Fractional Rates Of Protein Synthesis Research Articles

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Bed-rest and exercise remobilisation: concurrent adaptations in muscle glucose and protein metabolism

Bed rest (BR) of only a few days duration reduces muscle protein synthesis and induces skeletal muscle atrophy and insulin resistance, but the scale and juxtaposition of these events has not been investigated concurrently in the same individuals. Moreover, the impact of short-term exercise-supplemented remobilisation (ESR) on muscle volume, protein turnover and leg glucose uptake (LGU) in humans is unknown. Ten healthy males (24±1 years, body mass index (BMI) 22.7±0.6) underwent 3 days of BR, followed immediately by 3 days of ESR consisting of 5x30 maximal voluntary single leg isokinetic knee-extensions at 90o/sec each day. An isoenergetic diet was maintained throughout the study (30% fat,15% protein, 55% carbohydrate). Resting LGU was calculated from arteriovenous-venous difference across the leg and leg blood flow during the steady-state of a 3 hr hyperinsulinaemic euglycaemic clamp (60 mU/m2/min) measured pre BR, post BR and after remobilisation. Glycogen content was measured in Vastus Lateralis muscle biopsy samples obtained before and after each clamp. Leg muscle volume (LMV) was measured using magnetic resonance imaging pre BR, post BR and after remobilisation. Cumulative myofibrillar protein fractional synthesis rate (FSR), and whole-body muscle protein breakdown (MPB) were measured over the course of BR and remobilisation using deuterium oxide and 3-methylhistidine stable isotope tracers that were administered orally. Compared with pre BR, there was a 45% decline in insulin-stimulated LGU (p<0.05) after BR, which was paralleled by a reduction in insulin stimulated leg blood flow (p<0.01) and removal of insulin stimulated muscle glycogen storage. These events were accompanied by a 43% reduction in myofibrillar protein FSR (p<0.05) and a 2.5% decrease in LMV (p<0.01) during BR, along with a 30% decline in whole-body muscle myofibrillar protein breakdown after 2 days BR (p<0.05). Myofibrillar protein FSR and LMV were restored by 3 days of ERS (p<0.01, p<0.01; respectively), but not by ambulation alone. However, insulin stimulated LGU and muscle glycogen storage were not restored by ESR. Three days of BR caused concurrent reductions in LMV, myofibrillar protein FSR, myofibrillar protein breakdown and insulin stimulated LGU, leg blood flow and muscle glycogen storage in healthy, young volunteers. Resistance ESR restored LMV and myofibrillar protein FSR, but LGU and muscle glycogen storage remained depressed, highlighting divergences in muscle fuel and protein metabolism. Furthermore, ambulation alone did not restore LMV and myofibrillar protein FSR in the non-exercised contralateral limb, emphasising the importance of exercise rehabilitation following even short-term BR. This study was supported by the Biotechnology and Biological Sciences Research Council (BB/P005004/1), the Centre for Sport, Exercise and Osteoarthritis Research Versus Arthritis (grant no. 21595), and the National Institute of Health Research (NIHR) Nottingham Biomedical Research Centre. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of an immune challenge and two feed supplements on broiler chicken individual breast muscle protein synthesis rate

Optimization of broiler chicken breast muscle protein accretion is key for the efficient production of poultry meat, whose demand is steadily increasing. In a context where antimicrobial growth promoters use is being restricted, it is important to find alternatives as well as to characterize the effect of immunological stress on broiler chicken's growth. Despite its importance, research on broiler chicken muscle protein dynamics has mostly been limited to the study of mixed protein turnover. The present study aims to characterize the effect of a bacterial challenge and the feed supplementation of citrus and cucumber extracts on broiler chicken individual breast muscle proteins fractional synthesis rates (FSR) using a recently developed dynamic proteomics pipeline. Twenty-one day-old broiler chickens were administered a single 2H2O dose before being culled at different timepoints. A total of 60 breast muscle protein extracts from five experimental groups (Unchallenged, Challenged, Control Diet, Diet 1 and Diet 2) were analysed using a DDA proteomics approach. Proteomics data was filtered in order to reliably calculate multiple proteins FSR making use of a newly developed bioinformatics pipeline. Broiler breast muscle proteins FSR uniformly decreased following a bacterial challenge, this change was judged significant for 15 individual proteins, the two major functional clusters identified as well as for mixed breast muscle protein. Citrus or cucumber extract feed supplementation did not show any effect on the breast muscle protein FSR of immunologically challenged broilers. The present study has identified potential predictive markers of breast muscle growth and provided new information on broiler chicken breast muscle protein synthesis which could be essential for improving the efficiency of broiler chicken meat production. SignificanceThe present study constitutes the first dynamic proteomics study conducted in a farm animal species which has characterized FSR in a large number of proteins, establishing a precedent for biomarker discovery and assessment of health and growth status. Moreover, it has been evidenced that the decrease in broiler chicken breast muscle protein following an immune challenge is a coordinated event which seems to be the main cause of the decreased growth observed in these animals.

