Decreased insulin-like growth factor-1 expression in response to mechanical loading is associated with skeletal muscle anabolic resistance in cancer cachexia.

OBJECTIVE: Cachexia is a systemic metabolic syndrome characterized by loss of body weight and skeletal muscle mass during chronic wasting diseases, such as cancer. Skeletal muscle in cancer cachexia is less responsive to anabolic factors including mechanical loading; however, the precise molecular mechanism is largely unknown. In this study, we examined the underlying mechanism of anabolic resistance in skeletal muscle in a cancer cachexia model. METHODS: CD2F1 mice (male, 8 weeks old) were subcutaneously transplanted (1 × 106 cells per mouse) with a mouse colon cancer-derived cell line (C26) as a model of cancer cachexia. Mechanical overload of the plantaris muscle by synergist tenotomy was performed during the 2nd week and the plantaris muscle was sampled at the 4th week following C26 transplantation. RESULTS: The hypertrophic response of skeletal muscle (increased skeletal muscle weight/protein synthesis efficiency and activation of mechanistic target of rapamycin complex 1 signaling) associated with mechanical overload was significantly suppressed during cancer cachexia. Screening of gene expression profile and pathway analysis using microarray revealed that blunted muscle protein synthesis was associated with cancer cachexia and was likely induced by downregulation of insulin-like growth factor-1 (IGF-1) and impaired activation of IGF-1-dependent signaling. CONCLUSIONS: These observations indicate that cancer cachexia induces resistance to muscle protein synthesis, which may be a factor for inhibiting the anabolic adaptation of skeletal muscle to physical exercise in cancer patients.

Regulation of protein and oxidative energy metabolism are down-regulated in the skeletal muscles of Asiatic black bears during hibernation.

Hibernating animals exhibit an unexplained physiological characteristic of skeletal muscles being atrophy resistance, in which case muscle mass and strength remain almost unchanged both before and after hibernation. In this study, we examined the alterations in the regulatory systems of protein and energy metabolism in the skeletal muscles of Asiatic black bears during hibernation. Skeletal muscle samples (vastus lateralis muscle) were collected from identical individuals (n = 8) during the active (July) and hibernating (February) periods, while histochemical and biochemical analyses were performed. We observed no significant alterations in body weight, muscle fiber size, and fiber type composition during the active and hibernating periods, indicating that the skeletal muscles of bears are very well preserved during hibernation. In hibernating bear skeletal muscles, both regulatory pathways of muscle protein synthesis (Akt/mechanistic target of rapamycin and mitogen-activated protein kinase systems) and proteolysis (ubiquitin-proteasome and autophagy systems) were down-regulated. Gene expression levels of factors regulating oxidative metabolism were also decreased in hibernating bear skeletal muscles. This is likely an adaptive strategy to minimize the energy wasting of amino acids and lipids during hibernation, which is accompanied by a prolonged period of disuse and starvation.

The impact of visual cross-modal conflict with semantic and nonsemantic distractors on working memory task: A functional near-infrared spectroscopy study.

Cross-modal conflicts arise when information from multisensory modalities is incongruent. Most previous studies investigating audiovisual cross-modal conflicts have focused on visual targets with auditory distractors, and only a few studies have focused on auditory targets with visual distractors. Moreover, no study has investigated the differences in the impact of visual cross-modal conflict with semantic and nonsemantic competition and its neural basis. This cross-sectional study aimed to characterize the impact of 2 types of visual cross-modal conflicts with semantic and nonsemantic distractors through a working memory task and associated brain activities. The participants were 33 healthy, right-handed, young male adults. The paced auditory serial addition test was performed under 3 conditions: no-distractor and 2 types of visual distractor conditions (nonsemantic and semantic distractor conditions). Symbols and numbers were used as nonsemantic and semantic distractors, respectively. The oxygenated hemoglobin (Oxy-Hb) concentration in the frontoparietal regions, bilateral ventrolateral prefrontal cortex (VLPFC), dorsolateral prefrontal cortex, and inferior parietal cortex (IPC) were measured during the task under each condition. The results showed significantly lower paced auditory serial addition test performances in both distractor conditions than in the no-distractor condition, but no significant difference between the 2 distractor conditions. For brain activity, a significantly increased Oxy-Hb concentration in the right VLPFC was only observed in the nonsemantic distractor condition (corrected P = .015; Cohen d = .46). The changes in Oxy-Hb in the bilateral IPC were positively correlated with changes in task performance for both types of visual cross-modal distractor conditions. Visual cross-modal conflict significantly impairs auditory working memory task performance, regardless of the presence of semantic or nonsemantic distractors. The right VLPFC may be a crucial region to inhibit visual nonsemantic information in cross-modal conflict situations, and bilateral IPC may be closely linked with the inhibition of visual cross-modal distractor, regardless of the presence of semantic or nonsemantic distractors.

