#METABOLOMICS WORKBENCH elopezgonzalez_20250814_082207 DATATRACK_ID:6280 STUDY_ID:ST004126 ANALYSIS_ID:AN006839 PROJECT_ID:PR002594 VERSION 1 CREATED_ON 09-08-2025 #PROJECT PR:PROJECT_TITLE Glycerol-3-phosphate activates ChREBP, FGF21 transcription and lipogenesis in PR:PROJECT_TITLE Citrin Deficiency PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Citrin Deficiency (CD) is caused by inactivation of SLC25A13, a mitochondrial PR:PROJECT_SUMMARY membrane protein required to move electrons from cytosolic NADH to the PR:PROJECT_SUMMARY mitochondrial matrix in hepatocytes. People with CD do not like sweets. We PR:PROJECT_SUMMARY discovered that SLC25A13 loss causes accumulation of glycerol-3-phosphate (G3P), PR:PROJECT_SUMMARY which activates carbohydrate response element binding protein (ChREBP) to PR:PROJECT_SUMMARY transcribe FGF21, which acts in the brain to restrain intake of sweets and PR:PROJECT_SUMMARY alcohol, and to transcribe key genes of de novo lipogenesis. Mouse and human PR:PROJECT_SUMMARY data establish G3P-ChREBP as a new mechanistic component of the Randle Cycle PR:PROJECT_SUMMARY that contributes to metabolic dysfunction-associated steatotic liver disease PR:PROJECT_SUMMARY (MASLD) and forms part of a system that communicates metabolic states from liver PR:PROJECT_SUMMARY to brain in a manner that alters food and alcohol choices. The data provide a PR:PROJECT_SUMMARY framework for understanding FGF21 induction in varied conditions, suggest ways PR:PROJECT_SUMMARY to develop FGF21-inducing drugs, and drug candidates for both lean MASLD and PR:PROJECT_SUMMARY support of urea cycle function in CD. PR:INSTITUTE Beckman Research Institute of City of Hope PR:LAST_NAME Brenner PR:FIRST_NAME Charles PR:ADDRESS 1500 E. Duarte Road, Duarte, California, 91010, USA PR:EMAIL cbrenner@coh.org PR:PHONE 626-218-2750 PR:FUNDING_SOURCE National Institute of Health grant R01HL147545 PR:PUBLICATIONS Tiwari, V. et al Glycerol-3-phosphate activates ChREBP, FGF21 transcription and PR:PUBLICATIONS lipogenesis in Citrin Deficiency doi: 10.1101/2024.12.27.630525 PR:DOI http://dx.doi.org/10.21228/M8BN8K PR:CONTRIBUTORS Edwin Lopez Gonzalez, Vinod Tiwari and Olivia Sun #STUDY ST:STUDY_TITLE Glyc3P, hexose phosphates, and pentose phosphates measurements in citrin ST:STUDY_TITLE deficiency. ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY Citrin Deficiency (CD) is caused by inactivation of SLC25A13, a mitochondrial ST:STUDY_SUMMARY membrane protein required to move electrons from cytosolic NADH to the ST:STUDY_SUMMARY mitochondrial matrix in hepatocytes. In this study we used the mutant mouse ST:STUDY_SUMMARY models, with deletions of NADH shuttle systems, Slc25a13-/-, Gpd2-/-, and ST:STUDY_SUMMARY Slc25a13-/- Gpd2-/- to further characterize CD and ChREBP activation by ST:STUDY_SUMMARY measuring the liver levels of hexose phosphates, pentose phosphates, and ST:STUDY_SUMMARY glycerol-3-phosphate. The pentose phosphate xylulose-5-phosphate and hexose ST:STUDY_SUMMARY phosphates: glucose-6-phosphate and fructose-6-phosphate, were previously ST:STUDY_SUMMARY proposed activators of ChREBP. We provided ad libitum glycerol (5% w/v) to these ST:STUDY_SUMMARY mutant mice and compared the levels of these metabolites to determine their ST:STUDY_SUMMARY ability to activate ChREBP. Here we show that only G3P is elevated and ST:STUDY_SUMMARY accumulates in the liver of the mouse model of CD, and that ChREBP is activated ST:STUDY_SUMMARY in this model with transcription of FGF21 and activation of a lipogenic ST:STUDY_SUMMARY transcriptional program. Our data further show that G3P is a specific ligand of ST:STUDY_SUMMARY the ChREBP GSM, and suggest that features of the G3P-ChREBP activation mechanism ST:STUDY_SUMMARY can account for why fructose is more lipogenic than glucose, provide a unifying