Regulation of Novel Mitochondrial Uncoupling Proteins

最近的证据表明解偶联蛋白2（UCP2）的表达增强与II型糖尿病的发展之间存在联系。该提案的一个主要重点是研究UCP2功能和胰腺β细胞生物能量学，以澄清这种联系。我们将使用我们的UCP2酵母表达系统进行参考实验，这是确认UCP2固有的特定特性所必需的。因此，将在体外用重组酵母表达的UCP2以及用从胰腺13细胞分离的线粒体进行实验。研究重组酵母表达的UCP2的底物需求，以确定UCP2介导的质子转运是否对脂肪酸链和饱和度有特殊要求。研究核苷酸结合和抑制UCP2，并确定其他天然和人工UCP2抑制剂。研究从用PPAR激动剂处理或未处理的胰腺β细胞中分离的线粒体的生物能量学。目标1和2将采用质子通量的重构和测量。他们还将包括一个假设的检验，即脂肪酸诱导的质子转运UCP2表现出辅酶Q10的绝对需求。目的3将利用灵敏和精确的耗氧量测量来检测β细胞线粒体中的UCP2活性。我们将尝试区分UCP2表达增加和UCP2在处理和未处理细胞线粒体中的生化调节改变的贡献。

DESCRIPTION (provided by applicant) Recent evidence suggests that there is a link between enhanced expression of uncoupling protein 2 (UCP2) and development of type II diabetes. A major focus of this proposal is to investigate UCP2 function and pancreatic Beta-cell bioenergetics in an effort to clarify this connection. We will employ our yeast expression system for UCP2 to perform the reference experiments, which are required to confirm that a particular property is inherent to UCP2. Thus, experiments will be performed in vitro with recombinant yeast-expressed UCP2 and also with mitochondria isolated from pancreatic 13-cells. The Specific Aims of the proposal are: To study substrate requirements of recombinant yeast-expressed UCP2 to determine whether UCP2-mediated proton transport has a particular requirement for fatty acid chain and saturation. To study nucleotide binding and inhibition of UCP2 and to identify other natural and artificial inhibitors of UCP2. To study the bioenergetics of mitochondria isolated from pancreatic beta-cells that have been treated or untreated with PPAR agonists. Aims 1 and 2 will employ reconstitutions and measurements of proton flux. They will also include a test of the hypothesis that fatty acid-induced proton transport by UCP2 exhibits an absolute requirement for coenzyme Q10. Aim 3 will utilize sensitive and precise measurements of oxygen consumption to detect UCP2 activity in Beta-cell mitochondria. We will attempt to distinguish between the contributions of increased UCP2 expression and altered biochemical regulation of UCP2 in mitochondria from treated and untreated cells.