Summary of Study ST003968

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002482. The data can be accessed directly via it's Project DOI: 10.21228/M8TC38 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003968
Study TitleMeasuring TCA cycle directional fluxes using 13C6-glucose
Study SummaryThis study aimed to investigate the directionality of carbon flux through the TCA cycle in cells experiencing OXPHOS dysfunction, with and without SDHAF2 elevation, using 13C6-glucose tracer metabolomics. 13C-enrichment in TCA cycle intermediates was significantly reduced in cells with either a genetic (UQCRC1KO and UQCRC1/SDHAF2DKO)Antimycin A based OXPHOS dysfunction regardless of the presence of SDHAF2. Further detailed analysis of the isotopomer distribution in key TCA cycle intermediates revealed a decrease in isotopomers associated with operation of a canonical cyclic TCA in which carbon backbones cycle multiple times (e.g. leading to M+2, M+4 and M+6 citrate isotopomers, M+2, M+4 malate isotopomers) with a concomitant increase in the unlabeled (M+0) isotopomer of all TCA intermediates. Although SDHBKO cells followed a similar trend, the impact was relatively modest and consistent with their modest basal respiration rates compared to UQCRC1KO.
Institute
University of Melbourne
DepartmentBiochemistry and Pharmacology
LaboratoryStroud Lab
Last NameRoopasingam
First NameKugapreethan
Address30 Flemington Road, Parkville Melbourne
Emailk.roopasingam@unimelb.edu.au
Phone0434297212
Submit Date2025-05-29
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2025-06-12
Release Version1
Kugapreethan Roopasingam Kugapreethan Roopasingam
https://dx.doi.org/10.21228/M8TC38
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002482
Project DOI:doi: 10.21228/M8TC38
Project Title:Complex II assembly drives metabolic adaptation to OXPHOS dysfunction.
Project Summary:During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse activity of succinate dehydrogenase (Complex II) maintains the redox state of Coenzyme Q by utilizing either fumarate or oxygen as terminal electron acceptors. The tendency for one over another has been suggested to be tissue-specific, but the underlying mechanism and consequence of this is unknown. Using quantitative proteomics to screen a panel of HEK293T knockout cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly factor that enhances the flavination of catalytic subunit SDHA, as critical for metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 during Complex III inhibition resulted in a reduction in Complex II F-site derived reactive oxygen species (ROS), a severe growth impairment, and a net reductive TCA cycle driven by an inability of mitochondria to support additional Complex II assembly. This in turn leads to use of fumarate as terminal electron acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being present. Thus, our study reveals how Complex II assembly controls a balance between protection of the Q-pool and ROS-meditated signaling during oxidative stress in cells reliant on mitochondrial OXPHOS.
Institute:University of Melbourne
Last Name:Roopasingam
First Name:Kugapreethan
Address:30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
Email:k.roopasingam@unimelb.edu.au
Phone:0434297212

Subject:

