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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13687/m_MTBLS13687_GC-MS_positive__metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13687/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13687/s_MTBLS13687.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13687/a_MTBLS13687_GC-MS_positive__metabolite_profiling.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS13687Stable isotope enrichment of metabolites was calculated as net labeling after correction for natural isotope abundance (PMID: 21112315; PMID: 8799277). For lipid synthesis, the retention time of palmitic acid derivative was at 14.2 min with detected m/z 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, and 329 for M0, M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M15, and M16 palmitate.MetaboLightsPublicGas Chromatography MS - positiveSamples were then transferred to GC vials for GC–MS analysis using an Agilent 8890 GC coupled to a 5977B mass spectrometer. One microliter of the derivatized sample was injected into an HP-5MS UI column (Agilent; 30 m × 0.25 mm, 0.25 µm film thickness). The oven temperature was held at 80 °C for 2 minutes, increased at 7 °C per minute to 280 °C, and maintained at 280 °C for the remainder of the 40-minute run. For lipid synthesis, GC-MS analysis was performed on an Agilent 7890B GC system equipped with a HP-5MS capillary column (30 m, 0.25 mm i.d., 0.25 μm-phase thickness; Agilent J&W Scientific, Santa Clara, CA). Samples were transferred to GC-MS vials and were ready for GC-MS analysis after brief centrifugation at 800 × g for 5 min. The injection volume was 1 μL, and samples were injected in split mode with a split ratio of 5:1. GC oven temperature was held at 80°C for one minute, increased to 280°C at 14°C/min, and held at 280°C for 9.5 min with a total run time of twenty five minutes.Aberrant accumulation of cyclin D1 rewires glutamine metabolism via loss of IDH3 generating metabolic vulnerability in ESCC and HNSCC.Shuyuan ZhangDepartment of Biochemistry, School of Medicine, Case Western Reserve UniversityESCC cellmass spectrometry assayCell pellets were spiked with 2 nmol norvaline as an internal standard and extracted twice with 0.8 mL cold methanol. The extracts were combined and dried completely under a stream of nitrogen gas.The dried residues were derivatized with 40 µL methoxylamine hydrochloride at 40 °C for 90 minutes, followed by 20 µL N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide at 80 °C for 30 minutes.  For lipid synthesis, The combined MeOH extracts were briefly centrifuged and dried completely under nitrogen gas. The dried residue was dissolved in 25% KOH ethanol solution with brief sonication. The total lipid was hydrolyzed into free fatty acids at 85°C for 3 h. The hydrolyzed fatty acid solution (200 μl) was acidified with 300 μl 6 M HCl. Fatty acids were extracted by adding 600 μl chloroform. After centrifugation at 800 × g for 10 min, the chloroform phase (400 μl) was transferred to a new Eppendorf via and was completely dried under nitrogen gas. The dried residue was derivatized with 50 μl MTBSTFA + 1% TBDMS at 60°C for 30 min. (PMID: 33321098)Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS13687Bartosz Mucha.Cecilia Martin.Shuyuan Zhang. sxz809@case.edu.Andres Klein-Szanto.Anil Rustgi.Guofang Zhang.Wendi Quinn O'Neill.Marganit Farago.Hiroshi Nakagawa.J. Alan Diehl. Case Western Reserve University. jad283@case.edu.Polly Phillips-Mason.Kwok-Kin Wong.Brian Henick.Data analysis was performed using Agilent MassHunter Quantitative Analysis software (version 10.1). The labeling of all measured metabolites in cells/islets treated with 13C-labeled substrates was corrected based on the natural isotope distribution assayed from cell samples treated with unlabeled carbon sources. The natural isotope distribution of each measured metabolite was assayed and averaged to build a matrix for correcting isotope labeling of metabolite in the tracer-treated samples.Glutamine concentrationsxz809@case.eduDay 0, 1.5x105 TE7 cells and 2x105 TE15 cells were seeded in a 6-well plate in 2 mL complete DMEM containing 25 mM glucose, 4 mM glutamine and 1xPenicillin-Streptomycin. Day 1, media was removed and the cells were washed twice with PBS. Add 2 mL DMEM containing 25 mM glucose, and 0.5/5 mM [U-13C5]glutamine. Day 2 and day 3, remove the media and wash with PBS twice, add 0.5/5 mM [U-13C5]glutamine. Day 4, (72 h