Morin improves dexamethasone-induced muscle atrophy by modulating atrophy-related genes and oxidative stress in female mice.

This study investigated the effect of morin, a flavonoid, on dexamethasone-induced muscle atrophy in C57BL/6J female mice. Dexamethasone (10 mg/kg body weight) for 10 days significantly reduced body weight, gastrocnemius and tibialis anterior muscle mass, and muscle protein in mice. Dexamethasone significantly upregulated muscle atrophy-associated ubiquitin ligases, including atrogin-1 and MuRF-1, and the upstream transcription factors FoxO3a and Klf15. Additionally, dexamethasone significantly induced the expression of oxidative stress-sensitive ubiquitin ligase Cbl-b and the accumulation of the oxidative stress markers malondialdehyde and advanced protein oxidation products in both the plasma and skeletal muscle samples. Intriguingly, morin treatment (20 mg/kg body weight) for 17 days effectively attenuated the loss of muscle mass and muscle protein and suppressed the expression of ubiquitin ligases while reducing the expression of upstream transcriptional factors. Therefore, morin might act as a potential therapeutic agent to attenuate muscle atrophy by modulating atrophy-inducing genes and preventing oxidative stress.

Astaxanthin Exerts Immunomodulatory Effect by Regulating SDH-HIF-1α Axis and Reprogramming Mitochondrial Metabolism in LPS-Stimulated RAW264.7 Cells.

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in cell membranes and mitochondria, which consist of the bilayer molecules. Targeting mitochondria to ameliorate inflammatory diseases by regulating mitochondrial metabolism has become possible and topical. Although AX has been shown to have anti-inflammatory effects in various cells, the mechanisms are quite different. In particular, the role of AX on mitochondrial metabolism in macrophages is still unknown. In this study, we investigated the effect of AX on mitochondria-mediated inflammation and its mechanisms in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. AX attenuated the mitochondrial O2- production and maintained the mitochondrial membrane potential, implying that AX preserved mitochondrial homeostasis to avoid LPS stimulation-induced mitochondrial dysfunction. Additionally, AX prevented the decrease in mitochondrial complexes I, II, and III, which were caused by LPS stimulation. Especially, AX inhibited the reduction in mitochondrial succinate dehydrogenase (SDH; complex II) activity and upregulated the protein and mRNA level of SDH complex, subunit B. Furthermore, AX blocked the IL-1ß expression by regulating the SDH-HIF-1α axis and suppressed the energy shift from an OXPHOS phenotype to a glycolysis phenotype. These findings revealed important effects of AX on mitochondrial enzymes as well as on mitochondrial energy metabolism in the immune response. In addition, these raised the possibility that AX plays an important role in other diseases caused by SDH mutation and metabolic disorders.

Balenine, Imidazole Dipeptide Promotes Skeletal Muscle Regeneration by Regulating Phagocytosis Properties of Immune Cells.

Balenine is one of the endogenous imidazole dipeptides derived from marine products. It is composed of beta-alanine and 3-methyl-L-histidine, which exist mainly in the muscles of marine organisms. The physiological functions of dietary balenine are not well-known. In this study, we investigated whether the supplementation of dietary balenine was associated with muscle function in a cardiotoxin-indued muscle degeneration/regeneration model. Through morphological observation, we found that the supplementation of balenine-enriched extract promoted the regeneration stage. In addition, the expression of regeneration-related myogenic marker genes, such as paired box protein 7, MyoD1, myogenin, and Myh3, in a group of mice fed a balenine-enriched extract diet was higher than that in a group fed a normal diet. Moreover, the supplementation of balenine-enriched extract promoted the expression of anti-inflammatory cytokines as well as pro-inflammatory cytokines at the degeneration stage. Interestingly, phagocytic activity in the balenine group was significantly higher than that in the control group in vitro. These results suggest that balenine may promote the progress of muscle regeneration by increasing the phagocytic activity of macrophages.

Morin attenuates dexamethasone-mediated oxidative stress and atrophy in mouse C2C12 skeletal myotubes.

