Runx2 Regulates Galnt3 and Fgf23 Expressions and Galnt3 Decelerates Osteoid Mineralization by Stabilizing Fgf23.

Runx2 (runt related transcription factor 2) is an essential transcription factor for osteoblast proliferation and differentiation. Uridine diphosphate (UDP)-N-acetylgalactosamine (GalNAc): polypeptide GalNAc-transferase 3 (Galnt3) prevents proteolytic processing of fibroblast growth factor 23 (Fgf23), which is a hormone that regulates the serum level of phosphorus. Runx2 and Galnt3 were expressed in osteoblasts and osteocytes, and Fgf23 expression was restricted to osteocytes in bone. Overexpression and knock-down of Runx2 upregulated and downregulated, respectively, the expressions of Galnt3 and Fgf23, and Runx2 directly regulated the transcriptional activity of Galnt3 in reporter assays. The expressions of Galnt3 and Fgf23 in osteoblast-specific Runx2 knockout (Runx2fl/flCre) mice were about half those in Runx2fl/fl mice. However, the serum levels of phosphorus and intact Fgf23 in Runx2fl/flCre mice were similar to those in Runx2fl/fl mice. The trabecular bone volume was increased during aging in both male and female Galnt3-/- mice, but the osteoid was reduced. The markers for bone formation and resorption in Galnt3-/- mice were similar to the control in both sexes. Galnt3-/- mice exhibited hyperphosphatemia and hypercalcemia, and the intact Fgf23 was about 40% that of wild-type mice. These findings indicated that Runx2 regulates the expressions of Galnt3 and Fgf23 and that Galnt3 decelerates the mineralization of osteoid by stabilizing Fgf23.

Short-term dietary magnesium deficiency downregulates the expression of bone formation-related genes in rats.

Dietary magnesium deficiency increases osteoclastic bone resorption and decreases osteoblastic bone formation. Increased bone resorption due to dietary magnesium deficiency can be explained by increased expression of the receptor activator of nuclear factor kB ligand. However, the detailed mechanisms underlying decreased bone formation remain unclear. Thus, in the present study, to determine the mechanism underlying decreased bone formation induced by dietary magnesium deficiency, we investigated the effects of short-term dietary magnesium deficiency on the mRNA expression of genes related to bone formation in rats. Male Wistar rats were fed a control or magnesium-deficient diet for eight days. The mRNA expression level of Runx2, Sp7, Bglap, Alpl, Col1a1, Igf1, and Bmp2 in the femur was significantly lower in magnesium-deficient rats than in control rats. These results suggest that short-term dietary magnesium deficiency decreases the gene expression of insulin-like growth factor-1 and bone morphogenetic protein 2, which, in turn, decreases osteoblastic bone formation through the downregulation of osteoblastogenesis-related gene expression.

Effects of dietary phosphorus concentration and phosphate salt form on renal tubule function in unilateral nephrectomized rats.

Background: Excessive consumption of phosphorus (P) impairs renal tubule function; however, the effects of different dietary phosphate salts on chronic kidney disease (CKD) are unclear. Aim: To examine the effects of potassium dihydrogen phosphate (KH2PO4) and potassium tripolyphosphate (K5P3O10) and P concentration on renal function in a rat model of early CKD. Methods: Male sham-operated Sprague-Dawley rats were fed a diet containing KH2PO4 with a normal P level. Kidney injury was induced by unilateral nephrectomy (UNx), and the rats were divided into four groups fed dietary KH2PO4 or K5P3O10 with a normal (UNx-NKH, UNx-NKP) or high (UNx-HKH, UNx-HKP) P concentration, respectively, for 21 days. Results: UNx-NKH rats showed significantly lower creatinine clearance (CCr) and higher albumin (ALB) compared with those of sham rats, confirming UNx-induced kidney injury. The urinary levels of liver-type fatty acid-binding protein (L-FABP) and ALB were significantly higher in UNx-HKP rats than in UNx-HKH rats. However, other markers of renal tubule function, such as CCr, serum creatinine (CRE), calcium (Ca), and hormones, only differed among groups according to the P concentration and not the dietary phosphate salt form. Histological examination showed higher incidence and severity of tubulointerstitial lesions, tubule regeneration, tubule dilation, and calcification in the high-phosphorus than in the normal-phosphorus UNx groups. These changes were more severe in the UNx-HKP group compared with the UNx-HKH group. Conclusion: This study highlights the importance of controlling dietary P intake in terms of both concentration and source to prevent the progression of CKD.

