Osteoporosis Prevention by -Cryptoxanthin - ACS Publications

Chapter 35

Osteoporosis Prevention by β-Cryptoxanthin

Downloaded by UNIV OF AUCKLAND on June 5, 2016 | http://pubs.acs.org Publication Date: September 19, 2008 | doi: 10.1021/bk-2008-0993.ch035

Masayoshi Yamaguchi and Satoshi Uchiyama Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan

Pharmacologic and nutritional factors may play a role in the prevention of osteoporosis with aging. β-Cryptoxanthin, a carotenoid, is abundant in Satsuma mandarin orange. Among various carotenoid including β-cryptoxanthin, lutein, lycopene, β-carotene, astaxanthin, and rutin, β-cryptoxanthin has been found to have a unique anabolic effect on bone calcification. β-Cryptoxanthin has stimulatory effects on osteoblastic bone formation and inhibitory effects on osteoclastic bone resorption in vitro. β-Cryptoxanthin has an effect on the gene expression of various proteins which are related to osteoblastic bone formation and osteoclastic bone resorption. Oral administration of β-cryptoxanthin has been shown to have the anabolic effect on bone components in young and aged rats, the preventive effect on bone loss in streptozotocin-diabetic rats and ovariectomy-induced bone loss. Moreover, the intake of β-cryptoxanthin-reinforced juice for longer periods has been shown to have stimulatory effects on bone formation and inhibitory effects on bone resorption in healthy human and postmenopausal women as estimated based on serum biochemical markers of bone metabolism in vivo. The intake of dietary β-cryptoxanthin may have a preventive effect on osteoporosis.

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© 2008 American Chemical Society Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

409 Aging induces a decrease in bone mass. Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. The most dramatic expression of the disease is represented by fractures of the proximal femur (7). Nutritional and pharmacologic factors may be important in preventing bone loss with increasing age (2). Isoflavones, which are contained in soybeans, menaquinone-7 (vitamin K ), which is abundant in fermented soybeans, or other food and plant factors have been demonstrated to stimulate osteoblastic bone formation and to inhibit osteoclastic bone resorption in vitro (3,4). The supplementation of these food factors has preventive effects on bone loss induced in animal model of osteoporosis and in humans. Food chemical factors thus play a role in bone health and may be important in the prevention of bone loss with aging. Meanwhile, retinol (vitamin A) is known to have a detrimental effect on bone at high doses. In laboratory animals, high levels of vitamin A lead to accelerated bone resorption, bone fractures, and osteoporotic bone lesions (5). More recent studies have shown the anabolic effect of P-cryptoxanthin, a kind of carotenoid which is abundant in Satsuma mandarin (Citrus unshiu MARC.) on bone metabolism. In this chapter, the role of P-cryptoxanthin in preventing osteoporosis is introduced.


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P-Cryptoxanthin Stimulates Bone Formation and Inhibits Bone Resorption In Vitro The chemical structure of p-cryptoxanthin is shown in Figure 1.

Figure 7. Chemical structure ofP-cryptoxanthin.

The effects of various carotenoids and rutin on calcium content and alkaline phosphatase activity in the femoral-diaphyseal (cortical bone) and metaphyseal

Shibamoto et al.; Functional Food and Health ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

410 (trabecular bone) tissues of young rats in vitro were investigated in vitro (6). Culture with P-cryptoxanthin (10" or 10' M) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. Lutein, lycopene, and rutin at 10" to 10' M did not have anabolic effects on calcium contents and alkaline phosphatase activity in rat femoral-diaphyseal and -metaphyseal tissues. Astaxanthin and P-carotene (10" or 10' M) did not have an effect on the femoral calcium contents. Alkaline phosphatase participates in mineralization in bone tissues. P-Cryptoxanthin had a unique anabolic effect on bone calcification in vitro. The effects of Pcryptoxanthin increasing bone components was completely prevented with cycloheximide, an inhibitor of protein synthesis, suggesting that the effect is needed newly protein synthesis (7). The effects of P-cryptoxanthin on bone resorption were investigated using bone tissues in vitro. Culture with the bone-resorbing factor parathyroid hormone (PTH) or prostaglandin E (PGE ) caused a significant decrease in calcium content in die diaphyseal and metaphyseal tissues (7). This decrease was completely inhibited by P-cryptoxanthin (10 - 10' M) (7). In addition, pcryptoxanthin completely inhibited the PTH- or PGE -induced increase in medium glucose consumption and lactic acid production by bone tissues (7). PCryptoxanthin had inhibitory effects on bone resorption in tissue culture in vitro. Thus p-cryptoxanthin was found to have stimulatory effects on bone formation and inhibitory effects on bone resorption in bone tissue culture in vitro, as shown in Figure 2. 7

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Figure 2. P-Cryptoxanthin stimulates bone formation and inhibits bone resorption, thereby increasing bone mass.


