Kanamycin inhibits daidzein metabolism and abilities of the metabolites to prevent bone loss in ovariectomized mice.

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.

Dietary zinc supplementation increased TNFα and IL1ß-induced RANKL expression, resulting in a decrease in bone mineral density in rats.

We investigated the effect of dietary zinc supplementation on bone metabolism in rats. Four-week-old male Wistar rats were fed a 30.0 mg zinc/kg diet (C), a 300.0 mg zinc/kg diet (HZ) or a 3,000.0 mg zinc/kg diet (EZ) for 4 weeks. The zinc content of the femur gradually increased in accordance with the gradual increase in the dietary zinc level. Although the mRNA expression of zinc transporters in bone did not differ between the groups, the mRNA expression of metallothioneins was increased in the HZ and EZ groups compared to the C group. Moreover, the bone mineral density was significantly decreased in the HZ and EZ groups compared to the C group. Furthermore, the mRNA expression of tumor necrosis factor α, Interleukin-1ß and osteoclastogenesis-related genes such as receptor for activator of nuclear factor-κB (NF-κB) ligand, tumor necrosis factor receptor-associated factor 6, and nuclear factor of activated T cells cytoplasmic 1 was significantly increased in the HZ and EZ groups compared to the C group. These findings suggested that dietary zinc supplementation reduced bone mineral density through the promotion of bone resorption via an increase in the expression of receptor for activator of NF-κB ligand induced by tumor necrosis factor α and Interleukin-1ß.

Zinc Deficiency Increases Serum Concentrations of Parathyroid Hormone through a Decrease in Serum Calcium and Induces Bone Fragility in Rats.

We hypothesized that a zinc-deficient diet alters the mineral (calcium, magnesium, and phosphorus) components of bones, as well as hormones related to bone remodeling, and negatively affects bone metabolism. Four-week-old male Wistar rats were randomly assigned to one of three groups for 4 wk: a zinc-adequate group (C, 30 ppm); a zinc-deficient group (ZD, 1 ppm); and a pair-fed group (PF, 30 ppm), which was pair-fed to the ZD group. Bone mineral density and bone mechanical properties were reduced in the ZD group compared to the C and PF groups. Compared with the C and PF groups, serum osteocalcin, a bone formation marker, was reduced in the ZD group. Conversely, urine deoxypyridinoline, a bone resorption marker, was increased in the ZD group compared to the C and PF groups. Calcium and phosphorus concentrations in bone were not different among all groups. The bone magnesium concentration was significantly higher in the ZD group than in the PF and C groups. Interestingly, compared with the C and PF groups, the ZD group showed a reduction in serum calcium concentration along with an increase in serum parathyroid hormone (PTH) concentration. Although serum 1,25-dihydroxycholecalciferol concentration was significantly higher in the ZD and PF groups than in the C group, the rate of apparent calcium absorption was significantly lower in the ZD group than in the C and PF groups. Therefore, zinc deficiency is suspected to cause an increase in serum PTH concentration owing to an inability to maintain calcium homeostasis, resulting in bone fragility.

Synergistic effect of isoflavone glycosides and fructooligosaccharides on postgastrectomy osteopenia in rats.

Fructooligosaccharides stimulate the growth of Bifidobacteria, which cleave isoflavone glycosides to yield corresponding aglycones, and convert metabolites by enhancing enterohepatic recirculation of isoflavones in rats. In the present study, we determined the synergistic effect of dietary isoflavone glycosides and fructooligosaccharides on postgastrectomy osteopenia in rats. Nine-week-old male Sprague-Dawley rats were gastrectomized (n = 20) or sham operated, (control, n = 5) and then randomly assigned to 5 diet groups: sham-a purified diet control, gastrectomized-control, gastrectomized-isoflavone (0.2% isoflavone glycosides), gastrectomized-fructooligosaccharides (7.5% fructooligosaccharides), and isoflavone and fructooligosaccharides (0.2% isoflavone glycosides + 7.5% fructooligosaccharides). After 6 weeks, the rats were killed and biological samples were collected. In gastrectomized rats, fructooligosaccharides prevented femoral bone fragility, but isoflavone without fructooligosaccharides did not inhibit postgastrectomy osteopenia. Isoflavone and fructooligosaccharides exhibited a synergistic in the distal metaphyseal trabecular bone, indicated by peripheral quantitative computed tomography. Moreover, fructooligosaccharides increased calcium absorption and equol production from daidzein in gastrectomized rats. These results indicate that isoflavone alone did not inhibit postgastrectomy osteopenia, but the combination of isoflavone and fructooligosaccharides improved the inhibition of trabecular bone loss by increasing calcium absorption and equol production through fructooligosaccharides supplementation.

