Eicosapentaenoic acid changes muscle transcriptome and intervenes in aging-related fiber type transition in male mice.

Age-related sarcopenia is associated with a variety of changes in skeletal muscle. These changes are interrelated with each other and associated with systemic metabolism, the details of which, however, are largely unknown. Eicosapentaenoic acid (EPA) is a promising nutrient against sarcopenia and has multifaceted effects on systemic metabolism. In this study, we hypothesized that the aging process in skeletal muscle can be intervened by the administration of EPA. Seventy-five-week-old male mice were assigned to groups fed an EPA-deprived diet (EPA-) or an EPA-enriched diet with 1 wt% EPA (EPA+) for 12 wk. Twenty-four-week-old male mice fed with normal chow were also analyzed. At baseline, the grip strength of the aging mice was lower than that of the young mice. After 12 wk, EPA+ showed similar muscle mass but increased grip strength compared with EPA-. EPA+ displayed higher insulin sensitivity than EPA-. Immunohistochemistry and gene expression analysis of myosin heavy chains (MyHCs) revealed fast-to-slow fiber type transition in aging muscle, which was partially inhibited by EPA. RNA sequencing (RNA-Seq) analysis suggested that EPA supplementation exerts pathway-specific effects in skeletal muscle including the signatures of slow-to-fast fiber type transition. In conclusion, we revealed that aging skeletal muscle in male mice shows lower grip strength and fiber type changes, both of which can be inhibited by EPA supplementation irrespective of muscle mass alteration.NEW & NOTEWORTHY This study demonstrated that the early phenotype of skeletal muscle in aging male mice is characterized by muscle weakness with fast-to-slow fiber type transition, which could be ameliorated by feeding with EPA-enriched diet. EPA induced metabolic changes such as an increase in systemic insulin sensitivity and altered muscle transcriptome in the aging mice. These changes may be related to the fiber type transition and influence muscle quality.

The Influence of Glucocorticoid Receptor on Sex Differences of Gene Expression Profile in Skeletal Muscle.

Skeletal muscle functions as a locomotory system and maintains whole-body metabolism. Sex differences in such skeletal muscle morphology and function have been documented; however, their underlying mechanisms remain elusive. Glucocorticoids are adrenocortical hormones maintaining homeostasis, including regulating whole-body energy metabolism in addition to stress response. In skeletal muscle, glucocorticoids can reduce the synthesis of muscle proteins and simultaneously accelerate the breakdown of proteins to regulate skeletal muscle mass and energy metabolism via a transcription factor glucocorticoid receptor (GR). We herein evaluated the related contributions of the GR to sex differences of gene expression profiles in skeletal muscle using GR-floxed (GRf/f) and skeletal muscle-specific GR knockout (GRmKO) mice. There were no differences in GR mRNA and protein expression levels in gastrocnemius muscle between males and females. A DNA microarray analysis using gastrocnemius muscle from GRf/f and GRmKO mice revealed that, although most gene expression levels were identical in both sexes, genes related to cholesterol and apolipoprotein synthesis and fatty acid biosynthesis and the immunological system were predominantly expressed in males and females, respectively, in GRf/f but not in GRmKO mice. Moreover, many genes were up-regulated in response to starvation in GRf/f but not in GRmKO mice, many of which were sex-independent and functioned to maintain homeostasis, while genes that showed sex dominance related to a variety of functions. Although the genes expressed in skeletal muscle may be predominantly sex-independent, sex-dominant genes may relate to sex differences in energy metabolism and the immune system and could be controlled by the GR.

GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

Glucocorticoids (GCs) potently induce T-cell apoptosis in a GC receptor (GR)-dependent manner and are used to control lymphocyte function in clinical practice. However, its downstream pathways remain controversial. Here, we showed that GC-induced transcript 1 (GLCCI1) is a novel downstream molecule of the GC-GR cascade that acts as an antiapoptotic mediator in thymic T cells. GLCCI1 was highly phosphorylated and colocalized with microtubules in GLCCI1-transfected human embryonic kidney QBI293A cells. GR-dependent up-regulation of GLCCI1 was associated with GC-induced proapoptotic events in a cultured thymocyte cell line. However, GLCCI1 knockdown in a thymocyte cell line led to apoptosis. Consistently, transgenic mice overexpressing human GLCCI1 displayed enlarged thymi that consisted of larger numbers of thymocytes. Further molecular characterization showed that GLCCI1 bound to both dynein light chain LC8-type 1 (LC8) and its functional kinase, p21-protein activated kinase 1 (PAK1), thereby inhibiting the kinase activity of PAK1 toward LC8 phosphorylation, a crucial event in apoptotic signaling. GLCCI1 induction facilitated LC8 dimer formation and reduced Bim expression. Thus, GLCCI1 is a candidate factor involved in apoptosis regulation of thymic T cells.-Kiuchi, Z., Nishibori, Y., Kutsuna, S., Kotani, M., Hada, I., Kimura, T., Fukutomi, T., Fukuhara, D., Ito-Nitta, N., Kudo, A., Takata, T., Ishigaki, Y., Tomosugi, N., Tanaka, H., Matsushima, S., Ogasawara, S., Hirayama, Y., Takematsu, H., Yan, K. GLCCI1 is a novel protector against glucocorticoid-induced apoptosis in T cells.

Correlation among body composition and metabolic regulation in a male mouse model of Cushing's syndrome.

Glucocorticoids play a critical role in the regulation of homeostasis, including metabolism. In patients with Cushing's syndrome, chronic glucocorticoid excess disrupts physiological internal milieu, resulting in central obesity, muscle atrophy, fatty liver, and insulin resistance. However, the relationship among various metabolic effects of glucocorticoids remains unknown. In the present study, we studied a male mouse model of Cushing's syndrome and indicated that glucocorticoid excess alters metabolic phenotype and body composition involving possible communication among skeletal muscle, liver, and adipose tissue.

LSD1 mediates metabolic reprogramming by glucocorticoids during myogenic differentiation.

The metabolic properties of cells are formed under the influence of environmental factors such as nutrients and hormones. Although such a metabolic program is likely initiated through epigenetic mechanisms, the direct links between metabolic cues and activities of chromatin modifiers remain largely unknown. In this study, we show that lysine-specific demethylase-1 (LSD1) controls the metabolic program in myogenic differentiation, under the action of catabolic hormone, glucocorticoids. By using transcriptomic and epigenomic approaches, we revealed that LSD1 bound to oxidative metabolism and slow-twitch myosin genes, and repressed their expression. Consistent with this, loss of LSD1 activity during differentiation enhanced the oxidative capacity of myotubes. By testing the effects of various hormones, we found that LSD1 levels were decreased by treatment with the glucocorticoid dexamethasone (Dex) in cultured myoblasts and in skeletal muscle from mice. Mechanistically, glucocorticoid signaling induced expression of a ubiquitin E3 ligase, JADE-2, which was responsible for proteasomal degradation of LSD1. Consequently, in differentiating myoblasts, chemical inhibition of LSD1, in combination with Dex treatment, synergistically de-repressed oxidative metabolism genes, concomitant with increased histone H3 lysine 4 methylation at these loci. These findings demonstrated that LSD1 serves as an epigenetic regulator linking glucocorticoid action to metabolic programming during myogenic differentiation.

Pressure overload inhibits glucocorticoid receptor transcriptional activity in cardiomyocytes and promotes pathological cardiac hypertrophy.

