IN VITRO AND IN VIVO STRUCTURE/FUNCTION ANALYSIS OF LPL AND HL

LPL和HL的体外和体内结构/功能分析

• 批准号: 2576774
• 负责人: S SANTAMARINA-FOJO
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2576774
• 关键词: Adenoviridae; blood lipoprotein metabolism; enzyme activity; hepatic lipase; laboratory mouse; lipoprotein lipase; protein structure function; recombinant proteins; recombinant virus

As the major enzyme involved in the hydrolysis of triglycerides and phospholipids present in intermediate and high density lipoproteins hepatic lipase (HL) plays a central role in lipoprotein metabolism. Classically, HL function has been primarily ascribed to lipolysis but recent studies have suggested a role of HL in lipoprotein metabolism independent of the hydrolytic function of the enzyme. To investigate the proposed role of hepatic lipase in mediating the clearance of lipoproteins independent of its lipolytic function in vivo, we used recombinant adenovirus (AdV) to express wild type HL (HL-WT, n=3), catalytically inactive HL (HL145G, n=8), and luciferase (Lucif, n=8) in HL deficient mice with increased plasma concentrations of HDL. Day 4 after i.v. injection of 6x10 to the eighth pfu, similar levels of HL-WT and mutants were detected by ELISA in post heparin plasma (HL-WT: 5.9 plus/minus 1.4 ug/ml; HL145G: 5.0 plus/minus 3.4) while post-heparin plasma HL activity was detected only in mice expressing HL-WT (35.5 plus/minus 6.7 u/mol/ml/min). Compared to baseline (day 0) values (mg/dl): HL deficient mice expressing lipolytically inactive HL145G (day 4) had decreased cholesterol (-35%, p<0.00001), phospholipids (-28%, p<0.00001), HDL-cholesterol (-44%, p<0.005). No significant (p>0.3) lipid changes were observed in mice injected with 6x10 to the eighth pfu of Lucif. HL-WT decreased baseline cholesterol (-85% plus/minus 7%), phospholipids -84 plus/minus 6%), HDL-cholesterol (-100 plus/minus 0%) and apoA-I (-82 plus/minus 7%) more significantly than HL145G. These studies demonstrate that adenovirus mediated expression of catalytically inactive HL145G significantly decreased plasma cholesterol, phospholipids and HDL-cholesterol in HL deficient mice. Thus, lipolysis of HDL lipids does not account for all of the HL-induced metabolic changes in HDL. Our study provides in vivo evidence for a role of HL in HDL metabolism independent of its lipolytic function, a process which may involve HL enhanced uptake of lipoproteins by proteoglycan and/or receptor-mediated mechanisms.

作为参与甘油三酯水解的主要酶， 存在于中密度和高密度脂蛋白中的磷脂 肝脂酶（HL）在脂蛋白代谢中起中心作用。 传统上，HL功能主要归因于脂解， 最近的研究表明HL在脂蛋白代谢中的作用 与酶的水解功能无关。 探讨 肝脂酶在介导清除 脂蛋白独立的脂解功能在体内，我们使用 重组腺病毒（AdV）表达野生型HL（HL-WT，n=3）， 无催化活性的HL（HL 145 G，n=8）和荧光素酶（Lucif，n=8） HDL血浆浓度升高的HL缺陷小鼠。 第4天 在静脉注射6 × 10 - 8 pfu后，HL-WT的水平相似， 通过ELISA检测肝素后血浆中的突变体（HL-WT：5.9 ± 1.4 ug/ml; HL 145 G：5.0 ± 3.4），而肝素后 仅在表达HL-WT的小鼠中检测到血浆HL活性（35.5 加/减6.7u/mol/ml/min）。与基线（第0天）值相比 (mg/dl）：表达脂解失活的HL 145 G的HL缺陷型小鼠（天 4)胆固醇（-35%，p<0.00001）、磷脂（-28%， p<0.00001），高密度脂蛋白胆固醇（-44%，p<0.005）。 无显著性（p>0.3） 在注射了6 × 10 ~ 8 PFU的小鼠中观察到脂质变化 关于Lucif HL-WT降低基线胆固醇（-85% ± 7%）， 磷脂-84 ± 6%），HDL-胆固醇（-100 ± 0%） apoA-I（-82 ± 7%）显著高于HL 145 G。 这些 研究表明，腺病毒介导的催化表达 非活性HL 145 G显著降低血浆胆固醇、磷脂 和HDL-胆固醇。因此，HDL脂质的脂解 并不能解释所有的HL诱导的HDL代谢变化。我们 一项研究为HL在HDL代谢中的作用提供了体内证据 独立于其脂解功能，这一过程可能涉及HL 蛋白聚糖和/或受体介导的脂蛋白摄取增强 机制等