Skip to main content
NCBI home page
Journal of Medicinal Food logoLink to Journal of Medicinal Food
. 2014 Mar 1;17(3):332–338. doi: 10.1089/jmf.2013.2763

Raspberry Ketone Promotes the Differentiation of C3H10T1/2 Stem Cells into Osteoblasts

Tomoyo Takata 1,,2,✉, Chie Morimoto 3
PMCID: PMC3961782  PMID: 24404978

Abstract

The decrease in the bone mass associated with osteoporosis caused by ovariectomy, aging, and other conditions is accompanied by an increase in bone marrow adipose tissue. The balance between osteoblasts and adipocytes is influenced by a reciprocal relationship. The development of modalities to promote local/systemic bone formation by inhibiting bone marrow adipose tissue is important in the treatment of fractures or metabolic bone diseases such as osteoporosis. In this study, we examined whether raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK], which is one of the major aromatic compounds of red raspberry and exhibits anti-obesity action, could promote osteoblast differentiation in C3H10T1/2 stem cells. Confluent C3H10T1/2 stem cells were treated for 6 days with 10–100 μg/mL of RK in culture medium containing 10 nM all-trans-retinoic acid (ATRA) or 300 ng/mL recombinant human bone morphogenetic protein (rhBMP)-2 protein as an osteoblast-differentiating agent. RK in the presence of ATRA increased alkaline phosphatase (ALP) activity in a dose-dependent manner. RK in the presence of rhBMP-2 also increased ALP activity. RK in the presence of ATRA also increased the levels of mRNAs of osteocalcin, α1(I) collagen, and TGF-βs (TGF-β1, TGF-β2, and TGF-β3) compared with ATRA only. RK promoted the differentiation of C3H10T1/2 stem cells into osteoblasts. However, RK did not affect the inhibition of early-stage adipocyte differentiation. Our results suggest that RK enhances the differentiation of C3H10T1/2 stem cells into osteoblasts, and it may promote bone formation by an action unrelated to adipocyte differentiation.

Key Words: : bone formation, C3H10T1/2 stem cells, osteoblast, raspberry ketone, TGF-βs

Introduction

Bone loss caused by aging and osteoporosis results in a shift in bone marrow composition to increased numbers of adipocytes, and a decline in osteoblasts.1,2 The balance between osteoblasts and adipocytes is characterized by a reciprocal relationship in which excess bone marrow adipose tissue is considered to cause osteopenia, the result of a negative risk factor due to an imbalance between osteogenesis and adipogenesis. In fact, studies suggest that osteoblasts and adipocytes are derived from a common precursor, multipotential mesenchymal stem cells in bone marrow. In addition, some specific reciprocal factors that regulate osteoblast and adipocyte differentiation were reported.3–8 Furthermore, experimental data suggest that mature osteoblasts can transdifferentiate into adipocytes or mature adipocytes can transdifferentiate into osteoblasts.9,10

Bone metabolism is known to proceed in relation to fat metabolism.11,12 Long-term epidemiological investigations have shown that high body weight, BMI, and body fat values are positively correlated with bone mass.13–17 This is because mechanical loading of the body weight has a positive effect on bone, and also as hormones derived from adipocytes of body fat, such as leptin and adiponectin, increase the bone density.13,18–21 These observations suggest the possibility of a therapeutic strategy by which the inhibition of adipogenesis in bone marrow and the control of fat metabolism will stimulate osteoblastogenesis, resulting in increased bone cells.

Raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK] is one of the major aromatic compounds of red raspberry (European red raspberry, Rubus idaeus),22 and it is used in products such as soft drinks and sweets. The structure of RK is similar to those of capsaicin and synephrine (Fig. 1), which lower adipose tissue weight.23,24 RK has also been reported to exert anti-obesity action and alter lipid metabolism.25,26 We, therefore, analyzed whether RK has a positive effect on osteogenesis, because there has been no report on its effect on bone formation.

FIG. 1.

FIG. 1.

Open in a new tab

Chemical structure of raspberry ketone [4-(4-hydroxyphenyl)butan-2-one; RK].

