UNIVERSITY OF CALIFORNIA, SAN DIEGO Regulation of Gene Expression by Peroxisome Proliferator Activated Receptors A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Isaac R. Mehl Committee in charge: Professor Marc R. Montminy; Chair Professor Ronald M. Evans Professor James R. Feramisco Professor Amy E. Pasquinelli Professor Bing Ren 2007 Copyright Isaac R. Mehl, 2007 All rights reserved. The dissertation of Isaac R. Mehl is approved, and it is acceptable in quality and form for publication on microfilm: ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ ____________________________________________ Chair University of California, San Diego 2007 iii Dedicated to my best friend Dinh Dinh Diep Without you this work would not be possible iv TABLE OF CONTENTS Signature Page ……………………………………………………………….. iii Dedication ……………………………………………………………………. iv Table of Contents …………………………………………………………….. v List of Figures and Tables ……………………………………………………. vi Acknowledgements …………………………………………………………... viii Curriculum Vitae……………………………………………………………… xi Abstract ………………………………………………………………………. xii I. Introduction …………………………………………………………………... 1 II. Adipose tissue is responsible for the HDL raising effect of PPARδ ligands………………………………………….…………………………………. 19 III. The role of PPARα and PPARδ in caloric restriction……..………..………. 36 IV. Conclusions………………………………………………………………….. 60 v LIST OF FIGURES AND TABLES Chapter II Figure II.1: GW501516 rapidly increases serum HDL cholesterol and apolipoprotein AI…………………………….……………… 29 Figure II.2: Apoa1 mRNA and protein are upregulated in white adipose tissue by PPARδ ligand treatment………………….…........ 30 Figure II.3: Apoa1 can be detected in adipose tissue by immunohistochemistry………………………………………. 31 Figure II.4: WAT transplantation can rescue the effect of PPARδ ligand in AZIP and PBN animals ……….……………..……….……... 33 Figure II.5. Apoa1 protein can be detected in apoa1 -/- animals with adipose transplant ……………………………………………. 34 Chapter III Figure III.1: PPARα null animals have decreased weight loss during CR.. 48 Figure III.2: Thyroid hormone levels are dysregulated in PPARα null animals on CR………………………………………………. 49 Figure III.3: Seminal vesicles are greatly increased in WT animals on CR …………….....………………………………..………… 50 Figure III.4: Changes in liver gene expression of PPARα -/- animals on CR……………………………………………......…………. 51 Figure III.5: Animals treated with PPARα ligand lose less weight after CR…………………………...………………………….….. 52 Table I: Metabolic consequences of CR with PPARα and PPARδ ligands………………………………………………………. 53 Figure III.6: The effect of WY treatment on weight loss is PPARα specific....................................................................…………. 54 vi Figure III.7: PPARα ligand treatment causes decreased fasting glucose but increased serum insulin when coupled with CR………….... 55 Figure III.8: Activation of PPARα increases fatty acid synthesis ……..... 56 vii ACKNOWLEDGEMENTS During my five years in the Evans’ lab almost everyone contributed either directly or indirectly to my growth as a scientist and a person. First of all, I would like to thank my advisor, Ron, for creating a lab environment which was extremely challenging but at the same time a comforting place. Over the years I have made many mistakes but I am thankful that Ron allowed me to mature in a safe environment. The experience I gained in this lab will surely translate into a much more successful and fruitful life in the future. I would specifically like to thank Dr. Chih-Hao Lee for all of his guidance and help. It was Lee who first suggested the role of PPAR delta ligands in cholesterol metabolism as a possible project and worked closely with me on this project ever since. Besides being “Mr. 100%” in the lab, Lee and I also shared many wonderful times outside of lab. Another mentor of mine in the Evans’ lab was Dr. Junichiro Sonoda. Unlike many people, Jun has maintained that insatiable, innate curiosity which causes most of us to pursue a career in science in the first place. That curiosity and his ridiculous work ethic were a great inspiration to me in the last stretch of my thesis work. Jun also has the habit of always saying good things about others while still being an extremely critical scientist. We collaborated closely on all of the work presented in chapter three of this thesis. It was amazing how quickly we acquired new data working together something that also rekindled my love of science. viii Several other members of the Evans’ lab were responsible for making my graduate career unforgettable. Peter Olson was the leader for all of our outdoor adventures. Because of him, I now have some of the greatest memories of hiking in the