Development and characterization of experimental β-cell senescence models revealing autophagy defects and altered...

Aging is a major contributor to β-cell dysfunction in type 2 diabetes, with cellular senescence increasingly recognized as an important underlying mechanism. Here, we established a doxorubicin (DOX)-induced senescence model using MIN6 mouse insulinoma β-cell line to elucidate the mechanisms by which senescence remodels organelle homeostasis and insulin secretion. Senescence was validated by senescence-associated β-galactosidase positivity, p16^INK4a/p21/p53 upregulation, and cell cycle arrest. Using mitochondrial MitoTimer and cytochrome c oxidase subunit 8-enhanced green fluorescent protein-mCherry reporters, along with a custom Insulin-Timer construct, we visualized organelle aging and assessed β-cell functions via glucose-stimulated insulin secretion, insulin content, mitochondrial membrane potential, Ca²⁺ imaging, and reactive oxygen species production analyses. Senescent β-cells accumulated mitochondria and insulin granules with prolonged residence time since synthesis and exhibited defective clearance and exaggerated mitochondrial hyperpolarization accompanied by altered Ca²⁺ influx and enhanced reactive oxygen species production upon glucose stimulation. Despite the reduced insulin content, secretion normalized to storage was disproportionately enhanced, suggesting remodeled stimulus-secretion coupling; similar findings were also observed in the p16^INK4a/p21/p53 overexpression model. This study provides evidence that DOX-induced β-cell senescence serves as a surrogate model linking DNA damage to impaired mitochondrial and insulin granule clearance. By mimicking aspects of age-related β-cell dysfunction, this model highlighted autophagy defects as drivers of organelle retention and provided insights into the mechanisms by which senescence reshapes stimulus-secretion coupling, thereby enhancing our understanding of β-cell senescence in diabetes. Importantly, this experimentally tractable model provides a platform to test interventions targeting senescent-cell burden or senescence-associated dysfunction and to dissect mechanisms of β-cell functional remodeling.