Cardiovascular pharmacology

Protective effects of sitagliptin on myocardial injury

and cardiac function in an ischemia/reperfusion rat model
Guanglei Chang, Peng Zhang, Lin Ye, Kai Lu, Ying Wang, Qin Duan, Aihua Zheng, Shu Qin
n
,
Dongying Zhang
nn
Department of Cardiology, The First Afliated Hospital of Chongqing Medical University, No.1 Yixueyuan Road, Chongqing 400016, PR China
a r t i c l e i n f o
Article history:
Received 20 March 2013
Received in revised form
28 August 2013
Accepted 4 September 2013
Available online 13 September 2013
Keywords:
DPP4 inhibitor
Sitagliptin
Ischemia/reperfusion
Myocardial injury
Cardiomyocyte apoptosis
Cardiac function
a b s t r a c t
The purpose of this study is to investigate the effects and the underlying mechanisms of sitagliptin
pretreatment on myocardial injury and cardiac function in myocardial ischemia/reperfusion (I/R) rat
model. The rat model of myocardial I/R was constructed by coronary occlusion. Rats were pretreated with
sitagliptin (300 mg/kg/day) for 2 weeks, and then subjected to 30 min ischemia and 2 h reperfusion.
The release of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), cardiac function and
cardiomyocyte apoptosis were evaluated. The levels of malondialdehyde (MDA), glutathione peroxidase
(GSH-Px) and superoxide dismutase (SOD) in heart and glucagon-like peptide-1 (GLP-1) level in plasma
were measured. Western blot analysis was performed to detect the target proteins of sitagliptin.
Our results showed that sitagliptin pretreatment decreased LDH and CK-MB release, and MDA level in I/R
rats. More importantly, we revealed for the rst time that sitagliptin pretreatment decreased cardio-
myocyte apoptosis while increased the levels of GSH-Px and SOD in heart. Sitagliptin also increased GLP-
1 level and enhanced cardiac function in I/R rats. Furthermore, sitagliptin pretreatment up-regulated
Akt
serine473
and Bad
serine136
phosphorylation, reduced the ratio of Bax/Bcl-2, and decreased expression
levels of cleaved caspase-3 and caspase-3. Interestingly, the above observed effects of sitagliptin were all
abolished when co-administered with GLP-1 receptor antagonist exendin-(9-39) or PI3K inhibitor
LY294002. Taken together, our data indicate that sitagliptin pretreatment could reduce myocardial
injury and improve cardiac function in I/R rats by reducing apoptosis and oxidative damage. The
underlying mechanism might be the activation of PI3K/Akt signaling pathway by GLP-1/GLP-1 receptor.
Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction
Myocardial infarction is a major cause of mortality and morbid-
ity of patients with diabetes mellitus (Acar et al., 2011). In order to
prevent the myocardium from further damage, the best therapeutic
strategy for myocardial infarction is to reestablish the blood ow
as earlier as possible. Nevertheless, ischemia/reperfusion (I/R) injury
such as cardiomyocyte apoptosis is inevitable. Cardiomyocyte
apoptosis induced by I/R plays an important role in causing
a gradual decline of cardiac function (Gottlieb, 2011). Therefore,
the exploration of new therapeutic agents that reduce I/R injury of
myocardial infarction patients has become very important.
Glucagon-like peptide-1 (GLP-1) is secreted by the entero-
endocrine L cells of the intestinal mucosa and released in response
to nutrient ingestion (Nauck et al., 1993). It exerts insulinotropic and
insulinomimetic effects via the G-protein-coupled GLP-1 receptor
(Verge and Lopez, 2010). The therapy based on the functions of GLP-
1 is currently used as a novel anti-diabetic approach (Doupis and
Veves, 2008; Garber, 2012). However, GLP-1 is rapidly degraded
by dipeptidyl peptidase-4 (DPP
4
) enzyme in the blood (Green et al.,
2006). The short half life time limited its clinical use. Thus, two
classes of drugs, including GLP-1 analogs (Garber, 2012)
(i.e. exenatide) and DPP4 inhibitors (Doupis and Veves, 2008)
(i.e. sitagliptin), have been recently used for treating type 2 diabetes.
Recently, growing evidences have demonstrated the benecial
effects of GLP-1 analogs during I/R injury in both animal models and
in clinical studies, such as limiting infarct, improving cardiac function
and enhancing myocardial glucose uptake (Bhashyam et al., 2010;
Chinda et al., 2012a; Lorber, 2012; Mundil et al., 2012). The mechan-
isms underlying the cardioprotective effects of GLP-1 analogs may
be both GLP-1 receptor dependent and independent pathways
(Ban et al., 2008; Chinda et al., 2012a). Unlike GLP-1 analogs,
evidences regarding the cardioprotective effects of DPP4 inhibitors
are scarce and controversial. Recently, more and more researchers
have paid close attention to the cardioprotective effects of DPP4
inhibitors. Chinda, et al. (2012b) reported that DPP4 inhibitor could
stabilize cardiac electrophysiology in a myocardial I/R pig model.
