#METABOLOMICS WORKBENCH silviaradenkovic_20230414_133420 DATATRACK_ID:3863 STUDY_ID:ST002561 ANALYSIS_ID:AN004222 PROJECT_ID:PR001653 VERSION 1 CREATED_ON April 18, 2023, 10:44 am #PROJECT PR:PROJECT_TITLE Metabolomic profiling of PMM2-CDG zebrafish in presence and absence of PR:PROJECT_TITLE epalrestat 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. Targeting AR with epalrestat decreases polyol levels and increases PR:PROJECT_SUMMARY GDP-mannose in vivo in pmm2 mutant zebrafish. PR:INSTITUTE Mayo Clinic PR:LAST_NAME Radenkovic PR:FIRST_NAME Silvia PR:ADDRESS 200 2nd Ave SW Rochester MN, USA PR:EMAIL radenkovic.silvia@mayo.edu PR:PHONE 507(77) 6-6107 PR:FUNDING_SOURCE NIH, KU Leuven PR:PUBLICATIONS Tracer metabolomics reveals the role of aldose reductase in glycosylation PR:CONTRIBUTORS Silvia Radenkovic, Anna N. Ligezka, Sneha S. Mokashi, Karen Driesen, Lynn PR:CONTRIBUTORS Dukes-Rimsky, Graeme Preston, Luckio F. Owuocha, Leila Sabbagh, Jehan Mousa, PR:CONTRIBUTORS Christina Lam, Andrew Edmondson, Austin Larson, Matthew Schultz, Pieter PR:CONTRIBUTORS Vermeersch, David Cassiman, Peter Witters, Lesa J. Beamer, Tamas Kozicz, Heather PR:CONTRIBUTORS Flanagan-Steet, Bart Ghesquière, Eva Morava #STUDY ST:STUDY_TITLE Metabolomic profiling of PMM2-CDG zebrafish in presence and absence of ST:STUDY_TITLE epalrestat 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. Targeting AR with epalrestat decreases polyol levels and increases ST:STUDY_SUMMARY GDP-mannose in vivo in pmm2 mutant zebrafish. 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 Fish SU:SUBJECT_SPECIES Danio rerio SU:TAXONOMY_ID 7955 SU:GENOTYPE_STRAIN WT/PMM2 mutant sa10150 SU:AGE_OR_AGE_RANGE 6 dpf #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 - MCF001027_wt1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt1 SUBJECT_SAMPLE_FACTORS - MCF001027_wt2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt2 SUBJECT_SAMPLE_FACTORS - MCF001027_wt3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt3 SUBJECT_SAMPLE_FACTORS - MCF001027_wt4 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt4 SUBJECT_SAMPLE_FACTORS - MCF001027_wt1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt1_ep10 SUBJECT_SAMPLE_FACTORS - MCF001027_wt2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt2_ep10 SUBJECT_SAMPLE_FACTORS - MCF001027_wt1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt1_ep40 SUBJECT_SAMPLE_FACTORS - MCF001027_wt2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt2_ep40 SUBJECT_SAMPLE_FACTORS - MCF001027_wt3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_wt3_ep40 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm1 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm2 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm3 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm4 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm4 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm1_ep10 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm2_ep10 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm1_ep40 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm2_ep40 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm3_ep40 SUBJECT_SAMPLE_FACTORS - MCF001027_pmm4_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=MCF001027_pmm4_ep40 SUBJECT_SAMPLE_FACTORS - wt1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt1 SUBJECT_SAMPLE_FACTORS - wt2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt2 SUBJECT_SAMPLE_FACTORS - wt3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt3 SUBJECT_SAMPLE_FACTORS - wt1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt1_ep10 SUBJECT_SAMPLE_FACTORS - wt2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt2_ep10 SUBJECT_SAMPLE_FACTORS - wt3_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt3_ep10 SUBJECT_SAMPLE_FACTORS - wt4_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt4_ep10 SUBJECT_SAMPLE_FACTORS - pmm1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm1 SUBJECT_SAMPLE_FACTORS - pmm2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm2 SUBJECT_SAMPLE_FACTORS - pmm3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm3 SUBJECT_SAMPLE_FACTORS - pmm4 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm4 SUBJECT_SAMPLE_FACTORS - pmm1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm1_ep10 SUBJECT_SAMPLE_FACTORS - pmm2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm2_ep10 SUBJECT_SAMPLE_FACTORS - pmm3_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm3_ep10 SUBJECT_SAMPLE_FACTORS - pmm4_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm4_ep10 SUBJECT_SAMPLE_FACTORS - wt4 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt4 SUBJECT_SAMPLE_FACTORS - wt5 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt5 SUBJECT_SAMPLE_FACTORS - wt1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt1_ep40 SUBJECT_SAMPLE_FACTORS - wt2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt2_ep40 SUBJECT_SAMPLE_FACTORS - wt3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=HiliC; RAW_FILE_NAME=wt3_ep40 SUBJECT_SAMPLE_FACTORS - pmm5 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm5 SUBJECT_SAMPLE_FACTORS - pmm6 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm6 SUBJECT_SAMPLE_FACTORS - pmm7 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm7 SUBJECT_SAMPLE_FACTORS - pmm8 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm8 SUBJECT_SAMPLE_FACTORS - pmm1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm1_ep40 SUBJECT_SAMPLE_FACTORS - pmm2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm2_ep40 SUBJECT_SAMPLE_FACTORS - pmm3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm3_ep40 SUBJECT_SAMPLE_FACTORS - pmm4_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=HiliC; RAW_FILE_NAME=pmm4_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_wt1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt1 SUBJECT_SAMPLE_FACTORS - MCF001395_wt2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt2 SUBJECT_SAMPLE_FACTORS - MCF001395_wt1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt1_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_wt2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt2_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_wt1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt1_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_wt2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt2_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_wt3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt3_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_wt4_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt4_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm1 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm2 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm1_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm2_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm1_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm2_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_wt3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt3 SUBJECT_SAMPLE_FACTORS - MCF001395_wt4 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt4 SUBJECT_SAMPLE_FACTORS - MCF001395_wt3_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt3_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_wt4_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_wt4_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm3 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm4 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm4 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm3_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm3_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm4_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm4_ep10 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm3_ep40 SUBJECT_SAMPLE_FACTORS - MCF001395_pmm4_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001395_pmm4_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_wt1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt1 SUBJECT_SAMPLE_FACTORS - MCF001428_wt2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt2 SUBJECT_SAMPLE_FACTORS - MCF001428_wt3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt3 SUBJECT_SAMPLE_FACTORS - MCF001428_wt1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt1_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_wt2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt2_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_wt3_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt3_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_wt4_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt4_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_wt1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt1_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_wt2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt2_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_wt3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio wildtype; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_wt3_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm1 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm1 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm2 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm2 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm3 Sample treatment:DMSO Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm3 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm1_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm1_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm2_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm2_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm3_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm3_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm4_ep10 