Strategies to Increase ß-Cell Mass Expansion

This electronic thesis or dissertation has been downloaded from the King’s Research Portal at <a href="/goto?url=https://kclpure.kcl.ac.uk/portal/" target="_blank">https://kclpure.kcl.ac.uk/portal/ </a>Strategies to increase -cell mass expansion Drynda, Robert Lech 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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Oct. 2021 Strategies to increase β-cell mass expansion A thesis submitted by Robert Drynda For the degree of Doctor of Philosophy from King’s College London Diabetes Research Group Division of Diabetes & Nutritional Sciences Faculty of Life Sciences & Medicine King’s College London 2017 Table of contents <a href="#3_0">Table of contents .................................................................................................. 2 </a><a href="#7_0">Abstract................................................................................................................. 6 </a><a href="#8_0">List of abbreviation</a><a href="#8_0">s</a><a href="#8_0">.</a><a href="#8_0">............................................................................................. 7 </a><a href="#12_0">List of figures....................................................................................................... 11 </a><a href="#14_0">List of tables........................................................................................................ 13 </a><a href="#15_0">Acknowledgements............................................................................................. 14 </a><a href="#16_0">Chapter </a><a href="#16_0">1</a><a href="#16_0">.</a><a href="#16_0">........................................................................................................... 15 </a><a href="#16_1">Introductio</a><a href="#16_1">n</a><a href="#16_1">.</a><a href="#16_1">........................................................................................................ 15 </a><a href="#17_0">1.1 </a><a href="#17_0">β</a><a href="#17_0">-cells and diabetes...................................................................................... 16 </a> 1.1.1Islets of Langerhans............................................................................. 16 1.1.2Insulin secretion and action.................................................................. 16 1.1.3Diabetes............................................................................................... 18 <a href="#21_0">1.2 </a><a href="#21_0">Hormonal control of β</a><a href="#21_0">-cell adaptation to pregnancy ..................................... 20 </a> 1.2.1Lactogenic hormones........................................................................... 21 1.2.2Hepatocyte Growth Factor signalling ................................................... 24 1.2.3Glucocorticoids..................................................................................... 25 1.2.4Oestradiol............................................................................................. 26 <a href="#28_0">1.3 Placenta functions ........................................................................................ 27 </a><a href="#29_0">1.4 The role of chemokines during pregnancy.................................................... 28 </a><a href="#0_0">1.5 G protein-coupled receptors ......................................................................... 30 </a> 1.5.1GPCR signalling................................................................................... 31 1.5.2G proteins............................................................................................. 34 1.5.3Proteins modulating GPCR functions................................................... 36 21.5.4Signalling independent of G proteins ................................................... 37 <a href="#0_4">1.6 WNT signalling ............................................................................................. 38 </a> 1.6.1WNT proteins and their receptors ........................................................ 38 1.6.2Canonical WNT/β-catenin signaling ..................................................... 38 1.6.3Regulation of the WNT pathway by R-spondins................................... 40 <a href="#0_1">1.7 Aims and objectives...................................................................................... 44 </a><a href="#0_1">Chapter </a><a href="#0_1">2</a><a href="#0_1">.</a><a href="#0_1">........................................................................................................... 45 </a><a href="#0_7">Materials and methods........................................................................................ 45 </a><a href="#0_8">2.1 Animals.........................................................................................................