Protein Synthesis In Myotubes Research Articles

Effect of synchronicity of amino acid supply on the synthesis of protein in C2C12 myotubes cultured in vitro.

Previous studies inferred that the synthesis rate/efficiency of protein in body tissue is probably affected by synchronicity of different amino acid (AA) supply in its metabolic pool. In order to further observe the influence of synchronicity of AA supply on the synthesis of protein in cell level, a cell culture experiment in vitro was conducted with C2C12 myotubes. C2C12 myotubes were cultured for 24 h, meanwhile the culture medium was replaced for each 8 h. Those myotubes were subjected to 3 treatments (1 for controlled and 2 for tested), control myotubes were cultured with same normal complete medium within the whole 24 h, and the 2 tested myotubes were cultured with asynchronous amino acid supply medium in which the levels of different AAs (Lysine, threonine, methionine, leucine, valine and glutamic acid) either increased and then decreased or decreased and then increased, at different replaced medium time point (at 0, 8, and 16 h). However, during the whole experiment period all the 3 treated myotubes received same amount of each AA. The sample of the myotubes were used for myotube morphology, protein, AA, and proteomic analysis. The results showed that asynchronous AA nutrition affect the synthesis and degradation of myotube proteins, and the AAAS in the medium increase, thus decreasing the synthesis rate of myotube proteins (p < 0.05) and decreasing the diameter of myotubes (p < 0.05). The process of reduced protein synthesis affects the PI3K-AKT and FoxO signaling pathway by downregulating the levels of IRS1 and EGFR, and the degradation amplitude is greater than the synthesis amplitude. Therefore, this study further revealed the effect of the asynchronous supply of amino acids on myotube protein synthesis and the underlying mechanism and provided a theoretical reference for the precision of nutrition to animals.

Circulating Amino Acid Concentration after the Consumption of Pea or Whey Proteins in Young and Older Adults Affects Protein Synthesis in C2C12 Myotubes.

As older adults tend to reduce their intake of animal-source proteins, plant-source proteins may offer valuable resources for better protein intake. The aim of this study was to assess whether the pea proteins can be used to achieve blood amino acid levels that stimulate muscle protein synthesis. We measured variations in plasma amino acid concentrations in young and older adults given pea (NUTRALYS® S85 Plus) or whey proteins either alone or in a standardized meal. The effect of amino acid concentrations on protein synthesis in C2C12 myotubes was determined. In terms of results, plasma amino acid concentrations reflected the difference between the amino acid contents of whey and pea proteins. Blood leucine showed a greater increase of 91 to 130% with whey protein compared to pea protein, while the opposite was observed for arginine (A greater increase of 147 to 210% with pea compared to whey). Culture media prepared with plasmas from the human study induced age-dependent but not protein-type-dependent changes in myotube protein synthesis. In conclusion, pea and whey proteins have the same qualities in terms of their properties to maintain muscle protein synthesis. Pea proteins can be recommended for older people who do not consume enough animal-source proteins.

Serine synthesis pathway enzyme PHGDH is critical for muscle cell biomass, anabolic metabolism, and mTORC1 signaling.

Cells use glycolytic intermediates for anabolism, e.g., via the serine synthesis and pentose phosphate pathways. However, we still understand poorly how these metabolic pathways contribute to skeletal muscle cell biomass generation. The first aim of this study was therefore to identify enzymes that limit protein synthesis, myotube size, and proliferation in skeletal muscle cells. We inhibited key enzymes of glycolysis, the pentose phosphate pathway, and the serine synthesis pathway to evaluate their importance in C2C12 myotube protein synthesis. Based on the results of this first screen, we then focused on the serine synthesis pathway enzyme phosphoglycerate dehydrogenase (PHGDH). We used two different PHGDH inhibitors and mouse C2C12 and human primary muscle cells to study the importance and function of PHGDH. Both myoblasts and myotubes incorporated glucose-derived carbon into proteins, RNA, and lipids, and we showed that PHGDH is essential in these processes. PHGDH inhibition decreased protein synthesis, myotube size, and myoblast proliferation without cytotoxic effects. The decreased protein synthesis in response to PHGDH inhibition appears to occur mainly mechanistic target of rapamycin complex 1 (mTORC1)-dependently, as was evident from experiments with insulin-like growth factor 1 and rapamycin. Further metabolomics analyses revealed that PHGDH inhibition accelerated glycolysis and altered amino acid, nucleotide, and lipid metabolism. Finally, we found that supplementing an antioxidant and redox modulator, N-acetylcysteine, partially rescued the decreased protein synthesis and mTORC1 signaling during PHGDH inhibition. The data suggest that PHGDH activity is critical for skeletal muscle cell biomass generation from glucose and that it regulates protein synthesis and mTORC1 signaling.NEW & NOTEWORTHY The use of glycolytic intermediates for anabolism was demonstrated in both myoblasts and myotubes, which incorporate glucose-derived carbon into proteins, RNA, and lipids. We identify phosphoglycerate dehydrogenase (PHGDH) as a critical enzyme in those processes and also for muscle cell hypertrophy, proliferation, protein synthesis, and mTORC1 signaling. Our results thus suggest that PHGDH in skeletal muscle is more than just a serine-synthesizing enzyme.

