Femoral-diaphyseal Tissues Research Articles

Role of Serum Albumin in fracture healing

Albumin is the most abundant plasma protein with molecular weight of 69 kd and it accounts for 55-60% measured in total serum protein, produced in liver. The multipotent progenitor cells of bone marrow differentiate into functional hepatocyte-like cells which expresses albumin. The 66kDa albumin protein is very prominent, and this protein component is present in the bone tissue as a major bone protein. The increase in IGF-I with fracture healing induce the production of albumin in the femoral-diaphyseal tissues and thus the osteoblastic proliferation takes place. The albumin also enhanced the bone calcium and DNA content that further promotes fracture healing, and its deficiency may be a significant determinant in healing process which may lead to fracture impairment. In this review we focused on the valuable significant role of albumin in fracture healing as well as the nonunion.

Oral Administration of Cal K2 Containing Menaquinone-4 (Vitamin K2) Enhances Serum r-Carboxylated Osteocalcin and Biochemical Components in the Femoral Tissues of Rats

The effects of menaquinone-4 (MK-4; vitamin K2) supplemented egg shell calcium (Cal K2) on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats was investigated. Cal K2 contained calcium carbonate (Ca 100 mg/g) and MK-4 (593 μg/g). Rats were orally administered a solution of Cal K2 (10, 25, or 50 mg/ml/100 g body weight) or placebo (without MK-4, 50 mg/ml/100 g body weight) once daily for 7 days. The serum r-carboxylated osteocalcin concentration, which is produced in osteoblastic cells, was significantly increased after the administration of Cal K2 (50 mg/100 g body weight) as compared with that in the control group or placebo-control group without MK-4. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal tissues were significantly increased after Cal K2 (25 or 50 mg/100 g) as compared with that in the placebo-control group. Femoral-metaphyseal calcium content was significantly increased after the administration of Cal K2 (25 or 50 mg/100 g) as compared with that in the control group. DNA content in the femoral-diaphyseal and -metaphyseal tissues was significantly increased after the administration of Cal K2 (50 mg/100 g) as compared with that in the control or placebo-controlled group. This study demonstrates that the oral administration of Cal K2 containing MK-4 has anabolic effects on bone components in rats. Supplemental Cal K2 may have a role in the prevention of bone loss with aging.

Oral Administration in Combination with Zinc Enhances .BETA.-Cryptoxanthin-Induced Anabolic Effects on Bone Components in the Femoral Tissues of Rats in Vivo

The effects of combined beta-cryptoxanthin and zinc on bone components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats in vivo were investigated. Rats were orally administered either vehicle, beta-cryptoxanthin (5 or 10 microg/100 g body weight), zinc sulfate (0.1 or 0.5 mg Zn/100 g), or their combination once a day for 7 d. Calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal tissues was not significantly altered by the administration of beta-cryptoxanthin (5 microg/100 g) or zinc (0.1 or 0.5 mg/100 g). Combined administration of beta-cryptoxanthin (5 microg/100 g) and zinc (0.1 or 0.5 mg/100 g) caused a synergistic increase in calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal tissues. The effect of beta-cryptoxanthin (5 or 10 microg/100 g) in increasing calcium and DNA contents in the metaphyseal tissues was significantly enhanced by the combined administration of zinc (0.1 or 0.5 mg/100 g), but did not have a significant effect on the metaphyseal components. The metaphyseal alkaline phosphatase activity was markedly increased by the combination of beta-cryptoxanthin (5 microg/100 g) and zinc (0.1 or 0.5 mg/100 g). This study demonstrates that the oral administration of the combination of zinc at lower doses synergistically enhances beta-cryptoxanthin-induced anabolic effects on bone components in the femoral tissues of rats in vivo.

