European Journal of Pharmacology 765 (2015) 591597

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Insulin sensitizing and cardioprotective effects of Esculetin and

Telmisartan combination by attenuating Ang II mediated vascular
reactivity and cardiac brosis
Almesh Kadakol, Anuradha Pandey, Santosh Kumar Goru, Vajir Malek,
Anil Bhanudas Gaikwad n
Laboratory of Molecular Pharmacology, Department of Pharmacy, Birla Institute of Technology and Science Pilani, Pilani Campus, Rajasthan 333031, India

art ic l e i nf o

a b s t r a c t

Article history:
Received 18 June 2015
Received in revised form
19 September 2015
Accepted 22 September 2015
Available online 25 September 2015

The combination of the angiotensin receptor blockers (ARBs) with other synthetic and natural molecules
has been reported to have better safety prole and therapeutic efcacy in prevention of diabetes and its
associated complications than their monotherapy. Driven by the aforementioned facts, this study was
conceived to evaluate the potential additive effect of combination of Telmisartan and Esculetin in prevention of insulin resistance and associated cardiac brosis. Recently, we have reported that Esculetin
prevented cardiovascular dysfunction associated with insulin resistance (IR) and type 2 diabetes. Insulin
resistance was developed by high fat diet (HFD) feeding to Wistar rats. Telmisartan and Esculetin were
administered at 10 mg/kg/day and 50 mg/kg/day doses (P.O, 2 weeks), respectively either alone or in
combination. Plasma biochemical analyses, vascular reactivity and immunohistochemical experiments
were performed to assess the benecial effect of Telmisartan, Esculetin and their combination on insulin
resistance and associated cardiac brosis. The study results showed that, co-administered Telmisartan
and Esculetin ameliorated the pathological features like metabolic perturbation, morphometric alterations, vascular hyper responsiveness, extracellular matrix accumulation and the expression of bronectin
and TGF- more effectively than monotherapy in HFD fed rats. Hence, the study urges us to conclude that
the solution to IR and associated cardiovascular dysfunction may lie in the Telmisartan and Esculetin
combination therapy.
& 2015 Elsevier B.V. All rights reserved.

Keywords:
Insulin resistance
Esculetin
Telmisartan
Cardiac brosis
Ang II
TGF-

1. Introduction
Insulin resistance (IR), involving attenuated responsiveness of tissues (adipocytes, muscle and liver) towards insulin leads to compensatory increment in its secretion culminating into pathogenesis of type
2 diabetes, cardiovascular diseases and non-alcoholic fatty liver disease
(Perry et al., 2014). IR triggers several downstream molecules (e.g.
insulin receptor substrate, PI3K, and Akt), activates renin angiotensin
system (RAS), augments oxidative stress, and immune response in
cardiovascular diseases (Kalupahana and Moustaid Moussa, 2012).
Thus, the use of angiotensin receptor blockers (ARBs) play an important role in controlling IR and associated complications like hypertension, obesity and diabetes (Kurtz and Pravenec, 2004).
Telmisartan, an AT1 receptor antagonist and PPAR- partial
agonist, controls inammatory cascades involved in progression of
diabetic complications (Yamagishi et al., 2007). However,
n

Corresponding author. Tel.: 91 1596 515717; fax: 91 1596 244183.

E-mail address: anil.gaikwad@pilani.bits-pilani.ac.in (A.B. Gaikwad).

http://dx.doi.org/10.1016/j.ejphar.2015.09.035
0014-2999/& 2015 Elsevier B.V. All rights reserved.

combination with other molecules improves the protective effects

of Telmisartan. For instance, Telmisartan and Aspirin combination
shows better renoprotective effect in type 1 diabetes by attenuating expression of inammatory markers than their monotherapy
(Mulay et al., 2010). A clinical trial demonstrated that combination
of Telmisartan with Ramipril enhanced its cardioprotective effect
by improving the left ventricular diastolic indices and reducing
plasma brain natriuretic peptide levels in the diabetic patients
(Symeonides et al., 2007). The prophylactic treatment with Telmisartan and Resveratrol combination was reported to enhance
the renoprotective effect by reducing blood pressure, proteinuria
and transforming growth factor- (TGF-) expression more effectively than monotherapy in the murine model of diabetic nephropathy (Abd Allah and El-Debakey, 2010). Telmisartan and
Amlodipine combination attenuated vascular dysfunction associated with type 1 diabetes (Mollnau et al., 2013) and Irbesartan
and Curcumin combination improves insulin sensitivity and reduces inammation associated with type 1 diabetes more efciently than monotherapy (Mahfouz, 2010).

