Regulation Of Muscle Protein Synthesis Research Articles

Gene structure, expression and function analysis of the MyoD gene in the Pacific white shrimp Litopenaeus vannamei

The Pacific white shrimp Litopenaeus vannamei is a representative species of decapod crustacean and an economically important marine aquaculture species worldwide. However, research on the genes involved in muscle growth and development in shrimp is still lacking. MyoD is recognized as a crucial regulator of myogenesis and plays an essential role in muscle growth and differentiation in various animals. Nonetheless, little information is available concerning the function of this gene among crustaceans. In this study, we identified a sequence of the MyoD gene (LvMyoD) with a conserved bHLH domain in the L. vannamei genome. Phylogenetic analysis revealed that both the overall protein sequence and specific functional sites of LvMyoD are highly conserved with those of other crustacean species and that they are evolutionarily closely related to vertebrate MyoD and Myf5. LvMyoD expression is initially high during early muscle development in shrimp and gradually decreases after 40 days post-larval development. In adults, the muscle-specific expression of LvMyoD was confirmed through RT–qPCR analysis. Knockdown of LvMyoD inhibited the growth of the shrimp in body length and weight. Histological observation and transcriptome sequencing of muscle samples after RNA interference (RNAi) revealed nuclear agglutination and looseness in muscle fibers. Additionally, we observed significant effects on the expression of genes involved in heat shock proteins, myosins, actins, protein synthesis, and glucose metabolism. These findings suggest that LvMyoD plays a critical role in regulating muscle protein synthesis and muscle cell differentiation. Overall, this study highlights the involvement of LvMyoD in myogenesis and muscle growth, suggesting that it is a potentially important regulatory target for shrimp breeding efforts.

Increasing Muscle Mass in Elders through Diet and Exercise: A Literature Review of Recent RCTs.

This study aimed to review the current evidence on the independent and combined effects of diet and exercise and their impact on skeletal muscle mass in the elderly population. Skeletal muscle makes up approximately 40% of total body weight and is essential for performing daily activities. The combination of exercise and diet is known to be a potent anabolic stimulus through stimulation of muscle protein synthesis from amino acids. Aging is strongly associated with a generalized deterioration of physiological function, including a progressive reduction in skeletal muscle mass and strength, which in turn leads to a gradual functional impairment and an increased rate of disability resulting in falls, frailty, or even death. The term sarcopenia, which is an age-related syndrome, is primarily used to describe the gradual and generalized loss of skeletal muscle mass (mainly in type II muscle fibers) and function. Multimodal training is emerging as a popular training method that combines a wide range of physical dimensions. On the other hand, nutrition and especially protein intake provide amino acids, which are essential for muscle protein synthesis. According to ESPEN, protein intake in older people should be at least 1 g/kgbw/day. Essential amino acids, such as leucine, arginine, cysteine, and glutamine, are of particular importance for the regulation of muscle protein synthesis. For instance, a leucine intake of 3 g administered alongside each main meal has been suggested to prevent muscle loss in the elderly. In addition, studies have shown that vitamin D and other micronutrients can have a protective role and may modulate muscle growth; nevertheless, further research is needed to validate these claims. Resistance-based exercise combined with a higher intake of dietary protein, amino acids, and/or vitamin D are currently recognized as the most effective interventions to promote skeletal muscle growth. However, the results are quite controversial and contradictory, which could be explained by the high heterogeneity among studies. It is therefore necessary to further assess the impact of each individual exercise and nutritional approach, particularly protein and amino acids, on human muscle turnover so that more efficient strategies can be implemented for the augmentation of muscle mass in the elderly.

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

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

Mechanotransduction for Muscle Protein Synthesis via Mechanically Activated Ion Channels

Cell mechanotransduction, the ability to detect physical forces and convert them into a series of biochemical events, is important for a wide range of physiological processes. Cells express an array of mechanosensors transducing physical forces into intracellular signaling cascades, including ion channels. Ion channels that can be directly activated by mechanical cues are known as mechanically activated (MA), or stretch-activated (SA), channels. In response to repeated exposures to mechanical stimulation in the form of resistance training, enhanced protein synthesis and fiber hypertrophy are elicited in skeletal muscle, whereas a lack of mechanical stimuli due to inactivity/mechanical unloading leads to reduced muscle protein synthesis and fiber atrophy. To date, the role of MA channels in the transduction of mechanical load to intracellular signaling pathways regulating muscle protein synthesis is poorly described. This review article will discuss MA channels in striated muscle, their regulation, and putative roles in the anabolic processes in muscle cells/fibers in response to mechanical stimuli.

