Publications by authors named "Takashi Ohira"

25 Publications

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Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice.

J Proteomics 2020 04 12;217:103686. Epub 2020 Feb 12.

Advanced Medical Research Center, Yokohama City University, Kanagawa, Japan. Electronic address:

Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. SIGNIFICANCE: The skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.
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http://dx.doi.org/10.1016/j.jprot.2020.103686DOI Listing
April 2020

Role of 72-kDa Heat Shock Protein in Heat-stimulated Regeneration of Injured Muscle in Rat.

J Histochem Cytochem 2019 11 24;67(11):791-799. Epub 2019 Jun 24.

Graduate School of Medicine, Osaka University, Osaka, Japan.

The regeneration of injured muscles is facilitated by intermittent heat stress. The 72-kDa heat shock protein (HSP72), the level of which is increased by heat stress, is likely involved in this effect, but the precise mechanism remains unclear. This study was conducted to investigate the localization and role(s) of HSP72 in the regenerating muscles in heat-stressed rats using immunohistochemistry. Heat stress was applied by immersion of the rat lower body into hot water (42C, 30 min, every other day) following injection of bupivacaine into the soleus muscles. After 1 week, we found that HSP72 was expressed at high levels not only in the surviving myofibers but also in the blood vessels of the regenerating muscles in heated rats. In addition, leukocytes, possibly granulocytes, expressing cluster of differentiation 43 within the blood capillaries surrounding the regenerating myofibers also highly expressed HSP72. In contrast, marked expression of HSP72 was not observed in the intact or regenerating muscles without heat stress. These results suggest that heat-stress-induced HSP72 within the myofibers, blood vessels, and circulating leukocytes may play important roles in enhancing regeneration of injured muscles by heat stress. Our findings would be useful to investigate cell-specific role(s) of HSP72 during skeletal muscle regeneration.
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http://dx.doi.org/10.1369/0022155419859861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824005PMC
November 2019

Correction to: Liver Safety of Fasiglifam (TAK-875) in Patients with Type 2 Diabetes: Review of the Global Clinical Trial Experience.

Drug Saf 2018 12;41(12):1431-1437

Takeda Pharmaceuticals, Takeda Development Center Americas, Inc., 1 Takeda Pkwy, Deerfield, IL, 60015, USA.

In the original publication of the article, the ALT and AST values in Fig. 5a-e were capped at 10× ULN, which did not accurately reflect the narrative provided for each case. In this correction, the original Fig. 5a-e (Fig. 1a-e) and the correct Fig. 5a-5e (Fig. 2a-e) are published.
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http://dx.doi.org/10.1007/s40264-018-0745-0DOI Listing
December 2018

Liver Safety of Fasiglifam (TAK-875) in Patients with Type 2 Diabetes: Review of the Global Clinical Trial Experience.

Drug Saf 2018 06;41(6):625-640

Takeda Pharmaceuticals, Takeda Development Center Americas, Inc., 1 Takeda Pkwy, Deerfield, IL, 60015, USA.

Introduction: Fasiglifam (TAK-875) is a G protein-coupled receptor 40 agonist that was being investigated for treatment of type 2 diabetes mellitus (T2DM). A development program was terminated late in phase III clinical trials due to liver safety concerns.

Methods: The liver safety of fasiglifam was assessed from data based on six phase II and nine phase III double-blind studies and two open-label studies with emphasis on pooled data from 15 double-blind studies from both global and Japanese development programs. Taking into consideration different daily doses of fasiglifam administered in clinical studies, the primary comparisons were between all patients exposed to fasiglifam (any dose) versus placebo, and, where applicable, versus the two active comparators, sitagliptin or glimepiride. A Liver Safety Evaluation Committee consisting of hepatologists blinded to treatment assignments evaluated hepatic adverse events (AEs) and serious AEs (SAEs) for causal relationship to study drug.

