Summary of Study ST002503
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 PR001617. The data can be accessed directly via it's Project DOI: 10.21228/M8PH89 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 | ST002503 |
Study Title | Endothelial Cell CD36 Regulates Membrane Ceramide Formation, Exosome Fatty Acid Delivery to Tissues and Circulating Fatty Acid Levels |
Study Type | Membrane ceramide and Fatty Acid Uptake |
Study Summary | Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we visualized FA transfer by ECs using click chemistry. Apical FA interaction with EC CD36 induces actin reorganization, caveolin-1 tyrosine 14 (Cav-1Y14) phosphorylation, and ceramide generation in caveolae, culminating in caveolae internalization into FA/CD36/ceramide vesicles that are secreted basolaterally as small extracellular vesicles, sEVs (exosomes). In transwells, ECs expressing fluorescent CD63 (exosomal marker) transfer FAs to underlying myotubes in CD63-labeled sEVs. In vivo FA-CD63-CD36 transfer from ECs to muscle is shown using a mouse with EC-specific expression of emGFP-CD63. CD36 depletion, actin-binding latrunculin-B, Src inhibition, Cav-1Y14 mutation, and membrane neutral sphingomyelinase inhibition by GW4869 helped map the FA-sEV pathway. Injecting GW4869 to mice reduced muscle FA uptake, raised plasma FAs and lowered glucose, mimicking prominent Cd36-/- phenotypes. The FA-sEV pathway we describe contributes to crosstalk between ECs and underlying cells and links regulation of membrane ceramide to blood FAs. |
Institute | Washington University in St. Louis |
Department | IM-Nutitrional Science |
Laboratory | Abumrad Lab |
Last Name | Palacios |
First Name | Hector |
Address | West Building 00201, St. Louis, Missouri, 63110, USA |
hectorp@wustl.edu | |
Phone | 314-362-5397 |
Submit Date | 2023-03-06 |
Raw Data Available | Yes |
Raw Data File Type(s) | mzML |
Analysis Type Detail | LC-MS |
Release Date | 2023-03-22 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
Project ID: | PR001617 |
Project DOI: | doi: 10.21228/M8PH89 |
Project Title: | Endothelial Cell CD36 Regulates Membrane Ceramide Formation, Exosome Fatty Acid Delivery to Tissues and Circulating Fatty Acid Levels |
Project Type: | MS quantitative analysis |
Project Summary: | Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we visualized FA transfer by ECs using click chemistry. Apical FA interaction with EC CD36 induces actin reorganization, caveolin-1 tyrosine 14 (Cav-1Y14) phosphorylation, and ceramide generation in caveolae, culminating in caveolae internalization into FA/CD36/ceramide vesicles that are secreted basolaterally as small extracellular vesicles, sEVs (exosomes). In transwells, ECs expressing fluorescent CD63 (exosomal marker) transfer FAs to underlying myotubes in CD63-labeled sEVs. In vivo FA-CD63-CD36 transfer from ECs to muscle is shown using a mouse with EC-specific expression of emGFP-CD63. CD36 depletion, actin-binding latrunculin-B, Src inhibition, Cav-1Y14 mutation, and membrane neutral sphingomyelinase inhibition by GW4869 helped map the FA-sEV pathway. Injecting GW4869 to mice reduced muscle FA uptake, raised plasma FAs and lowered glucose, mimicking prominent Cd36-/- phenotypes. The FA-sEV pathway we describe contributes to crosstalk between ECs and underlying cells and links regulation of membrane ceramide to blood FAs. |
Institute: | Washington University in St. Louis |
Department: | IM-Nutitrional Science |
Laboratory: | Abumrad Lab |
Last Name: | Palacios |
First Name: | Hector |
Address: | West Building 00201, St. Louis, Missouri, 63110, USA |
Email: | hectorp@wustl.edu |
Phone: | 314-362-5397 |
Subject:
Subject ID: | SU002600 |
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 | Treatment |
---|---|---|---|
SA251330 | KO3n_14_FA | Knock-out | Neg |
SA251331 | KO1n_13_FA | Knock-out | Neg |
SA251332 | KO3n_06_FA | Knock-out | Neg |
SA251333 | KO1n_21_FA | Knock-out | Neg |
SA251334 | KO3n_22_FA | Knock-out | Neg |
SA251335 | KO1n_05 | Knock-out | Neg |
SA251336 | KO3n_22 | Knock-out | Neg |
SA251337 | KO1n_21 | Knock-out | Neg |
SA251338 | KO1n_13 | Knock-out | Neg |
SA251339 | KO3n_06 | Knock-out | Neg |
SA251340 | KO3n_14 | Knock-out | Neg |
SA251341 | KO1n_05_FA | Knock-out | Neg |
SA251342 | KO1p_07_FA | Knock-out | Pos |
SA251343 | KO3p_08_FA | Knock-out | Pos |
SA251344 | KO3p_16_FA | Knock-out | Pos |
SA251345 | KO1p_15_FA | Knock-out | Pos |
SA251346 | KO1p_23_FA | Knock-out | Pos |
SA251347 | KO1p_15 | Knock-out | Pos |
SA251348 | KO3p_08 | Knock-out | Pos |
SA251349 | KO3p_16 | Knock-out | Pos |
SA251350 | KO3p_24 | Knock-out | Pos |
SA251351 | KO1p_07 | Knock-out | Pos |
SA251352 | KO3p_24_FA | Knock-out | Pos |
SA251353 | KO1p_23 | Knock-out | Pos |