Effects of eccentric, concentric and eccentric/concentric training on muscle function and mass, functional performance, cardiometabolic health, quality of life and molecular adaptations of skeletal muscle in COPD patients: a multicentre randomised trial

BackgroundChronic obstructive pulmonary disease (COPD) is the third cause of death worldwide. COPD is characterised by dyspnoea, limited exercise tolerance, and muscle dysfunction. Muscle dysfunction has been linked to dysregulation between muscle protein synthesis, myogenesis and degradation mechanisms. Conventional concentric cycling has been shown to improve several clinical outcomes and reduce muscle wasting in COPD patients. Eccentric cycling is a less explored exercise modality that allows higher training workloads imposing lower cardio-metabolic demand during exercise, which has shown to induce greater muscle mass and strength gains after training. Interestingly, the combination of eccentric and concentric cycling training has scarcely been explored. The molecular adaptations of skeletal muscle after exercise interventions in COPD have shown equivocal results. The mechanisms of muscle wasting in COPD and whether it can be reversed by exercise training are unclear. Therefore, this study aims two-fold: (1) to compare the effects of 12 weeks of eccentric (ECC), concentric (CONC), and combined eccentric/concentric (ECC/CONC) cycling training on muscle mass and function, cardiometabolic health, physical activity levels and quality of life in severe COPD patients; and (2) to examine the molecular adaptations regulating muscle growth after training, and whether they occur similarly in specific muscle fibres (i.e., I, IIa and IIx).MethodsStudy 1 will compare the effects of 12 weeks of CONC, ECC, versus ECC/CONC training on muscle mass and function, cardiometabolic health, levels of physical activity and quality of life of severe COPD patients using a multicentre randomised trial. Study 2 will investigate the effects of these training modalities on the molecular adaptations regulating muscle protein synthesis, myogenesis and muscle degradation in a subgroup of patients from Study 1. Changes in muscle fibres morphology, protein content, genes, and microRNA expression involved in skeletal muscle growth will be analysed in specific fibre-type pools.DiscussionWe aim to demonstrate that a combination of eccentric and concentric exercise could maximise the improvements in clinical outcomes and may be ideal for COPD patients. We also expect to unravel the molecular mechanisms underpinning muscle mass regulation after training in severe COPD patients.Trial Registry: Deutshches Register Klinischer Studien; Trial registration: DRKS00027331; Date of registration: 12 January 2022. https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00027331.

Initiation of muscle protein synthesis was unrelated to simultaneously upregulated local production of IGF-1 by amino acids in non-proliferating L6 muscle cells.

BackgroundIGF-1 is considered an important regulator of muscle protein synthesis. However, its role in stimulation of muscle protein synthesis by amino acids (AA) is not clear, despite pronounced alterations in IGF-1 mRNA expression and signaling in muscle tissues by feeding. This study evaluates the role of locally produced IGF-1 and IGF-1 signaling when skeletal muscle protein synthesis is activated by increased amino acid availability in confluent, non-proliferating cells.MethodsL6 skeletal muscle cells were subjected to amino acid starvation (24 h, 0.14 mM) followed by 18 h amino acid refeeding in Low AA (0.28 mM) or High AA concentrations (9 mM). Protein synthesis rates were estimated by L-[U-14C]-phenylalanine incorporation into cellular proteins. IGF-1 and IGF-1 receptor mRNA expression were quantified by real time PCR. SiRNA knockdown, antibodies and chemical inhibitors were used to attenuate muscle IGF-1 production and signaling.ResultsHigh AA concentrations (9mM) increased IGF-1 mRNA expression (+ 30%, p<0.05) and increased L-[U-14C]-phenylalanine incorporation compared to Low AA in confluent, non-proliferating muscle cells. Blocking IGF-1 signaling by chemical inhibitors reduced IGF-1 mRNA upregulation (~50%, p< 0.01), without decrease of protein synthesis. SiRNA knockdown of IGF-1 reduced protein synthesis, mainly explained by reduced cell proliferation. High AA or IGF-1 inhibitors did not change IGF-1 receptor mRNA expressions.ConclusionAmino acids increased IGF-1 mRNA expression and stimulated muscle protein synthesis. However, simultaneous upregulation of IGF-1 mRNA did not relate to increased protein synthesis by amino acids. The results indicate that increased IGF-1 mRNA expression is rather a covariate to amino acid initiation of protein synthesis in non-proliferating muscle cells; effects that may be related to unrecognized metabolic activities, such as transport of amino acids.

