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Equol, a metabolite of daidzein, binds to the estrogen receptor with greater affinity than daidzein and exhibits various biological properties. It exists as an enantiomer, either (S)-equol or (R)-equol.We have previously shown that the inhibitory effect of (S)-equol on bone fragility is stronger than that of racemic equol in ovariectomized (OVX) mice; however, the effect of (R)-equol has not been elucidated. The aim of this study was to compare the activities of equol enantiomers on bone metabolism in vitro and in vivo.Bone marrow cells (BMCs) and RAW 264.7 cells were treated with equol enantiomers. The number of osteoclasts and caspase-3/7 activity were measured. We examined the effect of equol enantiomers on osteoblast differentiation in MC3T3-E1 cells. In vivo, 8-wk-old female ddY mice were assigned to 4 groups: sham-operated (sham), OVX, OVX + 0.5 mg/d of (S)-equol (S-eq), and OVX + 0.5 mg/d of (R)-equol (R-eq). Four weeks after the intervention, femoral bone mineral density (BMD) and osteoclastic gene expression were analyzed, along with concentrations of equol enantiomers in the serum and tissues.(S)-equol and (R)-equol inhibited osteoclast differentiation in BMCs (97% and 60%, P 0.05) and RAW 264.7 cells (83% and 68%, P 0.05). (S)-equol promoted apoptosis of mature osteoclasts by inducing caspase-3/7 activity (29%, P 0.05) and enhanced osteoblast differentiation (29%, P 0.05). In OVX mice, BMD was ameliorated in (S)-equol-treated mice (11%, P 0.05), but not in (R)-equol-treated mice. The concentrations of (S)-equol were greater than those of (R)-equol in the serum, tibia, liver, and kidney (by 148%, 80%, 22%, and 139%, respectively).These results suggest that (S)-equol is more effective than (R)-equol in inhibiting osteoclast formation and enhancing osteoclast apoptosis in vitro, supporting the beneficial effect of (S)-equol to reduce estrogen deficiency-induced bone loss in OVX mice.

We evaluated the influence of pine bark extract (PBE) on organs, the cytochrome-P450 (CYP) activities in liver and estrogenic effects in normal and ovariectomized (OVX) female mice. The PBE did not affect organ weights and liver-function indexes (activities of alkaline phosphatase, aspartate amino transferase, and alanine amino transferase) at doses; 0.04%, 0.4%, and 2.0% PBE in the diet, in normal and OVX female mice. In the OVX mice, CYP1A1 activity was significantly higher in the 0.4% and 2.0% PBE groups than in the OVX control group, and in the 0.4% and 2.0% PBE groups were significantly higher than in the 0.04% PBE group. CYP1A2 and 3A4 activities were significantly higher in the 2.0% PBE group than in all other groups. The PBE did not affect uterine weight and femoral bone mineral density at all PBE doses. These results showed that the dose of PBE at the recommended human intake, had no toxic and estrogenic effects in normal female and OVX mice, however, it may need attention to use the excess intake of PBE with some drugs in postmenopausal women.

Female athlete triad (FAT) is an interrelationship between menstrual dysfunction, low energy availability with or without eating disorder, and decreased bone mineral density (BMD) in female athletes. The purpose of this study was to investigate whether isoflavone intake can prevent bone loss caused by voluntary wheel running under energy-restricted condition. We used a female rat model of osteoporosis for female athletes established previously. Fourteen female Sprague-Dawley rats (8-wk old) were fed ad libitum and had free access to wheels throughout the study. At 18 wk of age, the rats were divided randomly into the following groups: 1) running control (RC), 2) running energy restriction (RR), and 3) running energy restriction and isoflavone-fed (RR+Iso) groups. The RR group was 30% dietary restricted. The RR+Iso group was 30% dietary restricted and fed the diet containing 0.5% isoflavone powder (Fujiflavone P40). The experimental period lasted 31 wk. At the end of this experiment, BMD of the proximal femur in the RR group was significantly lower than that in the RC group. However, the BMD in the RR+Iso group was not significantly different from that in the RC group. Moreover, the plasma estradiol (E2) level in the RR and RR+Iso groups was significantly lower than that in the RC group. These findings suggest that isoflavone intake inhibited bone loss when the E2 level was low in female mature rat model. Our findings may reveal the possible novel role of isoflavone in osteoporosis among female athletes.

Numerous Foods with Function Claims that contain the extract of Pueraria flower (kudzu) isoflavones (PFI) are available in the Japanese market. These are labelled with function claims of reducing visceral fat. However, these foods have not undergone proper safety assessment such as the evaluation of their oestrogenic activity and effects on drug-metabolising enzymes (cytochrome P-450: CYP) in the liver. This study evaluated the estrogenic effect and the hepatic CYP activity and mRNA expression in normal female mice as a safety assessment of PFI (Experiment 1). In addition, the bone mineral density and visceral fat weight in ovariectomised mice (OVX) compared to soy isoflavones (SI) was evaluated to assess the efficacy of PFI (Experiment 2). OVX control fed a control diet, OVX fed a PFI diet (the recommended human intake of PFI), OVX fed a PFI20 diet (20- times the recommended PFI), OVX fed an SI diet (the recommended human intake of SI), and OVX fed an SI20 diet (20 -times the recommended intake of SI) for 28 days in Experiment 2. Body, liver, and visceral fat weights were not affected by the PFI, PFI20, SI, or SI20 diets. The hepatic CYP1A and CYP3A activities were elevated by the SI20 treatment. Ovariectomy-induced bone loss was inhibited by the SI20 treatment, but not by the PFI20 treatment. These results suggest that (1) PFI intake in human doses had no oestrogenic properties and did not affect CYP activity in the liver, (2) there was no evidence that PFI affects the amount of visceral fat in OVX mice.

Soybeans are a complete nutritional resource and soybean proteins are known to affect lipid metabolism via multiple mechanisms. Soybean meal (SBM) is produced after extraction of soybean oil and in this study, we investigated the ability whether the SBM could prevent high fat diet-induced obesity and understand the underlying mechanisms. Male Sprague-Dawley rats, aged 5 weeks, were randomly divided into three groups (n=8 each) and fed one of three diets for 28 days: Cont (AIN-93G), HFD (high fat diet with 40% of calories derived from fat) and HFD+SBM (HFD with 30% SBM). White adipose tissue weight as well as plasma and hepatic triglycerides were significantly decreased in HFD+SBM rats. Expression of hepatic SREBP-1 and its target genes was significantly decreased in HFD+SBM rats. Meanwhile, expression of SHP gene expression was significantly increased in HFD+SBM, and there was a negative correlation between SHP and SREBP-1 expression. Together these results suggest that hepatic SREBP-1 gene expression is negatively regulated by SHP. Expression of PPARG, the transcriptional factor that regulates SHP expression, was increased in HFD+SBM rats. Furthermore, expression of genes controlled by PPARG/SHP, such as those involved in hepatic gluconeogenesis, was also significantly decreased in HFD+SBM rats. Therefore, in addition to the previous findings of SBM on obesity here we show an additional mechanism which by changing the expression of genes involved in lipid metabolism via the PPARG/SHP pathway in the liver.

