Measurement of muscle protein degradation in live mice by accumulation of bestatin-induced peptides

1997 ◽  
Vol 273 (6) ◽  
pp. E1149-E1157 ◽  
Author(s):  
Violeta Botbol ◽  
Oscar A. Scornik
Keyword(s):  
Protein Degradation ◽  
Protein Turnover ◽  
Skeletal Muscles ◽  
Muscle Protein ◽  
Cellular Proteins ◽  
Protein Breakdown ◽  
Maximum Accumulation ◽  
Muscle Protein Breakdown ◽  
Muscle Protein Degradation ◽  
Acute Changes

Bestatin, an aminopeptidase inhibitor, permits the degradation of cellular proteins to di- and tripeptides but interferes with the further breakdown of these peptides to amino acids. We propose to measure instant rates of protein degradation in skeletal muscles of intact mice by the accumulation of bestatin-induced intermediates. Muscle protein was labeled by injection ofl-[guanidino-14C]arginine; 3 days later, maximum accumulation of intermediates was measured in abdominal wall muscles 10 min after the intravenous injection of 5 mg of bestatin. The peptides were partially purified and hydrolyzed in 6 N HCl, and the radioactivity in peptide-derived arginine was determined, after conversion to14CO2by treatment with arginase and urease. The measurement of bestatin-induced intermediates provides a unique tool for studying acute changes in muscle protein turnover in live mice. We observed a 62% increase in muscle protein breakdown after a 16-h fast, which was reversed by refeeding for 3.5 h, and a 38% increase after 3 days of protein depletion.

scholarly journals The effects of calcium on protein turnover in skeletal muscles of the rat

1982 ◽  
Vol 204 (1) ◽  
pp. 257-264 ◽  
Author(s):  
S E M Lewis ◽  
P Anderson ◽  
D F Goldspink
Keyword(s):  
Protein Synthesis ◽  
Cyclic Nucleotides ◽  
Protein Turnover ◽  
Skeletal Muscles ◽  
Extensor Digitorum Longus ◽  
Muscle Protein ◽  
Ionophore A23187 ◽  
Protein Breakdown ◽  
External Concentration ◽  
The Media

Several experimental procedures were used to increase the intracellular concentration of Ca2+ and determine its effects on protein turnover in isolated extensor digitorum longus and soleus muscle. These methods included the use of ionophore A23187, caffeine, dibucaine, thymol and procaine, all agents known to induce the release of calcium by acting either on the sarcolemma and/or on the sarcoplasmic reticulum. Another approach involved varying the external concentration of Ca2+ in the media in which the muscles were incubated. The changes in muscle Ca2+ concentrations after exposure to the various calcium-releasing agents were in keeping with accepted modes of action of these agents on muscle membranes. The findings suggest that increasing the sarcoplasmic concentration of Ca2+ inhibits protein synthesis and enhances protein breakdown. These catabolic effects of Ca2+ are compared with the changes induced in muscle protein turnover after exposure to insulin or cyclic nucleotides, and in myopathic muscle and situations of work overload. Attention is also drawn to some of the difficulties involved in definitively implicating Ca2+ as a factor involved in the normal regulation of protein turnover.

Exercise training and protein metabolism: influences of contraction, protein intake, and sex-based differences

2009 ◽  
Vol 106 (5) ◽  
pp. 1692-1701 ◽  
Author(s):  
Nicholas A. Burd ◽  
Jason E. Tang ◽  
Daniel R. Moore ◽  
Stuart M. Phillips
Keyword(s):  
Signaling Pathways ◽  
Protein Turnover ◽  
Muscle Hypertrophy ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Protein Breakdown ◽  
Muscle Protein Turnover ◽  
Muscle Protein Breakdown ◽  
Response To Exercise ◽  
Net Protein

Muscle contraction during exercise, whether resistive or endurance in nature, has profound affects on muscle protein turnover that can persist for up to 72 h. It is well established that feeding during the postexercise period is required to bring about a positive net protein balance (muscle protein synthesis − muscle protein breakdown). There is mounting evidence that the timing of ingestion and the protein source during recovery independently regulate the protein synthetic response and influence the extent of muscle hypertrophy. Minor differences in muscle protein turnover appear to exist in young men and women; however, with aging there may be more substantial sex-based differences in response to both feeding and resistance exercise. The recognition of anabolic signaling pathways and molecules are also enhancing our understanding of the regulation of protein turnover following exercise perturbations. In this review we summarize the current understanding of muscle protein turnover in response to exercise and feeding and highlight potential sex-based dimorphisms. Furthermore, we examine the underlying anabolic signaling pathways and molecules that regulate these processes.