Results: The analysis included data from 9139 patients with T2DM in 15 double-blind controlled studies who received either fasiglifam (n = 5359, fasiglifam group), fasiglifam and sitagliptin (n = 123), or a comparator agent (n = 3657, non-exposed group consisting of placebo and other antidiabetic agents). Exposure to treatment for more than 1 year ranged from 249 patients in the placebo arm, to 370 patients in the glimepiride arm and 617 patients in the fasiglifam 50 mg arm. The primary focus of the analysis was on the hepatic safety of fasiglifam. The overall safety profile based on treatment-emergent AEs (TEAEs), SAEs, deaths, and withdrawal due to AEs was similar between fasiglifam and placebo (excluding liver test abnormalities). However, there was an increased incidence rate of serum alanine aminotransferase (ALT) elevations > 3 × upper limit of normal (ULN), 5 × ULN, and 10 × ULN in fasiglifam-treated patients compared with those treated with placebo or active comparators. ALT elevations > 3 × ULN for fasiglifam were 2.7% compared with 0.8 and 0.5% for the active comparators and placebo. There did not appear to be a clear dose response in incidence of ALT elevations between patients receiving 25 or 50 mg daily. The cumulative incidence of elevations in serum ALT > 3 × ULN was higher in the first 6 months of treatment with fasiglifam compared with both placebo and the active comparators, but the rate of new ALT elevations appeared to be similar across all treatment groups thereafter. No demographic or baseline patient characteristics were identified to predict elevations exceeding ALT > 3 × ULN in fasiglifam-treated patients. The pattern of liver injury with fasiglifam was hepatocellular, and there were no reports of liver-related deaths, liver failure or life-threatening liver injury. Most fasiglifam-associated ALT elevations were asymptomatic and resolved promptly upon discontinuing treatment, but in two patients the recovery was prolonged. Importantly, three important serious liver injury cases were identified among fasiglifam-treated patients; one case was adjudicated to be a clear Hy's Law case and the two remaining cases were considered to closely approximate Hy's Law cases.

Conclusions: Although the incidence of overall AEs, SAEs, and deaths was similar between fasiglifam and placebo, a liver signal was identified based primarily on the difference in liver chemistry values in the fasiglifam group compared with the placebo and active comparator groups. Three serious liver injuries were attributed to fasiglifam treatment. Clinical development of fasiglifam was halted due to these liver safety concerns.
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http://dx.doi.org/10.1007/s40264-018-0642-6DOI Listing
June 2018

The effects of heat stress on morphological properties and intracellular signaling of denervated and intact soleus muscles in rats.

Physiol Rep 2017 Aug;5(15)

Space Biomedical Research Group, Japan Aerospace Exploration Agency, Tsukuba, Ibaraki, Japan.

The effects of heat stress on the morphological properties and intracellular signaling of innervated and denervated soleus muscles were investigated. Heat stress was applied to rats by immersing their hindlimbs in a warm water bath (42°C, 30 min/day, every other day following unilateral denervation) under anesthesia. During 14 days of experimental period, heat stress for a total of seven times promoted growth-related hypertrophy in sham-operated muscles and attenuated atrophy in denervated muscles. In denervated muscles, the transcription of ubiquitin ligase, atrogin-1/muscle atrophy F-box (), and muscle RING-finger protein-1 (), genes was upregulated and ubiquitination of proteins was also increased. Intermittent heat stress inhibited the upregulation of , but not transcription. And the denervation-caused reduction in phosphorylated protein kinase B (Akt), 70-kDa heat-shock protein (HSP70), and peroxisome proliferator-activated receptor coactivator-1 (PGC-1), which are negative regulators of and transcription, was mitigated. In sham-operated muscles, repeated application of heat stress did not affect and transcription, but increased the level of phosphorylated Akt and HSP70, but not PGC-1 Furthermore, the phosphorylation of Akt and ribosomal protein S6, which is known to stimulate protein synthesis, was increased immediately after a single heat stress particularly in the sham-operated muscles. The effect of a heat stress was suppressed in denervated muscles. These results indicated that the beneficial effects of heat stress on the morphological properties of muscles were brought regardless of innervation. However, the responses of intracellular signaling to heat stress were distinct between the innervated and denervated muscles.
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http://dx.doi.org/10.14814/phy2.13350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555886PMC
August 2017

Responses of skeletal muscles to gravitational unloading and/or reloading.

J Physiol Sci 2015 Jul 8;65(4):293-310. Epub 2015 Apr 8.

Space Biomedical Research Office, Japan Aerospace Exploration Agency, Tsukuba, Ibaraki, 305-8505, Japan.

Adaptation of morphological, metabolic, and contractile properties of skeletal muscles to inhibition of antigravity activities by exposure to a microgravity environment or by simulation models, such as chronic bedrest in humans or hindlimb suspension in rodents, has been well reported. Such physiological adaptations are generally detrimental in daily life on earth. Since the development of suitable countermeasure(s) is essential to prevent or inhibit these adaptations, effects of neural, mechanical, and metabolic factors on these properties in both humans and animals were reviewed. Special attention was paid to the roles of the motoneurons (both efferent and afferent neurograms) and electromyogram activities as the neural factors, force development, and/or length of sarcomeres as the mechanical factors and mitochondrial bioenergetics as the metabolic factors.
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http://dx.doi.org/10.1007/s12576-015-0375-6DOI Listing
July 2015

Retardation of C2C12 myoblast cell proliferation by exposure to low-temperature atmospheric plasma.

