Kyoichi Kishi, Arinobu Yamauchi, Fujiko Shizuka, Yasuhiro Kido, Takeshi Nikawa and Kazuhito Rokutan : Serotonin in the Central Nervous System and Periphery, --- Effect of macronutrient intakes on brain serotonin metabolite as measured by in vivo microdialysis in the rat ---, Elsevier, Tokyo, Apr. 1995.
15.
Nobuhiko Katunuma, Hisao Kakegawa, Y Matsunaga, Takeshi Nikawa and Eiki Kominami : Agents Actions:Proreases,Protease Inhibitors and Protease-Drived Peptides(JC Cheronis and JE repine,eds), --- Different functional share of individual lysosomal cathepsins in normal and pathological conditions ---, Birkhauser Verlag, May 1993.
学術論文(審査論文):
1.
Takayuki Uchida, Kohno Madoka, Chinami Itoh, Erika Yamano, Hiroki Matsuki, MIZANUR MD Rahman, ANAYT ULLA, Minori Suzuki, Takahiro Ishiguro and Takeshi Nikawa : Inhibitory Effect of Kori-tofu Protein on Denervation-Induced Atrogene Expression in Mouse Skeletal Muscle, Biological Sciences in Space, Vol.38, 1-7, 2024.
2.
Yurika Ito, Mari Yamagata, Takuya Yamamoto, Katsuya Hirasaka, Takeshi Nikawa and Takahiko Sato : The reciprocal regulation between mitochondrial-associated membranes and Notch signaling in skeletal muscle atrophy., eLife, Vol.12, RP89381, 2023.
(要約)
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.
Shintaro Inoue, Takahito Watanabe, Taiki Hamaguchi, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji and Taro Mito : Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus., PLoS ONE, Vol.18, No.5, 2023.
(要約)
Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.
Taishi Kondo, Tomoaki Ishida, Ke Ye, Marin Muraguchi, Yohei Tanimura, Masato Yoshida, Kan'ichiro Ishiuchi, Tomoki Abe, Takeshi Nikawa, Keisuke Hagihara, Hidetoshi Hayashi and Toshiaki Makino : Correction to: Suppressive effects of processed aconite root on dexamethasone-induced muscle ring finger protein-1 expression and its active ingredients., Journal of Natural Medicines, Vol.76, No.3, 594-604, 2023.
Luchuanyang Sun, Sangeun Kim, Ryoichi Mori, Nobuyuki Miyaji, Takeshi Nikawa and Katsuya Hirasaka : Astaxanthin Exerts Immunomodulatory Effect by Regulating SDH-HIF-1α Axis and Reprogramming Mitochondrial Metabolism in LPS-Stimulated RAW264.7 Cells., Marine Drugs, Vol.20, No.11, 660, 2022.
(要約)
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-1β 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.
ANAYT ULLA, Kanae Osaki, Mizanur Md Rahman, Reiko Nakao, Takayuki Uchida, Isafumi Maru, Kazuaki Mawatari, Tomoya Fukawa, Hiro-omi Kanayama, Iori Sakakibara, Katsuya Hirasaka and Takeshi Nikawa : Morin improves dexamethasone-induced muscle atrophy by modulating atrophy-related genes and oxidative stress in female mice., Bioscience, Biotechnology, and Biochemistry, Vol.86, No.10, 1448-1458, 2022.
(要約)
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.
Min Yang, Luchuanyang Sun, Yasunosuke Kawabata, Fumihito Murayama, Takahiro Maegawa, Takeshi Nikawa and Katsuya Hirasaka : Balenine, Imidazole Dipeptide Promotes Skeletal Muscle Regeneration by Regulating Phagocytosis Properties of Immune Cells., Marine Drugs, Vol.20, No.5, 313, 2022.
(要約)
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.
Yuichiro Adachi, Masashi Masuda, Iori Sakakibara, Takayuki Uchida, Yuki Niida, Yuki Mori, Yuki Kamei, Yosuke Okumura, Hirokazu Ohminami, Kohta Ohnishi, Hisami Okumura, Takeshi Nikawa and Yutaka Taketani : All-trans retinoic acid changes muscle fiber type via increasing GADD34 dependent on MAPK signal., Life Science Alliance, Vol.5, No.7, 2022.
(要約)
All-trans retinoic acid (ATRA) increases the sensitivity to unfolded protein response in differentiating leukemic blasts. The downstream transcriptional factor of PERK, a major arm of unfolded protein response, regulates muscle differentiation. However, the role of growth arrest and DNA damage-inducible protein 34 (GADD34), one of the downstream factors of PERK, and the effects of ATRA on GADD34 expression in muscle remain unclear. In this study, we identified ATRA increased the GADD34 expression independent of the PERK signal in the gastrocnemius muscle of mice. ATRA up-regulated GADD34 expression through the transcriptional activation of gene via inhibiting the interaction of homeobox Six1 and transcription co-repressor TLE3 with the MEF3-binding site on the gene promoter in skeletal muscle. ATRA also inhibited the interaction of TTP, which induces mRNA degradation, with AU-rich element on mRNA via p-38 MAPK, resulting in the instability of mRNA. Overexpressed GADD34 in C2C12 cells changes the type of myosin heavy chain in myotubes. These results suggest ATRA increases GADD34 expression via transcriptional and post-transcriptional regulation, which changes muscle fiber type.
Kitahata Kanako, Takayuki Uchida, Taniguchi Runa, Kato Ayano, Kosuke Sugiura, Iori Sakakibara, Oarada Motoko, Tomoya Fukawa, Junsoo Park, Inho Choi and Takeshi Nikawa : Additional effects of simultaneous treatment with C14-Cblin and celastrol on the clinorotation-induced rat L6 myotube atrophy, The Journal of Medical Investigation : JMI, Vol.69, No.1,2, 127-134, 2022.
(要約)
Two novel reagents, N-myristoylated Cbl-b inhibitory peptide (C14-Cblin) and celastrol, a quinone methide triterpene, are reported to be effective in preventing myotube atrophy. The combined effects of C14-Cblin and celastrol on rat L6 myotubes atrophy induced by 3D-clinorotation, a simulated microgravity model, was investigated in the present study. We first examined their effects on expression in atrogenes. Increase in MAFbx1/atrogin-1 and MuRF-1 by 3D-clinorotation was significantly suppressed by treatment with C14-Cblin or celastrol, but there was no additive effect of simultaneous treatment. However, celastrol significantly suppressed the upregulation of Cbl-b and HSP70 by 3D-clinorotation. Whereas 3D-clinorotation decreased the protein level of IRS-1 in L6 myotubes, C14-Cblin and celastrol inhibited the degradation of IRS-1. C14-Cblin and celastrol promoted the phosphorylation of FOXO3a even in microgravity condition. Simultaneous administration of C14-Cblin and celastrol had shown little additive effect in reversing the impairment of IGF-1 signaling by 3D-clinorotation. While 3D-clinorotation-induced marked oxidative stress in L6 myotubes, celastrol suppressed 3D-clinorotation-induced ROS production. Finally, the C14-Cblin and celastrol-treated groups were inhibited decrease in L6 myotube diameter and increased the protein content of slow-twitch MyHC cultured under 3D-clinorotation. The simultaneous treatment of C14-Cblin and celastrol additively prevented 3D-clinorotation-induced myotube atrophy than single treatment. J. Med. Invest. 69 : 127-134, February, 2022.
Taishi Kondo, Tomoaki Ishida, Ke Ye, Marin Muraguchi, Yohei Tanimura, Masato Yoshida, Kan'ichiro Ishiuchi, Tomoki Abe, Takeshi Nikawa, Keisuke Hagihara, Hidetoshi Hayashi and Toshiaki Makino : Suppressive effects of processed aconite root on dexamethasone-induced muscle ring finger protein-1 expression and its active ingredients., Journal of Natural Medicines, Vol.76, No.3, 594-604, 2022.
(要約)
values were 0.49 and 50 µM, respectively. The contents of higenamine and salsolinol in the decoctions of commercially available fourteen PA products are 0.12 and 14 µg/ml as the average values, and varied with the coefficient of variation (CV) values of 97 and 63%, respectively. Higenamine also significantly suppressed dexamethasone-induced mRNA expressions of muscle atrophy F-box protein (MAFbx)/atrogin1, casitas B-lineage lymphoma-b (Cbl-b), troponin, branched-chain amino acid aminotransferase 2 (BCAT2), and Bcl-2 binding and pro-apoptotic protein3 (Bnip3). Although the quality control of PA is regulated by the contents of diterpene alkaloids, salsolinol and higenamine can be used as the marker compounds to certificate the pharmacological activities of PA.
Madoka Kohno, ANAYT ULLA, Rina Taniguchi, Akane Ohishi, Kako Hirayama, Yuma Takemura, Shoichiro Takao, Yuki Kanazawa, Yuki Matsumoto, Masafumi Harada, Tomoya Fukawa, Hiro-omi Kanayama, Takayuki Uchida, Toshio Suzuki and Takeshi Nikawa : Daily Dietary Supplementation with Steamed Soybean Improves Muscle Volume and Strength in Healthy People Lacking Exercise., Journal of Nutritional Science and Vitaminology, Vol.68, No.6, 521-526, 2022.
(要約)
Various dietary protein supplements are used by the elderly and bedridden to maintain their skeletal muscle mass and functions. High-quality proteins act as an anabolic driver and help to improve muscle strength and performance. Previously, we reported that soy protein significantly attenuates denervation-induced loss of muscle mass and myofiber cross sectional area in mice with inhibition of ubiquitination and degradation of IRS-1 in tibialis anterior muscle. It also increased muscle volume and strength in bedridden patients. In the present study, we investigated the effects of dietary soybean supplementation on muscle functions in taxi drivers lacking vigorous physical exercise. We conducted a case-control study on 25 healthy, male taxi drivers between the ages of 36 and 71 y performing minimal physical exercise. They were divided into two dietary groups: the soybean diet group (n=13) who ate daily meals (dinner) supplemented with 50 g of steamed soybean for 30 d and the control diet group (n=12) who received no soybean supplement. Next, we measured the muscle cross-sectional area (CSA) and muscle strength and function in both the groups before and after 30 d of soybean intake. The body weights of both diet groups did not differ significantly over time. However, after 30 d of soybean supplementation, the soybean-fed group developed significantly higher muscle CSA and grip strength compared to the control groups. In conclusion, dietary soybean supplementation improved muscle function in taxi drivers who lacked exercise.
Oarada Motoko, Yuushi Okumura, Katsuya Hirasaka, Kosuke Sugiura, Tachibana Nobuhiko, Tsurusaki Yoshinori and Takeshi Nikawa : Dietary Sodium Nitrite Causes Similar Modifications to Splenic Inflammatory Gene Expression as a High-Fat Diet, Journal of Nutritional Science and Vitaminology, Vol.67, No.6, 404-416, 2021.
(要約)
Sodium nitrite (NaNO) is a widely used food additive. The present study compared the outcomes from intakes of dietary NaNO and a high-fat diet (HFD), and assessed their combined effects on inflammatory gene expression in the immune tissues of the mouse. In experiment I, mice were fed a standard low-fat diet (LFD) without or with NaNO (0.02 and 0.08%, w/w) for 11 wk. In experiment II, mice were fed an LFD without or with NaNO (0.02%) or HFD without or with NaNO (0.02%) for 11 wk. Inflammatory gene expression in the immune tissues was then measured. NaNO consumption and HFD feeding each resulted in increased splenic mRNAs for cell markers of neutrophils (Ngp, NE, Ly6g, Mpo) and eosinophils (Epo, Ear6), and an S100 family member (S100A8). In contrast, NaNO consumption and HFD feeding each resulted in decreased splenic mRNAs for cell markers of macrophages (Emr1, Itgax, CD68, CD206, Dectin-1, TLRs 4, 6, and 7), T- (CD3, CD4), NK- (CD56) and B-cells (CD20, CD40), pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-γ, IL-18, IL-10, TGF-β), interleukin receptor antagonists (IL1ra, IL6ra) and cell adhesion molecules (ICAM-1, VCAM-1). However, dietary NaNO combined with HFD feeding caused no further decrease in these transcript levels compared with dietary NaNO alone. These NaNO- or HFD-induced modifications were less profound in the liver and abdominal adipose tissues than in the spleen. These findings indicate that dietary NaNO has similar modulatory effects to HFD feeding on splenic inflammatory genes.
Reiko Nakao, Shen Weilin, Shimajiri Yasuka, Kainou Kumiko, Sato Yuki, Ulla Anayt, Kohta Ohnishi, Ninomiya Miyuki, Ohno Ayako, Takayuki Uchida, Tanaka Mitsuru, Akama Kazuhito, Matsui Toshiro and Takeshi Nikawa : Oral intake of rice overexpressing ubiquitin ligase inhibitory pentapeptide prevents atrophy in denervated skeletal muscle, NPJ Science of Food, Vol.5, No.1, 25, 2021.
(要約)
We previously reported that intramuscular injections of ubiquitin ligase CBLB inhibitory pentapeptide (Cblin; Asp-Gly-pTyr-Met-Pro) restored lost muscle mass caused by sciatic denervation. Here, we detected Cblin on the basolateral side of Caco-2 cells after being placed on the apical side, and found that cytochalasin D, a tight junction opener, enhanced Cblin transport. Orally administered Cblin was found in rat plasma, indicating that intact Cblin was absorbed in vitro and in vivo. Furthermore, transgenic Cblin peptide-enriched rice (CbR) prevented the denervation-induced loss of muscle mass and the upregulation of muscle atrophy-related ubiquitin ligases in mice. These findings indicated that CbR could serve as an alternative treatment for muscle atrophy.
Kiminori Yukata, Takeshi Nikawa, Mitsuhiko Takahashi and Natsuo Yasui : Overexpressed osteoactivin reduced osteoclastic callus resorption during distraction osteogenesis in mice, Journal of Pediatric Orthopaedics. Part B, Vol.30, No.5, 500-506, 2021.
(要約)
Distraction osteogenesis is a widely used surgical technique to treat bone deformity and shortening. Several biological treatments have been studied to enhance bone formation during distraction osteogenesis in animals. However, role of osteoactivin in the osseous tissues during distraction osteogenesis remains poorly understood. In this animal experimental study, we investigated the spatiotemporal expression of osteoactivin by immunohistochemistry and real-time PCR using a mouse model for tibial lengthening. Furthermore, to address the role of osteoactivin in bone lengthening, we subjected the osteoactivin-transgenic mice to distraction osteogenesis model. During the lag phase, the fibroblast-like cells (possible progenitors of the osteoblasts or chondrocytes), which mainly express osteoactivin, were infiltrated into the osteotomy site. Osteoactivin was ubiquitously expressed in the lengthened segment during the distraction and consolidation phases. Consistent with the immunohistochemical analysis, the levels of the osteoactivin transcripts in the tibias were significantly increased throughout the distraction osteogenesis process. The bone mineral content in the osteoactivin-transgenic mice calculated using peripheral quantitative computed tomography was also significantly increased at the remodeling zone. The histomorphometric analysis revealed that newly formed callus resorption in the remodeling zone was significantly reduced but bone formation was not altered in the osteoactivin-transgenic mice. We conclude that osteoactivin functions as an inhibitor of callus resorption during the consolidation phase of distraction osteogenesis.
(キーワード)
Animals / Bone Resorption / Bone and Bones / Eye Proteins / Membrane Glycoproteins / Mice / Mice, Transgenic / Osteogenesis, Distraction / Osteotomy / Tibia
Kazuhito Akama, Yasuka Shimajiri, Kumiko Kainou, Ryota Iwasaki, Reiko Nakao, Takeshi Nikawa and Akio Nishikawa : Functional rice with tandemly repeated Cbl-b ubiquitin ligase inhibitory pentapeptide prevents denervation-induced muscle atrophy in vivo, Bioscience, Biotechnology, and Biochemistry, Vol.85, No.6, 1415-1421, 2021.
(要約)
Ubiquitin ligase Casitas B-lineage lymphoma-b (Cbl-b) play a critical role in nonloading-mediated skeletal muscle atrophy: Cbl-b ubiquitinates insulin receptor substrate-1 (IRS-1), leading to its degradation and a resulting loss in muscle mass. We reported that intramuscular injection of a pentapeptide, DGpYMP, which acts as a mimic of the phosphorylation site in IRS-1, significantly inhibited denervation-induced skeletal muscle loss. In order to explore the possibility of the prevention of muscle atrophy by diet therapy, we examined the effects of oral administration of transgenic rice containing Cblin (Cbl-b inhibitor) peptide (DGYMP) on denervation-induced muscle mass loss in frogs. We generated transgenic rice seeds in which 15 repeats of Cblin peptides with a WQ spacer were inserted into the rice storage protein glutelin. A diet of the transgenic rice seeds had significant inhibitory effects on denervation-induced atrophy of the leg skeletal muscles in frogs, compared with those receiving a diet of wild-type rice.
Ulla Anayt, Takayuki Uchida, Miki Yukari, Kosuke Sugiura, Higashitani Atsushi, Kobayashi Takeshi, Ohno Ayako, Reiko Nakao, Katsuya Hirasaka, Iori Sakakibara and Takeshi Nikawa : Morin attenuates dexamethasone-mediated oxidative stress and atrophy in mouse C2C12 skeletal myotubes, Archives of Biochemistry and Biophysics, Vol.704, 108873, 2021.
(要約)
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.
Sun Luchuanyang, Miyaji Nobuyuki, Yang Min, Mills M Edward, Taniyama Shigeto, Takayuki Uchida, Takeshi Nikawa, Li Jifeng, Shi Jie, Tachibana Katsuyasu and Katsuya Hirasaka : Astaxanthin Prevents Atrophy in Slow Muscle Fibers by Inhibiting Mitochondrial Reactive Oxygen Species via a Mitochondria-Mediated Apoptosis Pathway, Nutrients, Vol.13, No.379, 2021.
(要約)
Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in the lipid bilayer. This study aimed to investigate the effects of AX on muscle-atrophy-mediated disturbance of mitochondria, which have a lipid bilayer. Tail suspension was used to establish a muscle-atrophied mouse model. AX diet fed to tail-suspension mice prevented loss of muscle weight, inhibited the decrease of myofiber size, and restrained the increase of hydrogen peroxide (HO) production in the soleus muscle. Additionally, AX improved downregulation of mitochondrial respiratory chain complexes I and III in the soleus muscle after tail suspension. Meanwhile, AX promoted mitochondrial biogenesis by upregulating the expressions of , , in the soleus muscle of tail-suspension mice. To confirm the AX phenotype in the soleus muscle, we examined its effects on mitochondria using Sol8 myotubes derived from the soleus muscle. We found that AX was preferentially detected in the mitochondrial fraction; it significantly suppressed mitochondrial reactive oxygen species (ROS) production in Sol8 myotubes. Moreover, AX inhibited the activation of caspase 3 via inhibiting the release of cytochrome c into the cytosol in antimycin A-treated Sol8 myotubes. These results suggested that AX protected the functional stability of mitochondria, alleviated mitochondrial oxidative stress and mitochondria-mediated apoptosis, and thus, prevented muscle atrophy.
Rie Mukai, Takashi Fukuda, Asami Ohnishi, Takeshi Nikawa, Mutsuki Furusawa and Junji Terao : Chocolate as a food matrix reduces the bioavailability of galloylated catechins from green tea in healthy women., Food & Function, Vol.12, No.1, 408-416, 2021.
(要約)
In this study, we evaluated the food matrix effects of chocolate on the absorption of green tea catechins (GTCs), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), in five healthy 22-year-old women. In the single-intake experiment, the plasma concentrations of ECg (P < 0.05, at 1.5 h) and EGCg (P < 0.05, at 6 h) but not those of EC and EGC were reduced by the chocolate matrix. Regardless of the chocolate matrix, ECg and EGCg were mainly present as their aglycones in the plasma, whereas EGC and EC were found mostly as conjugated metabolites. After daily intake of GTCs mixed with chocolate for 14 days followed by overnight fasting, ECg but not EGCg was detected in the plasma. To compare the plasma profiles of ECg and EGCg, a mixture containing approximately equal amounts of ECg and EGCg was administered to nine rats for 14 days. Following treatment and overnight food deprivation, the plasma content of ECg was higher than that of EGCg. After a single injection of the same mixture in seven rats, ECg levels were higher than those of EGCg, and a greater amount of conjugated metabolites of ECg than those of EGCg was detected in the plasma 10 h after administration. In conclusion, the chocolate matrix affects the plasma profiles of GTCs, particularly ECg. ECg appears to persist in the plasma for a longer period, regardless of the chocolate matrix.
Ohno Ayako, Nobuo Maita, Tabata Takanori, Nagano Hikaru, Arita Kyohei, Ariyoshi Mariko, Takayuki Uchida, Reiko Nakao, Ulla Anayt, Kosuke Sugiura, Koji Kishimoto, Teshima-Kondo Shigetada, Okumura Yuushi and Takeshi Nikawa : Crystal structure of inhibitor-bound human MSPL that can activate high pathogenic avian influenza., Life Science Alliance, Vol.4, No.6, e202000849, 2021.
(要約)
Infection of certain influenza viruses is triggered when its HA is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, whereas the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal α-helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.
Takeshi Nikawa and Kazumi Ishidoh : Ubiquitin ligase Cbl-b and inhibitory Cblin peptides, Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol.1868, No.11, 140495, 2020.
