By Supervised by

STUDY OF LEPTIN, ADIPONECTIN AND C-PEPTIDE WITH OXIDATIVE STRESS IN OBESE INDIVIDUALS

A Thesis Submitted to the Council of the College of Medicine, University of Tikrit, in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biochemistry

By Entedhar Riffat Sarhat M.SC. IN MEDICAL BIOCHEMESTRY (TUCOM) B.V. & S. (BAGHDAD)

Supervised by Professor Professor Dr. Salam S. Ahmed

2014 A.D A.H

Assist. Dr. Firas T. Ismail

1435

Acknowledgments Before all greatest thanks to the Merciful ALLAH. My great appreciation to Ministry of Higher Education and Scientific Research for giving me this chance to study. I would like to present my great gratitude to my supervisor Prof. Dr. Salam S. Ahmed and assist. professor Dr. Firas T. Ismail, for his valuable advices, continuous support, and endless cooperation and patience throughout my study. I'm honored to be one of his students. I am grateful to Dr. Wessam Suhail Najem, Dean of the Medicine College, Tikrit University for providing opportunity to do my work. I would like to express my indebtedness and sincere thanks to Prof. Dr.

Nazar Al-Nasiry

, Dr. Amina H. Ahmed, Dr. Khudhair Abaas and Asst. Lecture Hashim A.Al-Sattar for their unlimited cooperation and intensive teaching during the courses.My deep thanks to Dr. Shaker Mahde, for doing the well prepared statistics of my study.

I

I wish to thank my family for their support during period of study. Finally I wish to thank My best friend

Dr.Ban Ismail for their support .

II

-------------------------------------------------------------- List of Contents

List of Contents:Acknowledgments ............................................................................................... I Abstract .............................................................................................................II List of Contents.................................................................................................. V List of Tables ................................................................................................... XI List of Figures ............................................................................................... XIII List of Abbreviations ..................................................................................... XVI

Chapter One:- Introduction Introduction ....................................................................................................... 1 Aim of the study ................................................................................................. 4

Chapter Two:- Literature Review Literature Review: - ....................................................................................... ..5 2.1:- Obesity………………………………….……………………………………………………………..5 2.1.2: Classification of Overweight and Obesity……………………………………5 2.1.3:- Epidemiology …………………………………………………………………7 2.1.4:-Etiological Factors……………………………………………………………10 2..1.5:- Causes of obesity………………………………………………………….. 10 2.2:- Adipose Tissue……………………………………………………………………12 2.2.1:- Adipose Tissue Distribution………………………………………………..12 2.2.1.1:- White Adipose Tissue……………………………………………………. 12 2.2.1.2:- Brown Adipose Tissue……………………………………………………. 13 2.3:- Adipose derived hormones……………………………………………………...14 2.4:- Leptin…………………………………………………………………………… 14 2.4.1:- Physiology of leptin………………………………………………………….15 2.4.2:- Regulation of leptin secretion……………………………………………….17 2.4.3:- Leptin receptors signaling…………………………………………………..17 2.4.4:- Leptin and adenophosphate kinase…………………………………….18 V

-------------------------------------------------------------- List of Contents 2.4.5:- Leptin action in hypothalamous…………………………………………19 2.4:- Adiponectin ……………………………………………………………………20 2.4.1:- Molecular structure……………………………………………………………20 2.4.2:- Adiponectin receptor signaling ……………………………………………21 2.4.3:- Adiponectin signaling……………………………………………………… 23 2.4.4:- Hypoadiponectinaemia in obesity………………………………………….23 2.4.5:-Biological function of adiponectin…………………………………………23 2.4.6:-Metabolic effects……………………………………………………………….24 2.5:- C-peptide………………………………………………………………….. ..….25 2.5.1:- Definition of C-peptide……………………………………………..…………25 2.5.2:- Secretion of C-peptide……………………………………………….……..25 2.5.3:- Biological Role of C-peptide……………………………………………….26 2.6:- Oxidative Stress…………………………………………………………………27 2.6.1:- Free radicals………………………………………………………………… 28 2.6.2:- Generation of Free Radicals………………………………………………..29 2.6.3:- Mechanisms of Formation of Free Radicals during Obesity ………….29 2.6.4:- Reactive oxygen species……………………………………………………. 30 2.6.5:- Complications-generated Oxidative Stress in Obesity…………………. 31 2.6.6:- Antioxidant Systems………………………………………………………… 32 2.6.6.1:- Superoxide Dismutase…………………………………………………….. 33 2.6.6.2:- Catalase…………………………………………………………………… 33 2.6.6.3:- Reduced glutathione…………………………………………………….. 34 2.6.6.4:- Glutathione Peroxidase…………………………………………………..35 2.6.6.5:- Malondialdehyde (MDA) ………………………………………………..35 2.6.6.5.1:- Structure of Malondialdehyde……………………………………….. 36 2.7:- Paraoxonase……………………………………………………………………. 37 2.7.1:- Paraoxonase1 (PON 1)……………………………………………………37 2.7.2:- PON2 and PON3………………………………………………………………38

VI

-------------------------------------------------------------- List of Contents 2.7.3:- Role of Paraoxonase in oxidative stress……………………………………39 2.8:- Plasma lipid and Lipoprotein…………………………………………………40 2.8.1:- Metabolism of lipids………………………………………………………….41 2.8.2:- Relation of triglycerides and cholesterol with obesity……………………42

Chapter Three:- Materials and Methods Materials and Methods................................................................................... .44 3.1:- Subjects………………………………………………………………………. 44 3.2:- Inclusion Criteria……………………………………………………………. 44 3.3:- Exclusion criteria……………………………………………………………… 45 3.4:-Data collection…………………………………………………………………..45 3.5:-Measurements and examinations …………………………………………….45 3.5.1:-Anthropometric Measurements………………………………………………45 3.5.2:-Waist circumference………………………………………………………….. 46 3.5.3:-Body fat percentage ………………………………………………………….46 3.5.4:-Blood pressure ………………………………………………………………..46 3.6:-Samples collection……………………………………………………………… 46 3.7:-Materials…………………………………………………………………………. 47 3.8:-Chemicals………………………………………………………………………… 47 3.9:-Apparatus and equipment ……………………………………………………..48 3.10:- Biochemical Test………………………………………….………………….48 3.10.1:- Estimation of Serum Leptin…………………………….………………….. 48 3.10.2:- Estimation of Serum Adiponectin ………………………………………..49 3.10.3:- Estimation of Serum C-peptide ………………………………………….. 50 3.10.4:- Estimation of Serum Glucose………………………………………………50 3.10.5:- Estimation of Serum Cholesterol………………………………………… 51 3.10.6:-Estimation of The Serum Triglyceride………………….……………… 51 3.10.7:- Estimation of Serum HDL-Cholesterol……………….….……………52 VII

-------------------------------------------------------------- List of Contents 3.10.8:- Estimation of Serum VLDL-c…………………………………………….52 3.10.9:-Estimation of Serum Malondialdehyde………………………………….53 3.10.10:-Determination of serum glutathione peroxidase……………………… 54 3.10.11:-Determination of serum reduced glutathione …………………………54 3.10.12:-Determination of superoxide dismutase activity…………………….. 54 3.10.13:-Determination of serum catalase enzyme activity……………………. 55 3.10.14:-Determination of PON1 Activity………………………………………. 55 3.11:-Statistical analysis……………………………………………………………. 55

Chapter Four:- Results Results ................................................................................................ 56 4.1:-Demographic Distribution of Study populated.. .................................... 56 4.2:- Age……………………………. …………………..……………..………….. 56 4.3:- Body Mass Index………………………………………………..………….. 57 4.4:- Waist Circumference …………………………………………………………..58 4.5:- Body Fat…………………………………………………………………………. 59 4.6: Serum - Leptin……………………………………………….………………….59 4.7:- Serum Adiponectin……………………………………………………………… 60 4.8:- Serum C-peptide………………………………………………………………… 61 4.9:- Serum Malondialdehyde……………………………………………………… 62 4.10:- Serum Superoxide Dismutase………………………………...…………….. 63 4.11:- Serum Reduced Glutathione ………………………………………………. 64 4.12:- Serum Glutathione Peroxidase ……………………………………..……..64 4.13:- Catalase……………………………………………….…………………….. 65 4.14:- Serum Paraoxonase ………………………………….………………………66 4.15:-Serum Glucose …………………………………….……………………………67 4.16:- Serum Cholesterol ………………………………………..……….…………68 4.17:- Serum Triglycerides……………………………………………...……….. 69 VIII

-------------------------------------------------------------- List of Contents 4.18:- Serum High Density Lipoprotein………………………………………..… 70 4.19:- Serum Very Low Density Lipoprotein……………………………………… 71 4.20:- Correlation of Leptin to other parameters ………………………….……..72 4.21:- Correlation of adiponectin to other parameters ……….…………………74 4.22:- Correlation of C-peptide to other Parameters ……………….…………76 4.23:- Correlation of Paraoxonase to other parameters……………………...…. 78

