Attenuation of LPS-Induced Lung Injury by Benziodarone via Reactive Oxygen Species Reduction.

As overproduction of reactive oxygen species (ROS) causes various diseases, antioxidants that scavenge ROS, or inhibitors that suppress excessive ROS generation, can be used as therapeutic agents. From a library of approved drugs, we screened compounds that reduced superoxide anions produced by pyocyanin-stimulated leukemia cells and identified benzbromarone. Further investigation of several of its analogues showed that benziodarone possessed the highest activity in reducing superoxide anions without causing cytotoxicity. In contrast, in a cell-free assay, benziodarone induced only a minimal decrease in superoxide anion levels generated by xanthine oxidase. These results suggest that benziodarone is an inhibitor of NADPH oxidases in the plasma membrane but is not a superoxide anion scavenger. We investigated the preventive effect of benziodarone on lipopolysaccharide (LPS)-induced murine lung injury as a model of acute respiratory distress syndrome (ARDS). Intratracheal administration of benziodarone attenuated tissue damage and inflammation via its ROS-reducing activity. These results indicate the potential application of benziodarone as a therapeutic agent against diseases caused by ROS overproduction.

Effectiveness of Albumin-Fused Thioredoxin against 6-Hydroxydopamine-Induced Neurotoxicity In Vitro.

Parkinson's disease (PD) is a neurodegenerative disorder caused by oxidative stress-dependent loss of dopaminergic neurons in the substantia nigra and elevated microglial inflammatory responses. Recent studies show that cell loss also occurs in the hypothalamus in PD. However, effective treatments for the disorder are lacking. Thioredoxin is the major protein disulfide reductase in vivo. We previously synthesized an albumin-thioredoxin fusion protein (Alb-Trx), which has a longer plasma half-life than thioredoxin, and reported its effectiveness in the treatment of respiratory and renal diseases. Moreover, we reported that the fusion protein inhibits trace metal-dependent cell death in cerebrovascular dementia. Here, we investigated the effectiveness of Alb-Trx against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. Alb-Trx significantly inhibited 6-OHDA-induced neuronal cell death and the integrated stress response. Alb-Trx also markedly inhibited 6-OHDA-induced reactive oxygen species (ROS) production, at a concentration similar to that inhibiting cell death. Exposure to 6-OHDA perturbed the mitogen-activated protein kinase pathway, with increased phosphorylated Jun N-terminal kinase and decreased phosphorylated extracellular signal-regulated kinase levels. Alb-Trx pretreatment ameliorated these changes. Furthermore, Alb-Trx suppressed 6-OHDA-induced neuroinflammatory responses by inhibiting NF-κB activation. These findings suggest that Alb-Trx reduces neuronal cell death and neuroinflammatory responses by ameliorating ROS-mediated disruptions in intracellular signaling pathways. Thus, Alb-Trx may have potential as a novel therapeutic agent for PD.

Crosstalk of copper and zinc in the pathogenesis of vascular dementia.

Copper and zinc are essential for normal brain functions. Both are localized in presynaptic vesicles and are secreted into synaptic clefts during neuronal excitation. Despite their significance, excesses of copper and zinc are neurotoxic. In particular, excess zinc after transient global ischemia plays a central role in the ischemia-induced neurodegeneration and pathogenesis of vascular type senile dementia. We previously found that sub-lethal concentrations of copper remarkably exacerbated zinc-induced neurotoxicity, and we investigated the molecular pathways of copper-enhanced zinc-induced neurotoxicity. The endoplasmic reticulum stress pathway, the stress-activated protein kinases/c-|Jun amino-terminal kinases pathway, and mitochondrial energy production failure were revealed to be involved in the neurodegenerative processes. Regarding the upstream factors of these pathways, we focused on copper-derived reactive oxygen species and the disruption of calcium homeostasis. Because excess copper and zinc may be present in the synaptic clefts during ischemia, it is possible that secreted copper and copper-induced reactive oxygen species may enhance zinc neurotoxicity and eventually contribute to the pathogenesis of vascular type senile dementia.

FAM210A is a novel determinant of bone and muscle structure and strength.