(要約)
This review focuses on the Cbl-b muscle atrophy-associated ubiquitin ligase and its inhibitors. Herein, the role of E3 ubiquitin ligase-associated muscle atrophy genes (atrogenes), including MAFbx-1/agrogin-1 and MuRF-1, as well as another ubiquitin ligase, Cbl-b and its inhibitors, is discussed. Cbl-b plays an important role in unloading muscle atrophy caused by spaceflight and in bedridden patients: Cbl-b ubiquitinated and induced the degradation of IRS-1, a key intermediate in the IGF-1 signaling. Furthermore, a pentapetpide (DGpYMP), inhibited Cbl-b-mediated IRS-1 ubiquitination. This peptide-based Cbl-b inhibitor Cblin and its homologous peptides in foods presumably affect muscle atrophy under such conditions.
Luqman Khan, Katsumi Sato, Shinichi okuyama, Takeshi Kobayashi, Kazumasa Ohashi, Katsuya Hirasaka, Takeshi Nikawa, Kunio Takada, Atsushi Higashitani and Kenji Abiko : Ultra-high-purity iron is a novel and very compatible biomaterial, Journal of the Mechanical Behavior of Biomedical Materials, Vol.106, 103744, 2020.
(要約)
Metals and alloys are used widely in bone prosthetic materials, stents and dental tissue reconstructions. The most common materials are stainless steels and cobalt-chromium-nickel and titanium alloys. These alloys can be easily deformed but are hard to break. However, their affinity for cells and tissues is very low. In addition, they can sometimes provoke unexpected metal allergies. Iron is an abundant trace element essential for humans. However, excess amounts in particular of Fe ions are toxic. We previously succeeded in obtaining 99.9996% ultra-high-purity iron (ABIKO iron). The chemical properties of ABIKO iron are completely different from that of conventional pure iron. For example, the reaction rate in hydrochloric acid is very slow and there is barely any corrosion. Here, we found that, in the absence of any type of coating, mammalian cells could easily attach to, and normally proliferate and differentiate on, ABIKO iron. On the other hand, cell densities and proliferation rate of the surfaces of plates made from Co-Cr-Mo or Ti-6Al-4V were significantly reduced. In addition, several stress and iron response genes, HSP70, SOD1, ATM and IRP2 did not change in the cells on ABIKO iron, while these genes were induced with exogenous application of FeSO. Cells also secreted and fastened some organics on ABIKO iron. In vitro collagen binding assay showed that ABIKO iron binds higher amount of collagens. These findings highlight ABIKO iron as a novel biocompatible prosthetic material.
Nobuhiko Tachibana, Masanori Fukao, Tomoko Irie, Yusuke Irisawa, Hirotaka Shirono, Motoko Oarada, Takeshi Nikawa and Tetsuya Fukaya : A Diet Including Red Bell Pepper Juice and Soy Protein Suppress Physiological Markers of Muscle Atrophy in Mice, Journal of Nutritional Science and Vitaminology, Vol.66, No.5, 449-455, 2020.
Yuki Niida, Masashi Masuda, Yuichiro Adachi, Aika Yoshizawa, Hirokazu Ohminami, Yuki Mori, Kohta Ohnishi, Hisami Okumura, Takayuki Uchida, Takeshi Nikawa, Hironori Yamamoto, Makoto Miyazaki and Yutaka Taketani : Reduction of stearoyl-CoA desaturase (SCD) contributes muscle atrophy through the excess endoplasmic reticulum stress in chronic kidney disease, Journal of Clinical Biochemistry and Nutrition, Vol.67, No.2, 179-187, 2020.
(要約)
<p>Skeletal muscle atrophy is associated with mortality and poor prognosis in patients with chronic kidney disease (CKD). However, underlying mechanism by which CKD causes muscle atrophy has not been completely understood. The quality of lipids (lipoquality), which is defined as the functional features of diverse lipid species, has recently been recognized as the pathology of various diseases. In this study, we investigated the roles of the stearoyl-CoA desaturase (SCD), which catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, in skeletal muscle on muscle atrophy in CKD model animals. In comparison to control rats, CKD rats decreased the SCD activity and its gene expression in atrophic gastrocnemius muscle. Next, oleic acid blocked the reduction of the thickness of C2C12 myotubes and the increase of the endoplasmic reticulum stress induced by SCD inhibitor. Furthermore, endoplasmic reticulum stress inhibitor ameliorated CKD-induced muscle atrophy (the weakness of grip strength and the decrease of muscle fiber size of gastrocnemius muscle) in mice and the reduction of the thickness of C2C12 myotubes by SCD inhibitor. These results suggest that the repression of SCD activity causes muscle atrophy through excessive endoplasmic reticulum stress in CKD.</p>
Kanae Saitoh, Tomohiro Yoshimura1, Luchuanyang Sun, Min Yang, Yao Wang, Shigeto Taniyama, Kenji Hara, Fumihito Murayama, Takeshi Nikawa, Katsuyasu Tachibana and Katsuya Hirasaka : Effect of dietary fish oil on enhanced inflammation and disturbed lipophagy in white adipose tissue caused by a high fat diet., Fisheries Science, Vol.86, No.1, 187-196, 2020.
Tomohiro Yoshimura, Kanae Saitoh, Luchuanyang Sun, Yao Wang, Shigeto Taniyama, Kenichi Yamaguchi, Takayuki Uchida, Tsutomu Ohkubo, Atsushi Higashitani, Takeshi Nikawa, Katsuyasu Tachibana and Katsuya Hirasaka : Morin suppresses cachexia-induced muscle wasting by binding to ribosomal protein S10 in carcinoma cells., Biochemical and Biophysical Research Communications, Vol.506, No.4, 773-779, 2018.
(要約)
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.
Shigeru Hiramoto, Nobuhiro Yahata, Kanae Saitoh, Tomohiro Yoshimura, Yao Wang, Shigeto Taniyama, Takeshi Nikawa, Katsuyasu Tachibana and Katsuya Hirasaka : Dietary supplementation with alkylresorcinols prevents muscle atrophy through a shift of energy supply., The Journal of Nutritional Biochemistry, Vol.61, 147-154, 2018.
(要約)
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.
Chisato Tomida, Naoko Yakagishi, Hikaru Nagano, Takayuki Uchida, Ayako Maita, Katsuya Hirasaka, Takeshi Nikawa and Shigetada Kondo : VEGF pathway-targeting drugs induce evasive adaptation by activation of neuropilin-1/cMet in colon cancer cells., International Journal of Oncology, Vol.52, No.4, 1350-1362, 2018.
(要約)
Anti-angiogenic therapies targeting vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) are important treatments for a number of human malignancies, including colorectal cancers. However, there is increasing evidence that VEGF/VEGF-R inhibitors promote the adaptive and evasive resistance of tumor cells to the therapies. The mechanism by which the cancer cells become resistant remains unclear. One potential mechanism is that VEGF/VEGF-R blockers directly act on tumor cells independently of anti-angiogenic effects. In this study, the direct effects of an anti-VEGF antibody (bevacizumab) and a VEGF-R tyrosine kinase inhibitor (sunitinib) on the evasive adaptation of colon cancer cells were compared. HCT116 and RKO human colon cancer cell lines were chronically exposed (3 months) to bevacizumab or sunitinib in vitro to establish bevacizumab- and sunitinib-adapted cells, respectively. Transwell migration and invasion assays, western blotting, reverse transcription-quantitative polymerase chain reaction, co-immunoprecipitation analysis, cell survival assays and ELISAs were conducted to analyze the adapted cells. Compared with the control vehicle-treated cells, the two cell models exhibited increased migration and invasion activities to different degrees and through different mechanisms. The bevacizumab-adapted cells, but not in the sunitinib-adapted cells, exhibited redundantly increased expression levels of VEGF/VEGF-R family members, including VEGF-A, placental growth factor, VEGF-C, VEGF-R1 and VEGF-R3. In addition, the phosphorylation levels of VEGF-R1 and VEGF-R3 were increased in the bevacizumab-adapted cells compared with the control cells. Thus, the inhibition of VEGF-R1 and VEGF-R3 decreased the evasive activities of the cells, suggesting that they remained dependent on redundant VEGF/VEGF-R signaling. By contrast, the sunitinib-adapted cells exhibited increased neuropilin-1 (NRP1) expression levels compared with the control cells. In the sunitinib-adapted cells, NRP1 interacted with phosphorylated cMet, and the cMet activation was dependent on NRP1. Thus, NRP1 or cMet blockade suppressed the evasive activation of the sunitinib-adapted cells. These results suggest that the sunitinib-adapted cells switched from a VEGF-R-dependent pathway to an alternative NRP1/cMet-dependent one. The findings of the present study indicate that VEGF/VEGF-R inhibitors directly act on colon cancer cells and activate their evasive adaptation via different mechanisms.
Takayuki Uchida, Yoshihiro Sakashita, Kanako Kitahata, Yui Yamashita, Chisato Tomida, Yuki Kimori, Akio Komatsu, Katsuya Hirasaka, Ayako Maita, Reiko Nakao, Atsushi Higashitani, Akira Higashibata, Noriaki Ishioka, Toru Shimazu, Takeshi Kobayashi, Yuushi Okumura, Inho Choi, Motoko Oarada, M Edward Mills, Shigetada Kondo, Shin'ichi Takeda, Eiji Tanaka, Keiji Tanaka, Masahiro Sokabe and Takeshi Nikawa : Reactive oxygen species up-regulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells., American Journal of Physiology, Cell Physiology, Vol.314, No.6, C721-C731, 2018.
(要約)
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. et al. 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 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 treatment, such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr 1 and/or Egr 2 to stimulate Cbl-b expression through the ERK 1/2 pathway in L6 myoblasts, since treatment with Egr 1/2 siRNA and an ERK 1/2 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.
ODO BAYARSAIKHAN, Nobuhiko Kawai, Hiroyo Mori, Nao Kinouchi, Takeshi Nikawa and Eiji Tanaka : Effects of co-transfection with myostatin-targeting siRNA and ActRIIB-Fc fusion proein on skeletal muscle growth., Journal of Oral Health and Biosciences, Vol.30, No.1, 1-7, 2017.
Hidehiro Umehara, Shusuke Numata, Shinya Watanabe, Yutaka Hatakeyama, Makoto Kinoshita, Yukiko Tomioka, Kiyoshi Nakahara, Takeshi Nikawa and Tetsuro Ohmori : Altered KYN/TRP, Gln/Glu, and Met/methionine sulfoxide ratios in the blood plasma of medication-free patients with major depressive disorder., Scientific Reports, Vol.7, No.1, 4855, 2017.
(要約)
Capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS) is a comprehensive, quantitative, and high throughput tool used to analyze metabolite profiles. In the present study, we used CE-TOFMS to profile metabolites found in the blood plasma of 33 medication-free patients with major depressive disorder (MDD) and 33 non-psychiatric control subjects. We then investigated changes which occurred in the metabolite levels during an 8-week treatment period. The medication-free MDD patients and control subjects showed significant differences in their mean levels of 33 metabolites, including kynurenine (KYN), glutamate (Glu), glutamine (Gln), methionine sulfoxide, and methionine (Met). In particular, the ratios of KYN to tryptophan (TRP), Gln to Glu, and Met to methionine sulfoxide were all significantly different between the two groups. Among the 33 metabolites with altered levels in MDD patients, the levels of KYN and Gln, as well as the ratio of Gln to Glu, were significantly normalized after treatment. Our findings suggest that imbalances in specific metabolite levels may be involved in the pathogenesis of MDD, and provide insight into the mechanisms by which antidepressant agents work in MDD patients.
Hyunwoo Ju, Taewan Kim, Chan-Moon Chung, Junsoo Park, Takeshi Nikawa, Kyoungsook Park and Inho Choi : Metabolic Suppression by 3-Iodothyronamine Induced Muscle Cell Atrophy via Activation of FoxO-Proteasome Signaling and Downregulation of Akt1-S6K Signaling., Biological & Pharmaceutical Bulletin, Vol.40, No.5, 576-582, 2017.
(要約)
The homeostasis of muscle properties depends on both physical and metabolic stresses. Whereas physical stress entails metabolic response for muscle homeostasis, the latter does not necessarily involve the former and may thus solely affect the homeostasis. We here report that metabolic suppression by the hypometabolic agent 3-iodothyronamine (T1AM) induced muscle cell atrophy without physical stress. We observed that the oxygen consumption rate of C2C12 myotubes decreased 40% upon treatment with 75 µM T1AM for 6 h versus 10% in the vehicle (dimethyl sulfoxide) control. The T1AM treatment reduced cell diameter of myotubes by 15% compared to the control (p<0.05). The cell diameter was reversed completely by 9 h after T1AM was removed. The T1AM treatment also significantly suppressed the expression levels of heat shock protein 72 and αB-crystallin as well as the phosphorylation levels of Akt1, mammalian target of rapamycin (mTOR), S6K, forkhead box O1 (FoxO1) and FoxO3. In contrast, the levels of ubiquitin E3 ligase MuRF1 and chymotrypsin-like activity of proteasome were significantly elevated by T1AM treatment. These results suggest that T1AM-mediated metabolic suppression induced muscle cell atrophy via activation of catabolic signaling and inhibition of anabolic signaling.
Hiroko Ikeda, Hikaru Souda, Anggraeini Puspitasari, D Kathryn Held, Jun Hidema, Takeshi Nikawa, Yukari Yoshida, Tatsuaki Kanai and Akihisa Takahashi : Development and performance evaluation of a three-dimensional clinostat synchronized heavy-ion irradiation system., Life Sciences in Space Research, Vol.12, 51-60, 2017.
(要約)
Outer space is an environment characterized by microgravity and space radiation, including high-energy charged particles. Astronauts are constantly exposed to both microgravity and radiation during long-term stays in space. However, many aspects of the biological effects of combined microgravity and space radiation remain unclear. We developed a new three-dimensional (3D) clinostat synchronized heavy-ion irradiation system for use in ground-based studies of the combined exposures. Our new system uses a particle accelerator and a respiratory gating system from heavy-ion radiotherapy to irradiate samples being rotated in the 3D clinostat with carbon-ion beams only when the samples are in the horizontal position. A Peltier module and special sample holder were loaded on a static stage (standing condition) and the 3D clinostat (rotation condition) to maintain a suitable temperature under atmospheric conditions. The performance of the new device was investigated with normal human fibroblasts 1BR-hTERT in a disposable closed cell culture chamber. Live imaging revealed that cellular adhesion and growth were almost the same for the standing control sample and rotation sample over 48h. Dose flatness and symmetry were judged according to the relative density of Gafchromic films along the X-axis and Y-axis of the positions of the irradiated sample to confirm irradiation accuracy. Doses calculated using the carbon-ion calibration curve were almost the same for standing and rotation conditions, with the difference being less than 5% at 1Gy carbon-ion irradiation. Our new device can accurately synchronize carbon-ion irradiation and simulated microgravity while maintaining the temperature under atmospheric conditions at ground level.
Atsuko Sakai, Hisayo Hamada, Keiko Hara, Kyoko Mori, Takayuki Uchida, Takashi Mizuguchi, Jun Minaguchi, Kenji Shima, Shu Kawashima, Yasuhiro Hamada and Takeshi Nikawa : Nutritional counseling regulates interdialytic weight gain and blood pressure in outpatients receiving maintenance hemodialysis., The Journal of Medical Investigation : JMI, Vol.64, No.1,2, 129-135, 2017.
(要約)
Maintenance hemodialysis outpatients must limit salt and water intake to maintain electrolyte balance and blood pressure. In Kawashima Hospital, nationally registered dietitians provide hemodialysis patients with monthly nutritional counseling. We investigated whether nutritional counseling affects interdialytic weight gain (IDWG) and blood pressure. We investigated 48 hemodialysis patients whose monthly average IDWG ratio to dry weight exceeded 5.1% and who had not had a long-term hospital admittance of > 1 month. After the 48-month nutritional counseling period, the IDWG ratio had improved in 37 of the patients (77.1%), significantly decreasing from 6.0±0.7 to 5.3±0.9%. Estimated salt and water intake decreased significantly from 13.3±2.7 to 11.8±2.4 g/day and 2528±455 to 2332±410 ml/day, respectively. During the intervention period, normalized protein catabolic rate and body mass index did not change substantially. Pre-hemodialysis systolic and diastolic blood pressures had significantly decreased from 149±19 to 134±18 mmHg, and 82±13 to 75±10 mmHg for 48 months after study initiation, respectively. The dosage of antihypertensive drugs had significantly decreased in the group that experienced improvement in the IDWG ratio. Long-term nutritional counseling by nationally registered dietitians may improve the IDWG ratio and blood pressure of hemodialysis patients by decreasing their salt and water intake. J. Med. Invest. 64: 129-135, February, 2017.
(キーワード)
Adult / Ambulatory Care / Antihypertensive Agents / 血圧 (blood pressure) / カウンセリング (counseling) / Female / Humans / 高血圧 (hypertension) / Male / Middle Aged / Nutrition Therapy / Nutritional Status / Renal Dialysis / Weight Gain
Chisato Tomida, Hikaru Nagano, Naoko Yamagishi, Takayuki Uchida, Ayako Maita, Katsuya Hirasaka, Takeshi Nikawa and Shigetada Kondo : Regorafenib induces adaptive resistance of colorectal cancer cells via inhibition of vascular endothelial growth factor receptor., The Journal of Medical Investigation : JMI, Vol.64, No.3-4, 262-265, 2017.
(要約)
Recently, inhibition of tumor angiogenesis has become an important anti-cancer therapy. Tumor angiogenesis is regulated by multiple signaling pathways, including VEGF and VEGF receptor (VEGF-R), FGF and FGF receptor (FGF-R), and PDGF and PDGF receptor (PDGF-R) pathways. Thus, the antiangiogenic agents, such as regorafenib, simultaneously target those receptors on vascular endothelial cells. In addition to endothelial cells, cancer cells express the three receptors, suggesting that the antiangiogenic inhibitors affect tumor cells. In fact, we previously demonstrated that regorafenib directly acted on human colorectal cancer cells and accelerated their apoptosis resistance and migration capability. Thus, we here elucidated how regorafenib induced the malignant phenotypes in colorectal cancer cells. To identify the responsible receptor among the regorafenib-targeting proangiogenic receptors, we examined the effects of a potent selective inhibitor for VEGF-R, FGF-R or PDGF-R on apoptosis resistance and migration capability. We clarified that blockade of VEGF-R, but not FGF-R and PDGF-R, induced the malignant phenotypes. We confirmed that blocking of VEGF ligands derived from colorectal cancer cells also induced the phenotypes. These results suggest that regorafenib progressed the malignancy via prevention of autocrine and paracrine VEGF signaling in colorectal cancer cells. J. Med. Invest. 64: 262-265, August, 2017.
Chisato Tomida, Naoko Yamagishi, Hikaru Nagano, Takayuki Uchida, Ayako Maita, Katsuya Hirasaka, Takeshi Nikawa and Shigetada Kondo : Antiangiogenic agent sunitinib induces epithelial to mesenchymal transition and accelerates motility of colorectal cancer cells., The Journal of Medical Investigation : JMI, Vol.64, No.3-4, 250-254, 2017.
(要約)
Although vascular endothelial growth factor receptor (VEGF-R)-targeted antiangiogenic agents are important treatment for a number of human malignancies, there is accumulating evidence that the therapies may promote disease progression, such as invasion and metastasis. How tumors become to promote their evasiveness remains fully uncertain. One of possible mechanisms for the adaptation may be a direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. To elucidate a direct effect of VEGF-R-targeting drug (sunitinib), we established a human colorectal cancer cell model adapted to sunitinib. The sunitinib-conditioned cells showed a significant increase in cellular motility and migration activities, compared to the vehicle-treated control cells. Consistent with the phenotype, the sunitinib-conditioned cells decreased the expression levels of E-cadherin (an epithelial marker), while significantly increased the levels of Slug and Zeb1 (mesenchymal markers). Expression profiles of VEGF-R in the sunitinib-conditioned cells showed that only neuropilin-1 (NRP1) expression was significantly increased among all VEGF-R tested. Blockade of NRP1 using its antagonist clearly repressed the migration activation in sunitinib-conditioned cells, but not in the control cells. These results suggest that inhibition of VEGF-R on colorectal cancer cells can drive the epithelial-mesenchymal transition, leading to activation of cell motility in an NRP1-dependent manner. J. Med. Invest. 64: 250-254, August, 2017.
ODO BAYARSAIKHAN, Nobuhiko Kawai, Hiroyo Mori, Nao Kinouchi, Takeshi Nikawa and Eiji Tanaka : Co-Administration of Myostatin-Targeting siRNA and ActRIIB-Fc Fusion Protein Increases Masseter Muscle Mass and Fiber Size., Journal of Nutritional Science and Vitaminology, Vol.63, No.4, 244-248, 2017.
(要約)
Myostatin, a member of the TGF- superfamily, is a negative regulator of skeletal muscle cell growth and differentiation, and binds with high affinity to the activin type IIB receptor (ActRIIB). The soluble ligand-binding domain of ActRIIB fused to the Fc domain of IgG (ActRIIB-Fc) potently binds and inhibits TGF- family members in muscle, leading to rapid and marked muscle growth. The present study was designed to assess the effectiveness of the co-delivery of myostatin-targeting siRNA (Mstn-siRNA) and ActRIIB-Fc into skeletal muscle as a potential treatment of atrophic myopathies. Eleven-week-old, male C57BL/6 mice were injected with atelocollagen (ATCOL)-mediated Mstn-siRNA with/without ActRIIB-Fc locally into the masseter muscle twice a week. Inhibition of myostatin function by the combination of Mstn-siRNA and ActRIIB-Fc increased muscle weight and myofibril size in murine masseter muscle. Real-time RT-PCR analysis revealed significant downregulation of myostatin mRNA expression in both the Mstn-siRNA-treated and the combination treatment group. Furthermore, myogenin mRNA expression was upregulated in the combination treatment group, while MuRF-1 and Atrogin-1 mRNA expression was downregulated compared to administration of each compound alone. These findings suggest that double inhibition of myostatin is a potentially useful treatment strategy to increase muscle mass and fiber size and could be a useful treatment of patients with various muscle atrophies, including muscular dystrophy.
