Regulating GLUT4 Sorting into the Insulin-Responsive Compartment in Muscle Cells by Kevin Patrick Foley A thesis submitted in conformity with the requirements for the degree of Doctorate of Philosophy in Biochemistry Department of Biochemistry University of Toronto © Copyright by Kevin Patrick Foley 2014 ii Regulating GLUT4 Sorting into the Insulin-Responsive Compartment in Muscle Cells Kevin Patrick Foley Doctorate of Philosophy in Biochemistry Department of Biochemistry University of Toronto 2014 Abstract Skeletal muscle and adipose tissue serve as the major storage sites for glucose, and insulin is the major signal for glucose uptake into these tissues. Glucose transporter 4 (GLUT4) is responsible for the uptake of glucose into muscle and adipose tissues. This protein constitutively recycles between the plasma membrane and intracellular depots. Under resting conditions, most GLUT4 molecules are maintained in intracellular compartments. Insulin shifts this dynamic equilibrium towards the plasma membrane by recruiting a fraction of GLUT4 to the plasma membrane from insulin responsive vesicles. However, steady-state measurements of GLUT4 localization have failed to reveal the subcellular localization of these vesicles or how GLUT4 is sorted to them. By analyzing the sorting of GLUT4 as it internalizes from the cell surface, advances may be made in revealing how GLUT4 acquires insulin-responsiveness and the intracellular location in which this occurs. In L6 myoblasts stably expressing myc-tagged GLUT4, surface-labelled GLUT4myc that internalizes for 30 min accumulates in a Syntaxin-6 (Stx6)-positive perinuclear compartment and displays insulin-responsive exocytosis. Although Stx6 knockdown does not alter the perinuclear accumulation of internalized GLUT4myc, it does inhibit by ~50% the ability of internalized GLUT4myc to undergo insulin-responsive exocytosis. These results suggest that the Stx6-positive perinuclear compartment consists of at least two sub-compartments – one that serves as a dynamic retention compartment and one that is insulin-responsive. Microtubule iii disruption with nocodazole prevents internalized GLUT4myc from reaching the Stx6-positive perinuclear compartment and undergoing insulin-responsive exocytosis. Removing nocodazole allows internalized GLUT4myc to re-acquire insulin-responsive exocytosis in correlation with recovering its accumulation in the Stx6-positive perinuclear compartment. C2-ceramide, which induces insulin resistance, inhibits GLUT4 sorting into the Stx6-positive perinuclear compartment and insulin-responsive exocytosis independently of affecting insulin-stimulated Akt. I propose that internalized GLUT4 must sort through a Stx6-positive compartment that is normally perinuclear as a preamble to acquiring insulin-responsiveness. iv Acknowledgments I can honestly say that pursuing a PhD has been the most challenging undertaking of my life. I also know that, without a doubt, I would not have gotten this far without the tutelage and support of a number of people. First and foremost I would like to thank my supervisor, Dr. Amira Klip. Her guidance has been instrumental in shaping my growth into the scientist I have become. Equally important has been her unwavering support, particularly during my most trying times, which motivated me to push forward and to use those experiences as opportunities to learn instead of as moments to despair. I would also like to thank my supervisory committee members, Dr. David Williams and Dr. Daniela Rotin. Their input and encouragement, and not least of all tremendous patience, has helped guide me to where I am today. I have had the tremendous pleasure of working with some very intelligent, kind, and all around wonderful people during my 6 years in Amira’s lab. Everyone with whom I worked was positive and supportive and provided a great environment in which to pursue science – which was great since I spent a lot of time with them in the lab. I’d like to thank everyone, past and present, from the Klip lab for their friendship and support during my time in the lab. It truly was a better experience because of you all. In particular, Phil was always available for helpful discussions – whether it be to help solve my problem of the day or to distract me from my problem of the day with “hockey talk”. It was especially fun when “hockey talk” proceeded to the pub to watch a game. Zhi was there for help with any technical procedures I needed to learn in the lab and to make sure everything ran smoothly. It was also a great comfort to have my boy Timmy with me to share in this roller coaster of a journey. If it was a rainy day I could always count on him to have an umbrella….in his beverage. Nico later joined the lab and provided a great friendship and somehow always had a ridiculous scientific paper to show me. He also provided one thing that Timmy couldn’t – long blonde hair. Special thanks go to Yi who made sure I slept and stayed healthy and who made me delicious Chinese dumplings to cheer me up when I was working late on seemingly futile experiments. I’d also like to thank all of the other people I was able to work with outside of the Klip Lab. In particular, the Grinstein Lab was always good for a laugh or social outing, not to mention reagents and tremendous scientific advice. Special thanks goes to Jay and Ron who helped me work out some experimental issues that took over a year to decipher. v Finally, I would like to thank my friends and family who supported me during my PhD studies, even when I neglected to call or visit while I hibernated in the lab. My aunt put it best when she said that when I finish each of them will feel their part in this accomplishment, as they have invested much time and effort in supporting me through this process…and this is an understatement as to the encouragement and moral uplifting they were able to provide to me. This is especially true of my “brother from another mother” Rock (Andrew), my Aunt Julie, and lastly (and left to the end on purpose) my parents, Pat and Julie. The love and support my parents have given me during this time and during all of my previous years have been immeasurable and much appreciated. vi Preface The work presented in this Ph.D. thesis is the research conducted from January 2008 to August 2013 under the supervision of Dr. Amira Klip in the Program in Cell Biology, The Hospital for Sick Children, Toronto, ON, Canada. Financial support was provided by grants from the Canadian Institutes of Health Research and Canadian Diabetes Association. Financial stipend to Kevin Patrick Foley was provided by the National Sciences and Engineering Research Council (NSERC), the Banting and Best Diabetes Centre (BBDC), and the Canadian Diabetes Association (CDA). Results previously published in journals are reprinted in this thesis with the copyright permission from the respective journals. vii Table of Contents Contents Acknowledgments .......................................................................................................................... iv Preface ............................................................................................................................................ vi Table of Contents .......................................................................................................................... vii List of Tables ................................................................................................................................ xii List of Figures .............................................................................................................................. xiii List of Appendices ...................................................................................................................... xvii List of Abbreviations ................................................................................................................. xviii Chapter 1 ......................................................................................................................................... 1 1 Introduction ................................................................................................................................ 1 1.1 Regulated intracellular protein transport ............................................................................ 1 1.1.1 Compartmentalization of cellular functions ............................................................ 1 1.1.2 Proteins involved in regulating inter-endomembrane traffic .................................. 3 1.1.3 Retrograde transport pathways ............................................................................... 9 1.2 Glucose Transporter 4 (GLUT4) ...................................................................................... 13 1.2.1 Distribution of GLUT4 in intracellular compartments ......................................... 14 1.2.2 GLUT4 retention ................................................................................................... 18 1.2.3 GLUT4 mobilization in response to insulin .......................................................... 21 1.2.4 Dynamics of GLUT4 sorting ................................................................................ 24 1.2.5 GLUT4 traffic in models of insulin resistance ..................................................... 50 1.3 Rationale and hypotheses .................................................................................................. 51 Chapter 2 ......................................................................................................................................