Regulation of energy homeostasis roles of foxo1 in dopaminergic neurons and characterization of gallic acid as an ampk activator

Regulation of Energy Homeostasis: Roles of FoxO1 in Dopaminergic Neurons and Characterization of Gallic Acid as an AMPK Activator Doan Van Khanh Department of Global Medical Science The Graduate School Yonsei University Regulation of Energy Homeostasis: Roles of FoxO1 in Dopaminergic Neurons and Characterization of Gallic Acid as an AMPK Activator Directed by Professor Ki Woo Kim A Doctoral Dissertation Submitted to the Department of Global Medical Science, The Graduate School of Yonsei University In partial fulfillment of the requirements for the degree of Doctor of Philosophy Doan Van Khanh December 2015 ACKNOWLEDGEMENTS First, I would like to sincerely thank my advisor, Professor Ki Woo Kim, for his guidance, understanding, support, and most important, his friendship during my graduate study in Yonsei University Wonju College of Medicine It has been an honor to be his first Ph.D student His mentorship was paramount in providing a great experience in the field of biomedical science He has supported me by providing not only research guidance but also academic and emotional encouragement through the rough journey of the Ph.D program I am also grateful to Dr Yun-Hee Choi for her great guidance and suggestions in getting experimental designs and professional techniques I wish to acknowledge Professor Jae Won Choi and Professor Chang Mann Ko, the present and former Chairman of Department of Pharmacology, for their support during my research period in the Pharmacology Lab In addition, I take this opportunity to express my heartfelt gratitude to all the members of Department of Pharmacology These friends and co-workers were of much assistance and their humorous and friendly nature provided helped me easily overcome somewhat stressful laboratory environment My special thanks are extended to the members of my doctoral committee, Professor Kyu Sang Park, Professor Yangsik Jeong, Professor Seung-Kuy Cha and Professor Won-Gil Cho, for their accessibility, valuable inputs and suggestions I would also like to thank the International Associations of Graduate Students in Wonju College of Medicine, Yonsei University for their support and helping me settle down and adjust to life in Korea Specifically, I am very grateful to Dr Peninah M Wairagu, the former and senior Ph.D student in Department of Biochemistry, for her technical training and her great help on editing my thesis Finally, I wish to thank my wife, my son, my parents for their support, encouragement, and unwavering love during the past three years of my oversea studying December20th 2015 Doan Van Khanh TABLE OF CONTENTS LIST OF FIGURES vii LIST OF TABLES x ABBREVIATIONS xi ABSTRACT xviii Part I Role of Forkhead Transcriptional Factor O1 (FoxO1) in Dopaminergic Neurons in Regulation of Energy Homeostasis I INTRODUCTION 1.1 Dopaminergic neuron system in the central nervous system 1.2 Role of dopaminergic system in energy balance and obesity 10 1.2.1 Dopamine, food reward and obesity 10 1.2.2 Dopamine and energy expenditure 16 1.3 Modulation of dopaminergic system by metabolic hormones 19 1.4 Forkhead box transcriptional factor O1 22 1.5 Role of FoxO1 in peripheral regulation of metabolism 24 1.6 Role of FoxO1 in central regulation of metabolism 26 II HYPOTHESIS AND RESEARCH APPROACH 29 III MATERIALS AND METHODS 31 3.1 Ethics approval 31 3.2 Mice 31 i 3.3 Dissection of brain regions 31 3.4 Body weight and body composition 