AbstractThis thesis investigates the in vivo and in vitro ability of glucagon-related molecules isolated from a wide range of fish species including sea lamprey, dogfish, ratfish, paddlefish, zebrafish and trout, to exert antidiabetic actions by acting on multiple GPCR receptors in a mammalian system. Overall, the piscine peptides with the most potential were glucagon and GLP-1 from sea lamprey and paddlefish, and oxyntomodulin from dogfish. These proglucagon-derived molecules showed insulinotropic activities in rat clonal pancreatic beta cells (BRIN-BD11), human-derived pancreatic beta cells (1.1B4) and isolated mouse islets similarly to human GLP-1. The dual agonist properties of fish hormones were demonstrated in vitro using glucagon and GLP-1 receptors antagonists, GLP-1R-transfected CHL and glucagonR-transfected HEK293 cells as well as CRISPR/CAS--engineered cells bearing either GLP-1R or glucagonR knock-out. Acute administration of fish peptides to normal mice produced significant beneficial antihyperglycaemic and insulinotropic changes, however, the actions were limited due to rapid enzymatic degradation in plasma by DPP-IV.
Structural modifications including the stereochemical configuration and the addition of a γ-glutamyl spacer to the selective fish glucagon and GLP-1 sequences were used to develop novel enzymatically stable analogues namely [D-Ser2 ]-lamprey glucagon-Lys30-gamma-glutamyl-pal, [D-Ser2 ]-paddlefish glucagon-Lys30-gamma-glutamylpal, [D-Ala2 ]-lamprey GLP-1-Lys31-gamma-glutamyl-pal and [D-Ala2 ]-paddlefish GLP-1-Lys28-gamma-glutamyl-pal. The most promising analogues, [D-Ala2 ]-lamprey GLP-1-Lys31-gamma-glutamyl-pal and [D-Ser2 ]-paddlefish glucagon-Lys30-gammaglutamyl-pal retained the potent biological properties of the native peptides as indicated in acute in vitro and in vivo studies. Moreover, chronic administration of [D-Ala2 ]-lamprey GLP-1-Lys31-gamma-glutamyl-pal and [D-Ser2 ]-paddlefish glucagonLys30-gamma-glutamyl-pal analogues improved metabolic status, islet morphology and the expression genes involved in insulin secretion in diet-induced obese mice and showed positive effects on beta-cell transdifferentiation in genetically modified GluCreRosa26-YFP mice.
To conclude, this thesis outlines the ability of fish glucagon-derived peptides and their analogues to modulate simultaneously multiple receptor signalling pathways resulting in prominent antidiabetic effects and illustrates the potential of the peptides to be developed into therapeutic agents for the treatment of type 2 diabetes-obesity.
|Date of Award||Apr 2019|
|Supervisor||Peter Flatt (Supervisor), Yasser Abdel-Wahab (Supervisor) & Michael Conlon (Supervisor)|