Myofibrillar Protein Synthesis Research Articles

Collagen Peptide Supplementation during Training Does Not Further Increase Connective Tissue Protein Synthesis Rates.

Protein supplementation increases post-exercise muscle protein synthesis rates and, as such, supports exercise-induced muscle conditioning. Collagen protein has been suggested as the preferred protein source to stimulate muscle connective protein synthesis rates during recovery from exercise. Here we assessed the effects of hydrolyzed collagen peptide supplementation on both myofibrillar as well as muscle connective protein synthesis rates during one week of strenuous resistance exercise training. In a randomized, double-blind, parallel design, 25 young men (24 ± 3 y, 76.9 ± 6.4 kg) were selected to perform one week of intense resistance-type exercise training. Subjects were randomly assigned into two groups receiving either 15 g hydrolyzed collagen peptides (COL) or a non-caloric placebo (PLA) twice daily during the intervention. Subjects were administered deuterated water (2H2O) daily, with blood and skeletal muscle tissue samples being collected prior to and after the intervention to determine daily myofibrillar and muscle connective protein synthesis rates. Post-absorptive plasma glycine, proline, and hydroxyproline concentrations increased following collagen peptide supplementation (p < 0.05) and showed higher levels when compared to the placebo group (p < 0.05). Daily muscle connective protein synthesis rates during the intervention period exceeded myofibrillar protein synthesis rates (1.99 ± 0.38 versus 1.34 ± 0.23 %/d, respectively; p < 0.001). Collagen peptide supplementation did not result in higher myofibrillar or muscle connective protein synthesis rates (1.34 ± 0.19 and 1.97 ± 0.47 %/d, respectively) when compared to the placebo group (1.34 ± 0.27 and 2.00 ± 0.27 %/d, respectively; p > 0.05). Collagen peptide supplementation (2 x 15 g daily) does not increase myofibrillar or muscle connective protein synthesis rates during one week of intense resistance exercise training in young, recreational athletes.

Mitochondrial bioenergetics are not associated with myofibrillar protein synthesis rates.

Mitochondria represent key organelles influencing cellular homeostasis and have been implicated in the signalling events regulating protein synthesis. We examined whether mitochondrial bioenergetics (oxidative phosphorylation and reactive oxygen species (H2O2) emission, ROS) measured in vitro in permeabilized muscle fibres represent regulatory factors for integrated daily muscle protein synthesis rates and skeletal muscle mass changes across the spectrum of physical activity, including free-living and bed-rest conditions: n=19 healthy, young men (26±4years, 23.4±3.3kg/m2) and following 12weeks of resistance-type exercise training: n=10 healthy older men (70±3years, 25.2±2.1kg/m2). Additionally, we evaluated the direct relationship between attenuated mitochondrial ROS emission and integrated daily myofibrillar and sarcoplasmic protein synthesis rates in genetically modified mice (mitochondrial-targeted catalase, MCAT). Neither oxidative phosphorylation nor H2O2 emission were associated with muscle protein synthesis rates in healthy young men under free-living conditions or following 1week of bed rest (both P>0.05). Greater increases in GSSG concentration were associated with greater skeletal muscle mass loss following bed rest (r=-0.49, P<0.05). In older men, only submaximal mitochondrial oxidative phosphorylation (corrected for mitochondrial content) was positively associated with myofibrillar protein synthesis rates during exercise training (r=0.72, P<0.05). However, changes in oxidative phosphorylation and H2O2 emission were not associated with changes in skeletal muscle mass following training (both P>0.05). Additionally, MCAT mice displayed no differences in myofibrillar (2.62±0.22 vs. 2.75±0.15%/day) and sarcoplasmic (3.68±0.35 vs. 3.54±0.35%/day) protein synthesis rates when compared with wild-type mice (both P>0.05). Mitochondrial oxidative phosphorylation and reactive oxygen emission do not seem to represent key factors regulating muscle protein synthesis or muscle mass regulation across the spectrum of physical activity.

Post-absorptive Myofibrillar Protein Synthesis Differs Between Pre- and Post-menopausal Females

Post-absorptive Myofibrillar Protein Synthesis Differs Between Pre- and Post-menopausal Females

Free essential amino acid feeding improves endurance during resistance training via DRP1-dependent mitochondrial remodelling.

