#METABOLOMICS WORKBENCH FareseWalthersLab2022_20220917_145916 DATATRACK_ID:3464 STUDY_ID:ST002303 ANALYSIS_ID:AN003764 PROJECT_ID:PR001476 VERSION 1 CREATED_ON October 6, 2022, 9:40 am #PROJECT PR:PROJECT_TITLE Fitm2 is required for ER homeostasis and normal function of murine liver PR:PROJECT_SUMMARY The ER-resident protein fat-inducing transcript 2 (FIT2) catalyzes acyl-CoA PR:PROJECT_SUMMARY cleavage in vitro and is required for endoplasmic reticulum (ER) homeostasis and PR:PROJECT_SUMMARY normal lipid storage in cells. The gene encoding FIT2 is essential for the PR:PROJECT_SUMMARY viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase in PR:PROJECT_SUMMARY vivo and how this activity affects liver, where the protein was discovered, are PR:PROJECT_SUMMARY unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice PR:PROJECT_SUMMARY fed a chow diet exhibited elevated acyl-CoA levels, ER stress, and signs of PR:PROJECT_SUMMARY liver injury. These mice also had more triglycerides in their livers than PR:PROJECT_SUMMARY control littermates due, in part, to impaired secretion of triglyceride-rich PR:PROJECT_SUMMARY lipoproteins and reduced capacity for fatty acid oxidation. Challenging FIT2-LKO PR:PROJECT_SUMMARY mice with a high-fat diet worsened hepatic ER stress and liver injury, but PR:PROJECT_SUMMARY unexpectedly reversed the steatosis phenotype, similar to what is observed in PR:PROJECT_SUMMARY FIT2-deficient cells loaded with fatty acids. Our findings support the model PR:PROJECT_SUMMARY that FIT2 acts as an acyl-CoA diphosphatase in vivo and is crucial for normal PR:PROJECT_SUMMARY hepatocyte function and ER homeostasis in murine liver. PR:INSTITUTE Harvard University PR:DEPARTMENT Molecular Metabolism PR:LABORATORY FareseWalther's lab PR:LAST_NAME Bond PR:FIRST_NAME Laura PR:ADDRESS 655 Huntington Avenue, Building 1, Room 203, Boston, MA, 02115, USA PR:EMAIL laurabond44@gmail.com PR:PHONE +18573087183 #STUDY ST:STUDY_TITLE Fitm2 is required for ER homeostasis and normal function of murine liver ST:STUDY_SUMMARY The ER-resident protein fat-inducing transcript 2 (FIT2) catalyzes acyl-CoA ST:STUDY_SUMMARY cleavage in vitro and is required for endoplasmic reticulum (ER) homeostasis and ST:STUDY_SUMMARY normal lipid storage in cells. The gene encoding FIT2 is essential for the ST:STUDY_SUMMARY viability of mice and worms. Whether FIT2 acts as an acyl-CoA diphosphatase in ST:STUDY_SUMMARY vivo and how this activity affects liver, where the protein was discovered, are ST:STUDY_SUMMARY unknown. Here, we report that hepatocyte-specific Fitm2 knockout (FIT2-LKO) mice ST:STUDY_SUMMARY fed a chow diet exhibited elevated acyl-CoA levels, ER stress, and signs of ST:STUDY_SUMMARY liver injury. These mice also had more triglycerides in their livers than ST:STUDY_SUMMARY control littermates due, in part, to impaired secretion of triglyceride-rich ST:STUDY_SUMMARY lipoproteins and reduced capacity for fatty acid oxidation. Challenging