Measurement of the synthesis rates of collagens and total protein in rabbit muscle.
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ABSTRACT:
SUBMITTER: Palmer RM
PROVIDER: S-EPMC1162379 | biostudies-other | 1980 Nov
REPOSITORIES: biostudies-other
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Project description:Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.
| S-EPMC4701543 | biostudies-literature
Project description:Key pointsProtein ingestion and cooling are strategies employed by athletes to improve postexercise recovery and, as such, to facilitate muscle conditioning. However, whether cooling affects postprandial protein handling and subsequent muscle protein synthesis rates during recovery from exercise has not been assessed. We investigated the effect of postexercise cooling on the incorporation of dietary protein-derived amino acids into muscle protein and acute postprandial (hourly) as well as prolonged (daily) myofibrillar protein synthesis rates during recovery from resistance-type exercise over 2 weeks. Cold-water immersion during recovery from resistance-type exercise lowers the capacity of the muscle to take up and/or direct dietary protein-derived amino acids towards de novo myofibrillar protein accretion. In addition, cold-water immersion during recovery from resistance-type exercise lowers myofibrillar protein synthesis rates during prolonged resistance-type exercise training. Individuals aiming to improve skeletal muscle conditioning should reconsider applying cooling as a part of their postexercise recovery strategy.AbstractWe measured the impact of postexercise cooling on acute postprandial (hourly) as well as prolonged (daily) myofibrillar protein synthesis rates during adaptation to resistance-type exercise over 2 weeks. Twelve healthy males (aged 21 ± 2 years) performed a single resistance-type exercise session followed by water immersion of both legs for 20 min. One leg was immersed in cold water (8°C: CWI), whereas the other leg was immersed in thermoneutral water (30°C: CON). After water immersion, a beverage was ingested containing 20 g of intrinsically (l-[1-13 C]-phenylalanine and l-[1-13 C]-leucine) labelled milk protein with 45 g of carbohydrates. In addition, primed continuous l-[ring-2 H5 ]-phenylalanine and l-[1-13 C]-leucine infusions were applied, with frequent collection of blood and muscle samples to assess myofibrillar protein synthesis rates in vivo over a 5 h recovery period. In addition, deuterated water (2 H2 O) was applied with the collection of saliva, blood and muscle biopsies over 2 weeks to assess the effects of postexercise cooling with protein intake on myofibrillar protein synthesis rates during more prolonged resistance-type exercise training (thereby reflecting short-term training adaptation). Incorporation of dietary protein-derived l-[1-13 C]-phenylalanine into myofibrillar protein was significantly lower in CWI compared to CON (0.016 ± 0.006 vs. 0.021 ± 0.007 MPE; P = 0.016). Postexercise myofibrillar protein synthesis rates were lower in CWI compared to CON based upon l-[1-13 C]-leucine (0.058 ± 0.011 vs. 0.072 ± 0.017% h-1 , respectively; P = 0.024) and l-[ring-2 H5 ]-phenylalanine (0.042 ± 0.009 vs. 0.053 ± 0.013% h-1 , respectively; P = 0.025). Daily myofibrillar protein synthesis rates assessed over 2 weeks were significantly lower in CWI compared to CON (1.48 ± 0.17 vs. 1.67 ± 0.36% day-1 , respectively; P = 0.042). Cold-water immersion during recovery from resistance-type exercise reduces myofibrillar protein synthesis rates and, as such, probably impairs muscle conditioning.
