1

Ph.D Thesis

Amin Al-awar

University of Szeged

Doctoral School of Biology

SZEGED

2018

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Ph.D Thesis

Novel potentials of dipeptidyl peptidase-4 inhibitor sitagliptin

against ischemia-reperfusion (I/R) injury in normolipidemic

and hyperlipidemic animals

Amin Al-awar

Supervisors:

Dr. Krisztina Kupai

Dr. Csaba Varga

Doctoral School of Biology

Department of Physiology, Anatomy and Neuroscience

Faculty of Science and Informatics

University of Szeged

SZEGED

2018

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1. TABLE OF CONTENTS

1. TABLE OF CONTENTS ................................................................................................................ 3

2. LIST OF ABBREVIATIONS ........................................................................................................ 6

3. SUMMARY ....................................................................................................................................... 9

4. ÖSSZEFOGLALÁS....................................................................................................................... 12

5. INTRODUCTION .......................................................................................................................... 15

5.1. Ischemia- reperfusion injury .................................................................................................................. 15

5.2. Mechanisms of ischemia- reperfusion injury ........................................................................... 17 5.2.1. Mitochondrial dysfunction ........................................................................................ 17

5.2.2. Overproduction of reactive oxygen species (ROS) ...................................................... 18

5.2.3. Reduction of nitric oxide bioavailability .................................................................... 18

5.2.4. Cardiac remodelling and Cell death .......................................................................... 19

5.3. Hyperlipidemia ......................................................................................................................... 19

5.4. Cardioprotective strategies ...................................................................................................... 21 5.4.1. Ischemia post-conditioning and pre-conditioning ....................................................... 21

5.5. Diabetes Mellitus and Anti-diabetic drugs .............................................................................. 23

5.6. Dipeptidyl peptidase-4 (DPP-4) inhibitors ............................................................................... 26

5.7. Sitagliptin .................................................................................................................................. 27

5.8. Pleiotropic cardioprotective effects of sitagliptin .................................................................... 27

5.9. New targeting markers of gliptins (NOS, TRP channels and CGRP) ..................................... 30 5.9.1. Nitric oxide synthase system (NOS) ........................................................................... 30

5.9.2. Transient receptor potential (TRP) channels and Calcitonine gene-related peptide ..... 31

6. AIMS ................................................................................................................................................. 35

7. MATERIALS AND METHODS ................................................................................................ 36

7.1. Drug preparations .................................................................................................................... 36

7.2. Animals and experimental design ............................................................................................ 36

7.3. Tissue staining and infarct size measurement ......................................................................... 39

7.4. Serum cholesterol and triglyceride measurements .................................................................. 40

7.5. Cholesterol and triglyceride measurements from liver samples ............................................. 40

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7.6. DPP-4 activity test .................................................................................................................... 40

7.7. Nitric oxide synthase (NOS) activity ........................................................................................ 41

7.8. ELISA measurements (GLP-1, TRPV-1 and CGRP) .............................................................. 41

7.9. Calcium (Ca2+

) content test ...................................................................................................... 42

7.10. TRPC-1, e-NOS and DPP-4 (CD26) protein expression by western blotting normalized to β-

actin .......................................................................................................................................... 42

7.11. Protein determination .............................................................................................................. 43

7.12. Statistical analysis..................................................................................................................... 43

8. RESULTS ......................................................................................................................................... 44

8.1. Normolipidemic animals .......................................................................................................... 44

8.1.1. DPP-4i Decreased the infarct size in heart tissues of Sitg (50 mg) treated group ........... 44

8.1.2. Sitg (50 mg) normalized DPP-4 activity and enhanced GLP-1 level................................. 45

8.1.3. DPP-4i increased TRPV-1 and CGRP levels in heart tissues of Sitg (50 mg) .................. 45

8.1.4. DPP-4i augmented cardiac calcium (Ca2+

) content in hearts of Sitg (50 mg) group ....... 46

8.1.5. DPP-4i positively affected TRPC-1 protein expression...................................................... 47

8.1.6. DPP4-i upregulated cNOS activity and e-NOS protein expression in heart tissues of Sitg

(50mg)……………………………………………………………………………………………………………………………… 48

8.1.6.1. cNOS activity ..................................................................................................................... 48

8.1.6.2. e-NOS protein expression ................................................................................................. 48

8.1.7. L-NAME inhibited NOS- mediated cardioprotection against infarct .............................. 49

8.1.8. Capsazepine inhibited TRPV-1- mediated cardioprotection against infarct ................... 50

8.2. Hyperlipidemic animals ........................................................................................................... 51

8.2.1. DPP-4i decreased the infarct size (IS) in heart tissues of Sitg (50mg) group ................... 51

8.2.2. Serum cholesterol and triglycerides concentration ............................................................ 52

8.2.3. Liver cholesterol and triglycerides concentration .............................................................. 53

8.2.4. Effect of Sitg on heart tissue DPP-4 activity and GLP-1 level ........................................... 54

8.2.5. Sitg (50 mg) normalized high DPP-4 level in heart tissues and aortas of control group 55

8.2.6. DPP-4i treatment caused no change in DPP-4 protein expression.................................... 56

8.2.7. DPP-4i increased CGRP but not TRPV-1 levels................................................................. 56

8.2.8. Enhanced cardiac calcium (Ca2+

) content in Sitg (50 mg)- treated group........................ 57

8.2.9. TRPC-1 protein expression level .......................................................................................... 58

8.2.10. DPP4-i upregulated cNOS activity and e-NOS protein expression in heart tissues of Sitg

(50 mg) …………………………………………………………………………………………………………………………… 58

8.2.10.1. cNOS activity ................................................................................................................. 58

8.2.10.2. e-NOS protein expression ............................................................................................. 59

8.2.11. L-NAME Inhibited NOS-mediated Cardioprotection Against Infarct ............................ 60

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9. DISCUSSION .................................................................................................................................... 62

10. ACKNOWLEDGEMENT ................................................................................................................ 66

11. REFERENCE LIST .......................................................................................................................... 67

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2. LIST OF ABBREVIATIONS

AAR Area at risk

ABS control Absolute control

AMI Acute myocardial infarction

Ang-II angiotensin II

ASCVD Atherosclerotic cardiovascular disease

ATP Adenosine triphosphate

AUC Area under the plasma concentration-time curve

Ca2+

Calcium

cAMP Cyclic adenosine monophosphate

CAP Capsaicin

cGMP Cyclic guanosine monophosphate

CGRP Calcitonin gene-related peptide

CHD Coronary heart disease

Chol Cholesterol

CPCR G protein-coupled receptor

CREB cAMP response element-binding protein

CVD Cardiovascular disease

DAG Diacylglycerol

DMSO Dimethyl sulfoxide

DPP-4 Dipeptidyl peptidase-4

DPP-4i DPP-4 inhibitor

DRG Dorsal root ganglia

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

EGTA Ethylene glycol-bis (β-aminoethyl ether) tetraacetic acid

e-NOS Endothelial nitric oxide synthase

ERAD ER-associated protein degradation

ET-1 Endothelin-1

FAD Flavin adenine dinucleotide

FMN Flavin mononucleotide

GIP Glucagon induced polypeptide

GLP-1 Glucagon-like-peptide-1

FLU Fluorescence intensity

GLP-1RA GLP-1 receptor agonists

GPCRs Gaq/11-protein coupled receptors

GSK-3ß Glycogen synthase kinase 3ß

HEPES N-[2-hydroxyethyl] piperazine-N’- [2-ethanesulfonic acid]

HFD High fat diet

HR Heart rate

HRP Horseradish peroxidase

IGF-1R Insulin-like growth factor-1-receptor

IGF-2 Insulin-growth factor-2

IMT Intima-media thickness

iNOS Inducible nitric oxide synthase

i.p. Intraperitoneal

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IPC Ischemia pre-conditioning

IPO Ischemia post-conditioning

IPC Ischemia pre-conditioning

I/R Ischemia-reperfusion

IRI Ischemia-reperfusion injury

IS Infarct size

LAD left anterior descending

LDL Low density lipoprotein

L-NAME Nω-Nitro-L-arginine methyl ester

LNMMA NG-monomethyl- L-arginine

LV Left ventricular

LVSP Left ventricular systolic pressure

MAPK Mitogen activated protein kinase

MEK 1/2-Erk ½ Extracellular signal–regulated kinases

MI Myocardial infarction

mPTP Mitochondrial permeability transition pores

MS metabolic syndrome

NADPH Dihydronicotinamide-adenine dinucleotide phosphate

n-NOS Neuronal nitric oxide synthase

NO Nitric oxide

NOS Nitric oxide synthase

NPY Neuropeptide Y

O2- Superoxide

OD Optical densities

ONOO- Peroxynitrite

PAGE Polyacrylamide gel electrophoresis

PI3K/Akt Phosphatidylinositol-3-kinase/Akt

PBS Phosphate buffer saline

PCI Percutaneous coronary intervention

PKA Protein kinase A

PKC Protein kinase C

PKG Protein kinase G

PLC Phospholipase C

RIPA Ice-cold radio immunoprecipitation assay

RISK Reperfusion Injury Salvage Kinase

ROS Reactive oxygen species

RT Room temperature

RTK Receptor tyrosine kinase

SAFE Survival activating factor enhancement pathway

SDF-1 Stromal cell-derived factor 1

SDS Sodium dodecyl sulfate

Sitg Sitagliptin

SNO Protein-S-nitrosylation

SOCC Store-operated calcium channel

SP Substance P

SR Sarcoplasmic reticulum

STAT3 Jak/signal transducer and activator of transcription 3

T1DM Type 1 diabetes mellitus

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T2DM Type 2 diabetes mellitus

TC Total cholesterol

TG Triglycerides

TGF-β Ttransforming growth factor- β

THB Tetrahydro-L-biopterin dihydrochloride

TNF-α Tumor necrosis factor alfa

TRP channels Transient receptor potential channels

TRPM Transient receptor potential melastatin

TRPML Transient receptor potential mucolipin

TRPP Transient receptor potential polycystic

TRPV Transient receptor potential vanilloid

TTC Triphenyltetrazolium chloride

UPR Unfolding protein response

VSMC Vascular smooth muscle cells

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3. SUMMARY

Experimental animal models of myocardial ischemia serve two nearly opposing aims, both

worthy of investigation. The aim is to provide a better mechanistic insight from an experimental

study that cannot be obtained from a clinical situation. To achieve this aim, experimental studies

may be reductionist with low direct applicability to the clinical situation, and experimental

models must replicate the clinical setting as closely as possible.

The isolated perfused heart is a convenient and reproducible model to test cellular and metabolic

mechanisms of myocardial injury, and for screening drugs or interventions for cardioprotective

properties. This model is studied independently of circulating factors or neuroendocrine inputs

from other organs, but retains the function, composition, and architecture of the intact heart. In

the Langendorff mode, the perfusate enters the coronary arteries to perfuse and oxygenate the

heart, which continues to beat for several hours.

In our study, we isolated heart tissues of wistar rats fed with normal or three months with high-

fat diet (HF) and pretreated daily for 14 days with saline or different doses of sitagliptin (25, 50,

100 or 150 mg/kg) and assigned them into a Langendorff system through aorta cannulation,

using prolonged and brief reperfusion protocols, for infarct size (IS) and biochemical

measurements respectively.

The 50 mg dose of sitagliptin exhibited a significant decrease in infarct size in both

normolipidemic and hyperlipidemic animals, with an infarct size- limiting property, while no

significant change was observed in the animal groups treated with other doses.

Gliptins are well known for their anti-hyperglycemic and incretin homeostatic properties, namely

the glucagon-like peptide-1 (GLP-1). We considered the 50 mg dose treatment and heart tissues

subjected to brief interval of reperfusion for biochemical measurements, to reveal and bring out

the mechanims mediating the protective effect of this drug in both diets.

As a second part of our investigation, we included the Dipeptidyl peptidase-4 (DPP-4) activity

and GLP-1 measurements in normal and high-fat (HF) diet animals. DPP-4 activity decreased

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significantly in normolipidemic animals, while no significant change was observed in the

hyperlipidemic groups, after treatment with sitagliptin 50 mg dose. Similarly, a significant

increase in GLP-1 preceded by decrease in DPP-4 activity was clearly observed in normal

animals, but not in the hyperlipidemic ones.

According to our hypothesis that nitric oxide synthase (NOS) and transient receptor potential

(TRP) channels can offer a pivotal role against ischemia-reperfusion (I/R) injury and infarct size-

attenuating effect, we extended our measurements to cNOS activity, and endothelial nitric oxide

synthase (e-NOS) protein expression, followed by transient receptor potential vaniloid-1 (TRPV-

1) level and transient receptor potential canonical- 1 (TRPC-1) protein expression.

Interestingly and beyond our expectations, a significant upregulation in NOS activity was the

case in animals fed with either normal or high-fat (HF) diet. Obtained results of e-NOS blots

displayed a significant increase in e-NOS expression only in normal diet condition, but not in

animals fed with normal diet mixed with fats. Our findings convincingly showed that sitagliptin

contributed in augmented TRPV-1 levels and TRPC-1 expression in normal diet animals only,

however, in high-fat (HF) diet animals this upregulatory effect was abolished.

Since transient receptor potential vanilloid type-1 (TRPV-1) is an upstream regulator of

calcitonin gene-related peptide (CGRP), and TRPV-1 stimulation promotes the release of CGRP,

mediating cardioprotection and enhancing cardiac function. Based on this fact and obtained

TRPV-1 results, we studied the regulatory effect of CGRP. Interestingly, the peptide abundance

significantly increased in normolipidemic animals as well as in the hyperlipidemic groups after

treatment with sitagliptin. This indiactes that the cardioprtective effect of sitagliptin in case of

hyperlipidemia can be independent of TRPV-1, or high-fat (HF) diet can be an intrinsic factor in

blocking ion channels like TRPV-1.

We also invesitgated the response of sitagliptin against size of infarction by inhibiting nitric

oxide synthase with the Nω-nitro-L-arginine methyl ester (L-NAME), that is known to have a

high affinity for e-NOS too. Intraperitoneal (i.p) administration of L-NAME with a dose (25

mg/kg/day) and three hours’ post-oral administration of sitagliptin showed a significant increase

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in infarct size compared to the treatments with sitagliptin alone. The cardioprotective effect of

sitagliptin mediated by NOS was lost in both diet conditions.

The inhibitory effect of capsazepine, a selective TRPV-1 antagonist was studied in

normolipidemic animals only. The daily intraperitoneal (i.p) administration of capsazepine at a

dose 1.0 mg/kg/day and three hours’ post-oral administration of sitagliptin showed an increase in

size of infarction, blocking sitagliptin-mediated TRPV-1 cardioprotective action.

The aforementioned results of L-NAME and Capsazepine inhibitory effects reveals that

sitagliptin-induced cardioprotection is mediated by NOS in normal and high-fat diet condition,

and transient receptor potential channels (TRP), only in normolipidemic condition.

