Quercetin glycosides prevent dexamethasone-induced muscle atrophy in mice

Although quercetin has numerous biological benefits, including preventing muscle atrophy due to disuse, no reports have been published to date about the preventive effects and molecular mechanisms underlying drug-induced muscle atrophy. Highly soluble and bioavailable quercetin glycosides (QGs) were used to examine the inhibition of dexamethasone (DEX)-induced muscle atrophy in vivo. Male BALB/cCrSlc mice were treated with or without QGs for 7 days ad libitum, followed by addition of DEX to their drinking water for a further 7 days. The weight of gastrocnemius (GM) adjusted by body weight was significantly decreased on day 7 after DEX treatment. DEX-induced decrease of GM weight was improved by QG co-administration on day 7. The mRNA levels of muscle atrophy-related genes in the gastrocnemius were significantly lowered by QGs on day 1. In particular, the expression of myostatin, a master regulator of muscle mass homeostasis, was suppressed to that of the control level. In murine C2C12 myotubes, quercetin elevated the phosphorylation of Akt, which are downstream of the myostatin pathway, as well as expression of atrogenes. We demonstrated the protective effect of QGs in DEX-induced muscle atrophy, which might depend on the suppression of myostatin signaling.     

Involvement of tyrosine hydroxylase up regulation in dexamethasone-induced hypertension of rats

We investigated the effect of dexamethasone (DEX) on tyrosine hydroxylase (TH) mRNA level, and TH activity and catecholamine levels in the adrenal medulla of the rat. DEX (1 mg/kg/day, s.c.) was administered for 2 days, and a control group was given corn oil. DEX significantly increased systolic blood pressure. TH mRNA level, TH activity, epinephrine level, and norepinephrine level in the adrenal medulla of DEX-treated rats were significantly higher than those of control rats. Also, epinephrine and norepinephrine levels in plasma were significantly higher in DEX-treated rats than in controls. alpha-Methyl-p-tyrosine prevented the DEX-induced blood pressure increase. These results suggest that the catecholamine synthetic pathway may be involved in DEX-induced hypertension.     

Autophagic-lysosomal pathway functions in the masseter and tongue muscles in the klotho mouse, a mouse model for aging

Klotho mutant (kl/kl) mice, a type of short-lived mouse models, display several aging-related phenotypes. To investigate whether the atrophy of skeletal muscles is induced in these mice via activation of the ubiquitin-proteasomal pathway and/or the autophagic-lysosomal pathway through an alteration of insulin/IGF-I signaling, we analyzed the activity of the two pathways for protein degradation and components of the insulin/IGF signaling pathway in their skeletal muscles. The masseter, tongue, and gastrocnemius muscles in kl/kl showed marked reductions in muscle weight and in myofiber diameter compared with +/+. The autophagic-lysosomal pathway in kl/kl was activated in the masseter and tongue, but not in the gastrocnemius, compared with that in +/+, whereas the ubiquitin-proteasomal pathway in these three muscles of kl/kl was not altered. No marked difference in the phosphorylation levels of insulin/IGF-I signaling components, such as insulin/IGF-I receptor, Akt, and FoxO in three muscles studied were found between kl/kl and +/+, but the phosphorylation levels of signaling component at the downstream of mTOR such as 4E-BP1 and p70 S6K were suppressed in the masseter and tongue of kl/kl compared with +/+. Deficiency of essential amino acids is reported to activate the autophagy-lysosomal pathway through the down-regulation of mTOR, not through IGF-Akt-FoxO. The masseter and tongue seem to be more actively moved than limb muscles in kl/kl, because they are essential for survival activities such as mastication, swallowing, and respiration. Thus, the deficiency of amino acid by the active movement of the masseter and tongue seems to stimulate the autophagic-lysosomal pathway via the down-regulation of mTOR signalling pathway.     

