#METABOLOMICS WORKBENCH hossein_maleki_20190415_061958 DATATRACK_ID:1704 STUDY_ID:ST001266 ANALYSIS_ID:AN002103 PROJECT_ID:PR000851 VERSION 1 CREATED_ON October 15, 2019, 12:34 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, part ST:STUDY_TITLE I β-Cells 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 β-cells are a primary target for iAs and its metabolites and that ST:STUDY_SUMMARY impaired insulin secretion by β-cells 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:CELL_STRAIN_DETAILS INS-1 832/13 cell line #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - CB_1 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_10 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_11 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_12 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_13 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_14 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_15 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_16 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_17 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_18 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_19 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_2 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_20 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_21 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_22 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_23 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_24 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_25 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_26 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_27 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_28 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_29 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_3 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_30 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_31 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_32 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_33 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_34 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_35 Cell Type:INS-1 (beta cells) | Treatment:monomethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_36 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_37 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_38 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_39 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_4 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_40 Cell Type:INS-1 (beta cells) | Treatment:dimethylated arsenic SUBJECT_SAMPLE_FACTORS - CB_5 Cell Type:INS-1 (beta cells) | Treatment:No treatment SUBJECT_SAMPLE_FACTORS - CB_6 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_7 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_8 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - CB_9 Cell Type:INS-1 (beta cells) | Treatment:inorganic arsenic (sodium arsenite) SUBJECT_SAMPLE_FACTORS - SP_CB_1_1 Cell Type:Beta cell sample pool | Treatment:- SUBJECT_SAMPLE_FACTORS - SP_CB_1_2 Cell Type:Beta cell sample pool | Treatment:- SUBJECT_SAMPLE_FACTORS - SP_CB_2_1 Cell Type:Beta cell sample pool | Treatment:- SUBJECT_SAMPLE_FACTORS - SP_CB_2_2 Cell Type:Beta cell sample pool | Treatment:- SUBJECT_SAMPLE_FACTORS - SP_CB_3_1 Cell Type:Beta cell sample pool | Treatment:- SUBJECT_SAMPLE_FACTORS - SP_CB_3_2 Cell Type:Beta cell sample pool | Treatment:- SUBJECT_SAMPLE_FACTORS - SP_CB_4 Cell Type:Beta cell sample pool | Treatment:- #COLLECTION CO:COLLECTION_SUMMARY Cells were trypsinized for approximately 30 seconds. Once collected, the cells CO:COLLECTION_SUMMARY were centrifuged at 1200 rpm for 5 minutes to form a pellet. The cell pellet was CO:COLLECTION_SUMMARY washed using PBS. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY INS-1 832/13 cells were seeded at a density of 1 million cells per well on a TR:TREATMENT_SUMMARY 12-well plate, and exposed to 2 µM iAsIII (sodium arsenite, > 99% pure; TR:TREATMENT_SUMMARY Sigma-Aldrich, St. Louis, MO), 0.2 µM MAsIII (methylarsine oxide, > 98% pure), TR:TREATMENT_SUMMARY or 2 µM DMAsIII (Iododimethylarsine, > 98% Pure; Sigma Aldrich, St. Louis, MO) TR:TREATMENT_SUMMARY for 24h prior to glucose stimulated insulin secretion or metabolomics. Cells TR:TREATMENT_SUMMARY treated with medium without any kind of As were used as Control. TR:CELL_STORAGE -80 °C #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The frozen beta-cell samples were put on dry ice with a randomized order. A SP:SAMPLEPREP_SUMMARY volume of 400 µL methanol-water (80:20) was added to the cells and vortexed by SP:SAMPLEPREP_SUMMARY a multi-tube vortexer for 5 min at 5000 rpm. All contents in the tube were SP:SAMPLEPREP_SUMMARY transferred into a pre-labeled MagNaLyzer tube (with 10-15 beads inside). The SP:SAMPLEPREP_SUMMARY tubes were put on the bead homogenizer using quick run setting for bacterial SP:SAMPLEPREP_SUMMARY cells (2 ml) with speed at 6.30 m/s for 45 sec in 1 cycle. All samples were SP:SAMPLEPREP_SUMMARY centrifuged at 16,000 rcf for 5 min at 4°C. Supernatant from individual sample SP:SAMPLEPREP_SUMMARY was transferred into a pre-labeled 1.5 ml low-bind Eppendorf tube. All the SP:SAMPLEPREP_SUMMARY samples were centrifuged at 16,000 rcf for 20 min at 4°C. For quality control SP:SAMPLEPREP_SUMMARY purpose, 32 µl of the supernatant from individual sample was transferred into a SP:SAMPLEPREP_SUMMARY new 2-ml tube to make a sample pool (SP). Aliquots (300 µl) of supernatant from SP:SAMPLEPREP_SUMMARY the studied samples and SP were transferred into the pre-labeled 1.5 ml Low-bind SP:SAMPLEPREP_SUMMARY Eppendorf tubes. All samples, including studied samples and SPs, were dried SP:SAMPLEPREP_SUMMARY using speed-vac. For immediate analysis, 100 µL of Water-Methanol (95:5) was SP:SAMPLEPREP_SUMMARY added to the residue, and then thoroughly mixed on multiple tube vortexer for 10 SP:SAMPLEPREP_SUMMARY mins at 5000 rpm. After centrifuge at 4°C, 16000 rcf for 4 min, the supernatant SP:SAMPLEPREP_SUMMARY from individual sample was transferred to pre-labeled auto-sampler vial for SP:SAMPLEPREP_SUMMARY 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 ST001266_AN002103_Results.txt UNITS:Relative Intensity Has m/z:Yes Has RT:Yes RT units:Minutes #END