Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

Refeeding with a high-protein diet after a 48 h fast causes acute hepatocellular injury in mice.

Elucidating the effects of refeeding a high-protein diet after fasting on disease development is of interest in relation to excessive protein ingestion and irregular eating habits in developed countries. The objective of the present study was to address the hepatic effects of refeeding a high-protein diet after fasting. Mice were fasted for 48 h and then refed with a test diet containing 3, 15, 35, 40, 45 or 50 % casein. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and liver immediate-early gene expression levels were sequentially measured for the first 24 h after initiation of refeeding. Refeeding with a 50 % casein diet after 48 h of fasting led to a rapid (within 2-3 h) and abnormal elevation in serum ALT (P = 0·006) and AST (P = 0·001) activities and a marked increase in liver Finkel-Biskis-Jinkins (FBJ) osteosarcoma oncogene (P = 0·007) and nuclear receptor subfamily 4, group A, member 1 (P = 0·002) mRNA levels. In contrast, refeeding of the 3, 15 or 35 % casein diets produced no substantial increases in serum ALT and AST activities in mice. Refeeding of 40, 45 or 50 % casein increased serum ALT and AST activities in proportion to this dietary casein content. In mice refed the 3, 15 or 35, but not 50 %, casein diets, liver heat shock protein 72 transcript levels greatly increased. We conclude from these data that the consumption of a high-protein diet after fasting causes acute hepatocellular injury in healthy animals, and propose that careful attention should be paid to the use of such diets.

Additional effects of simultaneous treatment with C14-Cblin and celastrol on the clinorotation-induced rat L6 myotube atrophy.

Two novel reagents, N-myristoylated Cbl-b inhibitory peptide (C14-Cblin) and celastrol, a quinone methide triterpene, are reported to be effective in preventing myotube atrophy. The combined effects of C14-Cblin and celastrol on rat L6 myotubes atrophy induced by 3D-clinorotation, a simulated microgravity model, was investigated in the present study. We first examined their effects on expression in atrogenes. Increase in MAFbx1/atrogin-1 and MuRF-1 by 3D-clinorotation was significantly suppressed by treatment with C14-Cblin or celastrol, but there was no additive effect of simultaneous treatment. However, celastrol significantly suppressed the upregulation of Cbl-b and HSP70 by 3D-clinorotation. Whereas 3D-clinorotation decreased the protein level of IRS-1 in L6 myotubes, C14-Cblin and celastrol inhibited the degradation of IRS-1. C14-Cblin and celastrol promoted the phosphorylation of FOXO3a even in microgravity condition. Simultaneous administration of C14-Cblin and celastrol had shown little additive effect in reversing the impairment of IGF-1 signaling by 3D-clinorotation. While 3D-clinorotation-induced marked oxidative stress in L6 myotubes, celastrol suppressed 3D-clinorotation-induced ROS production. Finally, the C14-Cblin and celastrol-treated groups were inhibited decrease in L6 myotube diameter and increased the protein content of slow-twitch MyHC cultured under 3D-clinorotation. The simultaneous treatment of C14-Cblin and celastrol additively prevented 3D-clinorotation-induced myotube atrophy than single treatment. J. Med. Invest. 69 : 127-134, February, 2022.

MuRF1 deficiency prevents age-related fat weight gain, possibly through accumulation of PDK4 in skeletal muscle mitochondria in older mice.