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European Journal of Pharmacology
0014-2999/$ - see front matter Crown Copyright & 2013 Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.09.007
n
Corresponding author. Tel.: 86 13101345177; fax: 86 2389011562.
nn
Corresponding author. Tel.: 86 13650502588; fax: 86 2368055542.
E-mail addresses: Qinshu@21cn.com (S. Qin),
augustlei127@aliyun.com (D. Zhang).
European Journal of Pharmacology 718 (2013) 105113
In addition, DPP4 inhibitor has been shown to attenuate the infarct
size and improve the left ventricular function during myocardial I/R
injury (Chinda et al., 2012a; Jose and Inzucchi, 2012; Lenski et al., 2011;
Scheen, 2012). However, the correlation between its cardioprotective
effect and cardiomyocyte apoptosis during myocardial I/R is unclear.
Hereby, the purpose of this study is to investigate whether the
cardioprotective effects of sitagliptin, a DPP4 inhibitor, is relative to
its anti-apoptotic function and to explore the underlying mechanism.
We hypothesized that sitagliptin played the role of cardioprotection
in a myocardial I/R rat model by reducing cardiomyocyte apoptosis.
To test this hypothesis, we pretreated rats with sitagliptin for 2 weeks
before inducing myocardial I/R. Then the effects of sitagliptin on
myocardial injury and cardiomyocyte apoptosis were determined.
Finally, we used the GLP-1 receptor antagonist exendin-(9-39) to
assess the role of GLP-1 receptor-dependent pathway in the cardio-
protective effects of sitagliptin.
2. Materials and methods
2.1. Experimental animals and drugs
Male SpragueDawley rats aged between 6 and 8 weeks were
purchased from the Laboratory Animal Center of Chongqing Medical
University [certicate: SCXK (YU) 2007-0001]. Rats were housed
under optimal conditions with standard hygiene, temperature, photo-
periods (12L: 12D), standard rat chow and water ad libitum. All of
these conditions were conformed to the Guidelines for Care and Use of
Laboratory Animals. All procedures on animals were approved by the
Ethical Committee of the Chongqing Medical University.
The DPP4 inhibitor sitagliptin was purchased from Merck Sharp
& Dohme Italia SPA. The PI3K inhibitor LY294002 was purchased
from Santa Cruz Biotechnology, Inc. The GLP-1 receptor antagonist
exendin-(9-39) was purchased from Sigma, St. Louis, MO, USA.
2.2. Establishment of myocardial I/R injury model
Forty Male SpragueDawley rats were randomly divided into the
following ve groups (n8): the Sham group, the I/R group,
the sitagliptinI/R group (sitagliptin), the sitagliptinexendin-(9-
39)I/R group (sitagliptinE) and the sitagliptinLY294002I/R
group (sitagliptinL). Sitagliptin (300 mg/kg/day) was administrated
by intraperitoneal injection for 2 weeks. Exendin-(9-39) (45 g/kg/3
days) and LY294002 (0.3 mg/kg/3 days) were given by intraperitoneal
injection 30 min before sitagliptin injection. Sitagliptin, exendin-(9-39)
and LY294002 were all dissolved in dimethyl sulfoxide (DMSO). The
Sham group and the I/R group received the same volume of DMSO for
2 weeks.
After pretreatment with sitagliptin for 2 weeks, all rats were
anesthetized by chloral hydrate (concentration 3.5%, 10 ml/kg).
Tracheotomy was carried out for ventilation by a respirator
(ALC-V8B, Shanghai Alcott Biotech Co., Ltd.) with a stroke volume
of 28 ml/kg, air pressure of 10 mmHg, respiration rate of 1:1 and at
a rate of 86 strokes per minute. And the electrocardiogram of lead
II was monitored. Thoracotomy was performed and the left
anterior descending coronary artery was ligated by 6-0 silk. Then
the left anterior descending coronary artery was subjected to
30 min of ischemia followed by reperfusion for 2 h. Rats in the
Sham group were subjected to the same surgery process without
coronary artery ligation.
