THE LIPIDOMICS OF NON-ALCOHOLIC FATTY LIVER DISEASE

280 Pages·2017·9.28 MB·English

THE LIPIDOMICS OF NON-ALCOHOLIC
FATTY LIVER DISEASE: FROM IN
VITRO TO HUMAN STUDIES

Kang-Yu Peng

Submitted in total fulfilment of the degree of Doctor of Philosophy

May 2017
Department of Biochemistry and Molecular Biology
Faculty of Medicine, Dentistry and Health Sciences
University of Melbourne
and
Metabolomics Laboratory, Baker IDI Heart & Diabetes Institute
ABSTRACT
Non-alcoholic fatty liver disease (NAFLD) has become a major threat to the liver health of
modern humans due to the unhealthy diet and lifestyle. In this thesis, the lipidomic technology
was applied to study the effects of free fatty acid overload on the phospholipidome of rat
hepatocytes, as well as the differences in the liver and plasma lipid profiles of morbidly obese
control (CTRL), steatosis (NAFL) and steatohepatitis (NASH) patients.
In medium supplementation experiments, a combination of oleate:palmitate (2:1) was shown
to be capable of altering hepatocyte phospholipid profiles leading to an overall decrease in the
phosphatidylcholine to phosphatidylethanolamine ratio. This effect was not induced by oleate
or palmitate alone. Flux analysis and modulation of the hepatocyte culture conditions showed
that the increased production of phosphatidylethanolamine induced by oleate:palmitate (2:1)
was primarily attributable to the activation of the CDP-ethanolamine pathway, leading to the
reduced phosphatidylcholine to phosphatidylethanolamine ratio. Cultivation of hepatocytes in
ethanolamine-free media corrected the low phosphatidylcholine to phosphatidylethanolamine
ratio, but not the lipotoxic effect of the free fatty acids.
Hepatic specimens from the NAFL and NASH patients were found to have higher levels of
cholesterol ester, diacylglycerol and triacylglycerol as compared to the control (CTRL) group.
A number of phospholipid species were also present at significantly lower concentrations in
NAFL and NASH patient samples (including species consisting of docohexanoic acid (22:6
fatty acid, DHA)) while levels of several sphingolipid species were elevated under the disease
state. Lipids that were structurally or functionally related to mitochondria, namely acylcarnitine,
cardiolipin and ubiquinone, were all elevated in the NAFL and/or NASH groups. Acylcarnitine
and dihexosylceramide species may thus represent useful biomarkers for identifying NAFL
and NASH. In contrast, analysis of the plasma lipidomics of this patient cohort did not show
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significant differences between NAFLD and control groups. This study provides a detailed
view of the changes in lipid metabolism under NAFLD, and brings new insights into the
pathogenesis and potential therapeutic targets for this disease.

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DECLARATION
This thesis comprises only my original work toward the PhD, except where indicated in the
preface. Acknowledgements have been made in the text to all other materials used. The thesis
is fewer than 100,000 words in length, exclusive of tables, bibliographies and appendices.

Kang-Yu Peng
May, 2017

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PREFACE
The following persons provided technical and/or intellectual inputs into this thesis. I would like
to express my sincere appreciation here.
Associate Professor Peter Meikle and Dr Christopher Barlow helped me design my
experiments and supervised my work. The thesis manuscript has been reviewed by Associate
Professor Peter Meikle prior to submission.
Dr Christopher Barlow helped me set up the LC-mass-spectrometry method for the stable
isotopic flux analysis. Jacqui Weir, Natalie Mellett and Kevin Huynh helped develop the
profiling mass-spectrometry method I used and assisted me in resolving some technical issues
related to the LC-mass spectrometry. The method for oxidised lipid detection was from the
earlier work of Dr Aliki Rasmiena.
Dr Hélène Kammoun trained me to perform the rat hepatocyte isolation and helped me with
the animal ethics application.
Dr Kaushala Jayawardana and Dr Piyushkumar Mundra provided me necessary help and advice
for all the statistical analyses.
Dr Ruth Meex and Professor Matthew Watt provided me the liver and plasma specimens of the
NAFLD patients. Dr Ruth Meex also took part in the sample handling and lipid extraction for
these samples.