Glucocorticoid receptor (GR) is abundantly expressed in cardiomyocytes. However, the role of GR in regulating cardiac hypertrophy and heart failure in response to pressure overload remains unclear. Cardiomyocyte-specific GR knockout (GRcKO) mice, mineralocorticoid receptor (MR) knockout (MRcKO), and GR and MR double KO (GRMRdcKO) mice were generated using the Cre-lox system. In response to pressure overload, GRcKO mice displayed worse cardiac remodeling compared to control (GRf/f) mice, including a greater increase in heart weight to body weight ratio with a greater increase in cardiomyocytes size, a greater decline in left ventricular contractility, and higher reactivation of fetal genes. MRcKO mice showed a comparable degree of cardiac remodeling compared to control (MRf/f) mice. The worse cardiac remodeling in pressure overloaded GRcKO mice is not due to compensatory activation of cardiomyocyte MR, since pressure overloaded GRMRdcKO mice displayed cardiac remodeling to the same extent as GRcKO mice. Pressure overload suppressed GR-target gene expression in the heart. Although plasma corticosterone levels and subcellular localization of GR (nuclear/cytoplasmic GR) were not changed, a chromatin immunoprecipitation assay revealed that GR recruitment onto the promoter of GR-target genes was significantly suppressed in response to pressure overload. Rescue of the expression of GR-target genes to the same extent as sham-operated hearts attenuated adverse cardiac remodeling in pressure-overloaded hearts. Thus, GR works as a repressor of adverse cardiac remodeling in response to pressure overload, but GR-mediated transcription is suppressed under pressure overload. Therapies that maintain GR-mediated transcription in cardiomyocytes under pressure overload can be a promising therapeutic strategy for heart failure.

Effects of long-term intake of a yogurt fermented with Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131 on mice.

The gut is an extremely complicated ecosystem where micro-organisms, nutrients and host cells interact vigorously. Although the function of the intestine and its barrier system weakens with age, some probiotics can potentially prevent age-related intestinal dysfunction. Lactobacillus delbrueckii subsp. bulgaricus 2038 and Streptococcus thermophilus 1131, which are the constituents of LB81 yogurt, are representative probiotics. However, it is unclear whether their long-term intake has a beneficial influence on systemic function. Here, we examined the gut microbiome, fecal metabolites and gene expression profiles of various organs in mice. Although age-related alterations were apparent in them, long-term LB81 yogurt intake led to an increased Bacteroidetes to Firmicutes ratio and elevated abundance of the bacterial family S24-7 (Bacteroidetes), which is known to be associated with butyrate and propanoate production. According to our fecal metabolite analysis to detect enrichment, long-term LB81 yogurt intake altered the intestinal metabolic pathways associated with propanoate and butanoate in the mice. Gene ontology analysis also revealed that long-term LB81 yogurt intake influenced many physiological functions related to the defense response. The profiles of various genes associated with antimicrobial peptides-, tight junctions-, adherens junctions- and mucus-associated intestinal barrier functions were also drastically altered in the LB81 yogurt-fed mice. Thus, long-term intake of LB81 yogurt has the potential to maintain systemic homeostasis, such as the gut barrier function, by controlling the intestinal microbiome and its metabolites.

Role of skeletal muscle glucocorticoid receptor in systemic energy homeostasis.

Glucocorticoids (GCs) affect nearly every organ and tissue in the body, regulating diverse physiologic processes including energy homeostasis. The metabolic mission of GCs is to supply enough glucose into the circulation to fuel the brain and ensure survival of the organism under conditions of acute stress or starvation. Recent studies have revealed that GCs, via orchestration between multiple organs, physiologically elicit fine tuning of systemic energy metabolism.

Protective Effect of Sex Hormone-Binding Globulin against Metabolic Syndrome: In Vitro Evidence Showing Anti-Inflammatory and Lipolytic Effects on Adipocytes and Macrophages.

