#METABOLOMICS WORKBENCH hossein_maleki_20200130_134026_mwtab.txt DATATRACK_ID:1914 STUDY_ID:ST001314 ANALYSIS_ID:AN002188 PROJECT_ID:PR000892 VERSION 1 CREATED_ON February 3, 2020, 2:24 pm #PROJECT PR:PROJECT_TITLE Metabolomic Profiles of Pancreatic β-Cells and Islets Exposed to Arsenic PR:PROJECT_SUMMARY Type-2 diabetes (T2D) is a complex metabolic disorder that affects hundreds of PR:PROJECT_SUMMARY millions of people world-wide and is a growing public health concern. Despite PR:PROJECT_SUMMARY recent advances in T2D research, the etiology of this disease and the mechanisms PR:PROJECT_SUMMARY underlying the metabolic defects remain poorly understood. While obesity is PR:PROJECT_SUMMARY thought to be the main cause for the rising prevalence of T2D, obesity alone PR:PROJECT_SUMMARY cannot explain differences in the trends of T2D among different geographical PR:PROJECT_SUMMARY regions and populations. Growing evidence suggests that environmental exposures PR:PROJECT_SUMMARY to toxic and diabetogenic substances must play important roles. Inorganic PR:PROJECT_SUMMARY arsenic (iAs) is a naturally occurring toxic metalloid. Hundreds of millions of PR:PROJECT_SUMMARY people worldwide are exposed to unsafe levels of iAs in drinking water and food. PR:PROJECT_SUMMARY iAs is a potent carcinogen, but iAs exposure has also been linked to increase PR:PROJECT_SUMMARY risk of T2D. While the link between iAs exposure and T2D is well-established, PR:PROJECT_SUMMARY the mechanisms underlying the diabetogenic effects of iAs exposure remain PR:PROJECT_SUMMARY unclear. Results of our previously published and ongoing studies suggest that PR:PROJECT_SUMMARY pancreatic β-cells are a primary target for iAs and its metabolites and that PR:PROJECT_SUMMARY impaired insulin secretion by β-cells is the mechanism by which iAs exposure PR:PROJECT_SUMMARY leads to diabetes. The proposed project will use metabolomics to identify PR:PROJECT_SUMMARY metabolic pathways in β-cells and pancreatic islets that are targeted by iAs PR:PROJECT_SUMMARY and its metabolites, monomethyl-As (MAs) and dimethyl-As (DMAs). The PR:PROJECT_SUMMARY metabolomics data combined with results of our ongoing mechanistic studies will PR:PROJECT_SUMMARY provide a comprehensive picture of the metabolic dysfunction leading to the PR:PROJECT_SUMMARY development of diabetes in individuals exposed to iAs and of the molecular PR:PROJECT_SUMMARY mechanisms that underlie this dysfunction. Identifying the affected pathways and PR:PROJECT_SUMMARY mechanisms will ultimately help to improve strategies for prevention and/or PR:PROJECT_SUMMARY treatment of T2D associated with chronic exposure to iAs. PR:INSTITUTE University of North Carolina at Chapel Hill PR:LAST_NAME Styblo PR:FIRST_NAME Miroslav PR:ADDRESS Departmnet of Nutrition, CB# 7461, Chapel Hill, NC 27599-7461 PR:EMAIL miroslav_styblo@med.unc.edu PR:PHONE (919) 966-5721 #STUDY ST:STUDY_TITLE Metabolomic Profiles of Pancreatic β-Cells and Islets Exposed to Arsenic, ST:STUDY_TITLE Islets (part-II) ST:STUDY_SUMMARY Type-2 diabetes (T2D) is a complex metabolic disorder that affects hundreds of ST:STUDY_SUMMARY millions of people world-wide and is a growing public health concern. Despite ST:STUDY_SUMMARY recent advances in T2D research, the etiology of this disease and the mechanisms ST:STUDY_SUMMARY underlying the metabolic defects remain poorly understood. While obesity is ST:STUDY_SUMMARY thought to be the main cause for the rising prevalence of T2D, obesity alone ST:STUDY_SUMMARY cannot explain differences in the trends of T2D among different geographical ST:STUDY_SUMMARY regions and populations. Growing evidence suggests that environmental exposures ST:STUDY_SUMMARY to toxic and diabetogenic substances must play important roles. Inorganic ST:STUDY_SUMMARY