FIT2-LKO ST:STUDY_SUMMARY mice with a high-fat diet worsened hepatic ER stress and liver injury, but ST:STUDY_SUMMARY unexpectedly reversed the steatosis phenotype, similar to what is observed in ST:STUDY_SUMMARY FIT2-deficient cells loaded with fatty acids. Our findings support the model ST:STUDY_SUMMARY that FIT2 acts as an acyl-CoA diphosphatase in vivo and is crucial for normal ST:STUDY_SUMMARY hepatocyte function and ER homeostasis in murine liver. ST:INSTITUTE Harvard School of Public Health ST:LAST_NAME Bond ST:FIRST_NAME Laura ST:ADDRESS 665 Huntington Ave., Building 2, 3rd Floor, Room 311 | Boston, MA 02115 ST:EMAIL laurabond44@gmail.com ST:PHONE 8573087183 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus #FACTORS #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 - Blank Genotype:Blank RAW_FILE_NAME=blank SUBJECT_SAMPLE_FACTORS - FIT2Flox_01 Genotype:flox RAW_FILE_NAME=FIT2Flox_01 SUBJECT_SAMPLE_FACTORS - FIT2Flox_02 Genotype:flox RAW_FILE_NAME=FIT2Flox_02 SUBJECT_SAMPLE_FACTORS - FIT2Flox_03 Genotype:flox RAW_FILE_NAME=FIT2Flox_03 SUBJECT_SAMPLE_FACTORS - FIT2Flox_04 Genotype:flox RAW_FILE_NAME=FIT2Flox_04 SUBJECT_SAMPLE_FACTORS - FIT2Flox_05 Genotype:flox RAW_FILE_NAME=FIT2Flox_05 SUBJECT_SAMPLE_FACTORS - FIT2-LKO_01 Genotype:Liver Knockout RAW_FILE_NAME=FIT2-LKO_01 SUBJECT_SAMPLE_FACTORS - FIT2-LKO_02 Genotype:Liver Knockout RAW_FILE_NAME=FIT2-LKO_02 SUBJECT_SAMPLE_FACTORS - FIT2-LKO_03 Genotype:Liver Knockout RAW_FILE_NAME=FIT2-LKO_03 SUBJECT_SAMPLE_FACTORS - FIT2-LKO_04 Genotype:Liver Knockout RAW_FILE_NAME=FIT2-LKO_04 SUBJECT_SAMPLE_FACTORS - FIT2-LKO_05 Genotype:Liver Knockout RAW_FILE_NAME=FIT2-LKO_05 SUBJECT_SAMPLE_FACTORS - FIT2-LKO_06 Genotype:Liver Knockout RAW_FILE_NAME=FIT2-LKO_06 #COLLECTION CO:COLLECTION_SUMMARY All mice fasted 2 hours prior to sacrifice. Mice were euthanized with isoflurane CO:COLLECTION_SUMMARY and tissues were collected. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Liver (~100mg) was homogenized in 1 mL of PBS using a Bead Mill Homogenizer SP:SAMPLEPREP_SUMMARY (VWR). Lipids were extracted, according to the Folch method. SP:PROCESSING_STORAGE_CONDITIONS On ice SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Thermo Accucore C30, 2.1 x 250 mm, 3 μm #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Mass spectrometry data analysis was performed using LipidSearch version 4.1 SP MS:MS_COMMENTS (Thermo Fisher Scientific). The results were exported to R-Studio where quality MS:MS_COMMENTS control was performed using pairwise correlations between replicates, a MS:MS_COMMENTS principal component analysis comparing sample groups, as well as retention time MS:MS_COMMENTS plot analysis to verify elution clustering within lipid classes. 