Powell E E, Kroon P A
Department of Biochemistry, University of Queensland, Brisbane, Australia.
J Clin Invest. 1994 May;93(5):2168-74. doi: 10.1172/JCI117213.
The liver plays a key regulatory role in cholesterol metabolism. Two proteins are central in this role; the LDL receptor and 3-hydroxy-3-methylglutaryl CoA reductase (HMG CoA reductase), the rate-limiting enzyme in cholesterol biosynthesis. In the current investigation, we have used a sensitive nonradioactive method to study the regulation of LDL receptor and HMG CoA reductase mRNA levels in liver biopsy samples and freshly isolated mononuclear leukocytes from 13 patients who underwent cholecystectomy for gallstones. mRNA copy numbers were determined by PCR amplification of reverse-transcribed RNA using synthetic RNA as an internal standard. Incorporation of digoxigenin-11-dUTP during amplification allowed direct detection and quantitation of mRNA levels by chemiluminescence. These experiments showed that the average number of LDL receptor mRNA molecules in liver (21 +/- 3 x 10(4)/micrograms of RNA) and mononuclear leukocytes (24 +/- 3 x 10(4)/micrograms of RNA) are indistinguishable, whereas the number of HMG CoA reductase molecules in liver (107 +/- 15 x 10(4)/micrograms of RNA) is smaller than that in mononuclear leukocytes (158 +/- 21 x 10(4)/micrograms of RNA, P < 0.05). These numbers correspond to an average of 1-6 copies of LDL receptor mRNA and 5-42 copies of HMG CoA reductase mRNA per cell. There was a significant correlation between the numbers of LDL receptor (P = 0.0005) and HMG CoA reductase (P = 0.003) mRNA molecules in liver and mononuclear leukocytes. Furthermore, the numbers of copies of HMG CoA reductase and LDL receptor mRNA were correlated with each other in both liver (P = 0.02) and mononuclear leukocytes (P = 0.01), consistent with coordinate regulation. These data demonstrate that the mechanisms which regulate mRNA levels in liver and mononuclear cells are similar and suggest that freshly isolated mononuclear cells can be used to predict HMG CoA reductase and LDL receptor mRNA levels in liver.
肝脏在胆固醇代谢中起关键调节作用。两种蛋白质在这一作用中至关重要，即低密度脂蛋白受体和3-羟基-3-甲基戊二酰辅酶A还原酶（HMG CoA还原酶），后者是胆固醇生物合成中的限速酶。在本研究中，我们采用一种灵敏的非放射性方法，研究了13例因胆结石接受胆囊切除术患者的肝活检样本及新鲜分离的单核白细胞中低密度脂蛋白受体和HMG CoA还原酶mRNA水平的调节情况。以合成RNA作为内标，通过逆转录RNA的PCR扩增来测定mRNA拷贝数。扩增过程中掺入地高辛-11-dUTP，可通过化学发光直接检测和定量mRNA水平。这些实验表明，肝脏（21±3×10⁴/μg RNA）和单核白细胞（24±3×10⁴/μg RNA）中低密度脂蛋白受体mRNA分子的平均数量无显著差异，而肝脏中HMG CoA还原酶分子的数量（107±15×10⁴/μg RNA）少于单核白细胞（158±21×10⁴/μg RNA，P<0.05）。这些数量相当于每个细胞平均有1 - 6个低密度脂蛋白受体mRNA拷贝和5 - 42个HMG CoA还原酶mRNA拷贝。肝脏和单核白细胞中低密度脂蛋白受体（P = 0.0005）和HMG CoA还原酶（P = 0.003）mRNA分子数量之间存在显著相关性。此外，肝脏（P = 0.02）和单核白细胞（P = 0.01）中HMG CoA还原酶和低密度脂蛋白受体mRNA拷贝数相互关联，符合协同调节。这些数据表明，调节肝脏和单核细胞中mRNA水平的机制相似，并提示新鲜分离的单核细胞可用于预测肝脏中HMG CoA还原酶和低密度脂蛋白受体mRNA水平。
