#METABOLOMICS WORKBENCH silviaradenkovic_20230418_123415 DATATRACK_ID:3869 STUDY_ID:ST002565 ANALYSIS_ID:AN004226 PROJECT_ID:PR001653 VERSION 1 CREATED_ON April 18, 2023, 5:08 pm #PROJECT PR:PROJECT_TITLE Metabolomic profiling of PMM2-CDG after fructose tracer experiments PR:PROJECT_SUMMARY Abnormal polyol metabolism has been predominantly associated with diabetes, PR:PROJECT_SUMMARY where excess glucose is converted to sorbitol by aldose reductase (AR). PR:PROJECT_SUMMARY Recently, abnormal polyol metabolism has also been implicated in PR:PROJECT_SUMMARY phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), and PR:PROJECT_SUMMARY epalrestat, an AR inhibitor, proposed as a potential therapy for this disorder. PR:PROJECT_SUMMARY Given that the PMM enzyme is not closely connected to polyol metabolism, and, PR:PROJECT_SUMMARY unlike in diabetes, PMM2-CDG does not present with hyperglycemia in blood, the PR:PROJECT_SUMMARY increased polyol production, and the therapeutic mechanism of epalrestat in PR:PROJECT_SUMMARY PMM2-CDG remained largely elusive. PMM2-CDG is caused by deficiency of the PMM PR:PROJECT_SUMMARY enzyme and results in a depletion of mannose-1-P and guanosine diphosphate PR:PROJECT_SUMMARY mannose (GDP-mannose), which is essential for glycosylation. Here, we show that PR:PROJECT_SUMMARY apart from glycosylation abnormalities, PMM2 deficiency also leads to changes in PR:PROJECT_SUMMARY intracellular glucose flux, which results in an increase in intracellular PR:PROJECT_SUMMARY polyols. To investigate the influence of fructose on polyol abundance, and PR:PROJECT_SUMMARY glucose flux we performed 13C6 glucose and fructose study in patient and healthy PR:PROJECT_SUMMARY control fibroblasts. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Radenkovic PR:FIRST_NAME Silvia PR:ADDRESS 200 2nd Ave SW Rochester MN PR:EMAIL radenkovic.silvia@mayo.edu PR:PHONE 507(77) 6-6107 PR:FUNDING_SOURCE NIH, KU Leuven #STUDY ST:STUDY_TITLE Metabolomic profiling of PMM2-CDG after siRNA mediated KD of AKR1b1 - 13C6 ST:STUDY_TITLE glucose and fructose study ST:STUDY_SUMMARY Abnormal polyol metabolism has been predominantly associated with diabetes, ST:STUDY_SUMMARY where excess glucose is converted to sorbitol by aldose reductase (AR). ST:STUDY_SUMMARY Recently, abnormal polyol metabolism has also been implicated in ST:STUDY_SUMMARY phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG), and ST:STUDY_SUMMARY epalrestat, an AR inhibitor, proposed as a potential therapy for this disorder. ST:STUDY_SUMMARY Given that the PMM enzyme is not closely connected to polyol metabolism, and, ST:STUDY_SUMMARY unlike in diabetes, PMM2-CDG does not present with hyperglycemia in blood, the ST:STUDY_SUMMARY increased polyol production, and the therapeutic mechanism of epalrestat in ST:STUDY_SUMMARY PMM2-CDG remained largely elusive. PMM2-CDG is caused by deficiency of the PMM ST:STUDY_SUMMARY enzyme and results in a depletion of mannose-1-P and guanosine diphosphate ST:STUDY_SUMMARY mannose (GDP-mannose), which is essential for glycosylation. Here, we show that ST:STUDY_SUMMARY apart from glycosylation abnormalities, PMM2 deficiency also leads to changes in ST:STUDY_SUMMARY intracellular glucose flux, which results in an increase in intracellular ST:STUDY_SUMMARY polyols. Ssing tracer glucose studies, we demonstrate that AR inhibition diverts ST:STUDY_SUMMARY glucose flux away from polyol production towards the synthesis of sugar ST:STUDY_SUMMARY nucleotides. ST:INSTITUTE Mayo Clinic ST:LAST_NAME Radenkovic ST:FIRST_NAME Silvia ST:ADDRESS 200 2nd Ave SW Rochester MN, USA ST:EMAIL radenkovic.silvia@mayo.edu ST:PHONE 507(77) 6-6107 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN WT/PMM2-CDG SU:AGE_OR_AGE_RANGE 40-45 SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS P2 SR01 Genotype:PMM2-CDG | Treatment:13C glu + 12C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR01 SUBJECT_SAMPLE_FACTORS P2 SR02 Genotype:PMM2-CDG | Treatment:13C glu + 12C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR02 SUBJECT_SAMPLE_FACTORS P2 SR03 Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR03 SUBJECT_SAMPLE_FACTORS P2 SR04 Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR04 SUBJECT_SAMPLE_FACTORS P2 SR05 Genotype:PMM2-CDG | Treatment:12C fructose+13C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR05 SUBJECT_SAMPLE_FACTORS P2 SR06 Genotype:PMM2-CDG | Treatment:12C fructose+13C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR06 SUBJECT_SAMPLE_FACTORS P2 SR07 Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR07 SUBJECT_SAMPLE_FACTORS P2 SR08 Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR08 SUBJECT_SAMPLE_FACTORS P2 SR09 Genotype:PMM2-CDG | Treatment:5.5mM 13C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR09 SUBJECT_SAMPLE_FACTORS P2 SR10 Genotype:PMM2-CDG | Treatment:5.5mM 13C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR10 SUBJECT_SAMPLE_FACTORS P2 SR11 Genotype:PMM2-CDG | Treatment:5.5mM glucose + Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR11 SUBJECT_SAMPLE_FACTORS P2 SR12 Genotype:PMM2-CDG | Treatment:5.5mM glucose + Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR12 SUBJECT_SAMPLE_FACTORS C3 SR13 Genotype:WT | Treatment:13C glu + 12C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR13 SUBJECT_SAMPLE_FACTORS C3 SR14 Genotype:WT | Treatment:13C glu + 12C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR14 SUBJECT_SAMPLE_FACTORS C3 SR15 Genotype:WT | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR15 SUBJECT_SAMPLE_FACTORS C3 SR16 Genotype:WT | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR16 SUBJECT_SAMPLE_FACTORS C3 SR17 Genotype:WT | Treatment:12C fructose+13C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR17 SUBJECT_SAMPLE_FACTORS C3 SR18 Genotype:WT | Treatment:12C fructose+13C fructose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR18 SUBJECT_SAMPLE_FACTORS C3 SR19 Genotype:WT | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR19 SUBJECT_SAMPLE_FACTORS C3 SR20 Genotype:WT | Treatment:12C fructose + 12C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR20 SUBJECT_SAMPLE_FACTORS C3 SR21 Genotype:WT | Treatment:5.5mM glucose + Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR21 SUBJECT_SAMPLE_FACTORS C3 SR22 Genotype:WT | Treatment:5.5mM glucose + Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR22 SUBJECT_SAMPLE_FACTORS C3 SR23 Genotype:WT | Treatment:5.5mM 13C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR23 SUBJECT_SAMPLE_FACTORS C3 SR24 Genotype:WT | Treatment:5.5mM 13C glucose Organism=Homo sapiens; Tissue type=Fibroblasts; RAW_FILE_NAME=SR24 #COLLECTION CO:COLLECTION_SUMMARY Cells were washed with PBS, cells were incubated with extraction buffer for 2min CO:COLLECTION_SUMMARY before scraping and transferring to a fresh eppendorf. Samples were precipitated CO:COLLECTION_SUMMARY overnight at -80, then they were centrifuged at max rpm, 20min, 4 degrees C and CO:COLLECTION_SUMMARY supernatant transferred to an M/S vial. CO:SAMPLE_TYPE Fibroblasts CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with 12C/13C glucose +/- 12C/13C fructose #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted with 80% Methanol and IS (d27 myristic acid) SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY C18 iP REVERSE PHASE CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 100% water; 10mM tributylamine; 15mM acetic acid CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B CH:FLOW_GRADIENT until 2 min post injection. A linear gradient to 37% B was carried out until 7 CH:FLOW_GRADIENT min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient CH:FLOW_GRADIENT increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the CH:FLOW_GRADIENT gradient returned to 5% B. The chromatography was stopped at 40 min. CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS El-Maven polly, ThermoFisher Xcalibur; Metabolites were annotated using the MS:MS_COMMENTS inhouse standard metabolite library- elution time and m/z values. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples SR01 SR02 SR03 SR04 SR05 SR06 SR07 SR08 SR09 SR10 SR11 SR12 SR13 SR14 SR15 SR16 SR17 SR18 SR19 SR20 SR21 SR22 SR23 SR24 Factors Genotype:PMM2-CDG | Treatment:13C glu + 12C fructose Genotype:PMM2-CDG | Treatment:13C glu + 12C fructose Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Genotype:PMM2-CDG | Treatment:12C fructose+13C fructose Genotype:PMM2-CDG | Treatment:12C fructose+13C fructose Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Genotype:PMM2-CDG | Treatment:12C fructose + 12C glucose Genotype:PMM2-CDG | Treatment:5.5mM 13C glucose Genotype:PMM2-CDG | Treatment:5.5mM 13C glucose Genotype:PMM2-CDG | Treatment:5.5mM glucose + Genotype:PMM2-CDG | Treatment:5.5mM glucose + Genotype:WT | Treatment:13C glu + 12C fructose Genotype:WT | Treatment:13C glu + 12C fructose Genotype:WT | Treatment:12C fructose + 12C glucose Genotype:WT | Treatment:12C fructose + 12C glucose Genotype:WT | Treatment:12C fructose+13C fructose Genotype:WT | Treatment:12C fructose+13C fructose Genotype:WT | Treatment:12C fructose + 12C glucose Genotype:WT | Treatment:12C fructose + 12C glucose Genotype:WT | Treatment:5.5mM glucose + Genotype:WT | Treatment:5.5mM glucose + Genotype:WT | Treatment:5.5mM 13C glucose Genotype:WT | Treatment:5.5mM 13C glucose Sorbitol 9.11E+06 6.44E+06 1.53E+07 1.26E+07 9.77E+06 9.28E+06 1.62E+07 1.50E+07 1.12E+07 1.02E+07 1.03E+07 9.66E+06 6.51E+06 6.59E+06 9.50E+06 9.70E+06 8.00E+06 6.70E+06 7.77E+06 6.93E+06 9.42E+06 9.56E+06 7.11E+06 6.48E+06 D-Glucose 9.32E+08 1.10E+09 1.34E+09 1.49E+09 8.00E+08 9.57E+08 1.40E+09 1.23E+09 9.30E+08 8.82E+08 1.08E+09 1.26E+09 8.12E+08 8.71E+08 1.01E+09 1.00E+09 9.78E+08 9.53E+08 1.36E+09 1.19E+09 9.87E+08 1.09E+09 9.68E+08 1.16E+09 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name parent formula M/Z HMDB ID isotopeLabel isotope count Sorbitol C6H14O6 181.07164 HMDB00247 C12 PARENT m0-6 D-Glucose C6H12O6 179.056061 HMDB00122 C12 PARENT m0-6 METABOLITES_END #END