#METABOLOMICS WORKBENCH lincoln010_20210115_071855 DATATRACK_ID:2399 STUDY_ID:ST001670 ANALYSIS_ID:AN002725 VERSION 1 CREATED_ON 03-03-2022 #PROJECT PR:PROJECT_TITLE Reinforcing one-carbon metabolism via folic acid/Folr1 promotes beta-cell PR:PROJECT_TITLE formation PR:PROJECT_SUMMARY Diabetes can be caused by an insufficient beta-cell mass. Here, we performed a PR:PROJECT_SUMMARY genetic screen in a zebrafish model with beta-cell loss to identify pathways PR:PROJECT_SUMMARY promoting beta-cell regeneration. We found that both folate receptor 1 (folr1) PR:PROJECT_SUMMARY overexpression and treatment with folate intermediates, i.e. tetrahydrofolic PR:PROJECT_SUMMARY acid (THF) or folinic acid stimulated beta-cell differentiation in zebrafish. PR:PROJECT_SUMMARY Treatment with these folate intermediates also stimulated beta-cell PR:PROJECT_SUMMARY differentiation in neonatal pig islet cultures, showing the effect could be PR:PROJECT_SUMMARY translated to a mammalian system. In both zebrafish and neonatal pig islets, the PR:PROJECT_SUMMARY increased beta-cell differentiation originated from ductal cells. PR:PROJECT_SUMMARY Mechanistically, comparative metabolomics analyses of zebrafish in the basal PR:PROJECT_SUMMARY state vs following ?-cell ablation and in un-treated vs folinic acid-treated PR:PROJECT_SUMMARY zebrafish indicated beta-cell regeneration could be attributed to changes in the PR:PROJECT_SUMMARY pyrimidine, carnitine and serine pathways. Overall, our results suggest PR:PROJECT_SUMMARY evolutionary conserved and previously unknown roles of folic acid and one-carbon PR:PROJECT_SUMMARY metabolism in the generation of beta-cell . PR:INSTITUTE North Carolina State University PR:DEPARTMENT Molecular and Structural Biochemistry PR:LAST_NAME Duan PR:FIRST_NAME Likun PR:ADDRESS 120 W Broughton Dr. PR:EMAIL lduan2@ncsu.edu PR:PHONE 9195939853 PR:DOI http://dx.doi.org/10.21228/M80Q4N #STUDY ST:STUDY_TITLE Metabolomics characterization of zebrafish larvae ST:STUDY_TYPE MS analysis ST:STUDY_SUMMARY Four different treatment groups were used for metabolite characterization: 5 dpf ST:STUDY_SUMMARY larvae with/without beta-cell ablation and with/without folinic acid treatment. ST:INSTITUTE North Carolina State University ST:DEPARTMENT Molecular and Structural Biochemistry ST:LAST_NAME Duan ST:FIRST_NAME Likun ST:ADDRESS 120 W Broughton Dr. ST:EMAIL lduan2@ncsu.edu ST:PHONE 9195939853 ST:SUBMIT_DATE 2021-01-15 #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]Additional sample data SUBJECT_SAMPLE_FACTORS NA AA 02030 metabo control 001 Treatment:control RAW_FILE_NAME=AA 02030 metabo control 001; sample name(backup)=ND+MTZ-control A2 SUBJECT_SAMPLE_FACTORS NA AA 02030 metabo control 002 Treatment:control RAW_FILE_NAME=AA 02030 metabo control 002; sample name(backup)=ND+MTZ-control A3 SUBJECT_SAMPLE_FACTORS NA AA 02030 metabo control 003 Treatment:control RAW_FILE_NAME=AA 02030 metabo control 003; sample name(backup)=ND+MTZ-control A4 SUBJECT_SAMPLE_FACTORS NA AA 02030 metabo control 004 Treatment:control RAW_FILE_NAME=AA 02030 metabo control 004; sample name(backup)=ND+MTZ-control A5 SUBJECT_SAMPLE_FACTORS NA AA 02030 metabo control 005 Treatment:control RAW_FILE_NAME=AA 02030 metabo control 005; sample name(backup)=ND+MTZ-control A6 SUBJECT_SAMPLE_FACTORS Regulation CC 02030 metabo reg control 001 Treatment:control RAW_FILE_NAME=CC 02030 metabo reg control 001; sample name(backup)=Reg+MTZ-control C1 SUBJECT_SAMPLE_FACTORS Regulation CC 02030 metabo reg control 002 Treatment:control RAW_FILE_NAME=CC 02030 metabo reg control 002; sample name(backup)=Reg+MTZ-control C2 SUBJECT_SAMPLE_FACTORS Regulation CC 02030 metabo reg control 003 Treatment:control RAW_FILE_NAME=CC 02030 metabo