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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/m_MTBLS15385_LC-MS_positive_hilic_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/RESULT.xlsxftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/a_MTBLS15385_LC-MS_positive_hilic.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/a_MTBLS15385_LC-MS_alternating_hilic.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/s_MTBLS15385.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/ctrl_4.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/ctrl_1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/ctrl_4.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/CKO_9.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/CKO_8.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/CKO_14.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/CKO_7.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/CKO_10.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/CKO_10.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/CKO_9.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/ctrl_3.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/CKO_7.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/CKO_8.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/ctrl_2.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/ctrl_2.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/COA/ctrl_13.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/ctrl_3.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/ctrl_1.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/ctrl_13.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385/FILES/DERIVED_FILES/glutamine/CKO_14.mzMLprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS15385 Each metabolite was identified by matching its retention time and MS/MS fragmentation pattern against an in-house spectral library, as well as public databases HMDB. For metabolites with available authentic standards, identification was further confirmed by co-elution with the corresponding reference standard and comparison of at least two diagnostic fragment ions at normalized collision energies of 20-40 eV. Only metabolites matching both retention time (within ±0.2 min) and characteristic fragment ions were considered as confidently identified.MetaboLightsPublicLiquid Chromatography MS - alternating - hilicLiquid Chromatography MS - positive - hilicFor glutamine-derived metabolites, chromatographic separation was achieved on an UPLC HILIC Amide XBridge column (4.6 mm × 100 mm, Waters) maintained at 40 °C. The mobile phase consisted of A: 5% acetonitrile in water containing 20 mM ammonium hydroxide and 20 mM ammonium acetate, and B: acetonitrile. The gradient program was: 0–3.5 min, 85% to 32% B; 3.5–12 min, 32% to 2% B; 12–16.5 min, 2% B; 16.5–17 min, 2% to 85% B; 17–25.5 min, 85% B, at a flow rate of 0.4 mL/min. For CoA species, separation was performed on an ACQUITY UPLC BEH C18 column (2.1 × 100 mm, 1.7 μm; Waters) maintained at 30 °C. The mobile phase consisted of A: water containing 10 mM ammonium acetate and 0.05% (v/v) ammonium hydroxide, and B: 95% acetonitrile/water containing 10 mM ammonium acetate and 0.05% (v/v) ammonium hydroxide. The gradient was: 0–1.2 min, 3% B; 1.2–1.7 min, 3% to 11% B; 1.7–7 min, 11% to 13% B; 7–7.2 min, 13% to 20% B; 7.2–9.4 min, 20% to 100% B; 9.4–11.4 min, 100% B; 11.4–11.7 min, 100% to 3% B; 11.7–13 min, 3% B, at a flow rate of 0.2 mL/min. The injection volume was 5 μL for both methods, and the autosampler temperature was maintained at 10 °C. Solvent blanks were injected every 6–10 samples, and the injection order was randomized.Enhanced SIRT3 activity potentiates germinal center epigenetic modulation through α-ketoglutarate.Xinyu YangShanghai Jiao Tong University School of Medicinespleenmass spectrometry assayAfter incubation, cells were harvested, washed twice with ice-cold PBS, and immediately quenched by adding 400 μL of ice-cold 80% (v/v) methanol per sample. Samples were ultrasonicated and centrifuged at 18,000 g for 15 min at 4 °C. The supernatant was split into two equal portions and evaporated to dryness by centrifugal concentration. One portion was reconstituted in 100 μL of 80% methanol for analysis of glutamine-derived metabolites; the other was reconstituted in 100 μL of deionized water for CoA detection.Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS15385Xinyu Yang. Shanghai Jiao Tong University School of Medicine. mingxiaoyu1996@163.com.Jinke Cheng. Shanghai Jiao Tong University School of Medicine. jkcheng@shsmu.edu.cn.Data were acquired and processed with MassLynx v4.1 software.For isotope tracing, isotopologue mass distribution vectors (MDVs) were calculated as the fractional abundance of each isotopologue relative to the sum of all isotopologues for a given metabolite. Natural 13C and 15N abundance contributions were corrected by subtracting the isotopologue fractions measured in the unlabeled control samples analyzed in the same batch. Corrected MDVs were normalized to cell number, and results are presented as MDVs to compare pathway activities. All tracing experiments were performed with four independent biological replicates, and data are shown as means ± S.D.GENOTYPEmingxiaoyu1996@163.comPrimary splenic B cells were first activated with 2 μg/mL anti-CD40 and 40 ng/mL IL-4 in complete RPMI 1640 medium for 4 days as described above. Activated B cells were then washed twice and resuspended at 1 × 10^6 cells/mL in glutamine-free RPMI 1640 medium (Shanghai BasalMedia) supplemented with 1% penicillin/streptomycin, 1% sodium pyruvate, 1% MEM non-essential amino acids, 55 μM 2-mercaptoethanol, 2 μg/mL anti-CD40, 40 ng/mL IL-4, and 2 mM 13C5-L-glutamine (MCE), without FBS. Cells were cultured for 24 h. In parallel, an unlabeled control culture was identically set up but with 2 mM unlabeled L-glutamine instead, to provide natural isotope abundance backgrounds.MetabolomicsMetabolomicsWaters Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters Corporation)Mus musculusACQUITY-I UPLC / Xevo TQSWaters Xevo TQ-Sspleenuntargeted analysisWaters ACQUITY UPLC I-Class Systemglutamineexperimental sampleuntargeted metabolite profilingMitochondriaexperimental blankMetabolomicsWaters Xevo TQ-S Triple Quadrupole Mass Spectrometer (Waters Corporation)Mus musculusACQUITY-I UPLC / Xevo TQSWaters Xevo TQ-Sspleenuntargeted analysisWaters ACQUITY UPLC I-Class Systemglutamineexperimental sampleuntargeted metabolite profilingMitochondriaexperimental blankMetabolite analysis was performed by Metabo-Profile Biotechnology (Shanghai) Co., Ltd. Mass spectrometry was performed on a Waters Xevo TQ-S tandem quadrupole mass spectrometer coupled to the UPLC system. Electrospray ionization (ESI) was used in negative ion mode for glutamine-derived metabolites and positive ion mode for CoA species. The source parameters were set as follows: capillary voltage 2.5 kV (negative mode) or 3.0 kV (positive mode); source temperature 150 °C; desolvation temperature 500 °C; desolvation gas flow 1000 L/h. Data acquisition and processing were performed using MassLynx v4.1 software (Waters). The instrument was maintained and optimized every 48 h according to the manufacturer's recommendations. Mass distribution vectors (MDVs) were calculated for each metabolite as the fractional abundance of each isotopologue relative to the sum of all isotopologues detected. Natural 13C and 15N abundance contributions were corrected by subtracting the isotopologue fractions measured in unlabeled control samples processed in the same batch. Corrected MDVs were normalized to cell number. All tracing experiments were performed with four independent biological replicates, and data are presented as means ± S.D.Glutamic acidcis AcnoticLactateSerinePyruvateOxoglutaric acidAcetyl-CoAOxaloacetic acidCitric acidIsocitric acidMalateFumarateSussinateAspartic acidGlutaminefalseSIRT3-dependent glutaminolysis enhances germinal center epigenetic programming through α-ketoglutarateGerminal center (GC) B cells depend on sustained epigenetic modulation to maintain transcriptional identity and support affinity maturation; however, whether mitochondrial metabolism directly enhances this chromatin state remains unclear. Here, we show that enhanced SIRT3 activity promotes glutamine-derived α-ketoglutarate (αKG) accumulation and is associated with reduced H3K27me3 enrichment at the Bcl6 locus. Using SIRT3 K223R gain-of-function mice, metabolomic profiling, and stable isotope tracing, we show that SIRT3 activation increases glutamine-derived αKG accumulation without a uniform increase in downstream tricarboxylic acid (TCA) cycle labeling. Elevated αKG was associated with reduced H3K27me3 at tested Bcl6 regulatory regions, reinforced BCL6 and AID expression, sustained B cell proliferation, and qualitatively enhanced antibody affinity maturation. Glutamine supplementation partially phenocopied these effects, whereas inhibition of glutamine metabolism attenuated them. Importantly, sustained activation of this metabolic-epigenetic axis was associated with enhanced GC expansion accompanied by increased autoantibody levels and renal IgG deposition in a pristane-induced lupus model. Collectively, our findings indicate that enhanced SIRT3 activity potentiates a glutamine-αKG-chromatin axis that links mitochondrial glutamine metabolism to germinal center epigenetic modulation and humoral immune output.2026-09-042026-08-18MTBLS15385HMDB0000641HMDB0000148HMDB0000134HMDB0000744HMDB0000223HMDB0000190HMDB0000243HMDB0000254HMDB0000208HMDB0000094HMDB0000193HMDB0000072HMDB0000187HMDB0000191HMDB0001206