Lutein Enhances Bone Mass by Stimulating Bone Formation and Suppressing Bone Resorption in Growing Mice

Abstract

Lutein is a member of the xanthophyll family of carotenoids, which are known to prevent hypoxia-induced cell damage in the eye by removing free radicals. However, its role in other tissues is controversial, and the effects of lutein on bone tissues are unknown. To identify a possible role of lutein in bone tissues, we examined the effects of lutein on bone formation and bone resorption and on femoral bone mass in mice. Lutein enhanced the formation of mineralized bone nodules in cultures of osteoblasts. On the other hand, lutein clearly suppressed 1α, 25-dihydroxyvitamin D₃-induced bone resorption as measured by pit formation in organ culture of mouse calvaria. In co-cultures of bone marrow cells and osteoblasts, lutein suppressed 1α, 25-dihydroxyvitamin D₃-induced osteoclast formation. In cultures of bone marrow macrophages, lutein suppressed soluble RANKL, the receptor activator of nuclear factor-kappaB (NF-κB) ligand, induced osteoclast formation. When five-week-old male mice were orally administered lutein for 4 weeks, the femoral bone mass was clearly enhanced in cortical bone, as measured by bone mineral density in dual X-ray absorptiometry and micro computed tomography (µCT) analyses. The present study indicates that lutein enhances bone mass in growing mice by suppressing bone resorption and stimulating bone formation. Lutein may be a natural agent that promotes bone turnover and may be beneficial for bone health in humans.

Bone remodeling is regulated by osteoclastic bone resorption and new bone formation. Osteoclasts are the primary bone-resorbing cells and are differentiated from monocyte-macrophages by a mechanism involving the receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL), which is expressed on the cell surface of osteoblasts.¹⁾ On the surface of bone tissues, osteoblasts stimulate new bone formation, which accompanies the production of matrix protein and mineralization. In young individuals, bone formation occurs dominantly than bone resorption, and bone mass increases in an age-dependent manner. After sexual maturation, the balance of bone formation and bone resorption remains constant to maintain the bone mass.

Lutein is a member of the xanthophyll family of carotenoids, a group of 40-carbon hydroxylated compounds that are synthesized in green leafy plants such as spinach and kale, corn and egg yolks.²⁾ Lutein acts as a powerful antioxidant and filters high-energy blue light.^3,4) The dietary intake of lutein is mainly accumulated in the retina of the eye, where it protects against hypoxia-induced cell damage in the eye by removing free radicals such as reactive oxygen species (ROS). In addition to the eye, lutein also may act on various tissues to prevent age-related diseases. However, its role in bone tissues remains to be elucidated.

Epidemiological studies have found a positive correlation between bone mass and the intake of vegetables and fruits,⁵⁾ and some natural components have been identified as being beneficial for bone tissues. We reported that genistein, a typical soybean isoflavone, prevents bone loss due to estrogen deficiency in mice.⁶⁾ Citrus factors, such as β-cryptoxanthin and hesperidin, have been reported to attenuate bone loss due to aging and the loss of sex steroid.^7,8) We previously reported that nobiletin, a polymethoxy flavonoid, suppresses bone resorption and maintain bone mass in mice.⁹⁾ Recent studies have also suggested a relationship between the activity of anti-oxidants and bone remodeling.¹⁰⁾ It is therefore possible that lutein exerts beneficial effects on bone tissues.

In the present study, we evaluated the effects of lutein on bone formation and bone resorption, and on bone mass in growing male mice. We will suggest that vegetable-derived lutein may be a natural agent that is beneficial for bone health in humans.

