Summary of Study ST001666

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 IDST001666
Study TitleBranched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the mouse 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 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.
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 Date2021-02-09
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:SU001743
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090
Age Or Age Range:8 weeks
Gender:Male

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA15222934GFP
SA1522309GFP
SA15223126GFP
SA15223233GFP
SA15223310GFP
SA15223417GFP
SA15223518GFP
SA15223635SLC25A44
SA15223736SLC25A44
SA15223828SLC25A44
SA15223911SLC25A44
SA15224012SLC25A44
SA15224119SLC25A44
SA15224220SLC25A44
Showing results 1 to 14 of 14

Collection:

Collection ID:CO001736
Collection Summary:Fed male C57BL/6J mice 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:TR001756
Treatment Summary:SLC25A44 and GFP expression plasmids with a CMV promoter containing ITRs were used to prepare recombinant AAV9 by the UMass Gene Therapy Core. Adult 8-week-old male mice were injected intravenously with 100 µl of 5.1 x 1011 vector genomes per mouse.

Sample Preparation:

Sampleprep ID:SP001749
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 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 AN002718
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:CH002006
Instrument Name:Agilent 7890B
Column Name:Agilent HP5-MS (30m x 0.25mm, 0.25 um)
Chromatography Type:GC

MS:

MS ID:MS002515
Analysis ID:AN002718
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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