
progression of salt-sensitive HTN [21–23,51]. In a preclinical study,
overexpression of DDAH is associated with enhanced insulin
sensitivity [28]. In addition, pharmacological manipulation of
DDAH using FXR agonists is known to reduce ADMA [32,34]
and improve insulin sensitivity in humans (http://www.
interceptpharma.com/) suggesting that DDAH may be an attractive
target to improve insulin sensitivity [28,52]. In fact, we have been
carrying out efforts to discover and validate agents that enhance
DDAH activity using the method we described [53].
INT-747 does not favorably influence tissue DDAH
activity/circulating ADMA/NO levels, nor reverse salt-
sensitive HTN. In the present study, we evaluated the
pharmacological potential of INT-747 in regulating BP and
insulin sensitivity in Dahl rats; a widely used pre-clinical model for
HTN and IR [30,31]. As an FXR agonist and an analogue of a
primary human bile acid (chenodeoxycholic acid) and other cholic
acid derivatives which are involved in modulating lipid, glucose
and bile acid homeostasis, INT-747 has been clinically shown to
improve liver function in patients with primary biliary cirrhosis
(http://www.interceptpharma.com/) and is being evaluated for
the treatment of nonalcoholic steatohepatitis (NASH) (http://
www.interceptpharma.com/); a disease often associated with IR
[54]. In our study, HS-diet significantly increased BP. Treatment
with INT-747 did not significantly lower systemic or pulmonary
arterial (PA) pressure. This finding may not be surprising in light of
the lack of significant changes in tissue DDAH activity, circulating
ADMA and NO levels following INT-747 treatment. In addition,
we did not see significant INT-mediated improvement in the
cardiac and renal hypertrophy that was caused by HS-diet feeding.
INT-747 effects on renal function. It is known that Dahl
salt-sensitive animals develop nephropathy and albuminuria in
response to HS- diet (http://www.harlan.com/). We found that a
high-dose of INT-747 tended to improve renal function in HS
animals as demonstrated by a reduction in albuminuria and
urinary creatinine. Previous studies have demonstrated that INT-
Figure 5. Measurements of serum ADMA and NO in Dahl rats at baseline and 6-weeks after feeding high-salt diet in the presence of
vehicle or INT-747 treatment. NO and ADMA were measured as described in the text. Data is expressed as Mean6SEM. NO = nitric oxide;
ADMA = asymmetric dimethylarginine.
doi:10.1371/journal.pone.0060653.g005
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747 ameliorates nephropathy in animal models of diabetes (types I
& II), associated with reductions in hyperlipoproteinemia, fibrosis,
proteinuria, inflammation, and oxidative stress [55,56]. Moreover,
in 5/6 nephrectomized ApoE-deficient mice, INT-747 reduces
chronic kidney disease (CKD)-induced vascular calcification
independent of atherosclerosis progression [57]. However, our
study of renal histology showed comparable TMA between the
INT-747 and vehicle treated groups.
INT-747 favorably effects hepatic DDAH expression but
not activity. More intriguingly, we demonstrate that HS-diet
significantly downregulated hepatic DDAH1 expression (by about
50%). However, treatment with INT-747 protected the loss in
hepatic DDAH1. Our observations are similar to those in an animal
model of bile duct ligation (BDL) injury where treatment with INT-
747 increases hepatic DDAH1 expression and improves portal
pressure [34,58]. Indeed, a small clinical study evaluating the
therapeutic potential of INT-747 in regulating portal hypertension
in patients with alcoholic cirrhosis has just completed (http://www.
ukctg.nihr.ac.uk/trialdetails/ISRCTN22662520). Although INT-
747 induced hepatic DDAH expression likely due to the presence of
a putative FXR response element in the DDAH1 promoter [32], it
did not favorably influence DDAH activity, nor did it favorably
influence circulating ADMA and NO levels in this model.
