Fractional Rates Of Protein Synthesis Research Articles

Adjustments of Protein Metabolism in Fasting Arctic Charr, Salvelinus alpinus

Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (KS) in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems) were measured in the same fish. This study is the first to measure both KS and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills). KS was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins.

Metabolic rate and rates of protein turnover in food-deprived cuttlefish, Sepia officinalis (Linnaeus 1758).

To determine the metabolic response to food deprivation, cuttlefish (Sepia officinalis) juveniles were either fed, fasted (3 to 5 days food deprivation), or starved (12 days food deprivation). Fasting resulted in a decrease in triglyceride levels in the digestive gland, and after 12 days, these lipid reserves were essentially depleted. Oxygen consumption was decreased to 53% and NH4 excretion to 36% of the fed group following 3-5 days of food deprivation. Oxygen consumption remained low in the starved group, but NH4 excretion returned to the level recorded for fed animals during starvation. The fractional rate of protein synthesis of fasting animals decreased to 25% in both mantle and gill compared with fed animals and remained low in the mantle with the onset of starvation. In gill, however, protein synthesis rate increased to a level that was 45% of the fed group during starvation. In mantle, starvation led to an increase in cathepsin A-, B-, H-, and L-like enzyme activity and a 2.3-fold increase in polyubiquitin mRNA that suggested an increase in ubiquitin-proteasome activity. In gill, there was a transient increase in the polyubiquitin transcript levels in the transition from fed through fasted to the starved state and cathepsin A-, B-, H-, and L-like activity was lower in starved compared with fed animals. The response in gill appears more complex, as they better maintain rates of protein synthesis and show no evidence of enhanced protein breakdown through recognized catabolic processes.

No additional effect of different types of physical activity on 10-hour muscle protein synthesis in elderly men on a controlled energy- and protein-sufficient diet

PurposeThe elderly lose skeletal muscle mass with age, which may be detrimental for function and quality of life. Both inactivity and heavy resistance exercise are known to have marked but opposite effects upon muscle mass. However, the potential effects of daily physical activity upon muscle protein synthesis (MPS) are less investigated. The aim of this study was to determine the effects of daily physical activities upon MPS in elderly individuals. MethodsA total of 24 elderly men (70±1year) were recruited and randomly assigned: inactivity in form of bed-rest (IA), daily physical activities (DA), or heavy resistance exercise (RE). All groups undertook a normal eating routine containing carbohydrates (52 E%), fat (32 E%), and protein (16 E%). Ingestion of labeled milk protein ([1-13C]leucine-labeled whey and caseinate) served to maintain tracer enrichment for determination of 10-hour myofibrillar protein fractional synthesis rates (FSR), and typical prerequisites for calculating FSR were fulfilled. Physical activities were monitored, and venous blood and muscle biopsies collected. ResultsPhysical activity was highest in the DA compared to both the IA and RE groups. Nutrient ingestion increased insulin, leucine, and phenylalanine plasma concentrations in all groups. [1-13C]leucine enrichment was stable throughout the 10-hour FSR period. Myofibrillar protein FSR were similar for IA, DA, and RE groups, 0.055±0.003%/h, 0.058±0.006%/h, and 0.065±0.008%/h, respectively (means±SE, P=0.44). ConclusionsIn elderly males, inactivity, daily activities, and resistance exercise interventions result in equal 10-hour, whole day MPS during an energy- and protein-sufficient diet regimen.

Assessing the reproducibility of fractional rates of protein synthesis in muscle tissue measured using the flooding dose technique

The flooding dose technique of Garlick et al. (1980) has become the main method for measuring tissue and whole-animal rates of protein synthesis in ectotherms. However, single tissue samples are used to determine rates of protein synthesis and no studies have examined the pattern of flooding in large tissues such as the white muscle in fishes, which can comprise up to 55% of the wet body mass of a fish and which is poorly perfused. The present study has examined, for the first time, the patterns of flooding and measured rates of protein synthesis in five different regions of the white muscle in the Arctic charr Salvelinus alpinus ranging in size from 25g to 1.6kg following a flooding dose injection of L-[3H]-phenylalanine. The results indicate that the degree of flooding (i.e. free pool specific radioactivity relative to that of the injection solution) and elevation in free phenylalanine concentrations can vary between regions but the calculated fractional rates of protein synthesis were similar in four of the five regions studied. The variability in rates of protein synthesis increased with body size with greater variability observed between regions for fish >1kg in body mass. For consistency between studies, it is recommended that samples are taken from the epaxial muscle in the region below the dorsal fin when measuring fractional rates of white muscle synthesis in fishes.

