#METABOLOMICS WORKBENCH jwalejko_20210126_112125_mwtab.txt DATATRACK_ID:2424 STUDY_ID:ST001666 ANALYSIS_ID:AN002718 PROJECT_ID:000000 VERSION 1 CREATED_ON January 28, 2021, 11:16 am #PROJECT PR:PROJECT_TITLE Branched-chain alpha-ketoacids are preferentially reaminated and activate PR:PROJECT_TITLE protein synthesis in the heart PR:PROJECT_SUMMARY Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are PR:PROJECT_SUMMARY elevated in an array of cardiometabolic diseases. Here we demonstrate that the PR:PROJECT_SUMMARY major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in PR:PROJECT_SUMMARY the heart is reamination to valine. Activation of cardiac branched-chain PR:PROJECT_SUMMARY α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, PR:PROJECT_SUMMARY BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of PR:PROJECT_SUMMARY BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of PR:PROJECT_SUMMARY expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its PR:PROJECT_SUMMARY overexpression significantly lowers accumulation of [13C]-labeled valine from PR:PROJECT_SUMMARY [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in PR:PROJECT_SUMMARY obese rats increased increases phosphorylation of the translational repressor PR:PROJECT_SUMMARY 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a PR:PROJECT_SUMMARY doubling of total protein synthesis. These data suggest that elevated BCKA PR:PROJECT_SUMMARY levels found in obesity may contribute to pathologic cardiac hypertrophy via PR:PROJECT_SUMMARY chronic activation of protein synthesis. PR:INSTITUTE Duke University PR:DEPARTMENT Medicine PR:LAST_NAME Walejko PR:FIRST_NAME Jacquelyn PR:ADDRESS 300 N Duke St Durham NC 27701 PR:EMAIL jacquelyn.walejko@duke.edu PR:PHONE 6086097615 #STUDY ST:STUDY_TITLE Branched-chain alpha-ketoacids are preferentially reaminated and activate ST:STUDY_TITLE protein synthesis in the mouse heart ST:STUDY_SUMMARY Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are ST:STUDY_SUMMARY elevated in an array of cardiometabolic diseases. Here we demonstrate that ST:STUDY_SUMMARY sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due ST:STUDY_SUMMARY to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in ST:STUDY_SUMMARY the heart, as its overexpression significantly lowers accumulation of ST:STUDY_SUMMARY [13C]-labeled valine from [U-13C]KIV. ST:INSTITUTE Duke University ST:LAST_NAME Walejko ST:FIRST_NAME Jacquelyn ST:ADDRESS 300 N Duke St ST:EMAIL jacquelyn.walejko@duke.edu ST:PHONE 9194792304 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 8 weeks SU:GENDER Male #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 - 9 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 10 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 17 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 18 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 26 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 33 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 34 Treatment:GFP SUBJECT_SAMPLE_FACTORS - 11 Treatment:SLC25A44 SUBJECT_SAMPLE_FACTORS - 12 Treatment:SLC25A44 SUBJECT_SAMPLE_FACTORS - 19 Treatment:SLC25A44 SUBJECT_SAMPLE_FACTORS - 20 Treatment:SLC25A44 SUBJECT_SAMPLE_FACTORS - 28 Treatment:SLC25A44 SUBJECT_SAMPLE_FACTORS - 35 Treatment:SLC25A44 SUBJECT_SAMPLE_FACTORS - 36 Treatment:SLC25A44 #COLLECTION CO:COLLECTION_SUMMARY Fed male C57BL/6J mice were anesthetized with 5% isoflurane, and isolated hearts CO:COLLECTION_SUMMARY were perfused in the Langendorff mode at 