Loss of muscle strength and endurance with aging or in various conditions negatively affects quality of life. Resistance exercise training (RET) is the most powerful means to improve muscle mass and strength, but it does not generally lead to improvements in endurance capacity. Free essential amino acids (EAAs) act as precursors and stimuli for synthesis of both mitochondrial and myofibrillar proteins that could potentially confer endurance and strength gains. Thus, we hypothesized that daily consumption of a dietary supplement of nine free EAAs with RET improves endurance in addition to the strength gains by RET. Male C57BL6J mice (9weeks old) were assigned to control (CON), EAA, RET (ladder climbing, 3 times a week), or combined treatment of EAA and RET (EAA+RET) groups. Physical functions focusing on strength or endurance were assessed before and after the interventions. Several analyses were performed to gain better insight into the mechanisms by which muscle function was improved. We determined cumulative rates of myofibrillar and mitochondrial protein synthesis using 2H2O labelling and mass spectrometry; assessed ex vivo contractile properties and in vitro mitochondrial function, evaluated neuromuscular junction (NMJ) stability, and assessed implicated molecular singling pathways. Furthermore, whole-body and muscle insulin sensitivity along with glucose metabolism, were evaluated using a hyperinsulinaemic-euglycaemic clamp. EAA+RET increased muscle mass (10%, P<0.05) and strength (6%, P<0.05) more than RET alone, due to an enhanced rate of integrated muscle protein synthesis (19%, P<0.05) with concomitant activation of Akt1/mTORC1 signalling. Muscle quality (muscle strength normalized to mass) was improved by RET (i.e., RET and EAA+RET) compared with sedentary groups (10%, P<0.05), which was associated with increased AchR cluster size and MuSK activation (P<0.05). EAA+RET also increased endurance capacity more than RET alone (26%, P<0.05) by increasing both mitochondrial protein synthesis (53%, P<0.05) and DRP1 activation (P<0.05). Maximal respiratory capacity increased (P<0.05) through activation of the mTORC1-DRP1 signalling axis. These favourable effects were accompanied by an improvement in basal glucose metabolism (i.e., blood glucose concentrations and endogenous glucose production vs. CON, P<0.05). Combined treatment with balanced free EAAs and RET may effectively promote endurance capacity as well as muscle strength through increased muscle protein synthesis, improved NMJ stability, and enhanced mitochondrial dynamics via mTORC1-DRP1 axis activation, ultimately leading to improved basal glucose metabolism.

The effects of whey, pea, and collagen protein supplementation beyond the recommended dietary allowance on integrated myofibrillar protein synthetic rates in older males: a randomized controlled trial

BackgroundSkeletal muscle mass is determined predominantly by feeding-induced and activity-induced fluctuations in muscle protein synthesis (MPS). Older individuals display a diminished MPS response to protein ingestion, referred to as age-related anabolic resistance, which contributes to the progression of age-related muscle loss known as sarcopenia. ObjectivesWe aimed to determine the impact of consuming higher-quality compared with lower-quality protein supplements above the recommended dietary allowance (RDA) on integrated MPS rates. We hypothesized that increasing total protein intake above the RDA, regardless of the source, would support higher integrated rates of myofibrillar protein synthesis. MethodsThirty-one healthy older males (72 ± 4 y) consumed a controlled diet with protein intake set at the RDA: control phase (days 1–7). In a double-blind, randomized controlled fashion, participants were assigned to consume an additional 50 g (2 × 25g) of whey (n = 10), pea (n = 11), or collagen (n = 10) protein each day (25 g at breakfast and lunch) during the supplemental phase (days 8–15). Deuterated water ingestion and muscle biopsies assessed integrated MPS and acute anabolic signaling. Postprandial blood samples were collected to determine feeding-induced aminoacidemia. ResultsIntegrated MPS was increased during supplemental with whey (1.59 ± 0.11 %/d; P < 0.001) and pea (1.59 ± 0.14 %/d; P < 0.001) when compared with RDA (1.46 ± 0.09 %/d for the whey group; 1.46 ± 0.10 %/d for the pea group); however, it remained unchanged with collagen. Supplemental protein was sufficient to overcome anabolic signaling deficits (mTORC1 and rpS6), corroborating the greater postprandial aminoacidemia. ConclusionsOur findings demonstrate that supplemental protein provided at breakfast and lunch over the current RDA enhanced anabolic signaling and integrated MPS in older males; however, the source of additional protein may be an important consideration in overcoming age-related anabolic resistance.This trial was registered clinicaltrials.gov as NCT04026607.

Investigating the Effect of Rapamycin on Impaired Proteostasis in Aged Skeletal Muscle After Disuse Atrophy

Older individuals undergo loss of muscle mass and strength, which can be compounded during bed rest and extended hospital stays. Adult and aged individuals lose muscle mass at similar rates during periods of disuse; however, only aged skeletal muscle fails to fully recover once activity resumes. Current efforts to facilitate skeletal muscle recovery are aimed at increasing myofibrillar protein synthesis by stimulating mTORC1(mammalian target of rapamycin complex 1), a major regulator of protein synthesis. However, we showed that skeletal muscle in aged rats already has higher rates of myofibrillar protein synthesis and mTORC1 activity than adult rats. We also showed that aged skeletal muscle has an accumulation of insoluble ubiquitinated proteins compared to adult muscle, indicating impaired proteostasis and possibly impaired autophagy degradation. We hypothesized that mTORC1 inhibition via rapamycin administration would reduce protein synthesis and the accumulation of p62 aggregates, facilitating recovery in aged skeletal muscle after disuse. Methods: Adult (10 months) and old (26 months) OKC HET rats were hindlimb unloaded (HU) for 14-days to induce atrophy, followed by subsequent 14 days of reloading (RE) through normal ambulation. Animals were fed either a control or rapamycin-supplemented (42 ppm) diet. To determine bulk protein synthesis, rats were labeled with deuterium oxide (D2O) during weight bearing (WB), HU, and RE, (n=4-6 per time point per group). Finally, to assess the presence of autophagy related protein aggregates we immuno stained for Sequestosome 1/p62, an autophagy cargo receptor protein. Results: Our data showed that myofibrillar protein synthesis rates were elevated in the old muscle compared to the adult ( k = 0.029 1/day ±0.002 vs. 0.039 1/day ±0.002, p&lt;0.0001), and that protein synthesis rates were lower in the old rapamycin cohort (k = 1/day ±0.002 vs. 1/day ±0.003, p&lt;0.0001). Furthermore, p62 puncta was three-fold more abundant (p&lt;0.01) in aged skeletal muscle, compared to the adult muscle. Although the sample size is small, when comparing p62 puncta in the old rapamycin group to the old controls, there was no difference between the cohorts. Conclusion: We show that mTORC1 inhibition reduces protein synthesis in aged skeletal muscle. We also show that aged muscle has more p62 aggregates compared to adult skeletal muscle, indicating that autophagy related targets are not being degraded effciently with age. Further analyses will assess the effects of mTORC1 inhibition on other proteostatic mechanisms in atrophied skeletal muscle of aged rats. NIA Training Grant: 5T32AG052363-04VA I01 BX005592 APS SURF. 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.