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4. ÖSSZEFOGLALÁS

A gliptineknek, (pl.: sitagliptin), mint dipeptidil-peptidáz-4 (DPP-4) enzim támadáspontú

farmakonok az endogén GLP-1 szintet emelésén keresztül a 2 típusú diabétesz mellitusban

szenvedő betegeknél terápiásan választható gyógyszerkészítmények. Ezen hatóanyagok nem

csak a vércukorszintet képesek szabályozni, hanem számos pleiotróp hatással (szérum triglicerid

szint normalizálása) is rendelkeznek, melyek pontos hatásmechanizmusa nem ismert. Ismert,

hogy a GLP-1 inkretinnek kardioprotektív hatása van iszkémia-reperfúziós károsodás esetén,

valamint a GLP-1 mediált vazorelaxációban szerepet játszanak a gazotranszmitterek. A

gazotranszmitterek családjába kis, gáz halmazállapotú anyagok tartoznak, melyek szabadon

átjutnak a membránokon; szabályozottan, enzimatikus úton termelődnek; fiziológiás

koncentrációban jól meghatározott fiziológiás hatásuk van és specifikus molekuláris targetjeik

vannak a cél sejtekben. Az első azonosított gazotranszmitter a nitrogén-monoxid (NO) volt, mely

jelen PhD munka egyik fontos mért biokémiai paramétere. A NO-ot a nitrogén monoxid szintáz

(NOS) enzim termeli, melynek három izoenzime ismert: neuronális NOS (n-NOS), indukálható

NOS (i-NOS) és endoteliális NOS (e-NOS). A NO-ról azonban az is ismert, hogy

hiperlipidémiában, ami kardiovaszkuláris betegségek egyik fő rizikófaktora, O2--nal reagálva

peroxinitritet (ONOO) képez és nitrozatív-oxidatív stresszt okoz. Ismert tény az is, hogy az

endogén kardioprotektív mechanizmusok elvesznek hiperlipidémiában, elvész a szívizom

adaptációs képessége.

Munkám célja volt, hogy megvizsgáljuk és feltérképezzük a sitagliptin kardioprotektív

hatásmechanizmusát, képes e csökknteni az infarktusos terület nagyságát normolipidémiában és

hiperlipidémiában egyaránt. Hatásmechanizmus során vizsgáltuk különböző biokémiai

útvonalakat, különös tekintettel: DPP-4 enzim aktivitásra, expresszióra, GLP-1, Ca szintekre, e-

NOS és TRP csatornák expresszióját illetően.

Kísérletünkben a hiperlipidémiát 3 hónapos 40% zsírban gazdag diétával hoztuk létre, míg a

normolipidémiás patkányok normál tápot kaptak. A sitagliptin hatásának vizsgálatához a

normolipidémiás és hiperlipidémiás patkányokat 2 hétig per os 25, 50, 100, 150 mg/kg/nap

stiagliptinnel vagy vivőanyaggal kezeltük. Két hét előkezelés után a patkányok szívét izoláltuk,

Langendorff szerint retrográd perfundáltuk, majd a szíveket 10 perc perfúziónak 45 perc

regionális iszkémiának és 120 perc reperfúziónak tettük ki. A protokoll végén az infarktus

méretet TTC festéssel vizsgáltuk. A lipid szintek méréséhez a szív izolálással egy időben vért

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vettünk. A zsírban gazdag táp hatására szignifikánsan emelkedett a patkányok szérum koleszterin

és triglicerid szintje. Viszont a különböző dózisú sitagliptin kezelés hatására a triglicerid és

koleszterin szint nem változott szignifikánsan. Az 50 mg/kg/nap sitagliptin kezelés

szignifikánsan csökkentette az infarktus méretet, mind a normolipidémiás mind hiperlipidémiás

patkányok esetében.

Ezután a védő hatású sitagliptin dózissal kezelt patkányokból újabb szíveket izoláltunk

biokémiai analízisre. A szíveket izolálás után a korábbiakhoz hasonló módon perfundáltuk és 10

perc reperfúzió után folyékony nitrogénben lefagyasztottuk. A fagyasztott szív mintákból

meghatároztuk a fentebb listázott komponenseket.

Eredményeink alapján a DPP-4 enzim aktivitása és ennek megfelelően a GLP-1 szint

szignifikánsan csak normolipidémiában változott, míg hiperlipidémiába az aktivitás nem de a

szöveti fehérje expresszió szignifikánsan csökkent.

A cNOS izoenzim expressziója szignifikánsan emelkedett sitagliptin kezelés hatására mind

normo-, mind hiperlipidémiában, azonban az eNOS fehérje expressziója szignifikánsan csak

normolipidémiás szívekben.

Mivel a védő hatású sitagliptin hatására fokozódott a NO termelő enzimek aktivitása, ezért

vivőanyaggal és 50 mg/kg sitagliptinnel kezelt szíveket L-NAME-mel (NOS gátló, ip. 25

mg/kg/nap 2 hétig) kezeltünk. Az L-NAME kezelés kivédte a sitagliptin védő hatását mind

normolipidémiás, mind hiperlipidémiás patkányokban.

A TRP csatonák nagy mennyiségben expresszálódnak endotél sejteken, funkciójukat tekintve

számos biokémiai folyamatot regulálnak, illetve szabályozzák a Ca homeosztázist. Hipotézisunk

szerint a sitagliptin kezelés hatására fokozódik a csatornák másodlagos hírvivőjének tekintett

CGRP szöveti előfordulása és a szívizom Ca tartalma is. Eredményeim szerint a TRP csatornák

közül csak a TRPV-1 altípus szintje volt szignifikánsan magasabb normolipidémiában.

Feltételezéseink szerint a hiperlipidémia indukálta neurológiai károsodás egyik target fehérjéje

molekuláris szinten ezek a csatornák. Meglepő módon viszint a CGRP mindkét diéta esetében

sitagliptin kezelés hatására szignifikáns emelkedést mutatott. Abban az esetben, ha viszont

farmakológiailag legátoljuk ezen csatornákat (capsazepin kezelés) a sitagliptin kezelés mellett,

az előzetesen kimutatott sitagliptinnel kiváltott kardioprotekció elvész.

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Összefoglalva, munkánk során kimutattuk, hogy a sitagliptinnek direkt kardioprotektív hatása

van, és ebben a pleiotrop hatásában a számos biokémiai paraméter és alternatív sejtszinalizáció

utak mellett NO-nak, mint gazotranszmitternek illetve a TRP csatornáknak kitüntetett szerepet

tulajdoníthatunk.

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5. INTRODUCTION

5.1. Ischemia- reperfusion injury

The incidence of cardiovascular disorders continues to grow across worldwide, contributing to

the largest rate of mortality and morbidity each year, including ischemic heart disease [1, 2],

when the blood supply of heart is decreased followed by a decrease in oxygen and nutrient

supply [3], and reperfusion is necessary for the restoration of epicardial and microvascular blood

flow and the normal physiology of the heart, avoiding further damage to the myocardium [4],

however, exposing heart tissues to abrupt reperfusion, can lead to reactive oxygen species (ROS)

generation, inflammatory reactions, mitochondrial dysfunction, further myocardial damage, and

subsequently cell death [5]. As one of the major causes of death in industrialized countries,

cardiovascular diseases need to be more researched, rendering myocardial ischemia/reperfusion

(I/R) injury a hot field of research.

I/R injury occurs when circulation is abruptly restored following prolonged ischemia and it is

well known that high levels of calcium and tissue neutrophil accumulation cause cellular damage

and produce ROS during reperfusion and trigger I/R injury [6]. Myocardial reperfusion

following ischemia can be associated with tissue damage, due to pH normalization, Ca2+

overload, production of ROS, and mitochondrial opening of permeability transition pores

(mPTP) [7], leading to myocardial infarction (MI) [8]. MI can lead to progressive heart failure

due to developing of cardiac remodelling, and distortion of left ventricular (LV) shape, LV

dilatation, cardiomyocytes hypertrophy, scar formation and increased wall stress could lead to

LV systolic and diastolic dysfunction [9]. During myocardial ischemia, the absence of oxygen

switches cell metabolism to anaerobic respiration, resulting in the production of lactate and a

drop in intracellular pH. This induces the Na+-H

+ exchanger to extrude H

+, resulting in

intracellular Na+

overload, which activates the Na+-Ca

2+ exchanger to function in reverse to

extrude Na+ and leads to intracellular Ca

2+ overload. The Na

+-K

+ ATPase stops functioning in

ischemia, exacerbating intracellular Na+ overload. Acidic conditions during ischemia prevent the

opening of the mPTP, making cardiomyocyte to hypercontract. The electron transport chain is

reactivated during reperfusion phase, generates ROS, in addition to the other sources of ROS

from xanthine oxidase (endothelial cells) and nicotinamide-adenine dinucleotide phosphate

(NADPH) oxidase (neutrophils). ROS induces mPTP opening, attracts neutrophils and mediates

dysfunction of the sarcoplasmic reticulum (SR). This contributes to intracellular Ca2+

overload

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and damages the cell membrane by lipid peroxidation, inducing enzyme denaturation and

causing oxidative damage to DNA. The reactivation of the Na+-H

+ exchanger during reperfusion

results in washout of lactic acid, restoration of physiological pH, mPTP opening and

cardiomyocyte contracture. Restoration of the mitochondrial membrane potential drives calcium

into the mitochondria, also inducing mPTP opening. Several hours after the onset of myocardial

reperfusion, neutrophils accumulate in the infarcted myocardial tissue in response to the release

of the chemoattractants ROS, cytokines, and activated complement (Figure. 1).

Figure. 1. Schematic diagram illustrating the whole process of myocardial I/R injury in blood vessels and

cardiomyocytes (J Clin Invest. 2013; 123(1): 92-100).

In most conditions, I/R can retrieve the heart back to its normal condition and repair the damaged

structure, while in some cases I/R acts oppositely by exacerbating and worsening the cardiac

function and structural damage, respectively [10]. Myocardial infarction consititutes a major

cardiovascular disorder with an increasing prevalence due to many risk factors as

hyperlipidemia. According to the literature, thousands of drugs and other interventions that

render ischemic myocardium resistant to infarction have been studied, but all what we have

today is reperfusion therapy, with no one drug has been approved for limitation of infarct size in

patients with acute coronary syndrome. Those drugs failed to find their way into clinical practice,

either because clinical trials may have been inadequately designed, or animal hearts may not be

an appropriate model of the human heart. The majority of the studies addressed the effect of anti-

inflammatory agents, calcium channel blockers, or free radical scavengers, therefore, finding

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new protective agents and therapeutic strategies is substantial in the aim of decreasing the

incidence of cardiovascular events, limiting the extent of infarction during I/R. In our study we

hypothesized that sitagliptin can induce protective effect against ischemia-reperfusion injury, by

reducing the size of infarction and inducing the activation of new targeted mechanisms.

5.2. Mechanisms of ischemia- reperfusion injury

5.2.1. Mitochondrial dysfunction

The heart requires a continuous energy supply of adenosine triphosphate (ATP) produced from

mitochondrial oxidative phosphorylation, to meet the high energy demand needed for

contractility and diastolic relaxation [11], and mitochondrial dysfunction in the heart is an

etiological factor of myocardial ischaemia, causing loss in ATP synthesis and increase in ATP

hydrolysis, formation of ROS, and impaired ionic homeostasis. The mitochondrial inner

membrane is impermeable in its nature under physiological conditions, while becomes non-

selectively permeable under pathological conditions as hyperlipidemia and myocardial

ischaemia, causing uncontrolled opening of the inner mPTP, depolarization and uncoupling of

oxidative phosphorylation, leading to ATP hydrolysis and breakdown, and increase in

mitochondrial inorganic phosphate [12, 13]. Cellular metabolism is switched to anaerobic

glycolysis during ischemia with reduced intracellular pH (< 7.0), due to lactate accumulation

[14]. Lack of ATP and acidosis can lead to intracellular Ca2+

overload and mitochondrial

swelling, mediated by excess Na+ caused by the increase of intracellular proton accumulation-

induced activation of Na+/H

+ ion exchanger [15]. During reperfusion, cardiac cells undergoe pH

correction, causing an increase in mPTP opening, electrons flow, damage of electron transport

chain, and ROS production [16, 17] (Figure. 2). Preventing mPTP opening during the time of

reperfusion is a promising therapeutic approach to protect the heart from myocardial IRI.

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5.2.2. Overproduction of reactive oxygen species (ROS)

ROS such as superoxide anion, hydrogen peroxide, and hydroxyl radicals originating from ROS

producing enzymes, including NADPH oxidase, superoxide dismutase and peroxidase are

physiologically needed for normal cellular function and signalling; however, in case of

myocardial I/R injury (stress condition) a massive amount of generated ROS is observed during

the first minute of reperfusion (restoration of blood flow), from damaged mitochondria.

Excessive ROS production leads to calcium overloads, generation of peroxynitrite (ONOO-),

breakdown of proteins due to protein oxidation, disruption of cholesterol containing membranes

due to lipid peroxidation, decrease in NO bioavailability, and opening of mPTP, a process known

as oxidative stress, followed by cell defense (e.g. catalase, glutathione peroxidase, and

superoxide dismutase) [18-20].

5.2.3. Reduction of nitric oxide bioavailability

NO is a crucial signalling molecule and one of the most effective defense mechanisms in

myocardial IRI, mediating cardioprotective interventions. Mechanisms of cardioprotection

exerted by NO are several: 1) activation of NO-sensitive guanylyl cyclase, 2) inhibition of

mitochondrial Ca2+

influx and mitochondrial KATP channel, 3) activation of cGMP,4)

enhancement of cyclooxygenase and 5) abrogation of ONOO-

mediated lipid radical chain

Figure. 2. Schematic diagram of

mitochondrial dysfunction during I/R

injury. Ischaemia reperfusion increases

the opening of mPTP which elevates ROS

generation, decreases nitric oxide (NO)

bioavailability and disrupts intracellular

pH and Ca2+

, Na+

distribution, resulting in

cardiomyocyte death and irreversible

myocardial injury (Br J Anaesth. 2016;

117 (S2): ii44–ii62).

19

propagation [21]. Prolonged ischemia is characterized by low oxygen supply accompanied by

reduced NO release due to reduced NOS activity (e-NOS and n-NOS). Enhanced NO

bioavailability through exogenous application of NOS and L-arginie attenuates post-ischemic

infarction, while high levels of NO with increased superoxide anion is detrimental and

exacerbates post-ischemic myocardial injury, as a consequence of ONOO- formation [22].

5.2.4. Cardiac remodelling and Cell death

In case of myocardial infarction, inflammatory processes play a key role in cardiac remodelling,

involving the pro-inflammatory and profibrotic cytokine transforming growth factor- β (TGF-β),

recruiting the downstream mediators of TGF-β, phospho-p38 and phospho-p44/42 MAPKs,

promoting extracellular matrix deposition and fibroblast proliferation [23]. Formation of oxygen

free redical, calcium overload, and mitochondrila damage are considered as driving mechanisms

for cell death in myocardial I/R injury. Oxygen free redicals are vastly produced during I/R,

reinforcing the development of I/RI mediated by the destructive effect of lipids and nucleic acids

chain reactions. The restoration of blood flow in ischemic heart leads to an increase in Ca2+

cellular content and consequent cellular damage, causing calcium overload. Alteration of

mitochondrial membrane accompanied by mitochondrial accumulation of Ca2+

, promotes the

opening of the mPTP, release of cytochrome C into the cytoplasm and cell death upon caspase

activation [10]. Myocardial death during I/R is regulated by common signaling pathways:

phosphatidylinositol-3-kinase/Akt (PI3K/Akt), mitogen-activated protein kinases (MAPKs),

caspase, and cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG).