Supplementing cultured human myotubes with hibernating bear serum results in increased protein content by modulating Akt/FOXO3a signaling.

Hibernating bears remain in their dens for 5-7 months during winter and survive without eating or drinking while staying inactive. However, they maintain their physical functions with minimal skeletal muscle atrophy and metabolic dysfunction. In bears, resistance to skeletal muscle atrophy during hibernation is likely mediated by seasonally altered systemic factors that are independent of neuromuscular activity. To determine whether there are components in bear serum that regulate protein and energy metabolism, differentiated human skeletal muscle cells were treated with bear serum (5% in DMEM/Ham's F-12, 24 h) collected during active summer (July) and hibernating winter (February) periods. The serum samples were collected from the same individual bears (Ursus thibetanus japonicus, n = 7 in each season). Total protein content in cultured skeletal muscle cells was significantly increased following a 24 h treatment with hibernating bear serum. Although the protein synthesis rate was not altered, the expression of MuRF1 protein, a muscle-specific E3 ubiquitin ligase was significantly decreased along with a concomitant activation of Akt/FOXO3a signaling. Increased levels of insulin-like growth factor-1 (IGF-1) were also observed in hibernating bear serum. These observations suggest that protein metabolism in cultured human myotubes may be altered when incubated with hibernating bear serum, with a significant increase in serum IGF-1 and diminished MuRF1 expression, a potential target of Akt/FOXO3a signaling. A protein sparing phenotype in cultured muscle cells by treatment with hibernating bear serum holds potential for the development of methods to prevent human muscle atrophy and related disorders.

Voluntary exercise prevents abnormal muscle mitochondrial morphology in cancer cachexia mice.

This study aimed to examine the effects of voluntary wheel running on cancer cachexia-induced mitochondrial alterations in mouse skeletal muscle. Mice bearing colon 26 adenocarcinoma (C26) were used as a model of cancer cachexia. C26 mice showed a lower gastrocnemius and plantaris muscle weight, but 4 weeks of voluntary exercise rescued these changes. Further, voluntary exercise attenuated observed declines in the levels of oxidative phosphorylation proteins and activities of citrate synthase and cytochrome c oxidase in the skeletal muscle of C26 mice. Among mitochondrial morphology regulatory proteins, mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1) were decreased in the skeletal muscle of C26 mice, but exercise resulted in similar improvements as seen in markers of mitochondrial content. In isolated mitochondria, 4-hydroxynonenal and protein carbonyls were elevated in C26 mice, but exercise blunted the increases in these markers of oxidative stress. In addition, electron microscopy revealed that exercise alleviated the observed increase in the percentage of damaged mitochondria in C26 mice. These results suggest that voluntary exercise effectively counteracts mitochondrial dysfunction to mitigate muscle loss in cachexia.

Changes in liver microRNA expression and their possible regulatory role in energy metabolism-related genes in hibernating black bears.