(-)-Epicatechin and its colonic metabolite hippuric acid protect against dexamethasone-induced atrophy in skeletal muscle cells

Cocoa flavanols have been shown to improve muscle function and may offer a novel approach to protect against muscle atrophy. Hippuric acid (HA) is a colonic metabolite of (-)-epicatechin (EPI), the primary bioactive compound of cocoa, and may be responsible for the associations between cocoa supplementation and muscle metabolic alterations. Accordingly, we investigated the effects of EPI and HA upon skeletal muscle morphology and metabolism within an in vitro model of muscle atrophy. Under atrophy-like conditions (24h 100μM dexamethasone (DEX)), C2C12 myotube diameter was significantly greater following co-incubation with either 25μM HA (11.19±0.39μm) or 25μM EPI (11.01±0.21μm) compared to the vehicle control (VC; 7.61±0.16μm, both P < .001). In basal and leucine-stimulated states, there was a significant reduction in myotube protein synthesis (MPS) rates following DEX treatment in VC (P=.024). Interestingly, co-incubation with EPI or HA abrogated the DEX-induced reductions in MPS rates, whereas no significant differences versus control treated myotubes (CTL) were noted. Furthermore, co-incubation with EPI or HA partially attenuated the increase in proteolysis seen in DEX-treated cells, preserving LC3 α/β II:I and caspase-3 protein expression in atrophy-like conditions. The protein content of PGC1α, ACC, and TFAM (regulators of mitochondrial function) were significantly lower in DEX-treated versus. CTL cells (all P < .050). However, co-incubation with EPI or HA was unable to prevent these DEX-induced alterations. For the first time we demonstrate that EPI and HA exert anti-atrophic effects on C2C12 myotubes, providing novel insight into the association between flavanol supplementation and favourable effects on muscle health.

Proteasome- and Calpain-Mediated Proteolysis, but Not Autophagy, Is Required for Leucine-Induced Protein Synthesis in C2C12 Myotubes.

Muscle protein synthesis and proteolysis are tightly coupled processes. Given that muscle growth is promoted by increases in net protein balance, it stands to reason that bolstering protein synthesis through amino acids while reducing or inhibiting proteolysis could be a synergistic strategy in enhancing anabolism. However, there is contradictory evidence suggesting that the proper functioning of proteolytic systems in muscle is required for homeostasis. To add clarity to this issue, we sought to determine if inhibiting different proteolytic systems in C2C12 myotubes in conjunction with acute and chronic leucine treatments affected markers of anabolism. In Experiment 1, myotubes underwent 1-h, 6-h, and 24-h treatments with serum and leucine-free DMEM containing the following compounds (n = 6 wells per treatment): (i) DMSO vehicle (CTL), (ii) 2 mM leucine + vehicle (Leu-only), (iii) 2 mM leucine + 40 μM MG132 (20S proteasome inhibitor) (Leu + MG132), (iv) 2 mM leucine + 50 μM calpeptin (calpain inhibitor) (Leu + CALP), and (v) 2 mM leucine + 1 μM 3-methyladenine (autophagy inhibitor) (Leu + 3MA). Protein synthesis levels significantly increased (p < 0.05) in the Leu-only and Leu + 3MA 6-h treatments compared to CTL, and levels were significantly lower in Leu + MG132 and Leu + CALP versus Leu-only and CTL. With 24-h treatments, total protein yield was significantly lower in Leu + MG132 cells versus other treatments. Additionally, the intracellular essential amino acid (EAA) pool was significantly greater in 24-h Leu + MG132 treatments versus other treatments. In a follow-up experiment, myotubes were treated for 48 h with CTL, Leu-only, and Leu + MG132 for morphological assessments. Results indicated Leu + MG132 yielded significantly smaller myotubes compared to CTL and Leu-only. Our data are limited in scope due to the utilization of select proteolysis inhibitors. However, this is the first evidence to suggest proteasome and calpain inhibition with MG132 and CALP, respectively, abrogate leucine-induced protein synthesis in myotubes. Additionally, longer-term Leu + MG132 treatments translated to an atrophy phenotype. Whether or not proteasome inhibition in vivo reduces leucine- or EAA-induced anabolism remains to be determined.

