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<p>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<sup>INK4a</sup>/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, Ca<sup>2</sup>⁺ 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 Ca<sup>2</sup>⁺ 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<sup>INK4a</sup>/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.</p> Graphical Abstract <p></p>

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Development and characterization of experimental β-cell senescence models revealing autophagy defects and altered stimulus–secretion coupling

  • 期刊论文

  • 发表日期：

    2026年3月9日

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  • Nanxiang Yin,
  • Toshimasa Takahashi,
  • Hiroki Hayashi,
  • Taiki Sugimoto,
  • Heedong Jeon,
  • Weidong Liu,
  • Yu Guo,
  • Ziwei Wang,
  • Cheng Wang,
  • Akitoshi Hara,
  • Yoichi Takami,
  • Takashi Sakurai,
  • Hironori Nakagami,
  • Koichi Yamamoto

摘要

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, p16INK4a/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 p16INK4a/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.

Graphical Abstract