#METABOLOMICS WORKBENCH dandanwang2022_20231213_102952 DATATRACK_ID:4510 STUDY_ID:ST003007 ANALYSIS_ID:AN004938 PROJECT_ID:PR001873
VERSION             	1
CREATED_ON             	December 14, 2023, 9:02 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                   	15N BCAA tracing in brown adipocyte
ST:STUDY_SUMMARY                 	To determine the metabolic fate and nitrogen flux of BCAA in mouse brown
ST:STUDY_SUMMARY                 	adipocytes, we used 15N 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           	-	EV_T0_C_neg_1	Genotype:unlabeled control | Sample type:Cell	RAW_FILE_NAME=EV_T0_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_T0_C_neg_2	Genotype:unlabeled control | Sample type:Cell	RAW_FILE_NAME=EV_T0_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_T0_C_neg_3	Genotype:unlabeled control | Sample type:Cell	RAW_FILE_NAME=EV_T0_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_T0_C_neg_1	Genotype:unlabeled MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_T0_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_T0_C_neg_2	Genotype:unlabeled MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_T0_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_T0_C_neg_3	Genotype:unlabeled MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_T0_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_1h_C_neg_1	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_1h_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_1h_C_neg_2	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_1h_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_1h_C_neg_3	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_1h_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_6h_C_neg_1	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_6h_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_6h_C_neg_2	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_6h_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_6h_C_neg_3	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_6h_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_24h_C_neg_1	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_24h_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_24h_C_neg_2	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_24h_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	EV_24h_C_neg_3	Genotype:Control | Sample type:Cell	RAW_FILE_NAME=EV_24h_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_1h_C_neg_1	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_1h_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_1h_C_neg_2	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_1h_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_1h_C_neg_3	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_1h_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_6h_C_neg_1	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_6h_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_6h_C_neg_2	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_6h_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_6h_C_neg_3	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_6h_C_neg_3_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_24h_C_neg_1	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_24h_C_neg_1_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_24h_C_neg_2	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_24h_C_neg_2_.RAW
SUBJECT_SAMPLE_FACTORS           	-	Cre_24h_C_neg_3	Genotype:MBC-KO | Sample type:Cell	RAW_FILE_NAME=Cre_24h_C_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             	Tracing media and corresponding unlabeled media were prepared. Twelve hours
TR:TREATMENT_SUMMARY             	before the isotope switch, cell media was replaced with fresh unlabeled media.
TR:TREATMENT_SUMMARY             	After switching to the tracing media, the cell samples were collected at 0 h, 1
TR:TREATMENT_SUMMARY             	h, 6 h, 24 h.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The adipocytes were homogenized in a TissueLyser II (Qiagen) (15 min 30 Hz)
SP:SAMPLEPREP_SUMMARY            	at 4 °C. 200 μL of the extracts were mixed with 100 μL Milli-Q water and 200
SP:SAMPLEPREP_SUMMARY            	μL chloroform and centrifuged at 16000g for 5 min at 4 °C. Subsequently, 150
SP:SAMPLEPREP_SUMMARY            	μL 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 hydroxider
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 was quantified by Compound Discoverer 3.3.
MS:MS_RESULTS_FILE               	ST003007_AN004938_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END