#METABOLOMICS WORKBENCH tarling_vallim_BA_20241223_091537 DATATRACK_ID:5491 STUDY_ID:ST003641 ANALYSIS_ID:AN005979 PROJECT_ID:PR002251 VERSION 1 CREATED_ON 12-30-2024 #PROJECT PR:PROJECT_TITLE The Spatial Transcriptional Activity of Hepatic TCF7L2 Regulates Zonated PR:PROJECT_TITLE Metabolic Pathways that Contribute to Liver Fibrosis PR:PROJECT_SUMMARY Study used single nuclei genomics techniques to examine the spatial PR:PROJECT_SUMMARY transcriptional function of the transcription factor 7-like 2 (TCF7L2) in rodent PR:PROJECT_SUMMARY liver. Research aimed to determine the consequences of TCF7L2 transcriptional PR:PROJECT_SUMMARY inactivation on the metabolic architecture of the liver, and on the function of PR:PROJECT_SUMMARY key zonated metabolic pathways that influence the development of fibrotic liver PR:PROJECT_SUMMARY diseases. Dietary stress was investigated using the Gubra Amylin Nash (GAN) and PR:PROJECT_SUMMARY choline-deficient amino acid-defined high fat (CDAHFD) diets to investigate the PR:PROJECT_SUMMARY susceptibility of liver-specific TCF7L2 mutant mice (Hep-TCF7L2ÎDBD) compared PR:PROJECT_SUMMARY to control (TCF7L2LoxP/LoxP) mice in hepatic fibrosis. PR:INSTITUTE UT Health San Antonio PR:LAST_NAME Norton PR:FIRST_NAME Luke PR:ADDRESS 7703 Floyd Curl Drive, San Antonio, TX 78229 PR:EMAIL nortonl@uthscsa.edu PR:PHONE (210)-567-0739 PR:DOI http://dx.doi.org/10.21228/M8NV72 #STUDY ST:STUDY_TITLE The Spatial Transcriptional Activity of Hepatic TCF7L2 Regulates Zonated ST:STUDY_TITLE Metabolic Pathways that Contribute to Liver Fibrosis ST:STUDY_SUMMARY Background and Aims: The molecular mechanisms regulating the zonal distribution ST:STUDY_SUMMARY of metabolism in liver are incompletely understood. Here we used single nuclei ST:STUDY_SUMMARY genomics techniques to examine the spatial transcriptional function of the ST:STUDY_SUMMARY transcription factor 7-like 2 (TCF7L2) in rodent liver. We also determined the ST:STUDY_SUMMARY consequences of TCF7L2 transcriptional inactivation on the metabolic ST:STUDY_SUMMARY architecture of the liver, and on the function of key zonated metabolic pathways ST:STUDY_SUMMARY that influence the development of fibrotic liver diseases. Methods: Multimodal ST:STUDY_SUMMARY 10X single nuclei RNA- and ATAC-Seq were used to dissect the cell-type specific ST:STUDY_SUMMARY expression and DNA binding activity of TCF7L2 and transcriptional co-regulators ST:STUDY_SUMMARY across the liver lobule. The transcriptional activity of TCF7L2 was targeted by ST:STUDY_SUMMARY excising exon 11 of Tcf7l2, which encodes part of the DNA binding domain (DBD). ST:STUDY_SUMMARY The consequences of TCF7L2 inactivation on zonation was investigated with a ST:STUDY_SUMMARY focus on the disruption to zonated metabolic pathways, including those linked to ST:STUDY_SUMMARY ammonia detoxification and glutamine metabolism, cholesterol homeostasis and ST:STUDY_SUMMARY bile acid synthesis. The susceptibility of Hep-TCF7L2ÎDBD mice to hepatic ST:STUDY_SUMMARY fibrosis under dietary stress was investigated using the Gubra Amylin Nash (GAN) ST:STUDY_SUMMARY and choline-deficient amino acid-defined high fat (CDAHFD) diets. Results: ST:STUDY_SUMMARY Tcf7l2 mRNA expression was ubiquitous across the liver lobule, but accessibility ST:STUDY_SUMMARY of the consensus TCF/LEF DNA binding motif was restricted to pericentral (PC) ST:STUDY_SUMMARY hepatocytes in zone 3. PC hepatocyte-specific gene expression was lost in ST:STUDY_SUMMARY Hep-TCF7L2ÎDBD mice, which we link to alterations in the transcriptional ST:STUDY_SUMMARY activity of zonal repressors Tbx3 and Tcf7l1. The absence of a