An investigation of the atherogenicity of intermediate density lipoprotein in people with and without type 2 diabetes mellitus

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University of Wollongong Research Online University of Wollongong Thesis Collection 1954-2016 University of Wollongong Thesis

diabetes mellitus Ching-Loong (Yvonne) Cheung University of Wollongong Recommended Citation Cheung, Ching-Loong (Yvonne), An

of Philosophy thesis, School of Health Sciences, University of Wollongong, 2009. http://ro.uow.edu.

1 University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 2009 An investigation of the atherogenicity of intermediate density lipoprotein in people with and without type 2 diabetes mellitus Ching-Loong (Yvonne) Cheung University of Wollongong Recommended Citation Cheung, Ching-Loong (Yvonne), An investigation of the atherogenicity of intermediate density lipoprotein in people with and without type 2 diabetes mellitus, Doctor of Philosophy thesis, School of Health Sciences, University of Wollongong, Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: research-pubs@uow.edu.au

3 An Investigation of the Atherogenicity of Intermediate Density Lipoprotein in People with and without Type 2 Diabetes Mellitus A thesis submitted in fulfillment of the requirements for the award of the degree of Doctor of Philosophy from UNIVERSITY OF WOLLONGONG by Ching-Loong, Yvonne Cheung (BSc (Hons) UOW) School of Health Sciences University of Wollongong Northfields Ave, NSW March, 2010 i

4 CERTIFICATION I, Ching-Loong, Yvonne Cheung, declare that this thesis, submitted in partial fulfillment of the requirements for the award of Doctor of Philosophy, in the School of Health Sciences, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution. Ching-Loong, Yvonne Cheung Date ii

5 Table of Content Chapter Page Certification Table of content List of tables List of figures List of abbreviation Publications Abstract Acknowledgement 1 Introduction 1 2 Literature review 2.1 Lipids and lipoproteins definition and classification 2.2 The function and role of each lipoprotein class in lipid metabolism 2.3 Definition and physiochemical properties of IDL 2.4 Pathogenesis of atherosclerosis Definition and clinical manifestation Response to injury hypothesis Extracellular matrix Binding of lipoproteins to proteoglycans Site and mechanism responsible for interaction of LDL and proteoglycan Consequences of retention of lipoproteins in ECM Modification of lipoproteins Oxidised lipoprotein Glycated lipoprotein 2.5 The relationship of diabetes with atherosclerosis 2.6 Potential atherogenic factors in diabetes Altered lipoprotein metabolism Enhanced inflammatory response Altered structure of extracellular matrix Advanced glycation product 2.7 Role of intermediate density lipoprotein in atherosclerosis Triglyceride-rich lipoproteins Intermediate density lipoprotein as an atherogenic lipoprotein 2.8 Aim 2.9 Hypothesis 3 General methods 3.1 Materials 3.2 Blood collection and extraction of plasma 3.3 Isolation and preparation of IDL 3.4 Protein analysis of IDL samples 3.5 Separation of two IDL subpopulations by CS affinity chromatography 3.6 Analysis of plasma and IDL samples 3.7 Cell culture ii iii vi vii viii x xi xiv iii

6 3.8 Lipids loading 3.9 Cells lysis 3.10 Sterol extraction and HPLC analysis 4 Proliferation of THP-1 cells in different culture conditions 4.1 Introduction 4.2 Aims 4.3 Methods and materials Materials Method Cell culture Cell growth and survival Statistics 4.4 Results Effects of different serum concentration on THP-1 growth and viability Effects of growing THP-1 monocytes in medium other than RPMI 4.5 Discussion 5 IDL-induced lipid accumulation in macrophages from diabetics and controls 5.1 Introduction 5.2 Aim and hypothesis 5.3 Subjects Samples collection and preparation Loading of samples to macrophages and measurement of intracellular lipid accumulation Statistical analysis 5.4 Results Subject characteristics Intracellular lipid accumulation in cells treated with IDL and its subpopulations Other factors Comparison of the composition among IDL, unbound and bound IDL Compositions of IDL and its subpopulations in T2DM and control subjects Factors that possibly affect the composition of IDL and its sub-populations 5.5 Discussion iv

