#METABOLOMICS WORKBENCH jwalejko_20210126_095433 DATATRACK_ID:2423 STUDY_ID:ST001665 ANALYSIS_ID:AN002717 PROJECT_ID:PR001070 VERSION 1 CREATED_ON January 28, 2021, 11:13 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 rat 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 the ST:STUDY_SUMMARY major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in ST:STUDY_SUMMARY the heart is reamination to valine. Activation of cardiac branched-chain ST:STUDY_SUMMARY α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, ST:STUDY_SUMMARY BT2, does not impede the strong flux of [U-13C]KIV to valine. 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 Rattus norvegicus SU:TAXONOMY_ID 10116 SU:AGE_OR_AGE_RANGE 10 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 - 199 Treatment:Control SUBJECT_SAMPLE_FACTORS - 200 Treatment:Control SUBJECT_SAMPLE_FACTORS - 207 Treatment:Control SUBJECT_SAMPLE_FACTORS - 208 Treatment:Control SUBJECT_SAMPLE_FACTORS - 215 Treatment:Control SUBJECT_SAMPLE_FACTORS - 205 Treatment:BT-2 SUBJECT_SAMPLE_FACTORS - 206 Treatment:BT-2 SUBJECT_SAMPLE_FACTORS - 216 Treatment:BT-2 SUBJECT_SAMPLE_FACTORS - 217 Treatment:BT-2 SUBJECT_SAMPLE_FACTORS - 210 Treatment:LY SUBJECT_SAMPLE_FACTORS - 211 Treatment:LY SUBJECT_SAMPLE_FACTORS - 212 Treatment:LY SUBJECT_SAMPLE_FACTORS - 213 Treatment:LY SUBJECT_SAMPLE_FACTORS - 214 Treatment:LY SUBJECT_SAMPLE_FACTORS - 218 Treatment:LY #COLLECTION CO:COLLECTION_SUMMARY Fed male Wistar rats 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 All heart perfusions underwent an initial 15-minute equilibration period with TR:TREATMENT_SUMMARY Krebs Ringer bicarbonate buffer containing 119 mM NaCl, 4.8 mM KCl, 2.6 mM TR:TREATMENT_SUMMARY CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose, and 0.05 mM TR:TREATMENT_SUMMARY L-carnitine at a flow rate of 12 ml/minute. After the equilibrium period, hearts TR:TREATMENT_SUMMARY were perfused for 30 minutes with 3% BSA, 100 µU/mL insulin, 0.4 mM palmitate, TR:TREATMENT_SUMMARY physiologic concentrations of amino acids, and either DMSO (Veh), BT2, or TR:TREATMENT_SUMMARY LY3351337. #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 and plasma extracts were then centrifuged at 4°C and SP:SAMPLEPREP_SUMMARY 14400 x g for 20 minutes and the clarified aqueous phase was transferred to a SP:SAMPLEPREP_SUMMARY fresh Eppendorf and stored in -80°C until processing for GC-MS analysis. For SP:SAMPLEPREP_SUMMARY GC-MS analysis, the extracted tissue was dried under N2 gas-flow at 37°C using SP:SAMPLEPREP_SUMMARY an evaporator. Amino and organic acids were derivatized via methoximation and SP:SAMPLEPREP_SUMMARY silylation. Briefly, metabolites were resuspended in 25 μL of methoxylamine SP:SAMPLEPREP_SUMMARY hydrochloride (2% (w/v) in pyridine) and incubated at 40°C for 90 minutes on a SP:SAMPLEPREP_SUMMARY heating block. After brief centrifugation, 35 μL of MTBSTFA + 1% TBDMS was SP:SAMPLEPREP_SUMMARY added and the 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 199 200 207 208 215 205 206 216 217 210 211 212 213 214 218 Factors Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:BT-2 Treatment:BT-2 Treatment:BT-2 Treatment:BT-2 Treatment:LY Treatment:LY Treatment:LY Treatment:LY Treatment:LY Treatment:LY Valine 16.939 12.568 18.676 14.727 12.347 10.748 10.096 11.053 15.856 4.176 4.224 4.233 3.852 5.549 6.445 3-HIB 6.819 3.813 8.878 4.769 3.682 14.683 17.516 11.537 13.416 12.090 7.288 8.646 8.145 8.789 16.124 Citrate 0.132 0.061 0.095 0.129 0.151 0.816 0.564 0.437 0.327 0.085 0.102 0.158 0.072 0.103 0.113 Succinate 0.156 0.057 0.076 0.062 0.111 0.528 0.652 0.278 0.243 0.026 0.018 0.082 0.011 0.026 0.068 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG Valine C00183 3-HIB C06001 Citrate C00158 Succinate C00042 METABOLITES_END #END