Skeletal muscle ULK1 and ULK2 modulate protein synthesis impacting fiber size

Maintenance of skeletal muscle homeostasis is largely influenced by the processes of protein synthesis and degradation. However, the regulation of these processes remains incompletely understood particularly in a tissue or cell type-specific manner. We have previously demonstrated that Unc-51 like autophagy activating kinases 1 and 2 (Ulk1/2) are among the top 5 mostly enriched putative autophagy genes in skeletal muscle when compared to &gt;90 other mouse tissues and cell lines (Fuqua et al., FASEB J, 2019). However, because protein kinases commonly have several downstream targets, here we hypothesized that skeletal muscle ULK1 and ULK2 redundantly modulate other important cellular processes. To begin to address this question, we generated mice with skeletal muscle-specific knockout of ULK1 and ULK2 (i.e., ULK1/2 skmDKO) and studied those at different ages. Muscle basal autophagy flux was impaired in ULK1/2 skmDKO mice. However, contrary to other models of autophagy impairment, which commonly leads to muscle atrophy, adult ULK1/2 skmDKO mice had increased muscle size (15-25%, P≤0.01). Hypertrophy occurred in all fiber types (6-20% increase in fiber diameter, P≤0.05) and in response to an acute (4-week) deficiency of ULK1/2 (via electroporations of plasmids encoding ULK1/2miR), where TA fiber diameter increased by 9% (p&lt;0.05). Further studies using D2O labeling and cellular fractionation revealed that ULK1/2 skmDKO mice have increased myofibrillar protein synthesis rates (~5%/day, p&lt;0.05). Examination of the molecular mechanisms involved suggests that ULK1/2 skmDKO mice have enhanced mTORC1-dependent signaling, particularly in the fed state. Altogether, our findings reveal for the first time that ULK1 and ULK2 collectively stimulate protein degradation and inhibit protein synthesis in skeletal muscle potentially impacting muscle mass and function in conditions of muscle atrophy and hypertrophy. Supported by the University of Iowa Fraternal Order of Eagles Diabetes Research Center (UIowa FOEDRC). 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.

Acute effects of a ketone monoester, whey protein, or their coingestion on mTOR trafficking and protein-protein colocalization in human skeletal muscle.

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their coingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their coingestion on mechanistic target of rapamycin (mTOR)-related protein-protein colocalization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2 ± 4.1 yr) ingested either: 1) 0.36 g·kg-1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET + PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120 and 300 min in the postprandial period for immunofluorescence assessment of protein translocation and colocalization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P < 0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) colocalization at 120 min versus basal; however, the decrease was sustained at 300 min versus basal (P < 0.0001) only in KET + PRO. PRO and KET + PRO increased (Interaction: P < 0.0001) mTOR-Rheb colocalization at 120 min versus basal; however, KET + PRO resulted in a sustained increase in mTOR-Rheb colocalization at 300 min that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) colocalization at 120 and 300 min (Time: P = 0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle.NEW & NOTEWORTHY We explored the effects of a ketone monoester (KET), whey protein (PRO), or their coingestion (KET + PRO) on mTOR-related protein-protein colocalization and intracellular trafficking in human muscle. All treatments decreased TSC2-Rheb colocalization at 120 minutes; however, KET + PRO sustained the decrease at 300 min. Only PRO and KET + PRO increased mTOR-Rheb colocalization; however, the increase at 300 min was greater in KET + PRO. Treatment intake increased mTOR-WGA colocalization, suggesting translocation to the fiber periphery. Ketone bodies influence the spatial regulation of mTOR.

Plant Protein Blend Ingestion Stimulates Postexercise Myofibrillar Protein Synthesis Rates Equivalently to Whey in Resistance-Trained Adults.