Chapter Five:- Discussion Discussion ....................................................................................................... 93 5.1:- Leptin ……………………………………………………………………….93 5.1.1:- Leptin and Lipid profile ………………………………………………..95 5.1.2:- Leptin and C-peptide ……………………………………………………. 97 5.1.3:- Leptin and Glucose ………………………………………………………..98 5.2:- Adiponectin ………………………………………………………………….. 99 5.2.1:- Adiponectin and Lipid profile ……………………………………………100 5.2.2:- Adiponectin and Glucose ………………………………………………. 102 5.2.3:- Adiponectin with Leptin ………………………………………………….103 5.3:- C-peptide ………………………………………………………………………104 5.3.1:- C-peptide with Adiponectin ……………………………………………….105 5.4:- Oxidative stress and obesity …………………………………………………107 5.4.1:- Lipid peroxidation ………………………………………………………… 109 5.4.2:- Glutathione peroxidase…………………………………………………… 110 5.4.3:- Catalase ……………………………………………………………………..112 5.4.4:- Superoxide dismutase ………………………………………………………112 5.4.5:- Reduced glutathione……………………………………………………… 115 5.4.6:- Dyslipidemic profiles and oxidative stress ……………………………….116 5.4.7:- Leptin and oxidative stress………………………………………………… 117 5.4.8:- Adiponectin and oxidative stress ………………………………………….118 IX

-------------------------------------------------------------- List of Contents 5.4.9:- C-peptide & oxidative stress………………………………………………. 119 5.5.1:- paraoxonase and leptin …………………………………………………...125 5.5.2:- paraoxonase and adiponectin …………………………………………..126 5.5.3:- Paraoxonase with Oxidative stress………………………………………. 127

Chapter Six:6.1:- Conclusions .......................................................................................... 128 6.2:- Recommendations ................................................................................. 130 6.3:- References ............................................................................................. 131 Appendices..................................................................................................... 161

X

Abbreviations ACC

Acetyl-CoA carboxylase

ACO

Acyl Coenzyme A oxidase

AdipoR1

Adiponectin Isoforms 1

AdipoR2

Adiponectin Isoforms2

AGRP

Agouti gene related peptide

AMPK

Adenosin 5’ monophosphate- activated protein kinase

ANT

Adenosine nucleotide translocator

ApN

Adiponectin

apoA-I

Apolipoprotein A-I

ARC

Arcuate nucleus

AT

Adipose tissue

ATP

Adenosine TriPhosphate

BAT

Brown Adipose Tissue

BF

Body fat

BHT

Butylated hydroxytoluene

BMI

Body Mass Index

CAT

Catalase

CD36

Cluster of differentiation

CETP

Cholesteryl-ester transfer protein

CHD

Coronary heart disease

CNS

central nervous system

CVD

Cardiovascular diseases

DTNB

5,5-dithiobis(2-nitro benzoic acid)

EDTA

Ethylene diamine tetra acetic acid

EGF

Epidermal growth factor

ELISA

Enzyme linked immunosorbent assay

XVI

eNO

Endothelial nitric oxide

eNOS

Endothelial nitric oxide synthase

ER

Endoplasmic reticulum

FABPs

Fatty acid binding proteins

FFA

Free fatty acids

GBP

Gelatin binding protein

GH

Growth hormone

GLUT4

Glucose transporter

GPX

Glutathione peroxidase

GSH

Reduced glutathione

GSSG

Oxidized glutathione

H2O2

Hydrogen peroxide

HDL-c

High-density lipoproteins-cholesterol

HL

Hepatic lipase

HMW

High Molecular Weight

HRP

Horseradish peroxidase

IGF-1

Insulin like growth factor- one

IL

Interleukin

IDL-c

Intermediate- density lipoproteins-cholesterol

iNOS

Inducible nitric oxide synthase

IR

Insulin receptor

IRS

Insulin receptor substrate

JNK

N-terminal JUN kinase

Kg/m2

Kilograms/ meter 2

LDL

Low-density lipoprotein-cholesterol

Lep

Leptin gene

LepR

Leptin receptor gene

LOO•

Peroxyl radical XVII

LOOH

Organic hydroperoxides

LOOH

Lipid hydroperoxide

LPa

Lipoprotein (a)

LPL

Lipoprotein lipase

LPO

Lipid peroxidation

LPS

Lipopolysaccharide

MC3R

Melanocortin 3 receptors

MC4R

Melanocortin 4 receptor

MCP-1

Monocyte Chemoattractant Protein-1

MDA

Malondialdehyde

mg/dl

Milligram per deciliter

miR

MicroRNAs

mmol/L

Mill molar

mRNA

Messenger ribonucleic acid

MS

Metabolic syndrome

MSH

Melanocyte- stimulating hormone

MTB

Methyl thymol blue

NADPH

Nicotinamide adenine dinucleotide phosphate

NAFLD

Non-alcoholic fatty liver disease

NBT

Nitro blue tetrazolium

NF-kB

Nuclear factor-kB

NO

Nitric oxide

NPY

Neuropeptide Y

NS

Non-significant

OB-R

Leptin receptor

OS

Oxidative stress

PI-3K

Phosphatidylinositol 3-kinase

PKC

Protein kinase C XVIII

POMC

Proopiomelanocortin

PPARs

Peroxisome-proliferator-activated receptors

PVN

Paraventricular nucleus

RNS

Reactive nitrogen species

ROS

Reactive oxygen species

SD

Standard deviation

SNA

Sympathetic Nervous Activation

SOD

Superoxide dismutase

SPSS

Statistical Package for Social Sciences

T2DM

Type 2 diabetes mellitus

TBA

Thiobarbituric acid

TCA

Tricarboxylic acid cycle

TG

Triglycerides

TNF-α

Tumor necrosis factor-alpha

U/L

Unit/liter

VLDL

Very low-density lipoprotein

WAT

White Adipose Tissue

WC

Waist circumference

WHO

World Health Organization

WHR

Waist-to-hip ratio

μU/ml

Microunit/milliliter

XIX

----------------------------------------------------------------List of Tables List of Tables:Table (2.1):- Classification of overweight and obesity by different international organizations.......................................................................................................... 7 Table (2.2):- Sources of reactive oxygen and nitrogen species in biology and medicine……………………………………………………………………………..31 Table (3.1):- Chemicals Used In This Study with Their Supplier ....................... 47 Table(3.2):- Apparatus used in this study with their suppliers……………...48 Table( 4. 1):- Characteristics distribution of Study Population…………… 56 Table(4.2): Comparison between the groups for age………………………… 57 Table(4.3): Comparison between groups for BMI (Kg/m2)……………….. 58 Table(4.4):- Comparison between groups for waist circumference…….….. 58 Table(4.5):- Comparison between groups for body fat (%)…………………..59 Table(4.6):- Comparison between groups for Leptin……………………….. 60 Table(4.7):- Comparison between groups for Adiponectin (μg/ml)……….....61 Table(4.8): - Comparison between groups for C-peptide(ng/ml)…………….62 Table(4.9):- Comparison between groups for MDA (µM)………………….. 63 Table(4.10):- Comparison between groups for SOD (U/L)………………….. 63 Table(4.11):- Comparison between groups for GSH (mg/g Hb)……………..64 Table(4.12):- Comparison between groups for GPx (U/g Hb)……………… 65 Table(4.13): Comparison between groups for Catalase (K/ml )…………….66 Table(4.14): Comparison between groups for Paraoxonase (U/L )…………67 Table(4.15): Comparison between groups for Glucose (mmol/L)……………68 Table(4.16): Comparison between groups for Cholesterol (mmol/L)……….69 Table(4.17):- Comparison between groups for triglycerides (mmol/L)………70 Table(4.18):- Comparison between groups for HDL (mmol/L)………………70 Table(4.19):- Comparison between groups for VLDL (mmol/L)…..…………71 Table(4.20):- Correlation of Leptin to other parameters ………………….. 73

XI

----------------------------------------------------------------List of Tables Table(4.21):- Correlation of adiponectin to other parameters ………...….…75 Table(4.22):- Correlation of C-peptide to other Parameters……….……..... 77 Table(4.23):- Correlation of Paraoxonase to other Parameters ………...... 79

XII

------------------------------------------------------------------------List of Figures

List of Figures:Fig. (2.1):- Key concept map of obesity ................................................ 9 Fig. (2.2):- White fat cell and brown fat cell. ..................................... 13 Fig. (2.3):- Leptin structure................................................................. 15 Fig. (2.4):- Leptin regulation .............................................................. 16 Fig. (2.5):- Targets of Leptin action in the brain ................................ 20 Fig. (2.6):- Schematic structure multimerformation of adiponectin... 21 Fig. (2.7):- Schematic structure of adiponectin receptors and adaptor protein.. ................................................................................................ 22 Fig. (2.8):- Adiponectin exerts beneficial effects on multiple targets 24 Figure (2.9):- Biosynthesis of C-peptide……………………………….26 Figure (2.11):- Malondialdehyde structure………………………….. 36 Figure (2.12):- Overall structure of PON1……………………………39 Fig. (3.1): Reaction between MDA and TBA……………….……….…53 Fig (3-2):- Reaction between GSH and DTNB……………….……….54 Fig (4.1):- Correlation between leptin and adiponectin in obese individuals……………………….……………………….………………...80 Fig(4.2):- Correlation between leptin and cholesterol in obese individuals………………………………………….……………………...80 Fig(4.3):- Correlation between leptin and triglycerides in obese individuals………………………………………………………………....81 Fig(4.4):- Correlation between leptin and VLDL in obese individuals……………………………………………………………...….81

