Regulation Of Muscle Protein Synthesis Research Articles

Rapamycin and mTORC2 inhibition synergistically reduce contraction-stimulated muscle protein synthesis.

Muscle contractions increase protein synthesis in a mechanistic target of rapamycin (mTOR)-dependent manner, yet it is unclear which/how mTOR complexes regulate muscle protein synthesis. We investigated the requirement of mTOR Complex 2 (mTORC2) in contraction-stimulated muscle protein synthesis. mTORC2 inhibition by muscle-specific Rictor knockout (Rictor mKO) did not prevent contraction-induced muscle protein synthesis. Rapamycin prevented contraction-induced muscle protein synthesis in Rictor mKO but not wild-type mice. Protein synthesis increases following muscle contractions. Previous studies have shown that inhibition of the mechanistic target of rapamycin complex 1 (mTORC1) suppresses the early but not late muscle protein synthesis response, while inhibition of both mTORC1 and mTORC2 abolishes the two effects. Therefore, we hypothesized that mTORC2 regulates muscle protein synthesis following muscle contractions. To test this, we investigated the effect of mTORC2 inhibition by mouse muscle-specific Rictor knockout (Rictor mKO) on muscle protein synthesis 3h after contraction. The right gastrocnemius muscles of Rictor mKO and wild-type (WT) mice were isometrically contracted using percutaneous electrical stimulation, while the left gastrocnemius muscles served as controls. Vehicle or the mTORC1 inhibitor rapamycin (1.5mg/kg) was injected intraperitoneally 1h before contraction. Treatment of WT mice with rapamycin and Rictor mKO lowered protein synthesis in general, but the response to contractions was intact 3h after contractions in both conditions. Rapamycin treatment in Rictor mKO mice prevented contraction-stimulated muscle protein synthesis. Notably, signalling traditionally associated with mTORC1 was increased by muscle contractions despite rapamycin treatment. In rapamycin-treated Rictor mKO mice, the same mTORC1 signalling was blocked following contractions. Our results indicate that although neither rapamycin-sensitive mTOR/mTORC1 nor mTORC2 is necessary for contraction-induced muscle protein synthesis, combined inhibition of rapamycin-sensitive mTOR/mTORC1 and mTORC2 synergistically inhibits contraction-induced muscle protein synthesis.

Mechanisms Regulating Muscle Protein Synthesis in CKD.

CKD induces loss of muscle proteins partly by suppressing muscle protein synthesis. Muscles of mice with CKD have increased expression of nucleolar protein 66 (NO66), as do muscle biopsy specimens from patients with CKD or those undergoing hemodialysis. Inflammation stimulates NO66 expression and changes in NF-κB mediate the response. Subtotal nephrectomy created a mouse model of CKD with BUN >80 mg/dl. Crossing NO66flox/flox with MCK-Cre mice bred muscle-specific NO66 (MCK-NO66) knockout mice. Experiments assessed the effect of removing NO66. Muscle-specific NO66 knockout in mice blocks CKD-induced loss of muscle mass and improves protein synthesis. NO66 suppression of ribosomal biogenesis via demethylase activity is the mechanism behind these responses. In muscle cells, expression of NO66, but not of demethylase-dead mutant NO66, decreased H3K4me3 and H3K36me3 and suppressed pre-rRNA expression. Knocking out NO66 increased the enrichment of H3K4me3 and H3K36me3 on ribosomal DNA. In primary muscle cells and in muscles of mice without NO66, ribosomal RNA, pre-rRNA, and protein synthesis all increased. CKD suppresses muscle protein synthesis via epigenetic mechanisms that NO66 mediates. Blocking NO66 could suggest strategies that counter CKD-induced abnormal muscle protein catabolism.

Direct or indirect regulation of muscle protein synthesis by energy status?

Muscle protein synthesis (MPS) is a complex and finely-regulated mechanism that plays a key role in muscle homeostasis. Amino acid bioavailability is widely considered a major driver of MPS regulation via mTOR pathway activation. However, recent results suggest that amino acid bioavailability affects cellular energy status. Whatever the tool used to modulate energy status (amino acid depletion or mild mitochondrial uncoupling), a decrease in cellular energy status decreases MPS, without necessarily involving the mTOR pathway. Here we propose that energy status directly regulates one or several energy-consuming step(s) during MPS. This new paradigm modifies our vision of protein metabolism and raises prospects for new advances in therapeutics.

