Regulation of Lipoprotein Transport in the Metabolic Syndrome · structure, function and...

Regulation of Lipoprotein Transport in the

Metabolic Syndrome:

Impact of Statin Therapy

Esther M. M. Ooi

Bachelor of Science (Hons)

This thesis is presented for the degree of

Doctor of Philosophy of Medicine

of the University of Western Australia

School of Medicine and Pharmacology

Royal Perth Hospital

Medicine

2007

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ABSTRACT

The metabolic syndrome is characterized by cardiovascular risk factors including

dyslipidemia, insulin resistance, visceral obesity, hypertension and diabetes. The

dyslipidemia of the metabolic syndrome includes elevated plasma triglyceride and

apolipoprotein (apo) B levels, accumulation of small, dense low-density lipoprotein

(LDL) particles and low high-density lipoprotein (HDL) cholesterol concentration.

However, the precise mechanisms for this dyslipoproteinemia, specifically low plasma

HDL cholesterol, are not well understood. This thesis therefore, focuses on HDL, its

structure, function and metabolism. However, lipoprotein metabolism is a complex

interconnected system, which includes forward and reverse cholesterol transport

pathways. Hence, this thesis also examines and discusses the metabolism of apoB-

containing lipoproteins.

This thesis tests the general hypothesis that apolipoprotein kinetics are altered in the

metabolic syndrome, and that lipid regulating therapies can improve these kinetic

abnormalities. The aims were first, to compare and establish the clinical, metabolic and

kinetic differences between metabolic syndrome and lean subjects; and second, to

determine the regulatory effects of statin therapy, specifically, rosuvastatin on

lipoprotein transport in the metabolic syndrome. Five observation statements were

derived from the general hypothesis and examined in the studies described below. The

findings are presented separately as a series of original publications.

Study 1 Twelve men with the metabolic syndrome and ten lean men were studied in a

case-control setting. The kinetics apoB-100 and HDL apoA-I and apoA-II were

measured using D3-leucine, gas chromatography-mass spectrometry and

multicompartmental modeling. The kinetics of HDL subpopulations LpA-I and LpA-

I:A-II were also examined using a new compartment model. Compared with lean men,

men with the metabolic syndrome had higher concentrations of very-low-density

lipoprotein (VLDL), intermediate-density lipoprotein (IDL) and LDL-apoB (+78%,

+57% and +59%, respectively, p

-17%, respectively, p

dose-dependent reductions in LpA-I FCR, with no changes in LpA-I PR. These findings

explain the HDL raising effects of rosuvastatin in the metabolic syndrome.

Collectively, these studies suggest that the dyslipidemia of the metabolic syndrome

results from increased production rates of VLDL and LDL particles, reduced fractional

catabolic rates of these lipoproteins, together with accelerated catabolism of HDL

particles. Treatment with rosuvastatin increases the catabolic rates of all apoB-

containing lipoproteins and at a higher dose, decreases LDL apoB production. These

effects are consistent with inhibition of cholesterol synthesis leading to an upregulation

of LDL receptors. Rosuvastatin decreases the fractional catabolism of HDL particles.

The effects of rosuvastatin on HDL kinetics may be related to a reduction in triglyceride

concentration and cholesterol ester transfer protein activity. These findings are

consistent with the general hypothesis that apolipoprotein kinetics are altered in the

metabolic syndrome, and that statin therapy improves these kinetic abnormalities.