Hibernating bears survive up to 6 months without feeding while yet maintaining metabolic homeostasis. We previously reported expression changes in energy metabolism-related genes in the liver of hibernating Japanese black bears. The present study examined the role of microRNAs in the regulation of hepatic gene expression during hibernation. The quantitative analyses revealed significant increases in the expression of 4 microRNAs (miR-221-3p, miR-222-3p, miR-455-3p, and miR-195a-5p) and decreases of 2 microRNAs (miR-122-5p and miR-7a-1-5p) during hibernation. RNA sequencing and in silico target prediction regarding 3 upregulated microRNAs (miR-221-3p, miR-222-3p and miR-455-3p) found 13 target mRNAs with significantly decreased expression during hibernation. The transfection of microRNA mimics into cells showed that miR-222 and miR-455 reduced solute carrier family 16 member 4 (SLC16A4) and fatty acid synthase (FASN) mRNA expression, respectively. Our results suggest that the increased levels of hepatic miRNA during hibernation (miR-222-3p and miR-455-3p) negatively regulate the expression of targeted genes predicted to be involved in the transport of energy source and de novo fatty acid synthesis, is consistent with a regulatory role of these miRNAs in energy metabolism in hibernating black bears.

Personality Traits Modulate the Impact of Emotional Stimuli During a Working Memory Task: A Near-Infrared Spectroscopy Study.

The purpose of the present study was to examine the influence of personality traits on the impact of emotional stimuli focusing on n-back task performance and brain activity changes. Previous neuroimaging studies have reported that individual differences in emotional processing can be attributed to personality traits, which is linked to the hemisphere-specific activity of the dorsolateral prefrontal cortex (DLPFC) in response to emotional stimuli. Thirty right-handed healthy young male participants were recruited in this study and classified into two groups, the behavioral inhibition system (BIS) group and behavioral activation system (BAS) group, based on their scores on the BIS/BAS scale. Participants saw six emotional images (two each with negative, neutral, and positive valence), which were selected from the International Affective Picture System and validated in a preliminary experiment. Then, a dual 2-back task that simultaneously employed auditory-verbal and visuospatial stimuli was conducted. Additionally, the concentration of oxygenated hemoglobin (Oxy-Hb) changes in the DLPFC was measured during the image presentation and dual 2-back task by near-infrared spectroscopy (NIRS). The task performance showed a significantly increased reaction time (RT) in the negative valence independent of personality traits. The results of Oxy-Hb changes showed a significant interaction between personality traits and emotional valence. Further, the hemisphere-subgroup analysis revealed that the right DLPFC activity was significantly higher in the negative valence than in the neutral valence in the BIS group; the right DLPFC activity was also significantly higher in the BIS group than in the BAS group in the positive valence. There was no main effect or interaction in the left DLPFC activity. These findings suggest the importance of considering personality traits when examining the impact of emotional stimuli. Further studies with large sample sizes warranted to examine the influence emotional stimuli exert on working memory performance, considering the personality traits to better understand individual differences in emotional processing.

Transient activation of mTORC1 signaling in skeletal muscle is independent of Akt1 regulation.

The regulation of cellular protein synthesis is a critical determinant of skeletal muscle growth and hypertrophy in response to an increased workload such as resistance exercise. The mechanistic target of rapamycin complex 1 (mTORC1) and its upstream protein kinase Akt1 have been implicated as a central signaling pathway that regulates protein synthesis in the skeletal muscle; however, the precise molecular regulation of mTORC1 activity is largely unknown. This study employed germline Akt1 knockout (KO) mice to examine whether upstream Akt1 regulation is necessary for the acute activation of mTORC1 signaling in the plantaris muscle following mechanical overload. The phosphorylation states of S6 kinase 1, ribosomal protein S6, and eukaryotic translation initiation factor 4E-binding protein 1 which show the functional activity of mTORC1 signaling, were significantly increased in the skeletal muscle of both wildtype and Akt1 KO mice following an acute bout (3 and 12 hr) of mechanical overload. Akt1 deficiency did not affect load-induced alteration of insulin-like growth factor-1 (IGF-1)/IGF receptor mRNA expression. Also, no effect of Akt1 deficiency was observed on the overload-induced increase in the gene expressions of pax7 and myogenic regulatory factor of myogenin. These observations show that the upstream IGF-1/Akt1 regulation is dispensable for the acute activation of mTORC1 signaling and regulation of satellite cells in response to mechanical overload.

Skeletal muscles of hibernating black bears show minimal atrophy and phenotype shifting despite prolonged physical inactivity and starvation.

