Heat Stress Modulates Both Anabolic and Catabolic Signaling Pathways Preventing Dexamethasone‐Induced Muscle Atrophy In Vitro

It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid‐induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid‐induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel‐like factor 15 (KLF15). Heat stress recovered the dexamethasone‐induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress‐induced protection against glucocorticoid‐induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. J. Cell. Physiol. 232: 650–664, 2017. © 2016 The Authors. Journal of Cellular Physiology published by Wiley Periodicals, Inc.     

Insulin-like growth factor-1 and muscle wasting in chronic heart failure

Chronic heart failure is a clinical syndrome of cardiac origin, which affects various organ systems. It is associated with metabolic abnormalities leading to a catabolic syndrome in advanced stages of the disease. As in several other chronic diseases, skeletal muscle dysfunction and structural muscle abnormalities result in progressive muscle wasting and cachexia. These changes are accompanied by increased expression of proinflammatory cytokines, increased rate of apoptosis and activation of the proteolytic ubiquitin-proteasome pathway. Further, reduced expression of the local anabolic insulin-like growth factor-1 has been demonstrated in skeletal muscle of animals and patients with chronic heart failure. This suppression occurs in the presence of normal serum levels of insulin-like growth factor-1. In addition to catabolic effects of proinflammatory cytokines, these recent findings are consistent with reduced anabolism involving altered local insulin-like growth factor-1 levels in progressive muscle atrophy in chronic heart failure. This article describes local effects of insulin-like growth factor-1 on skeletal muscle function and morphology, its role in stem cell recruitment and muscle regeneration as well as its regulation in circumstances of muscle inflammation and wasting.     

Insulin action on skeletal muscle protein metabolism during catabolic states

Insulin plays a major role in the regulation of skeletal muscle protein turnover but its mechanism of action is not fully understood, especially in vivo during catabolic states. These aspects are presently reviewed. Insulin inhibits the ATP-ubiquitin proteasome proteolytic pathway which is presumably the predominant pathway involved in the breakdown of muscle protein. Evidence of the ability of insulin to stimulate muscle protein synthesis in vivo was also presented. Many catabolic states in rats, e.g. streptozotocin diabetes, glucocorticoid excess or sepsis-induced cytokines, resulted in a decrease in insulin action on protein synthesis or degradation. The effect of catabolic factors would therefore be facilitated. In contrast, the antiproteolytic action of insulin was improved during hyperthyroidism in man and early lactation in goats. Excessive muscle protein breakdown should therefore be prevented. In other words, the anabolic hormone insulin partly controlled the 'catabolic drive'. Advances in the understanding of insulin signalling pathways and targets should provide information on the interactions between insulin action, muscle protein turnover and catabolic factors.     

Altered REDD1, myostatin, and Akt/mTOR/FoxO/MAPK signaling in streptozotocin-induced diabetic muscle atrophy

Type 1 diabetes, if poorly controlled, leads to skeletal muscle atrophy, decreasing the quality of life. We aimed to search highly responsive genes in diabetic muscle atrophy in a common diabetes model and to further characterize associated signaling pathways. Mice were killed 1, 3, or 5 wk after streptozotocin or control. Gene expression of calf muscles was analyzed using microarray and protein signaling with Western blotting. We identified translational repressor protein REDD1 (regulated in development and DNA damage responses) that increased seven- to eightfold and was associated with muscle atrophy in diabetes. The diabetes-induced increase in REDD1 was confirmed at the protein level. This result was accompanied by the increased gene expression of DNA damage/repair pathways and decreased expression in ATP production pathways. Concomitantly, increased phosphorylation of AMPK and dephosphorylation of the Akt/mTOR/S6K1/FoxO pathway of proteins were observed together with increased protein ubiquitination. These changes were especially evident during the first 3 wk, along with the strong decrease in muscle mass. Diabetes also induced an increase in myostatin protein and decreased MAPK signaling. These, together with decreased serum insulin and increased serum glucose, remained altered throughout the 5-wk period. In conclusion, diabetic myopathy induced by streptozotocin led to alteration of multiple signaling pathways. Of those, increased REDD1 and myostatin together with decreased Akt/mTOR/FoxO signaling are associated with diabetic muscle atrophy. The increased REDD1 and decreased Akt/mTOR/FoxO signaling followed a similar time course and thus may be explained, in part, by increased expression of genes in DNA damage/repair and possibly also decrease in ATP-production pathways.     

