Summary of Study ST003980
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 PR002492. The data can be accessed directly via it's Project DOI: 10.21228/M8HV6F This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003980 |
| Study Title | Glutamate-mediated TCA anaplerosis is increased in septic mitochondria |
| Study Summary | Mitochondria rely on three main respiratory substrates, i.e. pyruvate, free fatty acids via beta-oxidation or glutamate. Since pyruvate metabolism and free fatty acid oxidation are impaired in sepsis, we wanted to investigate the presence of enhanced glutaminolysis in sepsis by performing a 13C glutamate tracer experiment on isolated liver mitochondria from sham (control) and cecal ligation and puncture (CLP, sepsis) mice. This showed enhanced 13C incorporation in all tricarboxylic acid (TCA) metabolites in CLP mitochondria compared to sham mitochondria, proving the occurrence of enhanced glutamate-mediated TCA anaplerosis in sepsis. |
| Institute | Ghent University |
| Last Name | Libert |
| First Name | Claude |
| Address | Technologiepark-Zwijnaarde 71, 9052 Ghent, Belgium |
| claude.libert@irc.vib-ugent.be | |
| Phone | 09/331.37.00 |
| Submit Date | 2025-06-04 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML, raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-08-14 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002492 |
| Project DOI: | doi: 10.21228/M8HV6F |
| Project Title: | Unraveling mitochondrial pyruvate dysfunction to mitigate hyperlactatemia and lethality in sepsis |
| Project Summary: | Sepsis, killing 11 million people yearly, is associated with increased lactate production - a metabolite mechanistically linked to mortality - complicating glucose administration in sepsis. To understand the mechanism behind hyperlactatemia, we applied the cecal ligation and puncture (CLP) model and studied all pyruvate processing routes in liver mitochondria during acute sepsis. Our data suggest that mitochondrial pyruvate-driven respiration is nearly nonexistent in sepsis, not due to insufficient pyruvate uptake or carboxylation but due to a dysfunctional pyruvate dehydrogenase complex (PDC). Septic mitochondria compensate by glutamate-mediated TCA anaplerosis, simultaneously converting some pyruvate into alanine via enhanced mitochondrial glutamic pyruvate transaminase (GPT2) activity. Notably, PDC dysfunction is not caused by PDC inactivation per se but by a shortage of its cofactor, thiamine pyrophosphate (TPP). TPP supplementation restores pyruvate oxidation both ex vivo and in vivo and protects mice from sepsis. TPP also allows safe glucose administration in mice, leading to a novel, robust TPP-plus-glucose therapy. |
| Institute: | Ghent University |
| Department: | Inflammation research center - VIB |
| Laboratory: | Mouse Genetics in Inflammation |
| Last Name: | Libert |
| First Name: | Claude |
| Address: | Technologiepark-Zwijnaarde 71, 9052 Ghent, Belgium |
| Email: | claude.libert@irc.vib-ugent.be |
| Phone: | 09/331.37.00 |
Subject:
| Subject ID: | SU004117 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment condition | Tracer condition |
|---|---|---|---|---|
| SA454322 | MCF001895_LN01 | Liver mitochondria | CLP | 13C5 glutamate |
| SA454323 | MCF001895_LN04 | Liver mitochondria | CLP | 13C5 glutamate |
| SA454324 | MCF001895_LN05 | Liver mitochondria | CLP | 13C5 glutamate |
| SA454325 | MCF001895_LN07 | Liver mitochondria | CLP | 13C5 glutamate |
| SA454326 | MCF001895_LN09 | Liver mitochondria | CLP | 13C5 glutamate |
| SA454327 | MCF001895_LN11 | Liver mitochondria | CLP | Buffer |
| SA454328 | MCF001895_LN02 | Liver mitochondria | Sham | 13C5 glutamate |
| SA454329 | MCF001895_LN03 | Liver mitochondria | Sham | 13C5 glutamate |