Changes in PKB/Akt and calcineurin signaling during recovery in atrophied soleus muscle induced by unloading

Protein kinase B [PKB, also known as Akt (PKB/Akt)] and calcineurin (CaN) are postulated to play important roles in integrating intracellular signaling in skeletal muscle in response to disuse and increased muscle loading. These experiments investigated changes in signal transduction of the downstream pathways of PKB/Akt and CaN during recovery following disuse-induced muscle atrophy. A 10-day period of hindlimb unloading (HLU) via tail suspension (male rats) was used to produce soleus muscle atrophy. Muscle recovery was achieved by returning animals to normal ambulation for 3-10 days. HLU resulted in significant muscle atrophy and a slow-to-fast fiber transition as revealed by appearance of type IId/x and IIb myosin heavy chain (MHC) isoforms. Muscle mass in HLU animals recovered to control (Con) levels after 10 days of reloading, but the fast-to-slow shift in muscle MHC was incomplete, as indicated by the continued presence of type IId/x MHC. Ten days of HLU resulted in a significant decrease (-43%) in muscle levels of phosphorylated PKB/Akt. In contrast, muscle levels of phosphorylated PKB/Akt were greater (+56%) in HLU than in Con animals early after the onset of reloading (3 days). Soleus levels of phosphorylated p70S6K were significantly higher (+26%) in HLU animals after 3 days of muscle reloading. Muscle levels of phosphorylated PKB/Akt and phosphorylated p70S6K returned to Con levels by day 10 of recovery. Moreover, muscle CaN levels were significantly higher than Con levels after 10 days of muscle reloading. These findings are consistent with the hypothesis that PKB/Akt and its downstream mediators are active in the regrowth of muscle mass during the early periods of recovery from muscle atrophy. Our data support the concept that CaN is involved in muscle remodeling during the later phases of recovery from disuse muscle atrophy.     

Myosin isoform expression and MAFbx mRNA levels in hibernating golden-mantled ground squirrels (Spermophilus lateralis)

Hibernating mammals present many unexplored opportunities for the study of muscle biology. The hindlimb muscles of a small rodent hibernator (Spermophilus lateralis) atrophy slightly during months of torpor, representing a reduction in the disuse atrophy commonly seen in other mammalian models. How torpor affects contractile protein expression is unclear; therefore, we examined the myosin heavy-chain (MHC) isoform profile of ground squirrel skeletal muscle before and after hibernation. Immunoblotting was performed first to identify the MHC isoforms expressed in this species. Relative percentages of MHC isoforms in individual muscles were then measured using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The soleus and diaphragm did not display differences in isoforms following hibernation, but we found minor fast-to-slow isoform shifts in MHC protein in the gastrocnemius and plantaris. These subtle changes are contrary to those predicted by other models of inactivity but may reflect the requirement for shivering thermogenesis during arousals from torpor. We also measured mRNA expression of the Muscle Atrophy F-box (MAFbx), a ubiquitin ligase important in proteasome-mediated proteolysis. Expression was elevated in the hibernating gastrocnemius and the plantaris but was not associated with atrophy. Skeletal muscle from hibernators displays unusual plasticity, which may be a combined result of the intense activity during arousals and the reduction of metabolism during torpor.     

Effects of insulin-like growth factor 1 on muscle atrophy and motor function in rats with brain ischemia