Mayumi Takeya, Yuushi Okumura and Takeshi Nikawa : Modulation of cutaneous extracellular collagen contraction by phosphorylation status of p130Cas., The Journal of Physiological Sciences, Vol.67, No.5, 613-622, 2016.
(要約)
Skin can respond to various types of internal and/or external mechanostimuli, such as excessive tension caused by body growth or decompression due to weight loss, which significantly affect skin morphology. Mechanosensors, including p130Cas, are reported to play a role in deformation and subsequent recovery of various tissues including skeletal muscles and blood vessels. However, the role of mechanotransduction via p130Cas in the regulation of skin size remains unclear. In this report, p130Cas activation was manipulated using a fibroblast-embedded collagen gel model or mouse skin contraction model. Inhibition or activation of Src family kinase-mediated phosphorylation of p130Cas significantly depressed and accelerated collagen gel contraction, respectively. The results also demonstrated age-dependent depression of cutaneous p130Cas activation in vivo. Inhibition of p130Cas signaling in our mouse model significantly suppressed recovery from cutaneous deformation. Taken together, our study highlighted the important role of p130Cas in cutaneous mechanotransduction for skin homeostasis.
Rie Mukai, Hitomi Horikawa, Pei-Yi Lin, Nao Tsukumo, Takeshi Nikawa, Tomoyuki Kawamura, Hisao Nemoto and Junji Terao : 8-Prenylnaringenin promotes recovery from immobilization-induced disuse muscle atrophy through activation of the Akt phosphorylation pathway in mice., American Journal of Physiology. Regulatory, Integrative and Comparative Physiology, Vol.311, No.6, R1022-R1031, 2016.
(要約)
8-Prenylnaringenin (8-PN) is a prenylflavonoid that originates from hop extracts and is thought to help prevent disuse muscle atrophy. We hypothesized that 8-PN affects muscle plasticity by promoting muscle recovery under disuse muscle atrophy. To test the promoting effect of 8-PN on muscle recovery, we administered an 8-PN mixed diet to mice that had been immobilized with a cast to one leg for 14 days. Intake of the 8-PN mixed diet accelerated recovery from muscle atrophy, and prevented reductions in Akt phosphorylation. Studies on cell cultures of mouse myotubes in vitro demonstrated that 8-PN activated the PI3K/Akt/P70S6K1 pathway at physiological concentrations. A cell-culture study using an inhibitor of estrogen receptors and an in vivo experiment with ovariectomized mice suggested that the estrogenic activity of 8-PN contributed to recovery from disuse muscle atrophy through activation of an Akt phosphorylation pathway. These data strongly suggest that 8-PN is a naturally occurring compound that could be used as a nutritional supplement to aid recovery from disuse muscle atrophy.
(キーワード)
Animals / Cell Line / Enzyme Activation / Flavanones / Hindlimb Suspension / Male / Mice / Mice, Inbred C57BL / Muscle Proteins / Muscle, Skeletal / Muscular Atrophy / Oncogene Protein v-akt / リン酸化 (phosphorylation) / Phytoestrogens / Protein Biosynthesis / Recovery of Function / シグナル伝達 (signal transduction) / Treatment Outcome
Katsuya Hirasaka, EM Mills, Haruna Marie, Bando Aki, Ikeda Chika, Tomoki Abe, Kohno Shohei, SM Nowinski, CU Lago, K Akagi, H Tochio, Ohno Ayako, Teshima-Kondo Shigetada, Yuushi Okumura and Takeshi Nikawa : UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion, Biochemical and Biophysical Research Communications, Vol.472, No.1, 108-113, 2016.
(要約)
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+).
(キーワード)
Animals / Calcium / HEK293 Cells / Humans / Ion Channels / Mice / Mitochondria / Mitochondrial Membranes / Mitochondrial Proteins / Models, Molecular / Nuclear Magnetic Resonance, Biomolecular / Protein Conformation / Protein Interaction Domains and Motifs / Proteins / Recombinant Fusion Proteins / Two-Hybrid System Techniques
Ayako Ohno, Arisa Ochi, Nobuo Maita, Tatsuya Ueji, Aki Bando, Reiko Nakao, Katsuya Hirasaka, Tomoki Abe, Shigetada Teshima-Kondo, Hisao Nemoto, Yuushi Okumura, Akira Higashibata, Sachiko Yano, Hidehito Tochio and Takeshi Nikawa : Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin., Archives of Biochemistry and Biophysics, Vol.594, 1-7, 2016.
(要約)
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.
Rie Mukai, Naoko Matsui, Yutaka Fujikura, Norifumi Matsumoto, De-Xing Hou, Noriyuki Kanzaki, Hiroshi Shibata, Manabu Horikawa, Keiko Iwasa, Katsuya Hirasaka, Takeshi Nikawa and Junji Terao : Preventive effect of dietary quercetin on disuse muscle atrophy by targeting mitochondria in denervated mice., The Journal of Nutritional Biochemistry, Vol.31, 67-76, 2016.
(要約)
Quercetin is a major dietary flavonoid in fruits and vegetables. We aimed to clarify the preventive effect of dietary quercetin on disuse muscle atrophy and the underlying mechanisms. We established a mouse denervation model by cutting the sciatic nerve in the right leg (SNX surgery) to lack of mobilization in hind-limb. Preintake of a quercetin-mixed diet for 14days before SNX surgery prevented loss of muscle mass and atrophy of muscle fibers in the gastrocnemius muscle (GM). Phosphorylation of Akt, a key phosphorylation pathway of suppression of protein degradation, was activated in the quercetin-mixed diet group with and without SNX surgery. Intake of a quercetin-mixed diet suppressed the generation of hydrogen peroxide originating from mitochondria and elevated mitochondrial peroxisome proliferator-activated receptor- coactivator 1 mRNA expression as well as NADH dehydrogenase 4 expression in the GM with SNX surgery. Quercetin and its conjugated metabolites reduced hydrogen peroxide production in the mitochondrial fraction obtained from atrophied muscle. In C2C12 myotubes, quercetin reached the mitochondrial fraction. These findings suggest that dietary quercetin can prevent disuse muscle atrophy by targeting mitochondria in skeletal muscle tissue through protecting mitochondria from decreased biogenesis and reducing mitochondrial hydrogen peroxide release, which can be related to decreased hydrogen peroxide production and/or improvements on antioxidant capacity of mitochondria.
Katsuya Hirasaka, Shinobu Saito, Saki Yamaguchi, Riho Miyazaki, Yao Wang, Marie Haruna, Shigeto Taniyama, Atsushi Higashitani, Junji Terao, Takeshi Nikawa and Katsuyasu Tachibana : Dietary Supplementation with Isoflavones Prevents Muscle Wasting in Tumor-Bearing Mice., Journal of Nutritional Science and Vitaminology, Vol.62, No.3, 178-184, 2016.
(要約)
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--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- 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.
Uncoupling protein 3 (UCP3) and pyruvate dehydrogenase kinase 4 (PDK4) in skeletal muscle are key regulators of the glucose and lipid metabolic processes that are involved in insulin resistance. Medium-chain fatty acids (MCFAs) have anti-obesogenic effects in rodents and humans, while long-chain fatty acids (LCFAs) cause increases in body weight and insulin resistance. To clarify the beneficial effects of MCFAs, we examined UCP3 and PDK4 expression in skeletal muscles of mice fed a MCFA- or LCFA-enriched high-fat diet (HFD). Five-week feeding of the LCFA-enriched HFD caused high body weight gain and induced glucose intolerance in mice, compared with those in mice fed the MCFA-enriched HFD. However, the amounts of UCP3 and PDK4 transcripts in the skeletal muscle of mice fed the MCFA- or LCFA-enriched HFD were similar. To further elucidate the specific effects of MCFAs, such as capric acid (C10:0), on lipid metabolism in skeletal muscles, we examined the effects of various FAs on expression of UCP3 and PDK4, in mouse C2C12 myocytes. Although palmitic acid (C16:0) and lauric acid (C12:0) significantly induced expression of both UCP3 and PDK4, capric acid (C10:0) upregulated only UCP3 expression via activation of peroxisome proliferator-activated receptor-δ. Furthermore, palmitic acid (C16:0) disturbed the insulin-induced phosphorylation of Akt, while MCFAs, including lauric (C12:0), capric (C10:0), and caprylic acid (C12:0), did not. These results suggest that capric acid (C10:0) increases the capacity for fatty acid oxidation without inhibiting glycolysis in skeletal muscle.
M Sara Nowinski, Ashley Solmonson, E Joyce Rundhaug, Okkyung Rho, Jiyoon Cho, U Cory Lago, L Christopher Riley, Sunhee Lee, Shohei Kohno, K Christine Dao, Takeshi Nikawa, B Shawn Bratton, W Casey Wright, M Susan Fischer, John DiGiovanni and M Edward Mills : Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis., Nature Communications, Vol.6, 2015.
(要約)
To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling.
Chieko Shiota, Tomoki Abe, Nobuhiko Kawai, Ayako Ohno, Shigetada Teshima-Kondo, Hiroyo Mori, Junji Terao, Eiji Tanaka and Takeshi Nikawa : Flavones Inhibit LPS-Induced Atrogin-1/MAFbx Expression in Mouse C2C12 Skeletal Myotubes., Journal of Nutritional Science and Vitaminology, Vol.61, No.2, 188-194, 2015.
(要約)
Muscle atrophy is a complex process that occurs as a consequence of various stress events. Muscle atrophy-associated genes (atrogenes) such as atrogin-1/MAFbx and MuRF-1 are induced early in the atrophy process, and the increase in their expression precedes the loss of muscle weight. Although antioxidative nutrients suppress atrogene expression in skeletal muscle cells, the inhibitory effects of flavonoids on inflammation-induced atrogin-1/MAFbx expression have not been clarified. Here, we investigated the inhibitory effects of flavonoids on lipopolysaccharide (LPS)-induced atrogin-1/MAFbx expression. We examined whether nine flavonoids belonging to six flavonoid categories inhibited atrogin-1/MAFbx expression in mouse C2C12 myotubes. Two major flavones, apigenin and luteolin, displayed potent inhibitory effects on atrogin-1/MAFbx expression. The pretreatment with apigenin and luteolin significantly prevented the decrease in C2C12 myotube diameter caused by LPS stimulation. Importantly, the pretreatment of LPS-stimulated myoblasts with these flavones significantly inhibited LPS-induced JNK phosphorylation in C2C12 myotubes, resulting in the significant suppression of atrogin-1/MAFbx promoter activity. These results suggest that apigenin and luteolin, prevent LPS-mediated atrogin-1/MAFbx expression through the inhibition of the JNK signaling pathway in C2C12 myotubes. Thus, these flavones, apigenin and luteolin, may be promising agents to prevent LPS-induced muscle atrophy.
Ichiro Tonogai, Mitsuhiko Takahashi, Kiminori Yukata, Ryosuke Sato, Takeshi Nikawa, Natsuo Yasui and Koichi Sairyo : Osteoactivin attenuates skeletal muscle fibrosis after distraction osteogenesis by promoting extracellular matrix degradation/remodeling., Journal of Pediatric Orthopaedics. Part B, Vol.24, No.2, 162-169, 2015.
(要約)
The aim of this study was to determine whether osteoactivin attenuated skeletal muscle fibrosis caused by distraction osteogenesis. Tibial osteotomies were performed on wild-type and osteoactivin-transgenic (OA-Tg) mice, and tibiae were distracted for 2 weeks. Ankle plantar flexion torque and the gastrocnemius muscles were analyzed. The amount and area of collagenous tissue and the passive torque were reduced in the OA-Tg group at 8 weeks after osteotomy. Transcript levels of matrix metalloprotease (mmp)-3 and MMP-9 were upregulated, and MMP-3 and MMP-9 proteins were increased in the OA-Tg group. Osteoactivin-mediated increase in MMPs may attenuate skeletal muscle fibrosis.
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 120M, 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.
Nobuhiko Kawai, Katsuya Hirasaka, Tasuku Maeda, Marie Haruna, Chieko Shiota, Arisa Ochi, Tomoki Abe, Shohei Kohno, Ayako Ohno, Sigetada Teshima-Kondo, Hiroyo Mori, Eiji Tanaka and Takeshi Nikawa : Prevention of skeletal muscle atrophy in vitro using anti-ubiquitination oligopeptide carried by atelocollagen., Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol.1853, No.5, 873-880, 2015.
(要約)
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.
Rie Hashimoto, Atsuko Sakai, Masumi Murayama, Arisa Ochi, Tomoki Abe, Katsuya Hirasaka, Ayako Ohno, Shigetada Teshima-Kondo, Hiroaki Yanagawa, Natsuo Yasui, Mikiko Inatsugi, Daisuke Doi, Masanori Takeda, Rie Mukai, Junji Terao and Takeshi Nikawa : Effects of dietary soy protein on skeletal muscle volume and strength in humans with various physical activities, The Journal of Medical Investigation : JMI, Vol.62, No.3, 177-183, 2015.
(要約)
Background: In recent years, the number of bedridden people is rapidly increasing due to aging or lack of exercise in Japan. This problem is becoming more serious, since there is no countermeasure against it. In the present study, we designed to investigate whether dietary proteins, especially soy, had beneficial effects on skeletal muscle in 59 volunteers with various physical activities. Methods: We subjected 59 volunteers with various physical activities to meal intervention examination. Persons with low and high physical activities were divided into two dietary groups, the casein diet group and the soy diet group. They ate daily meals supplemented with 7.8 g of powdered casein or soy protein isolate every day for 30 days. Bedridden patients in hospitals were further divided into three dietary groups: the no supplementation diet group, the casein diet group and the soy diet group. They were also subjected to a blood test, a urinalysis, magnetic resonance imaging analysis and muscle strength test of the knee before and after the meal intervention study. Results: Thirty-day soy protein supplementation significantly increased skeletal muscle volume in participants with low physical activity, compared with 30-day casein protein supplementation. Both casein and soy protein supplementation increased the volume of quadriceps femoris muscle in bedridden patients. Consistently, soy protein significantly increased their extension power of the knee, compared with casein protein. Although casein protein increased skeletal muscle volume more than soy protein in bedridden patients, their muscle strength changes by soy protein supplementation were bigger than those by casein protein supplementation. Conclusions: The supplementation of soy protein would be one of the effective foods which prevent the skeletal muscle atrophy caused by immobilization or unloading. J. Med. Invest. 62: 177-183, August, 2015
Chisato Tomida, Kana Aibara, Naoko Yamagishi, Chiaki Yano, Hikaru Nagano, Tomoki Abe, Ayako Ohno, Katsuya Hirasaka, Takeshi Nikawa and Shigetada Teshima-Kondo : The malignant progression effects of regorafenib in human colon cancer cells., The Journal of Medical Investigation : JMI, Vol.62, No.3-4, 195-198, 2015.
(要約)
A number of anti-angiogenic drugs targeting vascular endothelial growth factor receptors (VEGF-R) have developed and enabled significant advances in cancer therapy including colorectal cancer. However, acquired resistance to the drugs occurs, leading to disease progression, such as invasion and metastasis. How tumors become the resistance and promote their malignancy remains fully uncertain. One of possible mechanisms for the resistance and the progression may be the direct effect of VEGF-R inhibitors on tumor cells expressing VEGF-R. We investigated here the direct effect of a VEGF-R-targeting agent, regorafenib, which is the first small molecule inhibitor of VEGF-Rs for the treatment of patients with colorectal cancer, on phenotype changes in colon cancer HCT116 cells. Treatment of cells with regorafenib for only 2 days activated cell migration and invasion, while vehicle-treated control cells showed less activity. Intriguingly, chronic exposure to regorafenib for 90 days dramatically increased migration and invasion activities and induced a resistance to hypoxia-induced apoptosis. These results suggest that loss of VEGF signaling in cancer cells may induce the acquired resistance to VEGF/VEGF-R targeting therapy by gaining two major malignant phenotypes, apoptosis resistance and activation of migration/invasion.
Chisato Tomida, Naoko Yamagishi, Kana Aibara, Chiaki Yano, Takayuki Uchida, Tomoki Abe, Ayako Ohno, Katsuya Hirasaka, Takeshi Nikawa and Shigetada Teshima-Kondo : Chronic exposure of VEGF inhibitors promotes the malignant phenotype of colorectal cancer cells., The Journal of Medical Investigation : JMI, Vol.62, No.1-2, 75-79, 2015.
(要約)
VEGF-targeting anti-angiogenic drugs have enabled significant advances in cancer therapy. However, acquired resistance to VEGF-targeting drugs occurs, leading to disease progression. How tumors become the resistance remains fully uncertain. One of possible mechanisms for the resistance may be the direct effect of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGF-R). We investigated here the direct effect of chronic VEGF inhibition on phenotype changes in cancer cells. To chronically inhibit cancer cell-derived VEGF, human colon cancer HCT116 cells were chronically exposed (3 months) to anti-VEGF neutralizing monoclonal antibody (HCT/mAb cells, blockade of VEGF alone) or VEGF-R tyrosine kinase inhibitor foretinib (HCT/fore cells, blockade of all VEGF family). HCT/mAb cells redundantly increased VEGF family member (VEGF, PlGF, VEGF-B, VEGF-R1 and VEGF-R2) and induced a resistance to hypoxia-induced apoptosis. By contrast, HCT/fore cells did not show the redundant increase in VEGF family member, but significantly increased a VEGF-independent pro-angiogenic factor FGF-2. HCT/fore cells showed increased migration and invasion activities in addition to a resistance to hypoxia-induced apoptosis. The resistance to apoptosis was significantly suppressed by inhibition of hypoxia-inducible factor-1 in HCT/mAb cells, but not in HCT/fore cells. These findings suggest that chronic inhibition of VEGF/VEGF-R accelerates malignant phenotypes of colon cancer cells. J. Med. Invest. 62: 75-79, February, 2015.
Motoko Oarada, Azusa Takahashi-Nakaguchi, Tomoki Abe, Takeshi Nikawa, Takashi Miki and Tohru Gonoi : Refeeding with glucose rather than fructose elicits greater hepatic inflammatory gene expression in mice., Nutrition, Vol.31, No.5, 757-765, 2014.
(要約)
Fasting for 46 h up-regulated the liver expression of endogenous ligands for TLRs (HspA5, Hsp90 aa1, and Hspd1). Refeeding with agar gel containing α-cornstarch or glucose increased the liver expression of Tlr2, proinflammatory genes (Cxcl2, Cxcl10, Cxcl1, Nfkb1, Nfkb2, RelB, Sectm1α, Il1β), stress response genes (Atf3, Asns, Gadd45 a, Perk, Inhbe), detoxification genes (Hmox1, Gsta1, Abca8b), genes involved in tissue regeneration (Gdf15, Krt23, Myc, Tnfrsf12a, Mthfd2), and genes involved in tumor suppression (p53, Txnrd1, Btg2). This refeeding also moderately but significantly elevated the serum levels of alanine aminotransferase. These effects were attenuated in mice refed with agar gel containing sucrose or fructose.
Hirokazu Shiheido, Takane Aoyama, Honami Takahashi, Kaori Hanaoka, Tomoki Abe, Emi Nishida, Chen Chen, Orie Koga, Masaki Hikida, Yoshio Shibagaki, Akimichi Morita, Takeshi Nikawa, Seisuke Hattori, Takeshi Watanabe and Jun Shimizu : Novel CD3-specific antibody induces immunosuppression via impaired phosphorylation of LAT and PLC1 following T-cell stimulation., European Journal of Immunology, Vol.44, No.6, 1770-1780, 2014.
(要約)
The activation of T cells is known to be accompanied by the temporary downmodulation of the TCR/CD3 complex on the cell surface. Here, we established a novel monoclonal antibody, Dow2, that temporarily induces downmodulation of the TCR/CD3 complex in mouse CD4(+) T cells without activating T cells. Dow2 recognized the determinant on CD3; however, differences were observed in the binding mode between Dow2 and the agonistic anti-CD3 Ab, 145-2C11. An injection of Dow2 in vivo resulted in T-cell anergy, and prolonged the survival of cardiac allografts without a marked increase in cytokine release. The phosphorylated forms of the signaling proteins PLC-1 and LAT in Dow2-induced anergic T cells were markedly decreased upon stimulation. However, the levels of phosphorylated LAT and PLC1 in Dow2-induced anergic T cells could be rescued in the presence of the proteasome inhibitor MG-132. These results suggest that proteasome-mediated degradation is involved in hypophosphorylated LAT and PLC1 in Dow2-induced anergic T cells. The novel CD3-specific Ab, Dow2, may provide us with a unique tool for inducing immunosuppression.
Taesik Gwag, Kyoungsook Park, Eunjung Kim, Chaeyeon Son, Junsoo Park, Takeshi Nikawa and Inho Choi : Inhibition of C2C12 myotube atrophy by a novel HSP70 inducer, celastrol, via activation of Akt1 and ERK1/2 pathways., Archives of Biochemistry and Biophysics, Vol.537, No.1, 21-30, 2013.