Osteoporosis and sarcopenia are common comorbid diseases, yet their shared mechanisms are largely unknown. We found that genetic variation near FAM210A was associated, through large genome-wide association studies, with fracture, bone mineral density (BMD), and appendicular and whole body lean mass, in humans. In mice, Fam210a was expressed in muscle mitochondria and cytoplasm, as well as in heart and brain, but not in bone. Grip strength and limb lean mass were reduced in tamoxifen-inducible Fam210a homozygous global knockout mice (TFam210a-/- ), and in tamoxifen-inducible Fam210 skeletal muscle cell-specific knockout mice (TFam210aMus-/- ). Decreased BMD, bone biomechanical strength, and bone formation, and elevated osteoclast activity with microarchitectural deterioration of trabecular and cortical bones, were observed in TFam210a-/- mice. BMD of male TFam210aMus-/- mice was also reduced, and osteoclast numbers and surface in TFam210aMus-/- mice increased. Microarray analysis of muscle cells from TFam210aMus-/- mice identified candidate musculoskeletal modulators. FAM210A, a novel gene, therefore has a crucial role in regulating bone structure and function, and may impact osteoporosis through a biological pathway involving muscle as well as through other mechanisms.

Copper as a Collaborative Partner of Zinc-Induced Neurotoxicity in the Pathogenesis of Vascular Dementia.

Copper is an essential trace element and possesses critical roles in various brain functions. A considerable amount of copper accumulates in the synapse and is secreted in neuronal firings in a manner similar to zinc. Synaptic copper and zinc modulate neuronal transmission and contribute to information processing. It has been established that excess zinc secreted during transient global ischemia plays central roles in ischemia-induced neuronal death and the pathogenesis of vascular dementia. We found that a low concentration of copper exacerbates zinc-induced neurotoxicity, and we have demonstrated the involvement of the endoplasmic reticulum (ER) stress pathway, the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) signaling pathway, and copper-induced reactive oxygen species (ROS) production. On the basis of our results and other studies, we discuss the collaborative roles of copper in zinc-induced neurotoxicity in the synapse and the contribution of copper to the pathogenesis of vascular dementia.

Neurometals in the Pathogenesis of Prion Diseases.

Prion diseases are progressive and transmissive neurodegenerative diseases. The conformational conversion of normal cellular prion protein (PrPC) into abnormal pathogenic prion protein (PrPSc) is critical for its infection and pathogenesis. PrPC possesses the ability to bind to various neurometals, including copper, zinc, iron, and manganese. Moreover, increasing evidence suggests that PrPC plays essential roles in the maintenance of homeostasis of these neurometals in the synapse. In addition, trace metals are critical determinants of the conformational change and toxicity of PrPC. Here, we review our studies and other new findings that inform the current understanding of the links between trace elements and physiological functions of PrPC and the neurotoxicity of PrPSc.

Modulators of Fam210a and Roles of Fam210a in the Function of Myoblasts.

Fam210a is a novel protein regulating muscle mass and strength in mice in vivo. However, detailed effects of Fam210a on the function of myoblasts as well as modulators of Fam210a are still unknown. We, thus, investigated (1) the roles of Fam210a in myoblast differentiation, proliferation, apoptosis and degradation, and (2) the factors that regulate Fam210a expression in murine C2C12 cells. We found that the level of Fam210a mRNA was reduced during myoblast differentiation. Reduction in endogenous Fam210a levels by siRNA suppressed mRNA levels of myogenic factors (Pax7, Myf5, Myogenin, and Mhc) and a muscle degradation factor (Murf1). On the other hand, Fam210a siRNA did not affect mRNA encoding the apoptotic factors Bcl-2 and Bax and the extent of apoptosis as measured by ELISA in C2C12 cells. In contrast, Fam210a siRNA increased the mRNA level of Mmp-12, which induces osteoclastogenesis. Interestingly, insulin and 1,25(OH)2D, which are known to affect cell metabolism and muscle function, significantly increased the level of Fam210a mRNA in a dose-dependent manner. In addition, a PI3-kinase inhibitor and reduction in endogenous levels of the vitamin D receptor (VDR) by siRNA suppressed insulin- and 1,25(OH)2D-induced expression of Fam210a, respectively. In conclusion, Fam210a might enhance myoblast differentiation and proteolysis. Moreover, insulin and 1,25(OH)2D may induce myoblast differentiation and degradation by enhancing the expression of Fam210a.

