Functional analysis of the adrenal circadian clock [Elektronische Ressource] / Silke Kießling

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Functional Analysis of the Adrenal Circadian Clock Von der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades einer Doktorin der Naturwissenschaften Dr. rer. nat. genehmigte Dissertation von Dipl.-Biol. Silke Kießling geboren am 22. Juli 1980, in Bremen 2010 Referent: Prof. Dr. Stephan Steinlechner Korreferent: Prof. Dr. Gregor Eichele Tag der Promotion: 5.5.2010 Meinen Eltern “Zeit ist das, was wir haben, wenn wir unsere Uhren wegwerfen” Jürgen Aschoff (Begründer der Chronobiologie) 4 CONTENTS Contents Figure & table list 7 Summary 9 Zusammenfassung 11 Abbreviations 13 1. Introduction 15 1.1. The biological clock 15 1.2. Properties of biological oscillators 16 1.3. A central pacemaker in the brain – the suprachiasmatic nuclei 17 1.3. Clock input 17 1 Meularclokwork /TL 191.3.2Clock otpu gens 21 1.3.13. Clock mutants / Clock nockuts 21.3.2. SCN output 22 1.4. Peripheral oscillators 23 1.4.1. Synchronization between SCN and peripheral clocks 24 1.4.1.2. SCN-adrenal connections 26 1.5. Glucocortoicoids (GCs) 27 1.5.1.

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Functional Analysis of the Adrenal Circadian Clock



Von der Naturwissenschaftlichen Fakultät der
Gottfried Wilhelm Leibniz Universität Hannover
zur Erlangung des Grades einer
Doktorin der Naturwissenschaften
Dr. rer. nat.
genehmigte Dissertation
von
Dipl.-Biol.
Silke Kießling
geboren am 22. Juli 1980, in Bremen

2010























Referent: Prof. Dr. Stephan Steinlechner
Korreferent: Prof. Dr. Gregor Eichele
Tag der Promotion: 5.5.2010




































Meinen Eltern












“Zeit ist das, was wir haben, wenn wir unsere Uhren wegwerfen”
Jürgen Aschoff (Begründer der Chronobiologie)















4 CONTENTS 
Contents

Figure & table list 7
Summary 9
Zusammenfassung 11
Abbreviations 13

1. Introduction 15

1.1. The biological clock 15
1.2. Properties of biological oscillators 16
1.3. A central pacemaker in the brain – the suprachiasmatic nuclei 17
1.3. Clock input 17 1 Meularclokwork /TL 19
1.3.2Clock otpu gens 21
1.3.13. Clock mutants / Clock nockuts 2
1.3.2. SCN output 22

1.4. Peripheral oscillators 23
1.4.1. Synchronization between SCN and peripheral clocks 24
1.4.1.2. SCN-adrenal connections 26

1.5. Glucocortoicoids (GCs) 27
1.5.1. Glucocorticoids and the circadian clock 27
1.52. Adrenal gand antomy 28
1.5.21. Cortex 2
1.52.3 Medula 9
1.5.3Glucocorticoid biosynthesis 29
1.5.3.1. Supply of cholesterol 29
1.5.32.Glucocorticoid biosynthesis pathway 30
1.5.3.4. Regulation of glucocorticoid biosynthesis 31

1.6 Clock disordes 31
1.6.1. Metabolic effects 31
1.6.2. Jet lag 31

1.7. Aims 2

5 CONTENTS 
2. Results 34

2.1. Publication: “The circadian rhythm of glucocorticoids
is regulated by a gating mechanism residing in the
adrenal cortical ock” 34

2.2. Publication: “A role for adrenal glucocorticoids in the
circadian resynchronization during jet lag” 67

2.3 Aditonal dat 93
2.3.1. Surgery and age related effects on jet lag 93
2.3.2. Impact of adrenal clock function on period length
in constant conditions 94
2.3.3. Impact of adrenal clock function on activity distribution in LD 98
2.3.4. Impact of adrenal clock function on resetting during jet lag 102
2.3.4.1. Activity monitoring 102
2.3.4.2. Corticosterone excretion rhythms 103


3. Conclusion & perspectives 107

3.1. The role of the adrenal clock in the generation of GC rhythms 107
3.2. Photic entrainment of peripheral oscillators 109
3.3. Stabilizing feedback from the adrenal to the SCN 110
3.4. The adrenal clock has an impact on resetting 112
3.5. Outlook: adrenal corticoids and other clocks 113


