#METABOLOMICS WORKBENCH michaelsa93_20170623_104303 DATATRACK_ID:1079 STUDY_ID:ST000656 ANALYSIS_ID:AN001002 PROJECT_ID:PR000461 VERSION 1 CREATED_ON June 26, 2017, 10:32 am #PROJECT PR:PROJECT_TITLE Omega-6 and omega-3 oxylipins are implicated in soybean oil-induced obesity in PR:PROJECT_TITLE mice PR:PROJECT_SUMMARY In this study we have compared the metabolic effects of conventional soybean oil PR:PROJECT_SUMMARY to those of genetically modified Plenish soybean oil, that is low in linoleic PR:PROJECT_SUMMARY acid and high in oleic acid. This work builds on our previous study showing that PR:PROJECT_SUMMARY soybean oil, rich in polyunsaturated fats, is more obesogenic and diabetogenic PR:PROJECT_SUMMARY than coconut oil, rich in saturated fats (PMID: 26200659). Here, in order to PR:PROJECT_SUMMARY elucidate the mechanisms responsible for soybean oil induced obesity, we have PR:PROJECT_SUMMARY performed the first ever metabolomics (in plasma and liver) and proteomics on PR:PROJECT_SUMMARY the livers of mice fed the two soybean oil diets (plus those fed a high coconut PR:PROJECT_SUMMARY oil and Viv chow diet). Our results show that the new high oleic soybean oil PR:PROJECT_SUMMARY induces less obesity and adiposity than conventional soybean oil, but can cause PR:PROJECT_SUMMARY hepatomegaly and liver dysfunction. Metabolomic analysis reveals that the PR:PROJECT_SUMMARY hepatic and plasma metabolic profiles differ considerably between the two PR:PROJECT_SUMMARY soybean oils. Hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3 PR:PROJECT_SUMMARY (ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the PR:PROJECT_SUMMARY cytochrome P450/soluble epoxide hydrolase pathway were found to correlate PR:PROJECT_SUMMARY positively with obesity. PR:INSTITUTE University of California, Riverside PR:DEPARTMENT Cell Biology and Neuroscience PR:LAST_NAME Sladek PR:FIRST_NAME Frances PR:ADDRESS 2115 Biological Sciences Building,University of California, Riverside, CA PR:ADDRESS 92521-0314 PR:EMAIL frances.sladek@ucr.edu PR:PHONE 951-827-2264 #STUDY ST:STUDY_TITLE Omega-6 and omega-3 oxylipins are implicated in soybean oil-induced obesity in ST:STUDY_TITLE mice (part III) ST:STUDY_SUMMARY In this study we have compared the metabolic effects of conventional soybean oil ST:STUDY_SUMMARY to those of genetically modified Plenish soybean oil, that is low in linoleic ST:STUDY_SUMMARY acid and high in oleic acid. This work builds on our previous study showing that ST:STUDY_SUMMARY soybean oil, rich in polyunsaturated fats, is more obesogenic and diabetogenic ST:STUDY_SUMMARY than coconut oil, rich in saturated fats (PMID: 26200659). Here, in order to ST:STUDY_SUMMARY elucidate the mechanisms responsible for soybean oil induced obesity, we have ST:STUDY_SUMMARY performed the first ever metabolomics (in plasma and liver) and proteomics on ST:STUDY_SUMMARY the livers of mice fed the two soybean oil diets (plus those fed a high coconut ST:STUDY_SUMMARY oil and Viv chow diet). Our results show that the new high oleic soybean oil ST:STUDY_SUMMARY induces less obesity and adiposity than conventional soybean oil, but can cause ST:STUDY_SUMMARY hepatomegaly and liver dysfunction. Metabolomic analysis reveals that the ST:STUDY_SUMMARY hepatic and plasma metabolic profiles differ considerably between the two ST:STUDY_SUMMARY soybean oils. Hepatic C18 oxylipin metabolites of omega-6 (ω6) and omega-3 ST:STUDY_SUMMARY (ω3) fatty acids (linoleic and α-linolenic acid, respectively) in the ST:STUDY_SUMMARY cytochrome P450/soluble epoxide hydrolase pathway were found to correlate ST:STUDY_SUMMARY positively with obesity. ST:INSTITUTE University of California, Davis ST:DEPARTMENT Genome and Biomedical Sciences Facility ST:LABORATORY WCMC Metabolomics Core ST:LAST_NAME Fiehn ST:FIRST_NAME Oliver ST:ADDRESS 1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, ST:ADDRESS CA 95616 ST:EMAIL ofiehn@ucdavis.edu ST:PHONE (530) 754-8258 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57/BL6N SU:GENDER Male SU:ANIMAL_HOUSING SPF facility SU:ANIMAL_LIGHT_CYCLE 12:12 h light-dark cycle SU:ANIMAL_FEED Ad libitum SU:ANIMAL_WATER Ad libitum #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - CSH_BioRec_01_pos mode_MS_07212014.d GROUP_DESCRIPTION:N/A | TIME_POINT:N/A | Genotype:N/A SUBJECT_SAMPLE_FACTORS - CSH_BioRec_02_pos mode_MS_07212014.d GROUP_DESCRIPTION:N/A | TIME_POINT:N/A | Genotype:N/A SUBJECT_SAMPLE_FACTORS - CSH_BioRec_03_pos mode_MS_07212014.d GROUP_DESCRIPTION:N/A | TIME_POINT:N/A | Genotype:N/A SUBJECT_SAMPLE_FACTORS - CSH_PM D3-17 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-17 e2_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-17 e3reinject_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-17 e4_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-18 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-18 e2reinject_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-18 e3_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D3-18 e4_pos mode_MS_07212014.d GROUP_DESCRIPTION:LA-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-16 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-16 e2_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-16 e3_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-16 e4_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-17 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-17 e2_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-18 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM D4-18 e4_pos mode_MS_07212014.d GROUP_DESCRIPTION:HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-4 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-4 e3_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-4 e4_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-5 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-5 e2_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-5 e3_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-6 e1_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_PM PL-6 e4_pos mode_MS_07212014.d GROUP_DESCRIPTION:PL-HFD | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-1_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-2_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-4_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-5_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-6_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-7_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-8_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 SUBJECT_SAMPLE_FACTORS - CSH_Viv-9_pos mode_MS_07212014.d GROUP_DESCRIPTION:Viv