Masakazu Notsu

Advanced glycation end product 3 (AGE3) suppresses the mineralization of mouse stromal ST2 cells and human mesenchymal stem cells by increasing TGF-β expression and secretion.

In diabetic patients, advanced glycation end products (AGEs) cause bone fragility because of deterioration of bone quality. We previously showed that AGEs suppressed the mineralization of mouse stromal ST2 cells. TGF-β is abundant in bone, and enhancement of its signal causes bone quality deterioration. However, whether TGF-β signaling is involved in the AGE-induced suppression of mineralization during the osteoblast lineage remains unknown. We therefore examined the roles of TGF-β in the AGE-induced suppression of mineralization of ST2 cells and human mesenchymal stem cells. AGE3 significantly (P < .001) inhibited mineralization in both cell types, whereas transfection with small interfering RNA for the receptor for AGEs (RAGEs) significantly (P < .05) recovered this process in ST2 cells. AGE3 increased (P < .001) the expression of TGF-β mRNA and protein, which was partially antagonized by transfection with RAGE small interfering RNA. Treatment with a TGF-β type I receptor kinase inhibitor, SD208, recovered AGE3-induced decreases in osterix (P < .001) and osteocalcin (P < .05) and antagonized the AGE3-induced increase in Runx2 mRNA expression in ST2 cells (P < .001). Moreover, SD208 completely and dose dependently rescued AGE3-induced suppression of mineralization in both cell types. In contrast, SD208 intensified AGE3-induced suppression of cell proliferation as well as AGE3-induced apoptosis in proliferating ST2 cells. These findings indicate that, after cells become confluent, AGE3 partially inhibits the differentiation and mineralization of osteoblastic cells by binding to RAGE and increasing TGF-β expression and secretion. They also suggest that TGF-β adversely affects bone quality not only in primary osteoporosis but also in diabetes-related bone disorder.

Activation of AMP-activated protein kinase protects against homocysteine-induced apoptosis of osteocytic MLO-Y4 cells by regulating the expressions of NADPH oxidase 1 (Nox1) and Nox2

BACKGROUND Elevated plasma homocysteine (Hcy) level is associated with the risk of osteoporotic fracture. While Hcy increases oxidative stress, AMP-activated protein kinase (AMPK) activation ameliorates it. This study aimed to investigate whether Hcy induces apoptosis of osteocytic MLO-Y4 cells through regulating expressions of oxidant and anti-oxidant enzymes and determine the effects of AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and metformin on the Hcy-induced apoptosis of the cells. RESULTS DNA fragment ELISA and TUNEL staining assays showed that Hcy treatments (0.1-5.0 mM) induced apoptosis of MLO-Y4 cells in a dose-dependent manner. The detrimental effect of Hcy was partly but significantly reversed by an antioxidant (N-acetylcysteine) and NADPH oxidase (Nox) inhibitors (apocynin and diphenyleneiodonium). In addition, treatment with AICAR (0.05-0.1 mM) and metformin (10-100 μM) ameliorated Hcy-induced apoptosis of the cells. The favorable effect of metformin on Hcy-induced apoptosis was completely canceled by an AMPK inhibitor Ara-A. Hcy increased the expression levels of Nox1 and Nox2, while it had no effects on the expressions of Nox4 or the anti-oxidant enzymes, superoxide dismutase 1 and 2. Hcy-induced increases in the expressions of Nox1 and Nox2 decreased significantly by treatments with AICAR. CONCLUSION These findings suggest that Hcy induces apoptosis of osteocytes by increasing the expressions of Nox1 and Nox2, and AMPK activation by AICAR and metformin effectively prevents the detrimental reactions. Thus, AMPK activation may be a potent therapeutic candidate for preventing Hcy-induced osteocyte apoptosis and the resulting bone fragility.

Visceral fat obesity increases serum DPP-4 levels in men with type 2 diabetes mellitus.