SA251354 | WT3n_10_FA | Wild-type | Neg |
SA251355 | WT3n_18_FA | Wild-type | Neg |
SA251356 | WT1n_17_FA | Wild-type | Neg |
SA251357 | WT1n_09_FA | Wild-type | Neg |
SA251358 | WT1n_01_FA | Wild-type | Neg |
SA251359 | WT3n_02 | Wild-type | Neg |
SA251360 | WT1n_17 | Wild-type | Neg |
SA251361 | WT1n_09 | Wild-type | Neg |
SA251362 | WT1n_01 | Wild-type | Neg |
SA251363 | WT3n_18 | Wild-type | Neg |
SA251364 | WT3n_10 | Wild-type | Neg |
SA251365 | WT3n_02_FA | Wild-type | Neg |
SA251366 | WT1p_11 | Wild-type | Pos |
SA251367 | WT1p_03 | Wild-type | Pos |
SA251368 | WT3p_20 | Wild-type | Pos |
SA251369 | WT3p_20_FA | Wild-type | Pos |
SA251370 | WT1p_03_FA | Wild-type | Pos |
SA251371 | WT1p_19_FA | Wild-type | Pos |
SA251372 | WT3p_12_FA | Wild-type | Pos |
SA251373 | WT3p_04 | Wild-type | Pos |
SA251374 | WT3p_04_FA | Wild-type | Pos |
SA251375 | WT1p_11_FA | Wild-type | Pos |
SA251376 | WT1p_19 | Wild-type | Pos |
SA251377 | WT3p_12 | Wild-type | Pos |
Showing results 1 to 48 of 48 |
Collection:
Collection ID: | CO002593 |
Collection Summary: | Samples were collected and aliquots representing the same EV counts were dried under nitrogen, and resuspended in 2:2:1 acetonitrile:methanol:H2O (v/v) (for polar metabolites, eg. fatty acids, phospholipids) or in 100% isopropanol (for less-polar metabolites, eg. ceramides). The samples were mixed on an orbital shaker (360 rpm) for 1 min at room temperature before the LC/MS analysis. |
Sample Type: | Cultured cells |
Treatment:
Treatment ID: | TR002612 |
Treatment Summary: | Myotube Treatment with hMEC Generated FA-sEV: hMECs grown to confluence were serum-starved and treated with OA:BSA (100µM:50µM) or BSA (50µM controls). The sEVs were isolated from media collected over 48h and particle number and protein content determined. |
Sample Preparation:
Sampleprep ID: | SP002606 |
Sampleprep Summary: | Samples were collected and aliquots representing the same EV counts were dried under nitrogen, and resuspended in 2:2:1 acetonitrile:methanol:H2O (v/v) (for polar metabolites, eg. fatty acids, phospholipids) or in 100% isopropanol (for less-polar metabolites, eg. ceramides). The samples were mixed on an orbital shaker (360 rpm) for 1 min at room temperature before the LC/MS analysis. |
Combined analysis:
Analysis ID | AN004112 | AN004113 | AN004114 |
---|---|---|---|
Analysis type | MS | MS | MS |
Chromatography type | Reversed phase | HILIC | HILIC |
Chromatography system | Agilent 1290 Infinity II | Thermo Vanquish | Thermo Vanquish |
Column | Waters CORTECS UPLC C18 (150 x 2.1mm,1.6um) | Waters CORTECS HILIC (100 x 2.1mm,1.6um) | Waters CORTECS HILIC (100 x 2.1mm,1.6um) |
MS Type | ESI | ESI | ESI |
MS instrument type | QTOF | Orbitrap | Orbitrap |
MS instrument name | Agilent 6545 QTOF | Thermo Orbitrap ID-X tribrid | Thermo Orbitrap ID-X tribrid |
Ion Mode | POSITIVE | POSITIVE | NEGATIVE |
Units | Peak Area | Peak Area | Peak Area |
Chromatography:
Chromatography ID: | CH003047 |
Methods Filename: | LC_Method.pdf |
Instrument Name: | Agilent 1290 Infinity II |
Column Name: | Waters CORTECS UPLC C18 (150 x 2.1mm,1.6um) |
Column Temperature: | 60 |
Flow Gradient: | 0-2 min, 30% B; 17 min, 75% B; 20 min, 85% B; 23-26 min, 100% B; 26 min, 30% B followed by a re-equilibration phase of 5 min. |
Flow Rate: | .25ml/min |
Solvent A: | A: 60% acetonitrile, 40% water, 0.1% formic acid, 10 mM ammonium formate, 2.5 mM medronic acid |
Solvent B: | 90% 2-propanol, 10% acetonitrile, 0.1% formic acid, 10 mM ammonium formate |
Chromatography Type: | Reversed phase |
Chromatography ID: | CH003048 |
Methods Filename: | LC_Method.pdf |
Instrument Name: | Thermo Vanquish |
Column Name: | Waters CORTECS HILIC (100 x 2.1mm,1.6um) |
Column Temperature: | 45 |
Flow Gradient: | 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B followed by a re-equilibration phase of 4 |
Flow Rate: | .25ml/min |
Solvent A: | A: 95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide solution (25% ammonia in water), 2.5 μM medronic acid |
Solvent B: | B: 95% acetonitrile, 5% water, 2.5 μM medronic acid |
Chromatography Type: | HILIC |
MS:
MS ID: | MS003859 |
Analysis ID: | AN004112 |
Instrument Name: | Agilent 6545 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | - |
Ion Mode: | POSITIVE |
Analysis Protocol File: | MS_Method.pdf |
MS ID: | MS003860 |
Analysis ID: | AN004113 |
Instrument Name: | Thermo Orbitrap ID-X tribrid |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | - |
Ion Mode: | POSITIVE |
Analysis Protocol File: | MS_Method.pdf |
MS ID: | MS003861 |
Analysis ID: | AN004114 |
Instrument Name: | Thermo Orbitrap ID-X tribrid |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | - |
Ion Mode: | NEGATIVE |
Analysis Protocol File: | MS_Method.pdf |