J Physiol Sci 2014 Sep 18;64(5):365-75. Epub 2014 Jul 18.

Department of Health and Sports Sciences, Graduate School of Medicine, Osaka University, 1-17 Machikaneyama-cho, Toyonaka, Osaka, 560-0043, Japan,

As the first step in evaluating the possibility of low-temperature atmospheric plasma for clinical applications in the treatment of rhabdomyosarcoma (RMS), we determined the effects of plasma exposure on C2C12 myoblasts. The low-temperature atmospheric plasma was generated through an electrical discharge in argon gas. One minute of plasma exposure every 24 h inhibited the cell proliferation, whereas myoblast differentiation was not affected. Plasma exposure increased the phosphorylation of ERK and JNK at 30 min after the exposure, but the phosphorylation of both was decreased to less than control levels at 1 and 4 h after the exposure. Plasma exposure increased the percentage of cells in the G2/M phase at 8 h after the exposure. In conclusion, plasma exposure retarded the proliferation of C2C12 myoblasts by G2/M arrest. Therefore, plasma exposure can be a possible treatment for the anti-proliferative effects of malignant tumors, such as RMS, without affecting differentiated skeletal muscle cells.
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http://dx.doi.org/10.1007/s12576-014-0328-5DOI Listing
September 2014

Effects of gravitational loading levels on protein expression related to metabolic and/or morphologic properties of mouse neck muscles.

Physiol Rep 2014 Jan 13;2(1):e00183. Epub 2014 Jan 13.

Research Center for Adipocyte and Muscle Science, Doshisha University, Kyotanabe City, 610-0394, Kyoto, Japan.

The effects of 3 months of spaceflight (SF), hindlimb suspension, or exposure to 2G on the characteristics of neck muscle in mice were studied. Three 8-week-old male C57BL/10J wild-type mice were exposed to microgravity on the International Space Station in mouse drawer system (MDS) project, although only one mouse returned to the Earth alive. Housing of mice in a small MDS cage (11.6 × 9.8-cm and 8.4-cm height) and/or in a regular vivarium cage was also performed as the ground controls. Furthermore, ground-based hindlimb suspension and 2G exposure by using animal centrifuge (n = 5 each group) were performed. SF-related shift of fiber phenotype from type I to II and atrophy of type I fibers were noted. Shift of fiber phenotype was related to downregulation of mitochondrial proteins and upregulation of glycolytic proteins, suggesting a shift from oxidative to glycolytic metabolism. The responses of proteins related to calcium handling, myofibrillar structure, and heat stress were also closely related to the shift of muscular properties toward fast-twitch type. Surprisingly, responses of proteins to 2G exposure and hindlimb suspension were similar to SF, although the shift of fiber types and atrophy were not statistically significant. These phenomena may be related to the behavior of mice that the relaxed posture without lifting their head up was maintained after about 2 weeks. It was suggested that inhibition of normal muscular activities associated with gravitational unloading causes significant changes in the protein expression related to metabolic and/or morphological properties in mouse neck muscle.
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http://dx.doi.org/10.1002/phy2.183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967672PMC
January 2014

Anti-interleukin-6 receptor antibody (MR16-1) promotes muscle regeneration via modulation of gene expressions in infiltrated macrophages.

Biochim Biophys Acta 2014 Oct 15;1840(10):3170-80. Epub 2014 Jan 15.

Graduate School of Medicine, Osaka University, Japan; Graduate School of Frontier Bioscience, Osaka University, Japan. Electronic address:

Background: Although rat anti-mouse IL-6 receptor (IL-6R) antibody (MR16-1) has been reported to effectively ameliorate various tissue damages, its effect on skeletal muscle regeneration has not been determined. Moreover, the localization, persistence and duration of action of this reagent in damaged tissues after systemic administration have not been assessed.

Methods: The MR16-1 was administered i.p. immediately after cardiotoxin (CTX)-induced muscle damage on mice.

Results: MR16-1 administered i.p. was observed only to the damaged muscle. This delivered MR16-1 was dramatically decreased from 3 to 7days post-injury concomitantly with a reduction of IL-6R expression. This reduction of the MR16-1 level in the damaged muscle was not rescued by additional administration of MR16-1, suggesting the short half-life of MR16-1 was not the factor for the remaining levels. In addition, a significant inhibitory effect of MR16-1 on phosphorylation of the signal transducer and activator of transcription 3 was observed in the macrophage-enriched area of damaged muscle 3days after injury. Finally, the acceleration of muscle regeneration observed at day 7 post-injury following MR16-1 treatment was associated with reduced expression of fibrosis-related genes, such as interleukin-10 and arginase, in the infiltrated macrophages.

