Within the pancreatic β-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in β-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from β-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the β-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in β-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 β-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the β-cell environment in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.

中文翻译：

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荧光计时器报告器可以按年龄对胰岛素分泌颗粒进行分类。


在胰腺 β 细胞内，胰岛素分泌颗粒 (SG) 存在于功能不同的池中，显示出运动性以及对接和融合能力的变化。目前增加胰岛素分泌的疗法没有考虑这些不同的 SG 池的存在。因此，这些方法仅在短期内有效，并且血糖恶化与β细胞功能的持续下降相关。胰岛素颗粒年龄作为选择胰岛素颗粒分泌的决定因素并未得到充分重视，并且可以解释为什么新合成的胰岛素优先从 β 细胞分泌。在这里，我们使用一种新型荧光计时器蛋白，旨在研究胰岛素分泌的优先分泌模型，并确定β细胞环境变化（例如高血糖）如何影响颗粒老化。我们演示了荧光定时器构建体 Syncollin-dsRedE5TIMER 的使用，该构建体在显微镜和荧光辅助细胞器分选技术中在 18 小时内将其荧光从绿色变为红色。我们确认 SG 靶向结构定位于 β 细胞中的胰岛素颗粒，并且不会干扰正常的胰岛素 SG 行为。我们观察了 MIN6 和 INS1 β 细胞系以及原代 C57BL/6J 小鼠和非糖尿病人胰岛细胞中胰岛素 SG 的老化行为。最后，我们将年轻和年老的胰岛素 SG 分开，揭示了在葡萄糖刺激的胰岛素分泌中，较年轻的颗粒优先分泌。我们还表明，SG 群体年龄受到 db/db 小鼠胰岛体内 β 细胞环境的调节，以及暴露于不同葡萄糖环境的 C57BL/6J 胰岛的离体 β 细胞环境的调节。