Summary of Study ST003961

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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003961
Study TitleMeasuring carbon flux into TCA cycle using 13C5-glutamine tracer metabolomics
Study SummaryThis study investigates carbon flux into the TCA cycle in HEK293T cell lines with and without SDHAF2 elevation during OXPHOS dysfunction using targeted GC-MS analysis. The investigation of percentage labeling and isotopologue distribution pattern of glutamate was assessed by labeling cells with 2 mM 13C5-glutamine for 8 hours followed by targeted GC-MS. The results indicated increased glutaminolysis and flux into the TCA cycle in cells with defective Complex III regardless of the presence or the absence of SDHAF2.
Institute
University of Melbourne
Last NameRoopasingam
First NameKugapreethan
Address30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia.
Emailk.roopasingam@unimelb.edu.au
Phone0434297212
Submit Date2025-05-29
Raw Data AvailableYes
Raw Data File Type(s)gqd
Analysis Type DetailGC-MS
Release Date2025-06-11
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:SU004098
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
SA4527914_HEK_A-A_SCAN+_001_UNK-0022_16042025_22HEK_A-A_001 HEK_A-A
SA4527924_HEK_A-A_SCAN+_002_UNK-0018_16042025_18HEK_A-A_002 HEK_A-A
SA4527934_HEK_A-A_SCAN+_003_UNK-0020_16042025_20HEK_A-A_003 HEK_A-A
SA4527795_HEKSDHAF2_SCAN+_001_UNK-0029_16042025_29HEKSDHAF2KO_001 HEKSDHAF2KO
SA4527805_HEKSDHAF2_SCAN+_002_UNK-0024_16042025_24HEKSDHAF2KO_002 HEKSDHAF2KO
SA4527815_HEKSDHAF2_SCAN+_003_UNK-0027_16042025_27HEKSDHAF2KO_003 HEKSDHAF2KO
SA4527826_HEKSDHAF2_A-A_SCAN+_001_UNK-0035_16042025_35HEKSDHAF2KO_A-A_001 HEKSDHAF2KO_A-A
SA4527836_HEKSDHAF2_A-A_SCAN+_002_UNK-0031_16042025_31HEKSDHAF2KO_A-A_002 HEKSDHAF2KO_A-A
SA4527846_HEKSDHAF2_A-A_SCAN+_003_UNK-0033_16042025_33HEKSDHAF2KO_A-A_003 HEKSDHAF2KO_A-A
SA4527858_SDHAF2_CIIIKO_A-A_SCAN+_001_UNK-0048_16042025_48HEKSDHAF2KO_CIIIKO_A-A_001 HEKSDHAF2KO_CIIIKO_A-A
SA4527868_SDHAF2_CIIIKO_A-A_SCAN+_002_UNK-0043_16042025_43HEKSDHAF2KO_CIIIKO_A-A_002 HEKSDHAF2KO_CIIIKO_A-A
SA4527878_SDHAF2_CIIIKO_A-A_SCAN+_003_UNK-0045_16042025_45HEKSDHAF2KO_CIIIKO_A-A_003 HEKSDHAF2KO_CIIIKO_A-A
SA4527887_SDHAF2_SDHAF2FLAG_SCAN+_001_UNK-0041_16042025_41HEKSDHAF2_SDHAF2FLAG_001 HEKSDHAF2_SDHAF2FLAG
SA4527897_SDHAF2_SDHAF2FLAG_SCAN+_002_UNK-0037_16042025_37HEKSDHAF2_SDHAF2FLAG_002 HEKSDHAF2_SDHAF2FLAG
SA4527907_SDHAF2_SDHAF2FLAG_SCAN+_003_UNK-0039_16042025_39HEKSDHAF2_SDHAF2FLAG_003 HEKSDHAF2_SDHAF2FLAG
SA4527943_HEKWT_SCAN+_001_UNK-0016_16042025_16HEK_WT_001 HEK_WT
SA4527953_HEKWT_SCAN+_002_UNK-0012_16042025_12HEK_WT_002 HEK_WT
SA4527963_HEKWT_SCAN+_003_UNK-0014_16042025_14HEK_WT_003 HEK_WT
Showing results 1 to 18 of 18

Collection:

Collection ID:CO004091
Collection Summary:HEK293Tvcells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10 % (v/v) fetal bovine serum (FBS, CellSera), 50 µg/mL uridine (Sigma), and a mixture of 100 µg/mL streptomycin and 100 units/mL penicillin (Thermo Scientific).
Sample Type:Cultured cells

Treatment:

Treatment ID:TR004107
Treatment Summary:The relevant cell lines were treated with doxycycline and Antimycin A for 24 and 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:SP004104
Sampleprep Summary:Polar metabolites were extracted from snap-frozen cells using 600 µL of HPLC grade methanol:chloroform mixture (9:1; [v/v]), along with internal standards (1.66μM 13C5,15N-valine, 1.66μM 13C6-sorbitol), as described above. The clarified supernatants and pooled biological quality controls (PBQC’s) were dried using a CentriVap concentrator (Labconco).

Chromatography:

Chromatography ID:CH004944
Instrument Name:Shimadzu GC-2010
Column Name:Agilent DB-5 GC column (30 m x 0.25 mm, 1 µm)
Column Temperature:100℃
Flow Gradient:N/A
Flow Rate:1 mL/min
Solvent A:N/A
Solvent B:N/A
Chromatography Type:GC

Analysis:

Analysis ID:AN006509
Analysis Type:MS
Chromatography ID:CH004944
Num Factors:18
Num Metabolites:5
Units:Relative abundance
  logo