Identifying the Molecular Pathways Regulating Glucose-dependent Insulin Secretion

确定调节葡萄糖依赖性胰岛素分泌的分子途径

• 批准号: 8408842
• 负责人: David Altshuler
• 金额: $ 4.35万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8408842
• 关键词: 2-cyclopentyl-5-(5-isoquinolylsulfonyl)-6-nitro-1H-benzo(D)imidazole; Affect; Agonist; American; Beta Cell; Biological; Biological Assay; Biology; Blood; Cell Line; Cell physiology; Cells; Characteristics; Chemicals; Chronic; Collaborations; Defect; Dependence; Development; Diabetes Mellitus; Diagnosis; Disease; Dose; Ensure; Environment; Enzyme-Linked Immunosorbent Assay; Epidemic; Exhibits; Extravasation; FDA approved; Functional disorder; Generations; Glucose; Goals; Human; Human Genetics; Hypoglycemia; Inflammation; Inherited; Inhibitory Concentration 50; Institutes; Insulin; Lead; Luciferases; Measures; Membrane Potentials; Molecular; Molecular Probes; Mus; Nature; Non-Insulin-Dependent Diabetes Mellitus; Observational Study; Pathogenesis; Pathway interactions; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Physiological; Play; Prediabetes syndrome; Predisposing Factor; Protocols documentation; Proxy; Research; Research Personnel; Resistance; Risk; Role; Secretory Cell; Secretory Vesicles; Stress; Structure of beta Cell of islet; Structure-Activity Relationship; Testing; Therapeutic; Therapeutic Agents; Variant; Work; base; burnout; compliance behavior; enzyme activity; follow-up; global health; glucose metabolism; high throughput screening; impaired glucose tolerance; improved; incretin hormone; inhibitor/antagonist; insulin secretion; islet; mitochondrial membrane; novel; prevent; programs; response; small molecule; sugar; therapeutic development; tool

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.

描述（由申请人提供）：β细胞功能障碍在2型糖尿病（T2 D）的发病机制中起着核心作用。1即使在诊断之前，葡萄糖耐量受损的患者（即，糖尿病前期（“前驱糖尿病”）缺乏葡萄糖刺激的胰岛素分泌的第一阶段，并表现出对肠促胰岛素激素的放大作用的抗性，肠促胰岛素激素通常在进餐时引发β细胞。2虽然胰岛素分泌不足现在被认为是T2 D发作和进展的先决条件，但调节这种关键生理反应的分子途径在很大程度上仍然未知。此外，目前没有治疗方法可用于预防糖尿病特征性β细胞下降的进行性性质，3并且不存在FDA批准的以葡萄糖依赖性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性胰岛素分泌的分子途径的化学探针，并鉴定葡萄糖-依赖性治疗，我们提出的项目将在近刺激性葡萄糖条件下采用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，筛选以鉴定胰岛素分泌的调节剂是不可行的;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测定法，其中荧光素酶靶向β细胞的分泌囊泡，在刺激时与胰岛素共分泌。荧光素酶作为胰岛素的密切代表，酶活性适当地响应于已知的促分泌素和胰岛素分泌抑制剂，并且如通过ELISA测量的与胰岛素密切相关（r2 = 0.96）。发现在接近刺激性葡萄糖浓度下增加荧光素酶分泌的化合物将在不存在葡萄糖的情况下进行反筛选，以鉴定表现出葡萄糖依赖性作用的那些化合物。这些化合物的活性将在使用胰岛素ELISA的二级测定中确认。此后，将使用ATP水平和线粒体膜电位测定探索β细胞内受影响的途径。最后，将测试热门歌曲对以下方面的影响： 分离的人类胰岛以确认跨物种相关性。如果成功，这种表型筛选将确定以葡萄糖依赖性方式调节β细胞功能的途径的新探针，提高我们对2型糖尿病致病机制的理解。此外，这种有价值的小分子工具箱应该被证明对研究人员探索β细胞对已知致病性损伤的反应有用，包括ER应激，炎症和糖脂毒性。最后，我们的筛选可以确定先导化合物，用于开发安全治疗糖尿病而无低血糖风险的治疗药物。