238 Leucine Supplementation Enhances Protein Synthesis in Lean Tissue Through a Sestrin2-GATOR2 and Raga-mTORC1 Signaling Pathway in a Preterm Piglet Model

Abstract Extrauterine growth restriction is common in preterm infants and is associated with reduced lean growth and long-term morbidities. We previously showed that leucine (Leu) acts as a nutrient signal to stimulate protein synthesis, and that in neonatal pigs born at term intermittent parenteral Leu pulses during continuous feeding increases skeletal muscle mTORC1 signaling and protein synthesis. The objective of this study was to determine the validity of the hypothesis that leu pulsing during continuous feeding enhances mTORC1 signaling via a Sestrin-mediated pathway to increase protein synthesis in lean tissue in the preterm piglet. Pigs delivered by cesarean section at 105 d gestation were continuously fed a protein and energy balanced milk-replacer diet [195 kcal ME and 13.5 g protein (kg body weight; BW)-1·d-1)]. Pigs (n = 11-12) were randomly assigned to Leu (LEU) or Alanine (ALA, isonitrogenous control) groups and treatments were administered as a pulse (1.6 mmol·kg BW-1·h-1) for 1 h every 4 h for 21 d. Indices of amino acid signaling and mTORC1 activation were determined postprandially, 60 min after initiation of the last pulse on day 24. Final body weight was greater (P < 0.05) and longissimus muscle weight was heavier in LEU than ALA pigs (P = 0.01). Upstream of mTORC1 activation, the abundance of the Sestrin2-GATOR2 complex was less and the RagA-mTORC1 complex was greater in longissimus muscle and gastrocnemius muscle of LEU compared with ALA pigs (P < 0.05). Downstream of mTORC1, the phosphorylation of S6K1 and 4EBP1 and abundance of the eIF4E-eIF4G complex were greater in longissimus muscle and gastrocnemius muscle of LEU compared with ALA pigs (P < 0.05). Fractional rates of protein synthesis in longissimus muscle and gastrocnemius muscle were greater in LEU compared with ALA pigs (P < 0.05). These results show that leucine supplementation enhances mTORC1-activated translation initiation in skeletal muscle of the preterm piglet via a Leu-sensing pathway mediated bySestrin2 and the Rag proteins. This leucine-mediated cascade promotes skeletal muscle and body weight gain by increasing muscle protein synthesis.

Lower muscle protein synthesis in humans with obesity concurrent with lower expression of muscle IGF1 splice variants.

This study tested the hypothesis that expression of insulin-like growth factor 1 (IGF-1) protein and mRNA splice variants is lower in skeletal muscle of humans with obesity who have a lower mixed-muscle protein fractional synthesis rate (MMP-FSR) when compared with individuals without obesity. The study included nine participants with obesity (OB, mean [SD], BMI = 35 [3] kg/m2 , MMP-FSR = 0.06%/h [0.02%/h]) and nine participants without obesity (W-OB, BMI = 24 [3] kg/m2 , MMP-FSR = 0.08%/h [0.02%/h]; for both BMI and MMP-FSR p < 0.05). MMP-FSR and mitochondrial protein FSR were measured following an overnight fast. Along with lower MMP-FSR, OB participants displayed lower mitochondrial protein FSR (p = 0.03) compared with W-OB participants. Expression of IGF-1 (p = 0.04) and IGF-1 receptor (p < 0.01) proteins was lower in muscle of OB participants. In addition, OB participantshad lower (p < 0.05) mRNA expression of IGF1 variants Eb and Ec. This study demonstrates that lower protein synthesis in muscle of humans with obesity occurs concurrently with lower expression of muscle IGF-1 and IGF-1 receptor proteins, as well as lower mRNA expression of the IGF1 splice variants. These findings indicate that lower protein synthesis observed in muscle of humans with obesity may result from diminished muscle IGF1 gene expression.