Oral Administration of <I>Saldi tierra</I> Containing Various Trace Elements Has Anabolic Effects on Bone Component in the Femoral Tissues of Rats

The effects of Saldi tierra containing various trace elements on bone components in the femoral-diaphyseal and -metaphyseal tissues of rats was investigated. Saldi tierra (sodium salt or potassium salt) was obtained from natural zeolite using the method of ion exchange with sodium chloride or potassium chloride solutions. Saldi tierra contained more than 20 elements. Rats were orally administered a solution of Saldi tierra once daily for 7 days. Calcium content, alkaline phosphatase activity and DNA content in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues of rats were significantly increased with the administration of Saldi tierra sodium salt (100 mg/100 g body weight). Calcium content and alkaline phosphatase activity in the femoral-diaphyseal tissues of rats were significantly increased with the administration of Saldi tierra sodium salt (50 mg/100 g). Saldi tierra (50 mg) contained 3.155 mg of calcium. Femoral components were not significantly altered by the administration of calcium chloride (Ca 3.155 mg/100 g body weight). Moreover, the administration of Saldi tierra potassium salt (50 or 100 mg/100 g) caused a significant increase in bone components in the femoral-diaphyseal or -metaphyseal tissues of rats. Potassium salt of Saldi tierra had a potent effect on bone components as compared with its sodium salt. Bone components were not significantly increased with the administration of an elemental mixture containing sodium (26.8 mg), potassium (1.660 mg), magnesium (0.431 mg), chloride (43.86 mg), silicon (11.1 µg), calcium (6.310 mg), zinc (0.42 µg), and manganese (1.32 mg/100 g), which are contained in Saldi tierra (100 mg). This study demonstrates that the oral administration of Saldi tierra containing various elements has anabolic effects on bone component in rats. The intake of Saldi tierra may have a role in the prevention of bone loss with aging.

Enhancement of albumin expression in bone tissues with healing rat fractures.

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.

Great increase in bone 66 kDa protein and osteocalcin at later stages with healing rat fractures: Effect of zinc treatment

Fracture healing has been demonstrated to increase production of bone growth factors, and this elevation has been shown to be enhanced by zinc treatment. Moreover, the effect of zinc treatment on production of bone osteocalcin, which is a kind of Ca2+-binding protein localized in bone matrix, at the later stages with bone fracture was investigated. Rats were sacrificed 7 (earlier stage) or 21 (later stage) days after fracture of femoral diaphysis. Femoral-diaphyseal tissues with fracture healing were cultured in a serum-free medium for 24 h. Many proteins in the bone tissues were released into the medium. Bone protein production was markedly elevated 21 days after bone fracture as compared with that of 7 days. A approximately 66 kDa protein molecule, a major protein component which was produced by the diaphyseal tissues during fracture healing, was predominantly increased at the later stages with fracture healing. Bone osteocalcin production was significantly increased during fracture healing. This increase was enhanced at the later stages with fracture healing. The presence of zinc acexamate (10(-4) M) in culture medium caused a significant increase in bone protein and osteocalcin production at 7 or 21 days after bone fracture. The effect of zinc acexamate in increasing bone total protein and osteocalcin production was remarkable at the later stages with fracture healing. Moreover, zinc treatment caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium content in the femoral-diaphyseal tissues of the later stages with fracture healing in vitro. The present study demonstrates that bone protein production is markedly increased at the later stages with fracture healing, and that zinc treatment can enhance production of bone protein components including osteocalcin in vitro. Zinc treatment may stimulate the healing of femoral fracture at earlier and later stages.

Role of endogenous regucalcin in bone metabolism: Bone loss is induced in regucalcin transgenic rats

The role of endogenous regucalcin in the regulation of bone metabolism was investigated by using regucalcin transgenic (TG) rats. The expression of regucalcin mRNA in the femoral-diaphyseal and -metaphyseal tissues of normal (wild-type) rats was shown by using reverse transcription-polymerase chain reaction (RT-PCR) with a specific primer of regucalcin cDNA. Regucalcin protein was detected in the femoral-diaphyseal and -metaphyseal tissues of normal (wild-type) rats using Western analysis. Regucalcin levels were significantly increased in the femoral-metaphyseal tissues of regucalcin TG male rats and in the diaphyseal and metaphyseal tissues of the TG female rats. The morphologic change in the femoral-diaphyseal and -metaphyseal tissues of regucalcin TG rats was demonstrated by using a peripheral quantitative computed tomography (pQCT); morphologic change was great in the femoral tissues of female rats as compared with that of male rats. Mineral content, mineral density and polar strength strain index in the femoral-diaphyseal and -metaphyseal tissues were markedly reduced in regucalcin TG female rats. A significant decrease in cortical thickness was seen in the femoral diaphysis of regucalcin TG female rats. Calcium content in the femoral-diaphyseal and -metaphyseal tissues was significantly decreased in regucalcin TG male and female rats; a remarkable decrease was seen in female rats. Femoral-metaphyseal alkaline phosphatase activity was significantly lowered in regucalcin TG female rats. The enzyme activity was not significantly changed in the femoral-diaphyseal tissues of the TG female rats. In the diaphyseal tissue of male rats, the enzyme activity was significantly decreased in the TG rats. A significant decrease in deoxyribonucleic acid (DNA) content was seen in the metaphyseal tissue of regucalcin TG male rats and in the diaphyseal and metaphyseal tissues of the TG female rats. This study demonstrates that bone loss is induced in the femoral tissue of regucalcin transgenic rats, and that a remarkable decrease in bone morphologic index and biochemical component was seen in the female rats. Regucalcin may be involved in the regulation of bone metabolism.

