Newly isolated Penicillium oxalicum A592-4B secretes enzymes that degrade milled rice straw with high efficiency.

An enzyme producing micro-organism, which can directly saccharify rice straw that has only been crushed without undergoing the current acid or alkaline pretreatment, was found. From the homology with the ITS, 28S rDNA sequence, the strain named A592-4B was identified as Penicillium oxalicum. Activities of the A592-4B enzymes and commercial enzyme preparations were compared by Novozymes Cellic CTec2 and Genencore GC220. In the present experimental condition, activity of A592-4B enzymes was 2.6 times higher than that of CTec2 for degrading milled rice straw. Furthermore, even when a quarter amount of A592-4B enzyme was applied to the rice straw, the conversion rate was still higher than that by CTec2. By utilizing A592-4B enzymes, improved lignocellulose degradation yields can be achieved without pre-treatment of the substrates; thus, contributing to cost reduction as well as reducing environmental burden.

Sodium dodecyl sulfate and sodium dodecyl benzenesulfonate are ligands for peroxisome proliferator-activated receptor γ.

Sodium dodecyl sulfate (SDS) and sodium dodecyl benzenesulfonate (SDBS) are widely used anionic surfactants in household, industrial, and institutional cleaners. Although there are many reports of their toxic effects, few studies have focused on the pharmacological properties of these surfactants. Peroxisome proliferator-activated receptor (PPAR) γ is a transcriptional factor belonging to the nuclear receptor superfamily. The ligands of PPARγ regulate its transcriptional activity and modulate many biological functions, including adipocyte differentiation and lipid metabolism. In this study, we investigated the ligand activities of SDS and SDBS for nuclear receptors using time-resolved fluorescence resonance energy transfer-based coactivator recruitment assays. SDS and SDBS showed selective ligand activities for PPARγ and these ligand activities were eliminated by a PPARγ antagonist. SDS and SDBS also promoted adipocyte differentiation accompanied by upregulation of adipocyte-specific gene expression in 3T3-L1 preadipocytes. These findings reveal novel actions of anionic alkyl surfactants as PPARγ ligands.

Nobiletin improves obesity and insulin resistance in high-fat diet-induced obese mice.

Nobiletin (NOB) is a polymethoxylated flavone present in citrus fruits and has been reported to have antitumor and anti-inflammatory effects. However, little is known about the effects of NOB on obesity and insulin resistance. In this study, we examined the effects of NOB on obesity and insulin resistance, and the underlying mechanisms, in high-fat diet (HFD)-induced obese mice. Obese mice were fed a HFD for 8 weeks and then treated without (HFD control group) or with NOB at 10 or 100mg/kg. NOB decreased body weight gain, white adipose tissue (WAT) weight and plasma triglyceride. Plasma glucose levels tended to decrease compared with the HFD group and improved plasma adiponectin levels and glucose tolerance. Furthermore, NOB altered the expression levels of several lipid metabolism-related and adipokine genes. NOB increased the mRNA expression of peroxisome proliferator-activated receptor (PPAR)-γ, sterol regulatory element-binding protein-1c, fatty acid synthase, stearoyl-CoA desaturase-1, PPAR-α, carnitine palmitoyltransferase-1, uncoupling protein-2 and adiponectin, and decreased the mRNA expression of tumor necrosis factor-α and monocyte chemoattractant protein-1 in WAT. NOB also up-regulated glucose transporter-4 protein expression and Akt phosphorylation and suppressed IκBα degradation in WAT. Taken together, these results suggest that NOB improves adiposity, dyslipidemia, hyperglycemia and insulin resistance. These effects may be elicited by regulating the expression of lipid metabolism-related and adipokine genes, and by regulating the expression of inflammatory makers and activity of the insulin signaling pathway.

Fargesin, a component of Flos Magnoliae, stimulates glucose uptake in L6 myotubes.