(要約)
Celastrol (CEL) is known as a potent inducer of heat shock protein (HSP) in non-muscle cells and exhibits cytoprotective function and inhibitory effects on proteasome and glucocorticoid receptor activities. To investigate an anti-atrophic effect of CEL on skeletal muscle cells, C2C12 myotubes were treated with 150 M dexamethasone (DEX) for 24h and 1.5 M CEL was added for the last 6h during the 24h DEX treatment. Compared to the control, the myotube diameter was reduced by a factor of 0.30 by DEX, but CEL treatment almost abrogated the DEX-induced atrophy. CEL treatment also increased expression of HSP72 and phosphorylation of heat shock transcription factor 1 (p-HSF1) 11-fold and 3.4-fold, respectively, as well as accumulation of p-HSF1 in the nucleus. Furthermore, CEL treatment elevated activities of Akt1, p70/S6K and ERK1/2 2.0- to 4.4-fold whereas DEX had no effect on these signaling activities. Inhibition of Akt1 and ERK1/2 pathways by specific inhibitors confirmed CEL-induced anti-atrophic effect. Moreover, DEX-mediated downregulation of FoxO3 phosphorylation and upregulation of MuRF1 expression and proteasome activity were abrogated by CEL treatment. These results demonstrate a novel anti-atrophic function of CEL in muscle cells via both activation of protein anabolic signals and suppression of catabolic signaling activities.
Tomoki Abe, Shohei Kohno, Tomonari Yama, Arisa Ochi, Takuro Suto, Katsuya Hirasaka, Ayako Ohno, Shigetada Teshima-Kondo, Yuushi Okumura, Motoko Oarada, Inho Choi, Rie Mukai, Junji Terao and Takeshi Nikawa : Soy Glycinin Contains a Functional Inhibitory Sequence against Muscle-Atrophy-Associated Ubiquitin Ligase Cbl-b., International Journal of Endocrinology, Vol.2013, 907565, 2013.
(要約)
Background. Unloading stress induces skeletal muscle atrophy. We have reported that Cbl-b ubiquitin ligase is a master regulator of unloading-associated muscle atrophy. The present study was designed to elucidate whether dietary soy glycinin protein prevents denervation-mediated muscle atrophy, based on the presence of inhibitory peptides against Cbl-b ubiquitin ligase in soy glycinin protein. Methods. Mice were fed either 20% casein diet, 20% soy protein isolate diet, 10% glycinin diet containing 10% casein, or 20% glycinin diet. One week later, the right sciatic nerve was cut. The wet weight, cross sectional area (CSA), IGF-1 signaling, and atrogene expression in hindlimb muscles were examined at 1, 3, 3.5, or 4 days after denervation. Results. 20% soy glycinin diet significantly prevented denervation-induced decreases in muscle wet weight and myofiber CSA. Furthermore, dietary soy protein inhibited denervation-induced ubiquitination and degradation of IRS-1 in tibialis anterior muscle. Dietary soy glycinin partially suppressed the denervation-mediated expression of atrogenes, such as MAFbx/atrogin-1 and MuRF-1, through the protection of IGF-1 signaling estimated by phosphorylation of Akt-1. Conclusions. Soy glycinin contains a functional inhibitory sequence against muscle-atrophy-associated ubiquitin ligase Cbl-b. Dietary soy glycinin protein significantly prevented muscle atrophy after denervation in mice.
Naoko Yamagishi, Shigetada Kondo, Kiyoshi Masuda, Kensei Nishida, Yuki Kuwano, Duyen T Dang, Long H Dang, Takeshi Nikawa and Kazuhito Rokutan : Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells, BMC Cancer, Vol.13, 229, 2013.
(要約)
Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.
Tomoki Abe, Katsuya Hirasaka, Sachiko Kagawa, Shohei Kohno, Arisa Ochi, Kenro Utsunomiya, Atsuko Sakai, Ayako Maita, Shigetada Teshima-Kondo, Yuushi Okumura, Motoko Oarada, Yoichi Maekawa, Junji Terao, Edward M. Mills and Takeshi Nikawa : Cbl-b is a critical regulator of macrophage activation associated with obesity-induced insulin resistance in mice., Diabetes, Vol.62, No.6, 1957-1969, 2013.
(要約)
We previously reported the potential involvement of casitas B-cell lymphoma-b (Cbl-b) in aging-related murine insulin resistance. Because obesity also induces macrophage recruitment into adipose tissue, we elucidated here the role of Cbl-b in obesity-related insulin resistance. Cbl-b(+/+) and Cbl-b(-/-) mice were fed a high-fat diet (HFD) and then examined for obesity-related changes in insulin signaling. The HFD caused recruitment of macrophages into adipose tissue and increased inflammatory reaction in Cbl-b(-/-) compared with Cbl-b(+/+) mice. Peritoneal macrophages from Cbl-b(-/-) mice and Cbl-b-overexpressing RAW264.7 macrophages were used to examine the direct effect of saturated fatty acids (FAs) on macrophage activation. In macrophages, Cbl-b suppressed saturated FA-induced Toll-like receptor 4 (TLR4) signaling by ubiquitination and degradation of TLR4. The physiological role of Cbl-b in vivo was also examined by bone marrow transplantation and Eritoran, a TLR4 antagonist. Hematopoietic cell-specific depletion of the Cbl-b gene induced disturbed responses on insulin and glucose tolerance tests. Blockade of TLR4 signaling by Eritoran reduced fasting blood glucose and serum interleukin-6 levels in obese Cbl-b(-/-) mice. These results suggest that Cbl-b deficiency could exaggerate HFD-induced insulin resistance through saturated FA-mediated macrophage activation. Therefore, inhibition of TLR4 signaling is an attractive therapeutic strategy for treatment of obesity-related insulin resistance.
Motoko Oarada, Takashi Miki, Shohei Kohno, Kanae Sakai, Takeshi Nikawa, Mitsutoshi Yoneyama and Tohru Gonoi : Refeeding with a standard diet after a 48-h fast elicits an inflammatory response in the mouse liver., The Journal of Nutritional Biochemistry, Vol.24, No.7, 1314-1323, 2013.
(要約)
Unhealthy eating behaviors increase the risk of metabolic diseases, but the underlying mechanisms are not fully elucidated. Because inflammation contributes to the pathogenesis of metabolic diseases, it is important to understand the effects of unhealthy eating on the inflammatory state. The objective of our present study was to address the effects of a fasting-refeeding regime, a model of irregular eating, on the hepatic inflammatory responses in mouse. The animals were fasted for 48 h and then refed either a standard or low-carbohydrate/high-fat diet. Inflammatory gene expression in the liver was then sequentially measured for the first 17 h after initiation of refeeding. To assess the roles of dietary carbohydrates and toll-like receptor 2 (TLR2) in the refeeding-induced inflammatory changes, gene expression levels in mice refed only carbohydrates (-corn starch and sucrose) at different doses and in TLR2-deficient mice refed a standard diet were also analyzed. Refeeding with a standard diet increased the liver expression of Tlr2, proinflammatory mediators (Cxcl10, Cxcl1, Cxcl2, Icam-1) and negative regulators of TLR-signaling (A20 and Atf3). These increases were attenuated in mice refed a low-carbohydrate/high-fat diet. Refeeding only -corn starch and sucrose also increased the expression of these inflammatory pathway genes depending on the doses. TLR2 deficiency significantly attenuated the refeeding-induced increase in the liver expression of Cxcl10, Cxcl1, Icam-1 and A20. These findings suggest that an irregular eating behavior can elicit a liver inflammatory response, which is at least partly mediated by TLR2, and that dietary carbohydrates play critical roles in this process.
Katsuya Hirasaka, Tasuku Maeda, Chika Ikeda, Marie Haruna, Arisa Ochi, Rie Mukai, Motoko Oarada, Shigetada Kondo, Ayako Ohno, Yuushi Okumura, Junji Terao and Takeshi Nikawa : Isoflavones derived from soy beans prevent MuRF1-mediated muscle atrophy in C2C12 myotubes through SIRT1 activation., Journal of Nutritional Science and Vitaminology, Vol.59, No.4, 317-324, 2013.
(要約)
Proinflammatory cytokines are factors that induce ubiquitin-proteasome-dependent proteolysis in skeletal muscle, causing muscle atrophy. Although isoflavones, as potent antioxidative nutrients, have been known to reduce muscle damage during the catabolic state, the non-antioxidant effects of isoflavones against muscle atrophy are not well known. Here we report on the inhibitory effects of isoflavones such as genistein and daidzein on muscle atrophy caused by tumor necrosis factor (TNF)- treatment. In C2C12 myotubes, TNF- treatment markedly elevated the expression of the muscle-specific ubiquitin ligase MuRF1, but not of atrogin-1, leading to myotube atrophy. We found that MuRF1 promoter activity was mediated by acetylation of p65, a subunit of NFB, a downstream target of the TNF- signaling pathway; increased MuRF1 promoter activity was abolished by SIRT1, which is associated with deacetylation of p65. Of interest, isoflavones induced expression of SIRT1 mRNA and phosphorylation of AMP kinase, which is well known to stimulate SIRT1 expression, although there was no direct effect on SIRT1 activation. Moreover, isoflavones significantly suppressed MuRF1 promoter activity and myotube atrophy induced by TNF- in C2C12 myotubes. These results suggest that isoflavones suppress myotube atrophy in skeletal muscle cells through activation of SIRT1 signaling. Thus, the efficacy of isoflavones could provide a novel therapeutic approach against inflammation-related muscle atrophy.
Kenro Utsunomiya, Kanako Owaki, Yuushi Okumura, Momoko Yano, Takahiro Oto, Eri Suzuki, Seiko Tamura, Tomoki Abe, Shohei Kohno, Ayako Maita, Katsuya Hirasaka, Shigetada Kondo and Takeshi Nikawa : An intracellular fragment of osteoactivin formed by ectodomain shedding translocated to the nucleoplasm and bound to RNA binding proteins., Bioscience, Biotechnology, and Biochemistry, Vol.76, No.12, 2225-2229, 2012.
(要約)
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.
(キーワード)
Active Transport, Cell Nucleus / Amino Acid Sequence / Animals / COS Cells / Cell Membrane / Cell Nucleus / Cercopithecus aethiops / Endoplasmic Reticulum / HEK293 Cells / Humans / Intracellular Space / Membrane Glycoproteins / Molecular Sequence Data / Peptide Fragments / Protein Binding / Protein Structure, Tertiary / Proteolysis / RNA-Binding Proteins
Rie Mukai, Hitomi Horikawa, Yutaka Fujikura, Tomoyuki Kawamura, Hisao Nemoto, Takeshi Nikawa and Junji Terao : Prevention of disuse muscle atrophy by dietary ingestion of 8-prenylnaringenin in denervated mice, PLoS ONE, Vol.7, No.9, e45048, 2012.
(要約)
Flavonoids have attracted considerable attention in relation to their effects upon health. 8-Prenylnaringenin (8-PN) is found in the common hop (Humulus lupulus) and assumed to be responsible for the health impact of beer consumption. We wanted to clarify the effects of prenylation on the physiological functions of dietary flavonoids by comparing the effects of 8-PN with that of intact naringenin in the prevention of disuse muscle atrophy using a model of denervation in mice. Consumption of 8-PN (but not naringenin) prevented loss of weight in the gastrocnemius muscle further supported by the lack of induction of the protein content of a key ubiquitin ligase involved in muscle atrophy, atrogin-1, and by the activation of Akt phosphorylation. 8-PN content in the gastrocnemius muscle was tenfold higher than that of naringenin. These results suggested that, compared with naringenin, 8-PN was effectively concentrated into skeletal muscle to exert its preventive effects upon disuse muscle atrophy. It is likely that prenylation generates novel functions for 8-PN by enhancing its accumulation into muscle tissue through dietary intake.
Shohei Kohno, Yui Yamashita, Tomoki Abe, Katsuya Hirasaka, Motoko Oarada, Ayako Ohno, Shigetada Teshima-Kondo, Akira Higashibata, Inho Choi, Edward M. Mills, Yuushi Okumura, Junji Terao and Takeshi Nikawa : Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages., Journal of Applied Physiology, Vol.112, No.10, 1773-1782, 2012.
(要約)
Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor- and interleukin-1, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.
C U. Lago, S M. Nowinski, J E. Rundhaug, M E. Pfeiffer, K Kiguchi, Katsuya Hirasaka, X Yang, E M. Abramson, S B. Bratton, O Rho, R Colavitti, M A. Kenaston, Takeshi Nikawa, C Trempus, J Digiovanni, S M. Fischer and E M. Mills : Mitochondrial respiratory uncoupling promotes keratinocyte differentiation and blocks skin carcinogenesis., Oncogene, Vol.31, No.44, 4725-4731, 2012.
(要約)
Decreased mitochondrial oxidative metabolism is a hallmark bioenergetic characteristic of malignancy that may have an adaptive role in carcinogenesis. By stimulating proton leak, mitochondrial uncoupling proteins (UCP1-3) increase mitochondrial respiration and may thereby oppose cancer development. To test this idea, we generated a mouse model that expresses an epidermal-targeted keratin-5-UCP3 (K5-UCP3) transgene and exhibits significantly increased cutaneous mitochondrial respiration compared with wild type (FVB/N). Remarkably, we observed that mitochondrial uncoupling drove keratinocyte/epidermal differentiation both in vitro and in vivo. This increase in epidermal differentiation corresponded to the loss of markers of the quiescent bulge stem cell population, and an increase in epidermal turnover measured using a bromodeoxyuridine (BrdU)-based transit assay. Interestingly, these changes in K5-UCP3 skin were associated with a nearly complete resistance to chemically-mediated multistage skin carcinogenesis. These data suggest that targeting mitochondrial respiration is a promising novel avenue for cancer prevention and treatment.Oncogene advance online publication, 23 January 2012; doi:10.1038/onc.2011.630.
Deficiency of the Cbl-b ubiquitin ligase gene activates macrophages in mice. This study aimed to elucidate the pathophysiological roles of macrophages in muscle degeneration/regeneration in Cbl-b-deficient mice. We examined immune cell infiltration and cytokine expression in cardiotoxin-injected tibialis anterior muscle of Cbl-b-deficient mice. Ablation of the Cbl-b gene expression delayed regeneration of cardiotoxin-induced skeletal muscle damage compared with wild-type mice. CD8-positive T cells were still present in the damaged muscle on day 14 after cardiotoxin injection in Cbl-b-deficient mice, but there was dispersal of the same cells over that time-frame in wild-type mice. Infiltrating macrophages in Cbl-b-deficient mice showed strong expression of RANTES (regulated-on-activation, normal T cell expressed and secreted), a chemokine for CD8-positive T cells. In turn, a neutralizing antibody against RANTES significantly suppressed the infiltration of CD8-positive T cells into the muscle, resulting in restoration of the disturbed muscle regeneration. Cbl-b is an important regulatory factor for cytotoxic T-cell infiltration via RANTES production in macrophages.
Rie Mukai, Reiko Nakao, Hironori Yamamoto, Takeshi Nikawa, Eiji Takeda and Junji Terao : Quercetin Prevents Unloading-Derived Disused Muscle Atrophy by Attenuating the Induction of Ubiquitin Ligases in Tail-Suspension Mice., Journal of Natural Products, Vol.73, No.10, 1708-1710, 2010.
(要約)
The effects of quercetin (1) were investigated on disused muscle atrophy using mice that underwent tail suspension. Periodic injection of 1 into the gastrocnemius muscle suppressed muscle weight loss and ubiquitin ligase expression. Compound 1 reduced the enhancement of lipid peroxidation in the muscle. Injection of N-acetyl-l-cysteine, but not flavone (2), also prevented muscle weight loss and enhancement of lipid peroxidation. These findings demonstrate that 1 can prevent disused muscle atrophy by attenuating the expression of ubiquitin ligases and that such prevention originates from its antioxidant activity.
Motoko Oarada, Miki Igarashi, Tsuyoshi Tsuzuki, Katsuhiko Kamei, Katsuya Hirasaka, Takeshi Nikawa, Teruo Miyazawa, Kiyotaka Nakagawa and Tohru Gonoi : Effects of a high-protein diet on host resistance to Paracoccidioides brasiliensis in mice, Bioscience, Biotechnology, and Biochemistry, Vol.74, No.3, 620-626, 2010.
(要約)
We investigated the effects of high protein intake on host resistance to Paracoccidioides brasiliensis. Two-d fasted mice were infected with P. brasiliensis and refed on diets with three different levels (54%, 20%, and 5%) of casein. The mice refed the 54% casein diet showed reduced antifungal activity in the spleen and liver as compared with the mice refed the 5% or the 20% casein diet. After infection, increases in spleen and liver mRNA levels of myeloperoxidase, cathepsin-G, and elastase-2 were more profound in the mice refed the 54% casein diet as compared with the mice refed the 5% or the 20% casein diet. Infected mice refed the 54% casein diet exhibited greater interferon (IFN)-gamma production in the spleen and liver and higher levels of thiobarbituric acid reactive substances (TBARSs) in the liver as compared with those refed the 5% casein diet. These results indicate that high protein intake impairs host resistance to P. brasiliensis.
Motoko Oarada, Miki Igarashi, Tsuyoshi Tsuzuki, Nobuyuki Kurita, Tohru Gonoi, Takeshi Nikawa, Katsuya Hirasaka, Teruo Miyazawa, Kiyotaka Nakagawa and Katsuhiko Kamei : Effect of dietary oils on host resistance to fungal infection in psychologically stressed mice, Bioscience, Biotechnology, and Biochemistry, Vol.73, No.9, 1994-1998, 2009.
(要約)
Psychological stress can modulate host defense against invading pathogens. In this study, we investigated the effect of dietary oils on social isolation stress-induced modulation of host resistance to Paracoccidioides brasiliensis. In olive oil-fed mice, 3 weeks of isolation stress resulted in temporarily delayed clearance of this fungus in the liver compared with group-housed mice. By contrast, in soybean oil-fed mice, isolation stress had no significant effect on antifungal activity. The olive oil-fed mice showed greater liver interferon (IFN)-gamma and interleukin (IL)-6 production in response to infection as compared with the soybean oil-fed mice. In the olive oil-fed mice, isolation stress led to greater infection-induced IFN-gamma production in the liver compared with the group-housed animals. These results indicate that the modulatory effects of psychological stress on host resistance to P. brasiliensis can vary depending on dietary fatty acid composition.
Motoko Oarada, Katsuhiko Kamei, Tohru Gonoi, Tsuyoshi Tsuzuki, Takahito Toyotome, Katsuya Hirasaka, Takeshi Nikawa, Ayaka Sato and Nobuyuki Kurita : Beneficial effects of a low-protein diet on host resistance to Paracoccidioides brasiliensis in mice, Nutrition, Vol.25, No.9, 954-963, 2009.
(要約)
Although protein malnutrition impairs immune functions, several studies have recently shown that protein restriction without malnutrition is beneficial to host defenses against invading pathogens and cancer. In an effort to establish the optimum diet for host resistance, we investigated the effect of different dietary protein levels on host resistance to Paracoccidioides brasiliensis. Mice were fasted for 2 days and then infected with P. brasiliensis. Immediately after challenge with this fungus, mice were refed on diets with three different levels (0%, 1.5%, or 20%) of casein. On days 0-7 after infection, antifungal activity and levels of proinflammatory mediators in the spleen and liver were measured. Mice refed on the 1.5% casein diet showed higher antifungal activity in the spleen and liver compared with mice on the 20% casein diet. The antifungal activity in the spleens of mice refed on the 0% casein diet was intermediate between the antifungal activities of those refed the 1.5% and 20% casein diets. After infection, increases in spleen and liver levels of interleukin-6 and interferon-gamma, liver mRNA levels of antimicrobial proteins (myeloperoxidase, cathepsin-G, and elastase-2), and liver mRNA levels of proinflammatory mediators (interleukin-18, chemokine C-X-C motif ligand 10, nuclear factor-kappaB, inducible nitric oxide synthase, and granulocyte-macrophage colony-stimulating factor) were less profound in mice on the 1.5% or 0% casein diet compared with mice refed the 20% casein diet. The present results suggest that protein restriction without malnutrition could be beneficial to host resistance to P. brasiliensis.
Reiko Nakao, Katsuya Hirasaka, Jumpei Goto, Kazumi Ishidoh, Chiharu Yamada, Ayako Ohno, Yuushi Okumura, Ikuya Nonaka, Koji Yasutomo, KM Baldwin, Eiki Kominami, Akira Higashibata, Keisuke Nagano, Keiji Tanaka, Natsuo Yasui, EM Mills, Shinichi Takeda and Takeshi Nikawa : Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading., Molecular and Cellular Biology, Vol.29, No.17, 4798-4811, 2009.
(要約)
Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.
Ibrahim Dalia Ismaeil Hemdan, Katsuya Hirasaka, Reiko Nakao, Shohei Kohno, Sachiko Kagawa, Tomoki Abe, Akiko Harada, Yuushi Okumura, Yutaka Nakaya, Junji Terao and Takeshi Nikawa : Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes., The Journal of Medical Investigation : JMI, Vol.56, No.1-2, 26-32, 2009.
(要約)
Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy.
Mayumi Mikura, Ippei Yamaoka, Masako Doi, Yuichi Kawano, Mitsuo Nakayama, Reiko Nakao, Katsuya Hirasaka, Yuushi Okumura and Takeshi Nikawa : Glucose Infusion Suppresses Surgery-induced Muscle Protein Breakdown by Inhibiting Ubiquitin-proteasome Pathway in Rats, Anesthesiology, Vol.110, No.1, 81-88, 2009.