4. Material & methods 114

4.1 Animal handling and sample collection 114
4.1.1. Mouse strains 114
4.2. Animal experiments 115
4.2.1. Activity monitoring 115
4.2.1.1. Light/dark (LD) cycle entrainment 115
4.2.1.2. Free-running in constant darkness 116
4.2.1.3. gconstantlight
4.2.1.4. Shifted LD cycles – jet lag 116
4.2.2. Adrenal transplantation 117
4.2.3.Hormone measurements and pharmacological treatments 117
4.2.3.1. Corticosterone extraction from feces 117
4.2.3.2. Corticosterone and ACTH extraction from plasma 118
4.2.3.3. Quantification of hormone metabolites 118
4.2.4. Pharmacological treatments 118
4.2.5.Tissue collection 119
6 CONTENTS 
4.3. Molecular biological methods 119
4.3.1. Genotyping 119
4.3.1.1. DNA extraction 119
4.3.1.2. Polymerase chain reaction (PCR) protocols 119
4.3.2. RNA extraction and RNA purification 120
4.3.3.cDNA synthesis 121
4.3.3.1. denaturation
4.3.3.2. Revers transcription reaction mix 121
4.3.3.3. RNA template removal 121
4.3.4. Quantitative real-time PCR (qPCR)
4.3.4.1. Primer sequences
4.3.4.2. Standard curve efficiency estimation 122
4.3.4.3. Assay protocoll 122
4.3.4.4. Data analysis 122

4.4. Histological methods 123
4.4.1. Radioactive in situ hybridization (ISH) 123
4.4.4.1. cDNA templates 123
4.4.4.2. RNA probes 123
4.4.4.3. Tissue preparation and paraffin sections 124
4.4.4.4. Hybridization 125
4.4.4.5. Post-hybridization 125
4.4.4.6. Quantification 126

4.5. Immunological methods 127
4.5.1. Radio-immuno assay (RIA) 127
4.5.1.1. Corticosterone RIA 127
4.5.1.2. ACTH RIA 128

4.6 Dat anlysi 128
4.6.1. Sine wave fitting 128
4.6.2.Correlation analysis 129
4.6.3. Group comparisons


5. References 130

6. Curriculum vitae 142

7. Acknowledgemnts 143


7 FIGURE & TABLE LIST 
Figure List
1. Anatomical structure of the photorecptors of the retina. 17
2. Photic input and the signal transduction cascades in the SCN neuron. 18
3. Model of the mammalian circadian clockwork within an individual SCN neuron. 20
4. The efferent projections of the suprachiasmatic nucleus. 22
5. Interaction between peripheral tissues and SCN, an illustration of the
main pathways by which periphery and central clocks might communicate
with the central nervous system. 23
6. Hierarchical organization of circadian clocks. 25
7. Neural pathways in circadian control of glucocorticoid release. 26
8. Histological profile of the adrenal gland 28
9. Mechanism for ACTH induced glucocorticoid synthesis. 29
10. Steroid hormone synthesis. 30
11. Robust circadian expresson of clock genes in the murine adrenal cortex. 57
12. Per2/Cry1 mutant mice are defective in adrenal clock gene and
HPA axis rhytmicty. 58
13. A peripheral clockwork residing in the adrenal cortex gates the ability of
ACTH toevoke corticosterone rleas. 59
14. Circadian clock regulatd genes involved in the control of adrenal
corticosterone biosynthesis. 60
15. Rhythmic expression of genes encoding regulators of steroidogenesis. 61
16. The functionality of the adrenal clock does neither affect Per1 rhythms
in the SCN nor locomotor activity. 62
17. HPA axis regulation and adrenal clock gene expression in adrenorecipient mice. 63
18. Behavioral entrainment during jet lag. 85
19. Resetting of clock genes during jet lag in the SCN. 86
20. Clock gene resetting kinetics in several peripheral tissues following a
6 hrs LDphase advnce. 87
21.Influence of adrenal clock function on activity re-entrainment after a
6 hrs phase advance of the LD cycle. 88
22. Shifting corticosterone rhythms prior to jet lag affects behavioral
reseting kinetics. 89
23. Transient desynchronization within and between tissues during jet lag. 90
24. Surgery effects on behavioral resetting at different times after surgery 93
WT WT25. Activity profiles of auto-grafted wild-type animals (h /a ) and wild-type
WT P2/C1hosts with Per2/Cry1 mutant adrenals (h /a ). 95
WT WT WT P2/C126. Free-running period of h /a and h /a mice under constant conditions. 96
27. Unexpected activity of entrainment behavior during first days in LD. 97
28. Activity periodograms in constant light (LL). 98
29. Activity patterns in LD of the different cross-genotypes. 99
30. y profiles of the wild-type host cross-genotype and its controls in LD. 99
31. Activity profiles of the mutant host cross-genotype and its control group in LD. 100
32. Total, diurnal and nocturnal activity levels. 100
33. Diurnal distribution of running-wheel activity (in 3 hrs intervals) of the
different cross-genotypes. 101
34. Representative double-plotted actograms of the different crossgenotypes
before, during and after a 6 hrs phase advance of the LD cycle. 102
35. Resetting kinetics of activity onsets during jet lag. 103
36. Wild-type plasma ACTH concentrations at different days during jet lag. 104
37. asma corticosterone concentrations at different days during jet lag. 104
38. Wild-type fecal corticosterone excretion rhythms at different days
during jet lag. 105 8 FIGURE & TABLE LIST 
39. Fecal corticoid excretion rhythms in the different cross-genotypes
at different days during jet lag. 105
40. Resetting kinetics of corticosterone excretion maxima peak times
during jet lag. 106
41. Role of the adrenal clock in circadian glucocorticoid release. 108
42. Possible adrenal feedback pathways affecting SCN-controlled
activity behavior. 111
43. Targeted disruption of Per2 and Cry1 genes in the mouse. 116