Essential Amino Acid Regulation of Protein Turnover to Support Skeletal Muscle Health with Age

Skeletal muscle (SM) is vital for both long term health and quality of life. Recent research suggests an increase in catabolic signals with age triggers pathologic conditions, such as sarcopenia. Although results from in vitro studies model how essential amino acids (EAA) can regulate muscle protein synthesis (MPS), the relevance of these models to muscle protein breakdown (MPB), and the presence of physiological plasma amino acid concentrations (PAA) remain to be established. Therefore, the objective of this study was to determine the effects of EAA with (0.2, 1.0, and 3.0 x PAA) in a young (passages 2‐10) and aging (passages 16‐24) C2C12 murine muscle cell model. We hypothesized that increased levels of EAA will increase MPS in aging cells and suppress MPB via the mTORC1 pathway when compared to young cells. Myoblasts were seeded (1x105) into 6‐well plates and differentiated into myotubes when they reached 80% confluency. Myotubes were serum and AA starved for 24 hours before receiving one of the following treatments: control (CON), 0.2 x PAA, 1.0 x PAA, 3.0 x PAA with or without rapamycin (100 nm; RAP) for 1, 6, and 24 hours. All treatments were performed in triplicate and then each experiment was repeated three times, yielding nine wells per treatment. Proteins related to sensing of AA and protein synthesis and degradation were assessed using Western Blot. Rapamycin inhibited phosphorylation of p70 S6 kinase 1 (p70S6K1) and 4E binding protein 1 (4E‐BP1), downstream targets of mTORC1, in both CON and AA‐treated young and aged cells. Phosphorylation of 4EBP1 and p70S6K1 was significantly increased (p&lt;0.05) in both young and aged cells following all PAA treatments. However, there was no significance after 24 hours of AA supplementation in aged cells. Particularly, after 6 hours of treatment all doses significantly increased (p&lt;0.0001) phosphorylation of p70S6K1 in both young and aged cells. 0.2 x PAA and 3.0 x PAA elicited a significantly greater response in 4EBP1 phosphorylation in young cells compared to the CON after 6 hours of treatment, whereas a dose of 3.0 x PAA induced a similar response in aged cells. In conclusion, in a C2C12 myotube model, increasing EAA concentrations downregulates the expression of translation initiation factors in young and aging cells. Specifically, it takes a 3.0 x PAA to elicit a response in aging cells, whereas young cells respond to lower physiological concentrations of 0.2 x PAA and 1.0 x PAA. Therefore, it can be proposed increasing EAA may be effective for adjusting the rate of MPS and MPB via the mTORC1 pathway in both young and aging muscle cells.

Short communication: Leucine, but not muscle contractions, stimulates protein synthesis in isolated EDL muscles from golden geckos

Resistance exercise and protein ingestion stimulate muscle protein synthesis in mammals and the combination of both stimuli exert an additive effect. However, mechanisms regulating muscle mass may be different in ectothermic vertebrates because these animals are adapted to low energy consumption, short bouts of physical activity, and prolonged periods of inactivity. Here, we investigated the effects of administration of leucine and simulated resistance exercise induced by electrical stimulation (ES) on protein synthesis rate in isolated extensor digitorum longus muscle from golden geckos (Gekko badenii). Muscles were placed in Krebs-Ringer buffer equilibrated with O2 (97%) and CO2 (3%) at 30 °C. One muscle from each animal was subjected to one of three interventions: 1) administration of leucine (0.5 mM) at rest, 2) isometric contractions evoked by ES, or 3) a combination of contractions and leucine, while the contralateral muscle served as untreated control. The rate of protein synthesis was measured through pyromycin-labeling. Administration of leucine led to a 2.75 (±1.88)-fold rise in protein synthesis rate in inactive muscles, whereas isometric contractions had no effect (0.67 ± 0.37-fold). The combination of isometric contractions and leucine did not affect protein synthesis rate (1.02 ± 0.34-fold), suggesting that muscle contractions attenuated the positive influence of leucine. Our study identifies leucine as a potent positive regulator of muscle protein synthesis in golden geckos, but also demonstrates that muscle contraction is not. More studies should be conducted in other taxonomic groups of ectothermic vertebrates to identify whether this is a general pattern.

