Adrenalectomy Improves Body Weight, Glucose, and Blood Pressure Control in Patients With Mild Autonomous Cortisol Secretion: Results of an Randomized Controlled Trial by the Co-work of Adrenal Research (COAR) Study.

OBJECTIVE: To assess the metabolic effects of adrenalectomy in patients with mild autonomous cortisol secretion (MACS). BACKGROUND: Despite retrospective studies showing the association of adrenalectomy for MACS with beneficial metabolic effects, there have been only 2 randomized prospective studies with some limitations to date. METHODS: A prospective, multicenter study randomized 132 patients with adrenal incidentaloma without any features of Cushing syndrome but with serum cortisol >50 nmol/L after a 1 mg overnight dexamethasone suppression test into an adrenalectomy group (n = 66) or control group (n = 66). The primary outcomes were changes in body weight, glucose, and blood pressure (BP). RESULTS: Among the 118 participants who completed the study with a median follow-up duration of 48 months (range: 3-66), the adrenalectomy group (n = 46) exhibited a significantly higher frequency of improved weight control, glucose control, and BP control (32.6%, 45.7%, and 45.7%, respectively) compared with the control group (n = 46; 6.5%, P = 0.002; 15.2%, P = 0.002; and 23.9%, P = 0.029, respectively) after matching for age and sex. Adrenalectomy [odds ratio (OR) = 10.38, 95% CI = 2.09-51.52, P = 0.004], body mass index (OR = 1.39, 95% CI = 1.08-1.79, P = 0.010), and cortisol after a 1 mg overnight dexamethasone suppression test levels (OR = 92.21, 95% CI = 5.30-1604.07, P = 0.002) were identified as independent factors associated with improved weight control. Adrenalectomy (OR = 5.30, 95% CI = 1.63-17.25, P = 0.006) and diabetes (OR = 8.05, 95% CI = 2.34-27.65, P = 0.001) were independently associated with improved glucose control. Adrenalectomy (OR = 2.27, 95% CI = 0.87-5.94, P = 0.095) and hypertension (OR = 10.77, 95% CI = 3.65-31.81, P < 0.001) demonstrated associations with improved BP control. CONCLUSIONS: adrenalectomy improved weight, glucose, and BP control in patients with MACS.

Preoperative prediction of metastatic pheochromocytoma and paraganglioma using clinical, genetic, and biochemical markers: A cohort study.

BACKGROUND: The prevalence of metastatic pheochromocytoma and paraganglioma (PPGL) is approximately 15%-20%. Although there are indicators to assess metastatic risks, none of them predict metastasis reliably. Therefore, we aimed to develop and validate a scoring system using clinical, genetic, and biochemical risk factors to preoperatively predict the metastatic risk of PPGL. METHODS: In the cross-sectional cohort (n = 180), clinical, genetic, and biochemical risk factors for metastasis were identified using multivariate logistic regression analysis, and a novel scoring system was developed. The scoring system was validated and compared with the age, size of tumor, extra-adrenal location, and secretory type (ASES) score in the longitudinal cohort (n = 114). RESULTS: In the cross-sectional cohort, pseudohypoxia group-related gene variants (SDHB, SDHD, or VHL), methoxytyramine >0.16 nmol/L, and tumor size >6.0 cm were independently associated with metastasis after multivariate logistic regression. Using them, the gene variant, methoxytyramine, and size of tumor (GMS) score were developed. In the longitudinal cohort, Harrell's concordance index of the GMS score (0.873, 95% confidence interval [CI]: 0.738-0.941) was higher than that of the ASES score (0.713, 95% CI: 0.567-0.814, p = 0.007). In the longitudinal cohort, a GMS score ≥2 was significantly associated with a higher risk of metastasis (hazard ratio = 25.07, 95% CI: 5.65-111.20). A GMS score ≥2 (p < 0.001), but not ASES score ≥2 (p = 0.090), was associated with shorter progression-free survival. CONCLUSION: The GMS scoring system, which integrates gene variant, methoxytyramine level, and tumor size, provides a valuable preoperative approach to assess metastatic risk in PPGL.

Aortic carboxypeptidase-like protein, a putative myokine, stimulates the differentiation and survival of bone-forming osteoblasts.

