The role of the clock in lipid metabolism A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2016 Siobhán Anne Ahern School of Medical Sciences 1 Contents List of figures ................................................................................................................................. 6 List of tables .................................................................................................................................. 9 Abstract ....................................................................................................................................... 10 Declaration of copyright statement ............................................................................................ 11 Abbreviations .............................................................................................................................. 12 Acknowledgements ..................................................................................................................... 18 Contributions .............................................................................................................................. 19 Chapter 1: Introduction .............................................................................................................. 20 1.1 Introduction ...................................................................................................................... 21 1.2 Energy Balance .................................................................................................................. 23 1.3 Homeostatic control of feeding ........................................................................................ 25 1.4 Drive to eat ....................................................................................................................... 30 1.5 Energy Expenditure ........................................................................................................... 30 1.6 BAT thermogenesis ........................................................................................................... 33 1.7 Digestion and Absorption ................................................................................................. 35 1.8 Liver as a metabolic regulator ........................................................................................... 36 1.9 Glucose regulation ............................................................................................................ 36 1.10 Lipid Metabolism ............................................................................................................ 38 1.11 Fatty acid oxidation ......................................................................................................... 44 1.12 Fatty Acid Synthesis ........................................................................................................ 46 1.13 Triglyceride Synthesis ..................................................................................................... 46 1.14 Triglyceride Degradation ................................................................................................. 47 1.15 Ketone Bodies ................................................................................................................. 51 1.16 Cholesterol Biosynthesis ................................................................................................. 51 1.17 Transcriptional control of lipid metabolism .................................................................... 53 1.18 Adipose Tissue ................................................................................................................ 54 1.19 Adipogenesis ................................................................................................................... 55 1.20 Secretory role of WAT ..................................................................................................... 56 1.20.1 Leptin ....................................................................................................................... 56 1.20.2 Adiponectin .............................................................................................................. 57 1.20.3 Other adipokines ...................................................................................................... 58 1.21 Pathophysiology of obesity ............................................................................................. 59 1.22 Adipose Tissue Expansion ............................................................................................... 60 2 1.23 Adipose tissue inflammation .......................................................................................... 61 1.24 Insulin resistance in obesity ............................................................................................ 62 1.25 Endoplasmic Reticulum (ER) stress ................................................................................. 64 1.26 Hypoxia in fat expansion ................................................................................................. 64 1.27 PPAR regulation in obesity .............................................................................................. 65 1.28 Circadian clocks ............................................................................................................... 66 1.28.1 The Master Clock ..................................................................................................... 67 1.28.2 Peripheral Clocks ...................................................................................................... 69 1.28.3 Molecular clock ........................................................................................................ 71 1.29 Clock control ................................................................................................................... 