Measurement of plasma protein and whole body protein metabolism using [15N]glycine in a young adult man – a pilot study

ABSTRACT A novel simplified method is presented for the estimation of the metabolism of plasma proteins (albumin, fibrinogen, α, β and γ-globulin, glycoprotein) with regard to the whole body protein metabolism in a young male volunteer (22 years, 81 kg body mass). This method is based on multiple oral administration of [15N]glycine followed by measurement of 15N in plasma proteins, total free amino acids, urea and excreted urinary N. The fractional synthesis rate of albumin was estimated to 6.8 % d−1 based on amino acids and 3.3 % d−1 based on urea, respectively. The fractional synthesis rate of the other plasma proteins ranged from 4.3 % d−1 (γ-globulin) to 26.4 % d−1 (α-globulin, fibrinogen). We conclude that the simplified approach using [15N]glycine provides results which are similar to results based on the simultaneously applied 131I-human serum albumin technique as ‘gold standard’ and to those reported in literature. The compartmental analysis considering comprehensive tracer kinetic data ensures reliable data treatment and enables statistical evaluation. The analytical effort is minimal because the 15N enrichment of plasma protein after chemical digestion may be directly used. Therefore, the novel stable isotope 15N method is suitable for studies in clinical and nutritional research and practice.

Short-term repeated human biopsy sampling contributes to changes in muscle morphology and higher outcome variability.

Changes in skeletal muscle are an important aspect of overall health. The collection of human muscle to study cellular and molecular processes for research requires a needle biopsy procedure which, in itself, can induce changes in the tissue. To investigate the effect of repeat tissue sampling, we collected skeletal muscle biopsy samples from vastus lateralis separated by 7 days. Cellular infiltrate, central nucleation, enlarged extracellular matrix, and rounding of muscle fibers were used as indices to define muscle damage, and we found that 16/26 samples (61.5%) revealed at least two of these symptoms in the secondary biopsy. The presence of damage influenced outcome measures usually obtained in human biopsies. Damaged muscle showed an increase in the number of small fibers even though average fiber and fiber type-specific cross-sectional area (CSA) were not different. This included higher numbers of embryonic myosin heavy chain-positive fibers (P = 0.001) as well as elevated satellite cell number (P = 0.02) in the damaged areas and higher variability in satellite cell count in the total area (P = 0.04). Collagen content was higher in damaged (P = 0.0003) as well as nondamaged areas (P = 0.05) of the muscle sections of the damaged compared with the nondamaged group. Myofibrillar protein and ribonucleic acid (RNA) fractional synthesis rates were not significantly different between the damaged compared with the nondamaged group. Results indicate that common outcomes as well as outcome variability in human muscle tissue are affected by previous biopsies. Therefore, the extent of potential damage should be assessed when performing repeated biopsies.NEW & NOTEWORTHY Indices of damage can be found in repeated biopsy samples of nonintervened control legs. Variables, directly and not directly related to muscle damage or regeneration, were compromised in second biopsy. There is a need to determine potential damage within muscle tissue when repeated muscle sampling is part of the study design. Muscle biopsy sampling may be a source of increased heterogeneity in human muscle data.

Underfeeding Alters Brain Tissue Synthesis Rate in a Rat Brain Injury Model.