Although insulin-like growth factor 1 (IGF 1) has been used in immobilizated muscles to prevent muscle atrophy, its effects on muscle atrophy after brain ischemia are not known. This study aimed to determine the effects of IGF 1 on preventing muscle atrophy in rats with brain ischemia. Middle cerebral artery occlusion (MCAO) was used to induce the brain ischemia. In the first part of the study, rats were assigned to sham control, ischemic control, and ischemia with different dosages of IGF 1 injection groups to determine the optimal dosage of IGF 1 on preventing muscle atrophy after brain ischemia. In the second part of the study, rats were assigned to sham control, ischemic control, ischemia with IGF 1, or with IGF 1 receptor inhibitor (AG1024) injection groups to determine the specificity of IGF 1 on preventing muscle atrophy after brain ischemia. IGF 1 or AG1024 was injected locally to calf muscles and anterior tibialis (TA) starting from one day after brain ischemia and injections were carried out every other day for 4 times. Muscle weight and myosin heavy chain (MHC) expression in both red (red gastrocnemius and soleus) and white (white gastrocnemius and TA) muscles were significantly decreased after brain ischemia. With at least moderate-dosage (200 ng/100 microl PBS) IGF 1 injection, the muscle weight and MHC protein could be restored in both red and white muscles resulting in better motor performance. However, the high-dose injection of IGF 1 (400 ng/100 microl PBS) did not result in further effects. IGF 1 increased the expression of p-Akt, but such effects were prevented by AG1024 resulting in muscle atrophy and poor motor function. In conclusion, peripheral application of IGF 1 not only prevented muscle atrophy but also enhanced motor function in rats with brain ischemia. The IGF 1-induced PI3K/Akt pathways are important for preventing muscle atrophy induced by brain ischemia.     

Leucine Supplementation Improves Acquired Growth Hormone Resistance in Rats with Protein-Energy Malnutrition

Background

Protein-energy malnutrition (PEM) can lead to growth hormone (GH) resistance. Leucine supplementation diets have been shown to increase protein synthesis in muscles. Our study aimed at investigating if long-term leucine supplementation could modulate GH-insulin-like growth factor (IGF)-1 system function and mammalian target of rapamycin (mTOR)-related signal transduction in skeletal muscles in a rat model of severe malnutrition.

Methodology/Principal Findings

Male Sprague-Dawley rats (n = 50; weight, 302 ± 5 g) were divided into 5 treatment groups, including 2 control groups (a normal control group that was fed chow and ad libitum water [CON, n = 10] and a malnourished control group [MC, n = 10] that was fed a 50% chow diet). After undergoing a weight loss stage for 4 weeks, rats received either the chow diet (MC-CON, n = 10), the chow diet supplemented with low-dose leucine (MC-L, n = 10), or the chow diet supplemented with high-dose leucine (MC-H, n = 10) for 2 weeks. The muscle masses of the gastrocnemius, soleus, and extensor digitorum longus were significantly reduced in the MC group. Re-feeding increased muscle mass, especially in the MC-L and MC-H groups. In the MC group, serum IGF-1, IGF-binding protein (IGFBP)-3, and hepatic growth hormone receptor (GHR) levels were significantly decreased and phosphorylation of the downstream anabolic signaling effectors protein kinase B (Akt), mTOR, and ribosomal protein S6 kinase 1 (S6K1) were significantly lower than in other groups. However, serum IGF-1 and IGF binding protein (IGFBP)-3 concentrations and hepatic growth hormone receptor (GHR) levels were significantly higher in the MC-L and MC-H groups than in the MC-CON group, and serum IGFBP-1 levels was significantly reduced in the MC-L and MC-H groups. These changes were consistent with those observed for hepatic mRNA expression levels. Phosphorylation of the downstream anabolic signaling effectors Akt, mTOR, and S6K1 were also significantly higher in the MC-L and MC-H groups than in the MC-CON group.

Conclusion/Significance

Our data are the first to demonstrate that long-term supplementation with leucine improved acquired growth hormone resistance in rats with protein-energy malnutrition. Leucine might promote skeletal muscle protein synthesis by regulating downstream anabolic signaling transduction.     

Chronic paraplegia-induced muscle atrophy downregulates the mTOR/S6K1 signaling pathway.