73 1.30 Epigenetic control of circadian clock .............................................................................. 75 1.31 Linking the circadian clock to energy homeostasis ......................................................... 76 1.32 Circadian regulation of metabolism ................................................................................ 77 1.33 Circadian control of carbohydrate metabolism .............................................................. 78 1.34 Circadian control of lipid metabolism ............................................................................. 80 1.35 Role of Rev-erbα in lipid metabolism ............................................................................. 83 1.36 Overall Aims .................................................................................................................... 85 Chapter 2 - Materials and Methods ............................................................................................ 87 2.1 Animal maintenance ......................................................................................................... 88 2.2 Dietary Manipulation ........................................................................................................ 88 2.2.1 HFD feeding ................................................................................................................ 88 2.2.2 Fasting ........................................................................................................................ 88 2.3 Remote Telemetry ............................................................................................................ 89 2.4 Comprehensive Lab Animal Monitoring System (CLAMS) ................................................ 89 2.5 TSE Feeding Cages ............................................................................................................. 90 2.6 Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) ........................................ 90 2.7 In vivo lipogenic/lipolytic challenge .................................................................................. 90 2.8 Cold Exposure ................................................................................................................... 91 2.9 Serum collection and lipid analysis ................................................................................... 91 2.10 Ex vivo bioluminescence ................................................................................................. 91 2.11 RNA extraction, conversion, and quantitative Real Time PCR ........................................ 92 2.12 SDS-Page and Western Blot analyses ............................................................................. 94 2.13 WAT explant cultures ...................................................................................................... 96 2.14 Statistical Analysis ........................................................................................................... 96 3 Results ......................................................................................................................................... 97 Chapter 3 - High-fat diet disrupts molecular circadian rhythms in mice .................................... 97 3.1 Introduction ...................................................................................................................... 98 3.2 Methods ............................................................................................................................ 99 3.3 Results ............................................................................................................................... 99 3.3.1. Circadian behaviour of obese mice ........................................................................... 99 3.3.2. Molecular clock disruption in response to obesity ................................................. 100 3.3.3. Effect of obesity of lipid handling genes ................................................................. 102 3.3.4. Tissue specific reprogramming of the clock-metabolic regulators ......................... 107 3.3.5. Effect of obesity on glucose handling genes ........................................................... 107 3.3.6. Damping of gene rhythmicity in gWAT associated with the development of obesity and tissue inflammation ................................................................................................... 110 3.4. Summary ........................................................................................................................ 110 Results ....................................................................................................................................... 114 Chapter 4 - Loss of Rev-erbα results in an obese phenotype and severe dysregulation of lipid handling in the mouse .............................................................................................................. 114 4.1. Introduction ................................................................................................................... 115 4.2 Methods .......................................................................................................................... 116 4.3 Results ............................................................................................................................. 118 4.3.1. Rhythmicity in locomotor activity and local tissue clocks in Rev-erbα-/- mice ........ 118 4.3.2. Metabolic phenotype of Rev-erbα-/- mice ............................................................... 120 4.3.3 Fasting response in Rev-erbα−/− mice ....................................................................... 127 4.3.4 Adrenergic stimulation of Rev-erbα−/− mice ............................................................. 