Summary of Study ST002007
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 PR001272. The data can be accessed directly via it's Project DOI: 10.21228/M89402 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 | ST002007 |
| Study Title | Isotope tracing analysis of stress erythroid progenitors |
| Study Summary | Isotope tracing analysis to study the intracellular metabolic changes of progenitors during the expansion stage of stress erythropoiesis and assess the effect of 1400w treatment. |
| Institute | Pennsylvania State University |
| Department | Veterinary and Biomedical Sciences |
| Laboratory | Paulson Lab |
| Last Name | Ruan |
| First Name | Baiye |
| Address | 228 AVBS Building Shortlidge Road University Park, PA 16802 |
| bur27@psu.edu | |
| Phone | 814-863-6306 |
| Submit Date | 2021-12-02 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-12-12 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001272 |
| Project DOI: | doi: 10.21228/M89402 |
| Project Title: | Metabolic profiles of murine stress erythroid progenitors |
| Project Summary: | Inflammation alters hematopoiesis, skewing production to generate myeloid effector cells at the expense of steady state erythropoiesis. To compensate, stress erythropoiesis is induced to maintain homeostasis until the inflammation is resolved. Unlike the constant production of steady state erythropoiesis, stress erythropoiesis generates a bolus of new erythrocytes by first producing immature progenitor cells, which then transition to committed erythroid progenitors and differentiate. We hypothesize that the proliferation of early progenitor cells and their transition to differentiation is regulated by changes in metabolism. Metabolomics and isotope tracing analysis was performed to assess the intracellular metabolic profiles in proliferating progenitors isolated from in vitro stress erythropoiesis cultures. We observed an active engagement of glucose metabolism in glycolysis and anabolic biosynthesis, while the levels of TCA intermediates suggested that TCA cycle and mitochondrial respiration were blocked. Concomitantly, inducible nitric oxide synthase (iNOS) was induced in progenitor cells to increase the production of nitric oxide (NO), which was demonstrated to be crucial for proliferating progenitor metabolism. Inhibition or genetic mutation of iNOS decreased NO levels resulting in the suppression of progenitor proliferation in vitro and in vivo. As evaluated by RNA-seq, inhibition of iNOS suppressed cell proliferation-related pathways including cell cycle and nucleotide metabolism, while upregulating erythroid differentiation genes. These data suggest that iNOS-derived NO production establishes a metabolism that promotes the proliferation of progenitor cells while inhibiting their differentiation. In contrast, the transition to differentiation is marked by decreased Nos2 expression and a change in metabolism to support induction of the erythroid gene expression program. These data support a model where increased pro-inflammatory signals inhibit steady state erythropoiesis, while at the same time promoting stress erythropoiesis to maintain homeostasis. |
| Institute: | Pennsylvania State University |
| Department: | Veterinary and Biomedical Sciences |
| Laboratory: | Paulson Lab |
| Last Name: | Paulson |
| First Name: | Robert |
| Address: | 228 AVBS Building, Shortlidge Road, University Park, PA 16802 |
| Email: | rfp5@psu.edu |
| Phone: | 814-863-6306 |
Subject:
| Subject ID: | SU002088 |
| 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 | Mouse_strain | Culture_stage | Time_point(days) | Treatment |
|---|---|---|---|---|---|
| SA187821 | EM1-2 | wild type C57BL/6 | Expansion stage | 1 | untreated |
| SA187822 | EM1-1 | wild type C57BL/6 | Expansion stage | 1 | untreated |
| SA187823 | EM1-5 | wild type C57BL/6 | Expansion stage | 1 | untreated |
| SA187824 | EM1-3 | wild type C57BL/6 | Expansion stage | 1 | untreated |
| SA187825 | EM1-4 | wild type C57BL/6 | Expansion stage | 1 | untreated |
| SA187826 | EM3-5 | wild type C57BL/6 | Expansion stage | 3 | untreated |
| SA187827 | EM3-4 | wild type C57BL/6 | Expansion stage | 3 | untreated |
| SA187828 | EM3-2 | wild type C57BL/6 | Expansion stage | 3 | untreated |
| SA187829 | EM3-3 | wild type C57BL/6 | Expansion stage | 3 | untreated |
| SA187830 | EM3-1 | wild type C57BL/6 | Expansion stage | 3 | untreated |
| SA187831 | 1400w-3 | wild type C57BL/6 | Expansion stage | 5 | 1400w |
| SA187832 | 1400w-1 | wild type C57BL/6 | Expansion stage | 5 | 1400w |
| SA187833 | 1400w-2 | wild type C57BL/6 | Expansion stage | 5 | 1400w |
| SA187834 | 1400w-4 | wild type C57BL/6 | Expansion stage | 5 | 1400w |
| SA187835 | 1400w-5 | wild type C57BL/6 | Expansion stage | 5 | 1400w |
| SA187841 | EM5-3 | wild type C57BL/6 | Expansion stage | 5 | untreated |
| SA187842 | EM5-1 | wild type C57BL/6 | Expansion stage | 5 | untreated |
