1. Submitted
  2. In curation
  3. In review
  4. Public
MTBLS724: Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Abstract

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to influence the frequencies of mtDNA types in nature and human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four Drosophila mitotypes and assayed their frequency in population cages. The nuclear genome was standardised. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result was repeated when the laboratory diet was replaced by natural fruits having high and low P:C ratios and when the nuclear genome was permuted. Quaternary structural modelling, in vitro assays of electron transport chain protein complexes, and protein gels suggested a V161L mutation in the ND4 subunit of Complex I of Dahomey mtDNA was functionally deleterious and resulted in an increase in larval development time on the 1:2 P:C diet. Conversely, the 1:16 P:C diet resulted in an elegant remodelling of energy metabolism and relative reduction in development time of larvae harbouring Dahomey mtDNA. These data question the use of mtDNA as an assumed neutral maker. We posit that humans with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of first-world diseases including cardiovascular disease, diabetes, obesity and perhaps Parkinson’s Disease.

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 Authors: Russell Pickford

  Release date: 07-Nov-2018

 Status: Public

Organism(s)

Drosophila melanogaster

  Study Design

CHMO:gas chromatography-mass spectrometry

untargeted metabolites

NCIT:Diet

EFO:third instar larva stage

  Experimental Factors

genotype

diet

Protocol Description
Sample collection Drosophila melanogaster lines harboring distinct mitotypes were originally collected in nature (Alstonville, Dahomey) and maintained in cages.

4 isocaloric Protein:Carbohydrate (P:C) diets were prepared by varying yeast, treacle, semolina and sucrose content in a standard base containing 1% agar, 0.1% nipagen, 0.1% propionic acid and 0.001% phosphoric acid. The P:C ratios were 1:2, 1:4, 1:8 and 1:16. The final concentration of ingredients was set at 180 g/l. The 1:2 P:C diet contained 79.4 g of yeast, 73.2 g of semolina, 27.2 g treacle and 0.2 g of sucrose/l (so dietary sucrose comprised ~0.1% of the 1:2 P:C diet). The 1:4 P:C diet contained 46.5 g yeast, 42.4 g semolina, 89.16 g treacle and 1.4 g sucrose/l. The 1:8 P:C diet contained 25.2 g yeast, 23.2 g semolina, 127.5 g treacle and 4.1 g sucrose/l. The 1:16 P:C diet contained 12.9 g of yeast, 11.9 g of semolina, 145.9 g of treacle and 9.3 g sucrose/l (so dietary sucrose comprised ~5% of the 1:16 P:C diet). The autolysed yeast (MP Biomedicals, catalogue no. 103304), contains 45% protein, 24% carbohydrate, 21% indigestible fibre, 8% water and 2% acids, minerals and vitamins. The treacle (CSR, Vic, Australia) contained 0.4% protein, 71% carbohydrate and 0.17% of sodium. The semolina (Quality Food Services, Qld, Australia) contains 11.8% protein, 68.8% carbohydrate, 1.6% fat, 3.2% dietary fibre and 0.0037% sodium. A limitation of these diets is that we could not modify the sugars while maintaining the P:C ratio and caloric content for the high P:C diets.

The fruit diet was constructed by adding 150 g of passion fruit or banana in a standard base containing 1% agar, 0.1% nipagen, 0.1% propionic acid and 0.001% phosphoric acid. The final volume was set at 300 ml.

Metabolomics studies were performed on 4 samples groups - each line fed either 1:2 P:C or 1:16 P:C. Approximately 20 mg of female third instar wandering larvae were collected for each analysis.
Extraction Methanol was added to make up each larval sample to 20 mg/ml. Metabolites were extracted using an ultrasonic probe (30 s), 1 hr incubation at 4 °C and then centrifugation to remove particulates. 100 µl aliquots of the supernatant were derivatised as described previously[1].

