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Regulation of Endoplasmic Reticulum Stress in Adipose Tissue Metabolism By Philip Demos Voyias, BSc Hons A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Medical Sciences Division of Translational and Systems Medicine, Warwick Medical School, University of Warwick June 2015 1 Table of Contents Abbreviations ....................................................................................................... 8 List of tables ........................................................................................................ 15 List of figures ...................................................................................................... 15 Acknowledgements ............................................................................................. 18 Declaration .......................................................................................................... 20 List of publications ............................................................................................. 21 Abstract ............................................................................................................... 22 Chapter 1: Introduction .................................................................................... 23 Introduction .................................................................................................... 24 1.1 Type II diabetes mellitus .......................................................................... 25 1.1.1 Definition of diabetes mellitus ............................................................. 25 1.1.2 Clinical sequelae of type II diabetes mellitus ...................................... 25 1.1.3 Epidemiology of type II diabetes mellitus ........................................... 26 1.1.4 Obesity and type II diabetes mellitus ................................................... 28 1.2 Adipose tissue heterogeneity .................................................................... 29 1.2.1 Brown adipose tissue ........................................................................... 29 1.2.2 Distinct adipose tissue depots .............................................................. 30 1.2.3 Gender and race differences in adipose tissue distribution .................. 32 1.2.4 Adipose tissue composition ................................................................. 34 1.3 Integrative adipose tissue functions ........................................................ 35 1.3.1 Immune functions ................................................................................ 35 1.3.2 Endocrine functions ............................................................................. 36 2 1.3.3 Energy homeostasis ............................................................................. 39 1.4 Metabolism in human adipose tissue ...................................................... 40 1.4.1 Lipid metabolism ................................................................................. 41 1.4.2 Carbohydrate metabolism .................................................................... 45 1.4.3 Regulation of adipose metabolism ....................................................... 47 1.5 Adipogenesis .............................................................................................. 50 1.5.1 Progenitor origin and cell lineage ........................................................ 51 1.5.2 Adipogenesis regulation ...................................................................... 53 1.5.3 Wnt signalling in adipogenesis ............................................................ 56 1.6 Adipocyte endoplasmic reticulum stress ................................................ 59 1.6.1 Endoplasmic reticulum stress and the unfolded protein response ....... 60 1.6.2 Adipose tissue endoplasmic reticulum stress ...................................... 64 1.6.3 Obesity induces adipocyte endoplasmic reticulum stress .................... 65 1.7 Endoplasmic reticulum stress induced insulin resistance in adipose tissue ................................................................................................................ 66 1.7.1 IRE1 activation of JNK and NFB ................................................... 67 1.7.2 ER stress induced inflammation and subsequent insulin resistance .... 69 1.7.3 ER stress integration with metabolism ................................................ 73 1.7.4 ER stress in adipogenesis ..................................................................... 76 1.8 Translation to the clinic ........................................................................... 77 1.9 Summary ................................................................................................... 81 1.10 Research hypothesis, aims and objectives ............................................ 82 1.10.1 Research hypothesis ........................................................................... 82 1.10.2 Research aims .................................................................................... 82 1.10.3 Research objectives............................................................................ 82 3 Chapter 2: Subjects, methods and materials ................................................... 84 2.1 Ethical approval ....................................................................................... 85 2.1.1 Bariatric study ...................................................................................... 85 2.1.2 Human adipose tissue collection.......................................................... 85 2.2 Subjects ...................................................................................................... 85 2.2.1 Subjects who underwent bariatric surgery ........................................... 85 2.2.2 Adipose tissue collection ..................................................................... 87 2.3 Tissue culture ............................................................................................ 87 2.3.1 Extracting primary human preadipocytes ............................................ 87 2.3.2 Human preadipocyte ChubS7 cell line ................................................ 89 2.3.3 Cell culture media composition ........................................................... 89 2.3.4 Propagation and differentiation of preadipocytes ................................ 91 2.3.5 Treatments ........................................................................................... 92 2.3.6 Small interfering RNA transfection ..................................................... 92 2.3.7 Collection of conditioned media, protein and RNA ............................ 93 2.4 Analysis of samples ................................................................................... 94 2.4.1 Analysis of blood samples ................................................................... 94 2.4.2 RNA analysis ....................................................................................... 94 2.4.3 Protein analysis .................................................................................... 97 2.4.4 Agarose gel electrophoresis ................................................................. 98 2.4.5 Lipolysis assay ..................................................................................... 98 2.4.6 Adipocyte staining ............................................................................... 99 2.4.7 Glucose uptake assay ......................................................................... 100 2.4.8 Cell viability assay ............................................................................. 101 2.5 Statistical Analysis .................................................................................. 102 4 Chapter 3: Endoplasmic reticulum stress in human adipose tissue ............ 103 3.1 Introduction ............................................................................................ 104 3.2 Results ...................................................................................................... 106 3.2.1 Body composition, basal metabolic variables and sample analyses .. 106 3.2.2 Adipose tissue expression of UPR related genes and proteins were reduced post bariatric surgery ..................................................................... 109 3.2.3 Regulation of UPR markers was improved in adipose tissue 6 months post-surgery ................................................................................................ 112 3.2.4 Effect of surgery type on ER stress (Restrictive versus Malabsorptive) .................................................................................................................... 115 3.2.5 Adipose tissue inflammation and macrophage infiltration ................ 118 3.3 Discussion ................................................................................................ 120 3.4 Conclusions ............................................................................................. 124 Chapter 4: Regulation of