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UC San Diego UC San Diego Electronic Theses and Dissertations Title Exploring the role of Urocortin 2 and Urocortin 3 in energy metabolism Permalink https://escholarship.org/uc/item/4cj0p89r Author Falkenhagen, Katherine M. Publication Date 2012 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Exploring the Role of Urocortin 2 and Urocortin 3 in Energy Metabolism A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences by Katherine M. Falkenhagen Committee in charge: Professor Richard Lieber, Chair Professor Ronald Evans Professor Mark Geyer Professor Jerrold Olefsky Professor Simon Schenk Professor Nicolas Webster 2012 Copyright Katherine M. Falkenhagen, 2012 All rights reserved The dissertation of Katherine M. Falkenhagen is approved, and it is acceptable in quality and form for publication on microfilm and electronically: Chair University of California, San Diego 2012 iii Dedication This dissertation is dedicated to the memory of Dr. Wylie Walker Vale. iv Table of Contents Signature Page…………….………………………………………………………………....iii Dedication.……………………………………………………………………………………iv Table of Contents..……………………………………………………………………………v List of Abbreviations………………………………………………………………………..viii List of Figures…………………………………………………………………………………x List of Tables…………………………………………………………………………………xii Acknowledgements…………………………………………………………………...…….xiii Vita……………………………………………………………………………………………xv Abstract of the Dissertation………………………………………………………………..xvi Chapter 1: General Introduction…………………………………………………………….1 Dissertation Overview……………………………………………………………….2 Discovery of the CRF family of peptides and receptors…………………………3 Parameters of energy homeostasis………………………………………………..7 Skeletal muscle and energy homeostasis……………………………………….10 Objective…………………………………………………………………………….13 v Significance…………………………………………………………………………15 References………………………………………………………………………….16 Chapter 2: Metabolic characterization of Ucn 2/Ucn 3 double knockout mice…...…..21 Abstract……………………………………………………………………………...22 Introduction………………………………………………………………………….23 Methods……………………………………………………………………………..25 Results………………………………………………………………………………28 Discussion…………………………………………………………………………..31 Figures………………………………………………………………………………35 References………………………………………………………………………….39 Chapter 3: Effect of Ucn 2 deficiency on skeletal muscle fatigability and mitochondrial biogenesis in mice…………………………………………………………………………..42 Abstract……………………………………………………………………………...43 Introduction………………………………………………………………………….44 Methods……………………………………………………………………………..46 Results………………………………………………………………………………52 Discussion…………………………………………………………………………..55 vi Figures………………………………………………………………………………57 Supplemental Figure…………………………………………………………….....63 References………………………………………………………………………….64 Chapter 4: Ucn 2 mRNA expression in mouse skeletal muscle.………………………66 Abstract……………………………………………………………………………...67 Introduction………………………………………………………………………….68 Methods……………………………………………………………………………..70 Results………………………………………………………………………………75 Discussion…………………………………………………………………………..77 Figures………………………………………………………………………………80 Supplemental Figures……………………………………………………………...84 . References………………………………………………………………………….86 Chapter 5: General Discussion...………………………………………………………….88 References………………………………………………………………………….95 vii List of Abbreviations ACTH adrenocorticotropic hormone AICAR 5-Aminoimidazole-4-carboxyamide ribonucleoside Akt protein kinase B AMPK AMP-activated protein kinase ATP adenosine-5'-triphosphate cAMP cyclic adenosine monophsophate cDNA complementary deoxyribonucleic acid CPT1 carnitine palmitoyltransferase I CRF corticotropin releasing factor CRF-BP corticotropin releasing factor binding protein CRFR corticotropin releasing factor receptor CVS chronic variable stress dKO double knockout EDL extensor digitorum longus Epac cAMP-regulated guanine nucleotide exchange factor Erk1/2 extracellular-signal-regulated kinase 1/2 GLUT-4 glucose transporter 4 GPCR G-protein coupled receptor GTT glucose tolerance test HFD high fat diet HPA hypothalamic-pituitary-adrenal i.p. intraperitoneal ITT insulin tolerance test LFD low fat diet MAPK mitogen-activated kinase MHC myosin heavy chain mRNA messenger ribonucleic acid mtTFA mitochondrial transcriptional factor A NRF-1 nuclear respiratory factor 1 NRF-2 nuclear respiratory factor 2 viii OXPHOS oxidative phosphorylation system PDK4 pyruvate dehydrogenase lipoamide kinase isozyme 4 PGC-1α peroxisome proliferator-activated receptor gamma coactivator 1α SDH succinate dehydrogenase TA tibialis anterior Ucn urocortin WT wild type ix List of Figures Figure 2-1. Weight gain and food intake of Ucn 2/Ucn 3 dKO mice and WT controls on LFD or HFD………………………………………………………………………………35 Figure 2-2. Fasting glucose and insulin levels of Ucn 2/Ucn 3 dKO mice compared to WT controls………………………………………………………………………………36 Figure 2-3. Glucose homeostasis phenotypes of Ucn 2/Ucn3 dKO mice……………37 Figure 2-4. Lipid profiles of Ucn 2/Ucn 3 dKO mice after overnight fast…………..