Supplemental figure list

1. Clock gene expression in the kidneys of adrenorecipient mice. 64
2. Resetting of clock genes during jet lag in peripheral oscillators. 91
3. Adrenal control of resetting of corticosterone excretion during jet lag 92

Table list

WT WT1. τ in DD and LL of h /a 96
WTP2C1 2. τ /a 96
3. Primer pairs used for the quantification of clock gene mRNAs,
product size and Entrez Gene ID. 121
4. Step by step protocol and pipetting scheme to quantify
fecal and plasma corticosterone concentrations. 127
5. Step by step protocol and pipetting scheme to quantify plasma
ACTH concentrations. 128

Supplemental table list

1. Regulators of corticosterone biosynthesis showing a circadian
expression rhythm in the adrenal gland. 65
2. Primer pairs used for the generation of cDNA templates for in
situ hybridization probes and for qPCR. 669 SUMMARY 
Summary
Keywords: Adrenal Circadian Clock, Glucocorticoids, Jet lag

The daily rotation of the Earth around its axis influences all live on our planet, from
unicellular organisms to humans. Endogenous clocks have evolved to anticipate and
synchronize physiology and behavior to daily recurring environmental changes
(“entrainment”). In mammals, a central circadian pacemaker is localized in the
suprachiasmatic nuclei (SCN) of the hypothalamus, directly entrained by light. The SCN
orchestrate subordinate clocks found throughout the body – via both humoral and neuronal
pathways – to coordinate the overall entrainment of the organism. In the absence of
external time information circadian rhythms are sustained with an endogenous period length
of approximately 24 hrs (hence the term circadian: lat. “circa” = approximately and “dies” =
day). Environmental timing cues (so called Zeitgeber) like the light/dark cycle can phase shift
the endogenous oscillator to synchronize the animal to external time. Circadian clocks are
built from a set of so-called “clock genes” organized in a system of transcriptional/
translational feedback loops and creating stabilized transcriptional rhythms of ca. 24 hrs
period.
In the first project we characterize the function of the circadian clock in the adrenal, an
important endocrine gland that synchronizes physiological and metabolic rhythms via the
regulation of circadian glucocorticoid (GC) synthesis. Using a transgenic mouse model with a
combined disruption of specific “clock” genes (Per2 and Cry1) that lacks circadian clock
function, we analyzed the impact of either the SCN or the adrenal clock in the regulation of
GC rhythms. We show that the adrenal pacemaker, which itself is light entrainable,
modulates/gates rather than controls the glucocorticoid production in response to a
hormonal signal (ACTH), ultimately controlled by the SCN.
The second project addresses the molecular events underlying circadian disruption during
jet lag. Rapid travel across several time zones causes jet lag that is characterized by a
transient disturbance of numerous physiological parameters such as the sleep-wake cycle,
hormone levels and metabolism. To better understand the molecular manifestations of jet
lag, we monitored central and peripheral circadian clocks under jet lag conditions in an
attempt to identify factors that regulate the adaptation of the circadian timing system to a
new time zone. We show that jet lag in mice causes a transient disruption of multiple core
clock genes, on the level of the SCN itself and in a similar fashion in peripheral oscillators. In
addition we observed transient de-synchronization between different tissues. This global