MATERIALS AND METHODS

Animals and Reagents

Newborn and 5- and 6-week-old ddy mice were obtained from Japan SLC Inc. (Shizuoka, Japan). Lutein, (3R,3′R,6′R)-β,ε-carotene-3,3′-diol, is a member of the xanthophyll family of carotenoids (Fig. 1A). Five-week-old male mice were orally administered lutein (1% [w/w]) through laboratory chow containing 1.12% calcium and 1.07% phosphorus (Nippon Clea, Tokyo, Japan) for 4 weeks. Through this diet containing 1% lutein, mice were fed 66 mg lutein/d/mouse. All procedures were performed in accordance with the institutional guidelines for animal research. The in vitro studies used highly purified lutein (purity: 99.9%; CaroteNature Co., Ltd., Ostermundingen, Switzerland), and the animal studies used FloraGLO Lutein 10%VG TabGrade (lutein content: min. 10.0%; Kemin Industries, Inc., Des Moines, Iowa, U.S.A.).

Fig. 1. The Effects of Lutein on Bone Formation and Osteoclast Formation

(A) The chemical structure of lutein. (B) To examine the effects of lutein on bone formation in vitro, primary osteoblastic cells were cultured with bone-inducing factors (50 µg/mL of ascorbic acid and 10 mM β-glycerophosphate) in the presence of lutein (3, 10 µM) for 14 d. After culturing, cells were stained with alkaline phosphatase (blue) and alizarin red (red). The area of alizarin-positive cells was measured on NIH images. The data are expressed as the means±S.E.M. of 4 independent wells. The asterisks and hashes indicate a significant difference: ** p<0.01 vs. control, ^### p<0.001 vs. bone-inducing factors. (C) Mouse bone marrow cells and osteoblastic cells were co-cultured for 7 d in the presence of 1α,25(OH)₂D₃ (100 nM) with and without lutein (3, 10 µM). The cells were stained for TRAP to detect osteoclasts. The data are expressed as the means±S.E.M. of 4 independent wells. The asterisks and hashes indicate a significant difference: *** p<0.001 vs. control, ^### p<0.001 vs. 1α,25(OH)₂D₃. (D) Mouse calvariae were cultured for 24 h in BGJb containing 1 mg/mL of BSA. After 24 h, the calvariae were transferred to new media, and were cultured for 5 d in the presence of 1α,25(OH)₂D₃ (100 nM) with and without lutein (3, 10 µM). The absorbed pit areas in calvariae were examined by a micro-focus X-ray CT system, and quantified with imaging software (Image J). The data are expressed as the means±S.E.M. of 4 independent wells. The asterisks and hashes indicate a significant difference: * p<0.05 vs. control, ^# p<0.05 vs. 1α,25(OH)₂D₃. (E) Mouse bone marrow cells were cultured with M-CSF for 5 d to form macrophages. The bone marrow macrophages were cultured with soluble RANKL (sRANKL; 100 ng/mL) for 5 d with or without lutein (3, 10 µM), and the formed osteoclasts were stained for TRAP. The data are expressed as the means±S.E.M. of 4 independent wells. The asterisks and hashes indicate a significant difference: *** p<0.001 vs. control, ^## p<0.01 vs. sRANKL.

Bone Formation with Mineralization

Primary osteoblastic cells were isolated from newborn mouse calvariae after five routine sequential digestions with collagenase and dispase,¹¹⁾ and fractions 2–4 were combined and cultured in α minimum essential medium (αMEM) with 10% fetal calf serum (FCS). The osteoblastic cells were cultured for 14 d in αMEM with 10% FCS containing bone-inducing factors, ascorbic acid (50 µg/mL) and β-glycerophosphate (10 mM) to form calcified bone nodules. After culturing, cells were stained with alkaline phosphatase and alizarin red. The areas of the alizarin-positive cells were defined as mineralized bone nodules, and the alizarin-positive area was measured on NIH images.