37°C with non-recirculating perfusate. CO:COLLECTION_SUMMARY The hearts were allowed to beat spontaneously throughout the perfusion. At the CO:COLLECTION_SUMMARY end of each perfusion, hearts were immediately freeze-clamped in liquid nitrogen CO:COLLECTION_SUMMARY using the Wollenberger technique and stored at -80 oC for further analysis. CO:SAMPLE_TYPE Heart #TREATMENT TR:TREATMENT_SUMMARY SLC25A44 and GFP expression plasmids with a CMV promoter containing ITRs were TR:TREATMENT_SUMMARY used to prepare recombinant AAV9 by the UMass Gene Therapy Core. Adult TR:TREATMENT_SUMMARY 8-week-old male mice were injected intravenously with 100 µl of 5.1 x 1011 TR:TREATMENT_SUMMARY vector genomes per mouse. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Frozen tissues were pulverized under liquid nitrogen using a mortar and pestle. SP:SAMPLEPREP_SUMMARY Metabolites were then extracted using sequential 500 μL additions of -20°C SP:SAMPLEPREP_SUMMARY MeOH, chilled water, and chloroform. After each addition, tissue lysates were SP:SAMPLEPREP_SUMMARY prepared with a Tissue Lyser (Qiagen) for 60 seconds at 30Hz. Similarly, plasma SP:SAMPLEPREP_SUMMARY metabolites (20 μL) were extracted by sequential 500 μL additions of -20°C SP:SAMPLEPREP_SUMMARY MeOH, chilled water, and chloroform. After each addition, samples were vortexed SP:SAMPLEPREP_SUMMARY for 30 seconds. Tissue extracts were then centrifuged at 4°C and 14400 x g for SP:SAMPLEPREP_SUMMARY 20 minutes and the clarified aqueous phase was transferred to a fresh Eppendorf SP:SAMPLEPREP_SUMMARY and stored in -80°C until processing for GC-MS analysis. For GC-MS analysis, SP:SAMPLEPREP_SUMMARY the extracted tissue was dried under N2 gas-flow at 37°C using an evaporator. SP:SAMPLEPREP_SUMMARY Amino and organic acids were derivatized via methoximation and silylation. SP:SAMPLEPREP_SUMMARY Briefly, metabolites were resuspended in 25 μL of methoxylamine hydrochloride SP:SAMPLEPREP_SUMMARY (2% (w/v) in pyridine) and incubated at 40°C for 90 minutes on a heating block. SP:SAMPLEPREP_SUMMARY After brief centrifugation, 35 μL of MTBSTFA + 1% TBDMS was added and the SP:SAMPLEPREP_SUMMARY samples were incubated at 60°C for 30 minutes. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Agilent 7890B CH:COLUMN_NAME Agilent HP5-MS (30m x 0.25mm, 0.25 um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 5977A MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Masshunter used for data acquisition and processing. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration of 13C (uM) MS_METABOLITE_DATA_START Samples 9 10 17 18 26 33 34 11 12 19 20 28 35 36 Factors Treatment:GFP Treatment:GFP Treatment:GFP Treatment:GFP Treatment:GFP Treatment:GFP Treatment:GFP Treatment:SLC25A44 Treatment:SLC25A44 Treatment:SLC25A44 Treatment:SLC25A44 Treatment:SLC25A44 Treatment:SLC25A44 Treatment:SLC25A44 Valine 25.96852622 51.02941116 22.43086614 22.03059884 16.00755427 53.8469441 54.38184352 24.28032923 24.97836774 24.07146792 19.66722676 6.59658293 22.74963658 9.851328203 3-HIB 0.31517 1.0885 1.176 1.281 0.29691 0.26722 0.15322 0.75563 0.51422 0.92129 1.305 0.29686 0.068806 0.11108 Citrate 0.15829 1.0592 1.3274 2.2816 0.6118 0.27657 0.32249 1.0581 0.76082 0.44858 1.9505 0.22664 0.14061 0.06018 Succinate 0.021311 0.27581 0.33594 0.42484 0.12983 0.1546 0.087559 0.21051 0.13236 0.28306 0.44263 0.31734 0.15493 0.9575 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG Valine C00183 3-HIB C06001 Citrate C00158 Succinate C00042 METABOLITES_END #END