Oral Administration of Isovitexin, a Naturally Occurring Apigenin Derivative Showed Osteoanabolic Effect in Ovariectomized Mice: A Comparative Study with Teriparatide.

Isovitexin (apigenin-6C-glucopyranose) is found in several food items and medicinal plants. Recently, we showed that isovitexin stimulated osteoblast differentiation through mitochondrial biogenesis and respiration that required adiponectin receptors (AdipoRs). Here, we studied whether oral isovitexin has a bone anabolic effect in vivo. At first, using a femur osteotomy model in adult mice, we compared the bone regenerative effect of isovitexin and apigenin. Whereas isovitexin-stimulated bone formation at the osteotomy site at 2.5 mg/kg and 5 mg/kg dose, apigenin had no effect. Subsequently, we tested the effect of isovitexin (5 mg/kg) in ovariectomized (OVX) osteopenic mice and observed that it restored bone mass and architecture of trabecular bones (femur metaphysis and fifth lumbar vertebra/L5) and cortical bones (femur diaphysis). Isovitexin completely restored bone strength at L5 (compressive strength) and femur (bending strength) in OVX mice. The bone anabolic effect of isovitexin was demonstrated by the increased surface referent bone formation parameters, increased expression of osteogenic genes (Runx2, bone morphogenetic protein-2 and type 1 collagen) in bones, and increased serum procollagen type 1N-terminal propeptide in OVX mice and these were on a par with teriparatide. Isovitexin inhibited bone and serum sclerostin as well as the serum type I collagen cross-linked C-telopeptide in OVX mice. Isovitexin has an oral bioavailability of 14.58%. Taken together, our data show that isovitexin had a significant oral bioavailability that translated to osteoanabolic effect equivalent to teriparatide and inhibited bone resorption, which implied a durable effect over teriparatide.

ß-Sitosterol-D-Glucopyranoside Mimics Estrogenic Properties and Stimulates Glucose Utilization in Skeletal Muscle Cells.

Estrogenic molecules have been reported to regulate glucose homeostasis and may be beneficial for diabetes management. Here, we investigated the estrogenic effect of ß-sitosterol-3-O-D-glucopyranoside (BSD), isolated from the fruits of Cupressus sempervirens and monitored its ability to regulate glucose utilization in skeletal muscle cells. BSD stimulated ERE-mediated luciferase activity in both ERα and ERß-ERE luc expression system with greater response through ERß in HEK-293T cells, and induced the expression of estrogen-regulated genes in estrogen responsive MCF-7 cells. In silico docking and molecular interaction studies revealed the affinity and interaction of BSD with ERß through hydrophobic interaction and hydrogen bond pairing. Furthermore, prolonged exposure of L6-GLUT4myc myotubes to BSD raised the glucose uptake under basal conditions without affecting the insulin-stimulated glucose uptake, the effect associated with enhanced translocation of GLUT4 to the cell periphery. The BSD-mediated biological response to increase GLUT4 translocation was obliterated by PI-3-K inhibitor wortmannin, and BSD significantly increased the phosphorylation of AKT (Ser-473). Moreover, BSD-induced GLUT4 translocation was prevented in the presence of fulvestrant. Our findings reveal the estrogenic activity of BSD to stimulate glucose utilization in skeletal muscle cells via PI-3K/AKT-dependent mechanism.

A butanolic fraction from the standardized stem extract of Cassia occidentalis L delivered by a self-emulsifying drug delivery system protects rats from glucocorticoid-induced osteopenia and muscle atrophy.

We recently reported that a butanol soluble fraction from the stem of Cassia occidentalis (CSE-Bu) consisting of osteogenic compounds mitigated methylprednisone (MP)-induced osteopenia in rats, albeit failed to afford complete protection thus leaving a substantial scope for further improvement. To this aim, we prepared an oral formulation that was a lipid-based self-nano emulsifying drug delivery system (CSE-BuF). The globule size of CSE-BuF was in the range of 100-180 nm of diluted emulsion and the zeta potential was -28 mV. CSE-BuF enhanced the circulating levels of five osteogenic compounds compared to CSE-Bu. CSE-BuF (50 mg/kg) promoted bone regeneration at the osteotomy site and completely prevented MP-induced loss of bone mass and strength by concomitant osteogenic and anti-resorptive mechanisms. The MP-induced downregulations of miR29a (the positive regulator of the osteoblast transcription factor, Runx2) and miR17 and miR20a (the negative regulators of the osteoclastogenic cytokine RANKL) in bone was prevented by CSE-BuF. In addition, CSE-BuF protected rats from the MP-induced sarcopenia and/or muscle atrophy by downregulating the skeletal muscle atrogenes, adverse changes in body weight and composition. CSE-BuF did not impact the anti-inflammatory effect of MP. Our preclinical study established CSE-BuF as a prophylactic agent against MP-induced osteopenia and muscle atrophy.