5.3. Hyperlipidemia

Despite the several causes and risk factors, hyperlipidemia is considered as a significant reason

and major contributor in the development of ischemic heart disease, resulting in lipid

accumulation in the athermanous lesions, and primary endothelial injury which promotes the

process of atherosclerosis [24]. Hyperlipidemia is characterized by high levels of serum total

cholesterol (TC), triglycerides (TG) and lipoproteins, with high risk of coronary heart diseases

like myocardial infarction and atherosclerosis [24]. Hyperlipidemia can exacerbate cell apoptosis

after myocardial ischemia- reperfusion, by upregulating capsase-3 and Bax protein expression

and decreasing Bcl-2 activity [25].

20

Therapeutic strategies have been emerged and extensively studied in the aim of protection of the

ischemic myocardium in normolipidemic and hyperlipidemic conditions [8]. Long-term high-fat

diet (HFD) consumption is associated with an increased risk of MI and LV dysfunction by

inducing obese-insulin resistance, hypertension, and dyslipidemia in myocardial I/RI model [26-

28]. Diets containing high cholesterol can increase myocardial infarct size following an

ischemia-reperfusion insult. HFD can modulate lipid and carbohydrate metabolism, causing

cardiac remodeling, and decreasing cardiac tolerance towards I/RI. The majority of

cardiovascular IRI studies used healthy animal models, while clinicaly, unhealthy diets and

lifestyle are normally associated with increased risk of MI [29], and the deleterious effects of

high fat diet on cardiovascular system must be studied after incretin-based therapies. Recent

advances in revascularization of coronary arteries through percutaneous coronary intervention

(PCI) and coronary artery bypass grafting have had a dramatic improvement in the fate of

patients suffering with ischemic heart disease [30]. Reperfusion and blood flow reestablishment

is considered one of the conventional remedies in case of MI; however, its increased prevalence,

encouraged researchers and pharmacologists to investigate new therapeutic agents that can

mitigate MI and cardiac damage by I/RI, especially that pharmacologic agents for reducing

myocardial injury in hyperlipidemia patients are limited [31].

Dipeptidyl Peptidase-4 (DPP-4) is a widely expressed glycoprotein peptidase that exhibits

complex biological roles, including cell membrane associated activation of intracellular signal

transduction pathways, cell-to-cell interaction, and enzymatic activity, exhibited by both

membrane-anchored and soluble forms of the enzyme [32]. Inhibition of DPP-4 system

represents a new approach for the treatment of type-2 diabetes (T2DM), due to its effect on

prolonging the half-life of incretins such as glucagon-like-peptide-1 (GLP-1) and glucagon

induced polypeptide (GIP). In favorable to post-prandial glycemic profile, the elevation of

incretin hormones results in glycemic control [33]. A number of pharmaceutical products have

been developed to use the incretin effect of GLP-1 while avoiding the difficulties associated with

its rapid breakdown to an apparently inactive form [34]. These drugs are commercially available,

including DPP-4 inhibitors such as sitagliptin, (Januvia, Merck, Kenilworth, New Jersey),

saxagliptin (Onglyza, AstraZeneca, Macclesfield, United Kingdom), and vildagliptin (Galvus,

Novartis, Basel, Switzerland), all of which increase levels of native GLP-1; and DPP-4-resistant

GLP-1 receptor agonists (GLP-1RA) such as exenatide (Byetta, AstraZeneca, United Kingdom)

and liraglutide (Victoza, Novo Nordisk, Bagsvaerd, Denmark) [34].

21

5.4. Cardioprotective strategies

5.4.1. Ischemia post-conditioning and pre-conditioning

Treatment strategies for protecting the myocardium against the I/RI are required to improve

clinical outcomes in patients with coronary heart disease (CHD). The ability of the heart to

condition itself is considered a potent injury limiting strategy, and brief multiple cycles of

ischemia and reperfusion protects the myocardium from I/RI, either before (pre-conditioning),

during (per-conditioning), or after (post-conditioning) ischemia [35].

Ischemia post-conditioning (IPO) is an intervention of short alternative cycles of ischemia and

reperfusion, applied at the early minutes of reperfusion [36], accompanied with reduced infarct

size, less accumulation of neutrophiles, less myocardial oedema, reduced cell death and

improved endothelial function. The cardioprotective effect of IPO is via reperfusion injury

salvage kinase (RISK) pathway that involves PI3K/Akt and e-NOS, and the survival activating

factor enhancement pathway (SAFE) which involves Jak/signal transducer and activator of

transcription 3 (STAT3), and its initiation is mediated by the pro-inflammatory cytokine tumour

necrosis factor-α (TNF-α). Additional components of RISK pathway, such as Protein kinase C

(PKC), Protein kinase G (PKG), and p38 MAPK, are also implicated in IPO cardioprotection

[37] (Figure. 3). The increase in peroxynitrite-induced nitrosative stress after post-conditioning is

considered a key mechanism in post-conditioning-induced cardioprotection that can also

regulates the mitogen-activated protein kinases [38]; however, the absence of this mechanism

during hyperlipidemia contirbutes to the loss of post-conditioning in high-fat diet conditions

[39], in addition to the loss of infarct size-limiting effect of post-conditioning.

Ischemia pre-conditioning (IPC), is a repetitive short episodes of ischemia followed by short

episodes of reperfusion (5 minute coronary artery occlusions followed by a 5-minute period of

reperfusion before the onset of a 40-minute sustained occlusion of the coronary artery),

immediately prior to the infarct, and it can also protect the myocardium against ischemic insult

[40]. IPC is triggered by the 3 G-coupled protein receptor dependent pathways: Adenosin,

bradykinin, and opioids [41-43], that activate their downstream mediators which are mainly

protein kinases: PKC, Akt, tyrosine kinase, and the MAPK. Other signalling activators that work

in parallel with the PKC pathway in IPC: receptor tyrosine kinase, MEK1/2- Erk1/2, the Jak-

STAT pathways, Glycogen synthase kinase 3ß (GSK-3ß), and ROS [44]. During IPC, opening of

22

mitochondrial KATP channel inhibits mitochondrial Ca2+

overload and attenuates myocardial IRI,

in addition to the inhibition of mPTP opening through GSK-3ß, e-NOS, or the reperfusion injury

salvage kinase (RISK) pathway [45] (Figure. 4). The upregulation of cardiac 3-nitrotyrosine, a

marker of peroxynitrite formation, is an initiative factor in triggering the cardioprotective

mechanisms induced by pre-conditioning [46].

Conditioning treatment combined with drug therapies like DDP-4 inhibitors is more effective in

restoring the hemodynamic function of cardiac tissue after ischemia, in addition to its anti-

inflammatory and anti-oxidative properties [47]. However, pharmacological agents given before

the index ischemia or at reperfusion can mimick the effect of IPC and IPO, and in both cases of

conditioning, imposing ischemia is not a part of the clinical mind-set, with a limitation of IPC

that can be only applied before the ischemic event takes place and only when it is predictable

[48].

Figure. 3. Schematic of myocardial ischaemic pos-tconditioning cardioprotective mechanism. IPO confers

cardioprotection through the SAFE and RISK pathways. The two mechanisms involve the activation of Jak/STAT3

and PI3K/Akt, which subsequently decreases mPTP opening and increases mitochondrial KATP (Mito KATP) channel

opening, and attenuates myocardial I/R injury (Br J Anaesth. 2016; 117 (S2): ii44–ii62).

23

Figure. 4. Schematic of ischemic pre-conditioning induced cardioprotection. Cardioprotection is induced through

the RISK pathway that involves the activation of PI3K/Akt and MEK1/2, accompanied by the activation of PKA, e-

NOS, P70S6K and GSK-3β, leading to a decrease in mPTP opening and increase in mitochondrial KATP (Mito KATP)

channel opening, attenuating myocardial I/RI (Br J Anaesth. 2016; 117 (S2): ii44–ii62).

5.5. Diabetes Mellitus and Anti-diabetic drugs

Diabetes mellitus is well known as a chronic metabolic disease that is characterized by a relative

or absolute lack of insulin, resulting in hyperglycemia. A variety of complications arises from

chronic hyperglycemia such as neuropathy, nephropathy, and retinopathy and increased risk of

cardiovascular disease. The two most common types of diabetes mellitus are type 1 diabetes

(T1DM) and T2DM. T1DM is generally thought to be precipitated by an immune-associated, if

not directly immune-mediated, destruction of insulin-producing pancreatic 𝛽 cells, while T2DM

is associated with insulin resistance and a lack of adequate compensation by the beta cells which

lead to a relative insulin deficiency [49, 50]. The outgrowth and progression of diabetic

complications are affected by various factors including obesity, insulin resistance,

hyperglycemia, and hyperlipidemia, and the management of the disease via blood glucose

monitoring and exogenous insulin administration is arduous and costly, which in parallel with

24

the meticulous efforts to regulate blood glucose can result in hyper- and hypoglycemic events

associated with systemic comorbidities [51]. (Figure. 5)

Numerous anti-diabetic drugs exert pleiotropic cardioprotective effects in addition to their

glycaemia-controlling effect (Table. 1). Those drugs include the first-line antidiabetic drug

metformin, and a new class of drugs known as gliptins [27, 52]. Both, metformin and vildagliptin

were extensively studied and were found to improve cardiac function impaired by I/R injury. In

addition to its anti-diabetic effect, both metformin and gliptins can reduce cardiac remodelling

and improve the LV function in animal and clinical studies of diabetic and non-diabetic subjects

with MI [53, 54]. Gliptins like vildagliptin, alogliptin, and linagliptin exhibites cardioprotective

effects mediated by NOS upregulation, but its still ambigious whether sitagliptin exerts the same

protective effect. Metformin can act by reducing insulin resistance, thus metformin can be more

effective in diabetic subjects or with insulin-resistance profile, than in normal subjects with

myocardial infarction; However, mechanisms of protection are still unclear, and a protective

drug against infarct size is still unknown.

Figure. 5. Hyperlipidemia- induced

signalling mechanisms attenuate ischemia

preconditioning cardioprotective effect

(Perfusion. 2015; 30(2): 94-105).

25

Cardioprotective anti-hyperglycemic agents

Drug name Subfamily Mechanism of action

1. Biguanides Metformin

Decreases hepatic glucose production

Increases glucose uptake

Increases insulin sensitivity

2. Sulfonylureas

Tolbutamide

Tolazamide

Acetohexamide

Chlorpropamide

Glyburide

Glipizide

Glimepiride

Stimulate insulin secretion by

pancreatic β- cells by closing

KATP channels

3. α-Glucosidase Inhibitors (AGi)

Acarbose

Voglibose

Miglitol

Inhibit α-glucosidase enzymes

Reduce the rate of absorption of

carbohydrates

Reduce postprandial glucose levels

4. Benzoic Acid Derivatives

(Meglitinides or Glinides)

Repaglinide

Nateglinide

Mitiglinide

Insulin-secretagogue agents

Stimulate the insulin release from

pancreatic β- cells by closing

KATP channels

5. Thiazolidinediones (Glitazones)

Rosiglitazone

Pioglitazone

Troglitazone

Peroxisome proliferator-activated

receptor-γ activators Enhance skeletal muscle insulin sensitivity

Reduce hepatic glucose production

6. Incretin Mimetics/Enhancers

Glucagon-Like Peptide-1

Receptor Agonists

(GLP-1 Ras, Incretin mimetics)

Liraglutide

Lixisenatide

Semaglutide

Exenatide

Dulaglutide

Albiglutide

Stimulate insulin release

Dipeptidyl Peptidase-4 Inhibitors

(Gliptins, Incretin enhancers)

Sitagliptin

Saxagliptin

Alogliptin

Linagliptin

Vildagliptin

Inhibit the degradation of GLP-1

Stimulate insulin release

7. Sodium–Glucose Co-transporter

Type 2 inhibitors (SGLT-2i/

Gliflozins)

Empagliflozin

Canagliflozin

Dapagliflozin

Ertugliflozin

Prevent the resorption of glucose in kidney

Decrease plasma glucose levels

and deplete sodium

Increase insulin sensitivity and

glucose uptake in muscles

Decrease gluconeogenesis

Improve insulin release from β- cells

26

Table. 1. Approved oral anti-hyperglycemic pharmacological therapies used as cardioprotective agents

(Drugs. Manolis et al. 2018).

5.6. Dipeptidyl peptidase-4 (DPP-4) inhibitors

DPP-4 inhibitors, including vildagliptin and sitagliptin, are oral anti-diabetic drugs that inhibit

the DPP-4 enzyme, resulting in a prolonged action of the GLP-1 hormone, an incretin hormone

secreted from intestinal L-cells [55]. GLP-1 (9-36) is a peptide metabolite derived from native

GLP-1 (7-36) amide after cleavage by DDP-4 (Figure. 6) [56], regulating multiple signaling

pathways, including regulation of PI3-kinase and Akt through ligation of the GLP-1 receptor

[56] (Figure. 6). DPP-4 has three major functions; adenosine deaminase binding, peptidase

activity, and extracellular matrix binding, all of which can influence the activity of the immune

and endocrine systems [57]. Inhibition of DPP-4 enzyme activity modulates the activity of

cardioactive peptides such as brain natriuretic peptide, neuropeptide Y (NPY), and stromal cell-

derived factor-1 (SDF-1), through non-GLP-1 mechanism of action [58]. Dipeptidyl peptidase-4

inhibitors exhibit beneficial and pleiotropic effects on metabolic parameters and the heart [59,

60], including increase in plasma insulin and decrease in glucose levels in T2DM models and

non-diabetic animals with I/R injury [61, 62]. The effects of DPP-4 inhibition on cardiovascular

function is attributed to the increase in GLP-1 levels, GLP-1 mediated phenomena, and

inhibiting the degradation of substrates involved in the cardiovascular homeostasis, while much

is less known about the direct cardiovascular effects of DPP-4 enzyme inhibition. DPP-4 is

abundantly expressed in cardiovascular system and endothelial cells [63].

Sitagliptin

27

Figure. 6. Schematic illustration of the proteolytic cleavage of the native 30 amino acid long peptide hormone

Glucagon-like peptide-1 (GLP-1) by the proteolytic enzyme dipeptidyl peptidase-4 (DPP-4). DPP-4 cleaves the

peptide bond between Ala8-Glu

9 resulting in the abundant GLP-1 (9–36), and this degradation process is inhibited

by the DPP-4 inhibitor Sitagliptin (Endocrinology. 2001; 142: 521–527).