| SA454330 | MCF001895_LN06 | Liver mitochondria | Sham | 13C5 glutamate |
| SA454331 | MCF001895_LN08 | Liver mitochondria | Sham | 13C5 glutamate |
| SA454332 | MCF001895_LN10 | Liver mitochondria | Sham | Buffer |
| Showing results 1 to 11 of 11 |
Collection:
| Collection ID: | CO004110 |
| Collection Summary: | Mice were subjected to the cecal ligation and puncture (CLP) to induce polymicrobial sepsis and the sham (control) procedure. After 24h, liver mitochondria were isolated as described by Frezza et al. (Nature Protocols: 2007;2(2):287-95. doi: 10.1038/nprot.2006.478). Isolated liver mitochondria were spinned down and in mitochondrial assay solution (MAS, 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5mM MgCl2, 2 mM HEPES, 1 mM EGTA and 0.2% fatty acid-free BSA) with 13C5 glutamic acid (10 mM; Sigma 604860) or only MAS ) and were incubated for 30 minutes at 37°C in oxygenated conditions. |
| Sample Type: | Mitochondria |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR004126 |
| Treatment Summary: | Mice were subjected to the cecal ligation and puncture (CLP), as described by Rittirsch et al. (Nature Protocols: 2009;4(1):31-6. doi: 10.1038/nprot.2008.214), to induce polymicrobial sepsis and the sham (control) procedure. After 24h, liver mitochondria were isolated. |
Sample Preparation:
| Sampleprep ID: | SP004123 |
| Sampleprep Summary: | Isolated liver mitochondria were spinned down and in mitochondrial assay solution (MAS, 70 mM sucrose, 220 mM mannitol, 10 mM KH2PO4, 5mM MgCl2, 2 mM HEPES, 1 mM EGTA and 0.2% fatty acid-free BSA) with 13C5 glutamic acid (10 mM; Sigma 604860) or only MAS ) and were incubated for 30 minutes at 37°C in oxygenated conditions. Isolated liver mitochondria were analyzed by mass spectrometry (LC-MS) to determine total abundance, fractional contribution and 13C-mass isotopologue enrichment of a labeled nutrient (13C5 glutamic acid) to the metabolites of interest. A total injection volume of 10 ul/sample was used. |
| Sampleprep Protocol Filename: | Protocol_methods_13C5_glutamate.pdf |
Combined analysis:
| Analysis ID | AN006556 |
|---|---|
| Chromatography ID | CH004975 |
| MS ID | MS006255 |
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Agilent InfinityLab Poroshell 120 HILIC-Z (150 x 2.1mm, 2.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | NEGATIVE |
| Units | peak intensity |
Chromatography:
| Chromatography ID: | CH004975 |
| Methods Filename: | Protocol_methods_13C5_glutamate.pdf |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Agilent InfinityLab Poroshell 120 HILIC-Z (150 x 2.1mm, 2.7um) |
| Column Temperature: | 25°C |
| Flow Gradient: | A linear gradient was carried out starting with 90% solvent A and 10% solvent B. From 2 to 12 min the gradient changed to 60% B. The gradient was kept on 60% B for 3 min and followed by a decrease to 10% B. The chromatography was stopped at 25 min |
| Flow Rate: | 0.25 mL/min |
| Solvent A: | 100% acetonitrile |
| Solvent B: | 100% water; 10 mM Sodium acetate (pH 9.3) |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS006255 |
| Analysis ID: | AN006556 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | - Full scan acquisition in the m/z range 70–1050, spray voltage 2.8 kV, capillary temperature 320°C, sheath gas 45, auxiliary gas 10, AGC target 3.0E+006, resolution 70,000. - Data collected using Xcalibur software (Thermo Scientific). Metabolite peak integration, isotopologue analysis, and quantification performed following standard workflows for 13C-labeled metabolite profiling. - Xcalibur (Thermo Scientific) for raw data acquisition and processing |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Protocol_methods_13C5_glutamate.pdf |