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A. Kadakol et al. / European Journal of Pharmacology 765 (2015) 591597

Thus, the combination of molecules which target the pathogenesis of insulin resistance and associated cardiovascular dysfunction may be a more appropriate therapeutic approach than
the conventional monotherapy. Esculetin (6, 7-dihydroxycoumarin), a functional food ingredient found in medicinal plants [e.g.
Artemesia capillaries, Citrus limonia, and Euphorbia lathyris] is used
in various inammatory, allergic and infectious diseases (Chang
et al., 1996; Sim et al., 2015). It has been reported to have a potent
anti-oxidant (Kadakol et al., 2015a), anti-inammatory (Kim et al.,
2014) and anti-proliferative (Kim and Lee, 2015) activity. In addition, esculetin did not have any mutagenic or genotoxic effect in
cultured human lymphocytes (Maistro et al., 2015). Esculetin also
shows a potential therapeutic role in cancer (Cho et al., 2015),
obesity (Kim and Lee, 2015; Sim et al., 2015), and diabetes (Surse
et al., 2011). Recently, we have reported that Esculetin shows
cardioprotective effect by reversing post translational histone
modications associated with IR and type 2 diabetes (Kadakol
et al., 2015b). We have also demonstrated that Esculetin improved
vascular responsiveness and reduced the expression of Ang II receptors in thoracic aorta of hyperglycaemic and hyperinsulinemic
rats (Kadakol et al., 2015a). Hence, we hypothesized that Telmisartan and Esculetin combination may be the better therapeutic
approach than the individual drug treatment regimen to ameliorate IR and associated cardiovascular dysfunction.

2. Materials and methods

2.1. Chemicals
The spectrophotometric kits were purchased from Accurex
Biomedical Pvt. Ltd. (Mumbai, Maharashtra, India) and ultra-sensitive rat insulin kit was obtained from Crystal Chem (Downers
Grove, IL, USA). The antibodies against Fibronectin, and TGF-
were obtained from Santa Cruz Biotechnology (Dallas, TX, USA)
and Keap1 antibody was purchased from Cell Signaling Technology
(Danvers, MA, USA). All the other chemicals were purchased from
Sigma (St. Louis, MO, USA), unless otherwise mentioned.

2015a, 2015b). Hence, in the present study we have used lower

dose of Esculetin (50 mg/kg/day) to evaluate its additive effect
with combination of Telmisartan in prevention of IR and associated cardiac brosis. After 4 weeks HFD feeding, the HFD fed rats
were treated either with Esculetin (50 mg/kg/day, P.O) or Telmisartan (10 mg/kg/day, P.O) (Ohmura et al., 2012) or Telmisartan
and Esculetin combination or vehicle (0.5% w/v sodium carboxy
methyl cellulose) for 2 weeks, during the treatment period animals were allowed to fed on their respective dietary regimens. The
body weight, biochemical estimations and blood pressure measurements were performed at the end of the study.
2.4. Assessment of insulin resistance and diabetes
The blood samples were collected, plasma was separated and
fasting plasma glucose (PGL), triglycerides (PTGs), total cholesterol
(PTC), and insulin (PI) were estimated as per manufacturers instructions by using commercially available spectrophotometric
kits. Insulin was estimated by ultra-sensitive rat insulin kit (Kadakol et al., 2015a, 2015b).
2.5. Assessment of vascular reactivity
At the end of 2 weeks' treatment period, all the animals were
sacriced and vascular reactivity experiments were performed as
described previously (Kadakol et al., 2015a). Briey, aortic ring was
suspended using a pair of stainless steel hooks in water-jacketed
organ bath (UGO Basile, Varese, Italy) and cumulative contractile
responses to increasing concentrations of Ang II (1 nM to 30 M)
were measured isometrically by using force transducer (7005-F,
UGO Basile) and the concentration-response curve (CRC) was recorded. Acetylcholine mediated relaxation was studied by precontracting aortic rings with 100 nmol/L phenylephrine (PE) after
which the CRC to acetylcholine (1 nM to 30 mM) was recorded. The
responses were normalized to cross sectional area of the tissue
and the tension developed was calculated as described before
(Kadakol et al., 2015a).
2.6. Histopathology and immunohistochemistry (IHC)