reg control 003; sample name(backup)=Reg+MTZ-control C3 SUBJECT_SAMPLE_FACTORS Regulation CC 02030 metabo reg control 004 Treatment:control RAW_FILE_NAME=CC 02030 metabo reg control 004; sample name(backup)=Reg+MTZ-control C4 SUBJECT_SAMPLE_FACTORS Regulation CC 02030 metabo reg control 005 Treatment:control RAW_FILE_NAME=CC 02030 metabo reg control 005; sample name(backup)=Reg+MTZ-control C5 SUBJECT_SAMPLE_FACTORS Regulation CC 02030 metabo reg control 006 Treatment:control RAW_FILE_NAME=CC 02030 metabo reg control 006; sample name(backup)=Reg+MTZ-control C6 SUBJECT_SAMPLE_FACTORS NA BB 02030 metabo FA 001 Treatment:Folinic acid RAW_FILE_NAME=BB 02030 metabo FA 001; sample name(backup)=ND+MTZ-Folinic acid B2 SUBJECT_SAMPLE_FACTORS NA BB 02030 metabo FA 002 Treatment:Folinic acid RAW_FILE_NAME=BB 02030 metabo FA 002; sample name(backup)=ND+MTZ-Folinic acid B3 SUBJECT_SAMPLE_FACTORS NA BB 02030 metabo FA 003 Treatment:Folinic acid RAW_FILE_NAME=BB 02030 metabo FA 003; sample name(backup)=ND+MTZ-Folinic acid B4 SUBJECT_SAMPLE_FACTORS NA BB 02030 metabo FA 004 Treatment:Folinic acid RAW_FILE_NAME=BB 02030 metabo FA 004; sample name(backup)=ND+MTZ-Folinic acid B5 SUBJECT_SAMPLE_FACTORS NA BB 02030 metabo FA 005 Treatment:Folinic acid RAW_FILE_NAME=BB 02030 metabo FA 005; sample name(backup)=ND+MTZ-Folinic acid B6 SUBJECT_SAMPLE_FACTORS Regulation DD 02030 metabo reg FA 001 Treatment:Folinic acid RAW_FILE_NAME=DD 02030 metabo reg FA 001; sample name(backup)=Reg+MTZ-Folinic acid D2 SUBJECT_SAMPLE_FACTORS Regulation DD 02030 metabo reg FA 002 Treatment:Folinic acid RAW_FILE_NAME=DD 02030 metabo reg FA 002; sample name(backup)=Reg+MTZ-Folinic acid D3 SUBJECT_SAMPLE_FACTORS Regulation DD 02030 metabo reg FA 003 Treatment:Folinic acid RAW_FILE_NAME=DD 02030 metabo reg FA 003; sample name(backup)=Reg+MTZ-Folinic acid D4 SUBJECT_SAMPLE_FACTORS Regulation DD 02030 metabo reg FA 004 Treatment:Folinic acid RAW_FILE_NAME=DD 02030 metabo reg FA 004; sample name(backup)=Reg+MTZ-Folinic acid D5 SUBJECT_SAMPLE_FACTORS Regulation DD 02030 metabo reg FA 005 Treatment:Folinic acid RAW_FILE_NAME=DD 02030 metabo reg FA 005; sample name(backup)=Reg+MTZ-Folinic acid D6 #COLLECTION CO:COLLECTION_SUMMARY Four different treatment groups were used for metabolite characterization: 5 dpf CO:COLLECTION_SUMMARY larvae with/without beta-cell ablation and with/without folinic acid treatment. CO:COLLECTION_SUMMARY Please refer to the publication for more details. CO:SAMPLE_TYPE Larvae #TREATMENT TR:TREATMENT_SUMMARY Four different treatment groups were used for metabolite characterization: 5 dpf TR:TREATMENT_SUMMARY larvae with/without beta-cell ablation and with/without folinic acid treatment. TR:TREATMENT_SUMMARY Please refer to the publication for more details. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Four different treatment groups were used for metabolite characterization: 5 dpf SP:SAMPLEPREP_SUMMARY larvae with/without beta-cell ablation and with/without folinic acid treatment. SP:SAMPLEPREP_SUMMARY Metabolites were extracted from 5 pooled larvae, and 6 replicates were used per SP:SAMPLEPREP_SUMMARY condition. Metabolites were extracted as described previously36. Some of the SP:SAMPLEPREP_SUMMARY replicates were used to optimize the analysis of polar metabolites and folate SP:SAMPLEPREP_SUMMARY derivatives using Vanquish UHPLC (Thermo Fisher Scientific). Please refer to the SP:SAMPLEPREP_SUMMARY publication for more details. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY For this polar metabolite analysis, a hydrophilic interaction chromatography CH:CHROMATOGRAPHY_SUMMARY method (HILIC) with an Xbridge amide column (100 x 2.1 mm i.d., 3.5 µm; Waters) CH:CHROMATOGRAPHY_SUMMARY was used for compound separation at 25 °C. Mobile phase A: water with 5 mM CH:CHROMATOGRAPHY_SUMMARY ammonium acetate (pH 6.8), and mobile phase B: 100 % acetonitrile. Linear CH:CHROMATOGRAPHY_SUMMARY gradient was: 0 min, 85% B; 1.5 min, 85% B; 5.5 min, 35% B; 6.9 min, 35% B; 10.5 CH:CHROMATOGRAPHY_SUMMARY min, 35% B; 10.6 min, 10% B; 12.5 min, 10% B; 13.5 min, 85% B; 17.9 min, 85% B; CH:CHROMATOGRAPHY_SUMMARY 18 min, 85% B; 20 min, 85% B. The flow rate was: 0-5.5 min, 0.11 ml/min; CH:CHROMATOGRAPHY_SUMMARY 6.9-10.5 min, 0.13 ml/min; 10.6-17.9 min, 0.25 ml/min; 18-20 min, 0.11 ml/min. CH:INSTRUMENT_NAME Vanquish UHPLC (Thermo Fisher Scientific). CH:COLUMN_NAME Waters Xbridge amide HILIC (100 x 2.1 mm i.d., 3.5 um) CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 480 MS:INSTRUMENT_TYPE Ion trap MS:MS_TYPE ESI MS:MS_COMMENTS Xcalibur 4.4 from Thermo Scientific for data acquisition and we use Sieve 2.2 MS:MS_COMMENTS for chromatographic alignment and peak integration.To analyze polar metabolites, MS:MS_COMMENTS the mass spectrometer was equipped with a HESI probe and operated in the MS:MS_COMMENTS positive/negative switching mode. The relevant parameters were as listed: MS:MS_COMMENTS vaporizer temperature, 350 °C; ion transfer tube temperature, 300 °C; sheath MS:MS_COMMENTS gas, 35; auxiliary gas, 7; sweep gas, 1; spray voltage, 3.5 kV for positive mode MS:MS_COMMENTS and 2.5 kV for negative mode; RF-lens (%), 30. The resolution was set at 60,000 MS:MS_COMMENTS (at m/z 200). Automatic maximum injection time (max IT) and automated gain MS:MS_COMMENTS control (AGC) were used. MS:ION_MODE UNSPECIFIED #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples AA 02030 metabo control 001 AA 02030 metabo control 002 AA 02030 metabo control 003 AA 02030 metabo control 004 AA 02030 metabo control 005 CC 02030 metabo reg control 001 CC 02030 metabo reg control 002 CC 02030 metabo reg control 003 CC 02030 metabo reg control 004 CC 02030 metabo reg control 005 CC 02030 metabo reg control 006 BB 02030 metabo FA 001 BB 02030 metabo FA 002 BB 02030 metabo FA 003 BB 02030 metabo FA 004 BB 02030 metabo FA 005 DD 02030 metabo reg FA 001 DD 02030 metabo reg FA 002 DD 02030 metabo reg FA 003 DD 02030 metabo reg FA 004 DD 02030 metabo reg FA 005 Factors Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:control Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid 2-Hydroxyglutarate 6690079.0000 4253612.0000 5116746.0000 4736280.0000 3341251.0000 5361647.0000 3208305.0000 4721407.0000 4038608.0000 4226186.0000 4495506.0000 5041303.0000 4542172.0000 4430560.0000 4750040.0000 2679111.0000 5666072.0000 4615401.0000 4550775.0000 4578641.0000 3608272.0000 2-Methylcitric acid 2140049.0000 2122360.0000 1715945.0000 2019078.0000 1395908.0000 1530103.0000 1547196.0000 1705737.0000 1932343.0000 1647738.0000 1848029.0000 1664141.0000 1582984.0000 1780491.0000 1776569.0000 1389072.0000 2200368.0000 1933632.0000 1520456.0000 1658518.0000 1232244.0000 2-METHYLGLUTARATE 1549880.0000 1253381.0000 998834.0000 2503415.0000 1033425.0000 1424453.0000 1586007.0000 1273770.0000 1153241.0000 1264712.0000 1235367.0000 1233084.0000 1443467.0000 1448658.0000 1122395.0000 627238.0000 1171838.0000 1202090.0000 1269458.0000 1082232.0000 1122431.0000 2-PHOSPHOGLYCERATE/3-PHOSPHOGLYCERATE 7645229.0000 8181792.0000 7309472.0000 7496042.0000 4542252.0000 6119082.0000 6095593.0000 7819420.0000 6287776.0000 7839866.0000 7263630.0000 6636571.0000 6783830.0000 7656576.0000 7736440.0000 4485343.0000 6189936.0000 8154675.0000 6567473.0000 6230443.0000 4161181.0000 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 2-Hydroxyglutarate 2-Methylcitric acid 2-METHYLGLUTARATE 2-PHOSPHOGLYCERATE/3-PHOSPHOGLYCERATE METABOLITES_END #END