Brain injuries (BI) are highly disruptive, often having long lasting effects. Inadequate standard of care (SOC) energy support in the hospital leads to dietary energy deficiencies in BI patients. However, it is unclear how underfeeding (UF) affects protein synthesis post-BI. Therefore, in a rat model, we addressed the issue of UF on the protein fractional synthesis rate (fSR) post-BI. Compared to ad libitum (AL)-fed animals, we found that UF decreased protein synthesis in hind-limb skeletal muscle and cortical mitochondrial and structural proteins (p ≤ 0.05). BI significantly increased protein synthesis in the left and right cortices (p ≤ 0.05), but suppressed protein synthesis in the cerebellum (p ≤ 0.05) as compared to non-injured sham animals. Compared to underfeeding alone, UF in conjunction with BI (UF+BI) caused increased protein synthesis rates in mitochondrial, cytosolic, and whole-tissue proteins of the cortical brain regions. The increased rates of protein synthesis found in the UF+BI group were mitigated by AL feeding, demonstrating that caloric adequacy alleviates the effects of BI on protein dynamics in cortical and cerebellar brain regions. This research provides evidence that underfeeding has a negative impact on brain healing post-BI and that protein reserves in uninjured tissues are mobilized to support cortical tissue repair following BI.

Vitamin D deficiency contributes to overtraining syndrome in excessive trained C57BL/6 mice.

Overtraining syndrome is a condition resulting from excessive training load associated with inadequate recovery and poor sleep quality, leading to performance decrements and fatigue. Here we hypothesized that vitamin D (VitD) deficiency is a lead factor in the development of the overtraining syndrome. To test this hypothesis, two groups of 60-week-old C57BL/6 mice followed a 16-week excessive eccentric-based overtraining by excessive downhill running with or without dietary VitD depletion (EX and EX-D- groups). Two control groups were trained by uphill running at the same load with or without VitD depletion (CX and CX-D- groups). Handgrip strength decreased throughout the protocol for all groups but the decrease was sharper in EX-D- group (VitD × training, p = 0.0427). At the end of the protocol, the mass of Triceps brachii muscle, which is heavily stressed by eccentric contractions, was reduced in eccentric-trained groups (training effect, p = 0.0107). This atrophy was associated with a lower concentration of the anabolic myokine IL-15 (training effect, p = 0.0314) and a tendency to a higher expression of the atrogene cathepsin-L (training effect, p = 0.0628). VitD depletion led to a 50% decrease of the fractional protein synthesis rate in this muscle (VitD effect, p = 0.0004) as well as decreased FGF21 (VitD effect, p = 0.0351) and increased osteocrin (VitD effect, p = 0.038) concentrations that would lead to metabolic defects. Moreover, the proportion of anti-inflammatory Th2 lymphocytes was significantly decreased by the combination of eccentric training with VitD depletion (vitD × training, p = 0.0249) suggesting a systemic inflammation. Finally, exploratory behavior time of mice was decreased by VitD depletion (VitD effect, p = 0.0146) suggesting a cognitive dysfunction. Our results suggest that VitD deficiency exacerbates the effects of overtraining.

Short-term disuse does not affect postabsorptive or postprandial muscle protein fractional breakdown rates.

The decline in postabsorptive and postprandial muscle protein fractional synthesis rates (FSR) does not quantitatively account for muscle atrophy during uncomplicated, short-term disuse, when atrophy rates are the highest. We sought to determine whether 2days of unilateral knee immobilization affects mixed muscle protein fractional breakdown rates (FBR) during postabsorptive and simulated postprandial conditions. Twenty-three healthy, male participants (age: 22±1year; height: 179±1cm; body mass: 73.4±1.5kg; body mass index 22.8±0.5kg·m-2 ) took part in this randomized, controlled study. After 48h of unilateral knee immobilization, primed continuous intravenous l-[15 N]-phenylalanine and l-[ring-2 H5 ]-phenylalanine infusions were used for parallel determinations of FBR and FSR, respectively, in a postabsorptive (saline infusion; FAST) or simulated postprandial state (67.5mg·kg body mass-1 ·h-1 amino acid infusion; FED). Bilateral m. vastus lateralis biopsies from the control (CON) and immobilized (IMM) legs, and arterialized-venous blood samples, were collected throughout. Amino acid infusion rapidly increased plasma phenylalanine (59±9%), leucine (76±5%), isoleucine (109±7%) and valine (42±4%) concentrations in FED only (all P<0.001), which was sustained for the remainder of infusion. Serum insulin concentrations peaked at 21.8±2.2mU·L-1 at 15min in FED only (P<0.001) and were 60% greater in FED than FAST (P<0.01). Immobilization did not influence FBR in either FAST (CON: 0.150±0.018; IMM: 0.143±0.017%·h-1 ) or FED (CON: 0.134±0.012; IMM: 0.160±0.018%·h-1 ; all effects P>0.05). However, immobilization decreased FSR (P<0.05) in both FAST (0.071±0.004 vs. 0.086±0.007%·h-1 ; IMM vs CON, respectively) and FED (0.066±0.016 vs. 0.119±0.016%·h-1 ; IMM vs CON, respectively). Consequently, immobilization decreased net muscle protein balance (P<0.05) and to a greater extent in FED (CON: -0.012±0.025; IMM: -0.095±0.023%·h-1 ; P<0.05) than FAST (CON: -0.064±0.020; IMM: -0.072±0.017%·h-1 ). We conclude that merely 2days of leg immobilization does not modulate postabsorptive and simulated postprandial muscle protein breakdown rates. Instead, under these conditions the muscle negative muscle protein balance associated with brief periods of experimental disuse is driven near exclusively by reduced basal muscle protein synthesis rates and anabolic resistance to amino acid administration.