Effect of the lysosomotropic agent chloroquine on mTORC1 activation and protein synthesis in human skeletal muscle

BackgroundPrevious work in HEK-293 cells demonstrated the importance of amino acid-induced mTORC1 translocation to the lysosomal surface for stimulating mTORC1 kinase activity and protein synthesis. This study tested the conservation of this amino acid sensing mechanism in human skeletal muscle by treating subjects with chloroquine—a lysosomotropic agent that induces in vitro and in vivo lysosome dysfunction.MethodsmTORC1 signaling and muscle protein synthesis (MPS) were determined in vivo in a randomized controlled trial of 14 subjects (10 M, 4 F; 26 ± 4 year) that ingested 10 g of essential amino acids (EAA) after receiving 750 mg of chloroquine (CHQ, n = 7) or serving as controls (CON, n = 7; no chloroquine). Additionally, differentiated C2C12 cells were used to assess mTORC1 signaling and myotube protein synthesis (MyPS) in the presence and absence of leucine and the lysosomotropic agent chloroquine.ResultsmTORC1, S6K1, 4E-BP1 and rpS6 phosphorylation increased in both CON and CHQ 1 h post EAA ingestion (P < 0.05). MPS increased similarly in both groups (CON, P = 0.06; CHQ, P < 0.05). In contrast, in C2C12 cells, 1 mM leucine increased mTORC1 and S6K1 phosphorylation (P < 0.05), which was inhibited by 2 mg/ml chloroquine. Chloroquine (2 mg/ml) was sufficient to disrupt mTORC1 signaling, and MyPS.ConclusionsChloroquine did not inhibit amino acid-induced activation of mTORC1 signaling and skeletal MPS in humans as it does in C2C12 muscle cells. Therefore, different in vivo experimental approaches are required for confirming the precise role of the lysosome and amino acid sensing in human skeletal muscle.Trial registration NCT00891696. Registered 29 April 2009.

5-Hydroxy-7-methoxyflavone derivatives from Kaempferia parviflora induce skeletal muscle hypertrophy.

Skeletal muscle plays a critical role in locomotion and energy metabolism. Maintenance or enhancement of skeletal muscle mass contributes to the improvement of mobility and prevents the development of metabolic diseases. The extracts from Kaempferia parviflora rhizomes contain at least ten methoxyflavone derivatives that exhibit enhancing effects on ATP production and glucose uptake in skeletal muscle cells. In the present study, we investigated the effects of ten K. parviflora‐derived methoxyflavone derivatives (six 5,7‐dimethoxyflavone (DMF) derivatives and four 5‐hydroxy‐7‐methoxyflavone (HMF) derivatives) on skeletal muscle hypertrophy. Murine C2C12 myotubes and senescence‐accelerated mouse‐prone 1 (SAMP1) mice treated with methoxyflavones were used as experimental models to determine the effects of HMF derivatives on myotube diameter and size and muscle mass. The four HMF derivatives, but not the six DMF derivatives, increased myotube diameter. The 5‐hydroxyflavone, 7‐methoxyflavone, and 5,7‐dihydroxyflavone had no influence on myotube size, a result that differed from HMF. Dietary administration of the mixture composed of the four HMF derivatives resulted in increase in the soleus muscle size and mass in SAMP1 mice. HMF derivatives also promoted protein synthesis in myotubes, and treatment with the intracellular Ca2+ chelator BAPTA‐AM, which depletes intracellular Ca2+ levels, inhibited this promotion. Furthermore, BAPTA‐AM inhibited HMF‐promoted protein synthesis even when myotubes were incubated in Ca2+‐free medium. These results indicate that HMF derivatives induce myotube hypertrophy and that both the 5‐hydroxyl group and the 7‐methoxy group in the flavones are necessary for myotube hypertrophy. Furthermore, these results suggest that HMF‐induced protein synthesis requires intracellular Ca2+, but not extracellular Ca2+.

