Jonas S.G. Silvander Keratins in the endocrine pancreas - Novel regulators of cellular processes in β-cells This Ph.D. thesis describes the role of keratins in the endocrine pancreas. It shows that keratins are maintaining normal insulin levels by involvement Jonas S.G. Silvander | Keratins 2018 in the endocrine pancreas| Jonas S.G. Silvander in β-cell mitochondrial ATP production and insulin vesicle morphology. On systemic level, keratins in β-cells regulate basal blood glucose levels, most Keratins in the endocrine pancreas likely in combination with insulin sensitive tissues. In addition, keratins are crucial for β-cell stress - Novel regulators of cellular processes in β-cells protection against chemically induced T1D in mice. These novel findings on insulin production and cell stress protection in β-cells, shed light on the potential role of keratins in diabetes susceptibility and progression. The author graduated from Ålands Lyceum, Marie- hamn, in 2007. He recieved his M.Sc. in Biomedical Imaging from Åbo Akademi University in May 2013. Since August 2013, he has been working as a Ph.D. student in Diana Toivola’s Epithelial Biology Labora- tory at Åbo Akademi University. 9 789521 236648 ISBN 978-952-12-3664-8 Keratins in the endocrine pancreas - Novel regulators of cellular processes in β-cells Jonas S.G. Silvander Cell biology Faculty of Science and Engineering Åbo Akademi University Turku, Finland 2018 The research projects were conducted at Cell biology, Faculty of Science and Engineering, Åbo Akademi University. Supervised by Diana Toivola, Ph.D. Cell biology Faculty of Science and Engineering Åbo Akademi University Finland Reviewed by Emilia Peuhu, Ph.D. Turku Center for Biotechnology University of Turku Finland Assistant Professor Damaris Lorenzo Department of Cell biology and Physiology University of North Carolina at Chapel Hill USA Opponent Professor Yassemi Capetanaki Vice-Director Center of Basic Research Biomedical Research Foundation Academy of Athens Greece ISBN 978-952-12-3664-8 (Printed) ISBN 978-952-12-3665-5 (PDF) Painosalama Oy, Turku, Finland 2018 To my wonderful family and friends. “Never, never, never give up.” Winston Churchill TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT .............................................................................................................. vi SWEDISH ABSTRACT/SVENSKT ABSTRAKT .................................................. vii LIST OF ORIGINAL PUBLICATIONS AND MANUSCRIPTS .......................... viii ABBREVIATIONS ................................................................................................... ix INTRODUCTION ..................................................................................................... 1 REVIEW OF THE LITERATURE ............................................................................ 2 1 Pancreas ........................................................................................................... 2 1.1 Endocrine pancreas and the Islet of Langerhans ........................................... 3 1.1.1 Cell types in the Islets of Langerhans ................................................... 3 1.1.2 Glucose homeostasis .............................................................................. 5 1.1.3 Insulin production in β-cells ................................................................. 6 1.1.4 Glucose-stimulated insulin secretion (GSIS) in β-cells ..................... 7 1.2 Diabetes ............................................................................................................... 9 1.2.1 Type 1 diabetes ....................................................................................... 9 1.2.2 Type 2 diabetes ....................................................................................... 9 1.2.3 Models for the study of diabetes ......................................................... 10 1.2.3.1 Streptozotocin-induced diabetes ............................................ 10 1.2.3.2 The non-obese diabetic mouse model ................................... 11 1.2.3.3 High-fat diet .............................................................................. 11 2 Cytoskeleton and intermediate filaments .................................................... 12 2.1 Intermediate filaments .................................................................................... 12 2.1.1 Structure of intermediate filaments ................................................... 13 2.2 Simple epithelial keratins ................................................................................ 14 2.2.1 Functions of simple epithelial keratins .............................................. 15 2.2.2 Keratins in human diseases ................................................................. 16 2.3 Cytoskeleton and β-cells in pancreas ............................................................ 17 3 Mitochondria ................................................................................................ 18 3.1 Mitochondrial structure and function .......................................................... 18 3.1.1 Oxidative phosphorylation.................................................................. 19 3.2 Mitochondrial dynamics ................................................................................. 21 3.2.1 Fusion of the mitochondrial network ................................................ 22 3.2.2 Fission of the mitochondrial network ............................................... 23 3.2 Mitochondria and intermediate filaments ................................................... 23 3.3 Mitochondria in β-cells and diabetes ............................................................ 25 OUTLINE AND AIMS OF THE THESIS ............................................................... 26 iv TABLE OF CONTENTS EXPERIMENTAL PROCEDURES AND MATERIAL ........................................... 27 1 Methods .......................................................................................................... 27 2 Experimental animals .................................................................................... 28 3 Antibodies ...................................................................................................... 28 RESULT AND DISCUSSION .................................................................................. 30 1 K8 and K18 are the major IFs of β-cells, with low expression of K7 (Study I + II) ................................................................................................... 30 2 Keratins in blood glucose regulation and diabetes susceptibility (Study I + II) ................................................................................................... 31 2.1 Keratins are involved in blood glucose regulation (Study I + II) ............... 31 2.2 Keratins participate in the regulation of insulin vesicle morphology and insulin content (Study I + II + III) ......................................................... 33 2.3 Delayed STZ-induced diabetes in K8−/− mice, but increased exocrine damage after low-dose STZ-treatment (Study I) ......................................... 34 2.4 K8−/−-induced GLUT2 mislocalization in β-cells correlates with STZ resistance (Study I + II) ................................................................................... 35 2.5 High-fat diet induces transiently higher blood glucose levels in K8+/– mice (Study II) .................................................................................................. 36 2.6 Upregulation of keratins after diabetes and chronic stress (Study I + II) . 36 3 Keratins interact with mitochondria in β-cells which might regulate insulin production (Study III) ....................................................................... 37 3.1 β-cell mitochondrial fusion is promoted by keratin-mediated stabilization of trichoplein-mitofusin complexes (Study III) ..................... 37 3.2 Keratins regulate mitochondrial complexes to maintain normal membrane potential, ATP levels and insulin levels in β-cells (Study III). 38 3.3 Keratin-mitochondria-association stabilizes mitochondrial movements in β-cells (Study III) .................................................................... 40 CONCLUDING REMARKS .................................................................................... 41 FUTURE PROSPECTS ............................................................................................ 43 ACKNOWLEDGEMENTS....................................................................................... 44 REFERENCES .......................................................................................................... 46 ORIGINAL PUBLICATIONS AND MANUSCRIPT ............................................. 57 v ABSTRACT ABSTRACT Keratins (K) are the intermediate filament (IFs) proteins of epithelial cells and they are involved in multifaceted cellular functions, such as stress protection, organelle/protein-targeting, cell signaling and epithelial polarity. Keratin mutations have been linked to the cause of, or susceptibility to, several diseases, such as skin and liver diseases. Type 1 (T1D) and Type 2 (T2D) diabetes are dramatically increasing throughout the world. The mechanisms of T1D and T2D development are not fully understood, but include defective insulin production in β-cells. Still there is no cure for diabetes. However, T1D and T2D can be managed by insulin injections or insulin enhancing drugs. The roles of keratins in insulin-producing pancreatic β-cells