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Hedgehog and Insulin Signalling in Β-Cell Biology and Glucose Metabolism

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Hedgehog and Insulin Signalling in Β-Cell Biology and Glucose Metabolism TECHNISCHE UNIVERSITÄT MÜNCHEN Fakultät für Medizin Hedgehog and insulin signalling in β-cell biology and glucose metabolism Felizitas Maria Schmitz Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation Vorsitzender: Prof. Dr. Matthias Tschöp Prüfer der Dissertation: 1. Prof. Dr. Heiko Lickert 2. Prof. Dr. Martin Hrabě de Angelis Die Dissertation wurde am 31.08.2017 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 21.02.2018 angenommen. Contents Contents 1 List of abbreviations ............................................................................................................................................. 1 2 Abstract ....................................................................................................................................................................... 7 3 Introduction .............................................................................................................................................................. 8 3.1 Relevance and implications of diabetes mellitus................................................................................... 8 3.2 The pancreas and its interplay with metabolic organs in the regulation of energy homeostasis .............................................................................................................................................................. 8 3.3 Pancreatic β-cell failure in the development of T2D ........................................................................ 11 3.4 Cilia biology and cilia-related disease ...................................................................................................... 11 3.5 Hedgehog signalling in vertebrates ........................................................................................................... 13 3.6 Insulin receptor and insulin like-growth factor receptor signalling ........................................ 16 3.6.1 The structure of INSR, IGF1R and IGF2R and their ligands ............................................... 16 3.6.2 Signal transduction and function of INSR, IGF1R and IGF2R ............................................ 17 3.6.3 Expression and physiological function of INSR and IGF1R ................................................ 19 3.6.4 Autocrine insulin signalling in the pancreatic β-cell ............................................................. 19 3.6.5 The function of the IGF2/CI-M6P receptor ................................................................................. 20 3.6.6 The metabolic regulator AMPK ......................................................................................................... 21 3.7 Endocytosis and trafficking of receptor tyrosine kinases .............................................................. 22 3.7.1 Receptor trafficking routes after endocytosis of RTKs ......................................................... 22 3.7.2 Receptor endocytosis pathways and COP vesicle-mediated transport of RTKs ..... 23 3.7.3 Regulation of RTK-mediated signalling through endocytic and trafficking processes ......................................................................................................................................................................... 25 3.8 The 5330417C22RIK gene: state of the art ........................................................................................... 26 3.9 Aims of the thesis ................................................................................................................................................ 27 4 Results ....................................................................................................................................................................... 28 4.1 Analysis of Hedgehog signalling in the islet of Langerhans and its impact on glucose metabolism ............................................................................................................................................................. 28 4.1.1 Hh signalling activity in pancreatic cells is differentially regulated in vitro and in vivo ................................................................................................................................................................. 28 4.1.2 Hh signalling elevates proliferation rate of endocrine islet cells in vitro .................... 34 4.1.3 Adult heterozygous Kif7 mice exhibit an insulin secretion defect ................................. 35 4.2 A novel Cre-inducible knock-in ARL13B-tRFP fusion cilium reporter.................................... 36 i Contents 4.2.1 Generation of the ARL13B-tRFP reporter mouse line ........................................................... 36 4.2.2 Functional analysis of the ARL13B-tRFP mouse strain ........................................................ 40 4.2.3 The ARL13B-tRFP reporter protein accurately localizes to cilia in mono-and multi- ciliated tissue............................................................................................................................................ 42 4.2.4 The ARL13B-tRFP reporter enables the monitoring of cilia assembly and disassembly in living cells ................................................................................................................. 45 4.3 Characterization of a putative modulator of the InsR/Igfr signalling system in vivo and in vitro ....................................................................................................................................................................... 47 4.3.1 Bioinformatic analysis of the 5330417C22RIK gene and protein .................................. 47 4.3.2 Generation of mono- and polyclonal Igfr-L antibodies......................................................... 56 4.3.3 Analysis of postnatal death of Igfr-L-/- mice ............................................................................... 60 4.3.4 Metabolic study of adult Igfr-L+/- male and female mice ..................................................... 63 4.3.5 The molecular function of Igfr-L ...................................................................................................... 78 5 Discussion ............................................................................................................................................................... 99 5.1 The ARL13B-tRFP cilia reporter is a valuable tool for the analysis of ciliary biology .... 99 5.2 Relevance of Hh signalling in the islet of Langerhans and its implication on glucose metabolism .......................................................................................................................................................... 100 5.3 Igfr-L, a putative modulator of the InsR/Igfr system and glucose homeostasis ............. 102 5.3.1 Potential causes for postnatal death of Igfr-L-/- pups ......................................................... 103 5.3.2 Influence of Igfr-L on energy homeostasis ............................................................................... 104 5.3.3 Igfr-L-mediated regulation of InsR/Igfr signalling .............................................................. 106 5.3.4 Protein structure and subcellular trafficking dynamics of Igfr-L ................................ 108 5.3.5 Regulation of cell surface location of Igfr-L by the presence of extracellular glucose ...................................................................................................................................................................... 110 5.3.6 Igfr-L endocytosis and trafficking by CME ............................................................................... 111 5.3.7 Mechanistical model of Igfr-L regulating InsR/Igfr signal transduction .................. 112 5.3.8 Igfr-L as potential therapeutic target.......................................................................................... 115 6 Material and methods .................................................................................................................................... 117 6.1 Material ................................................................................................................................................................. 117 6.1.1 Equipment................................................................................................................................................. 117 6.1.2 Consumables ............................................................................................................................................ 119 6.1.3 Kits and Mastermix .............................................................................................................................. 120 6.1.4 Chemicals................................................................................................................................................... 121 ii Contents 6.1.5 Buffers and solutions........................................................................................................................... 123 6.1.6 Solutions for cell culture.................................................................................................................... 125 6.1.7 Enzymes, inhibitors and growth factors ................................................................................... 126 6.1.8 Antibodies
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