Energy Deficit Attenuates Muscle Protein Synthetic Responses To Essential Amino Acids

BACKGROUND: Peripheral essential amino acid (EAA) concentrations regulate muscle protein synthesis (MPS). During energy balance, consuming ~9 g EAA doubles postprandial peripheral EAA concentrations and increases resting MPS by nearly 50%. During energy deficit, EAA requirements may be elevated due to a greater carbon skeleton requirement for energy metabolism. We examined if similar or greater increases in peripheral EAA concentrations during energy deficit stimulate the same magnitude increase in MPS as in energy balance. PURPOSE: Determine the effects of peripheral increases in EAA concentrations and the stimulation of MPS during energy balance and deficit. METHODS: Data were derived from two independent controlled studies assessing the effects of consuming high and low doses of EAA on peripheral EAA concentrations and MPS (2H5-phenylanine, direct incorporation method) during energy balance (BAL HIGH/LOW) and energy deficit (DEF HIGH/LOW). In BAL, measures were determined in 8 healthy adults (mean ± SD; 21.4 ± 2 y, 24.6 ± 3.2 kg/m2 ), ingesting in random order, either 4.3 ± 0.0 g or 8.6 ± 0.0 g EAA, separated by 7 d. In DEF, measures followed 5 days of controlled energy deficit (30 ± 4 %) in 19 healthy adults (22.9 ± 5 y, 25.4 ± 2.7 kg/m2), ingesting either 7.8 ± 0.9 g or 23.5 ± 2.6 g EAA, in random order, separated by 14 d. RESULTS: Peak EAA concentrations were 36% higher in DEF HIGH compared to BAL HIGH (2219 ± 470 vs. 1634 ± 320 μmol/L; p<0.05), but not for the low doses. Peak EAA concentrations were higher (p<0.05) in HIGH doses for both energy states compared to LOW doses. Independent of EAA dose, postprandial MPS for BAL (0.78 ± 0.36 %/h) and DEF (0.58 ± 0.15 %/h) were 81% and 26% greater than postabsorptive MPS for BAL (0.43 ± 0.18 %/h) and DEF (0.46 ± 0.24 %/h), respectively (energy-by-fed state, p<0.05). Postprandial MPS was 25% lower in DEF than BAL (energy-by-state, p<0.05). CONCLUSION: Ingesting roughly triple the dose of EAA in DEF than BAL (23.5 g vs. 8.6 g) resulted in marked differences in peripheral concentrations; yet the anabolic stimulus was similar. The reduction in postprandial MPS during DEF, despite ingesting 2-3 times the amount of EAA compared to BAL, suggests that muscle is not the primary target for the greater rise in peripheral EAA. Supported by USAMRDC; authors’ views not official U.S. Army or DoD policy.

Notch Inhibition via GSI Treatment Elevates Protein Synthesis in C2C12 Myotubes.

The role of Notch signaling is widely studied in skeletal muscle regeneration but little is known about its influences on muscle protein synthesis (MPS). The purpose of this study was to investigate whether Notch signaling is involved in the regulation of MPS. C2C12 cells were treated with a γ-secretase inhibitor (GSI), to determine the effect of reduced Notch signaling on MPS and anabolic signaling markers. GSI treatment increased myotube hypertrophy by increasing myonuclear accretion (nuclei/myotube: p = 0.01) and myonuclear domain (myotube area per fusing nuclei: p < 0.001) in differentiating C2C12 cells. GSI treatment also elevated myotube hypertrophy in differentiated C2C12s (area/myotube; p = 0.01). In concert, GSI treatment augmented pmTOR Ser2448 (p = 0.01) and protein synthesis (using SUnSET method) in myotubes (p < 0.001). Examining protein expression upstream of mTOR revealed reductions in PTEN (p = 0.04), with subsequent elevations in pAKT Thr308 (p < 0.001) and pAKT Ser473 (p = 0.05). These findings reveal that GSI treatment elevates myotube hypertrophy through both augmentation of fusion and MPS. This study sheds light on the potential multifaceted roles of Notch within skeletal muscle. Furthermore, we have demonstrated that Notch may modulate the PTEN/AKT/mTOR pathway.

Protein blend and casein supplementations before inactive phase similarly activate mechanistic target of rapamycin signaling in rat skeletal muscle.