OBJECTIVE The relationship between serum DPP-4 level and visceral fat mass is still unclear in type 2 diabetes mellitus (T2DM). This study thus aimed to examine the association of visceral fat accumulation and metabolic syndrome with serum DPP-4 levels in T2DM. METHODS Visceral and subcutaneous fat areas were evaluated by performing computed tomography scan in 135 men with T2DM, who had never taken DPP-4 inhibitors or GLP-1 receptor agonists. We investigated the association between serum DPP-4 levels and visceral fat area as well as the presence of metabolic syndrome. RESULTS Multiple regression analysis adjusted for age, duration of T2DM, body mass index, serum creatinine, and HbA1c showed that serum DPP-4 levels were positively associated with visceral fat area (β=0.25, p=0.04), but not subcutaneous fat area (β=-0.18, p=0.13). In logistic regression analyses adjusted for the confounding factors described above, serum DPP-4 levels were positively associated with visceral fat obesity and metabolic syndrome [odds ratio (OR)=1.63, 95% confidence interval (CI)=1.00-2.66 per standard deviation (SD) increase, p=0.04; OR=1.77, 95%CI=1.09-2.88 per SD increase, p=0.02, respectively]. CONCLUSIONS The present study showed that serum DPP-4 level was positively and specifically associated with accumulation of visceral fat and the presence of metabolic syndrome in men with T2DM.

Primary Aldosteronism as a Risk Factor for Vertebral Fracture

Context Some observational studies have revealed an association between excessive aldosterone levels and reduced bone mineral density (BMD). However, whether patients with primary aldosteronism (PA) are at higher risk of fracture than healthy individuals remains unclear. Objective This study aimed to clarify whether PA represents a risk factor for vertebral fracture (VF). Design and Patients We enrolled 56 patients with PA and 56 age- and sex-matched healthy individuals. Serum and urinary biological parameters, BMD, and presence of VFs were evaluated in both groups. We compared parameters between PA and control participants and performed multiple logistic regression analyses after adjustments for variables. Results Patients with PA showed higher systolic and diastolic blood pressure, higher hemoglobin A1c (HbA1c) and triglycerides, higher urinary calcium-to-creatinine ratio, and lower high-density lipoprotein cholesterol than controls (P < 0.05, each). Prevalence of VFs was significantly higher in patients with PA (44.6%) than in controls (23.2%, P < 0.05). Patients with PA showed severe fracture more frequently than controls. Multivariate logistic regression analyses adjusted for age, sex, and body mass index identified PA as being associated with the presence of VFs (odds ratio, 3.13; 95% confidence interval, 1.30 to 7.51; P < 0.05). This association remained statistically significant after further adjustment for systolic and diastolic blood pressure, HbA1c, triglycerides, and high-density lipoprotein cholesterol but not after adjustment for calcium-to-creatinine ratio and BMD. Conclusions We identified PA as a risk factor for VF, independent of blood pressure, HbA1c, and lipid profile. Fracture severity was significantly higher in patients with PA than in age- and sex-matched controls.

Simvastatin rescues homocysteine-induced apoptosis of osteocytic MLO-Y4 cells by decreasing the expressions of NADPH oxidase 1 and 2

Clinical studies have shown that hyperhomocysteinemia is associated with bone fragility. Homocysteine (Hcy) induces apoptosis of osteoblastic cell lineage by increasing oxidative stress, which may contribute to Hcy-induced bone fragility. Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, ameliorate oxidative stress by regulating oxidant and anti-oxidant enzymes. However, the effects of statins on Hcy-induced apoptosis of osteocytes are unknown. This study was thus aimed to investigate whether or not statins prevent Hcy-induced apoptosis of osteocytic MLO-Y4 cells and regulate NADPH oxidase (Nox) expression. TUNEL staining showed that 5 mM Hcy induced apoptosis of MLO-Y4 cells, and that co-incubation of 10(-9) or 10(-8) M simvastatin significantly suppressed the apoptotic effect. Moreover, we confirmed the beneficial effect of simvastatin against Hcys apoptotic effect by using a DNA fragment ELISA assay. However, TUNEL staining showed no significant effects of pravastatin, a hydrophilic statin, on the Hcy-induced apoptosis. Real-time PCR showed that Hcy increased the mRNA expressions of Nox1 and Nox2, whereas simvastatin inhibited the stimulation of Nox1 and Nox2 expressions by Hcy. In contrast, neither Hcy nor simvastatin had any effect on Nox4 expression. These findings indicate that simvastatin prevents the detrimental effects of Hcy on the apoptosis of osteocytes by regulating the expressions of Nox1 and Nox2, suggesting that statins may be beneficial for preventing Hcy-induced osteocyte apoptosis and the resulting bone fragility.

