Tag Archives: animal

Uridine Triphosphate Inhibits Bone Growth in Rat Cells In Vitro


Osteoblast responses to nucleotides increase during differentiation.

Accumulating evidence suggests that extracellular nucleotides, signaling through P2 receptors, play a role in modulating bone cell function. ATP and ADP stimulate osteoclastic resorption, while ATP and UTP are powerful inhibitors of bone formation by osteoblasts. We investigated changes in the expression of P2 receptors with cell differentiation in primary osteoblast cultures. Rat calvarial osteoblasts, cultured for up to 10 days, were loaded with the intracellular Ca(2+)-sensing fluorophore, Fluo-4 AM, and a fluorescence imaging plate reader was used to measure responses to nucleotide agonists. Peak responses occurred within 20 s and were evoked by ATP or UTP at concentrations as low as 2 microM. Osteoblast number doubled between day 4 and 10 of culture, but the peak intracellular Ca(2+) response to ATP or UTP increased up to 6-fold over the same period, indicating that osteoblast responsiveness to nucleotides increases as cell differentiation proceeds. The approximate order of potency for the most active nucleotide agonists at day 8 of culture was ATP > UTP and ATPgammaS > ADP > UDP, consistent with the expression of functional P2Y(2), P2X(2), P2Y(4), P2Y(1) and P2Y(6) receptors. Smaller responses were elicited by 2-MeSATP, Bz-ATP and alpha,beta-meATP, additionally suggesting the presence of functional P2X(1), P2X(3), P2X(5) and P2X(7) receptors. Expression of mRNA for the ATP- and UTP-selective P2Y(2) receptor increased strongly between day 6 and 15 in primary rat osteoblasts, whereas mRNAs for the P2Y(4) (also ATP/UTP selective) and P2Y(6) (UDP/UTP selective) receptors were highly expressed at intermediate time points. In contrast, mRNA for the cell-proliferation-associated P2X(5) receptor decreased to undetectable as osteoblasts matured, but mRNA for the cell-death-associated P2X(7) receptor was detected at all time points. Similar trends were evident using immunostaining and Western blotting for P2 receptors. Exposure to 10 muM ATP or UTP during days 10-14 of culture was sufficient to cause near-total blockade of the ‘trabecular’ bone nodules formed by osteoblasts; however, UDP and ADP were without effect. Our results show that there is a shift from P2X to P2Y expression during differentiation in culture, with mature osteoblasts preferentially expressing the P2Y(2) receptor and to a lesser extent P2Y(4) and P2Y(6) receptors. Taken together, these data suggest that the P2Y(2) receptor, and possibly the P2Y(4) receptor, could function as ‘off-switches’ for mineralized bone formation.

Orriss IR, Knight GE, Ranasinghe S, Burnstock G…
Bone Aug 2006
PMID: 16616882


ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling.

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the ‘universal’ agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.

Hoebertz A, Mahendran S, Burnstock G, Arnett TR
J. Cell. Biochem. 2002
PMID: 12210747


Regulation of the osteogenic and adipogenic differentiation of bone marrow-derived stromal cells by extracellular uridine triphosphate: The role of P2Y2 receptor and ERK1/2 signaling.

An imbalance in the osteogenesis and adipogenesis of bone marrow-derived stromal cells (BMSCs) is a crucial pathological factor in the development of osteoporosis. Growing evidence suggests that extracellular nucleotide signaling involving the P2 receptors plays a significant role in bone metabolism. The aim of the present study was to investigate the effects of uridine triphosphate (UTP) on the osteogenic and adipogenic differentiation of BMSCs, and to elucidate the underlying mechanisms. The differentiation of the BMSCs was determined by measuring the mRNA and protein expression levels of osteogenic- and adipogenic-related markers, alkaline phosphatase (ALP) staining, alizarin red staining and Oil Red O staining. The effects of UTP on BMSC differentiation were assayed using selective P2Y receptor antagonists, small interfering RNA (siRNA) and an intracellular signaling inhibitor. The incubation of the BMSCs with UTP resulted in a dose-dependent decrease in osteogenesis and an increase in adipogenesis, without affecting cell proliferation. Significantly, siRNA targeting the P2Y2 receptor prevented the effects of UTP, whereas the P2Y6 receptor antagonist (MRS2578) and siRNA targeting the P2Y4 receptor had little effect. The activation of P2Y receptors by UTP transduced to the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. This transduction was prevented by the mitogen-activated protein kinase inhibitor (U0126) and siRNA targeting the P2Y2 receptor. U0126 prevented the effects of UTP on osteogenic- and adipogenic-related gene expression after 24 h of culture, as opposed to 3 to 7 days of culture. Thus, our data suggest that UTP suppresses the osteogenic and enhances the adipogenic differentiation of BMSCs by activating the P2Y2 receptor. The ERK1/2 signaling pathway mediates the early stages of this process.