Osteoclast Formation in Co-cultures of Mouse Bone Marrow Cells and Osteoblasts and in Cultures of Mouse Bone Marrow Macrophages

Bone marrow cells (3×10⁶ cells) were isolated from 6-week-old mice and co-cultured with primary osteoblast cells (1×10⁴ cells) in αMEM containing 10% FCS.¹²⁾ After culturing for 7 d, the cells adhering to the well surface were stained for tartrate-resistant acid phosphatase (TRAP). The TRAP-positive multinucleated cells that contained three or more nuclei per cell were counted as osteoclasts. To examine the osteoclast differentiation from precursor cells, bone marrow macrophages were prepared by 5 d of culturing of bone marrow cells with macrophage colony-stimulating factor (M-CSF) (100 ng/mL), and cultured for 5 d with both M-CSF and soluble RANKL (sRANKL) to detect TRAP-positive osteoclasts.¹³⁾

Bone-Resorbing Activity in Organ Cultures of Mouse Calvaria

Calvariae were collected from newborn mice, dissected in half and cultured for 24 h in BGJb containing 1 mg/mL of bovine serum albumin.¹²⁾ After 24 h, the calvariae were transferred to new medium, and were cultured for another 5 d. The absorbed pit areas were examined by a micro-focus X-ray CT system (inspeXio SMX-90T; Shimadzu Co., Ltd., Kyoto, Japan), and the pit area was quantified with imaging software (Image J).

Measurement of Bone Mineral Density

The bone mineral density (BMD) of the mouse femurs was measured by dual X-ray absorptiometry (model DCS-600R; Aloka), as previously reported.¹²⁾ The bone mineral content of the femurs closely correlated with the ash weight. The BMD was calculated by dividing the bone mineral content of the measured area by the total area.

Micro Computed Tomography (CT) Analysis

CT of the femurs was performed using a micro-focus X-ray CT system (inspeXio SMX-90T; Shimadzu Co., Ltd., Kyoto, Japan). Three-dimensional microstructural image data were reconstructed using a cross-section of the distal femur for trabecular bone, or a cross-section of the central shaft for cortical bone. The structural indices were calculated for cortical bone (cortical bone volume/all bone volume [Cv/Av], cortical bone section area [Csa], external length [EX.L.]) and trabecular bone (bone volume/tissue volume [BV/TV], bone surface/bone volume [BS/BV], trabecular thickness [Tb.Th]) using the TRI/3D-BON software program (Ratoc System Engineering Co., Ltd., Tokyo, Japan).

Statistical Analyses

Data were analyzed using one-way ANOVA, followed by Tukey’s test for post hoc analysis. All data are presented as the means±standard error of the mean (S.E.M.), and all statistical analyses were performed using IBM SPSS Statistics Ver.23 software.

RESULTS

Effects of Lutein on Bone Formation and Bone Resorption

We examined the effects of lutein on bone formation in osteoblast cultures. When osteoblasts were cultured with a medium containing bone-inducing factors, alizarin-stained mineralized bone nodules were detected on day 14 (Fig. 1B). The addition of lutein, 3 and 10 µM, enhanced the formation of mineralized bone nodules in the cultures (Fig. 1B). In this experiment, the area stained by alkaline phosphatase, a typical enzyme of osteoblasts, were not influenced by adding lutein (data not shown), suggesting that lutein may act on mature osteoblasts to stimulate the process of mineralization. In the cultures of primary osteoblasts, lutein did not influence the cell growth of mouse osteoblasts (data not shown). In the co-cultures of bone marrow cells and osteoblasts, lutein suppressed osteoclast formation induced by 1α, 25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], a typical bone resorbing factor (Fig. 1C). Using ex vivo cultures, we examined the effects of lutein on 1α,25(OH)₂D₃-induced bone-resorbing activity in the mouse calvariae. The application of 1α,25(OH)₂D₃ markedly induced the absorbed pit areas, while lutein (10 µM) suppressed the pit area (Fig. 1D). In the calvarial cultures, the bone resorbing activity monitored by increased medium calcium was elevated by 1α,25(OH)₂D₃, and lutein significantly suppressed the 1α,25(OH)₂D₃-induced bone resorbing activity (data not shown). Bone marrow macrophages are known as the precursors of osteoclasts in bone tissues. To examine the osteoclast differentiation from the precursor cells, bone marrow macrophages were induced by M-CSF in bone marrow cultures, and cultured for 5 d with sRANKL and M-CSF to detect TRAP-positive osteoclasts. The RANKL-dependent osteoclast differentiation from the precursor cells was suppressed by adding 3 and 10 µM lutein (Fig. 1E). These results indicate that lutein acts on osteoblasts to stimulate bone formation and to suppress osteoclast formation. Lutein also acts on macrophages, osteoclasts precursors, to negatively regulate RANKL-dependent osteoclast formation in vitro.