Protein degradation in skeletal muscles after evisceration of fed or fasted rats

1986 ◽  
Vol 251 (4) ◽  
pp. E379-E384
Author(s):  
O. L. Smith
Keyword(s):  
Protein Degradation ◽  
Skeletal Muscles ◽  
Muscle Protein ◽  
Skeletal Muscle Protein ◽  
Peripheral Tissues ◽  
Muscle Protein Degradation ◽  
Plasma Tyrosine ◽  
Fasted Rats

To test the rate of protein degradation in muscles under more physiological conditions, in vitro methods were adapted for use in rats whose skeletal muscles had been isolated intact by an evisceration procedure. Under pentobarbital anesthesia the vessels to the gastrointestinal tract were ligated, the organs removed, and the liver left in situ. Normal rectal temperature was maintained, glucose was given to prevent hypoglycemia and the animals were studied for a 2-h period. For the present experiments, evisceration was carried out after protein synthesis was blocked with cycloheximide so that the accumulation of the amino acid tyrosine (not metabolized) in the plasma of the preparation could be used to indicate the rate protein was degraded in the peripheral tissues. When normal fed rats (270 g) were eviscerated, the concentration of plasma tyrosine increased postoperatively, but, if the rats were fasted 20 h and then eviscerated, tyrosine accumulation was significantly enhanced. After 2 h, it was 50% greater than in the fed controls. Refeeding for 3 h completely prevented this effect. The results suggest that the rate of overall skeletal muscle protein degradation can be measured by the use of eviscerated rats and that this rate is sensitive to short periods of food deprivation.

Decrease in human quadriceps muscle protein turnover consequent upon leg immobilization

1987 ◽  
Vol 72 (4) ◽  
pp. 503-509 ◽  
Author(s):  
J. N. A. Gibson ◽  
D. Halliday ◽  
W. L. Morrison ◽  
P. J. Stoward ◽  
G. A. Hornsby ◽  
...  
Keyword(s):  
Protein Turnover ◽  
Muscle Protein ◽  
Fibre Diameter ◽  
Quadriceps Muscle ◽  
Fibre Volume ◽  
Type I ◽  
Protein Breakdown ◽  
Muscle Protein Turnover ◽  
Muscle Protein Breakdown ◽  
Synthetic Rate

1. Quadriceps muscle protein turnover was assessed in the post-absorptive state in six men immediately after the end of unilateral leg immobilization (37 ± 4 days) in a plaster cast after tibial fracture. A primed-constant intravenous infusion of l-[1-13C]leucine was administered over 7 h. Quadriceps needle biopsies, taken bilaterally at the end of the infusion, were analysed for muscle protein leucine enrichment with 13C. 2. Quadriceps muscle protein synthetic rate, calculated from the fractional incorporation of [13C]leucine into protein compared with the average enrichment of blood α-ketoisocaproate, was 0.046 ±0.012%/h in the uninjured leg, but was only 0.034 ±0.007%/h in the quadriceps of the previously fractured leg (P > 0.05, means ± sd). 3. Muscle RNA activity (i.e. protein synthetic rate per RNA) fell from 0.27 ±0.08 μg of protein synthesized h−1 μg−1 of RNA in the control leg to 0.14 ±0.03 μg of protein synthesized h−1 μg−1 of RNA in the immobilized leg (P > 0.02). 4. Immobilization was associated with a significant atrophy of type I muscle fibres (mean diameter 69.5 ±21 μm immobilized, 81.1 ±18 μm control, P > 0.05), but no significant change occurred in type II fibre diameter. Mean quadriceps fibre volume calculated from the values for fibre diameter and percentage of each fibre type, was smaller in the injured leg by 10.6%; this value was near to the calculated difference in muscle thigh volume (calculated from thigh circumference and skin-fold thickness) which was less by 8.3%. 5. From estimated mean daily values for quadriceps protein synthetic rate (1.65 ±0.44%/day in the control legs and 1.22±0.28%/day in the injured legs) and change in fibre volume, mean daily muscle protein breakdown rates were calculated as 1.65%/ day and 1.53%/day respectively, suggesting that muscle protein breakdown was not enhanced and may have fallen. 6. The results suggest a decrease in muscle protein turnover during limb immobilization in man, with the decrement in muscle mass being due mainly to a substantial (25%) depression of muscle protein synthesis.