Conclusions: These results suggest that MR16-1 which was found primarily localized in infiltrated macrophages in the damaged muscle might facilitate muscle regeneration via immune modulation.

General Significance: These findings are deemed to provide further insight into the understanding not only of MR16-1 treatment on muscle regeneration, but also of the other anti-cytokine treatment on the cytokine-related disease.
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http://dx.doi.org/10.1016/j.bbagen.2014.01.014DOI Listing
October 2014

Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days.

PLoS One 2012 9;7(7):e40112. Epub 2012 Jul 9.

Behavioural Neuroscience Section, Cellular Biology and Neuroscience Department, Istituto Superiore di Sanità, Rome, Italy.

Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0040112PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3392276PMC
March 2013

The impact of long-term exposure to space environment on adult mammalian organisms: a study on mouse thyroid and testis.

PLoS One 2012 25;7(4):e35418. Epub 2012 Apr 25.

DIPTERIS, University of Genoa, Genova, Italy.

Hormonal changes in humans during spaceflight have been demonstrated but the underlying mechanisms are still unknown. To clarify this point thyroid and testis/epididymis, both regulated by anterior pituitary gland, have been analyzed on long-term space-exposed male C57BL/10 mice, either wild type or pleiotrophin transgenic, overexpressing osteoblast stimulating factor-1. Glands were submitted to morphological and functional analysis.In thyroids, volumetric ratios between thyrocytes and colloid were measured. cAMP production in 10(-7)M and 10(-8)M thyrotropin-treated samples was studied. Thyrotropin receptor and caveolin-1 were quantitized by immunoblotting and localized by immunofluorescence. In space-exposed animals, both basal and thyrotropin-stimulated cAMP production were always higher. Also, the structure of thyroid follicles appeared more organized, while thyrotropin receptor and caveolin-1 were overexpressed. Unlike the control samples, in the space samples thyrotropin receptor and caveolin-1 were both observed at the intracellular junctions, suggesting their interaction in specific cell membrane microdomains.In testes, immunofluorescent reaction for 3β- steroid dehydrogenase was performed and the relative expressions of hormone receptors and interleukin-1β were quantified by RT-PCR. Epididymal sperm number was counted. In space-exposed animals, the presence of 3β and 17β steroid dehydrogenase was reduced. Also, the expression of androgen and follicle stimulating hormone receptors increased while lutenizing hormone receptor levels were not affected. The interleukin 1 β expression was upregulated. The tubular architecture was altered and the sperm cell number was significantly reduced in spaceflight mouse epididymis (approx. -90% vs. laboratory and ground controls), indicating that the space environment may lead to degenerative changes in seminiferous tubules.Space-induced changes of structure and function of thyroid and testis/epididymis could be responsible for variations of hormone levels in human during space missions. More research, hopefully a reflight of MDS, would be needed to establish whether the space environment acts directly on the peripheral glands or induces changes in the hypotalamus-pituitary-glandular axis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035418PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338839PMC
September 2012

Effects of mechanical over-loading on the properties of soleus muscle fibers, with or without damage, in wild type and mdx mice.

PLoS One 2012 16;7(4):e34557. Epub 2012 Apr 16.

Graduate School of Frontier Biosciences, Osaka University, Toyonaka City, Osaka, Japan.

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (∼51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0034557PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327707PMC
August 2012

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.

PLoS One 2012 28;7(3):e33232. Epub 2012 Mar 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314659PMC
August 2012

Effects of hindlimb unloading on neurogenesis in the hippocampus of newly weaned rats.

Neurosci Lett 2012 Feb 22;509(2):76-81. Epub 2011 Dec 22.

Graduate School of Medicine, Osaka University, Toyonaka City, Osaka 560-0043, Japan.

Effects of hindlimb suspension (HS) and ambulation recovery on hippocampal neurogenesis of newly weaned rats were studied by using immunohistochemical techniques. The number of proliferating cell nuclear antigen-positive (PCNA(+)) cells in the subgranular zone (SGZ) markedly decreased during normal growth. However, neither HS nor subsequent recovery caused additional changes in the number of PCNA(+) cells. The number of doublecortin-positive (DCX(+)) neurons decreased gradually during normal growth. HS resulted in a further decrease in these neurons. However, DCX(+) cell numbers became identical to the levels in age-matched controls after 14 days of recovery. PCNA and DCX-double positive cells in the SGZ were also observed, and their cell numbers were not affected by HS and 14-day ambulation. Thus, HS suppressed the generation of DCX(+) neurons without affecting PCNA(+) cells in the SGZ of weaned rats. Taken together, hippocampal neurogenesis in weaned rats was not severely affected by HS while it decreased significantly as they had grown.
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http://dx.doi.org/10.1016/j.neulet.2011.12.022DOI Listing
February 2012

HSP25 can modulate myofibrillar desmin cytoskeleton following the phosphorylation at Ser15 in rat soleus muscle.