Glucose levels were measured with a blood glucose monitor
(Accu-Check
s
, Roche, Germany). Body weights of rats were weighted
after the establishment of the I/R model. At the end of hemodynamic
measurement, the blood plasma samples were collected from
the heart using the anticoagulant tube. The hearts were rapidly
excised and arrested in diastole in cold diethyl pyrocarbonate water
after the rats were euthanized. Then the heart was transected parallel
to the atrioventricular groove at the center of the ischemia area as
previously described (Li et al., 2010). The right ventricle and atria
were rapidly removed, and the left ventricle was weighed. The left
ventricular weight index was expressed as the ratio of left ventricular
weight to body weight. And the blood plasma samples and heart
tissue were collected immediately and stored at 80 1C.
2.3. Hemodynamic measurements
During the entire I/R period, the right common carotid artery
and left femoral artery were isolated. A polystyrene PE-20 catheter
was inserted into the left ventricle via right common carotid
artery, with one end connected to MPA-2000 multichannel phy-
siologic recorder. The left ventricular end-systolic pressure
(LVESP), left ventricular end-diastolic pressure (LVEDP) and the
rates of maximum positive and negative left ventricular pressure
development (7LVdp/dt max) were measured.
2.4. ELISA assay
Levels of active GLP-1 and creatine kinase-MB (CK-MB) in the
plasma were detected using ELISA kits according to the instructions
provided by the manufacturer (R&D Systems, Minneapolis, MN,
USA). Briey, plasma was centrifuged at 1600g for 10 min at 4 1C.
The supernatants were collected for the detection of GLP-1 and CK-
MB. Then the supernatants were incubated with the regents in kits.
Finally, the absorbance values were measured using a microplate
reader (Multiskan MK33, Thermolab systems, Helsinki, Finland). The
GLP-1 level was expressed as pmol/l. The CK-MB level was expressed
as U/l. The experiment of CK-MB was conducted for three times.
2.5. Colorimetry
The activity of lactate dehydrogenase (LDH) in plasma and the
concentrations of malondialdehyde (MDA), glutathione peroxidase
(GSH-Px) and superoxide dismutase (SOD) in heart homogenate were
determined by colorimetry. The experiment was performed using
commercially available kits, according to the manufacturer's instruc-
tions (Jiancheng Bioengineering Institute, Nanjing, China). Briey,
plasma was collected as above described. Heart tissues were collected
and lysed by cell lysis buffer. Then cell lysates were centrifuged at
1600g for 10 min at 4 1C. The supernatants of plasma and heart cell
lysates were collected for the detection of LDH, MDA, GSH-Px and
SOD. After incubation with the reagents in kits, the absorbance values
at 340 nm, 450 nm, 412 nm and 532 nm were measured using a
spectrophotometer (721D, Pudong Shanghai Physical Optical Instru-
ment Factory, Shanghai, China). The LDH level was expressed as U/ml.
The SOD and GSH-Px levels were expressed as U/mg protein. The MDA
levels were expressed as nmol/mg protein. The experiment of LDH
was performed for three times.
2.6. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin
nick end labeling (TUNEL) staining
TUNEL staining was performed with the TUNEL staining assay
kit according to the manufacturer's instructions (Boster Bio-
engineering Co., Ltd., Wuhan, China). Briey, after deparafniza-
tion, tissue sections were rst treated with hydrogen peroxide (3%)
and then digested with proteinase K (20 g/ml; pH 7.4) at 25 1C.
After digestion for 10 min, tissue sections were incubated with the
labeling buffer (1:18) at 37 1C. After incubation for 120 min, tissue
sections were incubated with biotinylated anti-digoxin antibody
(1:100) for 30 min at 37 1C. Then incorporated uorescein was
detected with streptavidinbiotin-peroxidase and subsequently
tissue sections were dyed with 3,3-diaminobenzidine (DAB).
G. Chang et al. / European Journal of Pharmacology 718 (2013) 105113 106
This assay detects apoptotic cells by labeling the 3-OH end
DNA fragments with digoxigenindeoxyuridine triphosphate
(DigdUTP) using terminal deoxynucleotidyl transferase. The
nuclei of apoptotic cells were stained brown and the nuclei of
normal cells were stained blue. Apoptotic index was determined
as the ratio of brown nuclei number to the total number of nuclei.
Nuclei in a total of 10 elds per tissue slice (n6) were included.
2.7. Flow cytometry analysis
Myocardial cells were isolated from heart homogenate by
ltration. After washing with ice-cold PBS, cells were double
stained with propidium iodide and FITC-coupled annexin V for
20 min. Flow cytometry was performed with a 488 nm laser
coupled to a FacsCalibur cell sorter (BD FACSvantage SE, Beckman
Coulter, America). Cells stained with both propidium iodide and
annexin V were considered necrotic and cells stained only with
annexin V were considered apoptotic.
2.8. Western blot analysis
Protein samples were isolated from the left ventricular myocar-
dium of I/R rats. Left ventricular myocardium lysates were prepared by
homogenization in cell lysis buffer (Beyotime Institute of Biotechnol-
ogy, China). Lysates were kept on ice for 45 min and total cardiac
proteins were isolated by centrifugation at 14,000g for 10 min at 4 1C.