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ACKNOWLEDGEMENTS
Finally the long journey has come to a full stop!
When I stepped into the field of lipidomics four years ago, I had very little knowledge about
what it was about. Everything was new to me, including the country Australia. Now I
completed a PhD thesis focusing on the lipidomics of fatty liver disease. I would like to
sincerely thank every individual who has helped me to achieve this.
I must first express my appreciation to my main supervisor Associate Professor Peter Meikle
for his supervision. All these would have not happened without him. Although I have not
always been the most responsible student, he still offered all the possible supports to help me
overcome the obstacles I faced during my PhD. I was lucky to be under his supervision. I would
also like to thank my co-supervisor Dr Christopher Barlow, who taught me so much about
lipidomics, lipid chemistry and how to master Excel. Chris had been a really good mentor for
the first two years of my study.
I also need to thank all the past and current members of the Metabolomics Laboratory at Baker
IDI. They have all been very supportive and always offered me timely help when I needed.
Jacqui Weir, Natalie Mellett and Kevin Huynh who have help me solved lots of mass-
spectrometric, analytical and technical problems throughout my PhD. Dr Kaushala
Jayawardana and Dr Piyush Mundra both have helped me with the statistical analysis. All the
other lab members, including Ricardo Tan, Dr Anmar Anwar, Dr Zahir Alshehry, Dr Michael
Christopher, Sudip Paul, Kevin Gunawan, Michelle Cinel, Dr Aliki Rasmiena, Dr Husna
Begum, Dr Gerard Wong and Dr Yuping Zhou have all given me a hand at certain points of
my PhD. I am really grateful that I have worked in such a great group.
I also received numerous trainings and advice from Dr Hélène Kammoun and Dr Graeme
Lancaster, both work at Baker IDI. They had no obligation to help me when I asked them for
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help, but they were both very willing to share with me their skills and knowledge. Helping
those in need has always been the good culture of Baker IDI and I am extremely fortunate to
work at such an awesome institute. Dr Ruth Meex offered me precious NAFLD specimens and
worked with me on the lipid extraction. I would like to express my appreciation here. I am also
grateful for the help from my committee members Professor Malcolm McConville (also my
co-supervisor) and Associate Professor Heung-Chin Cheng, who both had given me very useful
suggestions over the last four years.
I have made many new friends in this wonderful city of Melbourne and can not thank them all
here. However, I am glad that I befriended Yingying Liu and Pacific Huynh, who both worked
with me at Baker IDI and have chat and shared some good time with me for the past four years.
Yu-Ching Chiang and Li Xiao both played important parts in my life. I would like to thank
them for the great time they spent with me and the unforgettable memories. Jenny Su is a
wonderful girl I met in this great city where I studied. I would like to thank her for the warm
supports at the last stage of my PhD.
Finally, I would like to thank my family, my dear mother, father and brother for their love and
support. I can never achieve this without their support. I am fortunate to have the best family
in the world and I will continue to prosper and make them proud of me in the future.

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PRESENTATIONS
Nov. 2014, 2nd Australian Lipid meeting, University of Wollongong, Wollongong, New
South Wales (oral presentation).
SPEECH TITLE: Tracer Lipidomic Analysis Reveals Alterations in Rodent Hepatocyte
Phosphatidylcholine and Phosphatidylethanolamine Metabolisms After Fatty Acid Treatment.

Dec. 2014, 4th Mini Symposium of Lipids, Deakin University, Melbourne, Victoria (oral
presentation).
SPEECH TITLE: Tracer Lipidomic Analysis Reveals Alterations in Rodent Hepatocyte
Phosphatidylcholine and Phosphatidylethanolamine Metabolisms After Fatty Acid Treatment.

Oct. 2015, Gastro 2015, World Congress of Gastroenterology, Brisbane, Queensland (oral
presentation, Young Investigator Award Finalist).
SPEECH TITLE: Fatty Acid-Induced Hepatocyte Phospholipidome Changes and Their
Potential Roles in NAFLD.

Feb. 2016, APASL 2016, 25th Conference of the Asia Pacific Association for the Study of
the Liver, Tokyo, Japan (poster presentation).
POSTER TITLE: Fatty acid-Overloaded Steatotic Hepatocytes Display Altered
Phospholipidome and Membrane Integrity.

