#METABOLOMICS WORKBENCH dandanwang2022_20231213_133959 DATATRACK_ID:4513 STUDY_ID:ST003010 ANALYSIS_ID:AN004941 PROJECT_ID:PR001873
VERSION             	1
CREATED_ON             	December 14, 2023, 9:09 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                   	Media_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_M_0h_neg_1	Time:0h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_0h_neg_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_0h_neg_2	Time:0h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_0h_neg_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_0h_neg_3	Time:0h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_0h_neg_3.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_2h_neg_1	Time:2h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_2h_neg_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_2h_neg_2	Time:2h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_2h_neg_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_2h_neg_3	Time:2h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_2h_neg_3.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_6h_neg_1	Time:6h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_6h_neg_1.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_6h_neg_2	Time:6h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_6h_neg_2.RAW
SUBJECT_SAMPLE_FACTORS           	-	13C_EV_M_6h_neg_3	Time:6h	Sample type=Media; RAW_FILE_NAME=13C_EV_M_6h_neg_3.RAW
#COLLECTION
CO:COLLECTION_SUMMARY            	Twelve hours before the isotope switch, cell media was replaced with fresh
CO:COLLECTION_SUMMARY            	unlabeled media. After switching to the tracing media, the media samples were
CO:COLLECTION_SUMMARY            	collected at 0 h, 2 h, 6 h, 24 h.
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            	For the extraction of metabolites from media samples, after removing the cell
SP:SAMPLEPREP_SUMMARY            	debris by centrifuging the medium at 16,000 x g for 5 min at 4 °C, 200 μL
SP:SAMPLEPREP_SUMMARY            	clarified medium was transferred to the Eppendorf tube prefilled with 800 μL
SP:SAMPLEPREP_SUMMARY            	cold methanol. After mixing for 30s, the samples were incubated at -80 °C for 1
SP:SAMPLEPREP_SUMMARY            	hour and centrifuged at 16,000 x g at 4 °C for 15 min. Finally, 50 μL
SP:SAMPLEPREP_SUMMARY            	supernatant was transferred 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               	ST003010_AN004941_Results.txt	UNITS:Labeled peak areas	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END