Iron deficiency negatively regulates protein methylation via the downregulation of protein arginine methyltransferase.

Iron is an essential trace metal for all biological processes and plays a role in almost every aspect of body growth. Previously, we found that iron-depletion downregulated the expression of proteins, arginine methyltransferase-1 and 3 (PRMT1 and PRMT3), by an iron-specific chelator, deferoxamine (DFO), in rat liver FAO cell line using DNA microarray analysis (unpublished data). However, regulatory mechanisms underlying the association between iron deficiency and PRMT expression are unclear in vitro and in vivo. In the present study, we revealed that the treatment of cells with two iron-specific chelators, DFO and deferasirox (DFX), downregulated the gene and protein expression of PRMT1 and 3 as compared with the untreated cells. Subsequently, DFO and DFX treatments decreased protein methylation. Importantly, these effects were attenuated by a holo-transferrin treatment. Furthermore, weanling Wistar-strain rats were fed a control diet or an iron-deficient diet for 4 weeks. Dietary iron deficiency was found to decrease the concentration of hemoglobin and liver iron while increasing the heart weight. PRMT and protein methylation levels were also significantly reduced in the iron-deficient group as compared to the control group. To our knowledge, this is the first study to demonstrate that PRMT levels and protein methylation are reduced in iron-deficient models, in vitro and in vivo.

The effects of green kiwifruit combined with isoflavones on equol production, bone turnover and gut microflora in healthy postmenopausal women.

BACKGROUND AND OBJECTIVES: Isoflavone (daidzein and genistein) interventions in postmenopausal women have produced inconsistent skeletal benefits, partly due to population heterogeneity in daidzein metabolism to equol by enteric bacteria. This study assessed changes in microflora and bone turnover in response to isoflavone and ki-wifruit supplementation in New Zealand postmenopausal women. METHODS AND STUDY DESIGN: Healthy women 1-10 years post-menopause were randomly allocated to group A (n=16) or B (n=17) for a 16-week crossover trial. Two consecutive 6-week treatment periods had a 2-week lead-in period at intervention commencement and a 2-week washout period between treatments. Treatments prescribed either (1) daily isoflavone supplementation (50 mg/day aglycone daidzein and genistein) alone, or (2) with two green kiwifruit. At treatment baseline and end-point (four time points) the serum bone markers C Telopeptide of Type I collagen (CTx), undercarboxylated os-teocalcin (unOC), and serum and urinary daidzein and equol, were measured. Changes in gut microflora were monitored in a subgroup of the women. RESULTS: Equol producers made up 30% of this study population (equol producers n=10; non-equol producers n=23) with serum equol rising significantly in equol producers. Serum ucOC decreased by 15.5% (p<0.05) after the kiwifruit and isoflavone treatment. There were no changes in serum CTx or in the diversity of the gut microflora. CONCLUSIONS: 50 mg/day isoflavones did not reduce bone resorption but kiwifruit and isoflavone consumption decreased serum ucOC levels, possibly due to vitamin K1 and/or other bioactive components of green kiwifruit.

Iron deficiency induces autophagy and activates Nrf2 signal through modulating p62/SQSTM.

Iron is an essential trace metal in almost all organisms and plays an important role in the redox system. We previously reported that iron deficiency activated autophagy and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling for oxidative stress. However, regulatory mechanisms underlying the association between autophagy and Nrf2 signaling are unclear. In this study, we found that treatment of cells with an iron-specific chelator deferoxamine (DFO) increased reactive oxidative species (ROS) production by elevating the expression of p47phox and p67phox compared with that in untreated cells. The DFO treatment also induced protein aggregation and formed aggresome, which is a cellular response to misfolded protein. In addition, DFO treatment upregulated the expression of the autophagic gene p62/SQSTM1, which in turn activated intracellular proteolysis during autophagy. DFO treatment phosphorylated p62/SQSTM1 (Thr351) to activate Nrf2. However, silencing of p62/SQSTM1 followed by DFO treatment attenuated Nrf2 activation and resulted in the accumulation of carboxyl proteins compared with DFO treatment alone. These results indicated that iron deficiency activates Nrf2 signaling by modulating p62/SQSTM1 during autophagy.

