Ikuko Ezawa

The effect of ovariectomy on bone metabolism in rats.

It is well known that the ovariectomized rat is a good model for osteoporosis. Recently, it has been possible to measure the bone mineral density (BMD) of small animals accurately using dual energy X-ray absorptiometry (DXA). Therefore, in this study the change in the BMD in the different bone structures was examined in in vivo using aged and growing rats. The BMD of the lumbar spine and the tibial proximal metaphysis, which are mainly trabecular bone, in ovariectomized aged and growing rats with ovariectomized was significantly lower than those of the control (sham) group. However, the BMD of the tibial diaphysis, which is mainly cortical bone, showed no change nor had a tendency to decrease in the early stages of the experiment. These results suggest that it is important to take into account the age and the bone site in order to evaluate the experiment, which uses the rat model with ovariectomized osteoporosis.

The Advantage of Alfacalcidol Over Vitamin D in the Treatment of Osteoporosis

Abstract. Although alfacalcidol is widely used in the treatment of osteoporosis, its mechanism of action in bone is not fully understood. Alfacalcidol stimulates intestinal calcium (Ca) absorption, increases urinary Ca excretion and serum Ca levels, and suppresses parathyroid hormone (PTH) secretion. It remains to be clarified, especially under vitamin D-replete conditions, whether alfacalcidol exerts skeletal effects solely via these Ca-related effects, whether the resultant suppression of PTH is a prerequisite for the skeletal actions of alfacalcidol, and, by inference, whether alfacalcidol has an advantage over vitamin D in the treatment of osteoporosis. To address these issues, we (1) compared the effects of alfacalcidol p.o. (0.025–0.1 μg/kg BW) vis-à-vis vitamin D3 (50–400 μg/kg BW) on bone loss in 8-month-old, ovariectomized (OVX) rats as a function of their Ca-related effects, and (2) examined whether the skeletal effects of alfacalcidol occur independently of suppression of PTH, using parathyroidectomized (PTX) rats continuously infused with hPTH(1–34). The results indicate that (1) in OVX rats, alfacalcidol increases BMD and bone strength more effectively than vitamin D3 at given urinary and serum Ca levels: larger doses of vitamin D3 are required to produce a similar BMD-increasing effect, in the face of hypercalcemia and compromised bone quality; (2) at doses that maintain serum Ca below 10 mg/dl, alfacalcidol suppresses urinary deoxypyridinoline excretion more effectively than vitamin D3; and (3) alfacalcidol is capable of increasing bone mass in PTX rats with continuous infusion of PTH, and therefore acts independently of PTH levels. It is suggested that alfacalcidol exerts bone-protective effects independently of its Ca-related effects, and is in this respect superior to vitamin D3, and that the skeletal actions of alfacalcidol take place, at least in part, independently of suppression of PTH. Together, these results provide a rationale for the clinical utility of alfacalcidol and its advantage over vitamin D3 in the treatment of osteoporosis.

Maintenance of trabecular structure and bone volume by vitamin K(2) in mature rats with long-term tail suspension.

Abstract. Bone volume loss is one of the major health problems during long-term spaceflight. We examined the effects of vitamin K2 on bone abnormalities in tail-suspended mature male Sprague-Dawley rats (13 weeks old). In this model, increased bone resorption and sustained suppression of bone formation resulted in progressive bone loss in 4 weeks, which simulates bone changes in humans during spaceflight. A significant decrease in bone mineral density (BMD), as well as a decreased mineral apposition rate (MAR), increased number of osteoclasts per bone perimeter (N.Oc/B.Pm), and increased osteoclast surface per bone surface (Oc.S/BS) in the suspended group was effectively prevented by vitamin K2, given orally (menatetrenone, 22 mg/kg body weight). Microfocus computed tomography (CT) and node-strut analyses revealed that the volume and structure of trabecular bone were maintained near normal by the vitamin K2 treatment. A recent report has suggested the abnormal metabolism or action of vitamin K in a microgravity environment, and our data therefore suggest that vitamin K2 may be useful for the prevention of bone loss and for the maintenance of normal trabecular structure during spaceflight.

The effect of voluntary exercise on bone mineral density and skeletal muscles in the rat model at ovariectomized and sham stages.