5.7. Sitagliptin

Sitagliptin has neutral effects or may slow and/or attenuate progression of carotid intima-media

thickness (IMT), a surrogate marker of atherosclerotic CVD (ASCVD), defined as acute

coronary syndrome, a history of MI, stable or unstable angina, coronary or other arterial

revascularization, stroke, transient ischaemic attack or peripheral arterial disease [64]. The

pharmacokinetic properties of sitagliptin are similar in both healthy and diseased conditions,

with rapid absorption and peak plasma concentrations attained 1–4 h post- single dose of 100 mg

orally [65, 66], and exhibits potent and highly selective inhibition of DPP-4 [50 % inhibitory

concentration (IC50) 18 nmol/L], and more than 2600-fold greater for DPP-8 (IC50 48,000

nmol/L) and DPP-9 (>100000 nmol/L) [67]. The area under the plasma concentration-time curve

(AUC) from time zero to infinity increases in a dose-proportional manner with single doses of

sitagliptin 25–400 mg [66]. Single or multiple doses of sitagliptin (50-600 mg/day, ≤ 28 days) is

enough to induce a dose-dependent inhibition of DPP-4 (by ≥ 80 %, 24h postdose) and increase

the levels of GLP-1 and GIP two to three folds postprandialy in healthy, T2DM and non-diabetic

obese individuals [67]. The bioavailability of sitagliptin is 87 % and its oral absorption is not

affected by food [68], and metabolism plays a minor role in the elimination of sitagliptin, with

most (80 %) of an administered dose eliminated as unchanged drug in the urine [69].

5.8. Pleiotropic cardioprotective effects of sitagliptin

Activation of GLP-1 by DPP-4 inhibitors and GLP-1 analogs, limits myocardial infarct size (IS)

and protects cardiomyocytes from cell death, causing upregulation in intracellular cascades like

protein kinases, Akt/P-Akt, and ERK1/2 with protective profile against ischemia-reperfusion

injury [70, 71]. A change in myocardial glucose utilization may result in increased metabolic

efficiency and myocardial resistance to ischemia, thus limiting infarction, and this change in

myocardial glucose utilization is consistent with the physiological role of incretin hormone.

Vasodilation and reduction in systemic and/or pulmonary vascular resistance can also reduce

28

cardiac work and ATP demand during ischemia [34]. The vasodilatory effect of GLP-1 correlates

with an increase in cyclic guanosine monophosphate (cGMP) release and is attenuated by nitric

oxide synthase (NOS) inhibitors, suggesting that at least part of their vasodilatory mechanism is

nitric oxide (NO)/cGMP-dependent [56]. GLP-1 is an entero-hormone that enhances glucose-

dependent insulin secretion, inhibits glucagon secretion and slows gastric emptying [72]. The

two main routes of action of GLP-1 includes: (1) triggering glucose-induced insulin secretion

from pancreatic beta-cell and (2) binding to GLP-1R. Regulatory effects by GLP-1 depends on

the cAMP/PKA mediated induction of insulin-like growth factor-1 receptor (IGF-1R) expression

and increased activity of insulin-like growth factor 2 (IGF-2)/IGF-1R by autocrine mode of

action [73]. (Figure. 7)

Figure. 7. Schematic diagram of dipeptidyl-peptidase-4 inhibitors mechanism of action. DPP-4 inhibitors confer the

inhibition of DPP-4 activity with a subsequent increase in incretin peptides availability (glucagon-like peptide-1 and

gastric inhibitory polypeptide), inducing pleiotropic cardioprotective effects (Cell Signal. 2013; 25(9): 1799-1803).

Activation of GLP-1 receptors causes an increases in intracellular cAMP and calcium levels, and

activation of protein kinase A (PKA) and downstream phosphorylation of cyclic AMP response

element binding protein (CREB), were PKA is involved in the protection against IR injury by

29

upregulating p38 MAPK [70, 74] (Figure. 7). Incretin deficiency is among the key factors in the

pathophysiology of T2DM [75], and GLP-1R agonists are also considered good choice of

treatment in case of diabetes. GLP-1R is a member of the class B1 family of G-protein coupled

receptor detected in vascular and heart tissues of animals and human [76], and human coronary

artery endothelail cells. (Figure. 8)

Figure. 8. Schematic diagram showing the signaling pathways activated by the DPP-4 inhibitor sitagliptin, directly

or upon binding to GLP-1 receptors. The diagram represents the traditional signaling mechanisms involved in

cardioprotection, including cAMP/PKA, PI3K, Akt/P-Akt, ErK1/2, and cGMP, mediated NOS upregulation and e-

NOS production. It also clarifies the novelty of this study (Upregulation of TRP channels and CGRP mediated by

sitagliptin and GLP-1 (Int. J. Mol. Sci. 2018; 19: 3226).

30

5.9. New targeting markers of gliptins (NOS, TRP channels and CGRP)

5.9.1. Nitric oxide synthase system (NOS)

Nitric oxide synthases (NOSs) are enzymes that catalyzes the production of NO from L-arginine,

identified with 3 distinct isoforms: neuronal NOS (n-NOS), inducible (i-NOS), and endothelial

NOS (e-NOS) [77]. Nitric oxide originating from e-NOS isoform activates NO-cGMP-PKG

pathway and protein S-nitrosylation (SNO), possesses anti-atherosclerotic/anti-arteriosclerotic

actions by stimulating vasodilation, inhibition of vascular smooth muscle cell (VSMC)

proliferation, platelet aggregation, monocyte adhesion, vascular inflammation, and low density

lipoprotein (LDL) oxidation [78, 79]. e-NOS is highly abundant in endothelial cells, as well as in

cardiomyocytes, with a majority (80 %) localized in coronary endothelium and participating in

the relaxation of VSMC, while the rest (20 %) are located in cardiomyocytes [80, 81]. Several

mechanisms are involved in the in vivo regulation of e-NOS activity, including phosphorylation

of at S1176, resulting in increased enzymatic activity [82, 83]. In diabetic and hyperlipidemic

conditions, e-NOS phosphorylations becomes deficient, contributing to endothelial dysfunction.

Pharmacological studies on the different NOS isoforms involves the use of L-Arginine

analogues, including Nω-nitro- L-arginine methyl ester (L-NAME) and NG-monomethyl- L-

arginine (L-NMMA), as specific inhibitors. Oral treatment with L-arginine analogues leads to the

formation of coronary arteriosclerotic lesions in animals, the mechanism that had been attributed

to mediate the inhibition of e-NOS activity [84]. Endothelial dysfunction accompanied by loss of

endothelium derived NO and NO bioavailability occurs after ischemia-reperfusion injury [85],

triggering the pathophysiological events, such as up-regulation of adhesion molecules, leukocyte

adherence to endothelial cells of reperfused coronary arteries, transmigration of

polymorphonuclear cells and tissue damage due to apoptosis of reperfused myocardium [86].

Excessively produced NO derived from the activation of i-NOS contributes to the deterioration

of cardiac function through numerous mechanisms, including endothelial dysfunction, release of

inflammatory mediators, and overproduction of ROS [87]. Up-regulation of GLP-1 can restore

vascular NO bioavailability by the up-regulation of e-NOS, and blocking DPP-4 activity by

pharmacological inhibition or genetic deletion plays a role in the modulation of NOS enzymes

(Figure. 9) [58].

31

Figure. 9. Schematic diagram showing the mechanims of action of different NOS isoforms. Increased abundancy of

e-NOS contributes to enhanced endothelial function, decreased inflammation, and increased vasorelaxation in

endothelial and vascular smooth muscle cells. Excessive production of nitric oxide derived from the i-NOS

contributes to increased inflammation and endothelial dysfunction (Med Pregl. 2014; 67(9-10): 345-52).

In acute myocardial infarction, NO released by n-NOS prevents diastolic dysfunction, decreases

infarct size, activates β-adrenergic receptors, prevents cardiac hypertrophy, protects from

dysrhythmia, and reduces the synthesis of ROS from NADPH oxidase, enhancing the relaxation

of cardiac myocytes via cGMP/PKG pathway [88], while superoxide anion is formed instead of

NO due to e-NOS uncoupling as a consequence of oxidative stress and deficiency of cofactors

essential for NO synthesis [89]. Overexpression of i-NOS and NO overproduction in infarcted

cardiac cells mediates oxidative stress, inflammatory processes, and myocardial injury [90].

5.9.2. Transient receptor potential (TRP) channels and Calcitonine gene-related peptide

Transient receptor potential (TRP) channels are the most crucial Ca2+

permeable channels widely

expressed in vascular endothelium and cardiac tissue that regulate [Ca2+

] through its direct action

as Ca2+

entry channels in the plasma membrane, or by changing membrane potentials as a

modulator of the driving force for Ca2+

entry [91, 92] (Figure. 10). Their up-regulation in both

systems is contributed to its pathophysiology. TRP channels were firstly described in the

photoreceptor cells of Drosophila melanogaster as PLC allowing transmembrane calcium flux

Törölt:

32

[93], and gained vast attention as a superfamily of non-selective and non-voltage gated ion

channels functioning as cell signaling/sensory transducers [94], and its opening allows the

passage of Na+ and Ca

2+ ions, regulating several cellular functions [95]. Among the six

subfamilies of TRP channels: TRPA (ankyrin), TRPC (canonical), TRPV (vanilloid), TRPM

(melastatin), TRPP (polycistins), and TRPML (mucolipins), the canonical and vanilloid isoforms

are the most commonly localized and essential Ca2+

-permeable channels in vascular endothelial

cells, aorta, atria, ventricles, coronary blood vessels and sensory nerves innervating the heart and

involved in vascular tone regulation [91]. The TRPC family constitutes of seven members and

subdivided into two groups according to the structure and function: the TRPC-1/4/5 which are

non-sensitive to diacyglycerol (DAG), and TRPC-3/6/7 which are activated by DAG. Calcium

influx through cardiac TRPC channels (TRPC 1, 3, 4, 5 and 6) is essential for calcineurin

signaling and hypertrophic growth of hearts [96]. Depletion of calcium stores in endoplasmic

reticulum leads to the activation of these channels followed by calium influx [97], and ligand

binding to Gaq/11-protein coupled receptors (GPCRs) or receptor tyrosine kinases (RTKs) is

considered another mechanism of activation that induce phospholipase C to convert PIP2 to DAG

and activation of downstream molecules as protein kinase C [98]. The activation of TRPC

receptor by angiotensin II (Ang-II), and or endothelin-1 (ET-1) results in calcium pool depletion

in sarcoplasmic reticulum (SR) in cardiac cells, leading to up-regulation of TRPC-1, TRPC-4,

TRPC-5, opening of store-operated calcium channels (SOCC), and increase of Ca2+

influx, in

addition to the activation of TRPC-3, TRPC-6, and TRPC-7 [99]. TRPV channels are also

located on the heart sensory nerve endings, sciatic nerve and skeletal muscles [100]. TRPV-4-

mediated Ca2+

entry is involved in vasodilatation in response to arachidonic acid, an important

mediator of endothelium-derived hyperpolarizing-related vasodilation [101]. The characteristic

feature of TRPC channels is the six transmembrane domains and a pore-forming region

containing phenylalanine, tryptophan and leucine residues [102]. Transient receptor potential

vanilloid subtype 1 (TRPV-1) are localized in the sensory nerves that surrounds the

cardiovascular structure and in cardiomyocytes as well. The latter functions upon a chemical or

physical stimuli like capsaicin, proton and heat, releasing sensory neuropeptides functioning as

vasodilators, like calcitonin gene-related peptide (CGRP) and substance P [103], exhibiting

cardioprotective effects. CGRP is a neuropeptide that consitutes the sensory nerves and

synthesized in the dorsal root ganglia of the sensory nerves that terminates to the cardiovascular

system [104], mediating cardioprotection and regulating cardiac function [105], and reduction in

33

capsaicin sensitve neurons reduces myocardial CGRP and impairs cardiac function [106]. CGRP

is upstreamly regulated by the TRPV-1 that promotes its release upon stimulation (TRPV-1-

CGRP pathway) [107].

Activation of TRPV-1 by capsacin treatment exhibits cardioprotective effect against

ischemia/reperfusion injury, while TRPV-1 inhibition enhances the deleterious effects of

myocardial infarction [108], and this protective effect can be explained by the release of

neuropeptides (CGRP and substance P (SP)) that are known to protect against ischemic injury

[109]. CGRP is characterized by its cardiac regulatory function, enhancing myocardial

contarctility and increasing heart rate (HR) [110], and the reduction in HR and left ventricular

systolic pressure (LVSP) are key indicators of the insufficiency of CGRP activity in diabetic

animals, abrogating its inotropic and chronotropic effects on cardiomyocytes. Low levels of

CGRP in myocardial cells of diabetic heart impairs the calcium metabolism and KATP channels,

and affects diastolic function. Activation of e-NOS mediates the endothelium-dependent

vasodilatory actions induced by CGRP, and since e-NOS is Ca2+

/calmodulin-dependent, its

activity may be modulated by cytosolic Ca2+

levels [111]. Although TRPC and TRPV channels

play a fundamental role in mediating ischemia-reperfusion injury and regulating cardioprotective

signaling; however, the molecular mechanisms underlying the both types of channels are still

unknown, and further studies are needed to testify the significance and contribution of CGRP in

cardioprotection. (Figure. 10).

Figure. 10. Schematic diagram showing the activation of the non-selective cation channels, TRPC-1, increasing

calcium (Ca2+

) influx into the vascular smooth muscle cells (VSMC) and endothelial cells. Displayed data suggests

34

that calcium influx induces the upregulation of endothelial nitric oxide synthase (e-NOS) and nitric oxide (NO)

release, promoting endothelial protective effects (Int. J. Mol. Sci. 2018; 19: 3226).

35

6. AIMS

Due to the pleiotropic cardioprotective effect of gliptins as anti-diabetic drugs, we aimed to study

the effect of sitagliptin treatment on myocardial I/R injury in normal and high-fat diet animals,

using an ex-vivo model, and check whether high-fat diet can be a detrimental or an aiding factor

for sitagliptin.

To achieve our aim, we divided our study into 2 main parts:

1. Finding out the dose-limiting effect of sitagliptin on infarct size, in normolipidemic and

hyperlipidemic animals

2. Using heart tissues from the animal groups treated with the intended dose, to find out the

molecular mechanisms underlying the effect of sitagliptin

We hypothesized that:

1. Sitagliptin treatment can decrease the infarct size in both diet conditions

2. Protective effect of sitagliptin in normal and high-fat diet animals can be mediated by:

i. Nitric oxide synthase (NOS)

ii. Transient receptor potential channels (TRPC, TRPV)

iii. Calcitonin gene-related peptide (CGRP)

3. NOS and TRPV inhibition can block the cardioprotective effect of sitagliptin

36

7. MATERIALS AND METHODS

7.1. Drug preparations

Sitagliptin filmtablets (Januvia 100mg, Merk Sharp & Dohme Ltd., Hertfordshire EN11 9BU,

UK) were purchased and freshly dissolved in saline (0.9 %) on daily basis and before each

treatment. The anesthetic agent Thiopental (Tiobarbital Braun, 0.5 g, B. Braun Medical SA) was

also dissolved in saline (0.9 %). NOS-inhibitor (L-NAME), purchased from Sigma Aldrich was

dissolved in physiological saline (0.9 %), while TRPV-1 inhibitor (Capsazepine) was also

purchased from Sigma Aldrich and dissolved in dimethyl sulfoxide (DMSO).

