Monthly Archives: September 2013

CLA Isomers Promote Osteoblasts In Vitro

Abstract

Regulation of osteoblast and adipocyte differentiation from human mesenchymal stem cells by conjugated linoleic acid.

Conjugated linoleic acid (CLA) describes a group of isomers of linoleic acid and has variable effects on bone formation and adiposity in vivo and in vitro. The variability may be due to individual effects of the predominant bioactive 9cis,11trans (9,11) and 10trans,12cis (10,12) CLA isomers. Osteoblasts and adipocytes are derived from mesenchymal stem cells (MSCs), and bone loss is accompanied by an increase in marrow adiposity. Osteoblast differentiation from MSCs requires activation of Wnt/beta-catenin signaling by Wnt10b, which inhibits adipocyte differentiation by suppressing CCAAT/enhancer-binding protein (C/EBP) alpha. The objective of this study was to determine if 9,11 and 10,12 CLA affect osteoblast and adipocyte differentiation from MSCs and to determine whether any effects are associated with changes in Wnt10b and C/EBPalpha expression. Osteoblast differentiation was assessed by calcium deposition, alkaline phosphatase (ALP) activity, and the expression of Wnt10b, runx2 and osteocalcin. Adipocyte differentiation was assessed by oil red O staining and C/EBPalpha, PPARgamma and FABP4 expression. Compared to vehicle, 9,11 CLA decreased calcium deposition ( approximately 15%), increased oil red O staining ( approximately 21-28%) and increased FABP4 (AP2) expression ( approximately 58-75%). In contrast, 10,12 CLA increased calcium deposition ( approximately 12-60%), ALP activity ( approximately 2.1-fold) and the expression of Wnt10b ( approximately 60-80%) and osteocalcin ( approximately 90%), but decreased oil red O staining ( approximately 30%) and the expression of C/EBPalpha ( approximately 24-38%) and PPARgamma ( approximately 60%) (P<.05). Thus, our findings demonstrate isomer-specific effects of CLA on MSC differentiation, and suggest that 10,12 CLA may be a useful therapeutic agent to promote osteoblast differentiation from MSCs.

Platt ID, El-Sohemy A
J. Nutr. Biochem. Dec 2009
PMID: 19019668

CLA Prevents Bone Loss from Corticosteroids in Rats

Abstract

Conjugated linoleic acid prevents growth attenuation induced by corticosteroid administration and increases bone mineral content in young rats.

Corticosteroids are a common therapy in many disease states, despite frequent and potentially serious side effects. Nutritional supplementation with conjugated linoleic acid (CLA) has been shown to increase fat-free mass, whereas supplementation with n-3 and n-6 fatty acids has been shown to increase bone mineral density (BMD). To determine whether CLA can attenuate the side effects of 8 weeks of corticosteroid administration, we randomized twenty-four 5-week-old male Sprague-Dawley rats into 1 of 4 groups: control; control + methylprednisolone (7 mg.kg-1.week-1); CLA diet (1% CLA w/w); or CLA plus methylprednisolone. Body composition, bone mineral content (BMC), and BMD were assessed with dual-energy X-ray absorptiometry at the onset and at the end of the 8-week intervention. The mechanical properties of bone were determined using 3-point femur bending at the end of the intervention. Methylprednisolone resulted in an attenuation of the increase in body mass and lean mass over the 8 weeks (p < 0.05). CLA prevented the methylprednisolone-induced attenuation of body mass and lean mass accumulation. CLA also resulted in a greater increase in BMC (p < 0.05) in the lumbar spine. The energy at failure of the isolated femurs was increased with CLA (p < 0.05). Dietary CLA prevents many of the growth- and bone-related side effects arising from 8 weeks of corticosteroid administration, results in greater increases in BMC and BMD, and can contribute to an improvement in some of the mechanical properties of bone.

Roy BD, Bourgeois J, Rodriguez C, Payne E…
Appl Physiol Nutr Metab Dec 2008
PMID: 19088767

PQQ Inhibits Osteoclasts

Abstract

Inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ).

The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKL-stimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-κB and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN-β-mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN-β signaling due to augmented IFNAR expression.

