Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)-? treatment. In C2C12 myotubes, TNF-? treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NF?B, a downstream target of the TNF-? signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF-? in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.
Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.
We previously reported the potential involvement of casitas B-cell lymphoma-b (Cbl-b) in aging-related murine insulin resistance. Because obesity also induces macrophage recruitment into adipose tissue, we elucidated here the role of Cbl-b in obesity-related insulin resistance. Cbl-b(+/+) and Cbl-b(-/-) mice were fed a high-fat diet (HFD) and then examined for obesity-related changes in insulin signaling. The HFD caused recruitment of macrophages into adipose tissue and increased inflammatory reaction in Cbl-b(-/-) compared with Cbl-b(+/+) mice. Peritoneal macrophages from Cbl-b(-/-) mice and Cbl-b-overexpressing RAW264.7 macrophages were used to examine the direct effect of saturated fatty acids (FAs) on macrophage activation. In macrophages, Cbl-b suppressed saturated FA-induced Toll-like receptor 4 (TLR4) signaling by ubiquitination and degradation of TLR4. The physiological role of Cbl-b in vivo was also examined by bone marrow transplantation and Eritoran, a TLR4 antagonist. Hematopoietic cell-specific depletion of the Cbl-b gene induced disturbed responses on insulin and glucose tolerance tests. Blockade of TLR4 signaling by Eritoran reduced fasting blood glucose and serum interleukin-6 levels in obese Cbl-b(-/-) mice. These results suggest that Cbl-b deficiency could exaggerate HFD-induced insulin resistance through saturated FA-mediated macrophage activation. Therefore, inhibition of TLR4 signaling is an attractive therapeutic strategy for treatment of obesity-related insulin resistance.
"Hybrid exercise" utilizing combined electrical stimulation and voluntary muscle contraction has been developed as a muscle exercise method. Although our previous studies have confirmed the effectiveness of the procedure, the mechanisms of its efficacy still remain unclear. In the present study, we identified genes that are specifically expressed in disused muscles, using the semitendinosus muscle from patients who underwent anterior cruciate ligament (ACL) reconstruction. Preoperative exercise was performed by four ACL-injured patients, who were subjected either to hybrid exercise (n=2), electrical stimulation (n=1), or no electrical stimulation (n=1), in addition to standard weight training for 4 weeks. Cross-sectional area (CSA) of the semitendinosus muscle was measured before and after the exercise by magnetic resonance imaging (MRI). A piece of the semitendinosus muscle was isolated during the surgery, and comprehensive analysis of the gene expression in this sample was performed using DNA microarray analysis. CSA increased in size by 4.2 and 14.7%, respectively, after hybrid exercise, and by 1.4% after electrical stimulation. However it shrunk by 7.7% without electrical stimulation. DNA microarray analysis revealed that hybrid exercise was more effective at stimulating the expression of signal transduction-, transcription- and cytoskeleton-related genes in semitendinosus muscles than electrical stimulation alone. In particular, gene ontology analysis revealed that hybrid exercise induced significantly higher expression of eukaryotic translation initiation factor 5A (EIFSA), peroxisomal biogenesis factor 6 (PEX6) and histone cluster 1 H4 (HIST1H4), compared with electrical stimulation alone. The expression of signal transduction-, transcription- and cytoskeleton-related genes may play an important role in muscle bulk increasing mechanisms in hybrid exercise.
Influenza A virus (IAV) is one of the most common infectious pathogens in humans. Since the IVA genome does not have the processing protease for the viral hemagglutinin (HA) envelope glycoprotein precursors, entry of this virus into cells and infectious organ tropism of IAV are primarily determined by host cellular trypsin-type HA processing proteases. Several secretion-type HA processing proteases for seasonal IAV in the airway, and ubiquitously expressed furin and pro-protein convertases for highly pathogenic avian influenza (HPAI) virus, have been reported. Recently, other HA-processing proteases for seasonal IAV and HPAI have been identified in the membrane fraction. These proteases proteolytically activate viral multiplication at the time of viral entry and budding. In addition to the role of host cellular proteases in IAV pathogenicity, IAV infection results in marked upregulation of cellular trypsins and matrix metalloproteinase-9 in various organs and cells, particularly endothelial cells, through induced pro-inflammatory cytokines. These host cellular factors interact with each other as the influenza virus-cytokine-protease cycle, which is the major mechanism that induces vascular hyperpermeability and multiorgan failure in severe influenza. This mini-review discusses the roles of cellular proteases in the pathogenesis of IAV and highlights the molecular mechanisms of upregulation of trypsins as effective targets for the control of IAV infection. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.