32 3.5 Food intake and re-feeding experiments 32 3.6 Locomotor activity 33 3.7 Sucrose preference test 33 3.8 Metabolic analysis 34 3.9 Glucose and insulin tolerance tests 34 3.10 Body temperature measurement 35 3.11 Insulin and leptin measurements 35 3.12 Measurement of norepinephrine 36 3.13 Measurement of dopamine content 36 3.14 Cell culture 38 3.15 Vectors and siRNAs transfections 38 3.16 Western blot 39 3.17 Reverse transcription PCR and quantitative real-time PCR 43 3.18 Immunohistochemistry 47 3.19 Generation of luciferase reporter constructs 48 3.20 Measurement of promoter activity 49 3.21 Chromatin immunoprecipitation assays 49 3.22 Statistical analysis 54 ii IV RESULTS 55 4.1 Verification of DA-specific FoxO1 knockout mice model 55 4.2 Metabolic phenotypes of DA-specific FoxO1 knockout mice on chow diet 64 4.3 Metabolic phenotypes of DA-specific FoxO1 knockout mice on high fat diet 69 4.4 Food intake and feeding behaviors of DA-specific FoxO1 knockout mice 72 4.5 Increased energy expenditure in the DA-specific FoxO1 knockout mice 78 4.6 Increased catecholamine levels and interscapular brown adipose tissue thermogenesis in DA-specific FoxO1 knockout mice 83 4.7 Changes in dopamine D2 receptor and dopamine turnover in DA-specific FoxO1 knockout mice 89 4.8 FoxO1 directly regulates tyrosine hydroxylase expression in DA neurons 92 V DISCUSSION 99 VI CONCLUSION 108 VII REFERENCES 109 iii Part II Characterization of Gallic Acid as an AMPK Activator in Regulation of Glucose Homeostasis I INTRODUCTION 133 1.1 AMPK activation and metabolic benefits 134 1.1.1 Activation of AMPK signaling 134 1.1.2 AMPK activation and lipid metabolism 136 1.1.3 AMPK activation and mitochondrial biogenesis 136 1.1.4 AMPK activation and autophagy 138 1.1.5 AMPK activation and insulin sensitivity 139 1.2 AMPK activators - Role of polyphenols 139 1.2.1 AICAR 140 1.2.2 Biguanides 140 1.2.3 Thiazolidinediones 142 1.2.4 Polyphenols and other natural compounds 143 II SCREENING FOR AMPK ACTIVATORS AND RESEARCH HYPOTHESIS 150 III MATERIALS AND METHODS 152 3.1 Ethic approval 152 3.2 Materials 152 3.3 Cell culture 152 iv 3.4 Sirt1 gene silencing 153 3.5 GFP-LC3 localization 153 3.6 Oil Red O staining and triglyceride content 154 3.7 Nuclear extraction and immunoprecipitation 154 3.8 Animals 155 3.9 Glucose and insulin tolerance tests 156 3.10 Histological analysis 156 3.11 Western blot 157 3.12 RNA isolation and quantitative real-time PCR 157 3.13 Statistical analysis 158 IV RESULTS 159 4.1 GA activates AMPK signaling in vitro 159 4.2 GA induces autophagy 161 4.3 GA inhibits lipid accumulation 165 4.4 GA treatment increases the expression and activity of PGC1 and expression of mitochondrial biogenesis genes 167 4.5 Effect of GA on PGC1 is mediated via Sirt1 171 4.6 Activation of AMPK/Sirt1/PGC1 axis in vivo 173 4.7 GA treatment suppresses gluconeogenic genes expression 175 4.8 Beneficial metabolic effects of GA on diet-induced obese mice 177 v mitochondrial respiratory complex I: a mechanism for the action of berberine to activate AMP-activated protein kinase and improve insulin action Diabetes 2008;57:1414-8 66 Baur JA, Sinclair DA Therapeutic potential of resveratrol: the in vivo evidence Nat Rev Drug Discov 2006;5:493-506 67 Wang S, Moustaid-Moussa N, Chen L, Mo H, Shastri A, Su R, et al Novel insights of dietary polyphenols and obesity J Nutr Biochem 2014;25:1-18 68 Um JH, Park SJ, Kang H, Yang S, Foretz M, McBurney MW, et al AMP-activated protein kinase-deficient mice are resistant to the metabolic effects of 