A nutraceutical composition containing diosmin and hesperidin has osteogenic and anti-resorptive effects and expands the anabolic window of teriparatide.

A combination of diosmin and hesperidin (9:1 ratio) is marketed as a dietary supplement/nutraceutical for cardiovascular health. We studied the skeletal effect of this combination (90% diosmin and 10% hesperidin, henceforth named as DH). We showed that a) in rats with femur osteotomy, DH stimulated callus bone regeneration, b) in growing rats, DH promoted peak bone mass achievement and c) in OVX rats rendered osteopenic, DH completely restored femur trabecular bones and strength along with the increases in surface referent bone formation and serum osteogenic marker. Furthermore, DH suppressed bone resorption in OVX rats as well as in OVX rats treated with teriparatide (human parathyroid hormone 1-34) but did not affect the osteoanabolic effect of teriparatide. These data suggested that DH could prolong the anabolic window of teriparatide. To understand the mechanism of DH action, we performed pharmacokinetic studies and observed that upon its oral administration the only circulating metabolites was diosmetin (the aglycone form of diosmin) while none of the two input flavanones were detectable. Accordingly, subsequent experiments with diosmetin revealed that it was a selective estrogen receptor-ß agonist that stimulated osteoblast differentiation and suppressed sclerostin the anti-osteoblastogenic Wnt antagonist. Taken together, our study defined a positive skeletal effect of DH.

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/ß-catenin signaling.

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/ß-catenin pathway assessed by phosphorylation of GSK-3ß. Over-expressing of constitutively active GSK-3ß (caGSK-3ß) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3ß over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats.

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.

[6]-Gingerol induces bone loss in ovary intact adult mice and augments osteoclast function via the transient receptor potential vanilloid 1 channel.

SCOPE: [6]-Gingerol, a major constituent of ginger, is considered to have several health beneficial effects. The effect of 6-gingerol on bone cells and skeleton of mice was investigated. METHODS AND RESULTS: The effects of 6-gingerol on mouse bone marrow macrophages and osteoblasts were studied. 6-Gingerol-stimulated osteoclast differentiation of bone marrow macrophages but had no effect on osteoblasts. Capsazepine, an inhibitor of TRPV1 (transient receptor potential vanilloid 1) channel, attenuated the pro-osteoclastogenic effect of 6-gingerol or capsaicin (an agonist of TRPV1). Similar to capsaicin, 6-gingerol stimulated Ca(2) + influx in osteoclasts. The effect of daily feeding of 6-gingerol for 5 wk on the skeleton of adult female Balb/cByJ mice was investigated. Mice treated with capsaicin and ovariectomized (OVx) mice served as controls for osteopenia. 6-Gingerol caused increase in trabecular osteoclast number, microarchitectural erosion at all trabecular sites and loss of vertebral stiffness, and these effects were comparable to capsaicin or OVx group. Osteoclast-specific serum and gene markers of 6-gingerol-treated mice were higher than the OVx group. Bone formation was unaffected by 6-gingerol. CONCLUSION: Daily feeding of 6-gingerol to skeletally mature female mice caused trabecular osteopenia, and the mechanism appeared to be activation of osteoclast formation via the TRPV1 channel.

Estrogen receptor potentiates mTORC2 signaling in breast cancer cells by upregulating superoxide anions.

The estrogen receptor (ER) plays a cardinal role in estrogen-responsive breast carcinogenesis. It is, however, unclear as to how estrogen-ER interaction potentiates breast cancer progression. Compelling evidence supports estrogen-induced redox alterations, such as augmented reactive oxygen species (ROS) levels, as having a crucial role in breast carcinogenesis. Despite ER being a biological mediator of the majority of estrogen-induced cellular responses; its role in estrogen-induced tissue-specific ROS generation remains largely debatable. We examined a panel of human breast cancer specimens and found that ER-positive breast cancer specimens exhibited a higher incidence of augmented O(2)(•-) levels compared to matched normal tissue. ROS are known to function as signal transducers and ROS-mediated signaling remains a key complementary mechanism that drives carcinogenesis by activating redox-sensitive oncogenic pathways. Additional studies revealed that augmented O(2)(•-) levels in breast cancer specimens coincided with mammalian target of rapamycin complex 2 (mTORC2) hyperactivation. Detailed investigations using in vitro experiments established that 17ß-estradiol (E2)-stimulated breast cancer cells exhibited transiently upregulated O(2)(•-) levels, with the presence of ER being a crucial determinant for the phenomenon to take place. Gene expression, ER transactivation, and confocal studies revealed that the E2-induced transient O(2)(•-) upregulation was effected by ER through a nongenomic pathway possibly involving mitochondria. Furthermore, E2 treatment activated mTORC2 in breast cancer cells in a characteristically ER-dependent manner. Interestingly, altering O(2)(•-) anion levels through chemical/genetic methods caused significant modulation of the mTORC2 signaling cascade. Taken together, our findings unravel a novel nongenomic pathway unique to estrogen-responsive breast cancer cells wherein, upon stimulation by E2, ER may regulate mTORC2 activity in a redox-dependent manner by transiently modulating O(2)(•-) levels particularly within mitochondria. The findings suggest that therapies aimed at counteracting these redox alterations and/or resultant signaling cascades may complement conventional treatments for estrogen-responsive breast cancer.

