#METABOLOMICS WORKBENCH lincoln010_20210115_071855 DATATRACK_ID:2399 STUDY_ID:ST001670 ANALYSIS_ID:AN002726 PROJECT_ID:PR001073 VERSION 1 CREATED_ON February 1, 2021, 2:22 pm #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 #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 #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 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 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 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 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 The analysis of folic acid and its derivatives was performed using Luna column CH:CHROMATOGRAPHY_SUMMARY (100 x 3 mm i.d., 3 µm, C18(2), 100 Å, Phenomenex) at 25 °C. Mobile phase A: CH:CHROMATOGRAPHY_SUMMARY water with 0.1% formic acid, and mobile phase B: 100 % methanol. Linear gradient CH:CHROMATOGRAPHY_SUMMARY was: 0 min, 2% B; 1.5 min, 2% B; 3.0 min, 15% B; 5.5 min, 95% B; 14.5 min, 95% CH:CHROMATOGRAPHY_SUMMARY B; 15.0 min, 2% B; 22.0 min, 2% B. The flow rate was 0.150 ml/min. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish UHPLC CH:COLUMN_NAME Luna C18 LC (150 x 4.6 mm, 3µm) CH:CHROMATOGRAPHY_COMMENTS Luna® 3 µm C18(2) 100 Å, LC Column 150 x 4.6 mm, Ea #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 480 MS:INSTRUMENT_TYPE Ion trap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED 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 measure folate MS:MS_COMMENTS derivatives, Orbitrap Exploris 480 was operated in SIM mode (selected ion MS:MS_COMMENTS monitoring) targeting the following ions: folic acid (442.1470), MS:MS_COMMENTS 5-methyltetrahydrofolate (460.1939), and folinic acid (474.1732). #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 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 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 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:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid Treatment:Folinic acid 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 Folate 63243 103420 83131 82691 84651 93701 43257 42328 50610 44157 104392 49820 81253 58467 60879 54007 74655 66689 58274 52330 65491 Folinic acid 84996 36087 25231 51105 76171 1006124 185766 268170 159763 227629 33254 58233 97169 62200 77590 25840 411439 173715 174951 201294 242931 5-Methyltetrahydrofolate 56669 80299 90421 85669 91775 68142 71676 78651 81315 65435 102019 86394 86782 92486 100866 65445 123958 103408 103228 83485 68143 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Folate Folinic acid 5-Methyltetrahydrofolate METABOLITES_END #END