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: 8432446
• 负责人: JHEEM D MEDH
• 金额: $ 10.49万
• 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/8432446
• 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.

描述（由申请人提供）：该建议旨在了解脂蛋白脂肪酶（LPL）下调的机制和信号通路，从而提高了肌肉细胞中胰岛素敏感性的提高。在先前的研究中，证明了用噻唑烷二酮（TZDS）对L6大鼠肌管（TZDS）的慢性治疗大大降低了其LPL的表达，而葡萄糖摄取的同时增加。因此，TZD可以通过抑制LPL表达和活性至少部分地提高肌肉中胰岛素敏感性。与这一假设一致，使用LPL特异性siRNA对LPL消息和蛋白质水平进行直接下调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。如果减少肌肉LPL表达或活性可以改善肌肉细胞对胰岛素的总体代谢反应，则将具有很高的药物价值。短发夹RNA（SHRNA）质粒将用于产生稳定的LPL-KNOCK-OUT 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。 除脂肪溶解功能外，LPL还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。将确定胰岛素敏化作用是否需要下调LPL的脂解功能，其结合功能或两者兼而有之。这将通过使用特定的脂解抑制剂，四氢磷灰蛋白或使用肝素治疗来消除LPL的细胞表面结合来实现。另外，将在各种突变体形式的LPL表达后测量胰岛素敏感性，该突变体缺乏催化活性（突变体LPLC和S132T）或底物结合活性（突变体W390A/W393A/W394A）。 LPL不是传统的信号分子。因此，有趣的是，它的下调与葡萄糖摄取的增加是共同入射的。 PCR阵列是同时检查信号通路中涉及多个基因的表达的出色工具。初步PCR阵列数据表明，siRNA介导的LPL沉默会导致胰岛素信号传导所涉及的各种基因的表达模式发生了变化，包括GLUT4，PI3KIN酶，乙酰基COA羧化酶等。这些和其他相关基因的表达水平和其他相关基因在Lpl-Knock-nock-nock-nock-ofl-n6细胞中都会比较。这项研究将确定信号传导中间体将LPL表达与胰岛素灵敏度联系起来并绘制信号通路。 此处提出的实验将有助于采用胰岛素抵抗和II型糖尿病的开发方法。