Calcium restriction for 28 days markedly and negatively influences bone mineral density of the femur and lumbar vertebrae regardless of the high-fat diet ingestion in young adult male rats.

Calcium (Ca) is necessary for bone calcification, and Ca deficiency leads to decreased bone mineral density (BMD). Epidemiological studies have reported a correlation between Ca intake and BMD. Although the influences of Ca deficiency on BMD have been reported, the effects of Ca restriction on bone during high-fat diet ingestion remain unclear. Therefore, we hypothesized that high-fat diet ingestion would potentiate the negative effects of Ca restriction on bone. Sprague-Dawley strain male rats (aged 11 weeks) were divided into 4 groups: basic control diet (Cont.) (11% lipid energy rate, 0.5% calcium), basic control diet with Ca restriction (CaR) (11% lipid energy rate, 0.02% calcium), high-fat diet (HF) (40% lipid energy rate, 0.5% calcium), and high-fat diet with Ca restriction (HFCaR) (40% lipid energy rate, 0.02% calcium). At 28 days after starting the experimental diets, body weights were higher in the high-fat diet groups (HF and HFCaR) than in the standard-fat diet groups (Cont. and CaR) on 2-way analysis of variance. The apparent Ca absorption rate in the Ca-restricted groups (CaR and HFCaR) was higher than in the Ca-sufficient groups (Cont. and HF). BMD and bone strength parameters of the femur and lumbar vertebrae in the Ca-restricted groups were markedly lower than in the Ca-sufficient groups, whereas there were no significant differences between the standard-fat diet and HF diet groups. These results suggest that 28 days of Ca restriction increases the risk of bone fracture and osteoporosis.

Vitamin D restriction and/or a high-fat diet influence intestinal alkaline phosphatase activity and serum endotoxin concentration, increasing the risk of metabolic endotoxemia in rats.

Vitamin D insufficiency induces calcification disorder of bone or a decrease in bone mineral density, increasing the risk of fracture. Alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. It has also been suggested that ALP may prevent metabolic endotoxemia by dephosphorylating lipopolysaccharide. We hypothesized that vitamin D restriction and/or a high-fat diet influences ALP activity in each tissue and serum lipopolysaccharide concentrations and increases the risk of metabolic endotoxemia. Eleven-week-old female rats were divided into 4 groups: basic control diet (Cont.), basic control diet with vitamin D restriction (DR), high-fat diet (HF), and high-fat diet with vitamin D restriction (DRHF) groups. They were acclimated for 28 days. The results of 2-way analysis of variance showed that intestinal ALP activity, which may contribute to an improvement in phosphate/lipid metabolism and longevity, in the high-fat diet groups (HF and DRHF) was higher than in the low-fat diet groups (Cont. and DR). ALP activity in the vitamin D-restricted groups (DR and DRHF) was lower than in the vitamin D-sufficient groups (Cont. and HF). Furthermore, serum endotoxin concentrations were significantly higher in the high-fat diet groups (HF and DRHF) than in the low-fat diet groups (Cont. and DR). In the vitamin D-restricted groups (DR and DRHF), serum endotoxin concentrations were also significantly higher than in the vitamin D-sufficient groups (Cont. and HF). These results suggest that vitamin D restriction and/or a high-fat diet increases the risk of metabolic endotoxemia.

Characterization and Structure of Alternatively Spliced Transcript Variant of Human Intestinal Alkaline Phosphatase (ALPI) Gene.

Intestinal-type alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be a gut mucosal defense factor. In humans, a single gene (ALPI) for IAP has been isolated, and its transcription produces two kinds of alternatively spliced mRNAs (aAug10 and bAug10). Recently, we discovered that vitamin D up-regulated the expression of both types of human IAP alternative splicing variants in Caco-2 cells. However, the functional difference of protein encoded by the mRNA variants has remained elusive. In the present study, we aimed to provide further insight into the characterization and structure of IAP isoforms. To analyze the protein translated from the ALPI gene, we constructed two kinds of cDNA expression plasmids (aAug10 and bAug10), and the transfected cells were homogenized and assayed for alkaline phosphatase (ALP) activity. We also designed the homology-modeled 3D structures of the protein encoded by the mRNA variants (ALPI-aAug10 and ALPI-bAug10). The levels of ALP activity of COS-1 cells transfected with the aAug10 plasmid were increased significantly, while cells transfected with the bAug10 plasmid had undetectable ALP activity. The homology-modeled 3D structures revealed that the variant bAug10 lacks the central N-terminal α-helix and residue corresponding to Asp-42 of ALPI-aAug10 near the active site. This is the first report on the characterization and structure of alternatively spliced transcript variants of the human ALPI gene. Further studies on the regulation of aAug10 and/or bAug10 mRNA expression may identify novel physiological functions of IAP.

