#METABOLOMICS WORKBENCH gauravsarode143_20221222_105642 DATATRACK_ID:3668 STUDY_ID:ST002425 ANALYSIS_ID:AN003948 PROJECT_ID:PR001560 VERSION 1 CREATED_ON December 28, 2022, 8:51 am #PROJECT PR:PROJECT_TITLE Part 3 : Integrated gut microbiome and lipidomic analyses in animal models of PR:PROJECT_TITLE Wilson disease reveal a role of intestine ATP7B in copper-related metabolic PR:PROJECT_TITLE dysregulation PR:PROJECT_SUMMARY Although the main pathogenic mechanism of Wilson disease (WD) is related to PR:PROJECT_SUMMARY copper accumulation in the liver and brain, there is limited knowledge about the PR:PROJECT_SUMMARY role of ATP7B copper transporter in extra-hepatic organs, including the PR:PROJECT_SUMMARY intestine, and how it could affect metabolic manifestations of the disease. The PR:PROJECT_SUMMARY aims of the present study were to profile and correlate the gut microbiota and PR:PROJECT_SUMMARY lipidome in mouse models of WD, and to study the metabolic effects of PR:PROJECT_SUMMARY intestine-specific ATP7B deficiency in a newly generated mouse model. Animal PR:PROJECT_SUMMARY models of WD presented reduced gut microbiota diversity compared to mice with PR:PROJECT_SUMMARY normal copper metabolism. Comparative prediction analysis of the functional PR:PROJECT_SUMMARY metagenome showed the involvement of several pathways including amino acid, PR:PROJECT_SUMMARY carbohydrate, and lipid metabolisms. Lipidomic profiles showed dysregulated tri- PR:PROJECT_SUMMARY and diglyceride, phospholipid, and sphingolipid metabolism. When challenged with PR:PROJECT_SUMMARY a high-fat diet, Atp7bΔIEC mice confirmed profound deregulation of fatty acid PR:PROJECT_SUMMARY desaturation and sphingolipid metabolism pathways as well as altered APOB48 PR:PROJECT_SUMMARY distribution in intestinal epithelial cells. Gut microbiome and lipidomic PR:PROJECT_SUMMARY analyses reveal integrated metabolic changes underlying the systemic PR:PROJECT_SUMMARY manifestations of WD. Intestine-specific ATP7B deficit affects both intestine PR:PROJECT_SUMMARY and systemic response to high-fat challenge. WD is as systemic disease and PR:PROJECT_SUMMARY organ-specific ATP7B variants can explain the varied phenotypic presentations. PR:INSTITUTE University of California, Davis PR:DEPARTMENT Department of Internal Medicine, Division of Hepatology/Gastroenterology PR:LAST_NAME Sarode PR:FIRST_NAME Gaurav Vilas PR:ADDRESS 451 E. Health Sciences Dr. Genome and Biomedical Sciences Facility Room 6404A PR:ADDRESS Davis, CA 95616 PR:EMAIL gsarode@ucdavis.edu PR:PHONE 5307526715 PR:FUNDING_SOURCE National Institutes of Health grants R01DK104770 (V.M.) #STUDY ST:STUDY_TITLE Integrated gut microbiome and lipidomic analyses in animal models of Wilson ST:STUDY_TITLE disease reveal a role of intestine ATP7B in copper-related metabolic ST:STUDY_TITLE dysregulation ST:STUDY_SUMMARY Although the main pathogenic mechanism of Wilson disease (WD) is related to ST:STUDY_SUMMARY copper accumulation in the liver and brain, there is limited knowledge about the ST:STUDY_SUMMARY role of ATP7B copper transporter in extra-hepatic organs, including the ST:STUDY_SUMMARY intestine, and how it could affect metabolic manifestations of the disease. The ST:STUDY_SUMMARY aims of the present study were to profile and correlate the gut microbiota and ST:STUDY_SUMMARY lipidome in mouse models of WD, and to study the metabolic effects of ST:STUDY_SUMMARY intestine-specific ATP7B deficiency in a newly generated mouse model. Animal ST:STUDY_SUMMARY models of WD presented reduced gut microbiota diversity compared to mice with ST:STUDY_SUMMARY normal copper metabolism. Comparative prediction analysis of the functional ST:STUDY_SUMMARY metagenome showed the involvement of several pathways including amino acid, ST:STUDY_SUMMARY carbohydrate, and lipid metabolisms. Lipidomic profiles showed dysregulated tri- ST:STUDY_SUMMARY and diglyceride, phospholipid, and sphingolipid metabolism. When challenged with ST:STUDY_SUMMARY a high-fat diet, Atp7bΔIEC mice confirmed profound deregulation of fatty acid ST:STUDY_SUMMARY desaturation and sphingolipid metabolism pathways as well as altered APOB48 ST:STUDY_SUMMARY distribution in intestinal epithelial cells. Gut microbiome and lipidomic