Therapeutic effects of an orally administered edible seaweed-derived polysaccharide preparation, ascophyllan HS, on a Streptococcus pneumoniae infection mouse model.

Ascophyllan HS is a commercially available preparation of the edible brown alga Ascophyllum nodosum containing ascophyllan, a sulfated polysaccharide with diverse beneficial biological activities. In this study, the effects of ascophyllan HS were evaluated in a severe intranasal Streptococcus pneumoniae infection mouse model. The control untreated mice started to die on day 7 and 80% had died by day 14 post-infection. Continuous oral administration of ascophyllan HS before and after bacterial infection resulted in a remarkable increase in survival rate, with 90% of the low (167 mg/kg body weight/day) and 100% of the high (500 mg/kg body weight/day) dose ascophyllan HS-treated mice surviving at day 14 post-infection. Histopathological observation of the lungs of the infected mice revealed the induction of typical pneumonia features in the alveolar spaces of the untreated control mice, such as extensive infiltration of inflammatory cells, edema, and fibrin deposition. In contrast, notable levels of lung injuries or alterations were not observed in the ascophyllan HS-treated mice, and only a minor lesion was observed in one mouse. Furthermore, bacterial burdens in the lungs were significantly reduced in the ascophyllan HS-treated mice as compared to the control mice at day 4 post-infection. Significantly higher levels of IL-12 were detected in the serum of ascophyllan HS-treated mice than that of control mice measured at the end of the infection experiment (day 14). These results suggest that orally administered ascophyllan HS exerts a therapeutic effect on S. pneumoniae infection by activating the host defense systems. This is the first report of the therapeutic effect of an orally administered seaweed polysaccharide preparation on S. pneumoniae infection. Our findings suggest that ascophyllan HS has the potential to be developed as nutraceuticals and pharmaceuticals applicable for humans as well as a safe and promising therapeutic agent against S. pneumoniae infection.

Application of Bio-Impedance Analysis to Estimate the Condition of Yellowtail (Seriola quinqueradiata) Muscle at Different Storage Temperatures.

Changes in impedance at 2 kHz, adenosine triphosphate (ATP) content, and muscle contraction were evaluated in yellowtail during 0 (ice), 5, 10, 15, and 20°C storage. Histological changes during ice storage were also measured. At any temperature, although impedance increased with both rigor mortis and ATP consumption during early storage, it began to decrease rapidly when ATP was almost depleted. Moreover, temporarily increasing impedance had a strong relationship with ATP content; decreasing impedance had a significant correlation with storage temperature after ATP depletion. Furthermore, impedance increased with narrowing of intercellular spaces when sarcolemma was intact and decreased with expansion of intercellular spaces when sarcolemma was leaky. Meanwhile, changes of sarcolemma and intercellular spaces were accompanied by ATP change. Thus, ATP is one significant physiological factor for impedance change, and temperature greatly influenced impedance after depletion of ATP. Results suggest that impedance analysis can be used as a convenient and nondestructive method to diagnose condition of tissue at different storage temperatures. Bioelectromagnetics. 2019;40:488-497. © 2019 Bioelectromagnetics Society.

Dietary supplementation with alkylresorcinols prevents muscle atrophy through a shift of energy supply.

It has been reported that phytoextracts that contain alkylresorcinols (ARs) protect against severe myofibrillar degeneration found in isoproterenol-induced myocardial infarction. In this study, we examined the effect of dietary ARs derived from wheat bran extracts on muscle atrophy in denervated mice. The mice were divided into the following four groups: (1) sham-operated (control) mice fed with normal diet (S-ND), (2) denervated mice fed with normal diet (D-ND), (3) control mice fed with ARs-supplemented diet (S-AR) and (4) denervated mice fed with ARs-supplemented diet (D-AR). The intake of ARs prevented the denervation-induced reduction of the weight of the hind limb muscles and the myofiber size. However, the expression of ubiquitin ligases and autophagy-related genes, which is associated with muscle proteolysis, was slightly higher in D-AR than in D-ND. Moreover, the abundance of the autophagy marker p62 was significantly higher in D-AR than in D-ND. Muscle atrophy has been known to be associated with a disturbed energy metabolism. The expression of pyruvate dehydrogenase kinase 4 (PDK4), which is related to fatty acid metabolism, was decreased in D-ND as compared with that in S-ND. In contrast, dietary supplementation with ARs inhibited the decrease of PDK4 expression caused by denervation. Furthermore, the abnormal expression pattern of genes related to the abundance of lipid droplets-coated proteins that was induced by denervation was improved by ARs. These results raise the possibility that dietary supplementation with ARs modifies the disruption of fatty acid metabolism induced by lipid autophagy, resulting in the prevention of muscle atrophy.

