CIRCADIAN MECHANISMS of CALORIE RESTRICTION in DELAYING AGING KULDEEP MAKWANA Bachelor of Dental Surgery Pacific Dental College
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CIRCADIAN MECHANISMS OF CALORIE RESTRICTION IN DELAYING AGING KULDEEP MAKWANA Bachelor of Dental Surgery Pacific Dental College and General Hospital, Udaipur, India December 2010 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN REGULATORY BIOLOGY at the CLEVELAND STATE UNIVERSITY December 2018 © Copyright by Kuldeep Makwana 2018 We hereby approve this dissertation For Kuldeep Makwana Candidate for the Doctor of Philosophy in Regulatory Biology Degree for the Department of Biological, Geological and Environmental Sciences AND CLEVELAND STATE UNIVERSITY College of Graduate Studies by Date: 11/14/2018 Dr. Roman Kondratov, GRHD/BGES, Cleveland State University Major Advisor Date: 11/14/2018 Dr. Girish Shukla, GRHD/BGES, Cleveland State University Advisory Committee Member Date: 11/14/2018 Dr. Crystal Weyman, GRHD/BGES, Cleveland State University Advisory Committee Member Date: 11/14/2018 Dr. Justin Lathia, Department of Cellular and Molecular Medicine, CCF Advisory Committee Member Date: 11/14/2018 Dr. Aaron Severson, GRHD/BGES, Cleveland State University Internal Examiner Date: 11/14/2018 Dr. Yana Sandlers, Department of Chemistry, Cleveland State University External Examiner Student’s Date of Defense: 11/14/2018 DEDICATION I dedicate my work to my family and friends. To my parents for their support and faith, they have shown in all my decisions, I have ever taken to achieve my goals. To all my friends without whom this journey would not have been this eventful. ACKNOWLEDGEMENTS “A GOOD TEACHER CAN INSPIRE HOPE, IGNITE THE IMAGINATION, AND INSTILL A LOVE OF LEARNING” - BRAD HENRY First and foremost, I would like to acknowledge Dr. Roman Kondratov for being a mentor that epitomizes the sayings of Brad Henry. I would like to thank him for his support and believe he has shown in my abilities. He has been the guiding torch for all these years and played an important role in the development of my scientific intellect as well as interpersonal skills. Not only he guided me in my Ph.D. dissertation work but also taught about life lessons like a friend from time to time. He is one humble person and a very cool professor I have ever met. I’d like to acknowledge my parents. I’d always be indebted by their unconditional love and support. I’d like to extend my deepest regards and thanks to my advisory committee members: Dr. Shukla, Dr. Crystal Weyman, and Dr. Justin Lathia. They have always motivated me and helped me transform my project in various ways. At the end of every committee meeting, I have always learned something new which I will carry with me forever and implement in future as well. I’d also like to thank Dr. Aron Severson and Dr. Yana Sandlers for agreeing to serve on my committee as an internal and external reviewer. I’d like to thank all my lab mates, current and former, and friends in the BGES department for making this lengthy and difficult journey, at times, to be the one that will always remain a part of my life. I’d like to thank Sonal Patel for being a good friend and take all my stupid jokes lightly with no offense. Ravinder Kaur and Amra Ismail for being a family away from my family. Thanks to all these amazing people, going to the lab never felt like going to work. I thoroughly enjoyed and will cherish the time I spent in Dr. Roman Kondratov’s lab at Cleveland state university. Lastly, thanks to the almighty God for bestowing his blessing and love upon me. CIRCADIAN MECHANISMS OF CALORIE RESTRICTION IN DELAYING AGING KULDEEP MAKWANA ABSTRACT Calorie Restriction (CR) is a dietary intervention known to delay age associated pathologies and conditions. Its beneficial effects on the longevity are reported in variety of organisms ranging from unicellular to multi-cellular organisms like mammals. Various mechanisms have been proposed for the beneficial effect of CR on the lifespan. One of the proposed mechanisms by which CR brings about its beneficial effects on the lifespan is regulation of protein synthesis. Various studies have demonstrated an increase in protein synthesis under CR, some claimed inhibition of protein synthesis under CR, and some claimed no effect on protein synthesis under CR. In this work, using comprehensive circadian experimental setup, I have demonstrated inhibition of global protein synthesis under CR diet in mouse liver. Animals were subjected to two months of CR followed by polysome profiling of liver tissue. Protein translation was down-regulated in the liver of CR animals at all time points but after four hours of feeding, where it was found to be higher than AL animals. Transcripts associated with polysomes were isolated and mRNA-sequencing was performed. CR was found to