Proteins were separated by SDS-PAGE and transferred to membranes.
The membranes were blocked in 5% nonfat milk and incubated with
primary antibodies. The primary antibodies included anti-AKT anti-
body (1:1000, Cell Signaling Technology, Inc.), anti-phospho-
AKT
serine473
antibody (1:1000, Cell Signaling Technology, Inc.), anti-
cleaved caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.),
anti-caspase-3 antibody (1:1000, Cell Signaling Technology, Inc.), anti-
phospho-Bad
serine136
antibody (1:500, Santa Cruz Biotechnology, Inc.),
anti-Bcl-2 antibody (1:1000, Cell Signaling Technology, Inc.), anti-Bax
antibody (1:1000, Cell Signaling Technology, Inc.) and anti-GAPDH
antibody (1:1000, Beyotime Institute of Biotechnology, China). Then
the membranes were incubated with secondary antibodies (Beyotime
Institute of Biotechnology, China). The signals were detected with the
ECL system (Beyotime Institute of Biotechnology, China). Blots were
scanned using Bio-Rad gel imaging system (Bio-Rad Company, USA)
and bands were quantied with the Quantity One software.
2.9. Statistical analysis
The SPSS 17.0 software was used for statistical analysis. Data
were presented as mean7standard deviation (S.D.). Grouped data
were analyzed using a one-way analysis of variance (ANOVA)
followed by the StudentNewmanKeuls (SNK) test. When the
equal variance test failed, a MannWhitney Rank Sum test was
used. A P value of less than 0.05 was considered statistically
signicant.
3. Results
3.1. Sitagliptin does not affect the glucose level, body weight, left
ventricular weight and left ventricular weight index
In order to roll out the possible side effects of sitagliptin, we
measured the basic clinical features of rats after sitagliptin treatment.
The basic clinical features included the blood glucose level, body
weight, left ventricular weight and left ventricular weight index.
The blood glucose levels were measured at 2 weeks before inducing
I/R. Statistically, the differences in the blood glucose levels among the
ve groups were not signicant (data not shown). Body weight, left
ventricular weight and left ventricular weight index were measured
after the inducement of I/R model. Similarly there were no signicant
differences among the ve groups (data not shown).
3.2. Sitagliptin upregulates the plasma GLP-1 level after myocardial
injury in I/R rats
To examine the effect of sitagliptin on plasma GLP-1, we measured
the plasma level of GLP-1 after inducing I/R by ELISA assay. Our
results (data not shown) were consistent with previous reports. We
found that the level of GLP-1 was signicantly decreased in I/R group,
when compared with that in Sham group (P0.006). In contrast,
the level of GLP-1 was signicantly increased in sitagliptin group
compared with that in I/R group (Po0.001). Meanwhile, the levels of
GLP-1 in sitagliptinE group and sitagliptinL group were not
signicantly different from the level of GLP-1 in sitagliptin group
(P40.05).
3.3. Sitagliptin reduces the LDH and CK-MB release after myocardial
injury in I/R rats
LDH and CK-MB are the diagnostic markers of myocardial tissue
damage. Thus, we examined the effects of sitagliptin on LDH and CK-
MB levels in plasma. The changes in LDH and CK-MB levels in this
study (data not shown) were also consistent with previous reports.
Statistically, the levels of LDH (Po0.001) and CK-MB (Po0.001) in
I/R group were signicantly higher than those in Sham group.
Compared with those in I/R group, the levels of LDH (Po0.001)
and CK-MB (Po0.001) in sitagliptin group were signicantly lower.
In addition, the LDH levels in sitagliptinE group (Po0.001) and
sitagliptinL group (Po0.001) were signicantly higher than those
in sitagliptin group. Meanwhile, the CK-MB levels in sitagliptin
E group (Po0.001) and sitagliptinL group (Po0.001) were also
signicantly higher than those in sitagliptin group.
3.4. Sitagliptin increases SOD and GSH-Px and decreases MDA after
myocardial injury in I/R rats
GSH-Px, SOD and catalase are important enzymes of the rst
line cellular defense against oxidative injury. Therefore, we exam-
ined the effects of sitagliptin on levels of SOD, GSH-Px and MDA in
myocardial tissue. As shown in Fig. 1A, the concentrations of SOD
in Sham group were signicantly higher than those in I/R group
(Po0.001). Compared with those in I/R group, the concentrations
of SOD in sitagliptin group were signicantly increased (Po0.001).
