Manipulating /Preventing Mitochondrial Oxidative Stress

描述（由申请方提供）：高血压是糖尿病大血管疾病的主要独立风险因素。内皮细胞暴露于高血糖症的后果是明确的。然而，关于脂肪细胞如何对急性和慢性暴露于生理水平的高血糖反应知之甚少。我们最近的观察突出了脂肪细胞中ROS水平增加的显著影响。我们已经确定，高血糖症导致ROS水平的线粒体产生增加。这导致脂肪细胞中炎症反应、ROS诱导的DNA损伤和总体胰岛素抵抗的普遍诱导。重要的是，我们已经证明，我们可以通过改变线粒体二羧酸转运蛋白mDIC的表达水平，直接操纵线粒体ROS的产生，从而独立于高血糖症来操纵炎症和胰岛素抵抗。该转运蛋白作为脂肪细胞中电子传递链底物可用性的限速因子。此外，我们已经发现强有力的证据表明，PPARgamma激动剂对脂肪细胞中的线粒体产生快速的转录非依赖性作用，导致ROS水平的剂量依赖性降低。这可能通过与称为mitoNEET的17kD线粒体膜蛋白的直接相互作用发生。在这里，我们的目的是确定的mDIC在控制脂肪细胞中的ROS产生的作用，PPARgamma激动剂对线粒体膜电位的影响的特点，并确定mitoNEET是否是一个关键的球员在TZDs对脂肪细胞中的ROS的影响。具体而言，我们的目标是：SA 1）通过从小鼠脂肪细胞中遗传去除mDIC基因（"脂肪细胞特异性无效"），降低体内mDIC水平以及在脂肪细胞中过表达mDIC，证明mDIC在ROS产生中起关键作用。SA 2）确定PPARgamma激动剂对脂肪细胞中线粒体的直接作用。SA 3）分析mitoNEET作为脂肪细胞中的PPARgamma激动剂靶标的贡献。脂肪细胞中ROS的增加对细胞胰岛素敏感性有深远的影响。ROS诱导的损伤在这些非常长寿的细胞中随着时间的推移而积累，并显示出"高血糖记忆"，因为ROS诱导的损伤不容易逆转。该提案旨在突出两种特定候选分子mDIC和mitoNEET对脂肪细胞中ROS产生的贡献，从而提供两种新的有希望的治疗靶点。

DESCRIPTION (provided by applicant): Hyperglycemia is a major independent risk factor for diabetic macrovascular disease. The consequences of exposure of endothelial cells to hyperglycemia are well established. However, little is known about how adipocytes respond to both acute as well as chronic exposure to physiological levels of hyperglycemia. Our recent observations have highlighted dramatic effects of increased ROS levels in adipocytes. We have established that hyperglycemia leads to increased mitochondrial production of ROS levels. This leads to a general induction of the inflammatory response, ROS-induced DNA damage and overall insulin resistance in adipocytes. Importantly, we have demonstrated that we can manipulate inflammation and insulin resistance independent of hyperglycemia through direct manipulation of mitochondrial ROS generation by altering the expression levels of the mitochondrial dicarboxylate transporter, mDIC. This transporter acts as a rate-limiting factor for substrate availability for the electron transport chain in adipocytes. Furthermore, we have found strong evidence indicating that PPARgamma agonists exert rapid transcription-independent effects on mitochondria in adipocytes that lead to a dose-dependent reduction of ROS levels. This may occur through a direct interaction with a 17kD mitochondrial membrane protein called mitoNEET. Here, we aim to determine the role of the mDIC in the control of ROS production in adipocytes, characterize the effects of PPARgamma agonists on mitochondrial membrane potential, and determine whether mitoNEET is a critical player in the effects of TZDs on ROS in adipocytes. Specifically, we aim to: SA 1) Demonstrate that mDIC is critically involved in ROS generation by genetically removing the mDIC gene from mouse adipocytes ("adipocyte-specific null"), reducing the levels of mDIC in vivo as well as overexpressing it in adipocytes. SA 2) Define the direct effects of PPARgamma agonists on mitochondria in adipocytes. SA 3) Analyze the contributions of mitoNEET as a PPARgamma agonist target in adipocytes. The increased presence of ROS in adipocytes has a profound impact on cellular insulin sensitivity. ROS-induced damage in these very long-lived cells accumulates over time and displays a "hyperglycemic memory" in that ROS-induced damage is not readily reversible. This proposal aims to highlight the contribution of two specific candidate molecules, mDIC and mitoNEET, towards the generation of ROS in adipocytes, thereby providing two new promising therapeutic targets.