A new target that stimulates bone formation is needed to overcome limitations of current anti-osteoporotic drugs. Myokines, factors secreted from muscles, may modulate it. In this study, we investigated the role of aortic carboxypeptidase-like protein (ACLP), which is highly expressed in skeletal muscles, on bone formation. MC3T3-E1 cells and/or calvaria osteoblasts were treated with recombinant N-terminal mouse ACLP containing a signal peptide [rmACLP (N)]. The expression and secretion of ACLP were higher in skeletal muscle and differentiated myotube than in other tissues and undifferentiated myoblasts, respectively. rmACLP (N) increased bone formation, ALP activity, and phosphorylated p38 mitogen-activated protein (MAP) kinase in osteoblasts; reversal was achieved by pre-treatment with a TGF-ß receptor inhibitor. Under H2 O2 treatment, rmACLP (N) increased osteoblast survival, phosphorylated p38 MAP kinase, and the nuclear translocation of FoxO3a in osteoblasts. H2 O2 treatment caused rmACLP (N) to suppress its apoptotic, oxidative, and caspase-9 activities. rmACLP (N)-stimulated osteoblast survival was reversed by pre-treatment with a p38 inhibitor, a TGF-ß-receptor II blocking antibody, and a FoxO3a shRNA. Conditioned media (CM) from muscle cells stimulated osteoblast survival under H2 O2 treatment, in contrast to CM from ACLP knockdown muscle cells. rmACLP (N) increased the expressions of FoxO3a target anti-oxidant genes such as Sod2, Trx2, and Prx5. In conclusion, ACLP stimulated the differentiation and survival of osteoblasts. This led to the stimulation of bone formation by the activation of p38 MAP kinase and/or FoxO3a via TGF-ß receptors. These findings suggest a novel role for ACLP in bone metabolism as a putative myokine.

N-3 Polyunsaturated Fatty Acids Protect against Alcoholic Liver Steatosis by Activating FFA4 in Kupffer Cells.

Supplementation with fish oil rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) effectively reduces acute and chronic alcohol-induced hepatic steatosis. We aimed to find molecular mechanisms underlying the effects of n-3 PUFAs in alcohol-induced hepatic steatosis. Because free fatty acid receptor 4 (FFA4, also known as GPR120) has been found as a receptor for n-3 PUFAs in an ethanol-induced liver steatosis model, we investigated whether n-3 PUFAs protect against liver steatosis via FFA4 using AH7614, an FFA4 antagonist, and Ffa4 knockout (KO) mice. N-3 PUFAs and compound A (CpdA), a selective FFA4 agonist, reduced the ethanol-induced increase in lipid accumulation in hepatocytes, triglyceride content, and serum ALT levels, which were not observed in Ffa4 KO mice. N-3 PUFAs and CpdA also reduced the ethanol-induced increase in lipogenic sterol regulatory element-binding protein-1c expression in an FFA4-dependent manner. In Kupffer cells, treatment with n-3 PUFA and CpdA reversed the ethanol-induced increase in tumor necrosis factor-α, cyclooxygenase-2, and NLR family pyrin domain-containing 3 expression levels in an FFA4-dependent manner. In summary, n-3 PUFAs protect against ethanol-induced hepatic steatosis via the anti-inflammatory actions of FFA4 on Kupffer cells. Our findings suggest FFA4 as a therapeutic target for alcoholic hepatic steatosis.

Free Fatty Acid 4 Receptor Activation Attenuates Collagen-Induced Arthritis by Rebalancing Th1/Th17 and Treg Cells.

Dietary supplementation with n-3 polyunsaturated fatty acids (PUFA) has been found to be beneficial in rodent rheumatoid arthritis models and human trials. However, the molecular targets of n-3 PUFAs and their beneficial effects on rheumatoid arthritis are under-researched. Free fatty acid receptor 4 (FFA4, also known as GPR120) is a receptor for n-3 PUFA. We aim to investigate whether FFA4 activation reduces collagen-induced rheumatoid arthritis (CIA) by using an FFA4 agonist, compound A (CpdA), in combination with DBA-1J Ffa4 gene wild-type (WT) and Ffa4 gene knock-out (KO) mice. CIA induced an increase in the arthritis score, foot edema, synovial hyperplasia, pannus formation, proteoglycan loss, cartilage damage, and bone erosion, whereas the administration of CpdA significantly suppressed those increases in Ffa4 WT mice but not Ffa4 gene KO mice. CIA increased mRNA expression levels of pro-inflammatory Th1/Th17 cytokines, whereas CpdA significantly suppressed those increases in Ffa4 WT mice but not Ffa4 gene KO mice. CIA induced an imbalance between Th1/Th17 and Treg cells, whereas CpdA rebalanced them in spleens from Ffa4 WT mice but not Ffa4 gene KO mice. In SW982 synovial cells, CpdA reduced the LPS-induced increase in pro-inflammatory cytokine levels. In summary, the present results suggest that the activation of FFA4 in immune and synovial cells could suppress the characteristics of rheumatoid arthritis and be an adjuvant therapy.

