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

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=9）， 无催化活性的HL（HL 145 G，n=8）和荧光素酶（Lucif，n=8）， 血浆apoB浓度升高的apoE缺陷小鼠 含有脂蛋白。静脉注射1x 10至 第八pfu，通过ELISA检测到相似水平的HL-WT和突变体 在肝素后血浆中（HL-WT：7+/-3 μ g/ml; HL 145 G：7+/-4），而 肝素后血浆HL活性仅在表达 HL-WT（34+/-14 micromol/ml/min）。与基线（第0天）值相比 (mg/dl）：表达脂解失活的HL 145 G的apoE缺陷型小鼠 (day 4）具有降低的胆固醇（-51%，p<0.01），磷脂（-38%，p<0.01）， p<0.001），非HDL-胆固醇（-46%，p<0.005）。无显著性（p>0.3） 在注射1 × 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（-36%，p <$0.01）， 甘油三酯（-78%，p<0.001）磷脂-49，p<0.01）， 非HDL-胆固醇（36，p<0.01%）。这些研究证明 腺病毒介导的催化失活HL 145 G的表达 显著降低血浆胆固醇、甘油三酯、磷脂 和非HDL-胆固醇浓度。因此，在本发明中， 脂肪分解不能解释HL诱导的所有代谢变化 在含载脂蛋白B的脂蛋白中。我们的研究提供了体内证据， HL在脂蛋白代谢中的作用不依赖于其脂解作用， 功能，这一过程可能涉及HL增强脂蛋白的摄取 通过蛋白聚糖和/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.