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SM(d17:1/16:0) 0 240000000 258000000 270000000 277000000 290000000 342000000 404000000 355000000 322000000 340000000 353000000 SM(d18:1/16:0) 0 2610000000 2660000000 2200000000 2910000000 3050000000 3680000000 4390000000 3850000000 3580000000 3760000000 3980000000 SM(d18:1/24:1) 0 574000000 657000000 735000000 788000000 856000000 804000000 618000000 541000000 654000000 707000000 712000000 SM(d18:1/24:3) 0 1300000000 1280000000 1260000000 1290000000 1280000000 1450000000 1710000000 1350000000 1390000000 1360000000 1450000000 SM(d18:1/26:4) 0 1510000000 1100000000 1380000000 1450000000 1540000000 1560000000 1650000000 1700000000 1300000000 1540000000 1420000000 SM(d20:0/18:1) 0 1270000000 1180000000 1410000000 1120000000 1160000000 1870000000 2300000000 1930000000 1760000000 1900000000 1740000000 SM(d20:0+pO/20:0) 0 14500000 8200000 24800000 9710000 14300000 932000 12400000 9410000 11900000 11600000 11200000 SM(d22:0/18:2) 0 673000000 742000000 897000000 671000000 623000000 588000000 768000000 563000000 663000000 584000000 657000000 SM(d22:1/18:1) 0 1270000000 1220000000 1720000000 1020000000 1120000000 1100000000 1580000000 1230000000 1260000000 1390000000 1180000000 SM(d22:1/20:1) 0 12100000000 8660000000 11000000000 11200000000 11600000000 14000000000 14000000000 14700000000 11200000000 12700000000 12000000000 SM(d22:1/20:2) 0 1380000000 1040000000 1310000000 1210000000 1330000000 1450000000 1450000000 1340000000 1160000000 1200000000 1210000000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name LPC(14:0) LPC(15:0) LPC(16:0) LPC(16:0e) LPC(16:1) LPC(17:0) LPC(18:0) LPC(18:0e) LPC(18:0p) LPC(18:1) LPC(18:2) LPC(18:3) LPC(19:0) LPC(20:0) LPC(20:1) LPC(20:2) LPC(20:3) LPC(20:4) LPC(20:5) LPC(22:1) LPC(22:4) LPC(22:5) LPC(22:6) LPE(16:0) LPE(16:1) LPE(17:0) LPE(18:0) LPE(18:1) LPE(18:2) LPE(19:0) LPE(20:0) LPE(20:0e) LPE(20:1) LPE(20:3) LPE(20:4) LPE(20:5) LPE(22:5) LPE(22:6) LPG(22:6) LPI(16:0) LPI(18:0) LPI(18:1) LPI(20:3) LPI(20:4) LPS(18:0) LPS(20:4) LPS(22:6) PA(16:0/18:1) PA(16:0/18:2) PC(14:0/22:6) PC(15:0/18:2) PC(15:0/20:5) PC(15:0/22:4) PC(16:0/16:0) PC(16:0/16:1) PC(16:0/18:1) PC(16:0/18:2) PC(16:0/18:3) PC(16:0/20:4) PC(16:0/20:5) PC(16:0/22:6) PC(16:1/16:1) PC(16:1/18:2) PC(16:1/20:5) PC(16:1/22:6) PC(17:0/18:1) PC(17:0/18:2) PC(17:0/20:3) PC(17:0/20:4) PC(17:0/20:5) PC(17:0/22:5) PC(17:0/22:6) PC(17:1/16:0) PC(17:1/18:1) PC(17:1/18:2) PC(17:1/20:4) PC(18:0/13:0) PC(18:0/16:0) PC(18:0/18:1) PC(18:0/18:2) PC(18:0/20:2) PC(18:0/20:3) PC(18:0/20:4) PC(18:0/20:5) PC(18:0/22:5) PC(18:0/22:6) PC(18:0e/20:4) PC(18:0p/16:0) PC(18:0p/20:4) PC(18:1/18:1) PC(18:1/18:2) PC(18:1/20:3) PC(18:1/20:4) PC(18:1/22:5) PC(18:1/22:6) PC(18:2/18:2) PC(18:2/20:4) PC(18:2/22:6) PC(18:2p/16:0) PC(18:2p/18:1) PC(18:3/18:2) PC(18:3/20:5) PC(18:3/22:6) PC(18:4/18:0) PC(18:4/18:1) PC(19:0/18:1) PC(19:0/18:2) PC(19:0/20:3) PC(19:0/20:4) PC(19:0/22:6) PC(19:1/18:1) PC(20:0/18:2) PC(20:0/20:3) PC(20:0/20:4) PC(20:0/22:5) PC(20:0/22:6) PC(20:1/18:1) PC(20:1/20:3) PC(20:1/22:5) PC(20:1/22:6) PC(20:2/22:6) PC(20:3/20:4) PC(20:3/22:6) PC(20:4/22:6) PC(20:5/22:6) PC(22:0/18:2) PC(22:1/18:2) PC(22:6/22:6) PC(24:0/18:2) PE(14:0/20:4) PE(14:0