Summary of Study ST003454
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 PR002128. The data can be accessed directly via it's Project DOI: 10.21228/M8JN72 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003454 |
| Study Title | Branched chain alpha-ketoacids impact pulmonary artery smooth muscle cell metabolism |
| Study Summary | Hypoxia-inducible factor 1α (HIF1α) is a master regulator of biological processes in hypoxia. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in normal (primary) cells, remain elusive. Here, we show that HIF1α signalling is activated in several human primary vascular cells in normoxia, and in vascular smooth muscle cells (VSMCs) of normal human lungs. Mechanistically, aerobic HIF1α activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress PHD2 activity via direct inhibition and via LDHA-mediated generation of L-2-hydroxyglutarate. BCKA-mediated HIF1α signalling activation stimulated glycolytic activity and governed a phenotypic switch of pulmonary artery SMCs, which correlated with BCKA metabolic dysregulation and pathophenotypic changes in pulmonary arterial hypertension patients and male rat models. We thus identify BCKAs as novel signalling metabolites that aerobically activate HIF1α and that the BCKA-HIF1α pathway modulates VSMC function, an effect that may be relevant to pulmonary vascular pathobiology. |
| Institute | Peking University |
| Department | Department of Toxicology |
| Last Name | Xiao |
| First Name | Wusheng |
| Address | 38 Xueyuan Rd, Haidian District, Beijing, Beijing, 100191, China |
| wxiao@bjmu.edu.cn | |
| Phone | +1-010-82801628 |
| Submit Date | 2023-08-07 |
| Analysis Type Detail | MALDI |
| Release Date | 2024-09-19 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002128 |
| Project DOI: | doi: 10.21228/M8JN72 |
| Project Title: | Aerobic activation of hypoxia-inducible factor 1alpha in vascular cells. |
| Project Summary: | This project aims to delineate the possible mediators of aerobic activation of HIF1alpha in normal primary cells and its biological consequences related to pulmonary vascular pathobiology. |
| Institute: | Peking University |
| Department: | Department of Toxicology |
| Laboratory: | WXIAO |
| Last Name: | Xiao |
| First Name: | Wusheng |
| Address: | 38 Xueyuan Rd, Haidian District, Beijing, Beijing, 100191, China |
| Email: | wxiao@bjmu.edu.cn |
| Phone: | +1-010-82801628 |
Subject:
| Subject ID: | SU003582 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male and female |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment | Sample source |
|---|---|---|---|
| SA382027 | BCKAs 01 | BCKAs | Vascular smooth muscle cells |
| SA382028 | BCKAs 02 | BCKAs | Vascular smooth muscle cells |
| SA382029 | BCKAs 03 | BCKAs | Vascular smooth muscle cells |
| SA382030 | BCKAs 04 | BCKAs | Vascular smooth muscle cells |
| SA382031 | BCKAs 05 | BCKAs | Vascular smooth muscle cells |
| SA382032 | BCKAs 06 | BCKAs | Vascular smooth muscle cells |
| SA382033 | Ctrl 01 | control | Vascular smooth muscle cells |
| SA382034 | Ctrl 02 | control | Vascular smooth muscle cells |
| SA382035 | Ctrl 03 | control | Vascular smooth muscle cells |
| SA382036 | Ctrl 04 | control | Vascular smooth muscle cells |
| SA382037 | Ctrl 05 | control | Vascular smooth muscle cells |
| SA382038 | Ctrl 06 | control | Vascular smooth muscle cells |
| Showing results 1 to 12 of 12 |
Collection:
| Collection ID: | CO003575 |
| Collection Summary: | intracellular metabolites were collected and stored at -80 degree before LC-MS analysis. |
| Sample Type: | Vascular smooth muscle cells |
| Collection Method: | -80 C precold 80% LC-MS grade methanol was used to collect cell metabolites by scraping and centrifugation |
| Collection Location: | on dry ice |
| Collection Duration: | 1 min |
| Storage Conditions: | -80℃ |
| Collection Tube Temp: | -100 C |
| Tissue Cell Identification: | Pulmonary artery smooth muscle cells |
Treatment:
| Treatment ID: | TR003591 |
| Treatment Summary: | human pulmonary artery smooth muscle cells were treated with branched chain alpha-ketoacids at 50-100 micromolar concentration for 8 hours. |