J Appl Physiol (1985) 2012 Jan 13;112(1):176-86. Epub 2011 Oct 13.

Graduate School of Medicine, Osaka University, Toyonaka City, Osaka, Japan.

The main purpose of the present study was to investigate the role(s) of 25-kDa heat shock protein (HSP25) in the regulation and integration of myofibrillar Z-disc structure during down- or upregulation of the size in rat soleus muscle fibers. Hindlimb unloading by tail suspension was performed in adult rats for 7 days, and reloading was allowed for 5 days after the termination of suspension. Interaction of HSP25 and Z-disc proteins, phosphorylation status, distribution, and complex formation of HSP25 were investigated. Non- and single-phosphorylated HSP25s were generally expressed in the cytoplasmic fraction of normal muscle. The level of total HSP25, as well as the phosphorylation ratio, did not change significantly in response to atrophy. Increased expressions of HSP25, phosphorylated at serine 15 (p-Ser15) and dual-phosphorylated form, were noted, when atrophied muscles were reloaded. Myofibrillar HSP25 was also noted in reloaded muscle. Histochemical analysis further indicated the localization of p-Ser15 in the regions with disorganization of Z-disc structure in reloaded muscle fibers. HSP25 formed a large molecular complex in the cytoplasmic fraction of normal muscle, whereas dissociation of free HSP25 with Ser15 phosphorylation was noted in reloaded muscle. The interaction of p-Ser15 with desmin and actinin was detected in Z-discs by proximity ligation assay. Strong interaction between p-Ser15 and desmin, but not actinin, was noted in the disorganized areas. These results indicated that HSP25 contributed to the desmin cytoskeletal organization following the phosphorylation at Ser15 during reloading and regrowing of soleus muscle.
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http://dx.doi.org/10.1152/japplphysiol.00783.2011DOI Listing
January 2012

Responses of HSC70 expression in diencephalon to iron deficiency anemia in rats.

J Physiol Sci 2011 Nov 3;61(6):445-56. Epub 2011 Aug 3.

Section of Applied Physiology, Graduate School of Medicine, Osaka University, Toyonaka, Osaka, 560-0043, Japan.

A powdered diet containing 100 or 3 ppm Fe was fed to rats starting at the age of 3 weeks. The voluntary activity level was checked using a wheel in the cage during the 17th week after the beginning of supplementation. Significantly less activity was seen in the 3 ppm Fe group during both light and dark periods. After 20 weeks, the blood and diencephalon were sampled from both groups. Lower hematocrit and blood hemoglobin content was observed in the 3 ppm Fe group. The level of 70 kDa heat shock cognate (HSC70) expression was greater in the diencephalon of the 3 ppm Fe group. In addition, the distribution of HSC70 was determined by proximity ligation assay. More HSC70-positive as well as total cells were noted in several areas of the diencephalon of the iron-deficient rats. The altered expression and distribution of HSC70 might play some role in the neurological changes.
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http://dx.doi.org/10.1007/s12576-011-0164-9DOI Listing
November 2011

Region-specific responses of adductor longus muscle to gravitational load-dependent activity in Wistar Hannover rats.

PLoS One 2011 22;6(6):e21044. Epub 2011 Jun 22.

Graduate School of Frontier Biosciences, Osaka University, Toyonaka City, Osaka, Japan.

Response of adductor longus (AL) muscle to gravitational unloading and reloading was studied. Male Wistar Hannover rats (5-wk old) were hindlimb-unloaded for 16 days with or without 16-day ambulation recovery. The electromyogram (EMG) activity in AL decreased after acute unloading, but that in the rostral region was even elevated during continuous unloading. The EMG levels in the caudal region gradually increased up to 6th day, but decreased again. Approximately 97% of fibers in the caudal region were pure type I at the beginning of experiment. Mean percentage of type I fibers in the rostral region was 61% and that of type I+II and II fiber was 14 and 25%, respectively. The percent type I fibers decreased and de novo appearance of type I+II was noted after unloading. But the fiber phenotype in caudal, not rostral and middle, region was normalized after 16-day ambulation. Pronounced atrophy after unloading and re-growth following ambulation was noted in type I fibers of the caudal region. Sarcomere length in the caudal region was passively shortened during unloading, but that in the rostral region was unchanged or even stretched slightly. Growth-associated increase of myonuclear number seen in the caudal region of control rats was inhibited by unloading. Number of mitotic active satellite cells decreased after unloading only in the caudal region. It was indicated that the responses of fiber properties in AL to unloading and reloading were closely related to the region-specific neural and mechanical activities, being the caudal region more responsive.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021044PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3120817PMC
November 2011

Effects of creatine and its analog, β-guanidinopropionic acid, on the differentiation of and nucleoli in myoblasts.

