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Adipose Tissue As a Target Organ of the GIPR: Identification of Potential Pharmacodynamic Mrna Biomarkers for GIPR Agonism

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Adipose Tissue As a Target Organ of the GIPR: Identification of Potential Pharmacodynamic Mrna Biomarkers for GIPR Agonism Roskilde University Department of Science and Environment Adipose tissue as a target organ of the GIPR: identification of potential pharmacodynamic mRNA biomarkers for GIPR agonism Master’s thesis in Medicinal and Molecular biology - Sebastian Møller Heimbürger In Collaboration with GLP-1 Biology, Novo Nordisk A/S In vivo Pharmacology, Novo Nordisk A/S Academic Supervisors: Roskilde University: Louise Torp Dalgaard Novo Nordisk: Robert Augustin Submitted on: June 28, 2017 0 Preface This master’s thesis concludes the master’s degree in Medicinal and Molecular Biology at the Department for Science and Environment, Roskilde University Denmark. The experimental work and daily guidance were carried out at Novo Nordisk A/S in the department of Incretin/GLP-1 Biology in Måløv, under the supervision of Robert Augustin and co-supervision by Klaus Stensgaard Frederiksen. Internal supervision at Roskilde University was conducted by Louise Torp Dalgaard. The master’s thesis consists of experiments and knowledge gathered during the 5. September 2016 – 28. June 2017. The aim of this master’s thesis was to understand the pharmacology of GIPR agonism with adipose tissue as the target organ to identify potential mRNA biomarkers for GIPR agonism. The GIPR agonism specific mRNAs will be applied as acute pharmacodynamic biomarkers to investigate and establish the pharmacology and pharmacokinetics/pharmacodynamics (PK/PD) of GIP analogues as compared to GLP-1 analogues and GIP/GLP-1 dual-agonists. The master’s thesis contains three steps: 1) Identification of potential GIPR agonist relevant genes by RNA-seq of epididymal white adipose tissue from diet-induced obese mice treated with GIP, GLP-1 and GLP-1/GIP analogues. 2) Confirmation of the identified mRNA in mesenteric, retroperitoneal, inguinal, and brown adipose tissue and b) in 3T3-L1 adipocytes in vitro. 3) In vitro validation of GIPR agonism specific mRNA for direct effects in adipocytes by testing their dose- responsive regulation in 3T3-L1 adipocytes (Figure 1). Identification Confirmation Validation RNA-seq Fluidigm qRT-PCR . In vivo: . In vivo: Adipose . In vitro: 3T3-L1 Epididymal depots dose- adipose tissue responsiveness . In vitro: 3T3-L1 Figure 1: Overview of the thesis: 1) Identification of genes specifically regulated by GIPR agonism by RNA-seq of epididymal adipose tissue from DIO mice treated with the GLP-1R agonist (liraglutide), GIPR agonist (GIPα), and GLP-1R/GIPR agonist (co-agonist), 2) Confirmation of GIPR regulated genes in adipose tissue depots (in vivo treatment) and 3T3-L1 adipocytes (in vitro 1 treatment) applying the Fluidigm® technology. 3) Validation of mRNA for their direct and adipocyte specific regulation by GIPR agonism in 3T3-L1 adipocytes by qRT-PCR. Acknowledgements I would like to thank my internal supervisor Louise Torp Dalgaard for entrusting me with the opportunity to carry out my master’s thesis at Novo Nordisk A/S, and for giving me excellent academic supervision. I would also like to thank my external supervisor Robert Augustin, for all his support and daily guidance at Novo Nordisk A/S, for his extraordinary scientific and experimental guidance. Without Louise and Robert this collaboration would not have been possible. I am truly grateful for the confidence and trust they have given me. Furthermore I would like to thank the Dalgaard group, (Roskilde University) for allowing me to present my work and for academic sparring. In addition, I would like to thank the people from the department of GLP-1 biology for providing an excellent working environment and for being great colleagues. I also greatly appreciate the support Klaus Stensgaard Frederiksen, (Novo Nordisk A/S) has put into my master’s thesis, and sharing of data supporting my thesis. The Director of GLP-1 Biology at Novo Nordisk A/S, Siff Groth Rønn allowed me to attend various academic meetings, which provided insights into the pharmaceutical industry, for which I am very thankful. Thanks to everybody who helped and supported me in performing my master’s thesis, I am truly grateful for all the time and effort you have given me during the past 9 months. Sebastian Møller Heimbürger Roskilde University, Denmark June 2017 2 Abstract The glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR) have been implicated with diseases us as type 2-diabetes and obesity. However, the exact role of GIP in pathophysiology is uncertain and pharmacological interventions are exploring the therapeutic potential of both GIPR agonists and antagonists. The GIPR is predominantly expressed in the pancreas, adipose tissue, gut, heart, pituitary gland, adrenal cortex and the