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

93 pages
DissertationMichaela KeuperUlm UniversityThe role of fat cell apoptosis during obesity-associated adipose tissue inflammation2011The role of fat cell apoptosis during obesity-associated adipose tissue inflammation Dissertationzur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Naturwissenschaften der Universität Ulmvorgelegt von Michaela Keuper aus Hilden Ulm 2011 Prof. Dr. Axel GroßAmtierender Dekan: _________________________________Prof. Dr. Klaus-Dieter Spindler1. Gutachter: PD Dr. Frank Rosenau2. 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) CONTENTSContentsAbbreviations ....................................................................................................... iv1 Introduction.................................................................................................... 11.1 Obesity.................................................................................
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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 dalton
KD cells knock down cells
KO mice knock out mice
MacCM macrophage-conditioned medium
MAGL monoacylglyceride lipase
MAPK mitogen-activated protein kinase
MCP-1 monocyte chemotactic protein 1
MES 2-(N-morpholino) ethanesulfonic acid
MFI mean fluorescent intensity
MOI multiplicity of infection
iv
NEABBREVIATIONS
MOPS 3-(N-morpholino) propanesulfonic acid
MonCM monocyte-conditioned medium
NF- B nuclear factor ’kappa-light-chain-enhancer’ of activated B-cells
nSREBP-1c nuclear form of sterol regulatory element-binding protein-1c
o/n over night
p70S6K 70-kDa S6 kinase
PBS phosphate buffered saline
PCR polymerase chain reaction
qPCR quantitative real-time PCR
PDC pyruvate dehydrogenase complex
PGC-1 PPAR coactivator 1
PI3K phosphatidylinositol 3-kinase
PKB protein kinase B (Akt)
PPAR peroxisome proliferator-activated receptor gamma
RNA Ribonucleic Acid
RNAi RNA interference
ROS reactive oxygen species
RSK ribosomal S6 kinase
RT room temperature
SD standard deviation
SDHA succinate dehydrogenase complex subunit A
SEM standard error of mean
SGBS simpsom golabi behmel syndrome
shRNA short hairpin RNA
Sir silent information regulator
tBid truncated BH3-interacting domain death agonist
TBS Tris-buffered saline
TBS-T Tris-buffered saline and Tween 20
TG triglyceride
TNF tumor necrosis factor
TRAIL TNF-related apoptosis inducing ligand
UCP-1 uncoupling protein-1
WAT white adipose tissue
WHO world health organisation
v
NINTRODUCTION
1 Introduction
1.1 Obesity
Obesity is defined as an abnormal or excessive accumulation of body fat that
presents a risk to health (WHO, 2011). A very simplified and commonly used
measurement for obesity is the body mass index (BMI), a person’s weight (in
kilograms) divided by the square of the height (in meters). The World Health
Organization (WHO) defines "overweight" as a BMI equal to or more than 25
kg/m², and "obesity" as a BMI equal to or more than 30 kg/m² (WHO, 2007).
The prevalence of obesity is increasing in an epidemic manner. Formerly just
associated with high-income countries, obesity found its way to low- and
middle-income countries. According to the WHO in 2008, 1.5 billion adults were
overweight, of these more than 200 million men and almost 300 million women
were obese (WHO, 2011).
The excessive accumulation of adipose tissue gives rise to a variety of
diseases, particularly cardiovascular diseases, diabetes mellitus type 2, hepatic
steatosis, obstructive sleep apnea, cancer, and osteoarthritis (Blüher, 2009). As
a result, obesity reduces life expectancy (Symonds, 2010) and at least 2.8
million people die each year as a result of being overweight or obese (WHO,
2011). Although obesity is preventable, it is worldwide a leading cause of death.
Obesity is increasing in adults and children, therefore it is regarded as the most
serious public health problem of this century (Wilkin and Voss, 2004).
The cause for obesity besides medical or psychiatric (Barness et al., 2007)
reasons might be a genetic predisposition. Twin studies, for instance, estimated
the average heritability of obesity in the range between 40-75% (O’Rahilly and
Farooqi, 2008). Additionally, genome-wide association studies revealed several
genes with single nucleotide polymorphisms (SNPs) that associate with obesity.
However, results are only consistent for some genes, e.g. melanocortin receptor
4 (MC4R) (Vaisse et al., 1998; Yeo et al., 1998) or fat mass and obesity
associated (FTO) (Frayling et al., 2007). Nevertheless, the fundamental cause
1

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