Subject ID:SU004105
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Treatment
SA45306901_WT_UL_101_WT_UL_1 Unlabeled control_1
SA45307002_WT_UL_202_WT_UL_2 Unlabeled control_2
SA45307103_WT_UL_303_WT_UL_3 Unlabeled control_3
SA45307204_WT_L_104_WT_L_1 Labeled Control_1
SA45307305_WT_L_205_WT_L_2 Labeled Control_2
SA45307406_WT_L_306_WT_L_3 Labeled Control_3
SA45307507_AF2KO_107_AF2KO_1 SDHAF2KO_001
SA45307608_AF2KO_208_AF2KO_2 SDHAF2KO_002
SA45307709_AF2KO_309_AF2KO_3 SDHAF2KO_003
SA45307810_BKO_110_BKO_1 SDHBKO_001
SA45307911_BKO_211_BKO_2 SDHBKO_002
SA45308012_BKO_312_BKO_3 SDHBKO_003
SA45308113_UQKO_113_UQKO_1 UQCRC1KO_001
SA45308214_UQKO_214_UQKO_2 UQCRC1KO_002
SA45308315_UQKO_315_UQKO_3 UQCRC1KO_003
SA45308416_DKO_116_DKO_1 UQCRC1/SDHAF2DKO_001
SA45308517_DKO_217_DKO_2 UQCRC1/SDHAF2DKO_002
SA45308618_DKO_318_DKO_3 UQCRC1/SDHAF2DKO_003
SA45308728_WT_DMSO_128_WT_DMSO_1 Labeled Control with DMSO_1
SA45308829_WT_DMSO_229_WT_DMSO_2 Labeled Control with DMSO_2
SA45308930_WT_DMSO_330_WT_DMSO_3 Labeled Control with DMSO_3
SA45309031_WT_AA_131_WT_AA_1 Labeled Control with treatment_1
SA45309132_WT_AA_232_WT_AA_2 Labeled Control with treatment_2
SA45309233_WT_AA_333_WT_AA_3 Labeled Control with treatment_3
SA45309334_AF2KO_AA_134_AF2KO_AA_1 Labeled SDHAF2KO with treatment_1
SA45309435_AF2KO_AA_235_AF2KO_AA_2 Labeled SDHAF2KO with treatment_2
SA45309536_AF2KO_AA_336_AF2KO_AA_3 Labeled SDHAF2KO with treatment_3
Showing results 1 to 27 of 27

Collection:

Collection ID:CO004098
Collection Summary:Cells were seeded at 50% confluency in 6-well dishes containing DMEM media (Thermo Scientific) 24 hours prior to the experiment.
Sample Type:HEK cells

Treatment:

Treatment ID:TR004114
Treatment Summary:For stable isotopologue tracing of 13C6-glucose, 12C containing medium was replaced with glucose-free DMEM (Thermo Scientific) supplemented with 25 mM of 13C6-glucose (Merck), 1 mM Sodium pyruvate (Thermo Scientific), 10% FBS, 50 µg/mL uridine, and a mixture of 100 µg/mL streptomycin and 100 units/mL penicillin. After 8 hours of incubation in the tracer media, cells underwent polar metabolite extraction, as described in the subsequent sections. In relevant experiments, Antimycin A was introduced 8 hours prior to the addition of tracer and maintained until the completion of the experiment, respectively. Cells were then washed with PBS and snap-frozen by direct addition of liquid nitrogen.

Sample Preparation:

Sampleprep ID:SP004111
Sampleprep Summary:Polar metabolites were extracted by adding 600 µL of HPLC grade methanol:chloroform mixture (9:1; [v/v]), containing internal standards 1.66μM 13C5,15N-valine and 1.66μM 13C-sorbitol mixture for LC-MS analysis. After 10 minutes incubation on ice, cells were harvested, and lysates centrifuged at 16,100 x g at 4°C for 5 minutes. For LC-MS a stream of N2 used to dry samples and were subsequently subjected to LC-MS analysis

Chromatography:

Chromatography ID:CH004956
Instrument Name:Orbitrap ID-X Tribrid mass spectrometer (Thermo Scientific)
Column Name:Merck SeQuant ZIC-HILIC (150 x 4.6 mm, 5 μm)
Column Temperature:25℃
Flow Gradient:time (t) = 0.0 min, 80% B; t = 0.5 min, 80% B; t = 15.5 min, 50% B; t = 17.5 min, 30% B; t = 18.5 min, 5%; t = 21.0 min, 5% B; t = 23–33 min, 80%
Flow Rate:300 μL/min
Solvent A:100% Water; 20 mM Ammonium carbonate
Solvent B:100% Acetonitrile
Chromatography Type:HILIC

Analysis:

Analysis ID:AN006529
Analysis Type:MS
Chromatography ID:CH004956
Num Factors:27
Num Metabolites:481
Units:Relative abundance
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