During overnight sleep, the longest postabsorptive and inactive phase of the day causes protein catabolism and loss. However, the daytime ingestion of dairy proteins has been shown to stimulate muscle protein synthesis and growth. This study compared the effects of pre-sleep supplementation of a protein blend (PB) composed of micellar casein (MCa) and whey protein (1:1) versus isolate MCa on the plasma levels of branched-chain amino acids (BCAAs) and the activation of the mechanistic target of rapamycin (mTOR) signaling, a critical intracellular pathway involved in the regulation of muscle protein synthesis. After 10 h of fasting during the active phase, rats were fed with a single dose of PB or MCa (5.6 g protein/kg of body mass) by gavage, and samples of blood and gastrocnemius muscle were collected at 30, 90, and 450 min. PB and MCa supplementations induced an increase (~3-fold, P < 0.001) of plasma BCAAs at 30 and 90 min. Most importantly, the stimulatory phosphorylation levels of mTOR and its downstream target p70 ribosomal protein S6 kinase (p70S6K) were similarly higher (~2.5-fold, P < 0.001) 30 and 90 min after MCa and PB. Plasma levels of leucine, isoleucine, valine, and overall BCAAs were correlated with the activation of mTOR (P < 0.001) and p70S6K (P < 0.001). MCa and PB supplementations before the inactive phase of rats resulted in an anabolic milieu in the skeletal muscle by inducing a transient increase in plasma BCAAs and a similar activation of the mTOR/p70S6K axis.

Resistance Exercise-induced Regulation of Muscle Protein Synthesis to Intraset Rest.

We aimed to determine if any mechanistic differences exist between TRD and CLU signaling events associated with muscle anabolism. In randomized crossover trials, eight resistance-trained participants (23 ± 1 yr, 81 ± 4.7 kg, body fat: 18% ± 1.9%; 1 repetition maximum [1RM], 150 ± 9.1 kg) performed an acute bout of CLU (4 sets × (2 × 5) repetitions, 30-s intraset rest, 90-s interset rest) and TRD (4 sets × 10 repetitions, 120-s interset rest) barbell back squats at approximately 70% 1RM with total volume load equated during primed continuous L-[ring-C6]phenylalanine infusions. Blood and muscle biopsy samples were collected at rest and after exercise at 0, 2, and 5 h. There was no difference in postexercise MPS between the CLU and TRD condition (P > 0.05) and no changes in phosphorylation of mTORC1 downstream targets (p70S6K and 4EBP1). Total and phosphorylated yes-associated protein on Ser127 transiently increased (P < 0.01) immediately after exercise (t = 0) in CLU (~2.1-fold) and TRD condition (~2.2-fold). Our results show that CLU is a viable anabolic option by preserving power output with similar MPS stimulation when compared with the TRD condition in trained young adults.

The Importance of mTOR Trafficking for Human Skeletal Muscle Translational Control.

The mechanistic target of rapamycin (mTOR) is a central regulator of muscle protein synthesis, and its activation has long been attributed to its translocation to the lysosome. Here, we present a novel model of mTOR activation in skeletal muscle where the translocation of mTOR and the lysosome toward the cell membrane is a key process in mTOR activation.

PL - 033 A translational model of muscle protein synthetic bioactivity in vitro, ex vivo and in vivo