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.

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.

"Hybrid exercise" prevents muscle atrophy in association with a distinct gene expression pattern.

"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.

Awa (Tokushima) lactate-fermented tea as well as green tea enhance the effect of diet restriction on obesity in rats.

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.

Subcutaneous injection, from birth, of epigallocatechin-3-gallate, a component of green tea, limits the onset of muscular dystrophy in mdx mice: a quantitative histological, immunohistochemical and electrophysiological study.

Dystrophic muscles suffer from enhanced oxidative stress. We have investigated whether administration of an antioxidant, epigallocatechin-3-gallate (EGCG), a component of green tea, reduces their oxidative stress and pathophysiology in mdx mice, a mild phenotype model of human Duchenne-type muscular dystrophy. EGCG (5 mg/kg body weight in saline) was injected subcutaneously 4x a week into the backs of C57 normal and dystrophin-deficient mdx mice for 8 weeks after birth. Saline was injected into normal and mdx controls. EGCG had almost no observable effects on normal mice or on the body weights of mdx mice. In contrast, it produced the following improvements in the blood chemistry, muscle histology, and electrophysiology of the treated mdx mice. First, the activities of serum creatine kinase were reduced to normal levels. Second, the numbers of fluorescent lipofuscin granules per unit volume of soleus and diaphragm muscles were significantly decreased by about 50% compared to the numbers in the corresponding saline-treated controls. Third, in sections of diaphragm and soleus muscles, the relative area occupied by histologically normal muscle fibres increased significantly 1.5- to 2-fold whereas the relative areas of connective tissue and necrotic muscle fibres were substantially reduced. Fourth, the times for the maximum tetanic force of soleus muscles to fall by a half increased to almost normal values. Fifth, the amount of utrophin in diaphragm muscles increased significantly by 17%, partially compensating for the lack of dystrophin expression.

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.

Effects of single administration of Rokumi-gan (TJ-87) on serum amino acid concentration of 6 healthy Japanese male volunteers.

Rokumi-gan (TJ-87) has beneficial effects on renal diseases, including pollakisuria, dysuria and edema. We previously reported that its long-term administration clinically improved serum protein concentration and edema in renal failure. In this study, we focused on amino acid/protein contents in Rokumi-gan as one of its effectors. Commercially prepared Rokumi-gan contained arginine, aspartate and glutamate at the high levels, alanine, phenylalanine and serine at the moderate levels, and glycine, histidine, isoleucine, leucine, lysine and valine at the low levels. To examine effects of Rokumi-gan on serum amino acid concentrations, 6 healthy Japanese volunteers were treated with commercially prepared Rokumi-gan, an amino acid mixture, and lactose. In subjects treated with an amino acid mixture containing similar amounts of amino acids in Rokumi-gan (10 g), or lactose, serum amounts of many amino acids, except for arginine, gradually and significantly decreased until 6 hr after their treatments. In contrast, a single treatment with Rokumi-gan (10 g) increased serum levels of several amino acids, alanine, arginine, glutamate, glycine and serine. Serum concentrations of almost of all tested amino acids showed the peak value 1-2 hr after administration, and they were sustained at the basal level even 6 hr after the treatment. Our present results suggest that Rokumi-gan may be a beneficial amino acid supplier, because it could sustain serum amino acid concentration at the higher level than an amino acid mixture supplement.

Soy protein diet prevents hypermethioninemia caused by portacaval shunt in rats.