Biosci Biotechnol Biochem 2011 13;75(6):1085-9. Epub 2011 Jun 13.

Department of Health and Sports Sciences, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

The effects of supplementation with creatine (Cr) and its analog, β-guanidinopropionic acid (β-GPA), on the differentiation of myoblasts and the numbers of nucleoli were studied in C2C12 cells. The cells were cultured in differentiation medium for 4 d. Then Cr (1 mM) or β-GPA (1 mM) was added to the cells, and the mixture was cultured for an additional 2 d. Although the number of myotubes was not different among the groups, myotube diameters and nuclear numbers in myotubes were increased by Cr and β-GPA treatment respectively. The expression of differentiation marker proteins, myogenin, and the myosine heavy chain, was increased in the β-GPA group. Supplementation with β-GPA also increased the percentage of p21 (inhibitor for cell cycle progression)-positive myoblasts. Supplementation with Cr inhibited the decrease in nucleoli numbers, whereas β-GPA increased nucleolar sizes in the myotubes. These results suggest that β-GPA supplementation stimulated the differentiation of myoblasts into multi-nucleated myotubes through induction of p21 expression.
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http://dx.doi.org/10.1271/bbb.100901DOI Listing
October 2011

Mechanical stretch activates signaling events for protein translation initiation and elongation in C2C12 myoblasts.

Mol Cells 2010 Dec 14;30(6):513-8. Epub 2010 Oct 14.

Section of Applied Physiology, Department of Health and Sports Sciences, Graduate School of Medicine, Osaka University, Osaka 560-0043, Japan.

It has been proposed that mechanically induced tension is the critical factor in the induction of muscle hypertrophy. However, the molecular mechanisms involved in this process are still under investigation. In the present study, the effect of mechanical stretch on intracellular signaling for protein translation initiation and elongation was studied in C2C12 myoblasts. Cells were grown on a silicone elastomer chamber and subjected to 30-min of 5 or 15% constant static or cyclic (60 cycles/min) uniaxial stretch. Western blot analyses revealed that p70 S6 kinase (p70S6K) and eukaryotic elongation factor 2 (eEF2), which are the markers for translation initiation and peptide chain elongation, respectively, were activated by both static and cyclic stretch. The magnitude of activation was greater in response to the 15% cyclic stretch. Cyclic stretch also increased the phosphorylation of MAP kinases (p38 MAPK, ERK1/2 and JNK). However, the pharmacological inhibition of MAP kinases did not block the stretch-induced activation of p70S6K and eEF2. An inhibitor of the mammalian target of rapamycin (mTOR) blocked the stretch-induced phosphorylation of p70S6K but did not affect the eEF2 activation. A broad-range tyrosine kinase inhibitor, genistein, blocked the stretch-induced activation of p70S6K and eEF2, whereas Src tyrosine kinase and Janus kinase (JAK) inhibitors did not. These results suggest that the stretch-induced activation of protein translation initiation and elongation in mouse myoblast cell lines is mediated by tyrosine kinase(s), except for Src kinase or JAK.
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http://dx.doi.org/10.1007/s10059-010-0147-3DOI Listing
December 2010

Role(s) of gravitational loading during developing period on the growth of rat soleus muscle fibers.

J Appl Physiol (1985) 2010 Mar 7;108(3):676-85. Epub 2010 Jan 7.

Graduate School of Medicine, Osaka University, Toyonaka City, Osaka 560-0043, Japan.

Effects of gravitational loading or unloading on the gain of the characteristics in soleus muscle fibers were studied in rats. The tail suspension was performed in newborn rats from postnatal day 4 to month 3, and the reloading was allowed for 3 mo in some rats. Single expression of type I myosin heavy chain (MHC) was observed in approximately 82% of fibers in 3-mo-old controls, but the fibers expressing multiple MHC isoforms were noted in the unloaded rats. Although 97% of fibers in 3-mo-old controls had a single neuromuscular junction at the central region of fiber, fibers with multiple nerve endplates were seen in the unloaded group. Faster contraction speed and lower maximal tension development, even after normalization with fiber size, were observed in the unloaded pure type I MHC fibers. These parameters generally returned to the age-matched control levels after reloading. It was suggested that antigravity-related tonic activity plays an important role in the gain of single neural innervation and of slow contractile properties and phenotype in soleus muscle fibers.
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http://dx.doi.org/10.1152/japplphysiol.00478.2009DOI Listing
March 2010

Analysis of development of lesions in mice with serine palmitoyltransferase (SPT) deficiency -Sptlc2 conditional knockout mice-.