Ref:
[1] Roessner U, Wagner C, Kopka J, Trethewey RN, Willmitzer L. Technical advance: simultaneous analysis of metabolites in potato tuber by gas chromatography-mass spectrometry. Plant J. 2000;23(1):131-42. Epub 2000/08/06. doi:10.1046/j.1365-313x.2000.00774.x. PMID:10929108
Chromatography GC-MS analysis was performed using an Agilent tandem GC/MSD system (Agilent Technologies, CA, USA) which consisted of a 7683 Series autosampler-injector assembly and a 6890 Agilent GC oven interfaced with a 5973 MSD detector. Chromatographic separation was performed on a 30m SH-RXi-5Sil MS column (Shimadzu, NSW, Australia) with 0.25 mm internal diameter and 0.25 µm film thickness. The oven temperature was programmed at 70 °C for 2 min, then ramped at 15 °C/min to 320 °C, and held 8 min.
Mass spectrometry Mass spectrometric data was collected with an Agilent 5973 GC/MSD system (Agilent Technologies, CA, USA) controlled by Chemstation software. Electron ionisation mass spectra were recorded at 1.4 scans/s over the range m/z 50–700. The MSD auxiliary temperature, source temperature, and quadrupole temperature were set to 280 °C, 230 °C, and 150 °C, respectively.
Data transformation No internal standard was employed. Normalisation was performed by maintaining a constant sample mass per volume. Peak areas were log-transformed and statistical analysis used the limma package. Benjamini-Hochberg’s correction was used to control the FDR.
Metabolite identification Analytes were identified using the NIST 2011-Wiley Mass Spectra Library. GC peaks containing mass spectra with a match quality (spectral purity) of more than 70% were considered to be tentatively identified.
Source Name Organism Organism part Protocol REF Sample Name genotype diet
blank Drosophila melanogaster larva Sample collection 2015091702 blank blank
Alstonville 1:2 PC Drosophila melanogaster larva Sample collection 2015091703 Alstonville 1:2 P:C
Alstonville 1:2 PC Drosophila melanogaster larva Sample collection 2015091704 Alstonville 1:2 P:C
Alstonville 1:2 PC Drosophila melanogaster larva Sample collection 2015091705 Alstonville 1:2 P:C
Alstonville 1:2 PC Drosophila melanogaster larva Sample collection 2015091706 Alstonville 1:2 P:C
Alstonville 1:2 PC Drosophila melanogaster larva Sample collection 2015091707 Alstonville 1:2 P:C
Alstonville 1:16 PC Drosophila melanogaster larva Sample collection 2015091708 Alstonville 1:16 P:C
Alstonville 1:16 PC Drosophila melanogaster larva Sample collection 2015091709 Alstonville 1:16 P:C
Alstonville 1:16 PC Drosophila melanogaster larva Sample collection 2015091710 Alstonville 1:16 P:C
Alstonville 1:16 PC Drosophila melanogaster larva Sample collection 2015091711 Alstonville 1:16 P:C
Alstonville 1:16 PC Drosophila melanogaster larva Sample collection 2015091712 Alstonville 1:16 P:C
Dahomey 1:2 PC Drosophila melanogaster larva Sample collection 2015091713 Dahomey 1:2 P:C
Dahomey 1:2 PC Drosophila melanogaster larva Sample collection 2015091714 Dahomey 1:2 P:C
Dahomey 1:2 PC Drosophila melanogaster larva Sample collection 2015091715 Dahomey 1:2 P:C
Dahomey 1:2 PC Drosophila melanogaster larva Sample collection 2015091716 Dahomey 1:2 P:C
Dahomey 1:2 PC Drosophila melanogaster larva Sample collection 2015091717 Dahomey 1:2 P:C
Dahomey 1:16 PC Drosophila melanogaster larva Sample collection 2015091718 Dahomey 1:16 P:C
Dahomey 1:16 PC Drosophila melanogaster larva Sample collection 2015091719 Dahomey 1:16 P:C
Dahomey 1:16 PC Drosophila melanogaster larva Sample collection 2015091720 Dahomey 1:16 P:C
Dahomey 1:16 PC Drosophila melanogaster larva Sample collection 2015091721 Dahomey 1:16 P:C
Dahomey 1:16 PC Drosophila melanogaster larva Sample collection 2015091722 Dahomey 1:16 P:C

