#METABOLOMICS WORKBENCH Codreags00_20220603_070514 DATATRACK_ID:3293 STUDY_ID:ST002186 ANALYSIS_ID:AN003580 PROJECT_ID:PR001393 VERSION 1 CREATED_ON June 3, 2022, 8:34 am #PROJECT PR:PROJECT_TITLE An early-life microbiota metabolite protects against obesity via intestinal PR:PROJECT_TITLE PPAR-Gamma PR:PROJECT_TYPE Untargeted Metabolomics analysis PR:PROJECT_SUMMARY The mechanisms by which the early-life microbiota protects against environmental PR:PROJECT_SUMMARY factors that promote childhood obesity remain largely unknown. Using a mouse PR:PROJECT_SUMMARY model in which young mice are simultaneously exposed to antibiotics and a PR:PROJECT_SUMMARY high-fat diet, we show that Lactobacillus species, predominant members of the PR:PROJECT_SUMMARY small intestine microbiota, regulate intestinal epithelial cells (IECs) to limit PR:PROJECT_SUMMARY diet-induced obesity during early-life. A Lactobacillus-derived metabolite, PR:PROJECT_SUMMARY phenyllactic acid (PLA), protected against metabolic dysfunction caused by PR:PROJECT_SUMMARY early-life exposure to antibiotics and a high-fat diet by increasing the PR:PROJECT_SUMMARY abundance of peroxisome proliferator activated receptor gamma (PPAR-gamma) in PR:PROJECT_SUMMARY the small intestine IECs. Therefore, PLA is a microbiota-derived metabolite that PR:PROJECT_SUMMARY activates protective pathways in the small intestine epithelium to regulate fat PR:PROJECT_SUMMARY absorption and prevent obesity during early life. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Chemistry PR:LABORATORY Center for Innovative Technology PR:LAST_NAME Codreanu PR:FIRST_NAME Simona Gabriella PR:ADDRESS 1234 Stevenson Center Lane PR:EMAIL SIMONA.CODREANU@VANDERBILT.EDU PR:PHONE 16158758422 #STUDY ST:STUDY_TITLE An early-life microbiota metabolite protects against obesity via intestinal ST:STUDY_TITLE PPAR-gamma ST:STUDY_SUMMARY The mechanisms by which the early-life microbiota protects against environmental ST:STUDY_SUMMARY factors that promote childhood obesity remain largely unknown. Using a mouse ST:STUDY_SUMMARY model in which young mice are simultaneously exposed to antibiotics and a ST:STUDY_SUMMARY high-fat diet, we show that Lactobacillus species, predominant members of the ST:STUDY_SUMMARY small intestine microbiota, regulate intestinal epithelial cells (IECs) to limit ST:STUDY_SUMMARY diet-induced obesity during early-life. A Lactobacillus-derived metabolite, ST:STUDY_SUMMARY phenyllactic acid (PLA), protected against metabolic dysfunction caused by ST:STUDY_SUMMARY early-life exposure to antibiotics and a high-fat diet by increasing the ST:STUDY_SUMMARY abundance of peroxisome proliferator activated receptor gamma (PPAR-gamma) in ST:STUDY_SUMMARY the small intestine IECs. Therefore, PLA is a microbiota-derived metabolite that ST:STUDY_SUMMARY activates protective pathways in the small intestine epithelium to regulate fat ST:STUDY_SUMMARY absorption and prevent obesity during early life. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME Codreanu ST:FIRST_NAME Simona Gabriella ST:ADDRESS 1234 Stevenson Center Lane ST:EMAIL SIMONA.CODREANU@VANDERBILT.EDU ST:NUM_GROUPS 4 ST:TOTAL_SUBJECTS 20 ST:NUM_MALES 20 ST:STUDY_TYPE untargeted metabolomics analysis ST:PHONE 6158758422 #SUBJECT SU:SUBJECT_TYPE Other organism SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6N mice SU:AGE_OR_AGE_RANGE 5 weeks SU:GENDER Male #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 35.1 LF_351_S01 Treatment:Low Fat (LF) diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LF_351_S01; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 35.2 LF_352_S02 Treatment:LF diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LF_352_S02; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 35.3 LF_353_S03 Treatment:LF diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LF_353_S03; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 35.4 LF_354_S04 Treatment:LF diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LF_354_S04; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 35.6 LF_356_S05 Treatment:LF diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LF_356_S05; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 36.1 LFP_361_S06 Treatment:LF diet + low dose penicillin (LFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LFP_361_S06; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 36.2 LFP_362_S07 Treatment:LF diet + low dose penicillin (LFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LFP_362_S07; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 36.3 LFP_363_S08 Treatment:LF diet + low dose penicillin (LFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LFP_363_S08; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 36.4 LFP_364_S09 Treatment:LF diet + low dose penicillin (LFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LFP_364_S09; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 36.5 LFP_365_S10 Treatment:LF diet + low dose penicillin (LFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_LFP_365_S10; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 37.1 HF_371_S11 Treatment:High fat (HF) diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HF_371_S11; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 37.2 HF_372_S12 Treatment:High fat (HF) diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HF_372_S12; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 37.4 HF_374_S13 