Recent studies show that muscle mass and metabolic function are interlinked. Muscle RING finger 1 (MuRF1) is a critical muscle-specific ubiquitin ligase associated with muscle atrophy. Yet, the molecular target of MuRF1 in atrophy and aging remains unclear. We examined the role of MuRF1 in aging, using MuRF1-deficient (MuRF1-/- ) mice in vivo, and MuRF1-overexpressing cell in vitro. MuRF1 deficiency partially prevents age-induced skeletal muscle loss in mice. Interestingly, body weight and fat mass of more than 7-month-old MuRF1-/- mice were lower than in MuRF1+/+ mice. Serum and muscle metabolic parameters and results of indirect calorimetry suggest significantly higher energy expenditure and enhanced lipid metabolism in 3-month-old MuRF1-/- mice than in MuRF1+/+ mice, resulting in suppressed adipose tissue gain during aging. Pyruvate dehydrogenase kinase 4 (PDK4) is crucial for a switch from glucose to lipid metabolism, and the interaction between MuRF1 and PDK4 was examined. PDK4 protein levels were elevated in mitochondria from the skeletal muscle in MuRF1-/- mice. In vitro, MuRF1 interacted with PDK4 but did not induce degradation through ubiquitination. Instead, SUMO posttranscriptional modification (SUMOylation) of PDK4 was detected in MuRF1-overexpressing cells, in contrast to cells without the RING domain of MuRF1. MuRF1 deficiency enhances lipid metabolism possibly by upregulating PDK4 localization into mitochondrial through prevention of SUMOylation. Inhibition of MuRF1-mediated PDK4 SUMOylation is a potential therapeutic target for age-related dysfunction of lipid metabolism and muscle atrophy.

Rantes secreted from macrophages disturbs skeletal muscle regeneration after cardiotoxin injection in Cbl-b-deficient mice.

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.

Dietary Sodium Nitrite Causes Similar Modifications to Splenic Inflammatory Gene Expression as a High-Fat Diet.

Sodium nitrite (NaNO2) is a widely used food additive. The present study compared the outcomes from intakes of dietary NaNO2 and a high-fat diet (HFD), and assessed their combined effects on inflammatory gene expression in the immune tissues of the mouse. In experiment I, mice were fed a standard low-fat diet (LFD) without or with NaNO2 (0.02 and 0.08%, w/w) for 11 wk. In experiment II, mice were fed an LFD without or with NaNO2 (0.02%) or HFD without or with NaNO2 (0.02%) for 11 wk. Inflammatory gene expression in the immune tissues was then measured. NaNO2 consumption and HFD feeding each resulted in increased splenic mRNAs for cell markers of neutrophils (Ngp, NE, Ly6g, Mpo) and eosinophils (Epo, Ear6), and an S100 family member (S100A8). In contrast, NaNO2 consumption and HFD feeding each resulted in decreased splenic mRNAs for cell markers of macrophages (Emr1, Itgax, CD68, CD206, Dectin-1, TLRs 4, 6, and 7), T- (CD3, CD4), NK- (CD56) and B-cells (CD20, CD40), pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-1ß, IFN-γ, IL-18, IL-10, TGF-ß), interleukin receptor antagonists (IL1ra, IL6ra) and cell adhesion molecules (ICAM-1, VCAM-1). However, dietary NaNO2 combined with HFD feeding caused no further decrease in these transcript levels compared with dietary NaNO2 alone. These NaNO2- or HFD-induced modifications were less profound in the liver and abdominal adipose tissues than in the spleen. These findings indicate that dietary NaNO2 has similar modulatory effects to HFD feeding on splenic inflammatory genes.

Effects of a high-protein diet on host resistance to Paracoccidioides brasiliensis in mice.

We investigated the effects of high protein intake on host resistance to Paracoccidioides brasiliensis. Two-d fasted mice were infected with P. brasiliensis and refed on diets with three different levels (54%, 20%, and 5%) of casein. The mice refed the 54% casein diet showed reduced antifungal activity in the spleen and liver as compared with the mice refed the 5% or the 20% casein diet. After infection, increases in spleen and liver mRNA levels of myeloperoxidase, cathepsin-G, and elastase-2 were more profound in the mice refed the 54% casein diet as compared with the mice refed the 5% or the 20% casein diet. Infected mice refed the 54% casein diet exhibited greater interferon (IFN)-gamma production in the spleen and liver and higher levels of thiobarbituric acid reactive substances (TBARSs) in the liver as compared with those refed the 5% casein diet. These results indicate that high protein intake impairs host resistance to P. brasiliensis.

A Diet Including Red Bell Pepper Juice and Soy Protein Suppress Physiological Markers of Muscle Atrophy in Mice.

