The impact and regulatory function of Tribbles on metabolic homeostasis By Zabran Ilyas A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Medicine, Dentistry and Health Department of Infection, Immunity & Cardiovascular Disease (IICD) March 2018 i In memory of my beloved Father, Mohammed Ilyas. ‘This PhD is dedicated to my Father, Mohammed Ilyas (May his soul rest in peace), who has been a powerful influence in my life. His discipline, support and love gave me the ability and strength to prosper.’ ii Publications arisen from work presented in this thesis Conference abstracts Ilyas Z , Angyal A, Szilli D, Johnston J and Kiss-Toth E. (2016) MIRNA202 is a Novel Regulator of Tribbles-1 Expression. Heart 101(4) Ilyas Z , Al-Ghamdi J, Tan A and Kiss-Toth E. (2016) Trib3 is a novel regulator of lipid metabolism. Heart 18(3) Angyal A, Ilyas Z, Hadadi E, Johnston J, Ariaans M, Kraus R, Wilson H, Bauer R, Rader D, Francis S and Kiss-Toth E. (2015) Does myeloid expression of TRIB1 regulate plasma lipid levels? Atherosclerosis 241(1), 34 Reviews Johnston J, Basatvat S, Ilyas Z, Francis S & Kiss-Toth E (2015) Tribbles in inflammation. Biochemical Society Transactions , 43(5), 1069-1074 Oral presentations ‘Trib3 a novel regulator of lipid handling’. Tribbles 2nd conference- Beijing ‘miR202 novel regulator of TRIB1’- Singapore iii Abstract Metabolic syndrome significantly increases the risk of developing chronic inflammatory diseases including coronary artery disease, stroke and type 2 diabetes. Obesity is a major risk factor for many facets of metabolic diseases which lead to significant morbidity and mortality worldwide. The family of Tribbles pseudokinases, including Trib3 have been implicated in the development of many chronic inflammatory diseases. Previous human genetic studies have shown a Q84R polymorphism of TRIB3 to be associated with insulin resistance, dyslipidaemia and increased risk of developing diabetes. Furthermore, patients with type 2 diabetes (T2DM) were shown to have elevated levels of TRIB3 in the pancreas compared with healthy patients. TRIB3 was also implicated in the diabetic atherosclerosis process. Silencing Trib3 in apolipoprotein E (apoE)/LDR receptor (LDLR) double-knockout (ApoE–/–/LDLR–/–) mice with diabetes reduced atherosclerotic burden and stabilised the plaque in mice. All the evidence above indicates that Trib3 is an important regulator of insulin resistance and cardiovascular disease. However, the mechanisms of how Trib3 plays a part in chronic inflammatory diseases are unexplored. Development of metabolic syndrome is driven by a combination of different metabolic tissues becoming dysfunctional. While it is clear that the various organs may be affected differently, our insights into the role of Trib3 in various organs remains limited. This study is aimed to use a systemic approach, for the first time on a whole body Trib3ko mice to decipher a role for Trib3 in metabolic dysfunction. Full body Trib3ko mice were created using the gene-trap system. Male Trib3ko mice were obese with elevated plasma levels of HDL and total cholesterol. These knockout mice also displayed a fatty liver phenotype, an increased macrophage influx in the liver and dysregulated proliferation in the adipose tissue. Comparative gene expression microarray analysis was performed on the liver, adipose and muscle tissues from Trib3ko and WT littermates. Gene Ontology and Pathway Analysis were performed using Ingenuity Pathway Analysis software. This analysis revealed multiple metabolic pathways in the liver, adipose and muscle tissues were altered, suggesting a dysregulated inter organ communication underpinning obesity. Key signalling regulators, such as PPARα, CEBPα and Akt were altered in the liver of Trib3ko compared with WT mice as evidenced by qPCR and western blots. Interestingly, a concurrent increase in GLUT2 levels were detected in Trib3ko liver, pointing to a possible cross- regulation between liver lipid and glucose metabolisms via Trib3. Although Trib3 is implicated in lipid metabolism and insulin resistance, the isoform Trib1 has also been described as a regulator of inflammatory signalling and lipid metabolism. A genome-wide association study (GWAS) in a human patient had shown Trib1 to be associated with hyperlipidaemia and increase the risk of developing coronary artery disease (CAD) Trib1 has also shown to be an important negative regulator of inflammation and acts to lower plasma lipid levels. The half-life of Trib1 mRNA is <1 h, so the aim of the second part of the project was to find ways to stabilise Trib1 mRNA levels which would, therefore, have beneficial effects on lipogenesis. The liver is the primary organ to modulate systemic cholesterol levels, so the aim was to