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.
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.
The goal of this procedure is to develop enriched mitochondrial fractions that yield enough mitochondrial metabolites for metabolomics studies using Drosophila melanogaster. In our experience, metabolomics analysis using whole cellular extraction methods are unable to detect subtle mitochondrial metabolite changes in Drosophila. However, mitochondrial fractioning prior to metabolomics analysis increases the sensitivity to identify mitochondrial metabolite shifts.
Mitochondria are cellular organelles responsible for providing 90% of the energy that cells need for normal function1. In recent years it has been recognized that mitochondria play a much more dynamic role in cellular and organismal function than merely producing adenosine triphosphate(ATP), and are now recognized as hubs for the regulation of metabolic homeostasis2,3. Mitochondria are the result of an endosymbiotic process in which distinct microbial lineages merged ~1.5 billion years ago4. As mitochondria evolved into true organelles, genes from the endosymbiont were incorporated in the emerging nuclear genome. In animals today, approximately 1,500 mitochondrial proteins are nuclear-encoded while 37 genes remain in the mtDNA, 13 of which encode mitochondrial proteins that are subunits of the enzyme complexes of oxidative phosphorylation5. Coordination between mitochondria and nuclear compartments is needed to maintain proper mitochondrial function.
Using the methods described here we were able to detect mitochondrial metabolic changes in Drosophila that result from manipulation of the coordination between mitochondrial and nuclear genomes. We used a strain of Drosophila in which mtDNA from its sister species D. simulans was placed on a single D. melanogaster nuclear background6. This ‘disrupted’ mitonuclear genotype was compared to the ‘native’, or co-evolved mitonuclear genotype of D. melanogaster carrying the same nuclear genome with its native D. melanogaster mtDNA. The D. melanogaster and D. simulans mtDNAs differ by ~100 amino acids and >500 synonymous substitutions that affect mitonuclear communication7,8. We generated whole fly extracts and mitochondrial enriched extracts to study metabolite shifts in response to pharmacological stress. Here we show that when using mitochondrial enriched fractions we detect pronounced shifts in mitochondrial metabolites between the native, co-evolved genotype carrying the D. melanogaster mtDNAs and the disrupted genotype carrying D. simulans mtDNA. In contrast, the metabolite changes between these two genotypes are subtle using normal methods that utilize whole fly extract. Therefore, this method provided a path to understand how mtDNAs mediate mitochondrial changes in response to different drugs.
1. Reagents and Solutions
2. Rearing of the Drosophila Strains
3. Isolation of Mitochondrial Fractions
Using the protocol explained above, we performed metabolomic analysis on mitochondrial enriched fractions and whole animal extracts to test the effect of the drug rapamycin on divergent mtDNAs 7. We delivered 200 µM of rapamycin by dissolving the drug in the fly food. Flies were exposed to rapamycin for 10 days. Metabolites from whole fly extracts and from mitochondrial extracts were obtained by using gas chromatography mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) using standard solvent extraction methods.
Figure 1 shows an enrichment of the membrane transport protein porin as indicator of mitochondrial enrichment in the mitochondrial fractions. Porin protein was undetectable in the cytosolic fractions. Conversely, tubulin protein was not detected in the mitochondrial fraction, suggesting little contamination of cytosolic proteins. Figure 2 and Figure 3 show principle components analysis (PCA) of the complete metabolite profiles from the ‘native’ D. melanogaster strain (OreR) and the ‘disrupted’ strain of flies harboring mtDNA from D. simulans and nuclear DNA from D. melanogaster strain OreR (sm21). Both strains were exposed to the drug rapamycin. The data are presented as a bi-plot of principle component axes 1 and 2 to visualize the effect of treatment and mtDNAs7,9. Metabolites obtained using a standard whole fly extract are shown in Figure 2A. Figure 2B shows a similar PCA plot for metabolites in the mitochondrial enriched extracts. For whole fly extracts (Figure 2A), rapamycin treatment sifts the samples to significantly lower values of PC2. Yet, changes in metabolites due to mtDNA genotype are subtle, since OreR and sm21 mtDNAs exposed to rapamycin or control vehicle are closely adjacent or overlap in PCA space. However, when using enriched mitochondrial extracts, mtDNAs show distinct rapamycin effects on mitochondrial metabolite profiles. In particular, rapamycin has a strong effect on the metabolite profiles of the OreR strain, shown by the displacement along the PC1 axis. But, on the ‘disrupted’ sm21 mtDNA genotype, the metabolite profile for the control-food condition is displaced from that of the native OreR strain (red and black polygons, respectively, in Figure 2B). As a result, the rapamycin treatment has noticeably less of an effect on the shift of the metabolite landscapes in the disrupted mtDNA genotype, sm21 (compare red and blue polygons in Figure 2B).