7 6 Interaction of IDL with extracellular matrix in diabetic and non-diabetic subjects 6.1 Introduction 6.2 Study design Subjects recruitment Samples collection and preparation CS experiment Blood HbA1c, plasma lipids and glucose analysis Protein analysis Composition analysis Statistical analysis 6.3 Results Subjects characteristics and plasma lipids, glucose and HbA1c Affinity of IDL to CS in diabetics and controls Composition of unbound, low-and high-bound fractions Recovery 6.4 Discussion 7 Formation of advanced glycation end products in diabetics and controls 7.1 Introduction 7.2 Aims 7.3 Methods Study design Preparation and isolation of VLDL, IDL and LDL samples Preparation, isolation and detection of CMC and CML in lipoprotein samples Samples Preparation Samples Derivatisation Detection of CMC and CML in lipoprotein samples 7.4 Results Subject characteristics AGE accumulation in controls and diabetics Correlation of AGE to other parameters AGE accumulation in males and females 7.5 Discussion 7.6 Conclusion 8 General Discussion 8.1 Discussion on the overall findings 8.2 Limitations of this thesis Conclusion References v

8 LIST OF TABLES Table Description Page 2.1 Physical properties of and dominant apolipoproteins present in all 5 lipoprotein classes 2.2 Composition of all proteoglycans found in ECM Type of medium, serum and other supplements used in various culture 37 conditions for the growth of THP-1 monocytes 5.1 Subjects characteristics Comparison of lipid and glucose profile between control and diabetic 51 groups 5.3 Total amount of CE accumulated in macrophages after loading the cells 53 with IDL, unbound IDL or bound IDL from control or diabetic group 5.4 A comparison of CE accumulated in macrophages after loading the cells 54 with IDL, unbound IDL or bound IDL from control or diabetic groups 6.1 Subject Characteristics Lipid and glucose profiles of diabetic and control groups Subject Characteristics The profile of lipid, glucose and glycaemic control in control and T2DM 79 groups 7.3 The amount of S-(carboxylmethyl)cysteine (CMC) and N(epsilon)- 79 (carboxylmethyl)lysine (CML) accumulated in all lipoprotein fraction 7.4 Correlation of CMC or CML in each lipoprotein fraction to other 82 demographic or metabolic variables 7.5 The level of CMC and CML accumulated in VLDL, IDL and LDL fraction among males and females 83 vi

9 LIST OF FIGURES Figure Description Page 3.1 Typical HPLC trace a Comparison of the cell growth of THP-1 monocytes grown in RPMI 38 supplemented with 10% FBS (control group), 2% FBS or no serum supplementation 4.1b Comparison of the cell viability of THP-1 monocytes grown in RPMI 39 supplemented with 10% FBS (control group), 2% FBS or no serum supplementation. 4.2a Comparison of the cell growth of THP-1 monocytes grown in RPMI with 39 10% or 2% NBS or no supplementation of serum 4.2b Comparison of the cell viability of THP-1 monocytes grown in RPMI 40 supplemented with 10% FBS (control group), 10% or 2% NBS or no serum supplementation 4.3a Comparison of the cell growth of THP-1 monocytes grown in TMEM with 41 2% FBS or NBS or in the absence of serum to the control condition 4.3b Comparison of the cell viability of THP-1 monocytes grown in TMEM with 41 2% FBS or 2% NBS or in the absence of serum to the control condition 4.4a Comparison of the cell growth of THP-1 monocytes grown in DMEM with 42 10%, or 2% FBS or in the absence of serum to the control condition 4.4b Comparison of the cell viability of THP-1 monocytes grown in DMEM with 43 2% FBS or 2% NBS or in the absence of serum to the control condition 4.5a Comparison of the cell growth of THP-1 monocytes grown in XtenGo(serum 44 free medium) to the control condition 4.5b Comparison of the cell viability of THP-1 monocytes grown in XtenGo the 44 serum free medium to the control condition 5.1 The amount of TG in THP-1 macrophages induced by IDL from control or 52 T2DM (diabetic) group or AcLDL or NL cells 5.2 The amount of TG in THP-1 macrophages induced by unbound IDL from 52 control or T2DM (diabetic) group or AcLDL or NL cells 5.3 The amount of TG in THP-1 macrophages induced by bound IDL from 53 control or T2DM (diabetic) group or AcLDL or NL cells 5.4 Comparison of the composition of IDL, unbound and bound IDL between 55 control and diabetic (T2DM) group 5.5 Comparison of the composition of IDL, unbound and bound IDL between 56 control and diabetic (T2DM) group after adjusting for apo B concentration 6.1 Comparison of the composition of unbound fraction isolated from control and 66 diabetic (T2DM) groups 6.2 Binding pattern of diabetic or control IDL to CS A comparison of the composition of low bound fraction between control and 67 diabetic groups 6.4 A comparison of the composition of high-bound fraction between control and 67 diabetic groups 6.5 Comparison of the composition of unbound IDL, low-bound IDL and high bound IDL 68 vii