A novel quercetin analogue from a medicinal plant promotes peak bone mass achievement and bone healing after injury and exerts an anabolic effect on osteoporotic bone: the role of aryl hydrocarbon receptor as a mediator of osteogenic action.

We recently reported that extracts made from the stem bark of Ulmus wallichiana promoted peak bone mass achievement in growing rats and preserved trabecular bone mass and cortical bone strength in ovariectomized (OVX) rats. Further, 6-C-ß-D-glucopyranosyl-(2S,3S)-(+)-3',4',5,7-tetrahydroxyflavanol (GTDF), a novel flavonol-C-glucoside isolated from the extracts, had a nonestrogenic bone-sparing effect on OVX rats. Here we studied the effects of GTDF on osteoblast function and its mode of action and in vivo osteogenic effect. GTDF stimulated osteoblast proliferation, survival, and differentiation but had no effect on osteoclastic or adipocytic differentiation. In cultured osteoblasts, GTDF transactivated the aryl hydrocarbon receptor (AhR). Activation of AhR mediated the stimulatory effect of GTDF on osteoblast proliferation and differentiation. Furthermore, GTDF stimulated cAMP production, which mediated osteogenic gene expression. GTDF treatments given to 1- to 2-day-old rats or adult rats increased the mRNA levels of AhR target genes in calvaria or bone marrow stromal cells. In growing female rats, GTDF promoted parameters of peak bone accrual in the appendicular skeleton, including increased longitudinal growth, bone mineral density, bone-formation rate (BFR), cortical deposition, and bone strength. GTDF promoted the process of providing newly generated bone to fill drill holes in the femurs of both estrogen-sufficient and -deficient rats. In osteopenic OVX rats, GTDF increased BFR and significantly restored trabecular bone compared with the ovaries-intact group. Together our data suggest that GTDF stimulates osteoblast growth and differentiation via the AhR and promotes modeling-directed bone accrual, accelerates bone healing after injury, and exerts anabolic effects on osteopenic rats likely by a direct stimulatory effect on osteoprogenitors. Based on these preclinical data, clinical evaluation of GTDF as a potential bone anabolic agent is warranted.

Methoxylated isoflavones, cajanin and isoformononetin, have non-estrogenic bone forming effect via differential mitogen activated protein kinase (MAPK) signaling.

Following a lead obtained from stem-bark extract of Butea monosperma, two structurally related methoxyisoflavones; cajanin and isoformononetin were studied for their effects in osteoblasts. Cajanin had strong mitogenic as well as differentiation-promoting effects on osteoblasts that involved subsequent activation of MEK-Erk and Akt pathways. On the other hand, isoformononetin exhibited potent anti-apoptotic effect in addition to promoting osteoblast differentiation that involved parallel activation of MEK-Erk and Akt pathways. Unlike genistein or daidzein, none of these two compounds appear to act via estrogen receptors in osteoblast. Once daily oral (by gavage) treatment for 30 consecutive days was given to recently weaned female Sprague-Dawley rats with each of these compounds at 10.0 mg kg(-1) day(-1) dose. Cajanin increased bone mineral density (BMD) at all skeletal sites studied, bone biomechanical strength, mineral apposition rate (MAR) and bone formation rate (BFR), compared with control. BMD levels at various anatomic positions were also increased with isoformononetin compared with control however, its effect was less potent than cajanin. Isoformononetin had no effect on the parameters of bone biomechanical strength although it enhanced MAR and BFR compared with control. Isoformononetin had very mild uterotrophic effect, whereas cajanin was devoid of any such effect. Our data suggest that cajanin is more potent than isoformononetin in accelerating peak bone mass achievement. To the best of our knowledge, this work represents the first attempt to elucidate structure-activity relationship between the two methoxylated isoflavones regarding their effects in osteoblasts and bone formation.