Odkhuu E, Koide N, Haque A, Tsolmongyn B…
Immunol. Lett. Feb 2012
PMID: 22193059

MSM Enhances Osteoblasts and GH Signaling In Vitro

Abstract

MSM enhances GH signaling via the Jak2/STAT5b pathway in osteoblast-like cells and osteoblast differentiation through the activation of STAT5b in MSCs.

Methylsulfonylmethane (MSM) is a naturally occurring sulfur compound with well-known anti-oxidant properties and anti-inflammatory activities. But, its effects on bone are unknown. Growth hormone (GH) is regulator of bone growth and bone metabolism. GH activates several signaling pathways such as the Janus kinase (Jak)/signal transducers and activators of transcription (STAT) pathway, thereby regulating expression of genes including insulin-like growth factor (IGF)-1. GH exerts effects both directly and via IGF-1, which signals by activating the IGF-1 receptor (IGF-1R). In this study, we investigated the effects of MSM on the GH signaling via the Jak/STAT pathway in osteoblasts and the differentiation of primary bone marrow mesenchymal stem cells (MSCs). MSM was not toxic to osteoblastic cells and MSCs. MSM increased the expression of GH-related proteins including IGF-1R, p-IGF-1R, STAT5b, p-STAT5b, and Jak2 in osteoblastic cells and MSCs. MSM increased IGF-1R and GHR mRNA expression in osteoblastic cells. The expression of MSM-induced IGF-1R and GHR was inhibited by AG490, a Jak2 kinase inhibitor. MSM induced binding of STAT5 to the IGF-1R and increased IGF-1 and IGF-1R promoter activities. Analysis of cell extracts by immunoprecipitation and Western blot showed that MSM enhanced GH-induced activation of Jak2/STAT5b. We found that MSM and GH, separately or in combination, activated GH signaling via the Jak2/STAT5b pathway in UMR-106 cells. Using siRNA analysis, we found that STAT5b plays an essential role in GH signaling activation in C3H10T1/2 cells. Osteogenic marker genes (ALP, ON, OCN, BSP, OSX, and Runx2) were activated by MSM, and siRNA-mediated STAT5b knockdown inhibited MSM-induced expression of osteogenic markers. Furthermore, MSM increased ALP activity and the mineralization of MSCs. Taken together, these results indicated that MSM can promote osteogenic differentiation of MSCs through activation of STAT5b.

Joung YH, Lim EJ, Darvin P, Chung SC…
PLoS ONE 2012
PMID: 23071812 | Free Full Text

CLA No Help in Athletes

Abstract

Effects of conjugated linoleic acid supplementation during resistance training on body composition, bone density, strength, and selected hematological markers.

Conjugated linoleic acids (CLA) are essential fatty acids that have been reported in animal studies to decrease catabolism, promote fat loss, increase bone density, enhance immunity, and serve as an antiatherogenic and anticarcinogenic agent. For this reason, CLA has been marketed as a supplement to promote weight loss and general health. CLA has also been heavily marketed to resistance-trained athletes as a supplement that may help lessen catabolism, decrease body fat, and promote greater gains in strength and muscle mass during training. Although basic research is promising, few studies have examined whether CLA supplementation during training enhances training adaptations and/or affects markers of health. This study evaluated whether CLA supplementation during resistance training affects body composition, strength, and/or general markers of catabolism and immunity. In a double-blind and randomized manner, 23 experienced, resistance-trained subjects were matched according to body mass and training volume and randomly assigned to supplement their diet with 9 g;pdd(-1) of an olive oil placebo or 6 g;pdd(-1) of CLA with 3 g;pdd(-1) of fatty acids for 28 days. Prior to and following supplementation, fasting blood samples, total body mass, and dual-energy X-ray absorptiometry (DEXA) determined body composition, and isotonic bench press and leg press 1 repetition maximums (1RMs) were determined. Results revealed that although some statistical trends were observed with moderate to large effect sizes, CLA supplementation did not significantly affect (p > 0.05) changes in total body mass, fat-free mass, fat mass, percent body fat, bone mass, strength, serum substrates, or general markers of catabolism and immunity during training. These findings indicate that CLA does not appear to possess significant ergogenic value for experienced resistance-trained athletes.