INT-747 improves insulin sensitivity. Salt loading aggra-
vates insulin resistance in Dahl rats; an animal model that
inherently develops IR at weaning and prior to salt-loading
[39,40]. INT-747 enhanced insulin sensitivity in these animals as
demonstrated by the reduction in IR index. It is possible that this
effect is mediated in part by an increase in hepatic DDAH protein
expression. However, the improvement in insulin sensitivity may
be independent of any effect of INT-747 on DDAH. Indeed, it is
likely that INT-747 enhances insulin sensitivity in part by
regulating glucose homeostasis through other FXR-mediated
effects [59-61]. Therefore, further mechanistic studies are justified
to delineate the precise contribution of the DDAH pathway to bile
acid-mediated glucose homeostasis system in this model. Mean-
while, data from a double blinded placebo controlled clinical study
indicates that INT-747 improves insulin sensitivity (http://www.
interceptpharma.com/) in nonalcoholic fatty liver disease
(NAFLD) patients with type II diabetes.
Conclusion. The pharmacological potential of INT-747 was
evaluated in an animal model of both dietary salt-induced
hypertension and IR. This study reveals that it is unlikely for
INT-747 to be a dual- or poly- pharmacologic agent for the
treatment of systemic hypertension or pulmonary hypertension
and IR. Our study provides a valuable insight in that INT-747
may not be simultaneously used as an insulin sensitizer and
antihypertensive agent. However, it has a promising potential to
enhance insulin sensitivity in hypertensive patients.
Supporting Information
Figure S1 Measurement of organ weights for: a) heart
b) lungs c) liver, and d) kidneys. Data is normalized organ
Figure 6. Blood glucose and insulin measurements to assess insulin sensitivity: Measurement of a) blood glucose \and b) plasma
insulin concentration over time during GTT in Dahl rats fed with low or high-salt diet for 5-weeks prior to the glucose challenge
test. Values are Mean 6SEM for: Control (n = 6); Vehicle (n = 7); INT-10 mg/kg/day (n = 5) and INT-30 mg/kg/day (n= 9). *p,0.05 versus high-salt
diet data. ANOVA followed by Bonferroni post-test. GTT = glucose tolerance test. In c), the effect of INT-747 treatment on insulin sensitivity is shown.
Insulin Resistance (IR) index was calculated as described in the text. Data is expressed as Mean6SEM. (*p,0.05 versus low-salt diet data. ANOVA
followed by Bonferroni post-test). LS = low salt; HS = high salt; V = vehicle; INT-10 = INT-747 at 10 mg/kg/day and INT-30 = INT-747 at 30 mg/kg/day.
doi:10.1371/journal.pone.0060653.g006
Upregulation of DDAH and Insulin Sensitivity
PLOS ONE | www.plosone.org 7 April 2013 | Volume 8 | Issue 4 | e60653
weight to the respective body weight at time of sacrifice. Lungs and
kidneys weight was combined total weight for the right and left
tissues. Data is expressed as Mean6SEM. (*p,0.05 versus low-salt
diet data. ANOVA followed by Bonferroni post-test).
(TIF)
Figure S2 Assessing the effect of INT-747 on Pulmonary
Arterial (PA) pressure. Animals were intubated and catheter
was inserted into the PA for pressure measurement in low salt,
vehicle, INT-747 (10 mg/kg/day) and INT-747 (30 mg/kg/day)
groups. Data is expressed as Mean6SEM.
(TIF)
Figure S3 Assessment of renal function by measuring
urinary creatinine and albumin. The urinary creatinine and
albumin values were normalized to 24 h urine output and the
urinary-albumin-to-creatinine-ratio (UACR) was calculated as
described in the text. Data is expressed as Mean6SEM. (*p,0.05
versus low-salt diet data. ANOVA followed by Bonferroni post-test).
(TIF)
Figure S4 Assessment of tissue DDAH activity using a
stable-isotope assay in liver lysates of Dahl rats
following: LS-(n = 9) or HS- diet and administration of
vehicle (n = 8) or INT-747 at 10 mg/kg/day (n = 8) or at
30 mg/kg/day (n = 9) for 6 weeks. Data is from triplicate
experiments and is expressed as Mean6SEM. (p.0.05 among all
groups. ANOVA followed by Bonferroni post-test).