Although muscle atrophy can be caused by disuse and lifestyle-related syndromes, it may be possible to prevent this condition through dietary intervention. We hypothesized that a diet including red bell pepper juice (RBPJ) and soy protein isolate (SPI) would prevent muscle atrophy. Accordingly, an experimental diet containing RBPJ and/or SPI was administered for 18 d to normal C57BL/6J mice. The control group was administered a casein diet. Four days before the end of the test period, denervation-induced muscle atrophy and/or sham operation were performed. Anterior tibialis muscle samples were then obtained to assess muscle degradation and perform metabolome analysis. Under the denervation condition, the 20% SPI diet did not alter the mRNA expression levels of muscle atrophy marker genes compared with the 20% casein group. Although the diet comprising RBPJ and 20% casein did not prevent muscle atrophy compared with the control group, the diet containing RBPJ and 20% SPI did. Metabolome analysis revealed that a diet including RBPJ and SPI induced a greater than 1.5-fold change in the levels of 20 muscle atrophy-related metabolites. In particular, the level of S-adenosylmethionine, which concerned with energy metabolism and lifespan, showed a strong positive correlation with the muscle atrophy marker. These findings suggest that a diet including RBPJ and soy protein suppress gene expressions related with muscle atrophy. Further research in humans is needed to confirm whether a combination of RBPJ and SPI can indeed prevent muscle atrophy.

Effect of dietary oils on host resistance to fungal infection in psychologically stressed mice.

Psychological stress can modulate host defense against invading pathogens. In this study, we investigated the effect of dietary oils on social isolation stress-induced modulation of host resistance to Paracoccidioides brasiliensis. In olive oil-fed mice, 3 weeks of isolation stress resulted in temporarily delayed clearance of this fungus in the liver compared with group-housed mice. By contrast, in soybean oil-fed mice, isolation stress had no significant effect on antifungal activity. The olive oil-fed mice showed greater liver interferon (IFN)-gamma and interleukin (IL)-6 production in response to infection as compared with the soybean oil-fed mice. In the olive oil-fed mice, isolation stress led to greater infection-induced IFN-gamma production in the liver compared with the group-housed animals. These results indicate that the modulatory effects of psychological stress on host resistance to P. brasiliensis can vary depending on dietary fatty acid composition.

Effects of dietary fish oil on lipid peroxidation and serum triacylglycerol levels in psychologically stressed mice.

OBJECTIVE: The intake of omega-3 polyunsaturated fatty acids and psychological stress can each induce tissue lipid peroxidation. In our present study, we investigated their combined effects on the oxidative status of mouse tissues. METHODS: Mice were group-housed (four mice/cage) and fed a diet containing fish oil (as a source of omega-3 polyunsaturated fatty acids), soybean oil, or olive oil for 3 wk. These animals were then 1) housed under the same conditions (four per cage, control group) or 2) individually housed to generate psychological stress conditions (isolation stress). After 2 wk of isolation stress, the levels of thiobarbituric acid-reactive substances (an index of lipid peroxidation) and antioxidants in the liver and kidney and the serum levels of triacylglycerol were measured. RESULTS: Fish oil-fed mice showed increased levels of thiobarbituric acid-reactive substances in their livers and kidneys compared with soybean oil- or olive oil-fed mice. These increases in thiobarbituric acid-reactive substance levels in the fish oil-fed mice were less profound under isolation stress conditions when compared with the group-housed animals on the same diet. In the fish oil-fed mice, isolation stress led to an increase in liver vitamin E levels when compared with their group-housed counterparts. The fish oil-fed mice exhibited lower serum triacylglycerol levels compared with the soybean oil- or olive oil-fed mice, and this decrease was more profound under conditions of isolation stress when compared with group-housing conditions. CONCLUSION: Dietary fish oil combined with isolation stress results in lower levels of lipid peroxidation in the liver and kidney compared with dietary fish oil alone.

Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-kappa B signaling.