L-Citrulline Supplementation Enhances Fetal Growth and Protein Synthesis in Rats with Intrauterine Growth Restriction

Background: Intrauterine growth restriction (IUGR) results from either maternal undernutrition or impaired placental blood flow, exposing offspring to increased perinatal mortality and a higher risk of metabolic syndrome and cardiovascular disease during adulthood. L-Citrulline is a precursor of L-arginine and nitric oxide (NO), which regulates placental blood flow. Moreover, L-citrulline stimulates protein synthesis in other models of undernutrition.Objective: The aim of the study was to determine whether L-citrulline supplementation would enhance fetal growth in a model of IUGR induced by maternal dietary protein restriction.Methods: Pregnant rats were fed either a control (20% protein) or a low-protein (LP; 4% protein) diet. LP dams were randomly allocated to drink tap water either as such or supplemented with L-citrulline (2 g · kg−1 · d−1), an isonitrogenous amount of L-arginine, or nonessential L-amino acids (NEAAs). On day 21 of gestation, dams received a 2-h infusion of L-[1-13C]-valine until fetuses were extracted by cesarean delivery. Isotope enrichments were measured in free amino acids and fetal muscle, liver, and placenta protein by GC–mass spectrometry.Results: Fetal weight was ∼29% lower in the LP group (3.82 ± 0.06 g) than in the control group (5.41 ± 0.10 g) (P < 0.001). Regardless of supplementation, fetal weight remained below that of control fetuses. Yet, compared with the LP group, L-citrulline and L-arginine equally increased fetal weight to 4.15 ± 0.08 g (P < 0.05) and 4.13 ± 0.1 g (P < 0.05 compared with LP), respectively, whereas NEAA did not (4.05 ± 0.05 g; P = 0.07). Fetal muscle protein fractional synthesis rate was 35% lower in the LP fetuses (41% ± 11%/d) than in the control (61% ± 13%/d) fetuses (P < 0.001) and was normalized by L-citrulline (56% ± 4%/d; P < 0.05 compared with LP, NS compared with control) and not by other supplements. Urinary nitrite and nitrate excretion was lower in the LP group (6.4 ± 0.8 μmol/d) than in the control group (17.9 ± 1.1 μmol/d; P < 0.001) and increased in response to L-citrulline or L-arginine (12.1 ± 2.2 and 10.6 ± 0.9 μmol/d; P < 0.05), whereas they did not in the LP + NEAA group.Conclusion: L-Citrulline increases fetal growth in a model of IUGR, and the effect may be mediated by enhanced fetal muscle protein synthesis and/or increased NO production.

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs.

Neonatal pigs are used as a model to study and optimize the clinical treatment of infants who are unable to maintain oral feeding. Using this model, we have shown previously that pulsatile administration of leucine during continuous feeding over 24 h via orogastric tube enhanced protein synthesis in skeletal muscle compared with continuous feeding alone. To determine the long-term effects of leucine pulses, neonatal piglets (n = 11-12/group) were continuously fed formula via orogastric tube for 21 days, with an additional parenteral infusion of either leucine (CON + LEU; 800 μmol·kg-1·h-1) or alanine (CON + ALA) for 1 h every 4 h. The results show that body and muscle weights and lean gain were ∼25% greater, and fat gain was 48% lower in CON + LEU than CON + ALA; weights of other tissues were unaffected by treatment. Fractional protein synthesis rates in longissimus dorsi, gastrocnemius, and soleus muscles were ∼30% higher in CON + LEU compared with CON + ALA and were associated with decreased Deptor abundance and increased mTORC1, mTORC2, 4E-BP1, and S6K1 phosphorylation, SNAT2 abundance, and association of eIF4E with eIF4G and RagC with mTOR. There were no treatment effects on PKB, eIF2α, eEF2, or PRAS40 phosphorylation, Rheb, SLC38A9, v-ATPase, LAMTOR1, LAMTOR2, RagA, RagC, and LAT1 abundance, the proportion of polysomes to nonpolysomes, or the proportion of mRNAs encoding rpS4 or rpS8 associated with polysomes. Our results demonstrate that pulsatile delivery of a leucine supplement during 21 days of continuous enteral feeding enhances lean growth by stimulating the mTORC1-dependent translation initiation pathway, leading to protein synthesis in skeletal muscle of neonates.