(TIF)
Figure S5 The effect of INT-747 treatment on the
expression of c-JNK isoforms 1 and 2 in the Liver. Animals
were fed low (control)- or high-salt diet and treated with vehicle or
INT-747 at 10 or 30 mg/kg/day for 6 weeks. Liver lysates were
compared for c-JNK1 and 2 expression by Western blot. The c-JNK
expression was normalized to b-Actin (ACTB). C-JNK = c-Jun N-
terminal Kinase. Data is expressed as Mean6SEM. (*p,0.05
versus the data of low-salt or high-salt diet and INT-747 treated
with either dose. ANOVA followed by Bonferroni post-test).
(TIF)
Acknowledgments
The authors are grateful to Intercept Pharmaceuticals for kindly providing
INT-747 for the study. We are also grateful to the laboratory of Dr Philip
Tsao (Stanford) for providing us access to their glucometer and tissue
homogenizer equipments. We thank Stanford University Cardiovascular
Institute for overall support and the Department of Comparative Medicine
for immunohistochemical stainings.
Author Contributions
Conceived the idea, designed experiments, involved in data collection, data
analysis and interpretation: YTG KY JPC. Involved in data collection: JCL
CLCJ RA. Involved in data collection and critical revision of manuscript:
DA MA RHB. Involved in data collection and data analysis: JPH. Provided
financial support and final approval of manuscript: AJP JPC.. Conceived
and designed the experiments: YTG KY JPC. Performed the experiments:
YTG KY RA CLCJ JCL RA DA MA JPH. Analyzed the data: YTG KY
DA MA RA JPH JPC. Contributed reagents/materials/analysis tools:
YTG KY DA MA RHB JPH AJP JPC. Wrote the paper: YTG JPC.
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Upregulation of DDAH and Insulin Sensitivity
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    • "While early FXR Activation Limits Intestinal IRI translational studies were focused on the central role of FXR as a chief regulator of bile acid and lipid metabolism, the hepatic involvement of FXR and the beneficial effects of FXR agonists on hepatic inflammation and fibrosis were subsequently illustrated in both animal models of non-alcoholic fatty liver disease and toxic cirrhosis, amongst others [23][24][25][26]. Since then, the body of literature on FXR has rapidly expanded to extrahepatic compartments, such as vasculature (more specifically systemic and liver sinusoidal endothelium), lung (pulmonary arterial hypertension), kidney and the immune system [16,25,[27][28][29][30][31][32][33]. Comparably, only a relatively small number of studies assessed the function of FXR in the intestine [8,10,13,[34][35][36][37]. Recent studies revealed that FXR-knockout mice display a chronic IBD-like phenotype with impaired intestinal mucosal integrity leading to increased intestinal permeability and bacterial translocation from the gut [10,35,37]. "
    [Show abstract] [Hide abstract] ABSTRACT: Introduction The farnesoid X receptor (FXR) is abundantly expressed in the ileum, where it exerts an enteroprotective role as a key regulator of intestinal innate immunity and homeostasis, as shown in pre-clinical models of inflammatory bowel disease. Since intestinal ischemia reperfusion injury (IRI) is characterized by hyperpermeability, bacterial translocation and inflammation, we aimed to investigate, for the first time, if the FXR-agonist obeticholic acid (OCA) could attenuate intestinal ischemia reperfusion injury. Material and Methods In a validated rat model of intestinal IRI (laparotomy + temporary mesenteric artery clamping), 3 conditions were tested (n = 16/group): laparotomy only (sham group); ischemia 60min+ reperfusion 60min + vehicle pretreatment (IR group); ischemia 60min + reperfusion 60min + OCA pretreatment (IR+OCA group). Vehicle or OCA (INT-747, 2*30mg/kg) was administered by gavage 24h and 4h prior to IRI. The following end-points were analyzed: 7-day survival; biomarkers of enterocyte viability (L-lactate, I-FABP); histology (morphologic injury to villi/crypts and villus length); intestinal permeability (Ussing chamber); endotoxin translocation (Lipopolysaccharide assay); cytokines (IL-6, IL-1-β, TNFα, IFN-γ IL-10, IL-13); apoptosis (cleaved caspase-3); and autophagy (LC3, p62). Results It was found that intestinal IRI was associated with high mortality (90%); loss of intestinal integrity (structurally and functionally); increased endotoxin translocation and pro-inflammatory cytokine production; and inhibition of autophagy. Conversely, OCA-pretreatment improved 7-day survival up to 50% which was associated with prevention of epithelial injury, preserved intestinal architecture and permeability. Additionally, FXR-agonism led to decreased pro-inflammatory cytokine release and alleviated autophagy inhibition. Conclusion Pretreatment with OCA, an FXR-agonist, improves survival in a rodent model of intestinal IRI, preserves the gut barrier function and suppresses inflammation. These results turn FXR into a promising target for various conditions associated with intestinal ischemia.