ST:STUDY_SUMMARY mechanism for nonalcoholic and alcoholic hepatic steatogenesis, resolve ST:STUDY_SUMMARY paradoxes of FGF21 expression, and explain key aspects of CD pathogenesis ST:STUDY_SUMMARY including lean MASLD, the favorable effects of MCTs, and severe urea cycle ST:STUDY_SUMMARY dysfunction. ST:INSTITUTE Beckman Research Institute of City of Hope ST:LAST_NAME Brenner ST:FIRST_NAME Charles ST:ADDRESS 1500 E. Duarte Road, Duarte, California, 91010, USA ST:EMAIL cbrenner@coh.org ST:PHONE 626-218-2750 ST:SUBMIT_DATE 2025-08-14 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 8-16 Weeks SU:GENDER Male SU:ANIMAL_ANIMAL_SUPPLIER Dr. Saheki and the Citrin Foundation SU:ANIMAL_HOUSING Single-sex group-housed SU:ANIMAL_LIGHT_CYCLE 12:12-hour light-dark cycle SU:ANIMAL_FEED ad libitim LabDiet irradiated PicoLab Rodent Diet 20, 5053 SU:ANIMAL_WATER ad libitim water #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - M039 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GG_39.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_39.mzML SUBJECT_SAMPLE_FACTORS - M040 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GW_40.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GG_40.mzML SUBJECT_SAMPLE_FACTORS - M092 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GG_92.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_92.mzML SUBJECT_SAMPLE_FACTORS - M093 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GG_93.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_93.mzML SUBJECT_SAMPLE_FACTORS - M095 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GG_95.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GG_95.mzML SUBJECT_SAMPLE_FACTORS - M035 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GW_35.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_35.mzML SUBJECT_SAMPLE_FACTORS - M036 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GW_36.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_36.mzML SUBJECT_SAMPLE_FACTORS - M090 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GW_90.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_90.mzML SUBJECT_SAMPLE_FACTORS - M091 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GW_91.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=N/A SUBJECT_SAMPLE_FACTORS - M094 Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=GW_92.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_GW_94.mzML SUBJECT_SAMPLE_FACTORS - M118 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DG_118.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DG_118.mzML SUBJECT_SAMPLE_FACTORS - M119 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DG_119.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DG_119.mzML SUBJECT_SAMPLE_FACTORS - M186 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DG_186.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DG_186.mzML SUBJECT_SAMPLE_FACTORS - M191 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DG_191.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DG_191.mzML SUBJECT_SAMPLE_FACTORS - M195 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DG_195.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DG_195.mzML SUBJECT_SAMPLE_FACTORS - M105 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DW_105.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DW_105.mzML SUBJECT_SAMPLE_FACTORS - M124 Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=DW_124.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_DW_124.mzML SUBJECT_SAMPLE_FACTORS - M009 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SG_9.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SG_009.mzML SUBJECT_SAMPLE_FACTORS - M010 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SG_10.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SG_10.mzML SUBJECT_SAMPLE_FACTORS - M011 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SG_11.