4

TABLE OF CONTENT

ABSTRACT 2

TABLE OF CONTENTS 5

LIST OF TABLES 14

LIST OF FIGURES 17

LIST OF ABBREVIATIONS 21

PERSONAL CONTRIBUTION BY THE CANDIDATE 25

PUBLICATIONS AND COMMUNICATIONS 28

ACKNOWLEDGMENTS 33

CHAPTER 1 LITERATURE REVIEW 35

1.1 Overview of the Metabolic Syndrome 36

1.1.1 Definition of the metabolic syndrome 36

1.1.2 Prevalence of the metabolic syndrome 37

1.1.3 Components of the metabolic syndrome 40

1.1.3.1 Insulin resistance 40

1.1.3.2 Obesity and insulin resistance 41

1.1.3.3 Dyslipidemia and insulin resistance 42

1.1.3.4 Hypertension and insulin resistance 42

1.1.3.5 Inflammation, prothrombosis and insulin resistance 43

1.1.4 Significance of the metabolic syndrome 44

5

1.1.4.1 Association between the metabolic syndrome and cardiovascular

disease 44

1.1.4.2 Association between the metabolic syndrome and diabetes 46

1.1.5 Prevention and management of the metabolic syndrome 46

1.1.5.1 Therapeutic lifestyle intervention 47

1.1.5.2 Pharmacotherapy 47

1.2 Lipoprotein metabolism 50

1.2.1 Definition and classification of lipids 50

1.2.2 Lipoprotein metabolism 50

1.2.3 The lipoproteins 52

1.2.3.1 Chylomicrons 52

1.2.3.2 Very low density lipoprotein 52

1.2.3.3 Intermediate density lipoprotein 54

1.2.4.4 Low density lipoprotein 54

1.2.2.5 High density lipoprotein 56

1.2.2.6 Lipoprotein (a) 60

1.2.4 Apolipoproteins 62

1.2.4.1 Apolipoprotein A-I 62

1.2.4.1 a Structure and composition 62

1.2.4.1 b Metabolism 63

1.2.4.1 c Biological role and significance 64

1.2.4.1 d Genetics 65

6

1.2.4.2 Apolipoprotein A-II 66





1.2.4.3 Apolipoprotein A-IV 69

1.2.4.4 Apolipoprotein A-V 69

1.2.4.5 Apolipoprotein B 70





1.2.4.6 Apolipoprotein C 75

1.2.4.6 a ApoC-I 75

1.2.4.6 b ApoC-II 75

1.2.4.6 c ApoC-III 76

1.2.4.7 Apolipoprotein E 77

1.2.4.8 Apolipoprotein (a) 78

1.2.5 Receptors, transport proteins, enzymes and transfer proteins 81

1.2.5.1 Receptors and transport proteins 81

1.2.5.1 a LDL receptor 81

1.2.5.1 b LDL receptor related protein 82

7

1.2.5.1 c Scavenger Receptor Class B Type 1 83

1.2.5.1 d ATP binding cassette transporters 83

1.2.5.2 Enzymes 84

1.2.5.2 a Lipoprotein lipase 84

1.2.5.2 b Hepatic lipase 85

1.2.5.3 c Endothelial lipase 86

1.2.5.3 Cholesterol esterification and lipid transfer protein 86

1.2.5.3 a Lecithin-cholesterol acyltransferase 86

1.2.5.3 b Cholesteryl ester transfer protein 87

1.2.5.3 c Phospholipid transfer protein 88

1.3 Management of dyslipidemia 90

1.3.1 Lipid regulating drugs 91

1.3.2 HMG CoA reductase inhibitors 93

1.3.2.1 Overview 93

1.3.2.2 Rosuvastatin 95

1.4 Kinetic Studies and Lipoprotein Metabolism 103

1.4.1 Background 103

1.4.2 Principles of stable isotope methodology 104

1.4.3 Laboratory and mathematical methods of stable isotope studies 106

1.4.3.1 Tracer administration 106

1.4.3.2 Laboratory methodology 106

1.4.3.3 Compartmental modeling 106

8

1.4.3.4 Definitions in kinetic analysis 108

1.4.4 Overview of lipoprotein kinetic studies 109

1.4.4.1 Kinetic studies of apoB 109

1.4.4.1 a Obesity and apoB kinetics 109

1.4.4.1 b Nutritional interventions on apoB kinetics 111

1.4.4.1.c Effects of statins on apoB kinetics 111

1.4.4.2 Kinetic studies of apoA-I and apoA-II 115

1.4.4.2 a ApoA-I and apoA-II kinetics in normolipidemic and

insulin resistant subjects 115

1.4.4.2 b Genetic disease and apoA-I and apoA-II kinetics 116

1.4.4.2 c Nutritional and hormonal interventions on apoA-I and

apoA-II kinetics 119

1.4.4.2 d Effect of statins on apoA-I and apoA-II kinetics 119

1.4.4.3 Kinetic studies of other apolipoproteins and lipids 122

1.4.4.3 a Kinetic studies of other apolipoproteins 122

1.4.4.3 b Kinetic studies of triglycerides and cholesterol 122

CHAPTER 2 HYPOTHESIS, AIMS AND STUDY DESIGN 124

2.1 Philosophical Perspective 125

2.2 Hypothesis 125

2.3 General Hypothesis 127

2.3.1 Study 1: Apolipoprotein B-100 and A-I Kinetics in the Metabolic Syndrome

127

2.3.2 Study 2: High-Density Lipoprotein Apolipoprotein A-I Kinetics in Obesity

9

128

2.3.3 Study 3: Plasma Phospholipid Transfer Protein Activity, a Determinant of

HDL Kinetics In Vivo 128

2.3.4 Study 4: Dose-ranging Effect of Rosuvastatin on Apolipoprotein B-100

Kinetics in the Metabolic Syndrome 129

2.3.5 Study 5: Dose-Dependent Improvement in High-Density Lipoprotein

Metabolism with Rosuvastatin in the Metabolic Syndrome 130

CHAPTER 3 STUDY DESIGN, SAMPLE SIZE AND POWER CALCULATIONS

131

3.1 Overview of Studies 132

3.2 Studies Details 132

3.3 Sample Size and Power Calculations 134

CHAPTER 4 MATERIALS AND METHODS 143

4.1 Subjects 144

4.2 Method of recruitment 147

4.3 Protocol for screening 148

4.4 Anthropometric measurements 149

4.5 Blood pressure 149

4.6 Urinalysis 150

4.7 Assessment of nutrient intake 150

4.8 Protocol for blood sampling 150

4.9 Preparation of D3-leucine solution 151

4.10 Stable Isotope Injection Protocol 151

10

4.11 Collection and storage of buffy coat (white cells) protocol 152

4.12 Collection and Storage of Cholesterol Efflux and Preβ 152

4.13 Collection of samples for genetic analysis 153

4.14 Biochemical assays 153

4.15 Kinetic Analyses 162

4.15.1 Isolation and Measurement of Isotopic Enrichment of Apolipoproteins

and Plasma Leucine 162

4.15.1.1 ApoB-100 165

4.15.1.2 Apolipoprotein A-I and Apolipoprotein A-II 166

4.15.1.3 Plasma Leucine (Oxazolinone Method) 167

4.15.2 Derivatization of leucine 167

VLDL, IDL, LDL-apoB, HDL-apoA-I, apoA-II and plasma leucine eluate

4.16 Mathematical modeling 168

4.16.1 Model for VLDL, IDL and LDL-apoB 168

4.16.2 Model for apoA-I in HDL LpA-I and LpA-I:A-II particles and apoA-II

173

4.17 Statistical analyses 178

4.17.1 Measures of variability 178

4.17.2 Coefficient of variation 178

4.17.3 Distribution of data 179

4.17.4 Independent t-test 179

4.17.5 Correlational analyses 180

11

4.17.6 Regression analyses 181

4.17.7 The mixed model: an extension of the general linear model 184

CHAPTER 5 Apolipoprotein B-100 and A-I Kinetics in the Metabolic Syndrome 187

CHAPTER 6 High-Density Lipoprotein Apolipoprotein A-I Kinetics in Obesity 203

CHAPTER 7 Plasma Phospholipid Transfer Protein Activity, a Determinant of HDL

Kinetics In Vivo 213

CHAPTER 8 Dose-ranging Effect of Rosuvastatin on Apolipoprotein B-100 Kinetics in

the Metabolic Syndrome 222

CHAPTER 9 Dose-Dependent Improvement in High-Density Lipoprotein Metabolism

with Rosuvastatin in the Metabolic Syndrome 245

CHAPTER 10 Conclusion: Overview, Limitations, Implications and Future Research

267

10.1 Overview 268

10.2 Study Limitations 274

10.3 Implications 276

10.3.1 Dyslipidemia, metabolic syndrome and cardiovascular disease 276

10.3.2 Management and treatment of dyslipidemia in the metabolic syndrome

277

10.4 Synthesis: Perspectives for Future Research 278

10.4.1 Study population 278

10.4.2 Lipid substrate availability 278

10.4.3 VLDL and LDL subspecies 279

10.4.4 HDL subspecies 279

10.4.5 Cholesterol efflux 279

12

10.4.6 ApoC-III, apoA-IV, apoA-V and apoE 280

10.4.7 Genetic polymorphisms 280

10.4.8 Other interventions 280

10.5 Conclusion 286

REFERENCES 287

APPENDICES

Appendix 1 High-Density Lipoprotein Apolipoprotein A-I Kinetics: Comparison of

Radioactive and Stable Isotope Studies 344

Appendix 2 High-Density Lipoprotein (HDL) Transport in the Metabolic Syndrome:

Application of a New Model for HDL Particle Kinetics 352

Appendix 3 Relationships between Plasma Lipids Transfer Proteins and Apolipoprotein B-

100 Kinetics during Fenofibrate Treatment in the Metabolic Syndrome 360

Appendix 4 Fish oils, phytosterols and weight loss in the regulation of lipoprotein transport

in the metabolic syndrome: lessons from stable isotope tracer studies 368

Appendix 5 Dietary Plant Sterols Supplementation Does Not Alter Lipoprotein Kinetics in

Men with the Metabolic Syndrome 375

13

LIST OF TABLES

Chapter 1

Table 1.1 Clinical definitions of the metabolic syndrome 38

Table 1.2 Therapeutic interventions in the metabolic syndrome 49

Table 1.3 Classification and properties of major human plasma

lipoproteins

61

Table 1.4 Characteristics of the major apolipoproteins 79

Table 1.5 Lipid regulating drugs 92

Table 1.6 LDL reduction by percentage change according to statin and

daily dose (summary estimates from 164 randomized placebo

controlled trials)

94

Table 1.7 Summary of clinical efficacy studies with rosuvastatin 98

Table 1.8 Studies of apoB kinetics in normolipidemic and obese subjects 110

Table 1.9 Effects of statins on apoB kinetics 113

Table 1.10 Studies of apoA-I and apoA-II kinetics in normolipidemic and

insulin resistant subjects

117

Table 1.11 Effects of statins on apoA-I and apoA-II kinetics 121

Chapter 5

Table 1 Clinical and biochemical characteristics of the subjects with the

metabolic syndrome and the lean subjects

200

Table 2 Kinetic characteristics of VLDL, IDL and LDL-apoB in

subjects with the metabolic syndrome and lean subjects

201

14

Table 3 Kinetic characteristics of LpA-I, LpA-I:A-II, apoA-II and

plasma apoA-I in subjects with the metabolic syndrome and

lean subjects

202

Chapter 6

Table 1 Stable isotope studies included in the summary analysis 206

Table 2 Clinical and biochemical characteristics of lean and

overweight-obese subjects

206

Table 3 Associations of HDL apoA-I FCR and PR with other variables

in lean subjects

207


in overweight-obese subjects

208


from data pooled from both lean and overweight-obese group

209

Table 6 Multiple regression model of age, sex, BMI, triglycerides, and

HOMA score as predictors of HDL apoA-I FCR in pooled data

from lean and overweight-obese subjects

209

Table 7 Multiple regression model of age, sex, BMI, triglycerides, and

HOMA score as predictors of HDL apoA-I PR in pooled data

from lean and overweight-obese subjects

210

Chapter 7

Table 1 Clinical and biochemical characteristics of participants studied 216

Table 2 HDL parameters of the participants studied 217

Table 3 Multivariate regression analysis of the relationship between

HOMA score and plasma PLTP activity adjusting for age,

217

15

triglycerides, waist circumference and CETP activity

Table 4 Associations (Pearson correlation coefficients) of plasma PLTP

activity with clinical and biochemical characteristics of

participants studied

217

Table 5 Associations between plasma PLTP activity and HDL

parameters

217

Table 6 Multivariate regression analyses of the relationship between

plasma PLTP activity and LpA-I concentration, LpA-I

production rate and LpA-I fractional catabolic rate, adjusting

for HOMA score, triglycerides and CETP activity

218

Chapter 8

Table 1 Plasma lipids, lipoproteins and apolipoproteins in placebo, 10

mg/day rosuvastatin and 40 mg/day rosuvastatin

239

Table 2 Body weight, blood pressure, measure of insulin resistance and

nutrient intake on placebo and rosuvastatin treatments

240

Table 3 VLDL, IDL and LDL apoB-100 kinetics in placebo, 10 mg/day

rosuvastatin and 40 mg/day rosuvastatin

241

Chapter 9

Table 1 Plasma lipids and apolipoproteins during treatment with

placebo, 10 mg/day rosuvastatin and 40 mg/day rosuvastatin

261

Table 2 ApoA-I, apoA-II, LpA-I and LpA-I:A-II kinetic parameters

during placebo and rosuvastatin treatments

262

16

LIST OF FIGURES

Chapter 1

Figure 1.1 Insulin resistance, the metabolic syndrome, and cardiovascular

disease risk.

44

Figure 1.2 Typical Structure of a lipoprotein particle 51

Figure 1.3 Schematic diagram of lipid-free apoA-I 62

Figure 1.4 Role of apoA-I in the reverse cholesterol transport pathway 65

Figure 1.5 Schematic representation of the apo A-II gene, APOA2 68

Figure 1.6 Schematic diagram of the structure of apoB-100 on the surface

of LDL.

71

Figure 1.7 Schematic diagram of the pentapartite structural model, NH3-

ß 1-ß1- 2-ß2- 3-COOH, for apoB-100

71

Figure 1.8 Schematic representation of the pathway for assembly and

secretion of hepatic apoB containing lipoproteins

73

Figure 1.9 Independent role of apoB and apoA-I in predicting fatal

myocardial infarction in men.

74

Figure 1.10 Model of human apoE 77

Figure 1.11 Overview of lipoprotein metabolism 80

Figure 1.12 Schematic illustrating the uptake of lipoprotein particles by the

LDLR

82

Figure 1.13 A schematic representation of the structural model of human

PLTP

89

Figure 1.14 Cholesterol biosynthesis pathway 93

Figure 1.15 Molecular structure of rosuvastatin 95

17

Figure 1.16 The balancing feedback loop 103

Figure 1.17 Principle of tracer methodology 105

Chapter 2

Figure 2.1 Philosophical construct employed in this thesis 126

Chapter 4

Figure 4.1 Quantification of LpA-I concentration by differential

electroimmunoassay using Hydragel LpA-I kit

159

Figure 4.2 Derivatization of leucine with TFA/TFAA 168

Figure 4.3 Compartmental model describing apoB tracer kinetics 172

Figure 4.4 Isotopic enrichment of VLDL, IDL and LDL-apoB in a

representative metabolic syndrome subject

173

Figure 4.5 Compartment model describing apoA-I in LpA-I and LpA-I:A-

II particles, apoA-II and apoA-I tracer kinetics.

176

Figure 4.6 Isotopic enrichment for apoA-I in LpA-I and LpA-I:A-II

particles, apoA-II and apoA-I with D3-leucine in a

representative subject.

