#METABOLOMICS WORKBENCH tetsuya326_20221130_231541 DATATRACK_ID:3616 STUDY_ID:ST002377 ANALYSIS_ID:AN003874 PROJECT_ID:PR001529 VERSION 1 CREATED_ON December 1, 2022, 9:57 am #PROJECT PR:PROJECT_TITLE Hepatic Phosphatidylcholine Catabolism Driven by PNPLA7 and PNPLA8 Supplies PR:PROJECT_TITLE Endogenous Choline to Replenish the Methionine Cycle with Methyl Groups PR:PROJECT_SUMMARY Choline supplies methyl groups for regeneration of methionine and the methyl PR:PROJECT_SUMMARY donor S-adenosylmethionine in the liver. Here we demonstrate that the catabolism PR:PROJECT_SUMMARY of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine PR:PROJECT_SUMMARY (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables PR:PROJECT_SUMMARY endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PR:PROJECT_SUMMARY PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several PR:PROJECT_SUMMARY metabolites related to the methionine cycle, accompanied by various signs of PR:PROJECT_SUMMARY methionine insufficiency including growth retardation, hypoglycemia, PR:PROJECT_SUMMARY hypolipidemia, increased energy consumption, reduced adiposity, increased FGF21, PR:PROJECT_SUMMARY and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient PR:PROJECT_SUMMARY mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a PR:PROJECT_SUMMARY methionine/choline-deficient diet, both knockout strains display a decreased PR:PROJECT_SUMMARY hepatic triglyceride likely via reductions of lipogenesis and GPC-derived PR:PROJECT_SUMMARY glycerol flux. Collectively, our findings highlight the biological importance of PR:PROJECT_SUMMARY phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and PR:PROJECT_SUMMARY triglyceride synthesis in the liver. PR:INSTITUTE Tokyo Metropolitan Institute of Medical Science PR:LAST_NAME Hirabayashi PR:FIRST_NAME Tetsuya PR:ADDRESS 2-6-1 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan PR:EMAIL hirabayashi-tt@igakuken.or.jp PR:PHONE +81-3-5316-3100 #STUDY ST:STUDY_TITLE Hepatic Phosphatidylcholine Catabolism Driven by PNPLA7 and PNPLA8 Supplies ST:STUDY_TITLE Endogenous Choline to Replenish the Methionine Cycle with Methyl Groups ST:STUDY_TITLE (Pnpla7-knockout) ST:STUDY_SUMMARY Choline supplies methyl groups for regeneration of methionine and the methyl ST:STUDY_SUMMARY donor S-adenosylmethionine in the liver. Here we demonstrate that the catabolism ST:STUDY_SUMMARY of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine ST:STUDY_SUMMARY (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables ST:STUDY_SUMMARY endogenous choline stored in hepatic PC to be utilized in methyl metabolism. ST:STUDY_SUMMARY PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several ST:STUDY_SUMMARY metabolites related to the methionine cycle, accompanied by various signs of ST:STUDY_SUMMARY methionine insufficiency including growth retardation, hypoglycemia, ST:STUDY_SUMMARY hypolipidemia, increased energy consumption, reduced adiposity, increased FGF21, ST:STUDY_SUMMARY and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient ST:STUDY_SUMMARY mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a ST:STUDY_SUMMARY methionine/choline-deficient diet, both knockout strains display a decreased ST:STUDY_SUMMARY hepatic triglyceride likely via reductions of