#METABOLOMICS WORKBENCH nicholas_lesner_20220822_163028 DATATRACK_ID:3426 STUDY_ID:ST002314 ANALYSIS_ID:AN003781 PROJECT_ID:PR001484 VERSION 1 CREATED_ON October 13, 2022, 11:26 am #PROJECT PR:PROJECT_TITLE Differential requirements for mitochondrial electron transport chain components PR:PROJECT_TITLE in the adult murine liver PR:PROJECT_SUMMARY Mitochondrial electron transport chain (ETC) dysfunction due to mutations in the PR:PROJECT_SUMMARY nuclear or mitochondrial genome is a common cause of metabolic disease in PR:PROJECT_SUMMARY humans, and displays striking tissue specificity depending on the affected gene. PR:PROJECT_SUMMARY The mechanisms underlying tissue specific phenotypes are not understood. Complex PR:PROJECT_SUMMARY I (cI) is classically considered the entry point for electrons into the ETC, and PR:PROJECT_SUMMARY in vitro experiments indicate that cI is required for basal respiration and PR:PROJECT_SUMMARY maintenance of the NAD+/NADH ratio, an indicator of cellular redox status. This PR:PROJECT_SUMMARY finding has largely not been tested in vivo. Here, we report that mitochondrial PR:PROJECT_SUMMARY complex I (cI) is dispensable for homeostasis of the adult mouse liver; animals PR:PROJECT_SUMMARY with hepatocyte-specific loss of cI function display no overt phenotypes or PR:PROJECT_SUMMARY signs of liver damage, and maintain liver function, redox and oxygen status. PR:PROJECT_SUMMARY Further analysis of cI-deficient livers did not reveal significant proteomic or PR:PROJECT_SUMMARY metabolic changes, indicating little to no compensation is required in the PR:PROJECT_SUMMARY setting of complex I loss. In contrast, complex IV (cIV) dysfunction in adult PR:PROJECT_SUMMARY hepatocytes results in decreased liver function, impaired oxygen handling, PR:PROJECT_SUMMARY steatosis, and liver damage, accompanied by significant metabolomic and PR:PROJECT_SUMMARY proteomic perturbations. Metabolomic analysis suggests that the electron PR:PROJECT_SUMMARY transfer flavoprotein complex constitutes a major route for electron entry into PR:PROJECT_SUMMARY the hepatic ETC. Our results support a model whereby complex I loss is tolerated PR:PROJECT_SUMMARY in the mouse liver because hepatocytes use alternative electron donors to fuel PR:PROJECT_SUMMARY the mitochondrial ETC. PR:INSTITUTE The University of Texas Southwestern Medical Center at Dallas PR:DEPARTMENT Children's Research Institute PR:LABORATORY Prashant Mishra PR:LAST_NAME Lesner PR:FIRST_NAME Nicholas PR:ADDRESS 6000 Harry Hines BLVD PR:EMAIL nicholas.lesner@pennmedicine.upenn.edu PR:PHONE 2146483784 #STUDY ST:STUDY_TITLE Differential requirements for mitochondrial electron transport chain components ST:STUDY_TITLE in the adult murine liver - Lactate/Pyruvate Tolerance Test ST:STUDY_SUMMARY Hepatic Cox10 knockout mice were injected with a lactate/pyruvate (10:1) ST:STUDY_SUMMARY solution with 40% of the mixture labeled with [U-13C]. After 30 minutes, plasma ST:STUDY_SUMMARY and liver tissue was harvested. Metabolites were extracted, dried, and ST:STUDY_SUMMARY derivatized to form methoxime-TBDMS adducts. ST:INSTITUTE The University of Texas Southwestern Medical Center at Dallas ST:DEPARTMENT Children's Research Institute ST:LABORATORY Prashant Mishra ST:LAST_NAME Lesner ST:FIRST_NAME Nicholas ST:ADDRESS 6000 Harry Hines BLVD ST:EMAIL nicholas.lesner@pennmedicine.upenn.edu ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 9 ST:PHONE 2146483784 #SUBJECT SU:SUBJECT_TYPE Mammal 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 - 263N