#METABOLOMICS WORKBENCH xliu68_20220330_125443_mwtab.txt DATATRACK_ID:3145 STUDY_ID:ST002119 ANALYSIS_ID:AN003469 PROJECT_ID:PR001344 VERSION 1 CREATED_ON March 31, 2022, 8:01 am #PROJECT PR:PROJECT_TITLE MNK2 deficiency potentiates beta-cell regeneration via hypertranslation PR:PROJECT_SUMMARY Regenerating pancreatic beta-cells is a potential curative approach for PR:PROJECT_SUMMARY diabetes. We previously identified the small molecule CID661578 as a potent PR:PROJECT_SUMMARY inducer of beta-cell regeneration but its target and mechanism of action have PR:PROJECT_SUMMARY remained unknown. We now screened 257 million yeast clones and determined that PR:PROJECT_SUMMARY CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an PR:PROJECT_SUMMARY interaction we genetically validated in vivo. CID661578 increased beta-cell PR:PROJECT_SUMMARY neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and PR:PROJECT_SUMMARY human pancreatic ductal organoids. Mechanistically, we found that CID661578 PR:PROJECT_SUMMARY boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in PR:PROJECT_SUMMARY the translation initiation complex at the mRNA cap. Unexpectedly, this blocking PR:PROJECT_SUMMARY activity augmented eIF4E-phosphorylation depending on MNK1 and bolstered the PR:PROJECT_SUMMARY interaction between eIF4E and eIF4G, which is necessary for both PR:PROJECT_SUMMARY hypertranslation and beta-cell regeneration. Taken together, our findings PR:PROJECT_SUMMARY demonstrate a targetable role of MNK2-controlled translation in beta-cell PR:PROJECT_SUMMARY regeneration, a role that warrants further investigation in diabetes. PR:INSTITUTE North Carolina State University PR:LAST_NAME Liu PR:FIRST_NAME Xiaojing PR:ADDRESS Polk Hall, RM 128 PR:EMAIL xliu68@ncsu.edu PR:PHONE 9195154387 #STUDY ST:STUDY_TITLE Metabolomics analysis of zebrafish response to CID661578 treatment ST:STUDY_SUMMARY zebrafish larvae were treated with DMSO or CID661578 for 24 hours prior to ST:STUDY_SUMMARY global metabolomics analysis (n=6). Metabolites were extracted from pools of 10 ST:STUDY_SUMMARY zebrafish larvae at 5 dpf using a 80% methanol-based extraction method. Samples ST:STUDY_SUMMARY were dried in speed vac and stored in -80C freezer until ready for LC-MS ST:STUDY_SUMMARY analysis. ST:INSTITUTE North Carolina State University ST:LAST_NAME Liu ST:FIRST_NAME Xiaojing ST:ADDRESS Polk Hall, RM 128 ST:EMAIL xliu68@ncsu.edu ST:PHONE 9195154387 #SUBJECT SU:SUBJECT_TYPE Fish SU:SUBJECT_SPECIES Danio rerio SU:TAXONOMY_ID 7955 #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 - DMSO1 Treatment:Control RAW_FILE_NAME=DMSO1.raw SUBJECT_SAMPLE_FACTORS - DMSO2 Treatment:Control RAW_FILE_NAME=DMSO2.raw SUBJECT_SAMPLE_FACTORS - DMSO3 Treatment:Control RAW_FILE_NAME=DMSO3.raw SUBJECT_SAMPLE_FACTORS - DMSO4 Treatment:Control RAW_FILE_NAME=DMSO4.raw SUBJECT_SAMPLE_FACTORS - DMSO5 Treatment:Control RAW_FILE_NAME=DMSO5.raw SUBJECT_SAMPLE_FACTORS - DMSO6 Treatment:Control RAW_FILE_NAME=DMSO6.raw SUBJECT_SAMPLE_FACTORS - CID1 Treatment:CID661578 RAW_FILE_NAME=CID1.raw SUBJECT_SAMPLE_FACTORS - CID2 