Li W, Wei S, Liu C, Song M…
Int. J. Mol. Med. Jan 2016
PMID: 26531757 | Free Full Text

Protein Intake Above the Current RDA May Help Prevent Fractures


Dietary Protein Intake above the Current RDA and Bone Health: A Systematic Review and Meta-Analysis.

Dietary intake of protein is fundamental for optimal acquisition and maintenance of bone across all life stages; however, it has been hypothesized that intakes above the current recommended dietary allowance (RDA) might be beneficial for bone health. We utilized the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines when preparing and reporting this systematic review and meta-analysis. A literature search strategy through April 11, 2017, was developed for the following 3 databases: PubMed, Ovid Medline, and Agricola. Included studies were those randomized controlled trials and prospective cohort studies among healthy adults ages 18 and older that examined the relationships between varying doses of protein intake at or above the current U.S. RDA (0.8 g/kg/d or 10%-15% of total caloric intake) from any source on fracture, bone mineral density (BMD)/bone mineral content (BMC), and/or markers of bone turnover. Twenty-nine articles were included for data extraction (16 randomized controlled trials [RCTs] and 13 prospective cohort studies). Meta-analysis of the prospective cohort studies showed high vs low protein intakes resulted in a statistically significant 16% decrease in hip fractures (standardized mean difference [SMD] = 0.84, 95% confidence interval [CI], 0.73, 0.95; I(2) = 36.8%). Data from studies included in these analyses collectively lean toward the hypothesis that protein intake above the current RDA is beneficial to BMD at several sites. This systematic review supports that protein intakes above the current RDA may have some beneficial role in preventing hip fractures and BMD loss. There were no differences between animal or plant proteins, although data in this area were scarce. Larger, long-term, and more well-controlled clinical trials measuring fracture outcomes and BMD are needed to adequately assess whether protein intake above the current RDA is beneficial as a preventative measure and/or intervention strategy for osteoporosis. Key teaching points: Bone health is a multifactorial musculoskeletal issue, and optimal protein intakes are key in developing and maintaining bone throughout the life span. Dietary protein at levels above the current RDA may be beneficial in preventing hip fractures and BMD loss. Plant vs animal proteins do not seem to differ in their ability to prevent bone loss; however, data in this area are scarce. Larger, long-term RCTs using women not using hormone replacement therapy (HRT) are needed to adequately assess the magnitude of impact that protein intakes above the RDA have on preventing bone loss.

Wallace TC, Frankenfeld CL
J Am Coll Nutr Aug 2017
PMID: 28686536

CoQ10 Increases Osteoblasts and Decreases Resorption in Rats


Coenzyme Q10 promotes osteoblast proliferation and differentiation and protects against ovariectomy-induced osteoporosis.

Coenzyme Q10 (CoQ10) is a fat‑soluble vitamin‑like substance used for the treatment of a variety of disorders, including osteoporosis. The exact mechanism underlying CoQ10‑mediated protection against osteoporosis remains to be elucidated. The present study aimed to evaluate the effect of CoQ10 on osteoblastic cell proliferation and differentiation, and therapeutic effects on a rat model of osteoporosis. Following treatment with different concentrations of CoQ10, cell proliferation and differentiation of rat bone marrow stromal cells (BMSCs), and expression of osteoblastogenic markers, were measured. Rats with osteoporosis subjected to ovariectomy (OVX) were treated with different concentrations of CoQ10. Serum levels of estrogen and bone metabolism markers were measured. Micro computed tomography scans were used to analyze morphological changes in bones. In addition, mRNA and protein levels of phosphatidylinositol 3,4,5‑trisphosphate 3‑phosphatase and dual‑specificity protein phosphatase PTEN (PTEN)/phosphatidylinositol 4,5‑bisphosphate 3‑kinase (PI3K)/RAC‑alpha serine/threonine‑protein kinase(AKT), were determined. CoQ10 significantly increased the proliferation and osteogenic differentiation of BMSCs in a dose‑dependent manner, with an increased expression of osteogenic markers. CoQ10 significantly decreased bone resorption but exhibited no effect on serum E2 levels in vivo. CoQ10 markedly enhanced bone formation. Furthermore, the abundance of p‑PI3K and p‑AKT increased while PTEN levels decreased in a dose‑dependent manner following administration of CoQ10. CoQ10 stimulates the proliferation and differentiation of BMSCs and is effective for the treatment of OVX‑induced osteoporosis in rats. The above effects of CoQ10 may be mediated by activation of the PTEN/PI3K/AKT pathway.

