Biol. Pharm. Bull. 40, 716–721 (2017)
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Lutein Enhances Bone Mass by Stimulating Bone Formation and Suppressing Bone Resorption in Growing Mice Hiroshi Takeda,a Tsukasa Tominari,b,c Michiko Hirata,b Kenta Watanabe,b Chiho Matsumoto,b Florian M. W. Grundler,c,d Masaki Inada,a,b,c and Chisato Miyaura*,a,b,c a
Cooperative Major of Advanced Health Science, Tokyo University of Agriculture and Technology; 2–24–16 Nakacho, Koganei, Tokyo 184–8588, Japan: b Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology; 2–24–16 Nakacho, Koganei, Tokyo 184–8588, Japan: c Institute of Global Innovation Research, Tokyo University of Agriculture and Technology; 2–24–16 Nakacho, Koganei, Tokyo 184–8588, Japan: and d Institute of Crop Science and Resource Conservation, University of Bonn; Karlrobert-Kreiten Str. 13, 53115, Bonn, Germany. Received November 18, 2016; accepted February 23, 2017 Lutein is a member of the xanthophyll family of carotenoids, which are known to prevent hypoxiainduced 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. Key words
bone mass; bone formation; bone resorption; growing mouse; lutein
Bone remodeling is regulated by osteoclastic bone resorption and new bone formation. Osteoclasts are the primary bone-resorbing cells and are differentiated from monocytemacrophages 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 antioxidants 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-weekold 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). Fiveweek-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%; Caro-
* To whom correspondence should be addressed. e-mail:
[email protected] © 2017 The Pharmaceutical Society of Japan
Biol. Pharm. Bull. Vol. 40, No. 5 (2017)717
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 m M 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
