An investigation of the mechanisms by which down-regulation of lipoprotein lipase

描述(由申请人提供)：本提案旨在了解下调脂蛋白脂肪酶(LPL)导致肌肉细胞胰岛素敏感性改善的机制和信号通路。以往的研究表明，用噻唑烷二酮(TZDS)慢性处理大鼠L6肌管后，L6肌管LPL的表达显著降低，同时葡萄糖摄取增加。因此，TZDS可能至少部分通过抑制LPL的表达和活性来增加肌肉中的胰岛素敏感性。与这一假设一致的是，使用LPL特异性siRNA直接下调LPL消息和蛋白质水平导致胰岛素依赖的葡萄糖摄取同时增加。LPL沉默对胰岛素调节的其他代谢过程的影响将被检测。如果降低肌肉LPL的表达或活性能够改善肌肉细胞对胰岛素的整体代谢反应，将具有很大的药学价值。短发夹状RNA(ShRNA)质粒将被用来建立稳定的LPL基因敲除的L6细胞系，并研究胰岛素调节的代谢途径，包括脂肪酸的氧化和合成，以及糖原的合成。除了脂解功能外，LPL也是脂蛋白受体的配体，这是LPL促进细胞脂质摄取的另一种机制。将确定胰岛素增敏效应是否需要下调LPL的脂解功能、其结合功能，或者两者兼而有之。这将通过使用特定的脂解抑制剂四氢脂素，或通过使用肝素酶处理取消LPL的细胞表面结合来实现。此外，胰岛素敏感性将在缺乏催化活性(突变体LPLC和S132T)或缺乏底物结合活性(突变体W390A/W393A/W394A)的各种LPL突变形式过表达后进行测定。LPL不是一种传统的信号分子。因此，有趣的是，它的下调与葡萄糖摄取的增加是同时发生的。聚合酶链式反应阵列是同时检测信号通路中涉及的多个基因表达的极佳工具。初步的PCR芯片数据表明，siRNA介导的LPL沉默导致了参与胰岛素信号转导的各种基因的表达模式的变化，包括GLUT4、PI3Kinase、乙酰辅酶A羧基酶等。这些基因和其他参与胰岛素调节代谢途径的相关基因的表达水平将在LPL敲除细胞和基础L6细胞中进行比较。这项研究将确定将LPL表达与胰岛素敏感性联系起来的信号中间体，并绘制一条信号通路。这里提出的这项实验将有助于开发针对LPL的方法来管理胰岛素抵抗和II型糖尿病。

DESCRIPTION (provided by applicant): This proposal is aimed at understanding the mechanisms and signaling pathways by which down- regulation of lipoprotein lipase (LPL) leads to improved insulin sensitivity in muscle cells. In previous studies, it was demonstrated that chronic treatment of L6 rat myotubes with Thiazolidinediones (TZDs) drastically reduced their expression of LPL with a co-incident increase in glucose uptake. Thus, TZDs may increase insulin sensitivity in muscle, at least partly, by repressing LPL expression and activity. Consistent with this hypothesis, a direct down-regulation of LPL message and protein levels using LPL-specific siRNA resulted in a concurrent increase in insulin-dependent glucose uptake. The effect of LPL silencing on other insulin-regulated metabolic processes will be examined. It will be of great pharmaceutical value if decreasing muscle LPL expression or activity can improve the overall metabolic response of muscle cells to insulin. Short hairpin RNA (shRNA) plasmids will be used to generate a stable LPL-knock-out L6 cell line and study insulin-regulated metabolic pathways including the oxidation and synthesis of fatty acids, and the synthesis of glycogen. In addition to its lipolytic function, LPL is also a ligand for lipoprotein receptors, This is an additional mechanism by which LPL contributes to cellular lipid uptake. It will be determined if the insulin-sensitizing effect requires the down-regulation of LPL's lipolytic function, its binding function, or both. This will be accomplished by using a specific lipolysis inhibitor, tetrahydrolipostatin, or by abolishing cell surface binding of LPL using heparinase treatment. Additionally, insulin sensitivity will be measured after the over- expression of various mutant forms of LPL that either lack catalytic activity (mutants LPLC and S132T), or substrate binding activity (mutant W390A/W393A/W394A). LPL is not a traditional signaling molecule. Thus, it is interesting that its down-regulation is co- incident with increased glucose uptake. PCR arrays are excellent tools to simultaneously examine the expression of multiple genes involved in a signaling pathway. Preliminary PCR array data indicate that siRNA mediated silencing of LPL results in a change in the expression pattern of various genes involved in insulin signaling including Glut4, PI3Kinase, acetyl coA carboxylase, etc. The expression levels of these and other relevant genes involved in insulin-regulated metabolic pathways will be compared in LPL-knock- out and basal L6 cells. This study will identify signaling intermediates that link LPL expression to insulin sensitivity and map a signaling pathway. This experiments proposed here will help with the development LPL-targeted approaches for the management of insulin resistance and type II diabetes.