{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003008","ANALYSIS_ID":"AN004939","VERSION":"1","CREATED_ON":"December 14, 2023, 9:04 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":"13C BCAA tracing in differentiated brown adipocyte","STUDY_SUMMARY":"To determine the metabolic fate and carbon flux of BCAA in mouse brown adipocytes, we used 13C 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":"13C_EV_2h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_2h_neg_1.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_2h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_2h_neg_2.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_2h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_2h_neg_3.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_6h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_6h_neg_1.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_6h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_6h_neg_2.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_6h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_6h_neg_3.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_24h_neg_1",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_12h_neg_1.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_24h_neg_2",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_12h_neg_2.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_EV_24h_neg_3",
"Factors":{"Genotype":"Control"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_EV_12h_neg_3.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_2h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_2h_neg_1.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_2h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_2h_neg_2.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_2h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_2h_neg_3.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_6h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_6h_neg_1.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_6h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_6h_neg_2.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_6h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_6h_neg_3.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_24h_neg_1",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_12h_neg_1.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_24h_neg_2",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_12h_neg_2.RAW"}
},
{
"Subject ID":"-",
"Sample ID":"13C_Cre_24h_neg_3",
"Factors":{"Genotype":"MBC-KO"},
"Additional sample data":{"Sample type":"Cell","RAW_FILE_NAME":"13C_Cre_12h_neg_3.RAW"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"For the metabolite extraction from adipocytes, after aspirating the tracing media, the cells were immediately incubated with 500 μL cold methanol containing 1ug/ml internal standard (D8-Phe) for 5 min on dry ice and then scrapped into the Eppendorf tubes.","SAMPLE_TYPE":"Adipocytes"},

"TREATMENT":{"TREATMENT_SUMMARY":"Twelve hours before the isotope switch, cell media was replaced with fresh unlabeled media. After switching to the tracing media, the media and cell samples were collected at 0 h, 2 h, 6 h, 24 h."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The cells were homogenized in a TissueLyser II (Qiagen) (15 min 30 Hz) at 4 °C. 200 μL of the extracts were mixed with 100 μL Milli-Q water and 200 μL chloroform and centrifuged at 16000g for 5 min at 4 °C. Subsequently, 150 μL of the aqueous solution was centrifugally filtered through a 10-kDa cut-off filter (MRCPRT010, Millipore) to remove proteins. The filtrate 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 were quantified by Compound Discoverer 3.3.","MS_RESULTS_FILE":"ST003008_AN004939_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}