Summary of Study ST003010
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
| Study ID | ST003010 |
| Study Title | Media_13C BCAA tracing in differentiated brown adipocyte |
| Study Summary | To 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 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: | SU003124 |
| 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 | Time |
|---|---|---|
| SA326948 | 13C_EV_M_0h_neg_1 | 0h |
| SA326949 | 13C_EV_M_0h_neg_3 | 0h |
| SA326950 | 13C_EV_M_0h_neg_2 | 0h |
| SA326951 | 13C_EV_M_2h_neg_3 | 2h |
| SA326952 | 13C_EV_M_2h_neg_2 | 2h |
| SA326953 | 13C_EV_M_2h_neg_1 | 2h |
| SA326954 | 13C_EV_M_6h_neg_3 | 6h |
| SA326955 | 13C_EV_M_6h_neg_2 | 6h |
| SA326956 | 13C_EV_M_6h_neg_1 | 6h |
| Showing results 1 to 9 of 9 |
Collection:
| Collection ID: | CO003117 |
| Collection Summary: | Twelve hours before the isotope switch, cell media was replaced with fresh unlabeled media. After switching to the tracing media, the media samples were collected at 0 h, 2 h, 6 h, 24 h. |
| Sample Type: | Adipocytes |
Treatment:
| Treatment ID: | TR003133 |
| 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: | SP003130 |
| Sampleprep Summary: | For the extraction of metabolites from media samples, after removing the cell debris by centrifuging the medium at 16,000 x g for 5 min at 4 °C, 200 μL clarified medium was transferred to the Eppendorf tube prefilled with 800 μL cold methanol. After mixing for 30s, the samples were incubated at -80 °C for 1 hour and centrifuged at 16,000 x g at 4 °C for 15 min. Finally, 50 μL supernatant was transferred to the glass insert for LC-MS detection. |
Combined analysis:
| Analysis ID | AN004941 |
|---|---|
| Chromatography ID | CH003730 |
| MS ID | MS004684 |
| 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 | Labeled peak areas |
Chromatography:
| Chromatography ID: | CH003730 |
| 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 |
MS:
| MS ID: | MS004684 |
| Analysis ID: | AN004941 |
| 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 were quantified by Compound Discoverer 3.3. |
| Ion Mode: | NEGATIVE |