Twenty-eight days of vitamin D restriction and/or a high-fat diet influenced bone mineral density and body composition in young adult female rats.

BACKGROUND: Vitamin D deficiency is associated with the risk of osteoporosis, and also influences skeletal muscle functions. Recently, we reported that a high-fat diet with vitamin D restriction decreased bone mineral density (BMD) in young adult male rats. Therefore, we hypothesized that vitamin D restriction and/or a high-fat diet would influence BMD in young adult female rats. METHODS: Sprague-Dawley strain female rats (11-week-old) were divided into four groups: a basic control diet (Cont.), a basic control diet with vitamin D restriction (DR), a high-fat diet (F), and a high-fat diet with vitamin D restriction (FDR). RESULTS: At 28 days after starting the experimental diets, the weights of the soleus muscle and gastrocnemius muscle mass were significantly lower in the high-fat diet groups compared with the normal-fat diet groups. The cortical BMD of the femur was significantly lower in the DR group compared with the Cont. group, while no significant differences in the cortical, cancellous, and total BMD of the femur in the FDR were observed compared with the F group. Myogenin is one of the muscle-specific transcription factors. The levels of mRNA expression of myogenin in the gastrocnemius muscle from the DR and F groups were reduced compared with the Cont. group. CONCLUSIONS: We revealed the influences of vitamin D restriction and/or a high-fat diet on the BMD and muscle in young adult female rats. Further studies on vitamin D deficiency in the regulation of muscle as well as bone metabolism would provide valuable data for the prevention of osteoporosis and sarcopenia.

A high-fat diet in the presence of vitamin D deficiency status is associated with a negative influence on calcaneal quantitative ultrasound parameters in young adults: a cross-sectional study.

Vitamin D deficiency and a high-fat diet are considered health problems worldwide. The aims of this study were to examine the prevalence of vitamin D deficiency/insufficiency in young adults, factors related to the vitamin D status, and the influence of vitamin D deficiency and/or a high-fat diet on bone parameters. Here, we investigated the hypothesis that a high-fat diet in the presence of a vitamin D-deficient status would have a more negative influence on bone parameters than a normal-fat diet with such a status. In the present study, we targeted young Japanese adults aged 21-23 (n = 175). We conducted a diet survey based on 3-day food records, biochemical examination of serum, and quantitative ultrasound measurements at the calcaneus. As a result, the rates of vitamin D deficiency {serum 25-hydroxyvitamin D3 [25(OH)D] concentration less than 20 ng/mL} and insufficiency [serum 25(OH)D concentration less than 30 ng/mL but not less than 20 ng/mL] were 60.6 and 30.9%, respectively. A positive correlation was observed between the serum 25(OH)D level and serum bone-specific alkaline phosphatase level, which is a serum marker of bone formation (r = 0.253, P< .01) or the speed of sound (SOS) as an index of bone density (r = 0.259, P< .01). A negative correlation was observed between the ratio of fat intake to total energy intake (%E) and serum 25(OH)D levels (r = -0.206, P< .01). Furthermore, we revealed that a high-fat diet in the presence of a vitamin D deficient status reduced the SOS parameter compared with a normal-fat diet with a vitamin D-deficient status (P< .05).

Influence of dietary vitamin D deficiency on bone strength, body composition, and muscle in ovariectomized rats fed a high-fat diet.

