In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial proteins

Jaleel, Abdul; Short, Kevin R.; Asmann, Yan W.; Klaus, Katherine A.; Morse, Dawn M.; Ford, G. C.; Nair, K. Sreekumaran

doi:10.1152/ajpendo.90586.2008

Cited by 70 publications

(83 citation statements)

References 54 publications

(51 reference statements)

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“…Thus, the FBR measurement using 2 H 2 O will measure slow turnover protein rates even with some contamination of faster turnover proteins in the sample. This divergence between FSR and FBR measurements can be avoided only by isolating single proteins and measuring their turnover rate, similar to work by Jaleel and colleagues (24,25).…”

Section: Comparing the Fbrs With Existing Fsrsmentioning

confidence: 93%

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

Holm

O'Rourke²,

Ebenstein³

et al. 2013

American Journal of Physiology-Endocrinology and Metabolism

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. Determination of steadystate protein breakdown rate in vivo by the disappearance of proteinbound tracer-labeled amino acids: a method applicable in humans. Am J Physiol Endocrinol Metab 304: E895-E907, 2013. First published February 19, 2013; doi:10.1152/ajpendo.00579.2012.-A method to determine the rate of protein breakdown in individual proteins was developed and tested in rats and confirmed in humans, using administration of deuterium oxide and incorporation of the deuterium into alanine that was subsequently incorporated into body proteins. Measurement of the fractional breakdown rate of proteins was determined from the rate of disappearance of deuterated alanine from the proteins. The rate of disappearance of deuterated alanine from the proteins was calculated using an exponential decay, giving the fractional breakdown rate (FBR) of the proteins. The applicability of this protein-specific FBR approach is suitable for human in vivo experimentation. The labeling period of deuterium oxide administration is dependent on the turnover rate of the protein of interest. protein kinetics; protein degradation; fractional breakdown rate; deuterated water BODY PROTEIN POOLS UNDERGO CONTINUAL RENEWAL through a constant cycle of synthesis and degradation. Alterations in the balance between synthetic and degradative rates produce changes in protein pool size that are fundamental for the ability of a biological system to adapt to intrinsic and environmental factors (26). Therefore, accurate methods to assess directly the protein synthetic and breakdown rates are necessary to investigate and understand the regulation of protein balance. The rate of protein synthesis can be measured from the rate of isotope-labeled amino acid incorporation into proteins (45, 47). With technical advances in mass spectrometers and the combination of techniques, the determination of the synthesis rate of small quantities of individual proteins is now possible (21,24). The need for a comparable approach to measure proteinspecific breakdown rates is also important for in vivo human research. However, the assessment of protein breakdown is more troublesome (18). Currently, the in vivo approach used routinely relies on isotope dilution and arterial-venous catheterization (2, 6). More pools have been added to the method, improving validity (17), and the latest approach has been reworked and further refined by Zhang and colleagues (51-53). However, the same principle remains, i.e., to measure the dilution of free amino acid tracer enrichment at arterial, venous, and intracellular sites as a consequence of release of tracee amino acids when proteins degrade. Although this technique has substantial utility, the protein origin of the amino acids remains unknown. Hence, it does not permit estimation of the breakdown rate of specific proteins as it does for the direct tracer incorporation approach for protein synthesis measurements. For this reason we developed a fractional breakdown rate (FBR) method in rats, allowing the measurements of FBR of individu...

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Section: Comparing the Fbrs With Existing Fsrsmentioning

confidence: 93%

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

Holm

O'Rourke²,

Ebenstein³

et al. 2013

American Journal of Physiology-Endocrinology and Metabolism

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“…1. 13 C from [5,6,[7][8][9][10][11][12][13] C3]heptanoate metabolizes to CAC without the conversion to acetyl-CoA, and these data were used for analysis of the anaplerotic component to the CAC. NMR data were acquired on a Varian Direct Drive (VNMRS) 600-MHz spectrometer (Agilent Technologies) equipped with a Dell Precision T3500 Linux workstation running VNMRJ 3.2.…”

Section: Methodsmentioning

confidence: 99%

“…The anaplerotic contribution through propionyl-CoA from [5,6,[7][8][9][10][11][12][13] C3]heptanoate relative to acetyl-CoA entry was calculated by using several parameters obtained from the 13 C-NMR spectrum. The general scheme for the labeling pattern of glutamate is shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model

Kajimoto¹,

Ledee²,

Olson

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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]leucine) were systemically infused as metabolic markers for the final 60 min before left ventricular tissue extraction. Extracted tissues were analyzed for the 13 C-labeled and absolute concentrations of metabolites by nuclear magnetic resonance and gas chromatography-mass spectrometry. Octanoate produced markedly higher myocardial citrate concentration, and led to a higher [ATP]-to-[ADP] ratio compared with other groups. Unexpectedly, octanoate and heptanoate increased the flux of propionyl-CoA relative to acetyl-CoA into the CAC compared with control. MCFAs promoted increases in leucine oxidation, but were not associated with a difference in protein synthesis rate. In conclusion, octanoate provides energetic advantages to the heart over heptanoate. cardiac metabolism; extracorporeal circulation; fatty acids; pediatrics NEW & NOTEWORTHYOctanoate, an even-numbered medium-chain fatty acid, provides some energetic advantage for the heart, over odd-numbered heptanoate, during ventricular unloading by extracorporeal membrane oxygenation.

