The hydrolysis of triglycerides in triglyceride-rich lipoproteins by LPL is critical for the delivery of triglyceride-derived fatty acids to tissues, including heart, skeletal muscle, and adipose tissues. Physiologically active LPL is normally bound to the endothelial cell protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1), which transports LPL across endothelial cells, anchors LPL to the vascular wall, and stabilizes LPL activity. Disruption of LPL-GPIHBP1 binding significantly alters triglyceride metabolism and lipid partitioning. In this study, we modified the NanoLuc® Binary Technology split-luciferase system to develop a novel assay that monitors the binding of LPL to GPIHBP1 on endothelial cells in real time. We validated the specificity and sensitivity of the assay using endothelial lipase and a mutant version of LPL and found that this assay reliably and specifically detected the interaction between LPL and GPIHBP1. We then interrogated various endogenous and exogenous inhibitors of LPL-mediated lipolysis for their ability to disrupt the binding of LPL to GPIHBP1. We found that angiopoietin-like (ANGPTL)4 and ANGPTL3-ANGPTL8 complexes disrupted the interactions of LPL and GPIHBP1, whereas the exogenous LPL blockers we tested (tyloxapol, poloxamer-407, and tetrahydrolipstatin) did not. We also found that chylomicrons could dissociate LPL from GPIHBP1 and found evidence that this dissociation was mediated in part by the fatty acids produced by lipolysis. These results demonstrate the ability of this assay to monitor LPL-GPIHBP1 binding and to probe how various agents influence this important complex.

译文

LPL水解富含甘油三酸酯的脂蛋白中的甘油三酸酯对于将甘油三酸酯衍生的脂肪酸递送到包括心脏,骨骼肌和脂肪组织在内的组织至关重要。生理活性LPL通常与内皮细胞蛋白糖基磷脂酰肌醇锚定的高密度脂蛋白结合蛋白1 (GPIHBP1) 结合,该蛋白可跨内皮细胞运输LPL,将LPL锚定在血管壁上,并稳定LPL活性。LPL-GPIHBP1结合的破坏显著改变甘油三酯代谢和脂质分配。在这项研究中,我们修改了NanoLuc®二元技术裂解荧光素酶系统开发了一种新的检测方法,可实时监测LPL与内皮细胞GPIHBP1的结合。我们使用内皮脂肪酶和LPL的突变版本验证了该分析的特异性和敏感性,并发现该分析可靠且特异性地检测了LPL与gpihbp1之间的相互作用。然后,我们询问了LPL介导的脂解的各种内源性和外源性抑制剂是否具有破坏LPL与gpihbp1结合的能力。我们发现血管生成素样 (ANGPTL)4和ANGPTL3-ANGPTL8复合物破坏了LPL和GPIHBP1的相互作用,而我们测试的外源性LPL阻滞剂 (tyloxapol,poloxamer-407和四氢脂抑素) 则没有。我们还发现乳糜微粒可以将LPL与GPIHBP1解离,并发现证据表明这种解离部分是由脂解产生的脂肪酸介导的。这些结果证明了该测定法监测LPL-GPIHBP1结合和探测各种试剂如何影响该重要复合物的能力。

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