#METABOLOMICS WORKBENCH dandanwang2022_20231212_125005 DATATRACK_ID:4502 STUDY_ID:ST003004 ANALYSIS_ID:AN004935 VERSION 1 CREATED_ON 12-14-2023 #PROJECT PR:PROJECT_TITLE Uncoupling Metabolic Health from Thermogenesis via BCAA Flux in Brown Fat PR:PROJECT_TYPE MS quantitative analysis 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 BIDMC PR:LAST_NAME Wang PR:FIRST_NAME Dandan PR:ADDRESS 3 Blackfan Circle, Boston, MA, 02115, USA PR:EMAIL dandanwang2022@gmail.com PR:PHONE 5083733714 PR:DOI http://dx.doi.org/10.21228/M8MF0Z #STUDY ST:STUDY_TITLE Extracellular fluid metabolomics of BAT and eWAT ST:STUDY_SUMMARY We quantified metabolites of extracellular fluid samples from BAT and eWAT. ST:STUDY_SUMMARY Briefly, we collected the BAT_EF samples and eWAT_EF samples from 12 weeks chow ST:STUDY_SUMMARY diet C57BL/6J mice (n=5). We run the EF metabolomics using high ph HILIC method ST:STUDY_SUMMARY on Exploris 240. ST:INSTITUTE Harvard Medical School ST:LAST_NAME Wang ST:FIRST_NAME Dandan ST:ADDRESS 3 Blackfan Circle, Boston, MA, 02115, USA ST:EMAIL dandanwang2022@gmail.com ST:PHONE 5083733714 ST:SUBMIT_DATE 2023-12-12 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J SU:AGE_OR_AGE_RANGE 12 weeks SU:WEIGHT_OR_WEIGHT_RANGE 25-30g SU:GENDER Male #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - EF_BAT_neg_1 Tissue:BAT Sample type=EF; RAW_FILE_NAME=EF_BAT_neg_1.RAW SUBJECT_SAMPLE_FACTORS - EF_BAT_neg_2 Tissue:BAT Sample type=EF; RAW_FILE_NAME=EF_BAT_neg_2.RAW SUBJECT_SAMPLE_FACTORS - EF_BAT_neg_3 Tissue:BAT Sample type=EF; RAW_FILE_NAME=EF_BAT_neg_3.RAW SUBJECT_SAMPLE_FACTORS - EF_BAT_neg_4 Tissue:BAT Sample type=EF; RAW_FILE_NAME=EF_BAT_neg_4.RAW SUBJECT_SAMPLE_FACTORS - EF_BAT_neg_5 Tissue:BAT Sample type=EF; RAW_FILE_NAME=EF_BAT_neg_5.RAW SUBJECT_SAMPLE_FACTORS - EF_eWAT_neg_1 Tissue:eWAT Sample type=EF; RAW_FILE_NAME=EF_eWAT_neg_1.RAW SUBJECT_SAMPLE_FACTORS - EF_eWAT_neg_2 Tissue:eWAT Sample type=EF; RAW_FILE_NAME=EF_eWAT_neg_2.RAW SUBJECT_SAMPLE_FACTORS - EF_eWAT_neg_3 Tissue:eWAT Sample type=EF; RAW_FILE_NAME=EF_eWAT_neg_3.RAW SUBJECT_SAMPLE_FACTORS - EF_eWAT_neg_4 Tissue:eWAT Sample type=EF; RAW_FILE_NAME=EF_eWAT_neg_4.RAW SUBJECT_SAMPLE_FACTORS - EF_eWAT_neg_5 Tissue:eWAT Sample type=EF; RAW_FILE_NAME=EF_eWAT_neg_5.RAW #COLLECTION CO:COLLECTION_SUMMARY Animals were sacrificed immediately by cervical dislocation and tissues were CO:COLLECTION_SUMMARY rapidly extracted. Tissues were subjected to centrifugation (10 min, 800 g, CO:COLLECTION_SUMMARY 4°C) following placement in a 20 μm nylon mesh filter (EMD Millipore). CO:SAMPLE_TYPE Extracellular fluid #TREATMENT TR:TREATMENT_SUMMARY All mice were housed under a 12 h – 12 h light/dark cycle. Room-temperature TR:TREATMENT_SUMMARY mice were housed at 23˚C in ventilated cages with an ACH of 25. Mice were fed a TR:TREATMENT_SUMMARY standard diet (Lab Diet 5008) and had free access to food and water. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Animals were sacrificed immediately by cervical dislocation. Tissues were SP:SAMPLEPREP_SUMMARY subjected to centrifugation (10 min, 800 g, 4°C) following placement in a 20 SP:SAMPLEPREP_SUMMARY μm nylon mesh filter (EMD Millipore). Metabolites were extracted by adding SP:SAMPLEPREP_SUMMARY extraction buffer at a ratio of 1:100 interstitial fluid to methanol. Samples SP:SAMPLEPREP_SUMMARY were then centrifuged twice (5 min, 10,000 g, 4°C) and supernatant was SP:SAMPLEPREP_SUMMARY collected. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) CH:COLUMN_TEMPERATURE 25 °C 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:SOLVENT_A 100% water; 25mM ammonium acetate; 25mM ammonium hydroxide CH:SOLVENT_B 100% acetonitrile CH:CHROMATOGRAPHY_TYPE HILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo orbitrap exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI 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 data was analyzed by Compound Discoverer 3.3. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST003004_AN004935_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END