A case of empyema and septic arthritis due to Nocardia farcinica.

Septic arthritis due to Nocardia sp. should be suspected when a patient with risk factors such as pneumoconiosis or diabetes mellitus develops joint symptoms, especially if the patient has had nocardiosis in other sites.

Changes in circulating levels of fibroblast growth factor 23 induced by short-term dietary magnesium deficiency in rats.

Fibroblast growth factor 23 (FGF23) is a potent regulator of phosphorus (P) and vitamin D metabolism. Long-term dietary magnesium (Mg) deficiency increases circulating levels of FGF23, whereas the effects of short-term dietary Mg deficiency are unclear. Thus, the present study investigated whether short-term dietary Mg deficiency affects circulating levels of FGF23. We also assessed changes in renal mRNA expression of vitamin D metabolizing enzymes and type II sodium-phosphate (Na/Pi) cotransporters, since these are regulated by FGF23. Rats were fed a control diet (control group) or an Mg-deficient diet (Mg-deficient group) for 2, 4 or 7 days. Serum Mg levels were significantly lower in the Mg-deficient group than in the control group at all time points. Serum FGF23 levels were significantly higher in the Mg-deficient group than in the control group at day 7. The 25-hydroxyvitamin D-24-hydroxylase (24(OH)ase) mRNA levels were significantly higher in the Mg-deficient group than in the control group at day 7 . No significant differences in types IIa and IIc Na/Pi cotransporter mRNA levels were observed between the control and Mg-deficient groups. These results suggest that dietary Mg deficiency causes a rapid increase in circulating levels of FGF23 and renal 24(OH)ase mRNA levels.

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.

A combination of soy isoflavones and cello-oligosaccharides changes equol/O-desmethylangolensin production ratio and attenuates bone fragility in ovariectomized mice.

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.

A short-term zinc-deficient diet decreases bone formation through down-regulated BMP2 in rat bone.

We investigated the effects of a short-term dietary zinc deficiency on bone metabolism. Zinc deficiency increased the mRNA expression of zinc uptake transporters such as Zip1, Zip13, and Zip14 in bone. However, zinc deficiency might not maintain zinc storage in bone, resulting in a decrease in bone formation through downregulation of the expression levels of osteoblastogenesis-related genes.

Dietary zinc deficiency induces oxidative stress and promotes tumor necrosis factor-α- and interleukin-1ß-induced RANKL expression in rat bone.

We investigated the effects of dietary zinc deficiency on oxidative stress and bone metabolism. Four-week-old male Wistar rats were randomly assigned to one of three groups for 4 weeks: a zinc-adequate group (30 ppm); a zinc-deficient group (1 ppm); and a pair-fed group (30 ppm) that was pair-fed to the zinc-deficient group. The iron content and the thiobarbituric acid reactive substance level in bone were higher in the zinc-deficient group than in the zinc-adequate and pair-fed groups. The mRNA expression level of osteoblastogenesis-related genes such as bone morphogenetic protein 2 and runt-related transcription factor 2 was lower in the zinc-deficient group than in the zinc-adequate and pair-fed groups. In contrast, the mRNA expression levels of tumor necrosis factor-α, interleukin-1ß and osteoclastogenesis-related genes such as receptor activator of nuclear factor-κB ligand and nuclear factor of activated T cells cytoplasmic 1 were higher in the zinc-deficient group than in the zinc-adequate and pair-fed groups. These findings suggested that dietary zinc deficiency reduced osteoblastogenesis via a decrease in the expression of bone morphogenetic protein 2 and increased osteoclastogenesis via enhancement of the expression of receptor for activator of nuclear factor-κB ligand induced by oxidative stress-stimulated tumor necrosis factor-α and interleukin-1ß.

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.

Effect of Kiwifruit on Bone Resorption in Ovariectomized Mice.

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.

Effects of Dietary Calcium Supplementation on Bone Metabolism, Kidney Mineral Concentrations, and Kidney Function in Rats Fed a High-Phosphorus Diet.