wilderness. Also Russell Nofsinger was the first person I talked to in this lab and was also one of the biggest reasons I joined. Over the years I have enjoyed watching his metamorphosis from a typical Midwestern graduate student into a live-octopus-eating husband and postdoc. I have also had many good times with Mike Nelson especially during his brief stint of bachelorhood. Although we wasted many hours playing axis- and-allies together it was worth it knowing that the axis powers could not win. As a bonus he allowed me the experience of beating up on chubby 12-year-olds in LOTR TCG. Although often far away from me, my family has done much to support me through graduate school. Several times the trip to Columbus to visit my mother, Mike, Racheal, Jacob, Dad, and Noah was just the thing needed to de-stress. I also enjoyed the trips to the Bay to see my sister Sarah and her trips to SD. Hopefully we will be able to catch a lobster soon. One of the best things that could have happened to me was my brother Lev moving to SD. It has allowed us to recapture the friendship of our early childhood. Finally I cannot give enough thanks to my beautiful fiancé and soon-to-be wife Dinh. My graduate career started with meeting her and it is ending with our marriage and the beginning of a new adventure. The last few months especially would have been impossible without her help and affection. ix Dr. Chih-Hao Lee was a close collaborator on all of the work presented in chapter two of this thesis. Dr. Junichiro Sonoda was a close collaborator on all of the work presented in chapter three of this thesis. x CURRICULUM VITAE Graduate Education University of California San Diego 2001- Present Doctor of Philosophy in Biology Salk Research Fellows research talk award winner Salk mobile science lab volunteer National Institute of Health, CMG Training Grant 2002-2005 Teaching Assistant, Immunology 2006 Teaching Assistant, Genetics 2003 Teaching Assistant, Animal Physiology 2002 Undergraduate Education The Ohio State University 1996-2000 Bachelor of Science in Molecular Genetics Graduated with distinction in Molecular Genetics Graduated with honors in the Liberal Arts National Science Foundation REU participant 1999 Battelle scholarship recipient Denman Research Competition honorable mention 2nd place Biological Sciences Undergraduate Research Colloquium High School outreach program organizer Publications Mao, Y., I.R. Mehl, and M.T. Muller, Subnuclear distribution of topoisomerase I is linked to ongoing transcription and p53 status. Proc Natl Acad Sci U S A, 2002. 99(3): p. 1235-40. Lee, C.H., et al., Peroxisome proliferator-activated receptor delta promotes very low-density lipoprotein-derived fatty acid catabolism in the macrophage. Proc Natl Acad Sci U S A, 2006. 103(7): p. 2434-9. Lee, C.H., et al., PPARdelta regulates glucose metabolism and insulin sensitivity. Proc Natl Acad Sci U S A, 2006. 103(9): p. 3444-9. International Work Experience 2000-2001 Spoken English Foreign Expert, BinHai College, Qingdao China xi ABSTRACT OF THE DISSERTATION Regulation of Gene Expression by Peroxisome Proliferator Activated Receptors by Isaac R. Mehl Doctor of Philosophy in Biology University of California, San Diego, 2007 Professor Marc R. Montminy, Chair Nuclear hormone receptors are a model for ligand activated transcription. A subfamily of three nuclear receptors, the peroxisome proliferators-activated receptors, binds fatty acids to regulate a variety of biological processes. One of these, PPARδ, has a broad and even tissue distribution providing few clues as to its function. Recently, a high affinity synthetic ligand to this receptor has greatly facilitated the study of gene regulation by this receptor. xii One of the hallmarks of PPARδ activation is an increase in serum high-density lipoprotein cholesterol levels. Because low HDLc is a hallmark of the metabolic syndrome and a significant risk factor for cardiovascular disease we attempted to discover the mechanism by which ligand activation of PPARδ raises HDLc in mice. Surprisingly we found that PPARδ is able to regulate the main protein component of HDL, apolipoprotein AI, in adipose tissue. Amazingly, adipose tissue transplantation of AZIP/F1 or PPARδ knockout animals was able to rescue the effect of ligand. Recent studies have highlighted the role of PPARδ in oxidative metabolism. To study the effect of PPARδ activation in a state of increased oxidative metabolism we utilized a regimen of caloric restriction. Paradoxically PPARα and PPARδ ligand administration during caloric restriction decreased weight loss. Analysis of gene expression revealed that activation of the fatty acid synthesis pathway in