Hibernating mammals experience prolonged periods of torpor and starvation during winter for up to 5-7 months. Though physical inactivity and malnutrition generally lead to profound loss of muscle mass and metabolic dysfunction in humans, hibernating bears show limited muscle atrophy and can successfully maintain locomotive function. These physiological features in bears allow us to hypothesize that hibernating bears uniquely alter the regulation of protein and energy metabolism in skeletal muscle which then contributes to "muscle atrophy resistance" against continued physical inactivity. In this study, alteration of signaling pathways governing protein and energy metabolisms was examined in skeletal muscle of the Japanese black bear (Ursus thibetanus japonicus). Sartorius muscle samples were collected from bear legs during late November (pre-hibernation) and early April (post-hibernation). Protein degradation pathways, through a ubiquitin-proteasome system (as assessed by increased expression of murf1 mRNA) and an autophagy-dependent system (as assessed by increased expression of atg7, beclin1, and map1lc3 mRNAs), were significantly activated in skeletal muscle following hibernation. In contrast, as indicated by a significant increase in S6K1 phosphorylation, an activation state of mTOR (mammalian/mechanistic target of rapamycin), which functions as a central regulator of protein synthesis, increased in post-hibernation samples. Gene expression of myostatin, a negative regulator of skeletal muscle mass, was significantly decreased post-hibernation. We also confirmed that the phenotype shifted toward slow-oxidative muscle and mitochondrial biogenesis. These observations suggest that protein and energy metabolism may be altered in skeletal muscle of hibernating bears, which then may contribute to limited loss of muscle mass and efficient energy utilization.

High-intensity eccentric training ameliorates muscle wasting in colon 26 tumor-bearing mice.

Eccentric (ECC) contractions are used to maintain skeletal muscle mass and strength in healthy subjects and patients. Here we investigated the effects of ECC training induced by electrical stimulation (ES) on muscle wasting in colon 26 (C-26) tumor-bearing mice. Mice were divided into four groups: control (CNT), CNT + ECC, C-26, and C-26 + ECC. Cancer cachexia was induced by a subcutaneous injection of C-26 cells and developed for four weeks. In experiment 1, muscle protein synthesis rate and mammalian target of rapamycin complex (mTORC) 1 signaling were investigated six hours after one bout of ECC-ES (2 s contraction given every 6 s, 20°/s, 4 sets of 5 contractions). In experiment 2, ECC-ES training, a total of 14 sessions, was performed every other day starting one day after C-26 injection. Compared to the CNT mice, the gastrocnemius muscle weight was significantly decreased in the tumor-bearing mice. This change was accompanied by a reduction in protein synthesis rate and a marked increase in the expression levels of genes including regulated in development and DNA damage responses (REDD) 1, forkhead box protein O1 (FoxO1), muscle-specific E3 ubiquitin ligases atrogin-1, and muscle ring finger 1 (MuRF-1) mRNA. ECC-ES increased the protein synthesis rate and the phosphorylation levels of p70S6K (Thr389) and rpS6 (Ser240/244), markers for mTORC1 signaling, and reversed an upregulation of MuRF-1 mRNA in muscles from C-26 mice. Our findings suggest that ECC-ES training reduces skeletal muscle atrophy in C-26 tumor-bearing mice through activation of mTORC1 signaling and the inhibition of ubiquitin-proteasome pathway. Thus, ECC-ES training might be used to effectively ameliorate muscle wasting in patients with cancer cachexia.

Akt1 deficiency diminishes skeletal muscle hypertrophy by reducing satellite cell proliferation.