Objective The aim of this research was the development and validation of a translational model for the evaluation of exercise and nutrient stimulated muscle protein synthesis (MPS). To achieve this overall aim, three primary objectives had to be realised: (i) Development of an in vitro skeletal muscle cell bioassay to measure muscle growth and MPS; (ii) Development of an ex vivo model to evaluate the humoral effect on MPS in response to nutrient feeding and exercise; (iii) Use of a stable isotope technique to evaluate MPS in response to nutrient feeding and exercise in vivo.&#x0D; Methods To develop a novel in vitro skeletal muscle cell bioassay to measure muscle growth and MPS, C2C12 myoblasts were proliferated and subsequently differentiated to myotubes over 8 days in DMEM (2% HS). Changes in cell behavior and adhesion properties were monitored by measuring impedance via interdigitated microelectrodes using the xCELLigence system. MPS was measured by puromycin incorporation using the SUnSET technique, intracellular signalling measured by western blot, and myotube thickness by microscopy. To demonstrate the capability to monitor nutrient regulation of muscle growth, media was conditioned with a known potent regulator of MPS (leucine) in a dose response experiment (0.20 - 2.0 mM). To establish the ability of the bioassay to measure the humoral effect of MPS in response to feeding and exercise, media was conditioned by ex vivo human serum from fasted, rested, fed (protein and isonitrogenous non-essential amino acid (NEAA) control) and post-exercise conditions. To evaluate MPS in response to nutrient feeding and exercise in vivo, acute MPS (5 h) was assessed by measuring stable isotope deuterium oxide (D2O) incorporation into m. vastus lateralis skeletal muscle following consumption of either a Whey Protein (WP) or an isonitrogenous NEAA control combined with resistance exercise in resistance trained males.&#x0D; Results In vitro experiments observed a dose-response effect with a 32 % increase in cell index and a 27 % increase in cell thickness after 2 h in the presence of 2.0 mM leucine when compared with control myotubes. Ex vivo serum following ingestion of NEAA had no effect on protein signalling or MPS whereas WP fed serum significantly increased mTOR, P70S6K and 4E-BP1 phosphorylation (p&lt;0.01, p&lt;0.05) compared to fasted serum. Furthermore, the effect of WP fed serum on protein signalling and MPS was significantly increased (p&lt;0.01, p&lt;0.05) compared to NEAA fed serum. Ex vivo human serum following resistance exercise was also increased MPS (29 %) and phosphorylation of mTOR (6 %), p70S6K (12 %) and 4EBP1 (7 %), compared with resting serum. These ex vivo/in vitro findings translated to the in vivo model as myofibrillar fractional synthetic rates (myoFSR) (Basal 0.068±0.002%h-1 vs. WP 0.084±0.006 %h-1, p=0.033) and absolute synthetic rates (ASR) (Basal 10.34±1.01 vs. WP 13.18±0.71 g.day-1, p=0.026) were increased from basal levels only when resistance exercise was combined with WP ingestion and not the NEAA control (NEAA MPS 0.072±0.004%h-1, NEAA ASR 10.23±0.80 g.day-1). Thus, ingestion of WP in combination with resistance training augments acute MPS responses in resistance trained young men.&#x0D; Conclusions We have developed a translational model of muscle protein synthetic bioactivity using in vitro, ex vivo and in vivo methodologies. We have shown that we can impact MPS in vitro using ex vivo human serum to condition media, that MPS in vitro is differentially regulated by ex vivo serum containing bioactive WP compared to a non-bioactive NEAA control, and that this tranlates for resistance exercise combined with WP in humans when MyoFSR is measured using stable isotope technology. These experiments demonstrate that ex vivo/in vitro experiments translate to the in vivo model and these methods can be used to inform both exercise and nutrient human interventions.

The muscle anabolic effect of protein ingestion during a hyperinsulinaemic euglycaemic clamp in middle-aged women is not caused by leucine alone.

It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We compared the effects of ingesting protein or an amount of leucine equal to that in the protein during a hyperinsulinaemic-euglycaemic clamp (to eliminate potential confounding as a result of differences in the insulinogenic effect of protein and leucine ingestion) on muscle anabolic signalling and protein turnover in 28 women. We found that protein, but not leucine, ingestion increased muscle p-mTORSer2448 and p-p70S6KThr389 , although only protein, and not leucine, ingestion decreased muscle p-eIF2αSer51 and increased muscle protein synthesis. It has been suggested that leucine is primarily responsible for the increase in muscle protein synthesis (MPS) after protein ingestion because leucine uniquely activates the mTOR-p70S6K signalling cascade. We tested this hypothesis by measuring muscle p-mTORSer2448 , p-p70S6KThr389 and p-eIF2αSer51 , as well as protein turnover (by stable isotope labelled amino acid tracer infusion in conjunction with leg arteriovenous blood and muscle tissue sampling), in 28 women who consumed either 0.45g proteinkg-1 fat-free mass (containing 0.0513g leucinekg-1 fat-free mass) or a control drink (n= 14) or 0.0513g leucinekg-1 fat-free mass or a control drink (n= 14) during a hyperinsulinaemic-euglycaemic clamp procedure (HECP). Compared to basal conditions, the HECP alone (without protein or leucine ingestion) suppressed muscle protein breakdown by ∼20% and increased p-mTORSer2448 and p-p70S6KThr389 by >50% (all P< 0.05) but had no effect on p-eIF2αSer51 and MPS. Both protein and leucine ingestion further increased p-mTORSer2448 and p-p70S6KThr389 , although only protein, and not leucine, ingestion decreased (by ∼35%) p-eIF2αSer51 and increased (by ∼100%) MPS (all P< 0.05). Accordingly, leg net protein balance changed from negative (loss) during basal conditions to equilibrium during the HECP alone and the HECP with concomitant leucine ingestion and to positive (gain) during the HECP with concomitant protein ingestion. These results provide new insights into the regulation of MPS by demonstrating that leucine and mTOR signalling alone are not responsible for the muscle anabolic effect of protein ingestion during physiological hyperinsulinaemia, most probably because they fail to signal to eIF2α to initiate translation and/or additional amino acids are needed to sustain translation.

Regulation of muscle protein synthesis in an in vitro cell model using ex vivo human serum.