411 It has been reported that the serum concentration of P-cryptoxanthin due to consumption of vegetable juice in women is in the range of 1.3x10" to 5.3 xlO' M (8). p-Cryptoxanthin in the range of 10' to 10 M caused a significant anabolic effect on biochemical components in rat femoral tissues in vitro, suggesting a physiologic role in the regulation of bone metabolism. 7

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Action of p-Cryptoxanthin in Osteoblastic Cells To clarify the cellular mechanism by which p-cryptoxanthin stimulates bone formation in bone tissues, the effect of carotenoid was examined using osteoblastic cells. P-Cryptoxanthin was found to stimulate the proliferation of osteoblastic cells in subconfluent monolayers in a medium containing 10% FBS (P). Culture with P-cryptoxanthin also caused a significant increase in biochemical components of osteoblastic cells (P). The effects of p-cryptoxanthin in increasing protein content, alkaline phosphatase activity, and DNA content in osteoblastic cells are completely inhibited in the presence of DRB (5,6-dichloro1-beta-D-ribofuranosyl benzimidazole), an inhibitor of RNA polymerase II, suggesting that p-cryptoxanthin has a stimulatory effect on transcriptional activity in osteoblastic cells. Prolonged culture with p-cryptoxanthin was found to stimulate mineralization in osteoblastic cells (12). P-Cryptoxanthin was found to have stimulatory effects on cell differentiation and mineralization in osteoblastic cells. Culture with P-cryptoxanthin stimulates the expression of insulin-like growth factor (IGF-I) or transforming growth factor (TGF)-P-l mRNA in osteoblastic cells using RT-PCR analysis (P). This finding may support the view that p-cryptoxanthin has a stimulatory effect on transcriptional activity in osteoblastic cells. IGF-I or TGF-P-1, which is a bone growth factor, is produced from osteoblasts (10, 11). The anabolic effect of P-cryptoxanthin in osteoblastic cells may be partly mediated through the action of IGF-I or TGF-pi produced from the cells. P-Cryptoxanthin (10" or 10" M) has also been found to increase the expression of Runx2, a 1(1) collagen, and alkaline phosphatase mRNAs in osteoblastic MC3T3-E1 cells (12). Runx2 (Cbfa 1) is a member of the runt domain family of transcription factors, and it is involved in bone development (13). al(I) Collagen is a matrix protein that is related to bone formation and mineralization in osteoblast lineage cells. Alkaline phosphatase participates in the mineralization process in osteoblastic cells. p-Cryptoxanthin has a stimulatory effect on the expression of genes for proteins involved in osteoblasic bone formation. 7

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412 The effects of P-cryptoxanthin in stimulating Runx2, al(I) collagen, and alkaline phosphatase mRNA expression in osteoblastic MC3T3-E1 cells was found to be prevented completely in the presence of DRB (72), supporting the view that the carotenoid stimulates transcriptional activity in osteoblastic MC3T3-E1 cells. Vitamin A (retinol) may be able to bind to nuclear receptors in cells, pCryptoxanthin (10" or 10" M) caused a significant increase in alkaline phosphatase activity and protein content in osteoblastic cells. This effect was also seen in the presence of vitamin A (10" M) (72). Moreover, the stimulatory effect of p-cryptoxanthin on the expression of Runx2 type 1 and al (I) collagen mRNA was also observed in the presence of vitamin A (72). Vitamin A did not have a significant effect on Runx2 type 1 mRNA expression in osteoblastic MC3T3-E1 cells. Thus the mode of action of P-cryptoxanthin on gene expression in osteoblastic cells may differ from that of vitamin A, which is mediated through the RXR receptor in the nucleus of the cells (72). It is speculated that p-cryptoxanthin may be able to bind other receptors (including orphan receptors), and that the carotenoid may stimulate transcriptional activity in osteoblastic cells. The mechanism of P-cryptoxanthin action in stimulating mineralization in osteoblastic cells is summarized in Figure 3. 7