stabilise TRIB1 levels in the liver. miRNA202 was shown to be a novel small non-coding RNA which can modulate TRIB1 protein levels, therefore representing a target by which TRIB1 levels could be raised in vivo, providing a mechanism to augment lipid levels and therefore the anti- atherosclerotic effects of this protein. iv Declaration of Contribution I can confirm that majority of the work presented in this thesis is my own. Data interpretation and analysis have been carried out by myself. I would like to acknowledge those people that have contributed their work, who either came before me or during my PhD. Data carried out by others have been acknowledged in the relevant figure legends. I would like first to thank and appreciate Dr.Jihan Al Ghamdi who was a previous PhD student at Endre Kiss-Toth group for providing extensive lipid profile data for the Trib3ko mice vs WT littermates, including the glucose tolerance test and Insulin tolerance test data. Dr Steven Reynolds for the MRI scans of Trib3ko vs WT. Special appreciations go to Dr Li Liang and Tan Ming Jie who helped alongside myself carrying out the microarrays. The actual microarray was carried out at Denova sciences in Singapore. I would like to extend my gratitude to Dr Zoe Bichler for carrying out the metabolic cage experiments, and finally, I would like to thank Chiara Niespolo for the miRNA bioinformatics data, a PhD student in Endre Kiss-Toth group. v Acknowledgement I would like to express my gratitude and appreciation to my supervisors: Professor Endre Kiss-Toth and Professor Tan Nguan Soon who gave me the opportunity to work in their lab and the support and advice to complete my PhD. I am immensely grateful to them for giving me the chance to travel the world for scientific conferences. It has been an amazing experience meeting new people, exchanging ideas and making new scientific discoveries. Again, thank you for all this, I am eternally grateful. I would also like to especially thank Dr Adrienn Angyal and Dr Jessica Johnston, who have helped me in the Sheffield labs and have provided advice and support to me throughout my PhD journey. I am ever so grateful for their help, discussions and feedback. Furthermore, I would extend my wishes and thanks to the Kiss-Toth Group, past and present, whom I had enjoyed working with and had fruitful conversation with, especially at times when experiments were not working. Additionally, I would like to thank my colleagues in Tan Nguan Soon group in the Singapore labs; especially appreciations go to Dr Liang Li, Dr Penchang Chu, Dr Zhen Wei and Tan Ming Jie, who have made my Singapore trip amazing and provided the help and support I required upon my time in Singapore. I will cherish the memories in Singapore, and as promised we shall meet up in Sheffield and re-live the fun times. I would also like to thank Prof. Guillermo Velasco for giving me the opportunity to work in his lab and Dr Maria Lorento who supported me throughout my stay in Madrid. Thank you for showing me the ropes on Western blotting. Finally, I would take this opportunity to thank my family, especially my mum, my brothers, and my wife and son; Rayan, who have been a strong moral support for me when things got difficult. Their endless love and motivation had kept me going. It has not been an easy journey, but was well worth it. Undoubtedly, the journey has not only developed me as a scientist, but also made me stronger as a person. Without the teachings of my father, all this would not be possible; he gave me the ambition and the encouragement to believe that I can achieve anything in life. It was that believe and ambition which resulted in where I am today. vi Content page Abstract ................................................................................................................................................. iv Declaration of Contribution.................................................................................................................. v Acknowledgement ............................................................................................................................... vi Content page ....................................................................................................................................... vii List of Figures ...................................................................................................................................... xi List of Table .......................................................................................................................................... xi Abbreviations ....................................................................................................................................