Bell J J, Sargeant T E, Watson J A
J Biol Chem. 1976 Mar 25;251(6):1745-58.
Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.
与脂蛋白（全血清和人低密度脂蛋白）形成复合物的纯胆固醇，或与琥珀酸酯化（胆固醇琥珀酸酯）并与白蛋白结合的纯胆固醇，在短时间（4小时）孵育期间，能有效抑制在缺乏脂蛋白的血清培养基中生长的肝癌组织培养（HTC）细胞中的3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶活性。同时测量全血清和胆固醇琥珀酸酯中放射性未酯化胆固醇的摄取动力学、它们向脂质产物的转化以及酶活性的衰减，表明胆固醇诱导的抑制作用是由固醇本身介导的，而不是由其代谢产生的抑制性脂质产物介导的。几种胆固醇衍生物，如胆甾烯酮、7-酮胆固醇、7α-羟基胆固醇和25-羟基胆固醇，也能抑制HTC细胞中的还原酶活性，并且比纯胆固醇制剂的抑制作用明显更强。胆固醇及其衍生物引起的酶活性降低是浓度依赖性和特异性的。[1-14C]油酸掺入实验表明，在类固醇的抑制浓度下，HTC细胞中胆固醇酯的形成并未增加。这些数据表明，在HMG-CoA还原酶活性的反馈控制中，固醇酯的形成不是一个必需的过程。还原酶的半衰期（3至4小时）不受环己酰亚胺、全血清加或不加以及胆固醇琥珀酸酯的显著影响。相比之下，当HTC细胞与环己酰亚胺加最大浓度的25-羟基胆固醇、7-酮胆固醇或胆甾烯酮一起孵育时，半衰期会大大缩短，导致t1/2值分别为24、36和60分钟。全血清和胆固醇琥珀酸酯浓度的增加对酶失活的表观速率常数没有显著影响，而其表观合成速率分别降低了3倍和10倍。这些结果在氧化类固醇抑制剂作用下会逆转。随着培养基中25-羟基胆固醇、7-酮胆固醇和胆甾烯酮浓度的增加，还原酶的合成速率基本不变，而降解的表观速率常数分别增加了9倍、7倍和3倍。因此，HTC细胞中的HMG-CoA还原酶活性似乎受两种不同机制调节，其中类固醇结构很重要。全血清和胆固醇琥珀酸酯特异性降低酶的合成速率，而25-羟基胆固醇、7-酮胆固醇和胆甾烯酮增加还原酶的失活速率。