XIII

------------------------------------------------------------------------List of Figures

Fig (4.5):- Correlation between leptin and Paraoxonase in obese individuals………………………………………………………………….82 Fig(4.6):-

Correlation

between

leptin

and

MDA

in

obese

individuals………………………………………………………………….82 Fig(4.7):-

Correlation

between

leptin

and

SOD

in

obese

individuals…………………………………………………………………83 Fig (4.8):-Correlation between leptin and glutathione peroxidase in obese individuals…………………………………………………………..83 Fig(4.9):- Correlation between adiponectin and HDL in obese individuals……………………………………………………………….....84 Fig(4.10):- Correlation between adiponectin and glucose in obese individuals………………………...………………………………………..84 Fig(4.11): Correlation between adiponectin and triglycerides in obese individuals…………………………………………………………………..85 Fig(4.12):- Correlation between adiponectin and MDA in obese individuals………………………………………………………………....85 Fig (4.13):- Correlation between adiponectin and SOD in obese individuals………………………………………………………………….86 Fig(4.14):-Correlation between adiponectin and Paraoxonase in obese individuals……………………………………………………….…86 Fig(4.15):- Correlation between C-peptide and leptin in obese individuals……………………………………………………………...….87 Fig(4.16):- Correlation between C-peptide and adiponectin in obese individuals………………………………………………………………….87

XIV

------------------------------------------------------------------------List of Figures

Fig(4.17):- Correlation between C-peptide and glucose in obese individuals………………………………………………………………….88 Fig(4.18):- Correlation between C-peptide and cholesterol in obese individuals……………………………………………………………….....88 Fig. (4.19):- Correlation between C-peptide and HDL in obese individuals……………………………………………………………….…89 Fig. (4.20):- Correlation between C-peptide and glutathione peroxidase in obese individuals………………………………………...89 Fig. (4.21):- Correlation between C-peptide and MDA in obese individuals………………………………………………………………....90 Fig(4.22):- Correlation between C-peptide and SOD in obese individuals………………………………………………………………….90 Fig(4.23):- Correlation between C-peptide and catalase in obese individuals………………………………...……………………………….91 Fig(4.24):- Correlation between C-peptide and GSH in obese individuals……………………………………………………………..…..91 Fig.(4.25):-Correlation between C-peptide and Paraoxonase in obese individuals………………………………………………………………….92 Fig.(5.1):-Mechanisms modulating oxidant/antioxidant balance in obesity...............................................................................................108

XV

Chapter One--------------------------------------------- Introduction

CHAPTER ONE Introduction:In recent decades, the prevalence of obesity has increased alarmingly, making it a significant health problem in not only high-income countries, but low and middle-income countries as well

(1)

. Obesity is defined as

abnormal or excessive fat accumulation that presents a risk to human health (2). The current obesity plague is stimulated by the accessibility to high caloric food along with performing less physical activity(3,4). It is generally accepted that obesity is induced by a number of environmental and/or genetic factors. At the individual level, obesity is the result of excessive caloric intake in relation to the physical activity. However, the pathogenesis of obesity is far more complex than the simple imbalance between calorie intakes versus physical activity(5). The degree of obesity is usually estimated by the use of simple anthropometric parameters body mass index (BMI), waist circumference (WC),waist-to-hip ratio (WHR), and waist-to-height ratio (WHTR) or/and the use of percentage of body fat (%BF) mass (6). The majority of cells have limited ability to store excess lipids and to meet their own fuel needs during extended famine. As a result, specialized cells (adipocytes) have evolved to store fuel during caloric surplus and distribute lipids in times of need. When caloric intake exceeds caloric expenditure, adipocytes initially undergo hypertrophy. This process triggers adipose tissue paracrine signaling to stimulate adipogenesis in order to maintain adipose tissue physiological functions during increased energy storage (2).

1

Chapter One--------------------------------------------- Introduction There are also several compounds that appear to participate in the regulation of food intake, including circulating nutrients (e.g., glucose, amino acids, and fatty acids), metabolic compounds (e.g., lactate, pyruvate, and ketone bodies), and hormones (e.g., insulin, glucagon, cholecystokinin, leptin, and ghrelin) (7). Oxidative stress (OS) arising as a result of imbalance between free radical production and antioxidant defenses giving rise to a variety of toxic and reactive molecules . These molecules may cause severe damage and plays a key role in the pathogenesis of several human diseases

(8)

.

Biomarkers of oxidative damage are higher in individuals with obesity and correlate directly with BMI and the percentage of BF , in contrast, an inverse relationship between body fat, central adiposity, and antioxidant capacity has been suggested . Several processes are involved in obesity associated OS, caused by an overload of nutrients and in particular highfat and high-carbohydrate meals. An increment of fat levels corresponds to increased energy storage, mitochondrial oxidation of nutrients, and OS, caused by an imbalance between reactive oxygen species (ROS) generation and ROS elimination by the cellular defence systems . OS derives from an increase of plasmatic concentration of free fatty acid (FFA) and increases leptin level, and leads also to inflammation, subnormal vascular reactivity, and insulin resistance (9).

2

Chapter One--------------------------------------------- Introduction Aim of the study:To find out the liability of the apparently healthy obese individuals for diseases by detecting certain metabolic parameters.

Objectives of the study:1. Assess risk factors of obesity. 2. Determine adiponectin , leptin, and C-peptide in obese individuals and the control group. 3. Determine

the

malondialdehyde

changes (MDA),

SOD,

in

oxidative

catalase

(CAT),

stress reduced

glutathione (GSH), and glutathione peroxidase (GPx) in obese individuals and the control group. 4. Study the glucose and lipid profile (Cholesterol, HDL Cholesterol, LDL Cholesterol, VLDL Cholesterol and triglycerides in obese individuals and the control group. 5. Verify the impact of age, sex and BMI, on adiponectin , leptin, C-peptide and oxidative stress (MDA, SOD, CAT, GSH, and GPx) in obese individuals and the control group. 6. Investigate the relationship between serum adiponectin levels, and metabolic parameters including C-peptide, leptin, oxidative stress (MDA, SOD, CAT, GSH, and GPx), lipid profile , glucose, and BMI among obese individuals and the control group. To identify which among the metabolic parameters are closely associated with pathological levels of adiponectin. To assess the impact of BMI on adiponectin. 7. Investigate the associations between serum leptin levels, and metabolic parameters C-peptide, adiponectin, oxidative stress (MDA, SOD,

CAT, GSH, and GPx), lipid profile, glucose, and

BMI among obese individuals and the control group. To identify 3

Chapter One--------------------------------------------- Introduction which among the metabolic parameters are closely associated with pathological levels of leptin. 8. Find out the relation between C-peptide and metabolic parameters adiponectin, leptin, oxidative stress (MDA, SOD, CAT, GSH, and GPx), lipid profile, glucose, and BMI obese individuals and the control group. To identify which among the metabolic parameters are closely associated with pathological levels of C-peptide.

4

Chapter Two--------------------------------------------Literature Review

CHAPTER TWO Literature Review:2.1:- Obesity:Obesity is a serious nutritional problem and is defined as excessive fat accumulation that may impair health and increase mortality, as it increases the risk of morbidity from several pathologies, including hypertension, dyslipidemia, type 2 diabetes(T2DM), coronary heart disease(CVD), stroke ,non-alcoholic fatty liver disease(NAFLD), osteoarthritis, sleep apnea, and endometrial, breast, prostate, and colon cancers

(1)

. Excessive fat accumulation is a consequence of positive

energy balance that results from interaction, among several factors, including diet (increased intake of energy-dense foods and decreased intake of food rich in micronutrients and bioactive compounds) (10), decreased physical activity (sedentary lifestyle), nutritional and hormonal status in early life(11), as well as genetic, environmental, cultural, and economic factors (12). 2.1.2: Classification of Overweight and Obesity:The classification of overweight and obesity in adults using surrogate anthropometric measures proposed by different organizations are shown in Table (2.1), The WHO classification categories individuals into either being overweight or obesity based on the elevated BMI levels whereas the National Cholesterol Education Program (NCEP) and the International Diabetes Federation (IDF) classify individuals as obese and non-obese according to the waist circumference levels with purpose to define the metabolic syndrome (MS) (13). 5

Chapter Two--------------------------------------------Literature Review It is important to acknowledge that the WHO classification of BMI is based on the assumption that the association between BMI, morbidity and mortality follows a linear relation. However, on reviewing the literature it appears that although BMI is a strong predictor of metabolic risk factors, inconsistencies still remain with regard to the shape of the relation curve between this index of adiposity, morbidity and mortality(14). It is now well recognized that abdominal fat is a major risk for obesity-related diseases: indeed, visceral fat accumulation contributes to pro-oxidant and pro-inflammatory states, as well as to alterations in glucose and lipid metabolisms (1,15). Waist circumference or waist-to-hip ratio are useful indicators of visceral fat distribution: WC equal to or more than 80 cm (in women) or 94 cm (in men) and WHR above 0.90 for males and 0.85 for females are associated to high cardiometabolic risk in Europeans (16). According to a prediction by the WHO, there will be approximately 2.3 billion overweight adults of which 700 million will be clinically obese by 2015 (13).