scholarly journals Combined in vivo muscle mass, muscle protein synthesis and muscle protein breakdown measurement: a ‘Combined Oral Stable Isotope Assessment of Muscle (COSIAM)’ approach

GeroScience ◽  
2021 ◽  
Author(s):  
Jessica Cegielski ◽  
Daniel J. Wilkinson ◽  
Matthew S. Brook ◽  
Catherine Boereboom ◽  
Bethan E. Phillips ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Stable Isotope ◽  
Muscle Mass ◽  
Protein Turnover ◽  
Skeletal Muscle Mass ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Protein Breakdown ◽  
Muscle Protein Breakdown

AbstractOptimising approaches for measuring skeletal muscle mass and turnover that are widely applicable, minimally invasive and cost effective is crucial in furthering research into sarcopenia and cachexia. Traditional approaches for measurement of muscle protein turnover require infusion of expensive, sterile, isotopically labelled tracers which limits the applicability of these approaches in certain populations (e.g. clinical, frail elderly). To concurrently quantify skeletal muscle mass and muscle protein turnover i.e. muscle protein synthesis (MPS) and muscle protein breakdown (MPB), in elderly human volunteers using stable-isotope labelled tracers i.e. Methyl-[D3]-creatine (D3-Cr), deuterium oxide (D2O), and Methyl-[D3]-3-methylhistidine (D3-3MH), to measure muscle mass, MPS and MPB, respectively. We recruited 10 older males (71 ± 4 y, BMI: 25 ± 4 kg.m2, mean ± SD) into a 4-day study, with DXA and consumption of D2O and D3-Cr tracers on day 1. D3-3MH was consumed on day 3, 24 h prior to returning to the lab. From urine, saliva and blood samples, and a single muscle biopsy (vastus lateralis), we determined muscle mass, MPS and MPB. D3-Cr derived muscle mass was positively correlated to appendicular fat-free mass (AFFM) estimated by DXA (r = 0.69, P = 0.027). Rates of cumulative myofibrillar MPS over 3 days were 0.072%/h (95% CI, 0.064 to 0.081%/h). Whole-body MPB over 6 h was 0.052 (95% CI, 0.038 to 0.067). These rates were similar to previous literature. We demonstrate the potential for D3-Cr to be used alongside D2O and D3-3MH for concurrent measurement of muscle mass, MPS, and MPB using a minimally invasive design, applicable for clinical and frail populations.

scholarly journals Recent advances in measuring and understanding the regulation of exercise-mediated protein degradation in skeletal muscle

2021 ◽  
Author(s):  
Yusuke Nishimura ◽  
Ibrahim Musa ◽  
Lars Holm ◽  
Yu-Chiang Lai
Keyword(s):  
Skeletal Muscle ◽  
Quality Control ◽  
Protein Degradation ◽  
Protein Turnover ◽  
Degradation Rate ◽  
Protein Quality ◽  
Muscle Protein ◽  
Protein Quality Control ◽  
Muscle Protein Degradation ◽  
Protein Degradation Rate