lipogenesis and GPC-derived ST:STUDY_SUMMARY glycerol flux. Collectively, our findings highlight the biological importance of ST:STUDY_SUMMARY phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and ST:STUDY_SUMMARY triglyceride synthesis in the liver. ST:INSTITUTE Tokyo Metropolitan Institute of Medical Science ST:LAST_NAME Hirabayashi ST:FIRST_NAME Tetsuya ST:ADDRESS 2-6-1 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506, Japan ST:EMAIL hirabayashi-tt@igakuken.or.jp ST:PHONE +81-3-5316-3100 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6N SU:AGE_OR_AGE_RANGE 6-weeks old #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 - WT1 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT2 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT3 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT4 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT5 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - WT6 Genotype:Wild-type SUBJECT_SAMPLE_FACTORS - KO1 Genotype:Pnpla7-knockout SUBJECT_SAMPLE_FACTORS - KO2 Genotype:Pnpla7-knockout SUBJECT_SAMPLE_FACTORS - KO3 Genotype:Pnpla7-knockout SUBJECT_SAMPLE_FACTORS - KO4 Genotype:Pnpla7-knockout SUBJECT_SAMPLE_FACTORS - KO5 Genotype:Pnpla7-knockout SUBJECT_SAMPLE_FACTORS - KO6 Genotype:Pnpla7-knockout #COLLECTION CO:COLLECTION_SUMMARY Mice were deeply anesthetized and perfused intracardially with saline containing CO:COLLECTION_SUMMARY 1 mM MOPS (pH 7.4). Liver was rapidly removed, frozen on liquid nitrogen and CO:COLLECTION_SUMMARY stored at –80°C until further processing. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY No treatment in this study. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tissues with internal standards were homogenized with ice-cold methanol and SP:SAMPLEPREP_SUMMARY extracted by the Bligh–Dyer method using chloroform and methanol to separate SP:SAMPLEPREP_SUMMARY and recover the water-soluble fractions. The supernatant was filtered through a SP:SAMPLEPREP_SUMMARY 5-kDa cut-off filter (Ultrafree-MC-PLHCC, Human Metabolome Technologies) at SP:SAMPLEPREP_SUMMARY 9,100 × g for 2 h at 4 °C, dried in a centrifugal evaporator at 1,500 rpm SP:SAMPLEPREP_SUMMARY at 1,000 Pa, and resuspended in 50 μl of ultrapure water before metabolome SP:SAMPLEPREP_SUMMARY analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Agilent 7100 CE CH:COLUMN_NAME Fused silica capillary i.d. 50 μm × 80 cm #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6490 QQQ MS:INSTRUMENT_TYPE Other MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The spectrometer was scanned from m/z 50 to 1,000. Peaks were extracted using MS:MS_COMMENTS automatic integration software (MassHunter Quantitative Analysis B.06.00, MS:MS_COMMENTS Agilent Technologies). Putative metabolites were assigned based on the m/z value MS:MS_COMMENTS and migration time using Human Metabolomic Technologies’ standard library and MS:MS_COMMENTS known–unknown peak library. Peak areas were normalized to internal standards MS:MS_COMMENTS and sample amounts. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS nmol MS_METABOLITE_DATA_START Samples WT1 WT2 WT3 WT4 WT5 KO1 KO2 KO3 KO4 KO5 Factors Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Wild-type Genotype:Pnpla7-knockout Genotype:Pnpla7-knockout Genotype:Pnpla7-knockout Genotype:Pnpla7-knockout Genotype:Pnpla7-knockout 2-Hydroxybutyrate 157.61 238.51 149.62 209.49 126.01 426.58 145.14 278.66 217.27 183.06 2-Hydroxyglutarate 42.85 33.82 61.70 57.09 36.48 87.70 31.63 41.36 53.90 24.96 2-Oxobutyrate 68.87 25.83 31.90 76.70 91.05 92.64 37.86 60.34 33.55 109.03 3-(2-Hydroxyphenyl)propionate 13.91 12.31 21.32 23.68 6.16 17.66 4.70 8.19 17.33 7.27 3'-AMP 21.41 10.68 11.38 16.53 20.59 9.82 32.63 23.97 24.39 25.65 3-Hydroxy-3-methylglutarate 2.56 2.94 2.02 1.60 2.07 4.07 2.49 1.18 2.17 1.15 3-Hydroxybutyrate 798.76 1164.59 999.34 1021.46 677.49 981.38 510.99 636.69 756.49 815.49 3-Indoxyl sulfate 2.42 5.22 4.15 5.51 4.25 2.86 2.15 2.78 2.02 3.13 3-Phenyllactate 19.63 16.03 18.39 32.03 20.10 32.23 19.84 25.95 22.69 24.40 3-Ureidopropionate 101.04 154.98 50.55 67.85 74.94 94.21 30.71 16.68 45.59 41.43 3-Phosphoglycerate 44.57 65.55 25.98 46.71 56.20 78.09 34.58 16.46 73.76 26.34 4-Hydroxy-3-methoxymandelate 78.16 94.21 54.50 96.66 83.26 78.49 71.00 82.00 93.34 84.19 4-Methyl-2-oxopentanoate 17.13 10.93 9.18 13.57 24.70 13.14 18.82 19.34 14.24 32.13 4-Oxopentanoate 11.05 10.43 12.14 8.51 9.85 8.64 10.23 10.13 12.32 6.90 5-Oxoproline 503.54 427.83 323.82 390.82 440.38 318.53 347.04 489.47 380.88 547.19 6-Phosphogluconate 136.00 37.62 128.10 157.94 211.75 153.22 264.94 212.11 96.61 247.46 Adenosine 3',5'-diphosphate 4.08 18.90 13.18 44.25 45.78 12.76 N.D. N.D. 17.08 7.98 Adenylosuccinate 129.98 59.95 225.86 310.14 222.11 160.39 23.02 32.38 64.00 87.57 Adipate 6.52 8.76 10.48 10.81 8.51 10.11 11.65 12.26 8.88 6.16 ADP 193.03 371.53 179.78 243.12 265.52 352.63 154.31 126.18 369.83 138.73 ADP-glucose 25.61 28.44 24.06 23.14 22.05 17.10 14.30 19.58 22.19 14.85 ADP-ribose 4.00 36.47 29.78 36.40 11.84 37.36 13.05 8.43 16.42 5.24 AMP 1666.06 2612.32 2281.56 2168.97 1890.73 3821.44 1076.49 982.74 2570.31 732.80 ATP 5.65 50.05 18.87 22.65 43.43 20.06 3.45 2.69 32.88 11.92 Azelate 2.28 1.97 3.59 2.63 2.54 3.22 6.87 6.01 3.31 2.49 Biotin 73.75 58.88 41.18 38.42 65.14 22.00 36.00 40.58 46.58 54.63 Butanoate 69.29 33.66 25.87 29.90 21.90 N.D. 25.30 41.57 Carbamoylaspartate 2.50 3.14 8.45 6.39 1.33 9.76 N.D. N.D. 0.87 3.64 CDP 5.26 13.01 2.39 6.51 10.28 8.48 5.83 4.07 13.07 7.21 CDP-choline 91.81 65.82 53.14 61.87 64.08 55.19 45.31 64.91 82.29 73.40 cis-Aconitate 1.86 3.50 5.98 12.65 3.45 2.71 5.72 3.78 1.87 0.75 Citrate 21.43 44.76 127.47 395.65 97.83 39.71 146.92 73.83 17.03 9.48 CMP 113.50 86.60 112.33 139.68 103.37 104.28 131.35 114.56 127.88 166.88 Cysteine sulfinate 10.32 19.77 7.09 17.51 N.D. 20.29 7.47 N.D. 8.12 5.91 DHAP 9.81 7.36 23.51 18.66 21.19 7.67 6.98 23.45 7.51 45.26 Ethanolamine phosphate 1171.91 1047.42 656.58 869.52 1071.20 804.12 987.95 901.84 1128.23 1223.58 F1,6P 213.37 119.48 433.24 134.77 241.57 279.91 60.71 91.24 190.21 110.12 F6P 427.74 88.06 481.60 961.06 372.78 833.07 306.18 565.18 302.34 231.77 FAD 135.63 174.98 116.87 159.23 155.01 123.25 123.81 126.52 153.22 157.43 Fumarate 1231.22 435.21 635.73 943.69 775.46 957.44 555.59 1047.66 623.40 651.03 G1P (D-Glucose 1-phosphate) 401.92 119.39 386.98 608.51 455.55 546.49 286.36 417.07 462.71 272.71 G6P 674.06 128.18 1142.51 2705.05 1095.61 1538.63 821.07 1795.03 690.67 760.48 GDP-mannose 21.41 30.41 21.77 34.46 28.62 41.55 14.57 37.10 35.50 20.23 Glucuronate 