Flos Magnoliae (FM) is a commonly used Chinese medicinal herb for symptomatic relief of allergic rhinitis, sinusitis and headache. Although several FM species have been used as substitutes or adulterants for clinical use, possible differences in their pharmacological actions have not been reported. To confirm the effects of FM on skeletal muscle glucose metabolism, we tested the effects of several compounds isolated from FM on glucose uptake by L6 myotubes. We found that fargesin, a component of FM, dose-dependently stimulated glucose consumption in L6 myotubes, which was accompanied by enhanced glucose transporter (GLUT)-4 translocation to the cell surface. Fargesin-stimulated glucose uptake was blocked by wortmannin, a phosphatidylinositol-3 kinase (PI3 K) inhibitor. Fargesin stimulated Akt phosphorylation, a key component in the insulin signaling pathway, which was completely inhibited by wortmannin. Here, we demonstrated that fargesin, a bioactive component of Flos Magnoliae, increases basal glucose uptake and GLUT4 translocation in L6 myotubes by activating the PI3 K-Akt pathway.

Penicillium sp. strain that efficiently adsorbs lignosulfonate in the presence of sulfate ion.

Lignin is one of the major water insoluble substances that support the physical properties of plants and can be solubilized by sulfite or alkaline treatment in accordance with pulpification. The lignin derivatives produced by both the sulfite and the kraft processes are called lignosulfonate (LS) and kraft lignin (KL), respectively, and both derivatives show a broad spectrum of optical absorbance from ultraviolet to visible light. When the spore suspension of an isolated Penicillium sp., Penicillium sp. A, was inoculated to a medium containing 0.1% commercial LS, absorbance at 480 nm (A480) almost completely disappeared after 5 days of cultivation. Maximum decolorization of the culture broth was observed when the microbe was cultured in the 0.8% LS medium reaching 88%, and the amount of LS removed was calculated to be 7 g/L. In a similar assay with the dark-liquid containing KL, 80% of the A480 of a 20% (v/v) dark-liquid medium disappeared after 5 days of culturing and the amount of KL removed was calculated to be 2.9 g/L. These values significantly exceeded the previously reported amounts with respect to substrate concentration and decolorization. Furthermore, since similar results were obtained in the cases of both LS and KL, it is expected that the present strain is able to non-specifically adsorb a wide range of lignin derivatives, because most of the colored substances were recovered in the culture sediments. Thus, the strain can be used to clean up waste fluids containing water soluble lignin derivatives, adsorb lignin derivatives in waste fluids before dehydration.

Discovery of novel rules for G-quadruplex-forming sequences in plants by using bioinformatics methods.

The G-quadruplex is one of the most frequently studied secondary DNA structures and consists of 4 guanine residues that interact through Watson-Crick and Hoogsteen pairing. The G-quadruplex formation is thought to be a molecular switch for gene expression. Genome-wide analyses of G-quadruplexes have been published for many species; however, only one genome-wide analysis of G-quadruplexes in plants has been reported. Here, we propose a new approach involving a two-step procedure for identifying G-quadruplex-forming sequences (potential G4 DNA motif regions: G4MRs) and classifying positional relationships between G4MRs and genes. By using this approach, we exhaustively searched for G4MRs in the whole genomes of 8 species: Arabidopsis thaliana, Oryza sativa subsp. japonica, Populus trichocarpa, Vitis vinifera, Homo sapiens, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans. We classified genes on the basis of their positional relationships to their proximal G4MRs. We identified novel rules for G4MRs in plants, such as G4MR-enrichment in the template strands at transcription start sites (TSSs). Next, we focused on the template strands of TSSs and conducted gene ontology (GO) analysis of genes proximal to G4MRs. We identified GO terms such as chloroplast and nucleosome (or histone) in O. sativa. Although these terms were strongly associated in O. sativa, weak associations were identified in other plants. These results will be helpful for elucidating the functional roles of G4 DNA.

Fargesin improves lipid and glucose metabolism in 3T3-L1 adipocytes and high-fat diet-induced obese mice.

This study examined the effects of fargesin, a neolignan isolated from Magnolia plants, on obesity and insulin resistance and the possible mechanisms involved in these effects in 3T3-L1 adipocytes and high-fat diet (HFD)-induced obese mice. Fargesin promoted the glucose uptake in 3T3-L1 adipocytes. In HFD-induced obese mice, fargesin decreased the body weight gain, white adipose tissue (WAT), and plasma triglyceride, non-esterified fatty acid and glucose levels, and improved the glucose tolerance. Fargesin increased glucose transporter 4 (GLUT4) protein expression and phosphorylation of Akt, AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC) in both 3T3-L1 adipocytes and WAT of HFD-induced obese mice. Fargesin also decreased the mRNA expression levels of fatty acid oxidation-related genes, such as peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase-1 (CPT-1), uncoupling protein-2 (UCP-2) and leptin in WAT. Taken together, the present findings suggest that fargesin improves dyslipidemia and hyperglycemia by activating Akt and AMPK in WAT.

