Yasuhiro Akiyama

The inhibitory effect of vitamin K2 (menatetrenone) on bone resorption may be related to its side chain

Although the effects of vitamin K2 and vitamin K1 on bone metabolism have been reported, the difference between them has not been investigated. We now show the effects of menatetrenone, one of the vitamin K2 homologues, and vitamin K1 on bone resorption. Menatetrenone at greater than 3 x 10(-6) M significantly inhibited the calcium release from mouse calvaria induced by 3 x 10(-10) M of 1,25(OH)2D3 or 10(-7) M of prostaglandin E2, and it also inhibited osteoclast-like multinucleated cell (MNC) formation induced by 10(-8) M of 1,25(OH)2D3 in co-culture of spleen cells and stromal cells at the same concentrations. In contrast, the same doses of vitamin K1 had no effects on bone resorption and MNC formation in these in vitro systems. The inhibitory effect of menatetrenone on the calcium release from calvaria was not affected by the addition of 3 x 10(-5) M of warfarin, an inhibitor of vitamin K cycle. The same concentration of geranylgeraniol, the side-chain component of menatetrenone at the 3-position of the naphthoquinone, inhibited tartrate-resistant acid phosphatase (TRACP) activity and MNC formation to the same degree as menatetrenone. Phytol, the side-chain component of vitamin K1, did not affect TRACP activity at all doses tested, but weakly inhibited MNC formation. Moreover, multi-isoprenyl alcohols of two to seven units, except geranylgeraniol which contains four units, did not effect MNC formation. These findings suggest that the inhibitory effect of menatetrenone on bone resorption is not due to gamma-carboxylation and that the side chain of menatetrenone may play an important role in this inhibitory effect.

Effect of vitamin K2 (menatetrenone) on osteoclast-like cell formation in mouse bone marrow cultures.

The effects of menatetrenone, a vitamin K2 homologue, on osteoclast-like cell formation in mouse bone marrow culture were investigated. After 7 days of incubation, menatetrenone at 10(-6) M, 3 x 10(-6) M and 10(-5) M dose dependently inhibited the tartrate-resistant acid phosphatase-positive multinucleated cell formation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The addition of menatetrenone for the last 3 days of the 7-day incubation period was required to inhibit formation of multinucleated cells in response to 1,25(OH)2D3. Moreover, the addition of 1,25(OH)2D3 for the last 3 days was essential for multinucleated cell formation, and this activity was markedly inhibited by the simultaneous addition of menatetrenone. The inhibitory effects of menatetrenone on multinucleated cell formation may contribute to its ameliorative action on bone loss in vivo, and may indicate a new mechanism of vitamin K2 activity in bone metabolism.

Effects of menatetrenone on prednisolone-induced bone loss in rats

This study was carried out to evaluate the effect of menatetrenone, a vitamin K2 with 4 isoprene units, on prednisolone-induced bone loss. Three experiments were performed in rats which received menatetrenone as a dietary supplement. In experiment 1, a soluble form of prednisolone, dissolved in drinking water, was administered to rats at 7 mg/kg/day for 9 weeks. The length, dry weight, and bone density of femurs and tibiae, as well as urinary gamma-carboxyglutamic acid (Gla) content, were significantly lower in the prednisolone-control group than in the intact group. Menatetrenone (17 mg/kg/day) significantly inhibited the decrease in these bone parameters, especially in tibiae, and completely inhibited the decrease in urinary Gla content. In experiments 2 and 3, prednisolone (10 mg/kg), dissolved in cottonseed oil, was given to rats intramuscularly three times a week for 4 and 10 weeks, respectively. In experiment 2, bone length, bone strength and calcium content in the femur were reduced by 4-week prednisolone treatment. These reductions were significantly improved by menatetrenone (21 mg/kg/day). In experiment 3, 10-week prednisolone treatment reduced bone length and the calcium and hydroxyproline content of the femur. Menatetrenone (0.4, 10, and 50 mg/kg/day) significantly inhibited the reduction of calcium content in the femur. These results suggest that menatetrenone may inhibit the bone loss induced by corticosteroid treatment.