Sample treatment:10µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm4_ep10 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm1_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm1_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm2_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm2_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm3_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm3_ep40 SUBJECT_SAMPLE_FACTORS - MCF001428_pmm4_ep40 Sample treatment:40µM Epalrestat Organism part=whole organism; Organism type=zebrafish; Organism=Danio rerio PMM2 sa10150 mutant; Chromatography=Reverse phase IP; RAW_FILE_NAME=MCF001428_pmm4_ep40 #COLLECTION CO:COLLECTION_SUMMARY 10 embryos per sample were homogenized by sonication in extraction buffer (80% CO:COLLECTION_SUMMARY MeOH, IS). The metabolites were then precipitated at -80 °C overnight, after CO:COLLECTION_SUMMARY which the samples were centrifuged at 15,000 rpm, 20 min, 4 °C. Next, the CO:COLLECTION_SUMMARY supernatant was transferred to a fresh Eppendorf tube. The samples were CO:COLLECTION_SUMMARY centrifuged again at 15,000 rpm, 20 min, 4 °C to remove any leftover impurities CO:COLLECTION_SUMMARY and transferred to a fresh MS vial. Finally, the samples were analyzed by LC/MS CO:SAMPLE_TYPE Larvae CO:STORAGE_CONDITIONS -80? #TREATMENT TR:TREATMENT_SUMMARY Zebrafish were treated with vehicle or 10 or 40 microM epalrestat #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 10 embryos per sample were homogenized by sonication in extraction buffer (80% SP:SAMPLEPREP_SUMMARY MeOH, IS). The metabolites were then precipitated at -80 °C overnight, after SP:SAMPLEPREP_SUMMARY which the samples were centrifuged at 15,000 rpm, 20 min, 4 °C. Next, the SP:SAMPLEPREP_SUMMARY supernatant was transferred to a fresh Eppendorf tube. The samples were SP:SAMPLEPREP_SUMMARY centrifuged again at 15,000 rpm, 20 min, 4 °C to remove any leftover impurities SP:SAMPLEPREP_SUMMARY and transferred to a fresh MS vial. Finally, the samples were analyzed by LC/MS SP:PROCESSING_STORAGE_CONDITIONS -80? SP:EXTRACTION_METHOD 80 % MeOH with Internal standards (e.g. d27 myristic acid) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HILIC CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Agilent Infinity CH:COLUMN_NAME Poroshell 120 HILIC-Z PEEK Column 2.1x 150mm, 5 um CH:SOLVENT_A 100% acetonitrile CH:SOLVENT_B 100% water; 10 mM sodium acetate, pH 9.3 CH:FLOW_GRADIENT A linear gradient was carried out starting with 90% solvent A and 10% solvent B. CH:FLOW_GRADIENT From 2 to 12 min the gradient changed to 60% B. The gradient was kept on 60% B CH:FLOW_GRADIENT for 3 minutes and followed by a decrease to 10% B. The chromatography was CH:FLOW_GRADIENT stopped at 25 min CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Focus MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS El-Maven polly, ThemoFisher Xcalibur Metabolites were annotated using in-house MS:MS_COMMENTS metabolite library- elution time and m/z were considered #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples MCF001027_wt1 MCF001027_wt2 MCF001027_wt3 MCF001027_wt4 MCF001027_wt1_ep10 MCF001027_wt2_ep10 MCF001027_wt1_ep40 MCF001027_wt2_ep40 MCF001027_wt3_ep40 MCF001027_pmm1 MCF001027_pmm2 MCF001027_pmm3 MCF001027_pmm4 MCF001027_pmm1_ep10 MCF001027_pmm2_ep10 MCF001027_pmm1_ep40 MCF001027_pmm2_ep40 MCF001027_pmm3_ep40 MCF001027_pmm4_ep40 wt1 wt2 wt3 wt1_ep10 wt2_ep10 wt3_ep10 wt4_ep10 pmm1 pmm2 pmm3 pmm4 pmm1_ep10 pmm2_ep10 pmm3_ep10 pmm4_ep10 wt4 wt5 wt1_ep40 wt2_ep40 wt3_ep40 pmm5 pmm6 pmm7 pmm8 pmm1_ep40 pmm2_ep40 pmm3_ep40 pmm4_ep40 Factors Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:10µM Epalrestat Sample treatment:DMSO Sample treatment:DMSO Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:DMSO Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sample treatment:40µM Epalrestat Sorbitol 1.26E+06 1.40E+06 1.17E+06 9.85E+05 9.05E+05 1.35E+06 8.93E+05 8.26E+05 1.04E+06 1.49E+06 1.43E+06 1.48E+06 1.41E+06 1.42E+06 1.61E+06 1.28E+06 1.03E+06 1.50E+06 7.78E+05 3.06E+06 3.79E+06 2.92E+06 3.07E+06 3.04E+06 3.17E+06 2.84E+06 3.46E+06 3.50E+06 4.48E+06 3.08E+06 3.16E+06 2.98E+06 3.36E+06 2.95E+06 2.82E+06 2.91E+06 2.03E+06 1.90E+06 2.00E+06 2.43E+06 3.31E+06 2.77E+06 2.66E+06 2.19E+06 1.97E+06 1.80E+06 1.96E+06 GDP-mannose 3.03E+05 1.81E+05 1.76E+05 2.27E+05 3.16E+05 3.11E+05 2.82E+05 3.01E+05 3.46E+05 5.35E+04 9.92E+04 7.64E+04 6.00E+04 1.39E+05 1.44E+05 1.21E+05 1.44E+05 1.90E+05 2.99E+05 8.37E+05 8.28E+05 6.91E+05 7.77E+05 7.37E+05 7.90E+05 7.16E+05 5.19E+05 5.29E+05 3.44E+05 3.38E+05 5.50E+05 5.07E+05 5.30E+05 3.66E+05 6.76E+05 5.95E+05 5.19E+05 6.18E+05 6.81E+05 4.80E+05 3.77E+05 4.50E+05 4.25E+05 4.23E+05 4.58E+05 3.08E+05 3.63E+05 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 GDP-mannose C16H25N5O16P2 604.06897 HMDB01163 C12 PARENT m0 METABOLITES_END #END