Theriault A, Wang Q, Gapor A, Adeli K
Division of Medical Technology, University of Hawaii at Manoa, Honolulu, II 96822, USA.
Arterioscler Thromb Vasc Biol. 1999 Mar;19(3):704-12. doi: 10.1161/01.atv.19.3.704.
gamma-Tocotrienol (gamma-T3), a naturally occurring analog of tocopherol (vitamin E), has been shown to have a hypocholesterolemic effect in animals and humans. Unlike tocopherol, it has also been shown to reduce plasma apoB levels in hypercholesterolemic subjects. The aim of this study was to define the mechanism of action of gamma-T3 on hepatic modulation of apoB production using cultured HepG2 cells as the model system. HepG2 cells preincubated with gamma-T3 were initially shown to inhibit the rate of incorporation of [14C]acetate into cholesterol in a concentration- and time-dependent manner, with a maximum 86+/-3% inhibition at 50 micromol/L observed within 6 hours. gamma-T3, on the other hand, had no significant effect on the uptake of [14C]glycerol into pools of cellular triacylglycerol and phospholipid relative to untreated control. The rate of apoB synthesis and secretion was then studied by an [35S]methionine pulse-labeling experiment and quantified by immunoprecipitating apoB on chasing up to 3 hours. An average reduction of 24+/-3% in labeled apoB in the media was apparent with gamma-T3 despite a 60+/-2% increase in apoB synthesis. Fractionation of secreted apoB revealed a relatively denser lipoprotein particle, suggesting a less stable particle. Using a digitonin-permeabilized HepG2 cell system, the effects of gamma-T3 on apoB translocation and degradation in the endoplasmic reticulum were further investigated. The generation of a specific N-terminal 70-kDa proteolytic fragment proved to be a sensitive measure of the rate of apoB translocation and degradation. The abundance of this fragment increased significantly in gamma-T3-treated cells relative to untreated control cells (50+/-21%) after 2 hours of chase. In addition, the presence of gamma-T3 resulted in an average decrease of 64+/-8% in intact apoB. Taken together, the data suggest that gamma-T3 stimulates apoB degradation possibly as the result of decreased apoB translocation into the endoplasmic reticulum lumen. It is speculated that the lack of cholesterol availability reduces the number of secreted apoB-containing lipoprotein particles by limiting translocation of apoB into the endoplasmic reticulum lumen.
γ-生育三烯酚（γ-T3）是生育酚（维生素E）的一种天然类似物，已被证明在动物和人类中具有降胆固醇作用。与生育酚不同，它还被证明可降低高胆固醇血症患者的血浆载脂蛋白B（apoB）水平。本研究的目的是使用培养的HepG2细胞作为模型系统，确定γ-T3对肝脏apoB产生调节作用的机制。预先用γ-T3孵育的HepG2细胞最初显示出以浓度和时间依赖性方式抑制[14C]乙酸掺入胆固醇的速率，在6小时内50μmol/L时观察到最大抑制率为86±3%。另一方面，相对于未处理的对照，γ-T3对[14C]甘油摄取到细胞三酰甘油和磷脂池中的影响不显著。然后通过[35S]甲硫氨酸脉冲标记实验研究apoB的合成和分泌速率，并通过在长达3小时的追踪过程中免疫沉淀apoB进行定量。尽管apoB合成增加了60±2%，但γ-T3使培养基中标记的apoB平均减少了24±3%。对分泌的apoB进行分级分离发现了一种相对密度更高的脂蛋白颗粒，表明颗粒稳定性较差。使用洋地黄皂苷通透的HepG2细胞系统，进一步研究了γ-T3对apoB在内质网中的转运和降解的影响。特定N端70 kDa蛋白水解片段的产生被证明是apoB转运和降解速率的敏感指标。在追踪2小时后相对于未处理的对照细胞，γ-T3处理的细胞中该片段的丰度显著增加（50±21%）。此外，γ-T3的存在导致完整apoB平均减少64±8%。综上所述，数据表明γ-T3可能通过减少apoB向内质网腔的转运而刺激apoB降解。据推测，胆固醇供应不足通过限制apoB向内质网腔的转运减少了含apoB脂蛋白颗粒的分泌数量。
