Development of novel means to stimulate Ca2+-dependent exocytotic secretion

开发刺激 Ca2 依赖性胞吐分泌的新方法

• 批准号: 9155329
• 负责人: ZHE LU
• 金额: $ 40.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9155329
• 关键词: Adopted; Adverse effects; Affinity; Antidiabetic Drugs; Beta Cell; Blood Glucose; Bypass; Cell membrane; Cellular biology; Chronic; Coupled; Development; Diabetes Mellitus; Drug Targeting; Electrophysiology (science); Exocytosis; Genetically Engineered Mouse; Glucokinase; Glucose; Glyburide; Goals; Histology; Hormones; Hypoglycemia; Image; Insulin; Ions; Islets of Langerhans; Kinetics; Life Style; Mediating; Membrane; Membrane Proteins; Metabolism; Methods; Modeling; Molecular Biology; Mus; Mutation; Neurotransmitters; Pancreas; Pathogenesis; Patients; Pharmaceutical Preparations; Play; Protein Biochemistry; Proteins; Reagent; Research; Research Design; Resources; Role; Scientific Inquiry; Signal Transduction; Structure of beta Cell of islet; Sulfonylurea Compounds; Surface; Testing; Therapeutic; Time; Tissues; Transgenic Mice; Uncertainty; Venoms; cell type; combat; diabetic; gain of function mutation; glucose metabolism; glucose sensor; human disease; inhibitor/antagonist; innovation; insulin secretion; inward rectifier potassium channel; mouse model; neonatal diabetes mellitus; non-diabetic; novel; novel therapeutics; pandemic disease; prototype; stem; success; sulfonylurea receptor; syntaxin; voltage

The long-term goal of this project is to discover new methods to stimulate Ca2+-dependent exocytosis of neurotransmitters or hormones by tuning cellular excitability and thereby the intracellular Ca2+ signal. These new methods will then be used to develop useful therapeutic strategies to combat human diseases. Our immediate goal here is to develop specific reagents to increase membrane excitability to stimulate Ca2+- dependent exocytosis of insulin from pancreatic β cells. Elevated glucose concentrations increase metabolism and cytosolic ATP in β cells, which in turn inhibits the ATP-sensitive K+ (KATP) channel. Decreased KATP channel activity depolarizes the cell membrane, activating voltage-gated Ca2+ (CaV) channels. The CaV-mediated Ca2+ influx elevates intracellular Ca2+ concentration ([Ca2+]in), triggering insulin release. A KATP channel of the β cell consists of the inward-rectifier K+ channel Kir6.2 (KCNJ11) and the modulatory subunit sulphonylurea receptor, SUR1 (ABCC8). Sulphonylureas, a major class of antidiabetic drugs, are generally believed to stimulate insulin secretion by acting on SUR1 and thereby indirectly suppressing Kir6.2 activity. Conceptually, Kir6.2 itself should then represent a more direct, and potentially effective antidiabetic drug target. It is noteworthy that several studies suggest that SUR1 also plays other roles besides inhibiting Kir6.2, such as interacting with syntaxin, a key protein involved in exocytosis. Furthermore, the sulphonylurea glibenclamide appears to promote exocytosis of insulin also by KATP-independent mechanisms. Additionally, in the past decade, many gain-of-function mutations of Kir6.2 or SUR1 (lowering KATP's ATP sensitivity) were found to underlie most cases of permanent neonatal diabetes mellitus (PNDM). Most of these patients have since been successfully treated with sulphonylureas, instead of the traditional insulin therapy. However, some PNDM patients with certain mutations are unresponsive to sulphonylureas. In principle, these patients might be treatable with a suitable Kir6.2 inhibitor. Unfortunately, to date, there are no Kir6.2 inhibitors available. To obtain experimental evidence for judging, on scientific grounds, if serious resources should be invested in developing a clinically useful Kir6.2 inhibitor to treat diabetes, we propose to: i. develop a prototype specific Kir6.2 inhibitor and thereby demonstrate the feasibility of such an endeavor, ii. investigate whether a Kir6.2 inhibitor can stimulate adequate insulin secretion, and iii. apply this inhibitor to test the hypothesis that a sulphonylurea stimulates insulin secretion also via KATP-independent mechanisms. If this hypothesis is correct, a Kir6.2 inhibitor may then not have all the side effects of sulphonylureas. We will perform our studies with a combination of electrophysiology, Ca2+ imaging, protein biochemistry, molecular biology, cell biology, and histology. Our proposal constitutes an innovative, proof-of-concept study of a novel antidiabetic strategy; its significance is underscored by the fact that 350 million people worldwide have diabetes, a growing pandemic that calls for the development of new, effective therapeutic strategies.

该项目的长期目标是发现刺激Ca2+依赖性胞吐作用的新方法 神经递质或激素通过调节细胞刺激性，从而通过细胞内Ca2+信号。这些 然后，新方法将用于制定有用的治疗策略来打击人类疾病。我们的 这里的直接目标是开发特定的试剂以增加膜兴奋性以刺激Ca2+ - 来自胰腺β细胞的胰岛素依赖性胞吐作用。升高葡萄糖浓度会增加新陈代谢 β细胞中的胞质ATP，进而抑制对ATP敏感的K+（KATP）通道。减少Katp 通道活性使细胞膜去极化，激活电压门控Ca2+（CAV）通道。 CAV介导的Ca2+会影响细胞内Ca2+浓度（[Ca2+] IN），从而触发胰岛素释放。 β细胞的KATP通道由内向旋转剂K+通道KIR6.2（KCNJ11）和调节亚基磺酰脲受体SUR1（ABCC8）组成。磺酰尿菌是主要类抗糖尿病药物，通常是 认为可以通过对SUR1作用，从而刺激胰岛素分泌，从而间接抑制Kir6.2活性。 从概念上讲，Kir6.2本身应代表更直接且潜在的抗糖尿病药物靶标。 值得注意的是，一些研究表明，除了抑制Kir6.2之外，Sur1还扮演其他角色，例如 与语法素相互作用，该语法是参与胞吐作用的关键蛋白。此外，磺酰脲Glibenclamide 似乎也通过非依赖KATP的机制促进胰岛素的胞吐作用。另外，过去 十年来，发现Kir6.2或SUR1（降低KATP的ATP灵敏度）的许多功能收益突变被发现 基于大多数永久性新生儿糖尿病（PNDM）的病例。此后大多数已经 成功地用磺酰氟菌治疗，而不是传统的胰岛素治疗。但是，一些PNDM 某些突变的患者对磺酰尿不反应。原则上，这些患者可能是 可以用合适的Kir6.2抑制剂治疗。不幸的是，迄今为止，尚无KIR6.2抑制剂。到 如果应该投资认真的资源，以科学的理由获得实验证据来判断 我们建议：i。开发特定的原型 Kir6.2抑制剂，从而证明了这种努力的可行性，ii。调查Kir6.2是否 抑制剂可以刺激足够的胰岛素分泌，并且III。应用此抑制剂来检验一个假设 磺酰尿素也通过非KATP独立的机制刺激胰岛素分泌。如果这个假设是 正确，Kir6.2抑制剂可能没有磺酰尿菌的所有副作用。我们将进行学习 结合电生理学，CA2+成像，蛋白质生物化学，分子生物学，细胞生物学， 和组织学。我们的提案构成了一项新型抗糖尿病策略的创新，概念概念研究。 全世界有3.5亿人患有糖尿病，这是一个不断增长的事实，这一事实强调了它的意义 大流行要求发展新的有效的治疗策略。