Myofibrillar Fractional Synthesis Rate Research Articles

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.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Postprandial muscle protein synthesis rate is unaffected by 20-day habituation to a high protein intake: a randomized controlled, crossover trial.

During the last decade more researchers have argued in favor of an increased protein intake for older adults. However, there is a lack of knowledge on the long-term effects of conforming to such a high protein intake with regards to the basal and postprandial muscle protein turnover. The purpose of this study was to compare the postprandial synthesis response in muscle proteins, and the abundance of directly incorporated food-derived amino acids following habituation to high vs. recommended level of protein intake. In a double blinded crossover intervention 11 older male participants (66.6 ± 1.7years of age) were habituated for 20days to a recommended protein (RP) intake (1.1g protein/kg lean body mass (LBM)/day) and a high protein (HP) intake (> 2.1g protein/kg LBM/day). Following each habituation period, intrinsically labelled proteins were ingested as part of a mixed meal to determine the incorporation of meal protein-derived amino acids into myofibrillar proteins. Furthermore, the myofibrillar fractional synthesis rate (FSR) and amino acid kinetics across the leg were determined using gold standard stable isotope tracer methodologies. RT qPCR was used to assess the expression of markers related to muscle proteinsynthesis and breakdown. No impact of habituation was observed on skeletal muscle amino acid or protein kinetics. However, the shunting of amino acids directly from artery to vein was on average 2.9 [Formula: see text]mol/min higher following habituation to HP compared to RP. In older males, habituation to a higher than the currently recommended protein intake did not demonstrate any adaptions in the muscle protein turnover or markers hereof when subjected to an intake of an identical mixed meal. Journal number NCT02587156, Clinicaltrials.org. Date of registration: October 27th, 2015.

Divergent Anabolic Response to Exercise in Young and Older Adult Men-Dependency on Time Frame of Measurement.

Older adults’ skeletal muscle has shown to be less responsive to anabolic stimuli as compared to young both in vitro, in short and controlled in vivo settings and in long-term training studies. However, to translate controlled mechanistic findings to long-term adaptations intermediate measures allowing daily life routines with regard to activity and diet would be useful to evaluate physiological interventions. The purpose of this study was to investigate the exercise effect in young and older adults with 2 independent methods to measure muscle protein synthesis rate. Healthy young and old men were recruited to the study protocol where myofibrillar fractional synthesis rate was measured during 2 days allowing normal activities of daily living with D2O-labeled alanine and during 4 hours in the overnight fasted state with [13C6]phenylalanine infusion. During this period 1 leg completed an exercise session every day (exercise leg) while the contralateral leg was kept inactive (normal leg). Both legs were used for activities of daily living. Two-day myofibrillar fractional synthesis rate was significantly higher in the exercise leg in both young and old as compared to normal leg with no age difference. The 4-hour overnight fasted myofibrillar fractional synthesis rate showed that only young exercise leg was significantly higher than normal leg. The present findings support the notion that anabolic resistance exists in the skeletal muscle of healthy older men when evaluated in controlled settings. However, this response is not as clear when measured during daily life where variance is greater, which calls for further investigations in larger cohorts.

Dose-dependent beneficial effects of citrulline supplementation in short bowel syndrome in rats