Carnosine as a Possible Drug for Zinc-Induced Neurotoxicity and Vascular Dementia.

Increasing evidence suggests that the metal homeostasis is involved in the pathogenesis of various neurodegenerative diseases including senile type of dementia such as Alzheimer's disease, dementia with Lewy bodies, and vascular dementia. In particular, synaptic Zn2+ is known to play critical roles in the pathogenesis of vascular dementia. In this article, we review the molecular pathways of Zn2+-induced neurotoxicity based on our and numerous other findings, and demonstrated the implications of the energy production pathway, the disruption of calcium homeostasis, the production of reactive oxygen species (ROS), the endoplasmic reticulum (ER)-stress pathway, and the stress-activated protein kinases/c-Jun amino-terminal kinases (SAPK/JNK) pathway. Furthermore, we have searched for substances that protect neurons from Zn2+-induced neurotoxicity among various agricultural products and determined carnosine (ß-alanyl histidine) as a possible therapeutic agent for vascular dementia.

Amyloids: Regulators of Metal Homeostasis in the Synapse.

Conformational changes in amyloidogenic proteins, such as ß-amyloid protein, prion proteins, and α-synuclein, play a critical role in the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease, prion disease, and Lewy body disease. The disease-associated proteins possess several common characteristics, including the ability to form amyloid oligomers with ß-pleated sheet structure, as well as cytotoxicity, although they differ in amino acid sequence. Interestingly, these amyloidogenic proteins all possess the ability to bind trace metals, can regulate metal homeostasis, and are co-localized at the synapse, where metals are abundantly present. In this review, we discuss the physiological roles of these amyloidogenic proteins in metal homeostasis, and we propose hypothetical models of their pathogenetic role in the neurodegenerative process as the loss of normal metal regulatory functions of amyloidogenic proteins. Notably, these amyloidogenic proteins have the capacity to form Ca2+-permeable pores in membranes, suggestive of a toxic gain of function. Therefore, we focus on their potential role in the disruption of Ca2+ homeostasis in amyloid-associated neurodegenerative diseases.

Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis via NADPH Oxidase-Interleukin 1ß and 6 Pathway in Osteocyte-like Cells.

Homocysteine (Hcy) increases oxidation and inflammation; however, the mechanism of Hcy-induced bone fragility remains unclear. Because selective estrogen modulators (SERMs) have an anti-oxidative effect, SERMs may rescue the Hcy-induced bone fragility. We aimed to examine whether oxidative stress and pro-inflammatory cytokines such as interleukin (IL)-1ß and IL-6 are involved in the Hcy-induced apoptosis of osteocytes and whether bazedoxifene (BZA) inhibits the detrimental effects of Hcy. We used mouse osteocyte-like cell lines MLO-Y4-A2 and Ocy454. Apoptosis was examined by DNA fragmentation ELISA and TUNEL staining, and gene expression was evaluated by real-time PCR. Hcy 5 mM significantly increased expressions of NADPH oxidase (Nox)1, Nox2, IL-1ß, and IL-6 as well as apoptosis in MLO-Y4-A2 cells. Nox inhibitors, diphenyleneiodonium chloride and apocynin, significantly suppressed Hcy-induced IL-1ß and IL-6 expressions. In contrast, an IL-1ß receptor antagonist and an IL-6 receptor monoclonal antibody had no effects on Hcy-induced Nox1 and Nox2 expressions, but significantly rescued Hcy-induced apoptosis. BZA (1 nM-1 µM) and 17ß estradiol 100 nM significantly rescued Hcy-induced apoptosis, while an estrogen receptor blocker ICI 182,780 reversed the effects of BZA and 17ß estradiol. BZA also rescued Hcy-induced apoptosis of Ocy454 cell, and ICI canceled the effect of BZD. Moreover, BZA significantly ameliorated Hcy-induced expressions of Nox1, Nox2, IL-1ß, and IL-6, and ICI canceled the effects of BZA on their expressions. Hcy increases apoptosis through stimulating Nox 1 and Nox 2-IL-1ß and IL-6 expressions in osteocyte-like cells. BZA inhibits the detrimental effects of Hcy on osteocytes via estrogen receptor.