Skeletal muscle mass is determined by the net dynamic balance between protein synthesis and degradation. Although the Akt/mechanistic target of rapamycin (mTOR)-dependent pathway plays an important role in promoting protein synthesis and subsequent skeletal muscle hypertrophy, the precise molecular regulation of mTOR activity by the upstream protein kinase Akt is largely unknown. In addition, the activation of satellite cells has been indicated as a key regulator of muscle mass. However, the requirement of satellite cells for load-induced skeletal muscle hypertrophy is still under intense debate. In this study, female germline Akt1 knockout (KO) mice were used to examine whether Akt1 deficiency attenuates load-induced skeletal muscle hypertrophy through suppressing mTOR-dependent signaling and satellite cell proliferation. Akt1 KO mice showed a blunted hypertrophic response of skeletal muscle, with a diminished rate of satellite cell proliferation following mechanical overload. In contrast, Akt1 deficiency did not affect the load-induced activation of mTOR signaling and the subsequent enhanced rate of protein synthesis in skeletal muscle. These observations suggest that the load-induced activation of mTOR signaling occurs independently of Akt1 regulation and that Akt1 plays a critical role in regulating satellite cell proliferation during load-induced muscle hypertrophy.

TSC2/Rheb signaling mediates ERK-dependent regulation of mTORC1 activity in C2C12 myoblasts.

The enhanced rate of protein synthesis in skeletal muscle cells results in a net increase in total protein content that leads to skeletal muscle growth/hypertrophy. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent regulation of the activity of mechanistic target of rapamycin (mTOR) and subsequent protein synthesis has been suggested as a regulatory mechanism; however, the exact molecular processes underlying such a regulation are poorly defined. The purpose of this study was to investigate regulatory mechanisms involved in the MEK/ERK-dependent pathway leading to mTORC1 activation in skeletal muscle cells. Treatment with phorbol-12-myristate-13-acetate (PMA), a potent agonist of protein kinase C (PKC) and its downstream effector in the MEK/ERK-dependent pathway, resulted in the activation of mTORC1 signaling and phosphorylation of the upstream regulator tuberous sclerosis 2 (TSC2) in C2C12 myoblasts. PMA-induced activation of mTORC1 signaling was partially prevented by treatment with U0126 (a selective inhibitor of MEK1/2) or BIX-02189 (a selective inhibitor of MEK5) and completely blocked with BIM-I (a selective inhibitor of upstream PKC). TSC2 phosphorylation at Ser664 (an ERK-dependent phosphorylation site) was prevented with U0126, and BIM-I treatment blocked PMA-induced phosphorylation of TSC2 at multiple residues (Ser664, Ser939, and Thr1462). Overexpression of Ras homolog enriched in brain (Rheb), a downstream target of TSC2, and an mTORC1 activator, was sufficient to activate mTORC1 signaling. We also identified that PMA-induced activation of mTORC1 signaling was significantly inhibited in the absence of Rheb with siRNA knockdown. These observations demonstrate that the PKC/MEK/ERK-dependent activation of mTORC1 is mediated through TSC2 phosphorylation and its downstream target Rheb in C2C12 myoblasts.

Age-associated disruption of molecular clock expression in skeletal muscle of the spontaneously hypertensive rat.

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling.

The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 ß, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2­3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2.

Insulin like growth factor-1-induced phosphorylation and altered distribution of tuberous sclerosis complex (TSC)1/TSC2 in C2C12 myotubes.

Insulin like growth factor-1 (IGF-1) is established as an anabolic factor that can induce skeletal muscle growth by activating the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Although this signaling pathway has been the subject of much study, the molecular mechanisms linking IGF-1 binding to mTOR activation remain poorly defined in muscle. The present study aimed to test the hypothesis that IGF-1 activation of mTOR in C2C12 myotubes requires a phosphorylation-dependent, altered distribution of the tuberous sclerosis complex (TSC)1/TSC2 complex from the membrane to the cytosol. We found that IGF-1 treatment does not affect complex formation between TSC1 and TSC2, but rather IGF-1 induces an altered distribution of the TSC1/TSC2 complex in C2C12 myotubes. In response to IGF-1 treatment, there was a relative redistribution of the TSC1/TSC2 complex, composed of TSC1 and phosphorylated TSC2, from the membrane to the cytosol. IGF-1-stimulated TSC1/TSC2 phosphorylation and redistribution were completely prevented by the phosphoinositide 3-kinase inhibitor wortmannin, but were not with the downstream mTOR inhibitor, rapamycin. When a nonphosphorylatable form of TSC2 (S939A) was overexpressed, phosphorylation-dependent binding of the scaffold protein 14-3-3 to TSC2 was diminished and no redistribution of the TSC1/TSC2 complex was observed after IGF-1 stimulation. These results indicate that TSC2 phosphorylation in response to IGF-1 treatment is necessary for the altered distribution of the TSC1/TSC2 complex to the cytosol. We suggest that this translocation is likely critical for mTOR activation by dissociating the interaction between the GTPase activating protein activity of the TSC1/TSC2 complex and its downstream target, Ras homolog enriched in brain.