Indices of ACTH-stimulated adrenal venous sampling as predictors of postsurgical outcomes in primary aldosteronism.

OBJECTIVE: This study aimed to investigate the impact of indices of adrenal venous sampling (AVS) on postsurgical outcomes in patients with primary aldosteronism (PA). DESIGN AND PATIENTS: This retrospective study determined biochemical and clinical outcomes based on ACTH-stimulated AVS parameters (lateralisation index [LI], contralateral ratio [CLR], and ipsilateral ratio [ILR]) in 251 patients with PA at 3 months after surgery. RESULTS: Modified complete biochemical success was achieved in 8 of 12 (66.7%) patients with LI = 3-4, 39 of 47 (83.0%) with LI = 4-10, and 155 of 169 (91.7%) with LI ≥ 10 (p = .004 for trend). Modified complete biochemical success was achieved in 29 of 38 (76.3%) patients with CLR ≥ 1 and ILR ≤ 2, 73 of 86 (84.9%) with CLR = 0.25-1 and ILR > 2, and 100 of 104 (96.2%) with CLR < 0.25 and ILR > 2 (p = .001 for trend). After adjusting for confounders, modified complete biochemical success was associated with an LI ≥ 10 (odds ratio [OR] = 6.32; 95% confidence interval [CI] = 1.33-29.93) using LI = 3-4 as a reference and combined CLR < 0.25 and ILR > 2 (OR = 11.49; 95% confidence interval [CI] = 2.49-53.01) using combined CLR ≥ 1 and ILR ≤ 2 as a reference. Using combined CLR ≥ 1 and ILR ≤ 2 as a reference, complete clinical success was associated with combined CLR < 0.25 and ILR > 2 (OR = 3.10; 95% CI = 1.03-9.28) and combined CLR = 0.25-1 and ILR > 2 (OR = 4.92; 95% CI = 1.64-14.76). CONCLUSION: LI ≥ 10 may be appropriate for achieving biochemical success. With ILR > 2, CLR < 0.25, and CLR < 1 may be appropriate for achieving biochemical and clinical success, respectively.

Associations Between Plasma Growth and Differentiation Factor-15 with Aging Phenotypes in Muscle, Adipose Tissue, and Bone.

Growth and differentiation factor 15 (GDF-15) is associated with muscle, fat, and bone metabolism; however, this association has not been well characterized. Plasma GDF-15, appendicular skeletal muscle mass (ASM), fat mass (FM), and bone mineral density (BMD) were measured in 146 postmenopausal women. GDF-15 levels were higher in subjects with low Body Mass Index (BMI)-adjusted ASM than in those without (median [interquartile range] 831.3 [635.4-1011.4] vs. 583.8 [455.8-771.1] pg/mL, p = 0.018). The GDF-15 level was inversely correlated with BMI-adjusted ASM (r = - 0.377, p < 0.001) and BMD at femur neck (FN-BMD; r = - 0.201, p = 0.015), and positively correlated with percent FM (pFM; r = 0.328, p < 0.001). After adjusting for confounders, the GDF-15 level was inversely associated with BMI-adjusted ASM (ß = -0.250, p = 0.006) and positively associated with pFM (ß = 0.272, p = 0.004), and tended to be inversely associated with FN-BMD (ß = - 0.176, p = 0.076). The area under the receiver-operating characteristic curve of GDF-15 level > 618.4 pg/mL for sarcopenia was 0.706 (95% confidence interval (CI) 0.625-0.779) with a sensitivity of 83.3% and a specificity of 54.5%. Using a GDF-15 level of 618.4 pg/mL as a cut-off, the GDF-15 level was associated with a significantly greater likelihood of sarcopenia (odds ratio [OR] 2.35; 95% CI 1.00-5.51; p = 0.049), obesity (OR 3.28; 95% CI 1.48-7.27; p = 0.001), osteopenic obesity (OR 3.10; 95% CI 1.31-7.30; p = 0.010), and sarcopenic or osteosarcopenic obesity (OR 4.84; 95% CI 0.88-26.69; p = 0.070). These findings support the potential of GDF-15 as a biomarker for age-related changes in muscle, fat, and bone.