Insulin-Like Growth Factor-I Protects Against the Detrimental Effects of Advanced Glycation End Products and High Glucose in Myoblastic C2C12 Cells.

Previous studies suggested that advanced glycation end products (AGEs) and insulin-like growth factor-I (IGF-I) are involved in the mechanism of diabetes-induced sarcopenia. In this study, we examined effects of treatments with AGEs and/or IGF-I for 24 h on myogenic differentiation and apoptosis in mouse myoblastic C2C12 cells. Real-time PCR and Western blot were performed to investigate mRNA and protein expressions, and apoptosis was examined by using a DNA fragment detection ELISA kit. AGE3 significantly decreased mRNA and protein expressions of MyoD and Myogenin, whereas IGF-I significantly increased them and attenuated the effects of AGE3. AGEs significantly decreased endogenous IGF-I mRNA expression and suppressed IGF-I-induced Akt activation. High glucose (22 mM) significantly increased mRNA expression of Rage, a receptor for AGEs, while IGF-I significantly decreased it. DNA fragment ELISA showed that AGE2 and AGE3 significantly increased apoptosis of C2C12 cells, whereas IGF-I significantly suppressed the AGE2- and AGE3-induced apoptosis. In contrast, high glucose enhanced AGE3-induced apoptosis. IGF-I significantly attenuated the effects of high glucose plus AGE3 on the mRNA and protein expressions of MyoD and Myogenin as well as the apoptosis. These findings indicate that AGEs inhibit myogenic differentiation and increase apoptosis in C2C12 cells, and that high glucose increases RAGE and enhances the AGE3-induced apoptosis, suggesting that AGEs and high glucose might contribute to the reduction of muscle mass and function. Moreover, IGF-I attenuated the detrimental effects of AGEs and high glucose in myoblastic cells; thus, IGF-I-Akt signal could be a therapeutic target of DM-induced sarcopenia.

Overweight and underweight are risk factors for vertebral fractures in patients with type 2 diabetes mellitus.

The aim of this cross-sectional study was to examine the association between body mass index (BMI) and the prevalence of vertebral fracture (VF) in Japanese patients with type 2 diabetes (T2DM). A total of 798 patients with T2DM were enrolled. VF was determined semi-quantitatively using lateral X-ray films. The association between BMI quartiles (Q1: ≤ 21.2 kg/m2, Q2: 21.3-23.4 kg/m2, Q3: 23.5-25.8 kg/m2, Q4: 25.9≤ kg/m2) and the presence of VF was examined. Multiple logistic regression analyses adjusted for age, sex, diabetes duration, hemoglobin A1c (HbA1c), estimated glomerular filtration rate, and albumin showed that Q1, Q3, and Q4 were significantly associated with an increased VF risk compared to Q2, which served as a reference [Q1; odds ratio (OR) = 1.91, 95% confidence interval (CI) 1.24-2.95, p = 0.004, Q3; OR = 1.65, 95% CI 1.07-2.55, p = 0.023, and Q4; OR = 2.18, 95% CI 1.39-3.41, p < 0.001]. Moreover, these associations remained significant after additional adjustment for femoral neck T-score, a bone resorption marker, urinary N-terminal cross-linked telopeptide of type-I collagen, and use of insulin and thiazolidinedione. Our study shows for the first time that both overweight and underweight were associated with the bone mineral density (BMD)-independent risk of VF in patients with T2DM. Therefore, body weight control should be considered as a protective measure against diabetes-related bone fragility.

Osteoporosis and vertebral fracture are associated with deterioration of activities of daily living and quality of life in patients with type 2 diabetes mellitus.