OBJECTIVE: Vitamin D deficiency is associated with a greater risk for osteoporosis and also influences skeletal muscle functions. The aim of this study was to investigate the influence of vitamin D restriction on ovariectomized (OVX) rats fed a high-fat diet. METHODS: Twenty-four 13-wk-old female rats were ovariectomized, and another 6 received a sham operation (Sham). The OVX rats were divided into four groups and fed experimental diets: a basic control diet (OVX-Cont), a basic control diet with vitamin D restriction (OVX-DR), a high-fat diet (OVX-F), and a high-fat diet with vitamin D restriction (OVX-FDR). RESULTS: At 28 d after starting the experimental diets, the fat mass was significantly increased in the OVX-F and OVX-FDR groups compared with OVX-Cont group, whereas the muscle mass was significantly decreased in the OVX-F and OVX-FDR groups compared with the OVX-Cont group. Compared with the OVX-Cont group, the bone mineral content of the femur was significantly lower in the OVX-DR and OVX-FDR groups, and the bone mineral density of the femur was significantly lower in the OVX-DR group. Myogenin is one of the muscle-specific transcription factors. The levels of mRNA expression of myogenin in the soleus and gastrocnemius muscles from the OVX-DR and OVX-FDR groups were reduced markedly compared with those from the OVX-Cont group. CONCLUSION: We provided evidence that a high-fat diet with vitamin D restriction influences bone and muscle metabolism using OVX rats. Further studies on vitamin D deficiency in the regulation of muscle as well as bone metabolism would provide valuable data for the prevention of osteoporosis and sarcopenia.

Vitamin D-restricted high-fat diet down-regulates expression of intestinal alkaline phosphatase isozymes in ovariectomized rats.

Intestinal alkaline phosphatase (IAP) is expressed at a high concentration in the brush border membrane of intestinal epithelial cells. Intestinal alkaline phosphatase controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. Previously, we reported that IAP activity in the duodenum was significantly decreased in male rats receiving a high-fat diet with vitamin D restriction. Here, we tested the hypothesis that IAP is also regulated by a vitamin D-restricted high-fat diet in an animal model of menopause. Twenty-four female rats were ovariectomized (OVX), and another 6 female rats were sham operated. The OVX rats were divided into 4 groups and fed experimental diets: a basic control diet, a basic control diet with vitamin D restriction, a high-fat diet, and a high-fat diet with vitamin D restriction. After 28days of the experimental diets, the vitamin D-restricted high-fat diet decreased alkaline phosphatase activity in the duodenum of the OVX groups. The vitamin D-restricted high-fat diet down-regulated mRNA expressions of IAP isozymes in the duodenum of the OVX groups. These findings support the hypothesis that the expression of IAP is suppressed by a vitamin D-restricted high-fat diet in OVX rats. An adequate vitamin D intake and prevention of low vitamin D levels may be important for IAP expression in gut homeostasis.

1-alpha,25-Dihydroxyvitamin D3 up-regulates the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells and may be an important regulator of their expression in gut homeostasis.

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation, and development. The principal function of vitamin D in calcium homeostasis is to increase the absorption of calcium from the intestine, and the level of alkaline phosphatase (ALP) activity, a differentiation marker for intestinal epithelial cells, is regulated by vitamin D. Intestinal-type ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells, and is known to be affected by several kinds of nutrients. Recent reviews have highlighted the importance of intestinal-type ALP in gut homeostasis. Intestinal-type ALP controls bacterial endotoxin-induced inflammation by dephosphorylating lipopolysaccharide and is a gut mucosal defense factor. In this study, we investigated the influence of vitamin D on the expression of 2 types of alternative mRNA variants encoding the human alkaline phosphatase, intestinal (ALPI) gene in human Caco-2 cells as an in vitro model of the small intestinal epithelium. After treatment with 1-alpha,25-dihydroxyvitamin D3, the biologically active form of vitamin D3, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments showed that the increased ALP had properties of intestinal-type ALP. Reverse transcription-polymerase chain reaction analysis revealed that expression of the 2 types of alternative mRNA variants from the ALPI gene was markedly enhanced by vitamin D in Caco-2 cells. In conclusion, these findings agree with the hypothesis: vitamin D up-regulated the expression of 2 types of human intestinal alkaline phosphatase alternative splicing variants in Caco-2 cells; vitamin D may be an important regulator of ALPI gene expression in gut homeostasis.