177

Chapter 6

Figure 1 Association between HDL apoA-I FCR with apoA-I

concentration in lean and overweight-obese subjects

208

Figure 2 Association between HDL apoA-I PR with apoA-I

concentration in lean and overweight-obese subjects

208

18

Figure 3 Association between HDL apoA-I FCR and estimated HDL

particle size (HDL cholesterol:apoA-I ratio) for data pooled

from lean and overweight-obese groups

209

Chapter 7

Figure 1 Compartmental model describing apoA-I in LpA-I and LpA-

I:A-II particles, apoA-II and apoA-I tracer kinetics

216

Figure 2 Associations between plasma PLTP activity and (a) LpA-I

concentration, (b) LpA-I production rate, and (c) LpA-I

fractional catabolic rate

218

Figure 3 A proposed model of the relationships between insulin

resistance, PLTP and LpA-I kinetics in vivo

219

Chapter 8

Figure 1 Isotopic enrichment for VLDL (A), IDL (B) and LDL (C) apoB

with D3-leucine in a representative subject on placebo,

rosuvastatin 10 mg/day and rosuvastatin 40 mg/day

242

Figure 2 Fractional catabolic rates of VLDL (A), IDL (B) and LDL (C)

apoB on placebo, 10 mg rosuvastatin and 40 mg rosuvastatin

243

Figure 3 Associations between changes (Δ) in VLDL apoB FCR and

apoC-III concentration (A) and LDL apoB FCR and

lathosterol:cholesterol ratio (B) on 40mg/day rosuvastatin

relative to placebo

244

Chapter 9

Figure 1 Compartment model describing apoA-I in LpA-I and LpA-I:A-

II particles, apoA-II and apoA-I tracer kinetics.

264

19

Figure 2 Isotopic enrichment for HDL apoA-I (A) and apoA-II (B) with

D3-leucine in a representative subject on placebo, rosuvastatin

10 mg/day and rosuvastatin 40 mg/day

265

Figure 3 HDL particles fractional catabolic rate (FCR) (A) and

production rate (PR) (B) on placebo, 10 mg rosuvastatin and 40

mg rosuvastatin treatment

266

20

LIST OF ABBREVIATIONS AND DEFINITIONS OF TERMS

The following abbreviations and special terms are used in this thesis Abbreviation or special term Definition

ABC ATP-binding-cassette transporter

ACAT Acyl:cholesterol acyltransferase

ACEI Angiotensin Converting Enzyme Inhibitor

AF/Tex CAPS Ait Force/Texas Coronary Atheroma Prevention Study

ALT Alanine aminotrasferase

AP Alkaline phosphatase

Apo Apolipoprotein

AST Aspartate aminotransferase

ATP Adenosine triphosphate

ATP III Adult Treatment Panel III

ANOVA Analysis of variance

BMI Body mass index

BP Blood pressure

CAD Coronary artery disease

CARE Cholesterol and Recurrent Events

CB1 Cannabinoid 1

CE Cholesteryl ester

CETP Cholesteryl ester transfer protein

CK Creatine kinase

CHD Coronary heart disease

CI Confidence Interval

CM Chylomicrons

CR Chylomicron remnants

CRP C-reactive protein

CV Coefficient of variation

CVD Cardiovascular disease

DECODE European Diabetes Epidemiology Group

DGAT Diacylglycerol acyl transferase

ECG Electrocardiogram

EDTA Ethylene diamine tetra acetic acid

EL Endothelial Lipase

21

Abbreviation or special term Definition

ER Endoplasmic reticulum

FBE Full blood examination

FC Free cholesterol

FCR Fractional Catabolic Rate

FDB Familial defective apoB-100

FFA Free fatty acids

FH Familial hypercholesterolemia

FHA Familial hyperalphalipoproteinemia

FM Fat mass

FFM Free fat mass

FSR Fractional secretion or synthesis rate

GCMS Gas Chromatography - Mass Spectrometry

GLM General Linear Modeling

HBL Hypobetalipoproteinemia

HDL High-density lipoprotein

HL Hepatic lipase

HMG-CoA 3-Hydroxy-3-methylglutaryl-coenzyme A

HOMA Homeostasis model assessment

HPS Heart Protection Study

HSPG Heparan sulphate proteoglycans

IDF International Diabetes Federation

IDL Intermediate-density lipoprotein

IFG Impaired fasting glucose

IGT Impaired glucose tolerance

IL Interleukin

LCAT Lecithin:cholesterol acyltransferase

LDL Low-density lipoprotein

LDLR Low-density lipoprotein receptor

LFT Liver function test

LIPID Lipid Intervention with Pravastatin in Ischemic Disease

Lp Lipoprotein

LPL Lipoprotein lipase

LRP LDL receptor-related protein

LXR-α Liver X receptor

mg Milligram

22


MIXED Linear mixed-effects model

ML Maximum likelihood

mmol/L Millimoles per litre

mRNA Messenger ribonucleic acid

MTP Microsomal triglyceride transfer protein

NATA National Association of Testing Authorities

NCEP National Cholesterol Education Program

NCI Negative ion chemical ionisation

NEFA Nonesterified fatty acids

NHMRC National Health and Medical Research Council

NIDDM Non-insulin dependent diabetes mellitus

NPC1L1 Niemann-Pick C1 Like 1

PAI Plasminogen activator inhibitor

PBMC Peripheral blood mononuclear cells

PLTP Phospholipid transfer protein

PPAR Peroxisome proliferator-activated receptor

PR Production rate

preβ HDL Preβ subfraction of HDL (high density lipoprotein)

PRIME Prospective Epidemiological Study of Myocardial Infraction

PVD Peripheral vascular disease

RAP Receptor associated protein

RCT Reverse cholesterol transport

REML Restricted maximum likelihood

RIO Rimonabant in Obesity

rpm Revolutions per minute

RT Residence time

SAAM II Simulation, Analysis and Modelling Software II

SAE Serious adverse event

SD Standard deviation

SEM Standard error of the mean

4S Scandinavian Simvastatin Survival Study

SR Secretion or synthesis rate

SR-B1 Scavenger Receptor B1

SREBP Sterol regulatory element binding proteins

T4 Thyroxine

23


TC Total cholesterol

TFA Trifluoroacetic acid

TFAA Trifluoroacetic anhydride

TFT Thyroid function test

TG Triglyceride

TNF-α Tumour necrosis factor alpha

TNT Treat to New Targets

TRL Triglyceride-rich lipoprotein

TSH Thyroid stimulating hormone

TZD Thiazolidinedione

UKPDS UK Prospective Diabetes Study

ULN Upper limit of normal

VA-HIT Veterans Administration-High-Density Lipoprotein Intervention Trial

VLDL Very-low-density lipoprotein

WHO World Health Organisation

WOSCOPS West of Scotland Coronary Prevention Study

24

PERSONAL CONTRIBUTION BY THE CANDIDATE

My personal contributions to the thesis are outlined as follows. Contributions of other

individuals and organizations to the thesis are also acknowledged.

Clinical

I had the pleasure of performing the following aspects of the studies:

• Ethics preparation and write-up

• Preparation of Patient Information Sheet and Consent Form

• Clinical Study Protocol Crestor Trial 4522AS/0004

(with Dr Fiona Dunagan and Ms JoAnn Lyons of AstraZeneca Pty Ltd and

Professor Gerald Watts of School of Medicine and Pharmacology, Royal Perth

Hospital)

• Volunteer Recruitment

(with Research nurses, Ms Mary-Ann Powell, Ms Clare Haworth, Ms Sandy

Hamilton, Ms Estelle Zecchin, Ms Michelle Murphy and Professor Gerald

Watts)

• Participated in screening visits

• Participated in stable isotope studies

• Maintained patient records

• Analysis of food and exercise diaries

(with dietician, Ms Nicky Campbell, Ms Clare Jurczyk and Ms Amina

Currimbhoy)

Research nurses, Ms Mary-Ann Powell, Ms Clare Haworth, Ms Sandra Hamilton, Ms

Estelle Zecchin and Ms Michelle Murphy assisted in the recruitment of volunteers, the

measurement of anthropometric parameters (weight, height and waist circumference)

and provided excellent clinical expertise during the stable isotope injection visits. They

also counseled and informed subjects on health and weight reduction. All clinical

examinations, patient reviews and follow-up were performed by Professor Gerald Watts

and Dr Gerard Chew. Associate Professor Frank van Bockxmeer (PathWest Laboratory

Medicine, Royal Perth Hospital) performed the apoE genotyping for the determination

of the apoE2/E2 genotype. The Department of Pharmacy (Royal Perth Hospital)

prepared and dispensed D3-leucine and study medications.

25

Laboratory Analyses

I was responsible in carrying-out the following laboratory procedures:

• Collection and separation of plasma from subjects by centrifugation

• Isolation and storage of buffy coat from subjects

• Isolation of lipoprotein fractions VLDL, IDL, LDL and HDL by

ultracentrifugation (density gradient or use of heparin manganese treated

plasma)

• Delipidation and hydrolysis of apoB-100

• Isolation of HDL apoA-I and apoA-II by polyacrylamide gel and western

blotting

• Hydrolysis of HDL apoA-I and apoA-II

• Extraction of leucine by ion exchange chromatography

• Measuring plasma leucine, apoB-100, apo A-I and apo A-II enrichment with gas

chromatography-mass spectrometry (GCMS)

• Quantification of VLDL, IDL and LDL-apoB using the Lowry Method

• Quantification of LpA-I concentration by differential electroimmunoassay

• Isolation and snap-freezing of streptokinase treated plasma for cholesterol efflux

and preβ-HDL

Mr Kevin Dwyer, with the assistance of Dr Dick Chan, set-up the method for the

measurement of isotopic enrichment by GCMS at the School of Medicine and

Pharmacology, Royal Perth Hospital and verified (QC) all data acquired to ensure

authenticity. Ms Jock Ian Foo and Dr Juying Ji assisted with laboratory procedures of

studies. All biochemical analyses were carried out by NATA accredited laboratories at

the Division of Laboratory Medicine (PathWest Laboratory Medicine, Royal Perth

Hospital). Lipid transfer protein measurements were carried out by Professor Paul

Nestel, Dr Dmitri Sviridov and Ms Anh Hoang at the Baker Heart Research Institute,

Melbourne and Associate Professor Kerry-Anne Rye at the Heart Research Institute,

Sydney. I am familiar with the techniques applied in each these various procedures.