(要約)
It appears to have been well established that after surgery, protein catabolism is accelerated and glucose infusion suppresses the catabolic reactions. However, in the early postoperative period, the effects of surgical stress and glucose infusion on muscle protein catabolism and the related mechanisms remain unclear. Rats undergoing laparotomy were infused with acetated Ringer's solution (10 ml x kg(-1) x h(-1)) without glucose (control) or containing 1% or 5% glucose. The infusion was continued for a further 4 h after the surgical treatment. The catabolic index, excretion of urinary nitrogen and 3-methylhistidine, and release of tyrosine and 3-methylhistidine from isolated muscle were determined. Furthermore, muscular mRNA expression of proteolytic-related genes (atrogin-1/MAFbx, muscle ring finger-1, mu- and m-calpain, and cathepsin L and H) and phosphorylation of components of insulin signaling (forkhead box O3 and protein kinase B) were evaluated. Surgery increased the catabolic index, and this increase was suppressed by glucose infusion (both 1% and 5%). In the control group, mRNA expression of atrogin-1/MAFbx and muscle ring finger-1 was increased, and they were suppressed in the two glucose groups. Furthermore, insulin signaling (phosphorylation of protein kinase B and forkhead box O3) in muscles was stimulated by glucose infusion. The present study indicates that glucose infusion, even at a relatively low rate, suppresses muscle protein breakdown in the early postoperative period. The mechanism of this effect is related to the suppression of the ubiquitin-proteasome pathway, accompanied by activation of insulin signaling.
Masuhiro Nishimura, Mayumi Mikura, Katsuya Hirasaka, Yuushi Okumura, Takeshi Nikawa, Yuichi Kawano, Mitsuo Nakayama and Muneharu Ikeda : Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells, The Journal of Biochemistry, Vol.144, No.6, 717-724, 2008.
(要約)
We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.
Yuko Minami, Kyuichi Kawabata, Yoshiaki Kubo, Seiji Arase, Katsuya Hirasaka, Takeshi Nikawa, Noriko Bando, Yoshichika Kawai and Junji Terao : Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary beta-carotene in photoaging of hairless mouse skin., The Journal of Nutritional Biochemistry, Vol.20, No.5, 389-398, 2008.
(要約)
The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.
Yoshiko Nakae, Katsuya Hirasaka, J. Goto, Takeshi Nikawa, Masayuki Shono, M. Yoshida and PJ. Stowrd : 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., Histochemistry and Cell Biology, Vol.129, No.4, 489-501, 2008.
(要約)
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.
(キーワード)
epigallocatechin-3-gallate / green tea / mdx mice / histological
Katsuya Hirasaka, Tokuoka Kaori, Nakao Reiko, Yamada Chiharu, Oarada Motoko, imagawa Takahiko, Ishidoh Kazumi, Yuushi Okumura, Kyoichi Kishi and Takeshi Nikawa : Cathepsin C propeptide interacts with intestinal alkaline phosphatase and heat shock cognate protein 70 in human Caco-2 cells, The Journal of Physiological Sciences, Vol.58, No.2, 105-111, 2008.
(要約)
The oligomeric structure and the residual propeptide are distinct characteristics of cathepsin C from other members in the papain superfamily. In this study, we examined the physiological role of the cathepsin C propeptide. The stable overexpression of cathepsin C propeptide significantly decreased the activities of intestinal alkaline phosphatase (IAP) and sucrase in human Caco-2 intestinal epithelial cells, whereas it did not change the proliferation and cathepsin C activity. The overexpression of cathepsin C propeptide significantly decreased the amounts of IAP protein in differentiated Caco-2 cells, compared with the transfection of mock vector, whereas the amounts of IAP transcripts were not changed. Pulse-chase analysis confirmed that the reduction in IAP activity was due to an increase in IAP degradation, but not a decrease in IAP expression. For the mechanism of the enhanced IAP degradation, we identified proteins interacting with cathepsin C propeptide in Caco-2 cells by immunoprecipitation and mass spectrometry. Cathepsin C propeptide interacted with proteins with a molecular mass of approximately 70 kDa, including IAP and heat shock cognate protein 70. Our present results suggest that the propeptide of cathepsin C may stimulate the sorting to the lysosome, at least in part, contributing to the degradation of IAP in Caco-2 cells.
Oarada Motoko, Tsuzuki Tsuyoshi, Gonoi Tohru, Igarashi Miki, Kamei Katsuhiko, Takeshi Nikawa, Katsuya Hirasaka, Ogawa Takayuki, Miyazawa Teruo, Nakagawa Kiyotaka and Kurita Nobuyuki : Effects of dietary fish oil on lipid peroxidation and serum triacylglycerol levels in psychologically stressed mice, Nutrition, Vol.24, No.1, 67-75, 2008.
(要約)
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. 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. 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. 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.
Katsuya Hirasaka, S Kohno, J Goto, H Furochi, Kazuaki Mawatari, Nagakatsu Harada, Toshio Hosaka, Yutaka Nakaya, K Ishidoh, Toshiyuki Obata, Yousuke Ebina, H Gu, S Takeda, Kyoichi Kishi and Takeshi Nikawa : Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice., Diabetes, Vol.56, No.10, 2511-2522, 2007.
(要約)
c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. C57BL/6 (Cbl-b(+/+)) or Cbl-b-deficient (Cbl-b(-/-)) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. Elderly Cbl-b(-/-) mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b(-/-) mice than age-matched Cbl-b(+/+) mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b(-/-) mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b(-/-) macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b(-/-) macrophages compared with Cbl-b(+/+) macrophages. Treatment with a neutralizing anti-MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b(-/-) mice. Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.
Shunji Nakano, Takuya Mishiro, Sigeyuki Takahara, Hhiromiti Yokoi, Daisuke Hamada, Kiminori Yukata, Yoichiro Takata, Tomohiro Goto, Hiroshi Egawa, Susumu Yasuoka, H Furouchi, Katsuya Hirasaka, Takeshi Nikawa and Natsuo Yasui : Distinct expression of mast cell tryptase and protease activated receptor-2 in synovia of rheumatoid arthritis and osteoarthritis., Clinical Rheumatology, Vol.26, No.8, 1284-1292, 2007.
(要約)
The objective of this study is to examine the differential expression of mast cell tryptase and its receptor, protease-activated receptor-2 (PAR-2), in the synovium and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Biochemical and immunohistochemical analyses were performed to determine whether the trypsin-like protease in the synovium is identical to mast cell tryptase. The effects of mast cell tryptase on the proliferation of synovial fibroblast-like cells (SFCs) and the release of IL-8 thereof were evaluated by the [3H]-thymidine incorporation and ELISA, respectively. The trypsin-like protease in the synovium of RA patients was identical to human mast cell tryptase, which was composed of two subunits: 33 and 34 kDa. The 33- and 34-kDa proteins are different glycosylated forms of the 31-kDa protein, which was unglycosylated. Mast cell tryptase activity in RA synovial fluid was significantly higher than that in OA synovial fluid, while their activities and expression in the synovium were similar. Expression of PAR-2 mRNA in the synovium was higher in RA than in OA. Mast cell tryptase containing the unglycosylated 31-kDa subunit was the predominant form in synovial fluid. RA patients had higher amounts of this subunit in their synovial fluid than OA patients. Mast cell tryptase and PAR-2 activating peptide stimulated the proliferation of SFCs and release of IL-8 from these cells. Mast cell tryptase secretion into RA synovial fluid is higher than OA synovial fluid. Mast cell tryptase in synovial fluid stimulates the proliferation of SFCs and the release of pro-inflammatory cytokines via PAR-2, which may contribute to exacerbation of synovitis in RA.
Takayuki Ogawa, Harumi Furochi, Mai Mameoka, Katsuya Hirasaka, Yuko Onishi, Naoto Suzue, Motoko Oarada, Motoki Aakamatsu, Hiroshi Akima, Tetsuo Fukunaga, Kyoichi Kishi, Natsuo Yasui, Kazumi Ishidoh, Hideoki Fukuoka and Takeshi Nikawa : Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest., Muscle & Nerve, Vol.34, No.4, 463-469, 2006.
(要約)
In animal models, several ubiquitin ligases play an important role in skeletal muscle atrophy caused by unloading. In this study we examined protein ubiquitination and ubiquitin ligase gene expression in quadriceps femoris muscle from healthy volunteers after 20-day bedrest to clarify ubiquitin-dependent proteolysis in human muscles after unloading. During bedrest, thickness and cross-sectional area of the quadriceps femoris muscle decreased significantly by 4.6% and 3.7%, respectively. Ubiquitinated proteins accumulated in these atrophied human muscles. A real-time reverse transcription-polymerase chain reaction system showed that bedrest significantly upregulated expression of two ubiquitin ligase genes, Cbl-b and atrogin-1. We also performed DNA microarray analysis to examine comprehensive gene expression in the atrophied muscle. Bedrest mainly suppressed the expression of muscle genes associated with control of gene expression in skeletal muscle. Our results suggest that, in humans, Cbl-b- or atrogin-1-mediated ubiquitination plays an important role in unloading-induced muscle atrophy, and that unloading stress may preferentially inhibit transcriptional responses in skeletal muscle.
(キーワード)
Adaptor Proteins, Signal Transducing / Adult / Bed Rest / Body Weight / DNA / Gene Expression Profiling / Gene Expression Regulation, Enzymologic / Humans / Male / Muscle Proteins / Muscle, Skeletal / Muscular Atrophy / Oligonucleotide Array Sequence Analysis / Organ Size / Proteins / Proto-Oncogene Proteins c-cbl / SKP Cullin F-Box Protein Ligases / Ubiquitin-Protein Ligases
Tadatoshi Sato, Hironori Yamamoto, Naoki Sawada, Kunitaka Nashiki, Mitsuyoshi Tsuji, Kazusa Muto, H Kume, Hajime Sasaki, Hidekazu Arai, Takeshi Nikawa, Yutaka Taketani and Eiji Takeda : Restraint stress alters the duodenal expression of genes important for lipid metabolism in rat., Toxicology, Vol.227, No.3, 248-261, 2006.
(要約)
Stress, such as trauma and injury, is known to cause transcriptional changes in various tissues; however, there is little information on tissue-specific gene expression in response to stress. Here, we have examined duodenal gene expression in rats subjected to whole-body immobilization in order to elucidate the mechanism underlying the stress response in the duodenum--one of the tissues that is most sensitive to external stress. DNA microarray analysis revealed that the immobilization for 2 weeks caused great changes in gene expression in the rat duodenum: 165 genes exhibited more than a two-fold change in expression level (103 up-regulated; 62 down-regulated). In addition, functional classification of these genes showed that immobilization preferentially stimulated the expression of genes related to lipid metabolism, including genes encoding mitochondrial HMG-CoA synthase, a key enzyme in ketogenesis; solute carrier 27A2, a fatty acid transporter; and dienoyl CoA reductase, a key enzyme in beta-oxidation. To elucidate the factors mediating these immobilization-induced changes, we treated rats and small intestinal IEC-6 cells with dexamethasone and hydrogen peroxide. In both rats and IEC-6 cells, treatment with dexamethasone induced changes in gene expression that mimicked the immobilization-mediated increase in expression of the mitochondrial HMG-CoA synthase and dienoyl CoA reductase transcripts, suggesting that stress-induced synthesis of glucocorticoid hormones mediates, at least in part, the stress response in the duodenum. These results suggest that immobilization may alter lipid metabolism in the small intestine by modifying the expression of specific genes through which the small intestine may seek to protect itself from stress-induced damage.
Rie Shimooka, Yasuhiro Kido, Naoko Chiba, Junko Tanaka, Kazuhito Rokutan, Harumi Furochi, Katsuya Hirasaka, Takeshi Nikawa and Kyoichi Kishi : Soy protein diet prevents hypermethioninemia caused by portacaval shunt in rats., The Journal of Medical Investigation : JMI, Vol.53, No.3-4, 255-263, 2006.
(要約)
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.
Tadatoshi Sato, Hironori Yamamoto, Naoki Sawada, Kunitaka Nashiki, Mitsuyoshi Tsuji, Takeshi Nikawa, Hidekazu Arai, Kyoko Morita, Yutaka Taketani and Eiji Takeda : Immobilization decreases duodenal calcium absorption through a 1,25-dihydroxyvitamin D-dependent pathway., Journal of Bone and Mineral Metabolism, Vol.24, No.4, 291-299, 2006.
(要約)
Immobilization induces significant and progressive bone loss, with an increase in urinary excretion and a decrease in intestinal absorption of calcium. These actions lead to negative calcium balance and the development of disuse osteoporosis. The aims of this study were to evaluate the molecular mechanisms of decreased intestinal calcium absorption and to determine the effect of dietary 1,25-dihydroxyvitamin D [1,25(OH)2D] and a high-calcium diet on bone loss due to immobilization. The immobilized rat model was developed in the Bollman cage III to induce systemic disuse osteoporosis in the animals. There was a significant decrease in lumbar bone mineral density (BMD) and intestinal calcium absorption in the immobilized group compared with the controls. Serum 25-hydroxyvitamin D concentration did not change, but 1,25(OH)2D concentration decreased significantly. The mRNA levels of renal 25-hydoxyvitamin D 24-hydroxylase (24OHase) increased, whereas those of renal 25-hydroxyvitamin D 1-alpha hydroxylase (1alpha-hydroxylase), duodenal transient receptor potential cation channel, subfamily V, member 6 (TRPV6), TRPV5, and calbindin-D9k were all decreased. A high-calcium diet did not prevent the reduction in lumbar BMD or affect the mRNA expression of proteins related to calcium transport. Dietary administration of 1,25(OH)2D increased the intestinal calcium absorption that had been downregulated by immobilization. TRPV6, TRPV5, and calbindin-D9k mRNA levels were also upregulated, resulting in prevention of the reduction in lumbar BMD. Therefore, it is concluded that dietary 1,25(OH)2D prevented decreases in intestinal calcium absorption and simultaneously prevented bone loss in immobilized rats. However, it remains unclear that calcium absorption and expression of calcium transport proteins are essential for the regulation of lumbar BMD.
Naoto Suzue, Takeshi Nikawa, Yuko Onishi, Chiharu Yamada, Katsuya Hirasaka, Takayuki Ogawa, Harumi Furochi, Hirofumi Kosaka, Kazumi Ishidoh, Gu Hua, Shin'ichi Takeda, Naozumi Ishimaru, Yoshio Hayashi, Hironori Yamamoto, Kyoichi Kishi and Natsuo Yasui : Ubiqitin Ligase Cbl-b Downregulates Bone Formation Through Suppression of IGF-I Signaling in Osteoblasts During Denervation, Journal of Bone and Mineral Research, Vol.21, No.5, 722-734, 2006.
(要約)
Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.
(キーワード)
Animals / Base Sequence / Blotting, Western / Bone Development / Cells, Cultured / DNA Primers / Denervation / Down-Regulation / Hydrolysis / 免疫組織化学 (immunohistochemistry) / Insulin-Like Growth Factor I / Mice / Mice, Inbred C57BL / Osteoblasts / Protein Binding / Reverse Transcriptase Polymerase Chain Reaction / シグナル伝達 (signal transduction) / Ubiquitin / Ubiquitin-Protein Ligases
Yuko Onishi, Katsuya Hirasaka, Ibuki Ishihara, Motoko Oarada, Jumpei Goto, Takayuki Ogawa, Naoto Suzue, Shunji Nakano, Harumi Furochi, Kazumi Ishidoh, Kyoichi Kishi and Takeshi Nikawa : Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress, Biochemical and Biophysical Research Communications, Vol.336, No.3, 799-806, 2005.
(要約)
We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.
Takayuki Ogawa, Takeshi Nikawa, Harumi Furochi, Miki Kosyoji, Katsuya Hirasaka, Naoto Suzue, Koichi Sairyo, Shunji Nakano, Takashi Yamaoka, Mitsuo Itakura, Kyoichi Kishi and Natsuo Yasui : Osteoactivin upregulates expression of MMP-3 and MMP-9 in fibroblasts infiltrated into denervated skeletal muscle in mice, American Journal of Physiology, Cell Physiology, Vol.289, No.3, C697-C707, 2005.
(要約)
In this study, we examined pathophysiological roles of osteoactivin, a functionally unknown type I membrane glycoprotein, in mouse skeletal muscle atrophied by denervation (sciatic neurectomy). Denervation increased the amounts of osteoactivin, vimentin, matrix metalloproteinase-3 (MMP-3), and MMP-9 in mouse gastrocnemius muscle. Interestingly, immunohistochemical analysis revealed that vimentin, MMP-3, and MMP-9 were mainly present in fibroblast-like cells infiltrated into denervated mouse gastrocnemius muscle, whereas osteoactivin was expressed in the sarcolemma of myofibers adjacent to the fibroblast-like cells. On the basis of these findings, we reasoned that osteoactivin in myocytes was involved in activation of the infiltrated fibroblasts. To address this issue, we examined effects of osteoactivin on expression of MMPs in fibroblasts in vitro and in vivo. Overexpression of osteoactivin in NIH-3T3 fibroblasts induced expression of MMP-3, but not in mouse C(2)C(12) myoblasts, indicating that osteoactivin might functionally target fibroblasts. Treatment with recombinant mouse osteoactivin increased the amounts of collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts. The upregulated expression of these fibroblast marker proteins was significantly inhibited by heparin, but not by an integrin inhibitor, indicating that a heparin-binding motif in the extracellular domain might be an active site of osteoactivin. In osteoactivin-transgenic mice, denervation further enhanced expression of MMP-3 and MMP-9 in fibroblasts infiltrated into gastrocnemius muscle, compared with wild-type mice. Our present results suggest that osteoactivin might function as an activator for fibroblasts infiltrated into denervated skeletal muscles and play an important role in regulating degeneration/regeneration of extracellular matrix.
Katsuya Hirasaka, Takeshi Nikawa, Louis Yuge, Ibuki Ishihara, Akira Higashibata, Noriaki Ishioka, Atsuko Okubo, Takashi Miyashita, Naoto Suzue, Takayuki Ogawa, Motoko Oarada and Kyoichi Kishi : Clinorotation prevents differentiation of rat myoblastic L6 cells in association with reduced NF-κB signaling, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, Vol.1743, No.1-2, 130-140, 2005.
(要約)
In this study, we examined effects of the three-dimensional (3D)-clinorotation, a simulated-model of microgravity, on proliferation/differentiation of rat myoblastic L6 cells. Differentiation of L6 cells into myotubes was significantly disturbed in the 3D-clinorotation culture system, although the 3D-clinorotation had no effect on the proliferation. The 3D-clinorotation also suppressed the expression of myogenesis marker proteins, such as myogenin and myosin heavy chain (MHC), at the mRNA level. In association with this reduced differentiation, we found that the 3D-clinorotation prevented accumulation of ubiquitinated proteins, compared with non-rotation control cells. Based on these findings, we focused on the ubiquitin-dependent degradation of I kappa B, a myogenesis inhibitory protein, to clarify the mechanism of this impaired differentiation. A decline in the amount of I kappa B protein in L6 cells was significantly prevented by the rotation, while the amount of the protein in the non-rotated cells decreased along with the differentiation. Furthermore, the 3D-clinorotation reduced the NF-kappaB-binding activity in L6 cells and prevented the ubiquitination of I kappa B proteins in the I kappa B- and ubiquitin-expressing Cos7 cells. Other myogenic regulatory factors, such as deubiquitinases, cyclin E and oxygen, were not associated with the differentiation impaired by the clinorotation. Our present results suggest that simulated microgravity such as the 3D-clinorotation may disturb skeletal muscle cell differentiation, at least in part, by inhibiting the NF-kappa B pathway.
(キーワード)
Animals / Blotting, Western / COS Cells / 細胞分化 (cell differentiation) / Cell Line / Cell Line, Tumor / Cyclin E / I-kappa B Proteins / Immunoblotting / Immunoprecipitation / Muscle Fibers, Skeletal / Muscle, Skeletal / Muscles / Myogenin / Myosin Heavy Chains / NF-kappa B / Oxygen / RNA, Messenger / Rats / Reverse Transcriptase Polymerase Chain Reaction / シグナル伝達 (signal transduction) / Time Factors / Transfection / Ubiquitin / Weightlessness
Katsuya Hirasaka, Takeshi Nikawa, Yuki Asanoma, Harumi Furochi, Yuko Onishi, Takayuki Ogawa, Naoto Suzue, Motoko Oarada, Toru Shimazu and Kyoichi Kishi : Short-term hypergravity does not affect protein-ubiquitination and proliferation in rat L6 myoblastic cells, Biological Sciences in Space, Vol.19, No.1, 3-7, 2005.
(要約)
We previously reported that spaceflight (STS-90) and tail-suspension stimulated muscle protein ubiquitination and accumulated the degradation fragments. However, in space experiments the side-effects of hypergravity on samples are inevitable during the launch of a space shuttle into space or the reentry. To examine whether hypergravity also caused protein-ubiquitination in skeletal muscle cells, we exposed rat myoblastic L6 cells to various hypergravity conditions. Immunoblot analysis showed that the centrifugation at 2, 3, 30 or 100 G for 10 min did not increase the amount of ubiquitinated proteins in L6 cells, whereas the centrifugation at 100 G for 1 or 2 hrs significantly induced the protein-ubiquitination. In contrast, heat shock protein 70 (HSP70), another stress-responsive protein, in L6 cells was accumulated only by centrifugation at 100 G for more than 10 min. Short-term (10 min) hypergravity including 3 or 100 G did not affect the proliferation and morphological changes in L6 cells. Our present results suggest that the ubiquitination of muscle proteins is less sensitive to hypergravity than the induction of HSP70, and that the effect of hypergravity on protein-ubiquitination and proliferation of skeletal muscle cells may be negligible, as far as its duration is short-term.
(キーワード)
hypergravity / ubiquitination / HSP70 / rat myoblastic L6 cells
Takuya Mishiro, Shunji Nakano, Shigeyuki Takahara, Mari Miki, Yoichi Nakamura, Susumu Yasuoka, Takeshi Nikawa and Natsuo Yasui : Relationship Between Cathepsin B and Thrombin in Rheumatoid Arthritis, The Journal of Rheumatology, Vol.31, No.7, 1265-1273, 2004.