classic zone 3 ST:STUDY_SUMMARY gene expression program in TCF7L2 mutant mice led to hepatic cholesterol ST:STUDY_SUMMARY accumulation, impaired bile acid synthesis, and significant disruptions to ST:STUDY_SUMMARY glutamine and glutamate homeostasis. In human liver biopsy tissue, TCF7L2 ST:STUDY_SUMMARY expression declined as the severity of fibrosis progressed, and Hep-TCF7L2ÎDBD ST:STUDY_SUMMARY mice developed more severe hepatic fibrosis when fed the CDAHFD. Conclusion: ST:STUDY_SUMMARY TCF7L2 is key transcriptional regulator of the PC hepatocyte gene expression ST:STUDY_SUMMARY program in zone 3, and regulates multiple zonated metabolic pathways that may ST:STUDY_SUMMARY contribute to the development of fibrotic liver diseases. ST:INSTITUTE UT Health San Antonio ST:LAST_NAME Norton ST:FIRST_NAME Luke ST:ADDRESS 7703 Floyd Curl Drive, San Antonio, TX 78229 ST:EMAIL nortonl@uthscsa.edu ST:PHONE (210)-567-0739 ST:SUBMIT_DATE 2024-12-23 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 6697 10_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-10.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8005 11_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-11.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8015 12_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-12.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8152 13_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-13.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8156 14_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-14.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8160 15_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-15.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8164 16_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-16.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6682 9_B Sample source:Gallbladder | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-9.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8006 1_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-1.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8402 2_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-2_20220425171539.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8124 3_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-3.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8125 4_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-4.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8153 5_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-5.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8154 6_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-6.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8155 7_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-7.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8172 8_B Sample source:Gallbladder | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=1in2k-2ul-Norton-Bile-8.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6697 10_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-10_20220411222209.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8005 11_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-11.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8015 12_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-12.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8152 13_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-13.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8156 14_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-14.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8160 15_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-15.