Effects of Whey Protein Combined with Amylopectin/Chromium on the Muscle Protein Synthesis and mTOR Phosphorylation in Exercised Rats.

This study aimed to examine the impact of varying doses of whey protein (WP) and amylopectin/chromium complex (ACr) supplementation on muscle protein synthesis (MPS), amino acid and insulin levels, and the rapamycin (mTOR) signaling pathways in exercised rats. A total of 72 rats were randomly divided into nine groups: (1) Exercise (Ex), (2) Ex + WPI to (5) Ex + WPIV with various oral doses of whey protein (0.465, 1.55, 2.33, and 3.1g/kg) and (6) Ex + WPI + ACr to (9) Ex + WPIV + ACr with various doses of whey protein combined with 0.155g/kg ACr. On the day of single-dose administration, the products were given by oral gavage after exercise. To measure the protein fractional synthesis rate (FSR), a bolus dose of deuterium-labeled phenylalanine was given, and its effects were evaluated 1h after supplementation. Rats that received 3.1g/kg of whey protein (WP) combined with ACr exhibited the most significant increase in muscle protein synthesis (MPS) compared to the Ex group (115.7%, p < 0.0001). In comparison to rats that received the same dose of WP alone, those given the combination of WP and ACr at the same dosage showed a 14.3% increase in MPS (p < 0.0001). Furthermore, the WP (3.1g/kg) + ACr group exhibited the highest elevation in serum insulin levels when compared to the Ex group (111.9%, p < 0.0001). Among the different groups, the WP (2.33g/kg) + ACr group demonstrated the greatest increase in mTOR levels (224.2%, p < 0.0001). Additionally, the combination of WP (2.33g/kg) and ACr resulted in a 169.8% increase in 4E-BP1 levels (p < 0.0001), while S6K1 levels rose by 141.2% in the WP (2.33g/kg) + ACr group (p < 0.0001). Overall, supplementation with various doses of WP combined with ACr increased MPS and enhanced the mTOR signaling pathway compared to WP alone and the Ex group.

