#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