7.2. Animals and experimental design

The study conforms with the standards of the European Community guidelines for the Care and

Use of Laboratory Animals (2010/63/EU, and Hungarian law (XXVIII/1998). 40/2013, 14th

Governmental Decree. All procedures were performed according to the protocols approved by

the Institutional Ethical Animal Care and Use Committee of Szeged University, with the project

identification code and date of approval (XX. /4801/2015). Six to eight-week-old male Wistar

rats (body weight 200–300 g; Charles River, Hungary) were obtained and acclimatized for one

week before the commencement of treatments. All animals were housed in our temperature-

controlled animal facility (23+/- 2°C), maintained with a 12:12-h light–dark cycle, humidity

(55+/- 10%), with food and water provided ad libitum, fed either with standard rat chow only or

mixed with fats (High fat= HF) for 12 weeks to induce hyperlipidemia. All experiments were

conducted in compliance with the use of the 3Rs.

Animals were assigned into 4 different experiments:

Experiment 1. To determine the most effective dose (kg-1day) of Sitagliptin (Sitg), animals

were randomly divided into 5 groups: (Control (Saline), Sitg (25 mg), Sitg (50 mg), Sitg (100

mg) and Sitg (150 mg), n=8-16), in both normolipidemic and hyperlipidemic assigned animals.

The daily oral administration of different drug doses or its vehicle (Saline) lasted for two weeks.

At the end of the treatment, the whole-heart preparation and ischemia-reperfusion (I/R) injury

protocol started. Animals were anesthetized with thiopental (i.p. 100 mg/kg), heart tissues were

excised and immediately placed in ice-cold saline (0.9 %), mounted and ligated through the aorta

into the cannula (ex vivo) of a modified Langendorff Apparatus Working Heart System, and

37

perfused with 37°C Krebs buffer (118 mM NaCl, 4.70 mM KCl, 2.50 mM CaCl2, 1.18 mM

MgSO4, 1.18 mM KH2PO4, 5.50 mM glucose and 25 mM NaHCO3 and gassed with 95 % O2 and

5 % CO). Hearts were exposed to 10 min perfusion, 45 min prolonged regional ischemia,

followed by 120 min reperfusion (we couldn’t reach a 50-60% infarct size inside the risk zone

using the conventional 30 min coronary occlusion by left anterior descending (LAD) coronary

artery occlusion on our system, so we tried to use more prolonged coronary occlusion, and 45

min ischemia was effective to develop enough percentage of infarction, compared to results

obtained using 30 min of coronary occlusion). At the end of the experiment, LAD coronary

artery was religated, and the area at risk (AAR) was stained with Evans blue dye via the aortic

root. Hearts were weighed and stored at -20 oC for further triphenyltetrazolium chloride (TTC)

staining. The Sitg (50 mg)-dose was used in our further experiments, as the most effective dose

(Figure. 11a).

Experiment 2. For the purpose of in vitro laboratory measurements, another set of experiments

was carried out by assigning only two animal groups (Control (Saline) and Sitg (50 mg/kg/day),

n=10), under normolipidemic and hyperlipidemic conditions, and oral daily treatment with

sitagliptin lasted for two weeks. At the end of the treatment, the whole-heart preparation and IR

injury protocol were performed. Same anesthetization procedure and whole-heart preparation

process was carried out as in Experiment 1. Hearts were exposed to 10 min perfusion, 45 min

prolonged regional ischemia by occluding the LAD coronary artery, followed by a brief 10 min

reperfusion. At the end of the experiment, heart tissues were weighed, directly clamped and

stored at -80 oC for further biochemical analyses (Figure. 11b).

Experiment 3. To confirm the involvement of NOS in Sitg (50 mg)- mediated cardioprotection

against I/R injury, four different animal groups (Control (Saline), Sitg (50 mg), Control (Saline)

+ L-NAME, and Sitg (50 mg) + L-NAME, n=10-12) were studied in the normolipidemic

animals. The Control (Saline) and Sitg (50 mg) animal groups received the same daily oral

treatment as in Experiment 1, while the other two groups were co-treated intraperitoneally (i.p)

with a specific NOS inhibitor (L-NAME, 25 mg/kg/day) [112], three hours post-oral

administration of Saline and Sitg (50 mg). Similar animal groups and treatment procedures were

followed in the hyperlipidemic condition. At the end of the treatment, the same anesthesia, hearts

excision, whole-heart preparation and I/R injury protocols (10 min perfusion, 45 min prolonged

regional ischemia and 120 min reperfusion, ex vivo), coronary artery re-ligation, and cardiac

38

tissue staining procedures mentioned in Experiment 1 were performed for all groups (Figure.

11c).

Experiment 4. To evaluate the inhibitory effect of TRPV-1 on infarct size (IS), another four sets

of animals (Control (Saline) + DMSO, Sitg (50 mg) + DMSO, Control (Saline) + CAP, and Sitg

(50 mg) + CAP, n= 5-8) received the same daily oral treatments, and co-treated daily using

intraperitoneal (i.p) injections with either DMSO or TRPV-1 inhibitor (Capsazepine (CAP), 1

mg/kg/day) [113], three hours post-oral treatments, for two weeks, only under normolipidemic

conditions. DMSO was diluted with saline with a 1:4 ration, and a volume of 200 ul/ animal was

intraperitonealy injected (animals were daily checked, without addressing any side effects during

the 2 weeks i.p treatment). At the end of the treatment, the same anesthesia, heart excision,

whole-heart preparation and I/R injury protocols (10 min perfusion, 45 min prolonged regional

ischemia and 120 min reperfusion, ex vivo), coronary artery re-ligation, and cardiac tissue-

staining procedures mentioned in Experiment 1 were performed for all groups (Figure. 11d).

Figure. 11. Diagram illustrating 4 different experimental protocols. (a) Heart tissues from both diet animal groups

subjected to 45 min ischemia and 120 min of reperfusion, after 2 weeks of oral animal treatment with Saline and

different doses of Sitagliptin, for infarct size measurement. (b) Hearts from normolipidemic and hyperlipidemic

animals subjected to 45 min ischemia and 10 min brief reperfusion, afterwhich the animals received a 2 weeks’ oral

administration of Saline and Sitg (50 mg), for further biochemical measurements. Infarct size measurement of heart

39

tissues from normolipidemic and hyperlipidemic animals exposed to prolonged ischemia-reperfusion injury,

afterwhich the animals received a 2 weeks’ co-treatment of Saline, Sitg (50 mg) and intraperitoneal injection of

NOS-inhibitor (L-NAME) (c). In the 4th experimental protocol (d), the inhibitory effect of TRPV-1 against

ischemia-reperfusion (IR) injury was assessed by heart infarct size measurement at the end of a prolonged

reperfusion-injury, and after co-treating the normolipidemic animals intraperitoneally with Capsazepine (TRPV-1

inhibitor).

7.3. Tissue staining and infarct size measurement

At the end of each prolonged reperfusion phase (120 min), the LAD coronary artery was re-

ligated, and the risk zone was stained with Evans blue dye via the aortic root. Hearts were frozen,

transversely sectioned into (5-6 slices; 2-mm thickness) from the apex to the base, and incubated

in 1 % triphenyltetrazolium chloride (TTC) for 10 min at 37 °C. After incubation, tissue sections

were fixed for 10 min in 10 % formalin, and then placed for 30 min in phosphate buffer (pH 7.4).

All sections were mounted on glass slides, images were captured with a digital camera, and an

ImageJ 1.34 software was used to measure the infarcted areas. Infarcted areas were measured in

each section by an investigator who was blinded to the identity of the sections (Figure. 12). Same

staining procedure was used for normolipidemic and hyperlipidemic animals.

Figure. 12 (a) & (b). Representative photographs of transversely sectioned Evans-blue perfused, TTC-stained heart

tissues, outlining the area at risk (AAR; sum of white and red area); blue, healthy viable tissue; pale white, infarcted

(a) (b)

40

tissue. Myocardial infarct area (IS; white) was measured post-myocardial ischemia-reperfusion and TTC staining, in

different treated and control groups in normolipidemic (a) and hyperlipidemic (b) animals.

7.4. Serum cholesterol and triglyceride measurements

After heart tissue removal, blood was collected from the abdominal aorta of the hyperlipidemic

animals, centrifuged, and serum samples collected in eppendorffs and stored at -20 oC for

cholesterol (Chol) and triglycerides (TG) measurements. Chol and TG reagent kits

(Diagnosticum Zrt, Hungary) were used for both measurements. Quantitative determination of

cholesterol and triglycerides concentartion in serum based on enzymatic colorimetric method

(phenol + aminophenazone -PAP). Satndard and sample (10 ul) measurements at wavelength (λ=

490-550 nm) were carried in a 96-well plates, after 5-min incubation at 37 oC and according to

the protocols provided in the kit’s manual. Results of both measuremnts are expressed in

(mmol/l).

7.5. Cholesterol and triglyceride measurements from liver samples

Measured liver tissues harvested from hyperlipidemic animals were homogenized in ice-cold

modified phosphate buffer saline (PBS) by Ultra-Turrax T25 (13.500/s). Liver supernatants were

collected, and same chol and TG kits (Purchased from Diagnosticum Zrt) were used, follwoing

the same protocols with some modifications regarding dilutions and sample volume. Obtained

results are expressed in (mmol/l).

7.6. DPP-4 activity test

The cardiac DPP4 (CD26) activity in Control and Sitg (50 mg) treated normolipidemic and

hyperlipidemic animal groups was assessed using DPP4 activity assay kit and according to the

manufacturer’s guidelines (Sigma-Aldrich). A 10 mg of heart tissues were homogenized in ice-

cold DPP4 Assay Buffer, centrifuged at 13,000 xg, at 4°C for 10 min, and supernatants were

harvested. Standard and sample fluorescence intensity (FLU) measurements (λex=360/λem=460

nm) were carried out after five min of incubation at (37 o

C) in 96-well black plates specific for

fluorescence assays, using a fluorescence multiwell plate reader (Fluorometer). Incubation and

41

measuring cycles were repeated, until the most active sample was near to or greater than the

value of the highest standard (100 pmole/well). Results are expressed as microunit/ml.

7.7. Nitric oxide synthase (NOS) activity

NOS activity was measured in the 2 different conditioned diets, by quantifying the conversion of

[14

C]-labeled L-arginine to citrulline by a previously described method with some minor

modifications [114]. Heart tissues were homogenized with Ultra-Turrax T25 (13,500/s; twice for

30 sec) in ice-cold 10 mM N-[2-hydroxyethyl] piperazine-N’- [2-ethanesulfonic acid] (HEPES,

Sigma-Aldrich), 32 mM sucrose (Sigma-Aldrich), 1 mM dithiothreitol (DTT, Sigma-Aldrich),

0.1 mM ethylenediaminetetraacetic acid (EDTA), 10 μg/ml soybean trypsin inhibitor (Sigma-

Aldrich), 10 μg/ml leupeptin (Sigma-Aldrich), and 2 𝜇g/mL aprotinin (Sigma-Aldrich), at pH

7.4. Supernatants were collected by centrifugation for (30 min, 20000 xg, 4°C). Samples (40 μl)

were incubated for 10 min at 37°C with 100 μl of assay buffer (50 mM KH2PO4, 1.0 mM MgCl2,

50 mM L-valine, 0.2 mM CaCl2, 1.0 mM DTT, 1.0 mM L-citrulline, 15.5 nM L-arginine, 30 μM

flavin adenine dinucleotide (FAD), 30 μM flavin mononucleotide (FMN), 30 μM tetrahydro-L-

biopterin dihydrochloride (THB), 450 μM β-nicotinamide adenine dinucleotide phosphate (β-

NADPH), and 12 pM [14C]-L-arginine monohydrochloride (all from Sigma-Aldrich, Budapest,

Hungary). The reaction was terminated by addition of 0.5 ml of a 1:1 (v/v) suspension of ice-

cold DOWEX (Na+ form) in distilled water. The mixture was re-suspended by adding 850 μl of

ice-cold distilled water, supernatant (970 μl) was removed and radioactivity was determined by

scintillation counting. The Ca2+

dependence of the NOS activity was determined by addition of

10 μl of ethylene glycol-bis (β-aminoethyl ether) tetraacetic acid (EGTA; 1 mM, Sigma-

Aldrich). NOS activity was confirmed by inhibition with 10 μl of Nω-nitro-L-arginine methyl

ester (L-NNA; 3.7 mM, Sigma-Aldrich). The level of i-NOS was defined as the extent of

citrulline formation that was inhibited by L-NNA, but not by EGTA. The cNOS activity was

calculated from the difference between the extent of citrulline formation inhibited by EGTA and

the total activity. As the nature of the cNOS isoform (e-NOS or n-NOS) was not determined, this

activity is referred to as cNOS. NOS activity is expressed as (pmol/min/mg protein).

7.8. ELISA measurements (GLP-1, TRPV-1 and CGRP)

42

A double-antibody sandwich ELISA kits specific for rat GLP-1, TRPV-1 and CGRP

measurements were purchased from the same company (SunRed Biotechnology). Same

homogenization buffer (Phosphate Buffer Saline (PBS), PH 7.2-7.4) and homogenization

procedure (Homogenization by Ultra Turrax T8, 20 min centrifugation at 2000-3000 rpm). The

whole tissue sample preparation procedure was done on ice. The three parameters were measured

according to the manufacturer’s instructions and protocols, and optical densities (OD) were

determined at λ= 450 nm. Results were expressed in (ng/ml) for GLP-1 and TRPV-1, and (ng/L)

for CGRP. The three different measurements were done in animal groups from both diet

conditions.

7.9. Calcium (Ca2+

) content test

A Colorimetric Calcium Detection Assay Kit (Abcam) was used to determine the calcium (Ca2+

)

concentration in both diet conditions. Samples were homogenized on ice using PBS + 0.1% NP-

40, centrifuged at a maximum speed for 2-5 min. Supernatants were collected, and measurements

were performed according to the provided procedure. Optical densities (OD) were detected at

(λ= 575 nm). Results are expressed in (ng/mg protein).

7.10. TRPC-1, e-NOS and DPP-4 (CD26) protein expression by western blotting

normalized to β-actin

Measured heart tissues from both diet groups were homogenized by Ultra-Turrax T25 (13,500/s;

twice for 30 sec) with ice-cold radio immunoprecipitation assay (RIPA) buffer (containing a

protease inhibitor and TRITON-X-100), for DPP-4 (CD26) and TRPC-1 proteins, and Homo-

buffer (containing phosphatase inhibitor, vanadate (1:50)), for e-NOS. Homogenates were

centrifuged (10-15 min, 12000 rpm, 4°C). Proteins were resolved on an 8 % and 10 % sodium

dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE, 1 mm gel cassette), and

transferred into nitrocellulose membranes. Blots were probed overnight (4 oC, and 1 % milk)

with anti-TRPC-1 rabbit primary antibody (1:500, (ab192031) Abcam), and anti- eNOS mouse

primary antibody (1:250, (ab 76198) Abcam) respectively, 2 hours at room temperature with

anti-CD26 rabbit primary antibody (1:500, (ab129060) Abcam), and anti-beta actin mouse

primary antibody (1 % BSA, 1:4000, (ab 8226) Abcam). Membranes were then incubated for 1 h

at room temperature (RT) with secondary anti-rabbit antibody (1:1000, (sc-2370) Santa Cruz),

43

secondary anti-mouse antibody (1:5000, (A9044) Santa Cruz), secondary anti-rabbit (1:5000,

(sc-2370) Santa Cruz), and secondary anti-mouse antibody (1:2000, (A9044) Santa Cruz) for

TRPC-1, e-NOS, Cd-26 and β-actin, respectively. Secondary antibodies were conjugated with

horseradish peroxidase (HRP) enzyme. Signals were developed using an enhanced

chemiluminescent substrate for detection of HRP (ECL Western Blotting Substrate, Thermo

Scientific., Rockford, USA) and exposed to Hyperfilm. Films and protein bands densities were

analysed using the Image Quant Software (Amersham Pharmacia Biotech., Buckinghamshire,

UK) after scanning with Gel Analyst 3.01 Software (Iconix, Toronto, Canada), and were

normalized to housekeeping protein β-actin.