We investigated the effects of dietary calcium (Ca) supplementation on bone metabolism, kidney mineral concentrations, and kidney function in rats fed a high-phosphorus (P) diet. Wistar strain rats were randomly divided into 4 dietary groups and fed their respective diets for 21 d: a diet containing 0.3% P and 0.5% Ca (C), a diet containing 1.5% P and 0.5% Ca (HP), a diet containing 0.3% P and 1.0% Ca (HCa), or a diet containing 1.5% P and 1.0% Ca (HPCa). Compared to the C group, the high-P diet increased serum parathyroid hormone concentration, markers of bone turnover, receptor activator of NF-κB ligand mRNA expression of the femur, kidney Ca and P concentrations, urinary N-acetyl-ß-D-glucosaminidase activity, and urinary ß2-microglobulin excretion, and decreased bone mineral content and bone mineral density of the femur and tibia. Dietary Ca supplementation improved the parameters of bone metabolism and kidney function in rats fed the high-P diet, while there were no significant differences in kidney Ca or P concentrations between the HP and HPCa groups. These results suggest that dietary Ca supplementation prevented the bone loss and decline in kidney function induced by a high-P diet, whereas dietary Ca supplementation did not affect kidney mineral concentrations in rats fed the high-P diet.

Activation of Nrf2/Keap1 signaling and autophagy induction against oxidative stress in heart in iron deficiency.

We investigated the effects of dietary iron deficiency on the redox system in the heart. Dietary iron deficiency increased heart weight and accumulation of carbonylated proteins. However, expression levels of heme oxygenase-1 and LC3-II, an antioxidant enzyme and an autophagic marker, respectively, in iron-deficient mice were upregulated compared to the control group, resulting in a surrogate phenomenon against oxidative stress.

Effects of high phosphorus diet on bone metabolism-related gene expression in young and aged mice.

In this study, the effects of high phosphorus (P) diet on bone metabolism-related gene expression were investigated in young and aged mice. Twelve- and 80-week-old ddY male mice were divided into two groups, respectively, and fed a control diet containing 0.3% P or a high P diet containing 1.2% P. After 4 weeks of treatment, serum parathyroid hormone (PTH) concentration was significantly higher in the high P groups than in the control groups in both young and aged mice and was significantly higher in aged mice than in young mice fed the high P diet. High P diet significantly increased receptor activator of NF-κB ligand (RANKL) mRNA in the femur of both young and aged mice and significantly increased the RANKL/osteoprotegerin (OPG) mRNA ratio only in aged mice. High P diet significantly increased mRNA expression of transient receptor potential vanilloid type 6, calbindin-D9k, and plasma membrane Ca(2+)-ATPase 1b in the duodenum of both young and aged mice. These results suggest that high P diet increased RANKL mRNA expression in the femur and calcium absorption-related gene expression in the duodenum regardless of age. Furthermore, the high P diet-induced increase in PTH secretion might increase the RANKL/OPG mRNA ratio in aged mice.

Magnesium deficiency regulates vitamin D metabolizing enzymes and type II sodium-phosphate cotransporter mRNA expression in rats.

A magnesium (Mg) deficiency induces changes in calcium (Ca) and phosphorus (P) metabolism; however, the mechanisms responsible for these effects remain unclear. Since 1,25-dihydroxyvitamin D3 and type II sodium-phosphate (Na/Pi) cotransporters are essential regulators of Ca and P metabolism, this study examined the effects of Mg deficiency on the mRNA expression of vitamin D metabolizing enzymes (25-hydroxyvitamin D-1α-hydroxylase (1α(OH)ase) and 25-hydroxyvitamin D-24-hydroxylase (24(OH)ase)), and Na/Pi cotransporters (type IIa and IIc) in the rat kidney. Rats were divided into two groups and fed a control diet (Mg concentration: 0.05%) or a Mg-deficient diet (Mg concentration: Mg-free) for 21 days. 1α(OH)ase mRNA levels were significantly decreased in rats fed the Mg-deficient diet, while 24(OH)ase mRNA levels were significantly increased, compared to rats fed the control diet. Type IIa and IIc Na/Pi cotransporter mRNA levels in rats fed the Mg-deficient diet were significantly decreased compared to rats fed the control diet. These results suggest that Mg deficiency induces downregulation of 1α(OH)ase and type IIa and IIc Na/Pi cotransporters, and upregulation of 24(OH)ase in the kidney.

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.