Menaquinone-4 (vitamin K2) up-regulates expression of human intestinal alkaline phosphatase in Caco-2 cells.

Alkaline phosphatase (ALP) hydrolyzes several monophosphate esters into inorganic acid and alcohol. In humans, 4 kinds of ALP isozymes have been identified: tissue-nonspecific ALP, intestinal ALP, placental ALP, and germ cell ALP. Intestinal ALP is expressed at a high concentration in the brush border membrane of intestinal epithelial cells and is known to be affected by several kinds of nutrients, such as lipids, but the physiological function of intestinal ALP has remained elusive. Vitamin K is an essential cofactor for the posttranslational carboxylation of glutamate residues into γ-carboxy glutamate. Menaquinone-4 (MK-4) with 4 isoprene units, vitamin K2, has been shown to induce bone-type ALP activity and osteoblastogenesis in human bone marrow cells. In this study, we investigated the effects of MK-4 on the level of ALP activity and expression of ALP messenger RNA in the human colon carcinoma cell line Caco-2, which is known to differentiate into small intestinal epithelial cells in vitro. After treatment with MK-4, there were significant increases in the ALP activities of Caco-2 cells. Inhibitor and thermal inactivation experiments demonstrated that the increased ALP had properties of intestinal-type ALP. Semiquantitative reverse transcription-polymerase chain reaction analysis revealed that expressions of human intestinal ALP and sucrase-isomaltase, which are intestinal differentiation markers, were highly enhanced in Caco-2 cells by MK-4. This is the first report concerning ALP messenger RNA expression induced by vitamin K2 in Caco-2 cells. Further studies on the physiological functions of human intestinal ALP will provide useful data on the novel effects of vitamin K.

Influences of dietary vitamin D restriction on bone strength, body composition and muscle in rats fed a high-fat diet: involvement of mRNA expression of MyoD in skeletal muscle.

Vitamin D insufficiency is associated with a greater risk of osteoporosis and also influences skeletal muscle functions, differentiation and development. The present study investigated the influences of vitamin D restriction on the body composition, bone and skeletal muscle in rats fed a high-fat diet. Sprague-Dawley strain male rats (11weeks old) were divided into four groups and fed experimental diets: a basic control diet (Cont.), a basic control diet with vitamin D restriction (DR), a high-fat diet (F) and a high-fat diet with vitamin D restriction (FDR). At 28days after starting the experimental diets, the visceral fat mass was significantly increased in the F group compared with Cont. group, and the muscle mass tended to decrease in the DR group compared with Cont. group. The total volume of the femur was significantly lower in the DR group compared with Cont. group, and the bone mineral density (BMD) of the femur was significantly lower in the FDR group compared with F group. MyoD is one of the muscle-specific transcription factors. The levels of mRNA expression of MyoD of the gastrocnemius and soleus muscles from the DR group were reduced markedly compared with those from the Cont. group. In conclusion, our findings revealed the influences of a vitamin D-restricted high-fat diet on the bone strength, body composition and muscle. Further studies on vitamin D insufficiency in the regulation of muscle as well as fat and bone metabolism would provide valuable data for the prevention of lifestyle-related disorders, including osteoporosis and sarcopenia.

Effects of Fok-I polymorphism in vitamin D receptor gene on serum 25-hydroxyvitamin D, bone-specific alkaline phosphatase and calcaneal quantitative ultrasound parameters in young adults.