The changes of bone mineral density (BMD) and skeletal muscles were evaluated in the rat model at either sham or ovariectomized stages to attempt to make clear the effect of voluntary running exercise on bone metabolism. In comparison with the control groups within the ovariectomized (OVX) and the sham groups, in the running groups, (1) the urinary phosphorus (P) and creatinine (Cr) excretions showed an increase concurrently with the increase in the running distance; (2) the weight of the quadriceps femoris was significantly higher; and (3) the BMD of appendicular and axial bones was significantly greater. These results suggest that voluntary running exercise could effect the BMD, the weight of the skeletal muscles, and the acceleration of energy metabolism.

Adverse effects of an active fragment of parathyroid hormone on rat hippocampal organotypic cultures

Adverse effects of an active fragment of parathyroid hormone (PTH1–34), a blood Ca2+ level‐regulating hormone, were examined using rat hippocampal slices in organotypic culture. Exposure of cultured slice preparations to 0.1 μM PTH1–34 for 60 min resulted in a gradual increase in the intracellular Ca2+ concentration ([Ca2+]i); this effect was most obvious in the apical dendritic region of CA1 subfield. When PTH1–34 at a lower concentration (1 nM) was added to the culture medium and its toxic effects examined using a propidium iodide intercalation method, significant toxicity was seen 3 days after exposure and increased with time. Cells in the CA1 region seemed more vulnerable to the hormone than cells in other regions. At 1 week of exposure, the toxic effects were dose‐dependent over the range of 0.1 pM to 0.1 μM, the minimum effective dose being 10 pM. The adverse effects were not induced either by the inactive fragment, PTH39–84, or by an active fragment of PTH‐related peptide (PTHrP1–34), an intrinsic ligand of the brain PTH receptor. The PTH1–34‐induced adverse effects were significantly inhibited by co‐administration of 10 μM nifedipine, an L‐type Ca2+ channel blocker, but not by co‐administration of blockers of the other types of Ca2+ channel. The present study demonstrates that sustained high levels of PTH in the brain might cause degeneration of specific brain regions due to Ca2+ overloading via activation of dihydropyridine‐sensitive Ca2+ channels, and suggests that PTH may be a risk factor for senile dementia.

Estrogen deficiency and low-calcium diet increased bone loss and urinary calcium excretion but did not alter arterial stiffness in young female rats

Many epidemiological studies have reported that the severity of arterial diseases such as arterial calcification and stiffness is inversely related to bone loss, i.e., osteoporosis. However, the nature of this relationship is unclear. The purpose of the present study was to examine the influences of estrogen deficiency and/or low-calcium diet (0.1% Ca) on bone metabolism and calcium balance, as well as aortic wall composition and stiffness in young female rats. Twenty-eight 6-week-old female rats were randomized into four groups: OVX-Low calcium (OL) and OVX-Normal calcium groups (ON) were ovariectomized, and Sham-Low calcium (SL) and Sham-Normal calcium groups (SN) were sham-operated. After 12 weeks, the bone mineral density of the lumbar spine and tibial proximal metaphysis were significantly lower in ON than in SN, and also significantly lower in OL than in ON. Additionally, OL rats had significant higher (vs. SN and SL) urinary deoxypyridinoline, but not urinary calcium, excretion at 4 weeks after ovariectomy. However, at 12 weeks after ovariectomy, urinary calcium excretion was significantly higher in OL than in SL, with corresponding increases in two bone turnover markers, bone-type alkaline phosphatase and tartrate-resistant acid phosphatase. Neither estrogen deficiency nor low-calcium diet affected aortic stiffness or elastin degeneration and calcium deposition over the course of the present study, although changes of bone metabolism occurred rapidly. Taken together, these results show that bone loss and arterial stiffness did not progress simultaneously in the present experimental protocol.

The Effect of Different Amounts of Calcium Intake on Bone Metabolism and Arterial Calcification in Ovariectomized Rats

Reduced estrogen secretion and low calcium (Ca) intake are risk factors for bone loss and arterial calcification in female rodents. To evaluate the effects of Ca intake at different amounts on bone mass changes and arterial calcification, 8-wk-old female Wistar rats were randomly placed in ovariectomized (OVX) control and OVX with vitamin D3 plus nicotine (VDN) treatment groups. The OVX with VDN rats were then divided into six groups to receive different amounts of Ca in their diets: 0.01%, 0.1%, 0.3%, 0.6%, 1.2%, or 2.4% Ca. After 8 wk of administration, low Ca intake groups with 0.01% and 0.1% Ca diets had significantly reduced bone mineral density (BMD) and bone mechanical properties as compared with those of the other groups, whereas high Ca intake groups with 1.2% and 2.4% Ca diets showed no differences as compared with the 0.6% Ca intake group. For both the 0.01% and 2.4% Ca intake groups, Ca levels in their thoracic arteries were significantly higher as compared with those of the 0.6% Ca diet group, and that was highly correlated with serum PTH levels. An increase in relative BMP-2 mRNA expression in the arterial tissues of the 0.01% and 2.4% Ca diet groups was also observed. These results suggested that extremely low Ca intake during periods of estrogen deficiency may be a possible risk for the complications of reduced BMD and arterial calcification and that extremely high Ca intake may promote arterial calcification with no changes in BMD.