Skeletal muscle protein turnover plays a crucial role in controlling muscle mass and protein quality control, including sarcomeric (structural and contractile) proteins. Protein turnover is a dynamic and continual process of protein synthesis and degradation. The ubiquitin proteasome system (UPS) is a key degradative system for protein degradation and protein quality control in skeletal muscle. UPS-mediated protein quality control is known to be impaired in ageing and diseases. Exercise is a well-recognized non-pharmacological approach to promote muscle protein turnover rates. Over the past decades, we have acquired substantial knowledge of molecular mechanisms of muscle protein synthesis after exercise. However, there has been considerable gaps in the mechanisms of how muscle protein degradation is regulated at the molecular level. The main challenge to understand muscle protein degradation is due in part to the lack of solid stable isotope tracer methodology to measure muscle protein degradation rate. Understanding the mechanisms of UPS with the concomitant measurement of protein degradation rate in skeletal muscle will help identify novel therapeutic strategies to ameliorate impaired protein turnover and protein quality control in ageing and diseases. Thus, the goal of this present review is to highlight how recent advances in the field may help improve our understanding of exercise-mediated protein degradation. We discuss 1) the emerging roles of protein phosphorylation and ubiquitylation modifications in regulating proteasome-mediated protein degradation after exercise and 2) methodological advances to measure in vivo myofibrillar protein degradation rate using stable isotope tracer methods.

scholarly journals A comparison of methods for the measurement of protein turnover in vivo

1979 ◽  
Vol 184 (2) ◽  
pp. 473-476 ◽  
Author(s):  
M L MacDonald ◽  
S L Augustine ◽  
T L Burk ◽  
R W Swick
Keyword(s):  
Amino Acids ◽  
Steady State ◽  
Protein Turnover ◽  
Muscle Protein ◽  
Comparison Of Methods ◽  
Ornithine Aminotransferase ◽  
Protein Breakdown ◽  
Muscle Protein Breakdown ◽  
Fractional Rate

Steady-state rates of turnover of two single proteins were measured in vivo by two independent methods. The fractional rate of synthesis of liver ornithine aminotransferase, measured by a continuous infusion of L-[2,6-3H]tyrosine, was 0.42 day-1, whereas in the same animals the fractional rate of degradation measured by loss of radioactivity from amino acids labelled via [14C]bicarbonate was 0.40 day-1. The agreement between methods confirms the reliability of each method for the study of hepatic protein turnover. In contrast, [14C]bicarbonate-labelled amino acids are extensively reutilized in muscle, and are therefore unsuitable for measuring rates of muscle protein breakdown.

scholarly journals Effect of starvation or treatment with corticosterone on the amount of easily releasable myofilaments in rat skeletal muscles

1986 ◽  
Vol 234 (3) ◽  
pp. 659-664 ◽  
Author(s):  
B Dahlmann ◽  
M Rutschmann ◽  
H Reinauer
Keyword(s):  
Proteinase Inhibitor ◽  
Skeletal Muscles ◽  
Muscle Protein ◽  
Myofibrillar Proteins ◽  
Protein Breakdown ◽  
Degradative Pathway ◽  
Muscle Protein Breakdown ◽  
Glucocorticoid Administration ◽  
Slow Twitch ◽  
Fast Twitch

Treatment of isolated myofibrils with an ATP-containing relaxing solution results in the dissociation of a preformed quantity of myofilaments called ‘easily releasable myofilaments’. Van der Westhuyzen, Matsumoto & Etlinger [(1981) J. Biol. Chem. 256, 11791-11797] presented experimental evidence that these myofilaments represent intermediate products in the turnover of myofibrillar proteins. To investigate further this question, we measured the size of the fraction of easily releasable myofilaments in three different species of skeletal muscles from rats subjected to well-defined catabolic conditions, namely starvation or chronic glucocorticoid administration. The results were as follows: (1) The amount of easily releasable myofilaments was transiently increased about 2-3-fold during both experiments, and thus paralleled the known alterations in the rate of overall muscle protein breakdown rather than in those of synthesis. (2) These changes were observed in muscles containing predominantly fast-twitch fibres, but not in slow-twitch soleus muscle, a muscle that is known to be more resistant to catabolic conditions. (3) The starvation-induced increase of the size of the fraction of easily releasable myofilaments could be significantly reduced by treatment of the starving animals with the proteinase inhibitor E-64. These results are compatible with the idea that easily releasable myofilaments are intermediates in the degradative pathway of myofibrillar proteins and that a proteolytic step may be involved in the conversion of myofilaments into easily releasable myofilaments.

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