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.

针对标题为“细胞的营养调节”的RFA DK-94-023， 完整性和修复机制，”我们建议研究缺陷胰岛素 载脂蛋白（apo）B在肝细胞中生物合成的调节 Zucker肥胖大鼠，以阐明所涉及的分子缺陷 在胰岛素抵抗中的作用。我们建议研究 参与胰岛素受体信号转导的特定事件 微粒体事件参与载脂蛋白B生物发生，主要关注 活化磷脂酰肌醇3-激酶（PI 3-K）在 载脂蛋白B易位进入分泌途径。虽然大多数 研究涉及大鼠载脂蛋白B。试点研究将使用新技术， 受体介导的基因传递，以研究人载脂蛋白B构建体在原发性 大鼠肝细胞的培养物。提出了两个具体的目标来剖析 与PI 3-K脂质激酶和丝氨酸激酶活性相关的途径， 由胰岛素激活。目的是检验假设， 胰岛素引起PI 3-K的脂激酶定位和激活， 内质网（ERA膜，其导致快速积累 高极性磷脂的选择性。消极的方式。 调节apo B易位进入分泌途径。载脂蛋白B 转运并与脂质组装被困在ER膜内， 以降解为目标。这个过程负责观察到的 胰岛素剂量依赖性降低肝细胞的apo B分泌。的 胰岛素受体（IR）信号和PI 3-K激活对载脂蛋白B的作用 将在瘦大鼠和肥胖大鼠中评估分泌，后者是 胰岛素抵抗的载脂蛋白B途径，由于联合作用， 受体和受体后水平。- 目标2将检验以下假设： 胰岛素引起PI 3-K的丝氨酸激酶的活化， 定位于ER或通过随后的S6激酶活化。 磷酸化参与转运通道的蛋白质 载脂蛋白B易位或新合成的载脂蛋白B本身阻碍载脂蛋白B 易位随着持续翻译，载脂蛋白B不能 易位修饰正常的核糖体-ER相互作用， 细胞质apo B的产生，其不能在细胞分裂后 易位并靶向细胞内降解。研究采用 来自正常大鼠和瘦大鼠的原代和悬浮肝细胞 和肥胖Zucker大鼠，并包括IR信号传导，B亚单位， 磷酸化、IRS-I磷酸化、PI 3-K激活和PI 3-K质量 在亚细胞组分中。实验还采用体外载脂蛋白B 使用链球菌溶血素O透化肝细胞的易位测定。 拟议研究的结果将使人们更全面地了解 正常和有缺陷的载脂蛋白B通过肝脏生物合成， 高胆固醇血症发生的潜在机制 以及人肥胖症和胰岛素抵抗综合征如X综合征。