Mechanism of GLUT4 Inhibition by HIV Protease Inhibitor

描述(由申请人提供)：本项目的长期目标 是为了了解促进葡萄糖的分子机制 运输。最近发现的HIV蛋白水解酶抑制剂能够 选择性抑制主要的胰岛素反应葡萄糖GLUT4 Transporter，是第一个有可能选择性和 可逆性抑制单个促进性葡萄糖转运蛋白的活性 异构体。这种抑制发生的机制尚不清楚。这个 这项研究的具体目标是调查 HIV蛋白水解酶抑制剂对GLUT4的抑制作用 体外对GLUT4的影响可以在体内复制，导致急性和 可逆性胰岛素抵抗。首先，仔细地进行了动力学分析。 使用异源表达的GLUT4进行抑制过程 非洲爪哇卵母细胞。接下来，HIV蛋白水解酶抑制剂与GLUT4结合的位置 将通过对非洲爪哇卵母细胞转运蛋白的光标记来确定 和/或使用合成的反应性逆转录病毒蛋白酶的3T3-L1脂肪细胞 抑制剂衍生物。修饰蛋白质的表面增强分析 激光解吸电离(SELDI)质谱仪。最后，有能力 HIV蛋白酶抑制剂将急性和可逆地导致胰岛素抵抗 将通过测量正常血糖下的葡萄糖处置来研究活体 正常人群和糖尿病易感人群的高胰岛素钳夹状态 啮齿动物。更好地理解人类活动的机制 促进性葡萄糖转运蛋白可以以异构体的形式进行剧烈的调节 特定的方式将为研究血糖稳态提供一种新的手段。 正常人和有糖调节障碍的人，如 糖尿病。这项研究的结果也可能有助于 新型HIV蛋白水解酶抑制剂的开发 艾滋病毒治疗，同时避免其不利的新陈代谢后果。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the molecular mechanisms involved in facilitative glucose transport. The recent discovery that HIV protease inhibitors are capable of selectively inhibiting GLUT4, the major insulin responsive glucose transporter, is the first report that it is possible to selectively and reversibly inhibit the activity of a single facilitative glucose transporter isoform. The mechanism by which this inhibition occurs remains unknown. The specific goals of this research are to investigate the mechanistic basis for the inhibition of GLUT4 by HIV protease inhibitors and determine whether the in vitro effects on GLUT4 can be replicated in vivo, leading to acute and reversible insulin resistance. First, a careful kinetic analysis of the inhibition process will be conducted using GLUT4 heterologously expressed in Xenopus oocytes. Next, the site of HIV protease inhibitor binding to GLUT4 will be determined through photolabeling of the transporter in Xenopus oocytes and/or 3T3-L1 adipocytes using a synthesized reactive retroviral protease inhibitor derivative. Modified protein will be analyzed by Surface Enhanced Laser Desorption/Ionization (SELDI) mass spectrometry. Finally, the ability of HIV protease inhibitors to acutely and reversibly cause insulin resistance in vivo will be investigated by measuring glucose disposal under euglycemic hyperinsulinemic clamp conditions in both normal and diabetes susceptible rodents. A better understanding of the mechanism by which the activity of facilitative glucose transporters can be acutely modulated in an isoform specific manner will provide a new means of studying glucose homeostasis in normal individuals and those with disorders of glucose regulation, such as diabetes mellitus. The results of this research may also facilitate the development of newer HIV protease inhibitors that maintain their efficacy in HIV treatment while avoiding their adverse metabolic consequences.