2.2. Animal studies

The male adult Wistar rats (160180 g), procured from the
central animal facility, Birla Institute of Technology and Science
Pilani (BITS Pilani) were maintained under standard environmental conditions and provided with feed and water ad libitum.
The animal protocol [Approval Number: IAEC/RES/17/03/Rev-2/19/
35] is in accordance with the guidelines of the Committee for the
Purpose of Control and Supervision of Experiments on Animals
(CPCSEA), Ministry of Social Justice and Environment, Government
of India. Rules of CPCSEA are laid down as per Institute of Laboratory Animal Resources (Washington, DC, USA) guidelines and
prior permit was sought from the Institutional Animal Ethics
Committee (IAEC), BITS Pilani, for conducting the study.
2.3. Development of non-genetic animal model for insulin resistance
and drug treatment
Insulin resistance (IR) was induced by high fat diet (HFD)
feeding, as per the protocol described previously (Kadakol et al.,
2015a). Briey, the rats were allocated to two dietary regimens:
Normal control (NC); fed with normal pellet diet (NPD, n 6) and
insulin resistant group; fed with HFD (n 24) ad libitum for six
weeks, respectively. Recently, we have studied Esculetin at two
doses (50 and 100 mg/kg/day) against cardiovascular dysfunction
associated with IR and type 2 diabetes and it showed batter cardiovascular protective effect at 100 mg/kg/day dose (Kadakol et al.,

Histopathology and immunohistochemistry were performed as

per the protocol described earlier (Kadakol et al., 2015a, 2015b). In
brief, heart tissue samples from each rat were collected and xed
in 10% (v/v) formalin in phosphate buffered saline and embedded
in parafn. Section with 5 mm thick were taken and stained with
Picro-Sirius red (PSR) to quantify extracellular matrix (ECM) accumulation. Immunohistochemistry was performed using antikelch ECH associated protein 1 (Keap1), anti-transforming growth
factor-beta (TGF-) and anti-bronectin [rabbit, 1:50 (v/v) dilution] as primary antibody, and horseradish peroxidase (HRP)
conjugated anti-rabbit as secondary antibody. Finally detected by
chromogenic substance i.e. diaminobenzidine (DAB). For each
group at least 25 sections were observed under Olympus BX41
microscope (Melville, NY, USA) and images were captured. The
images were analyzed semi-quantitative by using Image J software
for measurement of % extracellular matrix (ECM) accumulation
(PSR) and % DAB positive area (IHC) and nal data were analyzed
by GraphPad 5 Prism software (Kadakol et al., 2015b).
2.7. Statistical analysis
Experimental values were expressed as mean 7 S.E.M. Statistical comparison between different groups was performed using
one way analysis of variance (ANOVA). If F value is signicant then
multiple comparisons was done by Tukey test using Prism software (version 5.0; GraphPad, San Diego, CA, USA) for Windows.

A. Kadakol et al. / European Journal of Pharmacology 765 (2015) 591597

Cumulative concentration response curves were analyzed for pD2

value and maximal contraction (Emax), and statistical differences
between the means were determined by one-way ANOVA followed by Tukey test. Data was considered statistically signicant if
p o0.05.

