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HEPATIC METABOLISM OF APOLIPOPROTEIN B

载脂蛋白 B 的肝脏代谢

基本信息

批准号：
2028118
负责人：
CHARLES Edward SPARKS
金额：
$ 23.73万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-04-01 至 1998-12-31
项目状态：
已结题

项目摘要

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.

目的是确定载脂蛋白肝生产的调节位点 B-脂蛋白。重点放在胰岛素在模型中的作用，改变的胰岛素状态，包括胰岛素引起的高胰岛素血症胰岛素泵、链脲佐菌素诱导的低胰岛素血症糖尿病和空腹- 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检查，在已建立的胰岛素改变动物模型中的肝细胞培养物行动上肝脏对载脂蛋白B产生的调节水平将被通过测量载脂蛋白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分泌以及载脂蛋白和脂质组成分泌性脂蛋白。研究将确定转录，翻译和分泌前事件。载脂蛋白E的评价将与载脂蛋白B相比。具体目标1：研究将调查在改变的胰岛素状态下apo B和apo E的转录调节。研究包括用以下方法测量肝载脂蛋白B mRNA（总、GLN、UAA）：与包括载脂蛋白E在内的其他蛋白质的mRNA进行比较。初步有证据表明，胰岛素可导致载脂蛋白B mRNA的增加，用apo的优先表达治疗低胰岛素血症糖尿病 B MRNA UAA（apo BL）。研究将探讨胰岛素是否在其他诱导apo B mRNA表达。全肝分析将与肝细胞和胰岛素的体外作用相比，将在延长至72小时的培养物中评估反调节激素。将使用以下方法在肝细胞中研究Apo BH、Apo BL和Apo E合成：稳态标记和脉冲追踪标记实验，以评估分泌途径蛋白质的翻译率将使用双标记技术结合亚细胞免疫沉淀分数具体目标2：研究将研究细胞内体外胰岛素状态改变时apo B的降解。细胞内使用氨基酸的脉冲追踪方案评价降解 label. 研究将包括使用蛋白酶抑制剂来区分溶酶体和非溶酶体途径。扰动的影响将是相对于apo B细胞内池进行评价。亚细胞分级分离研究将确定细胞内apo的动力学 B乐章。