SECRETION AND BIOSYNTHESIS OF INSULIN

胰岛素的分泌和生物合成

• 批准号: 8004800
• 负责人: MICHAEL L. MCDANIEL
• 金额: $ 4.2万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-11 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8004800
• 关键词: Acute; Address; Adult; Affect; Agonist; Amino Acids; Anabolism; Apoptosis; Bioenergetics; Cardiotonic Agents; Cell Cycle Progression; Cell Respiration; Cell physiology; Cells; Chronic; Complex; Cyclosporins; DNA biosynthesis; Data; Defect; Diabetes Mellitus; Diazoxide; Dose; Engraftment; Exhibits; Feedback; Genes; Genetic Transcription; Glucose; Glycogen Synthase Kinase 3; Goals; Growth; Growth Factor; Growth Factor Inhibition; Homeostasis; Human; Insulin; Insulin-Dependent Diabetes Mellitus; Islet Cell; Islets of Langerhans Transplantation; Knowledge; Lead; Leucine; Link; Lithium; Measures; Mediating; Medical; Metabolism; Mitochondria; Modeling; Molecular; Mus; Nitric Oxide; Nitric Oxide Synthase; Non-Insulin-Dependent Diabetes Mellitus; Nutrient; Oxidative Phosphorylation; Pathway interactions; Phosphotransferases; Physiological; Production; Protein Isoforms; Protein Kinase; Protein-Serine-Threonine Kinases; RNA Interference; Rattus; Regulation; Reperfusion Injury; Research Personnel; Rodent; Role; SCID Mice; Signal Pathway; Signal Transduction; Sirolimus; Small Interfering RNA; Structure of thyroid parafollicular cell; TSC1/2 gene; Testing; Transcriptional Activation; Transcriptional Regulation; Transgenic Organisms; Translations; Transplantation; Tuberous sclerosis protein complex; Up-Regulation; aminoguanidine; blood glucose regulation; cell growth; cell growth regulation; cellular targeting; detection of nutrient; diabetic; improved; inhibitor/antagonist; insight; insulin signaling; islet; mTOR protein; mutant; prevent; response; success; translational study; tumor

DESCRIPTION (provided by applicant): Both types 1 & 2 diabetes (T1DM, T2DM) result from the inability of ¿-cells to secrete sufficient insulin to maintain normal glucose homeostasis due to an acquired secretory defect and/or inadequate ¿-cell mass. Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from growth factors (GFs) & nutrients to regulate cell growth & proliferation. Recent studies have determined that inhibition of glycogen synthase kinase-3 (GSK-3) & tuberous sclerosis complex 2 (TSC2) are required for mTOR activation. Our studies have shown that GSK-3 inhibition stimulates mTOR-mediated DNA synthesis & cell cycle progression in rodent & human islets. The highly reproducible stimulation of mTOR-mediated DNA synthesis by lithium, a GSK-3 inhibitor, in human islets is remarkable since nutrients are rarely effective. Since GSK-3 inhibition is also integral to Wnt/2-catenin transcriptional regulation, we propose to evaluate possible interactions of this pathway with mTOR signaling. Our goal is to enhance the growth & proliferative capacity of adult ¿-cells by appropriately regulating both mTOR & the canonical Wnt signaling pathways using GSK-3 inhibitors, Wnt agonists & nutrients. Specific aim 1 is to establish the functions of the GSK-3/TSC/mTOR & the canonical Wnt/GSK-3/2-catenin pathways in primary adult rodent & human islets by A) identifying the molecular & cellular targets & the ability of GSK-3 inhibitors, Wnt agonists, nutrients & rapamycin to modulate these pathways, B) elucidating the role of these pathways in the growth, proliferation & function of ¿-cells & C) determining if a differential capacity to affect feedback inhibition of growth factor (GF) signaling distinguishes nutrients from GSK-3 inhibitors. Using isolated islets, we will utilize siRNA of GSK-3, ¿-catenin & mTOR, study Wnt- dependent TCF/LEF1 transcription using transgenic TOPGAL mice & evaluate GF signaling and GSK-3 activity after chronic nutrient or GSK-3 inhibitor treatment. Specific aim 2 is to determine if the mitochondrial transition pore (MTP) & nitric oxide (NO) are required for GSK-3 & mTOR signaling by A) examining if RNA interference of nitric oxide synthase (NOS) isoforms or MTP components, VDAC & ANT, alter the physiological responses of rodent & human islets to nutrients or GSK-3 inhibition, B) evaluating the ability of GSK-3 inhibitors, Wnt agonists or nutrients to regulate NOS expression & NO production & C) investigating how exogenous NO affects the MTP & ¿-cell growth, proliferation & function. Specific aim 3 is to determine whether manipulation of GSK-3 and mTOR signaling will promote ¿-cell function, growth & proliferation in a T1DM islet transplantation model by pre-culture of human or rodent islets with GSK-3 inhibitors or Wnt agonists 1 rapamycin prior to transplanting a "marginal dose" of islets into STZ-diabetic SCID mice. Understanding the co-regulation of the GSK-3/TSC/mTOR & Wnt/GSK-3/¿-catenin pathways will provide new strategies to enhance growth & proliferation of adult ¿-cells, serve important unmet medical needs of T1DM & T2DM & provide basic mechanistic knowledge of how nutrients affect these pathways.