Data collection

Drs Maryam Farvid, Simon Zilko and Michael Allen collected data for the pooled

analyses in Chapter 6 and Appendix 1.

26

Kinetic Analyses

I was responsible for the determination of the kinetics (tracer/tracee ratio, pool size and

production and fractional catabolic rates) for VLDL, IDL and LDL-apoB-100 and

apoA-I in LpA-I and LpA-I:A-II and apoA-II using multicompartmental modeling.

Professor P Hugh R Barrett (School of Medicine and Pharmacology, Royal Perth

Hospital) advised on the modeling of the kinetic data.

Database

I am personally responsible for the management of all laboratory databases. All

databases were password coded and accessible by authorized persons only. The research

nurse attached to the study and I, were responsible for the management of patient

database (personal details and patient records).

Statistical Analyses

I performed all statistical analyses using the statistical package SPSS 12.0 (Chicago,

Illinois, USA). Expert statistical advice and statistical verification (QC) was provided

by Dr Valerie Burke (Senior Research Officer, School of Medicine and Pharmacology,

Royal Perth Hospital). Statistical analyses using SAS (SAS Proc Mixed, SAS Institute)

were carried out by Dr Valerie Burke.

Publications

I conducted literature reviews and drafted all publications presented in this thesis

(Chapters 5, 6, 7, 8 and 9 and Appendix 1 and 5) including write-up, data presentation

of tables and production of figures. Revisions of drafts were performed following

comments and suggestions from co-authors. In the manuscripts included appendices 2, 3

and 4, I contributed to the clinical, laboratory and statistical analyses of the data.

Written declaration to support these contributions can be provided if required.

27

PUBLICATIONS AND COMMUNICATIONS

Publications arising from research conducted for this thesis at the time of submission

include:

Papers

1. Ooi EMM, Watts GF, Farvid MS, Chan DC, Allen MC, Zilko SR, Barrett PHR.

High-Density Lipoprotein Apolipoprotein A-I Kinetics in Obesity. Obesity

Research 2005; 13: 1008 – 1016 (Chapter 6)

2. Ooi EMM, Watts GF, Ji J, Rye KA, Johnson AG, Chan DC, Barrett PHR.

Phospholipid Transfer Protein Activity, a Determinant of HDL Kinetics In Vivo.

Clinical Endocrinology 2006; 65: 752 – 759 (Chapter 7)

3. Ooi EMM, Watts GF, Farvid MS, Chan DC, Allen MC, Zilko SR, Barrett PHR.

High-Density Lipoprotein Apolipoprotein A-I Kinetics: Comparison of

Radioactive and Stable Isotope Studies. European Journal of Clinical

investigation 2006; 36: 626 – 632 (Appendix 1)

4. Ji J, Watts GF, Johnson AG, Chan DC, Ooi EMM, Rye KA, Serone AP, Barrett

PHR. High-Density Lipoprotein (HDL) Transport in the Metabolic Syndrome:

Application of a New Model for HDL Particle Kinetics. Journal of Clinical

Endocrinology and Metabolism 2006; 91: 973 – 979 (Appendix 2)

5. Watts GF, Ji J, Chan DC, Ooi EMM, Johnson AG, Rye KA, Barrett PHR.

Relationships between Plasma Lipids Transfer Proteins and Apolipoprotein B-

100 Kinetics during Fenofibrate Treatment in the Metabolic Syndrome. Clinical

Science 2006; 111: 193 – 199 (Appendix 3)

6. Watts GF, Chan DC, Ooi EMM, Nestel PJ, Beilin LJ and Barrett PHR. Fish oils,

phytosterols and weight loss in the regulation of lipoprotein transport in the

metabolic syndrome: lessons from stable isotope tracer studies. Clinical and

Experimental Pharmacology and Physiology 2006; 33: 877 -882 (Appendix 4)

7. Ooi EMM, Barrett PHR, Chan DC, Nestel PN, Watts GF. Dose-Dependent

Effect of Rosuvastatin on Apolipoprotein B-100 Kinetics in the Metabolic

Syndrome (Chapter 8, accepted for publication in Atherosclerosis)

8. Ooi EMM, Watts GF, Barrett PHR, Chan DC, Ji J, Clifton PM, Nestel PJ.

Dietary Plant Sterols Supplementation Does Not Alter Lipoprotein Kinetics in

28

Men with the Metabolic Syndrome (Appendix 5, accepted for publication in

Asia Pacific Journal of Clinical Nutrition)

Manuscripts submitted

9. Ooi EMM, Watts GF, Nestel PN, Sviridov D, Hoang A, Barrett PHR. Dose-

Dependent Improvement of High Density Lipoprotein Metabolism with

Rosuvastatin in the Metabolic Syndrome (Chapter 9)

Published Abstracts

1. Ooi EMM, Watts GF, Nestel PN, Sviridov D, Barrett PHR. Dose-Dependent

Effect of Rosuvastatin on High Density Lipoprotein Kinetics in the Metabolic

Syndrome (American College of Cardiology 56th Annual Scientific Session,

New Orleans, Louisiana, USA)

2. Ooi EMM, Watts GF, Ji J, Rye KA, Johnson AG, Chan DC, Barrett PHR Role

of Plasma Phospholipid Transfer Protein Activity in Determining HDL Kinetics

In Vivo, XIV International Symposia of Atherosclerosis, Rome Italy, 2006

(Selected for Young Investigators Award)

3. Ooi EMM, Ji J, Watts GF, Rye KA, Johnson AG, Chan DC, Barrett PHR

Balancing Feedback and Lipoprotein Metabolism in the Metabolic Syndrome,

XIV International Symposia of Atherosclerosis, Rome Italy, 2006 (Selected for

Young Investigators Award)

4. Ji J, Watts GF, Johnson AG, Chan DC, Ooi EMM, Rye KA, Barrett PHR. High-

density lipoprotein transport in the metabolic syndrome: application of a new

model for HDL particle kinetics, XIV International Symposia of

Atherosclerosis, Rome Italy, 2006

5. Ooi EMM, Watts GF, Chan DC, Barrett PHR. HDL kinetics in overweight-

obese subjects with stable isotopy: Relative significance of catabolism and

production in determining apolipoprotein AI plasma concentration. Arterio

Thromb Vasc Biol 2005; 25: e74.

6. Ooi EMM, Watts GF, Barrett PHR, Clifton PM, Nestel PJ. Effects of

phytosterols on lipoprotein metabolism in subjects with the metabolic syndrome.

Arterio Thromb Vasc Biol 2005; 25: e75.

7. Ooi EMM, Watts GF, Chan DC, Barrett PHR. HDL kinetics in overweight-

obese subjects with stable isotopy: Relative significance of catabolism and

production in determining apolipoprotein AI plasma concentration.

29

Atherosclerosis Supplement 23-26 April 2005, 6(1S) (Selected for Young

Investigators)


phytosterols on lipoprotein metabolism in subjects with the metabolic syndrome.

Atherosclerosis Supplement 23-26 April 2005, 6(1S)

Conference Abstracts


Effect of Rosuvastatin on Apolipoprotein B Kinetics in the Metabolic

Syndrome, Australian Atherosclerosis Society Meeting, Couran Cove,

Queensland (2006)

2. Ooi EMM, Watts GF, Nestel PN, Sviridov D, Barrett PHR. Dose-Dependent

Effect of Rosuvastatin on High Density Lipoprotein Kinetics in the Metabolic

Syndrome, Australian Atherosclerosis Society Meeting, Couran Cove,

Queensland (2006)


Effect of Rosuvastatin (CRESTOR™) on Apolipoprotein B Kinetics in the

Metabolic Syndrome, School of Medicine and Pharmacology Research

Showcase, University Club (2006)

4. Ooi EMM, Watts GF, Ji J, Rye KA, Johnson AG, Chan DC, Barrett PHR. PLTP

Activity: A determinant of LpA-I Kinetics (Selected for Young Investigators

Competition), Australian Atherosclerosis Society Meeting, Darwin, Northern

Territory (2005)


Phytosterols on Lipoprotein Metabolism in Subjects with the Metabolic

Syndrome, School of Medicine and Pharmacology Research Showcase,

University Club, UWA (2005)

6. Ooi EMM, Watts GF, Chan DC, Barrett PHR. Kinetic Determinants of HDL

Apolipoprotein A-I in Lean and Overweight Subjects: Summary Analysis of

Stable Isotope Studies, Australian Atherosclerosis Society Meeting (2004)


Phytosterols on HDL kinetics in Overweight Subjects, Australian

Atherosclerosis Society Meeting (2004)

30


Phytosterols on HDL Kinetics in Overweight Subjects School of Medicine and

Pharmacology Annual Meeting, QEII, WA (2004)

Communications

1. Dose-Dependent Effect of Rosuvastatin on Apolipoprotein B Kinetics in the

Metabolic Syndrome, Australian Atherosclerosis Society Meeting, Couran

Cove, Queensland (2006) (Winner of the Young Investigator Award)

