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Molecular analysis of the HIV therapy associated lipodystrophy syndrome [Elektronische Ressource] / von Metodi Vasilev Stankov

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Molecular analysis of the HIV-therapy associated lipodystrophy-syndrome Der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität Hannover zur Erlangung des Grades Doktor der Naturwissenschaften Dr. rer. nat genehmigte Dissertation von Metodi Vasilev Stankov, Dipl. Mol. Biol. geboren am 24.01.1971 in Sofia, Bulgarien 2007 Referent: Prof Dr.rer.nat. Walter Müller Coreferent: Prof. Dr. Georg M.N. Behrens Day of Promotion: 09.02.2007 2 ABBREVIATIONS .......................................................................................................... 7 ABSTRACT.................................................................................................................... 11 ZUSAMMENFASSUNG ............................................................................................... 13 1 INTRODUCTION ....................................................................................................... 15 2 REVIEW OF THE LITERATURE ............................................................................. 16 2.1 HAART associated lipodystrophy syndrome ........................................................... 16 2.1.1 Morphological alterations...................................................................................... 16 2.1.2 Metabolic alterations.............................................................................................. 16 2.1.

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Published 01 January 2007
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Molecular analysis of the
HIV-therapy associated lipodystrophy-syndrome








Der Naturwissenschaftlichen Fakultät der Gottfried Wilhelm Leibniz Universität
Hannover