ObjectivesSupplementing diet with citrulline has proved an efficient means of preserving nitrogen balance and improving nutritional status after massive intestinal resection. The aim of this study was to model the action of citrulline in gut-resected rats using a dose-ranging study focused on skeletal muscle nitrogen homeostasis. MethodsForty-six rats were randomly assigned to one of the following groups: citrulline 0.5 g·kg·d–1 (n = 9), citrulline 1 g·kg·d–1 (n = 7), citrulline 2.5 g·kg·d–1 (n = 8), citrulline 5 g·kg·d–1 (n = 8), control (n = 6), and sham (n = 8). The sham group underwent transection and the other groups underwent resection of 80% of the small intestine. All rats were then fed enteral nutrition (EN; all diets were isocaloric and isonitrogenous). After 10 d, the rats were sacrificed to measure and analyze animal weight; duodenum, jejunum, and ileum weight; and muscle trophicity. Protein fractional synthesis rate (FSR) and mammalian target of rapamycin complex (mTORC)1 activation were measured in the tibialis muscle. ResultsThere was a significant dose-dependent association between rat weight and citrulline dose up to 2.5 g·kg·d–1 (P = 0.004). There was a significant improvement in tibialis weight correlated to plasma citrulline. Net protein FSR in the tibialis tended to be greater after resection and tended to return to baseline after citrulline supplementation. Citrulline supplementation significantly decreased the activated phosphorylated forms of S6 K1 (P = 0.003) and S6 RP (P = 0.003), with a significant positive association between myofibrillar FSR and activation of S6 K1 (r = 0.614; P = 0.02) and S6 RP (r = 0.601; P = 0.023). Jejunum weight was significantly positively correlated with plasma citrulline (r = 0.319; P = 0.0345). ConclusionCitrulline promotes body weight gain, preserves muscle trophicity, and enhances intestinal adaptation in a dose-dependent manner in a model of resected rats.

An exploration of the methods to determine the protein-specific synthesis and breakdown rates in vivo in humans.

The present study explores the methods to determine human in vivo protein‐specific myofibrillar and collagenous connective tissue protein fractional synthesis and breakdown rates. We found that in human myofibrillar proteins, the protein‐bound tracer disappearance method to determine the protein fractional breakdown rate (FBR) (via 2H2O ingestion, endogenous labeling of 2H‐alanine that is incorporated into proteins, and FBR quantified by its disappearance from these proteins) has a comparable intrasubject reproducibility (range: 0.09–53.5%) as the established direct‐essential amino acid, here L‐ring‐13C6‐phenylalanine, incorporation method to determine the muscle protein fractional synthesis rate (FSR) (range: 2.8–56.2%). Further, the determination of the protein breakdown in a protein structure with complex post‐translational processing and maturation, exemplified by human tendon tissue, was not achieved in this experimentation, but more investigation is encouraged to reveal the possibility. Finally, we found that muscle protein FBR measured with an essential amino acid tracer prelabeling is inappropriate presumably because of significant and prolonged intracellular recycling, which also may become a significant limitation for determination of the myofibrillar FSR when repeated infusion trials are completed in the same participants.

Beta2 -adrenoceptor agonist salbutamol increases protein turnover rates and alters signalling in skeletal muscle after resistance exercise in young men.