Several genes have been implicated as genetic determinants of osteoporosis. Vitamin D receptor (VDR) is an intracellular hormone receptor that specifically binds to the biologically active form of vitamin D, 1-alpha, 25- dihydroxyvitamin D3 [1, 25(OH)2D], and mediates its effects. One of the most frequently studied single nucleotide polymorphisms is the restriction fragment length polymorphism (RFLP) Fok-I (rs2228570). The presence of a Fok-I site, designated f, allows protein translation to initiate from the first ATG. An allele lacking the site (ATG>ACG: designated F), initiates from a second ATG site. In the present study, we explored the effect of the VDR Fok-I genotype on associations among serum bone-specific alkaline phosphatase (ALP), 25- hydroxyvitamin D3 [25(OH)D], 1, 25(OH)2D, and the dietary nutrient intake in healthy young Japanese subjects (n=193). Dietary nutrient intakes were calculated based on 3-day food records before the day of blood examinations. Quantitative ultrasound (QUS) parameters at the right calcaneus (heel bone) were measured. The allele frequencies were 0.622 for the F allele and 0.378 for the f allele in all subjects. Grouped by the VDR genotype, a significant positive correlation between the levels of serum bone-specific ALP and 25(OH)D was observed in the FF-type (p=0.005), but not in the ff-type. In addition, there was a significant positive correlation between the level of serum 25(OH)D and osteo-sono assessment index (OSI) in the FF-type (p=0.008), but not in the ff-type. These results suggest that the level of circulating 25(OH)D is an important factor when assessing the VDR Fok-I polymorphism to prevent osteoporosis.

Peyer's patches and mesenteric lymph nodes cooperatively promote enteropathy in a mouse model of food allergy.

BACKGROUND AND OBJECTIVE: To improve the efficacy and safety of tolerance induction for food allergies, identifying the tissues responsible for inducing intestinal inflammation and subsequent oral tolerance is important. We used OVA23-3 mice, which express an ovalbumin-specific T-cell receptor, to elucidate the roles of local and systemic immune tissues in intestinal inflammation. METHODS AND RESULTS: OVA23-3 mice developed marked enteropathy after consuming a diet containing egg white (EW diet) for 10 days but overcame the enteropathy (despite continued moderate inflammation) after receiving EW diet for a total of 28 days. Injecting mice with anti-IL-4 antibody or cyclosporine A confirmed the involvement of Th2 cells in the development of the enteropathy. To assess the individual contributions of Peyer's patches (PPs), mesenteric lymph nodes (MLNs), and the spleen to the generation of effector CD4+ T-cells, we analyzed the IL-4 production, proliferation in response to ovalbumin, and CD4+ T-cell numbers of these tissues. EW feeding for 10 days induced significant IL-4 production in PPs, the infiltration of numerous CD4+ T-cells into MLNs, and a decrease in CD4+ T-cell numbers in spleen. On day 28, CD4+ T-cells from all tissues had attenuated responses to ovalbumin, suggesting tolerance acquisition, although MLN CD4+ T-cells still maintained IL-4 production with proliferation. In addition, removal of MLNs but not the spleen decreased the severity of enteropathy and PP-disrupted mice showed delayed onset of EW-induced inflammatory responses. Disruption of peripheral lymphoid tissues or of both PPs and MLNs almost completely prevented the enteropathy. CONCLUSIONS: PPs and MLNs coordinately promote enteropathy by generating effector T-cells during the initial and exacerbated phases, respectively; the spleen is dispensable for enteropathy and shows tolerogenic responses throughout EW-feeding. The regulation of PPs may suppress the initiation of intestinal inflammation, subsequently restricting MLNs and inhibiting the progression of food-allergic enteropathy.

Effects of gamma-glutamyl carboxylase gene polymorphism (R325Q) on the association between dietary vitamin K intake and gamma-carboxylation of osteocalcin in young adults.

INTRODUCTION: It has been demonstrated that single nucleotide polymorphism (SNP) (R325Q, 974G>A) in the gamma-glutamyl carboxylase (GGCX) gene is associated with the bone mineral density (BMD). In the present study, we investigated the effect of GGCX polymorphism (974G>A) on the correlations among the vitamin K in-take, level of serum vitamin K, and ratio of undercarboxylated osteocalcin (ucOC) to intact osteocalcin (OC) in healthy young Japanese subjects. METHODS: Healthy young adult subjects (n=189) were genotyped for the poly-morphism, and we measured the levels of serum vitamin K, intact OC, ucOC, and dietary nutrient intakes. RESULTS: Dietary vitamin K intake from vegetables was significantly correlated with the level of serum phylloquinone (PK), and vitamin K intake from fermented beans, natto, was also significantly correlated with the level of serum menaquinone-7 (MK-7). Moreover, the total dietary vitamin K intake showed a significant negative correlation with the ratio of ucOC to intact OC. Interestingly, on grouping by the GGCX genotype, there was a significant interaction between the ratio of ucOC to intact OC with vitamin K intake in homozygotes (GG-type) and heterozygotes (GA-type) (p<0.001). These results suggest that an adequate nutritional strategy is necessary for people with high-risk genotypes (GG- or GA-type). CONCLUSIONS: We demonstrated the effects of SNP (974G>A) in the GGCX gene on the correlation between dietary vitamin K intake and gamma-carboxylation of serum OC. Our data may be useful for planning strategies to prevent osteoporosis.