However, the concentrations of SOD in sitagliptinE group
(Po0.001) and sitagliptinL group (Po0.001) were signicantly
lower than those in sitagliptin group. The effects of sitagliptin
on concentrations of GSH-Px are shown in Fig. 1B. Statistically,
the concentrations of GSH-Px in I/R group were signicantly
decreased than those in Sham group (Po0.001). And compared
with those in I/R group, the concentrations of GSH-Px in sitagliptin
group were signicantly increased (Po0.001). However, com-
pared with those in sitagliptin group, the concentrations of GSH-
Px in sitagliptinE group (Po0.001) and sitagliptinL group
(Po0.001) were signicantly decreased.
The effects of sitagliptin on concentrations of MDA are shown
in Fig. 1C. Statistically, the concentrations of MDA in I/R group
were signicantly higher than those in Sham group (Po0.001).
And compared with those in I/R group, the concentrations of MDA
in sitagliptin group were signicantly decreased (Po0.001).
However, compared with sitagliptin group, the concentrations of
MDA in sitagliptinE group (Po0.001) and sitagliptinL group
(Po0.001) were signicantly increased.
G. Chang et al. / European Journal of Pharmacology 718 (2013) 105113 107
3.5. Sitagliptin enhances left ventricular function after myocardial
injury in I/R rats
To determine the effects of sitagliptin on cardiac function in I/R
rats, hemodynamic measurements were performed during the entire
I/R period. As shown in Fig. 2, compared with those in Sham group,
I/R treatment signicantly decreased the LVdp/dt max (Po0.001),
LVdp/dt max (Po0.001), and LVESP (Po0.001) and signicantly
increased the LVEDP (Po0.001). Compared with those in I/R group,
sitagliptin signicantly enhanced the LVdp/dt max (Po0.001),
LVdp/dt max (Po0.001), and LVESP (P0.014) and signicantly
reduced the LVEDP (P0.003). However, the GLP-1 receptor antago-
nist exendin-(9-39) and the PI3K inhibitor LY294002 abolished the
effects of sitagliptin on LVdp/dt max (Po0.001, Po0.001), LVdp/
dt max (Po0.001, Po0.001), LVESP (P0.012, P0.03) and LVEDP
(P0.017, P0.009).
3.6. Sitagliptin inhibits cardiomyocyte apoptosis after myocardial
injury in I/R rats
We examined the effects of sitagliptin on cell apoptosis in
myocardial tissue by TUNEL assay and ow cytometry analysis. The
representative graphs of TUNEL assay and ow cytometry analysis
are shown in Fig. 3A and C, respectively. The apoptotic index of
TUNEL assay is shown in Fig. 3B and the apoptosis ratio of ow
cytometry analysis is shown in Fig. 3D. Representative
photomicrograph showed that TUNEL staining positive apoptotic
cells were more frequently observed in I/R group, sitagliptinE
group and sitagliptinL group as compared with Sham group and
sitagliptin group (Fig. 3A). Statistically, the apoptotic index in
sitagliptin group was signicantly lower than those in I/R group
(Po0.001), sitagliptinE group (Po0.001) and sitagliptinL group
(Po0.001).
As analyzed by ow cytometry, apoptotic cell ratio in I/R group
was signicantly increased compared with that in Sham group
(Po0.001). However, compared with that in I/R group, apoptosis
ratio was signicantly decreased in sitagliptin group (Po0.001).
Also apoptosis ratios in sitagliptinE group (Po0.001) and
sitagliptinL group (Po0.001) were signicantly increased com-
pared with those in sitagliptin group.
3.7. Sitagliptin increases expression of anti-apoptotic proteins and
inhibits expression of pro-apoptotic proteins after myocardial injury
in I/R rats
The effects of sitagliptin on pho-Akt
serine473
, pho-Bad
serine136
,
caspase-3, cleaved caspase-3, Bax and Bcl-2 in myocardial tissue
were analyzed by western blot (Fig. 4). The representative western
blot results are shown in Fig. 4A and the quantitative results are
shown in Figs. 4BE, G and H. And the ratio of Bax/Bcl-2 is shown in
Fig. 4F. Compared with those in Sham group, I/R treatment signi-
cantly decreased levels of pho-Akt
serine473
(P0.006), pho-Bad
serine136
Fig. 1. Effects of sitagliptin on levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats. Levels of SOD, GSH-Px and MDA in heart homogenate of I/R rats were
measured by colorimetry. (A) Levels of SOD in heart homogenate of I/R rats. Signicance was determined by ANOVA followed by the SNK test, F63.255. (B) Levels of GSH-Px
in heart homogenate of I/R rats. Signicance was determined by ANOVA followed by the SNK test, F53.189. (C) Levels of MDA in heart homogenate of I/R rats. Signicance
was determined by ANOVA followed by the SNK test, F74.287. SOD: superoxide dismutase; GSH-Px: glutathione peroxidase; and MDA: malondialdehyde. Values were
expressed as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinE: sitagliptin 300 mg/
kg/day and exendin-(9-39) 45 g/kg/3 days pretreatment group; sitagliptinL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N8 in each
group;
n
Po0.05 vs. Sham group;
#
Po0.05 vs. I/R group; and

Po0.05 vs. sitagliptin group.