Dietary L-arginine levels affect the liver protein turnover and alter the expression of genes related to protein synthesis and proteolysis of laying hens

Amino acids are considered to be anabolic factors that affect protein turnover. The aim of this study was to test the effects of dietary L-arginine (Arg) levels on protein metabolism in the liver of laying hens and the expression of genes related to protein synthesis and proteolysis. Xinyang black commercial laying hens (n = 864, 31 wk of age) were randomly allotted to 6 treatments with 4 replicates of 36 birds. The dietary treatments were corn-corn gluten meal based diets containing 0.64, 0.86, 1.03, 1.27, 1.42, and 1.66% L-Arg, respectively. Serum concentrations of total protein and albumin were maximized in the 1.27% L-Arg group, and serum concentration of urea acid was the lowest in the 1.27% L-Arg group. The 1.27% L-Arg group had the highest fractional protein synthesis rate and fractional protein gain rate in the liver. Consistent with the data on protein turnover, mRNA abundances of target of rapamycin (TOR) and ribosomal protein S6 kinase 1 increased in the liver of layers fed 1.27% L-Arg, while mRNA abundances of cathepsin B and 20S proteasome decreased at the same dietary L-Arg level. In conclusion, the dietary level of L-Arg increased the liver fractional protein synthesis rate and fractional protein gain rate of laying hens, and the action of an appropriate level of dietary L-Arg involves upregulating the gene expression of the TOR signaling pathway accompanied by suppressing the mRNA expression of cathepsin B and 20S proteasome in the liver.

The IGF-1/Akt/S6 Signaling Pathway is Age-Dependently Downregulated in the Chicken Breast Muscle.

The skeletal muscle mass is known to be controlled by the balance between protein synthesis and degradation. The fractional rate of protein synthesis has been reported to decrease age-dependently from 1 to 4 weeks of age in the chicken breast muscle (pectoralis major muscle). On the other hand, age-dependent change of the fractional protein degradation rate was reported to be less in the skeletal muscle of chickens. These findings suggest that protein synthesis is age-dependently downregulated in chicken muscle. We herein investigated the age-dependent changes in protein synthesis or proteolysis-related factors in the breast muscle of 7, 14, 28, and 49-day old broiler chickens. IGF-1 mRNA level, phosphorylation rate of Akt, and phospho-S6 content were coordinately decreased in an age-dependent manner, suggesting that IGF-1-stimulated protein synthesis is downregulated with age in chicken breast muscle. In contrast, atrogin-1, one of the proteolysis-related factors, gradually increased with age at mRNA levels. However, plasma Nτ-methylhistidine concentration, an indicator of skeletal muscle proteolysis, did not coordinately change with atrogin-1 mRNA levels. Taken together, our results suggest that the IGF-1/Akt/S6 signaling pathway is age-dependently downregulated in the chicken breast muscle.

Proteome-wide muscle protein fractional synthesis rates predict muscle mass gain in response to a selective androgen receptor modulator in rats.

Biomarkers of muscle protein synthesis rate could provide early data demonstrating anabolic efficacy for treating muscle-wasting conditions. Androgenic therapies have been shown to increase muscle mass primarily by increasing the rate of muscle protein synthesis. We hypothesized that the synthesis rate of large numbers of individual muscle proteins could serve as early response biomarkers and potentially treatment-specific signaling for predicting the effect of anabolic treatments on muscle mass. Utilizing selective androgen receptor modulator (SARM) treatment in the ovariectomized (OVX) rat, we applied an unbiased, dynamic proteomics approach to measure the fractional synthesis rates (FSR) of 167-201 individual skeletal muscle proteins in triceps, EDL, and soleus. OVX rats treated with a SARM molecule (GSK212A at 0.1, 0.3, or 1 mg/kg) for 10 or 28 days showed significant, dose-related increases in body weight, lean body mass, and individual triceps but not EDL or soleus weights. Thirty-four out of the 94 proteins measured from the triceps of all rats exhibited a significant, dose-related increase in FSR after 10 days of SARM treatment. For several cytoplasmic proteins, including carbonic anhydrase 3, creatine kinase M-type (CK-M), pyruvate kinase, and aldolase-A, a change in 10-day FSR was strongly correlated (r(2) = 0.90-0.99) to the 28-day change in lean body mass and triceps weight gains, suggesting a noninvasive measurement of SARM effects. In summary, FSR of multiple muscle proteins measured by dynamics of moderate- to high-abundance proteins provides early biomarkers of the anabolic response of skeletal muscle to SARM.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