In this study, we examined the effects of RK on osteoblast differentiation of C3H10T1/2 stem cells, multipotential mesenchymal stem cells, which are derived from mouse primary bone marrow stromal cells and can differentiate into osteoblasts, adipocytes, chondrocytes, and myocytes. In this experiment, we used all-trans-retinoic acid (ATRA) (Fig. 2) and recombinant human bone morphogenetic protein-2 (rhBMP-2) as differentiation agents to induce C3H10T1/2 stem cells into osteoblasts. Retinoic acid (RA) and BMP-2 have been reported to play roles in osteoblast differentiation and skeletal development.27–30 ATRA, which is one of the isomers of RA, has been reported to stimulate the activity and expression of alkaline phosphatase (ALP), an early osteoblast marker, but not osteocalcin, which is a late osteoblast marker, using C3H10T1/2 stem cells.31 BMP-2 has also been reported to induce ALP activity in C3H10T1/2 stem cells.27 Moreover, we explored whether the mechanism by which RK promoted bone formation depended on controlling the differentiation of mesenchymal stem cells into adipocytes, because osteoblasts and adipocytes have a reciprocal relationship, as mentioned earlier. TGF-βs inhibit adipocyte differentiation and also stimulate osteoblasts to synthesize extracellular matrix proteins in the earlier stage of osteoblast differentiation.32–34 BMP-2 can differentiate uncommitted mesenchymal stem cells into osteoblasts.27–30 Therefore, the expressions of TGF-βs and BMP-2 during osteoblast differentiation of C3H10T1/2 stem cells by RK were also examined.

FIG. 2.

FIG. 2.

Open in a new tab

Chemical structure of all-trans-retinoic acid (ATRA).

Materials And Methods

Dose-dependent effect of RK on viability of C3H10T1/2 stem cells

Cell viability assessment was performed according to the protocol for the CellTiter 96® Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA). The CellTiter 96 Aqueous One Solution Reagent contains a novel tetrazolium compound [MTS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] and an electron coupling reagent (phenazine ethosulfate [PES]). Cells were diluted to a density of 1×104 cells/mL in each medium containing five different doses of RK (10, 20, 50, 100, 200, and 500 μg/mL), and 100 μL of the cell dilutions were added to each well of 96-well tissue culture-treated plates. The plates were incubated at 37°C for 72 h in a humidified, 5% CO2 atmosphere. Twenty microliters' aliquots of the stock MTS reagent were added to each well containing 100 μL of medium and incubated with the cells for 1 h. After the incubation period, absorbance was recorded at 490 nm using a 96-well plate reader. The assay was performed in six wells with each concentration of RK (10, 20, 50, 100, 200, and 500 μg/mL).

Effect of RK under osteoblast differentiation conditions

C3H10T1/2 clone 8 stem cells (Health Science Research Resources Bank, Osaka, Japan) were grown to confluence in Dulbecco's-modified Eagle's medium containing 10% fetal bovine serum, 50 units/mL of penicillin, and 50 mg/mL of streptomycin (basal medium) in a humidified atmosphere containing 5% CO2. After achieving confluence, osteoblast differentiation was initiated with the basal medium that was supplemented with 10 nM ATRA (Sigma, St. Louis, MO, USA) or recombinant human BMP-2 (rhBMP-2) (R&D Systems, Inc., Minneapolis, MN, USA) at 300 ng/mL as a differentiating agent on pluripotential mesenchymal stem cells and 10–100 μg/mL raspberry ketone [4-(4-hydroxyphenyl)butan-2-one: RK] (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The medium was changed every 2 days.

Effect of RK under adipocyte differentiation conditions

C3H10T1/2 clone 8 stem cells were grown to confluence in the basal medium. Confluent cultures were treated for 2 days with the adipocyte differentiation inducer, which contained 0.5 mM 3-isobutyl-1-methylxanthine (MIX), 1 μM dexamethasone (DEX), and 1 μg/mL insulin (INS) in the basal medium. Two days later, the medium was replaced with the basal medium containing 1 μg/mL INS for 6 days to promote terminal adipocyte differentiation. To examine the effect of RK on inducing adipocyte differentiation at an early stage or toward promoting adipocyte differentiation at a late stage, 100 μg/mL RK was added to the basal medium that was supplemented with 0.5 mM MIX, 1 μM DEX, 1 μg/mL INS, or 1 μg/mL INS alone. The medium was changed every 2 days.