be involved in the temporal reprogramming of circadian rhythms in protein translation. Furthermore, the effect of CR on differential translation was studied. mRNA-Sequencing assayed 26,913 transcripts associated with polysomes, 0.1% of the total number of transcripts were found to be differentially abundant in the polysomes. My study has revealed, for the first, CR mediated induced expression of ACOT enzymes which are known to be involved fat metabolism. Thus, I demonstrated circadian mechanism of calorie restriction in vii regulating metabolism via controlling the gene expression at the level of translation in CR animals. viii TABLE OF CONTENTS Page ABSTRACT......................................................................................................................vii LIST OF TABLES ............................................................................................................xii LIST OF FIGURES .........................................................................................................xiii LISTOFABBREVIATIONS.............................................................................................xv CHAPTER I. INTRODUCTION 1.1. The Process Of Aging……………………………………..…………1 1.2. Calorie Restriction- A Dietary Regimen Known To Extend Lifespan.……………………………………………………………..6 1.3. Circadian Clocks………………………………………..…………..14 1.4. Protein Translation…………………………………….…..……......19 1.5. Fat/Lipid Metabolism And Aging…………………………..………30 1.6. Role Of ACOTs In Fat Metabolism……………………………..….39 II. MATERIALS AND METHODS 2.1. Animal Experiments…...…………………………………..……….45 2.2. Polyribosome Profiling…………………….…………….………....46 2.3. RNA Isolation…………………………………………….….……..46 2.4. RNA-Sequencing………………………………………………..….47 ix 2.5. Western Blotting………………………………………..…………..47 2.6 Quantitative RT-PCR………………………………...……………...47 2.7. KEGG Pathway Analysis…..……………………………………….48 2.8 Analysis Of mRNA Sequencing Library……………………..……..48 2.9. JTK_Cycle Analysis……………………………………………......49 2.10. Statistical Analysis…………………………………….………….49 III. CALORIE RESTRICTION INHIBITS GLOBAL PROTEIN TRANSLATION IN MOUSE LIVER……………………………………..50 3.1. Introduction…………………………………………………….…...50 3.2. Result………………………………………………………..……...51 3.3. Discussion……………………..……………………………………56 3.4. Conclusion………………….………………………..……..………58 IV. CALORIE RESTRICTION REPROGRAMS DIURNAL RHYTHMS IN PROTEIN TRANSLATION TO REGULATE METABOLISM…………………………………..…………………………60 4.1. Abstract…………………………..…………………………………60 4.2. Introduction……………………..…………………………………..61 4.3. Results…………………………………..…………………………..63 4.4. Discussion…………………….……………….……...………….....89 x 4.6. Acknowledgements………………………………………………....99 V. CALORIE RESTRICTION INDUCED ACOTs EXPRESSION IS BMAL1 DEPENDENT……………………………………………………..………..100 5.1. Introduction……………………………………………..…………100 5.2. Results………………………………………………………….….101 5.3. Discussion…………………………………………………………108 VI. CONCLUSION...…………………………………………….……………..106 BIBLIOGRAPHY………………………………………………………………………107 APPENDICES A. Supplemental Table………………....…………………………..…………..131 B. Supplemental Figure………...…...….……………………..……….…….…132 C. Supplemental Figure ……………………...………………….……..……....134 xi LIST OF TABLES Table Page 1. Substrate specificity of mouse peroxisomal ACOTs…………………...……………42 2. Primers for RT-qPCR………………………….…….…………..…………………...48 3. Phase of rhythmicity in translation and transcription for circadian clock genes…………………………………………………………...……………...131 xii LIST OF FIGURES Figure . Page 1-1. Beneficial Effects of Calorie Restriction………………………...……………..........7 1-2. Circadian clock organization ………………………………...…………………….16 1-3. Molecular clock………………………………………..…………………………...19 1-4. Initiation of translation………………………………..……………………………24 1-5. Mechanism of translation elongation…………………..…………………………..27 1-6. Mechanism of protein translation termination step……….…….………………….29 1-7. Mechanism of Acyl CoA shuttling inside the Mitochondria….……..………..........33 1-8. Mechanism of β-oxidation……………………………..……….…………………..35 1-9. Structure of animal FASI……………………………….…………………………..36 1-10. Subcellular localization of type I ACOTs………………………..……………….44 3-1. Inhibition of global protein translation in the mouse liver under CR…………...….53 3-2. Method for rate of protein translation quantification……………..………………..54 3-3. Rate of protein translation under 30% CR in mouse liver……………...…………..55 4-1. Schematic representation of experimental workflow………………..……………..64 4-2. Differential translation induced by CR diet………………...……………………....66 4-3. Validation of RNA-Seq data………………………...………………...…………....67 xiii 4-4. KEGG analysis of differentially abundant P-mRNAs under CR…………..………68 4-5. Rhythmic Translation of circadian core clock genes………………...……………..70 4-6. Rhythmic P-mRNAs in the liver polysomes of AL and CR animals……...……….72 4-7. KEGG analysis of rhythmic P-mRNAs…………………………………...………..73 4-8. KEGG analysis of rhythmic P-mRNAs