Summary of Study ST003007
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.
| Study ID | ST003007 |
| Study Title | 15N BCAA tracing in brown adipocyte |
| Study Summary | To determine the metabolic fate and nitrogen flux of BCAA in mouse brown adipocytes, we used 15N 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 Name | Wang |
| First Name | Dandan |
| Address | 3 Blackfan Circle |
| dandanwang2022@gmail.com | |
| Phone | 5083733714 |
| Submit Date | 2023-12-13 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-02-19 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU003121 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Sample type |
|---|---|---|---|
| SA326882 | EV_6h_C_neg_2 | Control | Cell |
| SA326883 | EV_6h_C_neg_1 | Control | Cell |
| SA326884 | EV_6h_C_neg_3 | Control | Cell |
| SA326885 | EV_24h_C_neg_2 | Control | Cell |
| SA326886 | EV_24h_C_neg_3 | Control | Cell |
| SA326887 | EV_1h_C_neg_3 | Control | Cell |
| SA326888 | EV_24h_C_neg_1 | Control | Cell |
| SA326889 | EV_1h_C_neg_1 | Control | Cell |
| SA326890 | EV_1h_C_neg_2 | Control | Cell |
| SA326891 | Cre_6h_C_neg_2 | MBC-KO | Cell |
| SA326892 | Cre_6h_C_neg_1 | MBC-KO | Cell |
| SA326893 | Cre_6h_C_neg_3 | MBC-KO | Cell |
| SA326894 | Cre_24h_C_neg_1 | MBC-KO | Cell |
| SA326895 | Cre_24h_C_neg_3 | MBC-KO | Cell |
| SA326896 | Cre_24h_C_neg_2 | MBC-KO | Cell |
| SA326897 | Cre_1h_C_neg_3 | MBC-KO | Cell |
| SA326898 | Cre_1h_C_neg_2 | MBC-KO | Cell |
| SA326899 | Cre_1h_C_neg_1 | MBC-KO | Cell |
| SA326903 | EV_T0_C_neg_2 | unlabeled control | Cell |
| SA326904 | EV_T0_C_neg_1 | unlabeled control | Cell |
| SA326905 | EV_T0_C_neg_3 | unlabeled control | Cell |
| SA326900 | Cre_T0_C_neg_2 | unlabeled MBC-KO | Cell |
| SA326901 | Cre_T0_C_neg_1 | unlabeled MBC-KO | Cell |
| SA326902 | Cre_T0_C_neg_3 | unlabeled MBC-KO | Cell |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO003114 |
| 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: | TR003130 |
| Treatment Summary: | Tracing media and corresponding unlabeled media were prepared. Twelve hours before the isotope switch, cell media was replaced with fresh unlabeled media. After switching to the tracing media, the cell samples were collected at 0 h, 1 h, 6 h, 24 h. |
Sample Preparation:
| Sampleprep ID: | SP003127 |
| Sampleprep Summary: | The adipocytes 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. |
Combined analysis:
| Analysis ID | AN004938 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Vanquish Horizon |
| Column | Waters ACQUITY UPLC BEH Amide (100 × 2.1mm, 1.7um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo orbitrap exploris 240 |
| Ion Mode | NEGATIVE |
| Units | Peak area |
Chromatography:
| Chromatography ID: | CH003727 |
| 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 hydroxider |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS004681 |
| Analysis ID: | AN004938 |
| Instrument Name: | Thermo orbitrap exploris 240 |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | ESI source parameters were set as follows: spray voltage, 3500 V or −2800 V, in positive or negative modes, respectively; vaporizer temperature, 350 °C; sheath gas, 50 arb; aux gas, 10 arb; ion transfer tube temperature, 325 °C. The full scan was set as: orbitrap resolution, 60,000; maximum injection time, 100 ms; scan range, 70–1050 Da. The ddMS2 scan was set as: orbitrap resolution, 30,000; maximum injection time, 60 ms; top N setting, 6; isolation width, 1.0 m/z; HCD collision energy (%), 30; Dynamic exclusion mode was set as auto. The metabolites was quantified by Compound Discoverer 3.3. |
| Ion Mode: | NEGATIVE |