Animal models have shown that beta2 -adrenoceptor stimulation increases protein synthesis and attenuates breakdown processes in skeletal muscle. Thus, the beta2 -adrenoceptor is a potential target in the treatment of disuse-, disease- and age-related muscle atrophy. In the present study, we show that a few days of oral treatment with the commonly prescribed beta2 -adrenoceptor agonist, salbutamol, increased skeletal muscle protein synthesis and breakdown during the first 5h after resistance exercise in young men. Salbutamol also counteracted a negative net protein balance in skeletal muscle after resistance exercise. Changes in protein turnover rates induced by salbutamol were associated with protein kinase A-signalling, activation of Akt2 and modulation of mRNA levels of growth-regulating proteins in skeletal muscle. These findings indicate that protein turnover rates can be augmented by beta2 -adrenoceptor agonist treatment during recovery from resistance exercise in humans. The effect of beta2 -adrenoceptor stimulation on skeletal muscle protein turnover and intracellular signalling is insufficiently explored in humans, particularly in association with exercise. In a randomized, placebo-controlled, cross-over study investigating 12 trained men, the effects of beta2 -agonist (6×4mg oral salbutamol) on protein turnover rates, intracellular signalling and mRNA response in skeletal muscle were investigated 0.5-5h after quadriceps resistance exercise. Each trial was preceded by a 4-day lead-in treatment period. Leg protein turnover rates were assessed by infusion of [13 C6 ]-phenylalanine and sampling of arterial and venous blood, as well as vastus lateralis muscle biopsies 0.5 and 5h after exercise. Furthermore, myofibrillar fractional synthesis rate, intracellular signalling and mRNA response were measured in muscle biopsies. The mean (95% confidence interval) myofibrillar fractional synthesis rate was higher for salbutamol than placebo [0.079(95% CI, 0.064 to 0.093) vs. 0.066(95% CI, 0.056 to 0.075%)×h-1 ] (P<0.05). Mean net leg phenylalanine balance 0.5-5h after exercise was higher for salbutamol than placebo [3.6(95% CI, 1.0 to 6.2nmol)×min-1 ×100 gLeg Lean Mass-1 ] (P<0.01). Phosphorylation of Akt2, cAMP response element binding protein and PKA substrate 0.5 and 5h after exercise, as well as phosphorylation of eEF2 5h after exercise, was higher (P<0.05) for salbutamol than placebo. Calpain-1, Forkhead box protein O1, myostatin and Smad3 mRNA content was higher (P<0.01) for salbutamol than placebo 0.5h after exercise, as well as Forkhead box protein O1 and myostatin mRNA content 5h after exercise, whereas ActivinRIIB mRNA content was lower (P<0.01) for salbutamol 5h after exercise. These observations suggest that beta2 -agonist increases protein turnover rates in skeletal muscle after resistance exercise in humans, with concomitant cAMP/PKA and Akt2 signalling, as well as modulation of mRNA response of growth-regulating proteins.

Accounting for non‐steady state conditions during long‐term deuterium oxide‐based measurement of protein turnover

IntroductionMost isotope calculations to measure synthesis rates require steady state conditions. In skeletal muscle, steady state refers to a period of time in which there is neither loss or gain of protein. Deuterium oxide (D2O) is a stable isotopic tracer that can measure protein synthesis over long periods of time. This is advantageous when measuring changes in protein synthesis in response to long‐term interventions such as exercise training or pharmacologic interventions. However, prolonging the period of measurement increases the likelihood of non‐steady state conditions. The purpose of this study was to demonstrate that non‐steady state conditions such as growth can change interpretation of tracer data and validate a model that accounts for non‐steady state conditions when assessing muscle protein synthesis. We hypothesized that use of the appropriate model can account for non‐steady state conditions when measuring muscle protein synthesis.MethodsTo demonstrate the potential implications of non‐steady state conditions, we used D2O to measure skeletal muscle protein synthesis over two weeks in three month old male Sprague Dawley rats with access to running wheels during a period of growth. Then we established a model to account for non‐steady state conditions and validated the model in cultured C2C12 myoblasts and myotubes during periods of cell growth and atrophy. Finally, we applied this model in vivo by measuring protein synthesis in four month old male CB6F1 mice with steady weight or weight loss over a month.ResultsDuring a period of growth, active rats gained less body mass than their sedentary counterparts. There was a significant (p&lt;0.05) positive relation between changes in body size and fractional synthesis rates (FSR) (R2 = 0.50, 0.475, 0.50 for myofibrillar, cytosolic, and mitochondrial FSR, respectively) that obscured any exercise‐related increases in protein synthesis. In vitro studies validated the model parameters that rely on a change in protein mass. We grouped mice based on weight maintenance versus weight loss during the period of measurement. We used our modeling parameters to estimate changes in protein synthesis during the steady and non‐steady conditions.ConclusionsNon‐steady state conditions occur with greater likelihood in long‐ versus short‐term protein synthesis studies and should be accounted for. We showed that a period of growth could account for ~50% of all variation in changes in protein synthesis in all subfractions of skeletal muscle, and this growth effect could mask more subtle effects that may be important for hypothesis testing. We have developed a model to account for non‐steady state conditions over long‐term interventions. We applied this model to a one month intervention to account for body mass changes in response to dietary restriction. By applying this model to future long‐term interventions, we can more accurately discern the effects of interventions on protein synthesis in non steady‐state conditions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Even effect of milk protein and carbohydrate intake but no further effect of heavy resistance exercise on myofibrillar protein synthesis in older men.