Honokiol and magnolol stimulate glucose uptake by activating PI3K-dependent Akt in L6 myotubes.

Honokiol and magnolol, ingredients of Magnolia officinalis, which is used in traditional Chinese and Japanese medicines, have been reported to have antioxidant, anticancer, and antiangiogenic effects. Effects of these compounds on glucose metabolism in adipocytes have also been reported. However, their effects on skeletal muscle glucose uptake and the underlying molecular mechanisms are still unknown. Here, we investigated the direct effects and signaling pathways activated by honokiol and magnolol in skeletal muscle cells using L6 myotubes. We found that honokiol and magnolol dose-dependently acutely stimulated glucose uptake without synergistic effects of combined administration in L6 myotubes. Treatment with honokiol and magnolol also stimulated glucose transporter-4 translocation to the cell surface. Honokiol- and magnolol-stimulated glucose uptake was blocked by the phosphatidylinositol-3 kinase inhibitor, wortmannin. Both honokiol and magnolol stimulated Akt phosphorylation, a key element in the insulin signaling pathway, which was completely inhibited by wortmannin. These results suggest that honokiol and magnolol might have beneficial effects on glucose metabolism by activating the insulin signaling pathway.

Berberine enhances defects in the establishment of leaf polarity in asymmetric leaves1 and asymmetric leaves2 of Arabidopsis thaliana.

Leaves develop as flat lateral organs from the indeterminate shoot apical meristem. The establishment of polarity along three-dimensional axes, proximal-distal, medial-lateral, and adaxial-abaxial axes, is crucial for the growth of normal leaves. The mutations of ASYMMETRIC LEAVES1 (AS1) and AS2 of Arabidopsis thaliana cause defects in repression of the indeterminate state and the establishment of axis formation in leaves. Although many mutations have been identified that enhance the adaxial-abaxial polarity defects of as1 and as2 mutants, the roles of the causative genes in leaf development are still unknown. In this study, we found that wild-type plants treated with berberine produced pointed leaves, which are often observed in the single mutants that enhance phenotypes of as1 and as2 mutants. The berberine-treated as1 and as2 mutants formed abaxialized filamentous leaves. Berberine, an isoquinoline alkaloid compound naturally produced in various plant sources, has a growth inhibitory effect on plants that do not produce berberine. We further showed that transcript levels of meristem-specific class 1 KNOX homeobox genes and abaxial determinant genes were increased in berberine-treated as1 and as2. Berberine treated plants carrying double mutations of AS2 and the large subunit ribosomal protein gene RPL5B showed more severe defects in polarity than did the as2 single mutant plants. We suggest that berberine inhibits (a) factor(s) that might be required for leaf adaxial cell differentiation through a pathway independent of AS1 and AS2. Multiple pathways might play important roles in the formation of flat symmetric leaves.

Detection of adiponectin and monocyte chemoattractant protein-1 using a calixcrown derivatives-coated protein chip.

We used a ProteoChip coated with a calixcrown derivative protein linker to measure adiponectin and monocyte chemoattractant protein-1 (MCP-1) levels and compared the results with commercial enzyme-linked immunosorbent assay (ELISA) kits. Adiponectin and MCP-1 levels in normal human serum and RAW264 cell supernatants, respectively, were measured. The ProteoChip quantification results correlated with those from the ELISA kits; however, the ProteoChip required less sample volume, exhibited higher sensitivity, and had a wider detection range. The ProteoChip was capable of detecting and quantifying small amounts of protein, possibly replacing ELISA kits in evaluating the levels of adiponectin and MCP-1.

Coptisine inhibits RANKL-induced NF-κB phosphorylation in osteoclast precursors and suppresses function through the regulation of RANKL and OPG gene expression in osteoblastic cells.

