Signalling, trafficking and glucoregulatory properties of glucagon‐like peptide‐1 receptor agonists exendin‐4 and lixisenatide

BACKGROUND AND PURPOSE: Amino acid substitutions at the N‐termini of glucagon‐like peptide‐1 (GLP‐1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub‐cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C‐termini of clinically approved GLP‐1 receptor agonists exendin‐4 and lixisenatide lead to similar phenomena. 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 fivefold less potent for cAMP signalling. Both peptides induced extensive GLP‐1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP‐1 receptor recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti‐hyperglycaemic and anorectic effects in mice with lixisenatide. N‐terminal substitution of His1 by Phe1 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‐1 receptor trafficking via mechanisms unrelated to GLP‐1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.