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.

Skeletal Muscle Protein Breakdown Remains Elevated in Pediatric Burn Survivors up to One-Year Post-Injury.

Acute alterations in skeletal muscle protein metabolism are a well-established event associated with the stress response to burns. Nevertheless, the long-lasting effects of burn injury on skeletal muscle protein turnover are incompletely understood. This study was undertaken to investigate fractional synthesis (FSR) and breakdown (FBR) rates of protein in skeletal muscle of pediatric burn patients (n = 42, >30% total body surface area burns) for up to 1 year after injury. Skeletal muscle protein kinetics were measured in the post-prandial state following bolus injections of C6 and N phenylalanine stable isotopes. Plasma and muscle phenylalanine enrichments were quantified using gas chromatography-mass spectrometry. We found that the FSR in burn patients was 2- to 3-fold higher than values from healthy men previously reported in the literature (P ≤ 0.05). The FBR was 4- to 6-fold higher than healthy values (P < 0.01). Therefore, net protein balance was lower in burn patients compared with healthy men from 2 weeks to 12 months post-injury (P < 0.05). These findings show that skeletal muscle protein turnover stays elevated for up to 1 year after burn, an effect attributable to simultaneous increases in FBR and FSR. Muscle FBR exceeds FSR during this time, producing a persistent negative net protein balance, even in the post-prandial state, which likely contributes to the prolonged cachexia seen in burned victims.

Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.)

For the first time, use of the flooding dose technique using 3H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of 3H-phenylalanine into the posterior adductor muscle. Incorporation of 3H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1–6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d−1 for the gill, 2.5±0.3%d−1 for the mantle and 2.6±0.3%d−1 for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day−1. The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

Preserved skeletal muscle protein anabolic response to acute exercise and protein intake in well-treated rheumatoid arthritis patients.

IntroductionRheumatoid arthritis (RA) is often associated with diminished muscle mass, reflecting an imbalance between protein synthesis and protein breakdown. To investigate the anabolic potential of both exercise and nutritional protein intake we investigated the muscle protein synthesis rate and anabolic signaling response in patients with RA compared to healthy controls.MethodsThirteen RA patients (age range 34–84 years; diagnosed for 1–32 years, median 8 years) were individually matched with 13 healthy controls for gender, age, BMI and activity level (CON). Plasma levels of C-reactive protein (CRP), interleukin (IL)-6 and tumor necrosis factor (TNF)-α were measured using enzyme-linked immunosorbent assay (ELISA) in resting blood samples obtained on two separate days. Skeletal muscle myofibrillar and connective tissue protein fractional synthesis rate (FSR) was measured by incorporation of the amino acid 13C6-phenylalanine tracer in the overnight fasted state for 3 hours (BASAL) and 3 hours after intake of whey protein (0.5 g/kg lean body mass) alone (PROT, 3 hrs) and in combination with knee-extensor exercise (EX) with one leg (8 × 10 reps at 70 % of 1RM; PROT + EX, 3 hrs). Expression of genes related to inflammatory signaling, myogenesis and muscle growth/atrophy were analyzed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).ResultsCRP was significantly higher in the RA patients (2.25 (0.50) mg/l) than in controls (1.07 (0.25) mg/l; p = 0.038) and so was TNF-α (RA 1.18 (0.30) pg/ml vs. CON 0.64 (0.07) pg/ml; p = 0.008). Muscle myofibrillar protein synthesis in both RA patients and CON increased in response to PROT and PROT + EX, and even more with PROT + EX (p < 0.001), with no difference between groups (p > 0.05). The gene expression response was largely similar in RA vs. CON, however, expression of the genes coding for TNF-α, myogenin and HGF1 were more responsive to exercise in RA patients than in CON.ConclusionsThe study demonstrates that muscle protein synthesis rate and muscle gene expression can be stimulated by protein intake alone and in combination with physical exercise in patients with well-treated RA to a similar extent as in healthy individuals. This indicates that moderately inflamed RA patients have maintained their muscle anabolic responsiveness to physical activity and protein intake.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0758-3) contains supplementary material, which is available to authorized users.

