{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002379","ANALYSIS_ID":"AN003878","VERSION":"1","CREATED_ON":"December 1, 2022, 10:24 am"},

"PROJECT":{"PROJECT_TITLE":"Pyruvate dehydrogenase kinase supports macrophage NLRP3 inflammasome activation during acute inflammation","PROJECT_TYPE":"Basic research","PROJECT_SUMMARY":"Activating macrophage NLRP3 inflammasome can promote excessive inflammation, with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages.","INSTITUTE":"Wake Forest School of Medicine","LAST_NAME":"Zhu","FIRST_NAME":"Xuewei","ADDRESS":"575 Patterson Ave","EMAIL":"xwzhu@wakehealth.edu","PHONE":"3367131445"},

"STUDY":{"STUDY_TITLE":"Glucose flux analysis of NLRP3 inflammasome activated macrophages","STUDY_TYPE":"Basic research","STUDY_SUMMARY":"Activating macrophage NLRP3 inflammasome can promote excessive inflammation, with severe cell and tissue damage and organ dysfunction. Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages.","INSTITUTE":"Wake Forest School of Medicine","LAST_NAME":"Zhu","FIRST_NAME":"Xuewei","ADDRESS":"575 Patterson Ave, Winston-Salem, NC 27101","EMAIL":"xwzhu@wakehealth.edu","PHONE":"3367131445"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Ctrl_C_1",
"Factors":{"Treatment":"No treatment"},
"Additional sample data":{"RAW_FILE_NAME":"Ctril_C_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_C_2",
"Factors":{"Treatment":"No treatment"},
"Additional sample data":{"RAW_FILE_NAME":"Ctril_C_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_C_3",
"Factors":{"Treatment":"No treatment"},
"Additional sample data":{"RAW_FILE_NAME":"Ctril_C_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_1",
"Factors":{"Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_2",
"Factors":{"Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_3",
"Factors":{"Treatment":"LPS"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_ATP_1",
"Factors":{"Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_ATP_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_ATP_2",
"Factors":{"Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_ATP_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_ATP_3",
"Factors":{"Treatment":"LPS+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_ATP_3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_JX06_ATP_1",
"Factors":{"Treatment":"JPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_JX06_ATP_1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_JX06_ATP_2",
"Factors":{"Treatment":"JPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_JX06_ATP_2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"LPS_JX06_ATP_3",
"Factors":{"Treatment":"JPS+JX06+ATP"},
"Additional sample data":{"RAW_FILE_NAME":"LPS_JX06_ATP_3.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Mouse bone marrow was cultured in low glucose DMEM supplemented with 30% L929 cell-conditioned medium, 20% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin for 6-7 days until the cells reached confluence. BMDMs were then reseeded in culture dishes overnight in RPMI-1640 medium containing 1% Nutridoma-SP medium (Sigma-Aldrich). LPS-primed BMDMs were stimulated with 5 mM ATP in the presence or absence of 10 uM JX06 for 30 min. Unstimulated macrophages were used as control.13C tracing started by replacing medium with glucose-free medium supplemented with 2.06 g/L [U-13C]-glucose (Cayman Chemical) for 90 min. Cells were washed with ice-cold saline and intracellular metabolites were extracted using cold methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). Here, we show that pharmacological or genetic inhibition of pyruvate dehydrogenase kinase (PDHK) significantly attenuates NLRP3 inflammasome activation in murine and human macrophages and septic mice by lowering caspase-1 cleavage and IL-1beta secretion. Inhibiting PDHK reverses NLRP3 inflammasome-induced metabolic reprogramming, enhances autophagy, promotes mitochondrial fusion over fission, preserves cristae ultrastructure, and attenuates mitochondrial ROS production. The suppressive effect of PDHK inhibition on the NLRP3 inflammasome is independent of its canonical role as a pyruvate dehydrogenase regulator. We suggest that PDHK inhibition improves mitochondrial fitness by reversing NLRP3 inflammasome activation in acutely inflamed macrophages.","SAMPLE_TYPE":"Macrophages","COLLECTION_METHOD":"Cells were washed with ice-cold saline and intracellular metabolites were extracted using cold methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich). The samples were pre-dissolved into 40 µL solution (LC-water, methanol, Acetonitrile 2:1:1) before injection.","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"LPS-primed BMDMs were stimulated with or without 5 mM ATP in the presence or absence of 10 uM JX06 for 30 min. 13C tracing started by replacing medium with glucose-free medium supplemented with 2.06 g/L [U-13C]-glucose (Cayman Chemical) for 90 min.","TREATMENT":"in vitro cell culture treatment","TREATMENT_COMPOUND":"LPS, ATP, JX06"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Macrophages were lysed, and polar metabolites were extracted using methanol and H2O (80:20; HPLC Grade; Sigma-Aldrich) (Liu et al., 2015). Extracts were dried in a vacuum concentrator at room temperature and stored at -80 freezer."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Ultimate 3000 UHPLC (Dionex) is coupled to Q Exactive Plus-Mass spectrometer (QE-MS, Thermo Scientific) for metabolite profiling.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Waters XBridge Amide (100 x 4.6mm, 3.5um)"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Jason Locosale","OPERATOR_NAME":"Likun Duan"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Plus Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"For each sample, 3 µL solution was injected into the LC-MS for analysis. The HPLC analysis of the isotope-labeled samples was performed using Ultimate 3000 UHPLC (Dionex) as described (Duan et al., 2022). The mass spectrometry analysis was performed using Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). The mass spectrometers were equipped with a HESI probe and operated in the positive/negative switching mode. When Q Exactive Plus mass spectrometer was used, the relevant parameters were as listed: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 3.0 kV; capillary temperature, 320°C; S-lens, 55. The resolution was set at 70,000 (at m/z 200). Maximum injection time (max IT) was set at 200 ms and automated gain control (AGC) was set at 3 × 106. The LC-MS peak extraction and integration of the raw data were performed using commercially available software Sieve 2.0 (Thermo Fisher Scientific). The integrated peak area was used to calculate 13C enrichment. Natural abundance correction was performed using software R with Bioconductor R package IsoCorrectoR."},