Comparative activities of the S-enantiomer and racemic forms of equol on bone fragility in ovariectomized mice.

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.

Absence of negative feedback on intestinal magnesium absorption on excessive magnesium administration in rats.

This study aimed to clarify the regulatory mechanism of Mg homeostasis on administration of excessive Mg in rats. Six-week-old male Wistar rats (n=30) were fed a Mg-deficient diet (D) or a control diet (M) in addition to which they received subcutaneous injections of saline (S) or additional Mg (M) for 14 d. Feces and urine were collected from the rats for 4 d every week. Between the MS and MM rats and the DS and DM rats, the injection of additional Mg increased Mg retention, but intestinal Mg absorption did not differ. Urinary Mg excretion in the MM rats was significantly greater than that in the MS rats, but fecal Mg excretion did not increase. Mg retention in the DM rats was approximately 30% of that in the MS rats, and urinary Mg excretion did not differ between the 2 groups, although the serum Mg in DM rats was low. There was no significant difference in the femoral Mg between the MM and MS groups. The physiological Mg pool in the bone appears to be limited. Therefore, there is no physiological Mg pool for the storage of excessive Mg, and there appears to be no negative feedback mechanism on intestinal Mg absorption upon administration of excessive Mg in the rats. In conclusion, it appears that the kidney is the only organ that regulates Mg in the body; apart from this, regulatory mechanisms corresponding to the physiological Mg requirement do not exist or are weak.

Effects of dietary ascorbic acid supplementation on lipid peroxidation and the lipid content in the liver and serum of magnesium-deficient rats.

We investigated the effects of ascorbic acid (AsA) supplementation on lipid peroxidation and the lipid content in the liver and serum of magnesium (Mg)-deficient rats. Eighteen 3-week-old male Sprague-Dawley strain rats were divided into 3 groups and maintained on a control diet (C group), a low-Mg diet (D group), or a low-Mg diet supplemented with AsA (DA group) for 42 d. At the end of this period, the final body weight, weight gain, and serum Mg concentrations were significantly decreased in the Mg-deficient rats. Further, dietary AsA supplementation had no effect on the growth, serum Mg concentration, Mg absorption, and Mg retention. The serum concentration of AsA was significantly lower in the D group than in the C group but was unaltered in the DA group. The levels of phosphatidylcholine hydroperoxide (PCOOH) in the serum and of triglycerides (TGs) and total cholesterol (TC) in the serum and liver were significantly higher in the D group than in the C group. The serum PCOOH, liver TG, and liver TC levels were decreased in the DA group. These results indicate that Mg deficiency increases the AsA requirement of the body and that AsA supplementation normalizes the serum levels of PCOOH and the liver lipid content in Mg-deficient rats, without altering the Mg status.

Citrus nobiletin suppresses bone loss in ovariectomized ddY mice and collagen-induced arthritis in DBA/1J mice: possible involvement of receptor activator of NF-kappaB ligand (RANKL)-induced osteoclastogenesis regulation.