(要約)
To investigate the pathophysiological significance of cathepsin B and thrombin in synovial fluid (SF) from patients with rheumatoid arthritis (RA). Thrombin and cathepsin B activities of samples from patients with RA and osteoarthritis (OA) were measured using fluorogenic synthetic substrates. The concentration of interleukin 8 (IL-8) in SF was measured by ELISA. The effect of thrombin on the proliferation of synovial fibroblast-like cells (SFC) was examined by measuring 3H-thymidine incorporation. The effect of thrombin on the release of IL-8 and cathepsin B from SFC was investigated. The expression of IL-8 mRNA in SFC after stimulation with thrombin was evaluated using real-time quantitative RT-PCR. The effect of recombinant IL-8 on the activation of cathepsin B was examined using the knee joints of rabbits. In SF supernatants, cathepsin B and thrombin-like activity was significantly higher in RA than in OA, and there was a significant correlation between them. Cathepsin B activity was also significantly higher in SF cells and synovial tissue extracts from RA patients than in those from OA patients. There was a significant correlation between cathepsin B activity and the concentration of IL-8 in RA SF. Thrombin enhanced the proliferation of SFC in a dose-dependent manner. Thrombin significantly enhanced the release of IL-8 from SFC as well as the expression of IL-8 mRNA in SFC. IL-8 induced activation of cathepsin B in the knee joints of rabbits. However, thrombin did not directly increase cathepsin B activity in SFC. In RA, thrombin was found to be related to the enhanced growth of SFC and the release of IL-8 from these cells; thus thrombin is probably related to worsening of inflammation through the recruitment of leukocytes (neutrophils), which release cathepsin B into the SF. Thrombin can induce activation of cathepsin B in SFC via increased expression of IL-8.
Takeshi Nikawa, Kazumi Ishidoh, Katsuya Hirasaka, Ibuki Ishihara, Madoka Ikemoto, Mihoko kano, Eiki Kominami, Ikuya Nonaka, Takayuki Ogawa, Gregory R. Adams, kenneth M. Baldwin, Natsuo Yasui, Kyoichi Kishi and Shinichi Takeda : Skeletal muscle gene expression in space-flown rats, The FASEB journal, Vol.18, No.3, 522-524, 2004.
(要約)
Skeletal muscles are vulnerable to marked atrophy under microgravity. This phenomenon is due to the transcriptional alteration of skeletal muscle cells to weightlessness. To further investigate this issue at a subcellular level, we examined the expression of approximately 26,000 gastrocnemius muscle genes in space-flown rats by DNA microarray analysis. Comparison of the changes in gene expression among spaceflight, tail-suspended, and denervated rats revealed that such changes were unique after spaceflight and not just an extension of simulated weightlessness. The microarray data showed two spaceflight-specific gene expression patterns: 1) imbalanced expression of mitochondrial genes with disturbed expression of cytoskeletal molecules, including putative mitochondria-anchoring proteins, A-kinase anchoring protein, and cytoplasmic dynein, and 2) up-regulated expression of ubiquitin ligase genes, MuRF-1, Cbl-b, and Siah-1A, which are rate-limiting enzymes of muscle protein degradation. Distorted expression of cytoskeletal genes during spaceflight resulted in dislocation of the mitochondria in the cell. Several oxidative stress-inducible genes were highly expressed in the muscle of spaceflight rats. We postulate that mitochondrial dislocation during spaceflight has deleterious effects on muscle fibers, leading to atrophy in the form of insufficient energy provision for construction and leakage of reactive oxygen species from the mitochondria.
Louis Yuge, Atsuko Okubo, Takashi Miyashita, Takanori Kumagai, Takeshi Nikawa, Shinichi Takeda, Masamoto Kanno, Yukio Urabe, Masanori Sugiyama and Katsuko Kataoka : Physical stress by magnetic force accelerates differentiation of human osteoblasts, Biochemical and Biophysical Research Communications, Vol.311, No.1, 32-38, 2003.
(要約)
We examined the effect of magnetic force on differentiation of cultured human osteoblasts. Magnetic microparticles (MPs) were introduced into the cytoplasm of a human osteoblast cell line and the cells were cultured in a magnetic field (MF) in group MP-MF. Three groups of controls were used: cells without MPs were cultured out of MF (group C), cells without MPs were cultured in MF (group MF), and cells with MPs were cultured out of MF (group MP). The cells in group MP-MF became larger and were elongated along the axis of the magnetic poles. Appearance of alkaline phosphatase (AlPase) activity, formation of bone nodules, and calcium deposition were accelerated depending on the intensity of the magnetic field. It takes longer culture in the other three groups to exhibit these changes. Core-binding factor A1 (Cbfa1: transcription factor for osteoblast differentiation) and osteocalcin (a bone-matrix protein involved in controlling osteogenesis) were expressed earlier or stronger in group MP-MF than the other groups. Then we compared phosphorylation of mitogen-activated protein kinase (MAPK) between group MP-MF and group C. Phosphorylation of p38(MAPK) (p38) was increased in group MP-MF, while total p38 as well as total and phosphorylated forms of MAPK/ERK 1/2 and SAPK/JNK were not changed between the two groups. When a p38 inhibitor, SB 203580, was added to the culture medium in group C, AlPase activity, formation of bone nodules, and calcium deposits were completely inhibited. On the other hand, they were inhibited only partially by a MAPK/ERK 1/2 inhibitor, U-0126. Based on these results, it is concluded that (1) osteoblast differentiation is accelerated by a magnetic force, (2) this acceleration is mainly attributed to the activation of p38 phosphorylation, and (3) the stimulus induced by a magnetic field offers a new approach to osteoblast differentiation.
Motoko Oarada, Tsuyoshi Tsuzuki, Toshihide Suzuki, Teruo Miyazawa, Takeshi Nikawa, Guan Hong-quan and Nobuyuki Kurita : Dietary supplementation with docosahexaenoic acid, but not with eicosapentaenoic acid, reduces host resistance to fungal infection in mice, Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1622, No.3, 151-160, 2003.
(要約)
The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-gamma concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.
Mihoko Kano, Takako Kitano, Madoka Ikemoto, Katsuya Hirasaka, Yuki Asanoma, Takayuki Ogawa, Shinichi Takeda, Ikuya Nonaka, Gregory R. Adams, Kenneth M. Baldwin, Motoko Oarada, Kyoichi Kishi and Takeshi Nikawa : Isolation and characterization of a novel gene sfig in rat skeletal muscle up-regulated by spaceflight (STS-90), The Journal of Medical Investigation : JMI, Vol.50, No.1-2, 39-47, 2003.
(要約)
We obtained the skeletal muscle of rats exposed to weightless conditions during a 16-day-spaceflight (STS-90). By using a differential display technique, we identified 6 up-regulated and 3 down-regulated genes in the gastrocnemius muscle of the spaceflight rats, as compared to the ground control. The up-regulated genes included those coding Casitas B-lineage lymphoma-b, insulin growth factor binding protein-1, titin and mitochondrial gene 16 S rRNA and two novel genes (function unknown). The down-regulated genes included those encoding RNA polymerase II elongation factor-like protein, NADH dehydrogenase and one novel gene (function unknown). In the present study, we isolated and characterized one of two novel muscle genes that were remarkably up-regulated by spaceflight. The deduced amino acid sequence of the spaceflight-induced gene (sfig) comprises 86 amino acid residues and is well conserved from Drosophila to Homo sapiens. A putative leucine-zipper structure located at the N-terminal region of sfig suggests that this gene may encode a transcription factor. The up-regulated expression of this gene, confirmed by Northern blot analysis, was observed not only in the muscles of spaceflight rats but also in the muscles of tail-suspended rats, especially in the early stage of tail-suspension when gastrocnemius muscle atrophy initiated. The gene was predominantly expressed in the kidney, liver, small intestine and heart. When rat myoblastic L6 cells were grown to 100% confluence in the cell culture system, the expression of sfig was detected regardless of the cell differentiation state. These results suggest that spaceflight has many genetic effects on rat skeletal muscle.
(キーワード)
Amino Acid Sequence / Animals / Base Sequence / Cell Line / DNA, Complementary / Gene Expression Profiling / Gene Expression Regulation / Immobilization / Leucine Zippers / Male / Molecular Sequence Data / Muscle Proteins / Muscle, Skeletal / Organ Specificity / Rats / Rats, Wistar / Sequence Homology, Amino Acid / Space Flight / Species Specificity / Subtraction Technique / Tail / Transcription Factors
Madoka Ikemoto, Yoshihito Okamura, Mihoko Kano, Katsuya Hirasaka, Reiko Tanaka, Taeko Yamamoto, Takahiro Sasa, Takayuki Ogawa, Koichi Sairyo, Kyoichi Kishi and Takeshi Nikawa : A Relative High Dose of Vitamin E Does Not Attenuate Unweighting-Induced Oxidative Stress and Ubiquitination in Rat Skeletal Muscle, Journal of Physiological Anthropology and Applied Human Science, Vol.21, No.5, 257-263, 2002.
(要約)
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.
Motoko Oarada, Takeshi Nikawa and Nobuyuki Kurita : Effect of timing of food deprivation on host resistance to fungal infection in mice, British Journal of Nutrition, Vol.88, No.2, 151-158, 2002.
(要約)
Mice were deprived of food for a period of 72 h at varying times relative to the time of infection with Paracoccidioides brasiliensis. Host resistance was diminished profoundly when the period of food deprivation was from 48 h before to 24 h after infection (group B). When food deprivation was initiated immediately after infection (group C), host resistance was reduced less profoundly. When food deprivation was initiated at 24 and 48 h post-infection, reductions in host resistance were only moderate or not observed respectively. These results suggest that the earlier in the course of infection starvation occurs, the more profoundly host resistance is impaired. When food deprivation was initiated 72 h before infection, finishing at the time of infection (group A), the reduction in host resistance was considerably less profound compared with group B mice, suggesting that refeeding initiated immediately after infection is responsible for rapid restoration of the antifungal resistance in starved mice. Infection-induced responses of corticosterone and interferon-gamma were changed according to the timing of food deprivation. Group A mice, similar to non-fasted controls, showed an infection-induced increase in serum corticosterone concentration, while groups B and C did not. Group C mice showed a substantially greater infection-induced increase in serum interferon-gamma compared with the other fasted and non-fasted control groups.
Takeshi Nikawa, Madoka Ikemoto, Takashi Sakai, Mihoko Kano, Takako Kitano, Tsukasa Kawahara, Shigetada Teshima, Kazuhito Rokutan and Kyoichi Kishi : Effects of a Soy Protein Diet on Exercise-Induced Muscle Protein Catabolism in Rats, Nutrition, Vol.18, No.6, 490-495, 2002.
(要約)
We examined effects of dietary soy protein isolate on muscle calpain activity and myosin heavy chain (MHC) degradation in rats performing an acute running exercise. In rats fed a 20% casein diet, the treadmill running exercise, fixed at 80 kg/m, transiently increased calpain activity in gastrocnemius muscles in parallel with the release of creatine kinase into plasma. The fixed running also caused an accumulation of immunoreactive degradation fragments of MHC in the muscle. Feeding a 20% soy protein isolate diet as opposed to the control casein diet to rats significantly suppressed the running-induced activation of mu- and m-calpains, fragmentation of MHC, and release of creatine kinase into plasma (P < 0.05). Rats fed the soy protein isolate diet had significantly higher calpastatin activity in gastrocnemius muscle than did rats fed the casein diet (P < 0.05), indicating that this increase inhibits the exercise-induced autoactivation of calpain. Activities of proteasome, cathepsin B + L, and antioxidant enzymes and the levels of glutathione and thiobarbituric acid-reactive substances in the muscle did not differ between the diet groups at the end of the exercise. Our results suggest that diets containing soy protein prevent exercise-induced protein degradation in skeletal muscle, possibly through inhibiting the calpain-mediated proteolysis.
(キーワード)
soy protein isolate / treadmill running / calpain / myosin heavy chain / creatine kinase
Takeshi Nikawa, Madoka Ikemoto, Chiho Watanabe, Takako Kitano, Mihoko Kano, Makoto Yoshimoto, Takae Towatari, Nobuhiko Katunuma, Fujiko Shizuka and Kyoichi Kishi : A Cysteine Protease Inhibitor Prevents Suspension-Induced Declines in Bone Weight and Strength in Rats, Journal of Physiological Anthropology and Applied Human Science, Vol.21, No.1, 51-57, 2002.
(要約)
In this study, we examined the effects of a potent cysteine protease inhibitor, N-(L-3-trans-carboxyoxirane-2-cabonyl)-L-leucine-4-aminobutylamide (E-64a), on bone weight and strength in tail-suspended rats. We first administered a vehicle or 4 or 8 mg/rat of E-64a to rats fed with a low calcium diet for 7 wks to determine effective doses of E-64a on bone resorption in vivo. Femoral cathepsin K-like activity and serum hydroxyproline level in rats fed with a low calcium diet were significantly higher than those in rats fed with a standard diet. The intraperitoneal injection of 8 mg/rat of E-64a to rats decreased their serum calcium and hydroxyproline concentrations after 3 to 6 hrs in parallel with changes in femoral cathepsin K-like activity, while 4 mg/rat of E-64a had weaker effects on these parameters. Based on these results, we injected 8 mg/rat of E-64a to tail-suspended rats twice a day for 2 wks and compared the results with those of treatment with 1 mg/rat of etidronate, a bisphosphonate, twice a week. In tail-suspended rats, femoral weight and strength, assessed by three-point bending test, significantly decreased from Day 5 to 21, while femoral cathepsin K-like activity and serum calcium and hydroxyproline concentrations did not change. E-64a inhibited femoral cathepsin K-like activity in tail-suspended rats, but etidronate did not. E-64a as well as etidronate significantly prevented the suspension-induced declines in bone weight and strength. However, more frequent injection and higher doses were required for E-64a to exhibit significant efficacy of antiresorption, compared with those of etidronate. Our results suggest that a cysteine protease inhibitor could improve suspension-induced osteopenia by inhibiting cathepsin K-like activity in bone; however, it needs several improvements in the effect as a clinical drug.
(キーワード)
Animals / Bone Resorption / Bone and Bones / Calcium / Cathepsin K / Cathepsins / Collagenases / Cysteine Proteinase Inhibitors / Etidronic Acid / Femur / Hydroxyproline / Leucine / Male / Organ Size / Rats / Rats, Wistar / Weightlessness
Tsukasa Kawahara, Yuki Kuwano, Shigetada Kondo, Tomoko Kawai, Takeshi Nikawa, Kyoichi Kishi and Kazuhito Rokutan : Toll-like receptor 4 regulates gastric pit cell responses to Helicobacter pylori infection, The Journal of Medical Investigation : JMI, Vol.48, No.3,4, 190-197, 2001.
(要約)
Gastric pit cells express mitogen oxidase1 (Mox1) and essential components for the phagocyte NADPH oxidase (p67-, p47-, p40-, and p22-phoxes). Helicobacter pylori (Hp) lipopolysaccharide (LPS) is a potent up-regulator of the Mox 1 oxidase. In this study, we examined the expression levels of several key members of the Toll-like receptor (TLR) family in primary cultures of guinea pig gastric pit cells. These cells expressed the TLR4 mRNA. Immunoblot analysis and immunofluorescence histochemistry with an anti-TLR4 antibody showed that gastric pit cells possessed significant amounts of TLR4 protein preferentially on the plasma membrane. In contrast, the cells did not express the TLR2 and TLR9 transcripts and did not contain detectable amounts of TLR2 protein. Neither peptidoglycan from Staphylococcus aureus nor Hp DNA with the CpG motif up-regulated Mox1 oxidase activity. Hp LPS activated nuclear factor-kappa B in association with the expression of cyclooxygenase II and tumor necrosis factor alpha transcripts. These findings suggest that TLR4 may play a crucial role in the initiation of inflammatory responses of gastric pit cells against Hp infection.
Tsukasa Kawahara, Yuki Kuwano, Shigetada Kondo, Toshiro Sugiyama, Tomoko Kawai, Takeshi Nikawa, Kyoichi Kishi and Kazuhito Rokutan : Helicobacter pylori lipopolysaccharide from type 1, but not type 2 strains, stimulates apoptosis of cultured gastric mucosal cells, The Journal of Medical Investigation : JMI, Vol.48, No.3,4, 167-174, 2001.
(要約)
The cag pathogenicity island (cag PAI) genes are a major determinant of virulence of Helicobacter pylori (Hp). Lipopolysaccharide (LPS) purified from the cag PAI-positive (type I) strains induced apoptosis of primary cultures of guinea pig gastric mucosal cells. Lipid A catalyzed this apoptosis. These cells expressed the Toll-like receptor 4 (TLR4) mRNA and its protein, and type I Hp LPS phosphorylated transforming growth factor beta-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in association with up-regulation of the TLR4 expressions, suggesting that type I Hp LPS evoked distinct TLR4 signaling. In contrast, Hp LPS from type II strains with complete or partial deletion of the cag PAI genes did not phosphorylate TAK1 and TAB1 and failed to induce apoptosis. Accelerated apoptosis of gastric epithelial cells is one of the important events relevant to chronic, atrophic gastritis caused by Hp infection. The difference in proapoptotic action of LPS between the type I and II strains may support an important role of the cag PAI genes in the pathogenesis of gastric lesions caused by Hp infection.
Takeshi Nikawa, Madoka Ikemoto, Mihoko Kano, Kaori Tokuoka, Katsuya Hirasaka, Shoji Uehara, Kiyoshi Takatsu, Kazuhito Rokutan and Kyoichi Kishi : Impaired Vitamin A-Mediated Mucosal IgA Response in IL-5 Receptor-Knockout Mice, Biochemical and Biophysical Research Communications, Vol.285, No.2, 546-549, 2001.
(要約)
To clarify actions of vitamin A on mucosal immunity associated with interleukin-5 (IL-5), we examined effects of vitamin A on mucosal IgA level in IL-5 receptor alpha-chain-knockout (IL-5Ralpha(-/-)) mice. Daily supplementation of retinyl acetate (1 mg/mouse) increased Th2 cytokine levels and a number of their positive cells in the small intestinal mucosa of IL-5Ralpha(-/-) mice, as observed in wild-type or IL-5Ralpha(+/-) mice. Wild-type and heterozygous mice increased the IgA level and a number of IgA-containing cells in the mucosa in response to the vitamin A treatment, but not in IL-5Ralpha(-/-) mice. Retinyl acetate increased anti-cholera toxin (CT) IgA level in the mucosa of wild-type mice, improving their survival rate after an exposure to 0.4 mg of CT. However, retinyl acetate failed to induce resistance to CT toxicity in IL-5Ralpha(-/-) mice. Our results suggest that IL-5 may play an important role in an action of vitamin A on mucosal IgA system.
(キーワード)
ビタミンA (vitamin A) / IgA / IL-4 / IL-5 / IL-6 / small intestinal mucosa / IL-5 receptor α-chain-deficient mice / cholera toxin
Madoka Ikemoto, Takeshi Nikawa, Shinichi Takeda, Chiho Watanabe, Takako Kitano, Baldwin M. Kenneth, Ryutaro Izumi, Ikuya Nonaka, Takae Towatari, Shigetada Teshima, Kazuhito Rokutan and Kyoichi Kishi : Space Shuttle flight (STS-90) enhances dagradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway, The FASEB journal, Vol.15, No.7, 1279-1281, 2001.
Takeshi Nikawa, Madoka Ikemoto, Kaori Tokuoka, Shigetada Teshima, Alpers H. David, Yoshihiro Masui, Kyoichi Kishi and Kazuhito Rokutan : Interleukin-1β enhances retinoic acid-mediated expression of bone-type alkaline phosphatase in rat IEC-6 cells, American Journal of Physiology, Gastrointestinal and Liver Physiology, Vol.280, No.3, 510-517, 2001.
(要約)
We previously showed that vitamin A upregulated the expression of bone-type alkaline phosphatase (ALP) in fetal rat small intestine and rat intestinal IEC-6 cells. In this study, we examined interactions between retinoic acid (RA) and several growth factors/cytokines on the isozyme expression in IEC-6 cells. Epidermal growth factor and interleukins (ILs)-2, -4, -5, and -6 completely blocked the RA-mediated increase in ALP activity. In contrast, IL-1beta markedly increased the activity, protein, and mRNA of the bone-type ALP only when RA was present. IL-1beta and/or RA did not change the type 1 IL-1 receptor transcript level, whereas IL-1beta enhanced the RA-induced expressions of retinoic acid receptor-beta (RAR-beta) and retinoid X receptor-beta (RXR-beta) mRNAs and RA-mediated RXR response element binding. The synergism of IL-1beta and RA on ALP activity was completely blocked by protein kinase C (PKC) inhibitors. Our results suggest that IL-1beta may modify the ALP isozyme expression in small intestinal epithelial cells by stimulating PKC-dependent, RAR-beta- and/or RXR-beta-mediated signaling pathways.
(キーワード)
Alkaline Phosphatase / Animals / Bone and Bones / Cell Line / DNA-Binding Proteins / Drug Synergism / Enzyme Inhibitors / Epidermal Growth Factor / Interleukin-1 / Interleukin-2 / Interleukin-4 / Interleukin-5 / Interleukin-6 / Intestine, Small / Isoenzymes / Protein Kinase C / RNA, Messenger / Rats / Receptors, Retinoic Acid / Transcriptional Activation / Tretinoin
Takeshi Nikawa, Kenji Odahara, Hiroyuki Koizumi, Yasuhiro Kido, Shigetada Teshima, Kazuhito Rokutan and Kyoichi Kishi : Vitamin A Prevents the Decline in Immunoglobulin A and Th2 Cytokine Levels in Small Intestinal Mucosa of Protein-Malnourished Mice1, Biochemical and Molecular Action of Nutrients, Vol.129, 934-941, 1999.