ALP assay in the cells

For assaying, confluent cells under the condition of osteoblast differentiation were harvested in 0.3 mL of 10 mM Tris-HCl buffer (pH 7.4) containing 0.1% Triton X and sonicated briefly at 0°C. The ALP activities in aliquots of the homogenate after being centrifuged were measured to analyze products generated from p-nitrophenyl phosphate as a substrate using a kit (Wako Pure Chemical Industries, Ltd.). Other aliquots of the homogenate were assayed for the amount of protein using the DC Protein Assay (Bio-Rad, Hercules, CA, USA). One unit of ALP activity was defined as that releasing 1 μmol of p-nitrophenol per 1 mg of protein for 1 min at 37°C.

Staining of ALP activity in the cells

Confluent cells treated with or without RK in culture medium containing 10 nM ATRA were fixed with 10% formalin in ethanol. After being fixed, cytochemical staining was performed with an AP-Red substrate kit (Zymed Laboratories, Inc., San Francisco, CA, USA), and the cells with ALP activity were dyed red (ALP activity-positive cells).

Measurement of triacylglycerol contents

To measure triacylglycerol (TG) contents of adipocyte differentiation-induced cells, the cells were harvested, sonicated, and centrifuged. Aliquots of the homogenate were used for protein and TG measurements. Protein was measured using the DC Protein Assay, and TG was measured using a Triglyceride Quantification Kit (Wako Pure Chemical Industries, Ltd.). TG contents are shown per milligram of protein.

Reverse transcription–polymerase chain reaction assay

Total RNA was extracted from confluent cells according to the manufacturer's protocol using the GeneElute™ mammalian Total RNA Kit (Sigma). A total of 1 μg of RNA-treated DNase I (Sigma) was reverse transcribed to cDNA using the Transcriptor First-Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA). One microliter of the product was used for polymerase chain reaction (PCR) amplification in a total volume of 25 μL containing 1×Taq reaction buffer, 0.2 mM dNTPs, 2 mM MgCl2, 1 μM of each primer, and 0.5 U of Takara Ex Taq® polymerase Hot Start Version (Takara, Otsu, Japan). The primers for α1(I) collagen, osteocalcin, transforming growth factor βs (TGF-β1, TGF-β2, and TGF-β3), and BMP-2 were synthesized by referring to already published articles.35,36 β-Actin37 was used as the housekeeping gene as an endogenous control. PCR cycles were performed with the following temperature profile: denaturation at 94°C for 1 min after initial denaturation for 5 min, primer annealing at 54°C–60°C for 1 min, and primer extension at 72°C for 1 min. After the initial denaturation for 5 min, we employed 22–32 cycles, followed by a final extension at 72°C for 5 min. PCR products was electrophoresed on 4% agarose gel and stained with ethidium bromide, and bands were visualized using a UV transilluminator.

Statistical analysis

All data are expressed as the mean±SE. Analyses of levels of ALP activity in the presence of ATRA, the TG content, and the cell viability were performed using one-way ANOVA, followed by Dunnett's test if significance was identified. Statistical analyses of levels of ALP activity in the presence of rhBMP-2 were analyzed using Student's two-tailed t-test. P values<.05 were considered significant.

Results

Dose-dependent effect of RK on cell viability

Figure 3 shows the dose-dependent effect of RK on C3H10T1/2 stem cell viability. From this study, we found that RK above a dose of 100 μg/mL caused a significant decrease in viability. So, RK of an approximate dose of 100 μg/mL was chosen for subsequent in vitro experiments.

FIG. 3.

FIG. 3.

Open in a new tab

Dose-dependent effects on cell viability. Dose-dependent effect of RK on cell viability. The MTS assay was carried out for this purpose. RK-0, RK-10, RK-20, RK-50, RK-100, RK-200, and RK-500: cell viability in RK-exposed C3H10T1/2 stem cells at a dose of 0, 10, 20, 50, 100, 200, and 500 μg/mL, respectively. Each point represents the mean±SE, n=6 (number of wells of 96-well tissue culture-treated plates). *P<.05 (vs. the value obtained from cultures treated with RK-0).