"MS_METABOLITE_DATA":{
"Units":"Fractional enrichment",

"Data":[{"Metabolite":"Aspartate","Ctrl_C_1":"0.501974129","Ctrl_C_2":"0.500588398","Ctrl_C_3":"0.496940099","LPS_1":"0.552796716","LPS_2":"0.550747014","LPS_3":"0.554508096","LPS_ATP_1":"0.728527353","LPS_ATP_2":"0.727321206","LPS_ATP_3":"0.718723128","LPS_JX06_ATP_1":"0.642121406","LPS_JX06_ATP_2":"0.642863874","LPS_JX06_ATP_3":"0.646908071"},{"Metabolite":"1[13C]Aspartate","Ctrl_C_1":"0.068509798","Ctrl_C_2":"0.06998721","Ctrl_C_3":"0.06837107","LPS_1":"0.040223885","LPS_2":"0.038462313","LPS_3":"0.040660272","LPS_ATP_1":"0.050028016","LPS_ATP_2":"0.048485326","LPS_ATP_3":"0.051964024","LPS_JX06_ATP_1":"0.050036639","LPS_JX06_ATP_2":"0.049182269","LPS_JX06_ATP_3":"0.049022698"},{"Metabolite":"2[13C]Aspartate","Ctrl_C_1":"0.251399598","Ctrl_C_2":"0.25334779","Ctrl_C_3":"0.253672986","LPS_1":"0.244887449","LPS_2":"0.248624285","LPS_3":"0.244503616","LPS_ATP_1":"0.147124351","LPS_ATP_2":"0.148095874","LPS_ATP_3":"0.155351321","LPS_JX06_ATP_1":"0.188349106","LPS_JX06_ATP_2":"0.187303587","LPS_JX06_ATP_3":"0.18627021"},{"Metabolite":"3[13C]Aspartate","Ctrl_C_1":"0.141846164","Ctrl_C_2":"0.141322062","Ctrl_C_3":"0.144298263","LPS_1":"0.129618123","LPS_2":"0.130350579","LPS_3":"0.129131105","LPS_ATP_1":"0.063022107","LPS_ATP_2":"0.064476718","LPS_ATP_3":"0.062792344","LPS_JX06_ATP_1":"0.098821038","LPS_JX06_ATP_2":"0.100314951","LPS_JX06_ATP_3":"0.097832114"},{"Metabolite":"4[13C]Aspartate","Ctrl_C_1":"0.036270312","Ctrl_C_2":"0.03475454","Ctrl_C_3":"0.036717582","LPS_1":"0.032473827","LPS_2":"0.031815808","LPS_3":"0.031196911","LPS_ATP_1":"0.011298172","LPS_ATP_2":"0.011620877","LPS_ATP_3":"0.011169183","LPS_JX06_ATP_1":"0.02067181","LPS_JX06_ATP_2":"0.020335318","LPS_JX06_ATP_3":"0.019966907"},{"Metabolite":"N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0.723301766","Ctrl_C_2":"0.653685643","Ctrl_C_3":"0.707759661","LPS_1":"0.636279874","LPS_2":"0.624716829","LPS_3":"0.616000559","LPS_ATP_1":"0.742562123","LPS_ATP_2":"0.730305855","LPS_ATP_3":"0.715430595","LPS_JX06_ATP_1":"0.63159895","LPS_JX06_ATP_2":"0.646392055","LPS_JX06_ATP_3":"0.634170605"},{"Metabolite":"1[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0.025296223","Ctrl_C_3":"0.006759818","LPS_1":"0.024093265","LPS_2":"0.022761538","LPS_3":"0.03101938","LPS_ATP_1":"0.055753185","LPS_ATP_2":"0.074959487","LPS_ATP_3":"0.06106321","LPS_JX06_ATP_1":"0.053690418","LPS_JX06_ATP_2":"0.057083471","LPS_JX06_ATP_3":"0.042852353"},{"Metabolite":"2[