Whey protein ingestion is typically considered an optimal dietary strategy to maximize myofibrillar protein synthesis (MyoPS) after resistance exercise. Although single-source plant protein ingestion is typically less effective, at least partly, due to less favorable amino acid profiles, this could theoretically be overcome by blending plant-based proteins with complementary amino acid profiles. We compared the postexercise MyoPS response after the ingestion of a novel plant-derived protein blend with an isonitrogenous bolus of whey protein. Ten healthy, resistance-trained, young adults (male/female: 8/2; age: 26 ± 6 yr; BMI: 24 ± 3 kg·m -2 ) received a primed continuous infusion of L-[ ring - 2 H 5 ]-phenylalanine and completed a bout of bilateral leg resistance exercise before ingesting 32 g protein from whey (WHEY) or a plant protein blend (BLEND; 39.5% pea, 39.5% brown rice, 21.0% canola) in a randomized, double-blind crossover fashion. Blood and muscle samples were collected at rest, and 2 and 4 h after exercise and protein ingestion, to assess plasma amino acid concentrations, and postabsorptive and postexercise MyoPS rates. Plasma essential amino acid availability over the 4 h postprandial postexercise period was ~44% higher in WHEY compared with BLEND ( P = 0.04). From equivalent postabsorptive values (WHEY, 0.042 ± 0.020%·h -1 ; BLEND, 0.043 ± 0.015%·h -1 ) MyoPS rates increased after exercise and protein ingestion (time effect; P < 0.001) over a 0- to 2-h period (WHEY, 0.085 ± 0.037%·h -1 ; BLEND, 0.080 ± 0.037%·h -1 ) and 2- to 4-h period (WHEY, 0.085 ± 0.036%·h -1 ; BLEND, 0.086 ± 0.034%·h -1 ), with no differences between conditions during either period or throughout the entire (0-4 h) postprandial period (time-condition interactions; all P > 0.05). Ingestion of a novel plant-based protein blend stimulates postexercise MyoPS to an equivalent extent as whey protein, demonstrating the utility of plant protein blends to optimize postexercise skeletal muscle reconditioning.

Postabsorptive and postprandial myofibrillar protein synthesis rates at rest and after resistance exercise in women with postmenopause.

Feeding and resistance exercise stimulate myofibrillar protein synthesis (MPS) rates in healthy adults. This anabolic characterization of "healthy adults" has been namely focused on males. Therefore, the purpose of this study was to examine the temporal responses of MPS and anabolic signaling to resistance exercise alone or combined with the ingestion of protein in postmenopausal females and compare postabsorptive rates with young females. Sixteen females [60 ± 7 yr; body mass index (BMI) = 26 ± 12 kg·m-2] completed an acute bout of unilateral resistance exercise before consuming either: a fortified whey protein supplement (WHEY) or water. Participants received primed continuous infusions of L-[ring-13C6]phenylalanine with bilateral muscle biopsies before and after treatment ingestion at 2 h and 4 h in nonexercised and exercised legs. Resistance exercise transiently increased MPS above baseline at 0-2 h in the water condition (P = 0.007). Feeding after resistance exercise resulted in a late phase (2-4 h) increase in MPS in the WHEY condition (P = 0.005). In both conditions, resistance exercise did not enhance the cumulative (0-4 h) MPS response. In the nonexercised leg, MPS did not differ at 0-2 h, 2-4 h, or 0-4 h of the measurement periods (all, P > 0.05). Likewise, there were no changes in the phosphorylation of p70S6K, AMPKα, or total and phosphorylated yes-associated protein on Ser127. Finally, postabsorptive MPS was lower in premenopausal versus postmenopausal females (P = 0.023). Our results demonstrate that resistance exercise-induced changes in MPS are temporally regulated, but do not result in greater cumulative (0-4 h) MPS in postmenopausal women.NEW & NOTEWORTHY An adequate quality and quantity of skeletal muscle is relevant to support physical performance and metabolic health. Muscle protein synthesis (MPS) is an established remodeling marker, which can be hypertrophic or nonhypertrophic. Importantly, protein ingestion and resistance exercise are two strategies that support healthy muscle by stimulating MPS. Our study shows postmenopause modulates baseline MPS that may diminish the MPS response to the fundamental anabolic stimuli of protein ingestion and resistance exercise in older females.

The muscle protein synthetic response following corn protein ingestion does not differ from milk protein in healthy, young adults

