Title Page
Abstract
Contents
Chapter 1. Introduction 10
1.1. Hypothalamic regulation of energy metabolism 11
1.2. Circadian regulation and molecular clock system 14
1.3. NAD biology 17
1.4. Extracellular NAMPT and intracellular NAMPT 19
1.5. Involvement of NAD biology in circadian regulation 20
Chapter 2. Materials and methods 22
2.1. Materials 23
2.2. Animals 23
2.3. Cell culture 24
2.4. Mouse circadian studies 24
2.5. Tissue NAD⁺ measurements 24
2.6. NAMPT ELISA and enzymatic activity assay 25
2.7. Monitoring of metabolic behaviors 26
2.8. FK866 osmotic pump study 26
2.9. Systemic Nampt siRNA study 26
2.10. NAMPT and NMN injection studies 27
2.11. Organotypic slice culture and real-time bioluminescence imaging 27
2.12. Neuronal activity study 28
2.13. Hypothalamic FOXO1 adenovirus study 29
2.14. Hypothalamic siRNA study 30
2.15. Measurement of mRNA expression 30
2.16. Immunoblotting 31
2.17. FISH and IF staining 32
2.18. Promoter assay 33
2.19. Human eNAMPT study 33
2.20. Blinded research 34
2.21. Statistical analysis 34
Chapter 3. Results 36
3.1. Nocturnal rhythmic elevation of hypothalamic NAD⁺ levels is in phase with that of plasma eNAMPT 37
3.1.1. Circadian oscillation in the MBH NAD⁺ levels and ARC NAD⁺ levels 37
3.1.2. Circadian oscillation of NAMPT protein in blood, MBH, liver, and adipose tissues and Nampt mRNA expression in leukocytes 39
3.1.3. Blood eNAMPT and MBH NAD⁺ levels under the light-shift and food-shift conditions 42
3.2. Plasma eNAMPT regulates circadian rhythms of locomotor activity and energy expenditure 45
3.2.1. Effects of systemic FK866 infusion on LMA, EE and tissue NAD⁺ levels 45
3.2.2. Reduced NAD⁺ synthesis in MBH and liver by systemic NAMPT siRNA injection 47
3.2.3. Increased MBH and liver NAD biosynthesis leading to increase LMA and EE through systemic administration of NAMPT/NMN 49
3.2.4. Increased MBH NAD⁺ level, LMA and EE through intracerebroventricular administration of NAMPT 51
3.3. Blood eNAMPT acts on hypothalamic POMC and NPY/AgRP neurons 53
3.3.1. Circadian oscillations of SCN clock under NAMPT treatment 53
3.3.2. Change of hypothalamic mRNA expression by i.c.v. NAMPT administration 55
3.3.3. Increased MBH and liver NAD biosynthesis leading to increase LMA and EE through systemic administration of NAMPT/NMN 57
3.4. Hypothalamic SIRT2-FOXO1 is a downstream mediator of eNAMPT actions 61
3.4.1. Hypothalamic SIRTs expression and circadian oscillation 61
3.4.2. SIRT2 acts as a downstream mediator of eNAMPT in the regulation of POMC and AGRP transcription 63
3.4.3. Blockade of NAMPT effects on LMA, EE and mRNA expression with knockdown of MBH Sirt2 65
3.4.4. Interaction of SIRT2 and FOXO1 in the hypothalamus 67
3.4.5. Regulation of LMA and EE through NAMPT-SIRT2-FOXO1 pathway 69
3.5. Obesity disrupts circadian fluctuations in plasma eNAMPT-hypothalamic NAD-FOXO1 axis 72
3.5.1. Disruption of circadian rhythm in obese mice 72
3.5.2. Disruption of plasma eNAMPT rhythm in obese human 74
Chapter 4. Discussion 76
Chapter 5. Conclusion 83
Reference 87
국문초록 93