What is the central question of this study? Can medium conditioned by ex vivo human serum regulate muscle protein synthesis in skeletal muscle cells in vitro? What is the main finding and its importance? This study demonstrates that medium conditioned by ex vivo human serum can regulate muscle protein synthesis in skeletal muscle cells in vitro via the mammalian Target of Rapomycin (mTOR) pathway, and this can be regulated differentially by fed and fasted ex vivo human serum. Human serum embodies the integrated systemic response to any condition or perturbation, which may regulate muscle protein synthesis (MPS). Conditioning of medium with human serum represents a physiologically relevant method of regulating MPS in vitro. The primary purpose of the study was the development of a model using ex vivo human serum to condition medium and regulate MPS in in vitro skeletal muscle cells. Four young healthy men reported to the laboratory after an overnight fast and were fed with 0.33g(kg body mass)-1 whey protein. Blood samples were taken before (Fasted) and 60min postprandial (Fed). Fully differentiated C2C12 skeletal muscle cells were nutrient and serum deprived for 1h and subsequently treated with medium conditioned with Fasted or Fed ex vivo human serum (20%) for 4h. The MPS was measured using the surface sensing of translation technique and activation of mTOR, P70S6K and 4EBP1 by Western blot. Fasted and fed ex vivo human serum increased MPS (P<0.05). Although a strong effect (ƞ2 =0.36) for increased MPS in Fed relative to Fasted was observed, this was not statistically significant (P>0.05). Activation of mTOR, P70S6K and 4EBP1 was significantly increased after treatment with Fed compared with Fasted ex vivo human serum (P<0.05). Here, we developed and optimized the conditions for culture of C2C12 skeletal muscle cells, measurement of MPS and signalling in medium conditioned by ex vivo human serum. Furthermore, the functionality of the model was demonstrated by comparison of the response to medium conditioned by Fasted and Fed ex vivo human serum.

Neuromuscular Electrical Stimulation Combined with Protein Ingestion Preserves Thigh Muscle Mass But Not Muscle Function in Healthy Older Adults During 5 Days of Bed Rest.

Short-term bed rest in older adults is characterized by significant loss in leg lean mass and strength posing significant health consequences. The purpose of this study was to determine in healthy older adults if the daily combination of neuromuscular electrical stimulation and protein supplementation (NMES+PRO) would protect muscle mass and function after 5 days of bed rest. Twenty healthy older adults (∼70 years) were subjected to 5 days of continuous bed rest and were randomized into one of two groups: NMES+PRO (n = 10) or control (CON) (n = 10). The NMES+PRO group received bilateral NMES to quadriceps (40 minutes/session, 3 × /day; morning, afternoon, and evening) followed by an interventional protein supplement (17 g). The CON group received an isocaloric equivalent beverage. Before and after bed rest, vastus lateralis biopsies occurred before and after acute essential amino acid (EAA) ingestion for purposes of acutely stimulating mechanistic target of rapamycin (mTORC1) signaling, a major regulator of muscle protein synthesis, in response to bed rest and NMES+PRO. Baseline (pre and post bed rest) muscle samples were also used to assess myofiber characteristics and gene expression of muscle atrophy markers. Thigh lean mass and muscle function were measured before and after bed rest. Five days of bed rest reduced thigh lean mass, muscle function, myofiber cross-sectional area, satellite cell content, blunted EAA-induced mTORC1 signaling, and increased myostatin and MAFbx mRNA expression. Interestingly, NMES+PRO during bed rest maintained thigh lean mass, but not muscle function. Thigh muscle preservation during bed rest with NMES+PRO may partly be explained by attenuation of myostatin and MAFbx mRNA expression rather than restoration of nutrient-induced mTORC1 signaling. We conclude that the combination of NMES and protein supplementation thrice a day may be an effective therapeutic tool to use to preserve thigh muscle mass during periods of short-term hospitalization in older adults. However this combined intervention was not effective to prevent the loss in muscle function.