Measurement of ALP activity and its staining

Figure 4A shows the ALP activity of RK with or without ATRA. RK with ATRA increased ALP activity in a dose-dependent manner at a concentration of 10 to 100 μg/mL, and the change in ALP activities with RK addition at concentrations of 20 to 100 μg/mL with ATRA was significantly higher compared with that on ATRA treatment alone. However, RK without ATRA was not able to increase ALP activity. Figure 4B shows the staining of ALP activity-positive cells treated with and without 50 μg/mL of RK under ATRA. In the cell cultures treated with ATRA alone, there were many ALP activity-positive cells but their staining intensity was low; however, cell cultures with RK addition under ATRA showed greater staining compared with ATRA treatment alone. This result corresponded with that of ALP activity.

FIG. 4.

FIG. 4.

Open in a new tab

Effect of various concentrations of RK on alkaline phosphatase (ALP) activity and ALP activity staining in the presence of ATRA. (A) Confluent cells were treated for 6 days with RK at the indicated concentrations in the presence or absence of 10 nM ATRA. The ALP activity was measured as described in Materials and Methods. Values given are the mean±SE for three experiments. *P<.05 (vs. the value obtained from cultures treated with ATRA alone.). (B) Confluent cells were treated for 6 days with/without 50 μg/mL of RK in the presence of 10 nM ATRA, fixed, and stained as described in Materials and Methods.

In addition, in the cell cultures treated with rhBMP-2 as an osteoblast-differentiating agent, when 100 μg/mL RK was added, ALP activity increased significantly (Fig. 5).

FIG. 5.

FIG. 5.

Open in a new tab

ALP activity by RK in the presence of recombinant human bone morphogenetic protein-2 (rhBMP-2) protein. The results indicated ALP activity of the confluent cells treated with or without RK for 5 days under 300 ng/mL rhBMP-2 protein. Values given are the mean±SE for three experiments. *P<.05 (vs. the value obtained from cultures treated with rhBMP-2 protein alone).

Effect of RK on mRNA expression of osteoblast differentiation markers

Figure 6 shows the levels of mRNAs of α1(I) collagen and osteocalcin as osteogenic markers in the simultaneous presence of RK and ATRA. An RK concentration higher than 10 μg/mL with 10 nM ATRA increased α1(I) collagen mRNA levels. On the other hand, a marked induction of osteocalcin, a marker of late osteoblast differentiation, was seen with RK higher than 50 μg/mL with 10 nM ATRA. In the presence of 10 nM ATRA, RK promoted terminal osteoblastic differentiation.

FIG. 6.

FIG. 6.

Open in a new tab

Reverse transcription–polymerase chain reaction (RT-PCR) analysis of the expression of osteoblast differentiation markers in C3H10T1/2 stem cells treated with RK in the presence of ATRA. Effect of RK on endogenous α1(I) collagen and osteocalcin expression as osteoblast differentiation markers, showing a dose-dependent relation. These are the results when confluent cells were treated for 6 days with RK at the indicated concentrations in the presence of 10 nM ATRA. V, vehicle; C, negative control [RT (−)].

Effect of RK on mRNA expression of BMP-2 and TGF-βs for osteoblast differentiation

Some BMPs are strong bone inducers in mesenchymal progenitor cells; BMP-2 appears to be the most potent inducer of bone formation. The mRNA expression of BMP-2 showed no difference with RK at concentrations from 10 to 100 μg/mL. The mRNA expressions of TGF-β1, TGF-β2, and TGF-β3 markedly increased in the presence of RK plus 10 nM ATRA compared with 10 nM ATRA only (Fig. 7). RK treatment resulted in a dose-dependent increase in TGF-β3 mRNA levels.

FIG. 7.

FIG. 7.

Open in a new tab

RT-PCR analysis of the expression of BMP-2 and TGF-β1, -β2, and -β3 by RK in the presence of ATRA. Confluent cells were treated for 6 days with RK at the indicated concentrations in the presence of 10 nM ATRA. C, negative control [RT (-)].

