Identifying the Molecular Pathways Regulating Glucose-dependent Insulin Secretion

描述(申请人提供)：β细胞功能障碍在2型糖尿病(T2D)的发病机制中起着核心作用。1即使在诊断之前，糖耐量受损(即“糖尿病前期”)的患者就缺乏葡萄糖刺激的胰岛素分泌的第一阶段，并对正常情况下在进餐时启动β细胞的胰岛素激素的放大作用表现出抵抗。2虽然胰岛素分泌不足现在被认为是T2D的发生和发展的先决条件，但调节这一关键生理反应的分子途径仍然很大程度上未知。此外，目前还没有可用的治疗方法来防止糖尿病特有的β细胞递减的进行性本质，3也不存在FDA批准的以葡萄糖依赖的方式直接增加胰岛素分泌的小分子，从而避免低血糖的风险。4为了开发调节葡萄糖依赖的胰岛素分泌的分子通路的化学探针，并确定葡萄糖依赖治疗的小分子先导，我们提议的项目将在近刺激性血糖条件下采用一种新的胰岛素分泌生物测定。从历史上看，由于缺乏合适的功能读数，识别胰岛素分泌调节器的筛查一直是不可行的；标准的胰岛素ELISA法是劳动密集型的，价格昂贵，并且限于96孔格式。为了克服这一瓶颈，我们开发了一种高通量发光胰岛素分泌试验，其中荧光素酶靶向于β细胞的分泌囊泡，并在刺激时与胰岛素共同分泌。荧光素酶是胰岛素的密切替代物，其活性与已知的促分泌剂和胰岛素分泌抑制剂有适当的反应，并与用酶联免疫吸附试验测得的胰岛素密切相关(R2=0.96)。在近刺激性葡萄糖浓度下发现增加荧光素酶分泌的化合物将在没有葡萄糖的情况下进行反筛选，以确定那些表现出葡萄糖依赖效应的化合物。这些化合物的活性将在使用胰岛素酶联免疫吸附试验的第二次测试中得到确认。此后，将使用三磷酸腺苷水平和线粒体膜电位的分析来探索β细胞内受影响的途径。最后，将测试热门歌曲在以下方面的效果 分离的人类胰岛以确认跨物种的相关性。如果成功，这一表型筛选将识别以葡萄糖依赖的方式调节β细胞功能的途径的新探针，提高我们对2型糖尿病病因疾病机制的理解。此外，这个有价值的小分子工具箱应该被证明对研究人员探索β细胞对已知病原性侮辱的反应有用，包括内质网应激、炎症和糖毒性。最后，我们的筛查可能会为开发安全治疗糖尿病而不存在低血糖风险的治疗药物识别先导化合物。

DESCRIPTION (provided by applicant): Beta-cell dysfunction plays a central role in the pathogenesis of type 2 diabetes (T2D).1 Even prior to diagnosis, patients with impaired glucose tolerance (i.e., "prediabetes") lack the first phase of glucose- stimulated insulin secretion and exhibit resistance to the amplifying effect of incretin hormones that normally prime the beta cell at meals.2 While deficient insulin secretion is now recognized as a prerequisite for both the onset and progression of T2D, the molecular pathways regulating this crucial physiologic response remain largely unknown. Moreover, no treatments are currently available to prevent the progressive nature of beta-cell decline characteristic of diabetes,3 and no small molecules exists that are FDA-approved to directly increase insulin secretion in a glucose-dependent manner, thereby avoiding the risk of hypoglycemia.4 To develop chemical probes of the molecular pathways regulating glucose-dependent insulin secretion and to identify small-molecule leads for glucose-dependent therapeutics, our proposed project wil employ a novel insulin secretion bioassay in the setting of near-stimulatory glucose conditions. Historically, a screen to identify modulators of insulin secretion has not been feasible, due to the lack of a suitable functional readout; the standard insulin ELISA is labor intensive, expensive, and limited to 96-well format. To overcome this bottleneck, we developed a high-throughput luminescent insulin secretion assay in which luciferase is targeted to the secretory vesicles of a beta cell an co-secreted with insulin upon stimulation. Luciferase serves as a close proxy for insulin, with enzyme activity responding appropriately to known secretagogues and inhibitors of insulin secretion, and in close correlation (r2 = 0.96) with insulin as measured by ELISA. Compounds found to increase luciferase secretion in near-stimulatory glucose concentrations will be counter- screened in the absence of glucose, to identify those exhibiting glucose-dependent effects. The activity of such compounds will be confirmed in a secondary assay using an insulin ELISA. Thereafter, the affected pathways within the beta cell will be explored using assays for ATP level and mitochondrial membrane potential. Lastly, top hits will be tested for their effect on dissociated human islets to confirm cross-species relevance. If successful, this phenotypic screen will identify new probes of pathways modulating beta-cell function in a glucose-dependent manner, improving our understanding of a causal disease mechanism for type 2 diabetes. In addition, this valuable toolbox of small molecules should prove useful to researchers exploring the response of the beta cell to known pathogenic insults, including ER stress, inflammation and glucolipotoxicity. Lastly, our screen may identify lead compounds for the development of therapeutics to safely treat diabetes without risk of hypoglycemia.