Differences in signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide

• Published in: bioRxiv
• Main Authors: Pickford, Phil, Lucey, Maria, Fang, Zijian, Bitsi, Stavroula, Broichhagen, Johannes, Hodson, David, Minnion, James, Rutter, Guy A, Bloom, Stephen R, Tomas, Alejandra, Jones, Ben
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 16.10.2019
• Subjects: Agonists; Amino acids; Blood glucose; C-Terminus; Fluorescence resonance energy transfer; Glucagon; Glucagon-like peptide 1; Insulin; Insulin secretion; Intracellular signalling; Ligands; Peptides; Secretion
• DOI: 10.1101/803833

Background and purpose: Amino acid substitutions at the N-termini of glucagon-like peptide-1 receptor agonist (GLP-1RA) peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here we aimed to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1RAs exendin-4 and lixisenatide lead to similar phenomena. We also sought to establish the impact of the C-terminus on signal bias resulting from modifications elsewhere in the molecule. Experimental approach: Exendin-4, lixisenatide, and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand, and their biased derivatives, were assessed in mice. Key results: Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was 5-fold less potent for cAMP signalling. Both peptides were rapidly endocytosed, but the GLP-1R recycled more slowly to the plasma membrane after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice. N-terminal substitutions to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. Conclusion and implications: Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1R trafficking via mechanisms unrelated to GLP-1R occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.