Kreider RB, Ferreira MP, Greenwood M, Wilson M…
J Strength Cond Res Aug 2002
PMID: 12173945

Review: PQQ Involved with Nitric Oxide in Bones?

Abstract

Is the antioxidant, anti-inflammatory putative new vitamin, PQQ, involved with nitric oxide in bone metabolism?

Our laboratory recently isolated free PQQ (2,7,9-tricarboxy-pyrroloquinoline quinone, methoxatin), a bacterial redox cofactor, from red cells, neutrophils, serum and milk and found free PQQ in CSF, synovial fluid and bile. The metabolism and functions of PQQ and ascorbate may be coupled. Physiologically, free PQQ catalyzes dioxygen-superoxide interconversion, and participates in both superoxide generation (respiratory burst) and scavenging (cell protection). Using a labeled aromatic o-diamine, superoxide formation by activated neutrophils was inhibited and the labeled phenazine adduct of PQQ could be isolated from the inhibited cells (Karnovsky et al., 1992). PQQ may convert xanthine oxidase to xanthine dehydrogenase (XD) and could be the physiological coenzyme of XD. PQQ plus copper, form a potent amine-oxidizing system. Shah et al., 1992 found that PQQ-Cu2+ catalyzes the oxidation of epsilon-amino groups in collagen and elastin. Rucker’s lab (Smidt et al., 1991) has found that PQQ may be a vitamin for mouse pups. Watanabe et al., 1988 and Nishigori et al., 1989, showed that injected PQQ protects animals against oxidative stress injury. PQQ’s in vivo antioxidant action, spares reduced glutathione. PQQ, as an actively transported organic anion, concentrates in cells. In other experiments (Aizenman et al., 1992), PQQ protected neurons against the neurotoxin action of the glutamate-receptor against NMDA. We shall consider possible roles for PQQ in the biosynthesis of nitric oxide (NO, endothelium-derived relaxing factor, EDRF) from L-arginine and in NO removal by superoxide. NO has now been linked to the inhibition of osteoclastic bone resorption.

Gallop PM, Paz MA, Flückiger R, Henson E
Connect. Tissue Res. 1993
PMID: 8403896

Naringin Reverses Bone Loss, Increasing Density, Volume, and Thickness in Ovariectomized Rats

Abstract

Naringin promotes osteoblast differentiation and effectively reverses ovariectomy-associated osteoporosis.

Osteoporosis is a common pathological condition that influences 20 % of women over 50 years of age. This condition decreases bone strength and increases the risk of bone fracture. Naringin is a major flavonoid found in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae). Studies have shown that naringin possesses many pharmacological effects. The current study evaluated the influence of naringin on osteoblastic cell differentiation and proliferation, and assessed its therapeutic effects on a rat osteoporosis model.
The proliferation, differentiation, and function of rat bone marrow stromal cells (BMSCs) were determined following treatment with various concentrations of naringin. Ovariectomy (OVX)-induced osteoporotic rats were orally administered naringin daily at low, medium, and high dosages, while a control group received PBS for 2 months. Femoral X-ray images and microCT scans were used for bone mineral density (BMD) and BV/TV (bone volume/total volume) analyses, and histological assessments of left tibiae were employed to check for changes in trabecular thickness (Tb.Th) and trabecular space (Tb.Sp) in the groups.
Naringin was effective at enhancing the proliferation and osteogenic differentiation of BMSCs, and a concentration of 10 μg/ml prompted the highest levels of osteocalcin expression among the in vitro study groups. There appeared to be a delayed response pattern of BMSCs to the naringin treatment. Naringin also effectively reversed OVX-induced bone loss via increasing BMD, bone volume, and trabecular thickness. The medium dose (300 mg/kg) appeared to be the optimal dosage for delivering satisfactory therapeutic effects.
Naringin promotes the proliferation and differentiation of BMSCs, and increases osteocalcin expression. Naringin also effectively reverses ovariectomy-induced osteoporosis in rats. The study suggests that naringin administration may represent an effective treatment for osteoporosis.

