Nanobiology Institute, Yale University School of Medicine, West Haven, CT, 06516 USA; Department of Pathology, Yale University School of Medicine, New Haven, CT, 06520 USA.
Nanobiology Institute, Yale University School of Medicine, West Haven, CT, 06516 USA; Department of Cell Biology, Yale University School of Medicine, New Haven, CT, 06520 USA.
Cell Calcium. 2023 Jul;113:102766. doi: 10.1016/j.ceca.2023.102766. Epub 2023 Jun 3.
High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.
目前的方法难以实现对第一和第二阶段胰岛素分泌动力学的高通量定量分析。独立的分泌阶段在代谢中发挥不同的作用，这一事实需要将它们分开，并进行高通量化合物筛选，以分别针对它们进行靶向治疗。我们开发了一种胰岛素纳米荧光素酶报告系统，以剖析参与胰岛素分泌的独立阶段的分子和细胞途径。我们通过遗传研究（包括敲低和过表达）、小分子筛选及其对胰岛素分泌的影响对该方法进行了验证。此外，我们证明该方法的结果与在活细胞上进行的单个囊泡胞吐实验的结果高度相关，为该方法提供了定量参考。因此，我们已经开发出一种用于筛选针对胰岛素分泌特定阶段的小分子和细胞途径的稳健方法，从而更好地了解胰岛素分泌，这反过来又将通过刺激内源性葡萄糖刺激的胰岛素分泌来实现更有效的胰岛素治疗。
