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Abstract Background Chronic renal failure induces bone mineral disorders and sarcopenia. Skeletal muscle affects other tissues, including bone, by releasing myokines. However, the effects of chronic renal failure on the interactions between muscle and bone remain unclear. Methods We investigated the effects of renal failure on bone, muscle, and myokines linking muscle to bone using a mouse 5/6 nephrectomy (Nx) model. Muscle mass and bone mineral density (BMD) were analysed by quantitative computed tomography 8 weeks after Nx. Results Nephrectomy significantly reduced muscle mass in the whole body (12.1% reduction, P

Chronic obstructive pulmonary disease (COPD) causes sarcopenia and osteoporosis. However, the mechanisms underlying muscle and bone loss as well as the interactions between muscle and bone in the COPD state remain unclear. Therefore, we herein investigated the effects of the COPD state on muscle and bone in mice intratracheally administered porcine pancreatic elastase (PPE). The intratracheal administration of PPE to mice significantly reduced trabecular bone mineral density (BMD), trabecular bone volume, trabecular number, cortical BMD and cortical area. It also significantly decreased grip strength, but did not affect muscle mass or the expression of myogenic differentiation-, protein degradation- or autophagy-related genes in the soleus and gastrocnemius muscles. Among the myokines examined, myostatin mRNA levels in the soleus muscles were significantly elevated in mice treated with PPE, and negatively related to grip strength, but not bone parameters, in mice treated with or without 2 U PPE in simple regression analyses. Grip strength positively related to bone parameters in mice treated with or without PPE. In conclusion, we showed that a PPE model of COPD in mice exerts dominant effects on bone rather than skeletal muscles. Increased myostatin expression in the soleus muscles of mice in the COPD state may negatively relate to a reduction in grip strength, but not bone loss.

Matrix vesicles (MtVs) are one of the extracellular vesicles (EVs) secreted by osteoblasts. Although MtVs have a classically-defined function as an initiator of ossification and recent findings suggest a role for MtVs in the regulation of bone cell biology, the effects of MtVs on bone repair remain unclear. In the present study, we employed collagenase-released EVs (CREVs) containing abundant MtVs from mouse osteoblasts. CREVs were administered locally in gelatin hydrogels to damaged sites after a femoral bone defect in mice. CREVs exhibited the characteristics of MtVs with a diameter

Abstract Background Osteoblasts and osteoclasts play important roles during the bone remodeling in the physiological and pathophysiological states. Although angiopoietin family Angiopoietin like proteins (Angptls), including Angptl1, have been reported to be involved in inflammation, lipid metabolism and angiogenesis, the roles of Angptl1 in bone have not been reported so far. Methods We examined the effects of Angptl1 on the osteoblast and osteoclast phenotypes using mouse cells. Results Angptl1 significantly inhibited the osteoclast formation and mRNA levels of tartrate-resistant acid phosphatase and cathepsin K enhanced by receptor activator of nuclear factor κB ligand in RAW 264.7 and mouse bone marrow cells. Moreover, Angptl1 overexpression significantly enhanced Osterix mRNA levels, alkaline phosphatase activity and mineralization induced by bone morphogenetic protein-2 in ST2 cells, although it did not affect the expression of osteogenic genes in MC3T3-E1 and mouse osteoblasts. On the other hand, Angptl1 overexpression significantly reduced the mRNA levels of peroxisome proliferator-activated receptor γ and adipocyte protein-2 as well as the lipid droplet formation induced by adipogenic medium in 3T3-L1 cells. Conclusions The present study first indicated that Angptl1 suppresses and enhances osteoclast formation and osteoblastic differentiation in mouse cells, respectively, although it inhibits adipogenic differentiation of 3T3-L1 cells. These data suggest the possibility that Angptl1 might be physiologically related to bone remodeling.

Abstract In bone tissues, metabolic turnover through bone resorption by osteoclasts and bone formation by osteoblasts, termed bone remodeling, is strictly controlled and maintains homeostasis. Fibrinolytic factors are expressed in osteoclasts and osteoblasts, and are involved in bone remodeling through bone resorption and formation. The repair/regeneration process after bone injury is divided into the acute inflammatory, repair, and remodeling stages. Osteoblasts, osteoclasts, chondrocytes, and macrophages involved in the bone repair process originate from hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stem cells (MSCs) in the bone marrow. Therefore, stem cells in the bone marrow may be strongly influenced by bone injury. The urokinase-type PA (u-PA)/plasminogen (Plg) system functions in macrophage accumulation/phagocytosis through chemokines in the acute inflammatory stage, and Plg increases blood vessel-related growth factor expression, being involved in vascularization in mice. Plasminogen activator inhivitor-1 (PAI-1) causes bone loss and delayed bone repair through the inhibition of osteoblast differentiation in a drug-induced diabetes model in mice. Plg is considered to induce transforming growth factor-β (TGF-β) production in macrophages in the bone repair process, TGF-β release from the extracellular matrix through the activation of matrix metalloproteinase-9 (MMP-9), and stromal cell-derived factor-1 (SDF-1) expression in endosteal preosteoblasts, leading to the induction of bone marrow HSPCs in mice. Based on the above, establishment of a fibrinolytic factor-targeting method efficiently promoting bone repair/regeneration and fracture healing, and development of a new osteoporosis treatment method and diagnostic marker are awaited.