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“…Gas chromatography and mass spectrometry (GC-MS) was performed to measure the absolute concentrations and 13 C enrichment of leucine, pyruvate, lactate, and CAC intermediates (citrate, ␣-ketoglutarate, succinate, fumarate, and malate) in LV myocardial tissue samples, as previously described (19,42,43). Protein fractional synthesis rates (FSRs) were calculated using the methods of Jaleel et al (19 Nuclear magnetic resonance. Metabolic analyses by 13 C-nuclear magnetic resonance (NMR) were performed on the swine heart as described previously for determination of specific carbon glutamate labeling (7).…”

Section: Metabolic Analysesmentioning

confidence: 99%

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

Ledee¹,

Kajimoto²,

Priddy

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

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-Extracorporeal membrane oxygenation (ECMO) provides mechanical circulatory support for infants and children with postoperative cardiopulmonary failure. Nutritional support is mandatory during ECMO although specific actions for substrates on the heart have not been delineated. Prior work shows that enhancing pyruvate oxidation promotes successful weaning from ECMO. Accordingly, we tested the hypothesis that prolonged systemic pyruvate supplementation activates pyruvate oxidation in an immature swine model in vivo. Twelve male mixed-breed Yorkshire piglets (age 30 -49 days) received systemic infusion of either normal saline (group C) or pyruvate (group P) during the final 6 h of 8 h of ECMO. Over the final hour, piglets received [2-13 C] pyruvate, as a reference substrate for oxidation, and [13 C6]-L-leucine, as an indicator for amino acid oxidation and protein synthesis. A significant increase in lactate and pyruvate concentrations occurred, along with an increase in the absolute concentration of the citric acid cycle intermediates. An increase in anaplerotic flux through pyruvate carboxylation in group P occurred compared with no change in pyruvate oxidation. Additionally, pyruvate promoted an increase in the phosphorylation state of several nutrient-sensitive enzymes, like AMP-activated protein kinase and acetyl CoA carboxylase, suggesting activation for fatty acid oxidation. Pyruvate also promoted O-GlcNAcylation through the hexosamine biosynthetic pathway. In conclusion, although prolonged pyruvate supplementation did not alter pyruvate oxidation, it did elicit changes in nutrient-and energy-sensitive pathways. Therefore, the observed results support the further study of pyruvate and its downstream effect on cardiac function. extracorporeal membrane oxygenation; leucine; pyruvate; metabolism EXTRACORPOREAL MEMBRANE OXYGENATION (ECMO) has been widely used in infants and children with life-threatening cardiopulmonary failure as both cardiac and respiratory support systems (16). Veno-arterial ECMO, especially, is frequently used as short-term extracorporeal life support for postoperative cardiopulmonary failure. However, unless adequate endogenous cardiac output and respiratory function ensue, patients are not able to be weaned from ECMO, and prolonged ECMO support can lead to higher morbidity and mortality (16,46). Thus ongoing research is attempting to identify nutritional supplements, which can improve recovery and prevent maladaptive cardiac remodeling. These potential supplements could enhance eventual weaning from mechanical circulation. Our prior work shows that successful adaptation and weaning after ECMO requires some metabolic flexibility (22). In particular, although ECMO independently promotes fatty acid oxidation (23), cardiac function after additional insult, such as ischemia reperfusion, can be improved by promoting oxidative flux through pyruvate dehydrogenase (11).Pyruvate serves as an important branch point of substrate metabolism (Fig. 1). However, the role of pyruvate as a metabolic substrat...

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In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial proteins

Abstract: Jaleel A, Short KR, Asmann YW, Klaus KA, Morse DM, Ford GC, Nair KS. In vivo measurement of synthesis rate of individual skeletal muscle mitochondrial proteins.

Cited by 70 publications

References 54 publications

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

Determination of steady-state protein breakdown rate in vivo by the disappearance of protein-bound tracer-labeled amino acids: a method applicable in humans

Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model

Pyruvate modifies metabolic flux and nutrient sensing during extracorporeal membrane oxygenation in an immature swine model

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