Characterization of the increase in bone 66 kDa protein component with healing rat fractures: Stimulatory effect of zinc

The characterization of protein components produced from bone tissues with fracture healing was investigated. Weanling rats were sacrificed between 1 and 7 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that many protein molecules were released from the diaphyseal tissues with fracture healing. Especially, a protein molecule of approximately 66 kDa was markedly increased by fracture healing. This protein molecule was significantly increased, when the diaphyseal tissues with fracture healing were cultured in the presence of zinc acexamate (10(-6)-10(-4) M). Zinc acexamate (10(-4) M)-induced increase in medium 66 kDa protein molecule was significantly inhibited in the presence of actinomycin D (10(-7) M) or cycloheximide (10(-6) M). The zinc effect was completely blocked in the presence of PD98059 (10(-5) M), an inhibitor of MAPK kinase, or staurosporine (10(-6) M), an inhibitor of protein kinase C. The medium 66 kDa protein molecule was significantly elevated in the presence of parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or transforming growth factor-beta (10(-11) M), while 17beta-estradiol (10(-9) M) did not have an effect. The effect of these bone-stimulating factors was equal to the zinc effect. Zinc did not significantly enhance the effect of insulin-like growth factor-I in increasing medium 66 kDa protein molecule. The present study demonstrates that fracture healing increases production of the approximately 66 kDa protein molecule which is a major component produced from femoral-diaphyseal tissues of weanling rats, and that this elevation is enhanced by zinc treatment.

Increase in bone growth factors with healing rat fractures: the enhancing effect of zinc.

The effect of zinc, a stimulator of bone formation, on bone protein components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed between 1 and 7 days after the femoral fracture, and the diaphyseal tissues were cultured in a serum-free Dulbecco's modified Eagle's medium for 24 h. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. The amount of protein in the medium cultured with the diaphyseal tissues obtained from fracture healing rats was markedly elevated as compared with that of normal rats, indicating that bone protein components were secreted into culture medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) showed that many protein molecules were secreted from the diaphyseal tissues with fracture healing. Especially, protein molecule of about 66 kDa was markedly secreted by fracture healing. The presence of zinc acexamate (10(-5) and 10(-4) M) in culture medium induced a significant elevation of medium protein content; the zinc effect was enhanced by culture with the diaphyseal tissues of fracture healing rats. Also, the culture of diaphyseal tissues with fracture healing caused a significant increase in insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta1) in culture medium. The production of IGF-I and TGF-beta1 from bone tissues with fracture healing was significantly enhanced in the presence of zinc acexamate (10(-6)-10(-4) M). Moreover, the addition of IGF-I (10(-8) M) or TGF-beta1 (10(-10) M) in a culture medium caused a significant elevation of protein content in the medium cultured with the femoral-diaphyseal tissues from normal and fracture healing rats. The effect of IGF-I or TGF-beta1 was significantly enhanced in the presence of zinc acexamate (10(-4) M). Also, deoxyribonucleic acid (DNA) content in the diaphyseal tissues from normal and fracture healing rats was significantly raised by the culture with IGF-I or TGF-beta1. The effect of IGF-I was significantly enhanced by zinc. The present study demonstrates that fracture healing increases production of bone IGF-I and TGF-beta1, and that this elevation is enhanced by zinc treatment.

Stimulatory effect of menaquinone-7 (vitamin K2) on osteoblastic bone formation in vitro.

Menaquinone-7, which is vitamin K2 (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10(-7)-10(-5) M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10(-7)-10(-5) M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10(-6) and 10(-5) M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.

Stimulatory effect of zinc and growth factor on bone protein component in newborn rats: enhancement with zinc and insulin-like growth factor-I.