| SA187843 | EM5-2 | wild type C57BL/6 | Expansion stage | 5 | untreated |
| SA187844 | EM5-4 | wild type C57BL/6 | Expansion stage | 5 | untreated |
| SA187845 | EM5-5 | wild type C57BL/6 | Expansion stage | 5 | untreated |
| SA187836 | Veh-4 | wild type C57BL/6 | Expansion stage | 5 | Vehicle (DMSO) |
| SA187837 | Veh-5 | wild type C57BL/6 | Expansion stage | 5 | Vehicle (DMSO) |
| SA187838 | Veh-3 | wild type C57BL/6 | Expansion stage | 5 | Vehicle (DMSO) |
| SA187839 | Veh-1 | wild type C57BL/6 | Expansion stage | 5 | Vehicle (DMSO) |
| SA187840 | Veh-2 | wild type C57BL/6 | Expansion stage | 5 | Vehicle (DMSO) |
| Showing results 1 to 25 of 25 |
Collection:
| Collection ID: | CO002081 |
| Collection Summary: | Unfractionated wild-type bone marrow cells were cultured in regular stress erythropoiesis expansion medium (SEEM). At indicated time point, stress erythroid progenitor cells were harvested, washed with PBS, and re-cultured in SEEM supplemented with 25 mM uniformly labeled [13C]-Glucose (Sigma-Aldrich) for 24 hrs. Cells were counted, washed with PBS and snap frozen in liquid nitrogen. |
| Sample Type: | Stem cells |
Treatment:
| Treatment ID: | TR002100 |
| Treatment Summary: | To assess the role of iNOS in progenitor metabolism, SEEM cultures were treated with 10 μM 1400w or vehicle (DMSO) at day 3 for 48 hrs, and then cells were re-cultured in SEEM with U-[13C]-Glucose for 24 hrs. |
| Treatment Compound: | 1400w |
| Treatment Dose: | 10 μM |
| Treatment Vehicle: | DMSO |
| Cell Media: | SEEM |
Sample Preparation:
| Sampleprep ID: | SP002094 |
| Sampleprep Summary: | Cell pellets were extracted with 1 ml pre-chilled 50:50 HPLC-grade water:methanol (v/v) containing 1 µM chlorpropamide as the internal standard. The samples were vortexed briefly followed by thorough homogenization. The samples were then snap frozen with liquid nitrogen and immediately thawed at room temperature. This step was repeated for three times followed by centrifuging for 10 min at 12,000 x g and 4 °C. The supernatants were transferred into fresh microfuge tubes. The remaining cell pellets were re-extracted with 0.5 ml 50% methanol containing 1 µM chlorpropamide, homogenized, frozen and thawed three times, spun down, and the supernatants were combined with the first extraction. Metabolites-containing supernatants were concentrated to dryness at room temperature in a SpeedVac concentrator and re-dissolved in 100 µl 97:3 water:methanol (v/v). After centrifuging for 10 min at 13000 × g and 4°C, 70 µl of supernatants were transferred into autosampler vials for LC-MS analysis. Two types of control were prepared in triplicates to run in concert with the experimental samples: the process blank control, and the pooled control containing an equal volume from each experimental sample. |
Combined analysis:
| Analysis ID | AN003270 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 |
| Column | Waters XSelect HSS C18 (250 x 4.6mm) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Exactive Plus Orbitrap |
| Ion Mode | NEGATIVE |
| Units | peak area |
Chromatography:
| Chromatography ID: | CH002414 |
| Chromatography Summary: | The sample run order was randomized to reduce bias from instrument drift. 10 µl sample was subjected to LC-MS analysis on a Exactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to an Ultimate 3000 UHPLC system (Thermo Fisher Scientific). Reversed-phase chromatography mode was used to separate compounds on a Xselect C18 HSS column (Waters) with solvent A (97:3 water:methanol (v/v), 10 mM tributylamine, and 15 mM acetic acid ) and solvent B (methanol). The flow rate was 200 µl/min, and the total run time was 25 min. The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. The mass spectrometer was operated in a negative-ion mode at a resolution of 140,000 at m/z 200 and with a scan range of 85 to 1000 m/z. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters XSelect HSS C18 (250 x 4.6mm) |
| Flow Gradient: | The gradient was 0 min, 0% B; 5 min, 20% B; 7.5 min, 55% B; 15 min, 65% B; 17.5 min, 95% B; and 21 min, 0% B. |
| Flow Rate: | 200 ul/min |
| Solvent A: | 97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine |
| Solvent B: | 100% methanol |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003042 |
| Analysis ID: | AN003270 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Raw data files were converted to .mzML file format using the ProteoWizard software, and they were analyzed by the MS-DIAL software. Metabolites were identified by comparison to an in-house reference library of pure metabolite standards which included mass-to-charge ratio (m/z) and retention time. For quantification of metabolite abundance, peak areas of identified metabolites were first normalized to the internal standard chlorpropamide, and then normalized to cell numbers from each sample. Data were analyzed using R and Cytoscape software. |
| Ion Mode: | NEGATIVE |