Excessive receptor activator of NF-κB ligand (RANKL) signaling causes enhanced osteoclast formation and bone resorption. The downregulation of RANKL expression and its downstream signals may be an effective therapeutic approach to the treatment of bone loss diseases such as osteoporosis. Here, we found that coptisine, one of the isoquinoline alkaloids from Coptidis Rhizoma, exhibited inhibitory effects on osteoclastogenesis in vitro. Although coptisine has been studied for its antipyretic, antiphotooxidative, dampness dispelling, antidote, antinociceptive, and anti-inflammatory activities in vitro and in vivo, its effects on osteoclastogenesis have not been investigated. Therefore, we evaluated the effects of coptisine on osteoblastic cells as well as osteoclast precursors for osteoclastogenesis in vitro. The addition of coptisine to cocultures of mouse bone marrow cells and primary osteoblastic cells with 10(-8) M 1α,25(OH)(2)D(3) caused significant inhibition of osteoclast formation in a dose-dependent manner. Reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed that coptisine inhibited RANKL gene expression and stimulated the osteoprotegerin gene expression induced by 1α,25(OH)(2)D(3) in osteoblastic cells. Coptisine strongly inhibited RANKL-induced osteoclast formation when added during the early stage of bone marrow macrophage (BMM) cultures, suggesting that it acts on osteoclast precursors to inhibit RANKL/RANK signaling. Among the RANK signaling pathways, coptisine inhibited NF-κB p65 phosphorylations, which are regulated in response to RANKL in BMMs. Coptisine also inhibited the RANKL-induced expression of NFATc1, which is a key transcription factor. In addition, 10 µM coptisine significantly inhibited both the survival of mature osteoclasts and their pit-forming activity in cocultures. Thus, coptisine has potential for the treatment or prevention of several bone diseases characterized by excessive bone destruction.

Biselyngbyaside, isolated from marine cyanobacteria, inhibits osteoclastogenesis and induces apoptosis in mature osteoclasts.

The mass and function of bones depend on the maintenance of a complicated balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An inhibitor of osteoclast differentiation and/or function is expected to be useful for treatment of bone lytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis into bone. Biselyngbyaside is a recently isolated macrolide compound from marine cyanobacteria Lyngbya sp. that shows wide-spectrum cytotoxicity toward human tumor cell lines. In this study, we investigated the effects of biselyngbyaside on osteoclast differentiation and function. Biselyngbyaside inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocytic RAW264 cells and primary bone marrow-derived macrophages at a low concentration. Similarly, biselyngbyaside suppressed osteoblastic cell-mediated osteoclast differentiation in cocultures. In the RANKL-induced signaling pathway, biselyngbyaside inhibited the expression of c-Fos and NFATc1, which are important transcription factors in osteoclast differentiation. In mature osteoclasts, biselyngbyaside decreased resorption-pit formation. Biselyngbyaside also induced apoptosis accompanied by the induction of caspase-3 activation and nuclear condensation, and these effects were negated by the pancaspase inhibitor z-VAD-FMK. Taken together, the present findings indicate that biselyngbyaside suppresses bone resorption via inhibition of osteoclastogenesis and induction of apoptosis. Thus, biselyngbyaside may be useful for the prevention of bone lytic diseases.

Harmine promotes osteoblast differentiation through bone morphogenetic protein signaling.

Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a ß-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related ß-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and Runx2 pathways. Our findings suggest that harmine has bone anabolic effects and may be useful for the treatment of bone-decreasing diseases and bone regeneration as a lead compound.

Osteogenic activity of diphenyl ether-type cyclic diarylheptanoids derived from Acer nikoense.