Serpinb1a, a serine protease inhibitor family protein, has been implicated in immunoregulation and several metabolic disorders, such as diabetes and obesity; however, its roles in bone remain unknown. Therefore, we herein investigated the physiological functions of Serpinb1a in osteoclastic and osteoblastic differentiation using mouse cell lines. Serpinb1a overexpression markedly reduced the number of tartrate-resistant acid phosphatase (TRAP)- and calcitonin receptor-positive multinucleated cells increased by receptor activator nuclear factor κB ligand (RANKL) in mouse preosteoclastic RAW 264.7 cells. Moreover, it significantly decreased the mRNA levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), TRAP and cathepsin K in these cells. Regarding osteoblasts, Serpinb1a overexpression significantly reduced the mRNA levels of alkaline phosphatase (ALP) and osteocalcin as well as ALP activity induced by bone morphogenetic protein-2 (BMP-2) in mouse mesenchymal ST2 cells, although it did not alter osteoblast differentiation in mouse osteoblastic MC3T3-E1 cells. Concerning the pathophysiological relevance of Serpinb1a, Serpinb1a mRNA levels were decreased in the soleus and gastrocnemius muscles of mice 4 weeks after bilateral sciatic nerve resection. In conclusion, we herein revealed for the first time that Serpinb1a inhibited osteoclast formation induced by RANKL in RAW 264.7 cells and suppressed BMP-2-induced ALP activity in ST2 cells.

The interactions between skeletal muscle and bone have been recently noted, and muscle-derived humoral factors related to bone metabolism play crucial roles in the muscle/bone relationships. We previously reported that extracellular vesicles from mouse muscle C2C12 cells (Myo-EVs) suppress osteoclast formation in mice. Although mechanical stress is included in extrinsic factors which are important for both muscle and bone, the detailed roles of mechanical stress in the muscle/bone interactions have still remained unknown. In present study, we examined the effects of fluid flow shear stress (FFSS) to C2C12 cells on the physiological actions of muscle cell-derived EV. Applying FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in mouse bone marrow cells in the presence of receptor activator nuclear factor κB ligand (RANKL). Moreover, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed mitochondria biogenesis genes during osteoclast formation with RANKL treatment. In addition, FFSS to C2C12 cells significantly enhanced muscle cell-derived EV-suppressed osteoclast formation and several osteoclast-related gene levels in Raw264.7 cells in the presence of RANKL. Small RNA-seq-analysis showed that FFSS elevated the expression of miR196a-5p and miR155-5p with the suppressive actions of osteoclast formation and low expression in mouse bone cells. On the other hand, muscle cell-derived EVs with or without FFSS to C2C12 cells did not affect the expression of osteogenic genes, alkaline phosphatase activity and mineralization in mouse osteoblasts. In conclusion, we first showed that FFSS to C2C12 cells enhances the suppressive effects of muscle cell-derived EVs on osteoclast formation in mouse cells. Muscle cell-derived EVs might be partly involved in the effects of mechanical stress on the muscle/bone relationships.

BackgroundTissue factor (TF) is the primary activator of the extrinsic coagulation protease cascade. Although TF plays roles in various pathological states, such as thrombosis, inflammatory diseases, cancer, and atherosclerosis, its involvement in bone metabolism remains unknown.Materials and methodsThe present study examined the roles of TF in delayed bone repair induced by a diabetic state in mice using wild-type (WT) and low TF-expressing (LTF) male mice. A diabetic state was induced by intraperitoneal injections of streptozotocin (STZ).ResultsA prolonged diabetic state significantly reduced total and trabecular bone mineral densities (BMD) as well as cortical bone thickness in WT and LTF mice; these BMD parameters were similar between WT and LTF mice treated with or without STZ. The diabetic state induced in WT mice delayed the repair of the femur following injury. The diabetic state induced in LTF mice was associated with further delays in bone repair. In in vitro experiments, TF significantly decreased receptor activator of nuclear factor-κB ligand-induced osteoclast formation and osteoclastogenic gene expression in RAW264.7 cells. However, it did not affect the gene expression levels of runt-related transcription factor 2 and osterix as well as alkaline phosphatase activity in mouse primary osteoblasts.ConclusionLow TF state was associated with enhanced bone repair delay induced by diabetic state in mice. The TF-induced suppression of bone remodeling may be a contributing factor to the protective effects of TF against delayed bone repair in a diabetic state.

Myonectin is a myokine, which is involved in the pathophysiology of diabetes and obesity, and various myokines are involved in the interactions between skeletal muscle and bone. However, roles of myonectin in bone have still remained unknown. We therefore examined the effects of myonectin on mouse osteoblast and osteoclast differentiation in vitro. Myonectin significantly suppressed the mRNA levels of osteogenic genes and alkaline phosphatase (ALP) activity in mouse osteoblasts. As for osteoclasts, myonectin significantly suppressed osteoclast formation as well as the mRNA levels of osteoclast-related genes enhanced by receptor activator nuclear factor κB ligand (RANKL) from mouse monocytic RAW264.7 cells. Moreover, myonectin significantly suppressed osteoclast formation from mouse bone marrow cells in the presence of macrophage-colony stimulating factor and RANKL. On the other hand, myonectin significantly suppressed RANKL-induced oxygen consumption rate and peroxisome proliferator-activated receptor γ coactivator-1β mRNA levels in RAW264.7 cells, although myonectin did not affect these mitochondrial biogenesis parameters in mouse osteoblasts. In conclusion, the present study demonstrated that myonectin suppresses the differentiation and ALP activity in mouse osteoblasts. Moreover, myonectin suppressed osteoclast differentiation from mouse bone marrow and RAW264.7 cells partly through an inhibition of mitochondrial biogenesis.

