EP393 - ECE_1290 - Modulation of mitochondrial function and Ca2⁺ dynamics by semaglutide in pancreatic beta cells

Abstract

GLP-1 receptor agonists such as semaglutide regulate multiple stimulus–secretion coupling components in pancreatic beta cells, integrating metabolic, ionic, and secretory pathways. These agents influence mitochondrial ATP production, membrane excitability, and Ca2⁺ handling, all of which are essential for insulin secretion. Despite their clinical relevance, the mechanistic links between GLP-1 signaling, mitochondrial remodeling, and intracellular Ca2⁺ dynamics remain incompletely defined. This study aimed to determine whether semaglutide alters mitochondrial morphology and function, oxygen consumption, Ca2⁺ dynamics, and the immunocontent of ion-transport proteins in INS-1E beta cells. Cells were treated with 0-200 nM semaglutide for 24–72 h and evaluated using fluorescence microscopy for mitochondrial morphology, Seahorse extracellular flux analysis for respiratory parameters, FURA-2 imaging for Ca2⁺ dynamics (in the presence or absence of extracellular Ca2⁺ and in the presence of specific inhibitors), Amplex Red assays for extracellular H₂O₂ production, and automated Western protein detection. Semaglutide did not affect cell viability. Basal and ATP-linked oxygen consumption decreased over time, whereas non-mitochondrial oxygen consumption increased in a dose-dependent manner, with no differences in glucose-stimulated H₂O₂ formation. Mitochondrial volume decreased over time, whereas sphericity and branching parameters increased independently of semaglutide concentration. Semaglutide increased cytosolic Ca2⁺ in a dose-dependent manner in the presence of extracellular Ca2⁺; this response was abolished under Ca2⁺-free conditions and by verapamil or glibenclamide, indicating dependence on VDCCs and upstream modulation of KATP channels. Inhibitors of mitochondrial Ca2⁺ transport (CGP and ruthenium red) eliminated the effect, and thapsigargin suppressed the signal, indicating contributions from both mitochondrial uptake and ER store mobilization. Store-operated calcium entry was reduced only at 200 nM semaglutide. MCU protein content increased exclusively at 200 nM, whereas NCLX remained unchanged. Together, these preliminary findings indicate that semaglutide integrates bioenergetic and Ca2⁺-signaling responses in beta cells through combined modulation of mitochondrial remodeling, respiratory activity, and organelle-dependent Ca2⁺ handling.