#METABOLOMICS WORKBENCH dandanwang2022_20231213_121950 DATATRACK_ID:4511 STUDY_ID:ST003008 ANALYSIS_ID:AN004939 PROJECT_ID:PR001873 VERSION 1 CREATED_ON December 14, 2023, 9:04 am #PROJECT PR:PROJECT_TITLE Uncoupling Metabolic Health from Thermogenesis via BCAA Flux in Brown Fat PR:PROJECT_SUMMARY Brown adipose tissue (BAT) is best known for thermogenesis. Whereas numerous PR:PROJECT_SUMMARY studies in rodents found tight associations between the metabolic benefits of PR:PROJECT_SUMMARY BAT and enhanced whole-body energy expenditure, emerging evidence in humans PR:PROJECT_SUMMARY suggests that BAT is protective against Type 2 diabetes independent of PR:PROJECT_SUMMARY body-weight. The underlying mechanism for this dissociation remained unclear. PR:PROJECT_SUMMARY Here, we report that impaired mitochondrial flux of branched-chain amino acids PR:PROJECT_SUMMARY (BCAA) in BAT, by deleting mitochondrial BCAA carrier (MBC, encoded by PR:PROJECT_SUMMARY Slc25a44), was sufficient to cause systemic insulin resistance without affecting PR:PROJECT_SUMMARY whole-body energy expenditure or body-weight. We found that brown adipocytes PR:PROJECT_SUMMARY catabolized BCAAs in the mitochondria as essential nitrogen donors for the PR:PROJECT_SUMMARY biosynthesis of glutamate, N-acetylated amino acids, and one of the products, PR:PROJECT_SUMMARY glutathione. BAT-selective impairment in mitochondrial BCAA flux led to elevated PR:PROJECT_SUMMARY oxidative stress and insulin resistance in the liver, accompanied by reduced PR:PROJECT_SUMMARY levels of BCAA-derived metabolites in the circulation. In turn, supplementation PR:PROJECT_SUMMARY of glutathione restored insulin sensitivity of BAT-specific MBC knockout mice. PR:PROJECT_SUMMARY Notably, a high-fat diet rapidly impaired BCAA catabolism and the synthesis of PR:PROJECT_SUMMARY BCAA-nitrogen derived metabolites in the BAT, while cold-induced BAT activity is PR:PROJECT_SUMMARY coupled with an active synthesis of these metabolites. Together, the present PR:PROJECT_SUMMARY work uncovers a mechanism through which brown fat controls metabolic health PR:PROJECT_SUMMARY independent of thermogenesis via BCAA-derived nitrogen carriers acting on the PR:PROJECT_SUMMARY liver. PR:INSTITUTE Harvard Medical School PR:LAST_NAME Wang PR:FIRST_NAME Dandan PR:ADDRESS 3 Blackfan Circle PR:EMAIL dandanwang2022@gmail.com PR:PHONE 5083733714 #STUDY ST:STUDY_TITLE 13C BCAA tracing in differentiated brown adipocyte ST:STUDY_SUMMARY To determine the metabolic fate and carbon flux of BCAA in mouse brown ST:STUDY_SUMMARY adipocytes, we used 13C labeled BCAA tracing (Leu (NLM-142-1, CIL), Ile ST:STUDY_SUMMARY (NLM-292-0.25, CIL) and Val (NLM-316-0.5, CIL)) followed by LC-MS analysis. ST:INSTITUTE Harvard Medical School ST:LAST_NAME Wang ST:FIRST_NAME Dandan ST:ADDRESS 3 Blackfan Circle ST:EMAIL dandanwang2022@gmail.com ST:PHONE 5083733714 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 13C_EV_2h_neg_1 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_2h_neg_1.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_2h_neg_2 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_2h_neg_2.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_2h_neg_3 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_2h_neg_3.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_6h_neg_1 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_6h_neg_1.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_6h_neg_2 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_6h_neg_2.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_6h_neg_3 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_6h_neg_3.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_24h_neg_1 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_12h_neg_1.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_24h_neg_2 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_12h_neg_2.RAW SUBJECT_SAMPLE_FACTORS - 13C_EV_24h_neg_3 Genotype:Control Sample type=Cell; RAW_FILE_NAME=13C_EV_12h_neg_3.