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 ID	ST001665
Study Title	Branched-chain alpha-ketoacids are preferentially reaminated and activate protein synthesis in the rat 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 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 Name	Walejko
First Name	Jacquelyn
Address	300 N Duke St
Email	jacquelyn.walejko@duke.edu
Phone	9194792304
Submit Date	2021-01-26
Analysis Type Detail	GC-MS
Release Date	2021-02-17
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:	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
SA152214	206	BT-2
SA152215	217	BT-2
SA152216	205	BT-2
SA152217	216	BT-2
SA152218	215	Control
SA152219	200	Control
SA152220	207	Control
SA152221	208	Control
SA152222	199	Control
SA152223	218	LY
SA152224	214	LY
SA152225	211	LY
SA152226	210	LY
SA152227	212	LY
SA152228	213	LY

Showing results 1 to 15 of 15

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.

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.

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