35.04 19.44 104.65 214.99 79.76 38.99 164.12 36.78 30.55 67.43 Glutarate 8.94 5.73 9.27 24.89 5.73 28.32 9.12 11.72 6.18 5.57 Glycerophosphate 4848.93 5798.37 2813.15 1716.25 2175.69 6261.09 4835.86 1831.14 5045.21 4585.23 Glycolate 110.32 106.74 119.92 176.21 90.22 153.62 72.89 120.17 99.56 55.65 GMP 370.59 493.92 377.63 428.21 361.08 578.04 237.44 205.33 556.69 142.17 Hippurate 16.57 13.69 52.44 19.29 6.96 42.62 5.22 12.85 9.03 9.27 IDP 28.82 48.19 27.59 33.81 33.89 46.36 19.73 18.36 48.87 21.11 IMP 617.46 552.88 612.48 546.19 755.74 462.15 714.74 228.62 965.02 252.37 Isethionate 32.15 36.43 16.48 27.67 3.72 26.32 74.45 66.51 62.38 61.71 Isocitrate 7.17 7.37 11.47 12.26 10.60 9.86 14.60 13.71 10.14 13.57 Lactate 27378.47 37633.46 32787.13 43985.57 34023.04 33050.32 23462.14 41933.53 28565.39 26826.79 Malate 1977.69 699.76 1554.29 1642.72 1253.77 1345.51 1488.18 2121.55 988.31 1722.73 Malonate 4.39 9.09 10.09 11.64 7.56 3.63 6.82 7.75 6.35 3.68 Mucate 3.20 2.48 2.09 3.03 4.57 2.07 4.79 2.93 3.13 6.11 N-Acetyl-beta-alanine 89.10 54.83 67.79 50.51 64.67 45.65 60.24 60.75 59.14 69.76 N-Acetylaspartate 6.14 5.59 3.95 7.38 3.51 6.96 8.13 7.13 6.46 7.51 N-Acetylglucosamine 1-phosphate 39.49 38.93 23.77 35.18 41.87 31.83 31.62 29.42 31.64 41.87 N-Acetylglucosamine 6-phosphate 31.96 37.87 23.13 29.62 39.08 27.67 36.13 18.93 29.12 34.11 N-Acetylglutamate 405.54 290.96 330.95 376.50 194.52 507.53 144.79 175.74 301.16 175.30 N-Acetylleucine 5.69 6.24 6.62 2.43 2.90 6.28 2.26 5.05 3.61 2.37 N-Acetylmethionine 15.83 18.60 20.13 8.51 7.55 11.95 9.60 16.54 13.88 10.68 N-Acetylneuraminate 31.65 31.88 25.84 31.14 35.67 20.11 23.95 32.27 28.76 35.73 NAD+ 70.11 103.61 66.82 78.23 73.42 84.68 82.00 70.39 91.57 76.72 NADP+ 41.42 60.73 30.33 40.39 59.01 45.46 33.17 24.51 55.34 41.84 O-Phosphoserine 2.55 1.46 1.05 2.04 2.57 1.14 1.46 1.56 2.03 2.36 Orotate 15.00 9.68 26.34 14.31 N.D. 28.72 6.58 9.62 7.56 Pantothenate 58.40 38.75 33.64 43.02 51.63 24.72 45.30 58.86 42.46 65.19 PEP(Phosphoenolpyruvate) 10.35 18.53 8.45 16.35 22.50 34.94 15.95 7.27 29.77 8.07 Pimelate 1.15 N.D. 3.23 N.D. 1.26 3.06 5.54 5.45 2.40 1.49 Propionate 104.17 63.98 64.97 123.56 117.82 N.D. 52.82 92.82 53.31 82.00 Pyruvate N.D. 103.4289432 N.D. N.D. 78.48542337 N.D. N.D. N.D. N.D. N.D. Ribulose 1,5-diphosphate 23.26 21.61 23.27 15.34 20.05 10.28 7.77 7.51 17.97 21.33 Ru5P(D-Ribulose 5-phosphate) 1998.21 2302.20 1223.79 1861.52 1715.22 2295.86 2126.95 1936.64 2605.50 2585.37 S7P(Sedoheptulose 7-phosphate) 93.20 78.15 104.00 247.18 190.84 163.64 168.60 175.97 158.43 127.66 Sebacate 1.19 0.56 0.89 0.66 0.67 1.05 3.63 3.02 1.27 1.24 Succinate 1168.06 801.21 880.36 1188.01 817.30 611.67 741.47 1118.02 664.51 1031.76 Tartrate 7.25 7.73 17.54 14.00 5.37 10.69 1.23 4.94 8.88 2.70 Taurocholate 701.35 128.06 620.13 263.71 327.71 909.42 870.32 151.71 515.42 288.40 Threonate 279.67 206.45 163.76 230.01 191.67 244.40 305.67 191.46 244.82 248.11 trans-4-Hydroxy-3-methoxycinnamate 31.26 23.78 23.99 46.32 27.52 21.59 19.22 25.76 16.30 27.29 trans-Aconitate 10.70 12.96 19.97 19.03 10.87 13.27 1.41 6.92 13.29 4.27 Trehalose 6-phosphate 39.17 62.49 32.16 48.82 38.58 49.74 30.75 35.07 44.71 31.00 UDP 35.18 66.87 29.05 49.66 49.82 51.51 31.80 22.69 70.75 20.35 UDP-glucose 389.50 918.38 293.80 276.13 825.59 258.08 