Rapamycin does not prevent increases in myofibrillar or mitochondrial protein synthesis following endurance exercise.

The present study aimed to investigate the role of the mechanistic target of rapamycin complex 1 (mTORC1) in the regulation of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis following endurance exercise. Forty-two female C57BL/6 mice performed 1 h of treadmill running (18 m min(-1) ; 5° grade), 1 h after i.p. administration of rapamycin (1.5 mg · kg(-1) ) or vehicle. To quantify skeletal muscle protein fractional synthesis rates, a flooding dose (50 mg · kg(-1) ) of l-[ring-(13) C6 ]phenylalanine was administered via i.p. injection. Blood and gastrocnemius muscle were collected in non-exercised control mice, as well as at 0.5, 3 and 6 h after completing exercise (n = 4 per time point). Skeletal muscle MyoPS and MitoPS were determined by measuring isotope incorporation in their respective protein pools. Activation of the mTORC1-signalling cascade was measured via direct kinase activity assay and immunoblotting, whereas genes related to mitochondrial biogenesis were measured via a quantitative RT-PCR. MyoPS increased rapidly in the vehicle group post-exercise and remained elevated for 6 h, whereas this response was transiently blunted (30 min post-exercise) by rapamycin. By contrast, MitoPS was unaffected by rapamycin, and was increased over the entire post-exercise recovery period in both groups (P < 0.05). Despite rapid increases in both MyoPS and MitoPS, mTORC1 activation was suppressed in both groups post-exercise for the entire 6 h recovery period. Peroxisome proliferator activated receptor-γ coactivator-1α, pyruvate dehydrogenase kinase 4 and mitochondrial transcription factor A mRNA increased post-exercise (P < 0.05) and this response was augmented by rapamycin (P < 0.05). Collectively, these data suggest that endurance exercise stimulates MyoPS and MitoPS in skeletal muscle independently of mTORC1 activation.

OR004: Protein Fractional Synthesis Rates (FSR) of Multiple Organs in a Pseudomonas aeruginosa (PM) Induced Hyperdynamic Sepsis Pig Model

Rationale: Survival in sepsis is related to loss of muscle mass and loss of gut mass, contributing to increased translocation of bacteria and sepsis. In clinical relevant models of sepsis, it is unknown in which organs or tissues protein synthesis is reduced in severe sepsis. Therefore we measured protein synthesis of the gut, muscle, liver, and lung in an acute live bacteria (PM) induced hyperdynamic severe sepsis catheterized pig model. Methods: We studied protein synthesis (FSR) of jejunum, jejunal mucosa scraping, ileum, liver, muscle, and lung in 13 pigs with intravenous PM induced hyperdynamic sepsis and 9 healthy controls, using primed-continuous infusion of Phenylalanine (C6) stable isotope. Eighteen hours after start of the sepsis induction, we collected blood samples and tissues. Enrichments of precursor pools and incorporated phenylalanine in protein were analyzed by LC-MS/MS. Data are mean (SE) (in %/hour); stats by unpaired t-test (Prism). Results: We found lower FSR in jejunum and jejunal mucosa in sepsis; Jejunum 0.98%/h (0.08) vs 1.35%/h (0.08) (p = 0.03) and jejunal mucosa 1.53%/h (0.08) vs 1.97%/h (0.1) (p = 0.003). Liver FSR tended to be higher 1.04%/h (0.1) vs 0.85%/h (0.05) (p = 0.091), but FSR in ileum, muscle and lung were not different. Conclusion: Sepsis induced by live bacteria reduces only jejunal, but not ileal, muscle and lung protein synthesis. We hypothesize that nutritional support specifically should aim at improving jejunal protein synthesis.