Bone resorption is known to accelerate during the onset of several disorders, including osteoporosis (OP) and rheumatoid arthritis (RA). Some epidemiological surveys have suggested that a high intake of vegetables and fruits has an inverse relation to such disease incidence, though the number of active constituents elucidated thus far is limited. In the present study, we examined the efficacy of various food phytochemicals using two animal models. First, female ddY mice were ovariectomized (OVX) or sham-operated (sham), after which five different compounds (phenethyl isothiocyanate, zerumbone, auraptene, 1'-acetoxychavicol acetate, and nobiletin) were administered separately to OVX mice with a mini-osmotic pump at doses of 0.25 or 0.5 mg/day for 4 weeks, with 17beta-estradiol (E_{2}, 0.03 microg/day) used as a positive control. Nobiletin, in contrast to the other tested phytochemicals, significantly (P<0.05) suppressed the reduction of whole bone mineral density by 61%, which was comparable to or higher than the efficacy of E_{2}. Next, nobiletin given as an i.p. administration at 20 mg/kg of body weight, but not 2 mg/kg, to male DBA/1J mice every 2 days for 12 days led to a marked decrease in type II collagen-induced arthritis by 45% (P < 0.05). Furthermore, the flavonoid (4-50 microM) attenuated receptor activator of nuclear factor kappaB ligand (RANKL)-induced osteoclastogenesis of RAW264.7 cells, as detected by tartarate-resistant acid phosphatase activity and microscopic observations. Of note, nobiletin also suppressed RANKL-activated extracellular signal-regulated kinase1/2, c-Jun N-terminal kinase1/2, and p38 mitogen-activated protein kinase activities, and thereby regulated the promoter activation of nuclear factor kappaB (NFkappaB) and activator protein-1, key transcription factors for differentiation. Together, our results suggest that nobiletin is a promising phytochemical for the prevention or treatment of osteoclastogenesis-related disorders, including OP and RA, with reasonable action mechanisms.

Effects of Oral Administration of Moringa oleifera Lam on Glucose Tolerance in Goto-Kakizaki and Wistar Rats.

Medicinal plants constitute an important source of potential therapeutic agents for diabetes. In the present study, we investigated the effects of Moringa oleifera (MO) Lam, Moringacea, on glucose tolerance in Wistar rats and Goto-Kakizaki (GK) rats, modeled type 2 diabetes. Major polyphenols in MO powder were quercetin glucosides, rutin, kaempferol glycosides and chlorogenic acids by HPLC analysis. As the results of glucose tolerance test, MO significantly decreased the blood glucose at 20, 30, 45and 60 min for GK rats and at 10, 30 and 45 min for Wistar rats (p<0.05) compared to the both controls after glucose administration. The area under the curve of changes in the blood glucose was significantly higher in the GK control group than in the GK plus MO group (p<0.05) in the periods 30-60 min and 60-120 min. Furthermore, MO significantly decreased stomach emptying in GK rats (p<0.05). The results indicated that MO has an ameliorating effect for glucose intolerance, and the effect might be mediated by quercetin-3-glucoside and fiber contents in MO leaf powder. The action of MO was greater in GK rats than in Wistar rats.

Effects of lowering food intake by high phosphorus diet on parathyroid hormone actions and kidney mineral concentration in rats.

We investigated whether lowering food intake by high phosphorus (P) diet influenced parathyroid hormone (PTH) actions, bone turnover markers, and kidney mineral concentration in rats. Rats in two of the three groups were respectively given free access to a control diet (C group) and a high P diet (HP group) for 21 days. Rats in another group (PF group) were pair-fed the control diet with the HP group. Compared to the C and PF groups, serum PTH concentration, urinary C-terminal telopeptide of type I collagen excretion, and kidney calcium and P concentrations were significantly higher in the HP group. Urinary excretion of cAMP was significantly lower in the HP group than in the C and PF groups. These results suggested that high P diet decreased PTH action in the kidney and increased bone resorption and kidney mineral concentrations independently of lowering food intake.

High-phosphorus diet stimulates receptor activator of nuclear factor-kappaB ligand mRNA expression by increasing parathyroid hormone secretion in rats.

The purpose of the present study was to clarify the manner by which the supplementation of high-P diet induces bone loss. Eighteen 4-week-old male Wistar-strain rats were assigned randomly to three groups and fed diets containing three P levels (0.3, 0.9, and 1.5 %) for 21 d. A lower serum Ca concentration was observed in the rats fed on the 1.5 % P diet than in the other two groups. Serum P and parathyroid hormone concentrations and urinary excretion of C-terminal telopeptide of type I collagen were elevated with increasing dietary P levels. Serum osteocalcin concentration was increased in the rats fed on the 1.5 % P diet than in the other two groups. Bone formation rate of the lumbar vertebra was significantly increased in the two high-P groups than in the 0.3 % P group. Osteoclast number was significantly increased with increasing dietary P levels. Bone mineral content and bone mineral density of the femur and lumbar vertebra and ultimate compression load of the lumbar vertebra were decreased with increasing dietary P levels. Additionally, ultimate bending load of the femur was decreased in the rats fed on the 1.5 % P diet than in the other two groups. Receptor activator of NF-kappaB ligand (RANKL) mRNA expression in the femur was significantly higher with increasing dietary P levels. These results suggest that secondary hyperparathyroidism due to a high-P diet leads to bone loss via an increase in bone turnover. Furthermore, an increase in osteoclast number was caused by increased RANKL mRNA expression.

