Summary of Study ST001665

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001070. The data can be accessed directly via it's Project DOI: 10.21228/M8D12Q This work is supported by NIH grant, U2C- DK119886.

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Study IDST001665
Study TitleBranched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the rat heart
Study SummaryBranched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine.
Institute
Duke University
Last NameWalejko
First NameJacquelyn
Address300 N Duke St
Emailjacquelyn.walejko@duke.edu
Phone9194792304
Submit Date2021-01-26
Analysis Type DetailGC-MS
Release Date2021-02-17
Release Version1
Jacquelyn Walejko Jacquelyn Walejko
https://dx.doi.org/10.21228/M8D12Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001070
Project DOI:doi: 10.21228/M8D12Q
Project Title:Branched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the heart
Project Summary:Branched-chain amino acids (BCAA) and their cognate α-ketoacids (BCKA) are elevated in an array of cardiometabolic diseases. Here we demonstrate that the major metabolic fate of uniformly-13C-labeled α-ketoisovalerate ([U-13C]KIV) in the heart is reamination to valine. Activation of cardiac branched-chain α-ketoacid dehydrogenase (BCKDH) by treatment with the BCKDH kinase inhibitor, BT2, does not impede the strong flux of [U-13C]KIV to valine. Sequestration of BCAA and BCKA away from mitochondrial oxidation is likely due to low levels of expression of the mitochondrial BCAA transporter SLC25A44 in the heart, as its overexpression significantly lowers accumulation of [13C]-labeled valine from [U-13C]KIV. Finally, exposure of perfused hearts to levels of BCKA found in obese rats increased increases phosphorylation of the translational repressor 4E-BP1 as well as multiple proteins in the MEK-ERK pathway, leading to a doubling of total protein synthesis. These data suggest that elevated BCKA levels found in obesity may contribute to pathologic cardiac hypertrophy via chronic activation of protein synthesis.
Institute:Duke University
Department:Medicine
Last Name:Walejko
First Name:Jacquelyn
Address:300 N Duke St Durham NC 27701
Email:jacquelyn.walejko@duke.edu
Phone:6086097615

Subject:

Subject ID:SU001742
Subject Type:Mammal
Subject Species:Rattus norvegicus
Taxonomy ID:10116
Age Or Age Range:10 weeks
Gender:Male

Factors:

Subject type: Mammal; Subject species: Rattus norvegicus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA152214206BT-2
SA152215217BT-2
SA152216205BT-2
SA152217216BT-2
SA152218215Control
SA152219200Control
SA152220207Control
SA152221208Control
SA152222199Control
SA152223218LY
SA152224214LY
SA152225211LY
SA152226210LY
SA152227212LY
SA152228213LY
Showing results 1 to 15 of 15

Collection:

Collection ID:CO001735
Collection Summary:Fed male Wistar rats were anesthetized with 5% isoflurane, and isolated hearts were perfused in the Langendorff mode at 37°C with non-recirculating perfusate. The hearts were allowed to beat spontaneously throughout the perfusion. At the end of each perfusion, hearts were immediately freeze-clamped in liquid nitrogen using the Wollenberger technique and stored at -80 oC for further analysis.
Sample Type:Heart

Treatment:

Treatment ID:TR001755
Treatment Summary:All heart perfusions underwent an initial 15-minute equilibration period with Krebs Ringer bicarbonate buffer containing 119 mM NaCl, 4.8 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose, and 0.05 mM L-carnitine at a flow rate of 12 ml/minute. After the equilibrium period, hearts were perfused for 30 minutes with 3% BSA, 100 µU/mL insulin, 0.4 mM palmitate, physiologic concentrations of amino acids, and either DMSO (Veh), BT2, or LY3351337.

Sample Preparation:

Sampleprep ID:SP001748
Sampleprep Summary:Frozen tissues were pulverized under liquid nitrogen using a mortar and pestle. Metabolites were then extracted using sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, tissue lysates were prepared with a Tissue Lyser (Qiagen) for 60 seconds at 30Hz. Similarly, plasma metabolites (20 μL) were extracted by sequential 500 μL additions of -20°C MeOH, chilled water, and chloroform. After each addition, samples were vortexed for 30 seconds. Tissue and plasma extracts were then centrifuged at 4°C and 14400 x g for 20 minutes and the clarified aqueous phase was transferred to a fresh Eppendorf and stored in -80°C until processing for GC-MS analysis. For GC-MS analysis, the extracted tissue was dried under N2 gas-flow at 37°C using an evaporator. Amino and organic acids were derivatized via methoximation and silylation. Briefly, metabolites were resuspended in 25 μL of methoxylamine hydrochloride (2% (w/v) in pyridine) and incubated at 40°C for 90 minutes on a heating block. After brief centrifugation, 35 μL of MTBSTFA + 1% TBDMS was added and the samples were incubated at 60°C for 30 minutes.

Combined analysis:

Analysis ID AN002717
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890B
Column Agilent HP5-MS (30m x 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5977A
Ion Mode UNSPECIFIED
Units Concentration of 13C (uM)

Chromatography:

Chromatography ID:CH002005
Instrument Name:Agilent 7890B
Column Name:Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Chromatography Type:GC

MS:

MS ID:MS002514
Analysis ID:AN002717
Instrument Name:Agilent 5977A
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:Masshunter used for data acquisition and processing.
Ion Mode:UNSPECIFIED
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