PGC-1α and PGC-1β Increase Protein Synthesis via ERRα in C2C12 Myotubes.

The transcriptional coactivators peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and PGC-1β are positive regulators of skeletal muscle mass and energy metabolism; however, whether they influence muscle growth and metabolic adaptations via increased protein synthesis is not clear. This study revealed PGC-1α or PGC-1β overexpression in C2C12 myotubes increased protein synthesis and myotube diameter under basal conditions and attenuated the loss in protein synthesis following the treatment with the catabolic agent, dexamethasone. To investigate whether PGC-1α or PGC-1β signal through the Akt/mTOR pathway to increase protein synthesis, treatment with the PI3K and mTOR inhibitors, LY294002 and rapamycin, respectively, was undertaken but found unable to block PGC-1α or PGC-1β’s promotion of protein synthesis. Furthermore, PGC-1α and PGC-1β decreased phosphorylation of Akt and the Akt/mTOR substrate, p70S6K. In contrast to Akt/mTOR inhibition, the suppression of ERRα, a major effector of PGC-1α and PGC-1β activity, attenuated the increase in protein synthesis and myotube diameter in the presence of PGC-1α or PGC-1β overexpression. To characterize further the biological processes occurring, gene set enrichment analysis of genes commonly regulated by both PGC-1α and PGC-1β was performed following a microarray screen. Genes were found enriched in metabolic and mitochondrial oxidative processes, in addition to protein translation and muscle development categories. This suggests concurrent responses involving both increased metabolism and myotube protein synthesis. Finally, based on their known function or unbiased identification through statistical selection, two sets of genes were investigated in a human exercise model of stimulated protein synthesis to characterize further the genes influenced by PGC-1α and PGC-1β during physiological adaptive changes in skeletal muscle.

Asymmetric cellular responses in primary human myoblasts using sera of different origin and specification.

For successful growth and maintenance of primary myogenic cells in vitro, culture medium and addition of sera are the most important factors. At present it is not established as to what extent sera of different origin and composition, supplemented in media or serum-free media conditions influence myoblast function and responses to different stimuli. By assessing markers of proliferation, differentiation/fusion, quiescence, apoptosis and protein synthesis the aim of the current study was to elucidate how primary human myoblasts and myotubes are modulated by different commonly used serum using FCS (foetal calf serum), (CS-FCS charcoal-stripped FCS, a manufacturing process to remove hormones and growth factors from sera), HS (horse serum) as well as in serum free conditions (DMEM). To characterise the biological impact of the different serum, myoblasts were stimulated with Insulin (100 nM) and Vitamin D (100 nM; 1α,25(OH)2D3, 1α,25-Dihydroxycholecalciferol, Calcitriol), two factors with characterised effects on promoting fusion and protein synthesis or quiescence, respectively in human myoblasts/myotubes. We demonstrate that sera of different origin/formulation differentially affect myoblast proliferation and myotube protein synthesis. Importantly, we showed that quantifying the extent to which Insulin effects myoblasts in vitro is highly dependent upon serum addition and which type is present in the media. Upregulation of mRNA markers for myogenic fusion, Myogenin, with Insulin stimulation, relative to DMEM, appeared dampened at varying degrees with serum addition and effects on p70S6K phosphorylation as a marker of protein synthesis could not be identified unless serum was removed from media. We propose that these asymmetric molecular and biochemical responses in human myoblasts reflect the variable composition of mitogenic and anabolic factors in each of the sera. The results have implications for both the reproducibility and interpretation of results from experimental models in myoblast cells/myotubes.

Acute myotube protein synthesis regulation by IL-6-related cytokines.