6

Chapter Two--------------------------------------------Literature Review Table (2.1): Classification of Overweight and Obesity by Different International Organizations (13). WHO

WHO 1995

NCEP 2001

IDF 2006

2000

Joint Statement 2009

BMI (kg/m2)

Underweight

< 18.5

Normal

18.5 - 24.9

Waist girth

Waist girth

Waist girth

(cm)

(cm)

(cm)

≥94/80

>102/88

weight Overweight

25.0 - 29.9

Obesity

≥ 30.0

men/women men/women

≥94/80 or

≥94/80 or

≥90/80

≥102/88

men/women

and ≥90/80 men/women

2.1.3:- Epidemiology:Obesity is considered the largest public health problem worldwide, especially in industrialized countries

(17)

. Obesity increases mortality

and the prevalence of CVD, DM, and colon cancer

(18)

. Substantial

literature has emerged that shows that overweight and obesity are major causes of co-morbidities, including T2DM, CVD, various cancers, and other health problems, which can lead to further morbidity and

7

Chapter Two--------------------------------------------Literature Review mortality. The related health-care costs are also substantial, therefore, a public health approach to develop population-based strategies for the prevention of excess weight gain is of great importance. However, public health intervention programs have had limited success in tackling the rising prevalence of obesity. The definition of overweight and obesity and variations regarding age and ethnicity and health consequences and factors contributing to the development of obesity, and presents a critical review of the effectiveness of current public health strategies for risk factor reduction and obesity prevention ,Key concept map of obesity as shown in Fig. (2.1) (19).

8

Chapter Two--------------------------------------------Literature Review

Fig. (2.1):-Key concept map of obesity (20).

9

Chapter Two--------------------------------------------Literature Review 2.1.4:- Etiological Factors:Fundamentally,

obesity is

the result of excessive energy

consumption compared with the energy expended; in children, increased consumption of fats and sugars and lack of physical activity have been linked with obesity. Changes in lifestyle and diet have resulted in an increase in the number of obese subjects; obesity has been regarded as an important factor in causing various health problem. Another theory for explaining the development of obesity is known as the fetal origins hypothesis of chronic diseases. This suggests that poor maternal nutrition and poor fetal growth are risk factors for developing chronic diseases that affect the programming of body structure, physiology, and metabolism . The central nervous system (CNS), by means of signals, regulates appetite, energy intake, and weight gain; obesity can result from a failure of these signaling pathways(21). Other etiological factors that are associated with obesity are some chromosomal aberrations (such as Prader-Willi syndrome), hormonal pathologies (such as Cushing’s disease), hypothalamic lesions or tumors, and drugs (such as steroids and antidepressants) (22). 2.2:- Causes of Obesity:1. Genetics: It is now evident that genetic mechanisms play a major role in

determining body weight. The importance of genetics as a determinant of obesity is indicated by the observation that children who are adopted usually show a body weight that correlate with their biologic rather than adoptive parents. Furthermore, identical twins have very similar BMI, whether reared together or apart, and their BMI are more similar than those of no identical, dizygotic twins. Rare, single gene mutations can

10

Chapter Two--------------------------------------------Literature Review cause human obesity. Mutation in the gene for the adipocyte hormone leptin or its receptor product hyperphagia and massive obesity, underscoring the importance of the leptin system in regulation of human body weight(20). 2. Diseases:

Diseases such as hypothyroidism, insulin resistance,

polycystic ovary syndrome and Cushing's syndrome, are also contributors to obesity(23). 3. Environmental and Behavioral Contributions:

The epidemic of obesity occurring over the last decade cannot be simply explained by changes in genetic factors, which are stable on this short time scale. Clearly, environmental factors, such as the ready availability of palatable, energy-dense foods, play a role in the increased prevalence of obesity, furthermore, sedentary lifestyles encouraged by TV watching, automobiles, computer usage, and energy-sparing devices in the workplace and at home, decreased physical activity and enhance the tendency to gain weight. Eating behaviors, such as snacking, portion size, variety of foods consumed, an individual's unique food preferences, and the number of people with whom one eats also influence food consumption. It is important to note, however, that in this same environment, many individuals do not become obese. The susceptibility to obesity appears to be explained, at least in part, by an interaction of an individual's genes and his or her environment, and can be influenced by additional factors such as maternal under- or over nutrition that may set the body regulatory systems to defend a higher or lower level of food fat(20).

11

Chapter Two--------------------------------------------Literature Review 4. Medications: Medications are documented to increase weight gain

include

antipsychotics

(phenothiazines,

butyrophenones),

antidepressants, antiepileptics, insulin and some oral hypoglycemics. Whereas most of these medications contribute modestly to obesity, the large doses of steroids sometimes used to treat autoimmune diseases or used as contraceptive can cause true obesity (24). 2.2:- Adipose Tissue:Adipose tissue (AT) or body fat or just fat is loose connective tissue composed of adipocytes (Adipocytes, also known as lipocytes and fat cells, are the cells that primarily compose adipose tissue, specialized in storing energy as fat(25). It is composed of roughly only 80% fat; fat in its solitary state exists in the liver and muscles. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. AT also serves as an important endocrine organ by producing hormones such as leptin, resistin, and the cytokine. Two types of AT exist: White Adipose Tissue (WAT) and Brown Adipose Tissue (BAT) (26)

.

2.2.1:- Adipose Tissue Distribution:2.2.1.1:- White Adipose Tissue:White Adipose Tissue is found in mammals (compared to brown adipose tissue). In humans, WAT composes as much as 20% of the body weight in men and 25% of the body weight in women. Its cells contain a single large fat droplet, which forces the nucleus to be squeezed into a thin rim at the periphery as shown in figure (2.2.A).They have receptors for insulin, growth hormones (GH) and

12

Chapter Two--------------------------------------------Literature Review norepinephrine. WAT tissue also acts as a thermal insulator, helping to maintain body temperature(27). 2.2.1.2:- Brown Adipose Tissue:Brown adipose tissue is found in mammals. It is especially abundant in newborns and in hibernating mammals. Its primary function is to generate body heat in animals or newborns that do not shiver. In contrast to white adipocytes (fat cells) as shown in figure (2.2.B), which contain a single lipid droplet, brown adipocytes contain numerous smaller droplets and a much higher number of mitochondria, which contain iron and make it brown. BAT also contains more capillaries than white fat, since it has a greater need for oxygen than most tissues(28).

A- White Adipose Tissue

B- Brown Adipose Tissue

Figure (2.2). White fat cell and brown fat cell(29).

13

Chapter Two--------------------------------------------Literature Review 2.3:- Adipose Derived Hormones(30):Adipose tissue is an important endocrine organ that secretes numerous protein hormones into circulation. These factors are generally referred to as adipocytokines or adipokines, however the strict definition of an adipokine is that it interacts with the immune system. In addition to secreting cytokines, AT secretes proteins that influence metabolism. Their relative roles in modifying appetite, insulin resistance and atherosclerosis are the subjects of intense research, as they may be modifiable causes of morbidity in people with obesity. Adipose tissue is responsive to both central and peripheral metabolic signals and is itself capable of secreting a number of proteins. These adipocyte-specific or enriched proteins, termed adipokines, have been shown to have a variety of local, peripheral, and central effects. These secreted proteins, which include tumor necrosis factor (TNF)-alpha, resistin, Interleukin-6 (IL-6), IL-8, leptin and adiponectin

seem to play important

regulatory roles in a variety of complex processes, including fat metabolism, feeding behavior, hemostasis, vascular tone, energy balance, and insulin sensitivity. 2.4:- Leptin:The name of Leptin derived from the Greek Word “Leptos” meaning thin, which was originally identified in 1994 by Jeffery M. Friedman

(31)

. It is a 16 KDa protein consisting of 167 amino

acid is mainly synthesized and secreted by adipocyte (32). Leptin is a four-helix bundle with one very short strand segment and two relatively long interconnected loops; this is consistent with a

14

Chapter Two--------------------------------------------Literature Review classification as a cytokine of four-helix bundle as shown in Fig.(2.3)

(33)

. Leptin contains an intra-chain disulfide bond that

appears to be necessary for its biological activity (34).

Figure (2.3):- Leptin structure(33).