The vestibular system controls balance, posture, blood pressure, and gaze. However, the roles of the vestibular system in energy and glucose metabolism remain unknown. We herein examined the roles of the vestibular system in obesity and impaired glucose metabolism using mice with vestibular lesions (VL) fed a high-sucrose/high-fat diet (HSHFD). VL was induced by surgery or arsenic. VL significantly suppressed body fat enhanced by HSHFD in mice. Glucose intolerance was improved by VL in mice fed HSHFD. VL blunted the levels of adipogenic factors and pro-inflammatory adipokines elevated by HSHFD in the epididymal white adipose tissue of mice. A β-blocker antagonized body fat and glucose intolerance enhanced by HSHFD in mice. The results of an RNA sequencing analysis showed that HSHFD induced alterations in genes, such as insulin-like growth factor-2 and glial fibrillary acidic protein, in the vestibular nuclei of mice through the vestibular system. In conclusion, we herein demonstrated that the dysregulation of the vestibular system influences an obese state and impaired glucose metabolism induced by HSHFD in mice. The vestibular system may contribute to the regulation of set points under excess energy conditions.

Glucocorticoids delay fracture healing and induce osteoporosis. However, the mechanisms by which glucocorticoids delay bone repair have yet to be clarified. Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of plasminogen activators and an adipocytokine that regulates metabolism. We herein investigated the roles of macrophages in glucocorticoid-induced delays in bone repair after femoral bone injury using PAI-1-deficient female mice intraperitoneally administered with dexamethasone (Dex). Dex significantly decreased the number of F4/80-positive macrophages at the damaged site two days after femoral bone injury. It also attenuated bone injury-induced decreases in the number of hematopoietic stem cells in bone marrow in wild-type and PAI-1-deficient mice. PAI-1 deficiency significantly weakened Dex-induced decreases in macrophage number and macrophage colony-stimulating factor (M-CSF) mRNA levels at the damaged site two days after bone injury. It also significantly ameliorated the Dex-induced inhibition of macrophage phagocytosis at the damaged site. In conclusion, we herein demonstrated that Dex decreased the number of macrophages at the damaged site during early bone repair after femoral bone injury partly through PAI-1 and M-CSF in mice.

We previously revealed that stromal cell-derived factor-1 (SDF-1) is involved in the changes in the number of bone marrow stem cells during the bone repair process in mice. Moreover, we reported that plasminogen (Plg) deficiency delays bone repair and the accumulation of macrophages at the site of bone damage in mice. We investigated the roles of Plg in the changes in bone marrow stem cells during bone repair. We analyzed the numbers of hematopoietic stem cells (HSC) and mesenchymal stem cells (MSCs) within bone marrow from Plg-deficient and wild-type mice after a femoral bone injury using flow cytometric analysis. Plg deficiency significantly blunted a decrease in the number of HSCs after bone injury in mice, although it did not affect an increase in the number of MSCs. Plg deficiency significantly blunted the number of SDF-1- and Osterix- or SDF-1- and alkaline phosphatase-double-positive cells in the endosteum around the lesion as well as matrix metalloprotainase-9 (MMP-9) activity and mRNA levels of SDF-1 and transforming growth factor–β (TGF-β) elevated by bone injury. TGF-β signaling inhibition significantly blunted a decrease in the number of HSCs after bone injury. The present study showed that Plg is critical for the changes in bone marrow HSCs through MMP-9, TGF-β, and SDF-1 at the damaged site during bone repair in mice. Keywords: Plasminogen, Bone repair, Hematopoietic stem cells, Stromal cell-derived factor-1, Transforming growth factor-β

Abstract Background Subchondral osteopenia is important for the pathophysiology of osteoarthritis (OA). Although previous studies suggest that plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, is related to bone metabolism, its role in OA remains unknown. We therefore investigated the roles of PAI-1 in the subchondral bone in OA model mice. Methods Wild type (WT) and PAI-1-deficient (KO) mice were ovariectomized (OVX), and then destabilization of the medial meniscus (DMM) surgery was performed. Results DMM and OVX significantly decreased the trabecular bone mineral density of the subchondral bone evaluated by quantitative computed tomography in PAI-1 KO mice. The effects of OVX and/or PAI-1 deficiency on the OARSI score for the evaluation of the progression of knee degeneration were not significant. PAI-1 deficiency significantly augmented receptor activator nuclear factor κB ligand mRNA levels enhanced by IL-1β in mouse primary osteoblasts, although it did not affect osteoblast differentiation. Moreover, PAI-1 deficiency significantly increased osteoclast formation from mouse bone marrow cells. Conclusion We showed that PAI-1 deficiency accelerates the subchondral osteopenia after induction of OA in mice. PAI-1 might suppress an enhancement of bone resorption and subsequent subchondral osteopenia after induction of OA in mice.

