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

• 批准号: 8227959
• 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
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8227959
• 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

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. PUBLIC HEALTH RELEVANCE: Type II diabetes is accompanied by insulin resistance. Previous studies show that reducing the levels of a fat metabolizing enzyme, lipoprotein lipase (LPL), results in improved utilization of glucose in muscle cells. In the absence of LPL, fats may become unavailable and muscle cells may be forced to use glucose, thereby helping with the management of hyperglycemia in diabetics. This project will clearly establish the regulatory relationship between LPL expression and insulin sensitivity, and will facilitate the development of novel approaches to manage insulin resistance in diabetes.

描述（由申请方提供）：本提案旨在了解脂蛋白脂肪酶（LPL）下调导致肌细胞胰岛素敏感性改善的机制和信号通路。在以前的研究中，它表明，长期治疗L 6大鼠肌管与噻唑烷二酮类（TZDs）大幅降低其LPL的表达与葡萄糖摄取的同时增加。因此，TZDs可能增加肌肉中的胰岛素敏感性，至少部分地，通过抑制LPL的表达和活性。与此假设一致，使用LPL特异性siRNA直接下调LPL信息和蛋白水平导致胰岛素依赖性葡萄糖摄取的同时增加。 将检查LPL沉默对其他胰岛素调节的代谢过程的影响。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，将具有重要的药用价值。短发夹RNA（shRNA）质粒将用于产生稳定的LPL敲除L 6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。 除了其脂解功能之外，LPL也是脂蛋白受体的配体，这是LPL有助于细胞脂质摄取的另一种机制。将确定胰岛素增敏作用是否需要下调LPL的脂解功能、其结合功能或两者。这将通过使用特异性脂解抑制剂四氢脂抑素或通过使用肝素酶处理消除LPL的细胞表面结合来实现。另外，在LPL的各种突变形式的过表达后测量胰岛素敏感性，所述LPL缺乏催化活性（突变体LPLC和S132 T）或底物结合活性（突变体W390 A/W393 A/W394 A）。 LPL不是传统的信号分子。因此，有趣的是，其下调与葡萄糖摄取增加一致。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。初步的PCR阵列数据表明，siRNA介导的LPL沉默导致参与胰岛素信号传导的各种基因（包括Glut 4、PI 3激酶、乙酰辅酶A羧化酶等）的表达模式发生变化。将在LPL敲除和基础L 6细胞中比较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。本研究将鉴定将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LPL的方法来管理胰岛素抵抗和II型糖尿病。 公共卫生相关性：II型糖尿病伴有胰岛素抵抗。以前的研究表明，降低脂肪代谢酶脂蛋白脂肪酶（LPL）的水平，可以改善肌肉细胞对葡萄糖的利用。在缺乏LPL的情况下，脂肪可能变得不可用，肌肉细胞可能被迫使用葡萄糖，从而有助于糖尿病患者高血糖的管理。该项目将明确LPL表达与胰岛素敏感性之间的调节关系，并将促进开发新的方法来管理糖尿病胰岛素抵抗。