We examined the cooperative effects of isoflavones and cello-oligosaccharides on daidzein metabolism and bone fragility in ovariectomized mice. Cello-oligosaccharides increased urinary equol and decreased O-desmethylangolensin. A combination of isoflavones and cello-oligosaccharides attenuated decreases in bone breaking force and stiffness caused by ovariectomy. Combination treatment with isofalvones and cello-oligosaccharides increases urinary equol/O-desmethylangolensin production ratio and prevents ovariectomy-induced abnormalities in bone strength.

Equol is a metabolite of the soya isoflavone (ISO) daidzein that is produced by intestinal microbiota. Equol has greater oestrogenic activity compared with other ISO, and it prevents bone loss in postmenopausal women. Resistant starch (RS), which has a prebiotic activity and is a dietary fibre, was reported to promote equol production. Conversely, the intestinal microbiota is reported to directly regulate bone health by reducing inflammatory cytokine levels and T-lymphocytes in bone. The present study evaluated the combined effects of diet supplemented with ISO and RS on intestinal microbiota, equol production, bone mineral density (BMD) and inflammatory gene expression in the bone marrow of ovariectomised (OVX) mice. Female ddY strain mice, aged 8 weeks, were either sham-operated (Sham, n 7) or OVX. OVX mice were randomly divided into the following four groups (seven per group): OVX control (OVX); OVX fed 0·05 % ISO diet (OVX+ISO); OVX fed 9 % RS diet (OVX+RS); and OVX fed 0·05 % ISO- and 9 % RS diet (OVX+ISO+RS). After 6 weeks, treatment with the combination of ISO and RS increased equol production, prevented the OVX-induced decline in trabecular BMD in the distal femur by modulating the enteric environment and altered OVX-induced inflammation-related gene expression in the bone marrow. However, there were no significant differences in bone parameters between the ISO+RS and ISO-alone groups in OVX mice. Our findings suggest that the combination of ISO and RS might alter intestinal microbiota and immune status in the bone marrow, resulting in attenuated bone resorption in OVX mice.

The present study aimed at investigating the effects of Puerarin (PR), a major isoflavonoid isolated from the Chinese medicinal herb Puerariae radix, on bone metabolism and the underlying mechanism of action. The in vivo assay, female mice were ovariectomized (OVX), and the OVX mice were fed with a diet containing low, middle, and high doses of PR (2, 4, and 8 mg·d(-1), respectively) or 17β-estradiol (E2, 0.03 μg·d(-1)) for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight, compared with the control. The total femoral bone mineral density (BMD) was significantly reduced by OVX, which was reversed by intake of the diet with PR at any dose, especially at the low dose. In the in vitro assay, RAW264.7 cells were used for studying the direct effect of PR on the formation of osteoclasts. PR reduced the formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in the RAW 264.7 cells induced by receptor activator for nuclear factor-κB Ligand (RANKL). MC3T3-E1 cells were used for studying the effects of PR on the expression of osteoprotegerin (OPG) and RANKL mRNA expression in osteoblasts. The expression of OPG mRNA and RANKL mRNA was detected by RT-PCR on Days of 5, 7, 10, and 12 after PR exposure. PR time-dependently enhanced the expression of OPG mRNA and reduced the expression of RANKL mRNA in MC3T3-E1 cells. In conclusion, our results suggest that PR can effectively prevent bone loss in OVX mice without any hyperplastic effect on the uterus, and the antiosteoporosis activity of PR may be related to its effects on the formation of osteoclasts and the expression of RANKL OPG in osteoblasts.

The intestinal microbiota may regulate bone metabolism by reducing levels of pro-inflammatory cytokines, and T cells in bone tissues of oestrogen-deficient mice have been reported. Resistant starch (RS) is a type of dietary fibre and results in changes in the composition of the gut microbiota. We evaluated the effects of RS supplemented in diets on intestinal microbial composition, bone mineral density, and inflammatory-gene expression in the colon and bone marrow of ovariectomised (OVX) mice. OVX mice were divided randomly into three groups: OVX control, OVX fed a 20% high amylose corn starch (HAS) diet, and OVX fed a 20% acid-hydrolysed HAS (AH-HAS) diet. HAS and AH-HAS diets contained 6.8% and 12% of RS, respectively. After 6 weeks, treatment with HAS or AH-HAS increased the abundance of Bifidobacterium spp. in faeces. The AH-HAS diet tended to upregulate mRNA expression of interleukin (IL)-10 in the colon, and downregulate expression of receptor activator of nuclear factor kappa-B ligand and IL-7 receptor genes in the bone marrow of OVX mice. AH-HAS treatment attenuated ovariectomy-induced bone loss. These findings suggest that AH-HAS might change the microbiota and immune status of the bone marrow, resulting in attenuated bone resorption in OVX mice.

New Zealand is a rich source of food components that may have bioactivity on bone. Docosahexaenoic acid (DHA) from fish oil has been shown to maintain bone in ovariectomised (OVX) rats. Kiwifruit, a source of fibre and carotenoids, may also affect bone via a prebiotic as well as direct cell-based mechanisms. We aimed to 1) ascertain the effects of DHA on two cell models, including interactions with soy isoflavones; 2) and investigate the specific effects of carotenoids from kiwifruit as well as whole kiwifruit in cell-based and rodent models as well as in a human study. RAW 264.7 mouse monocytes or mouse bone marrow was used to generate osteoclasts (OC). Cells were exposed to the agents between 5 and 21 d and formation and activity of OC measured, including molecular markers. DHA inhibited OC formation in both cell models, including expression of cathepsin K, NFATc1 as well as actin ring formation. Combination with isoflavones enhanced these effects. In OVX rats and mice fed with kiwifruit for 8 wk, green kiwifruit reduced the rate of bone loss after OVX, and in mice it reduced C-telopeptide of Type 1 collagen (CTX) levels and RANKL expression while in menopausal women, green kiwifruit affected blood lipids and bone markers positively.

In this study, we investigated the synergistic effects of daidzein (Dz) and kiwifruit on bone and equol production in ovariectomised (OVX) rats. Female Sprague-Dawley rats were randomly assigned to one of five groups: sham operated, OVX control, OVX fed 0.1% Dz-supplemented diet (OVX + Dz), OVX fed 0.1% Dz and green kiwifruit (GRK)-supplemented diet (OVX + Dz + GRK) and OVX fed 0.1% Dz and gold kiwifruit (GOK)-supplemented diet (OVX + Dz + GOK). There were no significant differences in whole body and femur bone mineral density (BMD) among groups at week 8. BMD in the OVX group significantly decreased at week 8; however, BMD in the OVX + Dz + GRK was not significantly different from baseline in the end of the study. However, supplementation with kiwifruit did not affect urinary equol concentrations, urinary ratios of equol to Dz and the composition of caecal microbiota. These results suggest that the combination of Dz and GRK may slightly reduce bone loss caused by oestrogen deficiency but does not affect equol production.