Ribosomal S6 kinase 1 (S6K1) is a downstream component of the mammalian target of rapamycin (mTOR) signaling pathway and plays a regulatory role in translation initiation, protein synthesis, and muscle hypertrophy. AMP-activated protein kinase (AMPK) is a cellular energy sensor, a negative regulator of mTOR, and an inhibitor of protein synthesis. The purpose of this study was to determine whether the hypertrophy/cell growth-associated mTOR pathway was downregulated during muscle atrophy associated with chronic paraplegia. Soleus muscle was collected from male Sprague-Dawley rats 10 wk following complete T(4)-T(5) spinal cord transection (paraplegic) and from sham-operated (control) rats. We utilized immunoprecipitation and Western blotting techniques to measure upstream [AMPK, Akt/protein kinase B (PKB)] and downstream components of the mTOR signaling pathway [mTOR, S6K1, SKAR, 4E-binding protein 1 (4E-BP1), and eukaryotic initiation factor (eIF) 4G and 2alpha]. Paraplegia was associated with significant soleus muscle atrophy (174 +/- 8 vs. 240 +/- 13 mg; P < 0.05). There was a reduction in phosphorylation of mTOR, S6K1, and eIF4G (P < 0.05) with no change in Akt/PKB or 4E-BP1 (P > 0.05). Total protein abundance of mTOR, S6K1, eIF2alpha, and Akt/PKB was decreased, and increased for SKAR (P < 0.05), whereas 4E-BP1 and eIF4G did not change (P > 0.05). S6K1 activity was significantly reduced in the paraplegic group (P < 0.05); however, AMPKalpha2 activity was not altered (3.5 +/- 0.4 vs. 3.7 +/- 0.5 pmol x mg(-1) x min(-1), control vs. paraplegic rats). We conclude that paraplegia-induced muscle atrophy in rats is associated with a general downregulation of the mTOR signaling pathway. Therefore, in addition to upregulation of atrophy signaling during muscle wasting, downregulation of muscle cell growth/hypertrophy-associated signaling appears to be an important component of long-term muscle loss.     

Denervation atrophy is independent from Akt and mTOR activation and is not rescued by myostatin inhibition

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.KEY WORDS: Skeletal muscle, Muscle atrophy pathophysiology, TGF-β signaling, Myostatin, Denervation atrophy     

Cortisone and dexamethasone inhibit myogenesis by modulating the AKT/mTOR signaling pathway in C2C12

Myogenesis occurs in both the prenatal and postnatal periods and the prenatal myogenesis is related to the postnatal myogenesis and the incidence of disease later in life. Glucocorticoids used as therapeutic agents for many diseases, but cause adverse effects on muscle homeostasis, including defects in fetal muscle development. The action of glucocorticoids on differentiated skeletal muscle was well studied, but their effects on myotube formation have not been well investigated. Dexamethasone (DEX) and cortisone (COR), two synthetic therapeutic glucocorticoids, suppress myotube formation in C2C12 cells. Both COR and DEX attenuated myotube formation through modulation of myogenic regulatory factors. In addition, they affected the IGF/PI3K/AKT/mTOR signaling pathway, resulting in increased proteolytic protein (atrogin-1 and MURF1) for muscle degradation and decreased ribosomal S6 phosphorylation. The current results conclude that COR and DEX inhibit myotube formation in C2C12 cells by modulating both the myogenic program via MRFs and protein metabolism via IGF/PI3K/AKT/mTOR signaling pathway.     

Tau phosphorylation and μ-calpain activation mediate the dexamethasone-induced inhibition on the insulin-stimulated Akt phosphorylation

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.     

Atrophy of S6K1(-/-) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control

The mammalian target of rapamycin (mTOR) and Akt proteins regulate various steps of muscle development and growth, but the physiological relevance and the downstream effectors are under investigation. Here we show that S6 kinase 1 (S6K1), a protein kinase activated by nutrients and insulin-like growth factors (IGFs), is essential for the control of muscle cytoplasmic volume by Akt and mTOR. Deletion of S6K1 does not affect myoblast cell proliferation but reduces myoblast size to the same extent as that observed with mTOR inhibition by rapamycin. In the differentiated state, S6K1(-/-) myotubes have a normal number of nuclei but are smaller, and their hypertrophic response to IGF1, nutrients and membrane-targeted Akt is blunted. These growth defects reveal that mTOR requires distinct effectors for the control of muscle cell cycle and size, potentially opening new avenues of therapeutic intervention against neoplasia or muscle atrophy.     