The effect of zinc and growth factor on bone protein component in newborn rats was investigated. The characterization of protein component in the femoral-diaphyseal and metaphyseal tissue of newborn rats (3-35 days old) was examined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The diaphyseal and metaphyseal tissues of 7 days-old rats were cultured for 24 or 48 h in a medium containing either vehicle, zinc sulfate (10-4 M) or dipicolinate (10-3 M), a chelator of zinc ion, in the presence or absence of insulin-like growth factor-I (IGF-I; 10-8 M) or transforming growth factor-beta (TFG-beta; 10-10 M) with an effective concentration. Many cellular protein molecules were present in the diaphyseal and metaphyseal tissues; potent bands were seen in protein molecules with about 66 and 46 kDa. Protein molecule of about 66 kDa was greatly released in the medium cultured with the diaphyseal and metaphyseal tissues. This protein in the medium was increased by culture with zinc, IGF-I or TGF-beta. Total protein content in the medium cultured with the diaphyseal and metaphyseal tissues was significantly increased in the presence of zinc, IGF-I or TGF-beta. The IGF-I-increased medium protein content was significantly enhanced by zinc. This enhancing effect was not seen in TGF-beta. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased by culture with zinc, IGF-I or TGF-beta. The effect of IGF-I was significantly enhanced by zinc, while it was not found in TGF-beta. The effect of IGF-I or TGF-beta in increasing the bone components was seen in the presence of dipicolinate. This study demonstrates that zinc, like IGF-I and TGF-beta, can increase protein components in the femoral-diaphyseal and metaphyseal tissues of new-born rats. Zinc may especially play a role in bone growth in collaboration with IGF-I.

Supplemental Intake of Isoflavones and Zinc-Containing Mineral Mixture Enhances Bone Components in the Femoral Tissue of Rats with Increasing Age.

The effect of the supplemental intake of isoflavone and a zinc-containing mineral mixture on bone components in the femoral tissue of rats was investigated. Rats (5 weeks old) were orally administered either vehicle, isoflavone glycoside (5mg/100g body weight), aglycone isofavone (5mg/100g, including 3.772mg daidzein, 0.395mg genistein, and 0.833mg glycitein), zinc-containing mineral mixture (0.833mg zinc, 50mg calcium, 25mg magnesium, and 0.208μg vitamin D3 per 100g body weight, respectively), or both aglycone isoflavone (5mg/100g) and zinc-containing mineral mixture for 7d. Administrations resulted in a significant increase in femoral dry weight, calcium content, alkaline phosphatase activity, and DNA content in the femoral-diaphyseal and metaphyseal tissues. Femoral zinc content was significantly increased by the administration of zinc-containing mineral mixtures with or without aglycone isoflavone. The effect of aglycone isoflavone in elevating bone components was significantly enhanced in combination with the zinc-containing mineral mixture. Moreover, the oral administration of both aglycone isoflavones and zinc-containing mineral mixture to aged rats (50 weeks old) for 21d resulted in a significant increase in femoral dry weight, zinc content, and bone components. This study demonstrates that supplemental intake of both aglycone isoflavone and a zinc-containing mineral mixture has an anabolic effect on bone components in aged rats, suggesting the role of nutrients in the prevention of osteoporosis with increasing age.

Increase in bone protein components with healing rat fractures: enhancement by zinc treatment.

The alteration in bone components in the femoral-diaphyseal tissues with fracture healing was investigated. Rats were sacrificed 7 and 14 days after the femoral fracture. Protein content in the femoral-diaphyseal tissues was markedly elevated by fracture healing. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that many protein molecules were induced in the diaphyseal tissues with fracture healing. Moreover, when the femoral-diaphyseal tissues with fracture healing were cultured for 24 and 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Also, the culture of the diaphyseal tissues with fracture healing caused a significant increase in bone alkaline phosphatase activity and deoxyribonucleic acid (DNA) content. Meanwhile, the presence of zinc acexamate (10-5 and 10-4 M), a stimulator of bone formation, in a culture medium induced a significant elevation of protein content and alkaline phosphatase activity in the diaphyseal tissues with fracture healing. Such an effect was completely abolished by the presence of cycloheximide (10-6 M), an inhibitor of protein synthesis. The present study suggests that fracture healing induces a newly synthesized bone protein component including stimulatory factor(s) for bone formation. Zinc supplementation may stimulate the healing of femoral fracture.