Background Daidzein is an isoflavone derived from soybeans that exerts preventive effects on bone loss in ovariectomized (OVX) animals. These effects have been correlated with increasing serum equol levels. In the present study, we investigated the effects of antibiotic intake on equol metabolism from daidzein, and the corresponding levels of bone loss in OVX mice. Methods Eight-week-old female ddY mice (n = 42) were either ovariectomized (OVX) or subjected to a sham operation (sham). OVX mice were then divided into six dietary subgroups: control diet (control), 0.3 % kanamycin diet (KN), 0.1 % daidzein diet (Dz), 0.1 % daidzein and 0.0375 % kanamycin diet (Dz+KN3.75), 0.1 % daidzein and 0.075 % kanamycin diet (Dz+KN7.5), and 0.1 % daidzein and 0.3 % kanamycin diet (Dz+KN30). The mice were fed their respective diets for 4 weeks. Results Uterine weight and femoral bone mineral density (BMD) were significantly lower in the OVX mice compared in the sham mice. No significant differences in uterine weight were observed among all OVX dietary subgroups. The Dz subgroup was found to exhibit higher plasma equol and O-desmethylangolensin (O-DMA) concentrations, as well as greater femoral BMD, compared to all other OVX subgroups. Furthermore, when compared to the Dz group, kanamycin intake decreased plasma equol and O-DMA concentrations, as well as femoral BMD in the OVX mice. Conclusions These results suggest that kanamycin intake inhibited the conversion of daidzein to equol and O-DMA, blocking the preventive effects of daidzein on bone loss in OVX mice. Therefore, the bone-protective effects of daidzein intake may be predominantly associated with increased plasma concentrations of either equol or O-DMA.

We compared the effects of the S-enantiomer and racemic forms of equol on bone using ovariectomized (OVX) mice. Femoral bone mineral density and bone strength decreased in the OVX mice, but not in OVX mice administered 0.5 mg/d S-equol. This, however, did not hold for racemic equol. Serum and urine S-equol concentrations were higher in the mice administered S-equol than in those administered racemic equol. These results suggest that the inhibitory effects of S-equol on bone fragility in OVX mice are greater than those of racemic equol.

AMP-activated protein kinase (AMPK) acts as an intracellular sensor to maintain the energy balance by phosphorylation of several downstream metabolic enzymes and certain transcription factors. We have identified a transcription factor named AREBP which is phosphorylated by AMPK in vitro. AREBP binds to the promoter element of PEPCK, a key enzyme of gluconeogenesis. Transient transfection experiments indicated AREBP repressed transcription of PEPCK gene in a phosphorylation dependent manner. To investigate the in vivo function of AREBP, we overexpressed AREBP in mice. Fasting-induced up-regulation of PEPCK gene expression was reduced by AICAR injection in AREBP mice. AICAR treatment repressed PEPCK gene expression in cultured hepatocytes derived from AREBP mice. Glucose output was reduced in AREBP mice after AICAR injection. Our results suggest AREBP is a key modulator of hepatic glucose production regulated by AMPK in vivo.

Both soy isoflavone and n-3 polyunsaturatedfatty acids are known to reduce the levels of bone-resorbingcytokines; however, the synergistic effects of these foodingredients have not been examined yet. This study wasperformed to elucidate the effect of concomitant intake ofsoy isoflavone and fish oil on bone mass in ovariectomizedmice. Eight-week-old ddY female mice were subjectedto ovariectomy (OVX) or sham surgery, and then fed anAIN-93G with safflower oil (So) as a control lipid source,isoflavone-supplemented safflower oil (So ? I), fish oilinstead of safflower oil (Fo) or isoflavone-supplemented fishoil (Fo ? I) for 4 weeks. Femoral bone mineral density wassignificantly decreased by OVX; however, this decrease wasinhibited by the intake of isoflavone and/or fish oil. Histomorphometricanalyses showed that bone volume and trabecularthickness in the distal femoral trabecular bone weresignificantly lower in the So group than in the sham group,but those were restored in the Fo ? I groups. The number ofosteoclasts was significantly decreased by isoflavone intake, Original article. オンライン版はSpringerLinkで The Japanese Society for Bone and Mineral Research and Springer 2010. 45p.

Equol binds to the estrogen receptor with greater affinity than its precursor, daidzein, an isoflavone found in soybeans. Inter-individual differences in the ability to produce equol may lead to differential effects of isoflavone intervention on human health. Here, we review previously published work from our laboratory on equol producer status and bone health in humans and in a mouse model of osteoporosis. We performed a 1-y, double-blind, randomized trial to compare the effects of isoflavone (75 mg of isoflavone conjugates/d; equivalent to 47 mg/d of the aglycone form) with those of placebo on bone mineral density (BMD), fat mass, and serum isoflavone concentrations in 54 early postmenopausal Japanese women classified by their equol-producer phenotype. Isoflavone intervention increased the serum equol concentration in equol producers but not in nonproducers (P < 0.04). The annualized changes in BMD in the total hip and intertrochanteric regions in the isoflavone-treated equol producers (-0.46 and -0.04%, respectively) were less than in the nonproducers (-2.28 and -2.61%, respectively). The annualized change in fat mass was lower in the equol producers compared with the nonproducers in the isoflavone group. The annualized changes in BMD and fat mass did not differ between the equol producers and nonproducers in the placebo group. Equol also inhibited bone loss and fat accumulation in estrogen-deficient osteoporotic mice. Our data suggest that prevention of bone loss and fat accumulation in early postmenopausal women by isoflavones may depend on an individual's equol-producing capacity.

Soy isoflavones, genistein, daidzein and its metabolite equol, as well as β-carotene have been reported to be effective for maintaining bone health. However, it remains to be elucidated whether combining soy isoflavones with β-carotene is beneficial to bone formation. This study investigated the combined effect of soy isoflavones and β-carotene on the differentiation of MC3T3-E1 preosteoblastic cells. Daidzein and genistein alone did not affect cell growth but increased alkaline phosphatase (ALP) activity. Beta-carotene alone inhibited cell growth and markedly enhanced ALP activity. Soy isoflavones combined with β-carotene resulted in higher ALP activity than treatment with isoflavones or β-carotene alone. We observed significant main effects of β-carotene on the enhanced expression of Runx2, ALP, and ostepontin mRNA, whereas there was a significant main effect of soy isoflavones on the expression of osterix mRNA. To investigate how β-carotene affected osteoblast differentiation, MC3T3-E1 cells were treated with retinoic acid receptor (RAR) pan-antagonist combined with β-carotene. Osteopontin and ALP mRNA expression levels, which were increased following treatment with β-carotene, were significantly suppressed by the RAR pan-antagonist. This suggests treatment with β-carotene enhanced early osteoblastic differentiation, at least in part via RAR signaling. These results indicate that a combination of isoflavones and β-carotene may be useful for maintaining a positive balance of bone turnover by inducing osteoblast differentiation.