The association between the type of anesthesia and hemodynamic instability during pheochromocytoma surgery: a retrospective cohort study.

BACKGROUND: Pheochromocytoma often carries a risk for perioperative hemodynamic instability (HDI). The aim of this study is to evaluate the risk factors of intraoperative HDI during minimally invasive posterior retroperitoneal adrenalectomy (PRA) for pheochromocytoma. MATERIALS AND METHODS: This retrospective study analyzed the prospectively collected data of 172 patients who underwent laparoscopic PRA or robotic PRA for pheochromocytoma between January 2014 and December 2020 at a single tertiary center. The patients were divided into two groups according to the intraoperative hypertensive event of systolic blood pressure (> 160 mmHg). The clinical manifestations and perioperative hemodynamic conditions were analysed. RESULTS: In the multivariate logistic regression analysis, the tumor size (> 3.4 cm) [OR 3.14, 95% confidence intervals (CI) (1.48-6.64), p = 0.003], type of preoperative alpha-blocker (selective type) [OR 3.9, 95% CI (1.52-10.02), p = 0.005], preoperative use of beta-blockers [OR 3.94, 95% CI (1.07-14.49), p = 0.039] and type of anesthesia [total intravenous anesthesia (TIVA) vs. balanced anesthesia (BA)] [OR 2.57, 95% CI (1.23-5.38), p = 0.012] were determined as independent risk factors of intraoperative hypertensive events during minimally invasive adrenalectomy. CONCLUSIONS: The type of anesthesia was independently associated with intraoperative HDI along with larger tumor size, type of preoperative alpha-blocker and the use of preoperative beta-blockers. TIVA increased the risk of intraoperative hypertensive events compared with BA. Thus, the consideration of the type of anesthesia prior to adrenal surgery for pheochromocytoma along with the use of preoperative non-selective alpha-blockers may be beneficial in minimizing the risk of intraoperative HDI.

Free Fatty Acid Receptor 4 (FFA4) Activation Ameliorates Imiquimod-Induced Psoriasis in Mice.

Dietary supplementation with n-3 polyunsaturated fatty acids (n-3 PUFA) has been used as an adjunct therapy for psoriasis due to its anti-inflammatory properties. Free fatty acid receptor 4 (FFA4 or GPR120) is a receptor-sensing n-3 PUFA. In the present study, we examined whether FFA4 acted as a therapeutic target for n-3 PUFA in psoriasis therapy. Experimentally, psoriasis-like skin lesions were induced by treatment with imiquimod for 6 consecutive days. A selective FFA4 agonist, Compound A (30 mg/kg), was used in FFA4 WT and FFA4 KO mice. Imiquimod-induced psoriasis-like skin lesions, which present as erythematous papules and plaques with silver scaling, as well as markedly elevated IL-17/IL-23 cytokine levels in skin tissues, were significantly suppressed by Compound A in FFA4 WT mice, but not in FFA4 KO mice. Enlarged lymph nodes and spleens, as well as imiquimod-induced, elevated IL-17/IL-23 cytokine levels, were also strongly suppressed by Compound A in FFA4 WT mice, but not in FFA4 KO mice. Imiquimod-induced increases in the CD4+IL-17A+ T cell population in lymph nodes and spleens were suppressed by Compound A treatment in FFA4 WT mice; however, this was not seen in FFA4 KO mice. Furthermore, compound A suppressed the differentiation of CD4+ naïve T cells from splenocytes into TH17 cells in an FFA4-dependent manner. In conclusion, we demonstrated that the activation of FFA4 ameliorates imiquimod-induced psoriasis, and the suppression of the differentiation of TH17 cells may partly contribute to its efficacy. Therefore, we suggest that FFA4 could be a therapeutic target for psoriasis therapy.