IL-6 and leukemia inhibitory factor (LIF), members of the IL-6 family of cytokines, play recognized paradoxical roles in skeletal muscle mass regulation, being associated with both growth and atrophy. Overload or muscle contractions can induce a transient increase in muscle IL-6 and LIF expression, which has a regulatory role in muscle hypertrophy. However, the cellular mechanisms involved in this regulation have not been completely identified. The induction of mammalian target of rapamycin complex 1 (mTORC1)-dependent myofiber protein synthesis is an established regulator of muscle hypertrophy, but the involvement of the IL-6 family of cytokines in this process is poorly understood. Therefore, we investigated the acute effects of IL-6 and LIF administration on mTORC1 signaling and protein synthesis in C2C12 myotubes. The role of glycoprotein 130 (gp130) receptor and downstream signaling pathways, including phosphoinositide 3-kinase (PI3K)-Akt-mTORC1 and signal transducer and activator of transcription 3 (STAT3)-suppressor of cytokine signaling 3 (SOCS3), was investigated by administration of specific siRNA or pharmaceutical inhibitors. Acute administration of IL-6 and LIF induced protein synthesis, which was accompanied by STAT3 activation, Akt-mTORC1 activation, and increased SOCS3 expression. This induction of protein synthesis was blocked by both gp130 siRNA knockdown and Akt inhibition. Interestingly, STAT3 inhibition or Akt downstream mTORC1 signaling inhibition did not fully block the IL-6 or LIF induction of protein synthesis. SOCS3 siRNA knockdown increased basal protein synthesis and extended the duration of the protein synthesis induction by IL-6 and LIF. These results demonstrate that either IL-6 or LIF can activate gp130-Akt signaling axis, which induces protein synthesis via mTORC1-independent mechanisms in cultured myotubes. However, IL-6- or LIF-induced SOCS3 negatively regulates the activation of myotube protein synthesis.

Cytokines IL‐6 and LIF can differentially regulate myotube protein synthesis

IL‐6 and LIF are members of the IL‐6 family of cytokines that have recognized paradoxical regulatory roles in skeletal muscle mass, but there are gaps in our understanding of the cellular mechanisms that contribute to this regulatory dichotomy. The IL‐6 family of cytokines can induce several upstream regulators of mTORC1, including PI3K/Akt, ERK1/2 and AMPK; these pathways have the ability to regulate muscle mTORC1 signaling and protein synthesis. However, mTORC1 signaling regulation of by the IL‐6 family of cytokines remains poorly understood. Therefore, we investigated the effects of IL‐6 and LIF dose and duration on mTORC1 signaling and protein synthesis in myotubes. C2C12 myotubes were treated with varying doses of IL‐6 or LIF for either a short (1–4h) or long duration (24h). Signaling through the gp130 receptor and downstream effectors, including PI3K/Akt, STAT3 and AMPK were investigated by specific siRNA or pharmaceutical inhibitors administration. The Akt‐mTORC1 signaling axis and protein synthesis was induced by short term IL‐6 or LIF administration, which was blocked by siRNA knock‐down of gp130 receptor or pharmaceutical inhibition of Akt signaling. Interestingly, inhibition of mTOR or STAT3 signaling decreased basal myotube protein synthesis, but did not block the protein synthesis induction by short term IL‐6 or LIF administration. Long term IL‐6 or LIF administration suppressed Akt‐mTORC1 signaling and protein synthesis, which coincided with increased AMPK phosphorylation. However, inhibition of AMPK signaling by siRNA knock‐down did not rescue the protein synthesis suppression by long term IL‐6 or LIF treatment. These results demonstrate that IL‐6 family of cytokines have the capacity to either activate or suppress myotube mTORC1 signaling and protein synthesis, and the ultimate effect depends on the degree of the cytokine exposure.Support or Funding InformationR01 CA121249 to J. A. Carson from the National Cancer Institute, P20 RR‐017698 to J. A. Carson from National Center for Research Resources Grant

