#METABOLOMICS WORKBENCH hectorp_20230306_152042 DATATRACK_ID:3778 STUDY_ID:ST002503 ANALYSIS_ID:AN004113 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 POSITIVE 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 8:0 Octanoic acid 6024318 5868495 4505258 4852857 4712712 5662254 5375819 5427124 11052060 11350732 10430580 12529638 13856810 11479179 11164885 11377403 7865014 8483268 7694214 7564237 6016050 7033731 6060782 5102615 14:0 Myristic acid 1185878 1032863 1780775 2435800 1134774 1426916 1572105 854415 2201592 2306945 2077278 5493065 7724039 3693623 2094948 3623004 1176171 1192817 4880511 2428226 2898656 2486526 1869929 884189 16:0 Palmitic acid 12127325 10622659 16030927 25341362 9438774 13117621 13019623 8381950 19197032 17101576 19305116 36367420 35722020 20583562 20127466 25109028 14660494 13951457 26796282 25597766 19798904 59079720 39484028 10015493 16:1 Palmitoleic acid 932776 734987 1721844 3514028 1117321 1314972 1930895 782311 2070416 2820426 1280194 8712184 13320464 4613169 2603465 5797492 693708 998516 7216673 3046189 4299951 2098857 1994622 699531 18:0 Stearic acid 5975823 5647972 7587838 15084091 4217518 6089621 6105756 4015148 9336024 7251828 8494870 16821188 12130309 7677414 10896469 11169005 8525398 6588690 11965555 14130922 7723477 42842516 33570108 4680781 18:1 Oleic acid 1288299 929850 2536738 5133695 1130065 1335842 1805759 886343 2875987 2752627 4115300 9288196 10812097 4397867 5059311 8966793 1257356 1680638 10383278 5176635 3423053 4402436 5260044 3357678 18:2 Linoleic acid 163272 131674 307290 823961 169590 215744 307718 144459 381450 396555 277073 1418888 2361871 704038 480006 793115 154802 324079 1118199 523763 657481 547389 453695 221166 20:4 Arachidonic acid 172214 91438 192771 156544 161808 56434 89148 81793 189254 134982 120572 154415 934768 154282 329201 223993 132348 141228 132988 56690 99388 161855 66536 113722 22:6 Docosahexaenoic acid 20777 28202 34056 50038 37277 18828 33066 36885 65188 74833 57527 82063 286728 77195 128374 112619 40312 59924 39742 17448 36379 59887 16871 31493 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name 8:0 Octanoic acid 14:0 Myristic acid 16:0 Palmitic acid 16:1 Palmitoleic acid 18:0 Stearic acid 18:1 Oleic acid 18:2 Linoleic acid 20:4 Arachidonic acid 22:6 Docosahexaenoic acid METABOLITES_END #END