128 4.3.5 HFD-feeding in Rev-erbα−/− mice .............................................................................. 133 4.3.6 In vivo insulin stimulation of Rev-erbα−/− mice ......................................................... 142 4.3.7 Ex vivo insulin stimulation of Rev-erbα−/− mice ........................................................ 146 4.4 Summary ......................................................................................................................... 149 Results ....................................................................................................................................... 151 Chapter 5 - Adipocyte-specific Bmal1 null mice showing attenuated feeding rhythms and sever lipid handling gene dysregulation whereas adipocyte specific loss of Rev-erbα has negligible physiological consequences ...................................................................................................... 151 5.1 Introduction .................................................................................................................... 152 5.2 Methods .......................................................................................................................... 153 5.3 Results ............................................................................................................................. 156 5.3.1 Confirmation of Adipose Specific Targeting............................................................. 156 4 5.3.2 Metabolic phenotype of AdipoCRE+Bmal1flox/flox mice................................................ 158 5.3.3 Fasting response in AdipoCREBmal1flox/flox ................................................................. 158 5.3.4 Cold challenge in AdipoCREBmal1flox/flox mice ............................................................ 161 5.3.5 Dysregulation of lipid handling genes in gWAT AdipoCREBmal1flox/flox mice .............. 165 5.3.6 Response of AdipoCREBmal1flox/flox mice to positive energy balance ......................... 169 5.3.7 Metabolic phenotype of AdipoCRERev-erbαflox/flox mice ............................................ 169 5.3.8 Fasting response in AdipoCRERev-erbαflox/flox ............................................................. 172 5.3.9 Expression of lipid handling genes in gWAT AdipoCRERev-erbαflox/flox mice ............... 177 5.3.10 Insulin sensitivity in AdipoCRERev-erbαflox/flox mice .................................................. 180 5. 4 Summary ........................................................................................................................ 180 Chapter 6 - Discussion ............................................................................................................... 184 6.1 Diet Induced Obesity disrupts behavioural and molecular circadian rhythms ............... 185 6.2 Loss of Rev-erbα results in severe disruptions to lipid handling .................................... 194 6.3 Adipocyte Specific loss of Bmal1 causes attenuated feeding rhythms and severe lipid handling gene dysregulation ................................................................................................. 205 6.4 Conclusion ....................................................................................................................... 209 References ................................................................................................................................ 212 Appendix 1 ................................................................................................................................ 256 Chappter 3 statistics ............................................................................................................. 256 Appendix 2 ................................................................................................................................ 267 Chapter 4 statistics ................................................................................................................ 267 Appendix 3 ................................................................................................................................ 276 Chapter 5 statistics ................................................................................................................ 276 5 List of figures Chapter 1 Figure 1.1 – Afferent gastrointestinal signals controlling food intake p.27 Figure 1.2 – Glucagon-signalling pathway p.39 Figure 1.3 – Biochemical representation of triglyceride breakdown P.41 Figure 1.4 – Reactions of beta-oxidation p.45 Figure 1.5 – Overview of lipid metabolism within the adipocyte p.49 Figure 1.6 – Reactions of ketogenesis p.52 Figure 1.7 – Schematic representation of the transcriptional feedback loops of the p.72 circadian clock Chapter 3 Figure 3.1 – Physiological changes in diet induced obesity. p.101 Figure 3.2 – Tissue-specific effects on the molecular clock associated with HFD p.103 feeding Figure 3.3 – Diurnal variation of lipid handling genes differentially affected by HFD p.106 feeding in different tissues Figure 3.4 – Disruption of downstream pathways is affected in a tissue-specific p.108 manner by High Fat Diet feeding Figure 3.5 – Diurnal variation of glucose handling genes differentially affected by p.109 HFD feeding in different tissues Figure 3.6 – Attenuation of the clock in gWAT was acutely affected by HFD p.111 feeding and associated with increased inflammation Chapter 4 Figure 4.1 – Circadian rhythmicity in Rev-erbα-/- mice p.119 Figure 4.2 – Physiological and behaviour changes in Rev-erbα-/- mice p.121 Figure 4.3 – Alterations in metabolic parameters in Rev-erbα-/- mice p.122 Figure 4.4 – Tissue specific effects of loss of Rev-erbα on lipid handling genes p.125 6 Figure 4.5 – Differential physiological responses to fasting in Rev-erbα-/- mice p.126 Figure 4.6 – Differential metabolic response to