arsenic (iAs) is a naturally occurring toxic metalloid. Hundreds of millions of ST:STUDY_SUMMARY people worldwide are exposed to unsafe levels of iAs in drinking water and food. ST:STUDY_SUMMARY iAs is a potent carcinogen, but iAs exposure has also been linked to increase ST:STUDY_SUMMARY risk of T2D. While the link between iAs exposure and T2D is well-established, ST:STUDY_SUMMARY the mechanisms underlying the diabetogenic effects of iAs exposure remain ST:STUDY_SUMMARY unclear. Results of our previously published and ongoing studies suggest that ST:STUDY_SUMMARY pancreatic islets are a primary target for iAs and its metabolites and that ST:STUDY_SUMMARY impaired insulin secretion by islets is the mechanism by which iAs exposure ST:STUDY_SUMMARY leads to diabetes. The proposed project will use metabolomics to identify ST:STUDY_SUMMARY metabolic pathways in β-cells that are targeted by iAs and its metabolites, ST:STUDY_SUMMARY monomethyl-As (MAs) and dimethyl-As (DMAs). The metabolomics data combined with ST:STUDY_SUMMARY results of our ongoing mechanistic studies will provide a comprehensive picture ST:STUDY_SUMMARY of the metabolic dysfunction leading to the development of diabetes in ST:STUDY_SUMMARY individuals exposed to iAs and of the molecular mechanisms that underlie this ST:STUDY_SUMMARY dysfunction. Identifying the affected pathways and mechanisms will ultimately ST:STUDY_SUMMARY help to improve strategies for prevention and/or treatment of T2D associated ST:STUDY_SUMMARY with chronic exposure to iAs. ST:INSTITUTE University of North Carolina at Chapel Hill ST:LAST_NAME Sumner ST:FIRST_NAME Susan ST:ADDRESS 500 Laureate Way, Kannapolis, NC 28081 ST:EMAIL Susan_sumner@unc.edu ST:PHONE (919)6224456 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Male SU:CELL_PRIMARY_IMMORTALIZED Islets isolated from pancreata of adult C57BL/J6 male mice SU:CELL_COUNTS 100 islets #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 - CI_29 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_29.RAW SUBJECT_SAMPLE_FACTORS - CI_22 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_22.RAW SUBJECT_SAMPLE_FACTORS - CI_15 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_15.RAW SUBJECT_SAMPLE_FACTORS - CI_33 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_33.RAW SUBJECT_SAMPLE_FACTORS - CI_14 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_14.RAW SUBJECT_SAMPLE_FACTORS - CI_20 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_20.RAW SUBJECT_SAMPLE_FACTORS - CI_17 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_17.RAW SUBJECT_SAMPLE_FACTORS - CI_19 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_19.RAW SUBJECT_SAMPLE_FACTORS - CI_36 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_36.RAW SUBJECT_SAMPLE_FACTORS - CI_21 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_21.RAW SUBJECT_SAMPLE_FACTORS - CI_8 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_8.RAW SUBJECT_SAMPLE_FACTORS - CI_39 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_39.RAW SUBJECT_SAMPLE_FACTORS - CI_38 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_38.RAW SUBJECT_SAMPLE_FACTORS - CI_3 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_3.RAW SUBJECT_SAMPLE_FACTORS - CI_40 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_40.RAW SUBJECT_SAMPLE_FACTORS - CI_27 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_27.RAW SUBJECT_SAMPLE_FACTORS - CI_6 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_6.RAW SUBJECT_SAMPLE_FACTORS - CI_9 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_9.RAW SUBJECT_SAMPLE_FACTORS - CI_18 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_18.RAW SUBJECT_SAMPLE_FACTORS - CI_35 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_35.RAW SUBJECT_SAMPLE_FACTORS - CI_23 