204 Awardee Talk: Recent Advances in Protein Nutrition in Horses

Abstract Protein has been recognized as an essential nutrient for animals for well over 100 years. Protein plays many important structural and metabolic roles, and some of its component amino acids have additional functions, including as regulatory molecules, as energy substrates and in the synthesis of other non-protein molecules. Skeletal muscle makes up approximately 50% of body weight in horses, with protein being the major non-water component. As an athletic species, the development and maintenance of muscle mass is of the utmost importance in horses. Because muscle mass is largely determined by the balance of rates of muscle protein synthesis and breakdown, understanding how these pathways are regulated and influenced by dietary protein and amino acid provision is essential. Historically, much research regarding protein nutrition in horses has focused on the protein digestibility of different feed ingredients, and the adequacy of different protein sources in supporting the growth and maintenance of horses. This presentation will focus on some of the current areas of active research relating to protein nutrition in horses: the activation of the signaling pathways that regulate muscle protein synthesis, amino acid supplementation in athletic horses, protein metabolism in aged and horses and those with insulin dysregulation, and amino acid and protein nutrition in predominantly forage-fed horses. There are many exciting opportunities for future research in the area of protein and amino acid nutrition in horses across the lifespan.

Investigation of niclosamide as a repurposing agent for skeletal muscle atrophy.

Skeletal muscle atrophy is a feature of aging (termed sarcopenia) and various diseases, such as cancer and kidney failure. Effective drug treatment options for muscle atrophy are lacking. The tapeworm medication, niclosamide is being assessed for repurposing to treat numerous diseases, including end-stage cancer metastasis and hepatic steatosis. In this study, we investigated the potential of niclosamide as a repurposing drug for muscle atrophy. In a myotube atrophy model using the glucocorticoid, dexamethasone, niclosamide did not prevent the reduction in myotube diameter or the decreased expression of phosphorylated FOXO3a, which upregulates the ubiquitin-proteasome pathway of muscle catabolism. Treatment of normal myotubes with niclosamide did not activate mTOR, a major regulator of muscle protein synthesis, and increased the expression of atrogin-1, which is induced in catabolic states. Niclosamide treatment also inhibited myogenesis in muscle precursor cells, enhanced the expression of myoblast markers Pax7 and Myf5, and downregulated the expression of differentiation markers MyoD, MyoG and Myh2. In an animal model of muscle atrophy, niclosamide did not improve muscle mass, grip strength or muscle fiber cross-sectional area. Muscle atrophy is also feature of cancer cachexia. IC50 analyses indicated that niclosamide was more cytotoxic for myoblasts than cancer cells. In addition, niclosamide did not suppress the induction of iNOS, a key mediator of atrophy, in an in vitro model of cancer cachexia and did not rescue myotube diameter. Overall, these results suggest that niclosamide may not be a suitable repurposing drug for glucocorticoid-induced skeletal muscle atrophy or cancer cachexia. Nevertheless, niclosamide may be employed as a compound to study mechanisms regulating myogenesis and catabolic pathways in skeletal muscle.

The Role of Coactivator‐associated Arginine Methyltransferase 1 (CARM1) During Sarcopenia

CARM1 is emerging as an important regulator of skeletal muscle plasticity. We recently reported reduced muscle mass, along with aberrant autophagic and atrophic signaling in CARM1 skeletal muscle-specific knockout (mKO) mice. Notably, CARM1 interacts with forkhead box O1 (FOXO1), a master transcriptional regulator of the atrophy program, and knockout of CARM1 in skeletal muscle results in elevated FOXO1 protein content. Although CARM1 appears to be indispensable for preserving muscle mass, the impact of CARM1 deletion during the sarcopenia of aging remains to be elucidated. The purpose of this study was to more comprehensively understand the function of CARM1 during the progressive loss of skeletal muscle mass and strength with age. Survival analysis revealed that mKO mice have a significantly shorter lifespan compared to their wild type (WT) littermates. For instance, the survival rate of mKO and WT animals at 22 months is 17% and 33%, respectively. In order to assess functional and physiological adaptations to aging, grip strength and tibialis anterior (TA) muscle weights were assessed in 3 month (young) and 22 month (old) animals. We detected main effects (p < 0.05) of aging and genotype on forelimb grip strength. In particular, grip strength was significantly lower by 24% in old mKO versus old WT mice. Main effects (p < 0.05) of aging and genotype were also observed for TA muscle weight normalized to body weight. Relative to old WT mice, TA muscle weight was significantly lower by 7% in old mKO animals. We also employed Western blot analyses to assess FOXO1Ser256 activation status (phosphorylated/total), as well as the mammalian target of rapamycin complex 1 (mTORC1)Ser2448, a key regulator of muscle protein synthesis. The phosphorylation status of FOXO1Ser256 was lower (p = 0.07) in old mKO versus old WT mice, suggesting that CARM1 deletion leads to greater FOXO1 nuclear entry and transcription of atrogenes with aging. Compared to old WT animals, phosphorylation levels of mTORC1Ser2448 were also lower (p < 0.05) in old mKO mice, suggesting that CARM1 is important for driving muscle protein synthesis throughout the lifespan. Collectively, these results highlight the importance of CARM1 for combating the progressive and generalized loss of skeletal muscle mass and strength that occurs with age. Manipulating the expression and/or activity of CARM1 in skeletal muscle may offer a viable therapeutic strategy for mitigating sarcopenia.

