胡椒碱对胰岛素抵抗糖代谢AMPK信号通路关键靶点的干预研究

Type 2 diabetes ,a deadly chronic disease ,shares a common set of physiopathologic characteristics with obesity, hyperlipidemia and hypertension. Insulin resistance (IR) is a main hallmark of these diseases.Previous research revealed that Alcohol Extract of Piper Longum.L (AEPL) was effective in improving IR in model rats with improving the insulin sensitivity index (ISI) and glucose tolerance significantly. Preliminary experiment results suggested that piperine(PIP) extracted, separated and purificated from piper Longum L.,showed similar effects as that of AEPL. And on that basis, this project is aim to investigate the effects and mechanism in improving IR of PIP, molecular and cell and animal to the overall. First,starting out by observing indicators of abnormal metabolism of glucose, hepatic activities of AMP-activated protein kinase(AMPK), adiponectin（Adi）， leptin(Lep), translocation of glucose transporter 4 (GLUT4),glycogen synthetase(GS)and ambulatory blood pressure monitoring in IR model rats ,to confirm the improving effects of PIP on IR and glucose metabolic disorder. Secondly,to explore the effects of PIP on activities of AMPK, Adi, Lep upstream and GLUT4 , GS downstream ,key targets in AMPK signiling pathway in IR model 3T3-Ll adpocytes. There is no report at home and abroad about the improving effects of PIP on glycometabolism disorder With IR.If the project is carried out,it will be conducive to development of new drugs from Yunnan ethnic crude medicinal herbs for treatment of type 2 diabetes, which will provide some valuable reference.

2型糖尿病是严重危害人民健康的慢性致死性疾病。胰岛素抵抗（IR）是2 型糖尿病和肥胖、高脂血症、高血压等疾病主要的病理生理学特征。前期研究工作证明荜茇醇提物具有改善IR模型大鼠的胰岛素敏感指数和糖耐量的作用。从荜茇醇提物分离纯化胡椒碱，初步实验证实胡椒碱同样具有改善糖代谢紊乱的作用。在此基础上，本项目拟从分子、细胞到整体动物水平上探索胡椒碱改善IR和糖代谢紊乱作用及机制。首先观察胡椒碱对胰岛素抵抗模型大鼠糖代谢、动态血压和肝脏AMPK、脂联素、瘦素、GLUT- 4等指标的影响，确证其改善IR和糖代谢紊乱的作用；其次，探索胡椒碱对IR模型3T3-L1脂肪细胞糖代谢AMPK信号通路关键靶点AMPK、上游脂联素、瘦素和下游GLUT- 4的干预作用，探讨机理。胡椒碱对IR糖代谢紊乱干预研究，国内外未见报道。实施本项目有助于开发云药，为治疗2 型糖尿病新药开发提供一些基础研究工作。

PIP是从民族常用药荜茇(Piper longum L.)中提取的一种桂皮酰胺类衍生物，本研究采用整体动物模型结合离体细胞模型的方法对PIP改善糖脂代谢异常的作用靶点进行系统的研究。从整体动物水平，成功建立SD 大鼠IR模型，较为全面的评价了PIP调节FBG、TG、FFA、FINS、ISI、BP、OGTT、ADI、AMPKα和GLUT4的影响。研究中发现，PIP能降低FBG、FINS、TG、FFA、BP水平，升高ISI和血清ADI水平，改善糖耐量异常。同时，PIP呈现明显上调p-AMPKα 的蛋白表达水平，升高AMP/ATP 比值，以及上调AMPK信号通路上游 ADI和下游GLUT4的mRNA和蛋白表达。实验研究结果证明PIP具有良好的调节血糖、改善IR状态活性，且作用机制确与AMPK信号通路有关。在此基础上，成功建立3T3-L1细胞IR模型、HepG2细胞IR模型和C2C12细胞的IR模型，从细胞水平上评价了PIP改善糖代谢紊乱和IR的作用，观察了PIP对AMPK糖代谢信号通路AMPKα和p-AMPKα活性及AMPK信号通路上游ADI、AdipoR、APPL1、LKB1和下游GLUT 4、TORC2、PGC-1α表达的影响。结果显示，PIP能显著增加3T3-L1细胞、HepG2细胞、C2C12细胞IR模型细胞的葡萄糖消耗量，改善糖代谢紊乱，升高3T3-L1细胞和HepG2细胞IR模型上清液中的ADI 水平；并呈现明显上调IR细胞模型AMPK信号通路AMPKα和p-AMPKα的活性。同时，PIP明显上调AMPK信号通路上游靶点ADI、AdipoR和脂联素转导AMPK信号通路AdipoR1、APPL1、LKB1以及下游靶点GLUT4的表达，下调HepG2细胞IR模型糖酵解通路TORC2、PGC-1α mRNA及蛋白表达量。本项目系统评价了PIP改善糖代谢紊乱活性及其机理，有助于开发治疗2型糖尿病的云药，具有良好的应用前景。