Metabolic Signal Transduction in Adipocytes

本申请中提出的工作目标是通过增加氧化来减少脂肪储存。这个 这种方法背后的假设是，阻止ROS诱导的呼吸抑制将导致 氧化增加，脂肪储存减少。基础以上的脂肪细胞呼吸受到抑制。然而， 活性氧(ROS)的清除剂，丙酮酸或N-乙酰半胱氨酸，可以恢复氧的消耗 并减少人类脂肪细胞中的脂肪储存。这只在完整的细胞中观察到，而不是 在分离的线粒体中，表明ROS的产生需要完整细胞的高脂肪环境。 我们已经开发出一种新型的固定化培养脂肪细胞的长效制剂，它允许 重复测量耗氧量和荧光分子的图像分析。 为了验证我们的假设，我们将使用分离的大鼠脂肪细胞和线粒体以及分化的人类 前脂肪细胞执行以下目标：目标1是确定清除ROS是否刺激 呼吸作用，减少大鼠和人脂肪细胞中的甘油三酯储存。这一预测将得到检验， 在ROS和TG之间存在着一种关系，即通过过度表达 超氧化物歧化酶、过氧化还蛋白或谷胱甘肽过氧化物酶会减少甘油三酯的储存。我们会 同时测量线粒体！膜电位(&W)和呼吸，以评估偶联。目标2 识别呼吸链中被ROS抑制的部位。为了了解该网站的身份， 我们将测量从底物进入呼吸链的脂肪细胞的氧气消耗 络合物I、III或IV。呼吸增加表示抑制解除。目标3是确定 O2“、H2O2、NO或其他ROS是否介导抑制呼吸。使用亚线粒体颗粒 我们将从脂肪细胞中鉴定抑制ROS的种类及其特定的作用部位。通量将 在存在Rosand特定底物的情况下进行测量。目标4将确定 低脂肪细胞的凋亡和更替，有证据表明线粒体中有很强的ROS作用。我们会 利用光激活技术绘制脂肪细胞线粒体网络图并检测网络改变的效果 通过基因敲除。我们预计，ROS和细胞凋亡之间的解偶联是一个必不可少的 脂肪细胞FFA处理的组成部分。 这些实验的预期结果是确定导致独一无二的 ROS对脂肪细胞呼吸的抑制及克服这种抑制的方法。脂肪细胞的呼吸作用 很少有人对此进行调查，部分原因是人们误以为这并不重要。这些实验 可以逆转节俭的表型，并可能使人们深入了解节俭的基因及其在 肥胖的发展。

The goal of the work proposed in this application is to decrease fat stores by increasing oxidation. The hypothesis underlying this approach is that preventing inhibition of respiration induced by ROS will lead to increased oxidation and decreased fat storage. Adipocyte respiration above basal is inhibited. However, scavengers of reactive oxygen species (ROS), pyruvate or N-acetyl cysteine, can restore O2 consumption in rat adipocytes and decrease fat stores in human adipocytes. This was observed only in intact cells and not in isolated mitochondria suggesting that ROS production required the high fat environment of the intact cell. We have developed a novel long-lasting preparation of immobilized cultured adipocytes that permits repeated measurement of O2 consumption and image analysis of fluorescent molecules. To test our hypothesis we will use isolated rat adipocytes and mitochondria and differentiated human preadipocytes to perform the following Aims: Aim 1 is to determine if ROS removal stimulates respiration and decreases TG stores in rat and human adipocytes. The prediction will be tested that there is a relationship between ROS and TG stores such that decreasing ROS through over-expression of superoxide dismutase, peroxiredoxin or glutathione peroxidase will decrease TG stores. We will simultaneously measure mitochondria! membrane potential (&W)and respiration to assess coupling. Aim 2 is to identify the site in the respiratory chain that is inhibited by ROS. To learn the identity of the site, we will measure O2 consumption in adipocytes from substrates that enter the respiratory chain through Complexes I, III or IV. Relief of inhibition will be indicated by increased respiration. Aim 3 is to determine whether O2", H2O2, NO or other ROS mediate inhibited respiration. Using submitochondrial particles derived from adipocytes we will identify the inhibitory ROS species and their specific site of action. Flux will be measured in the presence of ROSand site-specific substrates. Aim 4 will determine the mechanism of low adipocyte apoptosis and turnover amid evidence of strong ROSaction in mitochondria. We will map mitochondrial networking in adipocytes using photoactivation and test the effect of network alteration through gene knockdown. We anticipate that uncoupling between ROS and apoptosis is an essential component of adipocyte FFA handling. The anticipated outcome of these experiments is identification of the mechanism responsible for the unique inhibition of adipocyte respiration by ROS and ways to overcome this inhibition. Respiration in adipocytes has been little investigated, due in part, to the misconception that it is not important. These experiments could reverse the thrifty phenotype and possibly lend insight into the thrifty genotype and its role in the development of obesity.