Osteogenic activity of six diarylheptanoids, acerogenin A (1), (R)-acerogenin B (2), aceroside I (3), aceroside B(1) (4), aceroside III (5) and (-)-centrolobol (6) and two phenolic compounds; (+)-rhododendrol (7) and (+)-cathechin (8), isolated from the stem bark of Acer nikoense (Nikko maple) was evaluated using alkaline phosphatase (ALP) activity as a marker for early osteoblast differentiation. We found that the diphenyl ether-type cyclic diarylheptanoids 1-5 promoted ALP activity in mouse preosteoblastic MC3T3-E1 cells without affecting cell proliferation, but linear-type diarylheptanoid 6 and phenolic compounds 7 and 8 did not. Diphenyl ether-type cyclic diarylheptanoids 1-4 also increased protein production of osteocalcin, a late stage maker for osteoblast differentiation, and induced osteoblastic mineralization. Structure-activity relationships of these compounds demonstrated that the stimulative efficacy of aglycones was higher than that of its glycosides. Taken together, diphenyl ether-type cyclic diarylheptanoids promote early- and late-stage osteoblastogenesis, which may open the possibility for the development of novel osteogenic agents.

Honokiol enhances adipocyte differentiation by potentiating insulin signaling in 3T3-L1 preadipocytes.

Adipose tissue plays an essential role in energy homeostasis as a metabolic and endocrine organ. Accordingly, adipocytes are emerging as a major drug target for obesity and obesity-mediated metabolic syndrome. Dysfunction of enlarged adipocytes in obesity is involved in obesity-mediated metabolic syndrome. Adipocytokines, such as adiponectin released from small adipocytes, are able to prevent these disorders. In this study, we found that honokiol, an ingredient of Magnolia officinalis used in traditional Chinese and Japanese medicines, enhanced adipocyte differentiation in 3T3-L1 preadipocytes. Oil Red O staining showed that treatment with honokiol in the presence of insulin dose-dependently increased lipid accumulation in 3T3-L1 preadipoyctes although its activity was weak compared with rosiglitazone. During adipocyte differentiation, the expression of peroxisome proliferator-activated receptor γ2 (PPARγ2) mRNA and PPARγ target genes such as adipocyte protein 2 (aP2), adiponectin, and GLUT4 was induced by treatment with 10 µM honokiol. However, honokiol failed to show direct binding to the PPARγ ligand-binding domain in vitro. In preadipocytes, treatment with honokiol in the presence of insulin increased the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 protein and Akt protein, early insulin signaling pathways related to adipocyte differentiation, compared with insulin-only treatment. Taken together, our results suggest that honokiol promotes adipocyte differentiation through increased expression of PPARγ2 mRNA and potentiation of insulin signaling pathways such as the Ras/ERK1/2 and phosphoinositide-3-kinase (PI3K)/Akt signaling pathways.

Artepillin C, as a PPARγ ligand, enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigated the effects of artepillin C, an ingredient of Baccharis dracunculifolia, on adipogenesis and glucose uptake using 3T3-L1 cells. In PPARγ ligand-binding assays, artepillin C exhibited binding affinity toward PPARγ. Artepillin C dose-dependently enhanced adipocyte differentiation of 3T3-L1 cells. As a result of the artepillin C-induced adipocyte differentiation, the gene expression of PPARγ and its target genes, such as aP2, adiponectin and glucose transporter (GLUT) 4, was increased. These increases were abolished by cotreatment with GW9662, a PPARγ antagonist. In mature 3T3-L1 adipocytes, artepillin C significantly enhanced the basal and insulin-stimulated glucose uptake. These effects were decreased by cotreatment with a PI3K inhibitor. Although artepillin C had no effects on the insulin signaling cascade, artepillin C enhanced the expression and plasma membrane translocation of GLUT1 and GLUT4 in mature adipocytes. In conclusion, these findings suggest that artepillin C promotes adipocyte differentiation and glucose uptake in part by direct binding to PPARγ, which could be the basis of the pharmacological benefits of green propolis intake in reducing the risk of type 2 diabetes.

Effects of a Citrus depressa Hayata (shiikuwasa) extract on obesity in high-fat diet-induced obese mice.

