Patel D D, Pullinger C R, Knight B L
Biochem J. 1984 Apr 15;219(2):461-70. doi: 10.1042/bj2190461.
The true rate of cholesterogenesis in cultured monocyte-macrophages was determined from the incorporation of [2-14C]acetate into cholesterol, using the desmosterol (cholesta-5,24-dien-3 beta-ol) that accumulated in the presence of the drug triparanol to estimate the specific radioactivity of the newly formed sterols. It was shown that this procedure could be successfully adapted for use with cultured monocytes despite the accumulation of other unidentified biosynthetic intermediates. In cells maintained in 20% (v/v) whole serum approx. 25% of the sterol carbon was derived from exogenous acetate. Cholesterol synthesis was as high in normal cells as in cells from homozygous familial hypercholesterolaemic (FH) subjects and accounted for 50% of the increase in cellular cholesterol. The addition of extra low-density lipoprotein (LDL) reduced cholesterol synthesis, apparently through a decrease in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase). When incubated in lipoprotein-deficient serum some cells did not survive, but those that remained showed a normal increase in protein content; the amount of cellular protein and cholesterol in each well did not increase and cholesterol synthesis was reduced by over 80%. HMG-CoA reductase activity fell less dramatically and the proportion of sterol carbon derived from exogenous acetate increased, suggesting that the low rate of cholesterogenesis with lipoprotein-deficient serum was due to a shortage of substrate. The results indicate that under normal conditions monocyte-macrophages obtain cholesterol from endogenous synthesis rather than through receptor-mediated uptake of LDL, and that synthesis together with non-saturable uptake of LDL provides the majority of the cholesterol required to support growth.
通过[2-14C]乙酸掺入胆固醇来测定培养的单核细胞-巨噬细胞中胆固醇生成的真实速率，使用在药物三苯乙醇存在下积累的胆甾烯醇（胆甾-5,24-二烯-3β-醇）来估计新形成的固醇的比放射性。结果表明，尽管存在其他未鉴定的生物合成中间体的积累，但该方法仍可成功应用于培养的单核细胞。在含有20%（v/v）全血清的细胞中，约25%的固醇碳来自外源性乙酸。正常细胞中的胆固醇合成与纯合子家族性高胆固醇血症（FH）患者细胞中的一样高，占细胞胆固醇增加量的50%。额外添加低密度脂蛋白（LDL）可降低胆固醇合成，显然是通过降低3-羟基-3-甲基戊二酰辅酶A还原酶（HMG-CoA还原酶）的活性来实现的。当在无脂蛋白血清中孵育时，一些细胞无法存活，但存活的细胞蛋白质含量正常增加；每个孔中的细胞蛋白质和胆固醇含量没有增加，胆固醇合成减少了80%以上。HMG-CoA还原酶活性下降不那么显著，来自外源性乙酸的固醇碳比例增加，这表明无脂蛋白血清中胆固醇生成率低是由于底物短缺。结果表明，在正常条件下，单核细胞-巨噬细胞从内源性合成中获取胆固醇，而不是通过受体介导的LDL摄取，并且合成与LDL的非饱和摄取共同提供了支持生长所需的大部分胆固醇。