Plant-derived proteins are generally believed to possess lesser anabolic properties when compared with animal-derived proteins. This is, at least partly, attributed to the lower leucine content of most plant-derived proteins. Corn protein has a leucine content that is highest among most plant-derived proteins and it even exceeds the levels observed in animal-derived proteins such as whey protein. Therefore, this study aimed to compare muscle protein synthesis rates following the ingestion of 30 g corn protein and a 30 g blend of corn plus milk protein with 30 g milk protein. In a randomized, double blind, parallel-group design, 36 healthy young males (26 ± 4 y) received primed continuous L-[ring-13C6]-phenylalanine infusions and ingested 30 g corn protein (CORN), 30 g milk protein (MILK), or a 30 g proteinblend with 15 g corn plus 15 g milk protein (CORN + MILK). Blood and muscle biopsies were collected for 5 h following protein ingestion to assess post-prandial plasma amino acid profiles and myofibrillar protein synthesis rates. The results show that Ingestion of protein increased myofibrillar protein synthesis rates from basal post-absorptive values in all treatments(P < 0.001). Post-prandial myofibrillar protein synthesis rates did not differ between CORN vs MILK (0.053 ± 0.013 vs 0.053 ± 0.013%∙h−1, respectively; t-test P = 0.90), or between CORN + MILK vs MILK (0.052 ± 0.024 vs 0.053 ± 0.013%∙h−1, respectively; t-test P = 0.92). Ingestion of 30 g corn protein, 30 g milk protein, or a blend of 15 g corn plus 15 g milk protein robustly increases muscle protein synthesis rates in young males. The muscle protein synthetic response to the ingestion of 30 g corn-derived protein does not differ from the ingestion of an equivalent amount of milk protein in healthy, young males. Clinical Trial Registry number. NTR6548 (registration date: 27–06-2017) https://www.trialregister.nl/.

Acute ingestion of a ketone monoester, whey protein, or their co-ingestion in the overnight postabsorptive state elicit a similar stimulation of myofibrillar protein synthesis rates in young males: a double-blind randomized trial

BackgroundKetone bodies may have anabolic effects in skeletal muscle via their capacity to stimulate protein synthesis. Whether orally ingested exogenous ketones can stimulate postprandial myofibrillar protein synthesis (MyoPS) rates with and without dietary protein co-ingestion is unknown. ObjectivesThis study aimed to evaluate the effects of ketone monoester intake and elevated blood β-hydroxybutyrate (β-OHB) concentration, with and without dietary protein co-ingestion, on postprandial MyoPS rates and mechanistic target of rapamycin complex 1 (mTORC1) pathway signaling. MethodsIn a randomized, double-blind, parallel group design, 36 recreationally active healthy young males (age: 24.2 ± 4.1 y; body fat: 20.9% ± 5.8%; body mass index: 23.4 ± 2 kg/m2) received a primed continuous infusion of L-[ring-2H5]-phenylalanine and ingested one of the following: 1) the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET+PRO). Blood and muscle biopsy samples were collected during basal and postprandial (300 min) conditions to assess β-OHB, glucose, insulin, and amino acid concentrations, MyoPS rates, and mTORC1 pathway signaling. ResultsCapillary blood β-OHB concentration increased similarly during postprandial conditions in KET and KET+PRO, with both being greater than PRO from 30 to 180 min (treatment × time interaction: P < 0.001). Postprandial plasma leucine and essential amino acid (EAA) incremental area under the curve (iAUC) over 300 min was greater (treatment: both P < 0.001) in KET+PRO compared with PRO and KET. KET, PRO, and KET+PRO stimulated postprandial MyoPS rates (0–300 min) higher than basal conditions [absolute change: 0.020%/h; (95% CI: 0.013, 0.027%/h), 0.014%/h (95% CI: 0.009, 0.019%/h), 0.019%/h (95% CI: 0.014, 0.024%/h), respectively (time: P < 0.001)], with no difference between treatments (treatment: P = 0.383) or treatment × time interaction (interaction: P = 0.245). mTORC1 pathway signaling responses did not differ between treatments (all P > 0.05). ConclusionsAcute oral intake of a ketone monoester, 10 g whey protein, or their co-ingestion in the overnight postabsorptive state elicit a similar stimulation of postprandial MyoPS rates in healthy young males.This trial was registered at clinicaltrials.gov as NCT04565444 (https://clinicaltrials.gov/study/NCT04565444).

Resistance-only and concurrent exercise induce similar myofibrillar protein synthesis rates and associated molecular responses in moderately active men before and after training.

Aerobic and resistance exercise (RE) induce distinct molecular responses. One hypothesis is that these responses are antagonistic and unfavorable for the anabolic response to RE when concurrent exercise is performed. This thesis may also depend on the participants' training status and concurrent exercise order. We measured free-living myofibrillar protein synthesis (MyoPS) rates and associated molecular responses to resistance-only and concurrent exercise (with different exercise orders), before and after training. Moderately active men completed one of three exercise interventions (matched for age, baseline strength, body composition, and aerobic capacity): resistance-only exercise (RE, n = 8), RE plus high-intensity interval exercise (RE+HIIE, n = 8), or HIIE+RE (n = 9). Participants trained 3 days/week for 10 weeks; concurrent sessions were separated by 3 h. On the first day of Weeks 1 and 10, muscle was sampled immediately before and after, and 3 h after each exercise mode and analyzed for molecular markers of MyoPS and muscle glycogen. Additional muscle, sampled pre- and post-training, was used to determine MyoPS using orally administered deuterium oxide (D2 O). In both weeks, MyoPS rates were comparable between groups. Post-exercise changes in proteins reflective of protein synthesis were also similar between groups, though MuRF1 and MAFbx mRNA exhibited some exercise order-dependent responses. In Week 10, exercise-induced changes in MyoPS and some genes (PGC-1ɑ and MuRF1) were dampened from Week 1. Concurrent exercise (in either order) did not compromise the anabolic response to resistance-only exercise, before or after training. MyoPS rates and some molecular responses to exercise are diminished after training.