Muscle and bone masses are elevated by the increased mechanical stress associated with body weight gain in obesity. However, the mechanisms by which obesity affects muscle and bone remain unclear. We herein investigated the roles of obesity and humoral factors from adipose tissue in the recovery phase after reloading from disuse-induced muscle wasting and bone loss using normal diet (ND)- or high fat diet (HFD)-fed mice with hindlimb unloading (HU) and subsequent reloading. Obesity did not affect decreases in trabecular bone mineral density (BMD), muscle mass in the lower leg, or grip strength in HU mice. Obesity significantly increased trabecular BMD, muscle mass in the lower leg, and grip strength in reloading mice over those in reloading mice fed ND. Among the humoral factors in epididymal and subcutaneous adipose tissue, leptin mRNA levels were significantly higher in reloading mice fed HFD than in mice fed ND. Moreover, circulating leptin levels were significantly higher in reloading mice fed HFD than in mice fed ND. Leptin mRNA levels in epididymal adipose tissue or serum leptin levels positively correlated with the increases in trabecular BMD, total muscle mass, and grip strength in reloading mice fed ND and HFD. The present study is the first to demonstrate that obesity enhances the recovery of bone and muscle masses as well as strength decreased by disuse after reloading in mice. Leptin may contribute to the recovery of muscle and bone enhanced by obesity in mice.

Mechanical unloading simultaneously induces muscle and bone loss, but its mechanisms are not fully understood. The interactions between skeletal muscle and bone have been recently noted. Although canonical wingless-related integration site (Wnt)/β-catenin signaling is crucial for bone metabolism, its roles in the muscle and bone interactions have remained unknown. Here, we performed comprehensive DNA microarray analyses to clarify humoral factors linking muscle to bone in response to mechanical unloading and hypergravity with 3 g in mice. We identified Dickkopf (Dkk) 2, a Wnt/β-catenin signaling inhibitor, as a gene whose expression was increased by hindlimb unloading (HU) and reduced by hypergravity in the soleus muscle of mice. HU significantly elevated serum Dkk2 levels and Dkk2 mRNA levels in the soleus muscle of mice whereas hypergravity significantly decreased those Dkk2 levels. In the simple regression analyses, serum Dkk2 levels were negatively and positively related to trabecular bone mineral density and mRNA levels of receptor activator of nuclear factor-kappa B ligand (RANKL) in the tibia of mice, respectively. Moreover, shear stress significantly suppressed Dkk2 mRNA levels in C2C12 cells, and cyclooxygenase inhibitors significantly antagonized the effects of shear stress on Dkk2 expression. On the other hand, Dkk2 suppressed the mRNA levels of osteogenic genes, alkaline phosphatase activity and mineralization, and it increased RANKL mRNA levels in mouse osteoblasts. In conclusion, we showed that muscle and serum Dkk2 levels are positively and negatively regulated during mechanical unloading and hypergravity in mice, respectively. An increase in Dkk2 expression in the skeletal muscle might contribute to disuse- and microgravity-induced bone and muscle loss.

With the use of stable isotopes, this study aimed to compare the bioavailability of active absorbable algal calcium (AAACa), obtained from oyster shell powder heated to a high temperature, with an additional heated seaweed component (Heated Algal Ingredient, HAI), with that of calcium carbonate. In 10 postmenopausal women volunteers aged 59 to 77 years (mean ± S.D., 67 ± 5.3), the fractional calcium absorption of AAACa and CaCO3 was measured by a dual stable isotope method. 44Ca-enriched CaCO3 and AAACa were administered in all subjects one month apart. After a fixed-menu breakfast and pre-test urine collection (Urine 0), 42Ca-enriched CaCl2 was intravenously injected, followed by oral administration of 44Ca-enriched CaCO3 without carrier 15 minutes later, and complete urine collection for the next 24 hours (Urine 24). The fractional calcium absorption was calculated as the ratio of Augmentation of 44Ca from Urine 0 to Urine 24/ augmentation of 42Ca from Urine 0 to Urine 24. Differences and changes of 44Ca and 42Ca were corrected by comparing each with 43Ca. Fractional absorption of AAACa (mean ± S.D., 23.1 ± 6.4), was distinctly and significantly higher than that of CaCO3 (14.7 ± 6.4; p = 0.0060 by paired t-test). The mean fractional absorption was approximately 1.57-times higher for AAACa than for CaCO3. The serum 25(OH) vitamin D level was low (mean ± S.D., 14.2 ± 4.95 ng/ml), as is common in this age group in Japan. Among the parameters of the bone and mineral metabolism measured, none displayed a significant correlation with the fractional absorption of CaCO3 and AAACa. Higher fractional absorption of AAACa compared with CaCO3 supports previous reports on the more beneficial effect of AAACa than CaCO3 for osteoporosis.

