Dehydroepiandrosterone administration increased trabecular mass and dihydrotestosterone levels in the cancellous region of the tibia in young female rats.

The aim of the present study was to determine whether dehydroepiandrosterone (DHEA) administration affects bone mass and local sex hormone levels in the cancellous region of young female rats. Eleven female rats (6 weeks old) were randomly divided into 2 groups: control rats (CON, n=5) and rats treated with DHEA (DHEA, n=6). DHEA dissolved in sesame oil was administered to the DHEA group intraperitoneally at 20 mg DHEA/kg body weight, and the CON group was treated with vehicle only (sesame oil, 0.5 ml). The rats were treated with DHEA or vehicle for 3 consecutive days, followed by 1 day of no treatment. The experimental period was 8 weeks. According to dual-energy X-ray absorptiometry and high-resolution microcomputed tomography data, the DHEA group exhibited increased trabecular bone mineral density (BMD), bone volume, and tibial thickness compared to the findings in the CON group, whereas no effect was observed on cortical BMD or morphometry. The concentrations of free testosterone and estradiol in the cancellous region of the tibia did not differ between the 2 groups, but the DHT concentration was significantly higher in the DHEA group than in the CON group. These findings suggest that an increase in local DHT levels may stimulate an increase in trabecular bone mass during growth phases in female rats.

Voluntary running exercise attenuates the progression of endothelial dysfunction and arterial calcification in ovariectomized rats.

AIM: Loss of oestrogen synthesis capacity after menopause contributes to increases in arterial stiffness and calcification. Exercise training improves arterial stiffness and calcification. However, the mechanism of exercise training-induced improvement of arterial stiffness and calcification remains unclear. METHOD: We examined the mechanism by using aortas of sham-operated rats (sham control; SC), ovariectomized rats (OVX control; OC), OVX plus treatment with vitamin D(3) plus nicotine (VDN) rats (OV sedentary; OVSe), which is an animal model of endothelial dysfunction and arterial calcification, and voluntary running wheel exercise for 8 weeks plus OVX plus VDN rats (OV exercise; OVEx). RESULTS: The arterial tissue calcium and endothelin-1 (ET-1: a vasoconstrictor peptide and a potent regulator of arterial calcification) levels were significantly higher in OVSe rats compared with the SC and OC rats, whereas these levels in the OVEx rats were significantly lower than in the OVSe rats. Additionally, arterial expression of endothelial nitric oxide synthase (eNOS), which is an enzyme that produces nitric oxide (NO: a vasodilator substance), was reduced in OVSe rats. However, exercise training prevented the decrease in eNOS expression. Moreover, there was a significant positive correlation between arterial calcium level and arterial ET-1 level. CONCLUSION: These findings suggest that exercise training-induced improvement of ET-1 and NO prevents the impairment of endothelial function after menopause in females, and this improvement may result in less arterial calcification.

Change in calcium balance and bone mineral density during pregnancy in female rats.

This study investigated changes in Ca balance and BMD during pregnancy in female rats. During pregnancy, the intestinal Ca absorption increased significantly, and Ca accumulation was also markedly elevated. However, BMD values for the lumbar spine decreased significantly during pregnancy. Twenty female SD rats, 10 weeks of age (Japan SLC Co., Shizuoka, Japan) were acclimated for 2 weeks. Then, the rats were divided into two groups; the control (no pregnancy) group (n=10) and the pregnant group (n=10). The rats in the pregnant group were kept in a cage with a male rat for 5 days at 12 weeks of age, and all 10 were successfully impregnated. During the pregnancy period, the values for intestinal Ca absorption and the rate of the intestinal Ca absorption in the pregnant group were significantly greater than those of the control group. In addition, in spite of the significant increase in urinary Ca excretion in the pregnant group, the Ca accumulation was markedly elevated during the latter half of pregnancy. On the other hand, the BMD value in the lumbar spine for the pregnant group significantly decreased during pregnancy. These findings suggest that pregnancy accelerated intestinal Ca absorption and Ca accumulation in female rats, while the lumbar spine BMD decreased during pregnancy.

Phosphatidylinositol is essential determinant for K+ permeability involved in gastric proton pumping.