Treatment:CID661578 RAW_FILE_NAME=CID2.raw SUBJECT_SAMPLE_FACTORS - CID3 Treatment:CID661578 RAW_FILE_NAME=CID3.raw SUBJECT_SAMPLE_FACTORS - CID4 Treatment:CID661578 RAW_FILE_NAME=CID4.raw SUBJECT_SAMPLE_FACTORS - CID5 Treatment:CID661578 RAW_FILE_NAME=CID5.raw SUBJECT_SAMPLE_FACTORS - CID6 Treatment:CID661578 RAW_FILE_NAME=CID6.raw SUBJECT_SAMPLE_FACTORS - DMSO1_Pos Treatment:Control RAW_FILE_NAME=DMSO1_Pos.raw SUBJECT_SAMPLE_FACTORS - DMSO2_Pos Treatment:Control RAW_FILE_NAME=DMSO2_Pos.raw SUBJECT_SAMPLE_FACTORS - DMSO3_Pos Treatment:Control RAW_FILE_NAME=DMSO3_Pos.raw SUBJECT_SAMPLE_FACTORS - DMSO4_Pos Treatment:Control RAW_FILE_NAME=DMSO4_Pos.raw SUBJECT_SAMPLE_FACTORS - DMSO5_Pos Treatment:Control RAW_FILE_NAME=DMSO5_Pos.raw SUBJECT_SAMPLE_FACTORS - DMSO6_Pos Treatment:Control RAW_FILE_NAME=DMSO6_Pos.raw SUBJECT_SAMPLE_FACTORS - CID1_Pos Treatment:CID661578 RAW_FILE_NAME=CID1_Pos.raw SUBJECT_SAMPLE_FACTORS - CID2_Pos Treatment:CID661578 RAW_FILE_NAME=CID2_Pos.raw SUBJECT_SAMPLE_FACTORS - CID3_Pos Treatment:CID661578 RAW_FILE_NAME=CID3_Pos.raw SUBJECT_SAMPLE_FACTORS - CID4_Pos Treatment:CID661578 RAW_FILE_NAME=CID4_Pos.raw SUBJECT_SAMPLE_FACTORS - CID5_Pos Treatment:CID661578 RAW_FILE_NAME=CID5_Pos.raw SUBJECT_SAMPLE_FACTORS - CID6_Pos Treatment:CID661578 RAW_FILE_NAME=CID6_Pos.raw SUBJECT_SAMPLE_FACTORS - DMSO1_Neg Treatment:Control RAW_FILE_NAME=DMSO1_Neg.raw SUBJECT_SAMPLE_FACTORS - DMSO2_Neg Treatment:Control RAW_FILE_NAME=DMSO2_Neg.raw SUBJECT_SAMPLE_FACTORS - DMSO3_Neg Treatment:Control RAW_FILE_NAME=DMSO3_Neg.raw SUBJECT_SAMPLE_FACTORS - DMSO4_Neg Treatment:Control RAW_FILE_NAME=DMSO4_Neg.raw SUBJECT_SAMPLE_FACTORS - DMSO5_Neg Treatment:Control RAW_FILE_NAME=DMSO5_Neg.raw SUBJECT_SAMPLE_FACTORS - DMSO6_Neg Treatment:Control RAW_FILE_NAME=DMSO6_Neg.raw SUBJECT_SAMPLE_FACTORS - CID1_Neg Treatment:CID661578 RAW_FILE_NAME=CID1_Neg.raw SUBJECT_SAMPLE_FACTORS - CID2_Neg Treatment:CID661578 RAW_FILE_NAME=CID2_Neg.raw SUBJECT_SAMPLE_FACTORS - CID3_Neg Treatment:CID661578 RAW_FILE_NAME=CID3_Neg.raw SUBJECT_SAMPLE_FACTORS - CID4_Neg Treatment:CID661578 RAW_FILE_NAME=CID4_Neg.raw SUBJECT_SAMPLE_FACTORS - CID5_Neg Treatment:CID661578 RAW_FILE_NAME=CID5_Neg.raw SUBJECT_SAMPLE_FACTORS - CID6_Neg Treatment:CID661578 RAW_FILE_NAME=CID6_Neg.raw #COLLECTION CO:COLLECTION_SUMMARY zebrafish larvae were treated with DMSO or CID661578 for 24 hours prior to CO:COLLECTION_SUMMARY global metabolomics analysis (n=6). Metabolites were extracted from pools of 10 CO:COLLECTION_SUMMARY zebrafish larvae at 5 dpf using a 80% methanol/water-based extraction method. CO:COLLECTION_SUMMARY Detailed metabolite extraction was described previously using 80% methanol/water CO:COLLECTION_SUMMARY as solvent (PMID: 24410464, PMID: 24894601, PMID: 25795660) and dry pellets were CO:COLLECTION_SUMMARY stored in -80 °C freezer until ready for LC-MS analysis. Pellets were CO:COLLECTION_SUMMARY reconstituted into 30 μL of sample solvent (water:methanol:acetonitrile, 2:1:1, CO:COLLECTION_SUMMARY v/v), and 5 μL was injected into the LC-MS. CO:SAMPLE_TYPE new #TREATMENT TR:TREATMENT_SUMMARY Chemicals (DMSO orCID661578) were added to the E3 medium for larvae to reach TR:TREATMENT_SUMMARY final concentration of 10 uM. Pools of 10 wild-type (WT) larvae at 5 dpf were TR:TREATMENT_SUMMARY used for each of the 6 independent biological replicates for DMSO or CID661578 TR:TREATMENT_SUMMARY treatment from 4-5 dpf. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Intracellular metabolites were harvested as described previously using 80% SP:SAMPLEPREP_SUMMARY methanol/water as solvent (PMID: 24410464, PMID: 24894601, PMID: 25795660) and SP:SAMPLEPREP_SUMMARY dry pellets were stored in -80 °C freezer until ready for LC-MS analysis. For SP:SAMPLEPREP_SUMMARY metabolite analysis, pellets were reconstituted into 30 μL of sample solvent SP:SAMPLEPREP_SUMMARY (water:methanol:acetonitrile, 2:1:1, v/v), and 5 μL was injected into the SP:SAMPLEPREP_SUMMARY LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HILIC method is for general metabolomics analysis. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters Xbridge amide (100 × 2.1 mm i.d., 3.5 μm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS It was operated in the full-scan mode (positive ion mode only) with the MS:MS_COMMENTS resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 MS:MS_COMMENTS (Thermo Scientific), and the integrated area under metabolite peak was used to MS:MS_COMMENTS compare the relative abundance of each metabolite in different samples in the MS:MS_COMMENTS same batch. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples DMSO1_Pos DMSO2_Pos DMSO3_Pos DMSO4_Pos DMSO5_Pos DMSO6_Pos CID1_Pos CID2_Pos CID3_Pos CID4_Pos CID5_Pos CID6_Pos Factors Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:CID661578 Treatment:CID661578 Treatment:CID661578 Treatment:CID661578 Treatment:CID661578 Treatment:CID661578 acetyl-CoA 133063.821 125855.106 79763.07 68044.05 96745.416 78967.134 88387.719 79334.541 135358.299 91848.972 96118.131 71600.565 Propanoyl-CoA 31221.399 43384.341 48726.195 13642.125 23289.864 20121.597 55769.403 50613.171 90764.583 196322.619 58947.492 42712.101 crotonoyl-CoA 0 0 0 0 0 0 0 0 0 0 0 0 isobutyryl-CoA 26807.508 40590.513 48781.239 17954.34 35660.157 50901.837 109079.532 62423.67 138773.781 91589.316 149320.842 114692.787 Isovaleryl-CoA 28256.169 48897.501 58418.079 25756.686 49561.011 52287.744 148009.854 107519.577 184149.873 204500.922 235056.744 203805.444 malonyl-CoA 5929.911 5647.824 6359.547 10227.117 12042.84 1005.96 11133.408 13926.138 8915.946 2133.798 6277.836 6003.504 (S)-3-hydroxybutanoyl-CoA 0 0 0 0 0 0 0 0 0 0 0 0 Succinyl-CoA 42573.408 55697.07 57417.471 53847.744 51236.475 48101.928 72384.771 62165.325 89462.058 138883.08 63569.85 51982.62 Hydroxymethylglutaryl-CoA 0 0 0 0 0 0 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Chemical formula acetyl-CoA C23H38N7O17P3S Propanoyl-CoA C24H40N7O17P3S crotonoyl-CoA C25H40N7O17P3S isobutyryl-CoA C25H42N7O17P3S Isovaleryl-CoA C26H44N7O17P3S malonyl-CoA C24H38N7O19P3S (S)-3-hydroxybutanoyl-CoA C25H42N7O18P3S Succinyl-CoA C25H40N7O19P3S Hydroxymethylglutaryl-CoA C27H44N7O20P3S METABOLITES_END #END