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8164 16_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-16.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6682 9_I Sample source:Intestine | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-09.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8006 1_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-01.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8402 2_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-02.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8124 3_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-03.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8125 4_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-04.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8153 5_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-05.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8154 6_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-06.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8155 7_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-07.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 8172 8_I Sample source:Intestine | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=NORTON-INT-08.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=- SUBJECT_SAMPLE_FACTORS 6697 10_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-10.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-10.mzXML SUBJECT_SAMPLE_FACTORS 8005 11_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-11.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-11.mzXML SUBJECT_SAMPLE_FACTORS 8015 12_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-12.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-12.mzXML SUBJECT_SAMPLE_FACTORS 8152 13_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-13.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-13.mzXML SUBJECT_SAMPLE_FACTORS 8156 14_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-14.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-14.mzXML SUBJECT_SAMPLE_FACTORS 8160 15_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-15.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-15.mzXML SUBJECT_SAMPLE_FACTORS 8164 16_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-16.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-16.mzXML SUBJECT_SAMPLE_FACTORS 6682 9_L Sample source:Liver | Genotype:Mutant RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-9.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-09.mzXML SUBJECT_SAMPLE_FACTORS 8006 1_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-1.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-01.mzXML SUBJECT_SAMPLE_FACTORS 8402 2_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-2.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-02.mzXML SUBJECT_SAMPLE_FACTORS 8124 3_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-3.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-03.mzXML SUBJECT_SAMPLE_FACTORS 8125 4_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-4.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-04.mzXML SUBJECT_SAMPLE_FACTORS 8153 5_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-5.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-05.mzXML SUBJECT_SAMPLE_FACTORS 8154 6_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-6.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-06.mzXML SUBJECT_SAMPLE_FACTORS 8155 7_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-7.