#METABOLOMICS WORKBENCH rkuna_20240805_131509 DATATRACK_ID:5083 STUDY_ID:ST003421 ANALYSIS_ID:AN005620 PROJECT_ID:PR002116 VERSION 1 CREATED_ON August 20, 2024, 6:38 pm #PROJECT PR:PROJECT_TITLE Bempedoic acid improves diet-induced steatosis independent of hepatic ACLY PR:PROJECT_TYPE MS quantitative analysis PR:PROJECT_SUMMARY ATP citrate lyase (ACLY) synthesizes acetyl-CoA for de novo lipogenesis (DNL), PR:PROJECT_SUMMARY which is elevated in metabolic dysfunction-associated steatotic liver disease. PR:PROJECT_SUMMARY Hepatic ACLY is inhibited by the LDL-cholesterol lowering drug bempedoic acid PR:PROJECT_SUMMARY (BPA), which also improves steatosis in mice. Indeed, BPA potently suppresses PR:PROJECT_SUMMARY hepatic DNL and increases fat catabolism. However, it is unclear if ACLY is the PR:PROJECT_SUMMARY relevant molecular target in reducing liver triglyceride, particularly since the PR:PROJECT_SUMMARY acetyl-CoA synthetase ACSS2 can compensate for ACLY deficiency to provision PR:PROJECT_SUMMARY acetyl-CoA for DNL. We show that on a Western diet, loss of hepatic ACLY alone PR:PROJECT_SUMMARY or ACLY and ACSS2 together unexpectedly exacerbates steatosis, linked to reduced PR:PROJECT_SUMMARY hepatic abundance of endogenous PPARa (Peroxisome proliferator-activated PR:PROJECT_SUMMARY receptor alpha) ligands and lower expression of PPARa target genes controlling PR:PROJECT_SUMMARY fatty acid oxidation. Importantly, BPA treatment ameliorates Western PR:PROJECT_SUMMARY diet-mediated triglyceride accumulation in both WT and liver ACLY knockout mice, PR:PROJECT_SUMMARY indicating that its primary effects on hepatic lipid metabolism are independent PR:PROJECT_SUMMARY of ACLY. Together, these data indicate that hepatic ACLY plays an unexpected PR:PROJECT_SUMMARY role in restraining diet-dependent lipid accumulation, and that BPA improves PR:PROJECT_SUMMARY steatosis independent of ACLY. PR:INSTITUTE Salk Institute for Biological Studies PR:DEPARTMENT Molecular and Cell Biology Laboratory PR:LABORATORY Metallo Lab PR:LAST_NAME Kuna PR:FIRST_NAME Ramya PR:ADDRESS 10010 N Torrey Pines Rd, La Jolla, California, 92037, USA PR:EMAIL rkuna@salk.edu PR:PHONE 8582038321 #STUDY ST:STUDY_TITLE BPA regulates the abundance of endogenous PPARa ligands and fatty acid oxidation ST:STUDY_SUMMARY Given the regulation of PPARa(Peroxisome proliferator-activated receptor ST:STUDY_SUMMARY alpha)-dependent gene expression in the absence of ACLY, we postulated that ST:STUDY_SUMMARY endogenous PPARa ligand availability might be altered. Prior work has shown that ST:STUDY_SUMMARY phosphatidylcholines (PCs) can serve as PPARa ligands, and specifically, PC ST:STUDY_SUMMARY 16:0/18:1 has been established as an endogenous ligand. PC 16:0/18:1 abundance ST:STUDY_SUMMARY has also been found to be regulated in a circadian manner dependent on the fatty ST:STUDY_SUMMARY acid synthesis pathway. Since PCs as a class are suppressed in the liver ACLY KO ST:STUDY_SUMMARY mice, we asked if PC 16:0/18:1 is specifically reduced, finding that it its ST:STUDY_SUMMARY abundance is lower in the ACLY KO. ST:INSTITUTE Salk Institute for Biological Studies ST:LAST_NAME Kuna ST:FIRST_NAME Ramya ST:ADDRESS 10010 N Torrey Pines Rd, La Jolla, California, 92037, USA ST:EMAIL rkuna@salk.edu ST:PHONE 8582038321 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6J #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 - Alb_1974_1_WT Vehicle Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1974_1.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1978_5_WT Vehicle Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1978_5.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1983_10_WT Vehicle Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1983_10.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1996_23_WT Vehicle Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1996_23.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_3703_25_WT Vehicle Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-3703_25.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1976_3_WT BPA Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1976_3.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1982_9_WT BPA Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1982_9.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1988_15_WT BPA Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1988_15.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1991_18_WT BPA Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1991_18.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1995_22_WT BPA Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1995_22.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1997_24_WT BPA Genotype:Wild-type | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1997_24.