Activation of Free Fatty Acid Receptor 4 (FFA4) Ameliorates Ovalbumin-Induced Allergic Asthma by Suppressing Activation of Dendritic and Mast Cells in Mice.

Epidemiological and clinical studies have suggested that intake of n-3 polyunsaturated fatty acids (PUFA) reduces the incidence of allergic airway diseases and improves pulmonary function in patients with allergic asthma. However, the pharmacological targets of PUFA have not been elucidated upon. We investigated whether free fatty acid receptor 4 (FFA4, also known as GPR120) is a molecular target for beneficial PUFA in asthma therapy. In an ovalbumin (OVA)-induced allergic asthma model, compound A (a selective agonist of FFA4) was administrated before OVA sensitization or OVA challenge in FFA4 wild-type (WT) and knock-out (KO) mice. Compound A treatment of RBL-2H3 cells suppressed mast cell degranulation in vitro in a concentration-dependent manner. Administration of compound A suppressed in vivo allergic characteristics in bronchoalveolar lavage fluid (BALF) and lungs, such as inflammatory cytokine levels and eosinophil accumulation in BALF, inflammation and mucin secretion in the lungs. Compound A-induced suppression was not only observed in mice treated with compound A before OVA challenge, but in mice treated before OVA sensitization as well, implying that compound A acts on mast cells as well as dendritic cells. Furthermore, this suppression by compound A was only observed in FFA4-WT mice and was absent in FFA4-KO mice, implying that compound A action is mediated through FFA4. Activation of FFA4 may be a therapeutic target of PUFA in allergic asthma by suppressing the activation of dendritic cells and mast cells, suggesting that highly potent specific agonists of FFA4 could be a novel therapy for allergic asthma.

Lumican, an Exerkine, Protects against Skeletal Muscle Loss.

Exerkines are soluble factors secreted by exercised muscles, mimicking the effects of exercise in various organs, including the muscle itself. Lumican is reportedly secreted from muscles; however, its roles in skeletal muscle remain unknown. Herein, we found that lumican mRNA expression in the extensor digitorum longus was significantly higher in exercised mice than in unloading mice, and lumican stimulated myogenesis in vitro. Additionally, lumican knockdown significantly decreased muscle mass and cross-sectional area (CSA) of the muscle fiber in the gastrocnemius muscle of exercised mice. Lumican upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK) and a p38 inhibitor near completely blocked lumican-stimulated myogenesis. Inhibitors for integrin α2ß1 and integrin ανß3 also prevented lumican-stimulated myogenesis. Systemic lumican treatment, administered via the tail vein for 4 weeks, significantly increased relative muscle masses by 36.1% in ovariectomized mice. In addition, intramuscular lumican injection into unloaded muscles for 2 weeks significantly increased muscle mass by 8.5%. Both intravenous and intramuscular lumican treatment significantly increased muscle CSA. Our in vitro and in vivo experiments indicate that lumican is a muscle-secreted exerkine that affords protection against muscle loss by activating p38 MAPK via integrin receptors.

Associations of Circulating Levels of Sphingosine 1-Phosphate with the Trabecular Bone Score and Bone Mineral Density in Postmenopausal Women.

Despite the potential roles of sphingosine 1-phosphate (S1P) as a biomarker of osteoporotic fracture (OF), independent of bone mineral density (BMD) and clinical risk factors (CRFs), its association with bone microarchitecture, a key determinant of bone quality, have not been studied yet. We here investigated the association of S1P with the trabecular bone score (TBS), an index of the bone microarchitecture. The plasma S1P concentrations, TBS, and BMD were measured in the 339 postmenopausal women. The S1P level was inversely correlated with the TBS (γ=-0.096, p=0.049) and BMD at the femur neck (FN-BMD: γ=-0.122, p=0.025) and tended to be inversely correlated the BMD at the total hip (TH-BMD: γ=-0.096, p=0.079), but not at the lumbar spine (LS-BMD). After adjusting for fracture risk assessment tool probabilities of major OF from CRFs, the S1P level was inversely associated with the TBS (ß=-0.096, p=0.049) and FN-BMD (ß=-0.118, p=0.025) and tended to be inversely associated with the TH-BMD (ß=-0.092, p=0.083). Compared with subjects in the lowest S1P tertile, those in the highest S1P tertile had a significantly lower TBS (p=0.032) and BMD at femur (p=0.004-0.036). These findings indicated that a high S1P level in postmenopausal women was inversely associated with the both bone mass and microarchitecture, reflecting the compromised bone strength.