Associations between serum bone-specific alkaline phosphatase activity, biochemical parameters, and functional polymorphisms of the tissue-nonspecific alkaline phosphatase gene in a Japanese population.

INTRODUCTION: We had demonstrated that single nucleotide polymorphism (787T>C) in the tissue-nonspecific ALP (TNSALP) gene was associated with the bone mineral density (BMD). BMD was the lowest among TNSALP 787T homozygotes (TT-type) and highest among TNSALP 787T>C homozygotes (CC-type) in postmenopausal women. In the present study, we investigated the effects of the TNSALP genotype on associations among serum bonespecific alkaline phosphatase (BAP), serum calcium, and phosphorus in healthy young Japanese subjects. METHODS: Young healthy adult subjects (n=193) were genotyped for the polymorphism, and we measured the levels of serum BAP, serum calcium, and phosphorus. Dietary nutrient intakes were calculated based on 3-day food records before the day of blood examinations. RESULTS: Grouped by the TNSALP genotype, a significant negative correlation between serum BAP and phosphorus was observed in 787T>C homozygotes (CC-type), but not in heterozygotes (TCtype), nor in 787T homozygotes (TT-type). CONCLUSIONS: In the present study, we revealed that the single nucleotide polymorphism 787T>C in the TNSALP gene had effects on the correlation between serum BAP and phosphorus in young adult subjects. These results suggest that variation in TNSALP may be an important determinant of phosphate metabolism. Our data may be useful for planning strategies to prevent osteoporosis.

Retention of bone strength by feeding of milk and dairy products in ovariectomized rats: involvement of changes in serum levels of 1alpha, 25(OH)2D3 and FGF23.

The current study compared the effects of milk, yogurt or whey on the bone strength, body composition and serum biomarkers. Forty 12-week-old female Sprague-Dawley rats were ovariectomized (OVX), and another nine rats received a sham operation (Sham-Cont). After a 1-week recovery period, the OVX rats were divided into four dietary groups: OVX-control group (OVX-Cont), 17% skimmed milk powder diet group (OVX-Milk), 17% powdered fermented milk diet group (OVX-Yogurt) and 12% whey powder and 6% whey protein extract diet group (OVX-Whey) (n=10 in each group). The protein, nitrogen, fat, calcium and phosphorus contents of the experimental diets were adjusted to be similar to the control diet (AIN-93M). Eighty-four days after the beginning of the experimental diet, the total bone mineral density and bone mineral contents of lumbar vertebrae were significantly higher in the OVX-Milk and OVX-Whey groups than in the OVX-Cont group. Furthermore, the level of 1alpha, 25-dihydroxyvitamin D3 [1alpha, 25(OH)2D3] was significantly lower, while the serum level of FGF23 was significantly higher in the OVX-Milk, OVX-Yogurt and OVX-Whey groups than in the OVX-Cont group. These findings suggest that milk and the dairy products could improve bone metabolism in a postmenopausal animal model at least partly through changing the balance between 1alpha, 25(OH)2D3 and FGF23.

Spatiotemporal disorder in the axial skeleton development of the Mesp2-null mouse: a model of spondylocostal dysostosis and spondylothoracic dysostosis.