The responsiveness of older individuals' skeletal muscle to anabolic strategies may be impaired. However, direct comparisons within the same experimental setting are sparse. The aim of this study was to assess the resting and post-resistance exercise muscle protein synthesis rates in response to two types of milk protein and carbohydrate using a unilateral exercise leg model. Twenty-seven older (69 ± 1year, mean ± SE) men were randomly assigned one of three groups: Whey hydrolysate (WH), caseinate (CAS), or carbohydrate (CHO). By applying stable isotope tracer techniques (L-[15N]phenylalanine), the fasted-rested (basal) myofibrillar fractional synthesis rate (FSR) was measured. Hereafter, FSR was measured in the postprandial phase (0.45g nutrient/kg LBM) in both legs, one rested (fed-rest) and one exercised (10 × 8 reps at 70% 1RM; fed-exercise). In addition, the activity of p70S6K and venous plasma insulin, phenylalanine, and leucine concentrations were measured. Insulin, phenylalanine, and leucine concentrations differed markedly after intake of the different study drinks. The basal FSR in WH, CAS, and CHO were 0.027 ± 0.003, 0.030 ± 0.003, and 0.030 ± 0.004%/h, the fed-rested FSR were 0.043 ± 0.004, 0.045 ± 0.003, and 0.035 ± 0.004%/h, and the fed-exercised FSR were 0.041 ± 0.004, 0.043 ± 0.004, and 0.034 ± 0.004%/h, respectively. No significant differences were observed at any state between the groups. Fed-rested- and fed-exercised FSR were higher than basal (P < 0.001). 3h after exercise and feeding, no significant group differences were detected in the activity of p70S6K. Milk protein and carbohydrate supplementation stimulate myofibrillar protein synthesis in older men, with no further effect of heavy resistance exercise within 0-3h post exercise.

Blockade Of Mtor And Erk1/2 Resulted In Attenuated Protein Synthesis Rates In Differentiated C2c12 Myoblasts.

Fractional protein synthesis rates have long been used as in indicator of acute alterations in the anabolic state of various tissues. Through the use of a number of stable and isotopic tracer methodologies, the measurement of fractional synthesis rates (FSR) in vivo has become a staple of skeletal muscle physiology. Through the application of a deuterium oxide tracer, this project sought to measure pharmacological perturbations in fractional synthesis rates in culture in differentiated C2C12 murine myotubes. PURPOSE: To assess myofibrillar protein FSR in differentiated C2C12 murine myotubes following pharmacological inhibition of rapamycin-sensitive (mTOR) or -insensitive (ERK1/2) pathways. Furthermore, how signal transduction through these pathways impact FSR as compared to previous in vivo studies of pharmacological inhibition studies in skeletal muscle. METHODS: C2C12 murine myoblasts were cultured in collagen coated 6 well culture dishes, and grown to 60-70% confluency using a high glucose DMEM growth media (GM). Cultures were transitioned to a differentiation media (DM) upon reaching target confluency. DM was changed daily for 4 days to allow for complete differentiation to myotubes. Cultures were randomly assigned treatment conditions of cell control (CC), rapamycin inhibition (RAPA), ERK1/2 inhibition (ERK), and electrical stimulation (ESTIM). Cultures underwent treatment conditions for 24 hours with a 4% deuterium oxide GM supplement. Analysis was carried out using a gas chromatography mass spectrometer. RESULTS: Fractional rates of protein synthesis were significantly lower in the RAPA (p=0.028) and ERK (p=0.029) groups as compared to CC, with no differences between RAPA and ERK groups (p>0.05). Although statistics were not applied to the ESTIM group due to low sample size, electrical pulse stimulation shows promise for the stimulation of FSR in cultured myotubes. CONCLUSION: Diminished FSR in both RAPA and ERK groups are consistent with previous findings from in vivo rodent studies. These results may indicate comparable alterations in skeletal muscle anabolic signaling in cell culture as well as in vivo rodent models. Further investigations into anabolic signaling mechanisms related to the control of protein synthesis are needed.