Exp Anim 2009 Oct;58(5):515-24

Safety Assessment, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan.

Serine palmitoyltransferase (SPT) is the enzyme which catalyzes the first step of the biosynthesis of sphingolipids. However, the precise roles of SPT in vivo are not well understood, since complete knockout (KO) of genes which compose SPT results in a fetal lethal phenotype. A conditional KO (cKO) mouse of SPT long chain base 2 (Sptlc2) was therefore developed, and the effects of Sptlc2 deficiency were examined. Single cell necrosis in the epithelia of the crypts of the small and large intestines was observed as early as 24 h after induction of knockout. At 48 h after induction, decreases in spleen and thymus weights and decreases in numbers of reticulocytes and lymphocytes were observed in cKO mice, and single cell necrosis in the intestine became prominent. At 72 h after induction, decreases in body weight, spleen and thymus weights, and numbers of reticulocytes and lymphocytes became obvious in cKO mice. Histologically, atrophy of gastrointestinal mucosa and lymphoid necrosis as well as depletion of lymphoid and hematopoietic tissues were observed. These findings suggest that SPT plays important roles in the maintenance of the gastrointestinal mucosa, especially in the proliferation of the mucosal epithelial cells, and that deficiency of Sptlc2 induces necrotic lesions in gastrointestinal cells followed by atrophic change of the tissue in short term.
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http://dx.doi.org/10.1538/expanim.58.515DOI Listing
October 2009

Myonucleus-related properties in soleus muscle fibers of mdx mice.

Cells Tissues Organs 2010 18;191(3):248-59. Epub 2009 Sep 18.

Osaka University, Japan.

Distribution and total number of myonuclei in single soleus muscle fibers, sampled from tendon to tendon, were analyzed in mdx and wild-type (WT) mice. Apoptotic myonuclei and the microscopic structure around the myonuclei were also analyzed. Three types of muscle fibers of mdx mice with myonuclear distribution at either central, peripheral, or both central and peripheral regions were observed in the longitudinal analyses. All of the myonuclei were located at the peripheral region in WT mice. The total number of myonuclei counted in the whole length of fibers with peripheral myonuclei only was 17% less in mdx than in WT mice (p < 0.05). But the total myonuclear numbers in mdx mouse fibers with different distribution (peripheral vs. central) of myonuclei were identical, and the peripheral nucleus was noted where the central nucleus was missing. Myonuclei located between the center and peripheral regions were also seen in the cross-sectional analyses of muscle fibers. The cross-sectional area and length of fibers, sarcomere number, myonuclear size, myosin heavy chain expression, satellite cell number and neuromuscular junction were identical between each type of fiber. Apoptosis was not detected in any myonuclei located either in central or peripheral regions of muscle fibers. Thus, it was suggested that apoptosis-related loss of central myonuclei and regeneration-related new accretion at the peripheral region is not the cause of different distribution of myonuclei seen in muscle fibers in mdx mice. However, it was speculated that cross-sectional migration of myonuclei from central to peripheral regions may be induced in response to regeneration, because the total myonuclear numbers in fibers with different distribution of myonuclei were identical, and the peripheral nucleus was noted where the central nucleus was missing. Further, myonuclei located between the center and peripheral regions were also seen. However, the question remains as to how or why nuclei might migrate to the periphery in a regenerating muscle fiber, since there was no microscopic evidence of any structural changes around the myonuclei that may be responsible for the movement of the nucleus.
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http://dx.doi.org/10.1159/000240245DOI Listing
May 2010

Effects of reduced food intake on toxicity study parameters in rats.

J Toxicol Sci 2008 Dec;33(5):537-47

Tsukuba Safety Assessment Laboratories, Banyu Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan.

