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Title: The role of GPR56 and collagen III in islet functions
Author: Olaniru, Oladapo Edward
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2017
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Abstract:
G-protein coupled receptors (GPCRs) are surface membrane receptors that convey a diverse array of signals and control many physiological processes within cells. Several human disorders are as a result of mutations in these receptors and they are the targets of more than 40% of all drugs in modern medicine. This thesis is focused on GPR56, a member of the adhesion class of GPCRs that is the most abundantly expressed GPCR mRNA in human islets. GPR56 is also highly enriched in endocrine progenitors in the developing pancreas. Its function in islets, however, remains elusive. Previous studies have indicated that extracellular matrix collagen III is the ligand of GPR56 in the developing brain, where GPR56-collagen III interaction is important for maintaining pial basement membrane integrity and in the normal development of cerebral cortex. The aim of this project was to investigate the importance of GPR56 in islet functions. GPR56 mRNA was detected by RT-PCR in MIN6 β-cells and in mouse and human islets, while mRNA encoding collagen III was detected in islets but not in MIN6 β-cells. Islet GPR56 expression was confirmed by detection of a 70kDa immunoreactive protein and immunohistochemistry revealed that it was strongly expressed by ductal and β-cells and faintly by exocrine acini cells. In the developing mouse pancreas, GPR56 was expressed by PDX1+, NGN3+ and SOX9+ endocrine progenitor cells. GPR56 was strongly expressed at the early days of pancreas development and became downregulated as the cells differentiated. It was then upregulated at the stage of β-cell replication between E18 and P9. Immunostaining revealed that collagen III was present in islet capillaries but not in endocrine cells. Acute exposure of islets to 100nM collagen III increased insulin output in a GPR56-dependent manner. In addition, pre-exposure of islets to collagen III for 48 h exhibited enhanced insulin secretion at stimulatory glucose concentration, without affecting insulin content. The role of GPR56 in regulating islet mass and β-cell proliferation was determined using GPR56 knockout (KO) mice. Deletion of GPR56 did not affect mouse body weight. There was a trend of mild glucose intolerance in adult KO mice fed on normal diet compared with the WT mice, but there was no difference in islet insulin content. Quantification of islet size and proliferating β- cells in fixed, immunostained pancreas samples revealed that islets from adult, postnatal day 56 mice (P56) KO mice were smaller than WT islets and this was more pronounced in P9 mice, as a consequence of decreased β-cell proliferation as indicated by BrdU incorporation. Moreover, the number of β-cells actively replicating in the cell cycle, as determined by Ki67 staining, was greater in the WT islets compared to KO islets at P9, leading to less β-cells but higher numbers of α-cells in KO islets. However, there were no differences in islet capillary density or percentage islet nerve area. In addition, transient transfection of MIN6 β-cells, a mouse insulinoma cell line with functional similarity to mouse islets, with 1.2mg of GPR56 plasmids resulted in a 20 fold increase in GPR56 expression and this was associated with increased β- cell proliferation and reduced apoptosis. In summary, the data presented in this thesis have demonstrated that GPR56 is expressed by mouse and human islets and collagen III is localised to islet blood vessels, suggesting that GPR56 may be regulated in a paracrine manner by local interaction with its activating ligand. In addition, GPR56 is differentially expressed during mouse islet development, where it is required at key stages of proliferation and differentiation of endocrine cells. However, GPR56 is not required for islet innervation and vascularisation of the developing endocrine pancreas. Evidence is also provided for the role for GPR56 in islet mass regulation, as a consequence of increased β-cell proliferation and decreased apoptosis.
Supervisor: Persaud, Shanta Jean ; Jones, Peter Martin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.789115  DOI: Not available
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