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

Metodi Vasilev Stankov

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• Publié par: gottfried_wilhelm_leibniz_universitat_hannover
• Publié le: 01 janvier 2007
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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 Associat