Molecular mechanisms underpinning favourable physiological adaptations to exercise prehabilitation for urological cancer surgery.

Surgery for urological cancers is associated with high complication rates and survivors commonly experience fatigue, reduced physical ability and quality of life. High-intensity interval training (HIIT) as surgical prehabilitation has been proven effective for improving the cardiorespiratory fitness (CRF) of urological cancer patients, however the mechanistic basis of this favourable adaptation is undefined. Thus, we aimed to assess the mechanisms of physiological responses to HIIT as surgical prehabilitation for urological cancer. Nineteen male patients scheduled for major urological surgery were randomised to complete 4-weeks HIIT prehabilitation (71.6 ± 0.75 years, BMI: 27.7 ± 0.9 kg·m2) or a no-intervention control (71.8 ± 1.1 years, BMI: 26.9 ± 1.3 kg·m2). Before and after the intervention period, patients underwent m. vastus lateralis biopsies to quantify the impact of HIIT on mitochondrial oxidative phosphorylation (OXPHOS) capacity, cumulative myofibrillar muscle protein synthesis (MPS) and anabolic, catabolic and insulin-related signalling. OXPHOS capacity increased with HIIT, with increased expression of electron transport chain protein complexes (C)-II (p = 0.010) and III (p = 0.045); and a significant correlation between changes in C-I (r = 0.80, p = 0.003), C-IV (r = 0.75, p = 0.008) and C-V (r = 0.61, p = 0.046) and changes in CRF. Neither MPS (1.81 ± 0.12 to 2.04 ± 0.14%·day-1, p = 0.39) nor anabolic or catabolic proteins were upregulated by HIIT (p > 0.05). There was, however, an increase in phosphorylation of AS160Thr642 (p = 0.046) post-HIIT. A HIIT surgical prehabilitation regime, which improved the CRF of urological cancer patients, enhanced capacity for skeletal muscle OXPHOS; offering potential mechanistic explanation for this favourable adaptation. HIIT did not stimulate MPS, synonymous with the observed lack of hypertrophy. Larger trials pairing patient-centred and clinical endpoints with mechanistic investigations are required to determine the broader impacts of HIIT prehabilitation in this cohort, and to inform on future optimisation (i.e., to increase muscle mass).

The anabolic response to protein ingestion during recovery from exercise has no upper limit in magnitude and duration in vivo in humans

The belief that the anabolic response to feeding during postexercise recovery is transient and has an upper limit and that excess amino acids are being oxidized lacks scientific proof. Using a comprehensive quadruple isotope tracer feeding-infusion approach, we show that the ingestion of 100g protein results in a greater and more prolonged (>12 h) anabolic response when compared to the ingestion of 25g protein. We demonstrate a dose-response increase in dietary-protein-derived plasma amino acid availability and subsequent incorporation into muscle protein. Ingestion of a large bolus of protein further increases whole-body protein net balance, mixed-muscle, myofibrillar, muscle connective, and plasma protein synthesis rates. Protein ingestion has a negligible impact on whole-body protein breakdown rates or amino acid oxidation rates. These findings demonstrate that the magnitude and duration of the anabolic response to protein ingestion is not restricted and has previously been underestimated invivo in humans.

One Week of Single-Leg Immobilization Lowers Muscle Connective Protein Synthesis Rates in Healthy, Young Adults.

Short periods of limb immobilization lower myofibrillar protein synthesis rates. Within skeletal muscle, the extracellular matrix of connective proteins is recognized as an important factor determining the capacity to transmit contractile force. Little is known regarding the impact of immobilization and subsequent recovery on muscle connective protein synthesis rates. This study examined the impact of 1 wk of leg immobilization and 2 wk of subsequent ambulant recovery on daily muscle connective protein synthesis rates. Thirty healthy, young (24 ± 5 yr) men were subjected to 7 d of one-legged knee immobilization followed by 14 d of ambulant recovery. Deuterium oxide ingestion was applied over the entire period, and muscle biopsy samples were collected before immobilization, after immobilization, and after recovery to measure muscle connective protein synthesis rates and mRNA expression of key extracellular matrix proteins (collagen I, collagen III), glycoproteins (fibronectin, tenascin-C), and proteoglycans (fibromodulin, and decorin). A two-way repeated-measures (time-leg) ANOVA was used to compare changes in muscle connective protein synthesis rates during immobilization and recovery. During immobilization, muscle connective protein synthesis rates were lower in the immobilized (1.07 ± 0.30%·d -1 ) compared with the nonimmobilized (1.48 ± 0.44%·d -1 ; P < 0.01) leg. When compared with the immobilization period, connective protein synthesis rates in the immobilized leg increased during subsequent recovery (1.48 ± 0.64%·d -1 ; P < 0.01). After recovery, skeletal muscle collagen I, collagen III, fibronectin, fibromodulin, and decorin mRNA expression increased when compared with the postimmobilization time point (all P < 0.001). One week of leg immobilization lowers muscle connective protein synthesis rates. Muscle connective protein synthesis rates increase during subsequent ambulant recovery, which is accompanied by increased mRNA expression of key extracellular matrix proteins.