2.4.1:- Physiology of Leptin:Serum leptin circulates either in a free or a bound form. Free leptin is thought to be the biologically active form. Its main binding protein is a circulating soluble form of leptin receptor (soluble leptin receptor (sOB-R) which is produced by proteolytic cleavage of the short form of leptin receptor, and that sOB-R negatively regulates free leptin

(36)

. Leptin levels are

pulsatile and follow a circadian rhythm, with highest levels between midnight and early morning and lowest levels in the

15

Chapter Two--------------------------------------------Literature Review early- to mid-afternoon. The concentration of circulating leptin may be up to 75.6% higher during the night as compared to afternoon trough levels(37). Leptin production in adipocytes is regulated by indicators of acute nutritional status, such as insulin and counter- regulatory hormones, as well as by fat content. Adequacy of leptin levels suppresses feeding and the hypothalamic drive to produce glucocorticoids as well as permits the expenditure of energy by the reproductive, growth, and thyroid axes

(38)

.Leptin affects

these different functions either by direct action in peripheral tissues or through its action in the CNS as in figure (2.4) (39).

Figure (2.4). Leptin regulation(39).

16

Chapter Two--------------------------------------------Literature Review 2.4.2:- Regulation of Leptin Secretion:Leptin, is a peptide hormone that was initially reported to be synthesized and secreted exclusively from the adipocytes of white (40)

fat

. Nutrients, hormones, and neurotransmitters also seem to

play a major role in the regulation of leptin expression and secretion and

(41)

.Several Hormones can modulate leptin transcription

secretion,

the

most

important

being

insulin,

nor

epinephrine(41), dexamethasone, GH, triiodothyronine (T3) and insulin like growth factor-1 (IGF-1) (42).The changes in circulating leptin concentration in response to fasting, refeeding and macronutrient intake are likely mediated by insulin stimulated glucose metabolism in adipose tissue(32)

2.4.3:- Leptin Receptors Signaling:Leptin binds to leptin receptors (ObRs) located throughout the CNS and peripheral tissues, with at least six receptor isoforms identified (ObRa, ObRb, ObRc, ObRd, ObRe, and ObRf). These have homologous domains, but, due to alternative mRNA splicing, each receptor has a unique sequence and length. The short isoforms ObRa and ObRc are thought to have important roles in transporting leptin across the blood-brain barrier. The long isoform ObRb is ubiquitously expressed throughout the CNS and is primarily responsible for leptin signaling. The ObRb receptor is particularly important in the hypothalamus, where it regulates energy homeostasis and neuroendocrine function . In the db/db mouse model, the ObRb receptor is mutated and dysfunctional, resulting in the obese diabetic phenotype (35).

17

Chapter Two--------------------------------------------Literature Review 2.4.4:- Leptin and Adenophosphate Kinase:Adenosine 5’ monophosphate- activated protein kinase (AMPK) is a regulator of cellular and systemic energy homeostasis. It mediates some of the effects of peripheral hormones such as leptin and ghrelin also it is involved in the insulin- sensitizing role of the antidiabetic drug metformin, and has a central role in mediating the appetite- modulating and metabolic effects of many other hormones and substances, including the cannabinoids

(43,44)

. The first link

between leptin and AMPK was reported by Kahn's group showed that leptin increases AMPK activity in skeletal muscle, both directly and indirectly through stimulation the hypothalamosympathetic axis(45). Activation of AMPK and consequent inhibition of acetylcoenzyme A carboxylase (ACC) explains the effect of leptin on the inhibition of lipogenesis

and the increase in fatty acid

oxidation . Deficiency of leptin or of its receptor is associated with a decrease in AMPK activity in muscle and liver (46). Activation of AMPK in the hypothalamus leads to an increase in appetite. The effect of leptin on AMPK activity in the hypothalamus is the opposite of its effect in the periphery: AMPK activity is inhibited by leptin in the hypothalamus

(47)

.The leptin

and other agents such as ghrelin have opposite effects in different tissues. This might be the result of differing patterns of AMPK subunit expression in the various tissues, or to differential expression of the AMPK activators (43).

18

Chapter Two--------------------------------------------Literature Review 2.4.5:- Leptin Action in Hypothalamous:Leptin acts on the central nervous system by binding to its receptor (OBRb) to decrease food intake and increase energy expenditure as well as to regulate body weight and adipose tissue mass(48).Leptin

induces

production

and

release

of

proopiomelanocortin (POMC), a polypeptide precursor that is cleaved to generate α- and β- melanocyte- stimulating hormone (MSH). Interaction of MSH with melanocortin 4 receptor (MC4R) in the hypothalamus decrease food intake and increases energy expenditure, in contrast the MC4R antagonist agouti gene related peptide (AGRP) expenditure

(49.50)

increases food intake and decreases energy

. The regulation of leptin production by insulin and

counter- regulatory hormones suggests that leptin might serve as an indicator of energy balance (i.e. that increase energy stores yield increased leptin levels) So, Circulating leptin levels strongly correlate with body adiposity and changes in acute nutritional (38). The melanocortin system thus mediates the anorexigenic (inhibition of feeding) effects of leptin, reducing food intake and reducing energy expenditure, melanocortin 3 receptors (MC3R) is also located on POMC expression neurons in the arctuate nucleus (ARC), and may form part of a feedback loop which negatively regulates the anorexic tone of the POMC expressing neurons

(51)

.

Where melanocortin peptides from activated POMC neurons negatively auto regulate further POMC expression through their inhibitory actions at the MC3R. Recent evidence suggests that tyrosine kinase B receptor, brain derived neurotrophic factor, and nesfatin are critical mediators downstream of MC4R (52).

19

Chapter Two--------------------------------------------Literature Review Leptin also inhibits neurons co- expressing the orexigenic (feed promoting) neuropeptide Y (NPY) and AGRP in the ARC, which will otherwise promote feeding activity (53).

Figure(2.5): Targets of Leptin action in the brain (54). 2.4:- Adiponectin :2.4.1:- Molecular Structure:Adiponectin

(ApN)

is

also

named

Acrp30

(adipocyte

complement-related protein of 30kDa), AdipoQ (for its homology with the complement factor C1q), apM1 (adipose most abundant gene transcript 1) or GBP28 (gelatin binding protein (GBP) of 28 kDa). ApN, the most abundant adipokine, belongs structurally to the soluble defense collagen super-family sharing significant homology with collagen X, VIII and complement factor C1q. Full-length ApN protein is composed of a signal peptide, a variable N-terminal 20

Chapter Two--------------------------------------------Literature Review domain, followed by a collagenous domain and a C-terminal C1qlike globular domain. Full-length ApN can be processed by proteolytic cleavage into a smaller globular fragment (globular ApN). High Molecular Weight (HMW) form has been shown to possess the most potent insulin-sensitizing activity. ApN circulates in the concentration range of ~3–30μg/ml in healthy individuals, with lower levels in males compared to females, which is mainly attributed to lower amounts of HMW form. However, almost all ApN exists in plasma as full-length ApN, only a few globular ApN might circulate in human plasma as show in Fig.(2.6) (55,56).

Fig.(2.6): Schematic structure of adiponectin(56). 2.4.2:- Adiponectin Receptor Signaling :Two classical ApN receptors have been discovered and well described: adiponectin receptor 1 (AdipoR1) and receptor 2 (AdipoR2). Both AdipoR1 and AdipoR2 are surface membrane proteins with seven trans-membrane domains. AdipoR1 is ubiquitously expressed and most

21

Chapter Two--------------------------------------------Literature Review abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in mouse liver as show in Fig.(2.7)(55). Adiponectin automatically self-associates into larger structures. Initially, three adiponectin molecules bind together to form a homotrimer. The trimers continue to self-associate and form hexamers or decamers. Like the plasma concentration, the relative levels of the higher-order structures are sexually dimorphic, where females have increased proportions of the HMW forms. Adiponectin exerts some of its weight reduction effects via the brain. This is similar to the action of leptin, but the two hormones perform complementary actions, and can have additive effects(57). Overexpression of AdipoR1 activates AMPK, thereby reducing hepatic glucose production; on the contrary, AdipoR2 predominantly activates

peroxisome-proliferator-activated

receptors-α

signaling pathways, which induces fatty-acid oxidation

(58)

(PPAR-α) . AdipoR1

expression in AT is reduced in obese subjects and is increased after weight loss (59).

Figure(2- 7): Schematic structure of adiponectin receptors (56).

22

Chapter Two--------------------------------------------Literature Review 2.4.3:- Adiponectin Signaling:Results of various studies suggest the contribution of two major signaling pathways to ApN signal transduction

(60)

. In the skeletal

muscle and the liver, the modulation of the lipid and glucose metabolism by ApN is inhibited by blocking AMPK activation, indicating that stimulation of glucose utilization and fatty-acid oxidation by ApN occurs through activation of AMPK. In AT, ApN stimulates AMPK activation and inhibits LPS-stimulated nuclear factorkB(NF-kB) activation(55,56). Both AdipoRs are expressed in AT. As yet, the signaling mechanisms underlying the autocrine effects of ApN on AT are still largely unknown. One study showed that activation of AMPK mediates the stimulatory effect of ApN on glucose uptake in primary mice adipocytes (61). Another study found that suppressed NF-κB activation is involved in the inhibitory effect of ApN on LPS-stimulated release of TNF-α and IL-6 in adipocytes (62). 2.4.4:- Hypoadiponectinaemia in Obesity:Decreased mRNA expression and plasma levels of ApN are found in obese/diabetic mice and humans despite the increasing mass of AT. As the pro-inflammatory cytokines, OS and hyperinsulinaemia are largely induced by obesity and MS, their negative regulation on ApN transcription, multimerization and secretion could contribute to the hypoadiponectinaemia associated with obesity and MS (63). 2.4.5:-Biological Function of Adiponectin:The biological actions of ApN are implicated in multiple tissues as show in Fig.(2.8). Its pleiotropic properties play a role against various

23

Chapter Two--------------------------------------------Literature Review diseases including DM, NAFLD, CVD, obesity, and cancer ApN could thus be a therapeutic target for management of MS(64). 2.4.6:-Metabolic Effects:1.