Achilles Tendon Tissue Turnover Before and Immediately After an Acute Rupture

Background: An Achilles tendon rupture (ATR) is a frequent injury and results in the activation of tendon cells and collagen expression, but it is unknown to what extent turnover of the tendon matrix is altered before or after a rupture. Purpose/Hypothesis: The purpose of this study was to characterize tendon tissue turnover before and immediately after an acute rupture in patients. It was hypothesized that a rupture would result in pronounced collagen synthesis in the early phase (first 2 weeks) after the injury. Study Design: Cross-sectional study; Level of evidence, 3. Methods: The study included patients (N = 18) eligible for surgery after an ATR. At the time of inclusion, the patients ingested deuterium oxide (2H2O) orally, and on the day of surgery (within 14 days of the injury), they received a 3-hour flood-primed infusion of an 15N-proline tracer. During surgery, the patients had 1 biopsy specimen taken from the ruptured part of the Achilles tendon and 1 that was 3 to 5 cm proximal to the rupture as a control. The biopsy specimens were analyzed for carbon-14 (14C) levels in the tissue to calculate long-term turnover (years), incorporation of 2H-alanine (from 2H2O) into the tissue to calculate the fractional synthesis rate (FSR) of proteins in the short term (days), and incorporation of 15N-proline into the tissue to calculate the acute FSR (hours). Results: Both the rupture and the control samples showed consistently lower levels of 14C compared with the predicted level of 14C in a healthy tendon, which indicated increased tendon turnover in a fraction (48% newly synthesized) of the Achilles tendon already for a prolonged period before the rupture. Over the first days after the rupture, the synthesis rate for collagen was relatively constant, and the average synthesis rate on the day of surgery (2-14 days after the rupture) was 0.025% per hour, irrespective of the length of time after a rupture and the site of sampling (rupture vs control). No differences were found in the FSR between the rupture and control samples in the days after the rupture. Conclusion: Higher than normal tissue turnover in the Achilles tendon before a rupture indicated that changes in the tendon tissue preceded the injury. In addition, we observed no increase in tendon collagen tissue turnover in the first 2 weeks after an ATR. This favors the view that an increase in the formation of new tendon collagen is not an immediate phenomenon during the regeneration of ruptured tendons in patients. Registration: NCT03931486 (ClinicalTrials.gov identifier).

Evaluation of pea/rice and amylopectin/chromium as an alternative protein source to improve muscle protein synthesis in rats.

A preclinical study reported that the combination of an amylopectin/chromium complex (ACr) of branched-chain amino acids (BCAA) significantly enhanced muscle protein synthesis (MPS). This study was conducted to determine the effects of the addition of ACr complex to a pea/rice (PR) protein on MPS, insulin, muslin levels, and the mTOR pathway in exercised rats. Twenty-four rats were divided into three groups: (i) exercise (Ex); (ii) Ex + PR 1:1 blend (0.465g/kg BW); (iii) Ex + PR + ACr (0.155g/kg BW). On the day of single-dose administration, after the animals were exercised at 26/m/min for 2h, the supplement was given by oral gavage. The rats were injected with a bolus dose (250mg/kg BW, 25g/L) of deuterium-labeled phenylalanine to determine the protein fractional synthesis rate (FSR) one h after consuming the study product. The combination of PR and ACr enhanced MPS by 42.55% compared to the Ex group, while Ex + PR alone increased MPS by 30.2% over the Ex group (p < 0.0001) in exercised rats. Ex + PR plus ACr significantly enhanced phosphorylation of mTOR and S6K1 (p < 0.0001), and 4E-BP1 (p < 0.001) compared to the Ex (p < 0.0001). PR to ACr also significantly increased insulin and musclin levels (p < 0.0001) in exercised rats. Additionally, compared to Ex + PR alone, Ex + PR + ACr enhanced mTOR (p < 0.0001) and S6K1 (p < 0.0001) levels. These data suggested that PR + ACr may provide an alternative to animal proteins for remodeling and repairing muscle by stimulating MPS and mTOR signaling pathways in post-exercised rats. More preclinical and clinical human studies on combining pea/rice and amylopectin/chromium complex are required.

Mainly Muscle, Jejunum, And Lung Fractional Protein Synthesis Rates Are Related To Whole-Body Protein Synthesis In The Pig

Mainly Muscle, Jejunum, And Lung Fractional Protein Synthesis Rates Are Related To Whole-Body Protein Synthesis In The Pig

A novel dynamic proteomics approach for the measurement of broiler chicken protein fractional synthesis rate.