fasting in Rev-erbα-/- mice p.129 Figure 4.7 – Differential effects on lipid handling genes to a fast of Rev-erbα-/- p.131 mice. Figure 4.8 – In vivo adrenergic stimulation of Rev-erbα-/- mice. p.132 Figure 4.9 – Rev-erbα-/- mice show decreased sensitivity to adrenergic induction of p.134 HSL phosphorylation. Figure 4.10 – Differential effect of diet induced obesity on Reverbα-/- mice p.135 Figure 4.11 – Effects of diet induced obesity on Ppar genes in Rev-erbα-/- mice p.137 Figure 4.12 – Tissue specific effects of diet induced obesity on lipid handling genes p.138 in Rev-erbα-/- mice Figure 4.13 – Effects of diet induced obesity on lipid handling proteins in p.139 Rev-erbα- /- mice Figure 4.14 – Effects of diet induced obesity on glucose handling genes in p.140 Rev-erbα-/- mice Figure 4.15 – Effect of insulin administration on blood glucose and circulating p.141 triglycerides in Rev-erbα-/- mice Figure 4.16 – Transcript analysis of in vivo insulin stimulation of Rev-erbα-/- mice p.144 Figure 4.17 – Glucose and insulin tolerance tests in Rev-erbα-/- mice p.145 Figure 4.18 – Insulin dose response of WT gWAT explants p.147 Figure 4.19 – Ex vivo insulin stimulation of gWAT from Rev-erbα-/- mice p.148 Chapter 5 Figure 5.1 – Confirmation of adipose specific targeting p.157 Figure 5.2 – Body weight profiles of AdipoCREBmal1flox/flox mice p.159 Figure 5.3 – Food intake in AdipoCREBmal1flox/flox mice p.160 Figure 5.4 – Metabolic parameters of AdipoCREBmal1flox/flox mice p.162 Figure 5.5 – Fasting response of AdipoCREBmal1flox/flox mice p.164 Figure 5.6 – Metabolic response to fasting in AdipoCREBmal1flox/flox mice p.166 7 Figure 5.7 – Cold challenge of AdipoCREBmal1flox/flox mice p.167 Figure 5.8 – Transcript analysis of lipid handling genes in AdipoCREBmal1flox/flox mice p.168 Figure 5.9 – Ex vivo insulin stimulation of AdipoCREBmal1flox/flox mice p.170 Figure 5.10 – High Fat Diet challenge of AdipoCREBmal1flox/flox mice p.171 Figure 5.11 – Body weight profiles and food intake of AdipoCRERev-erbαflox/flox mice p.173 Figure 5.12 – High Fat Diet of AdipoCRERev-erbαflox/flox mice p.174 Figure 5.13 – Metabolic parameters of AdipoCRERev-erbαflox/flox mice p.175 Figure 5.14 – Fasting response of AdipoCRERev-erbαflox/flox mice p.176 Figure 5.15 – Fasting response AdipoCRERev-erbαflox/flox mice p.178 Figure 5.16 – Transcript analysis of lipid handling genes in AdipoCRERev-erbαflox/flox p.179 mice Figure 5.17 – Insulin Tolerance Test of AdipoCRERev-erbαflox/flox mice p.181 Chapter 6 Figure 6.1 – Confirmation of adipose specific targeting p.211 8 List of tables Chapter 2 Table 2.1 – Primer Sequences p.93 Table 2.2 – Antibody Weights and Concentrations p.95 9 Abstract The University of Manchester Submitted by Siobhán Anne Ahern for the degree of Doctor of Philosophy and entitled: The role of the clock in lipid metabolism. September 2016. In mammals, the circadian clock coordinates multiple behavioural and physical processes, including energy homeostasis. At the centre of these rhythms lies the circadian clock machinery, a precisely coordinated transcription-translation feedback system required to maintain the correct time. Metabolic homeostasis requires accurate and coordinated circadian timing within individual cells and tissues of the body. Moreover, recent evidence has shown that the coupling of circadian and metabolic circuits involves reciprocal regulatory feedback. In line with this, mounting evidence suggests that disruption of the clock contributes to the development of obesity and its comorbidities. This is particularly concerning given that modern lifestyles often undermine our bodies’ clock. However, the casual mechanisms which link circadian disruption to metabolic disease are not well defined. This work aims to gain a further understanding of clock control of metabolic homeostasis and especially regulation of lipid metabolism. This work uses dietary challenge to determine which peripheral clocks and downstream metabolic pathways are particularly susceptible to diet induced obesity (DIO). We demonstrate that although behavioural rhythmicity was maintained in DIO, gene expression profiling revealed tissue-specific alteration to the phase and amplitude of the molecular clockwork. Clock function was most significantly attenuated in visceral white adipose tissue (WAT) of DIO mice, and was coincident with elevated tissue inflammation, and dysregulation of clock-coupled metabolic regulators PPARα/γ. The rhythmic expression of Rev-erbα, a nuclear receptor involved in the circadian clock, was particularly affected in DIO mice. This study uses the Rev-erbα-/- mouse to explore clock-metabolic coupling, specifically lipid metabolism. In line with published work, Rev-erbα-/- mice exhibit an obese phenotype with associated upregulation in gWAT of lipogenic (Dgat2, Fasn) and fatty acid liberation (Lpl) genes. Differences in fat mobilization are observed as Rev-erbα-/- mice show a heightened insulin stimulated lipogenic drive and an attenuation of the lipolytic drive in the fasted state, suggesting an increased propensity for fat accumulation. The role of the clock was further investigated in adipose tissue by deletion of Bmal1 (clock ablation) or Rev-erbα (clock manipulation) specifically in adipocytes using Cre-Lox methodology. AdipoCREBmal1flox/flox mice showed attenuated feeding rhythms, indicating a direct effect of the adipocyte circadian clock on hypothalamic feeding centres and severe dysregulation of metabolic genes. However, AdipoCRERev-erbαflox/flox displayed very little phenotypic difference compared to control littermates, suggesting that global loss of Rev-erbα may have reinforcing metabolic consequences. This work suggests a key role of the clock in lipid handling and the pathogenesis of obesity. Insights into this link may lead to novel targets for treating both obesity and metabolic complications. 10