7.11. Protein determination

Aliquots (20 μl) from diluted samples (15- or 25-fold with distilled water) were mixed with 980

μl of distilled water, after which 200 μl of Bradford reagent was added to each sample. After

mixing and 10 min of incubation, samples were assayed spectrophotometrically at λ= 595 nm

with a commercial protein assay kit (Bio-Rad Labs, Budapest, Hungary). Protein levels were

expressed as (mg protein/ml).

7.12. Statistical analysis

All data are shown as mean ± SEM. Statistical comparisons were performed with Student’s two-

tailed unpaired t test and a multiple comparison test (Bonferroni) when necessary. Differences

were considered significant when P- values were less than 0.05 (P< 0.05).

44

8. RESULTS

8.1. Normolipidemic animals

8.1.1. DPP-4i Decreased the infarct size in heart tissues of Sitg (50 mg) treated group

After two weeks of daily oral administration of four different doses of the same DPP-4 inhibitor

(Sitagliptin), prior to ischemia-reperfusion injury and after subjecting the heart tissues to 45 min

of regional ischemia and 120 min of reperfusion, Sitg (50 mg/kg/day) treated group exhibited a

significant decrease in infarct size (22.20 ± 2.03 %) vs. Control (44.89 ± 4.02 %). Sitg (50 mg)

dose showed an infarct size-limiting effect, and the effective dose was used in further

experiments and measurements.

The area of infarction is expressed as the percentage of infarct size over the area at risk (Figure

13).

Control (

Salin

e)

Sitg

(25m

g)

Sitg

(50m

g)

Sitg

(100m

g)

Sitg

(150m

g)0

20

40

60

Infa

rct

size

/Are

a a

t ri

sk (

%)

Figure. 13. Effect of different doses of Sitagliptin (Sitg) on infarct size (expressed in %). Results are shown as

(Mean ± SEM); (n= 8-16 animals/group). Statistical significance: **P< 0.01 relative to the Control group. Sitg (50

mg) exhibited a cardioprotective effect against ischemia-reperfusion (IR) injury, while no significance was reported

in other doses (25, 100, and 150 mg/kg/day). Satistical comparisons were performed using the One-way ANOVA

multiple comparison test (Bonferroni).

**

45

8.1.2. Sitg (50 mg) normalized DPP-4 activity and enhanced GLP-1 level

Sitagliptin reduced DPP-4 activity (552.32 ± 100.02 microunits/ml) by 50 % in Sitg (50 mg/kg)

treated group, compared to the Control group (1005.92 ± 190.96 microunits/ml). Measurements

of GLP-1 level from heart tissues subjected to brief reperfusion (10-min), revealed a significant

increase (44.98 ± 4.02 ng/ml) in Sitg (50 mg) treated group compared to the Controls (22.20 ±

2.03 ng/ml). Results are shown in Figure 14.

Figure. 14. Changes in Dipeptidyl peptidase-4 (DPP-4) enzyme activity (expressed in microunits/mL x 102) and

Glucagon-like peptide 1 (GLP-1; expressed in ng/mL) in the heart tissues of Sitagliptin (Sitg)- 50 mg treated animal

groups. Data are represented as (Mean ± SEM); (n= 4-10 animals/group). Statistical significance: *P< 0.05

compared to the Control group.

8.1.3. DPP-4i increased TRPV-1 and CGRP levels in heart tissues of Sitg (50 mg)

Convincingly, a significant increase in TRPV-1 level (458.49 ± 27.62 ng/ml) was observed in

Sitg (50 mg) group compared to the Control (351.04 ± 17.40 ng/ml). Results of CGRP

measurements are in line with that of TRPV-1, showing a clear increase in CGRP level (16.91 ±

1.57 ng/mg protein) vs. (9.36 ± 0.65 ng/mg protein), in Sitg (50 mg) and Control groups

respectively. Results are displayed in Figure 15.

Contr

ol (Sal

ine)

Sitg

(50m

g)

Contr

ol (Sal

ine)

Sitg

(50m

g)0

5

10

15 DPP4 activity (microunits/mL) x 102

GLP-1 (ng/mL)

*

*

46

Figure. 15. Effect of Sitagliptin treatment on TRPV-1 (expressed in ng/mL) and CGRP (ng/mg protein) ischemic

cardiac tissue levels, compared to the Control animal group. A clear significant increase is observed in both proteins

levels observed comparing the treated group to the Control (**P< 0.01). Data are illustrated as (Mean ± SEM); (n=

5-10 animals/group).

8.1.4. DPP-4i augmented cardiac calcium (Ca2+

) content in hearts of Sitg (50 mg) group

To determine whether the ischemic cardiac calcium concentration was affected by the DPP-4

inhibitor (Sitagliptin) treatment, a colorimetric calcium detection assay kit was used, and

obtained findings indicated an increase in calcium content in heart tissues assigned to drug

therapy (72.23 ± 12.19 ng/mg protein) vs. Controls (39.55 ± 14.49 ng/mg protein) (Figure 16).

Contr

ol (Sal

ine)

Sitg

(50m

g)

Contr

ol (Sal

ine)

Sitg

(50m

g)0

20

40

60TRPV-1 (ng/mL) x 10

CGRP (ng/mg protein)

**

**

47

Control (Saline) Sitg (50mg)0

20

40

60

80

100C

alc

ium

co

nte

nt

(ng/m

g p

rote

in)

Figure 16. Changes in calcium content of cardiac tissues excised from the Sitagliptin (Sitg (50 mg); n= 7) treated

animals and the Control ones (n= 4). The bar chart displays an increase in calcium concentration in Sitg (50 mg)

group, and presented values are in terms of (Mean ± SEM).

8.1.5. DPP-4i positively affected TRPC-1 protein expression

The difference in TRPC-1 protein expression level between the control (Saline) and Sitg (50 mg)

treated groups normalized to β-actin is presented in Figures 17 & 19a. Sitg (50 mg) treated group

showed a 3- fold higher level of TRPC-1 expression (408.12 ± 16.29 Intensity x mm2) in

comparison with the Control group (129.38 ± 38.58 Intensity x mm2).

Control (Saline) Sitg (50mg)

0

200

400

600

800

TRP

C-1

pro

tein

exp

ress

ion

(In

ten

sity

x m

m2)

Figure 17. Upregulation of TRPC-1 protein expression level (expressed in Intensity x mm2) in the heart tissues of

Sitagliptin (Sitg (50 mg); n= 10) treated group vs. Control (n= 10). Data are in term of (Mean ± SEM). Statistical

significance: **P< 0.01.

**

_

48

8.1.6. DPP4-i upregulated cNOS activity and e-NOS protein expression in heart tissues of

Sitg (50 mg)

8.1.6.1. cNOS activity

Two weeks of daily oral treatment with Sitagliptin (50 mg), and brief reperfusion (45 min

occlusion and 10 min reperfusion) of the coronary artery showed a significant increase in heart

cNOS activity (260.87 ± 60.86 pmol/min/mg protein) relative to the Control group (96.47 ±

11.71 pmol/min/mg protein) (Figure 18a).

8.1.6.2. e-NOS protein expression

Protein expression of e-NOS isoform which is also known as nitric oxide synthase-3 (NOS-3) as

determined by Western blot and normalized to β-actin is shown in Figures 18b and 19b.

Obviously, e-NOS expression was significantly increased in heart tissues of Sitg (50 mg) treated

animals (979.38 ± 106.84 Intensity x mm2), in comparison with the Control ones (685.53 ± 60.26

Intensity x mm2).

Figures 18 (a) & (b). Increase in constitutive nitric oxide synthase (cNOS (n= 8)) enzyme activity and endothelial

nitric oxide synthase (e-NOS (n= 6-8)) protein expression in ischemic cardiac tissues from Sitagliptin (Sitg (50 mg))

treated group compared to Control (*P< 0.05). Values are expressed in (pmol/min/mg protein) and (Intensity x

mm2), for cNOS activity and e-NOS protein expression respectively. Presented data are (Mean ± SEM).

Control (Saline) Sitg (50mg)0

100

200

300

400

cNO

S a

ctiv

ity

(pm

ol/

min

/mg p

rote

in)

Control (Saline) Sitg (50mg)

0

200

400

600

800

e-N

OS

pro

tein

ex

pre

ssio

n

(In

ten

sit

y x

mm

2)

(a) (b)

* *

49

Figure 19 (a) & (b). Expression of e-NOS and TRPC-1 proteins in ischemic heart tissues treated with 50 mg

sitagliptin (Sitg (50mg)), compared to the control (Saline) group. The blots show that both proteins are significantly

expressed after sitagliptin treatment.

8.1.7. L-NAME inhibited NOS- mediated cardioprotection against infarct

To evaluate the cardioprotective mechanism of the DPP-4 inhibitor (Sitagliptin)- mediated by

nitric oxide synthase (NOS), rats were treated with L-NAME (NOS-inhibitor) and the size of

infarction was assessed. Myocardial infarct size quantifications as a percentage of the left

ventricle (LV) and the area at risk are shown in Figure 20. Results from Sitg (50 mg) treated

group matches the previous ones, showing 3-folds significant reduction (21.56 ± 2.41 %),

compared to the Controls (49.09 ± 4.60 %). However, this protective effect disappeared in Sitg

(50 mg) + L-NAME- treated animal group (36.99 ± 3.82 %) vs. the animal group treated with

Sitg (50 mg) alone (21.56 ± 2.41 %). L-NAME also decreased infarct size (34.18 ± 3.17 %) in

Control (Saline) + L-NAME group, compared to Control (Saline) (49.09 ± 4.60 %)

e-NOS

TRPC-1 (a)

(b)

β-actin

50

Figure. 20. Loss of cardioprotective effect mediated by NOS and increase in infarct size with intraperitoneal

injection of NOS- inhibitor (L-NAME), Infarct size expressed in (%). Comparing Control (Saline, n= 12) and

Sitagliptin (Sitg (50 mg), n= 10), shows a significant decrease in infarct size (***P< 0.001), while this protective

effect was abolished comparing the Sitg (50mg) + L-NAME (Sitagliptin 50 mg + L-NAME (i.p), n= 11) group

with the Sitg (50 mg, n= 10) treated group (#P< 0.05), which means that cardioprotective effect of Sitagliptin

against infarction is mediated through NOS. A significant decrease (†P< 0.05) in infarct size was also observed in

Control (Saline) + L-NAME (Saline L-NAME (i.p), n= 11) group, compared to Control (Saline). Statistical

analysis was performed using One-way ANOVA, as well as Two-way ANOVA when necessary. Data plotted as

(Mean ± SEM).

8.1.8. Capsazepine inhibited TRPV-1- mediated cardioprotection against infarct

The area of infarction was measured in the presence and absence of TRPV-1 inhibitor

(Capsazepine), to test whether TRPV-1 is directly implicated in DDP-4 inhibitor (Sitg)-

mediated cardioprotection. Quantification of infarct size as a percentage of the left ventricle (LV)

and the area at risk are shown in Figure 21, were the animal group treated with Sitg (50 mg) +

DMSO exhibited a clear decrease in infarct size (49 ± 2.50 %) vs. Control (Saline)+DMSO

group (60.08 ± 2.93 %). However, TRPV-1 inhibition with Capsazepine blocked this

cardioprotection in Sitg (50 mg) + CAP- treated group (63.01 ± 4.32 %) compared to the group

***

#

†

Control (

Salin

e)

Sitg

(50m

g)

Control (

Salin

e) + L-

NAME

Sitg

(50m

g) +

L-NAM

E0

20

40

60In

farc

t si

ze/A

rea

at r

isk

(%)

51

treated with Sitg (50 mg) + DMSO (49 ± 2.50 %). A significant difference in infarct size was

also observed in Saline + CAP group (64.81 ± 1.98 %), compared to Sitg (50mg) + DMSO (49 ±

2.50 %).

Figure. 21. Loss of cardioprotective effect mediated by TRPV-1 and increase in infarct size with i.p. injection of

TRPV1- inhibitor (Capsazepine; CAP), Infarct size expressed in (%). Comparing the 2 groups, Control (Saline) +

DMSO (Control (Saline) + DMSO (i.p), n= 7) and Sitg (50 mg) + DMSO (Sitg 50 mg + Dimethyl sulfoxide (i.p), n=

6), shows a significant decrease in infarct size (*P< 0.05), while this protective effect was abolished comparing the

Sitg (50 mg) + CAP (Sitagliptin 50 mg + CAP (i.p), n= 8) group with the Sitg (50 mg) + DMSO treated group (#P<

0.05), which means that cardioprotective effect of Sitg against infarction is mediated through TRPV-1. A significant

difference (††

P< 0.01) was observed in Sitg (50 mg) + DMSO (Sitg (50 mg) + DMSO (i.p), n= 6) group, compared

to Saline + CAP (Saline + CAP (i.p), n= 8). Statistical analysis was performed using One-way ANOVA, as well as

Two-way ANOVA when necessary. Data plotted as (Mean ± SEM).

8.2. Hyperlipidemic animals

8.2.1. DPP-4i decreased the infarct size (IS) in heart tissues of Sitg (50mg) group

Two weeks following the daily oral administration of different doses of the same DPP-4

inhibitor (Sitg) prior to ischemia-reperfusion injury, and after subjecting the hearts to 45 min of

* †

#

Control (

Salin

e) + D

MSO

Sitg

(50m

g) +

DM

SO

Salin

e + C

AP

Sitg

(50m

g) +

CAP

0

20

40

60

80

Infa

rct

size

/Are

a a

t ri

sk (

%)

52

regional ischemia and 120 min of reperfusion, Sitg (50 mg/kg)- treated group exhibited a

significant decrease in IS (19.99 ± 2.44 %) compared to the Control group (38.11 ± 1.82 %).

The area of infarction is expressed as the percentage of infarct size over the area at risk (Figure

22).

Figure. 22. Effect of different doses of Sitg on infarct size (expressed in %). Results are shown as (Mean ± SEM);

(n= 5-10 animals/group). Statistical analysis was performed using One-way ANOVA multiple comparison test

(Bonferroni), and statistical significance is represented as: *P< 0.05 relative to the Control group, and ##

P<0.01,

comparing HF+Sitg (50mg) and HF+Sitg (150mg) groups together. Sitg (50mg) exhibited a cardioprotective effect

against ischemia-reperfusion (I/R) injury, with no reported significance in other doses (25, 100, and 150 mg/g/day).