    Full-text · Article · Jan 2017
    • "GW4064 upregulates both DDAH-1 and the cationic transporter CAT-1, leading to increased ADMA cellular uptake and metabolism, and decreased serum ADMA concentrations [15,90]. Another FXR agonist, INT-747, can increase DDAH-1 expression [91,92], however this is not associated with significant changes in ADMA and NO concentrations [91]. The lipid lowering drug probucol also lowers ADMA concentrations [93] via up-regulation of both DDAH expression and activity, and down-regulation of PRMT-1 expression [78,94]. "
    [Show abstract] [Hide abstract] ABSTRACT: Dimethylarginine dimethylaminohydrolase (DDAH) is a highly conserved hydrolytic enzyme found in numerous species, including bacteria, rodents, and humans. In humans, the DDAH-1 isoform is known to metabolize endogenous asymmetric dimethylarginine (ADMA) and monomethyl arginine (l-NMMA), with ADMA proposed to be a putative marker of cardiovascular disease. Current literature reports identify the DDAH family of enzymes as a potential therapeutic target in the regulation of nitric oxide (NO) production, mediated via its biochemical interaction with the nitric oxide synthase (NOS) family of enzymes. Increased DDAH expression and NO production have been linked to multiple pathological conditions, specifically, cancer, neurodegenerative disorders, and septic shock. As such, the discovery, chemical synthesis, and development of DDAH inhibitors as potential drug candidates represent a growing field of interest. This review article summarizes the current knowledge on DDAH inhibition and the derived pharmacokinetic parameters of the main DDAH inhibitors reported in the literature. Furthermore, current methods of development and chemical synthetic pathways are discussed.
    Full-text · Article · May 2016
    • "HFD rats showed hyperinsulinemia as a result of impaired insulin sensitivity and CDCA reversed the hyperinsulinemia due to the improvement in the insulin resistance. Earlier reports show, FXR activation using other FXR agonists, INT-747 and GW4064 improved insulin peripheral sensitivity in mice and Zucker obese rats, respectively (Cariou et al., 2006; Ghebremariam et al., 2013). We further evaluated the effect of CDCA on Ser307 phosphorylation of Insulin receptor substrate 1 (IRS-1), a molecular marker of insulin resistance. "
    [Show abstract] [Hide abstract] ABSTRACT: Adipose tissue secretes adipokines that regulate insulin sensitivity in adipocytes and other peripheral tissues critical to glucose metabolism. Insulin resistance is associated with severe alterations in adipokines characterized by release of increased pro-inflammatory cytokines and decreased anti-inflammatory cytokines from adipose tissue. The role of Farnesoid X receptor (FXR) activation on adipokines in relation to adipose tissue inflammation and insulin resistance is not completely explored. For the first time, we have evaluated the ability of Chenodeoxycholic acid (CDCA), an endogenous FXR ligand, in restoring the disturbance in adipokine secretion and insulin resistance in palmitate treated 3T3-L1 cells and adipose tissues of High fat diet (HFD) rats. CDCA suppressed several of the tested pro-inflammatory adipokines (TNF-α, MCP-1, IL-6, Chemerin, PAI, RBP4, resistin, vaspin), and enhanced the major anti-inflammatory and insulin sensitizing adipokines (adiponectin, leptin). CDCA suppressed the activation of critical inflammatory regulators such as NF-κB and IKKβ which are activated by palmitate treatment in differentiated cells and HFD in rats. We show the altered adipokines in insulin resistance, its association with inflammatory regulators, and the role of CDCA in amelioration of insulin resistance by modulation of adipokines. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Full-text · Article · Jul 2015
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