10 In alphabetical order Name in Full 3-DG acldl AGE apob apocii apociii apoe ATCC BCA BMI BSA BSE CE CETP CMC CML CS CVD DMEM DS ECM FBS FC FFA GAG HbA1c HDL HDL-C HPLC HS ICAM-1 IDL LDL LDL-R LPDS LPL MCP-1 NBS NEFA PDGF OPA PL PMA SFM T2DM LIST OF ABBREVIATIONS Abbreviated for 3-deoxyglucosone Acetylated LDL Advanced Glycation Endproduct Apolipoprotein B Apolipoprotein CII Apolipoprotein CIII Apoliporotein E American Tissue Culture Collection Bicinchoninic acid Body Mass Index Bovine Serum Albumin Bovine Spongiform Encephalopathy Cholesterol ester cholesterol ester transfer protein S-(carboxymethyl)cysteine carboxylmethyllysine Chondroitin Sulphate Cardiovascular Disease Dulbecco's modified eagle medium Dermatan Sulphate Extracellular Matrix Foetal Bovine Serum Free Cholesterol Free Fatty Acid Glycoaminoglycan Glycated Hemoglobin High Density Lipoprotein HDL-cholesterol High Performance Liquid Chromatography Heparan Sulphate Intracellular Cell Adhesion Molecule Intermediate Density Lipoprotein Low Density Lipoprotein LDL-receptor Lipoprotein-Depleted Serum Lipoprotein Lipase Monocyte Chemoattractant Protein-1 Newborn Bovine Serum Non-Esterifed Fatty Acid Platelet Derived Growth Factor o- phthaldialdehyde Phospholipids Phorbal 12-Myristate, 13-Acetate Serum Free Medium Type 2 diabetes mellitus viii

11 Name in Full TC TG TMEM TRL VLDL WHO Abbreviated for Total Cholesterol Triglyceride Thermo Modified Eagle Medium Triglyceride-rich lipoprotein Very Low Density Lipoprotein World Health Organization ix

12 PUBLICATIONS Conference abstracts Cheung Y.C-L, Meyer B.J. Intermediate density lipoprotein isolated from diabetic or control subjects bound to chondroitin sulphate with similar affinities, Annual meeting of the Australian Atherosclerosis Society in Couran Cove Island, Queensland, 3-6 th of October, 2006 Cheung Y.C-L, Jessup W, Meyer B.J, Incubation of Intermediate density lipoprotein with macrophages induced triglyceride but not cholesterol accumulation in cells. Higher Degree Research Student Conference, University of Wollongong, September, 2005 x

13 ABSTRACT It is well-accepted that people with type 2 diabetes mellitus (T2DM) tend to have more adverse lipid profile than those without the disease. There is a lot of information on the adverse role played by low density lipoprotein (LDL) in atherosclerosis, however, much less is known on the intermediate density lipoprotein although both epidemiological evidence and animal and in vitro studies suggests it plays a role in atherosclerosis as well. Hence this thesis examines and compares the atherogenicity of intermediate density lipoprotein (IDL) between people with an without T2DM. In the first study the ability of IDL isolated from people with-and without- T2DM was investigated and the ability of IDL to induce lipid accumulation was compared. The results show that unlike LDL, IDL could be taken up by the macrophages in its unmodified form and the uptake was not likely to be receptormediated as it is mostly triglyceride (TG) (as opposed to CE) that was accumulated in the cells. It is believed that the triglyceride (TG) in IDL was first hydrolyzed into free fatty acids (FFA) by lipoprotein lipase outside the cells and these FFA were re-esterified to TG once inside the cells. Although there was no difference in the lipid accumulation between the T2DM and control groups, both groups accumulated significant amount of TG in the cells when their IDL was incubated with macrophages in vitro (5830±966 vs 5727±1241, sum of area under curve adjusted for cell protein, p>0.05). On the other hand, only negligible amount of cholesterol ester was accumulated in the cells in both groups and the cholesterol concentrations were similar to the concentration found in the non-loaded cells (21± 5 vs 19±3 vs 16±8, p>0.05). The second study investigated the atherogenicity of IDL by assessing the affinity of IDL to chondroitin sulphate (CS), which is a major proteoglycan found in vessel walls. The results show that IDL isolated from people with and without T2DM both bound to CS at similar affinities (86±0.2 % vs 90±2.0 %) and there were no differences in the composition of the unbound or bound fractions IDL between the two groups. The results also show that IDL bound to CS at two different affinities: lower affinity (lowbound IDL) and higher affinity (high-bound IDL). The high-bound IDL fraction contained significantly lower TG (0.64±0.06 vs 0.39±0.05 mmoll -1 /mg, p<0.01) and phospholipids (0.04±0.01 vs 0.03±0.01 mgdl -1 /mg, p<0.05) than the low-bound IDL fraction but both fractions contained similar concentration of total cholesterol xi