Genotype:WT Tissue=Liver; RAW_FILE_NAME=001 SUBJECT_SAMPLE_FACTORS - 263R Genotype:KO Tissue=Liver; RAW_FILE_NAME=002 SUBJECT_SAMPLE_FACTORS - 264N Genotype:WT Tissue=Liver; RAW_FILE_NAME=003 SUBJECT_SAMPLE_FACTORS - 264R Genotype:KO Tissue=Liver; RAW_FILE_NAME=004 SUBJECT_SAMPLE_FACTORS - 264L Genotype:WT Tissue=Liver; RAW_FILE_NAME=005 SUBJECT_SAMPLE_FACTORS - 268N Genotype:KO Tissue=Liver; RAW_FILE_NAME=006 SUBJECT_SAMPLE_FACTORS - 283N Genotype:WT Tissue=Liver; RAW_FILE_NAME=007 SUBJECT_SAMPLE_FACTORS - 283R Genotype:KO Tissue=Liver; RAW_FILE_NAME=008 SUBJECT_SAMPLE_FACTORS - 283L Genotype:KO Tissue=Liver; RAW_FILE_NAME=009 SUBJECT_SAMPLE_FACTORS - 263N_plasma Genotype:WT Tissue=Plasma; RAW_FILE_NAME=010 SUBJECT_SAMPLE_FACTORS - 263R_plasma Genotype:KO Tissue=Plasma; RAW_FILE_NAME=011 SUBJECT_SAMPLE_FACTORS - 264N_plasma Genotype:WT Tissue=Plasma; RAW_FILE_NAME=012 SUBJECT_SAMPLE_FACTORS - 264R_plasma Genotype:KO Tissue=Plasma; RAW_FILE_NAME=013 SUBJECT_SAMPLE_FACTORS - 264L_plasma Genotype:WT Tissue=Plasma; RAW_FILE_NAME=014 SUBJECT_SAMPLE_FACTORS - 268N_plasma Genotype:KO Tissue=Plasma; RAW_FILE_NAME=015 SUBJECT_SAMPLE_FACTORS - 283N_plasma Genotype:WT Tissue=Plasma; RAW_FILE_NAME=016 SUBJECT_SAMPLE_FACTORS - 283R_plasma Genotype:KO Tissue=Plasma; RAW_FILE_NAME=017 SUBJECT_SAMPLE_FACTORS - 283L_plasma Genotype:KO Tissue=Plasma; RAW_FILE_NAME=018 #COLLECTION CO:COLLECTION_SUMMARY Liver was frozen on liqN2. Liver was crushed on liqN2, and metabolites extracted CO:COLLECTION_SUMMARY with 80% meOH. MeOH was dried, and samples derivatized to form methoxime-TBDMS CO:COLLECTION_SUMMARY adducts and immediately injected onto the GCMS. CO:SAMPLE_TYPE Liver #TREATMENT TR:TREATMENT_SUMMARY Mice were IP injected with a lactate/pyruvate (10:1) solution. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 80% methanol containing metabolites was dried overnight in a speedvac. Dried SP:SAMPLEPREP_SUMMARY pellets were derivatized using methoxyamine and MTBSTFA. Samples were SP:SAMPLEPREP_SUMMARY immediately injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY 60C for 1 min then 10C/min to 320C for 1 min 9 min at 320C CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent HP5-MS (30m x 0.25mm, 0.25 um) CH:INJECTION_TEMPERATURE 60 CH:INTERNAL_STANDARD Norvaline CH:SAMPLE_INJECTION Splitless #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5977A MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Scan 75 - 1000 m/z Data processed using Agilent Enhanced Data Analysis Peaks MS:MS_COMMENTS were auto integrated and confirmed by hand. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 263N 263R 264N 264R 264L 268N 283N 283R 283L 263N 263R 264N 264R 264L 268N 283N 283R 283L Factors Genotype:WT Genotype:KO Genotype:WT Genotype:KO Genotype:WT Genotype:KO Genotype:WT Genotype:KO Genotype:KO Genotype:WT Genotype:KO Genotype:WT Genotype:KO Genotype:WT Genotype:KO Genotype:WT Genotype:KO Genotype:KO Pyruvate 791869 1117460 850403 861426 751009 800032 877829 765985 859064 1041533 1138034 1067730 1273782 981941 1231814 1250459 1497066 1187086 aHB 243754 137157 378211 201452 282049 141444 158133 100277 131135 151799 91533 220056 187999 185179 131451 89343 181646 120847 bHB 410001 319185 1189773 634399 866631 386551 560441 244156 344976 958969 711112 2394679 1919596 1828968 1120091 844942 230819 769859 Lactate 12810557 14653043 15216598 14727502 16680942 13175710 12331806 15508155 12310495 10164430 14041435 13316188 16139552 10685685 14807413 11444081 17769939 13282912 Alanine 7117885 15682563 6684575 8790009 