Regulation of mTOR and cell growth in response to energy stress by REDD1

The tuberous sclerosis tumor suppressors TSC1 and TSC2 regulate the mTOR pathway to control translation and cell growth in response to nutrient and growth factor stimuli. We have recently identified the stress response REDD1 gene as a mediator of tuberous sclerosis complex (TSC)-dependent mTOR regulation by hypoxia. Here, we demonstrate that REDD1 inhibits mTOR function to control cell growth in response to energy stress. Endogenous REDD1 is induced following energy stress, and REDD1-/- cells are highly defective in dephosphorylation of the key mTOR substrates S6K and 4E-BP1 following either ATP depletion or direct activation of the AMP-activated protein kinase (AMPK). REDD1 likely acts on the TSC1/2 complex, as regulation of mTOR substrate phosphorylation by REDD1 requires TSC2 and is blocked by overexpression of the TSC1/2 downstream target Rheb but is not blocked by inhibition of AMPK. Tetracycline-inducible expression of REDD1 triggers rapid dephosphorylation of S6K and 4E-BP1 and significantly decreases cellular size. Conversely, inhibition of endogenous REDD1 by short interfering RNA increases cell size in a rapamycin-sensitive manner, and REDD1-/- cells are defective in cell growth regulation following ATP depletion. These results define REDD1 as a critical transducer of the cellular response to energy depletion through the TSC-mTOR pathway.     

Krüppel样因子在肌肉组织中的功能研究进展

Krüppel样因子(Krüppel-like factors, KLFs)是一类C-末端含有3个C₂H₂锌指结构的转录因子,N-末端为转录调控结构域,能够结合多种特异蛋白质,介导转录调控。目前在人体基因组中共发现18种KLFs,它们在多种类型人类细胞的分化、表型维持和生理功能调控中发挥重要作用。多个KLFs参与了对人和动物的心肌、平滑肌和骨骼肌的发育和功能的调控。在心肌中,KLF4、KLF10、KLF11和KLF15参与心肌肥大的负调控,KLF6参与调控心脏纤维化,KLF13调控胚胎时期的心肌发育。在血管平滑肌中,KLF4受促增殖或促分化因子调控,介导调控血管平滑肌表型转换;KLF5促进血管平滑肌增殖,KLF8和KLF15抑制血管平滑肌增殖。在骨骼肌中,KLF2、KLF3、KLF4、KLF10和KLF15调控骨骼肌发育,此外,KLF15是肌肉组织能量代谢的调节因子。本文综述了KLFs在心肌、平滑肌和骨骼肌中的功能研究进展,为进一步揭示KLFs在肌肉组织中的作用和肌肉相关疾病的分子机制提供参考。     

Dexamethasone represses signaling through the mammalian target of rapamycin in muscle cells by enhancing expression of REDD1

The mammalian target of rapamycin (mTOR), a critical modulator of cell growth, acts to integrate signals from hormones, nutrients, and growth-promoting stimuli to downstream effector mechanisms involved in the regulation of protein synthesis. Dexamethasone, a synthetic glucocorticoid that represses protein synthesis, acts to inhibit mTOR signaling as assessed by reduced phosphorylation of the downstream targets S6K1 and 4E-BP1. Dexamethasone has also been shown in one study to up-regulate the expression of REDD1 (also referred to RTP801, a novel stress-induced gene linked to repression of mTOR signaling) in lymphoid, but not nonlymphoid, cells. In contrast to the findings of that study, here we demonstrate that REDD1, but not REDD2, mRNA expression is dramatically induced following acute dexamethasone treatment both in rat skeletal muscle in vivo and in L6 myoblasts in culture. In L6 myoblasts, the effect of the drug on mTOR signaling is efficiently blunted in the presence of REDD1 RNA interference oligonucleotides. Moreover, the dexamethasone-induced assembly of the mTOR regulatory complex Tuberin.Hamartin is disrupted in L6 myoblasts following small interfering RNA-mediated repression of REDD1 expression. Finally, overexpression of Rheb, a downstream target of Tuberin function and a positive upstream effector of mTOR, reverses the effect of dexamethasone on phosphorylation of mTOR substrates. Overall, the data support the conclusion that REDD1 functions upstream of Tuberin and Rheb to down-regulate mTOR signaling in response to dexamethasone.     

Molecular characterization of skeletal muscle atrophy in the R6/2 mouse model of Huntington's disease

Huntington's disease (HD), a neurodegenerative disorder caused by mutant huntingtin, is characterized by a catabolic phenotype. To determine the mechanisms underlying muscle wasting, we examined key signal transduction pathways governing muscle protein metabolism, apoptosis, and autophagy in R6/2 mice, a well-characterized transgenic model of HD. R6/2 mice exhibited increased adiposity, elevated energy expenditure, and decreased body weight and lean mass without altered food intake. Severe skeletal muscle wasting accounted for a majority of the weight loss. Protein synthesis was unexpectedly increased 19% in gastrocnemius muscle, which was associated with overactivation of basal and refeeding-stimulated mammalian target of rapamycin (mTOR) signaling, elevated Akt expression and Ser(473) phosphorylation, and decreased AMPK Thr(172) phosphorylation. Moreover, mRNA abundance of atrogenes muscle ring finger-1 and atrophy F-box, was markedly attenuated during fasting and refeeding, and the urinary excretion of 3-methylhistidine was decreased, arguing against a role for the ubiquitin proteasome-mediated proteolysis in the atrophy. In contrast, mRNA expression of several caspase genes and genes involved in the extrinsic or intrinsic apoptotic pathway, caspase-3/7, -8, and -9 activity, protein abundance of caspase-3 and -9, Fas, and Fadd, and cytochrome c release were elevated. Protein expressions of LC3B-I and -II, beclin-I, and atg5 and -7 in muscle were upregulated. Thus, mutant huntingtin in skeletal muscle results in increased protein synthesis and mTOR signaling, which is countered by activation of the apoptotic and autophagic pathways, contributing to an overall catabolic phenotype and the severe muscle wasting.     

