#METABOLOMICS WORKBENCH ap472_20230420_161055 DATATRACK_ID:3874 STUDY_ID:ST002696 ANALYSIS_ID:AN004369 VERSION 1 CREATED_ON 08-16-2023 #PROJECT PR:PROJECT_TITLE Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in PR:PROJECT_TITLE female mice PR:PROJECT_TYPE MS Untargeted Lipidomics PR:PROJECT_SUMMARY Genetic association studies have linked ATP10A and closely related type IV PR:PROJECT_SUMMARY P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, PR:PROJECT_SUMMARY such as atherosclerosis. In addition, prior studies of mice harboring large, PR:PROJECT_SUMMARY overlapping chromosomal deletions implicated Atp10A in the development of PR:PROJECT_SUMMARY diet-induced obesity and insulin resistance. Here, we generated gene-specific PR:PROJECT_SUMMARY Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not PR:PROJECT_SUMMARY gain excess weight relative to wild-type littermates. However, Atp10A-/- mice PR:PROJECT_SUMMARY displayed female-specific dyslipidemia characterized by elevated plasma PR:PROJECT_SUMMARY triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL PR:PROJECT_SUMMARY properties. We also observed increased circulating levels of several PR:PROJECT_SUMMARY sphingolipid species along with reduced levels of eicosanoids and bile acids. PR:PROJECT_SUMMARY The Atp10A-/- mice also displayed hepatic insulin resistance without PR:PROJECT_SUMMARY perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific PR:PROJECT_SUMMARY role in regulating plasma lipid composition and maintaining hepatic liver PR:PROJECT_SUMMARY insulin sensitivity in mice. PR:INSTITUTE Vanderbilt University PR:DEPARTMENT Biological Sciences PR:LABORATORY Graham PR:LAST_NAME Graham PR:FIRST_NAME Todd PR:ADDRESS 5260 Medical Research Building III BSB PR:EMAIL tr.graham@Vanderbilt.Edu PR:PHONE 615-343-1835 PR:PUBLICATIONS TBA PR:DOI http://dx.doi.org/10.21228/M83H7N #STUDY ST:STUDY_TITLE Deficiency of the lipid flippase ATP10A causes diet-induced dyslipidemia in ST:STUDY_TITLE female mice ST:STUDY_TYPE MS Untargeted Lipidomics ST:STUDY_SUMMARY Genetic association studies have linked ATP10A and closely related type IV ST:STUDY_SUMMARY P-type ATPases (P4-ATPases) to insulin resistance and vascular complications, ST:STUDY_SUMMARY such as atherosclerosis. In addition, prior studies of mice harboring large, ST:STUDY_SUMMARY overlapping chromosomal deletions implicated Atp10A in the development of ST:STUDY_SUMMARY diet-induced obesity and insulin resistance. Here, we generated gene-specific ST:STUDY_SUMMARY Atp10A knockout mice and show that Atp10A-/- mice fed a high-fat diet did not ST:STUDY_SUMMARY gain excess weight relative to wild-type littermates. However, Atp10A-/- mice ST:STUDY_SUMMARY displayed female-specific dyslipidemia characterized by elevated plasma ST:STUDY_SUMMARY triglycerides, free fatty acids and cholesterol, as well as altered VLDL and HDL ST:STUDY_SUMMARY properties. We also observed increased circulating levels of several ST:STUDY_SUMMARY sphingolipid species along with reduced levels of eicosanoids and bile acids. ST:STUDY_SUMMARY The Atp10A-/- mice also displayed hepatic insulin resistance without ST:STUDY_SUMMARY perturbations to whole-body glucose homeostasis. Thus, ATP10A has a sex-specific ST:STUDY_SUMMARY role in regulating plasma lipid composition and maintaining hepatic liver ST:STUDY_SUMMARY insulin sensitivity in mice. ST:INSTITUTE Vanderbilt University ST:DEPARTMENT Chemistry ST:LABORATORY Center for Innovative Technology ST:LAST_NAME May ST:FIRST_NAME Jody ST:ADDRESS 2301 Vanderbilt Place, Nashville, TN, 37235, USA ST:EMAIL jody.c.may@vanderbilt.edu ST:PHONE 615-875-8438 ST:SUBMIT_DATE 2023-04-20 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN Wild-type and ATP10A knockouts SU:AGE_OR_AGE_RANGE 16-20 weeks SU:WEIGHT_OR_WEIGHT_RANGE 23-28 grams SU:ANIMAL_HOUSING temperature and humidity-controlled facilities SU:ANIMAL_LIGHT_CYCLE 12 h light/dark cycles SU:ANIMAL_FEED standard chow or 60% HFD (D12492, Research Diets) ad