Osteocalcin is necessary and sufficient to maintain muscle mass in older mice

ObjectiveA decrease in muscle protein turnover and therefore in muscle mass is a hallmark of aging. Because the circulating levels of the bone-derived hormone osteocalcin decline steeply during aging in mice, monkeys and humans we asked here whether this hormone might regulate muscle mass as mice age. MethodsWe examined muscle mass and strength in mice lacking osteocalcin (Ocn−/−) or its receptor in all cells (Gprc6a−/−) or specifically in myofibers (Gprc6aMck−/−) as well as in 9 month-old WT mice receiving exogenous osteocalcin for 28 days. We also examined protein synthesis in WT and Gprc6a−/− mouse myotubes treated with osteocalcin. ResultsWe show that osteocalcin signaling in myofibers is necessary to maintain muscle mass in older mice in part because it promotes protein synthesis in myotubes without affecting protein breakdown. We further show that treatment with exogenous osteocalcin for 28 days is sufficient to increase muscle mass of 9-month-old WT mice. ConclusionThis study uncovers that osteocalcin is necessary and sufficient to prevent age-related muscle loss in mice.

Metabolic adaptation of skeletal muscle to hyperammonemia drives the beneficial effects of l-leucine in cirrhosis

Increased skeletal muscle ammonia uptake with loss of muscle mass adversely affects clinical outcomes in cirrhosis. Hyperammonemia causes reduced protein synthesis and sarcopenia but the cellular responses to impaired proteostasis and molecular mechanism of l-leucine induced adaptation to ammonia induced stress were determined. Response to activation of amino acid deficiency sensor, GCN2, in the skeletal muscle from cirrhotic patients and the portacaval anastomosis (PCA) rat were quantified. During hyperammonemia and l-leucine supplementation, protein synthesis, phosphorylation of eIF2α, mTORC1 signaling, l-leucine transport and response to l-leucine supplementation were quantified. Adaptation to cellular stress via ATF4 and its target GADD34 were also determined. Activation of the eIF2α kinase GCN2 and impaired mTORC1 signaling were observed in skeletal muscle from cirrhotic patients and PCA rats. Ammonia activated GCN2 mediated eIF2α phosphorylation (eIF2α-P) and impaired mTORC1 signaling that inhibit protein synthesis in myotubes and MEFs. Adaptation to ammonia induced stress did not involve translational reprogramming by activation transcription factor 4 (ATF4) dependent induction of the eIF2α-P phosphatase subunit GADD34. Instead, ammonia increased expression of the leucine/glutamine exchanger SLC7A5, l-leucine uptake and intracellular l-leucine levels, the latter not being sufficient to rescue the inhibition of protein synthesis, due to potentially enhanced mitochondrial sequestration of l-leucine. l-leucine supplementation rescued protein synthesis inhibition caused by hyperammonemia. Response to hyperammonemia is reminiscent of the cellular response to amino acid starvation, but lacks the adaptive ATF4 dependent integrated stress response (ISR). Instead, hyperammonemia-induced l-leucine uptake was an adaptive response to the GCN2-mediated decreased protein synthesis. Sarcopenia or skeletal muscle loss is the most frequent complication in cirrhosis but there are no treatments because the cause(s) of muscle loss in liver disease are not known. Results from laboratory experiments in animals and muscle cells were validated in human patients with cirrhosis to show that ammonia plays a key role in causing muscle loss in patients with cirrhosis. We identified a novel stress response to ammonia in the muscle that decreases muscle protein content that can be reversed by supplementation with the amino acid l-leucine.

Basal and Stretch‐induced Myotube Protein Synthesis Regulation by gp130 Signaling

Mechanical stimulation from muscle contractions or stretch can induce mTOR signaling and protein synthesis in cultured myotubes. Mechanical stimulation also induces muscle expression of IL‐6 family of cytokines, which can activate intracellular signaling pathways through glycoprotein 130 (gp130). Although several gp130 downstream targets, such as Akt and ERK1/2, can regulate mTOR activity, the role for gp130 initiated signaling in the stretch activation of mTOR signaling and protein synthesis has not been established. The purpose of this study was to investigate gp130 signaling on mTORC1 activation, and whether gp130 signaling is necessary for stretch‐induced protein synthesis in cultured myotubes. C2C12 myotubes were static stretched (5%) for 24h. Signaling was manipulated by gp130 siRNA transfection into cells or administration of neutralizing antibody in culture media. Phosphorylation of signaling molecules and puromycin incorporation (indicator of protein synthesis) were measured by western blot. We report that gp130 siRNA knock‐down decreased myotube basal p70S6K phosphorylation (T389) and protein synthesis. Activation of gp130 signaling by IL‐6 administration increased myotube Akt phosphorylation (T308) and protein synthesis, but not p70S6K phosphorylation. Static stretch for 24h increased myotube p70S6K phosphorylation, protein synthesis, and IL‐6 mRNA expression. Stretch in the presence of gp130 antibody attenuated the stretch‐induction of myotube p70S6K phosphorylation, and blocked the stretch‐induction of protein synthesis. These results demonstrate that gp130 dependent signaling has a role in the regulation of basal and stretch‐induced myotube protein synthesis, however, the cellular mechanism regulating protein synthesis differs between the 2 conditions.Support or Funding InformationR01 CA121249 to JAC from the National Cancer Institute

