Aus dem Paul-Langerhans Institut Dresden Direktor: Prof. Dr. Michele Solimena Delta cell reprogramming during mouse pregnancy Dissertationsschrift zur Erlangung des akademischen Grades Doktor der Biomedizin Doctor rerum medicinalium (Dr. rer. medic.) vorgelegt der Medizinischen Fakultät Carl Gustav Carus der Technischen Universität Dresden von Julia Katharina Panzer geb. Stertmann aus Münster Dresden 2018 1. Gutachter: 2. Gutachter: Tag der mündlichen Prüfung: gez.:____________________________ Vorsitzender der Promotionskomission Success is the ability to go from one failure to another with no loss of enthusiasm. – Winston Churchill Table of contents 1 Introduction .............................................................................................. 1 1.1 The Pancreas ...................................................................................................... 1 1.1.1 Anatomy ............................................................................................................................... 1 1.1.2 Development ....................................................................................................................... 2 1.2 Islets of Langerhans ............................................................................................ 6 1.2.1 Islet architecture .................................................................................................................. 6 1.2.2 Islet cell function .................................................................................................................. 7 1.3 Diabetes Mellitus................................................................................................ 9 1.3.1 Etiology of diabetes ............................................................................................................. 9 1.3.2 Therapeutic approaches .................................................................................................... 10 1.4 Plasticity of the pancreatic endocrine mass ....................................................... 12 1.4.1 Replication of preexisting beta cells .................................................................................. 12 1.4.2 Beta cell neogenesis........................................................................................................... 14 1.4.3 Transdifferentiation of non-beta cells ............................................................................... 15 1.5 Pregnancy as a physiological model system for endocrine cell plasticity ............. 17 1.5.1 The physiology of pregnancy ............................................................................................. 17 1.5.2 Adaptation of islets of Langerhans in response to pregnancy ........................................... 20 1.6 Aim .................................................................................................................. 23 2 Material and Methods ............................................................................. 24 2.1 Mice ................................................................................................................. 24 2.1.1 Genotyping of mice ............................................................................................................ 24 2.1.2 Mating and pregnancy control ........................................................................................... 26 2.2 Tamoxifen preparation and application ............................................................. 26 2.3 Experiments on isolated pancreatic islets .......................................................... 26 2.3.1 Pancreatic islet isolation .................................................................................................... 26 2.3.2 Culture of pancreatic islets ................................................................................................ 27 2.3.3 Cell dispersion .................................................................................................................... 27 2.3.4 FACS sorting ....................................................................................................................... 28 2.3.5 Quantification by qRT-PCR ................................................................................................. 28 2.4 Immunohistochemistry ..................................................................................... 29 2.4.1 Preparation of pancreatic tissue sections .......................................................................... 29 2.4.2 Preparation of pancreatic tissue slices .............................................................................. 30 2.4.3 Staining .............................................................................................................................. 30 2.4.4 Data aquisition ................................................................................................................... 32 2.4.5 Data analysis ...................................................................................................................... 32 2.5 Tissue slice perifusion ....................................................................................... 32 2.6 Hormonal profiling ........................................................................................... 33 2.6.1 Blood sampling and processing ......................................................................................... 33 2.6.2 Glucose tolerance test ....................................................................................................... 33 2.6.3 Plasma hormone levels ...................................................................................................... 34 2.7 Statistical analysis ............................................................................................ 34 3 Results .................................................................................................... 35 3.1 Metabolic characterization of pregnancy ........................................................... 35 3.1.1 Weight gain and glucose homeostasis ............................................................................... 35 3.1.2 Hormonal profile during mouse pregnancy ....................................................................... 38 3.2 Islet mass adaptations during pregnancy ........................................................... 39 3.2.1 Assessment of endocrine mass .......................................................................................... 39 3.2.2 Contribution of proliferation to endocrine mass expansion during pregnancy ................ 42 3.3 Influence of neogenesis on pregnancy ............................................................... 44 3.3.1 Ngn3 expression in the endocrine pancreas ...................................................................... 44 3.3.2 Increased Ngn3 promotor activity during early pregnancy ............................................... 45 3.3.3 Origin and fate of Ngn3-positive cells ................................................................................ 48 3.3.4 Occurrence of islet cell dedifferentiation during pregnancy ............................................. 50 3.3.5 Detection of Ngn3 mRNA levels in vitro ............................................................................ 53 3.3.6 Progesterone induces delta cell conversion in vitro .......................................................... 54 3.3.7 Delta cell dedifferentiation leads to increased beta cell function in vivo and in vitro ....... 56 4 Discussion .............................................................................................. 58 4.1 Functional adaptations precede islet mass expansion during pregnancy............. 59 4.2 Non-proliferative mechanisms contribute to the mass adaptations during pregnancy ................................................................................................................... 60 4.3 Recapitulation of Ngn3 promotor activity during early pregnancy ...................... 61 4.4 Onset of endogenous Ngn3 activity during early pregnancy is delta cell specific . 63 4.5 Early pregnancy induces partial loss of delta cell identity ................................... 64 4.6 Progesterone initiates cellular reprogramming of delta cells .............................. 66 4.7 Loss of delta cell identity leads to increased beta cell function ........................... 68 4.8 Conclusion and perspectives ............................................................................. 70 5 Summary ................................................................................................. 72 6 Zusammenfassung ................................................................................. 74 7 References .............................................................................................. 76 8 List of figures ......................................................................................... 91 9 List of tables ........................................................................................... 92 10 Acknowledgements ............................................................................ 93 Anlage 1 ........................................................................................................ 94 Anlage 2 ........................................................................................................ 95 List of abbreviations ADP adenosine diphosphate ATP adenosine triphosphate ARX aristaless