The study of protein synthesis in farm animals is uncommon despite its potential to increase knowledge about metabolism and discover new biomarkers of health and growth status. The present study describes a novel dynamic proteomics approach for the measurement of protein fractional synthesis rate (FSR) in broiler chickens. Chickens received a 10g/kg oral dose of 2 H2 O at day 21 of their life. Body water 2 H abundance was measured in plasma samples using a portable Fourier transform infrared spectrometer. Free and protein-bound amino acids (AAs) were isolated and had their 2 H enrichment measured by gas chromatography with mass spectrometry (GC/MS). Peptide 2 H enrichment was measured by proteomics analysis of plasma and muscle samples. Albumin, fibrinogen and muscle protein FSR were calculated from GC/MS and proteomics data. Ala appeared to be more enriched at the site of protein synthesis than in the AA free pools. Glu was found to be the AA closest to isotopic equilibrium between the different AA pools. Glu was used as an anchor to calculate n(AA) values necessary for chicken protein FSR calculation in dynamic proteomics studies. FSR values calculated using proteomics data and GC/MS data showed good agreement as evidenced by a Bland-Altman residual plot. A new dynamic proteomics approach for the measurement of broiler chicken individual protein FSR based on the administration of a single 2 H2 O oral bolus has been developed and validated. The proposed approach could facilitate new immunological and nutritional studies on free-living animals.

Insulin Treatment Affects Brain Protein Synthesis Rate in Streptozotocin-Induced Diabetic Rats

The purpose of this study was to determine whether insulin treatment affected the rate of brain protein synthesis in diabetic rats. Experiments were conducted on three groups of rats given streptozotocin without insulin treatment (diabetes), treated with streptozotocin + insulin (diabetes + insulin) or treated with neither streptozotocin nor insulin (control). The fractional rates of protein synthesis in brain, liver and kidney of diabetic rats treated with insulin were significantly greater than in diabetic rats without insulin treatment. In brain, liver and kidney, the RNA activity [g protein synthesized/(g RNA·d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that insulin treatment of diabetic rats is likely to increase the rate of protein synthesis in the brain, and that RNA activity is related to the fractional rate of brain protein synthesis.

Protein Synthesis Is Stimulated in Nutritionally Obese Rats

To investigate the tissue growth and the protein synthesis in vivo in nutritional obesity we used lipid-rich multichoice diet feeding. Young male rats of the Wistar strain were divided in two groups: control and obese. Control rats were fed pelleted nonpurified diet. Obese rats were fed a multichoice diet based on a variety of highly palatable energy-rich human foods for 30 d. Protein intake was kept equal in the groups to avoid its influence on protein turnover. The tissue growth pattern was evaluated by protein, DNA and RNA contents of liver, kidney, heart, skeletal muscles and small intestine. Protein synthesis in vivo was measured in these tissues by the phenylalanine flooding-dose technique. Rats fed the multichoice diet showed significantly greater body growth when compared with rats fed the nonpurified diet. Adipose and other tissue weights were significantly greater in the obese rats. The tissue growth pattern was characterized mainly by hyperplasia. In most tissues the net protein accretion found in obese rats resulted from an enhancement in the fractional rate of protein synthesis. The greater protein synthesis was due to an increase in the efficiency of ribosomes in kidney, heart and skeletal muscle and to an increase in the synthetic capacity in liver and small intestine. These data suggest that excess energy intake when protein intake is adequate stimulates tissue growth and protein synthesis in rats.

Relationship of Protein Synthesis to mRNA Levels in the Muscle of Chicks under Various Nutritional Conditions

The relationship between the rate of protein synthesis and the level of messenger RNA (mRNA) in the breast muscle (pectoralis major) was studied to examine the mechanisms regulating muscle protein synthesis in chicks under various nutritional conditions. The fractional synthesis rate (FSR) of protein was measured by a large-dose injection of L-[4-3H]phenylalanine. Poly(U)-Sepharose 4B affinity chromatography was used to extract Poly(A)+ RNA as mRNA. The first study examined the effect of dietary protein levels (0–60%) on protein synthesis rate and the contents of mRNA and total RNA. The fractional synthesis rate increased with increasing dietary protein levels from 0 to 20%. When chicks were fed high protein diets (40 and 60% of dietary protein), both the FSR and absolute synthesis rate (ASR) of protein were reduced compared with the 20% protein group. Multiple regression analysis indicated that for graded dietary protein levels below the dietary protein requirement, the response of muscle protein synthesis is largely regulated by variation in the ASR per unit RNA (the efficiency of RNA in synthesizing protein). At dietary protein levels above 20%, synthesis rates are also related to changes in the RNA:mRNA ratio (ribosome number relative to mRNA), the mRNA:DNA ratio (mRNA availability) and the DNA:protein ratio (DNA concentration). When chicks were food deprived for 2 d, FSR was reduced to about half that of well-fed controls. Upon refeeding of these chicks with the control diet, the FSR returned to the normal level within 0.5 h. When food-deprived chicks were refed with protein, carbohydrate or fat alone, the FSR returned to the control level within 0.5 h after consumption of each nutrient. The increase in muscle protein synthesis of unfed chicks following refeeding of various nutrients was explained by an increased ASR per unit RNA. Our results suggest that nutrient intake can change muscle protein synthesis rates in chicks largely by altering the ASR per unit RNA, in some cases associated also with changes in the levels of RNA, mRNA and DNA.

