The development of type 2 diabetes mellitus is closely associated with mitochondrial dysfunction of pancreatic β-cells, but the mechanisms by which glucagon-like peptide-1 receptor activation preserves mitochondrial homeostasis under glucotoxic conditions remain incompletely understood. Herein, we investigated whether Exendin-4 protects β-cells against chronic high glucose (HG)-induced mitochondrial injury by regulating the cAMP/PKA/Drp1 signaling pathway. INS-1 β-cells, pancreatic tissues from db/db mice, and isolated primary islets were used to assess oxidative stress, apoptosis, mitochondrial function and morphology, insulin secretion, and cAMP/PKA/Drp1 signaling. Prolonged HG exposure increased oxidative stress and apoptosis, impaired mitochondrial membrane potential, elevated mitochondrial ROS accumulation, reduced ATP content, and promoted mitochondrial fragmentation in INS-1 β-cells. These changes were accompanied by increased Drp1 expression, reduced cAMP levels and PKA activity, decreased inhibitory phosphorylation of Drp1 at Ser637, and increased Ser616 phosphorylation. Exendin-4 attenuated HG-induced oxidative stress and apoptosis, restored mitochondrial function, improved mitochondrial morphology, and partially restored Drp1 Ser637 phosphorylation, whereas it did not significantly affect HG-induced Ser616 phosphorylation. In db/db mice, Exendin-4 improved metabolic parameters and alleviated β-cell apoptosis, with partial recovery of Drp1 Ser637 phosphorylation in pancreatic islets. Furthermore, glucose-stimulated insulin secretion assays in isolated primary islets showed that Exendin-4 improved β-cell secretory function in islets isolated from db/db mice. Pharmacological inhibition of PKA with H89 attenuated Exendin-4-induced Drp1 Ser637 phosphorylation and mitochondrial protection. Collectively, these results suggest that Exendin-4 protects pancreatic β-cells against HG-induced mitochondrial dysfunction and β-cell injury by restoring PKA-associated inhibitory phosphorylation of Drp1 at Ser637 and improving mitochondrial dynamics.
