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

作为甘油三酯和甘油三酯的主要水解酶 中密度脂蛋白和高密度脂蛋白中的磷脂 肝脂酶(HL)在脂蛋白代谢中起着核心作用。 传统上，HL功能主要归因于脂解作用，但 最近的研究表明HL在脂蛋白代谢中起作用。 与酶的水解性无关。为了调查 肝脂酶在介导胆汁淤积清除中的作用 脂蛋白不依赖于其体内的脂解功能，我们使用 重组腺病毒(ADV)表达野生型HL(HL-WT，n=3)， 催化失活的HL(HL145G，n=8)和荧光素酶(Lucif，n=8) 高密度脂蛋白缺乏小鼠血浆高密度脂蛋白浓度升高。第4天 静脉注射后。向第8个PFU注射6x10，类似水平的HL-WT 用酶联免疫吸附试验检测肝素后血浆(HL-WT：5.9)中突变株 正/负1.4微克/毫升；HL145G：5.0正/负3.4) 仅在表达HL-WT(35.5)的小鼠中检测到血浆HL活性 加/减6.7u/mol/ml/min)。与基线(第0天)值比较 (mg/dl)：表达脂解性非活性HL145G的HL缺陷小鼠(天 4)降低了胆固醇(-35%，p&lt；0.00001)、磷脂(-28%， P&lt；0.00001)，高密度脂蛋白-胆固醇(-44%，P&lt；0.005)。没有重大意义(p&gt；0.3) 小鼠注射6x10至第8次PFU后的血脂变化 卢西夫。HL-WT降低了基线胆固醇(-85%正负7%)， 磷脂-84正/负6%)，高密度脂蛋白-胆固醇(-100正/负0%) ApoA-I(-82正/负7%)比HL145G更显著。这些 研究表明，腺病毒介导的催化表达 失活HL145G显著降低血浆胆固醇、磷脂 和HL缺陷小鼠的高密度脂蛋白-胆固醇。因此，高密度脂蛋白的脂解作用 并不能解释HL诱导的高密度脂蛋白的所有代谢变化。我们的 研究为HL在高密度脂蛋白代谢中的作用提供了体内证据 与其脂解功能无关，这一过程可能涉及HL 蛋白多糖和/或受体介导的脂蛋白摄取增强 机械装置。

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.