描述（由申请人提供）：1型和2型糖尿病（T1DM，T2DM）均由后天分泌缺陷和/或不足的胰岛细胞质量导致胰岛细胞不能分泌足够的胰岛素以维持正常的葡萄糖稳态引起。哺乳动物雷帕霉素靶点（mTOR）是一种蛋白激酶，它整合来自生长因子（GFs）和营养素的信号来调节细胞生长和增殖。最近的研究已经确定，糖原合成酶激酶-3（GSK-3）和结节性硬化症复合物2（TSC 2）的抑制是mTOR激活所必需的。我们的研究表明，GSK-3抑制剂刺激啮齿动物和人类胰岛中mTOR介导的DNA合成和细胞周期进程。锂（GSK-3抑制剂）对mTOR介导的DNA合成的高度可重复刺激在人类胰岛中是显著的，因为营养素很少有效。由于GSK-3抑制也是Wnt/2-catenin转录调控的组成部分，因此我们建议评估该途径与mTOR信号传导的可能相互作用。我们的目标是提高成人的生长和增殖能力， 通过使用GSK-3抑制剂、Wnt激动剂和营养素适当调节mTOR和经典Wnt信号通路来调节细胞。具体目标1是通过以下方式建立GSK-3/TSC/mTOR和经典Wnt/GSK-3/2-连环蛋白通路在初级成年啮齿动物和人类胰岛中的功能：A）鉴定GSK-3抑制剂、Wnt激动剂、营养素和雷帕霉素调节这些通路的分子和细胞靶点以及能力，B）阐明这些通路在胰岛细胞生长、增殖和功能中的作用，- 细胞和C）确定影响生长因子（GF）信号传导的反馈抑制的差异能力是否区分营养物与GSK-3抑制剂。使用分离的胰岛，我们将利用GSK-3、β-连环蛋白和mTOR的siRNA，使用转基因TOPGAL小鼠研究Wnt依赖的TCF/LEF1转录，并评估长期营养或GSK-3抑制剂处理后的GF信号传导和GSK-3活性。具体目标2是通过以下步骤确定线粒体过渡孔（MTP）和一氧化氮（NO）是否是GSK-3和mTOR信号传导所需的：A）检查一氧化氮合酶（NOS）同种型或MTP组分VDAC和ANT的RNA干扰是否改变啮齿动物和人胰岛对营养物或GSK-3抑制的生理反应，B）评估GSK-3抑制剂的能力，Wnt激动剂或营养素来调节NOS表达和NO产生& C）研究外源性NO如何影响MTP和细胞生长、增殖和功能。具体目标3是确定GSK-3和mTOR信号传导的操纵是否将在T1DM胰岛移植模型中通过在将"边缘剂量"的胰岛移植到STZ糖尿病SCID小鼠中之前用GSK-3抑制剂或Wnt激动剂1雷帕霉素预培养人或啮齿动物胰岛来促进胰岛细胞功能、生长和增殖。了解GSK-3/TSC/mTOR和Wnt/GSK-3/â-连环蛋白途径的共同调节将提供新的策略来增强成人â-细胞的生长和增殖，满足T1 DM和T2 DM未满足的重要医疗需求，并提供有关营养素如何影响这些途径的基本机制知识。