3. Results
3.1. Combination of Telmisartan with Esculetin ameliorated the
metabolic perturbation
The signicantly elevated levels of plasma glucose, triglycerides, total cholesterol and insulin in high fat diet (HFD) fed rats
compared with normal control rats were evidently suggest the
insulin resistance condition (Table 1). Esculetin and Telmisartan
monotherapy was unable to reduce the exacerbated level of PGL
and PTGs, while PTC level was signicantly reduced compared to
HFD fed rats by monotherapy. In contrast to this, increased PGL,
PTGs, and PTC levels were controlled to a remarkable extent by
Telmisartan and Esculetin combination. Further, it was noted that
Esculetin alone at 50 mg/kg/day dose did not show any signicant
reduction in insulin level, whereas treatment with Telmisartan
and Esculetin combination markedly reduced the insulin level
compared to HFD fed rats, indicating that a better insulin sensitivity can be achieved by combination therapy (Table 1).
3.2. Telmisartan and Esculetin combination prevented morphometric
alterations associated with insulin resistance
At the end of study animals were sacriced and body weight
(BW), heart weight (HW) and relative heart weight [(HW/BW)
*1000] were measured. The HFD fed rat showed signicant increased in BW and HW as compared to normal control. The rats
treated with Esculetin alone didnot show any signicant improvement in this morphometric alterations as compared to HFD
fed rats, while treatment with Telmisertan alone reduced BW
signicantly as compared to HFD fed rats. On other hand, it is
worth being noted that the Telmisartan and Esculetin combination
treatment showed a substantial reduction in animal body weight,
heart weight and the relative heart weight as compared to that of
HFD fed rats (Table 1). Moreover, the combination treatment signicantly reduced BW and HW as compared to Esculetin monotherapy, while only BW reduced signicantly by combination
treatment as compared to Telmisertan monotherapy.
3.3. Telmisartan and Esculetin combination restored the vascular
responsiveness to Ang II and acetylcholine mediated relaxation
The HFD fed rats showed hyper responsiveness to Ang II as depicted by upward shift of the cumulative concentration response

593

curves (CRCs), which indicates increased maximal contractile response to Ang II challenges as compared to normal control (Fig. 1A).
Telmisartan and Esculetin treatment ameliorated the exaggerated
vascular responsiveness to Ang II in HFD fed rats and even better
improvement was observed in case of the rats receiving Telmisartan
and Esculetin combination treatment regimen. The pD2 value remains unchanged among all the groups, which indicate similar
sensitivity of aortic rings from different groups to Ang II (Fig. 1A).
Furthermore, HFD fed rat showed impaired acetylcholine mediated
endothelial dependent relaxation compared to normal control, and
monotherapy with Telmisartan or Esculetin unable to make it better
while combination showed signicant improvement in acetylcholine
mediated vascular relaxation (Fig. 1B). These results clearly indicate
the additive effect of combination in attenuation of IR associated
vascular dysfunction.
3.4. Telmisartan and Esculetin combination alleviated oxidative
stress more efciently than monotherapy
The oxidative stress is one of the major promoting factors involved in the pathogenesis of the insulin resistance and associated
cardiac damage. The Keap1/Nrf2 complex is an important regulator of anti-oxidant genes expression (Tan et al., 2011). Hence,
we analyzed the Keap1 protein levels by IHC, and HFD fed rats
showed increased Keap1 level in heart as compared to NC rats. The
Esculetin and Telmisartan, both have the property of scavenging
free radicals to reduce the oxidative stress outburst (Lakshmanan
et al., 2011; Prabakaran and Ashokkumar, 2013). The Keap1 level
was found to be reduced by Esculetin and Telmisartan treatment;
however the combination drug regimen reduced the Keap1 expression more signicantly than the individual drugs (Fig. 2A).
3.5. Telmisartan and Esculetin combination reduced extracellular
matrix (ECM) accumulation associated with insulin resistance
To assess the ECM accumulation and cardiac brosis, histopathological examination of heart sections was done by Picro
Sirius Red (PSR) staining. The PSR staining demonstrated enhanced ECM accumulation in HFD fed rats' heart tissues as compared to normal control rats (Fig. 2B). The ECM deposition was
signicantly reduced by the administration of Telmisartan and
Esculetin. However, more pronounced reduction in brotic deposition was observed in case of the combination treated rats
compared to HFD fed rats (Fig. 2B).
3.6. Telmisartan and Esculetin combination attenuated cardiac brosis associated with insulin resistance
The condition of insulin resistance is intricately related with an
increased expression of brotic markers including TGF- and bronectin (Mulay et al., 2010). Hence, the expression of TGF- and