Citrus depressa Hayata (commonly known as shiikuwasa) is cultivated in the northern areas of Okinawa, Japan, and used as a juice. In this study, we examined the anti-obesity effects and mechanism of action of shiikuwasa peel extract (SE) using high-fat diet (HFD)-induced obese mice. Mice were fed a low-fat diet (LFD), HFD or HFD containing 1% or 1.5% (w/w) SE (HFD+1 SE and HFD+1.5 SE, respectively) for 5 weeks. The body weight gain and white adipose tissue weight were significantly decreased in the HFD+1.5 SE group compared with the HFD group. The plasma triglyceride and leptin levels were also significantly reduced in the HFD+1.5 SE group compared with the HFD group. Histological examinations showed that the sizes of the adipocytes were significantly smaller in the HFD+1.5 SE group than in the HFD group. The HFD+1.5 SE group also showed significantly lower mRNA levels of lipogenesis-related genes, such as activating protein 2, stearoyl-CoA desaturase 1, acetyl-CoA-carboxylase 1, fatty acid transport protein and diacylglycerol acyltransferase 1, than the HFD group. These results suggest that the anti-obesity effects of SE may be elicited by regulating the expressions of lipogenesis-related genes in white adipose tissue.

Harmine, a ß-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo.

Bone homeostasis is controlled by the balance between osteoblastic bone formation and osteoclastic bone resorption. Excessive bone resorption is involved in the pathogenesis of bone-related disorders such as osteoporosis, arthritis and periodontitis. To obtain new antiresorptive agents, we searched for natural compounds that can inhibit osteoclast differentiation and function. We found that harmine, a ß-carboline alkaloid, inhibited multinucleated osteoclast formation induced by receptor activator of nuclear factor-κB ligand (RANKL) in RAW264.7 cells. Similar results were obtained in cultures of bone marrow macrophages supplemented with macrophage colony-stimulating factor and RANKL, as well as in cocultures of bone marrow cells and osteoblastic UAMS-32 cells in the presence of vitamin D(3) and prostaglandin E(2). Furthermore, harmine prevented RANKL-induced bone resorption in both cell and bone tissue cultures. Treatment with harmine (10 mg/kg/day) also prevented bone loss in ovariectomized osteoporosis model mice. Structure-activity relationship studies showed that the C3-C4 double bond and 7-methoxy group of harmine are important for its inhibitory activity on osteoclast differentiation. In mechanistic studies, we found that harmine inhibited the RANKL-induced expression of c-Fos and subsequent expression of nuclear factor of activated T cells (NFAT) c1, which is a master regulator of osteoclastogenesis. However, harmine did not affect early signaling molecules such as ERK, p38 MAPK and IκBα. These results indicate that harmine inhibits osteoclast formation via downregulation of c-Fos and NFATc1 induced by RANKL and represses bone resorption. These novel findings may be useful for the treatment of bone-destructive diseases.

Palmatine attenuates osteoclast differentiation and function through inhibition of receptor activator of nuclear factor-κb ligand expression in osteoblast cells.

Osteoclasts are the only cell type capable of resorbing mineralized bone, and they act under the control of numerous cytokines produced by supporting cells such as osteoblasts and stromal cells. Among cytokines, receptor activator of nuclear factor-κB ligand (RANKL) was found to be a key osteoclastogenetic molecule that directly binds to its cognate receptor, RANK, on osteoclast precursor cells. In turn, RANKL, which is an essential factor for differentiation and activation of osteoclasts, is one of the major targets of anti-resorptive agents. In this study, we found that palmatine, an isoquinoline alkaloid originally isolated from Coptis chinensis, had an inhibitory effect on osteoclast differentiation and function in vitro. Palmatine inhibited osteoclast formation in the co-culture system with mouse bone marrow cells (BMC) and osteoblasts in the presence of 10 nM 1α,25-(OH)(2)D(3). Palmatine did not affect osteoclast formation induced by RANKL in the BMC cultures. Reverse-transcription polymerase chain reaction (RT-PCR) analysis showed that palmatine significantly inhibited the expression of 1α,25-(OH)(2)D(3)-induced expression of RANKL mRNAs in stromal cells without loss of cell viability. Moreover, palmatine suppressed resorption pit formation by mature osteoclasts on dentin slices and induced disruption of actin ring formation in mature osteoclasts with an impact on cell viability. Taken together, these results suggest that palmatine attenuates osteoclast differentiation through inhibition of RANKL expression in osteoblast cells, and its inhibitory effect on bone resorption is due to its disruptive effect on actin rings in mature osteoclasts. Therefore, palmatine might be an ideal candidate as an anti-resorptive agent for the prevention and treatment of bone disorders such as osteoporosis.