Gastric vesicles purified from acid-secreting rabbit stomach display K(+) permeability manifested by the valinomycin-independent proton pumping of H(+)-K(+)-ATPase as monitored by acridine orange quenching. This apparent K(+) permeability is attenuated by the treatment of the membrane with 5 mM Mg(2+), and this phenomenon has been attributed to membrane-bound phosphoprotein phosphatase. However, with the exception of the nonspecific inhibitor pyrophosphate, protein phosphatase inhibitors failed to inhibit the loss of K(+) permeability. Preincubation of the membrane with neomycin, a phospholipase C inhibitor, surrogated the effect of Mg(2+), whereas another inhibitor, U-73122, did not. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) restored the attenuated K(+) permeability by treatment with either Mg(2+) or neomycin. Furthermore, either phosphatidylinositol bound to phosphatidylinositol transfer protein or phosphatidylinositol 4,5,6-trisphosphate (PIP(3)) surrogated the effect of PIP(2). Mg(2+) and neomycin reduced K(+) permeability in the membrane as determined by Rb(+) influx and K(+)-dependent H(+) diffusion. Treatment with Mg(2+) reduced the contents of PIP(2) and PIP(3) in the membrane. These results suggest that PIP(2) and/or PIP(3) maintain K(+) permeability, which is essential for proton pumping in the apical membrane of the secreting parietal cell.

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

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 (1alpha(OH)D3) treatment was given to an osteoporotic rat model. In both 1alpha(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.

Effects of ouabain on the growth and DNA synthesis of PC12 cells.

We investigated the effects of ouabain and serum from salt-loaded Dahl salt-sensitive (S) rats, which contain abundant ouabain-like compounds, on the growth and DNA synthesis of rat pheochromocytoma PC12 cells. Ouabain decreased the growth of PC12 cells, as evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, in a concentration-dependent fashion. A moderate concentration (10(-7) M) of ouabain increased DNA synthesis, as measured by 5-bromo-2'-deoxyuridine incorporation, and induced transcription of the proto-oncogenes c-myc and c-fos. Serum from salt-loaded Dahl S rats also enhanced DNA synthesis, but serum from Dahl salt-resistant rats did not. Thus ouabain-like compounds may modify the growth or differentiation of neural tissues. This effect may contribute to the development of salt-induced hypertension in Dahl S rats.

[Phosphorus intake and osteoporosis].

Phosphorus (P) is one of the most important nutrients for bone metabolism, such as calcium. In general, P intake is usually adequate in our daily diet, and there is a risk of over-consumption from processed food. On the other hand, Ca intake is not always adequate from the Japanese daily diet. When Ca/P is taken from the daily diet at a level of 0.5 - 2.0, the P intake level dose not affect intestinal Ca absorption. Therefore, it is important not only to pay attention to preventing the over-consumption of P, but also to obtain a sufficient intake of Ca. For the prevention of osteoporosis, it is important to consume sufficient Ca and to maintain and appropriate Ca/P balance from diet.

Modulation of bone mass and turnover in growing rats by voluntary weight-bearing exercise and glucose supplementation.

Female Sprague-Dawley rats, 9 weeks of age, were assigned to four groups: Group 0 (n = 8) was dissected for base-line control, and the other three groups were fed for 3 mo: Group 1 (n = 9), sedentary controls; Group 2 (n = 6), running rats housed in a cage with a treadmill and pair-fed with Group 1; and Group 3 (n = 7), running rats, pair-fed and allowed free access to additional glucose. The distances of voluntary running did not significantly differ between Groups 2 and 3. Menstrual cycles in these rats were apparently maintained as observed from daily running distances. The amount of glucose taken by rats in Group 3 was 3.5 +/- 0.4 (mean and SE) g/d. Body weight (BW) at the end of the experiment for Groups 1, 2, and 3 were 295.0 +/- 7.9, 211.7 +/- 5.4 (p < 0.001 vs. Group 1), and 259.0 +/- 3.5 g (p < 0.01 vs. Group 2), respectively. The parameters of bone mass such as ash weights of the femur and bone mineral content of the lumbar spine and the tibia in Groups 1 and 2 did not differ, but the values were significantly greater in Group 3 than in Group 2. However, these parameter values corrected for BW were significantly greater in Group 2 than in Group 1 and did not significantly differ between Groups 2 and 3. The parameters of bone formation, such as serum bone alkaline phosphatase activity levels and trabecular bone formation rates corrected for BW, were significantly greater in Group 2 than in Group 1 but did not differ between Group 2 and 3. However, the parameters of bone resorption, such as serum tartrate resistant acid-phosphatase levels, were significantly less in Group 3 than in Group 2. These results suggest that voluntary running augments the age-dependent increase in bone mass by modulating the bone turnover when an adequate energy source is supplied under conditions of normal menstruation, and an adequate supply of energy could be necessary to enhance the age-dependent increase in bone mass.