G. Chang et al. / European Journal of Pharmacology 718 (2013) 105113 108
(P0.002) and Bcl-2 (Po0.001) expression. Meanwhile, compared
with those in Sham group, I/R treatment signicantly increased
the expression levels of caspase-3 (Po0.001), cleaved caspase-3
(Po0.001) and Bax (Po0.001). Compared with those in I/R group,
sitagliptin signicantly enhanced the levels of pho-Akt
serine473
(P0.01), pho-Bad
serine136
(Po0.001) and Bcl-2 (Po0.001). At the
same time, compared with those in I/R group, sitagliptin signicantly
reduced levels of caspase-3 (Po0.001), cleaved caspase-3 (P0.02)
and Bax (Po0.001). Also the Bax/Bcl-2 ratio was signicantly
decreased in sitagliptin group than that in I/R group (Po0.001).
However, the GLP-1 receptor antagonist exendin-(9-39) and the PI3K
inhibitor LY294002 attenuated the effects of sitagliptin on anti-
apoptotic and pro-apoptotic proteins. Statistically, compared with
sitagliptin group, exendin-(9-39) and LY294002 administration sig-
nicantly reduced the levels of pho-Akt
serine473
(P0.011, P0.007),
pho-Bad
serine136
(Po0.001, Po0.001) and Bcl-2 (Po0.001, Po0.001).
And compared with sitagliptin group, exendin-(9-39) and LY294002
administration signicantly increased the expression levels of caspase-
3 (Po0.001, Po0.001), cleaved caspase-3 (P0.001, P0.001) and
Bax (Po0.001, Po0.001).
4. Discussion
The major nding of our study was that sitagliptin pretreatment
could reduce myocardial injury and improve cardiac function by
inhibiting cardiomyocyte apoptosis in an I/R rat model. Though DPP4
inhibitors were reported to have cardioprotective effects during I/R,
this is the rst study to examine the effects of sitagliptin on
cardiomyocyte apoptosis and oxidative stress induced by myocardial
I/R. In addition, our data indicate that sitagliptin could decrease the
levels of pro-apoptotic proteins and increase the levels of
anti-apoptotic proteins. However, the above observed effects of
sitagliptin were all abolished when co-administered with GLP-1
receptor antagonist exendin-(9-39) or PI3K inhibitor LY294002. Thus
we speculate that sitagliptin protected the heart in I/R rats from
injury through the decrease of apoptosis and oxidative damage and
the activation of PI3K/Akt signaling pathway.
As reported by previous studies (Khalil et al., 2005; Sun et al.,
2012), myocardial I/R impaired cardiac function, increased the
release of LDH and CK-MB and the apoptotic rate of cardiomyo-
cyte. We found similar results in this study. Importantly, we found
that sitagliptin reduced LDH and CK-MB release in I/R rats. We
speculate that this effect might be ascribed to its potential to resist
against oxidative stress. Interestingly, we demonstrated that
sitagliptin signicantly increased the levels of SOD and GSH-
Px and decreased the level of MDA in myocardial tissues in I/R
rats. As we all know, SOD and GSH-Px are key antioxidant
enzymes, which constitute rst line cellular defense against
oxidative injury. MDA is one of the products of oxidative stress,
which reects the damage of cell caused by oxidative stress. To our
knowledge, our study demonstrated for the rst time that
Fig. 2. Effects of sitagliptin on left ventricular function in I/R rats. The cardiac functions of LVdp/dt max, LVdp/dt max, LVESP and LVEDP were measured by multichannel
physiologic recorder. (A) Values of LVdp/dt max (rates of maximum positive left ventricular pressure development). Signicance was determined by ANOVA followed by
the SNK test, F66.053. (B) Values of LVdp/dt max (rates of maximum negative left ventricular pressure development). Signicance was determined by ANOVA followed
by a MannWhitney Rank Sum test, F28.161. (C) Values of left ventricular end-systolic pressure (LVESP). Signicance was determined by ANOVA followed by the SNK test,
F25.952. (D) Values of left ventricular end-diastolic pressure (LVEDP). Signicance was determined by ANOVA followed by the SNK test, F24.460. Values were expressed
as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinE: sitagliptin 300 mg/kg/day and
exendin-(9-39) 45 g/kg/3 days pretreatment group; sitagliptinL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group; N8 in each group;
n
Po0.05 vs. Sham group;
#
Po0.05 vs. I/R group; and

Po0.05 vs. sitagliptin group.