Figure 3 shows PCA plots of metabolites involved in energy homeostasis (a subset of all metabolites, limited to cofactors, vitamins and Krebs cycle intermediates), often located inside of the mitochondria. Here again, metabolite shifts from enriched mitochondrial fractions show a different behavior than those from whole animal extracts.
These results illustrate how metabolic information obtained by mitochondrial enriched extract and whole animal extracts differ but complement each other, with the mitochondrial enriched extract being more sensitive to metabolic shifts in the mitochondria.
Figure 1. Western blot analysis showing effective separation of mitochondria in mitochondrial fractions. Antibodies used were against porin (a mitochondrial outer membrane protein) and tubulin (a cytosolic protein). Protein abundance of mitochondrial and cytosolic fractions were quantified using bicinchoninic acid (BCA) assay. 30 µg of total protein were loaded in each lane. Two independent extractions were performed.
Figure 2. Principle component analysis (PCA) of metabolites obtained using whole animal samples vs. mitochondrial enriched extracts on flies carrying OreR and sm21 mitochondrial haplotypes. (A) PCA of 210 metabolites identified in whole animal samples. (B) PCA of 230 metabolites detected on the mitochondrial extracts. Polygons surrounding points are intended to aid the visualization of the six replicate samples for each treatment. Please click here to view a larger version of this figure.
Figure 3. Principal component analysis of metabolites involved in energy homeostasis on flies carrying OreR and sm21 mitochondrial haplotypes. (A) PCA of 12 energy metabolites from whole fly extracts and 13 energy metabolites from mitochondrial extracts. (B) Polygons surrounding points are intended to aid the visualization of the six replicate samples for each treatment. Please click here to view a larger version of this figure.
The most critical steps in this protocol are: 1) rearing enough flies in abundant space. It is very important to not overpopulate the demography cages with more than 150 flies each; 2) changing the food of the cages frequently to avoid food competition and nutritional stress; and 3) maintaining all samples at 4 °C to ensure integrity during the isolation of the mitochondrial fraction. It is also recommended to chill the isolation buffer, the wash buffer, and the glass-teflon dounce homogenizer before use. To reduce cytosolic contamination of the enriched mitochondrial factions, the mitochondrial pellet from step 3.5 could be washed with wash buffer.
The extraction of metabolites from enriched mitochondrial fractions requires a high number of replicate samples (6 replicates/experimental condition). In total, 24 cages are used for Figures 1 and 2. We recommend performing the extraction of all the samples the same day to decrease experimental variability. Although feasible, this protocol entails careful planning and preparation of materials needed ahead of time. Labeling of the tubes ahead of time, and designing a plan to ensured consistency of waiting times between samples is needed to preserve sample quality and to decrease variation between replicates.
The buffers normally used to extract mitochondria contain sugars10,11; hence, the analysis of particular sugar metabolites may be affected by the extraction method. Although we have not used them in our analysis, KCl extraction buffers may be an alternative to mitochondrial isolation buffers that contain sugars12.
Due to the homeostatic nature of metabolic networks and a complex system of cellular signals that mediated this response, changes in mitochondrial metabolism could be detected by shifts in whole cell metabolite pools. In the case of mitochondrial regulation of such systems, subtle metabolite shifts may be hindered unless enough metabolites are extracted from mitochondrial enriched fractions. In model systems where plenty of mitochondria can be isolated from tissue, high resolution metabolomics has successfully detected changes in mitochondrial phenotypes13. However, in Drosophila melanogaster, harboring enough quality metabolites for a good composition of a sample may present a challenge. Using this protocol we obtained high quantity, quality and variety of metabolites to demonstrate that different extraction protocols reveal distinct metabolite shifts for whole-fly vs. mitochondrial fractions. Although the Western blot of mitochondrial and cytosolic proteins suggests little contamination of proteins in the two fractions, to confirm detailed quantitative measurements of mitochondrial metabolites, additional steps to limit contamination of cytosolic metabolites in the mitochondrial fraction could be applied. Measurement of metabolites before freezing the sample and comparison of these metabolites to those obtained after the freeze/thaw step may provide a more accurate quantification of mitochondrial specific metabolites. However, in this model where we manipulate the mitochondrial genome, the procedures we have used to prepare samples are particularly informative and may point to novel pathways of metabolic reprogramming mediated through mitochondria.
The authors have nothing to disclose.
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.
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 |