TECHNISCHE UNIVERSITÄT MÜNCHEN Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Experimentelle Genetik Developmental gene Pax6 in adult pancreas homeostasis and energy metabolism Nirav Florian Chhabra Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Wolfgang Wurst Prüfer der Dissertation: 1. Prof. Dr. Martin Hrabě de Angelis 2. Prof. Dr. Matrin Klingenspor Die Dissertation wurde am 14.06.2017 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 18.10.2017 angenommen. | I I. TABLE OF CONTENTS I. TABLE OF CONTENTS ------------------------------------------------------------------------- I II. TABLES AND FIGURES ---------------------------------------------------------------------- IV III. ABBREVIATIONS -------------------------------------------------------------------------- VII IV. SUMMARY/ZUSAMMENFASSUNG ---------------------------------------------------- X 1. INTRODUCTION --------------------------------------------------------------------------------- 1 1.1 β-cell identity and function --------------------------------------------------------------------------------------------- 1 1.1.1 Islets of Langerhans ----------------------------------------------------------------------------------------------------- 1 1.1.2 Pancreatic development and the endocrine lineage ---------------------------------------------------------------- 1 1.1.3 Insulin secretory mechanism ------------------------------------------------------------------------------------------ 3 1.1.3.1 Glucose stimulated insulin secretion --------------------------------------------------------------------------- 3 1.1.3.2 Incretin effects on insulin secretion ---------------------------------------------------------------------------- 4 1.1.4 Postnatal β-cell identity and regulation ------------------------------------------------------------------------------ 6 1.1.5 Type 2 Diabetes Mellitus ---------------------------------------------------------------------------------------------- 7 1.1.5.1 Overview ----------------------------------------------------------------------------------------------------------- 7 1.1.5.2 β-cell dysfunction in T2DM ------------------------------------------------------------------------------------- 8 1. 2 Central and peripheral metabolic pathways ----------------------------------------------------------------------- 9 1.2.1 Islet-liver axis ------------------------------------------------------------------------------------------------------------ 9 1.2.2 Hypothalamic control of metabolism ------------------------------------------------------------------------------- 13 1.3 PAX family of transcription factors--------------------------------------------------------------------------------- 14 1.3.1 PAX family -------------------------------------------------------------------------------------------------------------- 14 1.3.2 Paired box protein 6---------------------------------------------------------------------------------------------------- 16 1.3.2.1 PAX6: Master regulator ----------------------------------------------------------------------------------------- 16 1.3.2.2 PAX6 and metabolism------------------------------------------------------------------------------------------- 16 1.4 Aims and hypothesis --------------------------------------------------------------------------------------------------- 21 2. MATERIALS AND METHODS --------------------------------------------------------------22 2.1 Materials ----------------------------------------------------------------------------------------------------------------- 22 2.1.1 Buffers and solutions -------------------------------------------------------------------------------------------------- 22 2.1.2 In vitro insulin secretion assay materials --------------------------------------------------------------------------- 24 2.1.3 PCR Primers ------------------------------------------------------------------------------------------------------------ 24 2.1.3.1 Housekeeping genes --------------------------------------------------------------------------------------------- 24 2.1.3.2 Target genes ------------------------------------------------------------------------------------------------------- 25 2.1.4 Primary Antibodies ---------------------------------------------------------------------------------------------------- 26 2.1.4.1 Western blot and chromatin immunoprecipitation ---------------------------------------------------------- 26 2.1.4.2 Immunohistochemistry ------------------------------------------------------------------------------------------ 26 | II 2.1.5 Secondary Antibodies ------------------------------------------------------------------------------------------------- 27 2.1.5.1 Western blot ------------------------------------------------------------------------------------------------------- 27 2.1.5.2 Immunohistochemistry ------------------------------------------------------------------------------------------ 27 2.1.6 Chemicals and kits ----------------------------------------------------------------------------------------------------- 27 2.1.6.1 Consumables ------------------------------------------------------------------------------------------------------ 27 2.1.6.2 Chemicals ---------------------------------------------------------------------------------------------------------- 28 2.1.6.3 Reagents ----------------------------------------------------------------------------------------------------------- 28 2.1.6.4 Kits ----------------------------------------------------------------------------------------------------------------- 29 2.1.6.5 Laboratory devices and equipment ---------------------------------------------------------------------------- 29 2.2 Methods ------------------------------------------------------------------------------------------------------------------- 30 2.2.1 Mouse line maintenance ---------------------------------------------------------------------------------------------- 30 2.2.2 Metabolic and physiological methods ------------------------------------------------------------------------------ 30 2.2.2.1 Body composition and metabolic studies -------------------------------------------------------------------- 30 2.2.2.2 Dietary challenge test -------------------------------------------------------------------------------------------- 30 2.2.2.3 Leptin sensitivity assay ------------------------------------------------------------------------------------------ 30 2.2.2.4 Euglycemic-hyperinsulinemic clamps ------------------------------------------------------------------------ 31 2.2.2.5 Plasma collection and Clinical chemistry -------------------------------------------------------------------- 31 2.2.3 In vitro methods -------------------------------------------------------------------------------------------------------- 31 2.2.3.1 Islet isolation ------------------------------------------------------------------------------------------------------ 31 2.2.3.2 Insulin stimulatory studies -------------------------------------------------------------------------------------- 32 2.2.3.3 Hepatocyte isolation --------------------------------------------------------------------------------------------- 33 2.2.3.4 Primary hepatocyte sandwich culture ------------------------------------------------------------------------- 34 2.2.3.5 Glucose secretion assay ----------------------------------------------------------------------------------------- 35 2.2.3.6 Hepatocyte mitochondrial respiration------------------------------------------------------------------------- 35 2.2.3.7 Maintenance of βTC-3 cell line -------------------------------------------------------------------------------- 35 2.2.4 Molecular methods ----------------------------------------------------------------------------------------------------- 36 2.2.4.1 RNA isolation ----------------------------------------------------------------------------------------------------- 36 2.2.4.2 Microarray --------------------------------------------------------------------------------------------------------- 37 2.2.4.3 cDNA synthesis -------------------------------------------------------------------------------------------------- 38 2.2.4.4 Quantitative real-time Polymerase Chain Reaction (qRT-PCR) ----------------------------------------- 38 2.2.4.5 Protein isolation -------------------------------------------------------------------------------------------------- 39 2.2.4.6 Western blot ------------------------------------------------------------------------------------------------------- 40 2.2.4.7 Measurement of insulin and glucagon ------------------------------------------------------------------------ 40 2.2.4.8 FGF21 and Leptin ELISA -------------------------------------------------------------------------------------- 41 2.2.5 Histology ---------------------------------------------------------------------------------------------------------------- 41 2.2.5.1 Pancreatic tissue preparation for frozen sections ----------------------------------------------------------- 41 2.2.5.2 Hypothalamic tissue preparation for frozen sections ------------------------------------------------------- 41