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Pharmacol Rev 2012;64:166-87 123 Dulloo AG, Seydoux J, Girardier L, Chantre P, Vandermander J Green tea and thermogenesis: interactions between catechin-polyphenols, caffeine and sympathetic activity Int J Obes Relat Metab Disord 2000;24:252-8 124 Huang THW, Peng G, Kota BP, Li GQ, Yamahara J, Roufogalis BD, et al Anti-diabetic action of Punica granatum flower extract: Activation of 207 PPAR and identification of an active component Toxicol Appl Pharmacol 2005;207:160-9 125 Prasad CNV, Anjana T, Banerji A, Gopalakrishnapillai A Gallic acid induces GLUT4 translocation and glucose uptake activity in 3T3-L1 cells FEBS Letters 2010;584:531-6 208 ABSTRACT IN KOREAN 신체 에너지 항상성 조절에 관한 기전 연구: 도파민 뉴런에서 FoxO1의 에너지 항상성 조절 기전 연구 및 AMPK Activator 로써 Gallic Acid의 역할 두안 반 칸 연세대학교 대학원 글로벌의생명학과 전신의 에너지 항상성 유지는 조절 시스템들 (신경계, 내분비계, 면역계)의 기능적인 조화와 표적 기관 (간, 근육, 지방 조직 등)에 대한 즉각적인 반응이 요구된다 세포 및 분자 신호 수준에서 복잡하고 섬세한 조절 기전을 통해 중추 신경계에서는 정상적인 생리적 상태를 보존하기 위하여 말초 기관의 기능과 활성을 조절한다 209 조절 기전의 유지가 실패하거나 표적 기관에 대한 즉각적인 기능이 손상 되었을 때 비만과 당뇨와 같은 대사 질환을 유발될 수 있다 어떻게 신체가 순응하여 항상성을 유지하기 위해 에너지 균형을 조절하는가에 대한 이해는 대사 질환들의 관리에 있어 효율적인 치료 전략을 세우는데 중요한 핵심 요소가 된다 첫째 장에서, 본 연구진은 도파민 신경 FoxO1의 대사조절적 역할 조사를 통해 신체 항상성 조절의 이해를 확장하기 위한 연구를 진행하였다 시상하부에서 FoxO1은 렙틴, 인슐린과 같은 대사조절 호르몬의 기능을 중재하는데 있어 중요한 역할을 담당한다 하지만, 섭식행동, 음식 소비량, 에너지 균형을 조절하는 것으로 알려진 도파민 신경계에서 FoxO1의 항상성에 대한 역할은 조사되지 않았다 도파민 신경에서 FoxO1의 기능을 연구하기 위해, 본 연구진은 Cre-loxP system을 이용한 도파민 신경 선택적 FoxO1 결핍 쥐를 생산하였다 도파민 신경 선택적 FoxO1 결핍 쥐에서 에너지 소비량과 갈색지방 기능이 유의하게 증가되어 있었다 더욱이, FoxO1 결핍 쥐는 인슐린 민감성이 증가하였고 도파민 및 노르에피네피린 값이 또한 증가함을 보였다 또한 FoxO1은 직접적으로 티로신수산화효소의 전사를 억제한다는 것을 확인 할 수 있었다 이러한 발견은 도파민 신경에서 FoxO1이 섭식 행동뿐만 아니라 에너지 소비와 대사 항상성 210 조절에 중요한 역할을 함을 시사한다 또한, FoxO1에 의한 티로신수산화효소의 전사 조절의 규명은 신진대사와 도파민 계의 관련성을 제시한다 두 번째 장에서, 본 연구진은 천연으로부터 유래된 물질의 비만 및 당뇨 등 대사 질환의 예방과 치료를 목적으로 하는 약물 후보를 규명하고자 하였다 특히, 말초 기관 (간, 근육, 지방 조직)에서 AMPK 활성은 대사 조절에 많은 이로운 점을 보였기 때문에 AMPK 신호를 조절하는 파이토케미컬을 스크리닝 하였다 그 중 갈릭산이 AMPK를 강하게 활성시키는 것을 확인하였다 In vitro 및 in vivo 실험을 통하여, 갈릭산이 PGC1 및 Sirt1의 활성을 통해 미토콘드리아 생합성을 증진시키는 것을 확인하였다 또한, 비만 유도 쥐에서 갈릭산을 처치하였을 때 혈당 및 인슐린 항상성이 유의하게 향상되었고, 몸무게 증가가 억제되는 것을 확인하였다 더욱이, 에너지 소비와 관련된 UCP1과 및 여러 유전자들이 갈릭산 처치 쥐의 갈색지방에서 유의하게 증가됨을 관찰하였다 종합적으로, 이러한 결과들은 갈릭산이 AMPK/Sirt1/PGC1 경로의 활성과 갈색지방의 에너지 발생 관련 유전자들의 활성화를 통하여 대사에 이로운 효과를 보임을 시사한다 따라서, 본 연구는 갈릭산에 의한 AMPK/Sirt1/PGC1 경로를 활성 시킬 수 있는 물질 또는 그 파생물이 211 대사 질환에서 인슐린 저항성을 중재할 수 있는 잠재적 치료제가 될 수 있음을 시사한다 핵심되는 말 : 도파민 신경, Forkhead box O1 전사인자, 에너지 소비, 티로신수산화효소, 갈릭산, AMP-활성화 단백질 인산화효소, 포도당 항상성 212 PUBLICATION LIST Khanh DV et al Leptin and insulin signaling in dopaminergic neurons: relationship between energy balance and reward system Frontiers in Psychology 2014;5:846 Khanh DV et al Gallic Acid Regulates Body Weight and Glucose Homeostasis Through AMPK Activation Endocrinology 2015;156:157–168 Khanh DV et al FoxO1 in Dopaminergic Neurons Regulates Energy Homeostasis and Targets Communications In revision 213 Tyrosine Hydroxylase Nature .. .Regulation of Energy Homeostasis: Roles of FoxO1 in Dopaminergic Neurons and Characterization of Gallic Acid as an AMPK Activator Directed by Professor Ki Woo Kim A Doctoral... role of FoxO1 in regulation of energy balance has been established FoxO1 mediates the effects of insulin and leptin on AgRP and POMC neurons in the ARC as FoxO1 stimulates the transcription of. .. AMP-activated protein kinase, Glucose homeostasis xxi Part I Role of Forkhead Transcriptional Factor O1 (FoxO1) in Dopaminergic Neurons in Regulation of Energy Homeostasis Doan Van Khanh Department of Global