2. Dose-Dependent Effect of a Novel HMG CoA Reductase Inhibitor on

Apolipoprotein B-100 Kinetics in the Metabolic Syndrome, Clinical Pathology

and Biochemistry PathWest Laboratory Medicine, Royal Perth Hospital (2006)

3. Dose-Dependent Effect of Rosuvastatin (CRESTOR™) on Apolipoprotein B

Kinetics in the Metabolic Syndrome, School of Medicine and Pharmacology

Research Showcase 2006, UWA, Perth, WA Australia (2006)

4. Student perceptions of the effectiveness of writing medical prescriptions using

the national prescribing service case based education package, Teaching &

Learning Forum, UWA, Perth, WA, Australia (2006)

5. Effects of phytosterols on lipoprotein metabolism in subjects with the metabolic

syndrome, Satellite Symposium on Kinetics and Kinetic Modeling of

Lipoprotein, Lipid, and Sterol Metabolism in Systems Ranging from Human

Subjects to Cultured Cells, Washington D.C., USA (2005)

6. PLTP Activity: A determinant of LpA-I Kinetics (Selected for Young

Investigators Competition), Australian Atherosclerosis Society Meeting,

Darwin, NT, Australia (2005)

7. Effects of Phytosterols on HDL Kinetics in Overweight Subjects, Merck Sharp

& Dohme Young Investigators Day, Royal Perth Hospital, WA, Australia (2004)

8. Kinetics of Lipoprotein Metabolism in Chronic Renal Failure, Department of

Nephrology, Royal Perth Hospital, WA, Australia (2003)

Publications arising from work not related to this thesis

1. Ooi EMM, Arena G, Lake F, Joyce DA, Ilett KF. Evaluation of Student

Perceptions of Teaching Medical Prescription Writing Using a Web-based

Education Package.

31

Awards

1. Young Investigator Award, Australian Atherosclerosis Society Meeting, Couran

Cove, Queensland, Australia, 2006

2. Convocation Postgraduate Research Travel Award for 2007, UWA Graduates

Association, 2006

3. Japan Atherosclerosis Society Educational Travel Grant, XIV International

Symposia of Atherosclerosis (ISA), Rome, Italy, 2006

4. Australian Atherosclerosis Travel Grant, XIV International Symposia of

Atherosclerosis (ISA), Rome, Italy, 2006

5. UWA Postgraduate Student Association Conference Travel Award, XIV

International Symposia of Atherosclerosis (ISA), Rome, Italy, 2006

6. Australian Atherosclerosis Society Student Travel Award, Australian

Atherosclerosis Society (AAS) Meeting, Couran Cove, Queensland, Australia,

2006

7. Australian Atherosclerosis Society Student Travel Awards, European

Atherosclerosis Society (EAS), Prague, Czech Republic & 6th Annual

Atherosclerosis Thrombosis and Vascular Biology Meetings (ATVB),

Washington D.C., USA, 2005

8. National Heart Foundation Travel Grant, European Atherosclerosis Society

(EAS), Prague, Czech Republic & 6th Annual Atherosclerosis Thrombosis and

Vascular Biology Meetings (ATVB), Washington D.C., USA, 2005

9. University of Western Australia Graduate Research Student Travel Awards

European Atherosclerosis Society (EAS), Prague, Czech Republic & 6th Annual

Atherosclerosis Thrombosis and Vascular Biology Meetings (ATVB),

Washington D.C., USA, 2005


Atherosclerosis Society (AAS) Meeting, Darwin, NT, Australia, 2005

11. National Heart Foundation Travel Grant Australian Atherosclerosis Society

(AAS) Meeting, Darwin, NT, Australia, 2005


Atherosclerosis Society (AAS) Meeting, Barossa Valley, SA, Australia, 2004

13. National Heart Foundation Travel Grant, Australian Atherosclerosis Society

(AAS) Meeting, Barossa Valley, SA, Australia, 2004

32

ACKNOWLEDGEMENTS

First, I thank my principal supervisor Professor P Hugh R Barrett for his continuous

support in my research. Thank you for encouraging me to ask questions and to express

my ideas; for showing me different ways to approach a problem; for availing time to

instruct, listen and discuss; and for instilling a spirit of excellence towards research and

academia. Your mentorship truly encouraged creativity, built confidence and guided me

to discover how to learn on my own. I would also like to acknowledge your amazing

patience and fortitude in enduring daily “accidental” interruptions from me for the four

long years!

I would also like thank my co-supervisor, Professor Gerald Watts for his significant

contribution to my research. Thank you for emphasizing and imparting the value of

critical thinking, sound rationalization and self-reflection. Your creativity and intellect

continues to inspire learning and research.

I also thank Dr Dick Chan, our senior research fellow, for being an outstanding role

model and providing fantastic support. It is an honor to learn from you. I would also like

to acknowledge Professor Paul Nestel (Baker Heart Research Institute, Melbourne) for

his intellectual input and role in securing a research grant for the Crestor Study.

I am grateful to the nurses, Mary-Ann Powell, Sandy Hamilton, Claire Haworth, Estelle

Zecchin and Michelle Murphy, at the School of Medicine and Pharmacology for their

nursing assistance and Dr Gerard Chew for his clinical expertise.

I would also like to thank the laboratory staff at the Metabolic Research Centre, for their

assistance with laboratory analyses. A special thanks to Ms Jock Ian Foo, Mr Kevin

Dwyer and Dr Juying Ji for their excellent technical advice and constant support. Thank

you also to Dr Valerie Burke for invaluable statistical advice provided.

I wish to thank the Royal Perth Hospital Pharmacy for dispensing the isotope and study

medications, and PathWest Laboratory Medicine for routine analyses and apoE

genotyping.

33

I am grateful for the willing participation of the volunteers who undertook the stable

isotope studies.

I would also like to acknowledge the National Health and Medical Research Council of

Australia, the National Heart Foundation of Australia, the UWA Postgraduate

Association, AstraZeneca Pty. Ltd., the UWA Graduate Association, and the University

of Western Australia for financial support offered. In particular, I would like to

acknowledge Ms JoAnn Lyons from AstraZeneca (Australia), the study monitor

involved in the Crestor intervention study for her proficiency and encouragement.

I thank all my friends for their support, good cheer and constant encouragement.

Friendship adds a brighter radiance to prosperity and lightens the burden of adversity by

dividing and sharing it. A few special thanks: Alastair, the “big brother”, thank you for

tolerating the messy desk next to you the last 3 years; Lydia, my best friend, thank you

for your prayers, laughter and advice; and Doris and Wai, fellow PhD students, partners

in bubble tea and shopping, thank you for cheering me on through every challenge, for

sharing the good times, and laughing hysterically through the down times, and for

believing in me when I doubted myself.

To my family, thank you for your prayers, support, love and understanding; and finally,

to Sheng, thank you for your enduring patience, your steadfast prayers, your daily

encouragement, for all the calm you bring, and for your unconditional love that is all

sustaining. It must have been hard living with a high-strung and loud wife the past year.

Congratulations! You survived part one!

34

CHAPTER 1

Literature Review

35

1.1 OVERVIEW OF THE METABOLIC SYNDROME

1.1.1 Definition of the metabolic syndrome

The metabolic syndrome is characterized by a constellation of abnormalities that

includes glucose intolerance, insulin resistance, obesity, dyslipidemia, and hypertension.

These pathologies contribute to an increased risk of cardiovascular disease (CVD) and

type 2 diabetes. The clustering of metabolic abnormalities was first recognized in the

early 1920s by the Swedish physician Eskil Kylin. He defined this multifactorial disease

to include hypertension, hyperuricemia and hyperglycemia (Kylin 1923). A quarter

century later, Jean Vague, a professor at the University de Marseille established an

association between abdominal obesity with the risk of diabetes and CVD (Vague

1947). In the 1960s, Pietro Avogaro and Gaetano Crepaldi described the metabolic

syndrome as being accompanied by hyperlipidemia due to increased triglycerides (TG),

obesity, diabetes, hypertension, and a high risk of coronary artery disease (CAD)

(Avogaro et al 1965). 1n 1988, Gerald Reaven characterized the lipoprotein

abnormalities associated with the metabolic syndrome, and proposed the new term

“syndrome X”. This was followed by terms including “deadly quartet” (Kaplan 1989)

and “insulin resistance syndrome” (Ferrannini et al 1991). In 1998, the World Health

Organization (WHO) coined the term “metabolic syndrome”, which is the most widely

used description for this metabolic disorder.

The WHO definition of the metabolic syndrome includes insulin resistance, defined by

glucose intolerance, impaired fasting glucose or type 2 diabetes accompanied by at least

two of the following; hypertension, elevated TG, decreased high-density lipoprotein

(HDL) cholesterol, high body mass index (BMI) or microalbuminuria. The United

States National Cholesterol Education Program’s Adult Treatment Panel III (NCEP

ATP III) proposed a slightly different criteria that includes three out of five of the

following risk factors; abdominal obesity, elevated TG, decreased HDL cholesterol,

hypertension and elevated glucose (NCEP ATP III 2002) to define the metabolic

syndrome. The International Diabetes Federation (IDF) has since modified the NCEP

ATP III criteria to include central obesity as the major feature and fulfillment of two of

the other four factors. It also recommends an oral glucose tolerance test when the upper

36

limit of glucose concentration is exceeded and the waist circumference criterion comes

with cutpoints appropriate for different ethnic groups (Table 1.1).