14 (0.57±0.07 vs 0.44±0.07 mmoll -1 /mg, NS). This study have demonstrated that the IDL that bound to CS with a higher affinity was also the one with smaller size compared to the IDL bound to CS with lower affinity. Similar to LDL, IDL consists of different sizes and the smaller IDL particles bound to CS at a higher affinity than the larger IDL particles. In the third study, the concentration of glycation in IDL was assessed to determine the atherogenicity of IDL. The concentration of advanced glycation endproducts (AGE) accumulated in the IDL was also compared in T2DM and control groups. The concentration of two AGE, i.e. N-epsilon- (carboxyethyl)lysine (CML) and S-(carboxymethyl)cysteine (CMC), were measured respectively and the results indicate that the concentration of each was similar in both groups (CML: 0.005±0.003 vs 0.002±0.001 nmol/mg NS; CMC: 1.48±0.14 vs 1.45±0.23 nmol/mg, NS). The results also show that CMC in the IDL fraction was positively correlated with both plasma TG concentration (r=0.39, p=0.04) and body mass index (r=0.46, p=0.02). These findings suggest that the IDL is possibly be more glycated when the risk of diabetes increases. Interestingly the same data show that the CML concentration was similar in the T2DM and control groups. Together, this thesis demonstrated that IDL is atherogenic but the atherogenicity was not increased in the people with diabetes whose blood glucose concentrations were well controlled. xii

15 ACKNOWLEDGEMENT I owe a great debt of thanks to many people during my PhD studies in the University of Wollongong. First of all, I would like to say a huge thank you to my primary supervisor, Associate Professor Barbara Meyer, for her much kind patience and guidance to me throughout my PhD studies. I have to admit that there are times that we do not see things eyes in eyes yet nevertheless I would not forget her much kind understanding and patience in me, and being supportive and encouraging. I would also like to express my thanks to my co-supervisor, Associate Professor Wendy Jessup, for her advice in the results interpretation and my thesis in general. I would also like to take the opportunity to thank all my fellow students and other staff and academics for their lovely companies and support during my 5 years of studies and work in the School of Health Sciences (SHS), which was former known as the Department of Biomedical Science. I appreciate the friendships I received as well as the much laughter we shared there. In particular, I would never forget the high-pitch laughs generated by Gita Rajardo which is as awful yet as much enjoyable to hear. I would also never forget the many lunch-hours that Teresa Du Bois and I shared, lying and having lunch on the lovely lawn near our building. Also, I would also like to thank Todd Mitchell and Theresa Larkin, for both are my idols who I saw many qualities which I could learn from them. Furthermore, I would also not forget Associate Professor Nigel Taylor for his very good sense of humour and Associate Professor Arthur Jenkins for his great advice at all times. In all, SHS is a place full of memories and simply too difficult to forget. There are many others that I need to say thank you for making my PhD studies happen. First of all, I need to say thanks to all the volunteers that participated in my PhD projects. Without them, I would have no sample to collect and hence no results to generate. Thus, although they were not directly involved in my project, they were very important to the integrity of my project. Secondly, I would need to say thanks to all the staff that I worked with in Cardiovascular Research Laboratory in University of New South Wales (UNSW), in particular to Cecilia Sandoval and Dr Carmel Quinn, for without their help in teaching and assisting me in their laboratory I would not be able to complete the project. In fact I need to say thanks to Amanda Lane who also helped me out in completion of some of the lab work involved in this project. Thank you to her for replacing me to travel all the way from Wollongong to Sydney to do the experiment. xiii

16 Furthermore, I would like to thank Dr Bronwyn Brown from Heart Research Institute in University of Sydney (USYD) who is always so kind and helpful, who also taught and assisted me in some of the laboratory work in the glycation part of the project. Last but not least, I would like to give special thanks to both Associate Professor Wendy Jessup from UNSW and Professor Michael Davies from USYD for their collaboration in my project also for giving me the opportunity to see and learn in their laboratories. On the personal side, I have a lot to thank to my parents, especially my mum, for being so loving and supportive to me at all times. Thank you to them for giving me the chance to study in Australia, for otherwise I do not think I will be writing my PhD thesis here right now. I hope you both will be proud of me. Finally, I would also like to thank all my friends for their countless love and support to me throughout all these years. Thank you for being around for me and enduring my rather temperamental temper. All in all, although studying a PhD degree is much-more harder and painful than I could ever imagine, I am glad that I am through. I am thankful to God for giving me this challenging life lesson to me, not later, not before, but right there when it happened. In Him, nothing is impossible. xiv

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