Metabolic labeling unveils alterations in the turnover of HDL-associated proteins during diabetes progression in mice.

Several aspects of diabetes pathophysiology and complications result from hyperglycemia-induced alterations in the structure and function of plasma proteins. Furthermore, insulin has a significant influence on protein metabolism by affecting both the synthesis and degradation of proteins in various tissues. To understand the role of progressive hyperglycemia on plasma proteins, in this study, we measured the turnover rates of high-density lipoprotein (HDL)-associated proteins in control (chow diet), prediabetic [a high-fat diet (HFD) for 8 wk] or diabetic [HFD for 8 wk with low-dose streptozotocin (HFD + STZ) in weeks 5-8 of HFD] C57BL/6J mice using heavy water (2H2O)-based metabolic labeling approach. Compared with control mice, HFD and HFD + STZ mice showed elevations of fasting plasma glucose levels in the prediabetic and diabetic range, respectively. Furthermore, the HFD and HFD + STZ mice showed increased hepatic triglyceride (TG) levels, total plasma cholesterol, and plasma TGs. The kinetics of 40 proteins were quantified using the proteome dynamics method, which revealed an increase in the fractional synthesis rate (FSR) of HDL-associated proteins in the prediabetic mice compared with control mice, and a decrease in FSR in the diabetic mice. The pathway analysis revealed that proteins with altered turnover rates were involved in acute-phase response, lipid metabolism, and coagulation. In conclusion, prediabetes and diabetes have distinct effects on the turnover rates of HDL proteins. These findings suggest that an early dysregulation of the HDL proteome dynamics can provide mechanistic insights into the changes in protein levels in these conditions.NEW & NOTEWORTHY This study is the first to examine the role of gradual hyperglycemia during diabetes disease progression on HDL-associated protein dynamics in the prediabetes and diabetic mice. Our results show that the fractional synthesis rate of HDL-associated proteins increased in the prediabetic mice whereas it decreased in the diabetic mice compared with control mice. These kinetic changes can help to elucidate the mechanism of altered protein levels and HDL dysfunction during diabetes disease progression.

Metabolic and molecular responses of human patellar tendon to concentric- and eccentric-type exercise in youth and older age

Exercise training can induce adaptive changes to tendon tissue both structurally and mechanically; however, the underlying compositional changes that contribute to these alterations remain uncertain in humans, particularly in the context of the ageing tendon. The aims of the present study were to determine the molecular changes with ageing in patellar tendons in humans, as well as the responses to exercise and exercise type (eccentric (ECC) and concentric (CON)) in young and old patellar tendon. Healthy younger males (age 23.5 ± 6.1 years; n = 27) and older males (age 68.5 ± 1.9 years; n = 27) undertook 8 weeks of CON or ECC training (3 times per week; at 60% of 1 repetition maximum (1RM)) or no training. Subjects consumed D2O throughout the protocol and tendon biopsies were collected after 4 and 8 weeks for measurement of fractional synthetic rates (FSR) of tendon protein synthesis and gene expression. There were increases in tendon protein synthesis following 4 weeks of CON and ECC training (P < 0.01; main effect by ANOVA), with no differences observed between young and old males, or training type. At the transcriptional level however, ECC in young adults generally induced greater responses of collagen and extracellular matrix-related genes than CON, while older individuals had reduced gene expression responses to training. Different training types did not appear to induce differential tendon responses in terms of protein synthesis, and while tendons from older adults exhibited different transcriptional responses to younger individuals, protein turnover changes with training were similar for both age groups.