The role of fat cell apoptosis during obesity-associated adipose tissue inflammation [Elektronische Ressource] / Michaela Keuper

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• Publié par: universitat_ulm
• Publié le: 01 janvier 2011
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 3 Mo

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Dissertation
Michaela Keuper
Ulm University
The role of fat cell apoptosis
during obesity-associated
adipose tissue inflammation
2011The role of fat cell apoptosis
during obesity-associated
adipose tissue inflammation
Dissertation
zur Erlangung des Doktorgrades Dr. rer. nat.
der Fakultät für Naturwissenschaften
der Universität Ulm
vorgelegt von
Michaela Keuper
aus Hilden
Ulm 2011 Prof. Dr. Axel Groß
Amtierender Dekan: _________________________________
Prof. Dr. Klaus-Dieter Spindler
1. Gutachter:
PD Dr. Frank Rosenau
2. Gutachter: _________________________________
Prof. Dr. Martin WabitschWeitere Gutachter:
07.11.2011Tag der Promotion: _________________________________CONTENTS



































The work for this PhD-thesis was carried out in the




Division of Pediatric
Endocrinology and Diabetology
Department of Pediatrics and Adolescent Medicine
Ulm University




and it is based on the following publication:

Keuper, M. et al.
An inflammatory micro-environment promotes human adipocyte apoptosis.
Mol. Cell. Endocrinol 339, 105-113 (2011)


CONTENTS
Contents
Abbreviations ....................................................................................................... iv
1 Introduction.................................................................................................... 1
1.1 Obesity................................................................................................. 1
1.2 Adipose tissue ..................................................................................... 2
1.2.1 Adipose tissue - an endocrine organ ............................................ 2
1.2.2 Adipose tissue - a metabolic organ............................................... 4
1.2.2.1 Insulin-signaling in adipocytes 5
1.2.3 Adipose tissue - a dynamic organ................................................. 7
1.3 Apoptosis ............................................................................................. 8
1.3.1 Fat cell apoptosis........................................................................ 10
1.4 Chronic low grade inflammation......................................................... 11
1.5 Aim of the study ................................................................................. 13
2 Materials and Methods ................................................................................ 14
2.1 Materials ............................................................................................ 14
2.1.1 Biological material....................................................................... 14
2.1.2 Experimental subjects................................................................. 14
2.1.3 Equipment .................................................................................. 14
2.1.4 Reagents/ Chemicals/ growth factors ......................................... 15
2.1.5 Radioactive chemicals................................................................ 17
2.1.6 Enzymes/ Kits/ Vectors............................................................... 17
2.1.7 Cell Culture media ...................................................................... 17
2.1.7.1 SGBS media........................................................................ 17
2.1.7.2 THP-1 media ....................................................................... 18
2.1.8 Buffer solutions........................................................................... 19
2.1.9 Primers ....................................................................................... 20
2.1.10 Software ..................................................................................... 20
2.1.11 Antibodies................................................................................... 21
2.2 Methods............................................................................................. 22
2.2.1 Cell biology methods .................................................................. 22
2.2.2 Metabolic assays ........................................................................ 23
2.2.3 Protein biochemical methods...................................................... 24
iCONTENTS
2.2.4 Molecular-biological methods ..................................................... 26
2.2.5 Immunostaining procedures ....................................................... 28
2.2.6 Apoptosis detection .................................................................... 29
2.2.7 Statistics ..................................................................................... 30
3 Results.......................................................................................................... 31
3.1 Detection of macrophages and apoptotic adipocytes in vivo ............. 31
3.2 Establishment of an in vitro model for human adipose tissue
inflammation ................................................................................................. 33
3.3 Effects of an inflammatory micro-environment on adipocyte biology . 35
3.3.1 Effect of macrophage-secreted factors on adipogenic
differentiation ............................................................................................ 36
3.3.2 Effect of macrophage-secreted factors on insulin-sensitivity ...... 38
3.3.3ctors on insulin-signaling........ 40
3.4 Effect of an inflammatory micro-environment on fat cell apoptosis .... 43
3.4.1 Effect of macrophage-secreted factors on fat cell apoptosis ...... 43
3.4.2 Effect of direct co-culture on fat cell apoptosis ........................... 45
3.5 Inhibition of Insulin signaling (Akt) ..................................................... 49
3.5.1 Pharmacological inhibition of Akt................................................ 49
3.5.2 Genetic inhibition of Akt2 ............................................................ 50
4 Discussion.................................................................................................... 52
4.1 Inflammation decreases insulin sensitivity and increases apoptosis
sensitivity...................................................................................................... 52
4.2 Role of Akt2 ....................................................................................... 53
4.3 Macrophages create an inflammatory micro-environment that
promotes adipocyte apoptosis...................................................................... 54
4.4 Adipocyte death in vivo 55
4.5 In vitro model for human inflamed adipose tissue.............................. 56
4.5.1 Improvements of our human model system................................ 56
4.6 Apoptosis as initiator of adipose tissue inflammation?....................... 58
4.7 Conclusion ......................................................................................... 59
4.8 Perspective – clinical implications...................................................... 60
5 Summary....................................................................................................... 64
6 References.................................................................................................... 65
iiCONTENTS
List of Tables....................................................................................................... 76
List of Figures ..................................................................................................... 76
Appendix A .......................................................................................................... 78 B 80
Acknowledgements ............................................................................................ 83
Curriculum Vitae ................................................................................................. 84


iii ABBREVIATIONS
Abbreviations
ACC acetyl-CoA carboxylase
Apaf-1 apoptotic protease activating factor - 1
ATGL adipose triglyceride lipase
ATM e tissue macrophage
BAT brown adipose tissue
Bid BH3-interacting domain death agonist
BMI body mass index
bp base pare
C/EBP Ccaat-enhancer-binding proteins
Caspase cysteine-dependent aspartate-specific protease
cFLIP cellular FLICE-like inhibitory protein
Cyt C cytochrome C
DISC death-inducing signaling complex
DNA deoxyribonucleic acid
ERK extracellular-signal regulated kinase (p44/42 MAPK)
FA fatty acid
FABP4 fatty acid binding protein 4
FACS fluorescence-activated cell sorting
FADD Fas-associated death domain
FASN fatty acid synthase
GAPDH glyceraldehyde 3-phosphat dehydrogenase
GLUT4 glucose transporter type 4
GSK3 glycogen synthase kinase 3
HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
HRS hyper random sequence
HSL hormone sensitive lipase
IFN- interferon-
IGF insulin-like growth factor
IGF-1 e growth factor 1
IGF-1R insulin-like growth factor 1 receptor
IKK- IB kinase-beta
IL- interleukin-
Ins insulin
IR receptor
JNK1 c-Jun N-terminal kinase 1
kDa kilo