#METABOLOMICS WORKBENCH houyuanlong2020_20201209_193812 DATATRACK_ID:2346 STUDY_ID:ST001622 ANALYSIS_ID:AN002657 PROJECT_ID:000000 VERSION 1 CREATED_ON December 11, 2020, 10:20 am #PROJECT PR:PROJECT_TITLE Isotope Tracing Analysis in Intestinal Organoids PR:PROJECT_TYPE Isotope Tracing Analysis PR:PROJECT_SUMMARY To elucidate the mechanism by which increased fructose production favors ISC PR:PROJECT_SUMMARY proliferation, We performed untargeted metabolomics of intestinal organoids PR:PROJECT_SUMMARY treated with fructose (10 mM) for 24 hr. As expected,Typically, metabolites such PR:PROJECT_SUMMARY as glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), lactate, pyruvate, PR:PROJECT_SUMMARY citrate and glutamine were markedly enriched after fructose treatment.To gain PR:PROJECT_SUMMARY more functional insights, we further conducted isotope tracing analysis of PR:PROJECT_SUMMARY U-[13C]-fructose in intestinal organoids. [13C] tracing revealed that PR:PROJECT_SUMMARY fructose-treated organoids had higher percentage of fully labeled PR:PROJECT_SUMMARY F1P(M+6),lactate (M+3),G6P(M+6), F6P(M+6), supporting the enhancement of PR:PROJECT_SUMMARY glycolysis after fructose treatment Together, these results indicate that PR:PROJECT_SUMMARY fructose induces a metabolic reprogramming in intestinal organoids that favors PR:PROJECT_SUMMARY glycolysis. PR:INSTITUTE China Pharmaceutical University PR:DEPARTMENT School of Medicine PR:LABORATORY Metabonomics PR:LAST_NAME Hou PR:FIRST_NAME Yuanlong PR:ADDRESS tongjiaxiang, nanjing, jiangsu, 210000, China PR:EMAIL jian2103@163.com PR:PHONE 18851105337 PR:CONTRIBUTORS Xiao Zheng Haiping Hao #STUDY ST:STUDY_TITLE Isotope Tracing Analysis in Intestinal Organoids with fructose ST:STUDY_SUMMARY We isolated and cultured Intestinal Organoids from Mouse. To trace the ST:STUDY_SUMMARY metabolism of fructose, intestinal organoids were cultured in unlabeled ST:STUDY_SUMMARY fructose-containing DMEM/F12 medium (10 mM D-fructose, 0 mM D-glucose, 2.5 mM ST:STUDY_SUMMARY L-glutamine) for 12 hr, and then switched into 13C labeled fructose-containing ST:STUDY_SUMMARY medium (10 mM U-[13C] D-fructose, Sigma-aldrich) for 6 hr. ST:INSTITUTE China Pharmaceutical University ST:DEPARTMENT School of Medicine ST:LABORATORY Metabonomics ST:LAST_NAME Hou ST:FIRST_NAME Yuanlong ST:ADDRESS tongjiaxiang, nanjing, jiangsu, 210000, China ST:EMAIL jian2103@163.com ST:PHONE 18851105337 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 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 - 13C Fru1 Treatment:13C Fructose RAW_FILE_NAME=20200416-c13 FRUCTOSE1 SUBJECT_SAMPLE_FACTORS - 13C Fru2 Treatment:13C Fructose RAW_FILE_NAME=20200416-c13 FRUCTOSE2 SUBJECT_SAMPLE_FACTORS - 13C Fru3 Treatment:13C Fructose RAW_FILE_NAME=20200416-c13 FRUCTOSE3 SUBJECT_SAMPLE_FACTORS - Fru1 Treatment:Fructose RAW_FILE_NAME=20200416-FRUCTOSE1 SUBJECT_SAMPLE_FACTORS - Fru2 Treatment:Fructose RAW_FILE_NAME=20200416-FRUCTOSE2 SUBJECT_SAMPLE_FACTORS - Fru3 Treatment:Fructose RAW_FILE_NAME=20200416-FRUCTOSE3 #COLLECTION CO:COLLECTION_SUMMARY Isolation and Culture of Mouse Intestinal Organoids CO:SAMPLE_TYPE Intestine #TREATMENT TR:TREATMENT_SUMMARY For metabolic flux analysis of 13C-labeled glucose, intestinal organoids were TR:TREATMENT_SUMMARY pretreated with unlabeled fructose (10 mM) for 24 hr, washed with PBS, and then TR:TREATMENT_SUMMARY switched into 13C labeled glucose-containing DMEM/F12 medium (10 mM U-[13C] TR:TREATMENT_SUMMARY D-glucose, Cambridge Isotope Laboratories) for 6 hr. To trace the metabolism of TR:TREATMENT_SUMMARY fructose, intestinal organoids were cultured in unlabeled fructose-containing TR:TREATMENT_SUMMARY DMEM/F12 medium (10 mM D-fructose, 0 mM D-glucose, 2.5 mM L-glutamine) for 12 TR:TREATMENT_SUMMARY hr, and then switched into 13C labeled fructose-containing medium (10 mM U-[13C] TR:TREATMENT_SUMMARY D-fructose, Sigma-aldrich) for 6 hr. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY To harvest intracellular metabolites, organoid samples were washed with SP:SAMPLEPREP_SUMMARY pre-cooled PBS for three times and metabolism was quenched with 80% ice-cold SP:SAMPLEPREP_SUMMARY methanol. Pre-cooled methanol (1mL) with para-chlorophenylalanine (1 µM) as the SP:SAMPLEPREP_SUMMARY internal standard was added to each sample and incubated on ice for 10 min. SP:SAMPLEPREP_SUMMARY Sample lysates were then centrifuged at 4 °C for 15 min at 23,000 g. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME UPLC-Q-TOF-MS (Synapt G2si, waters) CH:COLUMN_NAME Waters XBridge Amide (100 x 4.6mm, 3.5um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt G2 Si QTOF MS:INSTRUMENT_TYPE GC-TOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS MS MassLynx v4.1 Quanlynx v4.1 #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS abundance MS_METABOLITE_DATA_START Samples Fru1 Fru2 Fru3 13C Fru1 13C Fru2 13C Fru3 Factors Treatment:Fructose Treatment:Fructose Treatment:Fructose Treatment:13C Fructose Treatment:13C Fructose Treatment:13C Fructose Fructose 6-Phosphate(M+0) 318734 293265 270236 96537 97564 108955 Fructose 6-Phosphate(M+1) 14012 12463 12974 8324 8304 9283 Fructose 6-Phosphate(M+2) 4852 4624 4007 6197 6261 5971 Fructose 6-Phosphate(M+3) 250 345 497 11197 12981 13175 Fructose 6-Phosphate(M+4) 184 214 131 7213 7846 7989 Fructose 6-Phosphate(M+5) 82 79 141 12276 14890 12295 Fructose 6-Phosphate(M+6) 100 100 100 77189 108767 80903 Glucose 6-Phosphate(M+0) 367399 389864 431494 148580 96667 142538 Glucose 6-Phosphate(M+1) 17803 17465 19004 12800 8929 12234 Glucose 6-Phosphate(M+2) 5325 5959 6558 8355 8137 6250 Glucose 6-Phosphate(M+3) 632 424 492 14609 13634 14043 Glucose 6-Phosphate(M+4) 230 287 211 9166 8061 8867 Glucose 6-Phosphate(M+5) 188 113 139 16590 18441 14581 Glucose 6-Phosphate(M+6) 388 259 308 126708 180706 91999 Fructose 1-Phosphate(M+0) 115938 94650 63186 85723 78094 71650 Fructose 1-Phosphate(M+1) 5398 4984 3630 5140 5397 4815 Fructose 1-Phosphate(M+2) 1089 1602 1977 2281 2416 1714 Fructose 1-Phosphate(M+3) 10 10 10 1924 3047 2389 Fructose 1-Phosphate(M+4) 130 20 10 1571 1866 1246 Fructose 1-Phosphate(M+5) 132 20 10 3028 3650 2770 Fructose 1-Phosphate(M+6) 169 220 100 28444 26499 33696 Lactate(M+0) 406188 396546 102658 102587 128709 129655 Lactate(M+1) 10006 9557 3952 7312 10131 8409 Lactate(M+2) 2868 2890 1552 10014 13233 11207 Lactate(M+3) 10 10 10 224952 313760 264830 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z HMDB ID Fructose 6-Phosphate(M+0) 8.82 259.0219 HMDB0000124 Fructose 6-Phosphate(M+1) 8.82 260.0252 HMDB0000124 Fructose 6-Phosphate(M+2) 8.82 261.0286 HMDB0000124 Fructose 6-Phosphate(M+3) 8.82 262.032 HMDB0000124 Fructose 6-Phosphate(M+4) 8.82 263.0353 HMDB0000124 Fructose 6-Phosphate(M+5) 8.82 264.0387 HMDB0000124 Fructose 6-Phosphate(M+6) 8.82 265.042 HMDB0000124 Glucose 6-Phosphate(M+0) 8.78 259.0219 HMDB0001401 Glucose 6-Phosphate(M+1) 8.78 260.0252 HMDB0001401 Glucose 6-Phosphate(M+2) 8.78 261.0286 HMDB0001401 Glucose 6-Phosphate(M+3) 8.78 262.032 HMDB0001401 Glucose 6-Phosphate(M+4) 8.78 263.0353 HMDB0001401 Glucose 6-Phosphate(M+5) 8.78 264.0387 HMDB0001401 Glucose 6-Phosphate(M+6) 8.78 265.042 HMDB0001401 Fructose 1-Phosphate(M+0) 8.7 259.0219 HMDB0001076 Fructose 1-Phosphate(M+1) 8.7 260.0252 HMDB0001076 Fructose 1-Phosphate(M+2) 8.7 261.0286 HMDB0001076 Fructose 1-Phosphate(M+3) 8.7 262.032 HMDB0001076 Fructose 1-Phosphate(M+4) 8.7 263.0353 HMDB0001076 Fructose 1-Phosphate(M+5) 8.7 264.0387 HMDB0001076 Fructose 1-Phosphate(M+6) 8.7 265.042 HMDB0001076 Lactate(M+0) 6.73 89.0239 HMDB0000190 Lactate(M+1) 6.73 90.0272 HMDB0000190 Lactate(M+2) 6.73 91.0306 HMDB0000190 Lactate(M+3) 6.73 92.0339 HMDB0000190 METABOLITES_END #END