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SG_11.mzML SUBJECT_SAMPLE_FACTORS - M025 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SG_25.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SG_25.mzML SUBJECT_SAMPLE_FACTORS - M026 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SG_26.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SG_26.mzML SUBJECT_SAMPLE_FACTORS - M187 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SW_187.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SW_187.mzML SUBJECT_SAMPLE_FACTORS - M188 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SW_188.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SW_188.mzML SUBJECT_SAMPLE_FACTORS - M189 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SW_189.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SW_189.mzML SUBJECT_SAMPLE_FACTORS - M190/20 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SW_190_20.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SW_190.mzML SUBJECT_SAMPLE_FACTORS - M191/20 Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=SW_191_20.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_SW_191.mzML SUBJECT_SAMPLE_FACTORS - M001 Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WG_1.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WG_1.mzML SUBJECT_SAMPLE_FACTORS - M002 Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WG_2.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WG_2.mzML SUBJECT_SAMPLE_FACTORS - M003 Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WG_3.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WG_3.mzML SUBJECT_SAMPLE_FACTORS - M004 Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WG_4.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WG_4.mzML SUBJECT_SAMPLE_FACTORS - M005 Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WG_5.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WG_5.mzML SUBJECT_SAMPLE_FACTORS - M2 Sample source:Liver | Genotype:Wild Type | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WtW2.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WW_2.mzML SUBJECT_SAMPLE_FACTORS - M3 Sample source:Liver | Genotype:Wild Type | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WtW3.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WW_3.mzML SUBJECT_SAMPLE_FACTORS - M4 Sample source:Liver | Genotype:Wild Type | Treatment:2 days water RAW_FILE_NAME(Glyc3P_Raw_File_Name)=WtW4.mzML; RAW_FILE_NAME(Pentose_and_Hexose_Phosphates_Raw_File_Name)=YEAST_WW_4.mzML #COLLECTION CO:COLLECTION_SUMMARY All mouse breeding and experiments were performed with protocols approved by the CO:COLLECTION_SUMMARY City of Hope Institutional Animal Care and Use Committee and Institutional CO:COLLECTION_SUMMARY Biosafety Committee. Sperm from a male Slc25a13 -/- Gpd2 -/- C57BL6/J mouse were CO:COLLECTION_SUMMARY a kind gift of Dr. Saheki and the Citrin Foundation. In vitro fertilization was CO:COLLECTION_SUMMARY performed with C57BL6/J eggs and a pseudopregnant C57BL6/J female recipient by CO:COLLECTION_SUMMARY Walter Tsark in the City of Hope Transgenic Mouse Core. Resulting diheterozygous CO:COLLECTION_SUMMARY offspring were intercrossed to generate wild-type, Slc25a13 -/-, Gpd2 -/-, and CO:COLLECTION_SUMMARY Slc25a13 -/- Gpd2 -/- mice of both sexes. At weaning, genotypes were determined CO:COLLECTION_SUMMARY by PCR of tail tissues (Transnetyx, Cordova, TN), and mice were single-sex CO:COLLECTION_SUMMARY group-housed until experimental use. Mice were maintained at 21°C under a CO:COLLECTION_SUMMARY standard 12:12-hour light-dark cycle and provided with ad libitim access to food CO:COLLECTION_SUMMARY and water. Mice were then euthanized by decapitation via guillotine without CO:COLLECTION_SUMMARY sedation, exsanguinated with a funnel for blood collection into 1.5 mL CO:COLLECTION_SUMMARY microcentrifuge tubes on ice, and livers were freeze-clamped in liquid nitrogen CO:COLLECTION_SUMMARY immediately after harvest. Sera were obtained by centrifuging blood at 8,000 g CO:COLLECTION_SUMMARY for 10 minutes at 4°C. Liver samples were pulverized with a mortar and pestle CO:COLLECTION_SUMMARY cooled by liquid nitrogen. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY 8-16 week old mice were single-housed with chow (LabDiet irradiated PicoLab TR:TREATMENT_SUMMARY Rodent Diet 20, 5053) and a single bottle of either water or 5% (w/v) glycerol TR:TREATMENT_SUMMARY for two days. TR:TREATMENT_COMPOUND Glycerol TR:TREATMENT_DOSE ad libitim 5% (w/v) glycerol solution #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For Glycerol-3-Phosphate quantification, 2.0 mg samples of pulverized frozen SP:SAMPLEPREP_SUMMARY liver were spiked with 13C3-Glycerol-3-Phosphate (2.28 µM final concentration, SP:SAMPLEPREP_SUMMARY Sigma Aldrich). Frozen samples were immediately processed with a boiling SP:SAMPLEPREP_SUMMARY buffered ethanol extraction (600 µL of 25% 10 mM HEPES Buffer and 75% Ethanol) SP:SAMPLEPREP_SUMMARY followed by vigorous vortexing. Samples were placed on a thermomixer for 5 min SP:SAMPLEPREP_SUMMARY at 55°C with shaking at 1,200 rpm, then cooled on ice for 30 seconds. Samples SP:SAMPLEPREP_SUMMARY were then placed in a water bath sonicator for 1 minute followed by an SP:SAMPLEPREP_SUMMARY additional 30 seconds on ice. Samples were clarified by centrifugation at 16,100 SP:SAMPLEPREP_SUMMARY g in a prechilled centrifuge at 4°C; the supernatant was transferred to a clean SP:SAMPLEPREP_SUMMARY tube followed by a second round of centrifugation. Clarified supernatants were SP:SAMPLEPREP_SUMMARY transferred to new tubes and dried for 5 hours in a vacuum centrifuge at 4°C. SP:SAMPLEPREP_SUMMARY Dried samples were resuspended in 80 µL of LC-MS water. Twenty-fold diluted SP:SAMPLEPREP_SUMMARY samples were transferred to MS vials for analysis on LC-MS/MS. For analysis of SP:SAMPLEPREP_SUMMARY hexose phosphates and pentose phosphates, 2.0 mg samples of pulverized frozen SP:SAMPLEPREP_SUMMARY liver were spiked with a 13C glucose-grown yeast extract (ISO101, Cambridge SP:SAMPLEPREP_SUMMARY Isotope Laboratories) as an internal standard and the same workup was performed. SP:PROCESSING_STORAGE_CONDITIONS Described in summary SP:EXTRACT_STORAGE Described in summary #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were analyzed on a Vanquish Horizon UHPLC with a tandem Thermo CH:CHROMATOGRAPHY_SUMMARY Scientific Orbitrap Fusion mass spectrometer. Vials were maintained in CH:CHROMATOGRAPHY_SUMMARY autosampler at 4°C. Instrument source parameters were held at 3 kV negative ion CH:CHROMATOGRAPHY_SUMMARY spray voltage, 300°C ion transfer tube temperature, 250°C vaporizer CH:CHROMATOGRAPHY_SUMMARY temperature, sheath gas of 20, auxiliary gas of 10, and sweep gas of 3. Liquid CH:CHROMATOGRAPHY_SUMMARY chromatography separation was carried out using an Acquity Premier HSS T3 column CH:CHROMATOGRAPHY_SUMMARY with VanGuard FIT, 1.8 µm, 2.1 x 150 mm with mobile phases A (5 mM CH:CHROMATOGRAPHY_SUMMARY tributylamine and 10 mM acetic acid with 5% v/v methanol in LC-MS grade water) CH:CHROMATOGRAPHY_SUMMARY and B (LC-MS grade methanol) at a constant flow rate of 0.5 ml/min. Separation CH:CHROMATOGRAPHY_SUMMARY was carried out with starting condition of 0% B; 0-10 min, 10.5% B; 10-18 min, CH:CHROMATOGRAPHY_SUMMARY 52.6% B; 18-19 min 52.6% B; 19-20 min, 0% B; 20-26 min, 0% B. Spectra for G3P CH:CHROMATOGRAPHY_SUMMARY were acquired using a targeted MS2 scan with parent ion of m/z 171.0058 with CH:CHROMATOGRAPHY_SUMMARY collision energy 25. Spectra for 13C3-G3P were acquired with parent ion m/z of CH:CHROMATOGRAPHY_SUMMARY 174.0165 with collision energy 25 with primary fragment ion of m/z 78.9588. CH:CHROMATOGRAPHY_SUMMARY Hexose phosphate spectra were acquired using a targeted MS2 scan of parent ion CH:CHROMATOGRAPHY_SUMMARY m/z 259.0198. 