Elucidating the roles of TCAP-1 on glucose transport and muscle physiology by Yani Chen A thesis submitted in conformity with the requirements for the degree of Masters of Science in Cell and Systems Biology Department of Cell and Systems Biology University of Toronto © Copyright by Yani Chen (2014) Elucidating the roles of TCAP-1 on glucose transport and muscle physiology Yani Chen For the degree of Masters of Science in Cell and Systems Biology (2014) Department of Cell and Systems Biology University of Toronto Abstract Teneurin C-terminal associated peptide (TCAP)-1 is a cleavable bioactive peptide on the carboxy terminus of teneurin proteins. Previous findings indicate that the primary role of TCAP-1 may be to regulate metabolic optimization in the brain by increasing the efficiency of glucose transport and energy utilization. The findings show that TCAP-1 administration in rats results in a 20-30% decrease in plasma glucose levels and an increase in 18F-2-deoxyglucose uptake into the cortex. In vitro, TCAP-1 also induces 3H-deoxyglucose transport into hypothalamic neurons via an insulin-independent manner. This is correlated with an increase in membrane GLUT3 immunoreactivity. A previously deduced pathway by which TCAP-1 signals in vitro was used to establish a link between the MEK-ERK1/2 pathway and glucose uptake as well as a connection between the MEK-ERK1/2 and AMPK pathways. Immunoreactivity studies indicate that the TCAP-1 system exists in muscle and may play a part in skeletal muscle metabolism and physiology. ii Acknowledgements Throughout the duration of my graduate studies, I have been blessed to have experienced so many opportunities to learn, mature as a person, and interact with so many wonderful people. The people I have met along the way have helped shape me into the researcher that I am. Firstly, I am truly grateful to Dr. David A. Lovejoy for the opportunity to complete my graduate studies under his supervision. His guidance, encouragement and patience have been essential to my success in the lab. He has also made my short stay in the lab a joy and has been an inspirational mentor for all of my endeavours in research. I would also like to thank Dr. Leslie Buck and Dr. Marius Locke for serving on my committee and taking time to talk with me on many occasions. I would also like to give a special thanks to all past and present lab members. Thank you to Lifang Song for her patience in teaching me during our time together. Thank you to Mei Xu for providing inspiration and basis for my project; Dr. Dhan Chand and Reuben de Almeida for all their guidance in the laboratory; Louise de Lannoy and Rebecca Woelfle for being great desk buddies; Choden Shrestha for being a great listener and moral supporter; Andrea D’Aquila for her assistance on many experiments and for her friendship. As well, my progress would not have been possible without help and moral support from the numerous undergraduates who have passed through the lab. Thank you to Ivan Gonzales, Mia Husic and Rebecca Crosier for their contributions to the metabolism project; Autumn Otchengco and Michael Colacci for making conferences even more enjoyable; Joseph James Vasquez for always putting a smile on everyone’s face; Alan Hsieh and Adam Hsieh for their assistantships with the in vivo studies; Jon del Castillo, Ola Michalec, and John Alton for their contributions to the lab. I would also like to acknowledge Dr. Dalia Barsyte-Lovejoy for her scientific insight on my project. As well, I give thanks to the Stephen Tobe lab, Les Buck lab, the CSB imaging facility, CBTC, TCP at Mount Sinai and MI in Ann Arbor for allowing me to use their facilities and services throughout my degree. I would like to extend my gratitude to Peggy Salmon and Ian Buglass for the wonderful T.A. experience that has allowed me to teach so many gifted students. As well, thank you to the members of the CSB Graduate Union for making my time serving the council such a pleasure. Lastly, I would like to thank the two people who brought me into this world; my mom and my dad for their unconditional love and support. I love you both and I will make you two proud. iii Table of Contents Abstract…………………………………………………………………………………………..ii Acknowledgements……………………………………………………………………………...iii Table of Contents………………………………………………………………………………..iv List of Abbreviations…………………………………………………………………………..viii List of Figures and Tables……………………………………………………………………...xii 1 Chapter One: Introduction………………………………………………………………….1 1.1 Peptides and signalling systems........................................................................................1 1.2 Discovery and functions of the Teneurin proteins..........................................................2 1.3 Evolution and structure of the Teneurin