Table 1
Effect of Telmisartan and Esculetin treatment on metabolic and morphometric alterations.
Groups

PGL (mmol/l)

TG (mg/dl)

PTC (mmol/l)

PI (pmol/ml)

BW (g)

HW (g)

(HW/BW)*1000

Normal Control (NC)

High Fat Diet Control (H)
High Fat Diet/Esculetin(50 mg) (H E)
High Fat Diet/Telmisartan(10 mg) (H T)
High Fat Diet/Telmisartan/Esculetin (H T E)

5.659 70.25
6.990 70.25a
6.346 70.22
6.138 70.14
6.001 70.12b

39.337 2.47
62.677 4.80a
54.177 4.23
53.43 7 2.51
41.577 3.06b

1.137 0.06
4.45 7 0.18a
3.177 0.17b
2.75 7 0.23b
1.25 7 0.24b,c,d

2.13 70.15
6.46 70.43a
6.3570.55
4.73 70.14b
2.61 70.19b,c,d

163.2 7 2.99
196.7 7 2.47a
190.3 7 2.02
181.3 7 3.07b
164.87 2.61b,c,d

0.60 70.03
0.80 70.04a
0.75 70.06
0.69 70.04
0.52 70.03b,c

3.69 7 0.26
4.08 7 0.19
3.95 7 0.17
3.81 7 0.23
3.127 0.13b

Note: Each data is represented as means 7 S.E.M.

a

n 6 rats/group vs NC.
n 6 rats/group vs H.
c
n 6 rats/group vs H E.
d
n6 rats/group vs H T.
b

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A. Kadakol et al. / European Journal of Pharmacology 765 (2015) 591597

Fig. 1. Telmisartan and Esculetin treatment restores vascular elasticity and responsiveness towards Ang II and Acetylcholine. (A) Ang II and (B) Acetylcholine mediated
Cumulative Response Curves (CRC) in aortic ring obtained from NC; Normal Control rats, H; HFD fed rats, H E; HFD fed rats treated with Esculetin, H T; HFD fed rats
treated with Telmisartan, H E T; HFD fed rats treated with both Esculetin and Telmisartan. Note: Each data is represented as means 7 S.E.M. (n 6). (*) Po 0.05 Vs NC; (#)
Po 0.05 Vs H; ($) P o0.05 Vs HE; (@) P o0.05 Vs H T.

bronectin in the heart was checked by using IHC. It was found

that TGF- and bronectin expression was increased in heart tissues under insulin resistance conditions (Fig. 3A and B), which was
attenuated by Esculetin and Telmisartan monotherapy. On the
other hand, the rats which were subjected to the combination
treatment showed more signicant reduction in TGF- and bronectin level in heart compared to HFD fed rats and single drug
treated rats (Fig. 3). These results proved that Telmisartan and

Esculetin combination improves insulin resistance and associated

cardiac brosis to a larger extent than the monotherapy.

4. Discussion
The present study was aimed to evaluate the effectiveness of Telmisartan and Esculetin combination in curbing insulin resistance (IR)

Fig. 2. Attenuation of oxidative stress and collagen deposition in insulin resistant heart tissue on treatment with Telmisartan and Esculetin combination. (A) Upper panel
images show light microscopic pictures and lower panel indicates inverted images of the respective photomicrographs illustrating the immunostaining and quantication of
% Keap1 positive area in the heart sections. (B) Images show Picro-Sirius red (PSR) staining and quantication of % brotic area showing extracellular matrix accumulation.
The scale bar represents 50 mm (original magnication,  100). Note: All values are represented as means 7 S.E.M. from at least 25 sections per group (n 6). (*) Po 0.05 Vs
NC; (#) Po 0.05 Vs H; ($) P o 0.05 Vs HE; (@) Po 0.05 Vs HT.