Zheng D, Cui C, Yu M, Li X…
Mol Med Rep Oct 2017
PMID: 29115467

Green Tea Extract is Bad for Growing Bones in Rats


Long-Term Intake of Green Tea Extract Causes Mal-Conformation of Trabecular Bone Microarchitecture in Growing Rats.

The purpose of this study was to examine the effects of green tea extract (GTE) intake on bone structural and physiological properties, such as bone mass, trabecular bone microarchitecture, cortical bone geometry, and bone mechanical strength, in growing rats. Four-week-old male Wistar rats were divided into the following four eenoups: standard diet feeding for 85 days (S-CON) or 170 days (L-CON), and GTE diet feeding for 85 days (S-GTE) or 170 days (L-GTE). At the end of the experiment, in addition to measurement of circulating bone formation/resorption markers, bone mass, trabecular bone microarchitecture, and cortical bone geometry were analyzed in the left femur, and bone mechanical strength of the right femur was measured. There was no difference in all bone parameters between the S-CON and S-GTE groups. On the other hand, the L-GTE group showed the decrease in some trabecular bone mass/microarchitecture parameters and no change in cortical bone mass/geometry parameters compared with the L-CON group, and consequently the reduction in bone weight corrected by body weight. There was no difference in bone formation/resorption markers and bone mechanical strength between the S-CON and S-GTE groups and also between the L-CON and L-GTE groups. However, serum leptin levels were significantly lower in the L-GTE group than in the L-CON group. Thus, the long-term GTE intake had negative effects on bone, especially trabecular bone loss and microarchitecture mal-conformation, in growing rats.

Minematsu A, Nishii Y, Imagita H, Sakata S
Calcif. Tissue Int. Nov 2017
PMID: 29103160

PQQ Stimulates Osteoblastic Bone Formation in Testosterone-Deficient Mice


Pyrroloquinoline quinone prevents testosterone deficiency-induced osteoporosis by stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption.

Accumulating evidences suggest that oxidative stress caused and deteriorated the aging related osteoporosis and pyrroloquinoline quinone (PQQ) is a powerful antioxidant. However, it is unclear whether PQQ can prevent testosterone deficiency-induced osteoporosis. In this study, the orchidectomized (ORX) mice were supplemented in diet with/without PQQ for 48 weeks, and compared with each other and with sham mice. Results showed that bone mineral density, trabecular bone volume, collagen deposition and osteoblast number were decreased significantly in ORX mice compared with shame mice, whereas PQQ supplementation largely prevented these alterations. In contrast, osteoclast surface and ratio of RANKL and OPG mRNA relative expression levels were increased significantly in ORX mice compared with shame mice, but were decreased significantly by PQQ supplementation. Furthermore, we found that CFU-f and ALP positive CFU-f forming efficiency and the proliferation of mesenchymal stem cells were reduced significantly in ORX mice compared with shame mice, but were increased significantly by PQQ supplementation. Reactive oxygen species (ROS) levels in thymus were increased, antioxidant enzymes SOD-1, SOD-2, Prdx I and Prdx IV protein expression levels in bony tissue were down-regulated, whereas the protein expression levels of DNA damage response related molecules including γ-H2AX, p53, Chk2 and NFκB-p65 in bony tissue were up-regulated significantly in ORX mice compared with shame mice, whereas PQQ supplementation largely rescued these alterations observed in ORX mice. Our results indicate that PQQ supplementation can prevent testosterone deficiency-induced osteoporosis by inhibiting oxidative stress and DNA damage, stimulating osteoblastic bone formation and inhibiting osteoclastic bone resorption.