High Circulating Sphingosine 1-Phosphate is a Risk Factor for Osteoporotic Fracture Independent of Fracture Risk Assessment Tool.

Circulating sphingosine 1-phosphate (S1P) levels may be a biomarker for osteoporotic fracture (OF). This study assessed whether the addition of S1P levels to the fracture risk assessment tool (FRAX) could improve predictability of OF risk. Plasma S1P concentrations and FRAX variables were measured in 81 subjects with and 341 subjects without OF. S1P levels were higher in subjects with than those without OF (3.11 ± 0.13 µmol/L vs. 2.65 ± 0.61 µmol/L, P = 0.001). Higher S1P levels were associated with a higher likelihood of OF (odds ratio [OR] = 1.33, 95% confidence interval [CI] = 1.05-1.68), even after adjusting for FRAX probabilities. Compared with the lowest S1P tertile, subjects in the middle (OR = 3.37, 95% CI = 1.58-7.22) and highest (OR = 3.65, 95% CI = 1.66-8.03) S1P tertiles had higher rates of OF after adjustment. The addition of S1P levels to FRAX probabilities improved the area under the receiver-operating characteristics curve (AUC) for OF, from 0.708 to 0.769 (P = 0.013), as well as enhancing category-free net reclassification improvement (NRI = 0.504, 95% CI = 0.271-0.737, P < 0.001) and integrated discrimination improvement (IDI = 0.044, 95% CI = 0.022-0.065, P < 0.001). Adding S1P levels to FRAX probabilities especially in 222 subjects with osteopenia having a FRAX probability of 3.66-20.0% markedly improved the AUC for OF from 0.630 to 0.741 (P = 0.012), as well as significantly enhancing category-free NRI (0.571, 95% CI = 0.221-0.922, P = 0.001) and IDI (0.060, 95% CI = 0.023-0.097, P = 0.002). S1P is a consistent and significant risk factor of OF independent of FRAX, especially in subjects with osteopenia and low FRAX probability.

Free fatty acid receptor 4 (FFA4) activation ameliorates 2,4-dinitrochlorobenzene-induced atopic dermatitis by increasing regulatory T cells in mice.

High dose intake of docosahexaenoic acid showed beneficial effects on atopic dermatitis in patients and was found to increase regulatory T cells in mice, but its molecular target has not been identified. Free fatty acid receptor 4 (FFA4, also known as GPR120) is a receptor sensing polyunsaturated long-chain fatty acids including docosahexaenoic acid. In the present study, we examined whether FFA4 acted as a therapeutic target of docosahexaenoic acid for treating atopic dermatitis. Experimental atopic dermatitis was induced in mice by 2,4-dinitrochlorobenzene (DNCB) sensitization on day 0, followed by repeated DNCB challenges from D7 to D48. The mice were treated with a selective agonist compound A (30 mg· kg-1· d-1, ip) from D19 to D48, and sacrificed on D49. We found that DNCB-induced atopic dermatitis-like skin lesions, i.e. hypertrophy and mast cell infiltration in skin tissues, as well as markedly elevated serum IgE levels. Administration of compound A significantly suppressed the atopic responses in ears and lymph nodes, such as hypertrophy and mast cell infiltration in the ears, enlarged sizes of lymph nodes, and elevated serum IgE and levels of cytokines IL-4, IL-13, IL-17, and IFN-γ in ear tissue. The therapeutic effects of compound A were abolished by FFA4 knockout. Similarly, increased CD4+Foxp3+ regulatory T-cell population in lymph nodes was observed in wide-type mice treated with compound A, but not seen in FFA4-deficient mice. In conclusion, we demonstrate that activation of FFA4 ameliorates atopic dermatitis by increasing CD4+Foxp3+ regulatory T cells, suggesting FFA4 as a therapeutic target for atopic dermatitis.

Coupling factors involved in preserving bone balance.

