Summary of Study ST003008

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001873. The data can be accessed directly via it's Project DOI: 10.21228/M8MF0Z This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003008
Study Title13C BCAA tracing in differentiated brown adipocyte
Study SummaryTo determine the metabolic fate and carbon flux of BCAA in mouse brown adipocytes, we used 13C labeled BCAA tracing (Leu (NLM-142-1, CIL), Ile (NLM-292-0.25, CIL) and Val (NLM-316-0.5, CIL)) followed by LC-MS analysis.
Institute
Harvard Medical School
Last NameWang
First NameDandan
Address3 Blackfan Circle
Emaildandanwang2022@gmail.com
Phone5083733714
Submit Date2023-12-13
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-02-19
Release Version1
Dandan Wang Dandan Wang
https://dx.doi.org/10.21228/M8MF0Z
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001873
Project DOI:doi: 10.21228/M8MF0Z
Project Title:Uncoupling Metabolic Health from Thermogenesis via BCAA Flux in Brown Fat
Project Type:MS quantitative analysis
Project Summary:Brown adipose tissue (BAT) is best known for thermogenesis. Whereas numerous studies in rodents found tight associations between the metabolic benefits of BAT and enhanced whole-body energy expenditure, emerging evidence in humans suggests that BAT is protective against Type 2 diabetes independent of body-weight. The underlying mechanism for this dissociation remained unclear. Here, we report that impaired mitochondrial flux of branched-chain amino acids (BCAA) in BAT, by deleting mitochondrial BCAA carrier (MBC, encoded by Slc25a44), was sufficient to cause systemic insulin resistance without affecting whole-body energy expenditure or body-weight. We found that brown adipocytes catabolized BCAAs in the mitochondria as essential nitrogen donors for the biosynthesis of glutamate, N-acetylated amino acids, and one of the products, glutathione. BAT-selective impairment in mitochondrial BCAA flux led to elevated oxidative stress and insulin resistance in the liver, accompanied by reduced levels of BCAA-derived metabolites in the circulation. In turn, supplementation of glutathione restored insulin sensitivity of BAT-specific MBC knockout mice. Notably, a high-fat diet rapidly impaired BCAA catabolism and the synthesis of BCAA-nitrogen derived metabolites in the BAT, while cold-induced BAT activity is coupled with an active synthesis of these metabolites. Together, the present work uncovers a mechanism through which brown fat controls metabolic health independent of thermogenesis via BCAA-derived nitrogen carriers acting on the liver.
Institute:Harvard Medical School
Last Name:Wang
First Name:Dandan
Address:3 Blackfan Circle, Boston, MA, 02115, USA
Email:dandanwang2022@gmail.com
Phone:5083733714

Subject:

Subject ID:SU003122
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Species Group:Mammals

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Genotype
SA32690613C_EV_24h_neg_2Control
SA32690713C_EV_24h_neg_3Control
SA32690813C_EV_2h_neg_1Control
SA32690913C_EV_6h_neg_3Control
SA32691013C_EV_24h_neg_1Control
SA32691113C_EV_2h_neg_2Control
SA32691213C_EV_6h_neg_1Control
SA32691313C_EV_2h_neg_3Control
SA32691413C_EV_6h_neg_2Control
SA32691513C_Cre_24h_neg_1MBC-KO
SA32691613C_Cre_24h_neg_2MBC-KO
SA32691713C_Cre_24h_neg_3MBC-KO
SA32691813C_Cre_6h_neg_3MBC-KO
SA32691913C_Cre_2h_neg_1MBC-KO
SA32692013C_Cre_2h_neg_2MBC-KO
SA32692113C_Cre_2h_neg_3MBC-KO
SA32692213C_Cre_6h_neg_1MBC-KO
SA32692313C_Cre_6h_neg_2MBC-KO
Showing results 1 to 18 of 18

Collection:

Collection ID:CO003115
Collection Summary:For the metabolite extraction from adipocytes, after aspirating the tracing media, the cells were immediately incubated with 500 μL cold methanol containing 1ug/ml internal standard (D8-Phe) for 5 min on dry ice and then scrapped into the Eppendorf tubes.
Sample Type:Adipocytes

Treatment:

Treatment ID:TR003131
Treatment Summary:Twelve hours before the isotope switch, cell media was replaced with fresh unlabeled media. After switching to the tracing media, the media and cell samples were collected at 0 h, 2 h, 6 h, 24 h.

Sample Preparation:

Sampleprep ID:SP003128
Sampleprep Summary:The cells were homogenized in a TissueLyser II (Qiagen) (15 min 30 Hz) at 4 °C. 200 μL of the extracts were mixed with 100 μL Milli-Q water and 200 μL chloroform and centrifuged at 16000g for 5 min at 4 °C. Subsequently, 150 μL of the aqueous solution was centrifugally filtered through a 10-kDa cut-off filter (MRCPRT010, Millipore) to remove proteins. The filtrate was transferred to the glass insert for LC-MS detection.

Chromatography:

Chromatography ID:CH003728
Instrument Name:Vanquish Horizon
Column Name:Waters ACQUITY UPLC BEH Amide (100 × 2.1mm, 1.7um)
Column Temperature:25 °C
Flow Gradient:The linear gradient eluted from 95% B (0.0–1 min), 95% B to 65% B (1–7.0 min), 65% B to 40% B (7.0–8.0 min), 40% B (8.0–9.0 min), 40% B to 95% B (9.0–9.1 min), then stayed at 95% B for 5.9 min.
Flow Rate:0.4 mL/min
Solvent A:100% water; 25mM ammonium acetate; 25mM ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC

Analysis:

Analysis ID:AN004939
Analysis Type:MS
Chromatography ID:CH003728
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST003008_AN004939_Results.txt
Units:Peak area
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