| Treatment Compound: | branched chain alpha-ketoacids KIC, KIV, and KMV |
| Treatment Route: | addition to medium |
| Treatment Dose: | 50-100 uM |
| Treatment Doseduration: | 8 hours |
| Treatment Vehicle: | water |
| Cell Growth Container: | 75 cm flasks |
| Cell Growth Config: | 1200 rpm |
| Cell Growth Rate: | 30-40 hours |
| Cell Media: | Complete vascular smooth muscle cell growth medium supplemented with growth factors and 5% FBS |
| Cell Envir Cond: | 37 C and 5% CO2 |
| Cell Pct Confluence: | 70-80% |
| Cell Media Lastchanged: | Every 48 hours |
Sample Preparation:
| Sampleprep ID: | SP003589 |
| Sampleprep Summary: | PASMCs were quickly washed with two volumes of ice-cold PBS, lysed for 15 min in pre-chilled 80% liquid chromatography-mass spectrometry (LC-MS)-grade methanol, and harvested by scraping on dry ice followed by centrifugation at maximal speed for 30 min at 4°C. For medium samples, 50 μL of medium were incubated in 200 μL of pre-cooled 100% LC-MS-grade methanol for 15 min on dry ice and then centrifuged as cell samples did. The supernatants of cell and medium samples were collected and evaporated to dryness on an Integrated SpeedVac System (Thermo Fisher Scientific; Waltham, MA) at 42°C. The resulting pellets were resuspended in 50 μL of LC-MS-grade water. Twenty microliters of sample were loaded in an autosampler vial for analysis. |
| Processing Storage Conditions: | Described in summary |
| Extract Storage: | Described in summary |
Combined analysis:
| Analysis ID | AN005675 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Thermo Vanquish |
| Column | Merck ZIC-pHILIC(150 mm × 2.1 mm,3.5um) |
| MS Type | MALDI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Thermo Q Exactive Focus |
| Ion Mode | UNSPECIFIED |
| Units | ion intensity |
Chromatography:
| Chromatography ID: | CH004310 |
| Chromatography Summary: | LC-MS analysis was performed on a Vanquish ultra-high performance liquid chromatography system coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific) that was equipped with an Ion Max source and HESI II probe. metabolites were separated using a ZIC-pHILIC stationary phase (150 mm × 2.1 mm × 3.5 mm; Merck) with a guard column. Mobile phase A contained 20 mM ammonium carbonate and 0.1% ammonium hydroxide. Mobile phase B was acetonitrile. The mobile phase flow rate was 100 μL/min and gradient (%B) was 0 min, 80%; 20 min, 20%; 20.5 min, 80%; 28 min, 80%; and 42 min, stop. The column effluent was introduced into the mass spectrometer with the following ionization source settings: sheath gas 40, auxiliary gas 15, sweep gas 1, spray voltage +3.0 or -3.1 kV, capillary temperature 275°C, S-lens RF level 40, and probe temperature 350°C. The mass spectrometer was operated in polarity-switching full scan mode from 70-1000 m/z. Resolution was set to 70,000, and the AGC target was 1×106 ions. Data were acquired and analyzed using TraceFinder 4.1 (Thermo) with peak identifications based on an in-house library of authentic metabolite standards previously analyzed utilizing this method. Missing values were imputed using random forest. Sample peak areas were normalized using probabilistic quotient normalization. |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Merck ZIC-pHILIC(150 mm × 2.1 mm,3.5um) |
| Column Temperature: | 4 |
| Flow Gradient: | gradient (%B) was 0 min, 80%; 20 min, 20%; 20.5 min, 80%; 28 min, 80%; and 42 min |
| Flow Rate: | 100 |
| Solvent A: | 100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS005399 |
| Analysis ID: | AN005675 |
| Instrument Name: | Thermo Q Exactive Focus |
| Instrument Type: | Triple quadrupole |
| MS Type: | MALDI |
| MS Comments: | The mass spectrometer was operated in polarity-switching full scan mode from 70-1000 m/z. Resolution was set to 70,000, and the AGC target was 1×106 ions. Data were acquired and analyzed using TraceFinder 4.1 (Thermo) with peak identifications based on an in-house library of authentic metabolite standards previously analyzed utilizing this method. Missing values were imputed using random forest. Sample peak areas were normalized using probabilistic quotient normalization |
| Ion Mode: | UNSPECIFIED |
