This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ A comparative analysis of G-protein- coupled receptor and peptide ligand expression within human and mouse islets of Langerhans, and its application in exploring the role of CXCL14 in islet function Hawkes, Ross Graham Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. 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Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 A comparative analysis of G-protein- coupled receptor and peptide ligand expression within human and mouse islets of Langerhans, and its application in exploring the role of CXCL14 in islet function A thesis submitted by Ross Graham Hawkes For the degree of Doctor of Philosophy from King’s College London Diabetes Research Group Division of Diabetes and Nutritional Sciences Faculty of Life Sciences and Medicine King’s College London Acknowledgements ACKNOWLEDGEMENTS I would firstly like to thank Professor Shanta Persaud and Professor Peter Jones for providing me with the opportunity to work for the Diabetes Research Group, and for creating a working environment that was perfect for undertaking a PhD in. The Diabetes Research Group at King’s College London is a unique and special place to work, and one that I will look back on fondly. An extended thank you to Professor Shanta Persaud for her guidance and support during my time at King’s, not only in relation to her supervisory role, but as someone who has lent an ear and provided sound advice in times that have required clarity and perspective. A very special thank you to Dr Stefan Amisten for the opportunity to undertake a PhD under his supervision. His patience, positivity, trust, contagious curiosity and open-mindedness have made these last years a truly interesting and enjoyable experience. I will be forever grateful to Stefan for this opportunity and look forward to celebrating over a slice of blueberry pie! In addition to my supervisors, I would also like to thank the entire Diabetes Research Group, past and present, especially Patricio Atanes, Amazon Austin, Zara Franklin, Zoheb Hassan, Robert Drynda, Alan Kerby, Ahmed Arzouni, Kerry Mclaughlin, Carolyn Johnson, Astrid Hauge-Evans, Bo Liu, Anastasia Tsakmaki, Inmaculada Ruz Maldonado, Thomas Hill and Jai David. Having fellow colleagues like these made every day that bit more enjoyable. Lastly, I would like to thank my wife Becky. Her continuous support during the ever changing course that my career has taken us on has been inspiring. Here’s to us and the future. 2 Abstract ABSTRACT G protein-coupled receptors (GPCRs) are a diverse super family of seven transmembrane spanning proteins whose primary function is to initiate the activation of intracellular signalling pathways following stimulation by extracellular stimuli, which include photons, amines, lipids, ions, peptides and proteins. Due to the ubiquitous expression of GPCRs throughout various tissues, they are implicated in the regulation of a variety of diverse physiological processes, such as secretion of the blood glucose controlling hormones insulin, glucagon and somatostatin from islets. As a result, GPCRs are being identified as therapeutic targets for the treatment of type-2 diabetes. Despite the large array of potential GPCR targets available, only a handful of GPCRs have proven to be successful clinical targets, which may partially be due to the lack of availability of suitable translational models that reflect the human GPCR landscape. The aim of the experiments described in this thesis was to compare the mRNA expression profiles of all GPCRs (the GPCRome) and all GPCR peptide ligands (the Secretome) in human and mouse islets in order to determine the suitability of using mouse islets as a translational model for predicting the role of islet GPCRs and GPCR peptide ligands in human islet function. In addition, some experiments demonstrate how the GPCRome and Secretome data were used to assess the role of CXCL14 in islet function. Quantitative real-time PCR (qPCR) was used to compare the mRNA expression profiles of 376 GPCRs and 159 GPCR peptide ligands in human islets with their orthologous counterparts in islets isolated from two strains of