ST:STUDY_SUMMARY analyses reveal integrated metabolic changes underlying the systemic ST:STUDY_SUMMARY manifestations of WD. Intestine-specific ATP7B deficit affects both intestine ST:STUDY_SUMMARY and systemic response to high-fat challenge. WD is as systemic disease and ST:STUDY_SUMMARY organ-specific ATP7B variants can explain the varied phenotypic presentations. ST:INSTITUTE University of California, Davis ST:DEPARTMENT Internal Medicine ST:LABORATORY Medici's Lab ST:LAST_NAME Sarode ST:FIRST_NAME Gaurav Vilas ST:ADDRESS 451 E. Health Sciences Dr. Genome and Biomedical Sciences Facility Room 6404A ST:ADDRESS Davis, CA 95616 ST:EMAIL gsarode@ucdavis.edu ST:PHONE 5307526715 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #FACTORS #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 WT-IEC9-1 Shibata022 Treatment:WT 5001 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata022_MX671911_Plasma_posCSH_1-001.d SUBJECT_SAMPLE_FACTORS WT-IEC9-2 Shibata016 Treatment:WT 5001 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata016_MX671911_Plasma_posCSH_2-002.d SUBJECT_SAMPLE_FACTORS WT-IEC9-3 Shibata008 Treatment:WT 5001 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata008_MX671911_Plasma_posCSH_3-003.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-1 Shibata018 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata018_MX671911_Plasma_posCSH_4-004.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-2 Shibata015 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata015_MX671911_Plasma_posCSH_5-005.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-3 Shibata005 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata005_MX671911_Plasma_posCSH_6-006.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-4 Shibata007 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata007_MX671911_Plasma_posCSH_7-007.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-5 Shibata012 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata012_MX671911_Plasma_posCSH_8-008.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-6 Shibata014 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata014_MX671911_Plasma_posCSH_9-009.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-7 Shibata017 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata017_MX671911_Plasma_posCSH_10-010.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-8 Shibata006 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Plasma; RAW_FILE_NAME=Shibata006_MX671911_Plasma_posCSH_11-011.d SUBJECT_SAMPLE_FACTORS WT-IEC9-1 Shibata021 Treatment:WT 5001 Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata021_MX671911_Liver_posCSH_12-012.d SUBJECT_SAMPLE_FACTORS WT-IEC9-2 Shibata009 Treatment:WT 5001 Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata009_MX671911_Liver_posCSH_13-013.d SUBJECT_SAMPLE_FACTORS WT-IEC9-3 Shibata013 Treatment:WT 5001 Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata013_MX671911_Liver_posCSH_14-014.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-1 Shibata002 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata002_MX671911_Liver_posCSH_15-015.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-2 Shibata004 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata004_MX671911_Liver_posCSH_16-016.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-3 Shibata020 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata020_MX671911_Liver_posCSH_17-017.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-4 Shibata003 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata003_MX671911_Liver_posCSH_18-018.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-5 Shibata010 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata010_MX671911_Liver_posCSH_19-019.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-6 Shibata011 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata011_MX671911_Liver_posCSH_20-020.