在代谢分析准备线粒体富集级的<em>果蝇</em
Summary
Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.
Abstract
Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions.
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.
Introduction
此过程的目标是开发能产生足够的线粒体代谢物用于使用果蝇代谢组学研究富集线粒体级分。根据我们的经验，采用全细胞提取方法代谢组学分析是无法检测果蝇微妙的线粒体代谢物的变化。然而，线粒体分馏之前代谢分析增加了灵敏度，以确定线粒体代谢物变化。
线粒体是负责提供90％的细胞所需的正常功能1的能量的细胞器。在最近几年，已经认识到，线粒体在细胞和生物体功能的更加动态的作用不仅仅是产生三磷酸腺苷（ATP）和现在被确认为枢纽的代谢平衡2,3的调节。线粒体是一种共生的过程中，存款保险计划的结果tinct微生物系1.5十亿年前4合并〜。由于线粒体演变成真正的细胞器，基因的共生细菌在新兴的核基因组被合并。在动物今天，约1500线粒体蛋白是核编码的，而37个基因保留在线粒体DNA，其中13个编码线粒体蛋白质，是氧化磷酸化5的酶复合物的亚基。线粒体和核车厢之间的协调是必要的，以保持适当的线粒体功能。
使用此处描述的方法，我们能够探测在果蝇线粒体代谢变化所造成的操纵线粒体和核基因组之间的协调。我们用果蝇的菌株，其中线粒体DNA，从它的姊妹品种D. simulans置于单个D.果蝇核背景6。这种“破坏”mitonuclear基因型相比D的 “天然”或共同进化mitonuclear基因型果蝇携带着其原生四相同的核基因组果蝇线粒体DNA。该D.果蝇和D. simulans线粒体DNA相差〜100个氨基酸和影响mitonuclear通信7,8> 500同义替换。我们生成的整个飞行提取物和线粒体富集提取物，研究代谢物的变化响应的药理压力。在这里，我们表明，使用线粒体富集的级分，当我们检测在本机，共同进化基因型携带D之间线粒体代谢物显着的变化果蝇线粒体DNA和破坏携带型D. simulans线粒体DNA。与此相反，这两种基因型之间的代谢变化是细微的使用，利用全动态提取正常的方法。因此，这种方法提供了一种路径理解线粒体DNA如何介导线粒体的变化响应于不同的药物。
Protocol
Representative Results
Discussion
在这个协议中最关键的步骤是：1）足够的饲养苍蝇充裕的空间。这是非常重要的不overpopulate的人口笼150多个苍蝇每一个; 2）改变笼经常避免食物竞争和营养应激的食物;和3）保持所有样品在4℃的线粒体级分的分离过程中，以确保完整性。此外，还建议以冷却该隔离缓冲器，洗涤缓冲液，并在使用前的玻璃 – 聚四氟乙烯Dounce匀浆。为了减少富集线粒体派别胞浆污染，从步骤3.5线粒体颗粒可以用洗涤…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
This work was supported by Adelphi University faculty development grant and grant R15GM113156 from NIGMS awarded to EVC, grant R01GM067862 from NIGMS and grant R01AG027849 from NIA awarded to DMR.
Materials
| 0.2% tegosept -methyl 4-hydroxybenzoate | VWR | AAA14289 | |
| Ethanol | Sigma-Aldrich | 792799 | |
| Mannitol | Sigma-Aldrich | M4125 | |
| Sucrose | Sigma-Aldrich | S9378 | |
| 3-(N-morpholino) propanesulfonic acid (MOPS) | Sigma-Aldrich | M1254 | |
| Ethylenediaminetetraacetic acid (EDTA) | Sigma-Aldrich | 38057 | |
| Bovine serum albumin (BSA) | Sigma-Aldrich | 5470 | |
| KCL | Sigma-Aldrich | P9333 | |
| Tris HCL | Sigma-Aldrich | RES3098T-B7 | |
| KH2PO4 | Sigma-Aldrich | 1551139 | |
| CO2 pads to anesthetize flies | Tritech Research | MINJ-DROS-FP | |
| 1 liter cage | Web Restaurant Store | 999RD32 | |
| 1 liter cage lid | Web Restaurant Store | 999LRD | |
| a glass-teflon dounce homogenizer | Fisher Scientific | NC9661231 | |
| Sodium hydroxide | Sigma-Aldrich | S8045 | |
| rapamycin | LC Laboratories | R-5000 | |
| anti-porin | MitoSciences | MSA03 | |
| anti-alpha tubulin | Developmental Studies Hybridoma Bank | 12G10 | |
| Pierce™ BCA Protein Assay Kit | Thermo Scientific | 23225 | |
| CO2 pad | Tritech Research, Inc | MINJ-DROS-FP | |
| filter flask | enasco | SB08184M | |
| rubber stopper | enasco | S08512M |
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