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.

Orchidectomy-induced alterations in volumetric bone density, cortical porosity and strength of femur are attenuated by dietary conjugated linoleic acid in aged guinea pigs

Age-related osteoporosis and sarcopenia are ascribed in part to reductions in anabolic hormones. Dietary conjugated linoleic acid (CLA) improves lean and bone mass, but its impact during androgen deficiency is not known. This study tested if CLA would attenuate the effects of orchidectomy (ORX)-induced losses of bone and lean tissue. Male guinea pigs (n=40; 70–72 weeks), were randomized into four groups: 1) SHAM+Control diet, 2) SHAM+CLA diet, 3) ORX+Control diet, 4) ORX+CLA diet. Baseline blood sampling and dual-energy X-ray absorptiometry (DXA) scans were conducted, followed by surgery 4days later with the test diets started 7days after baseline sampling. Serial blood sampling and DXA scans were repeated 2, 4, 8 and 16weeks on the test diets. Body composition and areal BMD (aBMD) of whole body, lumbar spine, femur and tibia were measured using DXA. At week 16, muscle protein fractional synthesis rate (FSR), volumetric BMD (vBMD), microarchitecture and bone strength were assessed. Body weight declined after SHAM and ORX surgery, with slower recovery in the ORX group. Dietary CLA did not affect weight or lean mass, but attenuated gains in fat mass. Lean mass was stable in SHAM and reduced in ORX by 2weeks with whole body and femur bone mineral content (BMC) reduced by 4weeks; CLA did not alter BMC. By week 16 ORX groups had lower free testosterone and myofibrillar FSR, yet higher cortisol, osteocalcin and ionized calcium with no alterations due to CLA. ORX+Control had higher prostaglandin E2 (PGE2) and total alkaline phosphatase compared to SHAM+Control whereas ORX+CLA were not different from SHAM groups. Femur metaphyseal vBMD was reduced in ORX+CTRL with the reduction attenuated by CLA. Femur cortical thickness (Ct.Th.) and biomechanical strength were reduced and cortical porosity (Ct.Po.) elevated by ORX and attenuated by CLA. This androgen deficient model with a sarcopenic-osteoporotic phenotype similar to aging men responded to dietary CLA with significant benefits to femur density and strength.

Anabolic responses to acute and chronic resistance exercise are enhanced when combined with aquatic treadmill exercise.

Aquatic treadmill (ATM) running may simultaneously promote aerobic fitness and enhance muscle growth when combined with resistance training (RT) compared with land-treadmill (LTM) running. Therefore, we examined acute and chronic physiological responses to RT, concurrent RT-LTM, and concurrent RT-ATM. Forty-seven untrained volunteers (men: n = 23, 37 ± 11 yr, 29.6 ± 4.6 kg/m(2); women: n = 24, 38 ± 12 yr, 27.53 ± 6.4 kg/m(2)) from the general population were tested for V̇o2max, body composition, and strength before and after training. All groups performed 12 wk of RT (2 wk, 3 × 8-12 sets at 60 to approximately 80% 1-repetition maximum). The RT-LTM and RT-ATM groups also performed 12 wk of LTM or ATM training (2 wk immediately post-RT and 1 wk in isolation, 60-85% V̇o2max, 250-500 kcal/session). Additionally, 25 subjects volunteered for muscle biopsy prior to and 24 h post-acute exercise before and after training. Stable isotope labeling (70% (2)H2O, 3 ml/kg) was utilized to quantify 24 h post-exercise myofibrillar fractional synthesis rates (myoFSR). Mixed-model ANOVA revealed that RT-ATM but not RT-LTM training produced greater chronic increases in lean mass than RT alone (P < 0.05). RT-LTM training was found to elicit the greatest decreases in percent body fat (-2.79%, P < 0.05). In the untrained state, acute RT-ATM exercise elicited higher 24-h myoFSRs compared with RT (+5.68%/day, P < 0.01) and RT-LTM (+4.08%/day, P < 0.05). Concurrent RT-ATM exercise and training elicit greater skeletal muscle anabolism than RT alone or RT-LTM.