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0.18969426","Ctrl_C_2":"0.225638617","Ctrl_C_3":"0.19076019","LPS_1":"0.22323286","LPS_2":"0.221034913","LPS_3":"0.234624577","LPS_ATP_1":"0.160901891","LPS_ATP_2":"0.141202715","LPS_ATP_3":"0.159752411","LPS_JX06_ATP_1":"0.196596833","LPS_JX06_ATP_2":"0.18929238","LPS_JX06_ATP_3":"0.208763261"},{"Metabolite":"3[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0.087003974","Ctrl_C_2":"0.090509766","Ctrl_C_3":"0.09472033","LPS_1":"0.094667925","LPS_2":"0.115657166","LPS_3":"0.101937731","LPS_ATP_1":"0.035896998","LPS_ATP_2":"0.04978177","LPS_ATP_3":"0.058145822","LPS_JX06_ATP_1":"0.101325584","LPS_JX06_ATP_2":"0.095136117","LPS_JX06_ATP_3":"0.092374966"},{"Metabolite":"4[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0.00486975","Ctrl_C_3":"0","LPS_1":"0.021726076","LPS_2":"0.015829554","LPS_3":"0.016417753","LPS_ATP_1":"0.004885804","LPS_ATP_2":"0.002488925","LPS_ATP_3":"0.005607963","LPS_JX06_ATP_1":"0.016788214","LPS_JX06_ATP_2":"0.012095977","LPS_JX06_ATP_3":"0.021838816"},{"Metabolite":"5[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0","Ctrl_C_3":"0","LPS_1":"0","LPS_2":"0","LPS_3":"0","LPS_ATP_1":"0","LPS_ATP_2":"0","LPS_ATP_3":"0","LPS_JX06_ATP_1":"0","LPS_JX06_ATP_2":"0","LPS_JX06_ATP_3":"0"},{"Metabolite":"6[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0","Ctrl_C_3":"0","LPS_1":"0","LPS_2":"0","LPS_3":"0","LPS_ATP_1":"0","LPS_ATP_2":"0","LPS_ATP_3":"0","LPS_JX06_ATP_1":"0","LPS_JX06_ATP_2":"0","LPS_JX06_ATP_3":"0"},{"Metabolite":"7[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0","Ctrl_C_3":"0","LPS_1":"0","LPS_2":"0","LPS_3":"0","LPS_ATP_1":"0","LPS_ATP_2":"0","LPS_ATP_3":"0","LPS_JX06_ATP_1":"0","LPS_JX06_ATP_2":"0","LPS_JX06_ATP_3":"0"},{"Metabolite":"8[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0","Ctrl_C_3":"0","LPS_1":"0","LPS_2":"0","LPS_3":"0","LPS_ATP_1":"0","LPS_ATP_2":"0","LPS_ATP_3":"0","LPS_JX06_ATP_1":"0","LPS_JX06_ATP_2":"0","LPS_JX06_ATP_3":"0"},{"Metabolite":"9[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0","Ctrl_C_3":"0","LPS_1":"0","LPS_2":"0","LPS_3":"0","LPS_ATP_1":"0","LPS_ATP_2":"0","LPS_ATP_3":"0","LPS_JX06_ATP_1":"0","LPS_JX06_ATP_2":"0","LPS_JX06_ATP_3":"0"},{"Metabolite":"10[13C]N(omega)-(L-Arginino)succinate","Ctrl_C_1":"0","Ctrl_C_2":"0","Ctrl_C_3":"0","LPS_1":"0","LPS_2":"0","LPS_3":"0","LPS_ATP_1":"0","LPS_ATP_2":"0.001261247","LPS_ATP_3":"0","LPS_JX06_ATP_1":"0","LPS_JX06_ATP_2":"0","LPS_JX06_ATP_3":"0"},{"Metabo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}

}