8.2.2. Serum cholesterol and triglycerides concentration

Cholesterol measurements from serum samples revealed significant increase in cholesterol level

in high-fat diet control animals (Control (HF+Saline); 2.72 ± 0.15 mmol/l), compared to normal

control animals (Control (Saline); 1.95 ± 0.17 mmol/l), while no significant change was observed

comparing the high-fat diet animals treated with sitagliptin (HF+Sitg (50 mg); 2.75 ± 0.10

mmol/l) compared to control animal group kept on high fat diet (Control (HF+Saline); 2.72 ±

0.15 mmol/l) Results are shown in Figure 23a. Similarly, serum triglyceride level was

significantly increased comparing the control animals from both diets (Control (HF+Saline) vs.

Control (Saline); 2.12 ± 0.13 vs. 0.72 ± 0.04 mmol/l), however, no significant change was

observed in triglyceride level in sitagliptin treated group from high-fat diet animals (HF+Sitg (50

*

Control (

HF+Sa

line)

HF+Si

tg (2

5mg)

HF+Si

tg (5

0 mg)

HF+Si

tg (1

00mg)

HF+Si

tg (1

50mg)

0

20

40

60

80

Infa

rct

size

/Are

a at

ris

k (%

)

**

53

mg); 2.09 ± 0.14 mmol/l), compared to the control animals kept on high-fat diet (Control

(HF+Saline); 2.12 ± 0.13 mmol/l) (Figure 23b).

Control (

Salin

e)

Control (

HF+Sa

line)

HF+Si

tg (5

0 mg)

0.0

0.5

1.0

1.5

2.0

2.5

Seru

m T

rigl

yce

rid

e le

vel

(mm

ol/

l)

Figure. 23 (a) & (b). Serum cholesterol (Figure 22a) and triglyceride (Figure 22b) levels (expressed in mmol/l) in

control animal groups kept on normal or high-fat diet, and high-fat diet animals treated with sitagliptin. Chol and TG

significantly increased after long-term feeding with high-fat diet, while no significant change was observed in both

parameters in animal groups kept on high-fat diet and treated with Sitg (50mg), compared to the high-fat control

ones (Control (HF+Saline)). Data are represented as (Mean ± SEM); (n= 5-8 animals/group). One-way ANOVA

multiple comparison test (Bonferroni) was used for statistical analysis, and results were considered significant when

(*P< 0.05).

8.2.3. Liver cholesterol and triglycerides concentration

Measurements from liver homogenates showed a significant decrease in cholesterol level in

HF+Sitg (50 mg) group compared to HF+Saline group and Absolute control group (2.64 ± 0.03

vs. 2.94 ± 0.04 mmol/l), and (2.64 ± 0.03 vs. 2.86 ± 0.04), respectively, while no change in

cholesterol profile was reported when comparing HF+Saline group (2.924 ± 0.044 mmol/l) to the

ABS Control group (2.982 ± 0.146 mmol/l) (Figure 24a). On the contrast, liver triglycerides

exhibited a significant increase in animals kept on high-fat diet (HF+Saline; 1.73 ± 0.03 mmol/l),

compared to the absolute control group (ABS control; 1.59 ± 0.03 mmol/l), however, no

significant change in hepatic triglyceride was observed in high-fat diet group treated with

sitagliptin (HF+Sitg (50 mg)) compared to high-fat diet control group (HF+Saline), (1.65 ± 0.04

mmol/l) vs. (1.73 ± 0.03 mmol/l), respectively. Results are shown in Figure 24b.

(a) (b)

Control (

Salin

e)

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

1

2

3

4

Seru

m C

ho

lest

ero

l le

vel

(mm

ol/

l)

** *** **

***

54

Figure. 24 (a) & (b). Liver cholesterol (Figure 23a) and triglyceride (Figure 23b) levels (expressed in mmol/l).

Illustrated results show no significant change in liver cholesterol comparing the high fat-diet control group

(HF+Saline) compared to the absolute control (ABS Control), while Cholesterol level decreased significantly in the

high-fat diet group treated with sitagliptin (HF+Sitg (50 mg)), compared to high-fat diet control group (HF+Saline).

Hepatic triglyceride level showed a significant change when comparing high fat-diet control group (HF+Saline) with

absolute controls (ABS Control), however, sitagliptin treatment (HF+Sitg (50 mg)) caused no significant change in

hepatic triglyceride, compared to the control group kept on high-fat diet (HF+Saline). Statistical significance was

obtained using the One-way ANOVA multiple comparison test (Bonferroni), and results were considered significant

when (*P< 0.05). Data are represented as (Mean ± SEM); (n= 4-5 animals/group).

8.2.4. Effect of Sitg on heart tissue DPP-4 activity and GLP-1 level

Measurements from heart tissues subjected to brief reperfusion (10-min) showed nearly

unchanged GLP-1 levels (8.72 ± 0.76 ng/ml vs. 7.21 ± 0.67 ng/ml) and DPP4 activity (5.41 ±

0.95 microunits/mL x102 vs. 3.64 ± 0.95 x10

2), in Sitg treated groups compared to Controls.

Results are represented in Figure 25.

ABS Control

HF+Saline

HF+Sitg (5

0mg)

0

1

2

3

4

Live

r Ch

oles

tero

l lev

el

(mm

ol/l

)

ABS Control

HF+Saline

HF+Sitg (5

0mg)

0.0

0.5

1.0

1.5

2.0

Live

r Tr

igly

ceri

de le

vel

(mm

ol/l

)

(a) (b)

***

* *

55

Contr

ol (HF+Sal

ine)

HF+Sitg

(50m

g)

Contr

ol (HF+Sal

ine)

HF+Sitg

(50m

g)0

2

4

6

8

10

DPP4 activity (microunits/mL) x 102

GLP-1 (ng/mL)

Figure. 25. Ischemic cardiac tissue level of Glucagon-like peptide 1 (GLP-1) and DPP-4 activity after 2 weeks of

animal treatment with Sitg (50 mg/kg/day). No any noticeable change is observed in Sitg- treated group compared to

the Controls in both parameters. Results are expressed in (ng/ml) and (microunits/mL x102) for GLP-1 and DPP-4

activity, respectively. Presented data are (Mean ± SEM); (n= 4-10 animals/group).

8.2.5. Sitg (50 mg) normalized high DPP-4 level in heart tissues and aortas of control

group

Results from heart tissues (Figure 26a) and aorta’s (Figure 26b) exhibited a significant reduction

in DPP-4 level (0.87 ± 0.09 vs. 1.22 ± 0.12 ng/mg protein) and (6.13 ± 0.55 vs. 8.52 ± 0.34

ng/mg protein) respectively, in Sitg (50 mg) group vs. Control (Saline).

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0.0

0.5

1.0

1.5

DP

P-4

(n

g/m

g p

rote

in)

Hea

rt t

issu

e

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

2

4

6

8

10

DP

P-4

(n

g/m

g p

rote

in)

Ao

rta

(a) (b)

* *

56

Figure 26 (a) & (b). Changes in DPP-4 level (expressed in ng/mg protein) in heart tissues (Figure 25a) and aorta

(Figure 25b) of Sitg (50 mg) treated animal group (HF+Sitg (50 mg)) compared to Control (HF+Saline). Data are

represented as (Mean ± SEM); (n= 5-7 animals/group). Statistical significance: *P< 0.05 compared to the Control

group.

8.2.6. DPP-4i treatment caused no change in DPP-4 protein expression

No significant difference in DPP-4 (CD26) protein expression was noticed in HF+Sitg (50 mg)

group (554.17 ± 136.91 Intensity x mm2) vs. HF+Control (Saline) group (581.03 ± 121.71

Intensity x mm2), after normalization with β-actin. Results are illustrated in Figures 27 & 33a.

Figure 27. Unchanged level of DPP-4 protein expression (expressed in Intensity x mm2) in heart tissues of (HF+Sitg

(50 mg), n= 6) group compared to Control (HF+Saline), n= 5) group. Data are presented in term of (Mean ± SEM).

8.2.7. DPP-4i increased CGRP but not TRPV-1 levels

No any marked change in heart TRPV-1 level was observerd in Sitg (50 mg)- treated group (4.08

± 0.28 ng/ml x10), compared to the Control (5.23 ± 0.31 ng/ml x10), while findings from CGRP

measurements displayed a clear augmentation in CGRP (16.65 ± 1.04 ng/mg protein) vs. (10.93

± 1.84 ng/mg protein), of Sitg (50 mg) vs. Control group respectively. Results are shown in

Figure 28.

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

200

400

600

800

DP

P4

pro

tein

exp

ress

ion

(H

eart

)

(In

ten

sity

x m

m2)

57

Figure. 28. Effect of 50 mg dose of Sitg on cardiac TRPV-1 and CGRP levels, after two weeks’ oral treatment. No

significant change was observed in TRPV-1, while CGRP significantly increased in Sitg (50 mg) treated group

(HF+Sitg (50 mg)) compared to Control (HF+Saline). Results are expressed in (ng/ml) and (ng/mg protein), for

TRPV-1 and CGRP, respectively. Data are illustrated as (Mean ± SEM); (n= 4-9 animals/group).

8.2.8. Enhanced cardiac calcium (Ca2+

) content in Sitg (50 mg)- treated group

To determine whether sitagliptin can have an influence on ischemic cardiac calcium

concentration in of hyperlipidemic state, a colorimetric calcium detection assay kit was used for

this purpose,

while obtained results showed unsignificant change in calcium content in heart tissues assigned

to drug therapy (61.52 ± 13.51 ng/mg protein) vs. Controls (22.79 ± 6.53 ng/mg protein), and

results are shown in Figure 29.

*

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

20

40

60

80

100

Cal

ciu

m c

on

ten

t

(ng/

mg

pro

tein

)

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

5

10

15

20

TRPV-1 (ng/mL) x 10

CGRP (ng/mg protein)

58

Figure 29. Changes in cardiac calcium content of tissues excised from the Sitg (50 mg) treated animals, compared

to the Control ones. The bar chart displays an increase in calcium concentration in Sitg (50 mg) group, and presented

values are in terms of (Mean ± SEM).

8.2.9. TRPC-1 protein expression level

The difference in TRPC-1 protein expression level between the control (HF+Saline) and

(HF+Sitg (50 mg)) treated groups is presented in Figures 30 & 33b. Unlike the results from the

normolipidemic animals, Sitg (50 mg)- treated group showed a slight but unsignificant decrease

(752.19 ± 40.11 Intensity x mm2) in TRPC-1 expression in comparison with the Control group

(862.77 ± 143.44 Intensity x mm2). The protein of interest was normalized to β-actin.

Figure 30. Unaffected TRPC-1 protein expression level (Intensity x mm2) in heart tissues from (HF+Sitg (50 mg);

n= 8) treated group vs. Control (HF+Saline) group (n= 8). Data are in term of (Mean ± SEM).

8.2.10. DPP4-i upregulated cNOS activity and e-NOS protein expression in heart tissues of

Sitg (50 mg)

8.2.10.1. cNOS activity

Two weeks of daily treatment with Sitagliptin (50 mg), followed by excision of heart tissues and

brief reperfusion (45 min occlusion and 10 min reperfusion) of the coronary artery, showed a

significant increase in cNOS activity (96.51 ± 13.75 pmol/min/mg protein) relative to the

Control group (52.38 ± 11.56 pmol/min/mg protein) (Figure 31).

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

500

1000

1500

TRP

C-1

pro

tein

exp

ress

ion

(In

ten

sity

x m

m2)

59

Figure 31. Significant increase (*P< 0.05) in constitutive nitric oxide synthase (cNOS) activity in heart tissues of

(HF+Sitg (50 mg), n= 7) group, compared to (Control (HF+Saline), n= 7) group. Values are expressed in

(pmol/min/mg protein). Presented data are (Mean ± SEM).

8.2.10.2. e-NOS protein expression

Expression of endothelial nitric oxide synthase (e-NOS) as determined by Western blot is shown

and normalized to β-actin is shown in Figures 32 & 33c. The level of expression was

insignificantly changed in HF+Sitg (50 mg)- treated animals (470.32 ± 73.79 Intensity x mm2),

compared to the

Controls (425.26 ±

31.77 Intensity x

mm2).

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

0

50

100

150cN

OS

acti

vity

(pm

ol/

min

/mg

pro

tein

)

*

Control (

HF+Sa

line)

HF+sit

g (5

0mg)

0

200

400

600

e-N

OS

pro

tein

ex

pre

ssio

n

(In

ten

sity

x m

m2)

60

Figure. 32. Unaffected level of e-NOS expression (expressed in Intensity x mm2) in heart tissues of (HF+Sitg (50

mg), n= 8) group compared to Control (HF+Saline), n= 6) group. Data are presented in term of (Mean ± SEM).

Figure 33 (a), (b) & (c). Expression of CD26, TRPC-1 and e-NOS proteins in ischemic heart tissues treated with 50

mg sitagliptin (Sitg (50 mg)), compared to the control (Saline) group. The blots show no significanct difference in

the measured proteins after sitagliptin treatment.

8.2.11. L-NAME Inhibited NOS-mediated Cardioprotection Against Infarct

The implication of NOS in DPP-4 inhibition- induced cardioprotection was confirmed by

treating the animals with NOS-inhibitor (L-NAME) and measuring the size of infarction.

Obtained results are in line with the previous ones in normolipidemic animals, showing a

significant reduction (18.47 ± 1.22 %) in Sitg (50 mg) group compared to Control (HF+Saline),

43.33 ± 1.86 %), but this protective effect was lost in HF+Sitg (50 mg) + L-NAME group (59.17

TRPC-1

CD26 (DPP-4) (a)

(b)

(c) e-NOS

β-actin

61

± 6.09 %) vs. animal group treated with Sitg (50 mg) alone (HF+Sitg (50 mg), 18.47 ± 1.22 %)

(Figure 34).

Figure. 34. Loss of Sitg (50 mg)- induced cardioprotection mediated by NOS, translated by increase in infarct size

(%), after intraperitoneal injection of NOS inhibitor (L-NAME). Comparing the two groups, (HF+Control (Saline),

n= 6) and (HF+Sitg (50 mg), n= 9) shows a significant decrease in infarct size (*P< 0.05), while this protective

effect was abrogated comparing the (HF+Sitg (50 mg) +L-NAME; High fat + Sitg (50 mg) + L-NAME (i.p), n= 5)

group with the (HF+Sitg (50 mg); High fat + Sitg (50 mg), n= 9) treated group (††

P< 0.01), which means that

cardioprotective effect of Sitagliptin against infarction is mediated through NOS. Statistical analysis were performed

using One-way ANOVA, as well as two-way ANOVA when necessary. Data plotted as (Mean ± SEM).