Combination use of vitamin K(2) further increases bone volume and ameliorates extremely low turnover bone induced by bisphosphonate therapy in tail-suspension rats.

Abstract. Bisphosphonate is a potent inhibitor of bone resorption, which results in the increase of bone volume. However, bisphosphonate treatment may lead to extremely low bone turnover and abnormal bone microstructure. In this study, we examined whether the combination of bisphosphonate with vitamin K2 treatment may have beneficial effects on bone turnover and trabecular microstructure as well as on bone volume loss by using tail-suspension model rats. In these model rats, bone mineral density (BMD) decreased with histological evidence of enhanced bone resorption and suppressed bone formation. By bisphosphonate treatment, BMD was increased compared with that of tail-suspended rats. Osteoclast surface per bone surface (Oc.S/BS) and number of osteoclasts per bone perimeter (N.Oc/B.Pm) were reduced and mineral apposition rate (MAR) decreased, suggesting extreme suppression of bone turnover. However, trabecular structure examined by microfocus CT was apparently abnormal. By contrast, combination of bisphosphonate with vitamin K2 leads to further increase of bone volume. MAR and BFR as well as Oc.S/BS and N.Oc/B.Pm were increased compared with those of the bisphosphonate-treated group. However, abnormal structure of trabeculae in secondary spongiosa was not completely ameliorated. These data suggested that concomitant use of vitamin K2 with bisphosphonate excessively ameliorates too much suppression of bone turnover while more efficiently preventing bone volume loss.

Phosphate depletion enhances bone morphogenetic protein-4 gene expression in a cultured mouse marrow stromal cell line ST2.

Alkaline phosphatases (ALPs) are glycosylated, membrane-bound enzymes that hydrolyze various monophosphate esters at an optimum high pH and are present in nearly all living organisms. In Escherichia coli, extracellular phosphate (Pi) limitation induces the ALP gene, indicating a role of extracellular Pi in ALP gene regulation. However, little is known about similar mechanisms in mammalian cells. Previously, we reported that Pi starvation increased the tissue-nonspecific ALP (TNSALP) activity and regulated its expression in the mouse stromal cell line ST2, derived from mouse bone marrow. In the present study, we further examined the effects of Pi starvation on the mechanism of TNSALP induction. The specific activity of TNSALP increased markedly after treatment by Pi starvation for 5 days and RT-PCR analysis revealed that the mRNA of the bone morphogenetic protein-4 (BMP-4) gene was highly stimulated. The combination of Pi depletion and mouse BMP-4 receptor IA/Fc chimera down-regulated the TNSALP activity. These results indicated that Pi depletion stimulates the TNSALP activity for the Pi supplementation, and that this system may involve the signaling pathway of the BMP-4 gene at the transcription level.

Effect of 1α-hydroxyvitamin D3 and egg-shell calcium on bone metabolism in ovariectomized osteoporotic model rats

Abstract Egg-shell calcium (Ca) is one of the effective Ca sources for bone metabolism. In the present study, we investigated whether egg-shell Ca had similar effects compared with calcium carbonate (CaCO3) when vitamin D3 (1α(OH)D3) treatment was given to an osteopo-rotic rat model. In both 1α(OH)D3-supplemented and -unsupplemented rats, the bone mineral density (BMD) of the lumber spine in the vitamin-supplemented group increased significantly compared with the unsupplemented group. In a Ca balance study, there were also significant differences in intestinal Ca absorption, urinary Ca and fecal Ca between the vitamin-supplemented and -unsupplemented groups. These results show that egg-shell Ca could have similar effects to CaCO3 on bone metabolism. In contrast with CaCO3, vitamin D3 supplementation did not significantly increase serum Ca levels in the egg-shell Ca group; however, the mechanism of Ca absorption is still unclear. Our results suggest that egg-shell Ca may be an effective nutrient in Ca metabolism for people treated with vitamin D3.