Assay 

Assay file name: a_ballard_metabolomics_metabolite_profiling_mass_spectrometry.txt
Measurement: metabolite profiling
Technology: mass spectrometry
Platform: Agilent 5973 GC-MS

Instrumentation

Sample Name Protocol REF Post Extraction Derivatization Extract Name Protocol REF Chromatography Instrument Autosampler model Column model Column type Labeled Extract Name Label Protocol REF Scan polarity Scan m/z range Instrument Ion source Mass analyzer MS Assay Name Raw Spectral Data File Protocol REF Normalization Name Derived Spectral Data File Protocol REF Data Transformation Name Metabolite Assignment File
2015091702 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091702 2015091702.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091703 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091703 2015091703.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091704 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091704 2015091704.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091705 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091705 2015091705.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091706 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091706 2015091706.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091707 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091707 2015091707.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091708 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091708 2015091708.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091709 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091709 2015091709.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091710 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091710 2015091710.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091711 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091711 2015091711.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091712 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091712 2015091712.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091713 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091713 2015091713.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091714 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091714 2015091714.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091715 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091715 2015091715.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091716 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091716 2015091716.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091717 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091717 2015091717.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091718 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091718 2015091718.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091719 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091719 2015091719.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091720 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091720 2015091720.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091721 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091721 2015091721.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091722 Extraction centrifuged and supernatant taken sylilation N/A Chromatography Agilent 6890 GC Agilent 7683 Series Automatic Liquid Sampler SH-Rxi-5Sil MS (0.25 µm, 0.25 mm x 30 m; Shimadzu) low polarity N/A Mass spectrometry positive 50-700 Agilent 5973 MSD electron ionization quadrupole 2015091722 2015091722.D.zip Data transformation Metabolite identification m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
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a_ballard_metabolomics_metabolite_profiling_mass_spectrometry.txt
i_Investigation.txt
s_Ballard Metabolomics.txt
2015091704.D.zip
2015091713.D.zip
2015091718.D.zip
2015091702.D.zip
2015091712.D.zip
2015091717.D.zip
2015091716.D.zip
2015091722.D.zip
2015091708.D.zip
2015091705.D.zip
2015091714.D.zip
2015091720.D.zip
2015091709.D.zip
2015091719.D.zip
m_ballard_metabolomics_metabolite_profiling_mass_spectrometry_v2_maf.tsv
2015091721.D.zip
2015091703.D.zip
2015091711.D.zip
2015091715.D.zip
2015091710.D.zip
2015091707.D.zip
2015091706.D.zip
metexplore_mapping.json

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PASSES Study Title MANDATORY STUDY OK
PASSES Study Description MANDATORY STUDY OK
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PASSES Sample(s) MANDATORY SAMPLES OK
PASSES Sample Name consistency check MANDATORY ASSAYS OK
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PASSES Minimal Experimental protocol MANDATORY PROTOCOLS OK
PASSES Comprehensive Experimental protocol OPTIONAL PROTOCOLS OK
PASSES Extraction protocol description MANDATORY PROTOCOLS OK
PASSES Data transformation protocol description MANDATORY PROTOCOLS OK
PASSES Metabolite Identification protocol description MANDATORY PROTOCOLS OK
PASSES Mass spectrometry protocol description MANDATORY PROTOCOLS OK
PASSES Chromatography protocol description MANDATORY PROTOCOLS OK
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PASSES Metabolite Identification File (MAF) content MANDATORY FILES OK
PASSES ISA-Tab investigation file check MANDATORY ISATAB OK

Pathways - Assay 



MetExplore Pathways Mapping

Name DB Identifier Mapped Metabolite(s)