#METABOLOMICS WORKBENCH hectorp_20230306_152042 DATATRACK_ID:3778 STUDY_ID:ST002503 ANALYSIS_ID:AN004114 PROJECT_ID:PR001617 VERSION 1 CREATED_ON March 9, 2023, 5:33 pm #PROJECT PR:PROJECT_TITLE Endothelial Cell CD36 Regulates Membrane Ceramide Formation, Exosome Fatty Acid PR:PROJECT_TITLE Delivery to Tissues and Circulating Fatty Acid Levels PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we PR:PROJECT_SUMMARY visualized FA transfer by ECs using click chemistry. Apical FA interaction with PR:PROJECT_SUMMARY EC CD36 induces actin reorganization, caveolin-1 tyrosine 14 (Cav-1Y14) PR:PROJECT_SUMMARY phosphorylation, and ceramide generation in caveolae, culminating in caveolae PR:PROJECT_SUMMARY internalization into FA/CD36/ceramide vesicles that are secreted basolaterally PR:PROJECT_SUMMARY as small extracellular vesicles, sEVs (exosomes). In transwells, ECs expressing PR:PROJECT_SUMMARY fluorescent CD63 (exosomal marker) transfer FAs to underlying myotubes in PR:PROJECT_SUMMARY CD63-labeled sEVs. In vivo FA-CD63-CD36 transfer from ECs to muscle is shown PR:PROJECT_SUMMARY using a mouse with EC-specific expression of emGFP-CD63. CD36 depletion, PR:PROJECT_SUMMARY actin-binding latrunculin-B, Src inhibition, Cav-1Y14 mutation, and membrane PR:PROJECT_SUMMARY neutral sphingomyelinase inhibition by GW4869 helped map the FA-sEV pathway. PR:PROJECT_SUMMARY Injecting GW4869 to mice reduced muscle FA uptake, raised plasma FAs and lowered PR:PROJECT_SUMMARY glucose, mimicking prominent Cd36-/- phenotypes. The FA-sEV pathway we describe PR:PROJECT_SUMMARY contributes to crosstalk between ECs and underlying cells and links regulation PR:PROJECT_SUMMARY of membrane ceramide to blood FAs. PR:INSTITUTE Washington University in St. Louis PR:DEPARTMENT IM-Nutitrional Science PR:LABORATORY Abumrad Lab PR:LAST_NAME Palacios PR:FIRST_NAME Hector PR:ADDRESS West Building 00201, St. Louis, Missouri, 63110, USA PR:EMAIL hectorp@wustl.edu PR:PHONE 314-362-5397 #STUDY ST:STUDY_TITLE Endothelial Cell CD36 Regulates Membrane Ceramide Formation, Exosome Fatty Acid ST:STUDY_TITLE Delivery to Tissues and Circulating Fatty Acid Levels ST:STUDY_TYPE Membrane ceramide and Fatty Acid Uptake ST:STUDY_SUMMARY Endothelial cell (EC) CD36 controls tissue fatty acid (FA) uptake. Here we ST:STUDY_SUMMARY visualized FA transfer by ECs using click chemistry. Apical FA interaction with ST:STUDY_SUMMARY EC CD36 induces actin reorganization, caveolin-1 tyrosine 14 (Cav-1Y14) ST:STUDY_SUMMARY phosphorylation, and ceramide generation in caveolae, culminating in caveolae ST:STUDY_SUMMARY internalization into FA/CD36/ceramide vesicles that are secreted basolaterally ST:STUDY_SUMMARY as small extracellular vesicles, sEVs (exosomes). In transwells, ECs expressing ST:STUDY_SUMMARY fluorescent CD63 (exosomal marker) transfer FAs to underlying myotubes in ST:STUDY_SUMMARY CD63-labeled sEVs. In vivo FA-CD63-CD36 transfer from ECs to muscle is shown ST:STUDY_SUMMARY using a mouse with EC-specific expression of emGFP-CD63. CD36 depletion, ST:STUDY_SUMMARY actin-binding latrunculin-B, Src inhibition, Cav-1Y14 mutation, and membrane ST:STUDY_SUMMARY neutral sphingomyelinase inhibition by GW4869 helped map the FA-sEV pathway. ST:STUDY_SUMMARY Injecting GW4869 to mice reduced muscle FA uptake, raised plasma FAs and lowered ST:STUDY_SUMMARY glucose, mimicking prominent Cd36-/- phenotypes. The FA-sEV pathway we describe ST:STUDY_SUMMARY contributes to crosstalk between ECs and underlying cells and links regulation ST:STUDY_SUMMARY of membrane ceramide to blood FAs. ST:INSTITUTE Washington University in St. Louis ST:DEPARTMENT IM-Nutitrional Science ST:LABORATORY Abumrad Lab ST:LAST_NAME Palacios ST:FIRST_NAME Hector ST:ADDRESS West Building 