Glucocorticoids are the drugs most commonly used to manage inflammatory diseases. However, they are prone to inducing muscle atrophy by increasing muscle proteolysis and decreasing protein synthesis. Various studies have demonstrated that antioxidants can mitigate glucocorticoid-induced skeletal muscle atrophy. Here, we investigated the effect of a potent antioxidative natural flavonoid, morin, on the muscle atrophy and oxidative stress induced by dexamethasone (Dex) using mouse C2C12 skeletal myotubes. Dex (10 µM) enhanced the production of reactive oxygen species (ROS) in C2C12 myotubes via glucocorticoid receptor. Moreover, Dex administration reduced the diameter and expression levels of the myosin heavy chain protein in C2C12 myotubes, together with the upregulation of muscle atrophy-associated ubiquitin ligases, such as muscle atrophy F-box protein 1/atrogin-1, muscle ring finger protein-1, and casitas B-lineage lymphoma proto-oncogene-b. Dex also significantly decreased phosphorylated Foxo3a and increased total Foxo3a expression. Interestingly, Dex-induced ROS accumulation and Foxo3a expression were inhibited by morin (10 µM) pretreatment. Morin also prevented the Dex-induced reduction of myotube thickness, together with muscle protein degradation and suppression of the upregulation of atrophy-associated ubiquitin ligases. In conclusion, our results suggest that morin effectively prevents glucocorticoid-induced muscle atrophy by reducing oxidative stress.

Suppressive effects of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on the production of NO and ROS in LPS-stimulated RAW264.7 cells.

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

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.

Relationship between the characteristics of rigor-mortis-related actomyosin and muscle fiber types in the ordinary muscle of various fishes.

BACKGROUND: Rigor mortis occurs when muscle extension vanishes through the irresistible coupling of actin and myosin by the consumption of adenosine triphosphate as energy. To clarify the cause of the differences in the progression of rigor mortis, seven fish species were used as samples. The superprecipitation reaction and Mg2+ -ATPase activity of actomyosin in dorsal ordinary muscle were measured, and the slope of the regression line between these two variables was calculated for each fish specimen. The fiber types of the dorsal ordinary muscle in each sample fish were discriminated by the stability of actomyosin ATPase at acid and alkaline preincubations. RESULT: Positive correlations were found between Mg2+ -ATPase activity and the superprecipitation reaction of actomyosin in all 27 fish specimens. The slopes of the regression lines were different not only between fish species but also in fish specimens within the same species. The area ratios of pink muscle fibers and the IIa and/or IIb subtypes of white muscle fibers in the dorsal ordinary muscle were also different between fish species, as well as in specimens within the same fish species. A positive correlation was found between the area ratios of pink muscle fibers in dorsal ordinary muscle and the slopes of the regression line. CONCLUSION: It was suggested that the differences in characteristics of rigor-mortis-related actomyosin of fish might have been caused by the differences in the interposition ratio of muscle fiber types, especially of the pink muscle fiber type, in the dorsal ordinary muscle. © 2019 Society of Chemical Industry.

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.

Morin suppresses cachexia-induced muscle wasting by binding to ribosomal protein S10 in carcinoma cells.

Cachexia, observed in most cancer patients, is a syndrome that includes wasting of bodily energy reserves and is characterized by muscle atrophy and fat loss. We have previously demonstrated that isoflavones, such as genistein and daidzein, prevent muscle wasting in tumor-bearing mice. In this study, we examined the effect of morin, a flavonoid, on cachexia. The wet weight and myofiber size of muscles in Lewis lung carcinoma (LLC) cell-bearing mice fed a normal diet were decreased, compared with those in control mice fed a normal diet. In contrast, intake of morin prevented the reduction of muscle wet weight and myofiber size. Moreover, the tumor weight in mice fed the morin diet was lower than that in mice fed the normal diet. Both cell viability and protein synthetic ability of LLC cells were reduced by treatment with morin, but C2C12 myotubes were not affected. Binding assay using morin-conjugated magnetic beads identified ribosomal protein S10 (RPS10) as a target protein of morin. Consistent with the result of morin treatment, knockdown of RPS10 suppressed LLC cell viability. These results suggest that morin indirectly prevents muscle wasting induced by cancer cachexia by suppressing cancer growth via binding to RPS10.