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_2h_neg_1 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_2h_neg_1.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_2h_neg_2 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_2h_neg_2.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_2h_neg_3 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_2h_neg_3.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_6h_neg_1 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_6h_neg_1.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_6h_neg_2 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_6h_neg_2.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_6h_neg_3 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_6h_neg_3.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_24h_neg_1 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_12h_neg_1.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_24h_neg_2 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_12h_neg_2.RAW SUBJECT_SAMPLE_FACTORS - 13C_Cre_24h_neg_3 Genotype:MBC-KO Sample type=Cell; RAW_FILE_NAME=13C_Cre_12h_neg_3.RAW #COLLECTION CO:COLLECTION_SUMMARY For the metabolite extraction from adipocytes, after aspirating the tracing CO:COLLECTION_SUMMARY media, the cells were immediately incubated with 500 μL cold methanol CO:COLLECTION_SUMMARY containing 1ug/ml internal standard (D8-Phe) for 5 min on dry ice and then CO:COLLECTION_SUMMARY scrapped into the Eppendorf tubes. CO:SAMPLE_TYPE Adipocytes #TREATMENT TR:TREATMENT_SUMMARY Twelve hours before the isotope switch, cell media was replaced with fresh TR:TREATMENT_SUMMARY unlabeled media. After switching to the tracing media, the media and cell TR:TREATMENT_SUMMARY samples were collected at 0 h, 2 h, 6 h, 24 h. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells were homogenized in a TissueLyser II (Qiagen) (15 min 30 Hz) at 4 SP:SAMPLEPREP_SUMMARY °C. 200 μL of the extracts were mixed with 100 μL Milli-Q water and 200 μL SP:SAMPLEPREP_SUMMARY chloroform and centrifuged at 16000g for 5 min at 4 °C. Subsequently, 150 μL SP:SAMPLEPREP_SUMMARY of the aqueous solution was centrifugally filtered through a 10-kDa cut-off SP:SAMPLEPREP_SUMMARY filter (MRCPRT010, Millipore) to remove proteins. The filtrate was transferred SP:SAMPLEPREP_SUMMARY to the glass insert for LC-MS detection. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Vanquish Horizon CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 × 2.1mm, 1.7um) CH:SOLVENT_A 100% water; 25mM ammonium acetate; 25mM ammonium hydroxide CH:SOLVENT_B 100% acetonitrile CH:FLOW_GRADIENT The linear gradient eluted from 95% B (0.0–1 min), 95% B to 65% B (1–7.0 CH:FLOW_GRADIENT min), 65% B to 40% B (7.0–8.0 min), 40% B (8.0–9.0 min), 40% B to 95% B CH:FLOW_GRADIENT (9.0–9.1 min), then stayed at 95% B for 5.9 min. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 25 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo orbitrap exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS ESI source parameters were set as follows: spray voltage, 3500 V or −2800 V, MS:MS_COMMENTS in positive or negative modes, respectively; vaporizer temperature, 350 °C; MS:MS_COMMENTS sheath gas, 50 arb; aux gas, 10 arb; ion transfer tube temperature, 325 °C. The MS:MS_COMMENTS full scan was set as: orbitrap resolution, 60,000; maximum injection time, 100 MS:MS_COMMENTS ms; scan range, 70–1050 Da. The ddMS2 scan was set as: orbitrap resolution, MS:MS_COMMENTS 30,000; maximum injection time, 60 ms; top N setting, 6; isolation width, 1.0 MS:MS_COMMENTS m/z; HCD collision energy (%), 30; Dynamic exclusion mode was set as auto. The MS:MS_COMMENTS metabolites were quantified by Compound Discoverer 3.3. MS:MS_RESULTS_FILE ST003008_AN004939_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END