Patients with type 2 diabetes mellitus (T2DM) have an increased risk of fragility fracture. However, whether diabetes-related osteoporosis independently contributes to the deterioration of activities of daily living (ADLs) and quality of life (QOL) is unclear. This cross-sectional study investigated the association between osteoporosis, ADLs, and QOL in 309 patients with T2DM. ADLs and QOL were assessed using Barthel Index (BI) and a SF-36 questionnaire. Multiple logistic regression analyses adjusted for age, gender, T2DM duration, body mass index, hemoglobin A1c, estimated GFR, diabetic neuropathy, retinopathy, nephropathy, cardiovascular disease, cerebrovascular disease, peripheral artery disease, and anti-diabetic treatments were conducted. The number of patients with osteoporosis or vertebral fracture was 166 (53.7%) and 118 (38.2%), respectively. Osteoporosis was significantly associated with lower general health (GH), social functioning (SF), and role emotional (RE) (OR 2.56, 1.79, and 1.92, respectively; all p values < 0.05 at least) and marginally associated with lower BI (OR 2.39, p = 0.068). Moreover, the presence of vertebral fracture grade 2 or 3 was significantly associated with lower BI, bodily pain (BP), GH, vitality, SF, and RE (OR 2.58, 2.01, 3.64, 1.99, 2.18, and 1.97, respectively; all p values < 0.05 at least). Osteoporosis and severe vertebral fracture were associated with the deterioration of ADLs and QOL independently of other diabetic complications. Therefore, the management of diabetes-related osteoporosis is an important strategy to avoid the deterioration of ADLs and QOL in T2DM.

Chemical modification-mediated optimisation of bronchodilatory activity of mepenzolate, a muscarinic receptor antagonist with anti-inflammatory activity.

The treatment for patients with chronic obstructive pulmonary disease (COPD) usually involves a combination of anti-inflammatory and bronchodilatory drugs. We recently found that mepenzolate bromide (1) and its derivative, 3-(2-hydroxy-2, 2-diphenylacetoxy)-1-(3-phenoxypropyl)-1-azoniabicyclo[2.2.2]octane bromide (5), have both anti-inflammatory and bronchodilatory activities. We chemically modified 5 with a view to obtain derivatives with both anti-inflammatory and longer-lasting bronchodilatory activities. Among the synthesized compounds, (R)-(-)-12 ((R)-3-(2-hydroxy-2,2-diphenylacetoxy)-1-(3-phenylpropyl)-1-azoniabicyclo[2.2.2]octane bromide) showed the highest affinity in vitro for the human muscarinic M3 receptor (hM3R). Compared to 1 and 5, (R)-(-)-12 exhibited longer-lasting bronchodilatory activity and equivalent anti-inflammatory effect in mice. The long-term intratracheal administration of (R)-(-)-12 suppressed porcine pancreatic elastase-induced pulmonary emphysema in mice, whereas the same procedure with a long-acting muscarinic antagonist used clinically (tiotropium bromide) did not. These results suggest that (R)-(-)-12 might be therapeutically beneficial for use with COPD patients given the improved effects seen against both inflammatory pulmonary emphysema and airflow limitation in this animal model.

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway.

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.

[ASBMR topics from clinical research(osteoporosis and sarcopenia).]

There are a lot of progressive topics about osteoporosis and sarcopenia in 2018 ASBMR Annual Meeting, involving an association between diabetes and bone microarchitecture, associations between atypical femoral fractures and bisphosphonate drug holidays as well as pre-treatment bone mineral density, an effect of combined denosumab and high-dose teriparatide on bone parameters, and relationships between muscle and deuterated creatine, a selective androgen receptor modulator, and high-dose vitamin D supplementation.

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.

Pyruvic acid prevents Cu2+/Zn2+-induced neurotoxicity by suppressing mitochondrial injury.