Differential regulation of myofibrillar and mitochondrial protein synthesis following acute endurance exercise (702.3)

Endurance exercise leads to rapid transcriptional‐ and translational‐mediated remodeling in skeletal muscle. The purpose of this study was to investigate the role of the mechanistic target of rapamycin (mTORC1) in the molecular regulation of myofibrillar and mitochondrial protein synthesis in the first 6h after endurance exercise. Female C57BL/6 mice performed one‐hour of treadmill running after administration of rapamycin or vehicle. A flooding dose of L‐[ring‐13C6]phenylalanine was administered via IP injection and blood and gastrocnemius muscle collected 0.5, 3 and 6h post exercise and in a separate, non‐exercising control group (n=4/time). Skeletal muscle was analyzed for phenylalanine incorporation into the myofibrillar and mitochondrial pools via GC‐C‐IRMS. Myofibrillar fractional synthesis rate (FSR) increased rapidly in the vehicle group post‐exercise and remained elevated at all post‐exercise times. Rapamycin had no effect on basal FSR, but attenuated the rise in myofibrillar protein FSR at 0.5 and 3h post exercise. Interestingly, rapamycin did not affect mitochondrial protein FSR, which was increased at 3h and 6h post exercise in both groups. Taken together our data suggest that the initial increase (0‐3h) in myofibrillar FSR after endurance exercise is mTORC1 mediated, whereas at latter time points (3‐6h) myofibrillar and mitochondrial FSR are both stimulated, independent of mTORC1.

Human myofibrillar fractional breakdown rate determined by the disappearance rate of water‐derived 2H alanine

We validate a newly developed method to determine the protein fractional breakdown rate (FBR) in a human setting. 19 males ingested a dose of 2H2O at day0. From 6 subjects frequent blood samples and muscle biopsies were obtained over 90 days to follow the tracer appearance. 8 subjects had 15N‐phe infused on day70 and had bilateral muscle biopsies taken at day80 and day94 to calculate the FBR over the 14‐d period based on both 2H‐ala and 15N‐phe tracers. At day94, ring‐13C6‐phe was infused to obtain comparable measures of the fractional synthesis rate (FSR). 5 subjects (preliminary) exercised one leg from day80–90 where they also had bilateral biopsies taken to calculate FBR in the sedentary and exercised muscles. At day90 they received an infusion of 1,2‐13C2‐leu to obtain measures of also the FSR. The intake of 2H2O and the infusion of 15N‐phe resulted in sufficient labeling of muscle proteins of 2H‐ala and 15N‐phe, respectively, but only free 2H‐ala had dropped to negligible levels during the period of FBR‐measurement. The myofibrillar protein FBR was 0.06±0.005 %/h with no difference between legs (p&gt;;0.05) and approximately similar to the myofibrillar protein FSR 0.08±0.01 %/h (p&lt;0.10). Preliminary results reveal that frequent resistance training did not affect the myofibrillar protein FBR. The 2H2O‐approach seems capable in humans to determine the muscle FBR and reveal results comparable to short‐term FSR.