libitum, #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS KO1-1n ko1-1n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=46_KO1-1_neg.mzML SUBJECT_SAMPLE_FACTORS KO1-1p ko1-1p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=17_KO1-1_pos.mzML SUBJECT_SAMPLE_FACTORS KO1-2n ko1-2n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=58_KO1-2_neg.mzML SUBJECT_SAMPLE_FACTORS KO1-2p ko1-2p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=27_KO1-2_pos.mzML SUBJECT_SAMPLE_FACTORS KO2-1n ko2-1n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=48_KO2-1_neg.mzML SUBJECT_SAMPLE_FACTORS KO2-1p ko2-1p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=09_KO2-1_pos.mzML SUBJECT_SAMPLE_FACTORS KO2-2n ko2-2n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=57_KO2-2_neg.mzML SUBJECT_SAMPLE_FACTORS KO2-2p ko2-2p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=23_KO2-2_pos.mzML SUBJECT_SAMPLE_FACTORS KO3-1n ko3-1n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=50_KO3-1_neg.mzML SUBJECT_SAMPLE_FACTORS KO3-1p ko3-1p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=14_KO3-1_pos.mzML SUBJECT_SAMPLE_FACTORS KO3-2n ko3-2n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=63_KO3-2_neg.mzML SUBJECT_SAMPLE_FACTORS KO3-2p ko3-2p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=29_KO3-2_pos.mzML SUBJECT_SAMPLE_FACTORS KO4-1n ko4-1n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=52_KO4-1_neg.mzML SUBJECT_SAMPLE_FACTORS KO4-1p ko4-1p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=13_KO4-1_pos.mzML SUBJECT_SAMPLE_FACTORS KO4-2n ko4-2n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=55_KO4-2_neg.mzML SUBJECT_SAMPLE_FACTORS KO4-2p ko4-2p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=20_KO4-2_pos.mzML SUBJECT_SAMPLE_FACTORS KO5-1n ko5-1n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=45_KO5-1_neg.mzML SUBJECT_SAMPLE_FACTORS KO5-1p ko5-1p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=11_KO5-1_pos.mzML SUBJECT_SAMPLE_FACTORS KO5-2n ko5-2n Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=59_KO5-2_neg.mzML SUBJECT_SAMPLE_FACTORS KO5-2p ko5-2p Genotype:ATP10A-knockout Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=22_KO5-2_pos.mzML SUBJECT_SAMPLE_FACTORS WT1-1n wt1-1n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=49_WT1-1_neg.mzML SUBJECT_SAMPLE_FACTORS WT1-1p wt1-1p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=16_WT1-1_pos.mzML SUBJECT_SAMPLE_FACTORS WT1-2n wt1-2n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=62_WT1-2_neg.mzML SUBJECT_SAMPLE_FACTORS WT1-2p wt1-2p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=25_WT1-2_pos.mzML SUBJECT_SAMPLE_FACTORS WT2-1n wt2-1n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=51_WT2-1_neg.mzML SUBJECT_SAMPLE_FACTORS WT2-1p wt2-1p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=10_WT2-1_pos.mzML SUBJECT_SAMPLE_FACTORS WT2-2n wt2-2n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=61_WT2-2_neg.mzML SUBJECT_SAMPLE_FACTORS WT2-2p wt2-2p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=26_WT2-2_pos.mzML SUBJECT_SAMPLE_FACTORS WT3-1n wt3-1n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=44_WT3-1_neg.mzML SUBJECT_SAMPLE_FACTORS WT3-1p wt3-1p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=12_WT3-1_pos.mzML SUBJECT_SAMPLE_FACTORS WT3-2n wt3-2n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=64_WT3-2_neg.mzML SUBJECT_SAMPLE_FACTORS WT3-2p wt3-2p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=28_WT3-2_pos.mzML SUBJECT_SAMPLE_FACTORS WT4-1n wt4-1n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=47_WT4-1_neg.mzML SUBJECT_SAMPLE_FACTORS WT4-1p wt4-1p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=18_WT4-1_pos.mzML SUBJECT_SAMPLE_FACTORS WT4-2n wt4-2n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=60_WT4-2_neg.mzML SUBJECT_SAMPLE_FACTORS WT4-2p wt4-2p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=21_WT4-2_pos.mzML SUBJECT_SAMPLE_FACTORS WT5-1n wt5-1n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=3; RAW_FILE_NAME=53_WT5-1_neg.mzML