1.1.2 Prevalence of the metabolic syndrome

Comparisons between published data on the prevalence of the metabolic syndrome are

confounded by the use of different definitions. Furthermore, the prevalence of the

metabolic syndrome differs according to age, sex and ethnicity. For example, the

prevalence of the metabolic syndrome is less than 10% for men and women in the 20 –

29 years age group, rising to 38% and 67% in the 60 – 69 years age group, respectively

(Azizi et al 2003). In addition, the National Heath and Nutrition Examination Survey

(NHANES III) reported that the prevalence of the metabolic syndrome increased from

7% in participants aged 20 – 29 years to 44% and 42% in the 60 – 60 years and 70 years

and over groups, respectively (Ford et al 2002).

Large epidemiological surveys have shown that the metabolic syndrome is common.

The NHANES III 1999 – 2002 estimated the age-adjusted prevalence of the metabolic

syndrome in the United States, aged 20 years and over to be between 34.6% (NCEP

ATP III) and 39.1% (IDF) (Ford 2005a). In the Australian population, data from the

AusDiab study between 1999 and 2000 demonstrated that the adjusted estimated

metabolic syndrome prevalence to be between 23.9% (NCEP ATP III) and 26.0%

(WHO) (Adams 2005).

37

Table 1.1 Clinical definitions of the metabolic syndrome

World Health Organization (WHO)

National Cholesterol Education Program (NCEP) (Adult Treatment Panel III)

International Diabetes Federation (IDF)

Criteria required

Hyperglycemia/insulin resistance plus two or more of four other criteria

Three or more of five criteria

Central obesity plus two or more of four other criteria

Central obesity

Waist/hip ratio > 0.90 (men), > 0.85 (women) and/or body mass index > 30 kg/m2

Waist circumference: Caucasian: ≥ 102 cm (men), ≥ 88 cm (women) Asian: ≥ 90 cm (men), ≥ 80 cm (women) Consider lower cut-offs (≥ 94 cm [men], ≥ 80 cm [women]) for some non-Asian adults with strong genetic predisposition to insulin resistance

Waist circumference (ethnic-specific): Europid, Sub-Saharan African, Eastern Mediterranean and Middle Eastern (Arab): ≥ 94 cm (men), ≥ 80 cm (women) South Asian, Chinese, South/Central American: ≥ 90 cm (men), ≥ 80 cm (women) Japanese: ≥ 85 cm (men), ≥ 90 cm (women)

Hyperglycemia Insulin resistance: diabetes, impaired fasting glucose, impaired glucose tolerance or hyperinsulinemic euglycemic clamp glucose uptake in lowest 25% of the population

Fasting plasma glucose level ≥ 5.6 mmol/L or current drug treatment for elevated glucose level

Fasting plasma glucose level ≥ 5.6 mmol/L or previous diagnosis of type 2 diabetes

38

Triglyceride levels ≥ 1.7 mmol/L or current drug treatment for hypertriglyceridemia

Triglyceride levels ≥ 1.7 mmol/L or current drug treatment for hypertriglyceridemia

Dyslipidemia† Triglyceride levels ≥ 1.7 mmol/L and/or HDL-cholesterol level < 0.9 mmol/L (men), < 1.0 mmol/L (women)

HDL-cholesterol level < 1.0 mmol/L (men), < 1.3 mmol/L (women) or current drug treatment for low HDL-cholesterol level

HDL-cholesterol level < 1.0 mmol/L (men), < 1.3 mmol/L (women) or current drug treatment for low HDL-cholesterol level

Elevated blood pressure

Blood pressure ≥ 140/90 mmHg

Blood pressure ≥ 130/85 mmHg, or current drug therapy for known hypertension

Blood pressure ≥ 130/85 mmHg, or current drug therapy for known hypertension

Other Microalbuminuria: urinary albumin excretion rate > 20 μg/min or Urinary albumin/creatinine ratio > 3.5 mg/mmol

• † Elevated triglycerides and low HDL cholesterol are considered separate criteria

in the NCEP and IDF definitions.

39

1.1.3 Components of the metabolic syndrome

The pathogenesis of the metabolic syndrome is still unclear, although it is evidently

multifactorial and influenced by environmental, genetic and epigenetic causes

(Magliano et al 2006). The following section discusses the various components of the

metabolic syndrome.

1.1.3.1 Insulin resistance

Insulin resistance underpins the spectrum of abnormalities of the metabolic syndrome. It

is classically defined as impaired insulin mediated glucose uptake by hepatic and

peripheral tissues (DeFronzo et al 1983). In the early stage of insulin resistance, a

compensatory increase in insulin concentration is observed. As a consequence,

hyperinsulinemia may result in overexpression of insulin action in tissues with normal

or minimally impaired insulin sensitivity. In the insulin resistant state, glucose tolerance

is impaired by several mechanisms including resistance to peripheral glucose uptake

and utilization, impaired hepatic glycogen synthesis, and increased hepatic

gluconeogenesis (Lewis et al 1997). Persistent stimulation of insulin secretion leads to

pancreatic β-cell failure and eventually the development of type 2 diabetes (Reaven

1988). Thus, the accentuation of some insulin actions and resistance to others, give rise

to the diverse clinical manifestations of the insulin resistance syndrome (McFarlane et al

2001).

A major contributor to the development of insulin resistance is free fatty acid (FFA).

FFA are derived from adipose tissue TG stores released via the action of the cyclic

adenosine monophosphate (cAMP) dependent enzyme hormone sensitive lipase (HSL),

and the lipolysis of TG-rich lipoproteins in tissues through the action of lipoprotein

lipase (LPL). Insulin stimulates postprandial uptake of FFA, inhibits HSL and

upregulates LPL. In insulin resistant states, there is an increase in FFA release from

adipose tissue concomitant with a decrease in FFA uptake by muscle tissues (Arner

2002). The vicious cycle may result in attenuated insulin signaling in these tissues,

increased FFA flux to the liver and exacerbation of insulin resistance (Dresner et al

1999, Shulman 1999). In addition, insulin controls the hepatic sterol regulatory element

binding protein (SREBP) expression, the key transcription factor in fatty acid regulation

40

and cholesterol biosynthesis. Chronic hyperinsulinemia may increase hepatic secretion

of SREBP-1c and hepatic lipogenic enzymes, leading to increased lipogenesis and

particle secretion of VLDL.

1.1.3.2 Obesity and insulin resistance

Obesity is a key component of the metabolic syndrome (Kahn and Flier 2000) and a

powerful risk factor for CVD and type 2 diabetes. It is a preventable condition with

multifactorial etiology, and defined as an accumulation of excess fat tissues from an

imbalance in energy intake and expenditure (WHO Expert Committee 2000). Several

methods can be applied to measure obesity including body mass index (BMI), waist

circumference, waist-to-hip ratio, underwater-weighing, bioelectrical impedance, dual

energy e-ray absorptiometry, and magnetic resonance imaging (MRI). The prevalence

of obesity (BMI >30kg/m2) is increasing in both developed and developing nations. In

the United States, the prevalence of obesity is 50.5% (Flegal et al 2002) while in

Australia, the prevalence for men and women is 27% and 39%, respectively (Cameron

et al 2003).

Obesity is strongly associated with insulin resistance, with studies reporting a positive

correlation between body fat mass and fasting or postprandial insulin concentration.

Furthermore, in most obese individuals, whole body insulin sensitivity is reduced

(Tappy et al 1991, Woo et al 2003). In particular, visceral obesity is highly associated

with insulin resistance (Despres and Lemieux 2006). Visceral fat accumulation,

increased expression of adrenergic receptors, increased catecholamine-mediated

lipolysis, and reduced insulin mediated antilypolysis contribute ultimately to increased

release of FFA. Visceral adipose tissues deliver FFA to the liver via the portal vein,

thereby inducing hepatic insulin resistance (Wajchenberg 2000, McGarry 2002).

Adipose tissues also secrete a diverse array of bioactive molecules known as

adipokines, including tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), visfatin,

leptin, adiponectin, and resistin. These adipokines through autocrine, paracrine, or

endocrine mechanisms, regulate energy metabolism in adipose tissue, and may play

important roles in the development of the metabolic syndrome (Ruan & Lodish 2004).

41

1.1.3.3 Dyslipidemia and insulin resistance

Atherogenic dyslipidemia is a cardinal feature of the metabolic syndrome and

characterized by increased fasting plasma TG, reduced HDL cholesterol (in particular

HDL2 cholesterol), elevated apolipoprotein (apo) B levels and the predominance of

small, dense low-density lipoprotein (LDL) particles. Plasma LDL cholesterol is usually

raised. These abnormalities are frequently associated with insulin resistance (Ginsberg

2000).

In insulin resistant states, there is increased release of FFA from peripheral fat tissue

that subsequently stimulates hepatic synthesis of VLDL particles. Insulin resistance also

decreases sensitivity to the inhibitory effects of insulin on apoB synthesis, the main

structural protein of VLDL. The availability of lipid substrates within the endoplasmic

reticulum (ER) lumen further stabilizes newly synthesized apoB that are normally

degraded by proteasomal and non-proteasomal pathways in a lipid poor state

(Sniderman and Cianflone 1993, Yao et al 1997). These, together with the upregulation

of microsomal triglyceride protein (MTP) and SREBP-1c expression contribute to an

enhanced VLDL-TG pool. In the presence an expanded VLDL-TG pool, plasma HDL

cholesterol concentration is decreased, as a result of increased neutral lipid exchange of

cholesterol esters for TG with VLDL, a process facilitated by cholesteryl ester transfer

protein (CETP) (Barter et al 2003a). The consequences of cholesterol ester depletion

and TG enrichment are increased hydrolysis of HDL particles through hepatic lipase

(HL) activity resulting in dissociation of apoA-I from HDL and hypercatabolism of

apoA-I by the kidney, and an increased proportion of small, dense HDL particles

(Barter et al 2003a). By a similar mechanism, LDL particles become TG-enriched and

are subject to further lipolysis by HL to form small, dense LDL particles (Baynes et al

1991). Hence, the dyslipidemia associated with insulin resistance is highly atherogenic

and can contribute to increased CVD risk in subjects with metabolic syndrome (Grundy

2006, Ninomiya et al 2004).

