Bone mass protective potential mediated by bovine milk basic protein requires normal calcium homeostasis in mice.

OBJECTIVES: Milk provide protective effects against bone loss caused by an impaired calcium balance. Although the effects of some elements have previously been confirmed, the involvement of milk basic protein (MBP) in bone mineral metabolism remains poorly characterized. Moreover, the importance of mineral nutrition sufficiency to establish the effect of MBP must be evaluated. METHODS: First, to evaluate the physiological conditions required for MBP activity, we examined the bone and mineral phenotypes of mice that suffer from insufficient calcium absorption due to a lack of intestinal vitamin D signaling. Second, to determine whether vitamin D signaling affects the effect of MBP on bone resorption, in vitro osteoclastogenesis were assessed using bone marrow cells. RESULTS: In mice with systemic vitamin D receptor (Vdr) inactivation, dietary MBP supplementation was unable to normalize hypercalcemia and hyperparathyroidism and failed to rescue bone mineralization impairments. In contrast, calcium and bone homeostasis responded to MBP supplementation when Vdr inactivation was restricted to the intestines. Hyperparathyroidism in intestine-specific Vdr knockout mice was also improved by MBP supplementation, along with a decrease in bone resorption in response to the level of serum tartrate-resistant acid phosphatase 5b. These results corresponded with a reduction in tartrate-resistant acid phosphatase-stained osteoclast numbers and the eroded surface on the tibia. MBP treatment dose-dependently suppressed osteoclastogenesis in cultured bone marrow macrophages regardless of vitamin D activity. These effects of MBP were blunted when parathyroid hormone was added to the culture medium, which is in line with the in vivo phenotype observed with systemic Vdr inactivation and suggests that severe hyperparathyroidism limits MBP activity in the bone. CONCLUSIONS: Therefore, adaptive calcium homeostasis is an essential requirement when MBP exerts protective effects through the inhibition of bone resorption.

[Bone and Nutrition. Vitamin D independent calcium absorption].

Vitamin D endocrine system is required for normal calcium and bone homeostasis. Trans-epithelial calcium absorption is initiated with calcium entry into the intestinal epithelial cells from luminal fluid through calcium permeable channels, and those expressions are strongly supported by vitamin D action. On the other hands, dietary treatment, mineral supplementation or restriction, successfully improves intestinal calcium absorption in global vitamin D receptor knock-out (VDR KO) mice, though vitamin D dependent active transport pathway is lacking. Dietary rescue of intestinal calcium absorption provided a positive calcium balance in this mouse model, and suggested that the major role of vitamin D function on calcium homeostasis was considered to be intestinal active absorption. To elucidate the entire process of intestinal calcium absorption, vitamin D independent calcium transport system was characterized into either trans-cellular or para-cellular process.

Role of local vitamin D signaling and cellular calcium transport system in bone homeostasis.

Mouse genetic studies have demonstrated that the 1,25-dihydroxyvitamin D [1,25(OH)2D] endocrine system is required for calcium (Ca(2+)) and bone homeostasis. These studies reported severe hypocalcemia and impaired bone mineralization associated with rickets in mutant mice. Specific phenotypes of these mice with an engineered deletion of 1,25(OH)2D cell signaling resemble the features observed in humans with the same congenital disease or severe 1,25(OH)2D deficiency. Decreased active intestinal Ca(2+) absorption because of reduced expression of epithelial Ca(2+) channels is a crucial mechanism that contributes to the major phenotypes observed in the mutant mice. The importance of intestinal Ca(2+) absorption supported by 1,25(OH)2D-mediated transport was further emphasized by the observation that Ca(2+) supplementation rescues hypocalcemia and restores bone mineralization in both patients and mice lacking 1,25(OH)2D signaling. This observation questions the direct role of 1,25(OH)2D signaling in bone tissue. Studies regarding tissue-specific manipulation of 1,25(OH)2D function have provided a consensus on this issue by demonstrating a direct action of 1,25(OH)2D on cells in bone tissue through bone metabolism and mineral homeostasis. In addition, movement of Ca(2+) from the bone as a result of osteoclastic bone resorption also provides a large Ca(2+) supply in Ca(2+) homeostasis; however, the system controlling Ca(2+) homeostasis in osteoclasts has not been fully identified. Transient receptor potential vanilloid (TRPV) 4 mediates Ca(2+) influx during the late stage of osteoclast differentiation, thereby regulating the Ca(2+) signaling essential for cellular events during osteoclast differentiation; however, the system-modifying effect of TRPV4 activity should be determined. Furthermore, it remains unknown how local Ca(2+) metabolism participates in systemic Ca(2+) homeostasis through bone remodeling. New insights are therefore required to understand this issue.

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.

Effect of dietary calcium: Phosphorus ratio on bone mineralization and intestinal calcium absorption in ovariectomized rats.

We investigated the effect of dietary calcium:phosphorus (Ca:P) ratio on bone mineralization and intestinal Ca absorption in ovariectomized (OVX) rat models of osteoporosis and sham-operated rats. Thirty 12-wk-old female Wistar rats were divided into three groups of OVX rats and three groups of sham rats. Thirty days after the adaptation period, OVX rats and sham rats were fed a diet formulated Ca:P, 1:0.5, 1:1 or 1:2 (each diet containing 0.5% Ca), respectively for 42 d. In both sham and OVX rats, serum osteocalcin, a marker of bone turnover, was increased by decreasing Ca:P ratio (1:2). In contrast, rats fed the Ca:P = 1:0.5 diet (dietary P restriction) suppressed the increased serum parathyroid hormone, osteocalcin and urinary deoxypyridinoline, and increased Ca absorption in both sham and OVX rats compared to the Ca:P = 1:1 and 1:2 diets. Especially, in OVX rats, the decreased bone mineral density of the fifth lumbar was also suppressed when rats were fed the Ca:P = 1:0.5 diet. These results indicated that the elevation of dietary Ca:P ratio may inhibit bone loss and increase intestinal Ca absorption in OVX rats.

Dose-responsive alteration in hepatic lipid peroxidation and retinol metabolism with increasing dietary beta-carotene in iron deficient rats.

Phosphatidylcholine hydroperoxide (PCOOH) levels are increased in the iron-deficient rat liver. We investigated the antioxidative effect of dietary beta-carotene and altered retinol metabolism in iron-deficient rats. Experiment 1: Male Wistar-strain rats were divided into six groups and fed a control diet, an iron-deficient diet, and iron-deficient diets with four different levels of dietary beta-carotene. The PCOOH concentration in the iron-deficient rat liver was decreased by supplementation with dietary beta-carotene. However, the beta-carotene dose response was not related to antioxidative potency. Hepatic and plasma beta-carotene concentrations were increased by iron deficiency. The hepatic retinol concentration was increased while the plasma retinol concentration was decreased in iron-deficient rats. Experiment 2: Male Wistar-strain rats were divided into two groups, with one group receiving a control diet with beta-carotene and the other an iron-deficient diet with beta-carotene. Intestinal iron was decreased and intestinal beta-carotene was unchanged in iron-deficient rats. The intestinal beta-carotene conversion ratio and beta-carotene cleavage enzyme activity were decreased in iron-deficient rats. Dietary beta-carotene played the role of an antioxidant in hepatic lipid peroxidation in the iron-deficient state, but there was no dose dependency. Moreover, intestinal beta-carotene cleavage and hepatic retinol release appear to be altered in iron-deficient rats.