(キーワード)
vitamin A / protein-malnutrition / Immunoglobulin A / Th2 cytokine / mice
109.
Shigetada Teshima, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Macrophage Colony-Stimulating Factor Stimulates Synthesis and Secretion of a Mouse Homolog of a Human IgE-Dependent Histamine-Releasing Factor by Macrophages In Vitro and In Vivo, The Journal of Immunology, Vol.161, No.11, 6356-6366, 1998.
110.
Shigetada Kondo, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Macrophage colony-stimulating factor stimulates synthesis and secretion of a mouse homolog of a human IgE-dependent histamine-releasing factor by macrophages in vitro and in vivo., The Journal of Immunology, Vol.161, No.11, 6356-6366, 1998.
(要約)
Treatment of murine resident peritoneal macrophages with macrophage-CSF (M-CSF) up-regulated the synthesis of a discrete set of proteins, including a 26-kDa protein (p26). The sequence of 20 NH2-terminal amino acids of the purified p26 was identical with the mouse homolog of a human IgE-dependent histamine-releasing factor (HRF). Among macrophage activators tested (M-CSF, granulocyte-macrophage-CSF, IL-3, TNF-alpha, IFN-gamma, and LPS), only M-CSF could up-regulate the p26 HRF synthesis by cultured macrophages. M-CSF not only increased the levels of p26 HRF mRNA and protein, but also stimulated the secretion of an N-glycosylated p26 HRF with a m.w. of 30 kDa. Repeated injections of M-CSF into mouse peritoneal cavity for 4 days elicited macrophages expressing abundant p26 HRF. A single i.p. injection of M-CSF failed to increase the p26 HRF level in peritoneal macrophages of thioglycollate-, LPS-, or adjuvant-treated mice, while M-CSF challenge to OVA-immunized mice caused macrophage infiltration and overproduction of p26 HRF, similarly as did OVA challenge. The Ag-specific priming for enhanced synthesis and secretion of p26 HRF by M-CSF was also demonstrated in cultured macrophages prepared from OVA-immunized mice. An i.p. injection of M-CSF or recombinant p26 HRF triggered eosinophil recruitment, even in the absence of the Ag, in the sensitized mice, but not in normal mice. Furthermore, recombinant p26 HRF could induce eosinophilia without marked macrophage and lymphocyte infiltrations. Our results suggest that p26 HRF secreted by M-CSF-stimulated macrophages may be an important mediator for the late phase allergic inflammation.
Shigetada Teshima, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system, Gastroenterology, Vol.115, No.5, 1186-1196, 1998.
(要約)
Superoxide anion (O2-) plays an important role in gastric pathophysiology. The aims of this study were to identify O2--producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. The amount of O2- was measured by the reduction of cytochrome c, and O2--producing cells were visualized by nitroblue tetrazolium reaction. Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox. Gastric pit cells, but not parietal cells, spontaneously released O2- at 50 nmol . mg protein-1 . h-1. NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity. p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O2- release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O2--dependent activation of nuclear factor κB occurred in these primed cells. Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity.
Kazuhito Rokutan, Shigetada Teshima, Mami Miyoshi, Tomoko Kawai, Takeshi Nikawa and Kyoichi Kishi : Glutathione depletion inhibits oxidant-induced activation of nuclear factor-kappa B,AP-1,and c-Jun/ATF-2 in cultured guinea-pig gastric epithelial cells, Journal of Gastroenterology, Vol.33, No.5, 646-655, 1998.
(要約)
The aim of this study was to reveal the role of intracellular glutathione in the oxidative stress responses of gastric epithelial cells. Metabolic radiolabeling with L-[35S]methionine and analysis of synthesized proteins by gel electrophoresis and fluorography showed that upon exposure to hydrogen peroxide (H2O2) or diamide, primary cultures of guinea-pig gastric epithelial cells rapidly induced several undefined proteins, as well as heat shock proteins. When intracellular glutathione was depleted to less than 10% of the control value by treatment with buthionine-[S,R]-sulfoximine, these inductions were completely inhibited. Gel mobility shift assay demonstrated that H2O2 and diamide rapidly activated nuclear factor-kappa B (NF-kappaB), and diamide activated activator protein (AP)-1, and c-Jun/activating transcription factor (ATF)-2, suggesting that the response may be coupled to these reduction-oxidation (redox)-sensitive transcription factors, as well as heat shock transcription factor 1. The activations of NF-kappaB, AP-1, and c-Jun/ATF-2 by the oxidants did not occur in glutathione-depleted cells. Northern blot analysis showed that glutathione depletion markedly or completely suppressed the diamide-induced expression of c-fos and c-jun mRNAs. These results suggest that intracellular glutathione redox may participate in the initiation of oxidative stress responses; thereby, it plays an important role in gastric mucosal defense.
Shigetada Kondo, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Cultured guinea pig gastric mucosal cells produce abundant superoxide anion through an NADPH oxidase-like system., Gastroenterology, Vol.115, No.5, 1186-1196, 1998.
(キーワード)
superoxide / gastoric cells / host defense
114.
Takeshi Nikawa, Kazuhito Rokutan, Kayo Nanba, Kaori Tokuoka, Shigetada Teshima, Michael J Engle, David H Alpers and Kyoichi Kishi : Vitamin A Up-Regulates Expression of Bone-Type Alkaline Phosphatase in a Rat Small Intestinal Crypt Cell Line and Fetal Rat Small Intestine, Biochemical and Molecular Roles of Nutrients, Vol.128, 1869-1877, 1998.
(キーワード)
vitamin A / liver/bone/kidney alkaline phosphatase / IEC-6 cells / retinoid receptors / fetal rat small intestine
115.
Kazuhito Rokutan, Shigetada Teshima, Mami Miyoshi, Takeshi Nikawa and Kyoichi Kishi : Oxidant-lnduced Activation of Nuclear Factor-Kappa B in Cultured Guinea Pig Gastric Epithelial Cells, Digestive Diseases and Sciences, Vol.42, No.9, 1880-1889, 1997.
Tetsuya Hirakawa, Kazuhito Rokutan, Takeshi Nikawa and Kyoichi Kishi : Geranylgeranylacetone induces heat shock proteins. in cultured guinea pig gastric mucosal cells and rat gastric mucosa, Gastroenterology, Vol.111, No.2, 345-357, 1996.
117.
Shigetada Teshima, Kazuhito Rokutan, Masayuki Takahashi, Takeshi Nikawa and Kyoichi Kishi : Induction of heat shock proteins and their possible roles in macrophages during activation by macrophage colony-stimulating factor, The Biochemical Journal, Vol.315, No.Pt 2, 497-504, 1996.
Arinobu Yamauchi, Fujiko Shizuka, Takashi Yamamoto, Takeshi Nikawa, Yasuhiro Kido, Kazuhito Rokutan and Kyoichi Kishi : Amino acids and glucose differentially increased extracellular 5-hydroxyindoleacetic acid of the rat brain., Journal of Nutritional Science and Vitaminology, Vol.41, No.3, 325-340, 1995.
(キーワード)
amino acid / serotonin / 5-HIAA / brain / glucose
119.
Takeshi Nikawa, A W. Schulz, E C. Vandenbrink, M Hanusch, P Vandersaag, W Stahl and H Sies : Efficacy of All-trans-β-Carotene, Canthaxanthin, and All-trans, 9-cis-, and 4-Oxoretinoic Acids in Inducing Differentiation of an F9 Embryonal Carcinoma RARβ-lacZ Reporter Cell Line, Archives of Biochemistry and Biophysics, Vol.316, No.2, 665-672, 1995.
120.
Shigetada Teshima, Kazuhito Rokutan, Masayuki Takahashi, Takeshi Nikawa, Yasuhiro Kido and Kyoichi Kishi : Alteration of the Respiratory Burst and Phagocytosis of Macrophages under Protein Malnutrition, Journal of Nutritional Science and Vitaminology, Vol.41, No.1, 127-137, 1995.
Takeshi Nikawa, G Schuch, G Wagner and H Sies : Interaction of ebselen with glutathione S-transferase and papain in vitro, Biochemical Pharmacology, Vol.47, No.6, 1007-1012, 1994.
(要約)
The interaction of ebselen(2-phenyl-1,2-benzisoselenazol-3(2H)-one) with rat liver cytosolic glutathione S-transferases (GSTs) and the plant cysteine protease, papain, was studied as cysteine residues are important for the activity of these enzymes. The capacity of GST 1-2 and 3-4 for ebselen binding is similar (1.5 mol ebselen/mol GST isozyme), while GST 2-2 and GST 7-7 bind 0.3 and more than 2.0 mol ebselen/mol GST isozyme, respectively. Ebselen does not bind to N-ethylmaleimide-treated GST, and its binding to GST is prevented by 5 mM thiols. Ebselen irreversibly inactivates the different GST isozymes with a second order rate constant ranging from 20 to 2250 M-1 sec-1 for the different subunits. GST inhibition by ebselen is partially restored by 5 mM thiols. Ebselen binds to untreated papain and to cysteine-treated papain at a ratio of about 0.1 and 0.75 mol ebselen/mol papain, respectively. Ebselen does not bind to N-ethylmaleimide-treated papain, and its binding to papain is interfered with by added thiols. Papain is inactivated by ebselen with a second order rate constant of 1800 M-1 sec-1 in the absence of thiols. However, in the presence of GSH, 2-mercaptoethanol or sodium borohydride, ebselen exerts an activating effect on papain. The binding of ebselen by a seleno-sulfide bond to cysteine residues of GSTs and papain leads to their inactivation.
Takeshi Nikawa, G Schuch, G Wagner and H Sies : Interaction of albumin-bound ebselen with rat liver glutathione S-transferase and microsomal proteins, Biochemistry and Molecular Biology International, Vol.32, No.2, 291-298, 1994.
(要約)
Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one) is a selenium-containing heterocyclic compound, transported bound to albumin in blood. In this report, we demonstrate the transfer of ebselen from its albumin complex to rat liver cytosolic glutathione-S-transferase (GST) and microsomal proteins, as compared to papain. Free ebselen binds to the sulfhydryl groups of proteins, such as GST and papain [Nikawa, T., Schuch, G., Wagner, G. & Sies, H. Biochem. Pharmacol. (in press)]. 2-Mercaptoethanol and N-ethylmaleimide interfere with ebselen binding to microsomal proteins, suggesting that ebselen also binds to the sulfhydryl groups of microsomal proteins. Ebselen is transferred from its albumin complex to rat liver cytosolic GST and microsomal proteins, but not to papain. These results suggest the possibility that ebselen is transferred from albumin to other proteins using their sulfhydryl groups in vivo.
Hisao Kakegawa, Takeshi Nikawa, Kahori Tagami, Hiroshi Kamioka, Koji Sumitani, Terushige Kawata, Marinka Drobnic-kosorok, Brigita Lenarcic, Vito Turk and Nobuhiko Katunuma : Participation of cathepsin L on bone resorption, FEBS Letters, Vol.321, No.2-3, 247-250, 1993.
124.
Takeyuki Ohshita, Takeshi Nikawa, Takae Towatari and Nobuhiko Katunuma : Effects of selective inhibition of cathepsin B and general inhibition of cysteine proteinases on lysosomal proteolysis in rat liver in vivo and in vitro, European Journal of Biochemistry, Vol.209, No.1, 2237-231, 1992.
Takeshi Nikawa, Takae Towatari and Nobuhiko Katunuma : Purification and characterization of cathepsin J from rat liver, European Journal of Biochemistry, Vol.204, No.1, 381-393, 1992.
127.
Takae Towatari, Takeshi Nikawa, Mitsuo Murata, Chihiro Yokoo, Masaharu Tamai, Kazunori Hanada and Nobuhiko Katunuma : Novel epoxysuccinyl peptides A selective inhibitor of cathepsin B, in vivo, FEBS Letters, Vol.280, No.2, 311-315, 1991.
Takeshi Nikawa, Takae Towatari, Yoshimasa Ike and Nobuhiko Katsunuma : Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants, FEBS Letters, Vol.255, No.2, 309-314, 1989.
Takeshi Nikawa, ANAYT ULLA and Iori Sakakibara : Polyphenols and Their Effects on Muscle Atrophy and Muscle Health, Molecules, Vol.26, No.16, 4887, Aug. 2021.
(要約)
Skeletal muscle atrophy is the decrease in muscle mass and strength caused by reduced protein synthesis/accelerated protein degradation. Various conditions, such as denervation, disuse, aging, chronic diseases, heart disease, obstructive lung disease, diabetes, renal failure, AIDS, sepsis, cancer, and steroidal medications, can cause muscle atrophy. Mechanistically, inflammation, oxidative stress, and mitochondrial dysfunction are among the major contributors to muscle atrophy, by modulating signaling pathways that regulate muscle homeostasis. To prevent muscle catabolism and enhance muscle anabolism, several natural and synthetic compounds have been investigated. Recently, polyphenols (i.e., natural phytochemicals) have received extensive attention regarding their effect on muscle atrophy because of their potent antioxidant and anti-inflammatory properties. Numerous in vitro and in vivo studies have reported polyphenols as strongly effective bioactive molecules that attenuate muscle atrophy and enhance muscle health. This review describes polyphenols as promising bioactive molecules that impede muscle atrophy induced by various proatrophic factors. The effects of each class/subclass of polyphenolic compounds regarding protection against the muscle disorders induced by various pathological/physiological factors are summarized in tabular form and discussed. Although considerable variations in antiatrophic potencies and mechanisms were observed among structurally diverse polyphenolic compounds, they are vital factors to be considered in muscle atrophy prevention strategies.
Furukawa Satoshi, Chatani Masahiro, Higashitani Atsushi, Akira Higashibata, Kawano Fuminori, Takeshi Nikawa, Numaga-Tomita Takuro, Ogura Toshihiko, Sato Fuminori, Sehara-Fujisawa Atsuko, Shinohara Masahiro, Shimazu Toru, Takahash Satoru and Watanabe-Takano Haruko : Findings from recent studies by the Japan Aerospace Exploration Agency examining musculoskeletal atrophy in space and on Earth, NPJ Microgravity, Vol.7, No.1, 18, May 2021.
(要約)
The musculoskeletal system provides the body with correct posture, support, stability, and mobility. It is composed of the bones, muscles, cartilage, tendons, ligaments, joints, and other connective tissues. Without effective countermeasures, prolonged spaceflight under microgravity results in marked muscle and bone atrophy. The molecular and physiological mechanisms of this atrophy under unloaded conditions are gradually being revealed through spaceflight experiments conducted by the Japan Aerospace Exploration Agency using a variety of model organisms, including both aquatic and terrestrial animals, and terrestrial experiments conducted under the Living in Space project of the Japan Ministry of Education, Culture, Sports, Science, and Technology. Increasing our knowledge in this field will lead not only to an understanding of how to prevent muscle and bone atrophy in humans undergoing long-term space voyages but also to an understanding of countermeasures against age-related locomotive syndrome in the elderly.
Tomoki Abe, Katsuya Hirasaka and Takeshi Nikawa : Involvement of Cbl-b-mediated macrophage inactivation in insulin resistance., World Journal of Diabetes, Vol.8, No.3, 97-103, Mar. 2017.
(要約)
Aging and overnutrition cause obesity in rodents and humans. It is well-known that obesity causes various diseases by producing insulin resistance (IR). Macrophages infiltrate the adipose tissue (AT) of obese individuals and cause chronic low-level inflammation associated with IR. Macrophage infiltration is regulated by the chemokines that are released from hypertrophied adipocytes and the immune cells in AT. Saturated fatty acids are recognized by toll-like receptor 4 (TLR4) and induce inflammatory responses in AT macrophages (ATMs). The inflammatory cytokines that are released from activated ATMs promote IR in peripheral organs, such as the liver, skeletal muscle and AT. Therefore, ATM activation is a therapeutic target for IR in obesity. The ubiquitin ligase Casitas b-lineage lymphoma-b (Cbl-b) appears to potently suppress macrophage migration and activation. Cbl-b is highly expressed in leukocytes and negatively regulates signals associated with migration and activation. Cbl-b deficiency enhances ATM accumulation and IR in aging- and diet-induced obese mice. Cbl-b inhibits migration-related signals and SFA-induced TLR4 signaling in ATMs. Thus, targeting Cbl-b may be a potential therapeutic strategy to reduce the IR induced by ATM activation. In this review, we summarize the regulatory functions of Cbl-b in ATMs.
Yoshihiro Sakashita, 内田 貴之, 二川 健 : [Impacts of physical exercise on remodeling and hypertrophy of skeletal muscle.], Clinical Calcium, Vol.27, No.1, 79-85, 2017年.
(要約)
The skeletal muscle has high sensitivity for the mechanical stress. Because it is enlarged by training, whereas it is easily withered by lack of exercise. When we exercise, skeletal muscle cells per se sense mechanical loading, and muscular remodeling and the muscular hypertrophy occur. It has been revealed that the intracellular signaling through PGC-1 participates in the remodeling of the skeletal muscle, while PGC-14, an isoform of PGC-1, and the dystrophin-glycoprotein complex play important roles in muscular hypertrophy. This review describes the impact of physical exercise gives on the remodeling and hypertrophy of muscle through the signaling.
Recently, muscle atrophy caused by unloading, such as spaceflight and bed rest has been becoming a social problem in Japan. However, the effective countermeasures against these disuse atrophy have not been developed. We have reviewed the mechanisms of disuse atrophy and its possible countermeasures.
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 28232649
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.
Chika Ikeda, Tomoki Abe, Atsuko Sakai, Katsuya Hirasaka and Takeshi Nikawa : [Space flight/bedrest immobilization and bone. Space flight and bed rest-mediated muscle atrophy]., Clinical Calcium, Vol.22, No.12, 1813-1820, Dec. 2012.
(要約)
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.
(キーワード)
Bed Rest / Bone and Bones / Humans / Immobilization / Muscle, Skeletal / Muscular Atrophy / Space Flight
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 23187073
近藤 茂忠, Ochi Arisa, Shohei Kohno, 安倍 知紀, Kenro Utsunomiya, Hikaru Nagano, Takuro Suto, Chisato Tomida, Naoko Yamagishi, Katsuya Hirasaka, 真板 綾子, Yushi Okumura, 二川 健 : [Space flight/bedrest immobilization and bone. Development of inhibitors for atrophy caused by unloading stress]., Clinical Calcium, Vol.22, No.12, 1879-1885, 2012年12月.
(要約)
Muscle atrophy caused by unloading stress is a serious problem in bed rest patients or astronauts. In our previous studies, we revealed that induction and activation of ubiquitin ligase Cbl-b played an important role in skeletal muscle atrophy caused by unloading stress. Under muscle atrophy conditions, Cbl-b interacted with and degraded IRS-1 (insulin receptor substrate 1) that is a central molecule in the IGF-1 signaling pathway. In addition, we developed a Cbl-b inhibitor (Cblin) that a pentapeptide mimetic of tyrosin608-phosphorylated IRS-1, DGpYMP. This Cblin peptide inhibited Cbl-b mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of MAFbx/atrogin-1. We are further developing Cbl-b inhibitors that are more effective than an original Cblin peptide.
(キーワード)
Bed Rest / Bone and Bones / Humans / Muscular Atrophy / Space Flight / Stress, Mechanical / Stress, Physiological
(文献検索サイトへのリンク)
● PubMed @ National Institutes of Health, US National Library of Medicine (PMID): 23187081
Kazuhito Rokutan, Tetsuya Hirakawa, Shigetada Teshima, Yoko Nakano, Mami Miyoshi, Tomoko Kawai, Emi Konda, Hiroko Morinaga, Takeshi Nikawa and Kyoichi Kishi : Implications of heat shock/stress proteins for medicine and disease, The Journal of Medical Investigation : JMI, Vol.44, No.3-4, 137-147, Feb. 1998.
(要約)
Heat shock/stress proteins (HSPs) are crucial for maintenance of cellular homeostasis during normal cell growth and for survival during and after various cellular stresses. The HSP70 family functions as molecular chaperones and reduces stress-induced denaturation and aggregation of intracellular proteins. In addition to the chaperoning activities, HSP70 has been suggested to exert its protective action by protecting mitochondria and by interfering with the stress-induced apoptotic program. The biochemical and functional properties of HSPs observed in cultured cells may be relevant to organs and tissues in whole animals. The activation of the hypothalamic-pituitary-adrenal axis and the sympathetic nerve system elicits the stress response in selected peripheral tissues; the HSP70 expression in the vasculature and stomach increases resistance against hemodynamic stress and stress-induced mucosal damage, respectively. Gastric mucosa pretreated with mild irritants acquires a tolerance against subsequent mucosal-damaging insults. This phenomenon is known as "adaptive cytoprotection". Transient ischemia also induces ischemic tolerance in the brain and heart, which is called "ischemic preconditioning". The heat shock response is believed to contribute to the acquisition of the tolerance. The therapeutic applications of chaperone inducers that induce HSPs without any toxic effect are also introduced.
Takeshi Nikawa : Our space biomedical experiments:From Myolab to Cell Gravisencing, 12th KoSBA Symposium 2024, Jan. 2024.
2.
Akira Takahashi, Katsuyuki Miyawaki, Kazuaki Mawatari, Takeshi Nikawa, Mutsumi Aihara, Fukushima Shiho, Akizawa Shinta, Yamashita Michiyo and Koi Yumena : Development of closed-circulation soybean cultivation system applicable to extreme environments, The 3rd Japan-France International Symposium on Space Nutrition/Medicine, Kyoto, Nov. 2023.
3.