A. Kadakol et al. / European Journal of Pharmacology 765 (2015) 591597

595

Fig. 3. Telmisartan and Esculetin combination reduces expression of brotic markers in insulin resistant heart tissues. Upper panel images show light microscopic pictures
and lower panel indicates inverted images of the respective photomicrographs illustrating the immunostaining and quantication of (A) % TGF- positive area and (B) %
Fibronectin positive area. The scale bar represents 50 mm (original magnication,  100). Note: All values are represented as means 7 S.E.M. from at least 25 sections per
group (n 6). (*) P o 0.05 Vs NC; (#) P o 0.05 Vs H; ($) P o 0.05 Vs H E; (@) Po 0.05 Vs HT.

Fig. 4. Proposed mechanism for better cardiovascular protective effect of Telmisartan and Esculetin combination under insulin resistance condition.

and associated cardiovascular complications. The study results showed

that in IR condition the combination of Telmisartan with Esculetin
alleviated the pathological features including the biochemical parameters, oxidative stress, cardiac brosis and vascular responsiveness

more efciently than their monotherapy (Fig. 4). This indicates that
application of this combination therapy can be more useful than the
treatment with individual molecule to control the pathogenesis of IR
and cardiac complications.

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A. Kadakol et al. / European Journal of Pharmacology 765 (2015) 591597

Insulin resistance, a common subclinical pathological state associated with reduced responsiveness to normal levels of circulating insulin involves accumulation of ectopic lipid metabolites,
activation of the unfolded protein response (UPR) pathway, innate
immune pathways, up-regulation of renin angiotensin system
(RAS) and augmented oxidative stress (Henriksen and Prasannarong, 2013; Samuel and Shulman, 2012). The lack of sensitivity
towards insulin in cardiovascular tissue leads to aberrant free fatty
acid metabolism responsible for lipotoxicity, a reduced nitric oxide
production which may culminate into endothelial dysfunction,
increased apoptosis, brosis, alterations in vascular elasticity and
myocardial structure. The cardiac alterations accompanied by external pressers like hypertension, tip the balance in the favor of
developing cardiomyopathy (Witteles and Fowler, 2008). The insulin resistance has also been found to be closely related to vascular stiffness and reduced vascular compliance (Chan et al., 2013).
Thus, the pathological changes occurring in the cardiovascular
system due to insulin resistance need to be controlled as early as
possible to reduced morbidity and mortality. The angiotensin receptor blockers (ARBs), due to their insulin sensitizing, free radical
scavenging and PPAR- partial agonistic property, are a very
popular choice among physicians to retard the progression of insulin resistance and its associated cardiovascular alterations. Furthermore, the existing literature shows that the protective effect of
Telmisartan (ARB) gets improved by its conjugation with another
molecule which acts through a different mechanism in order to
give an additive therapeutic effect (Abd Allah and El-Debakey,
2010; Mulay et al., 2010; Symeonides et al., 2007). Esculetin, a
natural coumarin derivative has anti-oxidant, anti-hypertrophic,
anti-inammatory and anti-allergic properties (Kadakol et al.,
2015a; Kim and Lee, 2015; Surse et al., 2011). Recently, we have
reported that Esculetin at 100 mg/kg/day dose improved insulin
sensitivity and vascular responsiveness, attenuated alterations in
RAS, reduced extracellular matrix (ECM) accumulation, and reversed post translational histone modications, eventually prevented cardiovascular dysfunction associated with hyperinsulinemia and hyperglycaemia (Kadakol et al., 2015a, 2015b).
Hence, Esculetin stands as a potential candidate which could be
clubbed with Telmisartan, to improve their individual therapeutic
effects.
Thus, in the search of a better therapeutic regimen, we conceived this study to check the effect of combination of Esculetin
and Telmisartan on the progression and pathogenesis of insulin
resistance and associated cardiac brosis. Insulin resistance was
developed in animals by high fat diet (HFD) feeding, which clinically mimics the pathological features of IR as observed in humans
(Srinivasan et al., 2005). The plasma biochemical analysis of the
rats fed with HFD showed clear features of IR like hyperinsulinemia and altered plasma lipid prole. The monotherapy
with Telmisartan or Esculetin could revert these alterations only
partially, which might be due to AT1 receptor antagonism or PPAR partial agonism (Benndorf et al., 2006), and reduction of oxidative stress and inammatory mediators, respectively (Kim and
Lee, 2015). In contrast, combination of Telmisartan and Esculetin
attenuated all these metabolic perturbations more signicantly in
IR condition, which might be due to combined effects of the two
molecules at different points of the pathogenic pathway, thus
obstructing pathogenesis more efciently. Furthermore, the morphometric analysis revealed that the combination treatment more
efciently reduced the heart weight, body weight and relative
heart weight than the monotherapy in IR rats.
An increase in the arterial stiffness is a major risk factor for the
adverse cardiovascular events in adults related with insulin resistance, obesity or type 2 diabetes (Urbina et al., 2012). Earlier, we
have reported that Esculetin treatment improved vascular responsiveness and endothelial functions in both IR and type