Kiwifruit is a good source of dietary components and has beneficial effects for health. In this study, we investigated the effects of two types of kiwifruit, green kiwifruit (GRK) and gold kiwifruit (GOK), on bone metabolism in ovariectomized (OVX) mice. Seven-week-old female Balb/c-strain mice were divided into four groups: sham-operated (sham) group, OVX group, and OVX mice that were fed a GRK-supplemented diet or GOK-supplemented diet. Freeze-dried GRK and GOK were prepared and added in the diet at a concentration of 3 g/100 g. After 9 wk, the mice were sacrificed, and the serum, uterus, and femurs were obtained. Final body weight did not differ significantly among the four groups. Compared to the sham group, uterine weight was significantly lower and serum C-terminal telopeptide of type I collagen (CTx) levels and receptor activator of NF-κB ligand (RANKL) mRNA expression of the whole femur were significantly higher in the OVX group. Compared to the OVX group, GRK, but not GOK, reduced serum CTx concentrations and RANKL mRNA expression of the whole femur without changes in uterine weight. These results suggest that the GRK inhibited bone resorption, which might be due to a decrease in RANKL mRNA expression in OVX mice.

It is well known that skeletal unloading induces bone loss. It has been shown that soybean isoflavones are effective in preventing bone loss in osteoporotic animals. We investigated the ability of isoflavones to protect bone loss induced by hindlimb unloading by using a tail-suspension mouse model. Eight-week-old female mice were divided into five groups: (1) normal housed group (Normal), (2) sham unloaded group fed a control diet (Sham-UL), (3) hindlimb unloaded group fed a control diet (UL-C), (4) hindlimb unloaded group fed a 0.25% isoflavone conjugates diet (UL-ISO 0.25), and (5) hindlimb unloaded group fed a 0.5% isoflavone conjugates diet (UL-ISO 0.5). After 3 weeks, bone mineral density (BMD) of the femur was significantly decreased in UL-C, and this bone loss was prevented by isoflavone treatment. Histomorphometric analysis revealed a decrease in the cancellous bone of the distal femur in the UL-C group, and isoflavone prevented this change. Serum corticosterone increased in the UL-C group, and isoflavones inhibited the elevation. These results suggest that isoflavones might be promising food components that provide protection from bone loss and normalize stress-induced serum corticosterone during skeletal unloading.

Puerariae radix (PR) is one of the earliest and most important crude herbs used in Chinese medicine for various medicinal purposes. PR contains a high amount of isoflavonoids, such as daidzein and genistein, which are known to prevent bone loss induced by estrogen deficiency. We have demonstrated that PR not only completely prevents bone loss but also significantly increases the bone mass at high doses in ovariectomized mice without exhibiting estrogenic action in the uterus. In this study, we examined whether PR exhibits effects on bone loss in androgen-deficient male mice similar to estrogen-deficient female mice. Male mice were orchidectomized (ORX) and fed a diet containing low, middle, and high doses (5%, 10%, and 20% of diet, respectively) of PR or normal diet with subcutaneous administration of 17beta-estradiol (E(2), 0.03 microg/d; Sigma, St Louis, Mo), for 4 weeks. In ORX mice, the seminal vesicle weight decreased markedly, and it was not affected by the administration of any doses of PR and E(2). The bone mineral density (BMD) of the whole femur was significantly decreased by ORX, and the decrease in BMD was completely prevented by intake of the diet with the low dose of PR. Intake of the diet with the middle dose of PR further normalized BMD in ORX mice. Furthermore, the high dose of PR administration (PR20) significantly increased BMD in ORX mice, and the potency was similar to that of E(2). Morphometric analysis of the femoral metaphysis showed that intake of the diet with the low dose of PR completely prevented the decrease in bone volume/tissue volume and trabecular number and restored the increase in trabecular separation in ORX mice. In addition, intake of the diet with the high dose of PR further increased bone volume/tissue volume and trabecular number and decreased trabecular separation in ORX mice. These results propose the possibility that estrogenic Chinese herbs such as PR can be one of the candidates for the treatment or prevention of osteoporosis in elderly men with hypogonadism.

Soybean isoflavones have structures similar to that of estrogen and have received attention as alternatives to hormone replacement therapy for the prevention of postmenopausal osteoporosis. Daidzein, a major isoflavone found in soybean, is metabolized to equol by gut microflora, and the metabolite exhibits a stronger estrogenic activity than daidzein. However, there is no direct evidence that equol affects bone metabolism. In this study, we examined the effect of equol on the inhibition of bone loss in ovariectomized (OVX) mice. Female mice (8 wk old) were assigned to 5 groups as follows: sham-operated (sham), OVX, OVX + 0.1 mg/d equol administration (0.1 Eq), OVX + 0.5 mg/d equol administration (0.5 Eq), and OVX + 0.03 microg/d 17beta-estradiol administration (E(2)). Equol and E(2) were administered s.c., using a mini-osmotic pump. At 4 wk after the intervention, uterine weight was less in the OVX mice than in sham-operated mice (P < 0.05). The weight was maintained in the E(2) group. In contrast, administration of equol at doses used in this study did not affect uterine atrophy in OVX mice. Bone mineral density (BMD) for the whole body in the OVX group measured by dual-energy X-ray absorptiometry was lower than that in the sham group, whereas administration of 0.5 mg/d Eq as well as E(2) maintained the BMD. The BMD of the femur and lumbar spine in the OVX group was also lower than those in the sham group, and treatment with 0.5 mg/d Eq maintained it. Notably, the BMD of the proximal femur in the 0.5 Eq group was the same as that of the sham group. E(2) inhibited bone loss from all regions induced by OVX. These results suggest that equol, a major metabolite of daidzein, inhibits bone loss apparently without estrogenic activity in the reproductive organs of OVX mice.

Exercise-induced bone gains are lost if exercise ceases. Therefore, continued exercise at a reduced frequency or intensity may be required to maintain these benefits. In this study, we evaluated whether 4 wk of reduced exercise after 4 wk of running exercise in growing male mice results in the maintenance of high bone mass. Five-week-old mice were divided into the following groups: 1) baseline control; 2) 4-wk control; 3) 4-wk exercise; 4) 8-wk control; 5) 4-wk exercise followed by 4-wk cessation of training; and 6) 4-wk exercise followed by reduced exercise at half the frequency. The regimen consisted of exercise 6 days/wk, and the reduced exercise regimen consisted of running 3 days/wk on a treadmill for 30 min/day, at 12 m/min on a 10° uphill slope. Running exercise significantly increased bone mineral density of the femur, periosteal mineral apposition rate, bone formation rate, percent labeled perimeter at the midfemur, and osteogenic activity of bone marrow cells. However, these parameters declined to the age-matched sedentary control after cessation of training. In contrast, the reduced exercise group had significantly higher mineral apposition rate compared with those of the sedentary control and cessation of training groups. Furthermore, bone mineral density for the reduced exercise group was significantly higher than those for the other groups. These results suggest that the high bone formation gained through exercise can be maintained, and bone mass was further increased by subsequent exercise even if the exercise frequency is reduced.

The purpose of this study was to improve the efficiency of the microbiological assays for vitamin B(6) and inositol by using lyophilized cells from Saccharomyces cerevisiae ATCC 9080. The use of lyophilized cells as an inoculum was assessed to avoid time-consuming processes like cell precultivation and washing. The authors also examined the effects of various protectants such as skim milk, lactose, maltose, and trehalose on the assay. The viable cell counts of the lyophilized cells were approximately equal. The standard curves for vitamin B(6) and inositol concentrations obtained using lyophilized cells with maltose and intact cells gave similar linear ranges. Furthermore, the measured vitamin concentrations of the standard reference material 1849 were in the range of the established values. Therefore, lyophilized cells with maltose are potential alternative inocula for the turbidimetric method. This will increase the overall convenience associated with microbiological assays.

