{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003009","ANALYSIS_ID":"AN004940","VERSION":"1","CREATED_ON":"December 14, 2023, 8:35 am"},

"PROJECT":{"PROJECT_TITLE":"Uncoupling Metabolic Health from Thermogenesis via BCAA Flux in Brown Fat","PROJECT_SUMMARY":"Brown adipose tissue (BAT) is best known for thermogenesis. Whereas numerous studies in rodents found tight associations between the metabolic benefits of BAT and enhanced whole-body energy expenditure, emerging evidence in humans suggests that BAT is protective against Type 2 diabetes independent of body-weight. The underlying mechanism for this dissociation remained unclear. Here, we report that impaired mitochondrial flux of branched-chain amino acids (BCAA) in BAT, by deleting mitochondrial BCAA carrier (MBC, encoded by Slc25a44), was sufficient to cause systemic insulin resistance without affecting whole-body energy expenditure or body-weight. We found that brown adipocytes catabolized BCAAs in the mitochondria as essential nitrogen donors for the biosynthesis of glutamate, N-acetylated amino acids, and one of the products, glutathione. BAT-selective impairment in mitochondrial BCAA flux led to elevated oxidative stress and insulin resistance in the liver, accompanied by reduced levels of BCAA-derived metabolites in the circulation. In turn, supplementation of glutathione restored insulin sensitivity of BAT-specific MBC knockout mice. Notably, a high-fat diet rapidly impaired BCAA catabolism and the synthesis of BCAA-nitrogen derived metabolites in the BAT, while cold-induced BAT activity is coupled with an active synthesis of these metabolites. Together, the present work uncovers a mechanism through which brown fat controls metabolic health independent of thermogenesis via BCAA-derived nitrogen carriers acting on the liver.","INSTITUTE":"Harvard Medical School","LAST_NAME":"Wang","FIRST_NAME":"Dandan","ADDRESS":"3 Blackfan Circle","EMAIL":"dandanwang2022@gmail.com","PHONE":"5083733714"},

"STUDY":{"STUDY_TITLE":"Media_15N BCAA tracing in brown adipocyte","STUDY_SUMMARY":"To determine the metabolic fate and nitrogen flux of BCAA in mouse brown adipocytes, we used 15N labeled BCAA tracing (Leu (NLM-142-1, CIL), Ile (NLM-292-0.25, CIL) and Val (NLM-316-0.5, CIL)) followed by LC-MS analysis.","INSTITUTE":"Harvard Medical School","LAST_NAME":"Wang","FIRST_NAME":"Dandan","ADDRESS":"3 Blackfan Circle","EMAIL":"dandanwang2022@gmail.com","PHONE":"5083733714"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"EV_M_1h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_1h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_1h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_1h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_1h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_1h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_6h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_6h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_6h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_6h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_6h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_6h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_12h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_12h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_12h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_12h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_12h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_12h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_24h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_24h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_24h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_24h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"EV_M_24h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"EV_M_24h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_1h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_1h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_1h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_1h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_1h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_1h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_6h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_6h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_6h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_6h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_6h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_6h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_12h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_12h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_12h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_12h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_12h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_12h_neg_3 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_24h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_24h_neg_1 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_24h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_24h_neg_2 .RAW"}
},
{
"Subject ID":"-",
"Sample ID":"Cre_M_24h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Media","RAW_FILE_NAME":"Cre_M_24h_neg_3 .RAW"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Twelve hours before the isotope switch, cell media was replaced with fresh unlabeled media. After switching to the tracing media, the media samples were collected at 0 h, 6 h, 12 h, 24 h.","SAMPLE_TYPE":"Adipocyte media"},

"TREATMENT":{"TREATMENT_SUMMARY":"Tracing media and corresponding unlabeled media were prepared. Twelve hours before the isotope switch, cell media was replaced with fresh unlabeled media. After switching to the tracing media, the media samples were collected at 0 h, 1 h, 6 h, 12h, 24 h."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"For the extraction of metabolites from media samples, after removing the cell debris by centrifuging the medium at 16,000 x g for 5 min at 4 °C, 200 μL clarified medium was transferred to the Eppendorf tube prefilled with 800 μL cold methanol. After mixing for 30s, the samples were incubated at -80 °C for 1 hour and centrifuged at 16,000 x g at 4 °C for 15 min. Finally, 50 μL supernatant was transferred to the glass insert for LC-MS detection."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Vanquish Horizon","COLUMN_NAME":"Waters ACQUITY UPLC BEH Amide (100 × 2.1mm, 1.7um)","SOLVENT_A":"100% water; 25mM ammonium acetate; 25mM ammonium hydroxide","SOLVENT_B":"100% acetonitrile","FLOW_GRADIENT":"The linear gradient eluted from 95% B (0.0–1 min), 95% B to 65% B (1–7.0 min), 65% B to 40% B (7.0–8.0 min), 40% B (8.0–9.0 min), 40% B to 95% B (9.0–9.1 min), then stayed at 95% B for 5.9 min.","FLOW_RATE":"0.4 mL/min","COLUMN_TEMPERATURE":"25 °C"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo orbitrap exploris 240","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"ESI source parameters were set as follows: spray voltage, 3500 V or −2800 V, in positive or negative modes, respectively; vaporizer temperature, 350 °C; sheath gas, 50 arb; aux gas, 10 arb; ion transfer tube temperature, 325 °C. The full scan was set as: orbitrap resolution, 60,000; maximum injection time, 100 ms; scan range, 70–1050 Da. The ddMS2 scan was set as: orbitrap resolution, 30,000; maximum injection time, 60 ms; top N setting, 6; isolation width, 1.0 m/z; HCD collision energy (%), 30; Dynamic exclusion mode was set as auto. The metabolites was quantified by Compound Discoverer 3.3.","MS_RESULTS_FILE":"ST003009_AN004940_Results.txt UNITS:Labeled peak areas (normalized) Has m/z:Yes Has RT:Yes RT units:Minutes"}

}