In hepatic disorders, abnormal plasma amino acid profiles are observed. In this study, we examined whether soy protein isolate (SPI) improved plasma methionine concentration in the model animals. Portacaval shunt (PCS) increased alanine aminotransferase (ALT) activity and methionine concentration in blood of rats fed a 40% casein diet supplemented with 0.6% methionine (casein-M diet). A 40% SPI diet supplemented with 1.28% methionine (SPI-M diet), which contained the same amount of methionine as that in 40% casein-M diet, normalized plasma ALT activity and methionine level in PCS rats. These effects of a SPI diet may be due to its amino acid composition, since an amino acid mixture diet mimicking a 40% SPI-M diet was also effective to hypermethioninemia of PCS rats. To find key enzymes for the beneficial effect of soy protein, we examined effects of a 40% SPI-M or casein-M diet on the activities of three methionine-metabolizing enzymes in liver of PCS rats. A SPI-M diet stimulated only the activity of cystathionine gamma-lyase, compared with a casein-M diet. A SPI diet has a preventive effect on hypermethioninemia, at least in part, by stimulating cystathionine gamma-lyase activity in liver and may be used for nutritional management of liver disorders with hypermethioninemia.

A relative high dose of vitamin E does not attenuate unweighting-induced oxidative stress and ubiquitination in rat skeletal muscle.

We previously reported that intragastric administration of cysteine could be beneficial to prevent unweighting-induced ubiquitination and degradation of muscle protein in association with redox regulation [Ikemoto et al., Biol. Chem., 383 (2002), 715-721]. In this study, we investigated whether vitamin E, another potent antioxidative nutrient, also had beneficial effects on the muscle protein catabolism. However, daily intragastric supplementation of 1.5 or 15 mg/rat of alpha-tocopherol did not prevent weight loss of hindlimb skeletal muscle in tail-suspended rats. To elucidate the reason for the non-effectiveness of vitamin E, we further examined concentrations of oxidative stress markers, ubiquitination of muscle proteins and fragmentation of myosin heavy chain in gastrocnemius muscle of rats daily treated with 15 mg of alpha-tocopherol. Unexpectedly, vitamin E increased concentrations of glutathione disulfide and thiobarbituric acid-reactive substance and decreased glutathione level in the muscle, compared with those of vehicle treatment, indicating that vitamin E enhanced unweighting-induced oxidative stress in skeletal muscle. The vitamin E supplementation did not suppress the ubiquitination of muscle proteins and fragmentation of myosin heavy chain caused by tail-suspension. Our results suggest that supplementation of a relative high dose of vitamin E could not inhibit ubiquitin-dependent degradation of muscle protein in tail-suspended rats possibly due to its prooxidant action.

Cysteine supplementation prevents unweighting-induced ubiquitination in association with redox regulation in rat skeletal muscle.

We have previously reported that spaceflight and tail suspension enhanced degradation of rat myosin heavy chain (MHC) in association with activation of a ubiquitin-dependent proteolytic pathway [Ikemoto et al., FASEB J. 15 (2001), 1279-1281]. To elucidate whether the ubiquitination is accompanied by oxidative stress, we measured markers for oxidative stress, such as thiobarbituric acid-reactive substance (TBARS) and glutathione disulfide (GSSG), in gastrocnemius muscle of tail-suspended rats. Glutathione (GSH) concentration in the muscle significantly decreased from day 5 and reached a minimum value on day 10. Tail suspension reciprocally increased concentrations of TBARS and GSSG in parallel with enhancement of protein ubiquitination, suggesting that oxidative stress may play an important role in protein ubiquitination caused by tail suspension. To prevent ubiquitination associated with oxidative stress, we also administered an antioxidative nutrient, cysteine, to tail-suspended rats. Intragastric supplementation of 140 mg/rat of cysteine for 2 weeks or longer normalized the ratio of GSH to GSSG in the muscle and suppressed protein ubiquitination and MHC fragmentation, compared with supplementation of the equimolar amount of alanine. The cysteine supplementation significantly suppressed the loss of hindlimb muscle mass. Our results suggest that supplementation of antioxidative nutrients, such as cysteine, may be beneficial for preventing ubiquitination of muscle proteins caused by unweighting.