Sex hormone-binding globulin (SHBG) is a serum protein released mainly by the liver, and a low serum level correlates with a risk for metabolic syndrome including diabetes, obesity, and cardiovascular events. However, the underlying molecular mechanism(s) linking SHBG and metabolic syndrome remains unknown. In this study, using adipocytes and macrophages, we focused on the in vitro effects of SHBG on inflammation as well as lipid metabolism. Incubation with 20 nM SHBG markedly suppressed lipopolysaccharide- (LPS-) induced inflammatory cytokines, such as MCP-1, TNFα, and IL-6 in adipocytes and macrophages, along with phosphorylations of JNK and ERK. Anti-inflammatory effects were also observed in 3T3-L1 adipocytes cocultured with LPS-stimulated macrophages. In addition, SHBG treatment for 18 hrs or longer significantly induced the lipid degradation of differentiated 3T3-L1 cells, with alterations in its corresponding gene and protein levels. Notably, these effects of SHBG were not altered by coaddition of large amounts of testosterone or estradiol. In conclusion, SHBG suppresses inflammation and lipid accumulation in macrophages and adipocytes, which might be among the mechanisms underlying the protective effect of SHBG, that is, its actions which reduce the incidence of metabolic syndrome.

[Homeostasis and Disorder of Musculoskeletal System.Influence of steroid therapy on muscuoskeletal system.]

Muscle atrophy occurs when glucocorticoid steroids are administered in pharmacological doses or in Cushing syndrome, and such pathological condition is termed as steroid myopathy. Its molecular mechanism is clarified from the study of the gene expression regulation mechanism mediated by steroid receptors in skeletal muscle, progressing to translational research and also addressing the mechanism by which steroids participate in the regulation of whole body energy metabolism via skeletal muscle.

The effects of bolus supplementation of branched-chain amino acids on skeletal muscle mass, strength, and function in patients with rheumatic disorders during glucocorticoid treatment.

OBJECTIVES: To test the effects of bolus supplementation of branched-chain amino acids (BCAA) on skeletal muscle mass, strength, and function in patients with rheumatic disorders taking glucocorticoid (GC). METHODS: Patients with rheumatic disorders treated with prednisolone (≥10 mg/day) were randomized to ingest additional daily 12 g of BCAA (n = 9) or not (n = 9) for 12 weeks. At baseline, and 4, 8, and 12 weeks, they underwent bioelectrical impedance analysis, muscle strength and functional tests, and computed tomography analysis for cross-sectional area of mid-thigh muscle. RESULTS: Disease activities of the patients were well controlled and daily GC dose was similarly reduced in both groups. Limb muscle mass was recovered in both groups. Whole-body muscle mass and muscle strength and functional mobility were increased only in BCAA (+) group. The effects of BCAA supplementation on recovering skeletal muscle mass were prominent in particular muscles including biceps femoris muscle. CONCLUSIONS: This trial is the first-in-man clinical trial to demonstrate that BCAA supplementation might be safe and, at least in part, improve skeletal muscle mass, strength, and function in patients with rheumatic disorders treated with GC.

[Novel role of skeletal muscle in systemic energy homeostasis -mechano-metabo coupling via muscle-liver-fat signaling axis.]

Skeletal muscle has a pleiotropic role in organismal energy metabolism, for example, by storing protein as an energy source, or by excreting endocrine hormones. Muscle proteolysis is an active process tightly controlled by specific signaling pathways and transcriptional programs, involving ubiquitin-proteasome and autophagy machineries. Glucocorticoid receptor(GR)transcriptionally controls expression levels of components in the both machineries, and modulates muscle-liver-fat signaling axis. This skeletal muscle-liver-fat signaling axis controls organismal energy distribution and may serve as a target for the development of therapies against various metabolic diseases, including obesity.

The role of Cas-L/NEDD9 as a regulator of collagen-induced arthritis in a murine model.