Glucose flux decreased gluconeogenesis.

2.

Increased glucose uptake.

3.

Lipid catabolism increase β-oxidation.

4.

Increase triglyceride clearance.

5.

Increase insulin sensitivity.

6.

Control of energy metabolism(65).

Figure (2.8): Adiponectin exerts beneficial effects on multiple targets(64).

24

Chapter Two--------------------------------------------Literature Review 2.5:- C-peptide: 2.5.1:- Definition of C-peptide:C-peptide is a polypeptide

originating from Proinsulin after its

cleavage in the B-cell. C-peptide is a 31 amino acid peptide that bridges the insulin A and B chains in the Proinsulin molecule as show in Fig.(2.9). It is secreted equimolarly with the other cleavage product, insulin, into the portal circulation. C-peptide has a half-life of about 20 to 30 minutes compared to insulin which is cleared through the liver and has a half-life of about 3 to 5 minutes

(66)

, it is metabolized in the

proximal renal tubule and is about 5-10% is execrated in the urine, it can be measured either in serum, plasma, or in urine(67).

2.5.2:- Secretion of C-peptide:Preproinsulin, the transcriptional product of the insulin gene, is produced in the endoplasmic reticulum of the beta cell. Microsomal enzymes cleave preproinsulin to proinsulin, which contains the insulin alpha and beta chains, linked by a connecting peptide, c-peptide. Proinsulin is then transported to the Golgi complex, where it is packaged within clathrin coated secretory granules

(68)

. C-peptide is essential for the correct folding of proinsulin by

forming two disulphide bridges between cysteine residues of the alpha and beta chains and one within the alpha chain. Following maturation, the secretory granule loses its clathrin coat and proinsulin undergoes proteolytic cleavage and further processing into insulin and C-peptide, which are cosecreted in equimolar amount into the portal circulation. Once c-peptide is cleaved, the terminal end of the beta chain can bind to the insulin receptor (69)

.The great interest in C-peptide is due to the limitations of the use of

25

Chapter Two--------------------------------------------Literature Review serum insulin as a measure of insulin secretion. After its secretion in to the portal vein, insulin passes through the liver where approximately 50% of the insulin delivered is extracted(66).

Figure (2.9) Biosynthesis of C-peptide(70) 2.5.3:- Biological Role of C-peptide:C-peptide has been shown to bind to the surface of a number of cell types such as neuronal, endothelial, fibroblast and renal tubular, at nanomolar concentrations to a receptor that is likely G-protein-coupled. The signal activates Ca2+-dependent intracellular signaling pathways eNOS and Na+ K+ ATPase activities(71). These two enzymes are known to have reduced activities in patients with type I diabetes and have been implicated in the development of long-term complications of type 1 diabetes such as peripheral and autonomic neuropathy. Studies in animal models of type1 DM have established that C-peptide administration results in significant improvements in nerve and kidney function. Thus, in animals with early signs of diabetes-induced neuropathy, C-peptide treatment in replacement dosage results in improved peripheral nerve function, as evidenced by increased nerve

26

Chapter Two--------------------------------------------Literature Review conduction velocity, increased nerve Na+,K+ ATPase activity, and significant amelioration of nerve structural changes (72).Likewise, Cpeptide administration in animals that had C-peptide deficiency (type 1 model) with nephropathy improves renal function and structure;

(73)

it

decreases urinary albumin excretion and prevents or decreases diabetesinduced

glomerular

changes

secondary

to

mesangial

matrix

expansion(73). The main roles for C-peptide testing are in the discrimination of diabetes subtypes, which in turn informs correct management and to monitor interventions aimed at preserving beta cell function (74) . C-peptide measurement has been used as an alternative, reliable surrogate for assessing pancreatic insulin secretion

(75)

. Diabetes Prevention Trial

(DPT-1) and other studies indicated a direct relationship between age and Cpeptide responses. Previously it was suggested that an increase in C-peptide responses occurs during puberty, but the data from the DPT-1 also showed that C-peptide increases with age in prepubertal children (76).

2.6:- Oxidative Stress:The term oxidative stress refers to a condition where the levels of reactive oxygen species significantly overwhelm the capacity of antioxidant defenses in a biological system. OS condition can be caused by either increased ROS formation or decreased activity of antioxidants or both in a biological system. Oxidative Stress condition is associated with oxidative damage to biomolecules, including proteins, lipids, and nucleic acids. Moderate oxidative stress may cause cell dysfunction, whereas overt oxidative stress usually causes cell death. It should be noted that increases in ROS levels in a biological system are not always

27

Chapter Two--------------------------------------------Literature Review associated with injury. Under certain circumstances, small transient increases in ROS levels can be employed as a signaling mechanism, leading to physiological cellular responses. In this context, the disease conditions in which OS plays a role usually involve sustained formation of relatively large amounts of ROS via various mechanisms, including activation of inflammatory cells(77). 2.6.1:- Free Radicals(78):Free radical is any chemical species capable of independent existence possessing one or more unpaired electrons. Biological free radicals are thus highly unstable molecules that have electrons available to react with various organic substrates. Free radicals accumulate in tissues due to intracellular and extracellular processes. Radical species may combine to form other more damaging or toxic species such as peroxy nitrite , a product of superoxide and NO radical reaction. Free radicals react with key organic substrates such as lipids, proteins, and DNA. Oxidation of these biomolecules can damage them, disturbing normal functions and may contribute to a variety of disease states. Oxidation and reduction are redox chemical reactions. Oxidation does not necessarily involve oxygen, after which it was named, but is most easily described as the loss of electrons from atoms and molecules. The inverse reaction, reduction, occurs when a molecule gains electrons. In biochemistry, the free radicals of interest are often referred to as ROS because the most biologically significant free radicals are oxygen centered.

28

Chapter Two--------------------------------------------Literature Review 2.6.2:- Generation of Free Radicals:Many free radicals are the result of naturally occurring processes such as oxygen metabolism and inflammatory processes. For example, when cells use oxygen to generate energy, free radicals are created as a consequence of ATP production by the mitochondria. The cellular redox environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals (78).

2.6.3:- Mechanisms of Formation of Free Radicals during Obesity:The increase in obesity-associated oxidative stress is probably due to the presence of excessive AT itself, because adipocytes and preadipocytes have been identified as a source of proinflammatory cytokines, including TNF-α, IL-1, and IL-6; thus, obesity is considered a state of chronic inflammation. These cytokines are potent stimulators for the production of reactive oxygen and nitrogen by macrophages and monocytes; therefore, a rise in the concentration of cytokines could be responsible for increased OS(79). Adipose tissue also has the secretory capacity of angiotensin II, which stimulates Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. NADPH oxidase comprises the major route for ROS production in adipocytes (21). Mitochondrial and peroxisomal oxidation of fatty acids are capable of producing free radicals in liver and, therefore, OS, which could result in mitochondrial DNA alterations in the oxidative phosphorylation that occurs in mitochondria, causing structural abnormalities and depletion

29

Chapter Two--------------------------------------------Literature Review of ATP. However, it is also possible that mitochondrial abnormalities are preexisting conditions that allow for overproduction of ROS (80). Obesity increases the myocardial metabolism; therefore, oxygen consumption is increased. One negative consequence of increased oxygen consumption is the production of ROS as superoxide, hydroxyl radical, and H2O2 derived from the increase in mitochondrial respiration and, of course, from the loss of electrons produced in the electron transport chain, resulting in the formation of superoxide radical. Excessive fat accumulation can cause cellular damage due to pressure effect from fat cells Cellular damage in turn leads to high production of cytokines such as TNF-α, which generates ROS in the tissues, increasing the LPO rate (21). Obesity takes place in disorders that affect mitochondrial metabolism, which favors ROS generation and the development of OS. On the other hand, another mechanism has been proposed that involves an effect of high triglyceride (TG) on the functioning of the mitochondrial respiratory chain, in which intracellular TG, which is also high, inhibits translocation of adenine nucleotides and promotes the generation of superoxide

(81)

. During obesity, an increase in FFA,

which is toxic to pancreatic cells that are sensitive to oxidation and inducing alterations in insulin release, may lead to the development of DM (81).

