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Title: The role of beta-cell glutamate receptors in pancreatic endocrine function and in the pathogenesis of type 1 diabetes mellitus
Author: Dwomoh, Louis
ISNI:       0000 0004 5992 9017
Awarding Body: University of the West of England
Current Institution: University of the West of England, Bristol
Date of Award: 2017
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Background: Kainate receptors (KARs) are one of the three classes of ionotropic glutamate receptors (iGluRs) expressed primarily in the central nervous system (CNS) where they mediate information transfer and neurotransmitter release. Very little is known about native KARs and their interacting partners outside the CNS. Aim: The aim of this study was to investigate systematically the molecular composition and functional properties of KARs in pancreatic endocrine cells and also to investigate the role of KARs in the pathogenesis of T1DM. Methods: The presence of KAR subunits was investigated in pancreatic clonal β-cells (MIN6 and INS-1), α-cells (α-TC) and primary rat islets of Langerhans using RT-PCR and immunoblotting. The effect of KAR activation on intracellular calcium concentration and insulin secretion in MIN6 was investigated using FURA-2AM epifluorescence imaging and Mercodia insulin assay respectively. The effect of glutamate, kainate and glutamate transporter inhibitor (dihydrokainic acid) on survival and viability of INS1, α-TC and neuroblastoma cells (SH-SY5Y) were investigated using MTT cell viability assay. The presence of autoantibodies against GluR subunits in serum of T1DM patients and controls was investigated using ELISA and immunoblotting. Results: RT-PCR identified mRNAs for GluK2-5 KAR subunits in clonal β-and α-cells and all five subunits (GluK1-5) in primary rat islets of Langerhans. The presence of these subunits was confirmed using immunoblotting with GluK2/3 and GluK5 antibodies. In addition, auxiliary KAR subunits Neto 1 and Neto 2 were also identified in all cell types. FURA-2AM epifluorescence imaging of cultured MIN6 β-cells showed that activation of KARs with kainate induced significant increase in intracellular calcium concentration and insulin secretion. These effects of kainate were blocked by KAR antagonist (NBQX; 30μM) but not by an antagonist (GYKI-53655 hydrochloride; 100μM) of other iGluRs. Chronic exposure to kainate (0.1-0.5mM), glutamate (0.25-12mM) and dihydrokainic acid (0.1mM) cause significantly reduced viability of pancreatic endocrine and neuronal cells. ELISA and immunoblotting showed that serum of T1DM patients and non-diabetic controls react against components of synaptosomal plasma membrane and also against overexpressed GluR subunits. Conclusion: Together, these results indicate that a range of functional KAR subunits and their interacting proteins are expressed in the endocrine pancreas. These KAR subunits identified could assemble as homomeric or heteromeric channels in both β-and α-cells of the pancreas. The activation of these receptors is likely to have an impact on pancreatic hormone secretion and viability of endocrine cells in the islets of Langerhans. Much work ought to be carried out to optimise the methods for identification of autoantibodies against GluR subunits in serum of T1DM patients. This will help to understand the potential role of GluRs in the pathogenesis of T1DM.
Supervisor: Not available Sponsor: University of the West of England
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available