Leucine improves growth performance of intrauterine growth retardation piglets by modifying gene and protein expression related to protein synthesis

ObjectiveLeucine has been reported to alter the gene expression of proteins, the activation of signaling components, and the fractional rates of protein synthesis in multiple organs of piglets. The aim of this study was to investigate the effects of leucine on molecular mechanisms regulating protein synthesis and degradation in skeletal muscle and determine how these adaptations affect body weight gain in intrauterine growth retardation (IUGR) pigs. MethodsThirty-two weaned piglets were randomly assigned to the following four experimental groups (n = 8 per group): normal birth weight, normal birth weight supplemented with leucine, IUGR, and IUGR supplemented with leucine. Piglets were fed from 14 to 35 d of age. Growth performance, major serum biochemical parameters, and enzyme activities were evaluated. The messenger RNA expression of muscle mammalian target of rapamycin (mTOR), muscle atrophy F-box (MAFbx), and muscle-specific ring finger-1 were investigated. Additionally, total and phosphorylated levels of mTOR and ribosomal protein S6 kinase 1 were measured in longissimus muscle. ResultsAverage daily weight gain and average daily feed intake were increased by leucine in IUGR pigs. At the end of the experiment, IUGR pigs had lower liver and kidney weight compared with the normal piglets. However, IUGR supplemented with leucine decreased serum concentration of urea. Leucine supplementation affected the concentrations of isoleucine, valine, lysine, and phenylalanine in serum. There was no significant difference in the expression of mTOR and muscle-specific ring finger-1 in IUGR piglets, whereas muscle atrophy F-box was reduced only by IUGR dependent of leucine. Compared with the IC group, dietary supplementation with 0.35% l-leucine increased the phosphorylated levels of mTOR and ribosomal S6 kinase 1 in IUGR piglets. ConclusionsThe present study identified a major role for leucine in the activation of the mTOR signaling pathway and reducing muscle atrophy in IUGR piglets, which contributed significantly to differences in body weight gain.

Fast-digestive protein supplement for ten days overcomes muscle anabolic resistance in healthy elderly men

Adequate protein intake is crucial to maintain muscle protein content in elderly subjects, but quality of dietary proteins should be considered. The aim was to determine whether soluble milk protein offers an original strategy to increase muscle anabolism in elderly subjects via a synergistic effect of fast-digesting proteins together with a unique essential AA content. We investigated the effect of a 10-day adequate-protein (AP) or high-protein (HP) diet together with the protein source as caseins (CAS) or soluble milk proteins (PRO) on specific muscle protein fractional synthesis rates (FSRs) in healthy elderly men (71.8±2.4yr, n=31). The isotopic study consisted of two periods of 4h each: a post-absorptive and a postprandial period. The fed state was defined by consumption of either 15g or 30g of PRO or CAS, given fractionally every 20min for 4h. Soluble milk proteins are produced using a membrane process directly from pasteurized milk. Specific muscle protein FSRs were measured during both postabsorptive and postprandial period using a continuous infusion of l-[1-(13)C]leucine. FSR of sarcoplasmic muscle proteins and actin did not increase significantly in the postprandial state compared to postabsorptive state, whereas myosin FSR rate was increased by feeding whatever the protein source in HP groups (0.024±0.005 vs 0.053±0.011%h(-1), P<0.05 and 0.026±0.004 vs 0.050±0.005%h(-1), P<0.004 for PRO HP and CAS HP) but only with the PRO meal in the AP groups (0.031±0.003 vs 0.062±0.009%h(-1), P<0.03 for PRO AP). Mitochondrial muscle protein FSR was also increased by feeding, irrespective of the protein quantity, but only in PRO meal groups (P<0.02). Fast-digesting soluble milk proteins improved postprandial muscle protein synthesis, especially mitochondrial muscle proteins and myosin fractional synthesis rates, in elderly subjects.