The Physiological and Metabolic Effects of Maternal Intake of EPA and DHA during Pregnancy and Lactation on Offspring's Muscle Development and Energy Homeostasis

EPA and DHA are long-chain fatty acids with health benefits on offspring underpinning the accumulative recommendations for their prenatal ingestion. Recent in vivo studies have emerged indicating a positive correlation of EPA and DHA with fetal BAT development, while other in vitro trials demonstrated their negative implication in promoting fetal myoblast reprogramming into adipoblasts. Therefore, the objective of this study was to test the effect of maternal EPA and DHA on fetal muscle development and energy balance. Two groups of Female C57BL/6 mice were assigned to different diets throughout the entire period of gestation and lactation; the first group was fed EPA and DHA enriched diet (FA); whereas, the other was fed a diet devoid of n-3 PUFA (CON). Embryos at day 13 of gestation and offspring at age of 1 and 21day were selected for sample collection and processing. No change in little number and body weight were observed between groups in newborns at day1, but a tendency to weigh loss in the FA group was detected after weaning. Transient increase in the expression levels of MYOD1, MYOG, MRF4, and MHC4 were observed at day 1 post-parturition (P<0.05). Except of IGF-1 P<0.05, the expression levels of genes regulating muscle protein synthesis and catabolism were comparable (P>0.05) between groups at day 1 and 21. Persistent increase in ISR expression (P<0.05) but not in GLUT-4 (P>0.05) were detected in day 1 and 21 age offspring. An increase in the expression of PPARγ and AP2 but not C/EBPα (P<0.05) at day 1 were observed. The three key regulators of basal adipogenesis were up-regulated in weaned mice (P<0.05). Up-regulation of PPARγ, AP2, and C/EBPα levels were accompanied by increasing intramuscular fat accumulation in day 1 and 21 samples. Considerable increase in the transcripts of genes regulating lipid catabolism and thermogenesis in liver including Cpt1α,Ehhadh, Mcad, Lcad, Acadvl, slc22a5, slc25a20, and Pparα (P<0.05) was noticed in (FA) group after 21 day of treatment; whereas, only the transcripts of Cpt1α andEhhadh increased in day1 neonates’ livers . Similarly, genes regulating lipolysis (ATGL and HSL, MGL and LPL) were up-regulated at day 21 of treatment only in (FO) group (P<0.05) while EPA and DHA maternal intake does not affect the expression of lipogenesis regulating genes (Fasn and Srebp1c) (P>0.05) in day 1 and 21 offspring. EPA and DHA treatment promoted BAT development and activity through increasing the expression of BAT signature genes including (UCP1, Cidea, Prdm16, Pgc1α, Dio2, Zic1, Fgf21, P2rx5, and PPARα(P<0.05). Also, maternal intake of EPA/ DHA enriched diet enhanced browning of sWAT by stimulating the expression of beige markers such as UCP1, Shox2, Tmem26, and PAT2 and thermogenesis regulating genes (PPARα, PGC1α, Cox7α1, and Cox8β) (P<0.05). Taken together, maternal ingestion of EPA and DHA may be suggested as a therapeutic option to encounter many pathophysiological disorders, mainly childhood obesity, and confer long-lasting metabolic benefits on offspring.

Muscle follistatin gene delivery increases muscle protein synthesis independent of periodical physical inactivity and fasting.

