Expression Of Muscle-specific Ubiquitin Ligases Research Articles

Ginsenoside Rg3 upregulates myotube formation and mitochondrial function, thereby protecting myotube atrophy induced by tumor necrosis factor-alpha

Ethnopharmacological relevanceGinsenoside Rg3 from Panax ginseng has reported to have multiple pharmacological activities including anti-diabetics, anti-inflammation and anti-cancer. However, the effect of ginsenoside Rg3 on myogenic differentiation and muscle atrophy is unknown. Aim to the studyIn this study, we investigated the myogenic effect and underlying molecular mechanisms of ginsenoside Rg3 on myotube atrophy induced by tumor necrosis factor-α (TNF-α). Materials and methodsC2C12 myoblasts were induced to differentiate for one day followed by the treatment of TNF-α along with vehicle or ginsenoside Rg3 for additional 2 days and subjected to immunoblotting, immunocytochemistry, quantitative RT-PCR and biochemical analysis for mitochondrial function. ResultsGinsenoside Rg3 promotes myogenic differentiation and multinucleated myotube formation through Akt activation in a dose-dependent manner, without any cytotoxicity. Ginsenoside Rg3 treatment restores myotube formation and increases myotube diameters under TNF-α-treated conditions. Ginsenoside Rg3 enhances Akt/mTOR (mammalian target of rapamycin) signaling that in turn stimulates muscle-specific gene expression such as myosin heavy chain (MHC) and Myogenin, and suppresses the expression of muscle-specific ubiquitin ligases. In addition, ginsenoside Rg3 in TNF-α-treated myotubes significantly inhibits the production of mitochondrial ROS and restores mitochondrial membrane potential (MMP) and ATP contents. Furthermore, ginsenoside Rg3 upregulates the activities and expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) and the mitochondrial biogenetic transcription factors, nuclear respiratory factor-1 (NRF1) and mitochondrial transcription factor A (Tfam) in TNF-α-induced myotube atrophy. ConclusionsThis study provides a mechanistic insight into the effect of ginsenoside Rg3 on myogenic differentiation and myotube atrophy, suggesting that ginsenoside Rg3 has a promising potential as a therapeutic or neutraceutical remedy to intervene muscle weakness and atrophy.

Osteolytic Breast Cancer Causes Skeletal Muscle Weakness in an Immunocompetent Syngeneic Mouse Model.

Muscle weakness and cachexia are significant paraneoplastic syndromes of many advanced cancers. Osteolytic bone metastases are common in advanced breast cancer and are a major contributor to decreased survival, performance, and quality of life for patients. Pathologic fracture caused by osteolytic cancer in bone (OCIB) leads to a significant (32%) increased risk of death compared to patients without fracture. Since muscle weakness is linked to risk of falls which are a major cause of fracture, we have investigated skeletal muscle response to OCIB. Here, we show that a syngeneic mouse model of OCIB (4T1 mammary tumor cells) leads to cachexia and skeletal muscle weakness associated with oxidation of the ryanodine receptor and calcium (Ca2+) release channel (RyR1). Muscle atrophy follows known pathways via both myostatin signaling and expression of muscle-specific ubiquitin ligases, atrogin-1 and MuRF1. We have identified a mechanism for skeletal muscle weakness due to increased oxidative stress on RyR1 via NAPDH oxidases [NADPH oxidase 2 (Nox2) and NADPH oxidase 4 (Nox4)]. In addition, SMAD3 phosphorylation is higher in muscle from tumor-bearing mice, a critical step in the intracellular signaling pathway that transmits TGFβ signaling to the nucleus. This is the first time that skeletal muscle weakness has been described in a syngeneic model of OCIB and represents a unique model system in which to study cachexia and changes in skeletal muscle.

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Dietary Supplementation with Isoflavones Prevents Muscle Wasting in Tumor-Bearing Mice.

Proinflammatory cytokines contribute to the progression of muscle wasting caused by ubiquitin-proteasome-dependent proteolysis. We have previously demonstrated that isoflavones, such as genistein and daidzein, prevent TNF-α-induced muscle atrophy in C2C12 myotubes. In this study, we examined the effect of dietary flavonoids on the wasting of muscle. Mice were divided into the following four groups: vehicle-injected (control) mice fed the normal diet (CN); tumor-bearing mice fed the normal diet (TN); control mice fed the isoflavone diet (CI); and tumor-bearing mice fed the isoflavone diet (TI). There were no significant differences in the intake of food or body weight gain among these four groups. The wet weight and myofiber size of gastrocnemius muscle in TN significantly decreased, compared with those in CN. Interestingly, the wet weight and myofiber size of gastrocnemius muscle in TI were nearly the same as those in CN and CI, although isoflavone supplementation did not affect the increased tumor mass or concentrations of proinflammatory cytokines, such as TNF-α and IL-6, in the blood. Moreover, increased expression of muscle-specific ubiquitin ligase genes encoding MAFbx/Atrogin-1 and MuRF1 in the skeletal muscle of TN was significantly inhibited by the supplementation of isoflavones. In parallel with the expression of muscle-specific ubiquitin ligases, dietary isoflavones significantly suppressed phosphorylation of ERK in tumor-bearing mice. These results suggest that dietary isoflavones improve muscle wasting in tumor-bearing mice via the ERK signaling pathway mediated-suppression of ubiquitin ligases in muscle cells.

