The eukaryotic translation initiation factor 2, a hero turned villain in β cells By Baroj Abdulkarim Université libre de Bruxelles Faculty of Medicine ULB Center for Diabetes Research Academic year 2016-2017 Jury Members: Dr. Ingrid Langer (President) Dr. Miriam Cnop (Promoter and secretary) Dr. Mariana Igoillo Esteve (Co-Promoter) Dr. Daniel Christophe Dr. Christophe Erneux Dr. Claudine Heinrichs Dr. Amar Abderrahmani Dr. Patrick Gilon Dedicated to my daughter Elîn 2 Contents Papers constituting this thesis .................................................................................... 4 Abbreviations .............................................................................................................. 5 Abstract ...................................................................................................................... 8 Résumé ...................................................................................................................... 9 Introduction ............................................................................................................... 10 Diabetes mellitus ................................................................................................... 10 How β cells work ................................................................................................ 11 Type 2 and monogenic diabetes ........................................................................ 12 Free fatty acids and diabetes ................................................................................ 14 Acute exposure to FFAs: enhanced β cell function ............................................ 15 Prolonged exposure to saturated FFAs: Lipotoxicity-induced ER stress ............ 17 The unfolded protein response ....................................................................... 17 Dysregulation of PERK/eIF2α pathway in diabetes ..................................... 22 β cell apoptosis ............................................................................................... 24 The extrinsic pathway .................................................................................. 26 The intrinsic pathway ................................................................................... 26 Aims of this thesis ..................................................................................................... 29 Results...................................................................................................................... 30 PAPER I ................................................................................................................ 30 PAPER II ............................................................................................................... 48 PAPER III .............................................................................................................. 62 Discussion ................................................................................................................ 84 Diseases caused by dysregulated ER stress signaling ......................................... 88 Not only β cells ...................................................................................................... 88 Models of PERK/eIF2α pathway ........................................................................... 90 Treating ER stress ................................................................................................. 91 Conclusions and perspectives .................................................................................. 92 Acknowledgements .................................................................................................. 95 References ............................................................................................................... 96 Supplementary data ............................................................................................... 111 3 Papers constituting this thesis 1. Cnop M, Abdulkarim B, Bottu G, Cunha DA, Masini M, Turatsinze JV, Griebel T, Igoillo-Esteve M, Bugliani M, Villate O, Ladriere L, Marselli L, Marchetti P, McCarthy MI, Sammeth M, Eizirik DL; RNA-sequencing identifies dysregulation of the human pancreatic islet transcriptome by the saturated fatty acid palmitate. Diabetes, 2013, 63, 6, 1978-1993 2. Abdulkarim B, Nicolino M, Igoillo-Esteve M, Daures M, Romero S, Philippi A, Senée V, Lopes M, Cunha DA, Harding HP, Derbois C, Bendelac N, Hattersley AT, Eizirik DL, Ron D, Cnop M, Julier C; A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly. Diabetes. 2015, 64, 11, 3951-3962. 3. Abdulkarim B,Hernangomez M, Igoillo-Esteve M, Ladriere L, Cunha DA, Marselli L, Marchetti P, Eizirik DL, Cnop M; Guanabenz sensitizes β-cells to endoplasmic reticulum stress-induced apoptosis. Endocrinology, 2017, Epub ahead of print. 