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

脂蛋白脂肪酶下调机制的研究

基本信息

批准号：
8626414
负责人：
JHEEM D MEDH
金额：
$ 10.88万
依托单位：
CALIFORNIA STATE UNIVERSITY NORTHRIDGE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-03-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8626414
关键词：
1-Phosphatidylinositol 3-Kinase 2,4-thiazolidinedione Acute Adenoviruses Binding Biological Assay Catalysis Catalytic Domain Cell Line Cell surface Cells Chronic Data Development Diabetes Mellitus Down-Regulation Enzymes Exhibits Fatty Acids Fatty acid glycerol esters Future Gene Expression Genes Genetic Transcription Genetically Engineered Mouse Glucose Glycogen Health Heparan Sulfate Proteoglycan Heparin Lyase Hyperglycemia Insulin Insulin Receptor Insulin Resistance Insulin Signaling Pathway Investigation Knock-out Ligands Link Lipids Lipolysis Lipoprotein Receptor Lipoproteins Maps Measures Mediating Metabolic Metabolic Pathway Metabolism Methods Molecular Muscle Muscle Cells Muscle Fibers Non-Insulin-Dependent Diabetes Mellitus Nonesterified Fatty Acids Palmitates Pathway interactions Pattern Pharmacologic Substance Phosphorylation Plasmids Play Proteins Publishing Rattus Repression Role Signal Pathway Signal Transduction Signaling Molecule Small Interfering RNA Subfamily lentivirinae Thiazolidinediones Triglycerides diabetic glucose uptake improved inhibitor/antagonist insulin receptor substrate 1 protein insulin sensitivity insulin signaling interest lipoprotein lipase mouse model mutant novel novel strategies orlistat oxidation prevent receptor binding research study response small hairpin RNA tool uptake

项目摘要

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.

描述（由申请人提供）：本提案旨在了解脂蛋白脂肪酶（LPL）下调导致肌肉细胞胰岛素敏感性改善的机制和信号通路。在先前的研究中，已经证明用噻唑烷二酮（TZDs）慢性治疗L6大鼠肌管可显著降低LPL的表达，同时增加葡萄糖摄取。因此，TZDs可能通过抑制LPL的表达和活性，至少部分地增加肌肉的胰岛素敏感性。与这一假设一致，使用LPL特异性siRNA直接下调LPL信息和蛋白质水平导致胰岛素依赖性葡萄糖摄取同时增加。LPL沉默对其他胰岛素调节代谢过程的影响将被研究。如果降低肌肉LPL的表达或活性可以改善肌肉细胞对胰岛素的整体代谢反应，将具有重要的药学价值。短发夹RNA （Short hairpin RNA, shRNA）质粒将用于生成稳定的ll敲除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。除了其溶脂功能，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。胰岛素增敏效应是否需要下调LPL的溶脂功能，其结合功能，或两者兼而有之，将被确定。这将通过使用一种特殊的脂解抑制剂，四氢脂抑素，或通过肝素酶处理消除LPL的细胞表面结合来实现。此外，在各种缺乏催化活性（突变体LPLC和S132T）或缺乏底物结合活性（突变体W390A/W393A/W394A）的LPL突变体过表达后，将测量胰岛素敏感性。LPL不是一种传统的信号分子。因此，有趣的是，它的下调是与葡萄糖摄取增加同时发生的。PCR阵列是同时检测参与信号通路的多个基因表达的极好工具。初步PCR阵列数据显示，siRNA介导的LPL沉默导致Glut4、PI3Kinase、乙酰辅酶a羧化酶等胰岛素信号通路相关基因的表达模式发生变化。我们将比较lpl敲除和基底L6细胞中这些和其他参与胰岛素调节代谢途径的相关基因的表达水平。本研究将确定将LPL表达与胰岛素敏感性联系起来的信号中间体，并绘制信号通路。本文提出的实验将有助于开发针对胰岛素抵抗和II型糖尿病的lpl靶向方法。