Exercise Mode Combined with Essential Amino Acid and Carbohydrate Supplementation Differentially Regulate Skeletal Muscle microRNA

Skeletal muscle microRNAs (myomiR) have been implicated in modulating intracellular regulation of muscle protein synthesis by negative inhibition of the mTORC1 pathway. The acute effects of endurance exercise mode and recovery essential amino acid and carbohydrate (EAA+CHO) nutrition on myomiR expression are not well defined. PURPOSE: Determine the effects of endurance exercise mode, with or without EAA+CHO ingestion on myomiR expression. METHODS: Twenty five adults (mean ± SD; 22 ± 2 y, 82 ± 11 kg, VO2peak 4.0 ± 0.5 L[BULLET OPERATOR]min-1) performed 90 min of metabolically-matched (2.2 ± 0.1 VO2 L[BULLET OPERATOR]m-1) load carriage (LC; performed on a treadmill wearing a vest equal to 30% of individual body mass; load carried 24 ± 3 kg) or cycle ergometry (CE) exercise, during which EAA+CHO (10 g EAA and 46 g CHO) or non-nutritive control (CON) drinks were consumed. Expressions of myomiR were determined using RT-qPCR in muscle samples obtained at rest (PRE), immediately post-exercise (POST) and after 3-hr recovery (REC). RESULTS: Relative to PRE, POST and REC expressions of miR-1-3p, miR-206, miR-208a-5 and miR-499 were lower (P < 0.05) for LC compared to CE, regardless of dietary treatment. Independent of exercise mode, miR-1-3p and miR-208a-5p expression were lower (P < 0.05) after ingesting EAA+CHO compared to CON. Expression of miR-206 was highest for CE+CON than any other treatment (exercise-by-drink, P < 0.05). CONCLUSIONS: These data show that myomiR expression is differentially regulated by endurance exercise mode and EAA+CHO nutrition. Weight bearing exercise downregulates myomiR expression, whereas myomiR expression appears to be upregulated after non-weight bearing exercise. Consuming EAA+CHO attenuated the increase in myomiR expression with non-weight bearing exercise, yet the suppression of myomiR expression with feeding was more pronounced when EAA+CHO were consumed during weight bearing exercise. These findings suggest that combining weight bearing exercise with protein and carbohydrate supplementation may facilitate muscle anabolic adaptations to exercise by lowering mTORC1 inhibition.

Whole Eggs and Egg Whites Ingestion Induce Similar Increases in Muscle Anabolic Signaling Phosphorylation after Resistance Exercise in Trained Young Men

The effect of nutrient‐dense food ingestion immediately after resistance exercise as a nutritional strategy to promote muscle anabolism has not been clearly established. Given that a coordinated anabolic response is necessary for the regulation of muscle protein synthesis with exercise and protein ingestion, our aim was to determine whether egg whites or whole eggs ingestion influenced post‐exercise mechanistic target of rapamycin complex 1 (mTORC1) and mitogen‐activated protein kinase (MAPK) signaling phosphorylation in human skeletal muscle. In crossover trials, 10 healthy resistance‐trained men (21±1 y; 88±3 kg; bodyfat: 16±1%; means±SD) completed lower body resistance exercise (4 sets of 10 repetitions at 80% of 10‐RM for both leg press and leg extensions) before ingesting whole eggs (18 g protein, 17 g fat) or egg whites (18 g protein, 0 g fat) in scrambled form. Muscle biopsies were obtained before exercise and at 2 and 5H after protein ingestion to assess the phosphorylation of candidate anabolic signaling molecules by Western blotting. The phosphorylation of mTORser2448 increased ~34% above fasted at 5H following whole egg and egg white consumption (time effect; P&lt;0.01) with no difference between conditions (P&gt;0.66). Downstream of mTORC1, 4E‐BP1Thr37/46 was elevated above fasted at 2H (~22–44%; P&lt;0.01) and was sustained above fasted at 5H (P&lt;0.01) with no differences between conditions (P&gt;0.14). There was no effect of food ingestion on S6K1Thr389 and eEF2Thr56. The phosphorylation of the MAPK ERK1/2Thr202/Tyr204 was similarly suppressed at 2 and 5H following feeding for the whole egg and egg white conditions (~430–780%; P&lt;0.001). Ingestion of isonitrogenous amounts of whole egg and egg white similarly stimulates anabolic signaling in young trained males. These findings would suggest a similar increase in mRNA translation initiation during recovery from resistance exercise in healthy young men. Identification of the intracellular localization of mTORC1 and its colocalization with positive (e.g. Rheb) and negative (e.g. TSC2) regulators via immunofluorescence is ongoing.