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1975_2_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1975_2.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1979_6_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1979_6.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1980_7_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1980_7.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1985_12_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1985_12.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1987_14_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1987_14.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1992_19_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1992_19.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1993_20_KO Vehicle Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1993_20.d; Group=Vehicle SUBJECT_SAMPLE_FACTORS - Alb_1977_4_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1977_4.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1981_8_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1981_8.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1984_11_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1984_11.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1986_13_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1986_13.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1989_16_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1989_16.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1990_17_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1990_17.d; Group=BPA SUBJECT_SAMPLE_FACTORS - Alb_1994_21_KO BPA Genotype:LACLYKO | Sample source:Mouse liver RAW_FILE_NAME(Raw file name)=Alb-1994_21.d; Group=BPA #COLLECTION CO:COLLECTION_SUMMARY Liver samples were collected using Wollenberger clamps pre-cooled to the CO:COLLECTION_SUMMARY temperature of liquid nitrogen and stored at -80 C until analysis. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY To test the effect of BPA on hepatic steatosis, we placed WT and LAKO mice on WD TR:TREATMENT_SUMMARY for 6 weeks, with 10 mg/kg BPA daily oral gavage over the last 3 weeks, a dose TR:TREATMENT_SUMMARY previously shown to be effective in reducing steatosis. Quantification of lipids TR:TREATMENT_SUMMARY by mass spectrometry demonstrated that ACLY deficiency increased abundance of PC TR:TREATMENT_SUMMARY 16:0/18:1. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Samples were extracted with 400 ul of methanol (-20 C), 100 ul of water SP:SAMPLEPREP_SUMMARY (ice-cold), 400 saline (ice-cold), and 1000 ul of chloroform (-20 C). #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Phenomenex Kinetex C8 (100 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.2% formic acid; 2 mM ammonium formate CH:SOLVENT_B 100% methanol; 0.2% formic acid; 1 mM ammonium formate CH:FLOW_GRADIENT 0 min, 82% B; 3 min, 82% B; 4 min, 90% B; 18 min, 99% B; 25 min, 99% B; 27 min, CH:FLOW_GRADIENT 82% B; 30 min, 82% B CH:FLOW_RATE 0.5 mL/min CH:COLUMN_TEMPERATURE 40˚C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS PC 16:0/18:1 species were analyzed by multiple reaction monitoring of the MS:MS_COMMENTS transition from precursor to product ions at associated optimized collision MS:MS_COMMENTS energies, and fragmentor voltages using Agilent Masshunter. The m/z values of MS:MS_COMMENTS the precursor and product ions are provided in the metabolite metadata section. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS relative abundance/mg tissue MS_METABOLITE_DATA_START Samples Alb_1974_1_WT Vehicle Alb_1978_5_WT Vehicle Alb_1983_10_WT Vehicle Alb_1996_23_WT Vehicle Alb_3703_25_WT Vehicle Alb_1976_3_WT BPA Alb_1982_9_WT BPA Alb_1988_15_WT BPA Alb_1991_18_WT BPA Alb_1995_22_WT BPA Alb_1997_24_WT BPA Alb_1975_2_KO Vehicle Alb_1979_6_KO Vehicle Alb_1980_7_KO Vehicle Alb_1985_12_KO Vehicle Alb_1987_14_KO Vehicle Alb_1992_19_KO Vehicle Alb_1993_20_KO Vehicle Alb_1977_4_KO BPA Alb_1981_8_KO BPA Alb_1984_11_KO BPA Alb_1986_13_KO BPA Alb_1989_16_KO BPA Alb_1990_17_KO BPA Alb_1994_21_KO BPA Factors Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:Wild-type | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver Genotype:LACLYKO | Sample source:Mouse liver PC 16:0/18:1 20.60182706 18.94930778 17.90380185 36.85803086 53.93359599 27.49417643 15.66643226 39.79689486 19.64593461 15.59198338 38.75060892 10.83606318 9.326991345 14.22350248 17.94759722 25.46370534 35.77078553 15.37137521 20.04589797 29.40065794 29.1614173 25.95053619 66.43297994 43.71141339 37.75430409 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion m/z Product Ion m/z PC 16:0/18:1 804.6 255.2 METABOLITES_END #END