Tropomyosin metabolism in muscles and its blood levels during physical exertion

Physical exercise induces phasic changes in the tropomyosin content and metabolism in muscles and its concentration in blood. The intensive catabolic processes (decrease of 14C-leicin inclusion and time of half-life) of muscle tropomyosin and its appearance in blood were shown 2-24 hours after the exercise. Intensive anabolic processes of muscle tropomyosin were found at the late period of rest (72-144 h). These results reveal the biochemical mechanism of muscle adaptation to physical exercise. Data on the tropomyosin content in blood permit recommending tropomyosin for development of the diagnostic test of functional condition of the skeletal muscle.     

Overexpression of interleukin-15 induces skeletal muscle hypertrophy in vitro: implications for treatment of muscle wasting disorders

Interleukin-15 (IL-15) is a novel anabolic factor for skeletal muscle which inhibits muscle wasting associated with cancer (cachexia) in a rat model. To develop a cell culture system in which the mechanism of the anabolic action of IL-15 on skeletal muscle could be examined, the mouse C2 skeletal myogenic cell line was transduced with a retroviral expression vector for IL-15 and compared to sister cells transduced with a control vector. Overexpression of IL-15 induced fivefold higher levels of sarcomeric myosin heavy chain and alpha-actin accumulation in differentiated myotubes. Secreted factors from IL-15-overexpressing myogenic cells, but not from control cells, induced increased myofibrillar protein accumulation in cocultured control myotubes. IL-15 overexpression induced a hypertrophic myotube morphology similar to that described for cultured myotubes which overexpressed the well-characterized anabolic factor insulin-like growth factor-I (IGF-I). However, in contrast to IGF-I, the hypertrophic action of IL-15 on skeletal myogenic cells did not involve stimulation of skeletal myoblast proliferation or differentiation. IL-15 induced myotube hypertrophy at both low and high IGF-I concentrations. Furthermore, in contrast to IGF-I, which stimulated only protein synthesis under these culture conditions, IL-15 both stimulated protein synthesis and inhibited protein degradation in cultured skeletal myotubes. These findings indicate that IL-15 action on skeletal myogenic cells is distinct from that of IGF-I. Due to the ability of IGF-I to stimulate cell division and its association with several forms of cancer, controversy exists concerning the advisability of treating cachexia or age-associated muscle wasting with IGF-I. Administration of IL-15 or modulation of the IL-15 signaling pathway may represent an alternative strategy for maintaining skeletal muscle mass under these conditions.     

Suppression of REDD1 attenuates oxygen glucose deprivation/reoxygenation-evoked ischemic injury in neuron by suppressing mTOR-mediated excessive autophagy

Cerebral ischemia/reperfusion (I/R) typically occurs after mechanical thrombectomy to treat ischemic stroke, generation of reactive oxygen species (ROS) after reperfusion may result in neuronal insult, ultimately leading to disability and death. Regulated in development and DNA damage responses 1 (REDD1) is a conserved stress response protein under various pathogenic conditions. Recent research confirms the controversial role of REDD1 in injury processes. Nevertheless, the role of REDD1 in cerebral I/R remains poorly defined. In the current study, increased expression of REDD1 was observed in neurons exposed to simulated I/R via oxygen glucose deprivation/reoxygenation (OGD/R) treatment. Knockdown of REDD1 enhanced OGD/R-inhibited cell viability, but suppressed lactate dehydrogenase (LDH) release in neurons upon OGD/R. Simultaneously, suppression of REDD1 also antagonized OGD/R-evoked cell apoptosis, Bax expression, and caspase-3 activity. Intriguingly, REDD1 depression abrogated neuronal oxidative stress under OGD/R condition by suppressing ROS, MDA generation, and increasing antioxidant SOD levels. Further mechanism analysis corroborated the excessive activation of autophagy in neurons upon OGD/R with increased expression of autophagy-related LC3 and Beclin-1, but decreased autophagy substrate p62 expression. Notably, REDD1 inhibition reversed OGD/R-triggered excessive neuronal autophagy. More importantly, depression of REDD1 also elevated the expression of p-mTOR. Preconditioning with mTOR inhibitor rapamycin engendered not only a reduction in mTOR activation, but also a reactivation of autophagy in REDD1 knockdown-neurons upon OGD/R. In addition, blocking the mTOR pathway muted the protective roles of REDD1 inhibition against OGD/R-induced neuron injury and oxidative stress. Together these data suggested that REDD1 may regulate I/R-induced oxidative stress injury in neurons by mediating mTOR-autophagy signaling, supporting a promising therapeutic strategy against brain ischemic diseases.