Analysis of physiologically relevant signalling events via GLP-1R in insulinoma cells
2016-11-29T04:36:08Z (GMT) by
Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted in response to nutrient ingestion following itâ€™s binding to the GLP-1 receptor (GLP-1R). Long-acting GLP-1R peptidomimetics have become an important class of therapeutics for treating type 2 diabetes (T2D) because of their gluco-regulatory actions. However, they have been associated with adverse side-effects. Pharmaceutical companies are actively pursuing development of small molecule ligands as alternatives, but with little success to date. GLP-1R ligands can stabilize distinct subsets of receptor conformations that can â€œtrafficâ€ stimulus to diverse functional outputs with varying prominence, a concept referred to as biased agonism. Allosteric modulators can also alter the signalling profiles of orthosteric ligands in a ligand-dependent manner, termed probe-dependence. While GLP-1R biased agonism and allosteric probe dependence are established, to date most studies are from recombinant systems overexpressing the GLP-1R. This thesis utilizes a reductionist (cAMP accumulation, glucose-stimulated insulin secretion (GSIS), ERK1/2 phosphorylation, proliferation and apoptosis) and non-reductionist approach (transcriptomics) to understand GLP-1R biased agonism and allosteric modulation in natively expressing cells that display glucose dependence, addressing some of the issues of translation from recombinant to more physiologically relevant systems mimicking Î²-cell physiology. Extensive and systemic analysis performed using GLP-1R peptides in INS-1 832/3 insulinoma cells, revealed that cAMP accumulation, proliferation and anti-apoptosis were glucose-independent, whereas, insulin secretion, [Ca2+]i mobilization and aspects of the ERK1/2 phosphorylation kinetics were glucose-dependent. Furthermore, attenuation of glucose-mediated activation of ERK1/2 at 5 min by GLP-1R peptides was a novel, unexpected observation. Assessment of ligand signalling profiles revealed that biased agonism occurred with distinct ligands; however the bias profile was different in two physiologically relevant glucose concentrations. One significant observation was a large degree of bias at the therapeutically relevant endpoints of proliferation and apoptosis, where for equivalent amounts of cAMP generated, GLP-1 was more efficacious compared to exendin-4 and oxyntomodulin. For small molecule ligands there was bias relative to GLP-1 between the amount of cAMP production and insulin secretion that was not observed with peptide ligands. In addition, both BETP and Compound 2 allosterically altered the signalling profiles of peptide ligands, in a peptide-dependent manner that differed depending on the glucose concentration assessed, observations that have clinical relevance for the development of allosteric drugs. In the final chapter, analysis of transcriptomics data identified a number of genes associated with functions of cell to cell signalling, signal transduction, proliferation, cell death and survival, along with number genes for GPCRs and GPCR ligands that were up or down-regulated following GLP-1R activation. In addition, a number of genes associated with T2D, but not previously with GLP-1R signalling also emerged. Exendin-4 and oxyntomodulin displayed bias relative to GLP-1 at the level of gene transcription with a number of differentially regulated genes identified. Thus, this thesis expands the existing knowledge around GLP-1R pharmacology and signalling. This will facilitate ligand profiling and better understanding of biased agonism in natively expressing systems that may be useful for future therapeutic development.