Effect on adipocyte differentiation of RK

Since the differentiations of osteoblasts and adipocytes are conflicting, we examined the effect of RK on the induction of adipocyte differentiation at an early stage and the promotion of adipocyte differentiation at a late stage (Fig. 8). The effect of RK during early-stage adipocyte differentiation resulted in significantly increased TG content compared with the untreated cultures. However, RK under late-stage adipocyte differentiation resulted in decreased TG content relative to the untreated cultures.

FIG. 8.

FIG. 8.

Open in a new tab

Triacylglycerol (TG) content in C3H10T1/2 stem cells with or without RK under adipocyte differentiation. Confluent cells were cultured treated with 100 μg/mL RK in MDI medium (for first 2 days) or in the basal medium, including insulin (INS) (for 6 days after the first 2 days). The TG content was measured as described in Materials and Methods. Values given are the mean±SE of three experiments. *P<.05 (vs. the value obtained from cultures not treated with RK.). MDI medium: 0.5 mM 3-isobutyl-1-methylxanthine (MIX), 1 μM dexamethasone (DEX), and 1 μg/mL INS in the basal medium.

Discussion

Our study investigated whether RK promotes osteoblast differentiation using C3H10T1/2 stem cells. The results of cell cultures demonstrate that RK more markedly increased ALP activity in pluripotential mesenchymal stem cells treated with ATRA, an osteoblast differentiating-inducing agent; and cultured cells showed high-intensity ALP activity staining. When rhBMP-2 as an osteoblast differentiation-inducing agent was added, ALP activity was increased by RK as well as by ATRA, and it was possible to promote osteoblast differentiation regardless of these inducing agents. Moreover, although the levels of mRNAs of α1(I) collagen and osteocalcin in the cultures treated with ATRA alone were very low, the simultaneous presence of RK with ATRA increased the levels of these two mRNAs. The expression of osteocalcin increased in a concentration-dependent manner after a 6-day culture. During the differentiation of osteoblasts, they express a characteristic pattern of genes.38,39 ALP and α1(I) collagen are molecular markers for the early stage of osteoblast differentiation.39 The expression of osteocalcin is a molecular marker for the late stage of osteoblast differentiation, and it is a non-collagenous protein that is produced in osteoblasts and exclusively expressed in the bone39 When C3H10T1/2 stem cells were cultured with 1 μM ATRA for only 6 days, it was reported that the expression of osteocalcin was not noted even if ALP activity underwent marked induction.40 Since the expression of osteocalcin was noted after RK addition with 10 nM ATRA, RK may enhance osteogenesis and promote the terminal osteoblastic differentiation of C3H10T1/2 stem cells.

TGF-βs that belong to the TGF-β superfamily are present at high levels in the bone and have been reported to be associated with the bone remodeling process as a “coupling factor” which links bone resorption to subsequent bone formation. TGF-βs also stimulates osteoblasts to synthesize extracellular matrix proteins in the earlier stage of osteoblast differentiation32 and inhibit adipocyte differentiation.32–34 Since BMP-2 can differentiate uncommitted mesenchymal stem cells into osteoblasts,27–30 the possibility of the therapeutic use of BMP-2 for osteogenesis has been studied.41,42 We questioned whether bone formation by RK occurred through the up-regulation of BMP-2; however, in cultures treated with RK and ATRA, the influence of RK addition on BMP-2 was very weak. However, the expressions of TGF-β1, TGF-β2, and TGF-β3 were increased by RK addition. The expression of TGF-β3 dose-dependently increased in proportion to RK. This result was different from the report that the expression of TGF-β1 and TGF-β2 increased and that of TGF-β3 decreased in C3H10T1/2 stem cells in the presence of ATRA only.40 In our study, RK promoted a further increase in the expression of TGF-β1 and TGF-β2, and it also increased that of TGF-β3, irrespective of the effect of ATRA. Based on the up-regulation of TGF-β1, TGF-β2, and TGF-β3 expression by RK, we observed the effect on adipocyte differentiation of RK. C3H10T1/2 stem cells were markedly induced to differentiate into adipocytes by a combination of RK and adipocyte differentiation inducers (MIX, DEX, and INS). However, when the cultures were treated with a combination of RK and INS for the purpose of promoting terminal adipocyte differentiation, RK decreased TG accumulation (Fig. 8). This seemed to have inhibited terminal differentiation into adipocytes, although RK did not inhibit early adipocyte differentiation. In previous studies, RK was reported to cause lypolysis in differentiated adipocytes and preadipocytes.25,26 Although our results may also support the promotion of lipolysis by RK, further investigation is needed to clarify the effect of RK on C3H10T1/2 stem cells and whether it was based on the promotion of lipolysis or an anti-INS effect. Collectively, the promotion of osteoblast differentiation by RK might not be mediated by TGF-βs.

