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Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart.

ABSTRACT: 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 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.

SUBMITTER: Walejko JM

PROVIDER: S-EPMC7960706 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Heart tissue was lysed by sonication in 5% SDS (w/v) in 50 mM TEAB (pH 8.5) 450 microg of each sample was reduced with 10 mM DTT at 80 degC for 10 min and alkylated with 25 mM iodoacetamide in the dark for 30 min at room temperature. SDS was removed, and samples were digested with 20 microg of Sequencing Grade Modified Trypsin (Promega) using an S-trap mini device (Protifi) at 47 degC for 2 h. Lyophilized peptides were reconstituted in 100 microl of 200 mM TEAB buffer and labeled with 41 TMT10 reagents (lot# UL292365). After 2 h, reactions were quenched with hydroxylamine and combined. TMT-labeled peptides were fractionated by high pH reversed-phase (HPRP) chromatography. Briefly, peptides were reconstituted in 400 microl of 20 mM ammonium formate, and 125 microl was fractionated using a 2.1 mm x 5 cm BEH C18 column (Waters) and Waters ACQUITY iClass UPLC. Separations utilized a flow rate of 0.4 ml/min and column temperature of 55 degC, and mobile phases consisted of 20 mM ammonium formate, pH 10 (MPA) and neat MeCN (MPB). Separations used a gradient as follows: 0 min, 3% B; 3 min, 7% B; 50 min, 50% B, 51 min, 90% B; 55 min, 90% B; 56 min, 3% B; 60 min, 3% B. Forty-eight equal fractions were collected over 52 minutes into a 96-well plate containing 10 microl of 20% TFA per well. This analysis was repeated 2 times total for fractionation of 2 mg peptides. The first 3 fractions of each injection were excluded, and the remaining samples were re-concatenated into 12 fractions. Approximately 5 microg of each fraction was separately aliquoted for analysis of the unenriched proteome, and samples were lyophilized. Phosphopeptide enrichment used 200 microg of each of the 12 HPRP fractions was reconstituted in 80% MeCN/1% TFA containing 1M glycolic acid (buffer A), phosphopeptides were enriched using GL Sciences p10 TiO tips. After loading, tips were washed twice with buffer A followed by 2 times with 80% MeCN/1% TFA before elution with 20% MeCN/5% aqueous ammonia. After addition of neat formic acid, samples were lyophilized. Finally, peptides were desalted using C18 Stage Tips, lyophilized and reconstituted in 12 microl of 10 mM citrate in 1% TFA/2% MeCN. The unenriched fractions were reconstituted in 10 microl of 1% TFA/2% MeCN. 1D-LC-MS/MS was performed on 4.5 microl of each of the phospho-enriched fractions or on 0.75 microg of unenriched fractions. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a coupled to a Exploris 480 high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source and FAIMS Pro Interface. Samples were first trapped on a Symmetry C18 180 microm x 20 mm trapping column (5 microl/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 microm Acquity HSS T3 C18 75 microm x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on was performed in data-dependent acquisition (DDA) mode with 3 FAIMS compensation voltages (-40, -60 and -80). Each CV used a 60,000 resolution full MS scan from m/z 375 to 1600 with a normalized AGC target of 300%, peptide monoisotopic peak determination, an intensity threshold of 5E3 ions, precursor fit of 70% with 0.7 m/z fit window, charge state of 2-5 and 40 s dynamic exclusion. MS/MS used a top6 method with 30,000 resolution and TurboTMT enabled, an isolation width of 0.7 m/z, NCE of 36, AGC target of 300% and maximum IT of 120 ms. Advanced precursor determination was enabled. The analysis of unenriched samples used a similar method except that a 1 s total scan time was used for each CV and MS/MS used an auto IT.

Project description:BACKGROUND:Protein ingestion increases muscle protein synthesis rates. However, limited data are currently available on the effects of branched-chain amino acid (BCAA) and branched-chain ketoacid (BCKA) ingestion on postprandial muscle protein synthesis rates. OBJECTIVE:The aim of this study was to compare the impact of ingesting 6 g BCAA, 6 g BCKA, and 30 g milk protein (MILK) on the postprandial rise in circulating amino acid concentrations and subsequent myofibrillar protein synthesis rates in older males. METHODS:In a parallel design, 45 older males (age: 71 ± 1 y; BMI: 25.4 ± 0.8 kg/m2) were randomly assigned to ingest a drink containing 6 g BCAA, 6 g BCKA, or 30 g MILK. Basal and postprandial myofibrillar protein synthesis rates were assessed by primed continuous l-[ring-13C6]phenylalanine infusions with the collection of blood samples and muscle biopsies. RESULTS:Plasma BCAA concentrations increased following test drink ingestion in all groups, with greater increases in the BCAA and MILK groups compared with the BCKA group (P < 0.05). Plasma BCKA concentrations increased following test drink ingestion in all groups, with greater increases in the BCKA group compared with the BCAA and MILK groups (P < 0.05). Ingestion of MILK, BCAA, and BCKA significantly increased early myofibrillar protein synthesis rates (0-2 h) above basal rates (from 0.020 ± 0.002%/h to 0.042 ± 0.004%/h, 0.022 ± 0.002%/h to 0.044 ± 0.004%/h, and 0.023 ± 0.003%/h to 0.044 ± 0.004%/h, respectively; P < 0.001), with no differences between groups (P > 0.05). Myofibrillar protein synthesis rates during the late postprandial phase (2-5 h) remained elevated in the MILK group (0.039 ± 0.004%/h; P < 0.001), but returned to baseline values following BCAA and BCKA ingestion (0.024 ± 0.005%/h and 0.024 ± 0.005%/h, respectively; P > 0.05). CONCLUSIONS:Ingestion of 6 g BCAA, 6 g BCKA, and 30 g MILK increases myofibrillar protein synthesis rates during the early postprandial phase (0-2 h) in vivo in healthy older males. The postprandial increase following the ingestion of 6 g BCAA and BCKA is short-lived, with higher myofibrillar protein synthesis rates only being maintained following the ingestion of an equivalent amount of intact milk protein. This trial was registered at Nederlands Trial Register (www.trialregister.nl) as NTR6047.

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Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart.

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