Flood‐primed continuous tracer infusion complies with the precursor‐labeling assumptions for measuring the muscle fractional synthesis rate using a single biopsy approach

In certain clinical settings it is allowed only to obtain one single tissue biopsy. Therefore, we developed a tracer administration approach that complies with assumptions for determining the fractional synthesis rate (FSR) by obtaining only one biopsy at the end of the infusion.12 males were recruited: 7 were given a flood‐primed (8.96 mmol, enriched 12.5% tracer:tracee ratio (TTR)) continuous (8 μmol/kg lean body mass (LBM)/h) infusion of ring‐13C6‐phe and muscle biopsies were obtained after 10, 30, 60, 120, and 360 min. 5 men were given a primed (9 μmol/kg LBM) continuous (9 μmol/kg LBM/h) infusion of 1,2‐13C2‐leu. After 120 and 300 min muscle biopsies were obtained to determine FSR. Hereafter, a flood‐primed continuous infusion of phe was given to mimic the protocol and a third muscle biopsy was obtained at 480 min to determine FSR for 3 h following the flood‐prime. Muscle free phe enrichment were up at steady state level from 10‐min after the flood‐prime: 6.98±0.70%, 6.87±0.71%, 6.42±0.58%, 6.58±0.70%, and 6.23±0.81% TTR at 10, 30, 60, 120, and 360 min, respectively. Basal myofibrillar FSR was 0.08±0.01 %/h and was unaffected by the flood‐primed continuous infusion of phe (0.07±0.01 %/h, p&gt;;0.05). The flood‐prime immediate introduced the phe tracer into the intramuscular pools and the continuous infusion kept the enrichment steady without affecting the muscle FSR. Funding: Danish Council of Independent Research.

The anabolic potential of dietary protein intake on skeletal muscle is prolonged by prior light-load exercise

Hyperaminoacidemia stimulates myofibrillar fractional synthesis rate (myoFSR) transiently in resting skeletal muscle. We investigated whether light-load resistance exercise can extent this responsiveness. Ten healthy males exercised one leg with a light-load resistance-like exercise at 16% of 1 repetition maximum and received oral protein boluses every hour for a 10-h period. Their myoFSR was determined by [1-(13)C]-leucine incorporation. Muscle biopsies were obtained from the resting (REST) and exercised (EXC) muscles every 2.5-h in the protein-fed period. Protein feeding significantly elevated plasma leucine and essential amino acids by an average of 39±9% (mean±SEM) and 20±4%, respectively, compared to the basal concentrations: 197±12μmolL(-1) and 854±35μmolL(-1), respectively. The myoFSR was similar in EXC and REST muscles in the first 8h (all time intervals p>0.05). After 8h the myoFSR dropped in the REST muscle to 0.041±0.005%·h(-1), which was 65±5% of the rate in EXC leg at the same time point (0.062±0.004%·h(-1)) and 80±14% of the level in REST leg from 0.5 to 8h (0.056±0.005%·h(-1)) (interaction p<0.05). Compared to rest, light-load exercise prolonged the stimulatory effect of dietary protein on muscle biosynthesis providing perspectives for a muscle restorative effect in clinical settings where strenuous activity is intolerable.

Concurrent resistance and aerobic exercise stimulates both myofibrillar and mitochondrial protein synthesis in sedentary middle-aged men