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Action of P-Cryptoxanthin in Osteoclastogenesis and Mature Osteoclasts The receptor activator of N F - K B ligand (RANKL) plays a pivotal role in osteoclastogenesis from bone marrow cells. RANKL expression is induced in osteoblastic cells and bone marrow stromal cells in response to osteoporotic factors, such as PTH, PGE , and 1,25-dihydroxy vitamin D (VD ), and combined treatment of hematopoietic cells with macrophage colony-stimulating factor (M-CSF), and the soluble form of RANKL (sRANKL) induces osteoclast differentiation in vitro (13). The receptor protein RANK is expressed on the surface of osteoclast progenitors. P-Cryptoxanthin (10" - 10" M) was shown to have a potent inhibitory effect on osteoclast-like cell formation in mouse marrow culture in vitro (14). The inhibitory effect of P-cryptoxanthin on osteoclast-like cell formation was seen at the later stage of osteoclast differentiation in bone marrow cultures. Culture with P-cryptoxanthin caused a marked inhibition of osteoblast-like cell formation induced in the presence of PTH, PGE , VD , LPS, or tumor necrosis factor-a (TNF-a). P-Cryptoxanthin had a significant inhibitory effect on osteoclast-like cell formation induced by RANKL (14). The inhibitory effect of P-cryptoxanthin was equal to that of 17 P-estradiol, calcitonin, genistein, and zinc sulfate, which can inhibit osteoclast-like cell formation induced by boneresorbing factors. 2

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Figure 3. The cellular mechanism by which P-cryptoxanthin (CRP) stimulates bone formation and mineralization in osteoblastic cells. CRP stimulates may bind to orphan receptors in nucleus and stimulates transcription activity for bone formation-related proteins.

The effects of P-cryptoxanthin on mature osteoclasts were investigated (16). M-CSF-dependent bone marrow macrophages were cultured in the presence of M-CSF and RANKL for 4 days (16). The osteoclastic cells formed were further cultured in medium containing P-cryptoxanthin with or without M-CSF and RANKL for 24-72 h. Osteoclastic cells were significantly decreased in culture with p-cryptoxanthin (10' or 10" M) with or without M-CSF and RANKL for 72 h. The p-cryptoxanthin-induced decrease in osteoclastic cells was significantly inhibited in the presence of caspase-3 inhibitor. Agarose gel electrophoresis showed the presence of low-molecular-weight DNA fragments of adherent cells cultured with P-cryptoxanthin, indicating that the carotenoid induces apoptotic cell death. Apoptosis-related gene expression was determined using RT-PCR (16). Culture with P-cryptoxanthin caused a significant increase in caspase-3 mRNA expression in the presence or absence of M-CSF and RANKL, while Bcl-2 and 7

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414 Apaf-2 mRNA expressions were not significantly increased in culture with pcryptoxanthin without M-CSF and RANKL. Akt-1 mRNA expression was not significantly changed with culture of the carotenoid. The expression of caspase-3 mRNA or Apaf-2, which involves apoptosis, in osteoclastic cells was found to stimulate when cultured with p-cryptoxanthin in the absence of M-CSF and RANKL (76). p-Cryptoxanthin stimulated caspase-3 mRNA expression in the presence of M-CSF and RANKL expression in the presence of M-CSF and RANKL (76). P-Cryptoxanthin-induced apoptotic cell death is partly mediated through caspase-3 expression in osteoclastic cells. In addition, the expression of Bcl-2 mRNA, which is involved in rescue of apoptosis, was significantly decreased in P-cryptoxanthin culture in the presence or absence of M-CSF and RANKL (76). However, Akt-1 mRNA expression is not significantly changed in culture with p-cryptoxanthin. The decrease in Bcl-2 mRNA expression may partly contribute to the effect of P-cryptoxanthin in stimulating the apoptotic cell death of osteoclastic cells. Culture with P-cryptoxanthin was found to have suppressive effects on tartrate-resistant acid phosphatase (TRACP) activity, TRACP, and cathepsin K mRNA expression in osteoclastic cells in the presence or absence of M-CSF and RANKL (76). Presumably, P-cryptoxanthin has inhibitory effects on the activation of mature osteoclasts. P-Cryptoxanthin has been demonstrated to have stimulatory effects on apoptotic cell death and suppressive effects on osteoclastic cell function. The action of P-cryptoxanthin on mature osteoclasts is summarized in Figure 4.

Preventive Effects of p-Cryptoxanthin against Bone Loss In Vivo p-Cryptoxanthin has been shown to have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption in vitro (6,9,12,14,16). Then, the anabolic effect of p-cryptoxanthin to exert preventive effects on osteoporosis was investigated using animal models. P-Cryptoxanthin (10, 25, or 50 pg/100 g body weight) was orally administered once daily for 7 days to young male rats (77). The administration of p-cryptoxanthin (25, or 50 pg/100 g body weight) caused a significant increase in calcium content, alkaline phosphatase activity, and DNA contents in the femoral-diaphyseal and -metaphyseal tissues. P-Cryptoxanthin has an anabolic effect on bone components in rats in vivo. Such an effect is also observed in the femoral tissues of aged (50-week-old) female rats (18).