</a><a href="#0_8"> </a><a href="#0_8">46 </a> 2.1.1Mice for pancreatic islet BrdU staining................................................. 46 2.1.2Mice for placental GPCR ligand secretome and GPCRome analysis .. 47 <a href="#0_11">2.2 Immunofluorescence stainin</a><a href="#0_11">g</a><a href="#0_11">.</a><a href="#0_11">......................................................................</a><a href="#0_11"> </a><a href="#0_11">47 </a> 2.2.1Analysis of pancreatic β-cell proliferation............................................. 48 2.2.2Analysis of pancreatic β-cell area ........................................................ 48 <a href="#0_4">2.3 Isolation of mouse pancreatic islet</a><a href="#0_4">s</a><a href="#0_4">.</a><a href="#0_4">............................................................. 49 </a><a href="#0_14">2.4 RNA isolation................................................................................................</a><a href="#0_14"> </a><a href="#0_14">49 </a> <a href="#0_15">2.4.</a><a href="#0_15">1</a><a href="#0_15">T</a><a href="#0_15">otal RNA isolation from mouse pancreatic islets and pancreatic MIN6 </a><a href="#0_15">β</a><a href="#0_15">-cells.</a><a href="#0_15">.</a><a href="#0_15">.</a><a href="#0_15">........................................................................................................ 50 </a>2.4.2Determination of RNA concentration.................................................... 50 <a href="#0_4">2.5 cDNA synthesis ............................................................................................ 51 </a><a href="#0_1">2.6 Quantitative real-time polymerase chain reaction (qRT-PCR</a><a href="#0_1">)</a><a href="#0_1">.</a><a href="#0_1">..................... 52 </a> 2.6.1Normalization against mouse housekeeping gene............................... 53 2.6.2Screening for mRNA expression.......................................................... 55 2.6.3DNA agarose gel electrophoresis......................................................... 56 2.6.4Gene expression quantification............................................................ 56 <a href="#0_1">2.7 Analysis of RSPO4 protein concentration in mouse plasma during </a><a href="#0_1">pregnancy……………………………………………………………………………….</a><a href="#0_1">57 </a>3<a href="#0_18">2.8 </a><a href="#0_18">Culturing MIN6 β</a><a href="#0_18">-cells .................................................................................. 57 </a><a href="#0_19">2.9 </a><a href="#0_19">MIN6 β</a><a href="#0_19">-cell proliferation assay ..................................................................... 58 </a><a href="#0_1">2.10 </a><a href="#0_1">MIN6 β</a><a href="#0_1">-cell apoptosis assa</a><a href="#0_1">y</a><a href="#0_1">.</a><a href="#0_1">...................................................................... 61 </a><a href="#0_1">2.11 Measuring insulin secretion from MIN6 cells............................................... 63 </a><a href="#0_20">2.12 Insulin radioimmunoassay........................................................................... 64 </a><a href="#0_21">2.13 Statistical analysis....................................................................................... 66 </a><a href="#0_1">Chapter </a><a href="#0_1">3</a><a href="#0_1">.</a><a href="#0_1">........................................................................................................... 67 </a><a href="#0_7">Analysis of mouse pancreatic β</a><a href="#0_7">-cell proliferation during pregnancy ................... 67 </a><a href="#0_8">3.1 Introduction...................................................................................................</a><a href="#0_8"> </a><a href="#0_8">68 </a><a href="#0_15">3.2 Methods........................................................................................................</a><a href="#0_15"> </a><a href="#0_15">69 </a><a href="#0_1">3.3 Results..........................................................................................................</a><a href="#0_1"> </a><a href="#0_1">70 </a> 3.3.1Changes in pancreatic β-cell replication during pregnancy.................. 70 3.3.2Loss of β-cells post-partum.................................................................. 73 <a href="#0_1">3.4 Discussion</a><a href="#0_1"> </a><a href="#0_1">.................................................................................................... 75 </a><a href="#0_1">Chapter </a><a href="#0_1">4</a><a href="#0_1">.</a><a href="#0_1">........................................................................................................... 79 </a><a href="#0_7">Analysis of the mouse placental GPCR ligand secretome and pancreatic islet </a><a href="#0_7">GPCRome during pregnanc</a><a href="#0_7">y</a><a href="#0_7">.