Soybean isoflavones exhibit selective effects on bone metabolism in postmenopausal women as well as in ovariectomized animals. Recently, the role of estrogen in bone metabolism in men has also received attention, because a man with a mutated estrogen receptor-alpha (ER(alpha)) gene will exhibit osteoporotic phenotypes. To examine the possible role of genistein, a soybean isoflavone, in bone marrow hemopoiesis and bone metabolism in men, male mice were orchidectomized (orx) and treated with genistein (0.4-0.8 mg/day) or 17beta-estradiol (E(2); 0.03 microg/day) subcutaneously for 3 weeks. In orx mice, seminal vesicle weight decreased markedly, and it was not affected by the administration of genistein or E(2). The number of bone marrow cells was markedly increased after orx, and the majority was B-220 weakly positive pre-B cells. Increased B-lymphopoiesis was restored completely by E(2) or genistein administration. In orx mice, bone mineral density of the femur decreased markedly, and this bone loss was prevented to a significant extent by treatment with genistein as well as E(2). Histomorphometry showed that the trabecular bone volume in the femoral distal metaphysis decreased markedly after orx, and genistein and E(2) prevented this bone loss. These results suggest that soybean isoflavones prevent bone loss due to androgen deficiency in males.

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3-E1 cells, but also up-regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1alpha,25(OH)2D3- or prostaglandin E2 (PGE2)-induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor kappaB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence-accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.

Genistein, an isoflavone abundantly present in soybeans, has structural similarity to estrogen, suggesting that genistein may act as a phytoestrogen. To examine the possible role of genistein in hemopoiesis and bone metabolism, female mice were either sham-operated or ovariectomized (OVX), and selected OVX mice were administered genistein for 2-4 weeks (0.1-0.7 mg/day) or 17beta-estradiol (E2; 0.01-0.1 microg/day) s.c., using a miniosmotic pump (Alza Corp., Palo Alto, CA). In OVX mice, uterine weight declined but was completely restored by E2 administration. In contrast, genistein did not demonstrate a reversal of the OVX-induced uterine atrophy. The number of bone marrow cells markedly increased, 2-4 weeks after OVX, and most of these were B220-weakly positive pre-B cells. The increased B-lymphopoiesis was completely restored, not only by E2 but also by genistein administration. In OVX mice, the trabecular bone volume of the femoral distal metaphysis, measured by microcomputed tomography scanning and dual-energy x-ray absorptiometry, was markedly reduced; and genistein restored this, as did E2. These results indicate that genistein exhibits estrogenic action in bone and bone marrow, to regulate B-lymphopoiesis and prevent bone loss, without exhibiting estrogenic action in the uterus. Phytoestrogens may be useful for preventing bone loss caused by estrogen deficiency in females.

The purpose of this study was to examine whether hesperidin inhibits bone loss in androgen-deficient male mice. Male ddY mice aged 7 weeks underwent either a sham operation or orchidectomy (ORX) and were divided into five groups: a sham-operated group fed a control diet (Sham) based on AIN-93G formulation with corn oil instead of soy bean oil, an ORX group fed the control diet (ORX), a group fed the control diet containing 0.5% hesperidin (ORX + H), a group fed the control diet containing 0.7% α-glucosylhesperidin (ORX + αG), and a group fed the control diet containing 0.013% simvastatin (ORX + St). Four weeks after intervention, ORX mice showed a striking decrease in seminal vesicle weight, which was not affected by the administration of hesperidin, α-glucosylhesperidin, or simvastatin. Femoral BMD was significantly reduced by ORX, and bone loss was inhibited by the administration of hesperidin, α-glucosylhesperidin or simvastatin. Histomorphometric analysis showed that the bone volume and trabecular thickness were significantly lower, and the osteoclast number was higher in the distal femoral cancellous bone in the ORX group than in the Sham group, and these were normalized in the ORX + H, ORX + αG and ORX + St groups. These results indicate that hesperidin inhibited bone resorption and hyperlipidemia, in ORX mice, and the preventive effect was stronger than that observed in ovariectomized mice in our previous study.

We investigated the effects of equol on adipogenesis by measuring lipid accumulation and analyzing the change in adipocyte-related gene expression in MC3T3-L1 cells. Treatment with 10 µM equol tended to increase adipocyte-related gene expression, whereas 100 µM equol reduced lipid accumulation and suppressed the expression of these genes and proteins. Our results suggest that equol regulated adipogenesis in a bi-phasic fashion.

Methylsulfonylmethane (MSM), which is one of the popular ingredients of so-called health foods in Japan, is expected to relieve inflammation in arthritis and allergies. However, there is no scientific evidence to confirm the efficacy and safety of MSM in detail. In this study, we examined the effects of MSM on cartilage formation in growing rats (G) and cartilage degradation in STR/Ort mice (A), an accepted human osteoarthritis (OA) model. For cartilage formation study, 6-week-old growing male Wister rats were assigned to four groups to receive a control or MSM-containing diet. To examine the efficacy of MSM on the cartilage of OA model mouse, 10-week-old male STR/OrtCrlj mice were assigned to three groups to receive a control or MSM-containing diet. The dosages used were amounts equal to the recommended supplements for humans [0.06 g/kg body weight (BW)/day: MSM1G and MSM1A], 10 fold higher (0.6 g/kg BW/day: MSM10G and MSM10A), and 100 fold higher (6 g/kg BW/day: MSM100G). Intake of MSM for 4 weeks did not affect cartilage formation in the knee joint in growing rats. Body, liver, and spleen weight in the MSM100G group were significantly lower than those in the control group. Intake of MSM for 13 weeks decreased degeneration of the cartilage at the joint surface in the knee joints in STR/Ort mice in a dose-dependent manner. These results suggest that appropriate intake of MSM is possibly effective in OA model mice; however, intake of large amounts of MSM induced atrophy of several organs.

Daidzein is metabolized to equol in the gastrointestinal tract by gut microflora. Equol has greater estrogenic activity than genistein and daidzein, with its production shown to be promoted by dietary fiber. It is known that resistant starch (RS) is not absorbed in the proximal intestine and acts as dietary fiber in the colon. In this study, we investigated the combined effects of daidzein and RS intake on equol production, bone mineral density, and intestinal microflora in ovariectomized (OVX) mice. Female mice of the ddY strain, aged 8 weeks, were either sham operated (n = 6) or OVX. The OVX mice were randomly divided into 5 groups: OVX control (n = 6), OVX fed 0.1% daidzein-supplemented diet (OVX + Dz, n = 8), OVX fed 0.1% daidzein- and 12% RS-supplemented diet (OVX + Dz + RS, n = 8), OVX fed 12% RS-supplemented diet (OVX + RS, n = 8), and OVX who received daily subcutaneous administration of 17 β-estradiol (n = 6). After 6 weeks, urinary equol concentration was significantly higher in the OVX + Dz + RS group than in the OVX + Dz group. The bone mineral density of the whole tibia was higher in the OVX + Dz +RS group compared with the OVX + Dz group. The occupation ratios of Bifidobacterium spp in the cecal microflora in groups fed RS were significantly higher than those in the other groups. The present study demonstrated that RS may increase the bioavailability of daidzein.