Cas-L/NEDD9 is a cytoplasmic docking protein downstream of ß1 integrin-mediated signaling pathway and is essential for cellular migration and ß1 integrin-mediated costimulation of T cells. We previously found that increased number of Cas-L positive leukocytes migrated into the inflamed joints of HTLV-I tax transgenic mice which spontaneously develop polyarthritis, suggesting a role of Cas-L in rheumatoid arthritis (RA) pathophysiology. Our current study expanded these findings on the role of Cas-L/NEDD9 in the development of RA by analyzing the pathophysiological changes in a Nedd9(-/-) mouse collagen-induced arthritis (CIA) model. Nedd9(-/-) mice exhibited a decrease in arthritis severity as compared to Nedd9(+/+) mice. In addition, as being conducted bone marrow transplantation experiments with a CIA model, Nedd9(-/-)→Nedd9(+/+) transplant showed a decrease in the incidence and severity score of arthritis, compared to those of Nedd9(+/+)→Nedd9(-/-) transplant. For analysis of serum levels of various cytokines, IL-1ß, IL-6, IL-17, TNF-α, IFN-γ and anti-collagen antibody were decreased, while IL-4 and IL-10 levels were increased, in Nedd9(-/-) mice as compared to those in Nedd9(+/+) mice. Furthermore, collagen-mediated cellular responses of lymphocytes isolated from spleen or affected lymph nodes of Nedd9(-/-) mice were reduced. Our results strongly suggest that Cas-L/NEDD9 plays a pivotal role in the pathophysiology of CIA, and that Cas-L/NEDD9 may be a potential molecular target for the treatment of RA.

Quantitative analysis of skeletal muscle mass in patients with rheumatic diseases under glucocorticoid therapy--comparison among bioelectrical impedance analysis, computed tomography, and magnetic resonance imaging.

OBJECTIVES: To determine the availability of bioelectrical impedance analysis (BIA), computed tomography (CT), and magnetic resonance imaging (MRI) for measurement of skeletal muscle mass in patients with rheumatic diseases and quantitatively assess skeletal muscle loss after glucocorticoid (GC) treatment. METHODS: The data from 22 patients with rheumatic diseases were retrospectively obtained. The muscle mass of body segments was measured with a BIA device in terms of skeletal muscle mass index (SMI). Cross-sectional area (CSA) was obtained from CT and MRI scans at the mid-thigh level using the image analysis program. We further assessed the data of three different measurements before and after GC treatment in 7 patients with rheumatic diseases. RESULTS: SMI of whole body was significantly correlated with estimated muscle volume and mid-thigh muscle CSA with CT and MRI (p < 0.01). Significant correlations between SMI and mid-thigh muscle CSA of each leg were also found (p < 0.01). All the three measurements were negatively correlated with GC dosage (p < 0.01). Significant decline in mid-thigh muscle CSA with CT and MRI was found after GC treatment in 7 patients (p < 0.02). Those patients showed significant decline in SMI of whole body after GC treatment, but not in SMI of each leg. On the other hand, significant correlations between mid-thigh muscle CSA with CT and MRI were found before and after GC treatment (p < 0.01). CONCLUSIONS: GC-related skeletal muscle loss could be quantitatively assessed with BIA, CT, or MRI in patients with rheumatic diseases, and CT and MRI appeared to be more accurate than BIA.

[Analysis of Musculoskeletal Systems and Their Diseases. Recent progress in molecular pathogensis and treatment for muscle disorders].

There are a variety of different skeletal muscle diseases, each of which carries its own unique set of symptoms. One of the most common characteristics affecting the skeletal muscles is muscular atrophy. Indeed, muscle atrophy occurs under a variety of pathologyical conditions, including sarcopenia, glucocortcoid treatment. Recently, its underlying molecular has been intensively studied and novel therapy is being developed.

Down-regulation of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor by HEXIM1 attenuates myocardial angiogenesis in hypoxic mice.

Pulmonary hypertension (PH) sustains elevation of pulmonary vascular resistance and ultimately leads to right ventricular (RV) hypertrophy and failure and death. Recently, proangiogenic factors hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) have been known to promote left ventricular myocardial angiogenesis and lead to cardiac hypertrophy, and this would be involved in RV hypertrophy of PH patients. Previously, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced cardiomyocyte hypertrophy and hypertrophic genes expression, and that cardiomyocyte-specific HEXIM1 transgenic mice ameliorates RV hypertrophy in hypoxia-induced PH model. Given these results, here we analyzed the effect of HEXIM1 on the expression of HIF-1α and VEGF and on myocardial angiogenesis of RV in PH. We revealed that overexpression of HEXIM1 prevented hypoxia-induced expression of HIF-1α protein and its target genes including VEGF in the cultured cardiac myocytes and fibroblasts, and that cardiomyocyte-specific HEXIM1 transgenic mice repressed RV myocardial angiogenesis in hypoxia-induced PH model. Thus, we conclude that HEXIM1 could prevent RV hypertrophy, at least in part, via suppression of myocardial angiogenesis through down-regulation of HIF-1α and VEGF in the myocardium under hypoxic condition.