This study comprehensively describes the effects of various levels of food reduction on a wide range of toxicological parameters in dietary-optimized rats (fed with approximately 75% of ad libitum food consumption daily; 16 g and 22 g/day for females and males, respectively) that has been established as a nutritionally appropriate and well-controlled animal model in conducting toxicity studies. Toxicological parameters, including general condition, ophthalmology, clinical pathology and anatomic pathology, were examined in dietary-optimized Crl:CD(SD) female and male rats fed 16 g and 22 g/day (control), 12 g and 17 g/day (75% group), 8 g and 11 g/day (50% group), or 4 g and 6 g/day (25% group), respectively for 2 weeks. There was mortality and morbidity including reddish urine in 25% group females. The reddish urine was identified as "hemoglobinuria" that resulted from extra/intra-vascular hemolysis induced by severe food reduction. Hemoconcentration, decreased leukocytes and platelets, decreases in nutritional elements (serum glucose, protein, and lipids), increased aspartate aminotransferase and alanine aminotransferase, imbalanced electrolytes, and/or decreased urinary pH were observed in all restriction groups. Histopathologically remarkable changes included erythrophagocytosis in the spleen/liver and renal tubular necrosis with hyaline cast/droplets in 25% group; in addition to bone marrow depletion, lymphoid depletion in thymus/spleen/lymph node, and/or decreased secretion in the prostate/seminal vesicle in all restriction groups. Most of these changes were considered attributable to nutritional deficiency, dehydration, accelerated protein catabolism, stress and/or hemolysis secondary to severe food reduction. These results will enable toxicologists to help distinguish primary drug-induced effects from secondary changes associated with decreases in food consumption.
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http://dx.doi.org/10.2131/jts.33.537DOI Listing
December 2008

Effects of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists on leucine-induced phosphorylation of translational targets in C2C12 cells.

Biochim Biophys Acta 2008 Oct 18;1780(10):1101-5. Epub 2008 Jun 18.

Department of Health and Sports Sciences, Graduate School of Medicine Bioscience, Osaka University, Osaka 560-0043, Japan.

Effect of peroxisome proliferator-activated receptor alpha (PPARalpha) agonists, WY-14,643 (WY) and/or clofibrate, on the leucine-induced phosphorylation of translational targets in C2C12 myoblasts was studied. C2C12 cells were treated with WY or clofibrate for 24 h prior to stimulation with leucine. Western blot analyses revealed that the leucine-induced phosphorylation of p70 S6 kinase (p70S6K), a key regulator of translation initiation, was significantly higher in WY-treated cells than in control and clofibrate-treated cells. Phosphorylation of extracellular-regulated kinase (ERK1/2) was higher in WY-treated cells. WY treatment also increased the leucine-induced phosphorylation of ribosomal protein S6 and eukaryotic initiation factor 4B. In contrast, eukaryotic elongation factor 2, a marker for peptide chain elongation process, was significantly activated (dephosphorylated) only in leucine-stimulated control cells. Pre-treatment of the cells with PD98059 (ERK1/2 kinase inhibitor) prevented the phosphorylation of ERK1/2 and decreased the leucine-induced phosphorylation of p70S6K. It is concluded that WY increased the leucine-induced phosphorylation of target proteins involving in translation initiation via ERK/p70S6K pathway, but impaired the signaling for elongation process, suggesting that p70S6K phosphorylation may be essential, but not sufficient for the activation of entire targets for protein translation in WY-treated cells.
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http://dx.doi.org/10.1016/j.bbagen.2008.06.002DOI Listing
October 2008

Essential role of satellite cells in the growth of rat soleus muscle fibers.

Am J Physiol Cell Physiol 2008 Aug 4;295(2):C458-67. Epub 2008 Jun 4.

Graduate School of Medicine, Osaka University, Osaka, Japan.

Effects of gravitational loading or unloading on the growth-associated increase in the cross-sectional area and length of fibers, as well as the total fiber number, in soleus muscle were studied in rats. Furthermore, the roles of satellite cells and myonuclei in growth of these properties were also investigated. The hindlimb unloading by tail suspension was performed in newborn rats from postnatal day 4 to month 3 with or without 3-mo reloading. The morphological properties were measured in whole muscle and/or single fibers sampled from tendon to tendon. Growth-associated increases of soleus weight and fiber cross-sectional area in the unloaded group were approximately 68% and 69% less than the age-matched controls. However, the increases of number and length of fibers were not influenced by unloading. Growth-related increases of the number of quiescent satellite cells and myonuclei were inhibited by unloading. And the growth-related decrease of mitotically active satellite cells, seen even in controls (20%, P > 0.05), was also stimulated (80%). The increase of myonuclei during 3-mo unloading was only 40 times vs. 92 times in controls. Inhibited increase of myonuclear number was not related to apoptosis. The size of myonuclear domain in the unloaded group was less and that of single nuclei, which was decreased by growth, was larger than controls. However, all of these parameters, inhibited by unloading, were increased toward the control levels generally by reloading. It is suggested that the satellite cell-related stimulation in response to gravitational loading plays an essential role in the cross-sectional growth of soleus muscle fibers.
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http://dx.doi.org/10.1152/ajpcell.00497.2007DOI Listing
August 2008