….38 Figure 3-1. Fatigue test of isolated EDL muscle from Ucn 2 KO mice and WT controls……………………………………………………………………………………….57 Figure 3-2. Nuclear gene expression of mitochondrial-related genes in Ucn 2 KO skeletal muscle relative to expression in WT controls…………………………………..58 Figure 3-3. Fiber type composition of Ucn 2 KO skeletal muscle and WT controls….59 Figure 3-4. Skeletal muscle mitochondrial density in Ucn 2 KO mice and WT controls……………………………………………………………………………………….60 Figure 3-5. Mitochondrial activity of Ucn 2 KO skeletal muscle and WT controls……61 Figure 3-6. Gene expression of PGC-1α and PDK4 in skeletal muscle of exercised mice relative to control……………………………………………………………………...62 Supplemental Figure 3-1. Body composition after 21-day voluntary exercise……….63 Figure 4-1. Effect of nutritional status on Ucn 2 mRNA expression in mouse skeletal muscle………………………………………………………………………………………..80 x Figure 4-2. Effect of hypoxia on Ucn 2 mRNA expression in mouse skeletal muscle………………………………………………………………………………………..81 Figure 4-3. Effect of exercise on Ucn 2 mRNA expression in mouse skeletal muscle………………………………………………………………………………………..82 Figure 4-4. Effect of AICAR on Ucn 2 mRNA expression in C2C12 myotubes and mouse skeletal muscle……………………………………………………………………..84 Supplemental Figure 4-1. Body weight and body composition of mice after HFD......85 Supplemental Figure 4-2. Blood glucose levels of fasted mice……..……………..…..84 xi List of Tables Table 5-1. Summary of metabolic phenotypes of genetically manipulated CRF family animal models……………………………………………………………………………….93 Table 5-2. Summary of metabolic phenotypes of genetically manipulated CRF family animal models after HFD…………………………………………………………………..94 xii Acknowledgements I am extremely grateful for all the people that have contributed to this dissertation in one way or another. I cannot possibly thank everyone, but I would like to acknowledge the people that have been most influential during my time as a graduate student. Firstly, I thank all past and current members of the Vale Lab. I especially thank Alissa Blackler, Anna Pilbrow, Cindy Donaldson, Elizabeth Flandreau, Eran Gershon, Ezra Wiater, Hannah Park, Joan Vaughan, Jonathon Kelber, Kathy Lewis, Louise Bilezikjan, Lykke “Li” Blaabjerg, Marilyn Perrin, Mark Huising Nick Justice, Peter Grey, Remy Manuel, Sandra Guerra, and Talitha van der Meulen. Each of you have helped me in some way, whether that be teaching me a technique, assisting me in an experiment, sharing reagents, providing feedback, or simply making me laugh. Thank you PBL-V. I have been very fortunate for all the collaborators who made the science in this dissertation possible. In the Montminy lab, I thank Naomi Goebel (metabolic cages), Jose Paz (Seahorse assays), Judith Altarejos (fat pads, lipid levels), Nina Miller (muscle homogenization), and Sam Van de Velde (islet isolation). I thank Shannon Bremner (SDH assay, fiber typing) and Caryn Urbanczyk (fatigue tests) of the Lieber lab. In the Evans Lab, I thank Weiwei Fan (mitochondrial copy number), Vihang Narkar (mitochondrial primers), and Michael Downes (AICAR). I thank the Powell lab for assistance with the hypoxic muscle studies. Finally, big thanks to the Schenk lab for numerous techniques including muscle extraction and xiii homogenization, glucose uptake assays, use of treadmills, citrate synthase assays and everything else. Of course I thank my committee members, Dr. Rick Lieber, Dr. Ron Evans, Dr. Mark Geyer, Dr. Jerrold Olefsky, Dr. Simon Schenk and Dr. Nick Webster for their guidance and wisdom over the past 4 years. It has been a pleasure working with you all. A special thanks goes out to Dr. Lieber for “adopting” me during that difficult time that was January 2012. Also, I am deeply grateful to Dr. Simon Schenk for his huge contributions to my work. Thank you for your endless support, for welcoming me into your lab, for teaching me so many techniques and for being so generous with your time. I cannot express my gratitude enough. Also, I send a special thanks halfway across the world to Dr. Alon Chen. Thank you so much for everything you have done to make this dissertation possible. While at the Salk Institute I received support in part from the National Institute of Diabetes and Digestive and Kidney Diseases Program Project Grant DK026741- 30-32, the National Skeletal Muscle Rehabilitation Research Center R24 HD050837- 04 (UCSD), the Elizabeth Keadle Fund, the Helen McLoraine Chair and the H.A. and