Stimulatory effect of zinc acexamate administration on fracture healing of the femoral-diaphyseal tissues in rats

The effect of zinc acexamate on fracture healing of the femoral-diaphyseal tissues in rats was investigated in vivo. Zinc acexamate (0.3 and 10.0 mg Zn/100 g body weight per day) was orally administered to rats (4 weeks old) surgically fractured the femoral diaphysis for 14 to 28 days. Calcium content and alkaline phosphatase activity in the femoral-diaphyseal tissues were significantly decreased in rats with fracture healing, while bone acid phosphatase activity and protein content were markedly increased. The administration of zinc acexamate (10.0 mg Zn/100 g) for 28 days caused a significant increase in calcium content, alkaline and acid phosphatases activities, protein and deoxyribonucleic acid (DNA) contents in the femoral-diaphyseal tissues of rats with fracture healing. With the lower dose (3.0 mg Zn/100 g), zinc compound had a partial effect on bone components. Femoral mineral density in rats with fracture healing was significantly increased by the administration of zinc acexamate (10.0 mg Zn/100 g) for 28 days. Femoral-diaphyseal zinc content was significantly decreased in rats with fracture healing. This decrease was completely restored by the administration of zinc acexamate (10.0 mg Zn/100 g) for 28 days. The present study suggests that the supplement of zinc compound stimulates fracture healing of the femoral-diaphyseal tissues in rats.

Anabolic Effect of Wasabi Leafstalk(Wasabia japonica MATSUM.) Extract on Bone Metabolism in Rats.

The effect of wasabi leafstalk (Wasabia japonica MATSUM.) extract on bone metabolism in vivo was investigated. The extract was obtained from a homogenate with 20% ethanol and was orally administered 7 times at 24-h intervals (1.0, 10, and 40 mg/100 g body weight) to young rats. Administration of wasabi extract (1.0 and 10 mg/100 g) caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in the femoral-metaphyseal tissues; the DNA content was significantly elevated by the dose of 40 mg/100 g. In the femoral-diaphyseal tissues, alkaline phosphatase activity and calcium content were significantly increased by the administration of 1.0 and 10 mg/100 g, while the DNA content was not appreciably altered. The present study demonstrates that wasabi leafstalk extract has an anabolic effect on bone metabolism in rats in vivo.

Alterations in Ca2+-ATPase activity and calcium-binding protein regucalcin mRNA expression in the kidney cortex of rats with saline ingestion.

The alteration in calcium metabolism in rats ingested with saline was investigated. Rats were freely given saline as drinking water for 2 and 7 days. Calcium concentration in the serum was significantly elevated by saline ingestion for 2 and 7 days, while serum inorganic phosphorus concentration was not altered. Serum urea nitrogen concentration was significantly increased by saline ingestion for 7 days. Calcium content in the femoral-diaphyseal and metaphyseal tissues was not altered by saline ingestion for 7 days. Calcium content in the kidney cortex was significantly elevated by saline ingestion for 7 days. Ca2+-ATPase activity in the basolsateral membranes of kidney cortex was clearly increased by saline ingestion for 2 and 7 days. The enzyme activity was not altered by the addition of sodium chloride (10(-3) and 10(-2) M), parathyroid hormone (10(-7) and 10(-6) M), and calcitonin (3 x 10(-8) and 3 x 10(-7) M) in the enzyme reaction mixture. A calcium-binding protein regucalcin mRNA expression in the kidney cortex was markedly suppressed by saline ingestion for 7 days, although such a suppression was not seen for 2 days. These results suggest that saline ingestion causes the disturbance of calcium transport system in the kidney cortex of rats, and that the renal disorder may induce hypercalcemia.

Zinc compounds inhibit osteoclast-like cell formation at the earlier stage of rat marrow culture but not osteoclast function.

