HEPATIC METABOLISM OF APOLIPOPROTEIN B

目的是确定载脂蛋白肝脏产生的调控部位。 B-脂蛋白。重点介绍了胰岛素在糖尿病模型中的作用。 胰岛素状态改变，包括由胰岛素引起的高胰岛素血症 泵，链脲佐菌素诱导的低血糖，以及空腹- 大鼠低胰岛素血症。胰岛素和胰岛素的相反作用 反调节激素将在体外进行初步检测 已建立的胰岛素改变动物模型的肝细胞培养 行动。肝脏调节载脂蛋白B产生的水平将是 通过测定载脂蛋白B mRNA(编辑和未编辑)，载脂蛋白B 合成包括载脂蛋白Bh(B100)和载脂蛋白Bl(B48)，载脂蛋白B降解， 载脂蛋白B池大小、载脂蛋白B分泌及载脂蛋白和脂组成 分泌型脂蛋白。研究将确定转录， 翻译性的和分泌性的事件。载脂蛋白E的评估将是 与载脂蛋白B比较具体目标1：研究将调查 载脂蛋白B和载脂蛋白E在胰岛素状态改变时的转录调控。 研究包括测定肝脏载脂蛋白B的mRNA(总、谷氨酰胺、UAA) 与包括载脂蛋白E在内的其他蛋白质的mRNA的比较 有证据表明，随着胰岛素的存在，载脂蛋白B的mRNA也会增加 载脂蛋白优势表达治疗低血糖性糖尿病 B基因UAA(Apo BL)。研究将探索胰岛素是否存在于其他 状态诱导载脂蛋白B基因表达。全肝分析将会是 与肝细胞比较及胰岛素和胰岛素的体外效应 抗呕吐激素将在持续到72小时的培养中进行评估。 在肝细胞中研究载脂蛋白BH、载脂蛋白B和载脂蛋白E的合成 稳态标记法和脉冲跟踪标记法评价 分泌途径。蛋白质的翻译率将用以下方法研究 双标记技术与亚细胞免疫沉淀相结合 分数。具体目标2：研究将调查细胞内 载脂蛋白B在胰岛素状态改变的体外降解细胞内 利用氨基酸的脉冲追逐方案来评估降解 标签。研究将包括使用蛋白酶抑制剂来区分 溶酶体途径和非溶酶体途径。扰动的影响将是 根据载脂蛋白B胞内池进行评估。亚细胞 分级研究将确定细胞内载脂蛋白的动力学 B运动。

The goal is to identify site of regulation of hepatic production of apo B-lipoproteins. Emphasis is placed on the role of insulin in models of altered insulin states including hyperinsulinemia produced by insulin pumps, streptozotocin-induced hypoinsulinemic diabetes, and fasting- hypoinsulinemia in rats. Insulin and the opposing effects of counterregulatory hormones will be examined in vitro in primary hepatocyte cultures in established animal models of altered insulin action. Levels of hepatic regulation of apo B production will be identified by measurement of apo B MRNA (edited and unedited), apo B synthesis including apo Bh (B100) and apo BL (B48), apo B degradation, apo B pool size, apo B secretion and apolipoprotein and lipid composition of secretory lipoproteins. Studies will identify transcriptional, translational, and pre-secretory events. Evaluation of apo E will be compared with apo B. Specific Aim 1: Studies will investigate transcriptional regulation of apo B and apo E in altered insulin states. Studies include measurement of hepatic apo B MRNA (total, GLN, UAA) with comparison to MRNA of other proteins including apo E. Preliminary evidence indicates that increased apo B MRNA are present with insulin treatment of hypoinsulinemic diabetes with preferential expression of apo B MRNA UAA (apo BL). Studies will explore whether insulin in other states induces apo B MRNA expression. Whole liver analysis will be compared to hepatocytes and in vitro effects of insulin and counterreulatory hormones will be assessed in cultures extending to 72h. Apo BH, apo BL and apo E synthesis will be studied in hepatocytes using steady-state labeling and pulse-chase labeling experiments to evaluate secretory pathways. Translation rates for proteins will be studied using dual label techniques combined with immunoprecipitation of subcellular fractions. Specific Aim 2: Studies will investigate intracellular degradation of apo B in altered insulin states in vitro. Intracellular degradation is evaluated using pulse-chase protocols with amino acid label. Studies will include use of protease inhibitors to distinguish lysosomal and non-lysosomal pathways. Effects of perturbations will be evaluated with respect to the apo B intracellular pool. Subcellular fractionation studies will determine the kinetics of intracellular apo B movement.