2 diabetic animals (Kadakol et al., 2015a). Angiotensin receptor

blockers are known to promote nitric oxide synthase uncoupling
by lowering the oxidative stress (Satoh et al., 2008). Telmisartan
demonstrated protective effect against endothelial dysfunction
and improved nitrate tolerance in Wistar rats with nitroglycerine
induced endothelial dysfunction (Knorr et al., 2011). Our results
showed that the exacerbated hyper responsiveness to Ang II and
altered endothelial dependent Acetylcholine mediated relaxation
was attenuated most signicantly by the Telmisartan and Esculetin
combination, which may be due to the additive effects of the two
molecules.
The HFD induced insulin resistance leads to an outburst of free
radicals, mitochondrial dysfunction and lipid accumulation, which
could be attributed to a reduction in the anti-oxidant enzyme
activities (Tan et al., 2011; Yuzefovych et al., 2013). Keap1/Nrf2
complex is a major component of cellular system, handling the
transcription of anti-oxidant genes (Tan et al., 2011). In the present
study, it was observed that there was a drastic upsurge in the
expression of Keap1 protein in the IR heart, which might attributed to the accompanying hyperinsulinemic condition that promote free radical generation. It was noted that increased expression of Keap1 in IR heart was markedly reduced in case of the
combination treatment than the individual treatments and this
observation can be justied based upon the fact that both, Telmisartan and Esculetin possesses anti-oxidant activities and their
combined regimen might show an additive anti-oxidant effect.
Similarly, we have demonstrated that animals receiving combination regimen showed more pronounced reduction in ECM accumulation in heart tissue as compared to that of the monotherapy by
histopathological examination. The pathogenesis of cardiac brosis
induced by insulin resistance is associated with an aberrant increased
in expression of brotic markers for example transforming growth
factor- (TGF-), and bronectin (Cavalera et al., 2014; Yan et al.,
2009). TGF-, a pleiotropic cytokine regulates an array of biological
processes including cell growth, differentiation, apoptosis, migration,
cell adhesion, and immune response (Yan et al., 2009). The current
study demonstrated an elevated expression of brosis marker protein like bronectin and TGF- in the HFD fed rats heart tissues. The
augmented expression of bronectin and TGF- was notably
abridged by the combination of Telmisartan and Esculetin than the
individual drug treatment. Thus, the mechanism through which a
better action is manifested in case of combination treatment may be
associated with a more efcient control over the TGF- mediated
brosis pathway, which was found to be activated in the insulin resistant heart (Fig. 4).
Based on the results derived from this study, it can be concluded that the combination of Telmisartan and Esculetin ameliorates the pathological features associated with insulin resistance
and cardiovascular dysfunction by attenuating the oxidative stress,
vascular stiffness, and TGF- activation (Fig. 4). Thus, the combination of Telmisartan and Esculetin may serve as a better therapeutic intervention for treatment of insulin resistance and associated cardiovascular dysfunction and further work needs to be
done in order to put this benecial combination of molecules to
clinical application.

Conict of interests
We declare that we have no conict of interests.

Acknowledgments
A.B.G sincerely acknowledges the nancial support obtained
from the Science & Engineering Research Board Department of

A. Kadakol et al. / European Journal of Pharmacology 765 (2015) 591597

Science & Technology (SERB-DST), Govt. of India (SB/EMEQ-053/

2013) and University Grant Commission Major Research Project
(UGC-MRP) [F.NO.42-702/2013 (SR)]for this work.

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