Lewis lung carcinoma regulation of mechanical stretch-induced protein synthesis in cultured myotubes.

Mechanical stretch can activate muscle and myotube protein synthesis through mammalian target of rapamycin complex 1 (mTORC1) signaling. While it has been established that tumor-derived cachectic factors can induce myotube wasting, the effect of this catabolic environment on myotube mechanical signaling has not been determined. We investigated whether media containing cachectic factors derived from Lewis lung carcinoma (LLC) can regulate the stretch induction of myotube protein synthesis. C2C12 myotubes preincubated in control or LLC-derived media were chronically stretched. Protein synthesis regulation by anabolic and catabolic signaling was then examined. In the control condition, stretch increased mTORC1 activity and protein synthesis. The LLC treatment decreased basal mTORC1 activity and protein synthesis and attenuated the stretch induction of protein synthesis. LLC media increased STAT3 and AMP-activated protein kinase phosphorylation in myotubes, independent of stretch. Both stretch and LLC independently increased ERK1/2, p38, and NF-κB phosphorylation. In LLC-treated myotubes, the inhibition of ERK1/2 and p38 rescued the stretch induction of protein synthesis. Interestingly, either leukemia inhibitory factor or glycoprotein 130 antibody administration caused further inhibition of mTORC1 signaling and protein synthesis in stretched myotubes. AMP-activated protein kinase inhibition increased basal mTORC1 signaling activity and protein synthesis in LLC-treated myotubes, but did not restore the stretch induction of protein synthesis. These results demonstrate that LLC-derived cachectic factors can dissociate stretch-induced signaling from protein synthesis through ERK1/2 and p38 signaling, and that glycoprotein 130 signaling is associated with the basal stretch response in myotubes.

Lewis Lung Carcinoma Regulation of Mechanical Stretch Induced Protein Synthesis in Cultured Myotubes

Mechanical stimuli, such as stretch, can activate muscle and myotube protein synthesis through mTORC1 signaling. However, it has not been determined if stretch can regulate protein synthesis in cac...

G-CSF treatment can attenuate dexamethasone-induced reduction in C2C12 myotube protein synthesis

Granulocyte-colony stimulating factor (G-CSF) has been demonstrated to enhance skeletal muscle recovery following injury and increases muscle function in the context of neuromuscular disease in rodent models. However, understanding of the underlying mechanisms used by G-CSF to mediate these functions remains poor. G-CSF acts on responsive cells through binding to a specific membrane spanning receptor, G-CSFR. Recently identified, the G-CSFR is expressed in myoblasts, myotubes and mature skeletal muscle tissue. Therefore, elucidating the actions of G-CSF in skeletal muscle represents an important prerequisite to consider G-CSF as a therapeutic agent to treat skeletal muscle. Here we show for the first time that treatment with moderate doses (4 and 40ng/ml) of G-CSF attenuates the effects of dexamethasone in reducing protein synthesis in C2C12 myotubes. However, a higher dose (100ng/ml) of G-CSF exacerbates the dexamethasone-induced reduction in protein synthesis. In contrast, G-CSF had no effect on basal or dexamethasone-induced protein degradation, nor did G-CSF influence the phosphorylation of Akt, STAT3, Erk1/2, Src, Lyn and Erk5 in C2C12 myotubes. In conclusion, physiologically relevant doses of G-CSF may attenuate reduced skeletal muscle protein synthesis during catabolic conditions, thereby improving recovery.

