Disuse muscle atrophy-improving effect of ninjin'yoeito in a mouse model.

Disuse syndrome indicates psychosomatic hypofunction caused by excess rest and motionless and muscle atrophy is termed disuse muscle atrophy. Disuse muscle atrophy-induced muscle weakness and hypoactivity further induces muscle atrophy, leading to a vicious cycle, and this is considered a factor causing secondary sarcopenia and subsequently frailty. Since frailty finally leads to a bedridden state requiring nursing, in facing a super-aging society, intervention for a risk factor of frailty, disuse muscle atrophy, is important. However, the main treatment of disuse muscle atrophy is physical therapy and there are fewer effective preventive and therapeutic drugs. The objective of this study was to search for Kampo medicine with a disuse muscle atrophy-improving effect. Ninjin'yoeito is classified as a qi-blood sohozai (dual supplement) in Chinese herbal medicine, and it has an action supplementing the spleen related to muscle. In addition, improvement of muscle mass and muscle weakness by ninjin'yoeito in a clinical study has been reported. In this study, the effect of ninjin'yoeito on disuse muscle atrophy was investigated. A disuse muscle atrophy model was prepared using male ICR mice. After surgery applying a ring for tail suspension, a 1-week recovery period was set. Ninjin'yoeito was administered by mixing it in the diet for 1 week after the recovery period, followed by tail suspension for 14 days. Ninjin'yoeito administration was continued until autopsy including the hindlimb suspension period. The mice were euthanized and autopsied immediately after completion of tail suspension, and the hindlimb muscles were collected. The food and water intakes during the hindlimb unloaded period, wet weight of the collected muscle, and muscle synthesis and muscle degradation-related factors in blood and muscle were evaluated. Ingestion of ninjin'yoeito inhibited tail suspension-induced reduction of the soleus muscle wet weight. In addition, an increase in the blood level of a muscle synthesis-related factor, IGF-1, and promotion of phosphorylation of mTOR and 4E-BP1 in the soleus muscle were observed. It was suggested that ninjin'yoeito has a disuse muscle atrophy-improving action. Promotion of the muscle synthesis pathway was considered the action mechanism of this.

Protein Supplementation Enhances the Effects of Intermittent Loading on Skeletal Muscles by Activating the mTORC1 Signaling Pathway in a Rat Model of Disuse Atrophy.

Inactivity leads to skeletal muscle atrophy, whereas intermittent loading (IL) during hind limb unloading (HU) attenuates muscle atrophy. However, the combined effects of IL and protein supplementation on disuse muscle atrophy are unclear. Therefore, we investigated the effects of IL and a high-protein oral nutritional supplement (HP) during HU on skeletal muscle mass and protein synthesis/breakdown. Male F344 rats were assigned to the control (CON), 14-day HU (HU), IL during HU (HU + IL), and IL during HU followed by HP administration (2.6 g protein/kg/day; HU + IL + HP) groups. Soleus and gastrocnemius muscles were sampled 30 min after the last IL and HP supplementation. HU decreased relative soleus and gastrocnemius muscle masses. Relative muscle masses and p70 ribosomal protein S6 kinase/ribosomal protein S6 phosphorylation in soleus and gastrocnemius muscles were higher in the HU + IL group than the HU group and further higher in the HU + IL + HP group than the HU + IL group in gastrocnemius muscle. Therefore, protein administration plus IL effectively prevented skeletal muscle atrophy induced by disuse, potentially via enhanced activation of targets downstream of mammalian target of rapamycin complex 1 (mTORC1) signaling pathway.

Countering disuse atrophy in older adults with low-volume leucine supplementation.