Zinc (Zn) is known as a co-factor for over 300 metalloproteins or enzymes, and has essential roles in many physiological functions. However, excessively high Zn concentrations are induced in pathological conditions such as interruption of blood flow in stroke or transient global ischemia-induced neuronal cell death. Furthermore, we recently found that copper (Cu2+) significantly exacerbates Zn2+ neurotoxicity in mouse hypothalamic neuronal cells, suggesting that Zn2+ interaction with Cu2+ is important for the development of neurological disease. Meanwhile, organic acids such as pyruvic acid and citric acid are reported to prevent neuronal cell death induced by various stresses. Thus, in this study, we focused on organic acids and searched for compounds that inhibit Cu2+/Zn2+-induced neurotoxicity. Initially, we examined the protective effect of various organic acids on Cu2+/Zn2+-induced neurotoxicity, and found that pyruvic acid clearly suppresses Cu2+/Zn2+-induced neurotoxicity in GT1-7 cells. Next, we examined the protective mechanisms of pyruvic acid against Cu2+/Zn2+-induced neurotoxicity. Specifically, we examined the possibilities that pyruvic acid chelates Cu2+ and Zn2+ or suppresses the ER stress response, but found that neither was suppressed by pyruvic acid treatment. In contrast, pyruvic acid significantly suppressed cytochrome c release into cytoplasm, an index of mitochondrial injury, in a dose-dependent manner. These results suggest that pyruvic acid prevents Cu2+/Zn2+-induced neuronal cell death by suppressing mitochondrial injury. Based on our results, we assume that pyruvic acid may be therapeutically beneficial for neurological diseases involving neuronal cell death such as vascular dementia.

Osteoblast AMP-activated protein kinase regulates glucose metabolism and bone mass in adult mice.

Previous 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 glucose metabolism and bone mass regulation in adult mice. Here, we used the inducible Cre system to control the onset of Ampk disruption after birth by removing doxycycline supplementation. We conditionally inactivated Ampk in osterix (Osx)-expressing cells in 3-week-old Ampk-/- mice. After 6 months of Ampk inactivation, the Ampk-/- mice displayed lower serum osteocalcin levels as well as glucose intolerance and insulin resistance, as indicated by glucose tolerance and insulin tolerance tests, respectively, when compared with wild-type mice. After 18 months of Ampk inactivation, micro computed tomography showed significant reductions in trabecular bone volume and cortical bone thickness in the femur of Ampk-/- mice when compared with wild-type mice. Moreover, bone stiffness was significantly lower in Ampk-/- mice than in wild-type mice. This is the first study to show that osteoblast AMPK plays an important roles in glucose metabolism and in maintaining trabecular bone volume, cortical thickness, and bone strength in adult mice.

Inhibition of adenosine monophosphate-activated protein kinase suppresses bone morphogenetic protein-2-induced mineralization of osteoblasts via Smad-independent mechanisms.

Previous studies showed that adenosine monophosphate-activated protein kinase (AMPK), which plays as an intracellular energy sensor, promotes the differentiation and mineralization of osteoblasts via enhancing expression of bone morphogenetic protein (BMP)-2, which is a potent inducer of osteoblastogenesis. Thus, the aim of this study was to examine the roles of AMPK in BMP-2-induced osteoblastogenesis. We used a murine osteoblastic cell line MC3T3-E1 and a murine marrow stromal cell line ST2. BMP-2 (50 and 100 ng/mL) stimulated alkaline phosphatase (ALP) activity and enhanced mineralization of MC3T3-E1 cells, while the effects of BMP-2 were partly abolished by an inhibitor of AMPK, ara-A (0.1 mM). Real-time PCR showed that BMP-2 significantly increased the mRNA expressions of Alp, osteocalcin (Ocn), Runx2, Osterix and Dlx-5 in MC3T3-E1 cells, while co-incubation of ara-A significantly decreased the BMP-2-stimulated expression of Alp, Ocn, and Runx2. Moreover, co-incubation of ara-A suppressed the BMP-2-induced upregulation of Alp and Ocn in ST2 cells. Western blot analysis showed that BMP-2 phosphorylated Smad1/5 although it did not affect AMPK phosphorylation in MC3T3-E1 cells. Furthermore, a BMP receptor inhibitor LDN-193189 inhibited the phosphorylation of Smad1/5, but did not affect AMPK. In addition, co-incubation of ara-A did not affect BMP-2-induced phosphorylation of Smad1/5. These findings suggest that the inhibition of AMPK activation reduces the osteo-inductive effects of BMP-2 by decreasing the expression of Alp, Ocn, and Runx2 through Smad-independent mechanisms in osteoblastic cells.