Effect of hypoxia exposure on the recovery of skeletal muscle phenotype during regeneration

Hypoxia impairs the muscle fibre-type shift from fast-to-slow during post-natal development; however, this adaptation could be a consequence of the reduced voluntary physical activity associated with hypoxia exposure rather than the result of hypoxia per se. Moreover, muscle oxidative capacity could be reduced in hypoxia, particularly when hypoxia is combined with additional stress. Here, we used a model of muscle regeneration to mimic the fast-to-slow fibre-type conversion observed during post-natal development. We hypothesised that hypoxia would impair the recovery of the myosin heavy chain (MHC) profile and oxidative capacity during muscle regeneration. To test this hypothesis, the soleus muscle of female rats was injured by notexin and allowed to recover for 3, 7, 14 and 28 days under normoxia or hypobaric hypoxia (5,500 m altitude) conditions. Ambient hypoxia did not impair the recovery of the slow MHC profile during muscle regeneration. However, hypoxia moderately decreased the oxidative capacity (assessed from the activity of citrate synthase) of intact muscle and delayed its recovery in regenerated muscle. Hypoxia transiently increased in both regenerated and intact muscles the content of phosphorylated AMPK and Pgc-1α mRNA, two regulators involved in mitochondrial biogenesis, while it transiently increased in intact muscle the mRNA level of the mitophagic factor BNIP3. In conclusion, hypoxia does not act to impair the fast-to-slow MHC isoform transition during regeneration. Hypoxia alters the oxidative capacity of intact muscle and delays its recovery in regenerated muscle; however, this adaptation to hypoxia was independent of the studied regulators of mitochondrial turn-over.     

MiR-122 Inhibits Cell Proliferation and Tumorigenesis of Breast Cancer by Targeting IGF1R

miRNAs are emerging as critical regulators in carcinogenesis and tumor progression. Recently, microRNA-122 (miR-122) has been proved to play an important role in hepatocellular carcinoma, but its functions in the context of breast cancer (BC) remain unknown. In this study, we report that miR-122 is commonly downregulated in BC specimens and BC cell lines with important functional consequences. Overexpression of miR-122 not only dramatically suppressed cell proliferation, colony formation by inducing G1-phase cell-cycle arrest in vitro, but also reduced tumorigenicity in vivo. We then screened and identified a novel miR-122 target, insulin-like growth factor 1 receptor (IGF1R), and it was further confirmed by luciferase assay. Overexpression of miR-122 would specifically and markedly reduce its expression. Similar to the restoring miR-122 expression, IGF1R downregulation suppressed cell growth and cell-cycle progression, whereas IGF1R overexpression rescued the suppressive effect of miR-122. To identify the mechanisms, we investigated the Akt/mTOR/p70S6K pathway and found that the expression of Akt, mTOR and p70S6K were suppressed, whereas re-expression of IGF1R which did not contain the 3′UTR totally reversed the inhibition of Akt/mTOR/p70S6K signal pathway profile. We also identified a novel, putative miR-122 target gene, PI3CG, a member of PI3K family, which further suggests miR-122 may be a key regulator of the PI3K/Akt pathway. In clinical specimens, IGF1R was widely overexpressed and its mRNA levels were inversely correlated with miR-122 expression. Taken together, our results demonstrate that miR-122 functions as a tumor suppressor and plays an important role in inhibiting the tumorigenesis through targeting IGF1R and regulating PI3K/Akt/mTOR/p70S6K pathway. Given these, miR-122 may serve as a novel therapeutic or diagnostic/prognostic-target for treating BC.