G. Chang et al. / European Journal of Pharmacology 718 (2013) 105113 109
pretreatment with sitagliptin could increase the concentrations of
antioxidant defense enzymes including GSH-Px and SOD, and
decrease the production of MDA in I/R rats.
We also found that sitagliptin administration signicantly
improved the cardiac function via increasing 7LVdp/dt max,
LVESP and limiting the increase of LVEDP. Similarly, Sauv et al.
(2010) observed that the left ventricular function was improved in
mice pretreated with sitagliptin for 12 h prior to aortic occlusion
and in DPP4 deleted mice after I/R injury. Ku et al. (2011) also
reported that DPP4 deciency could preserve cardiac function in
rats subjected to myocardial I/R. In a clinical study, single dose
sitagliptin treatment could improve the regional and global left
ventricular function in patients with coronary artery disease (Read
et al., 2010). However, a recent study demonstrated that in rats
Fig. 3. Effects of sitagliptin on cardiomyocyte apoptosis in I/R rats. Apoptosis were analyzed by TUNEL assay and ow cytometry analysis, respectively. (A) Representative
graphics of TUNEL staining. (B) Quantitative results of TUNEL staining (400). Apoptotic index was determined as the ratio of brown nuclei number to the total number of
nuclei. Nuclei in a total of 10 elds per tissue slice (N6) were included. Signicance was determined by ANOVA followed by the SNK test, F69.639. (C) Representative ow
cytometry results. (D) Quantitative results of ow cytometry results, N8. Apoptosis ratio was determined as the ratio of Annexin V positive and propidium iodide negative
cells to total cells analyzed. Signicance was determined by ANOVA followed by the SNK test, F70.412. Values were expressed as mean7S.D. Sham: Sham group; I/R:
ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinE: sitagliptin 300 mg/kg/day and exendin-(9-39) 45 g/kg/3 days
pretreatment group; sitagliptinL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group;
n
Po0.05 vs. Sham group;
#
Po0.05 vs. I/R group; and

Po0.05 vs. sitagliptin group.

G. Chang et al. / European Journal of Pharmacology 718 (2013) 105113 110
with ischemic heart failure, early or late treatment with vildaglip-
tin had no benecial effect on ventricular performance (Yin et al.,
2011). Meanwhile, Sauv et al. (2010) also reported that acute
treatment with sitagliptin for 20 min prior to I/R injury failed to
improve ventricular function. The discrepancy in these studies
might be attributed to the differences in the duration of treatment
time and differences in types of drug administration as well as the
differences in study models.
Cardiomyocyte apoptosis is one of the critical reasons of heart
failure after myocardial infarction. Many studies have conrmed
that blocking the apoptosis process could reduce the loss of
cardiomyocyte, minimize myocardial injury and improve ventri-
cular performance induced by I/R (Mughal et al., 2012). Given the
signicantly improved recovery of cardiac function, we examined
the effects of sitagliptin on preventing cardiomyocyte apoptosis in
I/R rats. We demonstrated that sitagliptin administration
Fig. 4. Effects of sitagliptin on expression levels of anti-apoptotic proteins and pro-apoptotic proteins in I/R rats. Expression levels of apoptosis related proteins were analyzed by
western blot. (A) Representative western blot results. (B) Ratios of phospho-AKT
serine473
to total AKT. Signicance was determined by ANOVA followed by a MannWhitney Rank Sum
test, F20.546. (C) Ratios of phospho-Bad
serine136
to GAPDH. Signicance was determined by ANOVA followed by the SNK test, F10.407. (D) Ratios of Bax to GAPDH. Signicance was
determined by ANOVA followed by a MannWhitney Rank Sum test, F212.404. (E) Ratios of Bcl-2 to GAPDH. Signicance was determined by ANOVA followed by a MannWhitney
Rank Sumtest, F75.357 (F) Ratios of Bax to Bcl-2. Signicance was determined by ANOVA followed by a MannWhitney Rank Sumtest, F84.528. (G) Ratios of Caspase-3 to GAPDH.
Signicance was determined by ANOVA followed by the SNK test, F70.092. (H) Ratios of cleaved caspase-3 to GAPDH. Signicance was determined by ANOVA followed by the SNK
test, F17.488. Values were expressed as mean7S.D. Sham: Sham group; I/R: ischemia/reperfusion group; sitagliptin: sitagliptin 300 mg/kg/day pretreatment group; sitagliptinE:
sitagliptin 300 mg/kg/day and exendin-(9-39) 45 g/kg/3 days pretreatment group; sitagliptinL: sitagliptin 300 mg/kg/day and LY294002 0.3 mg/kg/3 days pretreatment group;
N8 in every group;
n
Po0.05 vs. Sham group;
#
Po0.05 vs. I/R group; and

Po0.05 vs. sitagliptin group.