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-07.mzXML SUBJECT_SAMPLE_FACTORS 8172 8_L Sample source:Liver | Genotype:Wild-type RAW_FILE_NAME(RAW_File-Name-BileAcids)=Norton-Liver-8.mzXML; RAW_FILE_NAME(RAW_File-Name-C4)=C4-Rerun-10ul-NORTON-LIVER-08.mzXML #COLLECTION CO:COLLECTION_SUMMARY Tissues were collected from control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 CO:COLLECTION_SUMMARY mutant mice (Hep-TCF7L2ÎDBD) then stored at -80âC until further analysis was CO:COLLECTION_SUMMARY performed. CO:SAMPLE_TYPE Liver, Intestine, Gallbladder #TREATMENT TR:TREATMENT_SUMMARY Wild-type control (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice TR:TREATMENT_SUMMARY (Hep-TCF7L2ÎDBD) were fed methionine and choline deficient, 60% HFD diet TR:TREATMENT_SUMMARY (CDAHFD, Research Diets, Cat#: A06071302) for 8-weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Gallbladder, liver and intestine were isolated from Wild-type control SP:SAMPLEPREP_SUMMARY (TCF7L2LoxP/LoxP) and liver-specific TCF7L2 mutant mice (Hep-TCF7L2ÎDBD). Bile SP:SAMPLEPREP_SUMMARY acids were extracted from liver and intestine in Optima Methanol containing SP:SAMPLEPREP_SUMMARY recovery bile acid spikes (G-UDCA, G-CDCA) overnight at room temp. The following SP:SAMPLEPREP_SUMMARY day, samples were centrifuged at 4000rpm for 10min. Prior to analysis, bile was SP:SAMPLEPREP_SUMMARY diluted 1:1000 into Optima Methanol, then further diluted 1:2 into autosampler SP:SAMPLEPREP_SUMMARY vials. Liver supernatant was vortexed then filtered through a 0.45 µm SP:SAMPLEPREP_SUMMARY syringe-driven filter unit directly into autosampler vials. Intestine SP:SAMPLEPREP_SUMMARY supernatant was thoroughly vortexed, then an aliquot centrifuged at 15,000xg for SP:SAMPLEPREP_SUMMARY 5min. Intestine samples were then diluted 1:20 directly into autosampler vials. SP:SAMPLEPREP_SUMMARY For bile acid analysis, bile, intestine (2uL) and liver (5uL) were injected via SP:SAMPLEPREP_SUMMARY an Ultimate3000 autosampler module. For C4 analysis, 10uL of liver sample was SP:SAMPLEPREP_SUMMARY injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Separation and detection of bile acids using a UHPLC gradient method. Duration = CH:CHROMATOGRAPHY_SUMMARY 33min. Please see ETV-Lab-Bile-Acids-Methods.pdf for details. CH:METHODS_FILENAME ETV-Lab-Bile-Acids-Methods.pdf CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME ACE Excel C18-PFP (150 x 2.1mm, 2um, 100à ) CH:COLUMN_TEMPERATURE 50 CH:FLOW_GRADIENT 0.325 ml/min: 0min: 26% B, 74% A, 5min: 27.5% B, 72.5% A, 14min: 40% B, 60% A, CH:FLOW_GRADIENT 22min: 85% B, 15% A, 22.1min: 98% B, 2% A, 25.1min 98%, 2% A; 0.2 ml/min: 26min: CH:FLOW_GRADIENT 100% C, 27.9min: 100% C; 0.3 ml/min: 29min: 28.5% B, 71.5% A; 0.325 ml/min: CH:FLOW_GRADIENT 30-33min: 28.5% B, 71.5% A CH:FLOW_RATE 0.325 mL/min CH:INJECTION_TEMPERATURE 5 CH:SOLVENT_A Water 100%; 0.1% formic acid; 10 mM Ammonium acetate CH:SOLVENT_B 25% Methanol/75% Acetonitrile; 0.1% formic acid; Ammonium acetate (10mM) CH:CHROMATOGRAPHY_TYPE Reversed phase CH:SOLVENT_C 100% Methanol #ANALYSIS AN:LABORATORY_NAME Tarling-Vallim Lab, University of California, Los Angeles AN:ANALYSIS_TYPE MS AN:OPERATOR_NAME Madelaine Sholto #MS MS:INSTRUMENT_NAME Thermo TSQ Quantiva MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS Bile acid concentrations (nM) were quantified in Skyline-daily software MS:MS_COMMENTS (MacCross Lab Software) from standard curves of each individual bile acid MS:MS_COMMENTS generated from a mix of purified bile acid standards, with the exception of MS:MS_COMMENTS T-a-MCA, T-b-MCA, and T-w-MCA where T-CA was used. Total bile acid levels (nmol) MS:MS_COMMENTS per tissue were determined per intestine (g), whole liver weight (g), and MS:MS_COMMENTS gallbladder