In conclusion, our results indicate that RK has the ability to promote the differentiation of C3H10T1/2 stem cells into osteoblasts. RK also induced differentiation into adipocytes at an early stage but decreased the TG content of the cells at a late stage. Further investigations of the mechanisms leading to the effect of RK on osteoblast differentiation are required.

Acknowledgment

This work was supported by Grants in Aid for Scientific Research (16790862).

Author Disclosure Statement

No competing financial interests exist.

References

  • 1.Burkhardt R, Kettner G, Böhm W, Schmidmeir M, Schlag R, Frisch B, Mallmann B, Eisenmenger W, Glig TH: Changes in trabecular bone, hematopoesis and bone marrow vessels in aplastic anaemia, primary osteoporosis and old age: a comparative histomorphometric study. Bone 1987;8:157–164 [DOI] [PubMed] [Google Scholar]
  • 2.Meunier P, Aaron J, Edouard C, Vignon G: Osteoporosis and replacement of cell populations of the marrow by adipose tissue. Clin Orthop Relat Res 1971;80:147–154 [DOI] [PubMed] [Google Scholar]
  • 3.Gimble JM, Zvonic S, Floyd ZE, Kassem M, Nuttall ME: Playing with bone and fat. J Cell Biochem 2006;98:251–266 [DOI] [PubMed] [Google Scholar]
  • 4.Harada S, Rodan GA: Control of osteoblast function and regulation of bone mass. Nature 2003;423:349–355 [DOI] [PubMed] [Google Scholar]
  • 5.Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung U, Kubota N, Terauchi Y, Harada Y, Azuma Y, Nakamura K, Kadowaki T, Kawaguchi H: PPARγ insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest 2004;113:846–855 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Chang SL, Shao JS, Carlton-Kachigian N, Loewy AP, Towle DA: Msx2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem 2003;278:45969–45977 [DOI] [PubMed] [Google Scholar]
  • 7.Bäckesjö CM, Li Y, Lindgren U, Haldosén LA: Activation of Sirt1 decreases adipocyte formation during osteoblast differentiation of mesenchymal stem cells. J Bone Miner Res 2006;21:993–1002 [DOI] [PubMed] [Google Scholar]
  • 8.Hong JH, Hwang ES, McManus MT, Amsterdam A, Tian Y, Kalmukova R, Mueller E, Benjamin T, Spiegelman BM, Sharp PA, Hopkins N, Yaffe MB: TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 2005;309:1074–1078 [DOI] [PubMed] [Google Scholar]
  • 9.Song L, Tuan RS: Transdifferentiation potential of human mesenchymal stem cells derived from bone marrow. FASEB J 2004;18:980–982 [DOI] [PubMed] [Google Scholar]
  • 10.Schilling T, Nöth U, Klein-Hitpass L, Jakob F, Schütze N: Plasticity in adipogenesis and osteogenesis of human mesenchymal stem cells. Mol Cell Endocrinol 2007;271:1–17 [DOI] [PubMed] [Google Scholar]
  • 11.Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G: Stimulation of bone formation in vitro and in rodents by statins. Science 1999;286:1946–1949 [DOI] [PubMed] [Google Scholar]
  • 12.Cao JJ: Effects of obesity on bone metabolism. J Orthop Surg Res 2011;6:30–36 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhao LJ, Jiang H, Papasian CJ, Maulik D, Drees B, Hamilton J, Deng HW: Correlation of obesity and osteoporosis: effect of fat mass on the determination of osteoporosis. J Bone Miner Res 2008;23:17–29 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Reid IR, Ames R, Evans MC, Sharpe S, Gamble G, France JT, Lim TM, Cundy TF: Determinants of total body and regional bone mineral density in normal postmenopausal women-a key role for fat mass. J Clin Endocrinol Metab 1992;75:45–51 [DOI] [PubMed] [Google Scholar]
