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: 8017023
• 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/8017023
• 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; 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（由申请人提供）：该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传导途径。先前的研究表明，用噻唑烷二酮类 (TZD) 长期治疗 L6 大鼠肌管可显着降低其 LPL 表达，同时葡萄糖摄取增加。因此，TZD 可能至少部分通过抑制 LPL 表达和活性来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。 将检查 LPL 沉默对其他胰岛素调节代谢过程的影响。如果降低肌肉LPL表达或活性能够改善肌肉细胞对胰岛素的整体代谢反应，将具有巨大的药学价值。短发夹 RNA (shRNA) 质粒将用于生成稳定的 LPL 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。 除了其脂肪分解功能外，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。这将通过使用特定的脂肪分解抑制剂四氢脂肪抑制素或通过使用肝素酶处理消除细胞表面 LPL 结合来实现。另外，在缺乏催化活性（突变体LPLC和S132T）或底物结合活性（突变体W390A/W393A/W394A）的LPL的各种突变形式的过表达后测量胰岛素敏感性。 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同时发生。 PCR 阵列是同时检查信号通路中涉及的多个基因表达的优秀工具。初步PCR阵列数据表明，siRNA介导的LPL沉默会导致参与胰岛素信号转导的各种基因的表达模式发生变化，包括Glut4、PI3激酶、乙酰辅酶A羧化酶等。这些以及参与胰岛素调节代谢途径的其他相关基因的表达水平将在LPL敲除细胞和基底L6细胞中进行比较。这项研究将鉴定将 LPL 表达与胰岛素敏感性联系起来的信号中间体，并绘制信号通路图。 这里提出的实验将有助于开发针对 LPL 的胰岛素抵抗和 II 型糖尿病管理方法。 公众健康相关性：II 型糖尿病伴有胰岛素抵抗。先前的研究表明，降低脂肪代谢酶脂蛋白脂肪酶（LPL）的水平可以提高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控制。该项目将明确建立 LPL 表达与胰岛素敏感性之间的调节关系，并将促进管理糖尿病胰岛素抵抗的新方法的开发。