The role of the clock in lipid metabolism

280 Pages·2017·5.87 MB·English

by  Siobhan Ahern

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

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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
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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
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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
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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

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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

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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

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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

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List of tables

Chapter 2
Table 2.1 – Primer Sequences p.93

Table 2.2 – Antibody Weights and Concentrations p.95

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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.
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