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_23.RAW SUBJECT_SAMPLE_FACTORS - CI_25 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_25.RAW SUBJECT_SAMPLE_FACTORS - CI_32 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_32.RAW SUBJECT_SAMPLE_FACTORS - CI_13 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_13.RAW SUBJECT_SAMPLE_FACTORS - CI_37 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_37.RAW SUBJECT_SAMPLE_FACTORS - CI_26 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_26.RAW SUBJECT_SAMPLE_FACTORS - CI_2 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_2.RAW SUBJECT_SAMPLE_FACTORS - CI_12 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_12.RAW SUBJECT_SAMPLE_FACTORS - CI_11 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_11.RAW SUBJECT_SAMPLE_FACTORS - CI_34 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_34.RAW SUBJECT_SAMPLE_FACTORS - CI_31 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_31.RAW SUBJECT_SAMPLE_FACTORS - CI_10 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_10.RAW SUBJECT_SAMPLE_FACTORS - CI_28 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_28.RAW SUBJECT_SAMPLE_FACTORS - CI_1 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_1.RAW SUBJECT_SAMPLE_FACTORS - CI_24 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_24.RAW SUBJECT_SAMPLE_FACTORS - CI_5 Cell type:Islet | Treatment:No treatment RAW_FILE_NAME=CI_5.RAW SUBJECT_SAMPLE_FACTORS - CI_30 Cell type:Islet | Treatment:inorganic arsenic (sodium arsenite) RAW_FILE_NAME=CI_30.RAW SUBJECT_SAMPLE_FACTORS - CI_16 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_16.RAW SUBJECT_SAMPLE_FACTORS - CI_7 Cell type:Islet | Treatment:monomethylated arsenic RAW_FILE_NAME=CI_7.RAW SUBJECT_SAMPLE_FACTORS - CI_4 Cell type:Islet | Treatment:dimethylated arsenic RAW_FILE_NAME=CI_4.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_1_1 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_1_1.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_1_2 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_1_2.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_2_1 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_2_1.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_2_2 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_2_2.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_3_1 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_3_1.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_3_2 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_3_2.RAW SUBJECT_SAMPLE_FACTORS - SP_CL_4 Cell type:Islet sample pool | Treatment:Islet sample pool RAW_FILE_NAME=SP_CL_4.RAW #COLLECTION CO:COLLECTION_SUMMARY Pancreatic islets were isolated from adult male C57BL/6 mice (Charles River CO:COLLECTION_SUMMARY Laboratories, Wilmington, MA). All procedures involving mice were approved by CO:COLLECTION_SUMMARY the University of North Carolina Institutional Animal and Use Committee. Mice CO:COLLECTION_SUMMARY were sacrificed by cervical dislocation and pancreas was infused in situwith CO:COLLECTION_SUMMARY collagenase P (1 mg/ml, Roche Diagnostics Corp., Indianapolis, IN) via the CO:COLLECTION_SUMMARY common bile duct. Pancreas was then removed and digested in the collagenase CO:COLLECTION_SUMMARY solution for 12 min at 37 °C. The digestate was washed and islets were purified CO:COLLECTION_SUMMARY by centrifugation in a gradient of Ficoll PM 400 (GE Healthcare, Uppsala, CO:COLLECTION_SUMMARY Sweden) (Szot et al., 2007).The isolated islets were cultivated overnight at 37 CO:COLLECTION_SUMMARY °C with 5% CO2 in RPMI 1640 medium (Mediatech, Manassas, VA) with 10% fetal CO:COLLECTION_SUMMARY bovine serum, 10 mM Hepes, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 CO:COLLECTION_SUMMARY μg/ml streptomycin (all