</a><a href="#0_7">............................................................................. 79 </a><a href="#0_23">4.1 Introduction...................................................................................................</a><a href="#0_23"> </a><a href="#0_23">80 </a><a href="#0_1">4.2 Methods........................................................................................................</a><a href="#0_1"> </a><a href="#0_1">82 </a><a href="#0_24">4.3 Results..........................................................................................................</a><a href="#0_24"> </a><a href="#0_24">83 </a> 4.3.1Analysis of the mouse placenta GPCR ligand secretome during pregnancy..................................................................................................... 83 4.3.2Analysis of the mouse pancreatic islet GPCRome during pregnancy .. 87 <a href="#0_1">4.4 Discussion</a><a href="#0_1"> </a><a href="#0_1">.................................................................................................... 96 </a><a href="#0_1">Chapter </a><a href="#0_1">5</a><a href="#0_1">.</a><a href="#0_1">......................................................................................................... 109 </a><a href="#0_7">A potential role for R-</a><a href="#0_7">spondin 4 in β</a><a href="#0_7">-cell adaptations to pregnanc</a><a href="#0_7">y</a><a href="#0_7">.</a><a href="#0_7">................. 109 </a><a href="#0_26">5.1 Introduction.................................................................................................</a><a href="#0_26"> </a><a href="#0_26">110 </a><a href="#0_1">5.2 Methods......................................................................................................</a><a href="#0_1"> </a><a href="#0_1">112 </a>4<a href="#0_1">5.3 Results........................................................................................................</a><a href="#0_1"> </a><a href="#0_1">113 </a><a href="#0_1">5.4 Discussion</a><a href="#0_1"> </a><a href="#0_1">.................................................................................................. 121 </a><a href="#0_1">Chapter </a><a href="#0_1">6</a><a href="#0_1">.</a><a href="#0_1">......................................................................................................... 125 </a><a href="#0_7">General discussion ........................................................................................... 125 </a><a href="#0_23">6.1 General discussio</a><a href="#0_23">n</a><a href="#0_23">.</a><a href="#0_23">....................................................................................</a><a href="#0_23"> </a><a href="#0_23">126 </a><a href="#0_27">6.2 Future perspective</a><a href="#0_27">s</a><a href="#0_27">.</a><a href="#0_27">...................................................................................</a><a href="#0_27"> </a><a href="#0_27">130 </a><a href="#0_1">Appendix </a><a href="#0_1">I</a><a href="#0_1">.</a><a href="#0_1">........................................................................................................ 132 </a><a href="#0_1">Appendix I</a><a href="#0_1">I</a><a href="#0_1">.</a><a href="#0_1">....................................................................................................... 133 </a><a href="#0_1">Appendix II</a><a href="#0_1">I</a><a href="#0_1">.</a><a href="#0_1">...................................................................................................... 148 </a><a href="#0_1">Appendix IV....................................................................................................... 160 </a>Reference<a href="#0_1">s</a><a href="#0_1">…………………………………………………………………………….</a><a href="#0_1">184 </a>5Abstract Aim: Failure of the functional β-cell mass to adapt to compensate for peripheral insulin resistance leads to the development of type 2 diabetes and gestational diabetes. The overall aim of this thesis was to study mechanisms regulating β-cell mass expansion, using pregnancy in mice as an experimental model in which the β-cell mass increases during gestation and returns to normal levels post-partum. The mechanisms underlying this adaptation are not well understood, although placental signals are thought to be involved. The first objective of the project was to analyse changes in the β-cell mass during pregnancy, and post-partum. The second objective was to quantify the expression of islet β-cell G protein-coupled receptors (GPCRs) and their placental ligands to identify novel placental signals potentially involved in β-cell adaptation to pregnancy. The third objective was to examine the effects of one of the novel placental signals, R-spondin 4 (RSPO4), on β-cell function. Methods: β-cell proliferation was quantified using immunohistochemical staining of pancreatic sections labelled with 