The Role of Rapamycin‐Insensitive mTOR Signaling in the Regulation of Muscle Protein Synthesis in a Rat Model of Resistance Exercise

Resistance exercise (RE) activates signaling by the mammalian target of rapamycin (mTOR), and it has been suggested that rapamycin‐sensitive mTOR signaling is necessary for the increase in muscle protein synthesis (MPS) after RE. However, recent studies have reported that rapamycin does not completely inhibit the RE‐induced increase in MPS, suggesting that rapamycin‐sensitive mTOR‐independent (rapamycin‐insensitive mTOR and mTOR‐independent) mechanisms contribute to this event. The aim of this study was to investigate whether rapamycin‐insensitive mTOR signaling plays a role in the regulation of RE‐induced increase in MPS. The right gastrocnemius muscle of male Sprague‐Dawley rats aged 11 weeks was isometrically contracted via percutaneous electrical stimulation (100Hz, 5 sets of ten 3‐s contractions, 7‐s rest between contractions, 3 min rest between sets), while the left gastrocnemius muscle served as a control. Placebo, allosteric mTOR inhibitor rapamycin, or ATP‐competitive mTOR inhibitor AZD8055 was injected intraperitoneally 1 h before RE. Muscle samples were obtained 1 and 6 h after a bout of RE. RE increased MPS (measured by) at both time points after RE in placebo group. Rapamycin completely inhibited RE‐induced MPS 1 h, but not 6 h, after RE. On the other hand, AZD8055 completely inhibited RE‐induced MPS at both time points after RE. The phosphorylation of p70S6K at Thr389 and 4E‐BP1 at Thr37/46, which are downstream targets of mTORC1, was sensitive to both rapamycin and AZD8055. However, inhibition of 4E‐BP1 phosphorylation was more pronounced by AZD8055. Moreover, only AZD8055 inhibited the phosphorylation of Akt at Ser473, a downstream target of mTORC2. We conclude that RE‐induced increase in MPS is mTOR signaling‐dependent process, and furthermore, both rapamycin‐sensitive and ‐insensitive mTOR signaling regulate this event.Support or Funding InformationThis work was supported by JSPS KAKENHI Grant No. 267028.

Linking Cancer Cachexia-Induced Anabolic Resistance to Skeletal Muscle Oxidative Metabolism.

Cancer cachexia, a wasting syndrome characterized by skeletal muscle depletion, contributes to increased patient morbidity and mortality. While the intricate balance between protein synthesis and breakdown regulates skeletal muscle mass, the suppression of basal protein synthesis may not account for the severe wasting induced by cancer. Therefore, recent research has shifted to the regulation of “anabolic resistance,” which is the impaired ability of nutrition and exercise to stimulate protein synthesis. Emerging evidence suggests that oxidative metabolism can regulate both basal and induced muscle protein synthesis. While disrupted protein turnover and oxidative metabolism in cachectic muscle have been examined independently, evidence suggests a linkage between these processes for the regulation of cancer-induced wasting. The primary objective of this review is to highlight the connection between dysfunctional oxidative metabolism and cancer-induced anabolic resistance in skeletal muscle. First, we review oxidative metabolism regulation of muscle protein synthesis. Second, we describe cancer-induced alterations in the response to an anabolic stimulus. Finally, we review a role for exercise to inhibit cancer-induced anabolic suppression and mitochondrial dysfunction.

The role of myokines in muscle health and disease.

This article updates on the concept that muscle-derived cytokines (myokines) play important roles in muscle health and disease. Interleukin-6 (IL-6) is released from normal skeletal muscle in response to exercise, mediating both anti-inflammatory responses and metabolic adaptations, actions contradictory to the prevailing view that IL-6 is a proinflammatory cytokine that is inducing and propagating disease. The anti-inflammatory effects of IL-6 result from its trans-membrane signalling capability, via membrane-bound receptors, whereas its proinflammatory effects result instead from signalling via the soluble IL-6 receptor and gp130. IL-15 is elevated following exercise, promoting muscle fibre hypertrophy in some circumstances, while inducing fibre apoptosis in others. This functional divergence appears because of variations in expression of IL-15 receptor isoforms. Decorin, a recently described myokine, is also elevated following exercise in normal muscle, and promotes muscle fibre hypertrophy by competitively binding to, and thus inhibiting, myostatin, a negative regulator of muscle protein synthesis. Exercise-induced myostatin downregulation thus promotes muscle fibre growth, prompting recent trials of a biological myostatin inhibitor in inclusion body myositis. Myokines appear to exert diverse beneficial effects, though their mechanistic roles in myositis and other myopathologies remain poorly understood.

Erythropoietin Does Not Enhance Skeletal Muscle Protein Synthesis Following Exercise in Young and Older Adults.

