An In Vivo Screen for Genes Involved in Central Nervous System Control of Obesity Identifies Diacylglycerol Kinase as a Regulator of Insulin Secretion by Irene Trinh A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Irene Trinh, 2017. An In Vivo Screen for Genes Involved in Central Nervous System Control of Obesity Identifies Diacylglycerol Kinase as a Regulator of Insulin Secretion Irene Trinh Doctor of Philosophy Department of Molecular Genetics University of Toronto 2017 Abstract The increasing prevalence of obesity as well as its association with many chronic diseases has turned obesity into a major health concern worldwide. Obesity has many underlying environmental and genetic factors that disturb the balance between energy intake and energy expenditure, which is controlled by the central nervous system. The goal of my project is to help further our understanding of these CNS mechanisms by using the powerful tools available in Drosophila melanogaster to identify neuronal genes involved in energy homeostasis. Using the neuronal fru-Gal4 driver we performed an RNAi screen with 1748 genes and assayed for obese or lean phenotypes defined as increases or decreases in triacylglycerol (TAG) levels. After 3 rounds of screening I identified 25 hits that were reproducible and confirmed these phenotypes with independent RNAi lines. ii One of these hits was Diacylglycerol kinase (Dgk) whose mammalian homologues have been implicated in genome-wide association studies for metabolic defects. Manipulation of neuronal Dgk levels affects TAG and carbohydrate levels but these effects don’t seem to be mediated through Dgk’s regulation of DAG, PA levels or PKC activity. I hypothesized that Dgk acts in the insulin-producing cells (IPCs), a set of neurosecretory neurons that secrete Drosophila insulin-like peptides (dILPs) and are involved in the regulation of dILP secretion. Knockdown or overexpression of Dgk within the IPCs reproduces the same TAG, glucose and glycogen phenotypes seen with fru-Gal4. Moreover, overexpression of kinase-dead Dgk, but not wild- type, decreased circulating dILP2 and dILP5 levels resulting in lower insulin signalling activity. Conversely, despite having higher circulating dILP levels, Dgk RNAi flies have decreased pathway activity suggesting that they are insulin-resistant. Thus, after screening over 1700 genes in vivo I identified 25 genes as potential neuronal mediators of energy homeostasis. My results have also shown that one of these genes, Dgk, acts within the insulin-producing cells to regulate the secretion of dILPs and energy homeostasis in Drosophila. iii Acknowledgments This thesis has been a long time coming and wouldn’t have been possible without the people around me. First, I would like to thank Gabrielle for allowing me the opportunity to work in her lab. I believe that the environment here has helped shape me into a better scientist and critical thinker. I also want to thank her for the continuous guidance and support through the many ups and downs over the years that helped me to see this project through. I am grateful to my committee members Sabine Cordes and Helen McNeill for their helpful feedback through this whole process and for their support to the end (even reassuring me just before my defense). Thank you to all current and past members of the lab. I especially need to thank Oxana who, without her help, I would still not be done my thesis work and without her presence there, the lab would have been a less cheerful place. Thank you to Maeve, Tanya, Brenda, Dave and Mike who have offered help and advice even when my questions were less than intelligent. I also want to thank Alessandra and Sili who provided a sympathetic ear or a welcome distraction whenever I needed it. Lastly, I want to thank my family for their unconditional support and encouragement throughout grad school (even though they still don’t really understand what I’ve been studying all these years). iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................. ix List of Figures ..................................................................................................................................x List of Appendices ........................................................................................................................ xii List of Abbreviations ................................................................................................................... xiii 1 Introduction .................................................................................................................................1 1.1 Global obesity epidemic ......................................................................................................2 1.2 Etiology of obesity and metabolic disorders ........................................................................2 1.2.1 Genetics of human obesity .......................................................................................3 1.2.2 Approaches to identify obesity susceptibility genes ................................................3 1.3 Drosophila models of human diseases ................................................................................5 1.3.1 Drosophila models of metabolic disorders ..............................................................5 1.3.2 Utility of Drosophila in the study of energy homeostasis .......................................7 1.4 The central nervous system in energy homeostasis .............................................................9 1.4.1 The CNS in mammalian energy homeostasis ........................................................10 1.4.2 The CNS in Drosophila energy homeostasis .........................................................12 1.5 Insulin signalling in energy homeostasis ...........................................................................18 1.5.1 Insulin signalling pathway .....................................................................................18 1.5.2 Insulin functions in mammalian energy homeostasis ............................................21 1.5.3 Drosophila ILP signalling functions in energy homeostasis..................................23 1.5.4 Regulation of insulin secretion ..............................................................................25 1.6 Project rationale .................................................................................................................30 v 2 RNAi Screen to Identify Genes Involved in Central Nervous System Control of Energy Homeostasis ..............................................................................................................................32 2.1 Introduction ........................................................................................................................33 2.2 Materials and Methods .......................................................................................................33 2.2.1 Fly stocks and husbandry .......................................................................................33 2.2.2 TAG assay ..............................................................................................................34 2.2.3 Analysis of enriched annotation terms among screen hits .....................................34 2.2.4 Analysis of screen hits for homologues involved in human obesity .....................34 2.3 Results ................................................................................................................................34 2.3.1 Screen methodology and selection of a driver .......................................................34 2.3.2 First round of screening identifies 510 genes that have statistically significant changes in TAG when knocked down with fru-Gal4 ............................................36 2.3.3 Analysis of hits after first round of screen .............................................................38 2.3.4 Confirmation and validation of TAG phenotypes .................................................41 2.4 Discussion ..........................................................................................................................43 2.4.1 Use of fru-Gal4 as the driver for RNAi screen ......................................................44 2.4.2 Phospholipid metabolism and protein kinases in energy balance ..........................44 2.4.3 Identification of known metabolic genes by RNAi screen ....................................45 2.4.4 Conclusions ............................................................................................................46 3 Elucidating the Role of Diacylglycerol Kinase in Drosophila Energy Homeostasis ...............47 3.1 Introduction ........................................................................................................................48 3.1.1 The DGK gene family ............................................................................................49 3.1.2 DGK functions in metabolism ...............................................................................51