volume (uL). MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol/g dry weight (liver, intestine), nmol/uL (gallbladder) MS_METABOLITE_DATA_START Samples 10_B 11_B 12_B 13_B 14_B 15_B 16_B 9_B 1_B 2_B 3_B 4_B 5_B 6_B 7_B 8_B 10_I 11_I 12_I 13_I 14_I 15_I 16_I 9_I 1_I 2_I 3_I 4_I 5_I 6_I 7_I 8_I 10_L 11_L 12_L 13_L 14_L 15_L 16_L 9_L 1_L 2_L 3_L 4_L 5_L 6_L 7_L 8_L Factors Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Mutant Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Gallbladder | Genotype:Wild-type Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Mutant Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Intestine | Genotype:Wild-type Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Mutant Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type Sample source:Liver | Genotype:Wild-type a-MCA 0.1512 0.3220 0.1328 0.5790 0.3718 0.5451 0.7680 0.1338 0.2880 0.8235 0.6531 0.4290 0.5616 0.2756 0.2583 0.0915 13.5800 14.1000 16.1400 52.2000 30.7700 19.4900 18.3700 8.7000 32.7700 101.1000 44.7100 20.6600 28.3000 45.7500 50.8500 33.8100 0.4835 1.0739 1.1056 1.2206 0.7153 1.0009 0.7473 1.3250 1.2505 3.6050 1.3868 2.1842 1.4119 1.7671 1.2481 2.0817 b-MCA 8.4528 6.8860 2.4112 17.4330 7.7275 11.7277 20.4288 7.6061 4.8100 8.9400 15.0108 4.2207 6.6240 4.9192 7.1526 1.2345 339.7700 262.6400 184.4100 1007.9000 721.0800 306.9500 288.8300 374.5600 312.1700 767.9000 576.0800 124.3600 205.5000 710.9300 610.6700 431.0100 22.3355 65.2357 46.1104 66.6409 32.1744 47.6074 44.5454 101.6562 54.6153 117.2971 43.1952 33.6877 47.5472 99.2603 58.4948 106.8846 CA 3.7392 4.2040 2.5920 9.9210 8.9199 10.7249 14.8560 3.6009 3.4900 12.3000 5.8905 3.0536 7.3472 1.2584 5.1009 0.7380 954.9200 551.2800 801.9600 1210.4800 1501.0700 1119.2200 901.6100 594.6900 810.1700 2422.7400 1423.0100 372.1000 1037.1600 1709.8200 1888.9500 850.7000 6.8317 3.7633 3.8391 5.9821 4.2512 13.7068 5.9261 4.9392 2.5521 28.0903 12.5971 2.4382 3.5339 5.4738 3.9114 3.8110 CDCA 2.3800 4.0300 3.4700 3.3200 2.2100 3.0400 3.0500 1.6300 3.2900 11.7300 3.5000 0.3300 4.9800 8.4200 10.1300 3.6700 DCA 0.6516 0.5850 0.3717 0.4428 0.3396 0.6249 0.6631 0.3850 0.3070 0.3775 0.4061 0.2180 0.4435 0.7288 0.6926 0.4390 2.8100 0.6500 1.9600 5.2700 7.4800 1.5500 2.4300 2.3600 3.0400 7.5600 0.9800 1.3200 4.8000 5.0200 15.8900 5.2600 HDCA 0.9300 0.9400 1.4500 0.2800 0.1500 0.1900 0.8600 0.1200 1.0500 0.8700 LCA T-3a12b 0.3317 0.2548 0.2133 0.1920 0.4304 0.1624 0.4296 0.1518 0.1220 0.0367 0.2087 0.0650 0.2411 0.0127 0.1900 0.0295 3.1700 2.5000 6.1400 1.4500 3.8100 0.4200 1.5300 2.8200 0.3300 0.0600 1.0800 3.3600 2.2200 2.8700 3.6400 T-3a7k 0.1822 0.3492 0.3229 0.3090 0.4326 0.4326 0.4250 0.2856 0.2160 0.3578 0.4131 0.3530 0.7787 0.9493 0.6854 0.3312 1.2900 0.6800 6.6400 1.4400 1.7500 1.7600 2.3800 1.0700 4.5900 3.8900 2.6300 6.0100 6.1200 5.8900 4.4100 9.8800 T-aMCA 4.6284 9.9940 5.1528 6.2557 8.0883 8.5215 7.5012 6.4258 8.3300 8.8635 4.1496 8.6004 14.3792 23.0945 18.4737 7.4527 37.7200 23.0200 138.9400 43.2500 65.2800 72.7700 61.7600 51.2500 225.8300 196.9300 77.3600 148.4100 189.2000 242.8900 181.3100 274.7200 1.9646 1.2990 0.4910 1.2749 0.6756 1.9276 1.6378 4.0809 T-bMCA 317.5812 499.5860 217.1928 320.9385 384.1420 465.4706 494.3040 517.1686 216.0000 196.6358 180.5275 231.9273 383.3592 647.3870 554.6300 202.8060 3230.8100 2531.7100 6107.3600 2887.4800 3432.7500 4374.2500 4128.2700 4147.7300 5658.9700 4470.6300 3138.8900 4252.5300 5062.3000 6344.1200 5661.1900 7901.4800 74.3099 44.2671 107.5468 138.2425 77.1194 152.1425 44.7806 54.0400 178.3227 156.6287 199.4827 92.0721 89.0706 361.9574 148.5416 183.3252 T-CA 216.9876 310.2890 177.4600 