| S-EPMC7028023 | biostudies-literature
Project description:PURPOSE:To assess the impact of dietary protein ingestion on intramuscular connective tissue protein synthesis rates during overnight recovery from a single bout of resistance exercise. METHODS:Thirty-six healthy, young males were randomly assigned to one of three treatments. One group ingested 30 g intrinsically L-[1-C]-phenylalanine-labeled casein protein prior to sleep (PRO, n=12). The other two groups performed a bout of resistance exercise in the evening and ingested either a placebo (EX, n=12) or 30 g intrinsically L-[1-C]-phenylalanine-labeled casein protein prior to sleep (EX+PRO, n=12). Continuous intravenous infusions of L-[ring-H5]-phenylalanine and L-[1-C]-leucine were applied and blood and muscle tissue samples were collected to assess connective tissue protein synthesis rates and dietary protein-derived amino acid incorporation in the connective tissue protein fraction. RESULTS:Resistance exercise resulted in higher connective tissue protein synthesis rates when compared to rest (0.086±0.017 (EX) and 0.080±0.019 (EX+PRO) vs 0.059±0.016 (PRO) %·h; P<0.05). Post-exercise casein protein ingestion did not result in higher connective tissue protein synthesis rates when compared to post-exercise placebo ingestion (P=1.00). Dietary protein-derived amino acids were incorporated into the connective tissue protein fraction at rest, and to a greater extent during recovery from exercise (P=0.002). CONCLUSION:Resistance exercise increases intramuscular connective tissue protein synthesis rates during overnight sleep, with no further impact of post-exercise protein ingestion. However, dietary protein-derived amino acids are being utilized as precursors to support de novo connective tissue protein synthesis.
| S-EPMC7431152 | biostudies-literature
Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis. 4 samples of E. coli ribosome profiling and mRNA-seq, including biological replicates
2014-04-24 | E-GEOD-53767 | biostudies-arrayexpress
Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis.
2014-04-24 | GSE53767 | GEO
Project description:Tyrosine balance and protein synthesis were studied during the same incubation in isolated rabbit forelimb muscles. From these measurements, protein degradation was calculated. Isolated muscles were usually in a state of negative amino acid balance, principally as a result of the 75% decrease in protein synthesis. Muscles from rabbits starved for 18 h had lower rates of both protein synthesis and degradation compared with muscles from normally fed rabbits. Intermittent mechanical stretching and the addition of insulin at 100 microunits/ml increased rates of both protein synthesis and degradation. Increases in the rate of protein synthesis were proportionately greater in the muscles from starved animals. In muscles from both fed and starved donors, increases in protein-synthesis rates owing to intermittent stretching and insulin were proportionately greater than the increases in degradation rates. For example, insulin increased the rate of protein synthesis in the muscles from starved donors by 111% and the rate of degradation by 31%. Insulin also increased the rate of protein synthesis when added at a higher concentration (100 munits/ml); at this concentration, however, the rate of protein degradation was not increased. The suppressive effect of insulin on high rates of protein degradation in other skeletal-muscle preparations may reflect a non-physiological action of the hormone.
| S-EPMC1152594 | biostudies-other
Project description:Muscle loss is a severe complication of many medical conditions such as cancer, cardiac failure, muscular dystrophies, and nerve damage. The contribution of myofibrillar protein synthesis (MPS) to the loss of muscle mass after nerve damage is not clear. Using deuterium oxide (D2O) labeling, we demonstrate that MPS is significantly increased in rat m.tibialis anterior (TA) compared to control (3.23 ± 0.72 [damaged] to 2.09 ± 0.26%?day-1 [control]) after 4 weeks of nerve constriction injury. This is the case despite substantial loss of mass of the TA (350 ± 96 mg [damaged] to 946 ± 361 mg [control]). We also show that expression of regulatory proteins involved with MPS (p70s6k1: 2.4 ± 0.3 AU [damaged] to 1.8 ± 0.2 AU [control]) and muscle protein breakdown (MPB) (MAFbx: 5.3 ± 1.2 AU [damaged] to 1.4 ± 0.4 AU [control]) are increased in nerve damaged muscle. Furthermore, the expression of p70s6k1 correlates with MPS rates (r2 = 0.57). In conclusion, this study shows that severe muscle wasting following nerve damage is accompanied by increased as opposed to decreased MPS.