SUBJECT_SAMPLE_FACTORS WT5-1p wt5-1p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=1; RAW_FILE_NAME=15_WT5-1_pos.mzML SUBJECT_SAMPLE_FACTORS WT5-2n wt5-2n Genotype:Wild-type Treatment=OGTT; MS ion mode=negative; Batch=4; RAW_FILE_NAME=56_WT5-2_neg.mzML SUBJECT_SAMPLE_FACTORS WT5-2p wt5-2p Genotype:Wild-type Treatment=OGTT; MS ion mode=positive; Batch=2; RAW_FILE_NAME=24_WT5-2_pos.mzML #COLLECTION CO:COLLECTION_SUMMARY Plasma was collected, via a retroorbital bleed or cardiac puncture, from 5-hr CO:COLLECTION_SUMMARY fasted mice and 5-hr fasted mice that had undergone an OGTT. CO:SAMPLE_TYPE Blood (plasma) CO:COLLECTION_METHOD retroorbital bleed or cardiac puncture CO:STORAGE_CONDITIONS -20℃ #TREATMENT TR:TREATMENT_SUMMARY Mice were fasted for 5 hours (7AM-12PM). No additional treatments were TR:TREATMENT_SUMMARY administered. TR:TREATMENT WT vs KO TR:TREATMENT_COMPOUND n/a TR:TREATMENT_ROUTE n/a TR:TREATMENT_DOSE n/a TR:TREATMENT_DOSEVOLUME n/a TR:TREATMENT_DOSEDURATION n/a TR:TREATMENT_VEHICLE n/a TR:ANIMAL_VET_TREATMENTS n/a TR:ANIMAL_ANESTHESIA n/a TR:ANIMAL_ACCLIMATION_DURATION n/a TR:ANIMAL_FASTING 5 hour TR:ANIMAL_ENDP_EUTHANASIA n/a TR:ANIMAL_ENDP_TISSUE_COLL_LIST Plasma was collected, via a retroorbital bleed or cardiac puncture TR:ANIMAL_ENDP_CLINICAL_SIGNS n/a #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100 uL of plasma was collected from each mouse. An isotopically labeled lipid SP:SAMPLEPREP_SUMMARY mixture (SPLASH LIPIDOMIX, Avanti) was added as an internal standard to each SP:SAMPLEPREP_SUMMARY plasma sample. A liquid-liquid extraction was performed by adding 1 mL methyl SP:SAMPLEPREP_SUMMARY tert-butyl ether (MTBE), vortexing, and subsequently centrifuging for 10 minutes SP:SAMPLEPREP_SUMMARY at 10,000 rpm and 4˚C. The nonpolar, MTBE fraction (top liquid layer) SP:SAMPLEPREP_SUMMARY containing the lipophilic components was removed and dried under vacuum SP:SAMPLEPREP_SUMMARY centrifugation. Dried samples were stored at -80˚C until the day of MS SP:SAMPLEPREP_SUMMARY analysis. For LC-IM-MS analysis, dried samples were resuspended in 100 µL IPA SP:SAMPLEPREP_SUMMARY containing 40 µg/mL heptadecanoic acid and nonadecanoic acid, as well as 10 SP:SAMPLEPREP_SUMMARY µg/mL glucosyl(β) sphingosine and N-heptadecanoyl-D-erythrosphingosine. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACTION_METHOD MTBE SP:EXTRACT_ENRICHMENT vacuum centrifuge SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION 100 uL IPA SP:SAMPLE_DERIVATIZATION n/a SP:SAMPLE_SPIKING 14 heavy-labeled lipids prior to extraction; 4 odd-chain lipids during SP:SAMPLE_SPIKING reconstitution SP:SUBCELLULAR_LOCATION n/a #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY HPLC-IM-MS/MS on an Agilent 6560 mass spectrometer using a ZORBAX Extend-C18 CH:CHROMATOGRAPHY_SUMMARY RPLC column (Phase A: 0.1% formic acid and 10 mM NH4CHOO in water, Phase B: 0.1% CH:CHROMATOGRAPHY_SUMMARY formic acid and 10 mM NH4CHOO in 60:36:4 isopropanol:acetonitrile:water). CH:INSTRUMENT_NAME Agilent 6560 CH:COLUMN_NAME Agilent ZORBAX RRHD Extend-C18 (50 x 2.1mm,1.8um) CH:COLUMN_TEMPERATURE 40 CH:FLOW_GRADIENT 70% B for 1 min, 70-86% B in 2.5 min, 86% B for 6.5 min, 86-100% B in 1 min, CH:FLOW_GRADIENT 100% B for 6 min, 100-70% B in 0.1 min, and 70% B for 1.9 min. CH:FLOW_RATE 300 ul/min CH:SOLVENT_A Water (10mM Amm. Formate) 0.1% formic acid CH:SOLVENT_B 60:36:4 IPA:ACN:H2O (10mM Amm. Formate) 0.1% formic acid CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:LABORATORY_NAME Center for Innovative Technology AN:ANALYSIS_TYPE MS AN:ACQUISITION_DATE 1/20/2021 AN:SOFTWARE_VERSION MassHunter 10 AN:OPERATOR_NAME Bailey S. Rose #MS MS:INSTRUMENT_NAME Agilent 6560 Ion Mobility MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS Data alignment and biostatical analysis was performed using Progenesis QI MS:MS_COMMENTS (Waters). MS:ION_MODE POSITIVE MS:MS_RESULTS_FILE ST002696_AN004369_Results.txt UNITS:Ion Abundances Has m/z:Yes Has RT:Yes RT units:Minutes #END