The effect of zinc compounds on osteoclast-like cell formation in rat marrow culture in vitro was investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a well-known bone resorbing hormone (1, 25-dihydroxyvitamin D3 and parathyroid hormone [1-34]). Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of 1, 25-dihydroxyvitamin D3 (10(-8) M) or parathyroid hormone (PTH; 10(-8) M) induced a remarkable increase in osteoclast-like multinucleated cells (MNC). These increases were clearly inhibited by the presence of zinc sulfate or zinc-chelating dipeptide (beta-alanyl-L-histidinato zinc; AHZ) in the concentration range of 10(-7) to 10(-5) M. The inhibitory effect was seen at the earlier stage of osteoclast-like MNC formation. However, zinc compounds (10(-6) M) did not have an effect on PTH (10(-8) M)-induced osteoclast-like cell formation in the presence of EGTA (5 x 10(-4) M), dibucaine (10(-5) M) or staurosporine (10(-9) M). Moreover, when osteoclasts isolated from rat femoral-diaphyseal tissues were cultured for 24 h in the presence of zinc compounds (10(-7) to 10(-5) M), the compounds did not have an effect on cell numbers or lysosomal enzymes activity (acid phosphatase and beta-glucuronidase) in the cells. The present study clearly demonstrates that zinc compounds inhibit osteoclast-like cell formation at the earlier stage with differentiation of marrow cells.

Effect of insulin administration on bone formation is impaired in rats with skeletal unloading.

The effect of insulin administration on bone formation was investigated in the femoral diaphysis of rats with skeletal unloading. When the femoral-diaphyseal tissues from normal rats were cultured in the presence of insulin (10(-9) and 10(-8) M) for 48 h, the hormone produced a significant increase of alkaline phosphatase activity, collagen, deoxyribonucleic acid (DNA) and calcium contents in the bone tissues, indicating that it has a direct stimulatory effect on bone formation. Moreover, when normal rats received a subcutaneous administration of insulin (5.0 IU/100 g body weight) 5 times at 24-h intervals, the hormone caused a significant increase of alkaline phosphatase activity, DNA and calcium contents in the femoral diaphysis. Those increases were not seen by insulin administration to the skeletal-unloading rats with hindlimb hang for 4 d. The present results indicate that the stimulatory effect of insulin on bone formation is lost by skeletal unloading. The finding further supports the view that insulin impairment plays an important role in the deterioration of bone formation by microgravity.

Simulated weightlessness and bone metabolism: gravitational stimulation enhances insulin sensitivity

The effect of simulated weightlessness on bone metabolism was investigated in skeletal unloading for 4 days. Skeletal unloading was designed using the model of hindlimb hang in rats. Skeletal unloading with hindlimb hang cased a significant decrease of alkaline phosphatase activity, deoxyribonucleic acid (DNA) content, and glucose consumption in the femoral diaphysis, but not in the calvaria. When femoral-diaphyseal tissues were cultured in the presence of insulin (10(-8) M), the hormone produced a significant increase of alkaline phosphatase activity and decrease of glucose consumption in the femoral-diaphyseal tissues obtained from normal rats. This hormonal effect was not seen in the femoral diaphysis, but in the calvaria, of rats with skeletal unloading. However, insulin effect was seen in the femoral diaphysis obtained at 3 days after the removal of skeletal unloading. Meanwhile, the presence of other bone-regulating factors (10(-8) M parathyroid hormone [1-34] and 10(-4) M zinc sulfate) revealed an appreciable effect on alkaline phosphatase activity in the femoral diaphysis from rats with skeletal unloading. These results suggest that gravitational stimulation can directly enhance a specific insulin sensitivity in the regulation of bone metabolism.

Simulated weightlessness and bone metabolism: evidence for direct gravitational effect and its related insulin action

The effect of simulated weightlessness on bone metabolism was investigated in skeletal unloading for 4 days. Skeletal unloading was designed using the model of hind-limb hang in rats. In hypokinetic state, rats were fed while the right hind limb was weighed down and the left hind limb was unloaded (a state of weightlessness). Bone metabolism in normal rats did not vary significantly in the femoral diaphysis in right and left hind limb. Alkaline phosphatase activity and DNA content in the femoral diaphysis were not significantly altered by hypokinetic state without skeletal unloading, while the unloading caused an appreciable decrease in the enzyme activity and DNA content. However, femoral-diaphyseal zinc content and glucose consumption was significantly decreased by hypokinetic state with and without skeletal unloading. When femoral-diaphyseal tissues were cultured in the presence of insulin (10 nM), the hormone produced a significant increase of alkaline phosphatase activity and decrease of glucose consumption in the femoral-diaphyseal tissues obtained from normal and hypokinetic rats. This hormonal effect was not seen in bone tissues from hypokinetic rats with skeletal unloading. These results suggest that skeletal unloading-induced disorder of bone metabolism is directly related to gravitational effect and that gravitational stimulation may be involved in insulin action.