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=9)， 催化失活的HL(HL145G，n=8)和荧光素酶(Lucif，n=8) 载脂蛋白E缺陷小鼠血浆载脂蛋白B浓度升高 含有脂蛋白的。静脉注射后第4天。将1x10注射到 第八个PFU、HL-WT和突变体的ELISA值相近 肝素后血浆(HL-WT：7+/-3微克/毫升；HL145G：7+/-4)，而 肝素后血浆HL活性仅在表达 HL-WT(34+/-14微摩尔/毫升/分钟)。与基线(第0天)值比较 (mg/dl)：表达脂解失活HL145G的载脂蛋白E缺陷小鼠 (第4天)降低胆固醇(-51%，p&lt；0.01)、磷脂(-38%， P&lt；0.001)，非高密度脂蛋白胆固醇(-46%，P&lt；0.005)。没有重大意义(p&gt；0.3) 小鼠注射1x10至第8次PFU后的血脂变化 卢西夫。HL-WT可降低基线胆固醇(-36%，p&lt；0.01)， 甘油三酯(-78%，p&lt；0.001)， 非高密度脂蛋白-胆固醇(36，p&lt；0.01%)。这些研究表明， 腺病毒介导的催化失活HL145G的表达 显著降低血浆胆固醇、甘油三酯、磷脂 以及载脂蛋白E缺陷小鼠的非高密度脂蛋白-胆固醇浓度。因此， 脂解作用并不能解释HL引起的所有代谢变化 在含有载脂蛋白的载脂蛋白中。我们的研究提供了活体证据 HL在脂蛋白代谢中的作用不依赖于其脂解作用 功能，一个可能涉及HL增强脂蛋白摄取的过程 通过蛋白多糖和/或受体介导的机制。

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=9), catalytically inactive HL (HL145G, n=8), and luciferase (Lucif, n=8) in apoE deficient mice with increased plasma concentrations of apoB containing lipoproteins . Day 4 after i.v. injection of 1x10 to the eighth pfu, similar levels of HL-WT and mutants were detected by ELISA in post heparin plasma (HL-WT:7+/-3 microg/ml; HL145G:7+/-4) while post-heparin plasma HL activity was detected only in mice expressing HL-WT (34+/-14 micromol/ml/min). Compared to baseline (day 0) values (mg/dl): apoE deficient mice expressing lipolytically inactive HL145G (day 4) had decreased cholesterol (-51%, p<0.01), phospholipids (-38%, p<0.001),non-HDL-cholesterol (-46%, p<0.005). No significant (p>0.3) lipid changes were observed in mice injected with 1x10 to the eighth pfu of Lucif. HL-WT decreased baseline cholesterol (-36%,p<0.01), triglycerides (-78%, p<0.001) phospholipids -49, p<0.01), non-HDL-cholesterol (36, p<0.01%). These studies demonstrate that adenovirus mediated expression of catalytically inactive HL145G significantly decreased plasma cholesterol, triglycerides, phospholipids and non-HDL-cholesterol concentrations in apoE deficient mice. Thus, lipolysis does not account for all of the HL-induced metabolic changes in apoB containing lipoproteins. Our study provides in vivo evidence for a role of HL in lipoprotein metabolism independent of its lipolytic function, a process which may involve HL enhanced uptake of lipoproteins by proteoglycan and/or receptor-mediated mechanisms.