chow | TIME_POINT:24 weeks | Genotype:WT C57-Bl6 #COLLECTION CO:COLLECTION_SUMMARY Liver tissue from mice on the diets for 24 weeks for metabolomic analysis was CO:COLLECTION_SUMMARY collected, rinsed in cold PBS, excess fluid was blotted with a kim-wipe and CO:COLLECTION_SUMMARY tissue was immediately snap frozen in liquid nitrogen before storage at -80°C. CO:COLLECTION_SUMMARY Blood was collected by cardiac puncture and centrifuged at 9 rcf for 5 min at CO:COLLECTION_SUMMARY 4°C. Plasma was stored immediately at -20°C. #TREATMENT TR:TREATMENT_SUMMARY Male C57/BL6N mice weaned at 3weeks of age were randomly assigned to one of the TR:TREATMENT_SUMMARY four diets: 1) VIV chow- normal rodent chow , low in fat and high in fiber 2) TR:TREATMENT_SUMMARY HFD (referred to as CO in the manuscript) -40 kcal% high fat diet with 36 kcal% TR:TREATMENT_SUMMARY from coconut oil and 4 kcal% from conventional soybean oil 3) LA-HFD (referred TR:TREATMENT_SUMMARY to as SO+CO in the manuscript) - 40 kcal% high fat diet with 21 kcal% fat TR:TREATMENT_SUMMARY calories from coconut oil and 19 kcal% from conventional soybean oil, of which TR:TREATMENT_SUMMARY 10 kcal% were from LA 4) PL-HFD (referred to as PL+CO in the manuscript) TR:TREATMENT_SUMMARY -40kcal% high fat diet in which conventional soybean oil in LA-HFD was replaced TR:TREATMENT_SUMMARY on a per gram basis with the genetically modified (GM) High Oleic Soybean Oil , TR:TREATMENT_SUMMARY Plenish #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 1) Keep Specimen on Dry Ice 2) Transfer Tissue Contents into a new 1.5mL labeled SP:SAMPLEPREP_SUMMARY eppendorf tube; keep on dry ice at all times 3) Add three (3) 3mm metal grinding SP:SAMPLEPREP_SUMMARY balls to each sample; store in -80C for 10minutes 4) Homogenize the entire SP:SAMPLEPREP_SUMMARY tissue to fine powder using genogrinder; make sure that the metal grinding balls SP:SAMPLEPREP_SUMMARY are ice-cold prior to homogenization (step 3) 5) Upon completion of SP:SAMPLEPREP_SUMMARY homogenization, keep samples on dry ice 6) Weight out two (2) aliquots: a ~5mg SP:SAMPLEPREP_SUMMARY aliquot for CSH_lipidomics and a ~4mg aliquot for Primary Metabolites by GCTOF SP:SAMPLEPREP_SUMMARY a. Record the exact weight weighed out for each sample b. Keep all samples on SP:SAMPLEPREP_SUMMARY dry ice 7) KEEP remaining tissue specimen (>90mg) for analysis of Oxylipins SP:SAMPLEPREP_SUMMARY (store in -80C) 8) Analysis of Primary Metabolites (GCTOFMS) a. Add 1mL of SP:SAMPLEPREP_SUMMARY ice-cold “degassed” 3:3:2 ACN/IPA/H2O b. Vortex for 10seconds c. Shake on SP:SAMPLEPREP_SUMMARY shaker for 20min at -4C d. Centrifuge the samples for 2min at 14,000 rcf e. SP:SAMPLEPREP_SUMMARY Transfer two (2) 500μL aliquots to new 1.5mL eppendorf tubes; one for backup SP:SAMPLEPREP_SUMMARY the other to be dried to dryness using the SpeedVac f. IMPORTANT: The SP:SAMPLEPREP_SUMMARY precipitated protein will be used for analysis of the proteome, DO NOT DISCARD SP:SAMPLEPREP_SUMMARY THESE; Place these in a separate labeled box and store in -20C g. Keep all SP:SAMPLEPREP_SUMMARY samples on ice during extraction period h. Dry down one (1) 500μL aliquot to SP:SAMPLEPREP_SUMMARY complete dryness i. Perform cleanup on dried aliquot using 500μL of 50/50 v/v SP:SAMPLEPREP_SUMMARY ACN/H2O j. Transfer