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue. β-cell identity was confirmed by co-immunostaining for insulin and quantified by morphometric analysis. Islet GPCR and placental ligand mRNAs were quantified using a non-biased quantitative real time PCR (qRT-PCR) array approach. The effects of RSPO4 on insulin secretion, β-cell proliferation and survival were evaluated using radioimmunoassay, BrdU incorporation and caspase 3/7 apoptosis assays, respectively. Results: Mouse β-cells proliferated during pregnancy, peaking around gestational day 12, are the newly-formed β-cells which were not selectively lost post-partum. Placental expression of GPCR ligands was upregulated on day 12, and islet GPCR expression was differentially regulated during pregnancy. One candidate placental GPCR ligand, RSPO4, had pro-proliferative and insulinotropic effects in β-cells, consistent with an adaptive function during pregnancy. Conclusion: The placenta synthesizes many GPCR ligands, such as RSPO4, which have the potential to influence β-cell function during pregnancy, and which may have therapeutic potential in treating diabetes. 6List of abbreviations AKAP AKT (PKB) AP-2 A kinase anchoring protein Protein kinase B β2-adaptins ATP Adenosine triphosphate Bcl6 B-cell lymphoma 6 protein Body mass index BMI BrdU cAMP CaSR CCL11 CCL21 CCL7 CDK1 CDK4 CHOP C-MET C-MYC CNS 5-bromo-2'-deoxyuridine Cyclic adenosine monophosphate Calcium sensing receptor Eotaxin Chemokine (C-C motif) ligand 21 Chemotactic protein-3 Cyclin-dependent kinase 1 Cyclin-dependent kinase 4 CCAAT-enhancer-binding homologous protein Hepatocyte growth factor receptor V-Myc avian myelocytomatosis viral oncogene homolog Central nervous system CPT-1 CX3CL1 DAG Carnitine palmitoyltransferase 1 Fractalkine Diacylglycerol EDTA ELISA ER Ethylenediaminetetraacetate Enzyme-linked immunosorbent assay Endoplasmic reticulum ERK Extracellular signal–regulated kinase (MAPK) Fibroblast growth factor FGF FITC Fluorescein isothiocyanate 7FoxO1 GAD65 GAPs GDM Forkhead box protein O1 Glutamic acid decarboxylase GTPase-activating proteins Gestational diabetes mellitus GH Growth hormone GLP1 GLUT GPCR GR Glucagon-like peptide 1 receptor Glucose transporter G protein coupled receptor Glucocorticoid receptor GSIS Glucose stimulated insulin secretion Glycated haemoglobin HbA1c HCC-1 HCC-4 HGF Hemofiltrate CC chemokine-1 Hemofiltrate CC chemokine-4 Hepatocyte growth factor Human pituitary growth hormone Human placental growth hormone Hepatocyte nuclear factor-4α Human placental lactogen hGH-N hGH-V HNF-4α hPL HTR1D Serotonin receptor 1d (5-hydroksytryptamine receptor 1d) HTR2B Serotonin receptor 2b (5-hydroksytryptamine receptor 2b) IGF-1 IHC Insulin-like growth factor 1 Immunohistochemistry Interleukin 1β IL-1β INF-γ IPGTT IP3 Interferon γ Intraperitoneal glucose tolerance test Inositol 1,4,5-trisphosphate Insulin receptor substrate IRS 8Jak2 Janus kinase 2 LTB4 LTB4R1 MafA Leukotriene B4 Leukotriene B4 receptor 1 V-Maf masculoaponeurotic fibrosarcoma oncogene homologue A MAPK Mitogen activated protein kinase Macrophage-derived chemokine Major histocompatibility complex Macrophage inflammatory protein-1β Dual-specificity phosphoprotein phosphatase 1 Neurogenic differentiation 1 MDC MHC MIP-1β MKP-1 NeuroD1 NHERF2 NOD mice Na+/H+ exchanger regulatory factor 2 Non-obese diabetic mice (type 1 diabetes mellitus model) p18 Cyclin-dependend kinase 4 inhibitor C (CDKN2C) Cyclin-dependent kinase inhibitor 1 Cyclin-dependent kinase inhibitor 1B Cyclin-dependent kinase inhibitor 1C Pancreatic and duodenal homebox p21 p27 p57 Pdx-1 PGC-1α Peroxisome proliferator-activated receptor γ coactivator 1-α PA Phosphatidic acid PDZ Post-synaptic density of 95 kDa (PSD95)-disc large-zona occludens (PDZ) PI Phosphatidylinositol PIP2 PI3K PIBF PKA PKC Phosphatidylinositol 4,5-bisphosphate Phosphatidylinozytol 3-kinase Progesterone induced blocking factor 1 Protein kinase A Protein kinase C 9PL Placental lactogen PLD Phospholipase D PPARα PRL Peroxisome proliferator-activated receptor α Prolactin PRLR PTH1R PTH2R Rasd1 RSPO4 SAT Prolactin receptor Parathyroid hormone 1 receptor Parathyroid hormone 2 receptor Dexamethasone-induced ras-related protein 1 R-spondin 4 Subcutaneous adipose tissue Standard error of the means Sarcoendoplasmic reticulum Ca2+-ATPase 2 SH2-plekstrin homology domain Signal transducer and activator of transcription 3 Signal transducer and activator 5 Type 1 diabetes mellitus SEM SERCA2 SHC STAT3 STAT5 T1DM T2DM T3DM TGF-β1 TNF-α TPH1/2 TRB3 Tris Type 2 diabetes mellitus Type 3 diabetes mellitus Transforming growth factor β Tumour necrosis factor α Tryptophan hydroxylase 1 and 2 Tribble 3 Tris(hydroxymethyl)aminomethane Under-carboxylated osteocalcin Uncoupling protein 2

Strategies to Increase ß-Cell Mass Expansion

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