C-terminal-Associated Peptide (TCAP) family...................................................................................................................................3 1.4 TCAP-1 modulation of behaviour and neuroprotective actions....................................9 1.5 Central regulation of energy metabolism......................................................................11 1.6 Glucose transport and metabolism in the brain...........................................................13 1.7 Glucose metabolism in muscle........................................................................................18 1.8 The dystroglycans and the neuromuscular junction....................................................20 1.9 Thesis rationale and experimental design.....................................................................22 1.10 References.........................................................................................................................25 2 Chapter Two: Materials and Methods…………………………………………………….34 2.1 Objective #1: The roles of TCAP-1 on plasma glucose, muscle physiology and metabolic hormone levels in rats………………………………………………………34 2.1.1 Synthesis of mouse TCAP-1…………………………………………………..….34 2.1.2 In vivo TCAP-1 administration…………………………………………………..34 2.1.3 Glucose level determination……………………………………………………..35 2.1.4 Serum processing and haematology analysis………………………………….…35 2.1.5 Muscle and liver tissue processing and glycogen assays……………………..…36 2.1.6 Functional Positron Emission Tomography (fPET) ……………………………36 iv 2.2 Objective #2: The roles of TCAP-1 on the cellular glucose transport profiles in mHypoE-38 immortalized hypothalamic neurons and C2C12 myocytes….…………37 2.2.1 Culturing of the mHypoE-38 (N38) cell line …………………………………….37 2.2.2 2-Deoxy-D-Glucose Uptake Assays in mHypoE-38 cells………………………..37 2.2.3 Culturing of the C2C12 cell line…………………………………………………..38 2.2.4 2-Deoxy-D-Glucose Uptake Assays in C2C12 myocytes……………………….…39 2.2.5 Lactate assays…………………………………………………………………....40 2.2.6 Pyruvate assays……………………………………………………………….....40 2.2.7 Western blot analysis………………………………………………………….…41 2.3 Objective #3: The roles of TCAP-1 on the cellular glucose transport profiles in mHypoE-38 immortalized hypothalamic neurons and C2C12 myocytes….…………42 2.3.1 Sectioning of muscle tissue………………………………………………………42 2.3.2 FITC-TCAP-1 binding studies…………………………………………………..42 2.3.3 TCAP-1 antisera production……………………………………………………..43 2.3.4 Immunoreactivity studies………………………………………………………...44 2.3.5 Statistical analysis……………………………………………………………….44 3 Chapter Three: Results…………………………………………………………………….45 3.1 Objective #1: The roles of TCAP-1 on plasma glucose, muscle physiology and metabolic hormone levels in rats………………………………………………………45 3.1.1 The effect of acute TCAP-1 administration in adult Wistar rats on blood glucose…………………………………………………………………….45 3.1.2 The effect of acute TCAP-1 administration on glycogen storage in adult male Wistar rats…..........................................................................................................47 3.1.3 Functional Positron Emission Tomography (fPET) studies: The effect of acute TCAP-1 administration on 18F-2-deoxyglucose uptake in adult Wistar rats….....49 3.2 Objective #2: Effects of TCAP-1 on glucose transport and metabolic parameters in vitro………………………………………………………………………………………53 3.2.1 Effect of TCAP-1 on 3H-2-deoxyglucose uptake into mHypoE-38 immortalized embryonic hypothalamic neurons .……………………………………………....53 3 3.2.2 Effect of TCAP-1 on H-2-deoxyglucose uptake into C2C12 myocytes…………...56 3.2.3 Effect of TCAP-1 on membrane GLUT3 levels in mHypoE-38 immortalized embryonic hypothalamic neurons…......................................................................57 v 3.2.4 Effect of TCAP-1 treatments on 0 nM (vehicle) and 100 nM on intracellular pyruvate concentrations in mHypoE-38 immortalized embryonic hypothalamic neurons .................................................................................................................59 3.2.5 Effect of TCAP-1 treatments on 0 nM (vehicle) and 100 nM on intracellular lactate concentrations in mHypoE-38 immortalized embryonic hypothalamic neurons .................................................................................................................60 3.2.6 Effect of MEK inhibition on TCAP-1-induced increases on 3H-2-deoxyglucose uptake in mHypoE-38 immortalized embryonic hypothalamic neurons…............61 3.2.7 Effect of TCAP-1 treatments of 0 nM (vehicle) and 1 nM on MEK-dependent AMPK phosphorylation in mHypoE-38 immortalized embryonic hypothalamic neurons…...............................................................................................................62 3.3 Objective #3: Detecting the presence and