Treatment:High fat (HF) diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HF_374_S13; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 37.5 HF_375_S14 Treatment:High fat (HF) diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HF_375_S14; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 37.6 HF_376_S15 Treatment:High fat (HF) diet RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HF_376_S15; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 38.1 HFP_381_S16 Treatment:HF diet + LDP (HFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HFP_381_S16; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 38.2 HFP_382_S17 Treatment:HF diet + LDP (HFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HFP_382_S17; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 38.3 HFP_383_S18 Treatment:HF diet + LDP (HFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HFP_383_S18; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 38.4 HFP_384_S19 Treatment:HF diet + LDP (HFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HFP_384_S19; Timepoint=5 weeks SUBJECT_SAMPLE_FACTORS 38.6 HFP_386_S20 Treatment:HF diet + LDP (HFP) RAW_FILE_NAME=SC_20210114_RPLCp_FMS_HFP_386_S20; Timepoint=5 weeks #COLLECTION CO:COLLECTION_SUMMARY At the end of the experiment (5-weeks)after starting diet and antibiotic CO:COLLECTION_SUMMARY treatments, mice were humanely euthanized by CO2 administration. Afterwards, CO:COLLECTION_SUMMARY ileum (distal small intestine) content was collected. CO:SAMPLE_TYPE Feces CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Animals were fed either a 60% fat diet (HF) (OpenSource Diets, #D12492) or a 10% TR:TREATMENT_SUMMARY fat control diet (LF) (OpenSource Diets, #D12450J) for 5 weeks. Groups of LF or TR:TREATMENT_SUMMARY HF diet mice were also given low doses of penicillin (Sigma Aldrich #P1382)(LDP) TR:TREATMENT_SUMMARY (6.67 mg/L, (10)) in their drinking water throughout the experiment or clinical TR:TREATMENT_SUMMARY doses of penicillin (0.167 g/L, (35)) in their drinking water from days 0 – 5, TR:TREATMENT_SUMMARY 15 – 20, and 30 – 35. For long-term experiments, mice were fed a HF diet or TR:TREATMENT_SUMMARY given a HF diet and LDP for 5 weeks before being switched to either a LF or HF TR:TREATMENT_SUMMARY diet alone for an additional 5 weeks. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen mouse intestinal content samples (n=20, 5 biological replicates for each SP:SAMPLEPREP_SUMMARY sample group) were lysed in 500 µl ice-cold lysis buffer (1:1:2, v:v:v, SP:SAMPLEPREP_SUMMARY acetonitrile: methanol: ammonium bicarbonate 0.1M - pH 8.0) and sonicated SP:SAMPLEPREP_SUMMARY individually using a probe tip sonicator at 50% power (10 pulses). The lysis SP:SAMPLEPREP_SUMMARY buffer contained isotopically labeled standards (n=2) to determine sample SP:SAMPLEPREP_SUMMARY process variability. Homogenized samples were normalized by weight to the SP:SAMPLEPREP_SUMMARY smallest amount of tissue sample such that each sample contained an equal amount SP:SAMPLEPREP_SUMMARY of tissue. Proteins were precipitated from individual samples by addition of 800 SP:SAMPLEPREP_SUMMARY µL of ice-cold methanol followed by overnight incubation at -80°C. SP:SAMPLEPREP_SUMMARY Precipitated proteins were pelleted by centrifugation (15,000 rpm, 15 min) and SP:SAMPLEPREP_SUMMARY metabolite extracts were dried down in vacuo and stored at -80°C. Individual SP:SAMPLEPREP_SUMMARY samples were reconstituted in 100 μL of reverse phase liquid chromatography SP:SAMPLEPREP_SUMMARY reconstitution buffer (acetonitrile/water with 0.1% formic acid, 3:97, v/v) SP:SAMPLEPREP_SUMMARY containing isotopically labeled standards (n=2) to assess instrument SP:SAMPLEPREP_SUMMARY variability. A pooled quality control (QC) sample was prepared by pooling equal SP:SAMPLEPREP_SUMMARY volumes (10 μL) from each individual sample following reconstitution. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Following lysis and standard addition, protein precipitation was performed by SP:EXTRACTION_METHOD adding 800µL of ice-cold methanol (4x by volume). Samples were incubated at SP:EXTRACTION_METHOD -80°C overnight. Following incubation, samples were centrifuged at 10,000 rpm SP:EXTRACTION_METHOD for 10 min to eliminate proteins. The supernatants containing metabolites were SP:EXTRACTION_METHOD dried via speed-vacuum. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Prepared samples were analyzed by RPLC-HRMS/MS in the Vanderbilt Center for CH:CHROMATOGRAPHY_SUMMARY Innovative Technology (CIT) using a modified version of a reversed phase CH:CHROMATOGRAPHY_SUMMARY chromatography negative ionization method. Metabolites were separated on a CH:CHROMATOGRAPHY_SUMMARY Thermo Fisher Scientific (Waltham, MA) Hypersil Gold C18 column (100 x 2.1 mm, CH:CHROMATOGRAPHY_SUMMARY 1.9 μm particle size) using water/acetonitrile gradient with formic acid (0.1%) CH:CHROMATOGRAPHY_SUMMARY added to both mobile phases. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Thermo Hypersil Gold Hypersil Gold, 1.9 mm, 2.1 mm x 100 mm CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A 100% water, 0.1% Formic Acid CH:SOLVENT_B 80:20 acetonitrile:water, 0.1% Formic Acid #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE QTRAP MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Progenesis QI software MS:MS_RESULTS_FILE ST002186_AN003580_Results.txt UNITS:time_m/z Has m/z:Yes Has RT:Yes RT units:Minutes #END