  • 15.Felson DT, Zhang Y, Hannan MT, Anderson JJ: Effects of weight and body mass index bone mineral density in men and women: the Framingham study. J Bone Miner Res 1993;8:567–572 [DOI] [PubMed] [Google Scholar]
  • 16.Marcus R, Greendale G, Blunt BA, Bush TL, Sherman S, Sherwin R, Wahner H, Wells B: Correlates of bone mineral density in the postmenopausal estrogen/progestin interventions trial. J Bone Miner Res 1994;9:1467–1476 [DOI] [PubMed] [Google Scholar]
  • 17.Ravn P, Cizza G, Bjarnason NH, Thompson D, Daley M, Wasnich RD, Mcclung M, Hosking D, Yates AJ, Christiansen C: Low body mass index is an important risk factor for low bone mass and increased bone loss in early postmenopausal woman. J Bone Miner Res 1999;14:1622–1627 [DOI] [PubMed] [Google Scholar]
  • 18.Borer KT, Fogleman K, Gross M, La New JM, Dengel D: Walking intensity for postmenopausal bone mineral preservation and accrual. Bone 2007;41:713–721 [DOI] [PubMed] [Google Scholar]
  • 19.Dehority W, Halloran BP, Bikle DD, Curren T, Kostenuik PJ, Wronski TJ, Shen Y, Rabkin B, Bouraoui A, Morey-Holton E: Bone and hormonal changes induced by skeletal unloading in the mature male rat. Am J Physiol 1999;276:62–69 [DOI] [PubMed] [Google Scholar]
  • 20.Isaia GC, D'Amelio P, Di Bella S, Tamone C: Is leptin the link between fat and bone mass? J Endocrinol Invest 2005;28:61–65 [PubMed] [Google Scholar]
  • 21.Oshima K, Nampei A, Matsuda M, Iwaki M, Fukuhara A, Hashimoto J, Yoshikawa H, Shimomura I: Adiponectin increase bone mass by suppressing osteoclast and activating osteoblast. Biochem Biophys Res Commun 2005;331:520–526 [DOI] [PubMed] [Google Scholar]
  • 22.Gallois A: Quantitative evaluation of raspberry ketone using thin-layer chromatography. Sci Aliments 1982;2:99–106 [Google Scholar]
  • 23.Kawada T, Hagiwara K, Iwai K: Effects of capsaicin on lipid metabolism in rats fed a high fat diet. J Nutr 1986;116:1272–1278 [DOI] [PubMed] [Google Scholar]
  • 24.Haaz S, Fontaine KR, Cutter G, Limdi N, Perumean-Chaney S, Allison DB: Citrus aurantium and synephrine alkaloids in the treatment of overweight and obesity: an update. Obes Rev 2006;7:79–88 [DOI] [PubMed] [Google Scholar]
  • 25.Morimoto C, Satoh Y, Hara M, Inoue S, Tsujita T, Okuda H: Anti-obese action of raspberry ketone. Life Sci 2005;77:194–204 [DOI] [PubMed] [Google Scholar]
  • 26.Park KS: Raspberry ketone increases both lipolysis and fatty acid oxidation in 3T3-L1 adipocytes. Planta Med 2010;76:1654–1658 [DOI] [PubMed] [Google Scholar]
  • 27.Katagiri T, Yamaguchi A, Ikeda T, Yoshiki S, Wozney JM, Rosen V, Wang EA, Tanaka H, Omura S, Suda T: The non-osteogenic mouse pluropotent cells by recombinant human bone moephogenetic protein-2. Biochem Biophys Res Commun 1990;172:295–299 [DOI] [PubMed] [Google Scholar]
  • 28.Smith SM, Pang K, Sundin O, Wedden SE, Thaller C, Eichele G: Molecular approaches to vertebrate limb morphogenesis. Development 1989;107:121–131 [DOI] [PubMed] [Google Scholar]