Older adults are at increased risk of being bedridden and experiencing negative health outcomes including the loss of muscle tissue and functional capacity. We hypothesized that supplementing daily meals with a small quantity (3-4 g/meal) of leucine would partially preserve lean leg mass and function of older adults during bed rest. During a 7-day bed rest protocol, followed by 5 days of inpatient rehabilitation, healthy older men and women (67.8 ± 1.1 yr, 14 men; 6 women) were randomized to receive isoenergetic meals supplemented with leucine (LEU, 0.06 g/kg/meal; n = 10) or an alanine control (CON, 0.06 g/kg/meal; n = 10). Outcomes were assessed at baseline, following bed rest, and after rehabilitation. Body composition was measured by dual-energy X-ray absorptiometry. Functional capacity was assessed by knee extensor isokinetic and isometric dynamometry, peak aerobic capacity, and the short physical performance battery. Muscle fiber type, cross-sectional area, signaling protein expression levels, and single fiber characteristics were determined from biopsies of the vastus lateralis. Leucine supplementation reduced the loss of leg lean mass during bed rest (LEU vs. CON: -423 vs. -1035 ± 143 g; P = 0.008) but had limited impact on strength or endurance-based functional outcomes. Similarly, leucine had no effect on markers of anabolic signaling and protein degradation during bed rest or rehabilitation. In conclusion, providing older adults with supplemental leucine has minimal impact on total energy or protein consumption and has the potential to partially counter some, but not all, of the negative effects of inactivity on muscle health.NEW & NOTEWORTHY Skeletal muscle morphology and function in older adults was significantly compromised by 7 days of disuse. Leucine supplementation partially countered the loss of lean leg mass but did not preserve muscle function or positively impact changes at the muscle fiber level associated with bed rest or rehabilitation. Of note, our data support a relationship between myonuclear content and adaptations to muscle atrophy at the whole limb and single fiber level.

Resveratrol Improves Muscle Atrophy by Modulating Mitochondrial Quality Control in STZ-Induced Diabetic Mice.

SCOPE: In this study, we aim to determine the effects of resveratrol (RSV) on muscle atrophy in streptozocin-induced diabetic mice and to explore mitochondrial quality control (MQC) as a possible mechanism. METHODS AND RESULTS: The experimental mice were fed either a control diet or an identical diet containing 0.04% RSV for 8 weeks. Examinations were subsequently carried out, including the effects of RSV on muscle atrophy and muscle function, as well as on the signaling pathways related to protein degradation and MQC processes. The results show that RSV supplementation improves muscle atrophy and muscle function, attenuates the increase in ubiquitin and muscle RING-finger protein-1 (MuRF-1), and simultaneously attenuates LC3-II and cleaved caspase-3 in the skeletal muscle of diabetic mice. Moreover, RSV treatment of diabetic mice results in an increase in mitochondrial biogenesis and inhibition of the activation of mitophagy in skeletal muscle. RSV also protects skeletal muscle against excess mitochondrial fusion and fission in the diabetic mice. CONCLUSION: The results suggest that RSV ameliorates diabetes-induced skeletal muscle atrophy by modulating MQC.

Membrane lipid rafts are disturbed in the response of rat skeletal muscle to short-term disuse.

Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events that might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6-12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit or fluorescent sterols. In addition, resting intracellular Ca2+ level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na-K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid raft changes in control muscles but was ineffective in suspended muscles, which showed an initial loss of α2 Na-K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in the junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca2+ concentration only near the neuromuscular junction of muscle fibers. Our results provide evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na-K-ATPase. Lipid raft disturbance, accompanied by intracellular Ca2+ dysregulation, is among the earliest remodeling events induced by skeletal muscle disuse.

l-Carnitine supplement reduces skeletal muscle atrophy induced by prolonged hindlimb suspension in rats.

l-Carnitine was recently found to downregulate the ubiquitin proteasome pathway (UPP) and increase insulin-like growth factor 1 concentrations in animal models. However, the effect of l-carnitine administration on disuse muscle atrophy induced by hindlimb suspension has not yet been studied. Thus, we hypothesized that l-carnitine may have a protective effect on muscle atrophy induced by hindlimb suspension via the Akt1/mTOR and/or UPP. Male Wistar rats were assigned to 3 groups: hindlimb suspension group, hindlimb suspension with l-carnitine administration (1250 mg·kg-1·day-1) group, and pair-fed group adjusted hindlimb suspension. l-Carnitine administration for 2 weeks of hindlimb suspension alleviated the decrease in weight and fiber size in the soleus muscle. In addition, l-carnitine suppressed atrogin-1 mRNA expression, which has been reported to play a pivotal role in muscle atrophy. The present study shows that l-carnitine has a protective effect against soleus muscle atrophy caused by hindlimb suspension and decreased E3 ligase messenger RNA expression, suggesting the possibility that l-carnitine protects against muscle atrophy, at least in part, through the inhibition of the UPP. These observations suggest that l-carnitine could serve as an effective supplement in the decrease of muscle atrophy caused by weightlessness in the fields of clinical and rehabilitative research.