Coupling during bone remodeling refers to the spatial and temporal coordination of bone resorption with bone formation. Studies have assessed the subtle interactions between osteoclasts and osteoblasts to preserve bone balance. Traditionally, coupling research related to osteoclast function has focused on bone resorption activity causing the release of growth factors embedded in the bone matrix. However, considerable evidence from in vitro, animal, and human studies indicates the importance of the osteoclasts themselves in coupling phenomena, and many osteoclast-derived coupling factors have been identified. These include sphingosine-1-phosphate, vesicular-receptor activator of nuclear factor-κB, collagen triple helix repeat containing 1, and cardiotrophin-1. Interestingly, neuronal guidance molecules, such as slit guidance ligand 3, semaphorin (SEMA) 3A, SEMA4D, and netrin-1, originally identified as instructive cues allowing the navigation of growing axons to their targets, have been shown to be involved in the intercellular cross-talk among bone cells. This review discusses osteoclast-osteoblast coupling signals, including recent advances and the potential roles of these signals as therapeutic targets for osteoporosis and as biomarkers predicting human bone health.

SLIT2 inhibits osteoclastogenesis and bone resorption by suppression of Cdc42 activity.

Axon guidance molecules, originally found to mediate the positioning of axons during nerve development, have been receiving great attention due to their critical roles in regulating bone metabolism. Recently, SLITs, another group of neuronal guidance proteins, were found to be significantly expressed in bone cells. Furthermore, we had provided experimental evidence that SLIT3 is an osteoclast-secreted coupling factor playing an osteoprotective role. Therefore, we hypothesized that SLIT2, a member of the SLIT family, may also affect bone homeostasis. SLIT2 suppressed osteoclast differentiation in a dose-dependent manner and in vitro bone resorption by more than 80%. Consistently, the expression of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), was decreased by SLIT2. The migration and fusion of preosteoclasts were markedly reduced in the presence of SLIT2, suggesting that SLIT2 mainly functions in the early stage of osteoclastogenesis. SLIT2 suppressed Cdc42 activity among small GTPases, whereas Cdc42 overexpression almost completely reversed the SLIT2-mediated suppression of osteoclast differentiation. Among ROBO1-4, the SLIT receptors, ROBO1 and ROBO3 were known to be predominantly expressed in osteoclast lineages. A binding ELISA experiment in mouse bone marrow-derived macrophages showed that ROBO1, rather than ROBO3, was directly associated with SLIT2, and gene silencing with Robo1 siRNA blocked the SLIT2-mediated suppression of osteoclastogenesis. Taken together, our results indicated that SLIT2 inhibits osteoclastogenesis and the resultant bone resorption by decreasing Cdc42 activity, suggesting that this was a potential therapeutic target in metabolic bone diseases related to high bone turnover states.

The effects of myokines on osteoclasts and osteoblasts.

Recently, muscle has received much attention as an endocrine organ regulating other biological targets, including the pancreas, liver, and adipose tissue. Although there is a possibility that muscle-secreting factors biochemically affect bone metabolism in a paracrine manner, the net effects of myokines on the biology of osteoclasts and osteoblasts, particularly on bone mass in vivo, have not yet been thoroughly investigated. Therefore, we performed in vitro as well as animal experiments using conditioned media (CM) collected from C2C12 myoblast and myotube cultures to better understand the interactions between muscle and bone. Compared with non-CM (i.e., control) and myoblast CM, myotube CM markedly inhibited in vitro bone resorption through the suppression of osteoclast differentiation and resorptive activity of individual osteoclasts. Consistently, the expressions of osteoclast differentiation markers, such as tartrate-resistant acid phosphatase (Trap) and calcitonin receptor (Ctr), decreased with myotube CM. Myotube CM significantly stimulated preosteoblast viability and migration and reduced apoptosis, thereby resulting in an increase in calvaria bone formation. Importantly, systemic treatment with myotube CM for 4 weeks increased bone per tissue volume by 30.7% and 19.6% compared with control and myoblast CM, respectively. These results support the hypothesis that muscle plays beneficial roles in bone health via secretion of anabolic factors, in addition to mechanical stimuli, and importantly indicate that muscle-derived factors can be potential therapeutic targets against metabolic bone diseases.

Lack of Association Between Vitamin D and Hand Grip Strength in Asians: A Nationwide Population-Based Study.

