EFFECT OF DIABETES ON MITOCHONDRIAL ANION TRANSPORTERS

这个项目的长期目标是阐明分子 观察到线粒体阴离子转运体改变的基础 1型(胰岛素依赖型)糖尿病患者的功能。使用 链脲佐菌素模型的1型糖尿病，我们最近发现 肝脏线粒体柠檬酸转运蛋白的功能水平是 降低丙酮酸和二羧酸转运体的水平 与对照(即非糖尿病)动物相比增加。自.以来 这些转运蛋白对于相关的功能是必不可少的 代谢途径(即柠檬酸转运体：脂肪酸和甾醇 生物合成；丙酮酸转运蛋白：糖异生和脂肪酸 氧化；二羧酸转运体：糖异生作用)，而且由于 这些代谢途径的功能在链脲佐菌素中发生了改变-- 诱发糖尿病(即肝脏脂肪酸和可能的类固醇 生物合成减少，而糖异生和脂肪酸 氧化增加)，我们预测线粒体水平 阴离子转运蛋白的活性将与它们的 相关的代谢途径。我们的初步研究支持这一点。 假设。然后我们展示了对糖尿病动物的治疗 胰岛素逆转了观察到的转运蛋白功能的变化， 因此可以归因于胰岛素缺乏，这是这种疾病的特征 疾病。 我们现在建议阐明所观察到的改变的分子基础 线粒体柠檬酸和丙酮酸转运蛋白的功能 1型糖尿病。具体地说，利用获得的肝脏线粒体 来自对照、糖尿病和胰岛素治疗的糖尿病大鼠的实验 将进行：1)重组纯化这些转运蛋白 活性形成和比较内在的功能和分子/化学 纯化的转运蛋白的性质；2)定量 每种转运蛋白都存在于线粒体内膜内； 3)确定磷酸化反应调节的程度 体内、原位和体外的转运蛋白功能；以及4)确定 转运蛋白mRNAs的大小、稳态水平和合成速率。 这些研究将提供有关分子的第一批信息 线粒体转运蛋白功能的调控机制 无论是正常状态还是糖尿病状态。然后，这些信息将会 允许将来以特定的生化过程为目标 调节转运蛋白功能作为药理作用的可能部位 对糖尿病的干预。

The long-term objective of this project is to elucidate the molecular basis for observed alterations in mitochondrial anion transporter function in type 1 (insulin-dependent) diabetes mellitus. Using the streptozotocin model of type 1 diabetes, we have recently discovered that the functional level of the liver mitochondrial citrate transporter is decreased and the levels of the pyruvate and dicarboxylate transporters are increased relative to control (i.e., nondiabetic) animals. Since these transporters are essential for the functioning of associated metabolic pathways (i.e., citrate transporter: fatty acid and sterol biosyntheses; pyruvate transporter: gluconeogenesis and fatty acid oxidation; dicarboxylate transporter: gluconeogenesis), and since the functioning of these metabolic pathways is altered in streptozotocin-- induced diabetes (i.e., hepatic fatty acid and possibly sterol biosyntheses are decreased, whereas gluconeogenesis and fatty acid oxidation are increased), we predicted that the level of mitochondrial anion transporter activity would be regulated in coordination with their associated metabolic pathways. Our initial studies support this hypothesis. We then showed that treatment of diabetic animals with insulin reversed the observed alterations in transporter function, which can thus be ascribed to the insulin deficiency that characterizes this disease. We now propose to elucidate the molecular basis for the observed altered function of the mitochondrial citrate and pyruvate transport proteins in type 1 diabetes. Specifically, utilizing liver mitochondria obtained from control, diabetic, and insulin-treated diabetic rats, experiments will be conducted to: 1) purify these transporters in reconstitutively active form and compare the intrinsic functional and molecular/chemical properties of the purified transport proteins; 2) quantify the amount of each transport protein present within the mitochondrial inner membrane; 3) determine the extent to which phosphorylation reactions regulate transporter function in vivo, in situ, and in vitro; and 4) determine the size, steady-state levels and rates of synthesis of transporter mRNAs. These studies will provide the first information concerning the molecular mechanisms by which mitochondrial transporter function is regulated in both the normal and the diabetic states. This information will then permit the future targeting of specific biochemical processes that regulate transporter function as possible sites for pharmacological intervention in diabetes.