Macrophages play crucial roles in repair process of various tissues. However, the details in the role of macrophages during bone repair still remains unknown. Herein, we examined the contribution of the tissue fibrinolytic system to the macrophage functions in bone repair after femoral bone defect by using male mice deficient in plasminogen (Plg-/-), urokinase-type plasminogen activator (uPA-/-) or tissue-type plasminogen activator (tPA-/-) genes and their wild-type littermates. Bone repair of the femur was delayed in uPA-/- mice until day 6, compared with wild-type (uPA+/+) mice. Number of Osterix-positive cells and vessel formation were decreased in uPA-/- mice at the bone injury site on day 4, compared with those in uPA+/+ mice. Number of macrophages and their phagocytosis at the bone injury site were reduced in uPA-/- and Plg-/-, but not in tPA-/- mice on day 4. Although uPA or plasminogen deficiency did not affect the levels of cytokines, including TNF-α, IL-1β, IL-6, IL-4 and IFN-γ mRNA in the damaged femur, the elevation in CCL3 mRNA levels was suppressed in uPA-/- and Plg-/-, but not in tPA-/- mice. Neutralization of CCL3 antagonized macrophage recruitment to the site of bone injury and delayed bone repair in uPA+/+, but not in uPA-/- mice. Our results provide novel evidence that the tissue fibrinolytic system contributes to the induction of macrophage recruitment and CCL3 at the bone injury site, thereby, leading to the enhancement of the repair process.

Previous studies suggest that fracture healing is impaired in diabetes; however, the underlying mechanism remains unclear. Here, we investigated the roles of plasminogen activator inhibitor-1 (PAI-1) in the impaired bone repair process by using streptozotocin (STZ)-induced diabetic female wild-type (PAI-1+/+) and PAI-1-deficient (PAI-1-/-) mice. Bone repair and the number of alkaline phosphatase (ALP)-positive cells at the site of a femoral bone damage were comparable in PAI-1+/+ and PAI-1-/- mice without STZ treatment. Although the bone repair process was delayed by STZ treatment in PAI-1+/+ mice, this delayed bone repair was blunted in PAI-1-/- mice. The reduction in the number of ALP-positive cells at the site of bone damage induced by STZ treatment was attenuated in PAI-1-/- mice compared to PAI-1+/+ mice. On the other hand, PAI-1 deficiency increased the levels of ALP and type I collagen mRNA in female mice with or without STZ treatment, and the levels of Osterix and osteocalcin mRNA, suppressed by diabetic state in PAI-1+/+ mice, were partially protected in PAI-1-/- mice. PAI-1 deficiency did not affect formation of the cartilage matrix and the levels of types II and X collagen and aggrecan mRNA suppressed by STZ treatment, although PAI-1 deficiency increased the expression of chondrogenic markers in mice without STZ treatment. The present study indicates that PAI-1 is involved in the impaired bone repair process induced by the diabetic state in part through a decrease in the number of ALP-positive cells.

The effects of Enzamin on obesity-related metabolic disorders in obese db/db mice were examined to explore a novel agent for the prevention of insulin resistance. Db/db mice were treated with water containing Enzamin (0·1 and 1·0 %) for 8 weeks from 6 weeks of age. Enzamin treatment at 1·0 %, but not at 0·1 %, significantly decreased the fasting plasma glucose, serum total cholesterol and TAG levels in db/db mice, without affecting body weight gain and body fat composition. Furthermore, insulin sensitivity and glucose tolerance were improved by the treatment of db/db mice with 1·0 % Enzamin. Immunohistochemical studies and gene expression analysis showed that 1·0 % Enzamin treatment suppressed macrophage accumulation and inflammation in the adipose tissue. In addition, 1·0 % Enzamin treatment increased serum adiponectin in db/db mice. Treatment with 1·0 % Enzamin also significantly suppressed the expression of NADPH oxidase subunits, suggesting an antioxidative effect for Enzamin in the adipose tissue. Furthermore, in vitro experiments demonstrated that the lipopolysaccharide-induced inflammatory reaction was significantly suppressed by Enzamin treatment in macrophages. Enzamin treatment increased the expression of GLUT4 mRNA in muscle, but not GLUT2 mRNA in the liver of db/db mice. Enzamin also increased the mRNA expression of carnitine palmitoyltransferase 1a (CPT1a, muscle isoform) in db/db mice, whereas Enzamin treatment did not affect the mRNA expression of CPT1b (liver isoform) in db/db mice. In conclusion, our data indicate that Enzamin can improve insulin resistance by ameliorating impaired adipocytokine expression, presumably through its anti-inflammatory action, and that Enzamin possesses a potential for preventing the metabolic syndrome.

Mechanical-unloading-induced skeletal muscle atrophy results in physical frailty and disability. Elucidating its mechanism is required to establish effective countermeasures for this muscle adaptation. First, we analyzed the proteome profile in the gastrocnemius (Gast) and soleus muscles of space-flown mice raised under microgravity or artificial 1-g for 30 days, and found that the expression levels of fibrinolysis-related proteins were significantly elevated in the mechanical-unloaded muscles. Next, we investigated the roles of the fibrinolytic system in skeletal muscle atrophy induced by mechanical unloading on the ground. Eight-week-old male mice with plasminogen gene deficiency (Plg-/-) and their wild-type littermates were divided into control and hindlimb-suspended groups and were raised for 21 days. Plasminogen deficiency significantly enhanced the decrease in muscle mass at the lower limbs of mice following hindlimb unloading, and the Gast muscle atrophy was more prominent in Plg-/- mice. In addition, plasminogen deficiency significantly increased the expression of autophagy-related markers, beclin1 mRNA and LC3B protein, in the mechanical-unloaded Gast muscles, but did not affect the increase in the gene expression of ubiquitin ligases, atrogin-1 and MuRF1. Neither plasminogen deficiency nor hindlimb unloading affected the Akt/mechanistic target of rapamycin pathway in the Gast muscles. These results suggested that plasminogen deficiency might accelerate protein breakdown via the autophagy-lysosome, but not the ubiquitin-proteasome, system in the mechanicalunloaded Gast muscles. In conclusion, we first showed that plasminogen deficiency exacerbated the Gast muscle atrophy in hindlimb-unloaded mice. Plasminogen and the fibrinolysis system might play some protective roles against muscle atrophy induced by mechanical unloading in developing mice. [ABSTRACT FROM AUTHOR]