2.6.4:- Reactive Oxygen Species:Reactive oxygen species term can be simply defined as oxygencontaining reactive species. It is a collective term to include superoxide (O2.-), hydrogen peroxide (H2O2), hydroxyl radical (.OH), singlet oxygen (1O2), peroxyl radical (LOO.), alkoxyl radical (LO.), lipid 30

Chapter Two--------------------------------------------Literature Review hydroperoxide (LOOH), peroxynitrite (ONOO-), hypochlorous acid (HOCl), and ozone (O3). Similarly, the term reactive nitrogen species (RNS) has been coined to include NO, peroxynitrite, nitrogen dioxide (.NO2), and other oxides of nitrogen or nitrogen-containing reactive species. Utilization of molecular oxygen by aerobic organisms leads to the formation of ROS/RNS. ROS/RNS are generated from various endogenous cellular sources, such as NAD(P)H oxidases and mitochondria. ROS/RNS are also produced from a variety of exogenous sources, including physical agents, xenobiotics, and biologic agents(77).

Table (2.2). Sources of Reactive Oxygen and Nitrogen Species in Biology and Medicine(77): Source

Description

Endogenous Sources

NAD(P)H oxidases Mitochondria (electron transport chain and oxidases) Xanthine oxidoreductase Cytochrome P450 enzymes Uncoupled NOS, Peroxisomes

Exogenous Sources

Physical agents and Xenobiotics Biologic agents (bacteria and viruses)

2.6.5:- Complications-Generated Oxidative Stress in Obesity:Obesity and the consequent production of OS have been associated with the development of other pathologies (Table 2.3), the most straightforward of which is the MS. Diseases associated with obesity include: Insulin resistance and diabetes, systemic arterial hypertension,

31

Chapter Two--------------------------------------------Literature Review ischemic heart diseases, obstructive sleep apnea, asthma, gout, peripheral vascular disease, peripheral vascular disease, psychology problems, rheumatological and orthopedics, oncology problems, and liver failure(81). 2.6.6:- Antioxidant Systems:The term antioxidant is frequently used in biology and medicine, and has been defined in various ways in the literature. One common way to define this term is that antioxidant is any substance that can prevent, reduce, or repair the (ROS/RNS)-induced damage to a target biomolecule (82). Antioxidants are generally classified into two major categories based on their sources, as  Endogenous antioxidants  Exogenous antioxidants Endogenous antioxidants refer to those made by cells. Endogenous antioxidants can be further classified into two subgroups: (1) protein antioxidants and (2) non-protein antioxidants. Endogenous protein antioxidants include enzymes, such as superoxide dismutase (SOD) and catalase (CAT), and non-enzymes, such as ferritin and metallothionein. Examples of endogenous non-protein antioxidants include the reduced form of glutathione (GSH) and bilirubin(77). Exogenous antioxidants refer to those that are not made by cells. Exogenous antioxidants can be either derived from dietary sources or synthesized

in

laboratories.

Typical

examples

of

exogenous

antioxidants derived from the diet include vitamin C, vitamin E, and polyphenols(77).

32

Chapter Two--------------------------------------------Literature Review 2.6.6.1:- Superoxide Dismutase:The term superoxide dismutase(E.C.1.15.1.1) refers to a family of metalloenzymes that catalyze the dismutation of superoxide to H2O2 and molecular oxygen. There are three isozymes of SOD in mammals(77). There are three isozymes of SOD in mammals (77).  Copper, zinc superoxide dismutase (Cu,ZnSOD or SOD1)  Manganese superoxide dismutase (MnSOD or SOD2)  Extracellular superoxide dismutase (ECSOD or SOD3) All three isozymes of SOD catalyze dismutation of superoxide (O2 .-) to form H2O2 and molecular oxygen with a similar reaction rate constant of ~1.6 x 109M-1s-1 2O2 .- + 2H+ → H2O2 + O2 and thus are critical for protecting the cell against the toxic products of aerobic respiration(83). 2.6.6.2:- Catalase:Mammalian Catalase (E.C.1.11.1.6)

belongs to a family of iron-

protoporphyrin IX-containing proteins that include a variety of cytochromes, globins and peroxidases. Catalase is one of the best characterized antioxidant enzymes in mammalian tissues. It plays an important role in regulating intracellular H2O2levels in mammalian cells.Catalase is best known for its ability to catalyze the decomposition of H2O2 to form water and molecular oxygen (77). 2H2O2 Catalase

2H2O + O2

Catalase also possesses peroxidase or oxidase activities toward a number of substrates, including low molecular mass alcohols, the

33

Chapter Two--------------------------------------------Literature Review tryptophan precursor indole, and the neurotransmitter precursor phenylethylamine. In general, catalase exhibits the peroxidatic activity in the presence of low concentrations of H2O2(77). Overexpression of CAT attenuates oxidative stress, inflammation, and development of both type 1 and type 2 diabetes in experimental animals.

Catalase

overexpression

also

ameliorates

diabetic

complications, including diabetic cardiomyopathy. On the other hand, acatalasemic mice have been shown to be more susceptible to chemically-induced pancreatic-cell injury and DM

(84)

. Li X et all

reported that cytoplasmic catalase overexpression in nonobese mice accelerated diabetes development (85).

2.6.6.3:- Reduced Glutathione:Reduced glutathione is by far the most important antioxidant in most mammalian cells. This ubiquitous tripeptide, γ-Glu-Cys- Gly, performs many cellular functions. In particular, the thiol containing moiety is a potent reducing agent .GSH has the important function of destroying reactive oxygen intermediates and free radicals that are constantly formed in metabolism (83). Intracellular GSH is converted to (oxidized glutathione) GSSG by selenium- containing GPx, which catalyzes the reduction of H2O2 in the presence of GSH and GSH peroxidase is coupled with oxidation of glucose-6-phosphate and of 6-phosphogluconate, which provides NADPH for reduction of GSSG by GSSG reductase. This is a major pathway of H2O2 metabolism in many cells. It is thus important for the protection of membrane lipids against oxidation(83).

34

Chapter Two--------------------------------------------Literature Review 2.6.6.4:- Glutathione Peroxidase:Glutathione peroxidase(E.C.1.11.1.9)

is the general name for a

family of multiple isozymes that catalyze the reduction of H2O2 or organic hydroperoxides to water or corresponding alcohols using the GSH as the electron donor, in mammalian tissues, there are six GPx isozymes. All of the GPx isozymes are able to catalyze the reduction of H2O2 or organic hydroperoxides (LOOH) to water or corresponding alcohols (LOH) using GSH as the electron donor(77). Glutathione peroxidase an enzyme whose main biological role is to protect the organism from oxidative damage by free radicals, is key indicator of OS. The red cells has been a central focus of research on GPx because it is thought to undergo a high endogenous rate of H 2O2 production from hemoglobin autoxidation. GPx activity is considered to represent the initial protective response required for adjusting the H2O2 concentration under normal physiological conditions as well as after oxidative insult If GPx activity is decreased, more H2O2is present, which leads to direct tissue damage and activation of NF-KB- related inflammatory pathways(77).

2.6.6.5:- Malondialdehyde (MDA) :It is an end product of lipid peroxidation (LPO), which is a process where reactive oxygen species degrade polyunsaturated lipids. Oxygen free radicals react with all biological substances. However, the most susceptible ones are polyunsaturated fatty acids. Reactions with cell membrane constituents lead to LPO. Membrane phospholipids, specifically esterified polyunsaturated fatty acids, are converted by peroxidation to MDA(78). Therefore, measurement of malondialdehyde is widely used as an indicator of LPO(86). 35

Chapter Two--------------------------------------------Literature Review Lipid peroxidation is a well-established mechanism of cellular injury in both plants and animals, and is used as an indicator of oxidative stress in cells and tissues. MDA is a highly reactive three carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism(86). Malondialdehyde react with deoxyadenosine and deoxyguanosine in DNA forming DNA adducts

(87).

. Malondialdehyde is reactive and

potentially mutagenic (88) . 2.6.6.5.1:- Structure of Malondialdehyde:Malondialdehyde mainly exists in the enol form: CH2(CHO)2 → HOCH=CH-CHO In organic solvents, the cis-isomer is favored, whereas in water the trans-isomer predominates. MDA is a highly reactive compound that is not typically observed in pure form

(89)

. In the laboratory it can be

generated in situ by hydrolysis of 1,1,3,3-tetramethoxypropane, which is commercially available . It is easily deprotonated to give the sodium salt of the enolate (245°C). Malondialdehyde is generated from ROS, and as such is assayed in vivo as a biomarker of OS (90).

Figure (2.10) Malondialdehyde structure.