after replacing with tracer medium), the cells are ready for the extraction of metabolites:<ul><li>Remove medium and quickly wash two times with cold PBS</li><li>Add 1 ml cold methanol and scrape the cells and transfer to Eppendorf vial</li><li>Add another 0.5 ml cold methanol and do another sample extract and combine both extracts into the same Eppendorf vial</li></ul> Lipid synthesis experiment used the same sample collection protocol.Metabolomicsesophageal squamous cell carcinomaisocitrate dehydrogenase (NAD+) activityUniformly-labeled [13C]glutamineuntargeted metabolitesCyclin D1Glutaminehead and neck squamous cell carcinomaesophageal squamous cell carcinomaisocitrate dehydrogenase (NAD+) activityUniformly-labeled [13C]glutamineuntargeted metabolitesCyclin D1Glutaminehead and neck squamous cell carcinomaSamples were then transferred to GC vials for GC–MS analysis using an Agilent 8890 GC coupled to a 5977B mass spectrometer. Mass spectra were acquired in scan mode over an m/z range of 50–700. For lipid synthesis, GC-MS analysis was performed on an Agilent 7890B GC system equipped with a HP-5MS capillary column (30 m, 0.25 mm i.d., 0.25 μm-phase thickness; Agilent J&W Scientific, Santa Clara, CA), connected to an Agilent 5977A Mass Spectrometer operating under ionization by electron impact (EI) at 70 eV. Helium flow was maintained at 1 mL/min. The source temperature was maintained at 230°C, the MS quad temperature at 150°C, the interface temperature at 280°C, and the inlet temperature at 250°C. Mass spectra were recorded in mass scan mode with m/z from 50 to 700. (PMID: 33321098)M2 ProlineM1 MethionineM4 MethionineM2 SuccinateM4 FumarateM0 Glycerol-3-M3 4-AminobutyrateM5 2-HydroxyglutarateM3 CitrateM0 4-AminobutyrateM0 AspartateHistidineM1 MalateM2 MalateM3 MalateM0 MalateM3 LactateM2 GlycolateM4 MalateM5 GlutamineM0 SerineM2 AsparagineM0 GlyclateThreonineM2 SerineM3 CysteineM1 3-PhosphoglycerateM2 PyroglutamateM3 SuccinateM2 2-HydroxyglutarateM0 3-PhosphoglycerateM1 2-HydroxyglutarateM3 FumarateM3 GlutamateNorvaline (IS)M1 AspartateM4 GlutamineM3 3-HydroxybutyrateM6 CitrateM0 SuccinateGlycineM0 PyroglutamateM1 PyroglutamateM2 LactateM2 CitrateM2 LysineM1 AsparagineM0 FumarateM3 MethionineM2 FumarateM3 LysineM4/M5 ProlineM0 PhosphoenolpyruvateM2 2-KetoglutarateM1 SerineM1 PyruvateM3 AspartatePhenylalanineM5 2-KetoglutarateM1 PhosphoenolpyruvateM2 AlanineM4 GlutamateValineM1 GlutamateM1 ProlineM1 FumarateM4 AspartateM2 GlutamineM0 3-HydroxybutyrateM3 PyroglutamateM0 GlutamateM0 AsparagineM2 PhosphoenolpyruvateM3 PhosphoenolpyruvateM4 2-KetoglutarateM1 3-HydroxybutyrateM1 CysteineM1 2-KetoglutarateM5 CitrateM0 2-HydroxyglutarateM1 GlutamineM2 4-AminobutyrateM3 AlanineM2 MethionineM2 3-PhosphoglycerateM3 2-HydroxyglutarateM1 CitrateM1 LactateM4 2-HydroxyglutarateM3 3-PhosphoglycerateM2 CysteineM3 AsparagineM0 MethionineM3 PyruvateM0 ProlineLeucineM4 AsparagineM4 SuccinateM6 Valine (IS)M5 GlutamateM0 2-KetoglutarateM3 ProlineM3 2-KetoglutarateM2 GlutamateM0 LysineM1 LysineM2 PyruvateM4 3-HydroxybutyrateM0 AlanineM5 LysineM2 AspartateM4 LysineM4 CitrateM1AlanineM0 GlutamineIsoleucineM0 CysteineM4 PyroglutamateM3 SerineM0 CitrateM1 GlycolateM3 GlutamineM2 Glycerol-3-M1 Glycerol-3-M3 Glycerol-3-M0 LactateM2 3-HydroxybutyrateM0 PyruvateM1 4-AminobutyrateM4 4-AminobutyrateM5 PyroglutamateM1 SuccinatefalseAberrant accumulation of cyclin D1 rewires glutamine metabolism via loss of IDH3 generating metabolic vulnerability in ESCC and HNSCCExcessive cyclin D1 accumulation is a frequent oncogenic event in head and neck squamous cell carcinoma (HNSCC) and esophageal squamous cell carcinoma (ESCC). Although classically associated with enhanced proliferation and genomic instability, we reveal that cyclin D1 dysregulation induces unexpected metabolic dependencies. We define the resulting metabolic rewiring and the underlying mechanism in both established cell models and a newly developed HNSCC mouse model expressing degradation-resistant cyclin D1T286A mutant on a gain-of-function p53R172H background. We demonstrate that sustained nuclear retention of cyclin D1 drives metabolic reprogramming through activation of CDK4/6 and downregulation of mitochondrial isocitrate dehydrogenase 3 (IDH3). This metabolic alteration profoundly sensitizes cells to glutamine deprivation. In this study, we utilized GC-MS to analyze where glutamine is being used and the metabolite changes in cyclin D1 mutant cells.2026-08-242026-01-14MTBLS13687