Introduction and Objective: Dual incretin agonists have emerged as a promising therapeutic class for metabolic disorders. Tirzepatide, a first-in-class dual agonist, exhibits imbalanced activity towards the two incretin receptors, highlighting the need for cellular tools to allow mechanistic dissection of receptor-specific contributions. Methods: The EndoC-βH5® human pancreatic beta-cell line provides a physiologically relevant model, closely recapitulating the gene expression and functional properties of native human beta cells. To investigate GLP-1R-specific signalling, an EndoC-βH5® cell line with stable GLP1R knockdown was generated using a miRNA-based approach. Receptor gene silencing was confirmed by quantitative PCR. Cells were fully characterized in functional assays: glucose-stimulated insulin secretion assays and intracellular cyclic AMP (cAMP) measurements. Results: qPCR confirmed the complete loss of GLP1R expression in the knockdown model. In addition, stimulation with Exendin-4, even at concentrations 100-fold greater than the reported EC50, failed to elicit either cAMP accumulation or insulin secretion, confirming functional silencing. Comparative analysis of D-Ala²-GIP-induced responses between control and GLP1R knockdown cells revealed no differences in insulin secretion, confirming the specificity of the knockdown. Finally, evaluation of tirzepatide responses demonstrated that GLP1R depletion markedly diminished tirzepatide-induced cAMP production. Conclusion: These findings demonstrate the successful establishment of a unique EndoC-βH5® - based model that enables detailed investigation of the mechanisms underlying multireceptor agonist action through integrated analysis of cyclic AMP signaling and insulin secretion. This model will provide critical insights into the molecular basis of dual and multi-agonist therapies for obesity and type 2 diabetes, including the recruitment dynamics of β-arrestin and G proteins to assess potential signaling bias of candidate molecules. Disclosure H. Olleik: Employee; Current; Human Cell Design. B.C. Blanchi: None.
Olleik et al. (Fri,) studied this question.