| S-EPMC6127268 | biostudies-literature
Project description:Skeletal muscle plasticity is reflected by a dynamic balance between protein synthesis and breakdown, with basal muscle tissue protein synthesis rates ranging between 0.02 and 0.09%/h. Though it is evident that other musculoskeletal tissues should also express some level of plasticity, data on protein synthesis rates of most of these tissues in vivo in humans is limited. Six otherwise healthy patients (62±3 y), scheduled to undergo unilateral total knee arthroplasty, were subjected to primed continuous intravenous infusions with L-[ring-13C6]-Phenylalanine throughout the surgical procedure. Tissue samples obtained during surgery included muscle, tendon, cruciate ligaments, cartilage, bone, menisci, fat, and synovium. Tissue-specific fractional protein synthesis rates (%/h) were assessed by measuring the incorporation of L-[ring-13C6]-Phenylalanine in tissue protein and were compared with muscle tissue protein synthesis rates using a paired t test. Tendon, bone, cartilage, Hoffa's fat pad, anterior and posterior cruciate ligament, and menisci tissue protein synthesis rates averaged 0.06±0.01, 0.03±0.01, 0.04±0.01, 0.11±0.03, 0.07±0.02, 0.04±0.01, and 0.04±0.01%/h, respectively, and did not significantly differ from skeletal muscle protein synthesis rates (0.04±0.01%/h; P>0.05). Synovium derived protein (0.13±0.03%/h) and intercondylar notch bone tissue protein synthesis rates (0.03±0.01%/h) were respectively higher and lower compared to skeletal muscle protein synthesis rates (P<0.05 and P<0.01, respectively). Basal protein synthesis rates in various musculoskeletal tissues are within the same range of skeletal muscle protein synthesis rates, with fractional muscle, tendon, bone, cartilage, ligament, menisci, fat, and synovium protein synthesis rates ranging between 0.02 and 0.13% per hour in vivo in humans. Clinical trial registration: NTR5147.
| S-EPMC6837426 | biostudies-literature
Project description:Measurement of protein phosphorylation plays an essential role in delineating cell signaling pathways. Although the detection of a specific phosphoprotein has been largely accomplished by immunological methods, a specific and sensitive assay to measure total protein phosphorylation level in complex samples such as whole cell extracts has yet to be established. Here, we present a sensitive phosphorylation assay on a microwell plate to determine total protein phosphorylation level calibrated to a phosphoprotein standard. The core of the assay is a reagent termed pIMAGO that is multi-functionalized with titanium ions for its superior selectivity towards phosphorylated proteins and with fluorophores for quantification. The specificity, sensitivity, and quantitative nature of the assay were demonstrated with standard proteins and whole cell lysates. The method was then employed to measure the overall protein phosphorylation level of human cells under different treatments. At last, we investigated the practicability of the assay to serve as a sensitive tool to estimate the amount of phosphorylated samples prior to a mass spectrometry-based phosphoproteomic analysis.
| S-EPMC4425989 | biostudies-literature
Project description:In eight anaesthetized post-absorptive dogs we measured the concentration and specific radioactivity of phenylalanine and leucine in arterial and femoral-venous plasma, together with hindlimb flow during a continuous infusion of L-[ring-2,6-3H]phenylalanine and [1-14C]leucine. The femoral-venous plasma concentration was greater than arterial for both phenylalanine and leucine (P less than 0.05 for each). Despite net amino acid release there was a significant removal of both labelled phenylalanine and labelled leucine. Consequently, a significant dilution of specific radioactivity was observed between artery and vein for both radio-tracers. The uptake of leucine from the arterial circulation by the hindlimb exceeded by 2.6-fold that of phenylalanine; the measured molar ratio of leucine to phenylalanine in hindlimb muscle protein averaged 2.4 +/- 0.1. Since phenylalanine is neither synthesized nor degraded by muscle tissue, the measured removal of tracer and the dilution of tracer specific radioactivity across the hindlimb can be used to estimate rates of phenylalanine incorporation into, and release from, tissue protein. The estimated rate of protein synthesis by hindlimb averaged 644 +/- 250 nmol of phenylalanine/min. This was exceeded by the rate of tissue protein degradation (987 +/- 285 nmol of phenylalanine/min). The present results demonstrate that the dilution of the specific radioactivity of labelled phenylalanine can be readily measured across dog hindlimb. This measurement, coupled with an estimate of tissue blood flow, can provide a readily measured, non-destructive, method for estimation of protein turnover in specific muscle beds in vivo. Measurements can be made repeatedly over time in a single experiment, allowing the study of factors which regulate protein turnover. The method developed here in dogs can be readily extended to clinical studies.
| S-EPMC1148103 | biostudies-other