/22:6) PE(15:0/18:2) PE(15:0/20:4) PE(15:0/22:6) PE(16:0/16:0) PE(16:0/16:1) PE(16:0/18:1) PE(16:0/18:3) PE(16:0/20:1) PE(16:0/20:4) PE(16:0/20:5) PE(16:0/22:6) PE(16:0e/22:4) PE(16:0e/22:5) PE(16:0e/22:6) PE(16:0p/18:1) PE(16:0p/18:2) PE(16:0p/20:3) PE(16:0p/20:4) PE(16:0p/20:5) PE(16:0p/22:4) PE(16:0p/22:6) PE(16:1/16:1) PE(16:1/18:1) PE(16:1/18:2) PE(16:1/18:3) PE(16:1/20:4) PE(16:1/20:5) PE(16:1/22:6) PE(17:0/18:2) PE(17:0/20:3) PE(17:0/20:4) PE(17:0/20:5) PE(17:0/22:6) PE(17:1/16:0) PE(17:1/18:2) PE(17:1/20:4) PE(17:1/22:6) PE(18:0/16:0) PE(18:0/18:0) PE(18:0/18:1) PE(18:0/18:2) PE(18:0/20:0) PE(18:0/20:1) PE(18:0/20:2) PE(18:0/20:3) PE(18:0/20:4) PE(18:0/22:4) PE(18:0/22:5) PE(18:0/22:6) PE(18:0e/22:4) PE(18:0e/22:5) PE(18:0p/18:1) PE(18:0p/18:2) PE(18:0p/20:3) PE(18:0p/20:4) PE(18:0p/20:5) PE(18:0p/22:4) PE(18:0p/22:5) PE(18:0p/22:6) PE(18:1/18:1) PE(18:1/18:2) PE(18:1/18:3) PE(18:1/20:2) PE(18:1/20:3) PE(18:1/20:4) PE(18:1/22:5) PE(18:1/22:6) PE(18:1p/18:2) PE(18:1p/22:5) PE(18:1p/22:6) PE(18:2/18:2) PE(18:2/20:4) PE(18:2/22:6) PE(18:2p/18:2) PE(18:2p/20:4) PE(18:3/18:2) PE(18:3/20:5) PE(18:3/22:6) PE(18:4/22:6) PE(19:0/18:2) PE(19:0/20:3) PE(19:0/20:4) PE(19:0/22:5) PE(19:0/22:6) PE(19:1/20:4) PE(20:0/18:2) PE(20:0/20:4) PE(20:0/22:5) PE(20:0/22:6) PE(20:0e/22:6) PE(20:0p/18:2) PE(20:0p/20:4) PE(20:0p/22:6) PE(20:1/18:1) PE(20:1/22:5) PE(20:1/22:6) PE(20:1p/18:2) PE(20:1p/20:4) PE(20:2/22:6) PE(20:3/18:2) PE(20:3/20:3) PE(20:3/22:6) PE(20:4/22:6) PE(20:5/22:6) PE(22:0/20:4) PE(22:1/20:4) PE(22:5/18:2) PE(22:6/22:6) PG(16:0/18:1) PG(16:0/18:2) PG(16:0/20:4) PG(16:1/22:6) PG(18:0/18:1) PG(18:0/18:2) PG(18:0/20:3) PG(18:1/18:1) PG(18:1/18:2) PG(18:1/20:4) PG(18:1/20:5) PG(18:1/22:6) PG(18:2/18:2) PG(18:2/22:6) PG(18:3/22:6) PG(20:3/22:6) PG(20:4/22:6) PG(20:5/18:2) PG(20:5/22:6) PG(22:4/22:6) PG(22:5/18:2) PG(22:5/22:6) PG(22:6/22:6) PG(30:0/16:0) PG(30:1/16:0) PG(30:1/18:1) PI(14:0/20:4) PI(16:0/16:1) PI(16:0/18:1) PI(16:0/18:2) PI(16:0/18:3) PI(16:0/20:3) PI(16:0/20:4) PI(16:0/20:5) PI(16:0/22:6) PI(16:1/18:2) PI(16:1/20:5) PI(17:0/18:2) PI(17:0/20:4) PI(17:0/20:5) PI(17:1/18:2) PI(18:0/18:0) PI(18:0/18:1) PI(18:0/18:2) PI(18:0/20:2) PI(18:0/20:3) PI(18:0/20:4) PI(18:0/20:5) PI(18:0/22:4) PI(18:0/22:5) PI(18:0/22:6) PI(18:0p/20:4) PI(18:1/18:1) PI(18:1/18:2) PI(18:1/20:3) PI(18:1/20:4) PI(18:1/22:5) PI(18:1/22:6) PI(18:2/18:2) PI(18:2/20:4) PI(18:3/18:2) PI(18:3/20:4) PI(19:0/20:3) PI(19:0/20:4) PI(19:1/20:4) PI(20:0/20:3) PI(20:0/20:4) PI(20:4/20:4) PS(16:0/18:2) PS(16:0/20:3) PS(16:0/20:4) PS(16:0/20:5) PS(16:0/22:6) PS(17:0/22:6) PS(18:0/18:1) PS(18:0/18:2) PS(18:0/20:3) PS(18:0/20:4) PS(18:0/20:5) PS(18:0/22:4) PS(18:0/22:5) PS(18:0/22:6) PS(18:1/22:6) PS(18:2/18:2) PS(19:0/22:6) PS(20:0/22:6) PS(20:2/20:2) PS(20:4/22:6) PS(20:5/20:4) SM(d17:1/16:0) SM(d18:1/16:0) SM(d18:1/24:1) SM(d18:1/24:3) SM(d18:1/26:4) SM(d20:0/18:1) SM(d20:0+pO/20:0) SM(d22:0/18:2) SM(d22:1/18:1) SM(d22:1/20:1) SM(d22:1/20:2) METABOLITES_END #END