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TABLE OF CONTENTS
Abstract ................................................................................................................................................... ii
Declaration ............................................................................................................................................. iv
Preface .................................................................................................................................................... v
Acknowledgements ................................................................................................................................ vi
Presentations ....................................................................................................................................... viii
List of Tables .........................................................................................................................................xiv
List of Figures ........................................................................................................................................ xv
Abbreviations ....................................................................................................................................... xvii
1. Chapter I. Literature Review ........................................................................................................... 1
1.1. Non-alcoholic fatty liver disease .................................................................................................. 2
1.1.1. Introduction .......................................................................................................................... 2
1.1.2. NAFLD as an emerging global epidemic ................................................................................ 3
1.2. Molecular pathogenesis of NAFLD ............................................................................................... 4
1.2.1. Positive energy imbalance and insulin resistance fuel hepatosteatosis ............................... 4
1.2.2. NAFL progression to NASH: decompensation driven by multiple “hits” .............................. 7
1.2.2.1. Mitochondria and reactive oxygen species (ROS) ......................................................... 8
1.2.2.2. Endoplasmic reticulum stress ........................................................................................ 9
1.2.2.3. Cytokines and adipokines ............................................................................................ 10
1.2.2.4. Gut microbiota ............................................................................................................. 11
1.2.2.5. Revision of the “two hit” model based on emerging evidence ................................... 12
1.3. The role of lipids in NAFLD ......................................................................................................... 13
1.3.1. Free fatty acid ..................................................................................................................... 14
1.3.2. Diacylglycerol ...................................................................................................................... 15
1.3.3. Ceramide ............................................................................................................................. 16
1.3.4. Cholesterol .......................................................................................................................... 17
1.3.5. Phosphatidylcholine and phosphatidylethanolamine ........................................................ 18
1.4. Lipidomics .................................................................................................................................. 22
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1.5. Clinical lipidomics of NAFLD ....................................................................................................... 24
1.5.1. Early NAFLD lipidomics revealed changes in fatty acyl chain composition, lipid oxidation,
and eicosanoids metabolism ........................................................................................................ 25
1.5.2. Oxidative stress among NASH patients and the relevant lipidomic studies ....................... 27
1.5.3. Comparisons of the human studies to the animal models of NAFLD ................................. 28
1.5.4. NAFLD lipidomics: toward biomarker identification and disease monitoring .................... 29
1.5.5. Identification of new genetic polymorphisms predisposing NAFLD ................................... 32
1.5.6. Summary of the NAFLD lipidomic studies ........................................................................... 32
1.6. Hypotheses and Aims ................................................................................................................. 34
2. Chapter II. Materials and General Methods ................................................................................. 40
2.1. Isolation and culture of primary rat hepatocytes ...................................................................... 42
2.2. Preparation of fatty acid and BSA complex and subsequent cell treatment ............................. 43
2.3. Cell death determination ........................................................................................................... 43
2.4. Hepatocyte membrane phosphatidylethanolamine staining .................................................... 44
2.5. Sample preparation for lipid extraction ..................................................................................... 44
2.5.1. Rat hepatocyte cell lysates.................................................................................................. 44
2.5.2. Rat hepatocyte culture media ............................................................................................ 44
2.5.3. Human liver biopsies ........................................................................................................... 45
2.5.4. Plasma ................................................................................................................................. 45
2.5.5. Bicinchoninic acid (BCA) Assay ............................................................................................ 45
2.6. Samples, blanks and quality controls arrangements and general extraction protocol ............. 45
2.7. Lipidomics .................................................................................................................................. 47
2.7.1. Liquid chromatography. ...................................................................................................... 47
2.7.2. Mass spectrometric lipid analysis ....................................................................................... 49
2.7.3. Lipidomic data analysis ....................................................................................................... 50
2.8. Statistical analysis ...................................................................................................................... 50
3. Chapter III. The effect of free fatty acid on the rat hepatocyte phospholipidome: implications for
NAFLD .................................................................................................................................................... 54
3.1. Introduction ............................................................................................................................... 55
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