Retinol binding protein 4 (RBP4) is the transport protein that carries retinol in blood. RBP4 was described recently as a new adipokine that reduced insulin sensitivity. Mice lacking glucose transporter 4 (GLUT4) in adipocytes have enhanced Rbp4 gene expression; however, the molecular mechanism is unknown. We found a G4KA (GLUT4 knockdown-dependent transcriptional activation) element located approximately 1.3 kb upstream of the Rbp4 promoter. Mutations within the G4KA sequence significantly reduced expression of the Rbp4 promoter-reporter construct in G4KD-L1 (GLUT4 knockdown 3T3-L1) adipocyte cells. In a yeast one-hybrid screen of a G4KD-L1 cell cDNA library, using the G4KA element as bait, we identified subunits of the 20 S proteasome, PSMB1 and PSMA4, as binding partners. In chromatin immunoprecipitation assays, both subunits bound to the G4KA element; however, only PSMB1 was tightly bound in the GLUT4 knockdown model. PSMB1 RNA interference, but not PSMA4, significantly inhibited Rbp4 transcription. Nuclear transportation of PSMB1 was increased in G4KD-L1 cells. These results provide evidence for an exclusive proteasome subunit-related mechanism for transcriptional activation of RBP4 within a GLUT4 knockdown model.

Magnesium is related to a number of biological enzymatic reactions such as catalytic role for the reaction of kinases in ATP production. On the other hand, magnesium is one of the essential minerals for bone formation. In the magnesium-deficient rats, apparent bone loss caused by increase in bone resorption and decrease in bone formation was observed. Although, epidemiological studies suggest that magnesium deficiency is one of the risk factor for osteoporosis, a relationship between magnesium intake and bone mineral density is not clear. This may be due to the differences in the population, decrease in sex hormone secretion, and the possibility that magnesium-deficiency is also accompanied with another nutrient insufficiency, e.g., calcium.

Arbutin, a practically used skin-lightening agent, has been reported to possess a weak antioxidant activity compared to that of its precursor, hydroquinone. However, its antioxidant activity has not been systematically evaluated. Hence, this study reassessed its activity using five assay systems. Assays were first performed using model radicals, DPPH radical and ABTS(*+). Arbutin showed weak DPPH radical-scavenging activity compared to that of hydroquinone, but showed strong ABTS(*+)-scavenging activity. Its activity by ORAC assay was then evaluated using a physiologically relevant peroxyl radical. Arbutin exerted weak but long-lasting radical-scavenging activity and showed totally the same antioxidant activity as that of hydroquinone. Finally, it was shown that, in two cell-based antioxidant assays using erythrocytes and skin fibroblasts, arbutin exerted strong antioxidant activity comparable or even superior to that of hydroquinone. These findings indicate that the antioxidant activity of arbutin may have been under-estimated and suggest that it acts as a potent antioxidant in the skin.

Leukemia inhibitory factor/differentiation-stimulating factor (LIF/D-factor), expression of its mRNA, and possible roles in bone metabolism were studied in murine primary and clonal osteoblast-like cells. Local bone-resorbing factors such as IL-1, TNF alpha, and LPS strongly induced expression of LIF/D-factor mRNA in both clonal MC3T3-E1 cells and primary osteoblast-like cells. Neither parathyroid hormone nor 1 alpha,25-dihydroxyvitamin D3 stimulated expression of LIF/D-factor mRNA. LIF/D-factor per se did not stimulate expression of its own mRNA. Appreciable amounts of LIF/D-factor were detected in synovial fluids from rheumatoid arthritis (RA) patients but not in those with osteoarthritis (OA). Simultaneous treatment with LIF/D-factor, IL-1, and IL-6 at the concentrations found in synovial fluids from RA patients greatly enhanced bone resorption, though these cytokines did not stimulate bone resorption when separately applied. This suggests that LIF/D-factor produced by osteoblasts is in concert with other bone-resorbing cytokines such as IL-1 and IL-6 involved in the bone resorption seen in the joints of RA patients. LIF/D-factor specifically bound to MC3T3-E1 cells with an apparent dissociation constant of 161 pM and 1,100 binding sites/cell. LIF/D-factor dose-dependently suppressed incorporation of [3H]thymidine into MC3T3-E1 cells. In addition, it potentiated the alkaline phosphatase activity induced by retinoic acid, though LIF/D-factor alone had no effect on enzyme activity. These results suggest that LIF/D-factor is involved in not only osteoclastic bone resorption but also osteoblast differentiation in conjugation with other osteotropic factors.

We investigated the hypoglycemic and hypolipidemic effects of two hesperertin glycosides, namely, hesperidin and cyclodextrin (CD)-clathrated hesperetin, in Goto-Kakizaki (GK) weanling rats with type 2 diabetes. We demonstrated that hesperidin and CD-hesperetin normalized glucose metabolism by altering the activities of glucose-regulating enzymes and reducing the levels of lipids in the serum and liver of the GK rats. These effects of hesperidin glycosides were partly produced by altering the expression of genes encoding the peroxisome proliferator-activated receptors, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, and the low-density lipoprotein receptor.

Soybean isoflavones are structurally similar to estrogen, and exhibit weak estrogenic activity. Although, a number of observational studies confirm the findings from the animal studies, the results from intervention studies are still controversial. One of the potential reasons for these inconsistencies could be considered individual differences in the isoflavone metabolism. In this report, the current topics of research on isoflavones and their relationship to bone health will be introduced.

Soybean isoflavones are structurally similar to estrogen, bind to estrogen receptors, and exhibit weak estrogenic activity. It has been reported that isoflavones play an important role in the prevention of hormone-dependent diseases, including osteoporosis, cardiovascular diseases, cancer, and postmenopausal syndrome. There are many researches indicating isoflavones prevent bone loss caused by estrogen deficiency in animal models. Furthermore, it has been demonstrated that a combination of isoflavone treatment and exercise cooperatively prevented bone loss in the estrogen-deficient status. Epidemiological studies demonstrated the relationship between the lower incidence of osteoporosis in Asian women and a diet rich in soy foods. Although a number of observational studies confirm the findings from the animal studies, the results from intervention studies are still controversial. One of the potential reasons for these inconsistencies could be individual differences in the isoflavone metabolism. Recently, it has been suggested that the clinical effectiveness of isoflavones might partly depend on the ability to produce equol, a gut bacterial metabolite of daidzein showing stronger estrogenic activity than the predominant isoflavones. Several candidate bacteria responsible for equol production have been suggested, for example Lactococcus 20-92 strain. From these findings, food factors enhancing equol production have received great deal of attention recently. On the other hand, safety assessment of isoflavones has been conducted by the Japanese Food Safety Commission. Further studies are required to address the numerous questions on the potential benefits, mechanisms of action, and safety of isoflavones.

