INSULIN PI 3 KINASE AND APO B BIOGENESIS IN OBESE RATS

肥胖大鼠中胰岛素 PI 3 激酶和 APO B 生物发生

• 批准号: 2770537
• 负责人: JANET DEHOFF SPARKS
• 金额: $ 18.38万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2770537
• 关键词: adenosine triphosphate; apolipoprotein B; blood lipoprotein biosynthesis; endoplasmic reticulum; enzyme activity; exotoxins; hemolysin; immunoprecipitation; insulin; insulin receptor; laboratory rat; liver cells; microsomes; monoclonal antibody; obesity; phosphatidylinositol 3 kinase; phosphorylation; pore forming protein; protein sequence; protein transport; stoichiometry; tissue /cell culture; vesicle /vacuole

In response to RFA DK-94-023 entitled "Nutrient Modulation of Cell Integrity and Repair Mechanisms," we propose to study defective insulin regulation of apolipoprotein (apo) B biogenesis in hepatocytes derived from Zucker obese rats in order to elucidate the molecular defect involved in insulin resistance of this pathway. We propose to study the linkage of specific events involved in insulin receptor signal transduction with microsomal events involved in apo B biogenesis with primary focus on the regulatory role of activated phosphatidylinositol 3-kinase (PI 3-K) in the translocation of apo B into the secretory pathway. While the majority of studies involve rat apo B. pilot studies will use the new technology of receptor-mediated gene delivery to study human apo B constructs in primary cultures of rat hepatocytes. Two specific aims are proposed to dissect pathways related to PI 3-K lipid kinase and serine kinase activity both of which are activated by insulin. Aim l is to test the hypothesis that insulin causes localization and activation of lipid kinase of PI 3-K on endoplasmic reticulum (ERA membranes which results in rapid accumulation of highly polar phospholipids which selectively. in a negative fashion. regulate apo B translocation into the secretory pathway. Apo B that fails to translocate and assemble with lipid is trapped within ER membranes and targeted for degradation. This process is responsible for the observed insulin dose-dependent decrease in apo B secretion by hepatocytes. The role of insulin receptor (IR) signaling and PI 3-K activation on apo B secretion will be evaluated in both lean and obese rats, the latter being insulin-resistant in the apo B pathway due to combined effects at the receptor and post-receptor level. - Aim 2 will test the hypothesis that insulin causes activation of the serine kinase of PI 3-K which through localization to the ER or through subsequent activation of S6 kinase. phosphorylates either translocation channel component proteins involved in apo B translocation or newly synthesizing apo B itself hindering apo B translocation. With continued translation, the inability of apo B to translocate modifies normal ribosome-ER interaction and results in the generation of cytoplasmic apo B which is unable to post-translationally translocate and is targeted for intracellular degradation. Studies employ primary and suspended hepatocytes derived from normal rats and from lean and obese Zucker rats and include assays of IR signaling, B-subunit phosphorylation, IRS-I phosphorylation, PI 3-K activation and PI 3-K mass in subcellular fractions. Experiments also employ in vitro apo B translocation assays using streptolysin O permeabilized hepatocytes. Results of proposed studies will allow a more complete understanding of normal and defective apo B biogenesis by liver allowing insights into potential mechanisms involved in the development of hypertriglyceridemia and human obesity and insulin resistance syndromes such as Syndrome X.

响应RFA DK-94-023，标题为“细胞的营养调节 完整性和修复机制，“我们建议研究有缺陷的胰岛素 源自源自载脂蛋白（APO）B生物发生的肝细胞中的调节 从扎克肥胖大鼠来阐明涉及的分子缺损 在该途径的胰岛素抵抗中。我们建议研究 与胰岛素受体信号转导有关的特定事件 与Apo B生物发生有关的微粒体事件，主要关注 活化的磷脂酰肌醇3-激酶（PI 3-K）在调节中的作用 Apo B转移到分泌途径中。而大多数 研究涉及大鼠apoB。试验研究将使用新技术 受体介导的基因输送以研究原代的人apo b构建体 大鼠肝细胞的培养物。提出了两个具体目标以剖析 与Pi 3-K脂质激酶和丝氨酸激酶活性有关 被胰岛素激活。目的是检验以下假设 胰岛素引起PI 3-K的脂质激酶的定位和激活 内质网（ERA膜导致快速积累 高度极性磷脂的选择性。以消极的方式。 调节Apo B易位到分泌途径。失败的apo b 用脂质易位和组装被困在ER膜中，并且 针对降解的目标。这个过程是造成观察到的 胰岛素剂量依赖性降低肝细胞的APO B分泌。这 胰岛素受体（IR）信号传导和PI 3-K激活在APO B上的作用 分泌物将在瘦和肥胖的大鼠中进行评估，后者是 由于在 受体和受体后水平。 - AIM 2将检验以下假设 胰岛素引起PI 3-K的丝氨酸激酶的激活，该激酶通过 定位到ER或通过随后的S6激酶激活。 磷酸化的两种易位通道成分蛋白 apo b易位或新合成的apo b本身阻碍了apo b 易位。随着持续的翻译，apo b无法 转运器修饰正常的核糖体相互作用，并导致 细胞质Apo B的产生，无法在翻译后产生 易位并针对细胞内降解。研究 源自正常大鼠的原发性和悬浮肝细胞 和肥胖的Zucker大鼠，包括IR信号的测定，B-Subunit 磷酸化，IRS-I磷酸化，PI 3-K活化和PI 3-K质量 在亚细胞分数中。实验还采用体外APO B 使用链霉菌素O透化的肝细胞的转运测定。 拟议研究的结果将使人们对 肝脏正常和有缺陷的Apo B生物发生，可以洞悉 涉及高甘油三酯血症发展的潜在机制 以及人类肥胖和胰岛素抵抗综合征，例如综合征X。