We determined myofibrillar and mitochondrial protein fractional synthesis rates (FSR), intramuscular signaling protein phosphorylation, and mRNA expression responses after isolated bouts of resistance exercise (RE), aerobic exercise (AE), or in combination [termed concurrent exercise (CE)] in sedentary middle-aged men. Eight subjects (age = 53.3 ± 1.8 yr; body mass index = 29.4 ± 1.4 kg·m(2)) randomly completed 8 × 8 leg extension repetitions at 70% of one repetition-maximum, 40 min of cycling at 55% peak aerobic power output (AE), or (consecutively) 50% of the RE and AE trials (CE). Biopsies were obtained (during a primed, constant infusion of l-[ring-(13)C(6)]phenylalanine) while fasted, and at 1 and 4 h following postexercise ingestion of 20 g of protein. All trials increased mitochondrial FSR above fasted rates (RE = 1.3-fold; AE = 1.5; CE = 1.4; P < 0.05), although only CE (2.2) and RE (1.8) increased myofibrillar FSR (P < 0.05). At 1 h postexercise, phosphorylation of Akt on Ser(473) (CE = 7.7; RE = 4.6) and Thr(308) (CE = 4.4; RE = 2.9), and PRAS40 on Thr(246) (CE = 3.8; AE = 2.5) increased (P < 0.05), with CE greater than AE for Akt Ser(473)-Thr(308) and greater than RE for PRAS40 (P < 0.05). Despite increased phosphorylation of Akt-PRAS40, phosphorylation of mammalian target of rapamycin (Ser(2448)) remained unchanged (P > 0.05), while rpS6 (Ser(235/236)) increased only in RE (10.4) (P < 0.05). CE and AE both resulted in increased peroxisome proliferator receptor-γ coactivator 1-α (PGC1α) expression at 1 h (CE = 2.9; AE = 2.8; P < 0.05) and 4 h (CE = 2.6; AE = 2.4) and PGC1β expression at 4 h (CE = 2.1; AE = 2.6; P < 0.05). These data suggest that CE-induced acute stimulation of myofibrillar and mitochondrial FSR, protein signaling, and mRNA expression are equivalent to either isolate mode (RE or AE). These results occurred without an interference effect on muscle protein subfractional synthesis rates, protein signaling, or mRNA expression.

Effects of Estrogen Replacement and Lower Androgen Status on Skeletal Muscle Collagen and Myofibrillar Protein Synthesis in Postmenopausal Women

Our aim was to determine synthesis rate of myofibrillar and collagen proteins in 20 postmenopausal women, who were either nonusers (Controls) or users of estrogen replacement therapy (ERT) after hysterectomy/oophorectomy. Myofibrillar and muscle collagen protein fractional synthesis rate (FSR) were determined in a nonexercised leg and 24 hours after exercise in the contralateral leg. A significant interaction between treatment and mechanical loading was observed in myofibrillar protein FSR. At rest, myofibrillar protein FSR was found to be lower in ERT users than in Controls. Exercise enhanced myofibrillar protein FSR only in ERT users. Similarly, muscle collagen FSR tended to be lower in ERT users compared with Controls. In ERT participants, the androgen profile was reduced, whereas estradiol and sex hormone-binding globulin were higher. In conclusion, at rest, myofibrillar protein FSR was lower in hysterectomized/oophorectomized women using ERT compared with healthy postmenopausal women. Nevertheless, resistance exercise in combination with ERT seems to have a counteracting effect on myofibrillar FSR in hysterectomized/oophorectomized women.

Acute resistance exercise augments integrative myofibrillar protein synthesis

The purpose of this study was to determine whether an acute bout of high-intensity resistance exercise (RE) would augment integrative mixed muscle and myofibrillar protein fractional synthesis rates (FSRs) when total energy and macronutrient intake was controlled. Twelve healthy young men were studied over 24 hours and performed an acute bout of exhaustive (5 sets until volitional failure of their 85% 1-repetition maximum) unilateral leg press and knee extension exercise, such that one leg was exercised (EX) and the other served as a control (CON). 2H 2O (70%) was provided to measure mixed muscle and myofibrillar FSR, and muscle biopsies (vastus lateralis) were collected from the EX and CON legs 16 hours following the RE session. 2H-labeling of body water over the course of the experiment was 0.32 ± 0.01 mole percent excess. Interestingly, integrative mixed muscle FSR (percent per hour) was similar between the CON (0.76% ± 0.08%) and EX (0.69% ± 0.06%) legs. In contrast, upon determination of myofibrillar FSR, there was an RE effect (EX, 0.94% ± 0.16% vs CON, 0.75% ± 0.08%; P < .05). High-intensity RE without prior training impacts integrative myofibrillar 24-hour FSR, perhaps without altering total responses.