Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in
Drosophila |
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Authors: | Eugenia Villa-Cuesta David M Rand |
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Institution: | 1.Biology Department, Adelphi University;2.Department of Ecology and Evolutionary Biology, Brown University |
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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. |
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Keywords: | Developmental Biology Issue 103 Mitochondrial isolates metabolomics Drosophila mitochondrial DNAs mitochondrial physiology pharmacology |
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