Objective To investigate the effects of isoflavone on body weight, fat mass, and gene expression in relation to lipid metabolism. Methods Thirty-six female SD rats were ovariectomized or sham-operated and fed on a high-fat diet. Two months later, abdominal incision was made, blood was collected to separate serum, and the liver and adipose tissue were immediately collected and weighed. Some portions of these tissues were frozen in liquid nitrogen and stored at −80°C. Results Ovariectomy (OVX) with a high-fat diet could induce obesity in rats, while treatment with isoflavone significantly inhibited the increase in body weight and fat mass in abdomen. Serum total cholesterol and leptin were significantly decreased in isoflavone group, compared with the OVX group. The mRNA expression of liver fatty acid synthase (FAS) in the OVX group was significantly higher than that in sham-operated group, while this difference was not observed in the isoflavone group. The mRNA expression of liver hormone-sensitive lipase (HSL) in the OVX rats tended to be lower than that in the sham-operated rats. Furthermore, a large amount of isoflavone maintained the mRNA expression at a sham level. Conclusion Isoflavone may prevent obesity induced by ovariectomy with a high-fat diet, in part by modulating gene expression related to lipid metabolism.

Daidzein, a major isoflavone predominantly found in soybean, is mainly metabolized to equol and O-desmethylangolensin (O-DMA) by the human gut microflora. Equol exhibits a stronger estrogenic activity than daidzein, however, only approximately 30% of the population has been identified as equol-producers and there are too few direct evidences of the effects of the other major metabolite, O-DMA on estrogen-deficient status. The purpose of this study is therefore, to compare the effect of both O-DMA and equol on bone and lipid metabolism in vivo and in vitro. For the in vivo study, 8-week-old female mice were assigned to five groups as follows: sham-operated (sham), ovariectomized (OVX), OVX + 0.5 mg/day O-DMA (OVX + O-DMA), OVX + 0.5 mg/day equol (OVX + Eq), and OVX + 0.03 microg/day 17beta-estradiol (OVX + E2) administration. Three weeks after the intervention, O-DMA and equol did not affect uterine atrophy in OVX mice. The bone mineral density (BMD) of the femur was lower in the OVX group than in the sham group. The administration of equol but not O-DMA, maintained BMD through the intervention. Values of whole body fat mass and plasma lipids were lower in the equol and O-DMA treated OVX mice than those in OVX mice. In the in vitro study, equol significantly inhibited the osteoclast formation induced by 1alpha,25(OH)(2)D(3) in a dose-dependent manner in a co-culture system of mouse bone-marrow cells with primary osteoblastic cells. However, O-DMA slightly inhibited osteoclast formation, and the effect was not dose dependent. These results suggest that the effects of O-DMA on bone and lipid metabolism in OVX mice and osteoclast cell cultures are weaker than those of equol.

We examined the effects of vitamin D(3) on extracellular signal-related kinase (ERK). 1,25-(OH)(2)D(3), a form active in inducing transcription, caused rapid and transient ERK activation. 24R,25-(OH)(2)D(3), an inactive form, also activated ERK. In contrast, (22R)-22-methyl-20-epi-1,25-(OH)(2)D(3), a synthetic agonist of 1,25-(OH)(2)D(3), was not effective. These data provide evidence of selective roles of vitamin D(3) in nongenomic and genomic actions.

Soybean isoflavones show structural similarity to estradiol and therefore are known as phytoestrogen. Recently, isoflavones have received a great deal of attention for their preventive roles against hormone dependent diseases including postmenopausal osteoporosis, hyperlipidemia and cancer. Particularly, a number of studies have reported that soybean isoflavones inhibited bone loss in both female and male osteoporotic animal models as well as in postmenopausal women. On the other hand, since a kind of tablet with concentrated isoflavones has been recently applied for foods for specified health use (FOSHU), which are functional foods permitted to label a health claim by the Ministry of Health, Labor and Welfare in Japan, Food Safety Commission submitted a report the upper limit of daily isoflavone aglycon intake from FOSHU as well as from soybean products. This report demonstrates the effects of isoflavones on bone metabolism and safety.

There is a general agreement that isoflavones can be beneficial to health in adults. However, isoflavones are well known as endocrine-disrupting chemicals. It should be considered that soy foods might adversely affect the reproductive system and infants. The aim of this study was to evaluate the effects of genistein, an isoflavone, on dams and their offspring. Maternal rats were fed diets containing genistein at levels of 0 and 0.5 g/kg diet from pregnancy day 5 to postnatal day 13. No effects of genistein on the delivery, anogenital distance, reproductive organ weight, and body weight of the infants at birth were observed. There were no consistent effects on suckling pups after continuous genistein exposure during their fetal and suckling stages through their mothers, and there was no difference in effects according to the periods of exposure during pregnancy and lactation. We also observed no significant effect on the growth of offspring after weaning. Moreover, while we observed that the serum concentration of triiodothyronine (T3) in dams decreased, the result was a tendency, not a significant decrease. Our study suggested that maternal ingestion of genistein might have not induced serious adverse effects on dams, fetuses, infants or offspring during growth. However, the results indicated in many papers suggest the necessity of further study on the safety of genistein.

Few studies have examined the effects of isoflavones on bone formation during growth period in male and female animals. In this study, the effects of daidzein or genistein on bone formation were assessed in immature male and female mice. Five-week-old male and female mice were divided respectively into 3 groups (n = 8 per group) as follows: control group (C) fed a control diet (AIN-93G), daidzein group (D) fed a control diet containing 0.08% pure daidzein, and genistein group (G) fed a control diet containing 0.08% pure genistein. After 4 weeks, the male D group had a significantly higher bone mineral density (BMD) in whole body, lumbar spine, and femur than did the C group. On the contrary, BMD of the whole body and femur in the female D group was significantly lower than that in the C group. The BMD of the whole femur in the male G group also was significantly higher than for the C group. Histologic analysis revealed that the bone formation rate was significantly higher in the male D and G groups, and lower in the female D group compared with the C group. These results suggest that daidzein has a specific, sexually dimorphic effect on bone formation and BMD during growth period in mice.

The safety and effectiveness were examined of the spirulina alga on bone metabolism in ovariectomized estrogen-deficient rats and hindlimb-unloaded mice. The dosage range was from an amount equal to that recommended in so-called health foods for humans (0.08 g/kg BW/day) to a 100-fold higher dose. The bone mineral density (BMD) of the whole femur and tibia of ovariectomized rats in the any spirulina-treated groups was not significantly different from that of the ovariectomized group, although BMD of the distal femur and proximal tibia was significantly lower in the spirulina-treated groups than in the ovariectomized group after a 6 week-experimental period. BMD of the femur and tibia was not affected by treatment with any dose of spirulina in hindlimb-unloaded mice. These results suggest that the intake of spirulina decreased BMD in the trabecular bone of rodents under estrogen-deficient conditions.

