Proteomics of lipid accumulation and DGAT inhibition in HepG2 liver carcinoma cells. By Bhumika Bhatt-Wessel A thesis submitted to Victoria University of Wellington in fulfilment of the requirement for the degree of Doctor of Philosophy In Cell and Molecular Biology. Victoria University of Wellington 2017. i ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is a manifestation of the metabolic syndrome in the liver. It is marked by hepatocyte accumulation of triacylglycerol (TAG) rich lipid droplets. In some patients, the disease progresses to non-alcoholic steatohepatitis (NASH), characterized by cellular damage, inflammation and fibrosis. In some cases, cirrhosis and liver failure may occur. However, the pathogenesis of NAFLD is still unclear. The present project is based on the hypothesis that hepatocytes are equipped with mechanisms that allow them to manage lipid accumulation to a certain extent. Continued or increased lipid accumulation beyond this triggers molecular mechanisms such as oxidative stress, lipid peroxidation and cell death that aggravate the condition and cause disease progression. The aim of this project is to study the effects of lipid accumulation on the cells using proteomics approach to identify proteins involved in the disease progression. A cell culture model was used in the study. HepG2 cells, a human liver carcinoma cell line, were treated with a mixture of fatty acids (FA) to induce lipid accumulation. The lipid accumulation in HepG2 cells was measured with Oil red O assay and the effect of lipid accumulation on the proliferation of the cells was measured using an MTT cell proliferation assay. HepG2 cells treated with 1 mM FA mixture for 6 hours induced lipid accumulation 1.4 times of control with 90% of cell proliferation capacity of the control cells. The final and the only committed step in TAG biosynthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes. To investigate if limiting lipid accumulation in HepG2 cells would prevent molecular mechanisms of pathogenesis, inhibition of DGAT by small molecule inhibitors was performed. Among the three DGAT inhibitors (A922500, PF06424439 and PF04620110) tested, PF04620110 reduced the lipid accumulation to 1.2 fold of the control cells when they were treated with 100 µM of the inhibitor in the presence of 1 mM FA mixture for 6 h. Proteomic analyses were carried out for the control, FA-treated and inhibitor-treated cell groups to identify protein changes in the abundance. Functional analyses of the changed proteins identified suggest that lipid accumulation tends to adversely affect the functioning of the ER and the mitochondria. A complex interplay between the two organelles, possibly mediated by Ca2+ signalling may be vital in ensuring cell survival. PF04620110 was able to counter the FA induced changes in the abundance of some proteins involved in the i metabolic processes but it had limited effect on the ER chaperones whose abundance in the inhibitor-treated sample was comparable to that of the FA-treated sample. These data provided important information for future discoveries of biomarkers and molecular mechanisms involved in the progression of NAFLD. ii ACKNOWLEDGEMENTS The time spent working on this thesis has been a profound lesson in life summed up in three traits I had to use extensively - patience, resilience and fortitude. Patience to wait and not be tired of waiting, resilience in accepting unexpected outcomes and the fortitude to keep on going. I have been supported by the best of people in this journey and tested thoroughly by some. They have all helped me put the best of me in this work. Thank you to my supervisors Dr. Bill Jordan and Prof. John Miller for the encouragement, support and advice – this project would not have been possible without your help. Dr Lifeng Peng, my primary supervisor, has introduced me to proteomics and my work is a reflection of her training; thank you for your critiques and support with the LC-MS/MS that is at the heart of this work. The introduction in this thesis has excerpts from the manuscript of a review Dr. Peng and I have worked on. Her feedback has improved the quality of the work and I hope the review will be published soon. I must acknowledge Dr. Ian Vorster for the help with the MS analysis of A922500 and Dr. Christine Stockum and Sarah Corinder who initiated me into the lab and helped build my skills in Western blotting and tissue culture respectively. Thank you Leticia Castro for keeping a constant vigil against mycoplasma and, along with Remi Schneider, trying your luck at growing HepG2 cells when everything seemed to fail. A special thanks to Sushila Pillai - the confocal microscopy guru who has helped me with the imaging of immunolabelled cells. Thank you Dr. Lisa Woods for your insight into biostatistics. Over the course of this PhD, this beautiful city has become my home and the amazing friends I have made are the best surrogate family I could hope for. Nishad, Karan, Jasmine, Christina, Rose, Rosie, Remi, Dinido and the whole Chiasma crew – you guys are all right! There are perhaps not enough words to express the deep gratitude and love for my partner Ronald Wessel who has been as much part of this PhD as I have. He’s the only bridge engineer I know who can talk about lipid accumulation in the liver like he knows it. At the end of the day, I hope I can make my parents and mota mummy proud and fulfil the aspirations they have for me. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................................. iii LIST OF FIGURES ......................................................................................................................... ix LIST OF TABLES .......................................................................................................................... xii CHAPTER 1: INTRODUCTION ...................................................................................................... 1 1.1. Conception of this project................................................................................................... 1 1.2. Prevalence of non-alcoholic fatty liver disease .................................................................. 1 1.3. Progression from NAFLD to NASH ...................................................................................... 1 1.4. Factors affecting disease progression ................................................................................. 3 1.5. Molecular mechanism of disease progression ................................................................... 4 1.5.1. First hit: Accumulation of triglycerides in lipid droplets .............................................. 4 1.5.2. ER stress ....................................................................................................................... 5 1.5.3. Mitochondrial dysfunction ........................................................................................... 6 1.6. Role of DGATs in triglyceride synthesis .............................................................................. 7 1.6.1. DGAT genes .................................................................................................................. 7 1.6.2. Membrane topology .................................................................................................. 10 1.6.3. Source of substrate .................................................................................................... 11 1.6.4. DGAT Inhibitors .......................................................................................................... 12 1.7. Models used to study NAFLD ............................................................................................ 13 1.7.1. Clinical studies in humans .......................................................................................... 13 1.7.2. Animal models of NAFLD ........................................................................................... 14 1.7.3. Cell-culture based models.......................................................................................... 15 1.8. Proteomics approach to lipid accumulation ..................................................................... 15 1.8.1. Separation of peptides and mass spectrometry ........................................................ 16 1.8.2. Peptide sequencing and identification of proteins .................................................... 18 1.8.3. Quantitative proteomics ............................................................................................ 19 1.9. Aims of the thesis .............................................................................................................. 21 CHAPTER 2: MATERIALS & METHODS ...................................................................................... 22 2.1 Materials ............................................................................................................................ 22 2.2 Cell culture ......................................................................................................................... 23 iv 2.3 Lipid accumulation in HepG2 cells ...................................................................................... 24 2.3.1. Preparation of fatty-acid stock solutions ................................................................... 24 2.3.2. Optimization of treatment conditions to induce lipid accumulation ......................... 24 2.3.3. Quantification