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-7 Shibata019 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata019_MX671911_Liver_posCSH_21-021.d SUBJECT_SAMPLE_FACTORS iWT-IEChf9-8 Shibata001 Treatment:iWT 60% kcal fat Species=Mouse; Organ=Liver; RAW_FILE_NAME=Shibata001_MX671911_Liver_posCSH_22-022_002.d SUBJECT_SAMPLE_FACTORS - PoolQC001 Treatment:preShibata001 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=MtdBlank001_MX671911_Plasma_posCSH_preShibata001.d SUBJECT_SAMPLE_FACTORS - PoolQC002 Treatment:postShibata011 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=MtdBlank002_MX671911_Plasma_posCSH_postShibata011.d SUBJECT_SAMPLE_FACTORS - PoolQC003 Treatment:postShibata011 Species=Mouse; Organ=Liver; RAW_FILE_NAME=MtdBlank003_MX671911_Liver_posCSH_postShibata011.d SUBJECT_SAMPLE_FACTORS - PoolQC004 Treatment:postShibata022 Species=Mouse; Organ=Liver; RAW_FILE_NAME=MtdBlank004_MX671911_Liver_posCSH_postShibata022.d SUBJECT_SAMPLE_FACTORS - MtdBlank001 Treatment:preShibata001 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=PoolQC001_MX671911_Plasma_posCSH_preShibata001.d SUBJECT_SAMPLE_FACTORS - MtdBlank002 Treatment:postShibata010 Species=Mouse; Organ=Plasma; RAW_FILE_NAME=PoolQC002_MX671911_Plasma_posCSH_postShibata010.d SUBJECT_SAMPLE_FACTORS - MtdBlank003 Treatment:postShibata020 Species=Mouse; Organ=Liver; RAW_FILE_NAME=PoolQC003_MX671911_Liver_posCSH_postShibata020.d SUBJECT_SAMPLE_FACTORS - MtdBlank004 Treatment:postShibata022 Species=Mouse; Organ=Liver; RAW_FILE_NAME=PoolQC004_MX671911_Liver_posCSH_postShibata022.d #COLLECTION CO:COLLECTION_SUMMARY The liver was isolated. Blood samples were centrifuged at 8,000 rpm for 10 CO:COLLECTION_SUMMARY minutes and the plasma was aliquoted. All samples were stored at -80°C until CO:COLLECTION_SUMMARY further analysis. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY From 8 weeks of age, tx-j, KO, and Atp7bΔIEC mice, and their respective TR:TREATMENT_SUMMARY controls, were either continued on LabDiet 5001 diet or switched to a 60% kcal TR:TREATMENT_SUMMARY fat diet (D12492, Research Diets, Inc., New Brunswick, NJ). After 8 days, mice TR:TREATMENT_SUMMARY had body weights measured then were anesthetized with isoflurane, bled TR:TREATMENT_SUMMARY retro-orbitally into K3EDTA collection tubes, euthanized by cervical TR:TREATMENT_SUMMARY dislocation, and the liver weighed and flash-frozen in liquid nitrogen #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Combine 120 mL of chilled MeOH/QC mix with 400 mL of chilled MTBE/Cholesterol SP:SAMPLEPREP_SUMMARY Ester 22:1 in a clean 500 mL stock bottle. Mix thoroughly by swirling or SP:SAMPLEPREP_SUMMARY stirring the plate and store at -20°C until use. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6530 CH:COLUMN_NAME Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 60% acetonitrile/40% water; 0.1% formic acid; 10 mM ammonium formate CH:SOLVENT_B 90% isopropanol/10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate CH:FLOW_GRADIENT 0 min 15% (B), 0–2 min 30% (B), 2–2.5 min 48% (B), 2.5–11 min 82% (B), CH:FLOW_GRADIENT 11–11.5 min 99% (B), 11.5–12 min 99% (B), 12–12.1 min 15% (B), 12.1–15 CH:FLOW_GRADIENT min 15% (B) CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 65°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Data are analyzed in a four-stage process.First, raw data are processed in an MS:MS_COMMENTS untargeted (qualitative) manner by Agilent’s software MassHunterQual to find MS:MS_COMMENTS peaks in up to 300 chromatograms. Peak features are then imported MS:MS_COMMENTS intoMassProfilerProfessional for peak alignments to seek which peaks are present MS:MS_COMMENTS in multiplechromatograms, using exclusion criteria by the minimumpercentage of MS:MS_COMMENTS chromatograms in which these peaks arepositively detected. We usually use 30% as MS:MS_COMMENTS minimumcriterion. In a tedious manual process, these peaks arethen collated and MS:MS_COMMENTS constrained into a MassHunterquantification method on the accurate mass MS:MS_COMMENTS precursorion level, using the MS/MS information and theLipidBlast library to MS:MS_COMMENTS identify lipids with manualconfirmation of adduct ions and spectral MS:MS_COMMENTS scoringaccuracy. MassHunter enables back-filling ofquantifications for peaks MS:MS_COMMENTS that were missed in theprimary peak finding process, hence yielding data MS:MS_COMMENTS setswithout missing values. The procedure is given in thepanel to the left as MS:MS_COMMENTS workflow diagram MS:MS_RESULTS_FILE ST002425_AN003948_Results.txt UNITS:Peak hieght Has m/z:Yes Has RT:No RT units:No RT data #END