from Sigma-Aldrich, St. Louis, MO). The same medium was CO:COLLECTION_SUMMARY used in experiments in which the islets were exposed to iAsIII (sodium arsenite; CO:COLLECTION_SUMMARY Sigma-Aldrich), MAsIII(methylarsine oxide) or DMAsIII (iododimethylarsine). CO:SAMPLE_TYPE Pancreas CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY The isolated islets were incubated overnight at 37°C with 5% CO2 at 6-well TR:TREATMENT_SUMMARY plates in RPMI 1640 medium with 10% fetal bovine serum, 10 mM HEPES, 1 mM sodium TR:TREATMENT_SUMMARY pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Gibco). The TR:TREATMENT_SUMMARY islets were then exposed to 2 μM iAsIII, 0.25 μM MAsIII, or 0.5 μM DMAsIII TR:TREATMENT_SUMMARY for 48 hours. Control islets were incubated in the culture medium without TR:TREATMENT_SUMMARY arsenicals. TR:CELL_STORAGE -80 °C #SAMPLEPREP SP:SAMPLEPREP_SUMMARY With a randomized order, islet samples were put on dry ice before sample SP:SAMPLEPREP_SUMMARY preparation. A volume of 400 uL ice cold methanol-water (80:20) was added, and SP:SAMPLEPREP_SUMMARY then vortex for 5 min at 5000 rpm. All contents in the tube were transferred SP:SAMPLEPREP_SUMMARY into pre-labeled MagNaLyzer tubes (with 10-15 beads inside). The tubes were put SP:SAMPLEPREP_SUMMARY on bead homogenizer using quick run setting for bacterial cells (2 ml) with SP:SAMPLEPREP_SUMMARY speed at 6.30 m/s for 45 sec in 1 cycle. All samples were centrifuged at 16,000 SP:SAMPLEPREP_SUMMARY rcf for 20 min at 4°C. A volume of 300 µl supernatant were transferred into SP:SAMPLEPREP_SUMMARY pre-labeled 1.5 ml Low-bind Eppendorf tube. For quality control purpose, 32 µl SP:SAMPLEPREP_SUMMARY of the supernatant from individual sample was transferred and mixed in new 2-ml SP:SAMPLEPREP_SUMMARY tube to make a sample pool (SP). A volume of 300 µl SP was transferred into a SP:SAMPLEPREP_SUMMARY pre-labeled 2.0 ml Low-bind Eppendorf tubes. All samples, including study SP:SAMPLEPREP_SUMMARY samples and pools, were dried under speed-vac. For immediate analysis, 100 µL SP:SAMPLEPREP_SUMMARY of Water-Methanol (95:5) containing 500 ng/mL L-tryphtophan-d5 was added to the SP:SAMPLEPREP_SUMMARY residue, and then vortexed for 10 mins at 5000 rpm. After centrifuge at 4°C, SP:SAMPLEPREP_SUMMARY 16000 rcf for 4 min, the supernatant from individual sample was transferred to SP:SAMPLEPREP_SUMMARY pre-labeled auto-sampler vial for LC-MS analysis. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters Acquity BEH HSS T3 (100 x 2.1mm, 1.8um) CH:FLOW_RATE 0.4 ml/min CH:COLUMN_TEMPERATURE 50 CH:SOLVENT_A Water with 0.1% Formic Acid CH:SOLVENT_B Methanol with 0.1% Formic Acid CH:COLUMN_PRESSURE 6000-10000 psi CH:INJECTION_TEMPERATURE 8 CH:ANALYTICAL_TIME 22 min CH:CAPILLARY_VOLTAGE 3.75 KV CH:WEAK_WASH_SOLVENT_NAME 10:90 Methanol:Water with 0.1% FA solution CH:STRONG_WASH_SOLVENT_NAME 75:25 2-Propanol: Water with 0.1% FA solution CH:RANDOMIZATION_ORDER Yes #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME UNC-NRI Sumner Lab AN:OPERATOR_NAME Yuanyuan Li AN:DETECTOR_TYPE Orbitrap AN:SOFTWARE_VERSION Xcalibur 4.1 AN:DATA_FORMAT profile #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS We used DDA mode to acquire the MS and MS/MS data. Progenesis QI was used for MS:MS_COMMENTS peak picking, alignment, and normalization. MS:CAPILLARY_TEMPERATURE 275 °C MS:CAPILLARY_VOLTAGE 3.75 KV MS:COLLISION_ENERGY 10-35, ramp MS:COLLISION_GAS N2 MS:DRY_GAS_FLOW 45 MS:DRY_GAS_TEMP 325°C MS:FRAGMENTATION_METHOD CID MS:IONIZATION ES+ MS:MASS_ACCURACY 5ppm MS:DATAFORMAT Profile MS:DESOLVATION_GAS_FLOW 45 MS:DESOLVATION_TEMPERATURE 325°C MS:RESOLUTION_SETTING 120000 MS:SCAN_RANGE_MOVERZ 70-1050 m/z MS:SCANNING_RANGE 70-1050 m/z MS:MS_RESULTS_FILE ST001314_AN002188_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END