Prevention of skeletal muscle atrophy in vitro using anti-ubiquitination oligopeptide carried by atelocollagen.

Skeletal muscle atrophy occurs when the rate of protein degradation exceeds that of protein synthesis in various catabolic conditions, such as fasting, disuse, aging, and chronic diseases. Insulin-like growth factor-1 (IGF-1) signaling stimulates muscle growth and suppresses muscle protein breakdown. In atrophied muscles, ubiquitin ligase, Cbl-b, increases and stimulates the ubiquitination and degradation of IRS-1, an intermediate in IGF-1 signaling pathway, resulting in IGF-1 resistance. In this study, we evaluated the efficacy of atelocollagen (ATCOL)-transported anti-ubiquitination oligopeptide (Cblin: Cbl-b inhibitor) (consisting of tyrosine phosphorylation domain of IRS-1) in starved C2C12 myotubes. The amount of IRS-1 protein was lower in starved versus unstarved myotubes. The Cblin-ATCOL complex inhibited IRS-1 degradation in a concentration-dependent manner. Myotubes incubated with Cblin-ATCOL complex showed significant resistance to starvation-induced atrophy (p<0.01). Furthermore, the Cblin-ATCOL complex significantly inhibited any decrease in Akt phosphorylation (p<0.01) and localization of FOXO3a to the nucleus in starved myotubes. These results suggest that Cblin prevented starvation-induced C2C12 myotube atrophy by maintaining the IGF-1/Akt/FOXO signaling. Therefore, attachment of anti-ubiquitination oligopeptide, Cblin, to ATCOL enhances its delivery to myotubes and could be a potentially effective strategy in the treatment of atrophic myopathies.

UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.

A novel myogenic function residing in the 5' non-coding region of Insulin receptor substrate-1 (Irs-1) transcript.

BACKGROUND: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs). RESULTS: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes. CONCLUSIONS: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

N-myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle.

A DGpYMP peptide mimetic of tyrosine(608)-phosphorylated insulin receptor substrate-1 (IRS-1), named Cblin, was previously shown to significantly inhibit Cbl-b-mediated IRS-1 ubiquitination. In the present study, we developed N-myristoylated Cblin and investigated whether it was effective in preventing glucocorticoid-induced muscle atrophy. Using HEK293 cells overexpressing Cbl-b, IRS-1 and ubiquitin, we showed that the 50% inhibitory concentrations of Cbl-b-mediated IRS-1 ubiquitination by N-myristoylated Cblin and Cblin were 30 and 120 µM, respectively. Regarding the DEX-induced atrophy of C2C12 myotubes, N-myristoylated Cblin was more effective than Cblin for inhibiting the DEX-induced decreases in C2C12 myotube diameter and IRS-1 degradation. The inhibitory efficacy of N-myristoylated Cblin on IRS-1 ubiquitination in C2C12 myotubes was approximately fourfold larger than that of Cblin. Furthermore, N-myristoylation increased the incorporation of Cblin into HEK293 cells approximately 10-folds. Finally, we demonstrated that N-myristoylated Cblin prevented the wet weight loss, IRS-1 degradation, and MAFbx/atrogin-1 and MuRF-1 expression in gastrocnemius muscle of DEX-treated mice approximately fourfold more effectively than Cblin. Taken together, these results suggest that N-myristoylated Cblin prevents DEX-induced skeletal muscle atrophy in vitro and in vivo, and that N-myristoylated Cblin more effectively prevents muscle atrophy than unmodified Cblin.

Ubiquitin ligase Cbl-b and obesity-induced insulin resistance.

Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.