supernatant and dry to completeness k. Submit for SP:SAMPLEPREP_SUMMARY Derivatization 9) Analysis of Complex Lipids (LCQTOF) a. Add 225μL of ice-cold SP:SAMPLEPREP_SUMMARY “degassed” MeOH containing “ISTD mixture” to homogenized 5mg aliquot b. SP:SAMPLEPREP_SUMMARY Vortex for 10 seconds c. Add 750μL of ice-cold “degassed” MTBE containing SP:SAMPLEPREP_SUMMARY 22:1 CE ISTD d. Vortex for 10 seconds e. Shake on Orbital Mixer for 6min at 4C SP:SAMPLEPREP_SUMMARY f. Add 188μL of room temperature H2O g. Vortex for 20 seconds h. Centrifuge for SP:SAMPLEPREP_SUMMARY 2min at 14,000 rcf i. Transfer two (2) aliquots of 350μL of top layer, one for SP:SAMPLEPREP_SUMMARY backup stored in -20C, the other for analysis j. Keep bottom layer and store in SP:SAMPLEPREP_SUMMARY -20C k. Dry down one (1) 350μL aliquot to dryness using the Speedvac l. SP:SAMPLEPREP_SUMMARY Resuspend samples in 108.6μL of 50ng/mL CUDA m. Vortex and sonicate for SP:SAMPLEPREP_SUMMARY 5minutes n. Centrifuge for 2min at 14,000 rcf o. Transfer 90μL to an amber vial SP:SAMPLEPREP_SUMMARY with micro-insert (non-diluted) p. Transfer 10μL to a new 1.5mL eppendorf tube, SP:SAMPLEPREP_SUMMARY dilute 20X with 50ng/mL CUDA in 90:10 MeOH:Toluene (10μL + 190μL CUDA) and SP:SAMPLEPREP_SUMMARY transfer 100μL to amber vial with micro-insert (diluted for TGs) i. The SP:SAMPLEPREP_SUMMARY dilution is based off previous experiences with liver samples SP:SAMPLEPREP_PROTOCOL_FILENAME SP_Extraction_Protocol_for_liver_multi-omic.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 6550 CH:COLUMN_NAME Waters Acquity CSH C18 (100 x 2.1mm, 1.7um) CH:COLUMN_NAME 1.7um Pre-Column CH:FLOW_GRADIENT 15% B to 99% B CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 65 C CH:METHODS_FILENAME Data_Dictionary_Fiehn_laboratory_GCTOF_MS_primary_metabolism_10-15-2013_general.pdf CH:SOLVENT_A 60:40 Acetonitrile:Water +10mM Ammonium Acetate +10mM Acetic Acid CH:SOLVENT_B 9:1 Isopropanol:Acetonitrile +10mM Ammonium Acetate +10mM Acetic Acid CH:COLUMN_PRESSURE 450-850 bar CH:INJECTION_TEMPERATURE 4 C CH:INTERNAL_STANDARD See data dictionary CH:RETENTION_TIME See data dictionary CH:SAMPLE_INJECTION 5.0 uL CH:ANALYTICAL_TIME 13 min CH:CAPILLARY_VOLTAGE 3500 eV CH:TIME_PROGRAM 15 min CH:WEAK_WASH_SOLVENT_NAME Isopropanol CH:STRONG_WASH_SOLVENT_NAME Isopropanol CH:TARGET_SAMPLE_TEMPERATURE Autosampler temp 4 C CH:RANDOMIZATION_ORDER Excel #ANALYSIS AN:ANALYSIS_TYPE MS AN:DETECTOR_TYPE TOF MCP AN:SOFTWARE_VERSION Masshunter AN:DATA_FORMAT .d #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Agilent 6550 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:CAPILLARY_VOLTAGE 3500 eV MS:COLLISION_ENERGY 25 eV MS:COLLISION_GAS Nitrogen MS:DRY_GAS_FLOW 13L/min MS:DRY_GAS_TEMP 200 C MS:FRAGMENT_VOLTAGE 175 eV MS:FRAGMENTATION_METHOD Auto MS/MS MS:ION_SOURCE_TEMPERATURE 325 C MS:ION_SPRAY_VOLTAGE 1000 MS:IONIZATION Neg MS:PRECURSOR_TYPE Intact Molecule MS:REAGENT_GAS Nitrogen MS:SOURCE_TEMPERATURE 325 C MS:DATAFORMAT .d MS:DESOLVATION_GAS_FLOW 11 L/min MS:DESOLVATION_TEMPERATURE 350 C MS:NEBULIZER 35 psig MS:OCTPOLE_VOLTAGE 750 MS:RESOLUTION_SETTING Exteded Dyamic Range MS:SCAN_RANGE_MOVERZ 60-1700 Da MS:SCANNING_CYCLE 2 Hz MS:SCANNING_RANGE 60-1700 Da MS:SKIMMER_VOLTAGE 65 MS:MS_RESULTS_FILE ST000656_AN001002_Results.txt UNITS:Counts #END