Effects of Lutein on Bone Mass in Growing Male Mice

To examine the effects of lutein on bone mass in growing mice, lutein was orally administered to 5-week-old male mice for 4 weeks, and the femoral BMDs of total, proximal, distal and central areas were measured by DEXA (Fig. 2A). The total BMD and the central BMD were significantly enhanced in the femurs of male mice by the administration of lutein (Fig. 2B). The BMD in the area of proximal and distal showed a tendency to increase by the administration of lutein (Fig. 2B), which is consistent with the DEXA image shown in the right panel of Fig. 2A. In the µCT analysis of central area of the femurs, the Cv/Av and CSa of cortical bone was found to be elevated in male mice administered 1% lutein, and the EX.L. showed a tendency to increase by the administration of lutein (Fig. 3A). These results suggest that lutein stimulates cortical bone formation in growing male mice in vivo. In the µCT analysis of distal area of the femurs, the BV/TV and Tb.Th. of trabecular bone tended to be elevated by the administration of lutein in mice, but BS/BV showed a tendency to decrease by the administration of lutein (Fig. 3B). Body weight of male mice was not influenced by the administration of 1% lutein (data not shown). These results indicate that lutein clearly enhances cortical bone mass in growing mice.

Fig. 2. The Oral Administration of Lutein Enhances the Femoral Bone Mass in Growing Male Mice

To examine the effects of lutein on bone mass in vivo, 5-week-old male mice were fed a diet containing 1% lutein. After 4 weeks, their femurs were collected. (A) Dual X-ray absorptiometry (DEXA) images of the femurs. (B) Femoral bone mineral density (BMD) in each area (total, distal, central and proximal) was measured by DEXA. The data are expressed as the mean±S.E.M. of 8 mice. The asterisks indicate significant differences: * p<0.05, ** p<0.01 vs. control.

Fig. 3. The Effects of the Oral Administration of Lutein on the Femoral 3D μCT Reconstruction Image in Growing Mice

To examine the effects of lutein on bone mass in vivo, 5-week-old male mice were fed a diet containing 1% lutein. (A) A 3D μCT reconstruction image at the cortical bone region of the central femurs. The Cv/Av (Cortical bone volume/All bone volume; Cortical bone ratio), Csa (cortical bone section area) and EX.L. (External length) were calculated by a 3D μCT analysis. The data are expressed as the mean±S.E.M. of 7 mice. * p<0.05 vs. control. (B) A 3D μCT reconstruction image at the trabecular-rich region of the distal femurs. The BV/TV (bone volume/tissue volume), BS/BV (bone surface/bone volume) and Tb.Th (trabecular thickness) were calculated by a 3D μCT analysis. The data are expressed as the mean±S.E.M. of 7 mice. * p<0.05 vs. control.

DISCUSSION

Lutein stimulated bone formation and suppressed bone resorption in vitro (Fig. 1), and the administration of lutein significantly enhanced the femoral bone mass in growing male mice in vivo (Figs. 2, 3). In the in vitro experiment, the mRNA expression of osteocalcin, a typical marker of mature osteoblasts, was clearly induced by bone-inducing factors, but the expression was not influenced by adding lutein (data not shown). In the late stage of osteoblast differentiation, mature osteoblasts stimulate mineralization, and an osteoblast subpopulation could stat its differentiation into osteocytes embedded in mineralized bone.¹⁴⁾ It is possible that lutein may act on mature osteoblasts to stimulate the process of mineralization, and/or regulate the process of osteocyte differentiation, but further studies are needed to define the mechanism of action of lutein in bone formation. In the in vivo experiment, the increase in the total BMD may be due to the elevation of cortical bone mass, since both the BMD of central area and the cortical bone formation markers, such as Cv/Av and Csa, measured by μCT was significantly elevated by the administration of lutein. In growing mice, bone formation is much more pronounced than bone resorption. Therefore, the elevation of femoral bone mass by lutein in growing mice may be mainly due to the stimulation of bone formation in vivo. A detailed analysis is therefore necessary to determine the mode of action of lutein in bone turnover.