1.1.3.4 Hypertension and insulin resistance

Hypertension occurs in a third of those with metabolic syndrome and present in those

with evidence of insulin resistance (Ferranini et al 1987, Natali et al 1997). Insulin

42

resistance itself has been directly linked with hypertension (Ferranini et al 1987). The

possible mechanisms include impaired response to insulin-mediated vasodilation

(Steinberg et al 1994); impaired endothelial nitric oxide production (Montagnani et al

2002); increased sympathetic nervous system activity (Anderson et al 1991); sodium

retention (Natali et al 1993, Hall 1997); enhanced growth factor production and

activation, leading to proliferation of smooth muscle cells in the vessel wall, and

increased rates of intimal expansion (Trovati and Anfossi 2002); and more recently,

activation of the endothelin system (Sarafidis and Bakris 2006) and elevated

nonesterified free fatty acid (NEFA) concentration (Sarafidis and Bakris 2007).

1.1.3.5 Inflammation, pro-thrombosis and insulin resistance

Inflammation is an important feature of the metabolic syndrome. Evidence suggests that

insulin resistance is associated with chronic subclinical inflammation such as increased

C-reactive protein (CRP) concentration (Haffner 2006). Prospective studies showed that

CRP is also associated with increased risk of developing CVD and type 2 diabetes

(Ridker et al 2003, Santos et al 2005, Soinio et al 2006). Other studies further

demonstrated that elevated CRP is an independent predictor of atherosclerosis and that

other markers of the metabolic syndrome are significant determinants of CRP levels in

this population (Blackburn et al 2001, Linnemann et al 2006). Hence, it was suggested

that CRP should be included in future definitions of the metabolic syndrome, with a

CRP cutoff of 3 mg/L, a value thought to provide further prognostic information (Sattar

et al 2003).

A prothrombotic state is also frequently associated with the metabolic syndrome.

Plasminogen-activator inhibitor 1 (PAI-1) is synthesized in the liver and in adipose

tissues, and regulates thrombus formation by inhibiting the activity of tissue-type

plasminogen activator, an anticlotting factor. Hyperinsulinemia increases PAI-1 and

impairs fibrinolysis in normal human subjects, leading to increased risk of abnormal

coagulation and thrombosis (Calles-Escandon et al 1998). Insulin resistance is also

associated with increased platelet aggregation that may in part explain an altered

intracellular environment with elevated cytosolic Ca2+, enhanced thromboxane A2

synthesis, an increased number and/or function of complexes on platelet membranes,

43

and oxidative stress (Anfossi and Trovati 2006). These aberrations predispose to

atherogenesis and increase CVD risk in the metabolic syndrome.

Figure 1.1 Insulin resistance, the metabolic syndrome, and cardiovascular disease

risk.

Adapted from Avramoglu et al 2006

1.1.4 Significance of the metabolic syndrome

1.1.4.1 Association between the metabolic syndrome and CVD

The metabolic syndrome raises CVD risk at any given LDL cholesterol level. The

NCEP ATP III recognizes the metabolic syndrome as a secondary target, after LDL

cholesterol, for risk-reduction therapy for CVD. Several key studies have examined the

incidences of atherosclerosis and CVD in subjects with or without the metabolic

syndrome.

44

In the Kuopio Ischemic Heart Disease Risk Factor Study in Finland, (11 year follow up,

1209 Finnish men), middle-aged men with the metabolic syndrome as defined by the

WHO and NCEP had increased cardiovascular and overall mortality; this was

independent of the presence of CVD and diabetes at baseline, and also after adjustment

for conventional cardiovascular risk factors including smoking, alcohol consumption,

and serum LDL cholesterol levels (Lakka et al 2002). A larger study involving 4,483

subjects aged 35-70 years (the Botnia Study), showed that the risk for CVD and stroke

was increased three-fold and cardiovascular mortality six-fold in subjects with

metabolic syndrome (Isomaa et al 2001). Italian Bruneck Study, a prospective

population-based survey examining subjects aged 40-79 years, demonstrated that

subjects with the metabolic syndrome, as defined using WHO and NCEP criteria had

increased incidence of CHD during the five-year follow-up study (Bonora et al 2003).

The West of Scotland Coronary Prevention Study (WOSCOPS) reported that men with

four or five features of the metabolic syndrome had a 3.7-fold increase risk of CVD

(Sattar et al 2003). In the Turkish Adult Risk Factor Study (2398 men and women,

follow up three years), the metabolic syndrome was the major determinant of CVD

events, with the relative risk increased by approximately 70% (Onat et al 2002). In a

large multi-ethnic population study in Canada, individuals with metabolic syndrome had

a greater prevalence of CVD compared with those without (Anand et al 2003).

The DECODE Study examined the metabolic syndrome and its association with all-

cause and cardiovascular mortality in nondiabetic European men and women. The study

was based on 11 prospective European cohort studies comprising 6156 men and 5356

women without diabetes, aged 30-89 years, with a median follow up of 8.8 years. The

overall hazard ratios for all-cause and cardiovascular mortality in subjects with the

metabolic syndrome, compared with those without, were 1.44 (95% confidence interval

[CI], 1.17-1.84) and 2.26 (95% CI, 1.61-3.17) in men and 1.38 (95% CI, 1.02-1.87) and

2.78 (95% CI, 1.57-4.94) in women after adjustment for age, blood cholesterol levels,

and smoking. Nondiabetic subjects with the metabolic syndrome have an increased risk

of death from all causes and CVD (Hu et al 2004). The Atherosclerosis Risk in

Communities Study (ARIC) in the United States (14,502 black and white middle-age

subjects) showed that CHD prevalence was 7.4% among those with the metabolic

syndrome (NCEP) compared with 3.6% in control subjects. After adjustment for

established risk factors, subjects who had the metabolic syndrome were two times more

likely to have CHD than were those who did not (McNeill 2004).

45

Other studies that have examined the incidence of atherosclerosis and CVD in subjects

with the metabolic syndrome include the Women’s Health Study (WHS), the 4S Study,

the San Antonio Heart Study, the Air Force/Texas Coronary Atherosclerosis Prevention

Study (AFCAPS/TexCAPS) and the Framingham Offspring Study. As the studies

discussed previously, these studies confirmed that the metabolic syndrome increased

CVD risk and identified of a larger number of subjects at high risk of atherosclerosis

and cardiovascular events independent of cholesterol levels.

1.1.4.2 Association between the metabolic syndrome and diabetes

Several concurrent features of metabolic syndrome, including insulin resistance, are

observed in subjects with impaired glucose tolerance, impaired fasting glucose and type

2 diabetes (Haffner et al 2000). In the metabolic syndrome, insulin resistance leads to a

compensatory increase in insulin secretion; in those with inadequate pancreatic β-cell

insulin response, insulin resistance results in glucose intolerance and subsequently, the

development of type 2 diabetes (Kendall et al 2002). The associations between the

metabolic syndrome and diabetes are well established. In a population based cohort

study, middle-aged Finnish men with the metabolic syndrome, as defined by NCEP and

WHO have five to nine-fold (odds ratios = 5.0-8.8) increased likelihood of developing

diabetes (Laaksonen et al 2002). A summary analysis further reported that the metabolic

syndrome increased the risk of diabetes by 30-52%, which is higher than that for all-

cause mortality (6-7%) and CVD (12-17%) (Ford 2005b).

1.1.5 Prevention and management of the metabolic syndrome

First-line therapies for all lipid and non-lipid risk factors associated with the metabolic

syndrome are therapeutic lifestyle interventions, including weight reduction, increased

physical activity and dietary modification. Pharmacotherapies to improve insulin

sensitivity, dyslipidemia and hypertension are effective second-line strategies.

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1.1.5.1 Therapeutic lifestyle intervention

Therapeutic lifestyle interventions play important roles in managing excess body

weight, insulin resistance, dyslipidemia, hypertension and hyperglycemia seen in the

metabolic syndrome. Two studies reported that weight reduction in obese subjects, with

or without diabetes, is associated with reduced incidence of CVD; in those with IGT, it

is associated with decreased progression to type 2 diabetes mellitus (Tuomilehto et al

2001, Knowler et al 2002). Dietary modification, in particular, restricted carbohydrate

intake may lower blood glucose and TG levels, and increase insulin sensitivity (Volek

and Feinman 2005). Fish consumption, a rich source of n-3 fatty acids, was shown to

effectively raise HDL cholesterol and reduce TG in overweight hypertensive individuals

(Mori et al 2000). Additionally, moderate alcohol intake was associated with increased

HDL cholesterol and reduced CVD risk (Pearson 1996). Available evidence suggests

that increased physical activity is associated with decreased plasma TG, increased HDL

cholesterol levels, increased insulin sensitivity and decreased blood pressure (Carroll

and Dudfield 2004). These lifestyle modifications need to be promoted as first-line

therapies to subjects with the metabolic syndrome. More effort to integrate educational

and lifestyle interventions into the regular care of subjects with the metabolic syndrome

is essential. Success with such interventions will limit the need for pharmacotherapy,

and may provide added benefits if drug therapies are employed.