G. Chang et al. / European Journal of Pharmacology 718 (2013) 105113 111
signicantly decreased apoptosis ratio of cardiomyocytes as
revealed by TUNEL staining and ow cytometry analysis.
And sitagliptin administration signicantly reduced expression
levels of caspase-3 and cleaved caspase-3 in rats subjected to I/R
injury.
It has been reported that PI3K/Akt signaling pathway activation
could inhibit cardiomyocyte apoptosis after I/R injury (Fujio et al.,
2000; Matsui et al., 2001; Mullonkal and Toledo-Pereyra, 2007).
The mechanisms of the anti-apoptotic effect are acted through
various means, such as inhibiting caspase activation, inhibiting
death genes expression and regulating the activity of Bcl-2 family.
Bcl-2 family, the key regulators of apoptosis, consists of both cell
death promoters such as Bax and Bad, and cell death inhibitors
including Bcl-2, Bcl-x, etc. It is reported that the high ratio of Bax/
Bcl-2 was associated with great possibility to apoptotic activation
(Garca-Sez, 2012). In our study, we demonstrated that sitagliptin
could increase phosphorylation levels of Akt and Bad and decrease
expression levels of caspase-3, cleaved caspase-3 and Bax. Mean-
while, we also found that sitagliptin could up-regulate Bcl-2
expression, resulting in decreased Bax/Bcl-2 ratio in I/R rats. These
effects of sitagliptin were correlated with cardiomyocyte apoptosis
attenuation. Hence, we next examined whether the sitagliptin
exerted its anti-apoptotic action through activation of PI3K/Akt
pathway in rats subjected to I/R injury. The PI3K inhibitor
LY294002 was employed. We found that co-administration
of LY294002 and sitagliptin decreased phosphorylation levels of
Akt and Bad and increased expression levels of caspase-3, cleaved
caspase-3, Bax, and Bax/Bcl-2 ratio. These ndings suggested that
LY294002 could abolish the anti-apoptotic effects of sitagliptin.
The anti-apoptotic effects of sitagliptin were related to, at least in
part, activation of PI3K/Akt signaling pathway.
The function of DPP4 inhibitor is to inhibit the proteolytic
activity of DPP4 enzyme, postponing the myocardial degradation
of GLP-1 (Barnett, 2006). Our data showed that sitagliptin
administration resulted in signicant accumulation of plasma
GLP-1 in I/R rats. This result was similar to previous data reported
by Ku et al. (2011) and Ye et al. (2010). Moreover, our results
showed that higher levels of plasma GLP-1 were associated with
lower levels of cardiac injury markers, higher levels of antiox-
idant enzymes, lower ratio of cardiomyocyte apoptosis and better
ventricular performance in I/R rats. However, these effects were
attenuated by the GLP-1 receptor antagonist exendin-(9-39).
Exendin-(9-39) has been widely used to estimate the role of
receptor-dependent pathway. Thus we speculated that the car-
dioprotective effects of sitagliptin might be attributed to, at least
in part, the GLP-1 receptor-dependent pathway. In summary,
sitagliptin exerted its action via up-regulating the level of GLP-1,
which activated the PI3K/Akt signaling pathway via binding GLP-
1 receptor.
The prominent nding of this study was that sitagliptin pretreat-
ment could reduce myocardial injury and improve cardiac function in
an I/R rat model. The possible mechanisms might be relative to
decrease of apoptosis and oxidative damage, up-regulation of GLP-1
level (which activated the PI3K/Akt signaling pathway via binding
GLP-1 receptor), increase of Bad phosphorylation and decrease of
Bax/Bcl-2 ratio. However, exact cardioprotective mechanisms of DPP4
inhibitors need a further study. To sumup, we conclude that sitagliptin
administration has protective effects on myocardial I/R injury. Our
ndings could provide deeper insights into the treatment of heart
disease.
Acknowledgments
This work was supported by the National Natural Science Funds
for Youths (Grant no. 81100196), Natural Science Foundation
Project of CQ CSTC (Grant no. CSTC, 2011BB5133). Foundation
project of Traditional Chinese Medicine of Chongqing Municipal
Health Bureau (Grant no. 2012-2-125) and Pzer pharmaceutical
limited competition grants (Grant no. ws1790576). We greatly
appreciate Jianyong Wu and Dezhang Zhao (Institute of Life
Sciences, Chongqing Medical University) for their excellent tech-
nical support for the ow cytometry analysis.
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