Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila.
Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila.
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DOI：
10.3791/53149
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发表时间：
2015-09
期刊：
影响因子：
--
通讯作者：
Eugenia Villa-Cuesta;D. Rand
中科院分区：
文献类型：
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作者：
Eugenia Villa-Cuesta;D. Rand
由于线粒体在氨基酸代谢、碳水化合物代谢和脂肪酸氧化中发挥作用，线粒体功能缺陷往往危及患有这些复杂疾病的人的生命。检测线粒体代谢变化对于了解线粒体疾病和线粒体对药物的反应至关重要。虽然线粒体代谢是代谢调节的核心，但通过整个生物体或整个组织提取获得的其他细胞质代谢物的过度代表可能会阻碍检测线粒体代谢的细微变化。在这里，我们描述了一种检测果蝇线粒体代谢变化的分离方法，这种变化在全蝇和线粒体富集制剂之间是不同的。为了说明这种方法的敏感性，我们使用了一组具有遗传多样性线粒体dna （mtDNA）的果蝇，并将它们暴露于药物雷帕霉素中。使用这种方法，我们发现雷帕霉素以线粒体基因型依赖的方式改变线粒体代谢。然而，在代谢组学研究中，当从线粒体富集部分提取代谢物时，这些变化更加明显。相比之下，整个组织提取物只检测到由药物雷帕霉素介导的代谢变化，而不依赖于mtdna。
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.