zur Erlangung des Grades

Doktor der Naturwissenschaften
Dr. rer. nat


genehmigte Dissertation
von
Metodi Vasilev Stankov, Dipl. Mol. Biol.
geboren am 24.01.1971 in Sofia, Bulgarien
2007
Referent: Prof Dr.rer.nat. Walter Müller
Coreferent: Prof. Dr. Georg M.N. Behrens
Day of Promotion: 09.02.2007
2 ABBREVIATIONS .......................................................................................................... 7
ABSTRACT.................................................................................................................... 11
ZUSAMMENFASSUNG ............................................................................................... 13
1 INTRODUCTION ....................................................................................................... 15
2 REVIEW OF THE LITERATURE ............................................................................. 16
2.1 HAART associated lipodystrophy syndrome ........................................................... 16
2.1.1 Morphological alterations...................................................................................... 16
2.1.2 Metabolic alterations.............................................................................................. 16
2.1.3 Factors associated with the development of HAART-associated lipodystrophy .. 16
2.1.4 Proposed pathophysiological mechanisms of lipodystrophy syndrome ................ 17
2.1.4.1 Retinoic acid binding protein-1 (CRABP-1) mediated inhibition of adipocyte
differentiation.................................................................................................................. 17
2.1.4.2 Lamin A/C disturbances ..................................................................................... 17
2.1.4.3 Local changes in glucocorticoid concentrations................................................. 17
2.1.4.4 PI induced peripheral adipocyte apoptosis in HIV patients with lipodystrophy 18
2.1.4.5 PI inhibited differentiation.................................................................................. 18
2.1.4.6 NRTI induced mitochondrial toxicity................................................................. 18
2.1.4.6.1 Depletion of mtDNA ....................................................................................... 18
2.1.4.6.2 Depletion of mRNA......................................................................................... 20
2.1.4.6.3 Disturbances of the nucleotide pool 21
2.1.4.6.4 Mutations ......................................................................................................... 22
2.1.4.6.5 Physical interference........................................................................................ 22
2.1.4.7 Cytokines and adipocytokines ............................................................................ 22
2.1.4.8 HAART induced selective autonomic neuropathy ............................................. 24
2.2 Adipose tissue........................................................................................................... 24
2.2.1 Functions................................................................................................................ 24
2.2.1.1 Secretory function............................................................................................... 24
2.2.1.2 Metabolic function.............................................................................................. 25
2.2.2 Adipose tissue heterogeneity and plasticity........................................................... 26
2.2.3 Origin of adipocytes 26
2.2.4 Adipocyte differentiation....................................................................................... 26
2.3 Adipocyte and HAART ............................................................................................ 32
2.3.1 Effects of PI ........................................................................................................... 32
2.3.2 Effects of NRTI ..................................................................................................... 33
3 2.3.2.1 Effects of NRTI on adipogenesis........................................................................ 33
2.3.2.2 Effects of NRTI on mtDNA................................................................................ 33
2.3.2.3 Effeitochondria (besides mtDNA depletion).......................... 34
2.3.3 Other factors contributing to mitochondrial dysfunction in WAT ........................ 34
2.4 Metabolic complications........................................................................................... 34
2.4.1 Insulin resistance.................................................................................................... 34
2.4.2 Dyslipidemia.......................................................................................................... 35
3 AIMS OF THE STUDY .............................................................................................. 36
4 MATERIALS AND METODS ................................................................................... 37
4.1 MATERIALS............................................................................................................ 37
4.2 METODS.................................................................................................................. 51
4.2.1 DNA Isolation........................................................................................................ 51
4.2.2 Total RNA Isolation............................................................................................... 52
4.2.2.1 Total RNA Isolation from lipid tissues............................................................... 52
4.2.2.2 Total RNA isolation from no lipid tissues and cells........................................... 52
4.2.3 Reverse Transcription............................................................................................ 53
4.2.4 Real-Time PCR...................................................................................................... 54
4.2.5 ELISA .................................................................................................................... 57
4.2.6 Cell lines ................................................................................................................ 59
4.2.7 Oil Red O staining ................................................................................................. 63
4.2.8 Mitochondrial functions......................................................................................... 63
4.2.8.1 Membrane potential ............................................................................................ 63
4.2.8.2 Mitochondrial mass............................................................................................. 64
4.2.9 Propidium iodide staining...................................................................................... 64
4.2.10 Enzyme activities 65
4.2.11 Cloning................................................................................................................. 69
4.2.12 Animals................................................................................................................ 72
4.2.13 Drug administration 73
4.2.14 Assessment of body fat mass distribution............................................................ 73
4.2.15 Collection of tissues............................................................................................. 73
4.2.16 Glucose tolerance test (GTT)............................................................................... 73
34.2.17 H thymidine Assay 74
4.2.18 Statistical methods ............................................................................................... 74
5 RESULTS .................................................................................................................... 75
4 5.1 Relationship of mitochondrial DNA depletion and respiratory chain activity in
preadipocytes treated with nucleoside-analogue reverse transcriptase inhibitors .......... 75
5.1.1 Influence of NRTI on adipocyte morphology, differentiation, and triglycerol
accumulation................................................................................................................... 75
5.1.2 Molecular analysis of adipocyte differentiation. ................................................... 75
5.1.3 Effect of NRTI on adiponectin production ............................................................ 77
5.1.4 Effect of NRTI on mtDNA content in non-proliferating cells............................... 79
5.1.5 Effect of NRTI on mttent in differentiating or proliferating preadipocytes
........................................................................................................................................ 79
5.1.6 Effect of NRTI on mtDNA content in HUH 7 cell line......................................... 80
5.1.7. Association of mtDNA depletion with respiratory chain function....................... 82
5.1.8 Effect of NRTI on mtDNA content in human subcutaneous fibroblasts............... 83
5.1.9 Association of mtith respiratory chain function in.................... 84
5.1.10 NRTI effect on mitochondrial mass and function in primary human fibroblasts 85
5.1.11 Effect of NRTI on mtDNA content in human primary preadipocytes ................ 87
5.1.12 Association of mtDNA depletion with respiratory chain function in human
primary preadipocytes..................................................................................................... 89
5.2 Zidovudine inhibits clonal expansion and adipogenic differentiation of 3T3-L1 cells
........................................................................................................................................ 89
5.2.1 AZT perturbs adipocyte mass through an effect on differentiation-dependent TG
accumulation................................................................................................................... 89
5.2.2 Mitotic clonal expansion is affected by AZT. ....................................................... 90
5.2.3 Precursors proliferation is affected by AZT. ......................................................... 91
5.2.4 Effects of AZT on adipogenic marker expression................................................. 93
5.2.5 Impaired differentiation is confirmed by decrease in adipokines expression and
secretion.......................................................................................................................... 93
5.2.6 AZT effect on adipocyte viability.......................................................................... 94
5.3 Adipocyte viability and function but not preadipocyte differentiation is
compromised by IDV...................................................................................................... 96
5.3.1 IDV affects adipocyte mass but does not affect differentiation-dependent
accumulation of TG. ....................................................................................................... 96
5.3.2 IDV does not affect mitotic clonal expansion ....................................................... 96
5.3.3 Effects of IDV on adipogenic marker expression.................................................. 98
5.3.4 IDV perturbs adipocyte function ......................................................................... 100
5 5.3.5 IDV promotes loss of adipocyte viability............................................................ 100
5.3.6 In vivo analyse of HAART on adiponectin production. ...................................... 102
5.4 Lipoatrophy and ubiquitous mtDNA depletion in mice following long-term NRTI
traement ........................................................................................................................ 102
6.4.1 Effects of AZT and d4T on fat amount and distribution in treated mice............. 102
5.4.2 Effect of d4T and AZT on of body weight acquisition, in young male C57BL/6
mice............................................................................................................................... 104
5.4.3 Effect of d4T and AZT on water and food consumption..................................... 104
d4T and AZT ................................................................................................................ 104
5.4.4 Effects of therapeutic drug levels d4T on mtDNA content in different tissues... 104
5.4.5 Effect of d4T on adipokine production................................................................ 105
5.4.6 Effect of AZT and d4T on mouse insulin sensitivity........................................... 106
6 DISCUSSION............................................................................................................ 107
REFERENCES ............................................................................................................. 118
RESEARCH CONTRIBUTIONS ................................................................................ 142
PERSONAL DATA...................................................................................................... 144
ACKNOWLEDGEMENTS.......................................................................................... 146
Declaration on the dissertation ..................................................................................... 147