Transcriptional regulation of the Japanese flounder Cu,Zn-SOD (Jfsod1) gene in RAW264.7 cells during oxidative stress caused by causative bacteria of edwardsiellosis.

Edwardsiellosis is one of the most important bacterial diseases in fish, sometimes causing extensive economic losses in the aquaculture industry. Our previous studies demonstrated that the Cu,Zn-SOD (sod1) activity has significantly increased in Japanese flounder, Paralichthys olivaceus, hepatopancreas infected by causative bacteria of edwardsiellosis Edwardsiella tarda NUF251. In this study, NUF251 stimulated intracellular superoxide radical production in mouse macrophage RAW264.7 cells, which was reduced by N-acetylcysteine. This result suggests that NUF251 infection causes oxidative stress. To evaluate the regulatory mechanism of Jfsod1 at transcriptional levels under oxidative stress induced by NUF251 infection, we cloned and determined the nucleotide sequence (1124 bp) of the 5'-flanking region of the Jfsod1 gene. The sequence analysis demonstrated that the binding sites for the transcription factors C/EBPα and NF-IL6 involved in the transcriptional regulation of the mammalian sod1 gene existed. We constructed a luciferase reporter system with the 5'-flanking region (-1124/-1) of the Jfsod1 gene, and a highly increased transcriptional activity of the region was observed in NUF251-infected RAW264.7 cells. Further studies using several mutants indicated that deletion of the recognition region of NF-IL6 (-272/-132) resulted in a significant decrease in the transcriptional activity of the Jfsod1 gene in NUF251-infected RAW264.7 cells. In particular, the binding site (-202/-194) for NF-IL6 might play a major role in upregulating the transcriptional activity of the 5'-flanking region of the Jfsod1 gene in response to oxidative stress induced by NUF251 infection. These results could be provided a new insight to understand the pathogenic mechanism of causative bacteria of edwardsiellosis.

The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy.

Skeletal muscle atrophy and the inhibition of muscle regeneration are known to occur as a natural consequence of aging, yet the underlying mechanisms that lead to these processes in atrophic myofibers remain largely unclear. Our research has revealed that the maintenance of proper mitochondrial-associated endoplasmic reticulum membranes (MAM) is vital for preventing skeletal muscle atrophy in microgravity environments. We discovered that the deletion of the mitochondrial fusion protein Mitofusin2 (MFN2), which serves as a tether for MAM, in human induced pluripotent stem (iPS) cells or the reduction of MAM in differentiated myotubes caused by microgravity interfered with myogenic differentiation process and an increased susceptibility to muscle atrophy, as well as the activation of the Notch signaling pathway. The atrophic phenotype of differentiated myotubes in microgravity and the regenerative capacity of Mfn2-deficient muscle stem cells in dystrophic mice were both ameliorated by treatment with the gamma-secretase inhibitor DAPT. Our findings demonstrate how the orchestration of mitochondrial morphology in differentiated myotubes and regenerating muscle stem cells plays a crucial role in regulating Notch signaling through the interaction of MAM.

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.

An intracellular fragment of osteoactivin formed by ectodomain shedding translocated to the nucleoplasm and bound to RNA binding proteins.

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.

[Space flight/bedrest immobilization and bone. Space flight and bed rest-mediated muscle atrophy].

Muscle atrophy caused by unloading stress is a challenging problem for bed-rested patients or astronauts. However, countermeasures against these muscle atrophy have not been developed yet. Under unloading conditions, skeletal muscle mass is rapidly lost by the increase in protein breakdown and the decrease in protein synthesis. It has been shown that this enhancement of proteolysis in atrophying muscles results mainly from activation of the ubiquitin-proteasome proteolytic pathway. Previous our studies revealed that unloading stress led to skeletal muscle atrophy through the induction of ubiquitin ligase, Cbl-b (Casitas B-lineage lymphoma b) expression. Thus, Cbl-b inhibiters may be potent therapeutic and preventive sources against skeletal muscle atrophy caused by unloading stress.