REDD2 is enriched in skeletal muscle and inhibits mTOR signaling in response to leucine and stretch.

The protein kinase mammalian target of rapamycin (mTOR) is well established as a key regulator of skeletal muscle size. In this study, we determined that the stress responsive gene REDD2 (regulated in development and DNA damage responses 2) is a negative regulator of mTOR signaling and is expressed predominantly in skeletal muscle. Overexpression of REDD2 in muscle cells significantly inhibited basal mTOR signaling and diminished the response of mTOR to leucine addition or mechanical stretch. The inhibitory function of REDD2 on mTOR signaling seems to be mediated downstream or independent of Akt signaling and upstream of Rheb (Ras homolog enriched in brain). Knock down of tuberous sclerosis complex 2 (TSC2) using small interfering (si)RNA potently activated mTOR signaling and was sufficient to rescue REDD2 inhibition of mTOR activity, suggesting that REDD2 functions by modulating TSC2 function. Immunoprecipitation assays demonstrated that REDD2 does not directly interact with either TSC1 or TSC2. However, we found that REDD2 forms a complex with 14-3-3 protein and that increasing expression of REDD2 acts to competitively dissociate TSC2 from 14-3-3 and inhibits mTOR signaling. These findings demonstrate that REDD2 is a skeletal muscle specific inhibitory modulator of mTOR signaling and identify TSC2 and 14-3-3 as key molecular links between REDD2 and mTOR function.

Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals.

Growth and maintenance of skeletal muscle mass is critical for long-term health and quality of life. Skeletal muscle is a highly adaptable tissue with well-known sensitivities to environmental cues such as growth factors, cytokines, nutrients, and mechanical loading. All of these factors act at the level of the cell and signal through pathways that lead to changes in phenotype through multiple mechanisms. In this review, we discuss the animal and cell culture models used and the signaling mechanisms identified in understanding regulation of protein synthesis in response to mechanical loading/resistance exercise. Particular emphasis has been placed on 1) alterations in mechanical loading and regulation of protein synthesis in both in vivo animal studies and in vitro cell culture studies and 2) upstream mediators regulating mammalian target of rapamycin signaling and protein synthesis during skeletal muscle hypertrophy.

Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis.

Muscle growth is associated with an activation of the mTOR signaling pathway and satellite cell regulators. The purpose of this study was to determine whether 17 selected genes associated with mTOR/muscle protein synthesis and the satellite cells/myogenic program are differentially expressed in young and older human skeletal muscle at rest and in response to a potent anabolic stimulus [resistance exercise + essential amino acid ingestion (RE+EAA)]. Twelve male subjects (6 young, 6 old) completed a bout of heavy resistance exercise. Muscle biopsies were obtained before and at 3 and 6 h post RE+EAA. Subjects ingested leucine-enriched essential amino acids at 1 h postexercise. mRNA expression was determined using qRT-PCR. At rest, hVps34 mRNA was elevated in the older subjects (P < 0.05) while there was a tendency for levels of myoD, myogenin, and TSC2 mRNA to be higher than young. The anabolic stimulus (RE+EAA) altered mRNAs associated with mTOR regulation. Notably, REDD2 decreased in both age groups (P < 0.05) but the expression of Rheb mRNA increased only in the young. Finally, cMyc mRNA was elevated (P < 0.05) in both young and old at 6 h post RE+EAA. Furthermore, RE+EAA also increased expression of several mRNAs associated with satellite function in the young (P < 0.05), while expression of these mRNAs did not change in the old. We conclude that several anabolic genes in muscle are more responsive in young men post RE+EAA. Our data provide new insights into the regulation of genes important for transcription and translation in young and old human skeletal muscle post RE+EAA.