13C6-hexose phosphate spectra were acquired with a parent ion of CH:CHROMATOGRAPHY_SUMMARY m/z 265.0426 with collision energy of 20 and primary fragment ion m/z 96.9690. CH:CHROMATOGRAPHY_SUMMARY Pentose phosphate spectra were acquired using targeted MS2 scan for the parent CH:CHROMATOGRAPHY_SUMMARY ion m/z 229.0124 at collision energy 30 with a primary fragment ion m/z of CH:CHROMATOGRAPHY_SUMMARY 78.9588. CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Acquity Premier HSS T3 column with VanGuard FIT (150 x 2.1 mm, 1.8 µm) CH:COLUMN_TEMPERATURE 45°C CH:FLOW_GRADIENT Starting condition of 0% B; 0-10 min, 10.5% B; 10-18 min, 52.6% B; 18-19 min CH:FLOW_GRADIENT 52.6% B; 19-20 min, 0% B; 20-21, 0.5-0.8 mL/min; 21-23.9, 0.8 mL/min, 0% B; CH:FLOW_GRADIENT 23.9-24 min, 0.8-0.5 mL/min, 0% B. CH:FLOW_RATE 0.5 mL/min (unless specified in gradient) CH:SOLVENT_A 95% Water/5% Methanol; 5 mM tributylamine; 10 mM acetic acid CH:SOLVENT_B 100% Methanol CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Scientific Orbitrap Fusion MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:MS_COMMENTS Spectra for G3P were acquired using a targeted MS2 scan with parent ion of m/z MS:MS_COMMENTS 171.0058 with collision energy 25. Spectra for 13C3-G3P were acquired with MS:MS_COMMENTS parent ion m/z of 174.0165 with collision energy 25 with primary fragment ion of MS:MS_COMMENTS m/z 78.9588. Hexose phosphate spectra were acquired using a targeted MS2 scan of MS:MS_COMMENTS parent ion m/z 259.0198. 13C6-hexose phosphate spectra were acquired with a MS:MS_COMMENTS parent ion of m/z 265.0426 with collision energy of 20 and primary fragment ion MS:MS_COMMENTS m/z 96.9690. Pentose phosphate spectra were acquired using targeted MS2 scan for MS:MS_COMMENTS the parent ion m/z 229.0124 at collision energy 30 with a primary fragment ion MS:MS_COMMENTS m/z of 78.9588. Mass spectra data was analyzed using the open-source software MS:MS_COMMENTS Skyline. MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS millimoles/liter MS_METABOLITE_DATA_START Samples M039 M040 M092 M093 M095 M035 M036 M090 M091 M094 M118 M119 M186 M191 M195 M105 M124 M009 M010 M011 M025 M026 M187 M188 M189 M190/20 M191/20 M001 M002 M003 M004 M005 M2 M3 M4 Factors Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- Gpd2 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water Sample source:Liver | Genotype:Slc25a13 -/- | Treatment:2 days water Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Wild Type | Treatment:2 days 5% glycerol Sample source:Liver | Genotype:Wild Type | Treatment:2 days water Sample source:Liver | Genotype:Wild Type | Treatment:2 days water Sample source:Liver | Genotype:Wild Type | Treatment:2 days water Hexose Phosphates 0.6217 0.6279 0.6309 0.5993 0.3660 0.7480 0.7399 1.0509 0.8080 0.9236 0.4019 0.5087 0.5799 0.3934 0.7791 0.5856 0.6664 0.5938 0.5058 0.7001 0.8641 0.5912 0.7994 0.5445 0.6608 0.6800 1.0731 0.6946 0.4052 0.4694 1.0041 0.7870 0.7561 0.9184 Pentose Phosphates 1.1198 0.9426 1.6328 0.8218 0.6780 0.7968 1.4709 1.7133 1.1201 1.1282 1.1735 1.6935 1.3553 1.2092 2.2146 0.9496 0.8578 1.1686 0.6907 0.8501 1.2613 0.7874 0.8854 0.8599 1.2906 1.0484 1.8944 1.2248 0.5588 0.5430 1.8463 0.9191 0.9571 1.1238 sn-Glycero-3-Phosphate 2.2672 2.2913 1.8366 1.6954 1.5020 1.4428 2.2154 2.6618 2.2482 1.5658 1.5777 3.2106 1.3322 2.9547 1.0767 2.9753 0.9179 1.0390 1.3317 1.8695 1.1375 2.5311 1.7888 1.2711 2.1682 2.3498 1.7371 2.1911 2.0702 1.3155 1.4222 0.9490 1.0933 1.2613 0.8690 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant Hexose Phosphates N/A Pentose Phosphates N/A sn-Glycero-3-Phosphate 439162 C00093 METABOLITES_END #END