Identifying the Molecular Pathways Regulating Glucose-dependent Insulin Secretion

描述（由申请人提供）：β细胞功能障碍在2型糖尿病（T2D）的发病机制中起核心作用即使在诊断之前，糖耐量受损的患者（即“前驱糖尿病”）缺乏葡萄糖刺激胰岛素分泌的第一阶段，并且对通常在进餐时激活β细胞的肠促胰岛素激素的放大作用表现出抵抗虽然胰岛素分泌不足现在被认为是T2D发病和进展的先决条件，但调节这一关键生理反应的分子途径在很大程度上仍然未知。此外，目前还没有治疗方法可以预防糖尿病β细胞衰退的进行性特征，3也没有fda批准的小分子可以通过葡萄糖依赖的方式直接增加胰岛素分泌，从而避免低血糖的风险4为了开发调节葡萄糖依赖胰岛素分泌的分子途径的化学探针，并确定葡萄糖依赖治疗的小分子先导物，我们提出的项目将在近刺激葡萄糖条件下采用一种新的胰岛素分泌生物测定方法。从历史上看，由于缺乏合适的功能读数，筛选胰岛素分泌调节剂是不可行的；标准胰岛素ELISA是劳动密集型的，昂贵的，并且仅限于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β细胞的分泌囊泡，并在刺激下与胰岛素共同分泌。荧光素酶作为胰岛素的密切代表，其酶活性对已知的胰岛素分泌抑制剂和促分泌剂有适当的反应，并且通过ELISA检测与胰岛素密切相关（r2 = 0.96）。发现在接近刺激的葡萄糖浓度下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作用的化合物。这些化合物的活性将在使用胰岛素ELISA的二次分析中得到证实。此后，将使用ATP水平和线粒体膜电位测定来探索β细胞内受影响的途径。最后，将测试热门点击的效果

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.