Miyagawa Ryu, Asakura Takefumi, Nakamura Tomomi, Okada Hiromi, Iwaki Soichiro, Sobel Burton E, Fujii Satoshi
aDepartment of Molecular and Cellular Pathobiology and Therapeutics, Nagoya City University School of Pharmaceutical Sciences, Nagoya, Japan.
Coron Artery Dis. 2010 May;21(3):144-50. doi: 10.1097/MCA.0b013e328335790e.
Insulin increases, through several molecular mechanisms, expression of plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of fibrinolysis. This phenomenon has been implicated as a cause of accelerated coronary artery disease and the increased incidence of acute coronary syndromes associated with type 2 diabetes. We have previously reported that physiologic and pharmacologic concentrations of insulin induce PAI-1 synthesis in human HepG2 cells and that simvastatin can attenuate its effects. This study was performed to further elucidate mechanisms responsible for the insulin-induced PAI-1 production.
Concentrations of PAI-1 mRNA were determined by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-829 to +36 bp) activity was assayed with the use of luciferase reporter assays. The potential role of the 3'-untranslated region (UTR) in the PAI-1 gene was assayed with the use of luciferase constructs containing the 3'-UTR. Oxidative stress was measured by loading cells with carboxy-2,7 dichlorodihydrofluorescein.
Insulin increased PAI-1 promoter activity, PAI-1 mRNA, and accumulation of PAI-1 protein in the conditioned media. Insulin-inducible PAI-1 promoter activity was attenuated by simvastatin. Experiments performed with luciferase reporters containing the 3'-UTR showed that insulin increased luciferase activity through this region. Insulin also increased oxidative stress. Both insulin-inducible luciferase activity through the 3'-UTR and oxidative stress were attenuated by simvastatin.
Insulin can increase PAI-1 expression through multiple mechanisms including induction mediated by the 3'-UTR of the PAI-1 gene. Accordingly, beneficial pleiotropic effects of statins on coronary artery disease may be attributable, in part, to attenuation of overexpression of PAI-1 mediated by the 3'-UTR in syndromes of insulin resistance (such as the metabolic syndrome) and type 2 diabetes.
胰岛素通过多种分子机制增加纤溶酶原激活物抑制剂-1（PAI-1）的表达，PAI-1是纤维蛋白溶解的主要生理抑制剂。这一现象被认为是冠状动脉疾病加速以及2型糖尿病相关急性冠状动脉综合征发病率增加的原因。我们之前报道过，生理浓度和药理浓度的胰岛素可诱导人HepG2细胞合成PAI-1，且辛伐他汀可减弱其作用。本研究旨在进一步阐明胰岛素诱导PAI-1产生的机制。
通过实时聚合酶链反应（PCR）测定PAI-1 mRNA的浓度，并用蛋白质印迹法检测PAI-1蛋白。使用荧光素酶报告基因检测法测定PAI-1启动子（-829至+36 bp）的活性。使用含有3'非翻译区（UTR）的荧光素酶构建体检测PAI-1基因中3'UTR的潜在作用。通过用羧基-2,7二氯二氢荧光素加载细胞来测量氧化应激。
胰岛素增加了PAI-1启动子活性、PAI-1 mRNA以及条件培养基中PAI-1蛋白的积累。辛伐他汀减弱了胰岛素诱导的PAI-1启动子活性。用含有3'UTR的荧光素酶报告基因进行的实验表明，胰岛素通过该区域增加了荧光素酶活性。胰岛素还增加了氧化应激。辛伐他汀减弱了通过3'UTR的胰岛素诱导的荧光素酶活性以及氧化应激。
胰岛素可通过多种机制增加PAI-1表达，包括由PAI-1基因的3'UTR介导的诱导。因此，他汀类药物对冠状动脉疾病有益的多效性作用可能部分归因于在胰岛素抵抗综合征（如代谢综合征）和2型糖尿病中减弱由3'UTR介导的PAI-1过表达。