Early changes of muscle insulin‐like growth factor‐1 and myostatin gene expression in gastric cancer patients

Cachexia increases morbidity and mortality of cancer patients. The progressive loss of muscle mass negatively affects physical function and quality of life. We previously showed reduced muscle insulin-like growth factor-1 (IGF-1) expression and enhanced myostatin signaling in tumor-bearing animals. This study was aimed at investigating whether similar perturbations occur in gastric cancer patients. Early perturbations of myostatin and IGF-1 signaling (including the expression of muscle-specific ubiquitin ligases) were investigated in 16 gastric cancer patients and in 6 controls by analyzing muscle mRNA expression with semiquantitative reverse transcriptase polymerase chain reaction (PCR) and real-time PCR. In gastric cancer patients, muscle mRNA levels for IGF-1, myostatin, and atrogin-1 were reduced irrespective of weight loss (≤5% or >5%), whereas MuRF1 expression was unchanged. IGF-1 and myostatin mRNA levels are downregulated in gastric cancer patients who have minimal or no weight loss. These early alterations are particularly relevant in order to devise preventive and therapeutic strategies for cancer cachexia.

Calpain inhibition attenuated morphological and molecular changes in skeletal muscle of experimental allergic encephalomyelitis rats

Muscle weakness and atrophy are important manifestations of multiple sclerosis (MS). To investigate the pathophysiological mechanisms of skeletal muscle change in MS, we induced experimental autoimmune encephalomyelitis (EAE) in Lewis male rats and examined morphological and molecular changes in skeletal muscle. We also treated EAE rats with calpepetin, a calpain inhibitor, to examine its beneficial effects on skeletal muscle damage. Morphological changes in muscle tissue of EAE rats included smaller and irregularly shaped muscle fibers and fibrosis. Western blot analysis demonstrated increased calpain:calpastatin ratio, inflammation-related transcription factors (nuclear factor-κB:inhibitor of κB α ratio), and proinflammatory enzymes (cyclooxygenase-2). TUNEL-positive myonuclei in skeletal muscle cells of EAE rats indicated cell death. In addition, markers of apoptotic cell death (Bax:Bcl-2 ratio and caspase-12 protein levels) were elevated. Expression of muscle-specific ubiquitin ligases (muscle atrophy F-box and muscle ring finger protein 1), was upregulated in muscle tissue of EAE-vehicle animals. Both prophylactic and therapeutic treatment with calpeptin partially attenuated muscle changes noted in EAE animals. These results indicate that morphological and molecular changes including apoptotic cell death and protein breakdown develop in skeletal muscle of EAE animals and that these changes can be reversed by calpain inhibition.

Attenuation of proteolysis and muscle wasting by curcumin c3 complex in MAC16 colon tumour-bearing mice

Muscle wasting or cachexia is caused by accelerated muscle protein breakdown via the ubiquitin-proteasome complex. We investigated the effect of curcumin c3 complex (curcumin c3) on attenuation of muscle proteolysis using in vitro and in vivo models. Our in vitro data indicate that curcumin c3 as low as 0.50 microg/ml was very effective in significantly inhibiting (30 %; P < 0.05) tyrosine release from human skeletal muscle cells, which reached a maximum level of inhibition of 60 % (P < 0.05) at 2.5 microg/ml. Curcumin c3 at 2.5 microg/ml also inhibited chymotrypsin-like 20S proteasome activity in these cells by 25 % (P < 0.05). For in vivo studies, we induced progressive muscle wasting in mice by implanting the MAC16 colon tumour. The in vivo data indicate that low doses of curcumin c3 (100 mg/kg body weight) was able to prevent weight loss in mice bearing MAC16 tumours whereas higher doses of curcumin c3 (250 mg/kg body weight) resulted in approximately 25 % (P < 0.05) weight gain as compared with the placebo-treated animals. Additionally, the effect of curcumin c3 on preventing and/or reversing cachexia was also evident by gains in the weight of the gastrocnemius muscle (30-58 %; P < 0.05) and with the increased size of the muscle fibres (30-65 %; P < 0.05). Furthermore, curcumin inhibited proteasome complex activity and variably reduced expression of muscle-specific ubiquitin ligases: atrogin-1/muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MURF-1). In conclusion, oral curcumin c3 results in the prevention and reversal of weight loss. The data imply that curcumin c3 may be an effective adjuvant therapy against cachexia.