Li N, Jiang Y, Wooley PH, Xu Z…
J Orthop Sci May 2013
PMID: 23553541

Naringin Works Like Statins to Increase Bone Formation In Vitro

Abstract

Effect of naringin on bone cells.

Statin, a HMG-CoA reductase inhibitor, was shown to increase BMP-2 gene expression for bone formation, by blocking the mevalonate pathway in cholesterol production. We investigated the effect of naringin, a flavonoid available commonly in citrus fruits, which was also a HMG-CoA reductase inhibitor, in UMR 106 osteoblastic cell line in vitro. The control group consisted of cells cultured without any intervention for different time intervals (24 h, 48 h, and 72 h), whereas the experimental (naringin) group consisted of cells cultured with naringin of different concentrations (0.001 micromol/L, 0.01 micromol/L, and 0.1 micromol/L) for the same time intervals of the control. Colorimetric Tetrazolium (MTT) assay, total protein content assay, and alkaline phosphatase activity were used to measure the cellular activities. Results for the naringin group showed an increase in MTT assay compared with the control and the effect was dose dependent. At high concentration (0.1 micromol), the increases ranged from 60% to 80%. In the total protein content assay, naringin also showed an increase compared with control and the effect was also dose dependent. At high concentration (0.1 micromol), the increases ranged from 9% to 20%. In the alkaline phosphatase activity assay, naringin at high concentration (0.1 micromol) significantly increased the activity up to 20%. In conclusion, naringin significantly increased bone cell activities in vitro. This is the first study specifically attempted to investigate the effect of naringin on bone cell activities. Besides statin, this provided another example of mevalonate pathway blockage in the cholesterol production pathway by HMG-CoA reductase inhibition will increase the bone cell activities.

Wong RW, Rabie AB
J. Orthop. Res. Nov 2006
PMID: 16944474

Naringin and Neoeriocitrin Stimulate Osteoblasts In Vitro

Abstract

Stimulative activity of Drynaria fortunei (Kunze) J. Sm. extracts and two of its flavonoids on the proliferation of osteoblastic like cells.

The osteoblastic activity of extracts of Drynaria fortunei (Kunze) J. Sm. rhizome was assayed in the UMR106 cell line cultured in vitro. An ethanol extract and its fractions were added to the cell culture at different concentrations. Osteoblastic proliferation stimulating activity was determined using the MTT method. The ethanol extract, and its ethyl acetate and n-butanol fractions exhibited stimulating activity. Two active constituents were isolated from n-butanol fraction by bioassay-directed isolation, and identified as naringin and neoeriocitrin. The latter is reported for the first time from this herbal medicine.

Li F, Meng F, Xiong Z, Li Y…
Pharmazie Nov 2006
PMID: 17152991

Naringin Inhibits Osteoporosis Induced by Retinoic Acid in Rats

Abstract

Anti-osteoporosis activity of naringin in the retinoic acid-induced osteoporosis model.

Isoflavonoids isolated from plants have been confirmed to fight osteoporosis and promote bone health. However, few studies have been conducted to describe the anti-osteoporosis activity of botanical flavonone. Based on the experimental outcomes, we demonstrated the ability of naringin to fight osteoporosis in vitro. We developed a retinoic acid-induced osteoporosis model of rats to assess whether naringin has similar bioactivity against osteoporosis in vitro. After a 14-day supplement of retinoic acid to induce osteoporosis, SD rats were administered naringin. A blood test showed that naringin-treated rats experienced significantly lower activity of serum alkaline phosphatase and had higher femur bone mineral density, compared to untreated rats. All three dosages of naringin improved the decrease in bone weight coefficient, the length and the diameter of the bone, the content of bone ash, calcium, and phosphorus content induced by retinoic acid. The data of histomorphological metrology of naringin groups showed no difference as compared to normal control rats. These outcomes suggest that naringin offer a potential in the management of osteoporosis in vitro.

Wei M, Yang Z, Li P, Zhang Y…
Am. J. Chin. Med. 2007
PMID: 17708632