Natural Compounds Preventing Neurodegenerative Diseases Through Autophagic Activation.

Neurodegenerative diseases (NDDs) are a group of intractable diseases that significantly affect human health. To date, the pathogenesis of NDDs is still poorly understood and effective disease-modifying therapies for NDDs have not been established. NDDs share the common morphological characteristic of the deposition of abnormal proteins in the nervous system, including neurons. Autophagy is one of the major processes by which damaged organelles and abnormal proteins are removed from cells. Impairment of autophagy has been found to be involved in the pathogenesis of NDDs, and the regulation of autophagy may become a therapeutic strategy for NDDs. In recent years, some active compounds from plants have been found to regulate autophagy and exert neuroprotection against NDDs, including Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal and bulbar muscular atrophy, spinocerebellar ataxia 3, and amyotrophic lateral sclerosis, via activating autophagy. In this paper, we review recent advances in the use of active ingredients from plants for the regulation of autophagy and treatment of NDDs.

The non-thermal effects of pulsed ultrasound irradiation on the development of disuse muscle atrophy in rat gastrocnemius muscle.

This study examined the effects of therapeutic pulsed ultrasound (US) on the development of disuse muscle atrophy in rat gastrocnemius muscle. Male Wistar rats were randomly distributed into control, immobilization (Im), sham US, and US groups. In the Im, sham US and US groups, the bilateral ankle joints of each rat were immobilized in full plantar flexion with a plaster cast for a 4-wk period. The pulsed US (frequency, 1 MHz; intensity, 1.0 W/cm(2); pulsed mode 1:4; 15 min) was irradiated to the gastrocnemius muscle in the US group over a 4-wk immobilization period. The pulsed US irradiation delivered only non-thermal effects to the muscle. In conjunction with US irradiation, 5-bromo-2'-deoxyuridine (BrdU) was injected subcutaneously to label the nuclei of proliferating satellite cells 1 h before each pulsed US irradiation. Immobilization resulted in significant decreases in the mean diameters of type I, IIA and IIB muscle fibers of the gastrocnemius muscle in the Im, sham US and US groups compared with the control group. However, the degrees of muscle fiber atrophy for all types were significantly lower in the US group compared with the Im and sham US groups. Although the number of capillaries and the concentrations of insulin-like growth factor and basic fibroblast growth factor did not change in the muscle, the number of BrdU-positive nuclei in the muscle was significantly increased by pulsed US irradiation in the US group. The results of this study suggest that pulsed US irradiation inhibits the development of disuse muscle atrophy partly via activation of satellite cells.

ß-Hydroxy-ß-methylbutyrate (HMB) enhances the proliferation of satellite cells in fast muscles of aged rats during recovery from disuse atrophy.

