Parker R A, Pearce B C, Clark R W, Gordon D A, Wright J J
Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
J Biol Chem. 1993 May 25;268(15):11230-8.
Tocotrienols are natural farnesylated analogues of tocopherols which decrease hepatic cholesterol production and reduce plasma cholesterol levels in animals. For several cultured cell types, incubation with gamma-tocotrienol inhibited the rate of [14C]acetate but not [3H] mevalonate incorporation into cholesterol in a concentration- and time-dependent manner, with 50% inhibition at approximately 2 microM and maximum approximately 80% inhibition observed within 6 h in HepG2 cells. 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase total activity and protein levels assayed by Western blot were reduced concomitantly with the decrease in cholesterol synthesis. In HepG2 cells, gamma-tocotrienol suppressed reductase despite strong blockade by inhibitors at several steps in the pathway, suggesting that isoprenoid flux is not required for the regulatory effect. HMG-CoA reductase protein synthesis rate was moderately diminished (57% of control), while the degradation rate was increased 2.4-fold versus control (t1/2 declined from 3.73 to 1.59 h) as judged by [35S]methionine pulse-chase/immunoprecipitation analysis of HepG2 cells treated with 10 microM gamma-tocotrienol. Under these conditions, the decrease in reductase protein levels greatly exceeded the minor decrease in mRNA (23 versus 76% of control, respectively), and the low density lipoprotein receptor protein was augmented. In contrast, 25-hydroxycholesterol strongly cosuppressed HMG-CoA reductase protein and mRNA levels and the low density lipoprotein receptor protein. Thus, tocotrienols influence the mevalonate pathway in mammalian cells by post-transcriptional suppression of HMG-CoA reductase, and appear to specifically modulate the intracellular mechanism for controlled degradation of the reductase protein, an activity that mirrors the actions of the putative non-sterol isoprenoid regulators derived from mevalonate.
生育三烯酚是生育酚的天然法尼基化类似物，可降低动物肝脏胆固醇的生成并降低血浆胆固醇水平。对于几种培养的细胞类型，用γ-生育三烯酚孵育以浓度和时间依赖性方式抑制了[14C]乙酸盐掺入胆固醇的速率，但不抑制[3H]甲羟戊酸掺入胆固醇的速率，在HepG2细胞中，约2 microM时抑制率为50%，6小时内最大抑制率约为80%。通过蛋白质印迹法测定的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶的总活性和蛋白质水平随着胆固醇合成的减少而相应降低。在HepG2细胞中，尽管该途径的几个步骤被抑制剂强烈阻断，但γ-生育三烯酚仍能抑制还原酶，这表明类异戊二烯通量对于调节作用并非必需。通过对用10 microMγ-生育三烯酚处理的HepG2细胞进行[35S]甲硫氨酸脉冲追踪/免疫沉淀分析判断，HMG-CoA还原酶蛋白质合成速率适度降低（为对照的57%），而降解速率比对照增加了2.4倍（半衰期从3.73小时降至1.59小时）。在这些条件下，还原酶蛋白质水平的降低大大超过了mRNA的轻微降低（分别为对照的23%和76%）并且低密度脂蛋白受体蛋白质增加。相反，25-羟基胆固醇强烈共抑制HMG-CoA还原酶蛋白质和mRNA水平以及低密度脂蛋白受体蛋白质。因此，生育三烯酚通过转录后抑制HMG-CoA还原酶影响哺乳动物细胞中的甲羟戊酸途径，并且似乎特异性地调节还原酶蛋白质的受控降解的细胞内机制，该活性反映了源自甲羟戊酸的假定非甾醇类异戊二烯调节剂的作用。