Deficiency of the Cbl-b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl-b-deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin-injected tibialis anterior muscle of Cbl-b-deficient mice. Ablation of the Cbl-b gene expression delayed regeneration of cardiotoxin-induced skeletal muscle damage compared with wild-type mice. CD8-positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl-b-deficient mice, but there was dispersal of the same cells over that time-frame in wild-type mice. Infiltrating macrophages in Cbl-b-deficient mice showed strong expression of RANTES (regulated-on-activation, normal T cell expressed and secreted), a chemokine for CD8-positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8-positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl-b is an important regulatory factor for cytotoxic T-cell infiltration via RANTES production in macrophages.
Host cellular proteases induce influenza virus entry into cells by cleaving the viral surface envelope glycoprotein hemagglutinin (HA). However, details on the cellular proteases involved in this event are not fully available. We report here that ubiquitous type II transmembrane serine proteases, MSPL and its splice variant TMPRSS13, are novel candidates for proteases processing HA proteins of highly pathogenic avian influenza (HPAI) viruses, apart from the previously identified furin and proprotein convertases 5 and 6. HAs from all HPAI virus H5 and H7 strains have one of two cleavage site motifs, the R-X-K/R-R motif with R at position P4 and the K-K/R-K/T-R motif with K at position P4. In studies of synthetic 14-residue HPAI virus HA peptides with these cleavage site motifs, furin preferentially cleaved only HA peptides with the R-K-K-R motif in the presence of calcium and not peptides with the other motif, whereas MSPL and TMPRSS13 cleaved both types of HA peptides (those with the R/K-K-K-R motif) efficiently in the absence of calcium. Full-length recombinant HPAI virus HA with the K-K-K-R cleavage motif exhibited poor susceptibility to cleavage in the absence of MSPL or TMPRSS13 and the presence of furin in infected cells, but it was converted to mature HA subunits in transfected cells expressing MSPL or TMPRSS13, with membrane-fused giant-cell formation. This conversion and membrane fusion were suppressed by inhibitors of MSPL and TMPRSS13. Furthermore, infection with and multiplication of genetically modified live HPAI virus A/Crow/Kyoto/53/2004 (H5N1) with the K-K-K-R cleavage site motif were detected only in MSPL- and TMPRSS13-expressing cells.
Entry of enveloped viruses into host cells requires the activation of viral envelope glycoproteins through cleavage by either intracellular or extracellular proteases. In order to gain insight into the molecular basis of protease cleavage and its impact on the efficiency of viral entry, we investigated the susceptibility of a recombinant native full-length S-protein trimer (triSpike) of the severe acute respiratory syndrome coronavirus (SARS-CoV) to cleavage by various airway proteases.
Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.
Drinking tea is recommended for promoting health due to its bioactive nutrients, such as catechins and caffeine. In Tokushima area, we have a unique traditional tea, named Awa tea, which are fermented with Lactobacillus pentosus and Lactobacillus plantarum. The present study was designed to investigate anti-obesity effects of the Awa tea and compare with those of non-fermented green tea. Obese male Wistar rats (19 weeks of age) were given by low energy diets containing 3% of Awa and green tea extracts, respectively, or without any tea extracts (control), for 4 weeks. Awa tea contained smaller amount of catechins than green tea, although they contained similar amounts of polyphenols. This finding indicates that there are distinct kinds of polyphenols from catechins. The diets containing Awa and green tea extracts further decreased whole body weight, fat tissue mass and plasma leptin level, compared with control diet. In addition, their diets increased the daily amount of lipid excreted to feces and total 24-h-energy consumption, compared with the control group. However, there is no significant difference in these anti-obesity effects between Awa tea and green tea. Our results indicate that Awa lactate-fermented tea as well as green tea similarly enhance the effect of diet restriction on obesity, at least in part, through the increase in fat energy consumption and the decrease in fat absorption in rats.
Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy.
It appears to have been well established that after surgery, protein catabolism is accelerated and glucose infusion suppresses the catabolic reactions. However, in the early postoperative period, the effects of surgical stress and glucose infusion on muscle protein catabolism and the related mechanisms remain unclear.
Osteoactivin is a type I transmembrane protein upregulated by unloading stresses, including denervation, prolonged bed rest, and space flight, but the regulatory mechanisms of its expression and activation under these conditions remain undefined. Here we report that osteoactivin protein exists in two forms: an intact transmembrane form and a secreted form. The secreted form, the extracellular fragment of osteoactivin, was produced by ectodomain shedding and was released into a culture medium. Amino acid sequence analysis of the carboxy-terminal fragment of osteoactivin (OA-CTF) revealed that cleavage of osteoactivin by proteases occurred both at the cell surface and within the cell membrane. Localization analysis demonstrated translocalization of OA-CTF to the nucleus and the endoplasmic reticulum. Moreover, RNA binding proteins, which regulate pre-mRNA splicing, were identified as OA-CTF binding proteins. These results suggest that OA-CTF formed by ectodomain shedding is involved in the regulation of pre-mRNA splicing.
Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-? and interleukin-1?, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.