1.1.5.2 Pharmacotherapy

Pharmacological management currently treats individual components of the metabolic

syndrome including abnormal glucose tolerance, dyslipidemia, hypertension and

thrombosis (Table 1.2). In those with established diabetes or CVD, intensive glycemic

control is associated with reduced risk of microvascular disease, and to a lesser extent,

macrovascular complications (Jenkins et al 2004). Given that insulin resistance is an

etiologic factor, insulin sensitizers such as thiazolidinediones (TZD) and metformin

may be important treatment options (Colca 2006, Bhatia and Viswanathan 2006). These

interventions were associated with greater than 50% reduced risk of diabetes

(Tuomilehto et al 2001, Buchanan et al 2002) and hence, have significant potential to

limit CVD risk in the metabolic syndrome. Furthermore, metformin was shown to

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prevent vascular complications in those with type 2 diabetes (UK prospective Diabetes

Study, UKPDS, 1998).

Although there are no guidelines on the appropriate LDL cholesterol treatment target in

the metabolic syndrome, it is likely that the presence of additional traditional risk

factors (family history or smoking) or newer risk factors (elevated CRP levels), may

represent a CVD risk equivalent. Hence, the target of LDL cholesterol < 2.6 mmol/L

should be recommended and statin therapy is a appropriate choice (NCEP 2001). To

treat the atherogenic dyslipidemia (high TG, low HDL cholesterol), a fibrate or niacin

may be an suitable option. Combination therapy with statin-fibrate, statin-niacin, statin-

ezetimibe or ezetimibe-fibrate may further optimize the lipid profile of subjects with the

metabolic syndrome. In addition, a combination of fibrates with either metformin or

TZD may be beneficial as it would simultaneously address both insulin resistance and

dyslipidemia. Combined α and γ peroxisome proliferator-activated receptor (PPAR)

agonists that can simultaneously improve insulin resistance, glucose intolerance,

elevated TG and low HDL cholesterol levels, are also potential options (Pourcet et al

2006).

As inflammation and pro-thrombosis are major components of the metabolic syndrome,

anti-inflammatory and anti-thrombotic treatments would be beneficial. In particular,

aspirin has anti-inflammatory properties and an established role in preventing

atherothrombotic complications of CHD (Fuster et al 1993, Patrono et al 2005). Low-

dose aspirin administration for the prevention of ischemic events in CHD subjects is

now considered routine practice (Chapman 2006). Combination therapy with statin and

aspirin may be an effective and cost efficient secondary preventative measure to avoid

large numbers of premature deaths and cardiovascular events (Chapman 2006).

Angiotensin Converting Enzyme Inhibitors (ACEI) therapy may also be considered for

metabolic syndrome subjects with hypertension. Both β–blockers and angiotensin-2

receptor blockers can be considered appropriate alternatives to ACEI therapy in high-

risk patients (Komers and Komersova 2000). In addition, insulin sensitizers are also

beneficial for the management of hypertension observed in the metabolic syndrome

(Kurtz 2006).

48

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBG-4KWT6BY-1&_coverDate=09%2F15%2F2006&_alid=511648540&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5142&_sort=d&view=c&_acct=C000028118&_version=1&_urlVersion=0&_userid=554529&md5=9b3f8def7294322307d20cc3e6b93b4f#bib24#bib24http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TBG-4KWT6BY-1&_coverDate=09%2F15%2F2006&_alid=511648540&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5142&_sort=d&view=c&_acct=C000028118&_version=1&_urlVersion=0&_userid=554529&md5=9b3f8def7294322307d20cc3e6b93b4f#bib57#bib57

Table 1.2 Therapeutic interventions in the metabolic syndrome

Lifestyle interventions Weight loss

Physical exercise

Diet modifications in macro- and micro-nutrients

Insulin sensitisers Glitazones/ Thiazolidionediones

Metformines

Lipid modifiers Statins

Fibrates

Niacin

Ezetimibe

Anti-platelet/anti-inflammatory Aspirin

Anti-hypertensive Angiotensin converting enzyme inhibitors

Angiotensin receptor antagonists

Anti-obesity Sibutramine

Orlistat

Rimonabant

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1.2 LIPOPROTEIN METABOLISM

Dyslipidemia is an important feature of the metabolic syndrome. A better understanding

of lipoprotein metabolism may provide insight into the underlying mechanisms that

impact the development of the metabolic syndrome, and hence, improve management of

the syndrome.

1.2.1 Definition and Classification of Lipids

Lipids refer to a heterogeneous group of compounds that have ready solubility in

organic solvents and low solubility in water. Chemically, lipids are noted to be

compounds that yield fatty acids on hydrolysis or complex alcohols that couple with

fatty acids to form esters. The definition may include compounds related closely to fatty

acid derivatives through biosynthetic pathways (e.g. prostanoids, aliphatic ethers or

alcohols) or by their biochemical or functional properties (e.g. cholesterol). Complex

lipids contain additional non-fatty acid groups, which include amino acids, sulphates,

phosphoryl or sialic groups that increase lipid solubility in polar solvents.

The major lipids found in human plasma are triglycerides, phospholipids, fatty acids,

cholesterol, cholesterol esters and glycolipids. They have important physiological roles,

which include energy production, substrate storage and body absorption of fat-soluble

vitamins. Lipids are also major constituents of cells. Many structural components of the

cell membrane are lipids such as phospholipids and cholesterol. In addition, lipids such

as cholesterol are precursors to steroid hormones and bile acids.

1.2.2 Lipoprotein Metabolism

All lipids, with the exception of free fatty acids (FFA) are transported in plasma in the

form of lipoproteins. Lipoproteins are complex macromolecules of lipid and protein. All

lipoproteins consist of a non-polar lipid core, mainly triglyceride (TG) and cholesterol

esters (CE), surrounded by a polar monolayer of phospholipids, heads of free

cholesterol and apolipoproteins, with protruding hydroxide groups. Although they are

structurally similar, lipoproteins differ in the content of their non-polar lipid core, the

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proportion of the lipids within the core, and proteins found on their surface.

Lipoproteins also differ in their metabolic pathway, which is determined in part by the

apolipoproteins embedded in the surface monolayer. These apolipoproteins modulate

the activation of lypolytic enzymes and serve as ligands in receptor-mediated processes

(Kwiterovich 2000). Figure 1.2 illustrates a typical lipoprotein particle with a

hydrophobic core surrounded by a hydrophilic outer shell.

Lipoproteins are classified according to their density, electrophoretic mobility, particle

size, buoyancy (floatation rate) and chemical composition. Of the various means of

classification, electrophoretic mobility and density are the most widely used as the basis

for identification and isolation of lipoproteins. The five major classes of lipoproteins

according to increasing density isolated are: chylomicron (CM), very-low-density

lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein

(LDL) and high-density lipoprotein (HDL). CM are noted to be the largest particles that

contain the highest proportion of TG and lowest proportion of protein, while HDL are

the smallest, have the lowest proportion of lipids and are the most protein dense

lipoproteins.

Figure 1.2 Typical Structure of a lipoprotein particle

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1.2.3 The Lipoproteins

1.2.3.1 Chylomicrons (CM)

Chylomicrons (CM) are TG-rich lipoproteins, ranging between 100 to 1000nm with one

molecule of apoB-48 per particle (Young et al 1990). Following intestinal absorption,

dietary fat and cholesterol are esterified to form TG and CE within the enterocytes.

These lipids are packaged together with apoB-48, phospholipids, free cholesterol, apoE

and apoC to form nascent CM. CM enter the circulation via the thoracic lymph. TG in

CM are hydrolyzed by lipoprotein lipase (LPL), and FFA are subsequently released for

energy production and storage (Mahley et al 1984). As TG are removed, CM become

smaller CM and are removed from the circulation by hepatic receptors including LDL

receptors (LDLR) or the LDL receptor related protein (LRP). This cascade from dietary

lipids to removal of the remnants is known as the exogenous pathway (Figure 1.11).

Abnormalities in CM metabolism have been shown to be associated with increased risk

of coronary disease in subjects with insulin resistance (Ginsberg and Illingworth 2001).

Increased postprandial levels of TG and apoB-48, as well as abnormal retinyl palmitate

dynamics (a measure of CM metabolism), were found to be associated with increased

presence of CAD (Karpe et al 1994, Mero et al 2000). These abnormalities may be

related to decreased hepatic receptor activity and/or decreased LPL activity (Mamo et al

2001, Panarotto et al 2002). The insulin resistant state also results in increased levels of

apoC-III, an inhibitor of LPL, and impaired apoE-mediated receptor uptake of CM and

its remnants. Moreover, increased secretion of cytokines such as TNF-α and IL-6

inhibit LPL activity, this, contributing further to postprandial chylomicronemia (Kern et

al 1995, Yudkin et al 2000). Animal studies also support that the accumulation of CM

remnants in the metabolic syndrome may be due to oversecretion of intestinally derived

apoB-48 containing particles (Haidari et al 2002).

1.2.3.2 Very-low-density lipoprotein (VLDL)

Very-low density lipoprotein (VLDL) accounts for most of the TG in plasma and is an

energy source for extrahepatic tissues. It is synthesized in the liver and structurally

similar to CM. In the endogenous pathway, fatty acids that are returned to the liver from

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CM metabolism are re-esterified to form TG. These TG are then packaged together with

cholesterol, CE, phospholipids, apoB-100, apoC and apoE into nascent VLDL, which

are then secreted into the bloodstream (Bjorkegren et al 1998). Depending upon the

availability of TG, VLDL may differ in size and flotation rate. Compared with large

VLDL (Sf 60 to 400), smaller VLDL particles (Sf 20 to 60) are enriched in CE, depleted

in TG and have a low ratio of apoE and apoC to apoB. Large TG-rich VLDL are

secreted in situations where excess TG are synthesized, such as obesity (Egusa et al

1985). By contrast, small VLDL, and possibly IDL and LDL-like particles, are secreted

when TG availability is reduced (Ginsberg et al 1985).

T

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