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Figure 4. The cellular mechanism by which P-cryptoxanthin (CRP) has suppressive effects on mature osteoclasts. CRP stimulates apoptotic cell death and suppresses gene expression of bone resorption-related enzymes.

To determine whether p-cryptoxanthin has a preventive effect on bone loss in the pathphysiologic state, the effects of p-cryptoxanthin on bone components in streoptozotocin (STZ)-diabetic rats were determined (19). Young rats received a single subcutaneous administration of STZ (6.0 mg/100 g body weight), and then the animals were orally administered P-cryptoxanthin (5 or 10 pg/100 g body weight) once daily for 7 or 14 days. The administration of STZ caused a significant decrease in body weight and a significant increase in serum glucose, triacylglycerol, and calcium levels, indicating a diabetic state. These alterations were significantly prevented by the administration of p-cryptoxanthin (5 or 10 pg/100 g) for 14 days. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and -metaphyseal tissues were significantly decreased in STZ-diabetic rats. These decreases were significantly prevented by the administration of p-cryptoxanthin (5 or 10 pg/100 g) for 14 days. Thus the intake of p-cryptoxanthin was found to have a preventive effect on bone loss in STZ-diabetic rats.


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The effect of p-cryptoxanthin on ovariectomy (OVX)-induced bone loss was examined (20). p-Cryptoxanthin (5 or 10 pg/100 g body weight) was orally administered once daily for 3 months to OVX rats. The analysis using peripheral quantitative computed tomography (pQCT) showed that O V X induced a significant decrease in mineral content and mineral density in the femoraldiaphyseal and -metaphyseal tissues. These decreases were significantly prevented by the administration of P-cryptoxanthin (5 or 10 pg/100 g). Moreover, OVX induced a significant decrease in bone components. These decreases were completely prevented by the administration of p-cryptoxanthin (5 or 10 pg/100 g). P-Cryptoxanthin had preventive effects on OVX-induced bone loss in vivo.

Effects of p-Cryptoxanthin in Normal Individuals and Menopausal Women The effects of P-cryptoxanthin on bone metabolism in human have been investigated (21-23). The effects of prolonged intake of juice prepared from Satsuma mandarin (Citrus unshiu MARC.) containing p-cryptoxanthin on circulating biochemical markers of bone metabolism in subjects, including menopausal woman, were examined (23). Ninety volunteers, aged 27-65 years (19 men and 71 women), were enrolled in this study. The 71 females included 35 premenopausal women (ages, 27-50 years) and 36 menopausal women (ages, 46-65 years). Volunteers were divided into four groups; placebo juice without Pcyptoxanthin (5 men and 19 women), juice containing p-cyptoxanthin at 1.5 mg/200 ml of juice/day (4 men and 17 women), 3.0 mg/day (5 men and 17 women), and 6.0 mg/day (5 men and 18 women). Placebo or juice (200 mL) was ingested once a day for 28 or 56 days. Serum p-cryptoxanthin concentrations were significantly increased after the intake of juice containing P-cryptoxanthin (1.5, 3.0, or 6.0 mg/day) for 28 or 56 days, and the increases were dose-dependent. Serum bone-specific alkaline phosphatase and y-carboxylated osteocalcin are bone metabolic markers of bone formation, and serum bone TRACP and N telopeptides of type I collagen are metabolic markers of bone resorption. In ninety volunteers (aged 27-65 years), serum bone-specific alkaline phosphatase activity was significantly increased after the intake of juice containing Pcryptoxanthin (3.0 or 6.0 mg/day) for 56 days as compared with the value obtained before intake. y-Carboxylated osteocalcin concentration was significantly increased after the intake of juice containing p-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days as compared with the value obtained before intake or after the intake of placebo juice. Serum TRACP activity and type I collagen N-telopeptide concentration were significantly decreased after the intake of juice containing p-cryptoxanthin (3.0 or 6.0 mg/day) for 28 or 56 days