*

##

†††

Control (

HF+Sa

line)

HF+Si

tg (5

0mg)

HF+Contro

l (Sa

line)+

L-NAM

E

HF+Si

tg (5

0mg)

+L-NAM

E0

20

40

60

80

100

Infa

rct

size

/Are

a at

ris

k (%

)

62

9. DISCUSSION

Interestingly, in the present study, treatment with sitagliptin (50 mg) for 2 weeks successfully (i)

attenuated infarct size (IS), increased NOS activity, CGRP level, and calcium content in both

diets, (ii) reduced DPP-4 activity and DPP-4 level in normal and high-fat diet animals

respectively. The upregulation of GLP-1 and TRPV-1 levels, e-NOS and TRPC-1 proteins

expression in the normolipidemic groups, were abolished under hyperlipidemic condition.

However, taking into account the results of the ineffective doses of sitagliptin, this drug can be

considered clinicaly relevant for the treatment of ischemic diseases at a further level, after

clarifying the molecular mechanisms underlying these doses, and although sitagliptin therapy

seemed to be cardioprotective in normolipidemic animals and in some part in animals kept on

high-fat diet, this drug may lose its efficacy in hyperlipidemic condition, when patients suffer

from hyperlipidemia as a cardiovascular co-morbidity and risk factor, due to endothelial

dysfunction that occurs simultaneously with hyperlipidemia.

When the circulation is abruptly restored after a prolonged myocardial ischemia, this can lead to

cardiomyocyte damage, which is commonly referred to myocardial I/R injury, triggered by

neutrophil accumulation, causing ROS production and cellular damage [115]. Protein synthesis

is negatively regulated during myocardial I/R injury, activating ER-cytosol-nucleus stress-

induced signaling pathways, including unfolding protein response (UPR) and ER-associated

protein degradation (ERAD) due to misfolded or unfolded proteins buildup [116]. In the present

study, 45 min of regional ischemia and 120 min of reperfusion in sustained I/R injury, revealed a

significant percentage of infarction (50-60 %), that was checked using TTC staining.

Accordingly, developing cytoprotective pharmacological strategies in the frame of limiting

myocardial infarction by maintaining a proper blood flow to the ischemic myocardial region is

one of the main focuses of preclinical and clinical research [117]. Sitagliptin 50 mg (Sitg (50

63

mg)) showed a significant decrease in infarct size into 22 % and increase in cNOS activity in

normal diet animals, as well as after high fat diet enriched food. This DPP-4 inhibitor (DPP-4i)-

mediated cardioprotective effect was abrogated after NOS-inhibition by L-NAME, in both

animals subjected to normal and high fat diet as well. Similarly, and according to literature,

inhibition of cNOS activity also blunted its advantageous effect on myocardial infarction in

previous studies on animals fed with normal and high-fat diets [118].

Clinical investigations and experimental animal studies suggested that incretins, namely GLP-1

can exhibit pleiotropic cardioprotective potentials following myocardial ischemia (MI), via

preserving the cardiomyocytes viability, increasing metabolic efficiency, and inhibiting the

structural and functional cardiac remodeling [54]. DPP-4 is abundantly expressed in the

cardiovascular system and endothelial cells, and blocking its activity by DPP-4 inhibitors can

have advantageous cardiovascular outcomes, through up-regulation of GLP-1 levels, and

inhibition of substrates of cardiovascular homeostasis, in addition to its impact on glucose

metabolism [72].

DPP-4 inhibitors drugs were extensively studied in healthy animal models as a remedy against

cardiovascular disorders, while their interventional mechanisms were slightly addressed in

diseased animal models like hyperlipidemia. In our study, the effect of human- like

hyperlipidemia on development of myocardial infarction (MI) following a temporary coronary

occlusion (Ischemia/Reperfusion- injury) was studied using the high-fat diet (HFD) animals.

Treatment with sitagliptin (50 mg) showed no decrease in serum cholesterol and triglyceride

levels, however, a decrease in hepatic cholesterol was observed in HF+Sitg (50 mg) group

compared to the control (HF+Saline).

After 45 min of regional ischemia and 2 hrs of reperfusion, only 39 % of the area at risk (AAR)

became necrotic in hyperlipidemic vs. 44 % in normolipidemic animals, showing that high-fat

diet does not seem to increase the susceptibility of the myocardium to I/R injury, with the

importance of early reperfusion after acute myocardial infarction (AMI) in normal and high-fat

diet conditions. According to a study done by Chinda et al., acute administration of the DPP-4

inhibitor vildagliptin reduced infarct size by 44 %, and preserved heart function, as indicated by

Left Ventricular End Systolic Pressure (LVESP) and Stroke Volume assessments in a rat model

of ischemia-reperfusion [119].

64

GLP-1 displayed non-exclusive cardioprotective actions in ex vivo rodent Langendorff ischemic

heart, as well as in in vivo rat, rabbit, canine, swine models and most importantly in patients with

acute myocardial infarction [120, 121], and this peptide hormone was found to be highly

abundant in the heart, similarly to the gastrointestinal tract, while the administration of GLP-1 or

GLP-1 receptor agonists (GLP-1 RAs) can have beneficial effects in both organs [122, 123].

Treatment with Sitg (50 mg) exhibited a significant decrease in DPP-4 activity in heart tissues

from normolipidemic animals, and a significant decrease in DPP-4 level in heart tissues and

aorta’s of hyperlipidemic ones, while the expected increase in GLP-1 level was not the case in

animals fed with high-fat diet, compared to the normol diet animals that exhibited a significant

decrease in GLP-1 level. On the other hand, and taking into consideration the dietary factor

alone, a significant increase in GLP-1 level can be detected in hyperlipidemic (HF (Control))

group, relative to the normolipidemic (N (Control)) group, making high fat- diet a suspicious

factor in blocking the protective effect of sitagliptin. Independent of its insulin-potentiating

effects, GLP-1 has also been shown to have anti-apoptotic properties in cardiomyocytes through

the up-regulation of cyclic adenosine monophosphate (cAMP) and the phosphoinositide 3-kinase

(PI3K), which is considered a central component of the reperfusion injury salvage kinase (RISK)

pathway, via the GLP-1 receptor [124]. Former results addressed that oral and intraperitoneal

administration of sitagliptin at high doses, exerted a limiting effect on infarct size and triggered

pro-survival signaling cascades (PI3K-Akt and ERK1/2) by GLP-1 upregulation, as responses to

I/R injury (Figure. 8) [71, 125].

The deleterious consequences of I/R injury can be a major cause of endothelial dysfunction,

causing a reduction in endothelial nitric oxide synthase (e-NOS) expression, while maintaining

adequate level of e-NOS is cytoprotective [126]. Increased phosphorylation and activation of e-

NOS was addressed using the DPP-4 inhibitor alogliptin [127], and prolonged myocardial

ischemia was found to decrease cNOS activity and e-NOS (NOS-3) protein expression [128].

Findings from the present study showed an increase in e-NOS expression in ischemic hearts pre-

treated with 50 mg dose of sitagliptin in normolipidemic but not in hyperlipidemic animals. The

latter is the only isozyme considered to be constitutively expressed in cardiomyocytes, while

hypercholesterolemia is associated with impaired endothelial function in coronary circulation

[129], reduced nitric oxide (NO) production due to the increase in superoxide (O2-) and

peroxynitrite (ONOO-) formation [130], and decreased phosphorylation and e-NOS expression

that was observed in the heart of hypercholesterolemic rabbit model [131]. The correlative effect

65

between DPP-4 inhibitors and NOS system was depicted in few vulnerable studies; however, the

potential role of NOS remains unfostered in the ischaemic heart, and it is still unknown whether

sitagliptin exerts the same protective effect.

In our experiments, the cardioprotective action of sitagliptin- mediated by NOS was confirmed at

the level of infarct size, using L-NAME. The decrease in infarct size in Sitg (50 mg) treated

group, compared to the controls, was abrogated when compared to the group treated with

sitagliptin and L-NAME (Sitg (50 mg) + L-NAME), in normal and high-fat diet animals.

According to literature, inhibition of NOS activity also blunted the beneficial effects on

myocardial infarct size in a previous study done on high-fat diet-fed animal model [118].

Interestingly, L-NAME decreased the infarct size when treated alone with saline (Saline + L-

NAME), compared to the control (Saline) group, and this can be due to the effect of NOS

inhibitor (L-NAME) in protecting rat hearts from I/R injury by decreasing OONO- generation

[132].

Studies on transient receptor potential channels revealed that the upregulation of TRPC and

TRPV subfamilies contributes to the pathophysiology of vascular and cardiac tissues [133], with

direct implication of increased TRPC-1 levels in cardiac hypertrophy [134]. Our results are in

disagreement with these findings, showing a significant increase in TRPV-1 level and TRPC-1

expression post-ischemia-reperfusion injury, in cardiac tissues pretreated with 50 mg dose of

sitagliptin. Administration of TRP inhibitor (capsazepine), blocked the infarct size limiting effect

of sitagliptin, showing that this protection was mediated by these channels. Stimulation of

TRPV-1 promotes the release of CGRP, with accumulating data reporting the advantageous role

of CGRP in enhanced myocardial contractility and increased heart rate [135]. This protective

effect of TRPV-1 and CGRP is in concordance with measurements from our study. It was

previously suggested that hypercholesterolemia can partially block the ion channels and

membrane receptor downstream signaling by reducing membrane fluidity, leading to dysfunction

of cardiomyocyte [136]. This blocking effect was translated in the current study, since the

protective effect induced by sitagliptin and mediated by TRPV/TRPC upregulation in

normolipidemic animals, was lost after a long-term high fat- diet consumption. TRPV-1 level

and TRPC-1 expression were significantly increased in hyperlipidemic (HF (Control)) group,

compared to the normolipidemic (N (Control)) animals, suggesting that the blunted activity of

these channels might be a result of an indirect effect of high fat-diet on these channels. The level

of TRPV-1 downstream signaling peptide CGRP increased significantly after three months of

66

high fat diet regime and oral treatment with Sitg (50 mg). This upregulatory effect that was also

revealed in animals fed with standard normal diet as well. Without forgetting the activation of

this cascade (TRPV-1/CGRP) during cardioprotective brief episodes (Pre-conditioning and Post-

conditioning), against myocardial infarction in rat hearts [137, 138], but to the best of our

knowledge, this study was the first to show the upregulation of TRPV-1/CGRP axis in prolonged

ischemia-reperfusion injury, using sitagliptin as a new targeting therapy, in normal and high-fat

diet animals.

10. ACKNOWLEDGEMENT

First and foremost, I would like to praise and thank the “Almighty” who provided me vision,

health and strength, and my parents for their moral and financial support filled with affection,

tolerance, concern, cordial, and encouragement throughout my life.

My profound gratitude goes to the Tempus Public foundation and Szeged University for

providing me with the opportunity to do my PhD studies in Hungary, and the research funders

and supporters: GINOP 2.3.2-15-2016-00035, and EFOP-3.6.1-16-2016-00008.

I deeply and sincerely thank the Head of our lab and our department Dr. Csaba Varga for

granting me the opportunity to pursue my PhD research work in the department of Physiology,

Anatomy and Neuroscience, Faculty of Science and Informatics, University of Szeged.

I am profoundly grateful to my supervisor’s Dr. Krisztina Kupai and Dr. Csaba Varga for their

valuable guidance, help and constant support rendered throughout my PhD study period, and

during my PhD thesis.

I also would like to extend my thanks to all the lab members for their tremendous help during my

experimental work. My special thanks goes as well to all the faculty members and friends of the

Department of Physiology, Anatomy and Neuroscience.

67

I convey my gratitude to all my friends for joining their hands during my work and encouraged

me mentally during my difficult time.

Attestation of Authorship

I, Amin Al-awar hereby declare that this submisson is my own work and that to the best of my

knowledge and belief, it contains no material previously published or written by athor person.

Amin Al-awar

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LIST OF PUBLICATIONS (MTMT number: 10053167)

Publications related to thesis

Full papers (IF: 6.577)

Al-Awar A, Almási N, Szabó R, Takacs I, Murlasits Z, Szűcs G, Török S, Pósa A, Varga C,

Kupai K.

Novel Potentials of the DPP-4 Inhibitor Sitagliptin against Ischemia-Reperfusion (I/R) Injury

in Rat Ex-Vivo Heart Model. Int J Mol Sci. 2018 Oct 18;19(10). pii: E3226. doi:

10.3390/ijms19103226. IF: 3.687

79

Al-Awar A, Kupai K, Veszelka M, Szűcs G, Attieh Z, Murlasits Z, Török S, Pósa A, Varga C.

Experimental Diabetes Mellitus in Different Animal Models. J Diabetes Res.

2016;2016:9051426. doi: 10.1155/2016/9051426. IF: 2.89

Al-awar A, Almási N, Szabó R, Ménesi R, Szűcs G, Török S, Pósa A, Varga C, Kupai K.

Effect of DPP-4 inhibitor Sitagliptin against Ischemia-Reperfusion (I/R) injury in

hyperlipidemic animals (Under review in Acta Biol Hung). IF: 0.439

Publications not related to thesis

Kupai K, Szabó R, Veszelka M, Al-Awar A, Török S, Csonka A, Baráth Z, Pósa A, Varga C.

Consequences of exercising on ischemia-reperfusion injury in type 2 diabetic Goto-Kakizaki

rat hearts: role of the HO/NOS system. Diabetol Metab Syndr. 2015 Oct 6;7:85. doi:

10.1186/s13098-015-0080-x. IF: 2.413

Kupai K, Almási N, Kósa M, Nemcsók J, Murlasits Z, Török S, Al-Awar A, Baráth Z, Pósa A,

Varga C.

H2S confers colonoprotection against TNBS-induced colitis by HO-1 upregulation in rats.

Inflammopharmacology. 2018 Apr;26(2):479-489. doi: 10.1007/s10787-017-0382-8. IF: 3.304

Szabó R, Karácsonyi Z, Börzsei D, Juhász B, Al-Awar A, Török S, Berkó AM, Takács I, Kupai

K, Varga C, Pósa A.

Role of Exercise-Induced Cardiac Remodeling in Ovariectomized Female Rats. Oxid Med Cell

Longev. 2018 Feb 13;2018:6709742. doi: 10.1155/2018/6709742. IF: 4.936

Almási N, Pósa A, Al-awar A, Török S, Baráth Z, Nemcsók J, Murlasits Z, Nagy L.I, Puskás

G.L, Varga C and Kupai K.

80

Differentially expressed microRNAs and their relation to gasotransmitters in TNBS-induced

colitis in rat colon. Academia Journal of Scientific Research. September 2017, 5(9): 277-289,

doi: 10.15413/ajsr.2017.0136.

Al-awar A, Kupai K, Almási N, Murlasits Z, Török S, Bóta A, Krész M, Berkó A, Pósa A and

Varga C.

Effect of long-term physical exercise on metabolic risk parameters in Overweight/Obese

subjects: a network-based analysis approach. Academia Journal of Scientific Research. October

2017, 5(10): 419-427, doi: 10.15413/ajsr.2017.0149.

Al-awar A, Attieh Z and Balbaa M.

Mulberry leaves lower the enzymatic activity and expression of hepatic arylsulfatase B in

streptozotocin-induced diabetic rats. current topics in nutraceutical research. 2015, Vol. 13,

No. 3, pp. 121-128.

81

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