00201, St. Louis, Missouri, 63110, USA ST:EMAIL hectorp@wustl.edu ST:PHONE 314-362-5397 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS 5 KO1n_05 Genotype:Knock-out | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO1n_05.mzML SUBJECT_SAMPLE_FACTORS 13 KO1n_13 Genotype:Knock-out | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO1n_13.mzML SUBJECT_SAMPLE_FACTORS 21 KO1n_21 Genotype:Knock-out | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO1n_21.mzML SUBJECT_SAMPLE_FACTORS 7 KO1p_07 Genotype:Knock-out | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO1p_07.mzML SUBJECT_SAMPLE_FACTORS 15 KO1p_15 Genotype:Knock-out | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO1p_15.mzML SUBJECT_SAMPLE_FACTORS 23 KO1p_23 Genotype:Knock-out | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO1p_23.mzML SUBJECT_SAMPLE_FACTORS 6 KO3n_06 Genotype:Knock-out | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO3n_06.mzML SUBJECT_SAMPLE_FACTORS 14 KO3n_14 Genotype:Knock-out | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO3n_14.mzML SUBJECT_SAMPLE_FACTORS 22 KO3n_22 Genotype:Knock-out | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO3n_22.mzML SUBJECT_SAMPLE_FACTORS 8 KO3p_08 Genotype:Knock-out | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO3p_08.mzML SUBJECT_SAMPLE_FACTORS 16 KO3p_16 Genotype:Knock-out | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO3p_16.mzML SUBJECT_SAMPLE_FACTORS 24 KO3p_24 Genotype:Knock-out | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_KO3p_24.mzML SUBJECT_SAMPLE_FACTORS 1 WT1n_01 Genotype:Wild-type | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT1n_01.mzML SUBJECT_SAMPLE_FACTORS 9 WT1n_09 Genotype:Wild-type | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT1n_09.mzML SUBJECT_SAMPLE_FACTORS 17 WT1n_17 Genotype:Wild-type | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT1n_17.mzML SUBJECT_SAMPLE_FACTORS 3 WT1p_03 Genotype:Wild-type | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT1p_03.mzML SUBJECT_SAMPLE_FACTORS 11 WT1p_11 Genotype:Wild-type | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT1p_11.mzML SUBJECT_SAMPLE_FACTORS 19 WT1p_19 Genotype:Wild-type | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT1p_19.mzML SUBJECT_SAMPLE_FACTORS 2 WT3n_02 Genotype:Wild-type | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT3n_02.mzML SUBJECT_SAMPLE_FACTORS 10 WT3n_10 Genotype:Wild-type | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT3n_10.mzML SUBJECT_SAMPLE_FACTORS 18 WT3n_18 Genotype:Wild-type | Treatment:Neg Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT3n_18.mzML SUBJECT_SAMPLE_FACTORS 4 WT3p_04 Genotype:Wild-type | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT3p_04.mzML SUBJECT_SAMPLE_FACTORS 12 WT3p_12 Genotype:Wild-type | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT3p_12.mzML SUBJECT_SAMPLE_FACTORS 20 WT3p_20 Genotype:Wild-type | Treatment:Pos Batch=1a; RAW_FILE_NAME=HSS_Nada_Exo_Pos_IPA_WT3p_20.mzML SUBJECT_SAMPLE_FACTORS 1 WT1n_01_FA Genotype:Wild-type | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_01.mzML SUBJECT_SAMPLE_FACTORS 2 WT3n_02_FA Genotype:Wild-type | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_02.mzML SUBJECT_SAMPLE_FACTORS 3 WT1p_03_FA Genotype:Wild-type | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_03.mzML SUBJECT_SAMPLE_FACTORS 4 WT3p_04_FA Genotype:Wild-type | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_04.mzML SUBJECT_SAMPLE_FACTORS 5 KO1n_05_FA Genotype:Knock-out | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_05.mzML SUBJECT_SAMPLE_FACTORS 6 KO3n_06_FA Genotype:Knock-out | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_06.mzML SUBJECT_SAMPLE_FACTORS 7 KO1p_07_FA Genotype:Knock-out | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_07.mzML SUBJECT_SAMPLE_FACTORS 8 KO3p_08_FA Genotype:Knock-out | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_08.mzML SUBJECT_SAMPLE_FACTORS 9 WT1n_09_FA Genotype:Wild-type | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_09.mzML SUBJECT_SAMPLE_FACTORS 10 WT3n_10_FA Genotype:Wild-type | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_10.mzML SUBJECT_SAMPLE_FACTORS 11 WT1p_11_FA Genotype:Wild-type | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_11.mzML SUBJECT_SAMPLE_FACTORS 12 WT3p_12_FA Genotype:Wild-type | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_12.mzML SUBJECT_SAMPLE_FACTORS 13 KO1n_13_FA Genotype:Knock-out | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_13.mzML SUBJECT_SAMPLE_FACTORS 14 KO3n_14_FA Genotype:Knock-out | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_14.mzML SUBJECT_SAMPLE_FACTORS 15 KO1p_15_FA Genotype:Knock-out | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_15.mzML SUBJECT_SAMPLE_FACTORS 16 KO3p_16_FA Genotype:Knock-out | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_16.mzML SUBJECT_SAMPLE_FACTORS 17 WT1n_17_FA Genotype:Wild-type | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_17.mzML SUBJECT_SAMPLE_FACTORS 18 WT3n_18_FA Genotype:Wild-type | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_18.mzML SUBJECT_SAMPLE_FACTORS 19 WT1p_19_FA Genotype:Wild-type | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_19.mzML SUBJECT_SAMPLE_FACTORS 20 WT3p_20_FA Genotype:Wild-type | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_20.mzML SUBJECT_SAMPLE_FACTORS 21 KO1n_21_FA Genotype:Knock-out | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_21.mzML SUBJECT_SAMPLE_FACTORS 22 KO3n_22_FA Genotype:Knock-out | Treatment:Neg Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_22.mzML SUBJECT_SAMPLE_FACTORS 23 KO1p_23_FA Genotype:Knock-out | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_23.mzML SUBJECT_SAMPLE_FACTORS 24 KO3p_24_FA Genotype:Knock-out | Treatment:Pos Batch=1b; RAW_FILE_NAME=Nada_iHILIC_Exosomes_221_PNS_24.mzML #COLLECTION CO:COLLECTION_SUMMARY Samples were collected and aliquots representing the same EV counts were dried CO:COLLECTION_SUMMARY under nitrogen, and resuspended in 2:2:1 acetonitrile:methanol:H2O (v/v) (for CO:COLLECTION_SUMMARY polar metabolites, eg. fatty acids, phospholipids) or in 100% isopropanol (for CO:COLLECTION_SUMMARY less-polar metabolites, eg. ceramides). The samples were mixed on an orbital CO:COLLECTION_SUMMARY shaker (360 rpm) for 1 min at room temperature before the LC/MS analysis. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Myotube Treatment with hMEC Generated FA-sEV: hMECs grown to confluence were TR:TREATMENT_SUMMARY serum-starved and treated with OA:BSA (100µM:50µM) or BSA (50µM controls). TR:TREATMENT_SUMMARY The sEVs were isolated from media collected over 48h and particle number and TR:TREATMENT_SUMMARY protein content determined. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were collected and aliquots representing the same EV counts were dried SP:SAMPLEPREP_SUMMARY under nitrogen, and resuspended in 2:2:1 acetonitrile:methanol:H2O (v/v) (for SP:SAMPLEPREP_SUMMARY polar metabolites, eg. fatty acids, phospholipids) or in 100% isopropanol (for SP:SAMPLEPREP_SUMMARY less-polar metabolites, eg. ceramides). The samples were mixed on an orbital SP:SAMPLEPREP_SUMMARY shaker (360 rpm) for 1 min at room temperature before the LC/MS analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Waters CORTECS HILIC (100 x 2.1mm,1.6um) CH:SOLVENT_A A: 95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium CH:SOLVENT_A hydroxide solution (25% ammonia in water), 2.5 μM medronic