383 Effect of Feeding Ractopamine to Finisher Lard-Type Mangalica Pigs on Longissimus Muscle Fiber Myosin-Heavy Chain Isoform Expression and Cross-Sectional Area

Abstract Heritage breeds such as Mangalica pigs are gaining popularity in US niche markets and currently sold as high-quality meat due to greater meat quality characteristics. Research has demonstrated that feeding the β-adrenergic agonist ractopamine hydrochloride (RAC), stimulates hypertrophic growth of type-2, fast-twitch glycolytic muscle fibers via mTOR signaling resulting in greater myofibrillar protein synthesis. Therefore, the current study evaluated the effect of feeding RAC to Mangalica pigs during the finisher phase on longissimus muscle fiber myosin heavy-chain (MHC) isoform expression and cross-sectional area. Mangalica pigs were randomly assigned finisher diets for 21 d with 20 ppm (RAC; n = 8) or without (CTL; n = 6). Longissimus muscle samples were collected during carcass fabrication, flash-frozen in liquid nitrogen and stored at -80 °C. Immunofluorescence staining of 5-µm-thick cryosections to identify Type-1 (Developmental Studies Hybridoma Bank (DSHB) antibody Ba-D5), Type-2a (DSHB SC-71), and 2x (DSHB BF-35) MHC positive fibers. Type 2b fibers were determined by subtraction. Fibers in each image (≥ 150) were assessed for fiber type and their cross-sectional area (CSA) at 200X magnification. Data were analyzed as a one-way ANOVA using the GLIMMIX procedure of SAS V9.4. Means were separated with the PDIFF option and declared different when P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.1000. Feeding RAC decreased the density of Type 2a (P = 0.0136) and Type 2b (P = 0.0186) fibers in the longissimus muscle of Mangalica pigs. A tendency for RAC to reduce the density of Type 1, slow-twitch (6.61 vs. 16.14 fiber per mm2; P = 0.0505) fibers and increase the density of primarily oxidative fibers expressing a combination of Type 1 + Type 2a MHC isoforms (10.23 vs. 2.88%; P = 0.0524) was also observed. When analyzed as a proportion of total fibers, RAC reduced the proportion of fibers expressing only Type 2a (11.8 vs. 6.70%; P = 0.0468) and Type 2b (0.19 vs. 1.32%; P = 0.0101) MHC isoforms but increased the percentage of fibers expressing both Type 1 + Type 2a MHC isoforms (4.66 vs. 1.13%; P = 0.0280). Feeding RAC did not impact the density or proportion of fibers expressing Type 2x MHC (P ≥ 0.386). Mean longissimus muscle fiber CSA was not impacted by feeding Mangalica pigs RAC for 21 d (P = 0.4815). The CSA of the smallest and largest fibers nor fiber distribution were influenced by RAC inclusion in the diet of Mangalica pigs (P ≥ 0.484). In conclusion, feeding RAC Mangalica pigs impacted fiber MHC isoform expression in the longissimus muscle without altering fiber CSA. Future studies will explore the impact of RAC on the CSA of the various fiber types as well as the impact on other muscles in the Mangalica pig.

Protein from whole-body crayfish homogenate may be a high supplier of leucine or branched-chain amino acids - A call for validation on genus Procambarus sp.

Essential proteinogenic branched-chain amino acids (BCAA), particularly leucine (Leu) have been investigated for their role in enhancing human myofibrillar protein synthesis and biomedical research on tumor models. However, only a few protein sources in our current food system have high enough BCAA or Leu coefficients (% of total amino acids) to be considered as supplements for food, sport, or biomedical research. Mostly dairy-sourced proteins such as casein and whey or rarely plant source such as maize gluten are typically regarded as the gold standards. This study hypothesized that protein isolates derived from the whole-body homogenate (including the chitinous exoskeleton) of procambarid crayfish might exhibit unusually high BCAA and Leu content. The study provides open-access data on the amino acid compositions of two procambarid crayfish (Procambarus virginalis and P. clarkii), as well as a comparison with casein. The mentioned crayfish species could offer 6.36-7.39g Leu 100g-1 dry matter (at 43-48% protein only). Crayfish whole-body protein isolates exhibit a Leu coefficient (18.41±2.51% of total amino acids) and a BCAA coefficient (28.76±2.39% of total amino acids), which is comparable to or higher than of casein (Leu coefficient 8.65±0.08%; BCAA coefficient 20.03±0.73%). However, it is important to interpret these results with caution, due to the challenges associated with leucine and isoleucine separation, as well as potential interactions within the sample matrices. Hence, international validation of these findings is recommended. NOVELTY STATEMENT: Protein isolates from whole-body homogenate (including chitinous exoskeleton) of P. virginalis and/or P. clarkii are hypothesized to be dense in BCAA and Leu. For potential use in biomedical research or as additives in supplements for BCAA and Leu.