Blocking of myostatin and activins effectively counteracts muscle atrophy. However, the potential interaction with physical inactivity and fasting in the regulation of muscle protein synthesis is poorly understood. We used blockade of myostatin and activins by recombinant adeno-associated virus (rAAV)-mediated follistatin (FS288) overexpression in mouse tibialis anterior muscle. To investigate the effects on muscle protein synthesis, muscles were collected 7days after rAAV-injection in the nighttime or in the daytime representing high and low levels of activity and feeding, respectively, or after overnight fasting, refeeding, or ad libitum feeding. Muscle protein synthesis was increased by FS288 independent of the time of the day or the feeding status. However, the activation of mTORC1 signaling by FS288 was attenuated in the daytime and by overnight fasting. FS288 also increased the amount of mTOR colocalized with lysosomes, but did not alter their localization toward the sarcolemma. This study shows that FS288 gene delivery increases muscle protein synthesis largely independent of diurnal fluctuations in physical activity and food intake or feeding status, overriding the physiological signals. This is important for eg cachectic and sarcopenic patients with reduced physical activity and appetite. The FS288-induced increase in mTORC1 signaling and protein synthesis may be in part driven by increased amount of mTOR colocalized with lysosomes, but not by their localization toward sarcolemma.

Daily protein supplementation attenuates immobilization-induced blunting of postabsorptive muscle mTORC1 activation in middle-aged men.

Disuse-induced muscle atrophy is accompanied by a blunted postprandial response of the mammalian target of rapamycin complex 1 (mTORC1) pathway. Conflicting observations exist as to whether postabsorptive mTORC1 pathway activation is also blunted by disuse and plays a role in atrophy. It is unknown whether changes in habitual protein intake alter mTORC1 regulatory proteins and how they may contribute to the development of anabolic resistance. The primary objective of this study was to characterize the downstream responsiveness of skeletal muscle mTORC1 activation and its upstream regulatory factors, following 14 days of lower limb disuse in middle-aged men (45-60 yr). The participants were further randomized to receive daily supplementation of 20 g/d of protein (n = 12; milk protein concentrate) or isocaloric carbohydrate placebo (n = 13). Immobilization reduced postabsorptive skeletal muscle phosphorylation of the mTORC1 downstream targets, 4E-BP1, P70S6K, and ribosomal protein S6 (RPS6), with phosphorylation of the latter two decreasing to a greater extent in the placebo, compared with the protein supplementation groups (37% ± 13% vs. 14% ± 11% and 38% ± 20% vs. 25% ± 8%, respectively). Sestrin2 protein was also downregulated following immobilization irrespective of supplement group, despite a corresponding increase in its mRNA content. This decrease in Sestrin2 protein was negatively correlated with the immobilization-induced change in the in silico-predicted regulator miR-23b-3p. No other measured upstream proteins were altered by immobilization or supplementation. Immobilization downregulated postabsorptive mTORC1 pathway activation, and 20 g/day of protein supplementation attenuated the decrease in phosphorylation of targets regulating muscle protein synthesis.

Soluble Whey Protein Hydrolysate Ameliorates Muscle Atrophy Induced by Immobilization via Regulating the PI3K/Akt Pathway in C57BL/6 Mice.

Sarcopenia, a loss of skeletal muscle mass and function, is prevalent in older people and associated with functional decline and mortality. Protein supplementation is necessary to maintain skeletal muscle mass and whey protein hydrolysates have the best nutrient quality among food proteins. In the first study, C57BL/6 mice were subjected to immobilization for 1 week to induce muscle atrophy. Then, mice were administered with four different whey protein hydrolysates for 2 weeks with continuous immobilization. Among them, soluble whey protein hydrolysate (WP-S) had the greatest increase in grip strength, muscle weight, and cross-sectional area of muscle fiber than other whey protein hydrolysates. To investigate the molecular mechanism, we conducted another experiment with the same experimental design. WP-S significantly promoted the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway and inhibited the PI3K/Akt/forkhead box O (FoxO) pathway. In addition, it increased myosin heavy chain (MyHC) expression in both the soleus and quadriceps and changed MyHC isoform expressions. In conclusion, WP-S attenuated muscle atrophy induced by immobilization by enhancing the net protein content regulating muscle protein synthesis and degradation. Thus, it is a necessary and probable candidate for developing functional food to prevent sarcopenia.

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.