Purpose: Erythropoietin (EPO) is a renal cytokine that is primarily involved in hematopoiesis while also playing a role in non-hematopoietic tissues expressing the EPO-receptor (EPOR). The EPOR is present in human skeletal muscle. In mouse skeletal muscle, EPO stimulation can activate the AKT serine/threonine kinase 1 (AKT) signaling pathway, the main positive regulator of muscle protein synthesis. We hypothesized that a single intravenous EPO injection combined with acute resistance exercise would have a synergistic effect on skeletal muscle protein synthesis via activation of the AKT pathway.Methods: Ten young (24.2 ± 0.9 years) and 10 older (66.6 ± 1.1 years) healthy subjects received a primed, constant infusion of [ring-13C6] L-phenylalanine and a single injection of 10,000 IU epoetin-beta or placebo in a double-blind randomized, cross-over design. 2 h after the injection, the subjects completed an acute bout of leg extension resistance exercise to stimulate skeletal muscle protein synthesis.Results: Significant interaction effects in the phosphorylation levels of the members of the AKT signaling pathway indicated a differential activation of protein synthesis signaling in older subjects when compared to young subjects. However, EPO offered no synergistic effect on vastus lateralis mixed muscle protein synthesis rate in young or older subjects.Conclusions: Despite its ability to activate the AKT pathway in skeletal muscle, an acute EPO injection had no additive or synergistic effect on the exercise-induced activation of muscle protein synthesis or muscle protein synthesis signaling pathways.

Poor maternal nutrition during gestation alters the expression of genes involved in muscle development and metabolism in lambs.

Poor maternal nutrition during gestation can result in reduced muscle mass and increased adiposity of the muscle tissue in the offspring. This can have long-lasting consequences on offspring health and productivity. However, the mechanisms by which poor maternal nutrition affects postnatal muscle development are poorly understood. We hypothesized that poor maternal nutrition during gestation would alter expression of key pathways and genes involved in growth, development, and maintenance of the muscle of lambs. For this study, beginning at d 31 ± 1.3 of gestation, ewes were fed 100 (control), 60 (restricted), or 140% (overfed) of the NRC requirements. Within 24 h of birth, lambs were necropsied and semitendinosus muscle tissue was collected for gene expression analysis. Using RNA sequencing (RNA-seq) across dietary treatment groups, 35 and 10 differentially expressed genes were identified using the and reference annotations, respectively. Maternal overfeeding caused changes in the expression of genes involved in regulating muscle protein synthesis and growth as well as metabolism. Alternately, maternal nutrient restriction affected genes that are involved in muscle cell proliferation and signal transduction. That is, despite a similar phenotype, the genes identified differed between offspring born to restricted- or overfed, ewes indicating that the mechanism for the phenotypic changes in muscle are due to different mechanisms.

Loganin possesses neuroprotective properties, restores SMN protein and activates protein synthesis positive regulator Akt/mTOR in experimental models of spinal muscular atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disease characterized by motor neurons degeneration and muscular atrophy. There is no effective SMA treatment. Loganin is a botanical candidate with anti-inflammatory, anti-oxidant, glucose-lowering and anti-diabetic nephropathy activities. The aim of this study is to investigate the potential protective effects of loganin on SMA using two cellular models, SMN-deficient NSC34 cells and SMA patient fibroblasts, and an animal disease model, SMAο7 mice. In SMN-deficient NSC34 cells, loganin increased cell viability, neurite length, and expressions of SMN, Gemin2, SMN-Gemin2 complex, p-Akt, p-GSK-3β, p-CREB, BDNF and Bcl-2. However, both AG1024 (IGF-1 R antagonist) and IGF-1 R siRNA attenuated the protective effects of loganin on SMN level and cell viability in SMN-deficient NSC34 cells. In SMA patient fibroblasts, loganin up-regulated levels of SMN, FL-SMN2, and Gemins, increased numbers of SMN-containing nuclear gems, modulated splicing factors, and up-regulated p-Akt. Furthermore, in the brain, spinal cord and gastrocnemius muscle of SMAο7 mice, loganin up-regulated the expressions of SMN and p-Akt. Results from righting reflex and hind-limb suspension tests indicated loganin improved muscle strength of SMAο7 mice; moreover, loganin activated Akt/mTOR signal and inhibited atrogin-1/MuRF-1 signal in gastrocnemius muscle of SMAο7 mice. Loganin also increased body weight, but the average lifespan of loganin (20mg/kg/day)-treated SMA mice was 16.80±0.73 days, while saline-treated SMA mice was 10.91±0.96 days. In conclusion, the present results demonstrate that loganin provides benefits to SMA therapeutics via improving SMN restoration, muscle strength and body weight. IGF-1 plays an important role in loganin neuroprotection. Loganin can be therefore a valuable complementary candidate for treatment of neuromuscular diseases via regulation of muscle protein synthesis and neuroprotection.