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.

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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.

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Study IDST002503
Study TitleEndothelial Cell CD36 Regulates Membrane Ceramide Formation, Exosome Fatty Acid Delivery to Tissues and Circulating Fatty Acid Levels
Study TypeMembrane ceramide and Fatty Acid Uptake
Study SummaryEndothelial 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
DepartmentIM-Nutitrional Science
LaboratoryAbumrad Lab
Last NamePalacios
First NameHector
AddressWest Building 00201, St. Louis, Missouri, 63110, USA
Emailhectorp@wustl.edu
Phone314-362-5397
Submit Date2023-03-06
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-03-22
Release Version1
Hector Palacios Hector Palacios
https://dx.doi.org/10.21228/M8PH89
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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
SA251330KO3n_14_FAKnock-out Neg
SA251331KO1n_13_FAKnock-out Neg
SA251332KO3n_06_FAKnock-out Neg
SA251333KO1n_21_FAKnock-out Neg
SA251334KO3n_22_FAKnock-out Neg
SA251335KO1n_05Knock-out Neg
SA251336KO3n_22Knock-out Neg
SA251337KO1n_21Knock-out Neg
SA251338KO1n_13Knock-out Neg
SA251339KO3n_06Knock-out Neg
SA251340KO3n_14Knock-out Neg
SA251341KO1n_05_FAKnock-out Neg
SA251342KO1p_07_FAKnock-out Pos
SA251343KO3p_08_FAKnock-out Pos
SA251344KO3p_16_FAKnock-out Pos
SA251345KO1p_15_FAKnock-out Pos
SA251346KO1p_23_FAKnock-out Pos
SA251347KO1p_15Knock-out Pos
SA251348KO3p_08Knock-out Pos
SA251349KO3p_16Knock-out Pos
SA251350KO3p_24Knock-out Pos
SA251351KO1p_07Knock-out Pos
SA251352KO3p_24_FAKnock-out Pos
SA251353KO1p_23Knock-out Pos
SA251354WT3n_10_FAWild-type Neg
SA251355WT3n_18_FAWild-type Neg
SA251356WT1n_17_FAWild-type Neg
SA251357WT1n_09_FAWild-type Neg
SA251358WT1n_01_FAWild-type Neg
SA251359WT3n_02Wild-type Neg
SA251360WT1n_17Wild-type Neg
SA251361WT1n_09Wild-type Neg
SA251362WT1n_01Wild-type Neg
SA251363WT3n_18Wild-type Neg
SA251364WT3n_10Wild-type Neg
SA251365WT3n_02_FAWild-type Neg
SA251366WT1p_11Wild-type Pos
SA251367WT1p_03Wild-type Pos
SA251368WT3p_20Wild-type Pos
SA251369WT3p_20_FAWild-type Pos
SA251370WT1p_03_FAWild-type Pos
SA251371WT1p_19_FAWild-type Pos
SA251372WT3p_12_FAWild-type Pos
SA251373WT3p_04Wild-type Pos
SA251374WT3p_04_FAWild-type Pos
SA251375WT1p_11_FAWild-type Pos
SA251376WT1p_19Wild-type Pos
SA251377WT3p_12Wild-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
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