Long-term Efficacy and Safety of Sitagliptin in Elderly Patients with Type 2 Diabetes Mellitus

Objective We herein conducted a retrospective study to evaluate the long-term efficacy and safety of sitagliptin treatment in elderly patients with type 2 diabetes mellitus. Methods We analyzed the changes in glycemic control in 112 Japanese type 2 diabetes patients over 65 years of age treated with 50 mg/day sitagliptin. Hemoglobin A1c (HbA1c) levels, liver and kidney functions, and usage of hypoglycemic agents were recorded for 24 months. Results HbA1c levels were significantly decreased, and the significance of HbA1c reduction was maintained during the observation period [from 7.7±1.1% to 7.2±0.7% (p<0.001) at the end of observational period]. The %change in HbA1c levels was significantly and negatively correlated with the baseline HbA1c levels (r=-0.51, p<0.001), but not with age, duration of diabetes, or the estimated glomerular filtration rate (eGFR). No patient experienced severe hypoglycemia episodes, and aspartate transaminase, alanine transaminase, gamma-glutamyl transpeptidase, and the eGFR remained unchanged. The dose of sulfonylurea was finally decreased in 72% of patients treated with sulfonylurea. Conclusion Sitagliptin treatment continually decreases the HbA1c level for 24 months and is useful to reduce the dose of sulfonylurea in elderly patients with type 2 diabetes.

Activation of AMP-activated protein kinase decreases receptor activator of NF-κB ligand expression and increases sclerostin expression by inhibiting the mevalonate pathway in osteocytic MLO-Y4 cells

BACKGROUND AMP-activated protein kinase (AMPK) plays important roles in bone metabolism; however, little is known about its role in osteocytes. This study investigated the effects of AMPK activation on the expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in osteocytes. RESULTS Real-time PCR showed that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) significantly decreased the expression of Rankl in a dose- and time-dependent manner and significantly increased the expression of Sost, the gene encoding sclerostin, in osteocytic MLO-Y4 cells. Western blotting confirmed that AICAR decreased RANKL protein levels and increased sclerostin levels. In addition, suppression of AMPKα1 by siRNA significantly increased the expression of Rankl on 4 days after the transfection of siRNA, while Sost expression was not changed. Simvastatin, an inhibitor of HMG-CoA reductase, significantly decreased Rankl expression and increased Sost expression in MLO-Y4 cells. Supplementation with mevalonate or geranylgeranyl pyrophosphate, which are downstream metabolites of HMG-CoA reductase, significantly reversed the effects of AICAR. CONCLUSION These findings indicated that AMPK regulated RANKL and sclerostin expression through the mevalonate pathway in osteocytes.

Osteoblast AMP-Activated Protein Kinase Regulates Postnatal Skeletal Development in Male Mice

Studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in bone development. Thus, to investigate in vivo roles of osteoblast AMPK, we conditionally inactivated Ampk in osterix (Osx)-expressing cells by crossing Osx-Cre mice with floxed AMPKα1 to generate mice lacking AMPKα1 in osteoblasts (Ampk-/- mice). Compared with wild-type and Ampk+/- mice, Ampk-/- mice displayed retardation of postnatal bone development, although bone deformity was not observed at birth. Microcomputed tomography showed significant reductions in trabecular bone volume, cortical bone length, and density, as well as increased cortical porosity in femur as well as development defects of skull in 8-week-old Ampk-/- mice. Surprisingly, histomorphometric analysis demonstrated that the number of osteoclasts was significantly increased, although bone formation rate was not altered. Loss of trabecular network connections and mass, as well as shortened growth plates and reduced thickness of cartilage adjacent to the growth plate, was observed in Ampk-/- mice. In primary cultured osteoblasts from calvaria, the expressions of alkaline phosphatase, type 1 collagen, osteocalcin, bone morphogenetic protein 2, Runx2, and osterix were significantly inhibited in Ampk-/- osteoblasts, whereas the expression of receptor activator of nuclear κB ligand (RANKL) and the RANKL/osteoprotegerin ratio were significantly increased. These findings indicate that osteoblastic AMPK plays important roles in bone development in vivo and that deletion of AMPK in osteoblasts decreases osteoblastic differentiation and enhances bone turnover by increasing RANKL expression.