Decreased mRNA expression of the PTH/PTHrP receptor and type II sodium-dependent phosphate transporter in the kidney of rats fed a high phosphorus diet accompanied with a decrease in serum calcium concentration.

This study investigates the phosphorus (P) homeostasis in the process of an altered parathyroid hormone (PTH) action in the kidney of rats fed a high P diet. Four-week-old male Wistar strain rats were fed diets containing five different P levels (0.3, 0.6, 0.9, 1.2 and 1.5%) for 21 days. The serum PTH concentration and urinary excretion of P were elevated with increasing dietary P level. Compared to rats fed the 0.3% P diet, the serum calcium (Ca) concentration remained unchanged, while the serum 1,25(OH)(2)D(3) concentration and urinary excretion of cAMP were elevated with increasing dietary P level in rats fed the high P diets containing 0.6-0.9% P. On the other hand, a lower serum Ca concentration was observed in rats fed the high P diets containing 1.2% or greater P. The serum 1,25(OH)(2)D(3) concentration remained unchanged in rats fed the high P diets containing 1.2% or greater P, comparison with rats fed the 0.3% P diet. The urinary excretion of cAMP and PTH/PTH-related peptide (PTHrP) receptor and type II sodium-dependent phosphate transporter (NaPi-2) mRNA in the kidney were both decreased in rats fed the high P diets containing 1.2% or greater P. In conclusion, a high P diet with subsequent decrease in serum Ca concentration suppressed the PTH action in the kidney due to PTH/PTHrP receptor mRNA down-regulation. Furthermore, an increase in the urinary excretion of P might have been caused by decreased NaPi-2 mRNA expression without the effects of PTH and 1,25(OH)(2)D(3).

High phosphorus diet changes phosphorus metabolism regardless of PTH action in rats.

In this study, we ascertained whether the parathyroid hormone (PTH) dominantly regulated the effects of high phosphorus (P) intakes on urinary excretion of P and bone metabolism in rats. To maintain serum PTH level equally, parathyroidectomy (PTX) and sham-operated rats were constantly exposed to rPTH(1-34) and fed both control (0.3% P) and high P (1.2% P) diet for 7 days, respectively. Urinary excretions of P and C-terminal telopeptides of type I collagen were significantly increased in both PTX and sham rats by the high P diet. These results suggest that high P diet increased urinary P excretion while promoting bone resorption regardless of PTH-dependent regulation.

Effect of high phosphorus diet on phosphorus metabolism in parathyroidectomized rats.

To determine the parathyroid hormone (PTH) action on kidney and bone by high phosphorus (P) diet, this study investigated PTH/PTH-related peptide (PTHrP) receptor mRNA expression in 6-week-old parathyroidectomized (PTX) rats received constant amount of PTH. To maintain serum PTH levels equally to sham operated rats, PTX rats were constantly exposed to rPTH (1-34) and fed a control diet (0.3% P) and a high P diet (1.2% P) for 7 days, respectively. There were no significant differences in serum PTH (1-34) concentration in rats fed the control diet. In sham groups, serum PTH concentrations, both (1-84) and (1-34) fragments, were increased in rats fed the high P diet than in rats fed the control diet. Urinary excretions of P and C-terminal telopeptides of type I collagen were significantly increased in both PTX and sham rats by the high P diet. PTH/PTHrP receptor mRNA expression in kidney and femur was not changed in both PTX and sham rats by the high P diet. In conclusion, high P diet did not change PTH action in PTX rats and increased urinary excretion of P and bone resorption regardless of PTH action.