Spondylocostal dysostosis (SCDO) is a genetic disorder characterized by severe malformation of the axial skeleton. Mesp2 encodes a basic helix-loop-helix type transcription factor that is required for somite formation. Its human homologue, Mesp2, is a gene affected in patients with SCDO and a related vertebral disorder, spondylothoracic dysostosis (STDO). This work investigated how the loss of Mesp2 affects axial skeleton development and causes the clinical features of SCDO and STDO. We first confirmed, by three-dimensional computed tomography scanning, that Mesp2-null mice exhibited mineralized tissue patterning resembling the radiological features of SCDO and STDO. Histological observations and in situ hybridization probing for extracellular matrix molecules demonstrated that the developing vertebral bodies in Mesp2-null mice were extensively fused with rare insertions of intervertebral tissue. Unexpectedly, the intervertebral tissues were mostly fused longitudinally in the vertebral column, instead of exhibiting extended formation, as was expected based on the caudalized properties of Mesp2-null somite derivatives. Furthermore, the differentiation of vertebral body chondrocytes in Mesp2-null mice was spatially disordered and largely delayed, with an increased cell proliferation rate. The quantitative three-dimensional immunofluorescence image analyses of phospho-Smad2 and -Smad1/5/8 revealed that these chondrogenic phenotypes were associated with spatially disordered inputs of TGF-ß and BMP signaling in the Mesp2-null chondrocytes, and also demonstrated an amorphous arrangement of cells with distinct properties. Furthermore, a significant delay in ossification in Mesp2-null vertebrae was observed by peripheral quantitative computed tomography. The current observations of the spatiotemporal disorder of vertebral organogenesis in the Mesp2-null mice provide further insight into the pathogenesis of SCDO and STDO, and the physiological development of the axial skeleton.

A study of the association between serum bone-specific alkaline phosphatase and serum phosphorus concentration or dietary phosphorus intake.

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters into phosphoric acid and alcohol at a high optimum pH (pH 8-10). Human ALPs are classified into four types: tissue-non specific (TNSALP, liver/bone/kidney), intestinal, placental, and germ cell types. Based on studies of hypophosphatasia (HPP), which is a systemic bone disease caused by the presence of either one or two pathologic mutations in ALPL that encodes TNSALP, TNSALP was suggested to be indispensable for skeletal mineralization. In this study, we explored the possibility that dietary nutrients contribute to regulate serum bone-specific ALP (BAP) activity. Serum biochemical parameters, such as serum ALP, BAP, osteocalcin, and fibroblast growth factor 23 (FGF23), were measured in healthy young subjects (n=193). Dietary nutrient intakes were measured based on 3-d food records before the day of blood examinations. The presence of a carrier of the deletion of T at nucleotide 1559 (c.1559delT), which has been reported to be the most frequent in Japanese HPP, was not detected in any subject. By the analysis of BAP activity and other biochemical parameters or dietary nutrient intakes, we obtained significant correlations between BAP activity and serum phosphorus (r=-0.165, p=0.022), calcium intake (mg/1,000 kcal/d) (r=-0.186, p=0.010), or phosphorus intake (mg/1,000 kcal/d) (r=-0.226, p=0.002). Further study on the regulation of BAP activity and calcium and/or phosphorus homeostasis will provide useful data for improving skeletal health.

Vitamin K1 (phylloquinone) or vitamin K2 (menaquinone-4) induces intestinal alkaline phosphatase gene expression.

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters into inorganic acid and alcohol at a high optimum pH (pH 8-10). Previously, we identified a significant increase of intestinal ALP (IAP) activity in the rat intestine on long-term dietary vitamin K supplementation. However, it was unclear whether the induction of ALP gene expression was caused by vitamin K intake. In the present study, we examined the effects of vitamin K on IAP gene expression. A total of 21 male ICR strain mice (7 wk old) were divided into three groups: control, PK, and MK groups. Mice were orally administered a 0.1-mL solution of physiological saline in the control group, phylloquinone (3 mg/kg mouse) in the PK group, and menaquinone-4 (3 mg/kg mouse) in the MK group. Four hours after administration, we determined the ALP activity of the intestinal mucosa in three areas (duodenum, jejunum, and ileum). In the MK groups, the levels of ALP activity in the jejunum increased significantly compared with the control. Moreover, reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers revealed that IAP mRNA expression was significantly enhanced in the jejunum in both PK and MK groups. Interestingly, vitamin K administration also increased the expression of pregnane X receptor mRNA. This is the first report concerning IAP mRNA expression induced by oral administration of vitamin K. The results support the possible involvement of vitamin K in the regulation of IAP mRNA expression as a novel pharmacological effect of vitamin K.