25-Hydroxycholecalciferol Enhances Male Broiler Breast Meat Yield through the mTOR Pathway1–3

Background: In recent years, there has been a growing body of evidence indicating that replacing cholecalciferol (vitamin D3) with 25-hydroxycholecalciferol [25(OH)D3] through dietary supplementation enhances breast meat yield in broiler chickens. However, the underlying molecular mechanisms are still unknown.Objective: We investigated the effect of 25(OH)D3 on male broiler growth performance (body weight, feed intake, feed conversion ratio, and breast meat yield), muscle protein synthesis, and the potential underlying molecular mechanisms.Methods: Male Cobb 500 broiler chickens were divided into 4 body weight–matched groups and received a control diet with normal cholecalciferol (2760 IU/kg feed) for 42 d, a diet with high concentrations of cholecalciferol (5520 IU/kg feed) for 42 d, or a diet with 25(OH)D3 (5520 IU/kg feed) for 42 d (HyD-42). A fourth group consumed the HyD-42 for 21 d and then control feed for 21 d (HyD-21) (n = 360 birds, 12 replicates/treatment). Food and clean water were available for ad libitum consumption. At the end of the 42-d experiment, protein turnover was measured by phenylalanine flooding dose. Breast muscle tissues were collected and protein synthesis–related gene and protein expression were measured by real time polymerase chain reaction and Western blot, respectively. Functional studies were performed in vitro with the use of a quail myoblast (QM7) cell line. QM7 cells were treated with 2 doses (1 nM and 10 nM) of cholecalciferol or 25(OH)D3 alone or in combination with 100 nM rapamycin, and cell proliferation was determined by cell proliferation assay. Protein synthesis–related gene and protein expression were also determined.Results: The HyD-42 increased 25(OH)D3 circulating concentrations by 126% (P < 0.05), enhanced breast meat yield (P < 0.05), and increased the fractional rate of protein synthesis by 3-fold (P < 0.05) compared with the control diet. Molecular analyses revealed that breast muscle from chickens consuming the HyD-42 expressed significantly higher concentrations of vitamin D receptor (VDR), phospho mechanistic target of rapamycinSer2481, phospho ribosomal P70 S6 kinase (RPS6K)Thr421/Ser424, and antigen Ki-67 (Ki67) compared with the other groups. In line with the in vivo data, in vitro functional studies showed that cells treated with 25(OH)D3 for 24 h had increased VDR expression, and activated the mechanistic target of rapamycin (mTOR)/S6 kinase (S6K) pathway, enhanced Ki67 protein concentrations, and induced QM7 cell proliferation compared with untreated or cholecalciferol-treated cells. Blocking the mTOR pathway with rapamycin reversed these effects.Conclusion: Taken together, our findings provide evidence that the effects of 25(OH)D3 on male broiler breast muscle are likely mediated through the mTOR-S6K pathway.

Turnover rates of hepatic collagen and circulating collagen-associated proteins in humans with chronic liver disease.

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2–0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.

Application of high-resolution mass spectrometry to measure low abundance isotope enrichment in individual muscle proteins.

Stable isotope-labeled amino acids have long been used to measure the fractional synthesis rate of proteins, although the mass spectrometry platforms used for such analyses have changed throughout the years. More recently, tandem mass spectrometers such as triple quadrupoles have been accepted as the standard platform for enrichment measurement due to their sensitivity and the enhanced specificity offered by multiple reaction monitoring (MRM) experiments. The limit in the utility of such platforms for enrichment analysis occurs when measuring very low levels of enrichment from small amounts of sample, particularly proteins isolated from two-dimensional gel electrophoresis (2D-GE), where interference from contaminant ions impacts the sensitivity of the measurement. We therefore applied a high-resolution orbitrap mass spectrometer to the analysis of [ring-(13)C6]-phenylalanine enrichment in individual muscle proteins isolated with 2D-GE. Comparison of samples analyzed on both platforms revealed that the high-resolution MS has significantly improved sensitivity relative to the triple quadrupole MS at very low-level enrichments due to its ability to resolve interferences in the m/z dimension. At higher enrichment levels, enrichment measurements from the orbitrap platform showed significant correlation (R (2) > 0.5) with those of the triple quadrupole platform. Together, these results indicate that high-resolution MS platforms such as the orbitrap are not only as capable of performing isotope enrichment measurements as the more commonly preferred triple quadrupole instruments, but offer unparalleled advantages in terms of mass accuracy and sensitivity in the presence of similar-mass contaminants.