Burris T P, Pelton P D, Zhou L, Osborne M C, Cryan E, Demarest K T
Department of Drug Discovery, The R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869, USA.
Mol Endocrinol. 1999 Mar;13(3):410-7. doi: 10.1210/mend.13.3.0246.
Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, plays an essential role in the mediation of the actions of antidiabetic drugs known as thiazolidinediones (TZDs). PPARgamma activates many target genes involved in lipid anabolism including the adipocyte fatty acid binding protein (aP2). In this study, induction of aP2 gene expression by PPARgamma agonists was examined in both cultured cells and diabetic mice using branched DNA (bDNA)-mediated mRNA quantitation. bDNA technology allows for the direct measurement of a particular mRNA directly within cellular lysate using a 96-well plate format in a time frame comparable to a reporter gene assay. In cultured human subcutaneous preadipocytes, the TZDs, troglitazone and BRL-49653, both rapidly induced aP2 mRNA as detected with the bDNA method. In these cells, the effect of BRL-49653 on aP2 mRNA levels was detectable as early as 30 min after treatment (47% increase) and was maximal after 24 h of treatment (12-fold increase). The effects of troglitazone on aP2 mRNA induction were similar to those of BRL-49653 except that the maximal level of induction was consistently lower (e.g. 24 h treatment = 4-fold increase). Dose-response relationships for both of the TZDs were also determined using the 24-h treatment time point. EC50s for both BRL-49653 and troglitazone were estimated to be 80 nM and 690 nM, respectively. A natural PPARgamma ligand, 15-deoxy-delta12,14-PGJ2, was also active in this assay with a maximal induction of aP2 mRNA of approximately 5-fold when tested at 1 microM. Since the PPARgamma:retinoid X receptor (RXR) heterodimer has been characterized as a permissive heterodimer with respect to RXR ligands, the ability of 9-cis-retinoic acid (9-cis-RA) to induce aP2 mRNA was examined. Although 9-cis-RA had very low efficacy (2-fold induction), the maximal effect was reached at 100 nM. No synergism or additivity in aP2 mRNA induction was detected when 9-cis-RA was included with either of the TZDs used in this study. Significant induction of aP2 mRNA in bone marrow of db/db mice treated with either troglitazone or BRL-49653 was also detected, indicating that the bDNA assay may be a simple method to monitor nuclear receptor target gene induction in vivo.
过氧化物酶体增殖物激活受体γ（PPARγ）是核激素受体超家族的成员之一，在介导称为噻唑烷二酮类（TZDs）的抗糖尿病药物的作用中起着至关重要的作用。PPARγ激活许多参与脂质合成代谢的靶基因，包括脂肪细胞脂肪酸结合蛋白（aP2）。在本研究中，使用分支DNA（bDNA）介导的mRNA定量技术，在培养细胞和糖尿病小鼠中检测了PPARγ激动剂对aP2基因表达的诱导作用。bDNA技术允许在细胞裂解液中直接使用96孔板形式直接测量特定的mRNA，其时间框架与报告基因测定相当。在培养的人皮下前脂肪细胞中，bDNA方法检测到TZDs（曲格列酮和BRL-49653）均能快速诱导aP2 mRNA。在这些细胞中，BRL-49653对aP2 mRNA水平的影响在处理后30分钟即可检测到（增加47%），处理24小时后达到最大值（增加12倍）。曲格列酮对aP2 mRNA诱导的作用与BRL-49653相似，只是诱导的最大水平始终较低（例如，24小时处理增加4倍）。还使用24小时处理时间点确定了两种TZDs的剂量反应关系。BRL-49653和曲格列酮的半数有效浓度（EC50）分别估计为80 nM和690 nM。一种天然的PPARγ配体15-脱氧-Δ12,14-前列腺素J2（15-deoxy-delta12,14-PGJ2）在该测定中也具有活性，在1μM测试时，aP2 mRNA的最大诱导倍数约为5倍。由于PPARγ：视黄酸X受体（RXR）异二聚体已被表征为对RXR配体具有允许作用的异二聚体，因此研究了9-顺式视黄酸（9-cis-RA）诱导aP2 mRNA的能力。尽管9-顺式视黄酸的效力非常低（诱导2倍），但在100 nM时达到最大效应。当9-顺式视黄酸与本研究中使用的任何一种TZDs一起使用时，未检测到aP2 mRNA诱导中的协同作用或相加作用。在用曲格列酮或BRL-49653处理的db/db小鼠的骨髓中也检测到了aP2 mRNA的显著诱导，这表明bDNA测定可能是一种监测体内核受体靶基因诱导的简单方法。