Takayuki Uchida, Miki Yukari, Katsuya Hirasaka, Sato Takahiko, Teranishi Mika, Ishihara Naotada, Kobayashi Takeshi, Higashitani Atsushi, Sokabe Masahiro and Takeshi Nikawa : The elucidation of mechanism by MFN2-mediated induction of disuse muscle atrophy, International Symposium on Mechanobiology for Human Health:8 years progress in The AMED-CREST/PRIME project on mechanobiplogy, Mar. 2023.
4.
Takeshi Nikawa, Takata Miho, Kosuke Sugiura, Takayuki Uchida, Iori Sakakibara, Madoka Uezumi and Akiyoshi Uezumi : Functional analysis of Aconitase2 in fetal skeletal muscle, International Symposium on Mechanobiology for Human Health:8 years progress in The AMED-CREST/PRIME project on mechanobiplogy, Mar. 2023.
5.
Hitoshi Takagi, Antonio Norio Nakagaito, Kawakami Nozomi, Akira Takahashi and Takeshi Nikawa : Isolation of cellulose nanofibers from soybean waste, The 9th International Forum on Advanced Technologies and The 4th Japan-Taiwan International Engineering Forum (IFAT & JTIEF 2023), 50033_1-50033_2, Taipei, Mar. 2023.
6.
二川 健 : Food function on diseases, 第22回国際栄養学会議, 2022年12月.
7.
Takeshi Nikawa : Anti-muscle Atrophic Effects of Dietary Cricket Proteins, International Symposium on Research and Development for Future Food and Health in Moonshot Project, Nov. 2022.
8.
Takeshi Nikawa : Relevance of Protein Nutrition on the Moon, Joint Symposium : 33rd ISTS & 10th NSAT & 14th IAA LCPM, Mar. 2022.
9.
Takeshi Nikawa : Establishing an international institute for the development of next-generation space nutrition researchers, The 3rd International Moon Village Workshop & Symposium, Kyoto, Dec. 2019.
10.
Takeshi Nikawa : Functional space foods improve the QOL in moon village, The 3rd International Moon Village Workshop & Symposium, Tokyo, Dec. 2019.
11.
(名) Anaytulla, Takayuki Uchida, Miki Yukari, Sugiura Kohsuke, Ohno Ayako, Reiko Nakao and Takeshi Nikawa : Supplementation of Morin attenuates oxidative stress and muscle wasting in mouse C2C12 skeletal myotubes treated with dexamethasone, The 7th International Conference on Food Factors(ICoFF2019)/The 12th International Conference and Exhibition on Nutraceuticals and Functional Foods (ISNFF2019), Kobe, Dec. 2019.
12.
Takayuki Uchida, Sakashita Yoshihiro, Katsuya Hirasaka, Ohno Ayako, Reiko Nakao, Akira Higashibata, Kobayashi Takeshi, Sokabe Masahiro and Takeshi Nikawa : The influence of skeletal muscle thermogenesis on the regulation of body temperature during ketogenic diet-induced hypothermia, ADVANCED BIOMEDICAL ENGINEERING AND INSTRUMENTATION SUMMIT, San Francisco, Jun. 2019.
13.
Yukiko Tomioka, Hidehiro Umehara, Shinya Watanabe, Masahito Nakataki, Masuda Rumiko, Kazuaki Mawatari, Takeshi Nikawa, Akira Takahashi, Shusuke Numata and Tetsuro Ohmori : Altered plasma metabolites related to one-carbon metabolism in schizophrenia., WFSBP Asia Pacific Regional Congress of Biological Psychiatry, Kobe, Sep. 2018.
14.
Takeshi Nikawa, Takayuki Uchida, Ayako Maita and Reiko Nakao : Molecular mechanism and nutritional approach for unloading-mediated muscle atrophy, 2018 International Conference on Functional Food for improving Sarcopenia, Cachexia and Frailty, Mar. 2018.
15.
Takeshi Nikawa : Unloading induces reactive oxygen species associated signal transduction toward atrophy in skeletal muscle cells, 3rd International Symposium on Mechanobiology, Dec. 2017.
16.
Takeshi Nikawa : Unloading induces reactive oxygen species associated signal transduction toward atrophy in skeletal muscle cells, 14th Japan-Korea Joint Seminar on Space Environment Utilization Research, Aug. 2017.
17.
Tsugita Saki, Katsuya Hirasaka, Haruna Marie, M.Mills Edward, Bando Aki, Tomoki Abe, Shigetada Kondo, Yuushi Okumura and Takeshi Nikawa : Elucidation of the interaction between UCP3 and Hax-1, 11th Asian Microgravity Symposium, Oct. 2016.
18.
Takeshi Nikawa, Hashimoto Rie, Katsuya Hirasaka, Yano Chiaki, Aibara Kana, Takayuki Uchida, Tomoki Abe, Ayako Maita and Akama Kazuhito : Effects of dietary soy protein on unloading mediated muscle atrophy, 11th Asian Microgravity Symposium, Oct. 2016.
19.
Takayuki Uchida, Tomoki Abe, Ayako Maita, Reiko Nakao, Higashitani Atsushi, Kobayashi Takeshi, Sokabe Masahiro, Akira Higashibata and Takeshi Nikawa : Microgravity-induced signal transduction in skeletal muscle cells, 11th Asian Microgravity Symposium, Oct. 2016.
20.
Takeshi Nikawa : Microgravity-induced signal transduction in skeletal muscle cells, 13th Japan-Korea Joint Seminar on Space Environment Utilization Research, Oct. 2016.
21.
Ayako Maita, Tsugita Saki, Katsuya Hirasaka, Haruna Marie, Ikeda Chika, Akagi Ken-ichi, Tochio Hideto, Shigetada Kondo, Yuushi Okumura and Takeshi Nikawa : Calcium-Dependent Interactive between UCP3 and Hax-1, The 27th International Conference on Msgnetic Resonance in Biological Systems, Aug. 2016.
22.
Takeshi Nikawa, Hashimoto Rie, Katsuya Hirasaka, Yano Chiaki, Aibara Kana, Ayako Maita and Akama Kazuhito : Effects of dietary soy protein on skeletal muscle volume and strength in humans with various physical activities, Molecular Mechanisms Modulating Skeletal Muscle Development and Homeostasis in Health and Disease, Jun. 2016.
23.
Uchida Takayuki and Takeshi Nikawa : Nutritional Approarch to prevent unloading-mediated muscle atrophy, 12th Japan-Korea Joint Seminar on Space Environment Utilization Research, 韓国, Oct. 2015.
24.
Tomoki Abe and Takeshi Nikawa : Capric scid up-regulates UCP3 expression without PDK4 induction in mouse C2C12 myotubes, 44th European Muscle Conference, Sep. 2015.
25.
Takagi Marina and Takeshi Nikawa : A novel functional rice against skeletal muscle atrophy caused by unloading, 44th European Muscle Conference, Sep. 2015.
26.
Takeshi Nikawa, Katsuya Hirasaka and Yuushi Okumura : Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading, 30th International Symposium on Space Technology and Science, Jul. 2015.
27.
Shigetada Kondo, Yano Chiaki, Yamagishi Naoko, Tomida Chisato, Aibara Kana, Tomoki Abe, Ayako Maita and Takeshi Nikawa : Chemopreventive effects of polyphenols on malignant phenotypes induced by chronic inhibition of VEGF in colorectal cancer cells, 12th Asian Congress of Nutrition, May 2015.
28.
Takagi Marina, Ochi Arisa, Kitahata Kanako, Kishimoto Hikaru, Nakao Reiko, Ayako Maita, Shigetada Kondo, Tomoki Abe, Hisao Nemoto and Takeshi Nikawa : N-Myristoylated ubiquitin ligase Cbl-b inhibitor prevents on glucocorticoid-induced atrophy in mouse skeletal muscle, 12th Asian Congress of Nutrition, Vol.570, No.15, 23-31, May 2015.
Tomoki Abe, Katsuya Hirasaka, Takayuki Uchida, Marina Takagi, Ayako Ohno, Shigetada Kondo and Takeshi Nikawa : Cbl-b Is a Critical Regulator of Macrophage Activation Associated With Obesity-Induced Insulin Resistance in Mice, 12th Asian Congress of Nutrition, Vol.62, No.6, 1957-1969, May 2015.
Uchida Takayuki, Takeshi Nikawa, Tomoki Abe, Kohno Shohei, Katsuya Hirasaka, Ayako Maita, Shigetada Kondo, Yuushi Okumura and Mills M. Edward : Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading, 12th Asian Congress of Nutrition, May 2015.
31.
Rie Mukai, Naoko Matsui, Takeshi Nikawa and Junji Terao : Dietary quercetin prevents disuse muscle atrophy of denervated mice through regulation of mitochondrial biogenesis, 12th Asian congress of Nutrition, Yokohama, May 2015.
32.
Nobuhiko Kawai, Katsuya Hirasaka, Chieko Shiota, Takeshi Nikawa and Eiji Tanaka : Effectiveness Of Myostatin Knockdown On Jaw Muscle Activity, 2014 IADR/PER Congress, Dubrovnik, Croatia., Sep. 2014.
33.
Rie Mukai, Naoko Matsui, Takeshi Nikawa and Junji Terao : Pre-intake of quercetin slow the progression of disuse muscle atrophy by mitochondrial dysfunction., XXVIIth International Conference on Polyphenols &8th Tannin Conference, Nagoya, Sep. 2014.
34.
Tomoki Abe, Utsunomiya Kenro, Yuushi Okumura, Ayako Maita, Shigetada Kondo and Takeshi Nikawa : Inhibition of myostatin activation prevents dexamethasone-induced muscle atrophy in C2C12 myotubes., The 43rd European Muscle Conference, Sep. 2014.
35.
Rie Mukai, Naoko Matsui, N Matsumoto, N.T. Dang, Takeshi Nikawa, Hisao Nemoto and Junji Terao : Anti-Oxidative Flavonoid suppresses disuse muscle atrophy in denervation mice., XXVIIth International Conference on Polyphenols &8th Tannin Conference., 2014.
36.
Takeshi Nikawa, Arisa Ochi and Tomoki Abe : Anti-muscle atrophy peptides/proteins, 10th Japan-Korea Joint Seminar on Space Environment Utilization Research, Seoul, Sep. 2013.
37.
Marie Haruna, Katsuya Hirasaka, Aki Bando, Ayako Maita, Shigetada Kondo and Takeshi Nikawa : The role of uncoupling protein 3 regulating calcium ion uptake into mitochondria during sarcopenia, 10th Japan-Korea Joint Seminar on Space Environment Utilization Research, Seoul, Sep. 2013.
38.
Tomki Abe, Shohei Kohno, Katsuya Hirasaka, Ayako Maita, Shigetada Kondo, Yuushi Okumura and Takeshi Nikawa : Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages, The 29th International Symposium on Space Technology and Science, Nagoya, Jun. 2013.
39.
Arisa Ochi, Katsuya Hirasaka, Ayako Maita, Shigetada Kondo, Tomoyuki Kawamura, Hisao Nemoto, Rie Mukai, Junji Terao, Taesik Gwang, Inho Choi and Takeshi Nikawa : Development of anti-ubiquitination oligopeptide, Cblin:Cbl-b inhibitor that prevents unloading-induced muscle atorophy., The 29th International Symposium on Space Technology and Science, Nagoya, Jun. 2013.
40.
Takeshi Nikawa : Unloading-mediated signal transducation in skeletal muscle cells, The 22th International Rheumatology Symposium, kyoto, Apr. 2013.
41.
Rie Mukai, Takeshi Nikawa and Junji Terao : Antioxidative flavonoid quercetin suppresses disused muscle atrophy, Society for Free Radical Research International, London, Sep. 2012.
Katsuya Hirasaka, Edward M Mills, Kohno Shohei, Abe Tomoki, Shigetada Kondo, Ayako Maita, Yuushi Okumura and Takeshi Nikawa : Thioredoxin 2 Binding to Uncoupling Protein 3 is Processed by Inner Membrane Peptidase in the Mitochondrial Intermembrane Space and Attenuates Generation of Reactive Oxygen Species, 7th General Meeting of the International Proteolisis Society, 米国, Oct. 2011.
45.
Abe Tomoki, Katsuya Hirasaka, Kohno Shohei, Ochi Arisa, Yamashita Yui, Ayako Maita, Shigetada Kondo, Yuushi Okumura and Takeshi Nikawa : Cbl-b is a Crtical Regulator of Macrophage Activation Associated with Obesity-Induced Insulin Resistance in Mice, 7th General Meeting of the International Proteolisis Society, 米国, Oct. 2011.
46.
Kohno Shohei, Shigetada Kondo, Katsuya Hirasaka, Ayako Maita, Yuushi Okumura, Rie Mukai, Junji Terao, Akira Higashibata and Takeshi Nikawa : Regulation of the Gene Expression of Cbl-b Ubiquitin Ligase in Skeletal Muscle During Unloading Conditions., 7th General Meeting of the International Proteolisis Society, 米国, Oct. 2011.
47.
Yamashita Yui, Kohno Shohei, Abe Tomoki, Ayako Maita, Shigetada Kondo, Katsuya Hirasaka, Yuushi Okumura and Takeshi Nikawa : Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages, 40th European Muscle Conference, ドイツ, Sep. 2011.
48.
Ochi Arisa, Nakao Reiko, Ueji Tatsuya, Katsuya Hirasaka, Ayako Maita, Shigetada Kondo, Yuushi Okumura, Nagano Keisuke, Hisao Nemoto, Akama Kazuhito and Takeshi Nikawa : Development of anti-ubiquitination oligopeptide, Cblin:Cbl-b inhibitor that prevents unloading-induced skeletal muscle atrophy., 40th European Muscle Conference, ドイツ, Sep. 2011.
Tatsuya Ueji, Ayumi Shirakata, Shigetada Kondo, Keisuke Nagano, Ayako Maita, Nobuo Maita, Yuushi Okumura and Takeshi Nikawa : Crystal structure of Cbl-b TKB domain in complex with Cblin (Cbl-b inhibitor), Acta Crystallographica Section A, Vol.67, No.supplement, C304-C305, Madrid, Aug. 2011.
51.
Takeshi Nikawa, Nakao Reiko, Katsuya Hirasaka, Akira Higashibata, Inho Choi, Asashima Makoto, Edward M Mills and Yuushi Okumura : Molecular mechanism of unloading-induced muscle atrophy and development of its countermeasures, 2nd Annual Meeting of the Korean Microgravity Society,2011, Korea, May 2011.
52.
Rie Mukai, Takeshi Nikawa, Hisao Nemoto, Hironori Yamamoto, Eiji Takeda, Yoshichika Kawai and Junji Terao : Disuse muscle atrophy is suppressed by antioxidative flavonoid quercetin, International Symposium on Free Radical Research: Contribution to Medicine, Kyoto, Jan. 2011.
53.
Rie Mukai, Tomoyuki Kawamura, Yutaka Fujikura, Moemi Hayashi, Hisao Nemoto, Takeshi Nikawa, Hironori Yamamoto, Eiji Takeda and Junji Terao : Disuse muscle atrophy is prevented by flavonoids, 2010 International Chemical Congress of Pacific Basin Societies, Honolulu, Dec. 2010.
Takeshi Nikawa : Interim report of MyoLab experiment onboard ISS and its projection to the future, 7th Korea-Japan Joint Seminar on Space Environment Utilization Research, Sep. 2010.
56.
Takeshi Nikawa : Ubiquitin ligase Cbl-b is a negative regulator for IGF-1 signaling during muscle atrophy caused by unloading, ユビキチンカンファレンス, Aug. 2010.
57.
Rie Mukai, Hitoshi Ashida, Takeshi Nikawa and Junji Terao : Nuclear accumulation of flavonol aglycone in cultured cells detected with a fluorescence microscope, 4th International Conference on Polyphenol and Health, Harrogate, United Kingdom, Dec. 2009.
58.
Yoshiko Nakae, K. Hirasaki, J. Goto, Takeshi Nikawa, Masayuki Shono, M. Yoshida and P.J. Stoward : SUBCUTANEOUS INJECTION OF EPIGALLOCATECHIN GALLATE INTO DYSTROPHIN-DEFICIENT MDX MICE AMELIORATES MUSCULAR DYSTROPHY, USA, Hawaii, Aug. 2006.
59.
Takeshi Nikawa, Madoka Ikemoto, Takako Kitano, Mihoko Kano, Shigetada Teshima, Kazuhito Rokutan, Kyoichi Kishi, Shinichi Takeda, Ikuya Nonaka, Kenneth M. Baldwin, Ryutaro Izumi and Takae Towatari : Space Shuttle flight enhances degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway, 2nd iInternational Society of Proteolysis, Munich, Geramany, Oct. 2001.
60.
Takeshi Nikawa, Madoka Ikemoto, Takako Kitano, Mihoko Kano, Shigetada Teshima, Kazuhito Rokutan, Kyoichi Kishi, Shinichi Takeda, Ikuya Nonaka, Kenneth M. Baldwin, Ryutaro Izumi and Takae Towatari : Speaceflight and unweighting enhance degradation of rat myosin heavy chain in association with activation of ubiquitin-proteasome pathway and antiozidative nutrient prevents muscle protein degradation in tail-suspended rats, 17th International Congress of Nutrition, Wien, Aug. 2001.
61.
Takeshi Nikawa, Madoka Ikemoto, Chiho Watanabe, Takako Kitano, Shigetada Teshima, Kazuhito Rokutan, Kyoichi Kishi, Shinichi Takeda, Ikuya Nonaka, Kenneth M. Baldwin and Ryutaro Izumi : Tail-suspension stimulates expression of muscle proteases and cysteine supplementation prevents loss of muscle weight in tail-suspended rats, 21th Annual Gravitational Physiology Meeting, Nagoya, Apr. 2000.
62.
Takeshi Nikawa, K Tokuoka, Shigetada Teshima, Madoka Ikemoto, Takae Towatari, David H. Alpers, Kazuhito Rokutan and Kyoichi Kishi : Synergistic effects of retinoic acid and IL-1 on expression of the bone-type alkaline phosphatase and retinoid receptors in small intestinal epithelial IEC-6 cells, Annual Meeting of the American Association of the American Gastroenterological Association, Orlando, May 1999.
63.
Takeshi Nikawa, Kazuhito Rokutan, Shigetada Teshima, David H. Alpers and Kyoichi Kishi : Inflammatory cytokines are involved in retinoic acid-mediated maturation of small intestinal epithelial cells, 12th Federation of Asian and Oseanian Biochemists and Molecular Biologists, Tokushima, Oct. 1996.
64.
Takeshi Nikawa, Shigetada Teshima, David H. Alpers, Kyoichi Kishi and Kazuhito Rokutan : Involvement of inflammatory cytokines in retinoic acid-mediated maturation of small intestinal epithelial cells, Annual Meeting of the American Gastroenterological Association, San Francisco, May 1996.
65.
Takeshi Nikawa, Atsuko Sakai, M Emgle, David H. Alpers, Kazuhito Rokutan and Kyoichi Kishi : Stimulatory action of retinoic acid on differentiation of rat small intestinal cells, Annual Meeting of the American Gastroenterological Association, San Diego, May 1995.
66.
Takeshi Nikawa, A W. Schulz, E C. Vandenbrink, M Hanusch, P Vandersaag, W Stahl and H Sies : Efficacy of all-trans-β-carotene, canthaxanthin, and all-trans, 9-cis-, and 4-oxoretinoic acids in Inducing differentiation of an F9 embryonal carcinoma RARβ-lacZ reporter cell line, 2nd International Congress of Vitammins and Biofactors, San Diego, Feb. 1995.
67.
Takeshi Nikawa, G Schuch, G Wagner and H Sies : Interaction of ebselen with glutathione S-transferase and papain in vitro, Annual Autumu Meeting of German Biochemical Society, Dusseldorf, Germany, Sep. 1993.
68.
Takeshi Nikawa : Ebselen : its pharmaceutical role as an antioxidative agent, ドイツ フライブルク大学招待講演, Freiburg, Germany, Jul. 1993.
69.
Takeshi Nikawa, Takae Towatari and Nobuhiko Katunuma : A novel lysosomal cysteine protease:low sensitivity for E-64, 8th International Congress on Proteolysis,, Munich, Germany, Nov. 1990.
二川 健 : 骨格筋萎縮に対抗する新たな栄養学的アプローチ:タンパク質分解から酸化ストレスへ Novel nutritional approarches against skeletal muscle atrophy: From proteolysis to oxdative stress, International Symposium on Dietary Antioxidants and Oxidative Stress in Health, 2015年8月.
153.
二川 健 : ビタミンDと筋肉, 第1回Neo Vitamin D Workshop学術集会, 2015年8月.
154.
Floriane Rudwill and Takeshi Nikawa : Consequences of a 21-d bed rest on lipid metabolism in humans and effect of protein supplementation tested as a countermeasure, 第1回Neo Vitamin D Workshop学術集会, Aug. 2015.
Takeshi Nikawa : Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading, International Symposium on Mechanobiology 2014, May 2014.
Takeshi Nikawa, Madoka Ikemoto, Chiho Watanabe, Takako Kitano, Keeneth M. Baldwin, Ryutaro Izumi, Ikuya Nonaka, Shigetada Teshima, Kazuhito Rokutan, Shinichi Takeda and Kyoichi Kishi : Development of effective space food against weightlessness-induced muscle atrophy, The Progress Report of Ground Research Announcement for Space Utilization, Dec. 2001.
9.
二川 健, 池本 円, 加納 美保子, 平坂 勝也, 武田 伸一, Keeneth M. Baldwin, 埜中 征哉, 石堂 一巳 : Effects of space shuttle flight on gene expresson in rat skeletal muscle, Biol Sei Space, Vol.15, No.3, 312-313, 2001年10月.