Abstract Background The increasing incidence and prevalence of childhood overweight (OW) and obesity (OB) are major global health challenges. This study aimed to evaluate the effectiveness of a 2-year healthy lifestyle intervention program in reducing the incidence of OW and OB among schoolchildren aged 11–12 years in Vietnam. Methods Study design: Randomized controlled trial. Participants and interventions: In total, 733 students participated in this study. Participants were included from four schools in Hanoi based on a random cluster sampling method. Students’ health outcomes were assessed at baseline and after 2 years. Main outcomes: The primary outcomes were the incidence of OW and OB, and the secondary outcomes were the prevalence and remission of OW and OB. Generalized estimating equations (GEEs) were used to determine the outcomes and risk factors. Results After the intervention, the prevalence of OW decreased by 9.24% in the intervention group and 3.01% in the control group. After adjustment for age and sex, the odds of overweight were 36.7% lower in the intervention group than in the control group (OR = 0.633; 95% CI: 0.434–0.925; P = 0.018). The incidence of OW was 1.31% in the intervention group compared with 5.33% in the control group. After adjustment for age and sex, the odds of incident overweight in the intervention group were 77.4% lower than in the control group (OR = 0.226; 95% CI: 0.063–0.813; P = 0.023). The proportion of patients who achieved OW remission was 41.22% in the intervention group and 28.68% in the control group. After adjustment for age and sex, the odds of OW remission of overweight were 2.219 times greater in the intervention group than in the control group (OR = 2.219; 95% CI: 1.220–4.036; P = 0.009). For most OB-related factors, there were no significant differences between the intervention and control groups. Conclusion A multicomponent healthy lifestyle intervention was effective in reducing the incidence of OW; however, it had no significant effect on OB among children aged 11–12 years in Hanoi, Vietnam. Trial registration Retrospectively registered number: UMIN000014992, 20/08/2023.

“Cirriformia tentaculata” is considered to be distributed throughout Japanese waters and is used as an indicator species of organic pollution in coastal regions. However, previous studies indicated that “C. tentaculata” from Japan contained cryptic or sibling species. In order to solve this taxonomic problem, we collected specimens of Cirriformia Hartman, 1936 from throughout Japan and conducted taxonomic analyses. Molecular phylogenetic analyses showed that the Cirriformia worms collected could be divided into twelve phylogroups. We conducted detailed morphological analyses of each phylogroup and found morphological differences between each. Ten species were considered new to science, and the remaining two had the morphology according to the original description of C. tentaculata (Montagu, 1808). Physio-chemical characteristics of their habitats were not identical between each species, which suggested that the so-called “C. tentaculata” in Japanese waters is not adequate as a pollutant indicator.

The tubulanid palaeonemertean Parahubrechtia rayi Chernyshev, Polyakova, and Sun, 2022 has been only known from the type locality (4–6 m depth) in the Sea of Japan, Peter the Great Bay, Russia. Our nemertean faunal surveys at shallow subtidal zones yielded a couple of anterior body fragments of whitish tubulanids, each from i) Moroiso Cove (6 m depth) in Sagami Bay, middle Honshu, and ii) Akkeshi Bay (4 m depth), eastern Hokkaido. Partial 658-bp sequences of the mitochondrial cytochrome c oxidase subunit I gene from these specimens differed by up to 1.2% p-distance from four sequences of P. rayi from the type locality. This expands the species’ known distribution by ~1000 km eastward and southeastward to the Pacific coasts of Japan.

Abstract Aims/Introduction This study aimed to identify risk factors that contribute to the progression of slowly‐progressive type 1 diabetes by evaluating the positive predictive value (PPV) of factors associated with the progression to an insulin‐dependent state. Materials and Methods We selected 60 slowly‐progressive type 1 diabetes patients who tested positive for glutamic acid decarboxylase autoantibodies (GADA) at diagnosis from the Japanese Type 1 Diabetes Database Study. GADA levels in these patients were concurrently measured using both radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) techniques. Results Compared with the non‐progressor group (fasting C‐peptide [F‐CPR] levels maintained ≥0.6 ng/mL), the progressor group showed a younger age at diagnosis, lower body mass index (BMI), lower F‐CPR levels and a higher prevalence of insulinoma‐associated antigen‐2 autoantibodies (IA‐2A). The PPV of RIA‐GADA increased from 56.3 to 70.0% in the high titer group (≥10 U/mL), and further increased to 76.9, 84.2, 81.0 and 75.0% when combined with specific thresholds for age at diagnosis