Loss of myonuclei by apoptosis is thought to contribute to sarcopenia. We have previously shown, that the leucine metabolite, ß-hydroxy-ß-methylbutyrate (HMB) suppresses apoptotic signaling and the apoptotic index (the ratio of apoptotic positive to apoptotic negative myonuclei) during muscle disuse and during reloading periods after disuse in aged rats. However, it was not clear if the apoptotic signaling indexes were due only to preservation of myonuclei or if perhaps the total myogenic pool increased as a result of HMB-mediated satellite cell proliferation as this would have also reduced the apoptotic index. In this study, we tested the hypothesis that HMB would augment myogenic cells (satellite cells) proliferation during muscle recovery (growth) after a period of disuse in senescent animals. The hindlimb muscles of 34 month old Fisher 344 × Brown Norway rats were unloaded for 14 days by hindlimb suspension (HLS), and then reloaded for 14 days. The rats received either Ca-HMB (340 mg/kg body weight; n = 16), or the vehicle (n = 10) by gavage throughout the experimental period. HMB prevented the functional decline in maximal plantar flexion isometric force production during the reloading period, but not during HLS. HMB-treatment enhanced the proliferation of muscle stem cells as shown by a greater percentage of satellite cells that had proliferated (more BrdU positive, Pax-7 positive, and more Pax7/Ki67 positive nuclei) and as a result, more differentiated stem cells were present (more MyoD/myogenin positive myonuclei), relative to total myonuclei, in reloaded plantaris muscles as compared to reloaded muscles from vehicle-treated animals. Furthermore HMB increased the nuclear protein abundance of proliferation markers, inhibitor of differentiation-2 and cyclin A, as compared to vehicle treatment in reloaded muscles. Although HMB increased phosphorylated Akt during reloading, other mTOR related proteins were not altered by HMB treatment. These data show that HMB improved the proliferation of muscle stem cells in fast twitch plantaris muscles. Enhanced satellite cell proliferation leading to increased differentiated myonuclei should increase the transcriptional potential to support muscle hypertrophic changes and functional changes in sarcopenic muscles, and this could partly explain the reduced apoptotic index in HMB treated muscles. Indeed, muscle mass and fiber cross-sectional area were significantly greater in plantaris muscles from HMB-treated animal muscles after reloading as compared to vehicle-treated animals.

Prevention of muscle disuse atrophy by low-frequency electrical stimulation in rats.

When muscles lose neural drive, they atrophy rapidly. Neuromuscular electrical stimulation (NMS) has been used in attempts to prevent or reverse the atrophy, but optimal stimulation programs and parameters are not well defined. In this study, we investigated the effects of four different stimulation patterns on disuse atrophy produced in the tibialis anterior, lateral gastrocnemius, and soleus muscles of rats paralyzed with tetrodotoxin for seven days. Stimulation paradigms differed from one another by their stimulation frequency (2 or 10 pulses/s) and by their stimulation period (2 or 10 h a day). Results showed that stimulation with 2 pulses/s, paradigms were more effective at preventing disuse muscle atrophy than higher-frequency stimulation. The most marked difference was in the slow soleus muscle, which had only 10% mean atrophy when stimulated at 2 pulses/s for 10 h, compared to 26% atrophy when stimulated at 10 pulses/s for either 2 or 10 h and 32% atrophy in unstimulated, paralyzed controls. The level of atrophic change was not correlated with the levels of serum creatine kinase, used as an index of muscle damage. Results suggest that remediation of disuse atrophy may be accomplished using unphysiologically low rates of motor-unit activation despite the relatively low force produced by such unfused contractions. This may have significant implications for the design of therapies for muscle paralysis consequent to upper-motoneuron lesions.

L-Carnitine: a potential treatment for blocking apoptosis and preventing skeletal muscle myopathy in heart failure.

Skeletal muscle in congestive heart failure is responsible for increased fatigability and decreased exercise capacity. A specific myopathy with increased expression of fast-type myosins, myocyte atrophy, secondary to myocyte apoptosis triggered by high levels of circulating tumor necrosis factor-alpha (TNF-alpha) has been described. In an animal model of heart failure, the monocrotaline-treated rat, we have observed an increase of apoptotic skeletal muscle nuclei. Proapoptotic agents, caspase-3 and -9, were increased, as well as serum levels of TNF-alpha and its second messenger sphingosine. Treatment of rats with L-carnitine, known for its protective effect on muscle metabolism injuries, was found to inhibit caspases and to decrease the levels of TNF-alpha and sphingosine, as well as the number of apoptotic myonuclei. Staurosporine was used in in vitro experiments to induce apoptosis in skeletal muscle cells in culture. When L-carnitine was applied to skeletal muscle cells, before staurosporine treatment, we observed a reduction in apoptosis. These findings show that L-carnitine can prevent apoptosis of skeletal muscles cells and has a role in the treatment of congestive heart failure-associated myopathy.