Wu X, Li J, Zhang H, Wang H…
Am J Transl Res 2017
PMID: 28386349 | Free Full Text

PQQ Increases Osteoblasts in Bmi-1 Knockout Mice


Effect and mechanism of pyrroloquinoline quinone on anti-osteoporosis in Bmi-1 knockout mice-Anti-oxidant effect of pyrroloquinoline quinone.

Pyrroloquinoline quinone (PQQ), considered as an ROS scavenger,could protect mitochondrial activity from damage of oxidative stress. To determine the role of PQQ supplement in rescuing long bone osteoporosis in Bmi-1(-/-) mice. We fed Bmi-1 knockout mice a diet supplemented with PQQ (BKO+PQQ), BKO mice with normal diet (BKO) and wild type mice with normal diet (WT) as controls. We compared the differences of skeletal phenotype by means of imaging, histopathological and molecular biology methods in three groups of animals. Results showed that BKO+PQQ mice increased morphology of tibia, decreased X-ray transmittance, and increased bone density, thickness of cortical bone, width of growth plate and trabecular bone mass compared with BKO mice. Our study also investigated that, compared mice BKO, PCNA positive cells percentage of tibial growth plate areas significantly increased in BKO+PQQ mice, and TUNEL positive cells percentage was significantly decreased. To detect the effect of PQQ on osteoblast formation of tibiae. Our results showed, compared with BKO mice, osteogenic cell, osteoblast number areas, ALP, Col I and OCN positive areas significantly increased in tibia of BKO+PQQ mice. Further studies showed that supplemental PQQ played a role in anti-osteoporosis by up-regulating antioxidant capacity, inhibiting oxidative stress and reducing DNA damage, down-regulating CDKI proteins levels, and decreasing cell apoptosis. This study not only reveals the mechanism of PQQ supplementation in anti-osteoporosis, but also provides the experimental and theoretical basis for the clinical application of PQQ in osteoporosis.

Huang Y, Chen N, Miao D
Am J Transl Res 2017
PMID: 29118900 | Free Full Text

Hesperidin Improves Bone in Insulin-Treated Diabetic Rats


The ability of hesperidin compared to that of insulin for preventing osteoporosis induced by type I diabetes in young male albino rats: A histological and biochemical study.

Patients with type I diabetes are at increased risk of osteoporosis even after insulin therapy in adult stage. This study was conducted to compare the efficacy of hesperidin (hesp) therapy versus that of insulin alone in the alleviation of osteoporosis arising from type I diabetes mellitus (T1DM) in young rats.
Hesperidin was administered orally to STZ-induced diabetes. The animals were evaluated morphologically and biochemically and compared with that received daily SC injections of long-acting insulin.
Histologically, we observed the degeneration of osteoblasts and osteocytes, decreased collagen fibers, and disturbed bone turn over markers in untreated DM rats. Hesperidin+ insulin supplementation to diabetic rats caused significant improvement of most of the bone histological and morphometric parameters compared with the insulin-treated group. Furthermore, hesp treatment significantly reduced pro-inflammatory mediators TNFα and NF-κB and increased serum biochemical markers of bone turnover, including osteopontin (OPN), osteocalcin (OC) and decreased serum alkaline phosphatase (ALP).
These data demonstrated that hesp could be considered to be a beneficial drug for preventing diabetic osteoporosis in growing age.

Shehata AS, Amer MG, Abd El-Haleem MR, Karam RA
Exp. Toxicol. Pathol. Apr 2017
PMID: 28132802

SIRT1 Represses Sclerostin in Mice and may be Anabolic for Bone


Sirt1 is a regulator of bone mass and a repressor of Sost encoding for sclerostin, a bone formation inhibitor.

Sirt1, the mammalian ortholog of the yeast Sir2 (silent information regulator 2), was shown to play an important role in metabolism and in age-associated diseases, but its role in skeletal homeostasis and osteoporosis has yet not been studied. Using 129/Sv mice with a germline mutation in the Sirt1 gene, we demonstrate that Sirt1 haplo-insufficient (Sirt1(+/-)) female mice exhibit a significant reduction in bone mass characterized by decreased bone formation and increased marrow adipogenesis. Importantly, we identify Sost, encoding for sclerostin, a critical inhibitor of bone formation, as a novel target of Sirt1. Using chromatin immunoprecipitation analysis, we reveal that Sirt1 directly and negatively regulates Sost gene expression by deacetylating histone 3 at lysine 9 at the Sost promoter. Sost down-regulation by small interfering RNA and the administration of a sclerostin-neutralizing antibody restore gene expression of osteocalcin and bone sialoprotein as well as mineralized nodule formation in Sirt1(+/-) marrow-derived mesenchymal stem cells induced to osteogenesis. These findings reveal a novel role for Sirt1 in bone as a regulator of bone mass and a repressor of sclerostin, and have potential implications suggesting that Sirt1 is a target for promoting bone formation as an anabolic approach for treatment of osteoporosis.