acid CH:SOLVENT_B B: 95% acetonitrile, 5% water, 2.5 μM medronic acid CH:FLOW_GRADIENT 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B CH:FLOW_GRADIENT followed by a re-equilibration phase of 4 CH:FLOW_RATE .25ml/min CH:COLUMN_TEMPERATURE 45 CH:METHODS_FILENAME LC_Method.pdf #ANALYSIS AN:ANALYSIS_TYPE MS AN:ANALYSIS_PROTOCOL_FILE MS_Method.pdf #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS - #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples WT1n_01_FA WT3n_02_FA WT1p_03_FA WT3p_04_FA KO1n_05_FA KO3n_06_FA KO1p_07_FA KO3p_08_FA WT1n_09_FA WT3n_10_FA WT1p_11_FA WT3p_12_FA KO1n_13_FA KO3n_14_FA KO1p_15_FA KO3p_16_FA WT1n_17_FA WT3n_18_FA WT1p_19_FA WT3p_20_FA KO1n_21_FA KO3n_22_FA KO1p_23_FA KO3p_24_FA Factors Genotype:Wild-type | Treatment:Neg Genotype:Wild-type | Treatment:Neg Genotype:Wild-type | Treatment:Pos Genotype:Wild-type | Treatment:Pos Genotype:Knock-out | Treatment:Neg Genotype:Knock-out | Treatment:Neg Genotype:Knock-out | Treatment:Pos Genotype:Knock-out | Treatment:Pos Genotype:Wild-type | Treatment:Neg Genotype:Wild-type | Treatment:Neg Genotype:Wild-type | Treatment:Pos Genotype:Wild-type | Treatment:Pos Genotype:Knock-out | Treatment:Neg Genotype:Knock-out | Treatment:Neg Genotype:Knock-out | Treatment:Pos Genotype:Knock-out | Treatment:Pos Genotype:Wild-type | Treatment:Neg Genotype:Wild-type | Treatment:Neg Genotype:Wild-type | Treatment:Pos Genotype:Wild-type | Treatment:Pos Genotype:Knock-out | Treatment:Neg Genotype:Knock-out | Treatment:Neg Genotype:Knock-out | Treatment:Pos Genotype:Knock-out | Treatment:Pos 14:0 Myristic acid 5174412 4499299 7213572 9330013 4240502 5783439 6230174 3742426 7848177 9324360 7405458 19819936 27167100 13764636 7988237 12877522 5147082 4207413 17974282 8986743 10706074 9938686 7236720 3097104 16:0 Palmitic acid 39366300 34815072 55607364 88705360 31658364 41503328 41878436 26317482 65774520 58825428 66410664 118884016 113863360 70514784 65597904 81915792 48110024 46474204 93923640 83072136 65935236 193837904 136372752 33984828 16:1 Palmitoleic acid 3654700 3183822 6753332 12696920 4591224 4510049 6350590 2904515 8173620 9795025 5040198 29136504 46582192 16965616 9332579 20452512 3110572 4162394 27234002 10992311 15644136 8259623 7312306 2613202 18:0 Stearic acid 34629372 32159584 32261404 69251072 29829112 34720772 34972152 27852610 50106988 42594608 43390988 77338200 54581448 44347744 51928552 53519028 46769004 40467368 59319432 75947976 42862348 189841232 133486416 22137404 18:1 Oleic acid 6380513 4843296 9724976 19117218 5631393 6096055 7850957 4844458 12460580 9869190 16991828 32322382 39550304 17704512 17251704 33463742 6282863 8086649 38695932 21284118 14813989 18685788 19683022 12338788 18:2 Linoleic acid 997400 703669 1449890 3038347 967308 957268 1314698 761571 1512459 1852912 1173704 5165102 8380669 2959558 1778942 3025610 878234 1525928 4379050 1997698 2535115 2095674 1839744 1204400 18:3 alpha-Linolenic acid 223414 267130 289923 322422 162293 262378 250594 206119 249243 291429 281585 660271 736758 240545 540737 719003 268226 165244 544781 551440 198302 1267389 1108248 238126 20:4 Arachidonic acid 698245 389426 801037 625398 625513 291024 356511 341782 780878 642567 476001 613929 3467324 686537 1309384 911856 569848 597243 533066 253269 420401 727050 252215 495053 22:6 Docosahexaenoic acid 0 105589 134171 187493 144104 61058 120801 133265 248533 285394 216920 313820 1121396 291238 478154 421420 144324 231157 0 66033 133115 206941 0 118005 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 14:0 Myristic acid 16:0 Palmitic acid 16:1 Palmitoleic acid 18:0 Stearic acid 18:1 Oleic acid 18:2 Linoleic acid 18:3 alpha-Linolenic acid 20:4 Arachidonic acid 22:6 Docosahexaenoic acid METABOLITES_END #END