brain. While the insulino- and glucagonotropic activity of GIP has been well characterized, less is known on its physiological role and potential pharmacological relevance in adipose tissue. This thesis aimed to identify GIPR agonist specific RNA in adipose tissue applying RNA sequencing of epididymal adipose tissue from diet-induce obese (DIO) mice treated with agonists for the GIPR, Glucagon-like peptide 1 receptor (GLP-1R) and GIPR/GLP-1R. DIO mice were treated for 17 days with long-acting agonists and mRNA sequencing identified 210, 3006, and 2850 transcripts being significantly regulated by GIPR, GLP-1R, and GIPR/GLP-1R agonism, respectively. To confirm mRNAs that are specifically regulated by GIPR agonism, 74 transcripts were quantified by qRT-PCR in adipose tissue depots (retroperitoneal WAT, inguinal WAT, mesenteric WAT, intrascapular BAT) and in 3T3-L1 adipocytes treated with a GIPR agonist or liraglutide (GLP-1R agonist). Finally, for 48 transcripts dose-dependent regulation by GIPR agonism was tested in 3T3-L adipocytes. Transcripts for the vascular-adhesion molecule 1 (VCAM1) and the myosin regulatory light chain interacting protein (MYLIP) were identified, confirmed, and shown to be dose-dependently down- regulated by GIPR agonism suggesting a direct and specific effect in adipocytes caused by receptor activation. The transcript of interferon induced protein 44 (IFI44) was less consistent in its dose- responsiveness. The Gipr mRNA was dose-dependently down-regulated at higher concentrations of GIPR agonist, suggesting a potential desensitization. Future studies aim to translate these in vitro qualified mRNA towards their applicability as acute pharmacodynamic biomarkers for GIPR agonism in adipose tissue of mice - to support the pharmacology of GIPR agonism and its mechanism of action. 3 Resume Glucose-dependent insulinotropic polypeptide (GIP) og dets receptor (GIPR), er forbundet med livsstilsygdomme såsom type 2-diabetes og fedme, men den eksakte funktion i patofysiologien er stadig ukendt. Farmakologiske studier undersøger behandlingsmuligheder med både GIPR agonister og antagonister. GIPR er overvejende udtrykt i bugspytkirtlen, fedtvæv, tarm, hjertet, hypofysen, binyrebarken og hjernen. I modsætning til GIP’s insulin- og glukagonregulerende effekt, er der mindre viden omkring dens fysiologiske rolle og den potentielle farmakologiske relevans i fedtvæv. Formålet med dette speciale er at identificere specifikke GIP-regulerede gener på mRNA niveau i fedtvæv, ved brug af RNA sekvensering af epididymal fedtvæv fra diæt induceret fede (DIF) mus behandlet med agonister for GIPR, Glucagon-like peptide 1 receptor (GLP-1R) og GIPR/GLP-1R. DIF musene blev behandlet i 17 dage med langtidsvirkende agonister. RNA-sekvensering identificerede 210, 3006 og 2850 transkripter som var signifikant reguleret af henholdsvis GIPR, GLP-1R og GIPR/GLP-1R agonisme. For at bekræfte den specifikke regulering af GIPR agonisme blev 74 transkripter kvantificeret med qRT-PCR i forskellige fedtdepoter (retroperitonealt-, lyske- og mesenterisk hvidt fedtvæv, samt intraskapular brunt fedtvæv), og i 3T3-L1 adipocytter behandlet med GIPR agonist eller liraglutide (GLP-1R agonist). På baggrund af kvantificeringen blev 48 transkripter udvalgt og testet i 3T3-L1 adipocytter for dosisafhængig regulering af GIPR agonisme. Transkripter for vascular-adhesion molecule 1 (vCAM1) og myosin regulatory light chain interacting protein (MYLIP) var dosisafhængig nedreguleret af GIPR agonisme, hvilket indikerer en direkte effekt forårsaget af receptor aktivering. Reguleringen af interferon induced protein 44 (IFI44) var mindre konsistent i dets dosisafhængige regulering. GIPR var også dosis afhængig nedreguleret ved højere dosis af GIPR agonist, som indikerer en potentiel desensibilisering. Fremtidige studier vil undersøge anvendeligheden af disse in vitro kvalificerede mRNA som akutte farmakologiske dynamiske biomarkører for GIPR agonisme i fedtvæv fra mus, for at understøtte farmakologien og mekanismen bag GIPR agonisme. 4 Table of contents Preface ......................................................................................................................... 1 Acknowledgements ................................................................................................ 2 Abstract........................................................................................................................ 3 Resume ........................................................................................................................ 4 Abbreviations ............................................................................................................ 8 1 Introduction ......................................................................................................... 9 1.1 Incretins ............................................................................................................................
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