In this study, we examined effects of the three-dimensional (3D)-clinorotation, a simulated-model of microgravity, on proliferation/differentiation of rat myoblastic L6 cells. Differentiation of L6 cells into myotubes was significantly disturbed in the 3D-clinorotation culture system, although the 3D-clinorotation had no effect on the proliferation. The 3D-clinorotation also suppressed the expression of myogenesis marker proteins, such as myogenin and myosin heavy chain (MHC), at the mRNA level. In association with this reduced differentiation, we found that the 3D-clinorotation prevented accumulation of ubiquitinated proteins, compared with non-rotation control cells. Based on these findings, we focused on the ubiquitin-dependent degradation of I kappa B, a myogenesis inhibitory protein, to clarify the mechanism of this impaired differentiation. A decline in the amount of I kappa B protein in L6 cells was significantly prevented by the rotation, while the amount of the protein in the non-rotated cells decreased along with the differentiation. Furthermore, the 3D-clinorotation reduced the NF-kappaB-binding activity in L6 cells and prevented the ubiquitination of I kappa B proteins in the I kappa B- and ubiquitin-expressing Cos7 cells. Other myogenic regulatory factors, such as deubiquitinases, cyclin E and oxygen, were not associated with the differentiation impaired by the clinorotation. Our present results suggest that simulated microgravity such as the 3D-clinorotation may disturb skeletal muscle cell differentiation, at least in part, by inhibiting the NF-kappa B pathway.

Capric Acid Up-Regulates UCP3 Expression without PDK4 Induction in Mouse C2C12 Myotubes.

Uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle are key regulators of the glucose and lipid metabolic processes that are involved in insulin resistance. Medium-chain fatty acids (MCFAs) have anti-obesogenic effects in rodents and humans, while long-chain fatty acids (LCFAs) cause increases in body weight and insulin resistance. To clarify the beneficial effects of MCFAs, we examined UCP3 and PDK4 expression in skeletal muscles of mice fed a MCFA- or LCFA-enriched high-fat diet (HFD). Five-week feeding of the LCFA-enriched HFD caused high body weight gain and induced glucose intolerance in mice, compared with those in mice fed the MCFA-enriched HFD. However, the amounts of UCP3 and PDK4 transcripts in the skeletal muscle of mice fed the MCFA- or LCFA-enriched HFD were similar. To further elucidate the specific effects of MCFAs, such as capric acid (C10:0), on lipid metabolism in skeletal muscles, we examined the effects of various FAs on expression of UCP3 and PDK4, in mouse C2C12 myocytes. Although palmitic acid (C16:0) and lauric acid (C12:0) significantly induced expression of both UCP3 and PDK4, capric acid (C10:0) upregulated only UCP3 expression via activation of peroxisome proliferator-activated receptor-δ. Furthermore, palmitic acid (C16:0) disturbed the insulin-induced phosphorylation of Akt, while MCFAs, including lauric (C12:0), capric (C10:0), and caprylic acid (C12:0), did not. These results suggest that capric acid (C10:0) increases the capacity for fatty acid oxidation without inhibiting glycolysis in skeletal muscle.

Dietary supplementation with docosahexaenoic acid, but not with eicosapentaenoic acid, reduces host resistance to fungal infection in mice.

The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.

Short-term hypergravity does not affect protein-ubiquitination and proliferation in rat L6 myoblastic cells.

We previously reported that spaceflight (STS-90) and tail-suspension stimulated muscle protein ubiquitination and accumulated the degradation fragments. However, in space experiments the side-effects of hypergravity on samples are inevitable during the launch of a space shuttle into space or the reentry. To examine whether hypergravity also caused protein-ubiquitination in skeletal muscle cells, we exposed rat myoblastic L6 cells to various hypergravity conditions. Immunoblot analysis showed that the centrifugation at 2, 3, 30 or 100 G for 10 min did not increase the amount of ubiquitinated proteins in L6 cells, whereas the centrifugation at 100 G for 1 or 2 hrs significantly induced the protein-ubiquitination. In contrast, heat shock protein 70 (HSP70), another stress-responsive protein, in L6 cells was accumulated only by centrifugation at 100 G for more than 10 min. Short-term (10 min) hypergravity including 3 or 100 G did not affect the proliferation and morphological changes in L6 cells. Our present results suggest that the ubiquitination of muscle proteins is less sensitive to hypergravity than the induction of HSP70, and that the effect of hypergravity on protein-ubiquitination and proliferation of skeletal muscle cells may be negligible, as far as its duration is short-term.