Cohen-Kfir E, Artsi H, Levin A, Abramowitz E…
Endocrinology Dec 2011
PMID: 21952235

Oxytocin Associated with Bone Density in Postmenopausal Women


Oxytocin, a new determinant of bone mineral density in post-menopausal women: analysis of the OPUS cohort.

Oxytocin (OT), a neurohypophysial hormone regulated by estrogen and leptin, may play a role in bone metabolism in humans as suggested by animal studies. This study assessed the relationship between OT and bone status in a large population of postmenopausal women.
Subjects were included in the Osteoporosis and Ultrasound study, a 6-year prospective study in a population-based cohort. Final visit data were used for this cross-sectional study. OT, leptin, and estradiol serum levels were measured in 1097 postmenopausal women and compared with bone mineral density (BMD), fractures, and the bone turnover markers (BTMs) procollagen type 1 N-terminal propeptide, bone alkaline phosphatase, and C-telopeptide of type 1 collagen.
The median age was 70.8 years, 16% were osteoporotic, 48% were osteopenic, and 29% had at least one fracture. The OT serum level was related to spine (r = +0.12, P = .0002) and total hip BMD (r = +0.21, P < .0001) and with BTM (procollagen type 1 N-terminal propeptide: r = -0.13, P < .0001, bone alkaline phosphatase: r = -0.07, P = .02, C-telopeptide of type 1 collagen: r = -0.18, P < .0001). The relationship of OT with BMD was independent of BTM. After adjustment for confounding factors, the correlation between OT serum level and BMD remains significant at the hip in women with unmeasurable estradiol or leptin above the median value. There was no significant relationship between OT serum levels and fractures.
High OT levels are associated with high BMD, especially at the hip in women with low estradiol or high leptin serum levels. The mechanism may be explained by the effect of OT on bone turnover.

Breuil V, Panaia-Ferrari P, Fontas E, Roux C…
J. Clin. Endocrinol. Metab. Apr 2014
PMID: 24446658

Oxytocin is Lower in Osteoporosis


Oxytocin controls differentiation of human mesenchymal stem cells and reverses osteoporosis.

Osteoporosis constitutes a major worldwide public health burden characterized by enhanced skeletal fragility. Bone metabolism is the combination of bone resorption by osteoclasts and bone formation by osteoblasts. Whereas increase in bone resorption is considered as the main contributor of bone loss that may lead to osteoporosis, this loss is accompanied by increased bone marrow adiposity. Osteoblasts and adipocytes share the same precursor cell and an inverse relationship exists between the two lineages. Therefore, identifying signaling pathways that stimulate mesenchymal stem cells osteogenesis at the expense of adipogenesis is of major importance for developing new therapeutic treatments. For this purpose, we identified by transcriptomic analysis the oxytocin receptor pathway as a potential regulator of the osteoblast/adipocyte balance of human multipotent adipose-derived stem (hMADS) cells. Both oxytocin (OT) and carbetocin (a stable OT analogue) negatively modulate adipogenesis while promoting osteogenesis in both hMADS cells and human bone marrow mesenchymal stromal cells. Consistent with these observations, ovariectomized (OVX) mice and rats, which become osteoporotic and exhibit disequilibrium of this balance, have significant decreased OT levels compared to sham-operated controls. Subcutaneous OT injection reverses bone loss in OVX mice and reduces marrow adiposity. Clinically, plasma OT levels are significantly lower in postmenopausal women developing osteoporosis than in their healthy counterparts. Taken together, these results suggest that plasma OT levels represent a novel diagnostic marker for osteoporosis and that OT administration holds promise as a potential therapy for this disease.

Elabd C, Basillais A, Beaupied H, Breuil V…
Stem Cells Sep 2008
PMID: 18583541 | Free Full Text