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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST001666 |
| Study Title | Branched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the mouse heart |
| Study Summary | Branched-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 Name | Walejko |
| First Name | Jacquelyn |
| Address | 300 N Duke St |
| jacquelyn.walejko@duke.edu | |
| Phone | 9194792304 |
| Submit Date | 2021-01-26 |
| Analysis Type Detail | GC-MS |
| Release Date | 2021-02-09 |
| Release Version | 1 |
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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 |
|---|---|---|
| SA152229 | 34 | GFP |
| SA152230 | 9 | GFP |
| SA152231 | 26 | GFP |
| SA152232 | 33 | GFP |
| SA152233 | 10 | GFP |
| SA152234 | 17 | GFP |
| SA152235 | 18 | GFP |
| SA152236 | 35 | SLC25A44 |
| SA152237 | 36 | SLC25A44 |
| SA152238 | 28 | SLC25A44 |
| SA152239 | 11 | SLC25A44 |
| SA152240 | 12 | SLC25A44 |
| SA152241 | 19 | SLC25A44 |
| SA152242 | 20 | SLC25A44 |
| 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 |