Postmenopausal women as well as rodents after ovariectomy, which results in a lack of estrogen, can become obese. Ovariectomy-induced obesity in mice is associated with a decrease in oxygen consumption, indicating repressed energy expenditure. In this study, to elucidate the mechanism of weight gain after ovariectomy, we examined the expression patterns of genes related to energy expenditure and lipid metabolism, in mouse tissues including adipose tissue and skeletal muscle. In adipose tissue and skeletal muscle, at 2-4 wk after ovariectomy, levels of nuclear receptors and cofactors involved in energy expenditure such as ERR1, PPARalpha and PPARdelta, and PGC1alpha and PGC1beta were lower than in control mice. mRNA levels of their targets, medium-chain acyl coenzyme A dehydrogenase and acetyl CoA oxidase, enzymes for fatty acid beta-oxidation, were lower. In addition, the expression of PPARgamma and SREBP1, transcription factors important for lipogenesis, was decreased, as well as that of acetyl CoA carboxylase and fatty acid synthase, enzymes for fatty acid synthesis, and diacyl glycerol acetyl transferase 1 and 2, enzymes for triglyceride synthesis. These changes in gene expression are consistent with the obese phenotype in mice after ovariectomy. Thus a decrease in the expression of energy expenditure-related genes in adipose tissue and skeletal muscle could, in part, be responsible for obesity after ovariectomy.

Body fat accumulation and bone loss are both often associated with estrogen deficiency following menopause. In this study, we examined whether soy isoflavone, one of the phytoestrogens, and moderate exercise interventions exhibit cooperative effects on body composition and bone mass in ovariectomized (OVX) mice. Eight-week-old female mice were assigned to 6 groups: (1) sham-operated (sham); (2) OVX; (3) OVX with received a soy isoflavone diet (OVX+ISO); (4) OVX with exercised on a treadmill (OVX+EX); (5) OVX with given both isoflavone and exercise (OVX+ISOEX ); and (6) OVX with treated with 17 beta-estradiol subcutaneously (OVX+E2). Body composition and bone mineral density (BMD) were estimated by dual-energy x-ray absorptiometry (DXA). After the 6-week intervention, whole body fat (%) in the OVX group showed significantly higher than that in the sham group. Intervention of exercise and isoflavone alone partially inhibited OVX-induced body fat gain, and the combined intervention as well as E2 treatment completely restored fat mass to the sham level. Lean body mass in the whole body was not different in OVX group compared with that in OVX+ISO, OVX+EX, and OVX+E2 groups, but it was significantly higher in OVX+ISOEX than in other groups. BMD of the whole body, lumbar spine, or femur showed significantly reduced by OVX, and the bone loss was partially inhibited by intervention of exercise or isoflavone alone. However, the combined intervention completely restored the bone mass to the level of sham, as did E2. Serum total cholesterol was significantly increased by OVX, which was normalized by the combined intervention or E2 treatment. These results demonstrate that combined intervention of soybean isoflavone and exercise prevented body fat accumulation in the whole body with an increase in lean body mass and restoration of bone mass, and reduced high serum cholesterol in OVX mice.

The purpose of this study was to examine the effectiveness of collagen peptide intake on bone metabolism in growing (G) and calcium-deficient mature (M) rats. As for the dosages used, they were amounts equal to the recommended supplements for humans (0.166 g/kg body weight (BW) per day: Coll-1G and Coll-1M groups), 10-fold higher (1.66 g/kg BW per day: Coll-10G and Coll-10M groups), and 100-fold higher (16.6 g/kg BW per day: Coll-100G group). In growing male rats, bone mineral density (BMD) of the femur in the Coll-100G group was significantly higher than that in the other groups after the 4-week experimental period. On the other hand, kidneys in the rats from the Coll-100G group exhibited hypertrophy. To examine the effects of collagen peptide on bone metabolism in a calcium-deficient status, mature female rats were fed a 0.01% Ca diet for 9 weeks and then fed a diet with 0.2% calcium with or without collagen peptide (control, Coll-1M, and Coll-10M groups) or a 0.5% calcium diet (normal Ca) for 8 weeks. BMD of the whole femur in the Coll-10M group was significantly higher than that in the control and Coll-1M groups, and the level was similar to that in the normal Ca group. BMD of the lumbar spine in the Coll-10M group was significantly higher than their baseline value, as well as being significantly higher than that in the control and Coll-1M groups. These results suggest that orally administered collagen peptide may provide beneficial effects on bone metabolism, especially in the calcium-deficient condition, without obvious undesirable effects.

The purpose of this study was to examine whether hesperidin inhibits bone loss in ovariectomized mice (OVX), an animal model of postmenopausal osteoporosis. Forty 8-wk-old female ddY mice were assigned to five groups: a sham-operated group fed the control diet (AIN-93G), an OVX group fed the control diet, an OVX+HesA group fed the control diet containing 0.5 g/100 g hesperidin, and an OVX+HesB group fed the control diet containing 0.7 g/100 g alpha-glucosylhesperidin and an OVX+17beta-estradiol (E(2)) group fed the control diet and administered 0.03 micro g E(2)/d with a mini-osmotic pump. After 4 wk, the mice were killed and blood, femoral, uterine and liver were sampled immediately. Hesperidin administration did not affect the uterine weight. In OVX mice, the bone mineral density of the femur was lower than in the sham group (P < 0.05) and this bone loss was significantly prevented by dietary hesperidin or alpha-glucosylhesperidin. The Ca, P and Zn concentrations in the femur were significantly higher in the hesperidin-fed and E(2) groups than in the OVX group. Histomorphometric analyses showed that the trabecular bone volume and trabecular thickness in the femoral distal metaphysis were markedly decreased (P < 0.05) by OVX, and alpha-glucosylhesperidin significantly prevented this bone loss. Furthermore, hesperidin decreased the osteoclast number of the femoral metaphysis in OVX mice, as did E(2). Serum and hepatic lipids were lower in mice that consumed the hesperidin-containing diets (P < 0.05) than in the OVX group fed the control diet. These results suggest a possible role for citrus flavonoids in the prevention of lifestyle-related diseases because of their beneficial effects on bone and lipids.

Fructooligosaccharides (FOS) stimulate the growth of bifidobacteria, which cleave isoflavone conjugates to yield the corresponding aglycones and metabolites. In a previous study, FOS modified the absorption and enterohepatic recirculation of isoflavones in rats. In the present study, we determined the effect of the combination of dietary FOS and isoflavone conjugates on bone mass in ovariectomized (OVX) and surgical control mice. After undergoing OVX or sham operation, female ddY mice (8 wk old, n = 64) were randomly assigned to four groups: a purified control diet (AIN-93G) group, a FOS diet (AIN-93G + 5% FOS) group, an isoflavone diet (AIN-93G + 0.2% isoflavone conjugates) group, or a FOS and isoflavone diet (AIN-93G + 5% FOS + 0.2% isoflavone conjugates) group. After 6 wk, the mice were killed and the blood and femora were sampled immediately. In OVX mice, both isoflavone conjugates and FOS prevented femoral bone loss. An additive effect of dietary isoflavone conjugates and FOS was observed by dual-energy X-ray absorptiometry in the distal part of the femur and in trabecular bone, by peripheral quantitative computed tomography. Moreover, FOS increased cecal beta-glucosidase activity and equol production from daidzein in both OVX and surgical control mice fed isoflavone conjugates. These results suggest that FOS increase the bioavailability of isoflavones, leading to cooperative effects in the prevention of osteopenia in OVX mice.