178.3245 343.0526 372.5206 347.0160 268.3241 158.0000 124.6732 201.9339 134.9931 396.2336 424.5683 414.2219 151.1070 1738.2200 982.4900 2983.1200 786.5300 1881.2000 1912.3000 2251.4800 1749.2400 3377.2200 890.0700 1288.1700 2177.7100 3129.2600 2656.1500 2250.9500 3794.5000 80.8296 81.3671 132.0977 120.7526 85.6454 154.3249 78.3412 103.0713 152.1380 216.8989 138.1165 45.0709 110.5150 198.1758 125.1073 265.0472 T-CDCA 1.3800 2.3260 2.2784 1.4325 1.8898 2.6151 1.9752 1.5291 2.4200 1.8645 1.7598 2.1923 5.0736 4.3342 4.7565 2.2710 5.1500 40.1800 8.6100 3.7100 2.9500 57.9700 22.7300 2.1500 36.7200 47.3700 44.4300 30.8000 69.9600 2.6516 1.2426 4.6124 1.5636 2.5642 3.2271 2.8220 1.5955 5.8699 7.8818 2.7735 5.5875 5.5799 7.7581 6.4863 8.0406 T-DCA 1.2240 1.3260 1.8736 1.5870 2.5157 0.6693 1.8312 1.3969 1.5300 1.0605 0.2289 1.2320 5.5968 4.0872 6.4743 2.0100 13.7800 0.6800 9.8800 27.2500 17.0100 42.4300 16.2300 36.8300 49.1000 4.6012 3.9053 4.0019 4.7413 4.7911 3.2818 4.2852 4.0929 12.8882 11.2413 2.5051 6.8066 15.0487 18.5332 13.0709 12.4464 T-HDCA 0.1536 0.0440 0.4256 0.2040 0.1760 0.0230 0.3216 0.2341 0.3210 0.0255 0.2695 0.5328 1.1284 0.9933 0.4770 3.1200 0.9200 10.0200 4.8600 0.9100 2.1100 3.1400 2.4900 8.9400 2.4900 0.3100 8.5500 13.0900 11.5800 13.0500 20.0800 T-LCA 0.0151 0.0150 0.0392 0.0186 0.0219 0.0175 0.0158 0.0251 0.0138 0.0103 0.0033 0.0731 0.0575 0.0884 0.0343 0.4300 0.4600 0.6200 0.4800 0.5500 0.4300 0.4600 0.3600 0.6300 0.6700 0.3000 0.5500 0.8600 0.6900 0.8500 1.0800 0.0550 0.0475 0.0757 0.0216 0.0864 0.0784 0.0153 0.0672 0.0286 0.0630 0.1471 0.1638 0.0688 0.1652 T-UDCA 1.7640 3.6480 2.2512 2.2770 3.6630 3.3235 3.2016 3.0567 2.8800 2.1375 2.4927 2.2627 5.1344 7.5894 7.1190 2.8860 11.5200 4.3400 56.4800 3.4100 20.9100 14.0500 15.9200 17.0000 70.7300 37.9600 5.8000 32.1600 63.2300 63.2200 64.9600 95.8100 0.1872 0.3195 1.5465 0.9886 0.7828 1.7503 0.6271 1.6510 4.0579 3.8505 2.7556 0.5892 0.8962 4.4393 2.4962 1.6742 T-wMCA 16.4388 23.8450 12.2816 21.3607 27.9642 8.0626 24.9888 13.4870 18.9000 19.1663 3.7706 16.6155 35.4160 66.2207 64.8175 7.0537 165.5300 175.4200 364.9000 244.5900 386.8500 94.0900 265.0600 143.2000 629.0000 495.1300 72.6700 349.0600 544.5000 762.5500 815.3500 327.1500 3.1575 8.2418 4.5515 0.8936 4.2429 13.9935 3.3779 7.5497 28.0476 11.2970 UDCA 4.0200 4.4900 3.8100 8.8300 6.1600 1.9900 3.2300 5.1500 7.7700 16.9600 7.4100 3.5500 4.7200 14.5900 13.4400 9.1900 w-MCA 0.6960 0.5660 0.3088 1.9890 1.3354 0.3266 2.1480 0.4165 0.5370 1.7370 0.7581 0.7931 1.1184 0.6734 1.2600 0.0825 24.5300 19.8700 13.6100 106.9700 88.7000 8.6600 26.7000 16.7700 35.9300 133.2000 23.5300 14.9600 33.5700 92.7900 86.6500 21.2700 1.4038 2.7869 2.2112 4.0553 7.2473 1.8980 2.7802 2.5737 3.9430 11.9261 1.6104 2.3061 5.6052 5.6462 5.7394 3.4365 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 a-MCA 5283852 C17647                      407.28 b-MCA 5283853 C17726                      407.28 CA 221493 C00695                      407.28 CDCA 10133 C02528                      391.285 DCA 222528 C04483                      391.285 HDCA 5283820 C15517                      391.285 LCA 9903 C03990                      375.29 T-3a12b 498.289 T-3a7k 496.3 T-aMCA 13955653 514.284 T-bMCA 21124703 514.284 T-CA 6675 C05122                      514.284 T-CDCA 387316 C05465                      498.289 T-DCA 2733768 C05463                      498.289 T-HDCA 119046 498.289 T-LCA 439763 C02592                      482.42 T-UDCA 9848818 C16868                      498.289 T-wMCA 118703092 514.284 UDCA 31401 C07880                      391.285 w-MCA 5283851 C17727                      407.28 METABOLITES_END #END