mice (outbred ICR mice and inbred C57/BL6 mice). A reasonable degree of similarity in GPCR mRNA expression between human islets and islets from each mouse strain was found (r2 = 0.360 vs. ICR; r2 = 0.304 vs C57), with a highly similar expression profile observed between the ICR and the C57 mouse strains (r2 = 0.946). Regression analysis of GPCR peptide ligand mRNA expression revealed that human islets exhibit a reasonable degree of similarity compared to islets of both mouse strains (r2 = 0.245 vs. ICR; r2 = 0.225 vs. C57) with a highly similar expression profile observed between the two mouse strains (r2 = 0.968). In the process of quantifying the GPCR peptide ligand mRNA expression profiles, it was revealed that the orphan chemokine CXCL14 is expressed by both mouse and human islets. Studies have shown that CXCL14 knockout mice are protected from hyperglycaemia and hyperinsulinemia and they have improved insulin sensitivity. However, CXCL14’s role in islet function has yet to be explored. The experiments described in this thesis demonstrate that CXCL14 inhibits insulin secretion from mouse islets and the MIN6 mouse β-cell line by a 3 Abstract mechanism that is not transduced through a Gαi-mediated reduction in intracellular cAMP, but is likely to occur through an inhibition of glucose uptake or glucokinase activity. Further experiments designed to elucidate the target involved in CXCL14 function revealed that CXCL14 is neither an agonist nor an antagonist for the CXCR7 receptor and the putative CXCR4 receptor, and thus these receptors are not responsible for mediating CXCL14 function. In summary, the experiments described in this thesis reveal that human and mouse islets exhibit some degree of similarity in GPCR and GPCR peptide ligand mRNA expression, but the suitability of using mouse islets as surrogates for predicting human islet physiology is receptor/receptor family specific. This thesis also reveals how the GPCRome and Secretome data can be employed to investigate the role of particular ligands, such as CXCL14, and potential GPCRs responsible for mediating the actions of such ligands in islet function. 4 Abbreviations Abbreviation Definition 18S 18S ribosomal rRNA 2DG 2-deoxy-D-glucose α Alpha Ab Antibody ATP Adenosine triphosphate β Beta bp Base pair BrdU 5-bromo-2’-deoxyuridine BSA Bovine serum albumin oC Degrees centigrade Ca2+ Calcium cAMP Cyclic adenosine 3’, 5’ - monophosphate cDNA Complementary DNA CO2 Carbon dioxide Conc. Concentration cpm Counts per minute Ct Threshold cycle CXCL12 Chemokine (C-X-C motif) ligand 12 CXCL14 Chemokine (C-X-C motif) ligand 14 dH2O Distilled water DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulphoxide DNA Deoxyribonucleic acid ECM Extracellular matrix 5 Abbreviations EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay Emax Maximum efficacy ER Endoplasmic reticulum FBS Foetal bovine serum FLIPR Fluorescent light imaging plate reader FRET Fluorescence resonance energy transfer Fura-2-AM Fura-2-acetoxymethyl derivative γ Gamma GAPDH Glyceraldehyde 3-phosphate dehydrogenase G6P Glucose-6-phosphate G6PDH Glucose-6-phosphate dehydrogenase GDP Guanosine diphosphate GLP-1 Glucagon-like peptide-1 GLUT Glucose transporter GPCR G-protein-coupled receptor Grb2 Growth factor receptor-bound protein 2 GTP Guanosine triphosphate HbA1c Glycated haemoglobin A1c HCl Hydrochloric acid Hr Hour HTRF Homogeneous time-resolved fluorescence IBMX 3-isobutyl-1-methylxanthine 6 Abbreviations INS-1 Rat insulinoma cell line IP3 Inositol 1,4,5-triphosphate IR Insulin receptor IRS Insulin receptor substrate µ Micro µg Microgram µM Micromolar mg Milligram Min Minute MIN6 Mouse insulinoma cell line mM Millimolar ml Millilitre mRNA Messenger RNA MuLVRT Murine Leukaemia Virus Reverse Transcriptase Mwt Molecular weight NaOH Sodium hydroxide NCS Newborn calf serum ng Nanogram nM Nanonmolar NSB Non-specific binding PBS Phosphate-buffered saline PCR Polymerase chain reaction PEG Polyethylene glycol PI3K Phosphatidylinositol-3-kinase 7 Abbreviations PIP2 Phosphatidylinositol 4,5-biphosphate PKA Cyclic-AMP-dependent protein kinase A PKC Protein kinase C PLA2 Phospholipase A2 PLC Phospholipase C PLD Phospholipase D PPIA