Glucose uptake inhibition decreases expressions of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteocalcin in osteocytic MLO-Y4-A2 cells

Bone and glucose metabolism are closely associated with each other. Both osteoblast and osteoclast functions are important for the action of osteocalcin, which plays pivotal roles as an endocrine hormone regulating glucose metabolism. However, it is unknown whether osteocytes are involved in the interaction between bone and glucose metabolism. We used MLO-Y4-A2, a murine long bone-derived osteocytic cell line, to investigate effects of glucose uptake inhibition on expressions of osteocalcin and bone-remodeling modulators in osteocytes. We found that glucose transporter 1 (GLUT1) is expressed in MLO-Y4-A2 cells and that treatment with phloretin, a GLUT inhibitor, significantly inhibited glucose uptake. Real-time PCR and Western blot showed that phloretin significantly and dose-dependently decreased the expressions of RANKL and osteocalcin, whereas osteoprotegerin or sclerostin was not affected. Moreover, phloretin activated AMP-activated protein kinase (AMPK), an intracellular energy sensor. Coincubation of ara-A, an AMPK inhibitor, with phloretin canceled the phloretin-induced decrease in osteocalcin expression, but not RANKL. In contrast, phloretin suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, and treatments with the p38 inhibitor SB203580 and the MEK inhibitor PD98059, but not the JNK inhibitor SP600125, significantly decreased expressions of RANKL and osteocalcin. These results indicate that glucose uptake by GLUT1 is required for RANKL and osteocalcin expressions in osteocytes, and that inhibition of glucose uptake decreases their expressions through AMPK, ERK1/2, and p38 MAPK pathways. These findings suggest that lowering glucose uptake into osteocytes may contribute to maintain blood glucose levels by decreasing osteocalcin expression and RANKL-induced bone resorption.

Long-term efficacy and safety of vildagliptin add-on therapy in type 2 diabetes mellitus with insulin treatment

BACKGROUND The use of dipeptidyl peptidase (DPP)-4 inhibitors in patients with type 2 diabetes treated with insulin may be beneficial. However, the long-term efficacy and safety of vildagliptin add-on therapy in these patients remains unclear. SUBJECTS AND METHODS A total of 73 patients with type 2 diabetes treated with insulin were randomly assigned to receive either add-on therapy of vildagliptin (n=37) or conventional therapy without DPP-4 inhibitors (n=36) for glucose control. Hemoglobin A1c (HbA1c) levels, dose and number of insulin injections, number of hypoglycemia episodes, and liver and renal function were monitored for 2years. RESULTS The baseline characteristics of subjects, including age, dose of insulin injections, or HbA1c levels, did not differ between the two groups. In the vildagliptin group, HbA1c levels significantly decreased and the significance of HbA1c reduction was maintained for 24months (from 8.0±1.2% to 7.4±1.0%, p<0.05, at the end of observational period). In addition, the dose and number of insulin injections significantly reduced (-5.6units, p<0.01, and -0.9 times, p<0.001). However, these parameters were unchanged in the control group. The number of patients who experienced three or more episodes of hypoglycemia per year was significantly lower in the vildagliptin group (n=4) than in the control group (n=11) (odds ratio, 0.28; 95% confidence interval, 0.08-0.97; p<0.05). CONCLUSION Vildagliptin as an add-on to insulin treatment for 24months was well tolerated and led to sustained reductions in HbA1c, the dose and number of insulin injections, and the risk of hypoglycemia.