563.68 280.71 999.79 566.04 UDP-glucuronate 1.04 1.40 4.00 9.23 4.83 2.95 6.86 0.82 5.72 1.37 UDP-N-acetylglucosamine 683.99 760.28 637.07 569.08 623.74 581.31 333.00 328.13 520.38 253.81 UMP 561.13 453.43 688.38 817.83 480.54 806.40 437.72 387.11 718.19 253.20 Urate 11.51 20.49 8.81 14.83 16.10 17.72 20.35 62.47 14.81 15.63 XMP(Xanthosine 5'-phosphate) N.D. N.D. N.D. N.D. 2.23 N.D. N.D. N.D. N.D. 2.53 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z 2-Hydroxybutyrate C05984 103.0401 2-Hydroxyglutarate C02630,C01087,C03196 147.0299 2-Oxobutyrate C00109 101.0244 3-(2-Hydroxyphenyl)propionate C01198 165.0557 3'-AMP C01367 346.0558 3-Hydroxy-3-methylglutarate C03761 161.0455 3-Hydroxybutyrate C01089,C03197 103.0401 3-Indoxyl sulfate No ID 212.0023 3-Phenyllactate No ID 165.0557 3-Ureidopropionate C02642 131.0462 3-Phosphoglycerate C00197 184.9857 4-Hydroxy-3-methoxymandelate No ID 197.0455 4-Methyl-2-oxopentanoate C00233 129.0557 4-Oxopentanoate No ID 115.0401 5-Oxoproline C01879 128.0353 6-Phosphogluconate C00345 275.0174 Adenosine 3',5'-diphosphate C00054 426.0221 Adenylosuccinate C03794 462.0668 Adipate C06104 145.0506 ADP C00008 426.0221 ADP-glucose C00498,C00325 588.0750 ADP-ribose C00301 558.0644 AMP C00020 346.0558 ATP C00002 505.9885 Azelate No ID 187.0976 Biotin C00120 243.0809 Butanoate C00246 87.0452 Carbamoylaspartate No ID 175.0360 CDP C00112 402.0109 CDP-choline C00307 547.1212 cis-Aconitate C00417 173.0092 Citrate C00158 191.0197 CMP C00055 322.0446 Cysteine sulfinate No ID 152.0023 DHAP C00111 168.9908 Ethanolamine phosphate C00346 140.0118 F1,6P C00354 338.9888 F6P C05345,C00085 259.0224 FAD C00016 784.1499 Fumarate C00122 115.0037 G1P (D-Glucose 1-phosphate) C00103 259.0224 G6P C00092 259.0224 GDP-mannose C00394 604.0699 Glucuronate C00191 193.0354 Glutarate C00489 131.0350 Glycerophosphate C00093 171.0064 Glycolate C00160 75.0088 GMP C00144 362.0507 Hippurate C01586 178.0510 IDP C00104 427.0062 IMP C00130 347.0398 Isethionate C05123 124.9914 Isocitrate C00311 191.0197 Lactate C00186,C00256,C01432 89.0244 Malate C00149,C00497,C00711 133.0142 Malonate C00383 103.0037 Mucate No ID 209.0303 N-Acetyl-beta-alanine No ID 130.0510 N-Acetylaspartate No ID 174.0408 N-Acetylglucosamine 1-phosphate C04256 300.0490 N-Acetylglucosamine 6-phosphate C00357 300.0490 N-Acetylglutamate C00624 188.0564 N-Acetylleucine C02710 172.0979 N-Acetylmethionine C02712 190.0543 N-Acetylneuraminate C00270 308.0987 NAD+ C00003 662.1019 NADP+ C00006 742.0682 O-Phosphoserine C01005 184.0017 Orotate C00295 155.0098 Pantothenate C00864 218.1034 PEP(Phosphoenolpyruvate) C00074 166.9751 Pimelate C02656 159.0663 Propionate C00163 73.0295 Pyruvate C00022 87.0088 Ribulose 1,5-diphosphate C01182 308.9782 Ru5P(D-Ribulose 5-phosphate) C00199 229.0119 S7P(Sedoheptulose 7-phosphate) C05382 289.0330 Sebacate No ID 201.1132 Succinate C00042 117.0193 Tartrate C00898 149.0092 Taurocholate C05122 514.2844 Threonate C01620 trans-4-Hydroxy-3-methoxycinnamate No ID 193.0506 trans-Aconitate C02341 173.0092 Trehalose 6-phosphate C00689 421.0753 UDP C00015 402.9949 UDP-glucose C00029,C00052 565.0478 UDP-glucuronate C00167 579.0270 UDP-N-acetylglucosamine C00203,C00043 606.0743 UMP C00105 323.0286 Urate C00366 167.0211 XMP(Xanthosine 5'-phosphate) C00655 363.0347 METABOLITES_END #END