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Abstract
BackgroundApelin signalling pathways have important cardiovascular and metabolic functions. Recently, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)], were reported to function independent of the apelin receptor in vivo to produce beneficial metabolic effects without modulating blood pressure. We aimed to show that these peptides bound to the apelin receptor and to further characterise their pharmacology in vitro at the human apelin receptor. Methods[Pyr1]apelin-13 saturation binding experiments and competition binding experiments were performed in rat and human heart homogenates using [125I]apelin-13 (0.1 nM), and/or increasing concentrations of apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] (50pM-100μM). Apelin-36 and its analogues apelin-36-[F36A], apelin-36-[L28A], apelin-36-[L28C(30kDa-PEG)], apelin-36-[A28 A13] and [40kDa-PEG]-apelin-36 were tested in forskolin-induced cAMP inhibition and β–arrestin assays in CHO-K1 cells heterologously expressing the human apelin receptor. Bias signaling was quantified using the operational model for bias. ResultsIn both species, [Pyr1]apelin-13 had comparable subnanomolar affinity and the apelin receptor density was similar. Apelin-36, apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] competed for binding of [125I]apelin-13 with nanomolar affinities. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting β-arrestin. Bias analysis suggested that these peptides were G protein biased. Additionally, [40kDa-PEG]-apelin-36 and apelin-36-[F36A] retained nanomolar potencies in both cAMP and β-arrestin assays whilst apelin-36-[A13 A28] exhibited a similar profile to apelin-36-[L28C(30kDa-PEG)] in the β–arrestin assay but was more potent in the cAMP assay. ConclusionsApelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases.
Highlights
Apelin is the endogenous ligand of the previously orphaned G protein-coupled apelin receptor [1]
We observed that in both species [Pyr1]apelin-13 bound with comparable single (Hill slopes were close to 1; human 0.95 ± 0.11, rat 0.95 ± 0.03) subnanomolar affinities
We have demonstrated that apelin-36-[L28A] and apelin-36[L28C(30kDa-PEG)] do bind to the apelin receptor in human and rat heart where they competed for binding with [125I]apelin-13 with nanomolar affinities
Summary
Apelin is the endogenous ligand of the previously orphaned G protein-coupled apelin receptor [1]. Apelin increases insulin sensitivity by promoting increased glucose uptake in skeletal muscle [11], and apelin knockout mice develop hyperinsulinemia and insulin resistance without any apparent differences in body weight [17,18]. In both normal and dietinduced obese mice apelin decreased adiposity, serum insulin and triglycerides, without modulating food intake [19]. Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] inhibited forskolin-induced cAMP release, with nanomolar potencies but they were less potent compared to apelin-36 at recruiting β-arrestin. Conclusions: Apelin-36-[L28A] and apelin-36-[L28C(30kDa-PEG)] are G protein biased ligands of the apelin receptor, suggesting that the apelin receptor is an important therapeutic target in metabolic diseases
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