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Title: Characterisation and pharmacological regulation of GLP-1-mediated glucose homeostasis
Author: Yeung, Ho Yan
ISNI:       0000 0004 9359 9840
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2020
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Type 2 diabetes mellitus (T2DM) is characterised by the hormonal imbalance of insulin and glucagon, leading to dysfunctional glucose homeostasis. Glucagon-like peptide 1 (GLP-1), which is an incretin hormone, activates the predominantly Gαs-coupled glucagon-like peptide 1 receptor (GLP-1R), which is a class B G protein-coupled receptor (GPCR), to mediate glucose homeostasis. It does so by promoting glucose stimulated insulin secretion (GSIS) in the pancreatic β cells and inhibiting glucagon secretion in the pancreatic α cells. Given its proven clinical efficacy in reducing long term blood glucose level, GLP-1-based treatments, such as exenatide and liraglutide, have been widely used in T2DM patients. However, in contrast to the well-studied phenomenon of how GLP-1 enhances GSIS, the mechanism of how GLP-1 regulates glucagon secretion is still unclear. Therefore, the aim of this work is to shed new lights on how GLP-1 mediates its glucagonostatic action. To do so, the signalling properties of GLP-1 and its closely-related peptide hormones, namely oxyntomodulin (OXM), glucagon (GCG), glucose-dependent insulinotropic polypeptide (GIP), and its metabolite, GLP-1(9-36)NH2, were examined in recombinant cell lines and rodent clonal α and β cell lines using cAMP functional assaying technique. It was demonstrated that these glucagon-like peptides, including GLP-1(9-36)NH2 yet except GIP, can activate both GLP-1R and glucagon receptor (GCGR), which is struc- turally analogous to GLP-1R. Furthermore, GLP-1R, despite its very low expression in the mouse αTC1.6 cell line detected through semi-quantitative RT-PCR studies, is found to play a critical role in directly inhibiting glucagon secretion upon GLP-1 activation through performing glucagon secretion antagonism studies. More importantly, the physiologically abundant GLP-1 metabolite is discovered to play a glucagonostatic role in the mouse glucagonoma cell line via the direct actions of GLP-1R and GCGR, an observation that has not yet been documented. Therefore, this thesis provides evidence of how GLP-1 and its metabolite are actively involved in their glucagonostatic actions via direct activations of GLP-1R and GCGR. Another aim of this work is to identify viable pharmacological regulator of GLP-1- mediated glucose homeostasis through the action of positive allosteric modulator (PAM). Here, compound 249, which was identified previously as a small molecule GLP-1R PAM, was further pharmacologically validated using various signal transduction assaying techniques in recombinant cell lines. It was also demonstrated that compound 249 works independent of the cysteine-347 residue on the GLP-1R, an amino acid residue which has been previously shown to be instrumental for the actions of another GLP-1R agonist-PAMs. More importantly, compound 249 demonstrates robust potentiation of GLP-1 and OXM-augmented GSIS in the rat INS-1 832/3 insulinoma cell line and ex vivo isolated mouse islets, substantiating the potential of compound 249 to be further developed as a novel T2DM treatment. Overall this thesis presents new evidence on the direct involvement of GLP-1R on GLP-1-regulated glucagon secretion in the pancreatic α cells and illustrates compound 249 as a PAM to promote GLP-1 mediated GSIS. The findings in this thesis will be used for future design of safer and more efficacious T2DM treatments.
Supervisor: Ladds, Graham Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: G protein-coupled receptors ; Incretins ; Drug discovery ; Type 2 diabetes mellitus ; GLP-1