Lewis lung carcinoma cells regulation of stretch‐induced mTOR activation in C2C12 myotubes (1163.8)

Imbalances in rates of protein synthesis and degradation are responsible for cancer‐induced skeletal muscle wasting. Lewis lung carcinoma (LLC) cell implantation is a widely used explant mouse model for examining cancer cachexia and muscle wasting. We have demonstrated that trans‐signaling molecules from LLC cells resulted in mTOR suppression in C2C12 myotubes and muscle from mice implanted with LLC tumors. Mechanical overload activates mTOR signaling and increases protein synthesis rate, serving as a potential therapeutic strategy for skeletal muscle mass maintenance. The purpose of this study is to investigate the effect of mechanical stretch on C2C12 myotube protein synthesis regulation in the presence of trans‐signaling molecules from LLC cells. C2C12 myotubes were differentiated and then stretched on collagen‐coated silastic membranes. Chronic uniaxial stretch (5% for 24h) induced ERK (T202/Y204) and S6 ribosomal protein (S235/236) phosphorylation as well as rate of protein synthesis in control medium (DMEM with 2.5% FBS). However, the addition of LLC medium blocked the stretch induction of S6 ribosomal protein phosphorylation. These results suggest LLC can directly suppress mechanical activation of signaling that regualtes of skeletal muscle protein synthesis.

OP019 EXPLORATION OF WHAT LEVEL OF PROTEIN IS NEEDED FOR MUSCLE MASS ACCRETION DURING WEIGHT LOSS WITH RESISTANCE TRAINING IN OBESE ELDERLY

Rationale: Recent studies from our laboratory have indicated that glycine represents an effective anti-inflammatory agent that attenuates muscle loss in tumour-bearing mice. However, it remains to be established whether glycine affects skeletal muscle directly. The aim was to investigate how muscle cell health, metabolism and inflammation are affected by glycine. Methods: After differentiation, C2C12 myotubes were incubated with either 2.5mM glycine or 2.5mM alanine and exposed to 1) lipopolysaccharide (LPS, 1mg/mL) for 24 h to induce an inflammatory response and wasting; or 2) serum free media (SFM) for 48 h to induce wasting. Myotube diameter was assessed and changes in intracellular [Ca2+] were monitored by loading cells with Fluo-4/AM and imaging using confocal microscopy Protein synthesis was determined using SunSET methodology. Insulin-mediated glucose uptake was determined using fluorescent deoxyglucose and anabolic signalling was assessed using routine western blot analyses. One way ANOVA was used to identify differences between groups. Results: Serum withdrawal reduced protein synthesis in myotubes by 25% (P < 0.05). Interestingly, glycine treatment completely prevented this decrease. LPS-treated myotubes were 25% smaller, had an 80% higher intracellular [Ca2+], and a 40% reduction in insulin-mediated glucose uptake compared with control cells (P < 0.05). Glycine treatment reduced the loss of myotube diameter by 50% and improved insulinmediated glucose uptake and Akt phosphorylation by 40% and 10%, respectively (P < 0.05). Conclusion: Glycine protects muscle cells from various catabolic stimuli in vitro, thereby maintaining protein synthesis, anabolic signalling and insulin-mediated glucose transport. These findings suggest a direct protective effect of glycine on skeletal muscle.

The tumor suppressor protein PDCD4 is a critical regulator of muscle cell differentiation

The mammalian target of rapamycin complex 1/S6 ribosomal protein kinase 1 (mTORC1/S6K1) pathway is a critical regulator of mRNA translation and skeletal muscle mass. It does this in part by inhibiting the tumor suppressor protein, programmed cell death 4 (PDCD4). In C2C12 and L6 muscle cells, we showed that PDCD4 abundance was high on day 1 and then decreased as myoblasts differentiated into myotubes (p&lt;0.05). siRNA‐mediated knockdown of S6K1 reversed the decrease in PDCD4 abundance and significantly decreased myosin heavy chain 1 (MHC 1) protein abundance, suggesting that PDCD4 regulation was vital for differentiation. Indeed, cells depleted of PDCD4 had reduced MHC abundance, showed delayed myoblast fusion and abnormal myotube formation. On days 3 and 4 of differentiation, myotubes depleted of PDCD4 showed 40–60% reductions in myotube protein synthesis. This study unravels a link between PDCD4 and muscle cell differentiation, and suggests that this mTORC1/S6K1 substrate may be of therapeutic significance for muscle recovery following injury or atrophy. Funded by NSERC.