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Title: Beta-cell dedifferentiation following short term hypoxia and clinical islet transplantation
Author: Anderson, Scott James
ISNI:       0000 0004 7425 2497
Awarding Body: University of Newcastle upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2017
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Individuals with insulin-requiring type 1 diabetes account for around 10% of all diabetes cases in the UK. Administration of exogenous insulin to maintain blood glucose homeostasis can lead to life-threatening hypoglycaemia in a subset of people with type 1 diabetes, who have lost hypoglycaemic awareness. Islet transplantation is a life-saving therapy for these individuals who suffer recurrent, hospital requiring, episodes of hypoglycaemia. Despite improving outcomes, mainly due to an improved immunosuppression regimen, the functional duration of islet transplants still fails to match the gold standard in endocrine replacement therapy, whole pancreas transplant. During pancreas retrieval, islet isolation and post-transplantation, islets are subjected to prolonged periods of low oxygen (hypoxia). Islets are highly vascularised within the pancreas, providing sufficient oxygen for appropriate beta-cell glucose sensing and appropriate insulin secretion. Islet hypoxia is associated with decreased beta-cell function and worse transplant outcomes. Despite this, the processes which cause beta­cell dysfunction during periods of hypoxia are not fully understood. The aim of this thesis was to elucidate the mechanisms of hypoxia-induced beta-cell dysfunction. Using the MIN6 functional beta-cell line, hypoxic stress was modelled in vitro. MIN6, subjected to short-term hypoxia, demonstrated decreased viability, increased expression of hypoxia-associated genes and reduction in beta-cell specific gene expression. Gene expression changes were associated with decreased in vitro function, decreased insulin content and nuclear-to-cytoplasmic translocation of beta­cell transcription factors Pdxl and Nkx6.1. Using optimised isolation protocols, freshly isolated human islets were established in normal and hypoxic culture conditions. Hypoxic cultured islets demonstrated decreased expression of beta-cell genes, decreased function and viability. Further analysis of beta-cell phenotype by immunofluorescence identified decreased co­expression of end-differentiated marker urocortin-3 and insulin, alongside co­expression of insulin and the mesenchymal marker vimentin in hypoxia treated islets. This model suggests short-term hypoxic exposure causes changes in end- differentiated islet beta-cell phenotype. To determine if these phenotypes are present following clinical islet transplantation we performed immunofluorescence analysis of islets engrafted within recipient liver. Transplanted islet beta-cells in two recipients with excellent graft function demonstrated loss of mature phenotype, with all insulin positive cells lacking co­expression of urocortin-3. Transplanted islet beta-cells also contained cells co­expressing insulin and vimentin, suggesting mature end-differentiated phenotype is lost following transplantation. To determine whether hypoxia-induced HIF1α stabilisation plays a role in loss of end- differentiated beta-cell phenotype, HIF1α stabilisation and knockdown models were established in the MIN6 cells. Using cobalt chloride as a hypoxia mimic leading to HIF1α stabilisation led to changes in beta-cell gene expression and function comparable to those induced by hypoxia. These changes persisted following HIF la knockdown demonstrating that changes in beta-cell phenotype during hypoxia are HIF1α independent. These studies offer new insight into loss of mature beta-cell phenotype as a novel cause of beta-cell dysfunction during islet hypoxia.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available