Rhadinorhynchus Lühe, 1911 consists of acanthocephalans parasitizing migratory fishes. Of about 50 congeners currently assigned in this genus, 10 named and six unidentified species have previously been studied in molecular phylogenetic works; the intrageneric phylogeny and molecular diversity are thus still incompletely understood. In this study, we examined the phylogenetic positions of the two species R. seriolae (Yamaguti, 1963) and R. laterospinosus Amin, Heckmann, and Ha, 2011 based on the cytochrome c oxidase subunit I and 18S rRNA genes determined from material found in the intestines of the Pacific bluefin tuna Thunnus orientalis (Temminck and Schlegel, 1844) (new host) and the frigate tuna Auxis thazard (Lacepède, 1800), respectively. This is the first record of R. laterospinosus from Japan.

The chromodorid nudibranch Goniobranchus aureopurpureus (Collingwood, 1881) is characterized by numerous yellow spots on a white mantle that is bordered with purple spots. It is believed to be widely distributed in both the Northern and Southern Hemispheres in the West Pacific, showing color varieties in a more-or-less geographically coherent manner. Chromodoris variata Risbec, 1928 (type locality: New Caledonia) has been considered a synonym. In addition, three, similar-looking congeners have been proposed as synonymous with G. aureopurpureus: G. albopustulosus (Pease, 1860) (type locality: Hawaii), G. alius (Rudman, 1987) (type locality: Tanzania), and G. rufomaculatus (Pease, 1871) (type locality: French Polynesia). To test the synonymies proposed above, we performed maximum-likelihood and Bayesian-inference analyses utilizing partial sequences of the cytochrome c oxidase subunit I (COI) and 16S rRNA genes. The taxon sampling included a specimen of G. aureopurpureus newly collected in Hokkaido, Japan, along with other sequences of Goniobranchus deposited in public databases (31 specimens representing 22 species), including one identified as ‘Chromodoris aureopurpurea’ from New South Wales, Australia; G. albopustulosus from Hawaii; and two ‘Chromodoris rufomaculata’ from Guam and the Mariana Islands. Goniobranchus aureopurpureus from Japan and from Australia were sister taxa in the resulting trees but differed from each other by 8.6% in terms of 658-bp COI, suggesting that these two represent closely-related but different biological entities. For the Australian taxon, Goniobranchus cf. variatus comb. nov. is proposed herein. Goniobranchus albopustulosus and G. rufomaculatus were not closely related to the G. aureopurpureus–G. cf. variatus clade, rejecting the hypothesis that they are synonymous with either of these species. However, G. albopustulosus and G. rufomaculatus appeared to comprise a single biological entity, warranting further investigation of this potential synonymy. The placement of the Indian Ocean G. alius also remains to be tested.

An unidentified colonial ascidian called gaikotsu-panda-hoya in Japanese, literally meaning ‘skeleton panda ascidian,’ has been attracting SCUBA divers’ attention for the past few years since its strange appearance was introduced on the Internet by a diving shop in Kumejima Island, Japan. To confirm the taxonomic status of this species, fresh samples were collected from a diving point off the coast of Kumejima Island. Our morphological examination revealed that they represent a new species, herein described as Clavelina ossipandae sp. nov., which can be distinguished from 44 congeners in the genus Clavelina Savigny, 1816 by the combination of the following seven characteristics: i) colony consisting of completely free zooids, ii) zooids up to 20 mm in length, iii) in the living state, zooids transparent, with laterally elongated white patch between oral and atrial siphons, as well as four black markings, one between siphons, one mid-dorsally, and the other two situated laterally in a pair on the anterior part of the body, iv) transverse vessels white, v) endostyle black, vi) 10–14 stigmatal rows, and vii) two longitudinal muscular bands running from the abdomen to the endostyle on each side. Partial sequences (810 bp) of the cytochrome c oxidase subunit I gene from the holotype and one of the paratypes differed at 10 sites from each other (1.26% K2P distance) but were the same when translated into amino acids. A phylogenetic tree supported that this species is included in the genus Clavelina.

Abstract Background Previous studies have shown conflicting evidence regarding the incidence of cancer in patients with systemic lupus erythematosus (SLE) compared with that in healthy individuals. Calcineurin inhibitors (CNIs) such as cyclosporine and tacrolimus have been widely used to treat SLE; however, their effects on cancer risk remain unclear. We aimed to investigate the incidence of cancer in patients with SLE and determine the potential association between CNI use and cancer risk. Methods The standardized incidence ratio (SIR) of cancer among patients with lupus in the Lupus Registry of Nationwide Institutions (LUNA) was calculated based on the age-standardized incidence rate of cancer reported by Japan’s Ministry of Health, Labour and Welfare. We also examined the association between CNI exposure and cancer risk, while considering potential confounding factors. The analysis accounted for confounding variables such as age, sex, smoking history, maximum glucocorticoid dose, treatment history with cyclophosphamide, ongoing hydroxychloroquine, Systemic Lupus International Collaboration Clinics/American College of Rheumatology Damage Index (SDI) value (excluding cancer occurrence), comorbidity of diabetes mellitus, and smoking history. Results The study included 704 patients with SLE (625 females; 88.8%) with a median age of 44 years [interquartile range (IQR) = 34–55] years. The median past maximum glucocorticoid dose was 40 mg/day [IQR = 30–60 mg/day], and the SDI at registration was 1 [IQR = 0–2]. Among the patients, 246 (35.1%) had smoking histories, and 38 (5.4%) experienced cancer complications. Gynecological malignancies accounted for 63.2% of all cancers. The SIR of cancer in the LUNA cohort was 1.08 (95% confidence interval [CI] = 0.74–1.43). No statistically significant risks of cancer were found in relation to CNI treatment history; the odds ratio using multiple logistic regression was 1.12 (95% CI = 0.42–3.00), the risk ratio using standardization was 1.18 (95% CI = 0.47–2.16), and the risk ratio using inverse probability weighting was 1.8 (95% CI = 0.41–4.66). Conclusions The incidence of cancer in patients with SLE in the LUNA cohort did not significantly differ from that in the general population. These findings suggest that CNI treatment in this cohort did not pose a risk factor for cancer development.