  • 29.Canalis E, Economides AN, Gazzerro E: Bone morphogenetic proteins, their antagonists, and the skelton. Endocr Rev 2003;24:218–235 [DOI] [PubMed] [Google Scholar]
  • 30.Skillington J, Choy L, Derynck R: Bone morphogenetic protein and retinoic acid signaling cooperate to induce osteoblast differentiation of preadipocytes. J Cell Biol 2002;159:135–146 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Hisada K, Hata K, Ichida F, Matsubara T, Orimo H, Nakano T, Yatani H, Nishimura R, Yoneda T: Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes. J Bone Miner Metab 2012;31:53–63 [DOI] [PubMed] [Google Scholar]
  • 32.Dallas SL, Alliston T, Bonewald LF: Transforming growth factor-β. In: Principles of Bone Biology (Bilezikian JP, Raisz LG, Martin TJ, eds.) Academic Press, San Diego, 2008, pp. 1145–1166 [Google Scholar]
  • 33.Locklin RM, Oreffo ROC, Triffitt JT: Effects of TGFβ and βFGF on the differentiation of human bone marrow stromal fibroblasts. Cell Biol Int 1999;23:185–194 [DOI] [PubMed] [Google Scholar]
  • 34.Petruschke T, Röhrig K, Hauner H: Transforming growth factor beta (TGF-β) inhibits the differentiation of human adipocyte precursor cells in primary culture. Int J Obes Relat Metab Disord 1994;18:532–536 [PubMed] [Google Scholar]
  • 35.Zamurovic N, Cappellen D, Rohner D, Susa M: Coordinated activation of Notch, Wnt, and transforming growth factor-β signaling pathways in bone morphogenic protein 2-induced osteogenesis. J Biol Chem 2004;279:37704–37715 [DOI] [PubMed] [Google Scholar]
  • 36.Moerman EJ, Teng K, Lipschitz DA, Lecka-Czernik B: Aging activates adipogenic and suppresses osteogenic programs on mesenchymal marrow stroma/stem cells: the role of PPAR-γ2 transcription factor and TGF-β/BMP signaling pathways. Aging Cell 2004;3:379–389 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Song C, Guo Z, Ma Q, Chen Z, Liu Z, Jia H, Dang G: Simvastatin induces osteoblastic differentiation and inhibits adipocytic differentiation in mouse bone marrow stromal cells. Biochem Biophys Res Commun 2003;308:458–462 [DOI] [PubMed] [Google Scholar]
  • 38.Caetano-Lopes J, Canhão H, Fonseca JE: Osteoblats and bone formation. Acta Reumatol Port 2007;32:103–110 [PubMed] [Google Scholar]
  • 39.Cohen MM, Jr.: The new bone biology: pathologic, molecular, and clinical correlates. Am J Med Genet A 2006;140A:2646–2706 [DOI] [PubMed] [Google Scholar]
  • 40.Gazit D, Ebner R, Kahn AJ, Derynck R: Modulation of Expression and cell surface binding of members of the transforming growth factor-β superfamily during retinoic acid-induced osteoblastic differentiation of multipotential mesenchymal cells. Mol Endocrinol 1993;7:189–198 [DOI] [PubMed] [Google Scholar]
  • 41.Turgeman G, Zilberman Y, Zhou S, Kelly P, Moutsatsos IK, Kharode YP, Borella LE, Bex FJ, Komm BS, Bodine PVN, Gazit D: Systemically administered rhBMP-2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice. J Cell Biochem 2002;86:461–474 [DOI] [PubMed] [Google Scholar]
  • 42.Chang SCN, Chuang HL, Chen YR, Chen JK, Chung HY, Lu YL, Lin HY, Tai CL, Lou J: Ex vivo gene therapy in autologous bone marrow stromal stem cells for tissue-engineered maxillofacial bone regeneration. Gene Ther 2003;10:2013–2019 [DOI] [PubMed] [Google Scholar]

Articles from Journal of Medicinal Food are provided here courtesy of Mary Ann Liebert, Inc.

RESOURCES