4 Abbreviations APAF1 Apoptotic protease activating factor 1 ATF Activating transcription factor BH Bcl2 homology Bad Bcl2 associated agonist of cell death Bak Bcl2 antagonist/killer Bax Bcl2 associated X protein Bcl2 B-Cell lymphoma 2 Bid BH3 interacting domain death agonist Bim Bcl2 interacting mediator of cell death BiP Immunoglobulin heavy chain binding protein cAMP Cyclic AMP CHOP CCAAT/enhancer binding protein homologous protein CPE carboxypeptidase E CReP Constitutive repressor of eIF2α phosphorylation DP5 Death protein 5 eIF Eukaryotic translation initiation factor EPAC Exchange factor directly activated by cAMP ER Endoplasmic reticulum ERAD ER associated degradation ERO1 ER oxidoreductase 1 FACS Fluorescent activated cell sorting FADD Fas-associated death domain protein FFA Free fatty acid GADD34 Growth arrest DNA damage inducible 34 GATA6 GATA binding protein 6 GCK Glucokinase GCN2 General control nonderepressible 2 GLP-1 Glucagon like peptide 1 Glut Glucose transporter HNF1A Hepatocyte nuclear factor 1 alpha HRI Heme regulated initiation IDF International diabetes federation 5 INS Insulin gene Ini-Met Initiator Methionine IRE1 Inositol requiring 1 ISR Integrated stress response ISRIB ISR inhibitor JNK c-Jun N-terminal kinase + KATP ATP sensitive K channel KCNJ11 Potassium voltage gated channel subfamily J member 11 LC8 Light chain 8 Mcl1 Myeloid cell leukemia sequence 1 MODY Maturity onset diabetes of the young NeuroD1 Neuronal differentiation 1 NDM Neonatal diabetes mellitus NO Nitric Oxide Noxa phorbol-12-myristate-13-acetate-induced protein 1 Nrf2 nuclear factor erythroid-2-related factor-2 NRSF Neuronal-restrictive silencer factor ORF Open reading frame P58IPK 58 kDa inhibitor of PKR PDX1 Pancreatic and duodenal homeobox 1 PERK PKR-like endoplasmic reticulum kinase PKA Protein kinase A PKR Protein kinase R PP1 Protein phosphatase 1 PPAR Peroxisome proliferator-activated receptor PUMA P53 upregulated modulator of apoptosis Rap1 Ras-related protein 1 RNA-seq RNA sequencing RRP Readily releasable pool SERCA Sarcoendoplasmic reticulum Ca2+ ATPase SNAP25 Synaptosomal-associated protein 25 SNARE SNAP receptor TIRF Total internal reflection fluorescence TRAF2 Tumor necrosis factor receptor- associated factor 2 6 Trib3 Tribblespseudokinase 3 uORF Upstream ORF UPR Unfolded protein response VDCC Voltage dependent Ca2+ channel VAMP Vesicle associated membrane protein WFS1 Wolfram syndrome 1 XBP1 X-box binding protein 1 7 Abstract The prevalence of type 2 diabetes is increasing dramatically worldwide. Type 2 diabetes is a major health and socio-economic burden. Genetic predisposition and the obesity epidemic, due to sedentary life style and high caloric food intake, are associated with development of type 2 diabetes. Circulating free fatty acids (FFAs), in particular saturated FFAs, are linked with insulin resistance and β cell dysfunction. Following this background we performed RNA sequencing of human pancreatic islets treated with the saturated FFA palmitate to acquire a global image of the islet response to this insult. We identified several stress pathways induced by palmitate with a major induction of the endoplasmic reticulum (ER) stress response. The ER stress response, in particular the PKR-like ER kinase (PERK) branch, has been shown to be induced by saturated FFA. It leads to increased β cell apoptosis both in fluorescence activated cell sorter (FACS) purified rat β cells and human islets. We further clarified the role of this pathway by studying the involvement of the constitutive repressor of eIF2α phosphorylation (CReP) in a monogenic form of diabetes. CReP is a repressor of eukaryotic translation initiation factor 2α (eIF2α) phosphorylation. A direct target of PERK, eIF2α is involved in translational attenuation and induction of apoptosis. We have shown that CReP loss-of-function leads to a new syndrome of young onset diabetes, intellectual disability and microcephaly. The identified R658C mutation abrogated CReP activity leading to increased eIF2α phosphorylation and β cell apoptosis. To further demonstrate the importance of eIF2α dysregulation in β cell demise, we used guanabenz, a chemical inhibitor of growth arrest DNA damage inducible 34 (GADD34). GADD34 is an ER stress-induced repressor of eIF2α phosphorylation. Guanabenz potentiated FFA-mediated ER stress and apoptosis in clonal and primary rat β cells and in human islets through the activation of CCAAT/enhancer binding protein homologous protein (CHOP), downstream of eIF2α. Guanabenz administration in mice impaired glucose tolerance and led to β cell dysfunction. In ex vivo experiments guanabenz also induced β cell dysfunction