Despite the beneficial role and plausible mechanism of vitamin D on skeletal muscle in animal studies, its association in humans remains a controversial issue due to inconsistent clinical results, especially in older Asians. This was a population-based, cross-sectional study from the Korea National Health and Nutrition Examination Surveys, which enrolled 354 men aged ≥ 50 years and 328 postmenopausal women. Hand grip strength (HGS) was measured using a digital grip strength dynamometer. Low muscle strength was defined based on Korean-specific cut-off point of HGS. Serum 25-hydroxyvitamin D [25(OH)D] levels were 19.4 ± 6.7 and 17.1 ± 7.2 ng/mL in men and women, respectively. Among covariates including age, body mass index, lifestyle factors, and protein intake, age was inversely associated with HGS in both men and women, and protein intake (g/day) was positively associated with HGS only in men. However, the independent correlation between serum 25(OH)D and HGS was not observed, regardless of gender. When subjects were divided into three groups [deficient (25(OH)D < 20 ng/mL; 63.8%), insufficient (20 ≤ 25(OH)D < 30 ng/mL; 30.0%), or sufficient (25(OH)D ≥ 30 ng/mL; 6.2%)], there was no significant difference in HGS among these groups in both men and women. Consistently, serum 25(OH)D was not significantly different between subjects with and without low muscle strength, and there was no independent association of serum 25(OH)D with the risk of low muscle strength in both genders. These findings provide clinical evidence that protective role of vitamin D on human muscle metabolism may not be evident at least in older Asians.

The Local and Systemic Interactions Between Muscle and Bone in Postmenopausal Korean Women.

Despite many studies about local and systemic interactions between bone and muscle, the more dominant interaction remains unclear. We aimed to compare the association of skeletal muscle mass with bone mineral density (BMD) at the femur, which seemed more likely affected by local interaction, and the association of skeletal muscle mass with BMD at the lumbar spine (LS-BMD) and the trabecular bone score (TBS), which seemed more likely affected by systemic interaction. In 279 women, we measured the femoral neck BMD (FN-BMD), total hip BMD (TH-BMD), LS-BMD, and TBS. Appendicular skeletal muscle mass (ASM), lean mass (LM), and other LM (OLM; remaining LM excluding ASM) were measured using bioelectrical impedance analysis. ASM (ß = 0.008-0.014, p < 0.001-0.014), OLM (ß = 0.006-0.011, p < 0.001-0.044), and LM (ß = 0.004-0.007, p < 0.001-0.020) were positively associated with FN-BMD and TH-BMD, but not with LS-BMD or TBS. The positive association of ASM, but not of OLM, was stronger than that of LM (p = 0.023). Odd ratios (ORs) with 95% confidence intervals (95% CIs) for osteoporosis were statistically significant for ASM (OR 0.74, 95% CI 0.59-0.93) and marginally significant for OLM (OR 0.80, 95% CI 0.64-1.01) in the femur, but not in the LS. The direct and indirect (through OLM) effects of ASM on BMD were 69.1-72.2% and 27.8-30.9%, respectively. In the conclusion, ASM was more positively associated with FN-BMD, but not with LS-BMD and TBS, than OLM. This suggests stronger effects of local interaction than systemic interaction between muscle and bone.

Change of skeletal muscle mass in patients with pheochromocytoma.

The effects of catecholamine excess due to pheochromocytoma on body composition, including skeletal muscle mass, are unknown. Here, we investigated the effects of catecholamine metabolites on body composition in subjects with pheochromocytoma. After body compositions using bioelectrical impedance analysis, urinary metanephrine (UM), and urinary normetanephrine (UNM) were measured in 16 patients with pheochromocytoma and 224 patients with nonfunctioning adrenal incidentaloma (NFAI), we compared skeletal muscle mass and fat mass (FM) between the two groups. After adjustments for confounders, UM (ß = - 0.171, P = 0.006) and UNM (ß = - 0.249, P < 0.001) levels were correlated inversely with skeletal muscle mass index (SMI), but not FM or percentage FM (pFM), in all subjects. Patients with pheochromocytoma had lower ASM by 7.7% (P = 0.022) and SMI by 6.6% (P = 0.001) than patients with NFAI. Conversely, FM and pFM were not statistically different between the two groups. The odds ratio for low skeletal muscle mass in the presence of pheochromocytoma was 10.33 (95% confidence interval, 2.65-40.22). Our results indicate that patients with pheochromocytoma have a reduced skeletal muscle mass and suggest that catecholamine excess has adverse effects on skeletal muscle metabolism.