10970720 12348452 6038237 19146586 13932173 93241 250131 104769 168099 115372 211150 57855 630535 233519 Glycine 3565324 4284510 1892590 2532650 2769293 1946558 2548153 2548728 2563714 18153 18694 10890 2594546 15946 17563 9111 29265 20760 Valine 413238 495278 357567 328394 339003 359912 229439 235447 227456 110323 136328 97432 102356 106208 171896 48644 96311 86982 Leucine 744251 1077974 702844 642501 719201 761573 446463 456253 416679 179742 247752 182382 171495 185704 324437 73933 154255 119463 Isoleucine 324651 540641 295494 378312 261142 247662 285066 277631 291567 93786 148407 104267 115868 83342 125842 53395 102348 96487 Proline 236559 500708 189542 195251 192916 354370 179340 560180 232662 12627 27503 15217 22709 20506 25742 9361 45174 26472 Succinate 8137744 10824105 9468315 11236970 10912308 8860910 7736043 7532160 8591349 5134 5670 6764 11849 3488 4873 2735 3984 3172 Fumarate 2062620 4884304 2411659 2840175 3528839 3995576 2900317 2815725 3084503 5686 8111 6996 6975 5349 4910 3460 5640 5925 Methionine 73537 110854 64539 116471 64928 61039 65195 69972 100848 21027 55715 26723 40680 29732 56792 21125 73378 69464 Serine 859622 1825319 768805 875762 2108885 1330458 630287 736048 971744 37012 87490 48970 75153 50484 81391 25407 109959 93136 Threonine 195553 354316 189430 259329 409180 229137 137904 250972 294210 138178 319698 147531 230199 201786 315524 96853 333346 367927 Phenylalanine 262641 393919 257840 264562 263817 256566 222394 182942 190724 89034 141147 107834 134074 106583 165309 65127 123932 102596 aKG 15600 14015 22129 17201 34275 18671 18020 9014 11702 728 927 769 777 977 725 921 1335 1514 Malate 2114169 5594762 2688824 3262429 4525975 5346584 3492441 3620405 3283255 0 0 0 0 0 513 0 686 691 Aspartate 1764737 8450969 1184031 1774363 1876551 5627321 2548393 9389445 6636185 0 0 0 0 0 0 0 0 0 Cysteine 27030 65808 42556 72009 41008 40942 44655 39525 43063 4278 6208 7312 1024 7960 6910 5404 10147 9226 Glutamate 15483938 18894417 16596446 18638636 21209296 20628454 16829909 22524302 21299428 0 2094 0 1494 908 0 518 1764 1963 Lysine 352823 603718 484556 355228 596642 635846 275640 784263 499173 0 989 0 583 0 1006 0 2317 1162 Glutamine 2791540 3884541 3922823 5796034 7465299 6710807 4564018 9226501 7583914 6841 15836 11490 16867 14052 15419 6536 97230 23672 Histidine 575056 2348855 708015 779809 732139 1253717 763706 1687291 964398 1915 6025 3049 7581 2423 6905 1545 20878 7360 Citrate 606826 612976 792908 544029 1157214 1058971 1290050 263913 484387 19161 14235 16351 19980 19102 15940 19890 15141 17850 3PG 25349 50339 40001 37057 116615 30277 32929 21562 20815 0 0 0 0 0 0 0 0 0 Tyrosine 162418 495679 128872 238065 180967 377681 132932 505073 209333 30601 93965 26446 70766 36855 104389 18459 199882 75573 PEP 69860 161182 182059 144244 151546 96937 80352 50130 64970 0 0 0 0 0 0 0 0 0 Glucose 330508 208172 386567 328868 445338 294226 327412 248490 217457 540177 476708 525141 530942 513437 493169 521420 386827 510752 d4-BHB 134509 144653 139545 193352 340559 98038 149759 135945 207937 1553725 1624220 1667270 1616022 1443093 1442617 1499418 1048560 1503391 aKB 226312 309609 243327 103160 219449 209396 238984 128759 262103 260020 134525 258317 156714 267733 184986 163746 181276 201509 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Pyruvate aHB bHB Lactate Alanine Glycine Valine Leucine Isoleucine Proline Succinate Fumarate Methionine Serine Threonine Phenylalanine aKG Malate Aspartate Cysteine Glutamate Lysine Glutamine Histidine Citrate 3PG Tyrosine PEP Glucose d4-BHB aKB METABOLITES_END #END