Abstract The diagnostic criteria for slowly progressive type 1 diabetes (slowly progressive insulin‐dependent diabetes mellitus; SPIDDM) have been revised by the Committee on Type 1 Diabetes of the Japan Diabetes Society. All of the following three criteria must be met for ‘a definitive diagnosis of SPIDDM’: (1) presence of anti‐islet autoantibodies at some point in time during the disease course; (2) absence of ketosis or ketoacidosis at the diagnosis of diabetes with no requirement for insulin treatment to correct hyperglycemia immediately after diagnosis in principle; and (3) gradual decrease of insulin secretion over time, with insulin treatment required at more than 3 months after diagnosis, and the presence of severe endogenous insulin deficiency (fasting serum C‐peptide immunoreactivity

Polychaetes are typically found in marine environments with limited species adapting to semi-terrestrial habitats. The genus Stygocapitella comprises interstitial polychaetes dwelling in sandy beach areas around or above the high-water line. Based on molecular data, previous studies suggested the presence of multiple cryptic species in some different localities in the world lumped together as Stygocapitella subterranea. In Japan, reports on Stygocapitella were scarce, with only one species having been documented 40 years ago at Ishikari Beach in Hokkaido by the name of S. subterranea. We revisited these earlier findings and uncovered the presence of two distinct species in Stygocapitella. One of these species is herein named Stygocapitella itoi sp. nov., while the other corresponds to S. budaevae, originally described from the Russian Far East. Stygocapitella itoi sp. nov. possesses a chaetal pattern similar to that of S. australis, S. furcata and S. pacifica but can be distinguished from the congeners by two characters: a slightly forked pygidium and forked chaetae consisting of two teeth and two outer prongs. Our multi-locus phylogenetic analysis showed close relationships across the Pacific Ocean in two separated lineages in the genus, suggesting ancient dispersal or allopatric speciation after vicariance events.

Abstract Some cases of bronchial asthma are refractory to conventional therapies. As the pathogenesis of bronchial asthma has been clarified, new treatments, such as bronchial thermoplasty and biological drugs, have been developed. Tezepelumab, an anti‐thymic stromal lymphopoietin antibody, has been reported to inhibit the exacerbation of severe asthma; however, its adverse effects on glucose metabolism have not yet been reported. We encountered a case of weight gain and worsening glycemic management in a patient with type 2 diabetes and refractory bronchial asthma after the initiation of tezepelumab treatment. It has been reported that the overexpression of thymic stromal lymphopoietin in mice resulted in an enhanced release of free fatty acids from adipose tissues and the liver; thus, the administration of anti‐thymic stromal lymphopoietin antibodies in the present case might have caused obesity, fatty liver and lower glucose tolerance.

Fimbrora Monniot & Monniot, 1991, a macrophagous ascidian genus within the family Ascidiidae Adams & Adams, 1858, is currently monotypic, represented by F. calsubia Monniot & Monniot, 1991, a species previously recorded from the bottom of the South Pacific at depths of 1000–1860 m. The taxonomic status of Fimbrora has remained ambiguous because characteristics in its branchial papillae and neural-gland opening are incompletely known in previous studies, while these traits are essential for distinguishing other ascidiid genera. So far, no nucleotide sequence representing F. calsubia is available. In this study, we collected a single specimen of F. calsubia at a depth of 2027 m, about 400 km off the Pacific coast of Honshu, Japan. This is the deepest record, as well as the first report from the North Pacific, for the species. Our examination indicates that Fimbrora is morphologically similar to another ascidiid genus, Psammascidia Monniot, 1962, by having only secondary branchial papillae in the pharynx. Our phylogenetic analysis, based on the 18S ribosomal RNA and cytochrome c oxidase subunit I genes, along with those of 27 ascidian species available in public databases, showed that F. calsubia was more closely related to Ascidia zara Oka, 1935, Phallusia fumigata (Grube, 1864) and Phallusia mammilata (Cuvier, 1815) than to Ascidia ceratodes (Huntsman, 1912), Ascidiella aspersa (Müller, 1776) and Ascidiella scabra (Müller, 1776). Our results also indicated that acquisitions of macrophagous feeding by deep-sea members happened independently at least three times in the evolutionary history of the entire Ascidiacea.