INSULIN PI 3 KINASE AND APO B BIOGENESIS IN OBESE RATS

回应RFA DK-94-023《细胞的营养调节》 完整性和修复机制，“我们建议研究缺陷胰岛素 肝细胞载脂蛋白B生物合成的调控 从Zucker肥胖大鼠中分离出来，以阐明其中涉及的分子缺陷 在这一途径中的胰岛素抵抗。我们建议研究 胰岛素受体信号转导中的特定事件 与载脂蛋白B生物发生有关的微生物体事件 激活的磷脂酰肌醇3-激酶(PI 3-K)在细胞周期中的调节作用 载脂蛋白B转位到分泌途径。虽然大多数人 涉及大鼠载脂蛋白B的研究试点研究将使用新技术 受体介导的基因传递研究原代培养的人载脂蛋白B结构 大鼠肝细胞培养。提出了两个具体的目标来进行剖析 与等电点3-K脂蛋白激酶和丝氨酸激酶活性相关的通路 它们被胰岛素激活。目的L是为了检验这一假说 胰岛素引起磷脂酰肌醇3-K的定位和激活 内质网(ERA膜)，导致快速积累 有选择性的高极性磷脂。以消极的方式。 调节载脂蛋白B转位到分泌途径。失败的载脂蛋白B 与脂质的移位和组装被困在内质网的膜和 以降级为目标。这一过程是对观察到的 胰岛素剂量依赖性降低肝细胞载脂蛋白B的分泌。这个 胰岛素受体信号转导和PI3-K激活在载脂蛋白B中的作用 将评估瘦小和肥胖大鼠的分泌情况，后者是 载脂蛋白B途径中的胰岛素抵抗是由于 受体和受体后水平。-Aim 2将检验以下假设 胰岛素引起PI3-K丝氨酸激酶的激活 定位于内质网或通过随后激活S6激酶。 磷酸化任何一种参与转位的通道成分蛋白 载脂蛋白B易位或新合成载脂蛋白B本身阻碍载脂蛋白B 易位。随着继续翻译，载脂蛋白B无法 移位改变正常的核糖体-内质网相互作用并导致 不能翻译后的细胞质载脂蛋白B的产生 移位，并以细胞内降解为目标。研究人员 正常大鼠和瘦肉型大鼠的原代和悬浮肝细胞 和肥胖的Zucker大鼠，包括IR信号、B亚单位的分析 磷酸化、IRS-I磷酸化、PI 3-K激活和PI 3-K质量 以亚细胞形式存在。实验还采用了体外载脂蛋白B 链球菌素O穿透肝细胞的易位分析。 拟议的研究结果将使我们能够更全面地了解 正常和缺陷的载脂蛋白B通过肝脏的生物发生允许洞察 高甘油三酯血症发生的可能机制 以及人类肥胖和胰岛素抵抗综合征，如X综合征。

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.