Refeeding with glucose rather than fructose elicits greater hepatic inflammatory gene expression in mice.

OBJECTIVE: We previously reported that refeeding after a 48-h fast, used as a study model of starvation and refeeding, promotes acute liver inflammatory gene expression, which is at least partly mediated by toll-like receptor 2 (TLR2). We also previously demonstrated that dietary carbohydrates play critical roles in this process. The aim of this study was to compare the outcomes of refeeding with different carbohydrate sources. METHODS: Mice were fasted for 46 h and then refed with 1.5% (w/w) agar gel containing 19% carbohydrate (sources: α-cornstarch, glucose, sucrose, or fructose). The liver expression of inflammatory and other specific genes was then sequentially measured for the first 14 h after refeeding initiation. RESULTS: Fasting for 46 h up-regulated the liver expression of endogenous ligands for TLRs (HspA5, Hsp90 aa1, and Hspd1). Refeeding with agar gel containing α-cornstarch or glucose increased the liver expression of Tlr2, proinflammatory genes (Cxcl2, Cxcl10, Cxcl1, Nfkb1, Nfkb2, RelB, Sectm1α, Il1ß), stress response genes (Atf3, Asns, Gadd45 a, Perk, Inhbe), detoxification genes (Hmox1, Gsta1, Abca8b), genes involved in tissue regeneration (Gdf15, Krt23, Myc, Tnfrsf12a, Mthfd2), and genes involved in tumor suppression (p53, Txnrd1, Btg2). This refeeding also moderately but significantly elevated the serum levels of alanine aminotransferase. These effects were attenuated in mice refed with agar gel containing sucrose or fructose. CONCLUSION: Dietary glucose, rather than fructose, plays a critical role in refeeding-induced acute liver inflammatory gene expression and moderate hepatocyte destruction. Further studies are recommended regarding the role of these effects in liver inflammation and, consequently, liver dysfunction.

Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90).

We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.

Refeeding with a standard diet after a 48-h fast elicits an inflammatory response in the mouse liver.

Unhealthy eating behaviors increase the risk of metabolic diseases, but the underlying mechanisms are not fully elucidated. Because inflammation contributes to the pathogenesis of metabolic diseases, it is important to understand the effects of unhealthy eating on the inflammatory state. The objective of our present study was to address the effects of a fasting-refeeding regime, a model of irregular eating, on the hepatic inflammatory responses in mouse. The animals were fasted for 48 h and then refed either a standard or low-carbohydrate/high-fat diet. Inflammatory gene expression in the liver was then sequentially measured for the first 17 h after initiation of refeeding. To assess the roles of dietary carbohydrates and toll-like receptor 2 (TLR2) in the refeeding-induced inflammatory changes, gene expression levels in mice refed only carbohydrates (α-corn starch and sucrose) at different doses and in TLR2-deficient mice refed a standard diet were also analyzed. Refeeding with a standard diet increased the liver expression of Tlr2, proinflammatory mediators (Cxcl10, Cxcl1, Cxcl2, Icam-1) and negative regulators of TLR-signaling (A20 and Atf3). These increases were attenuated in mice refed a low-carbohydrate/high-fat diet. Refeeding only α-corn starch and sucrose also increased the expression of these inflammatory pathway genes depending on the doses. TLR2 deficiency significantly attenuated the refeeding-induced increase in the liver expression of Cxcl10, Cxcl1, Icam-1 and A20. These findings suggest that an irregular eating behavior can elicit a liver inflammatory response, which is at least partly mediated by TLR2, and that dietary carbohydrates play critical roles in this process.

Isoflavones derived from soy beans prevent MuRF1-mediated muscle atrophy in C2C12 myotubes through SIRT1 activation.

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)-α treatment. In C2C12 myotubes, TNF-α 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 NFκB, a downstream target of the TNF-α 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-α 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.

Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b.

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.

Cbl-b is a critical regulator of macrophage activation associated with obesity-induced insulin resistance 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.