36

Chapter Two--------------------------------------------Literature Review MDA-modified proteins may show altered physico-chemical behavior and antigenicity . The synergistic effect of antioxidants in human serum is known to provide greater protection against free radical aggression than any single antioxidant Alone(86). Obesity is associated with enhanced LPO, one of the most frequently used biomarkers providing an indication of LPO level is the plasma concentration of MDA, one of several by-products of LPO processes(91). 2.7:- Paraoxonase:The Paraoxonase (PON, aryldialkyl phosphatase, E.C. 3.1.8.1), is an Ca- dependent enzyme that is synthesized in liver. It is related to HDL-c and has 43-45 KDa molecular weight with glycoprotein structure. The Paraoxonase was coincided initially in HDL-c after electrophoresis of human serum in 1961 (92). Paraoxonase refers to a family of enzymes that includes three members in mammals, namely, PON1, PON2, and PON3. They all possess antioxidant properties, share 65% similarity at the amino acid level, and the genes are located in tandem on chromosome 7 in humans and on chromosome 6 in mice. Although the amino acid chains of three PON proteins are similar 65 % in proportion, their expressions in tissues and dispersions are different from each other(93,94). 2.7.1:- Paraoxonase1 (PON 1):Paraoxonase1 is a calcium-dependent esterase that was first described for its capacity to hydrolyze organophosphates and pesticides, including paraoxon. PON1 is a 43–45 kDa glycoprotein. It has a sixbladed β-propeller shape and two calcium ions are located in the central tunnel of the enzyme which are believed to be important for both structure stability and catalytic function (Fig 2.11.A and B) (95).

37

Chapter Two--------------------------------------------Literature Review PON1 is synthesized and secreted by liver, and is primarily associated with HDL-c in plasma.PON1 is an excellent example of amultitasking protein, displaying at least two important biochemical functions: (1) hydrolysis of organophosphates (pesticides and nerve agents) and (2) inhibition of lipoprotein oxidation by catalyzing degradation of oxidized lipids.PON1 effectively hydrolyzes a variety of organophosphate pesticides and nerve agents, including paraoxon, diazoxon, chlorpyrifosoxon, sarin, and soman. Hydrolysis of these organophosphates by PON1 leads to their detoxification(96). Serum PON1 activity is affected by diet, pregnancy, smoking, hormones, acute phase proteins and age. PON1 activity in newborns and premature infants is half of adult PON1 activity, only after a year it reaches its adult level. PON1 activity decreases with age , but enzyme activity does not vary by sexes (97). The important function of PON1 is its anti-atherogenic activity. HDL isolated from the PON1-deficient animals is unable to protect low density lipoprotein (LDL-c) from LPO, suggesting that PON1 mediates the antioxidant activities of HDL-c (98). 2.7.2:- PON2 and PON3:PON2 is an intracellular protein that is widely distributed in mammalian tissues. Similar to PON1, PON2 with a molecular mass of 44 KDa also acts as an antioxidant protecting against OS.PON3 (40 KDa) is primarily expressed in liver, and associated with HDL in plasma(99). The protective effects of both PON2 and PON3 in experimental atherosclerosis have been recently demonstrated. Despite lower levels of very low-density lipoprotein (VLDL-c) and LDL-c , mice deficient in

38

Chapter Two--------------------------------------------Literature Review PON2 develop significantly larger atherosclerotic lesions compared with their wild-type counterparts (100). Three potential mechanisms may account for the enhanced atherosclerotic lesions in PON2-deficient mice. They are: (1)enhanced inflammatory properties of LDL-c,(2) diminished anti-atherogenic capacity of HDL-c, and (3) a heightened state of OS coupled with an exacerbated inflammatory response from PON2-deficient macrophages (101)

.

Figure (2.11) Overall structure of PON1(95). 2.7.3:- Role of Paraoxonase in Oxidative Stress:High density lipoprotein associated PON1 is an antioxidant enzyme that protects LDL-c and HDL-c from LPO. And it is considered to be the main anti atherosclerotic (preclusive to atherosclerosis) component of HDL-c

(102)

. PON1 protects lipoproteins and arterial cells against

oxidation, probably by hydrolyzing lipid peroxides such as specific

39

Chapter Two--------------------------------------------Literature Review oxidized cholesteryl esters and phospholipids, and it was suggested to contribute to the antioxidant protection conferred by HDL-c on LDL-c oxidation

(103)

.HDL-associated PON was able to hydrolyze long-chain

oxidized phospholipids isolated from oxidized LDL or serve as a target for peroxides . PON1 through interactions between the enzyme-free sulfhydryl group and oxidized lipids. H2O2 is a major ROS produced by arterial wall cells during atherogenesis, and it is converted under OS into more potent hydroxyl radical leading to LDL oxidation (104). 2.8:- Plasma Lipid and Lipoprotein:The term lipid applies to a class of compounds that are soluble in organic solvents and nearly insoluble in water

(105)

. Plasma lipid consist

of triacylglycerol, phospholipids, cholesterol, and Cholesteryl esters and a much smaller fraction of long-chain fatty acids (free fatty acidsFFA) which are most metabolically active of the Plasma lipids(105). Lipids and cholesterol are transported through blood stream as macromolecular complexes of lipids and proteins known as lipoproteins. These consist of a central core of hydrophobic lipid (including

triacylglycerol,

and

Cholesteryl

esters)

encased

in

hydrophilic coat of polar phospholipids, free cholesterol, and apolipoprotein (106). There are six classes of lipoproteins: chylomicrons, VLDL-c, intermediate- density lipoproteins (IDL-c), LDL-c, and HDL-c. HDL-c can be divided further into two subpopulations: HDL2 and HDL3. The normal function of lipoproteins is to distribute and recycle cholesterol (105,107)

.

40

Chapter Two--------------------------------------------Literature Review Apolipoproteins are the protein moiety of a lipoprotein which carry out several roles: (1) they can form part of the structure

of the

lipoprotein, e.g., apo B; (2) they are enzyme cofactors, e.g, C-II for lipoprotein lipase (LPL); and (3) they act as ligands for interaction with lipoprotein receptors in tissues, e.g., apo B and apo E for the LDL receptor (108). 2.8.1:- Metabolism of Lipids: When dietary cholesterol and triglycerides are absorbed from the intestine, they are transported in the intestinal lymphatics

as

chylomicrons, the largest of the lipoprotein particles and of which approximately 80% of the lipid core and triglycerides

(107)

.

Chylomicrons (diameter 100-1000 mm) are transported in lymph and then blood to capillaries in muscle and adipose tissues. Here, TGs are hydrolyzed by LPL, and the tissues take up the resulting free fatty acids and glycerol (106). The cholesterol-rich chylomicron remnant is taken up by receptors on hepatocyte membrane, and in this way, dietary cholesterol is delivered to the liver and cleared from the circulation (107).Cholesterol liberated in hepatocytes is stored, oxidized to bile acids, secreted unaltered in bile or can enter the endogenous pathway with the newly synthesized TGs and the two are transported from the liver as nascent VLDL-c. This TGs-rich particle (55% by mass) contains apoB-100, apoE, and small amount of C Apolipoproteins on its surface (105,106). The TG content of VLDL-c is removed by LPL and forms IDL-c particles which may be transported to the liver or be further hydrolyzed,

41

Chapter Two--------------------------------------------Literature Review where about 50% of the IDL-c is removed by hepatocytes. Most of the remaining TGs are removed and become LDL-c (105,107). Low-density lipoprotein is major cholesterol-carrying particle in the plasma, provides cholesterol, bile acids and a precursor of steroid hormones to those cells that require it

(107)

.Cells in the extra hepatic

tissues take up LDL-c by endocytosis via LDL-c receptors that recognize LDL apolipoprotein and through scavenger receptors (105,106). Lipoprotein a (LPa) is a species of LDL-c that is strongly associated with atherosclerosis and contains a unique apoprotein, apo (a) with structural similarities to plasminogen. LPa competes with and inhibits the binding of LP(a) is that less plasmin is generated, fibrinolysis is inhibited and thrombosis promoted (106). High-density lipoproteins is formed from the unesterified cholesterol and phospholipids removed from peripheral tissues and the surface of TG-rich proteins. HDL-c mediate the return of

lipoprotein and

cholesterol from peripheral tissues to the liver for excretion in a process known as reverse cholesterol transport (105,107,108). 2.8.2:-Relation of Triglycerides and Cholesterol with Obesity:Cholesterol levels and LDL are increased in obesity due to the high fat intake of these individuals and their lack of movement, so they are at a high risk of heart disease. When LDL-c levels are too high, the LDL-c tends to stick to the lining of the blood vessels, leading to the stimulation of atherosclerosis or hardening of the arteries. Atherosclerosis plaques cause narrowing of the arteries and lead to heart attacks and strokes, whereas, increased levels of HDL-c are associated with a lower risk of heart disease. Thus, HDL-c appears 42

Chapter Two--------------------------------------------Literature Review to be a good protector against heart disease

(109)

. There is some

evidence that the HDL-c molecule scours the walls of blood vessels, and cleans out excess cholesterol which is transported to the liver for further processing(109). Obesity in most individuals is linked to an elevated level of TG. Excess TG in plasma is called hypertriglyceridemia. It is linked to the occurrence of CAD in some individuals. Elevated TG may be a consequence of other diseases, such as untreated DM, by accumulating in pancreatic β-cells and causing it`s dysfunction

(110)

. Like cholesterol,

increases in TG levels can be detected by plasma measurements. Very high TG can cause pancreatitis, an enlarged liver and spleen, and fatty deposits in the skin called xanthomas. It was reported that the levels of HDL-c decreases consistently with increasing BMI, while the levels of LDL-c and triglycerides and the ratio of total cholesterol to HDL-c increased steadily (111).

43