Proadrenomedullin N-terminal 20 peptide (PAMP) inhibits proliferation of human neuroblastoma TGW cells.

We investigated the effects of proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM) on the growth of human neuroblastoma TGW cells. Both PAMP and AM inhibited growth and DNA synthesis in neuroblastoma cells. Calcitonin gene-related peptide (CGRP)(8-37), an antagonist to CGRP, abolished the inhibitory effect of AM on growth and DNA synthesis of neuroblastoma cells but did not affect that of PAMP. AM(22-52), an antagonist to AM, also reversed the effect of AM. On the other hand, pertussis toxin (PTX) and omega-conotoxin GIVA blocked the effect of PAMP alone. Thus, PAMP inhibits the growth of neuroblastoma cells by inhibiting N-type Ca2+ channels through PTX-sensitive G protein-coupled receptors, which is different mechanism of AM-induced inhibition of the cell growth.

Expression of mRNA encoding intestinal type alkaline phosphatase in rat liver and its increase by fat-feeding.

The presence of types of alkaline phosphatase (ALP) other than the tissue non-specific type enzyme in rat liver and its increase by fat feeding are known. In order to examine expression of intestinal type ALP in liver, specific oligonucleotide primers corresponding to two types of mRNAs of rat intestinal ALP (RTIN-1 and -2) were designed and amplified by means of the reverse transcriptase-polymerase chain reaction (RT-PCR). It was found that RTIN-1 mRNA was expressed only in the intestine but not in the liver, while RTIN-2 mRNA was expressed both in the intestine and in the liver. By fat feeding, expression of RTIN-1 mRNA increased in the intestine and that of RTIN-2 mRNA increased both in the intestine and in the liver. Thus, it was concluded that rat liver expressed one of the intestinal type ALP (RTIN-2) which was enhanced by fat feeding.

Ovariectomy decreases osteogenetic activity in rat bone.

The purpose of this study was to investigate the effect of ovariectomy on osteoinductive activity in the bone in rat. Homograft implantation of decalcified humeral diaphysis from ovariectomized or sham-operated rats was performed and harvested after several time periods. A significant decrease in bone induction was found in terms of soft X-ray photography, alkaline phosphatase activity, mineral content and expression of osteocalcin (BGP; bone gla-protein) in the implants from the ovariectomized group in comparison to those from the sham-operated animals. This result suggested that the level of osteoinductive activity, probably due to bone morphogenetic protein, decreased in ovariectomized animals.

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.

Voluntary exercise increases osteogenetic activity in rat bones.

The purpose of this study was to investigate the effect of voluntary exercise on osteoinductive activity in rat bone. Sprague-Dawley male and female rats were allowed to exercise freely by running on a treadmill or kept as controls without exercise for 53 days. Decalcified humeral diaphyses from experimental and control rats were implanted intraperitoneally into host rats and harvested after 33 days. A significant increase in bone formation was confirmed in the implanted bone matrices from the running group in comparison with those from control animals by soft X-ray photography and determination of alkaline phosphatase activity and mineral content. Alkaline phosphatase activity in bone and serum was increased by exercise in both male and female animals. The results suggest that osteoinductive activity in the bone was probably due to increased levels of bone morphogenetic protein following voluntary exercise.

The nutritional evaluation of globin on maintenance of bone metabolism in ovariectomized osteoporotic rats.

In our previous study, globin was found to be an effective dietary source for increasing bone mineral density (BMD) and mechanical strength. In this study, the bioavailability of the globin preparation was examined to clarify the mechanism of increase in bone density and strength. Six-week-old Sprague-Dawley female rats were ovariectomized and were fed on a low Ca diet for 30 days to produce the experimental osteoporotic rats. Thereafter they were divided into two groups. The BMD and the mechanical strength of bone of the rat group, whose diet was supplemented with globin, were significantly higher than those of the control group. The levels of the serum calcitonin and the bone-type alkaline phosphatase (Alp) activity in serum and bone were also higher, and the tartrate-resistant acid phosphatase (Tr-Acp) activity in serum and bone was lower in the globin group. Moreover, the bone morphogenetic protein activity in bone in the globin group was found to be greater. From these results, it is concluded that alimentary globin is effective for the acceleration of bone formation and the prevention of bone resorption.