Effects of long-term vitamin K(1) (phylloquinone) or vitamin K(2) (menaquinone-4) supplementation on body composition and serum parameters in rats.

Vitamin K is a cofactor for γ-glutamyl carboxylase, which is an essential enzyme for the γ-carboxylation of vitamin K-dependent proteins such as osteocalcin and matrix Gla protein. Although it has been suggested that vitamin K plays an important role in the improvement of bone metabolism, the relationship between dietary vitamin K intake and bone metabolism has not been thoroughly investigated. Moreover, vitamin K is thought to have other actions beyond influencing the γ-carboxylation status. In the present study, we examined the effects of the long-term addition of phylloquinone (PK) or menaquinone-4 (MK-4) to a control diet on bone mineral density, bone strength, body composition, and serum parameters in rats. A total of 23 female Sprague-Dawley strain rats (6 weeks old) were divided into three groups: basic control diet group, PK diet (PK: 600mg/kg diet) group, and MK diet (MK-4: 600mg/kg diet) group. Three months after starting the experimental diet, the addition of PK to the basic control diet significantly increased the bone mineral density (BMD) of the femur (p<0.05). In the MK group, there was no significant difference in the BMD of the femur. However, two types of bone strength parameter: the minimum cross-sectional moment of inertia and the polar moment of inertia, were significantly higher in the MK group than in the control (p<0.05, respectively). Furthermore, the femoral bone parameters (the width, dry weight and ash weight, and cortical, cancellous, trabecular, and total bone mineral contents) in the MK group were increased significantly compared with the control. Interestingly, the addition of PK or MK-4 significantly decreased the total fat accumulation (p<0.01 and p<0.05, respectively), and serum triglycerides were reduced by 48% in the PK group and 29% in the MK group compared with the control. There were no significant differences in the levels of serum calcium, phosphorus, alkaline phosphatase, growth hormone, insulin-like growth hormone-1, insulin-like growth hormone binding protein-3, and cross-linked N-teleopeptide of type I collagen among the three groups. This is the first study to demonstrate the effect of the long-term addition of PK or MK-4 to the control diet on body composition and serum parameters in an in vivo system using rats. Further studies on the mechanism of vitamin K supplementation in the regulation of bone metabolism would provide valuable data on the prevention of lifestyle-related disorders, including osteoporosis.

[Development of oral function and eating habits of infants living in a city area of Japan: in relation to the results of dental health examinations of infants aged 14 months at public health centers].

OBJECTIVE: The aim of this study was to investigate the relationships between eruption of deciduous teeth and eating habits determined by health examinations of infants. METHODS: We verified eruption of deciduous teeth based on observations of 455 fourteen-month-old infants at health examinations in a ward of Tokyo, and performed a questionnaire survey involving their mothers regarding the hardness of infants' meals and their eating habits. We examined 420 infants excluding 17 whose births were 'pre-term delivery (born at or before 36 weeks)' and 18 whose questionnaire had excessive omissions. RESULTS: The percentage of infants who began a weaning diet at 5 to 6 months of age was 81.4%, and 71.2% of mothers considered their infant's age in months before starting a weaning diet. We divided the children into three stages: those not showing full eruption of the eight front deciduous teeth (stage I, 27.4%); those with full eruptions of the eight front deciduous teeth excluding the first primary molars (stage II, 61.9%); those with full eruptions of the first primary molars (stage III, 10.7%). Most mothers cooked meals considering the hardness of the gingival gums (stage I; 53.5%, stage II; 54.4%, stage III; 40.0%). The percentage of mothers who cooked meals considering the hardness of the primary molars was 14.0 and 15.1% in stages I and II, respectively. In addition, the percentage who cooked meals while considering the hardness in relation to adult meals was 7.0, 9.7, and 24.4% in stages I, II, and III, respectively. Moreover, the percentage considering the salt-taste in relation to adult meals was 13.2, 17.3, and 22.2% in stages I, II, and III, respectively. CONCLUSION: In the present study, we obtained valuable data showing that the timing deciduous teeth eruption varies among individuals. These results suggested that nutritional education on the appropriate quality of meals for infants based on their state of deciduous teeth eruption is necessary.