Prognosis of primary aldosteronism in Japan: results from a nationwide epidemiological study.

The Research Committee of Disorders of Adrenal Hormones, Japan, undertook a nationwide epidemiological study of primary aldosteronism (PA). The present study was undertaken as a part of this study to reveal the relationship between type of treatment and the prognosis of PA. In the primary survey, 4161 patients with PA during the period January 1, 2003-December 31, 2007 were reported from 3252 departments of internal medicine, pediatrics and urology. In the secondary survey, a questionnaire that requested detailed clinical information on individual patients was sent to those departments reporting patients in the primary survey. In total, data on 1706 patients with PA were available in the present study. Among patients with bilateral or unilateral aldosterone-producing adenoma, after adjustment for age at which prognosis was examined, sex, surgical treatment and medical treatment, surgical treatment was significantly associated with amelioration of hypertension (adjusted odds ratio [OR]: 0.47 [95% confidence interval (CI): 0.29-0.77]) and hypokalemia (adjusted OR: 0.17 [95% CI: 0.11-0.29]). No significant relationship was observed between medical treatment and such prognosis in this group of patients. Among patients with bilateral or unilateral adrenal hyperplasia, surgical, but not medical, treatment was significantly associated with amelioration of hypokalemia (adjusted OR: 0.23 [95% CI: 0.06-0.74]), while there was no relationship between surgical or medical treatment and the prognosis of hypertension. In conclusion, surgery offered a better prognosis of PA than medication with regards to hypertension and hypokalemia, with the limitation that a new anti-aldosterone drug, eplerenone, was not available during the study period.

[Glucocorticoids].

The established evidence of the rapid effects, along with the growing recognition of their disease-modifying properties has led us to reconsider the potential of glucocorticoids in the treatment of rheumatoid arthritis. Given their acceptable safety profile, especially when used at low dosages, several glucocorticoid-based therapeutic approaches have been explored in order to optimize their clinical benefits, while limiting the adverse effects. Encouraging results on the clinical and sub-clinical effects of low dosages are going to lead to a shift in usual daily practice. Optimizing the use of key non-biologic drugs including glucocorticoids may prolong disease control, thereby delaying the need for costly biologic therapies.

The crucial role of muscle glucocorticoid signaling in accelerating obesity and glucose intolerance via hyperinsulinemia.

Metabolic crosstalk from skeletal muscle to multiple organs is important for maintaining homeostasis, and its dysregulation can lead to various diseases. Chronic glucocorticoid administration often induces muscle atrophy and metabolic disorders such as diabetes and central obesity; however, the detailed underlying mechanism remains unclear. We previously reported that the deletion of glucocorticoid receptor (GR) in skeletal muscle increases muscle mass and reduces fat mass through muscle-liver-fat communication under physiological conditions. In this study, we show that muscle GR signaling plays a crucial role in accelerating obesity through the induction of hyperinsulinemia. Fat accumulation in liver and adipose tissue, muscle atrophy, hyperglycemia, and hyperinsulinemia induced by chronic corticosterone (CORT) treatment improved in muscle-specific GR-knockout (GR-mKO) mice. Such CORT-induced fat accumulation was alleviated by suppressing insulin production (streptozotocin injection), indicating that hyperinsulinemia enhanced by muscle GR signaling promotes obesity. Strikingly, glucose intolerance and obesity in ob/ob mice without CORT treatment were also improved in GR-mKO mice, indicating that muscle GR signaling contributes to obesity-related metabolic changes, regardless of systemic glucocorticoid levels. Thus, this study provides insight for the treatment of obesity and diabetes by targeting muscle GR signaling.