Key words: lipodystrophy, HIV, highly active antiretroviral therapy (HAART)
Schlagwörter: lipodystrophie, HIV, hochaktiven antiretroviralen therapie (HAART)
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ABBREVIATIONS
ATP Adenosine triphosphate
ADP Adenosine diphosphate
AMP Adenosine monophosphate
AMPK Adenosine monophosphate kinase
A-2-P L-Ascorbic acid 2-phosphate
sesquimagnesium salt hydrate

aP2 Fatty acid binding protein
Akt (PKB) Protein kinase B
AZT Zidovudine
BSA Bovine serum albumin
BCA Bicinchoninic acid
C/EBP CCAAT/enhancer-binding protein
CPT1 Carnitine palmitoyltransferase 1
CHD Coronary heart disease
Carbonyl cyanide m-CCCP
chlorophenylhydrazone

CD36 Transporter of fatty acids
d4T Stavudine
DHEA Dihydroepiandrosterone
dTTP Deoxythymidine triphosphate
Deoxycytosine triphosphate dCTP
dUTP Deoxyuridine triphosphate
dsDNA Double-stranded DNA
DMSO Dimethyl sulfoxide
DMEM Dulbecco's modified eagle medium
7 Deoxyribonucleotide triphosphate dNTP
DOC Dissolved organic carbon
ddC Zalcitabine
ELISA Enzyme-linked immunosorbent assay
EDTA Ethylenediaminetetraacetic acid
Earle's BSS Earle's balanced salt solution
EGTA Ethylene glycol bis( β-aminoethyl ether)-
N,N,N’,N-tetraacetate

ERK MAP-kinase
ECM Extracellular matrix
FAS Fatty acid synthase
FFA Free fatty acids
FBS Fetal bovine serum
FACS Fluorescence activated cell sorting
FP Prostaglandin F receptor
Fetal calf serum FCS
GH Growth hormone
HAART Highly active antiretroviral therapy
HDL High-density lipoprotein
HIV Human immunodeficiency virus
HBV Hepatitis B virus
HSL Hormone sensitive lipase
HCV Hepatitis C virus
IFN Interferon
IDV Indinavir
Interleukin IL
IBMX 3-Isobutyl-1-methylxanthine
8 Isopropyl- β-D-thiogalactopyranosid IPTG
IR Insulin resistance
IGF Insulin-like growth factor
JNK MAP kinase
LPL Lipoprotein lipase
LDH Lactate dehydrogenase
LB Luria-Bertani
LDL Low density lipoprotein
LRP Low-density lipoprotein receptor–like
protein

mtDNA Mitochondrial DNA
mRNA Messanger RNA
MRC Mitochondrial respiratory chain
MTR Mito Tracker Red
NNRTI Nonnucleoside reverse transcriptase
inhibitor

NFV Nelfinavir
NRTI Nucleoside reverse transcriptase inhibitor
NADH Reduced nicotinamide adenine
dinucleotide

OD Optical density
PI Protease inhibitor
PPAR Peroxisome proliferator-activated receptor
PAI1 Plasminogen activator inhibitor-1
PBMC Peripheral blood mononuclear cell
PCR Polymerase chain reaction
PBS Phosphate buffered saline
K-phosphoenolpyruvate PEP
9 Preadipocyte factor pref
PS Penicillin/streptomycin
PEPCK Phosphoenolpyruvate carboxykinase
PDGF Platelet-derived growth factor
PG Prostaglandin
Rpm Revelations per minute
PK Pyruvate kinase
RTV Ritonavir
ROS Reactive oxygen species
RA Retinoic acid
Reverse transcription RT
SREBP Sterol regulatory element-binding protein
SQV Saquinavir
SDS Sodium dodecyl sulfate
SCD Stearoyl-CoA desaturase
SAT Subcutaneous adipose tissue
TNF Tumor necrosis factor
TNF-R Tumor necrosis factor-reseptor
TZD Thiazolidinedione
TG Triglycerides
Thymidine kinase TK
TGF Transforming growth factor
WAT White adipose tissue
X-Gal 5-bromo-4-chloro-3-indolyl-beta-D-
galactopyranoside


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