The maintenance of peak bone mass is important in the prevention of bone diseases such as osteoporosis due to aging and sex steroids deficiency, since a high peak bone mass at a young age may reduce the risk of low bone mass in elderly individuals of both sexes.¹⁵⁾ In growing male mice, lutein enhanced bone mass in vivo. Administration of lutein in growing female mice also enhanced bone mass (data not shown). Lutein may therefore exert beneficial effects on bone tissues to enhance the peak bone mass in boys and girls and to prevent bone loss due to aging and/or menopause.

Lutein clearly suppressed the bone resorbing activity monitored by pit formation in the cultured mouse calvaria, and the osteoclast formation from the precursor cells. We showed the possible two mechanisms of action of lutein in osteoclast formation. Firstly lutein acts on macrophages, osteoclast precursor cells, and suppressed RANKL-dependent osteoclast formation. Secondly, lutein acts on osteoblasts and suppresses osteoclast differentiation by inhibiting the expression of RANKL in osteoblasts, since lutein suppressed the mRNA expression of RANKL induced by bone resorbing factor in osteoblasts (data not shown).

Sumantran et al.¹⁶⁾ have reported that lutein selectively induced apoptosis in transformed but not normal human mammary epithelial cells, and that lutein protected normal cells from apoptosis induced by the chemotherapy agents. Therefore, it is possible that lutein modulates cell growth and apoptosis in various cells. In the present study, lutein, at 3–10 µM, did not affect the cell growth of mouse osteoblastic cells (data not shown). In addition, the survival of macrophages was not influenced by lutein in the macrophage cultures for RANKL-induced osteoclast formation (data not shown). Therefore the suppression of osteoclast differentiation by adding lutein may not be due to the cell death or apoptosis in the cultures.

Epidemiological studies have shown a positive correlation between bone mass and the intake of vegetables and fruits,⁵⁾ and natural components have been suggested to exert beneficial effects on bone tissues in humans and animals. Postmenopausal osteoporosis is a typical bone disease in the world, and closely associated by increased osteoclast differentiation. Genistein, a soybean isoflavone, is known to prevent bone loss in ovariectomized (OVX) animal models.⁶⁾ Citrus factors, such as β-cryptoxanthin and hesperidin, have been reported to attenuate bone loss in OVX animal models. We also reported that nobiletin, which is found in citrus, clearly suppresses bone resorption and prevents bone loss due to estrogen deficiency in mice.⁹⁾ In OVX mice, the administration of lutein partly restored the bone loss due to estrogen deficiency (data not shown), but further studies are needed to define the possible role of lutein in the prevention of postmenopausal osteoporosis. Sugiura et al.^17,18) reported that the intake of a Japanese citrus containing β-cryptoxanthin was related to bone density in postmenopausal women. Lutein is synthesized and accumulated in green leafy plants such as spinach and kale. Further epidemiological studies are necessary to define the possible contribution of vegetable-derived lutein to bone mass in humans.

Acknowledgments

This work was supported by a Grant-in-Aid ‘A Scheme to Revitalize Agriculture and Fisheries in Disaster Area through Deploying Highly Advanced Technology’ from the Ministry of Agriculture, Forestry and Fisheries of Japan (CM). This work was partly supported by Institute of Global Innovation Research in TUAT (MI and FG).

Conflict of Interest

The authors declare no conflict of interest.

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