Ooyen C, Zecca A, Bersino A M, Catapano A L
Institute of Pharmacological Sciences, University of Milan, Italy.
Atherosclerosis. 1999 Jul;145(1):87-95. doi: 10.1016/s0021-9150(99)00018-0.
Intracellular cholesterol biosynthesis may play a key role in supplying cholesterol (as cholesteryl ester) for the neutral core of very low density lipoprotein (VLDL), thus modulating the secretion of apolipoprotein B-100 (apo B-100) from hepatocytes. The effect of compound NK-104 was studied, a new competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA-reductase), on apo B-100 synthesis and secretion from the human hepatoma cell line Hep G2. Cells were preincubated with NK-104 (0.01-5 microM) in the presence or absence of oleate (0.8 mM). Apo B-100 in the medium was determined by an enzyme-linked immunosorbent assay (ELISA). Incubation of Hep G2 with NK-104 resulted in a marked inhibition of cholesterogenesis (up to 95%), determined as incorporation of [14C]acetate into sterols, and decreased in a dose-dependent manner apo B-100 secretion, both in basal conditions (from 110 to 82 ng/mg cell protein, P < 0.01) and after incubation with oleate (from 227 to 165 ng/mg cell protein, P < 0.01). Density gradient for distribution of apo B-100 secreted, showed that this decrease was essentially due to a reduction of apo B-100 associated with lipoproteins in the density range of low density lipoproteins (LDL). Pulse chase experiment demonstrated that NK-104 did not affect the synthetic rate of apo B-100 but increased intracellular degradation of newly synthesized protein. The compound had only marginal effect on the mass of intracellular triglyceride but significantly decreased intracellular mass of free cholesterol and cholesteryl ester (P < 0.01). It is speculated that the ability of compound NK-104 to decrease apo B-100 secretion from Hep G2 cells is due to a decreased intracellular cholesterol availability.
细胞内胆固醇生物合成可能在为极低密度脂蛋白（VLDL）的中性核心提供胆固醇（作为胆固醇酯）方面发挥关键作用，从而调节载脂蛋白B - 100（apo B - 100）从肝细胞的分泌。研究了新型3 - 羟基 - 3 - 甲基戊二酰辅酶A还原酶（HMG CoA还原酶）竞争性抑制剂化合物NK - 104对人肝癌细胞系Hep G2中apo B - 100合成和分泌的影响。细胞在有或无油酸（0.8 mM）存在的情况下用NK - 104（0.01 - 5 microM）预孵育。通过酶联免疫吸附测定（ELISA）测定培养基中的apo B - 100。用NK - 104孵育Hep G2导致胆固醇生成显著抑制（高达95%，通过[14C]乙酸盐掺入甾醇来测定），并且在基础条件下（从110降至82 ng/mg细胞蛋白，P < 0.01）以及与油酸孵育后（从227降至165 ng/mg细胞蛋白，P < 0.01），apo B - 100分泌呈剂量依赖性降低。分泌的apo B - 100的密度梯度分布表明，这种降低主要是由于低密度脂蛋白（LDL）密度范围内与脂蛋白相关的apo B - 100减少。脉冲追踪实验表明，NK - 104不影响apo B - 100的合成速率，但增加了新合成蛋白质的细胞内降解。该化合物对细胞内甘油三酯质量只有轻微影响，但显著降低了细胞内游离胆固醇和胆固醇酯的质量（P < 0.01）。据推测，化合物NK - 104降低Hep G2细胞中apo B - 100分泌的能力是由于细胞内胆固醇可用性降低。