The effect of tochu bark on bone metabolism in the rat model with ovariectomized osteoporosis.

In this study, Tochu bark extract was examined to find out whether it is effective in preventing bone loss after menopause. Six-week-old Sprague-Dawley female rats were ovariectomized (OVX) or were sham-operated (sham). The rats in the OVX and the sham groups were fed a low-calcium diet (Ca 0.01%, P 0.3%) for 33 days. Thereafter, the rats in the OVX group were subdivided into two groups: a control group and a Tochu group. The diet for the Tochu group contained 2% Tochu bark extract (Ca 0.3%, P 0.3%). The diet for the control group and the sham group was the control diet; it contained 0.3% Ca and 0.3% P. The rats in each group were fed each experimental diet for the next 31 days. The bone mineral density (BMD) and the breaking strength of the control group were lower than those of the sham group. However, the BMD and the bone strength had improved in the Tochu group. These results suggest that the Tochu bark extraction could be effective in the prevention of osteoporosis. Moreover, in the Tochu group, intestinal Ca absorption increased. This means the Tochu bark extract accelerated the intestinal Ca absorption. This could possibly affect the increase of the BMD and the bone strength. Moreover, the muscle weight of the Tochu group was higher than that of the control group. These results suggest that Tochu bark extract is also effective for the improvement of the bone and the muscle metabolism in sedentary people with back pains and/or joint pains.

Evaluation of the effect of soybean milk and soybean milk peptide on bone metabolism in the rat model with ovariectomized osteoporosis.

We have reported that soybean milk is an excellent source for increasing bone mineral density (BMD) and mechanical bone strength. However, it is not known what kind of components in soybean milk affect bone metabolism. Consequently, the purpose of this study is to find the effective components in soybean milk on bone metabolism. It might be some peptide in soybean milk. Therefore, in this study the soybean milk was separated into two different preparations according to molecular weight: a high-molecular-weight soybean milk, and a low-molecular-weight soybean milk. Then, the effect of the peptides in soybean milk on bone metabolism was examined. After producing the experimental osteoporotic model rats, they were divided into four groups. The BMD and the mechanical bone strength of the three experimental groups, whose diet contained a soybean milk, a high-molecular soybean milk, or a low-molecular soybean milk (soybean milk peptide) were significantly higher than those of the control group. Moreover, the intestinal Ca absorption for the three experimental groups was significantly increased. From these results, the peptides in the soybean milk are effective for the acceleration of the intestinal Ca absorption. It is possible to assume that the mechanism for increasing the BMD and the mechanical strength in the three experimental groups was due to increasing the intestinal Ca absorption by alimenting the soybean milk, the high-molecular soybean milk or the low-molecular soybean milk.

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.

The effect of spiny lobster shell powder on bone metabolism in ovariectomized osteoporotic model rats.

Calcium has been found to be indispensable in the prevention of osteoporosis. Recently, there has been a great deal of research into the best way to consume calcium. In this study, the effect of "powdered lobster shell" on bone metabolism was examined in ovariectomized osteoporotic model rats. This powder has a good flavor and taste, and contains high quantities of calcium. Six-week-old SD-strain female rats were ovariectomized and were fed a low Ca diet (0.01% Ca and 0.3% P) for 32 days. Thereafter, the rats were divided into two groups; the control group was fed a control diet (0.3% Ca and 0.3% P) and an experimental group, the lobster group, was fed a lobster shell powder diet (0.3% Ca and 0.3% P) ad libitum for 30 days. The results were as follows: in comparison with the control group, the lobster group had significant increases in (1) bone mineral density [BMD (DEXA Hologic's QDR-1000] of lumbar spines and tibial proximal metaphyses, which are mainly trabecular bones, and BMD of tibial diaphyses, which is a mainly cortical bone, (2) the breaking force and energy of femur. These results suggests the lobster shell powder could be a valuable source of dietary calcium in increasing BMD, breaking force and energy in osteoporotic model rats.