417 as compared with the value obtained before intake or after intake of placebo juice, and significant decreases were also seen after the intake of 1.5 mg/day βcryptoxanthin as compared with the value obtained before intake. In menopausal women (36 volunteers), bone-specific alkaline phosphatase activity and γ -carboxylated osteocalcin concentration were significantly increased after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 56 days as compared with the value obtained after placebo intake. Also, this intake caused a significant decrease in bone TRACP activity and type I collagen N-telopeptide concentration. Meanwhile, serum calcium, inorganic phosphorous, and parathyroid hormone (intact) were not changed after the intake of β-cryptoxanthincontaining juice for 28 or 56 days. Other serum biochemical findings were not changed after the intake of juice containing β-cryptoxanthin (3.0 or 6.0 mg/day) for 56 days. We confirmed the safety of β-cryptoxanthin in humans. The prolonged intake of juice fortified with β-cryptoxanthin has been demonstrated to have stimulatory effects on bone formation and inhibitory effects on bone resorption in humans, and the intake has an effect in menopausal women. Thus the dietary intake of β-cryptoxanthin was demonstrated to have preventive effects on osteoporosis.

References 1. 2. 3. 4. 5.

Cooper, C.; Melton, J. III. Trends Endocrinol. Metab. 1995, 3, 224-229. Bonjour, J.-P.; Schurch, M.-A.; Rozzori, R. Bone. 1996, 18, 1395-1445. Yamaguchi, M. J. Health Sci. 2002, 48, 209-222. Yamaguchi, M. Yakugaku Zasshi, 2006, 126, 1117-1137. Promislow, J. H. E.; Goodman-Gruen, D.; Slymen, D. J.; Barret-Connor, E.

J. Bone Miner. Res. 2002, 17, 1349-1358. 6. 7. 8.

9. 10. 11. 12. 13.

14.

Yamaguchi, M.; Uchiyama, S. Biol. Pharm. Bull. 2003, 26, 1188-1191. Yamaguchi, M.; Uchiyama, S. Mol. Cell. Biochem. 2004, 258, 137-144. McEligot, A. J.; Rock, C. L.; Shanks, T. G.; Flatt, S. W.; Newman, V.; Farber, S.; Pierce, J. P. Cancer Epideminol. Biomarkers Prev. 1999, 8, 227231. Uchiyama, S.; Yamaguchi, M. Int. J. Mol. Med. 2005, 15, 675-681. Centrella, M.; McCarthy, T. L.; Canalis, E. Endocrinology 1990, 126, 39-44. Palcy, S.; Bolivar, I.; Goltzman, D. J. Bone Miner, Res. 2000, 15, 23522361. Uchiyama, S.; Yamaguchi, M. J. Cell. Biochem. 2005, 95, 1224-1234. Komori, T.; Yagi, H.; Nomura, S.; Yamaguchi, Α.; Sasaki, K.; Deguchi, K.; Shimizu, Y.; Bronson, R. T.; Gao, Y. H.; Inada, M.: Sato, M.; Okamoto, R.; Kitamura, Y.; Yoshiki, S.; Kishimoto, T.; Cell 1997, 89, 755-764. Uchiyama, S.; Yamaguchi, M. Biochem.Pharmacol. 2004, 67, 1297-1305.



418 15. Zaidi, M.; Blair, H. C.; Moonga, B. S.; Abe, E.; Huang, C. L. -H. J. Bone Miner. Res. 2003, 18, 599-609. 16. Uchiyama, S.; Yamaguchi, M . J. Cell. Biochem. 2006, 98, 1185-1195. 17. Uchiyama, S.; Sumida T.; Yamaguchi M . Biol. Pharm. Bull. 2004, 27, 232235. 18. Uchiyama, S.; Sumida, T.; Yamaguchi, M . J. Health Sci. 2004, 50, 491496. 19. Uchiyama, S.; Yamaguchi, M . Biol. Pharm. Bull. 2005, 28, 1766-1769. 20. Uchiyama, S.; Yamaguchi, M. Int. J. Mol. Med. 2006, 17,15-20. 21. Yamaguchi, M . ; Igarashi, A.; Uchiyama, S.; Morita, S.; Sugawara, K.; Sumida, K. J. Health Sci. 2004, 50, 619-624. 22. Yamaguchi, M . ; Igarashi, A.; Morita, S.; Sumida, T.; Sugawara, K. J. Health Sci. 2005, 51, 738-743. 23. Yamaguchi, M . ; Igarashi, A.; Uchiyama, S.; Sugawara, K.; Sumida, T.; Morita, S.; Ogawa, H.; Nishitani, M.; Kajimoto, Y. J. Health Sci. 2006, 52, 758-768.


Osteoporosis Prevention by -Cryptoxanthin - ACS Publications

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