Regulation of GPIHBP1-dependent and independent triglyceride clearance

项目总结 临床高甘油三酯血症很常见，对可用药物只有部分反应。相反， 甘油三酯过量输送到个别组织可导致骨骼肌胰岛素抵抗，心脏 脂毒性，或严重的炎症反应。考虑到适当传递甘油三酯的重要性，它是 当务之急是，我们必须了解管理这一过程的机制。正常情况下有两种蛋白质 清除甘油三酯所需的是脂蛋白脂酶(LPL)和GPIHBP1。LPL是必需的 血浆甘油三酯的实际水解物，释放脂肪酸供组织摄取。血管内皮细胞 蛋白GPIHBP1需要正确地将LPL定位于发生脂解的血管管腔。 拟议研究的长期目标是了解并最终调整 甘油三酯衍生的脂肪酸到特定的组织。此应用程序的目标是推进我们的 血管生成素样蛋白Angptl3和ANGPTL4如何调节 甘油三酯清除。Angptl3和ANGPTL4在体外均能抑制LPL，Angptl3或 ANGPTL4缺乏会导致血浆甘油三酯水平降低。然而，如果以及如何Angptl3和 ANGPTL4是否直接调节LPL/GPIHBP1介导的甘油三酯清除尚不清楚。此外， GPIHBP1和ANGPTL4基因缺陷小鼠的血浆甘油三酯水平表明ANGPTL4可能 调节未确定的、不依赖于GPIHBP1的甘油三酯清除途径。这些问题将是 通过追求两个具体目标来解决：1)定义Angptl3和ANGPTL4 调节依赖于GPIHBP1的甘油三酯清除；以及2)鉴定GPIHBP1的新机制- 独立清除甘油三酯。AIM 1中的研究使用了基于细胞培养的结合和脂肪酶 活性测定，以及体内注射重组蛋白和组织特异性基因敲除小鼠 ANGPTL蛋白与内皮细胞表面GPIHBP1/LPL复合体相互作用的研究 细胞。第二个目标将解决ANGPTL4-/-Gpihbp1-/-小鼠甘油三酯显著降低的问题。 甘油三酯清除和脂肪酸摄取试验将用于识别 甘油三酯被清除了。表达、蛋白质和脂肪酶活性分析将用于鉴定 负责这一清除的蛋白质。拟议研究中的方法是创新的。 因为他们将标准方法与新的鼠标模型相结合，以解决以前未经过测试的问题 关于甘油三酯清除如何受到监管的问题。拟议的研究具有重要意义，因为 他们将阐明Angptl3和ANGPTL4如何以及在哪里调节功能性LPL的水平 影响甘油三酯的传递，并将发现甘油三酯清除的新机制，而不是 需要血管LPL，最终允许设计新的策略来预防或治疗血脂异常。

PROJECT SUMMARY Clinical hypertriglyceridemia is common and is only partially responsive to available drugs. Conversely, excess triglyceride delivery to individual tissues can lead to skeletal muscle insulin resistance, cardiac lipotoxicity, or severe inflammatory responses. Given the importance of proper triglyceride delivery, it is imperative that we understand the mechanisms that regulate this process. Two proteins normally required for triglyceride clearance are lipoprotein lipase (LPL) and GPIHBP1. LPL is required for the actual hydrolysis of plasma triglyceides, liberating fatty acids for tissue uptake. The endothelial cell protein GPIHBP1 is required to properly localize LPL to the vascular lumen where lipolysis takes place. The long term goal of the proposed research is to understand and ultimately modulate the delivery of triglyceride-derived fatty acids to specific tissues. The objectives in this application are to advance our understanding of how the angiopoietin-like (ANGPTL) proteins, ANGPTL3 and ANGPTL4, modulate triglyceride clearance. ANGPTL3 and ANGPTL4 can both inhibit LPL in vitro, and ANGPTL3 or ANGPTL4 deficiency leads to lower plasma triglyceride levels. However, if and how ANGPTL3 and ANGPTL4 directly modulate LPL/GPIHBP1-mediated triglyceride clearance is not clear. Moreover, the plasma triglyceride levels in mice deficient in both GPIHBP1 and ANGPTL4 indicate that ANGPTL4 may regulate an uncharacterized, GPIHBP1-independent triglyceride clearance pathway. These issues will be addressed by pursuing two specific aims: 1) Define the mechanisms by which ANGPTL3 and ANGPTL4 modulate GPIHBP1-dependent triglyceride clearance; and 2) Identify novel mechanisms of GPIHBP1- independent triglyceride clearance. The studies in aim 1 employ cell culture–based binding and lipase activity assays, as well as in vivo injection of recombinant proteins and tissue-specific knockout mice to investigate how ANGPTL proteins interact with GPIHBP1/LPL complexes on the surface of endothelial cells. The second aim will address the remarkably lower triglycerides in Angptl4–/–Gpihbp1–/– mice. Triglyceride clearance and fatty acid uptake assays will be used to identify the tissues to which triglycerides get cleared. Expression, protein, and lipase activity analysis will be used to identify the proteins responsible for this clearance. The approaches in the proposed research are innovative because they combine standard methods with novel mouse models to address previously untested questions about how triglyceride clearance is regulated. The proposed studies are significant because they will elucidate how and where ANGPTL3 and ANGPTL4 modulate levels of functional LPL to influence triglyceride delivery and will uncover novel mechanisms of triglyceride clearance that do not require vascular LPL, ultimately permitting new strategies designed to prevent or treat dyslipidemia.