67 Effect of Different Dietary Protein Sources on Skeletal Muscle Growth Characteristics and Satellite Cell Mitotic Activity of Young Piglets

Abstract The neonatal period in pigs is distinguished by rapid growth as a function of extensive protein synthesis. Muscle satellite cells also referred to as stem cells (SC) have an essential role in postnatal hypertrophic muscle growth by increasing myofibrillar protein synthesis potential through fusion with existing myofibers to increase the DNA template available for transcription of these important muscle proteins. Thus, the impact of protein sources and differences in their amino acid availability on SC-mediated hypertrophic growth over time in neonatal pigs is of interest. To evaluate how different dietary protein sources fed to young piglets affect muscle fiber cross-sectional area (CSA), fiber density, and SC proliferative activity, a randomized complete block design experiment was conducted using piglets (n = 123) from 5 different farrowing groups. At birth, piglets were assigned to 1 of 4 treatments consisting of a control group and 3 liquid dietary treatments. The commercial milk replacer-based diets contained: whey only (MLK), MLK + spray dried plasma (SDP), and MLK + soy protein concentrate (SPC). After birth, all piglets were allowed to nurse at least 36-h on the sow to allow colostrum intake, assigned to 1 of 3 dietary treatments, transferred to Rescue Decks, and reared until tissue collection on d 6, 14, 19, and 25. The control group was left in the farrowing crate and nursed by the sow until sampling (SOW). On sampling day, pigs were injected with 5’-bromo-2’-deoxyuridine (BrdU) to label mitotically active cells. One-hour post-injection, pigs were euthanized and longissimus dorsi (LM) and biceps femoris (BF) muscle samples were collected for immunofluorescence microscopy analysis. Cell populations were classified as either mitotically inactive SC (MyoD+:BrdU-; Pax7+:BrdU-; MyoD+:Pax7+:BrdU-) or mitotically active SC (MyoD+:BrdU+; Pax7+:BrdU+; MyoD+:Pax7:BrdU+). Data were analyzed as a 2-way ANOVA using SAS V9.4 PROC GLIMMIX and means were separated using PDIFF at P ≤ 0.05. Tendencies were declared when 0.0501 &amp;lt; P ≤ 0.10. At d 14 and 19, MLK-fed piglets had the least BWG (P ≤ 0.0810). On d 14 and 19, MLK-fed piglet LM fibers had the smallest CSA and greatest density (P ≤ 0.0459) though they were similar in fiber density to SOW piglets on d 14. The MLK-fed piglets exhibited the least density of Pax7+:BrdU+ SC in BF (P = 0.0185) and the greatest density of MyoD+ SC in LM (P = 0.0831) on d 6 as well as the greatest density of MyoD+ SC in LM (P = 0.0551) and proliferative MyoD+:BrdU+ SC in BF (P = 0.0799) on d 19, though these differences did not persist through d 25. Overall, these data indicate that supplementation of different dietary protein sources can impact satellite cell-mediated hypertrophic muscle growth of neonatal piglets though the mechanism will require further investigation.

A single bout of prior resistance exercise attenuates muscle atrophy and declines in myofibrillar protein synthesis during bed-rest in older men.

Impairments in myofibrillar protein synthesis (MyoPS) during bed rest accelerate skeletal muscle loss in older adults, increasing the risk of adverse secondary health outcomes. We investigated the effect of prior resistance exercise (RE) on MyoPS and muscle morphology during a disuse event in 10 healthy older men (65-80years). Participants completed a single bout of unilateral leg RE the evening prior to 5days of in-patient bed-rest. Quadriceps cross-sectional area (CSA) was determined prior to and following bed-rest. Serial muscle biopsies and dual stable isotope tracers were used to determine rates of integrated MyoPS (iMyoPS) over a 7day habitual 'free-living' phase and the bed-rest phase, and rates of acute postabsorptive and postprandial MyoPS (aMyoPS) at the end of bed rest. Quadriceps CSA at 40%, 60% and 80% of muscle length significantly decreased in exercised (EX) and non-exercised control (CTL) legs with bed-rest. The decline in quadriceps CSA at 40% and 60% of muscle length was attenuated in EX compared with CTL. During bed-rest, iMyoPS rates decreased from habitual values in CTL, but not EX, and were significantly different between legs. Postprandial aMyoPS rates increased above postabsorptive values in EX only. The change in iMyoPS over bed-rest correlated with the change in quadriceps CSA in CTL, but not EX. A single bout of RE attenuated the decline in iMyoPS rates and quadriceps atrophy with 5days of bed-rest in older men. Further work is required to understand the functional and clinical implications of prior RE in older patient populations. KEY POINTS: Age-related skeletal muscle deterioration, linked to numerous adverse health outcomes, is driven by impairments in muscle protein synthesis that are accelerated during periods of disuse. Resistance exercise can stimulate muscle protein synthesis over several days of recovery and therefore could counteract impairments in this process that occur in the early phase of disuse. In the present study, we demonstrate that the decline in myofibrillar protein synthesis and muscle atrophy over 5days of bed-rest in older men was attenuated by a single bout of unilateral resistance exercise performed the evening prior to bed-rest. These findings suggest that concise resistance exercise intervention holds the potential to support muscle mass retention in older individuals during short-term disuse, with implications for delaying sarcopenia progression in ageing populations.