Vitamin K2 (menatetrenone) inhibits bone loss induced by prednisolone partly through enhancement of bone formation in rats

Vitamin K(2) (K(2), menatetrenone) has been reported to enhance bone formation and inhibit bone resorption in vitro. However, there is no evidence that K(2) enhances bone formation in vivo. The aim of this study was to characterize the effect of K(2) on bone formation in vivo. We carried out two experiments using a prednisolone (pred)-induced bone loss model in male (10-week-old) Fischer rats. Pred was orally administered three times a week. In experiment 1, we compared the degree of bone loss induced by a 4 week treatment (30 or 100 mg/kg) and an 8 week treatment (3, 10, or 30 mg/kg) with pred by peripheral quantitative computed tomography (pQCT). At 4 weeks, total bone mineral density (BMD) was decreased only with the 100 mg/kg pred treatment. At 8 weeks, total BMD was significantly reduced at >10 mg/kg pred. In experiment 2, we investigated the effect of K(2) on bone loss induced by 3 and 30 mg/kg pred. K(2) (15 mg/kg) was given to rats as a dietary supplement for 8 weeks. Intestinal calcium transport (S/M) and total, trabecular, and cortical BMD at the metaphysis and diaphysis were measured, and histomorphometry was performed in diaphysial cross sections. Pred treatment decreased total and trabecular BMD in the proximal metaphysis. A decrease in cortical BMD in the diaphysis was observed in the pred 30 mg/kg group. Pred treatment also reduced mineralizing surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS). The decrease in total and trabecular BMD in the proximal metaphysis, and in cortical BMD in the diaphysis, was inhibited by K(2) treatment. K(2) treatment also inhibited the decrease in MS/BS and BFR/BS induced by 30 mg/kg pred. These results suggest that K(2) prevents bone loss partly through the enhancement of bone formation.

Chronic hypertension induced by streptozotocin in rats

SummaryAn intravenous injection of 40 or 65 mg/kg streptozotocin induced not only diabetes but also severe hypertension in rats. Whereas the hyperglycemia developed fully within a few days after the injection of streptozotocin, the hypertension progressively advanced and reached maximum level several weeks after the treatment and lasted more than 20 weeks. Twenty mg/kg streptozotocin did not induce hyperglycemia but significantly increased blood pressure several weeks after the treatment. Arrest of growth, polyuria, glycosuria, hyperlipemia and lenticular cataracts developed in the animals treated with 40 or 65 mg/kg streptozotocin, but in none of the animals treated with 20 mg/kg. In histological examinations in the 24th week after the treatment, degranulation and necrosis in the pancreatic β-cells, and vacuolization and deposition of PAS-positive materials in the renal proximal tubules were found in the animals treated with 40 or 65 mg/kg streptozotocin.

Increased circulating levels of γ-carboxyglutamic acid-containing protein and decreased bone mass in children on anticonvulsant therapy

SummaryIn order to investigate the pathophysiology of anticonvulsant-induced osteopenia, circulating levels of bone γ-carboxyglutamic acid-containing protein (Bone Gla Protein: BGP) and urinary excretion of BGP were measured in 16 chidren on chronic anticonvulsant therapy and in 12 control children. Using microdensitometry analysis, osteopenia was found in 25% of the anticonvulsant therapy group, but it was not observed in the control group. Serum BGP and A1-P levels were significantly increased in the anticonvulsant group compared with the control group (P<0.05 andP<0.01, respectively), and a positive correlation was found between serum BGP and A1-P levels (P<0.05). Urinary excretion of BGP and hydroxyproline showed an increase in the anticonvulsant group, but it was not statistically significant. On the other hand, there was no significant difference between the two groups in serum levels of vitamin D metabolites, PTH, calcitonin, Ca, or P or in urinary excretion of Ca or P. It is suggested, therefore, that the increased BGP level in children receiving anticonvulsant therapy is a reflection of high bone turnover due to anticonvulsant drug complications.

Warfarin administration disrupts the assembly of mineralized nodules in the osteoid.

This study aimed to elucidate the ultrastructural role of Gla proteins in bone mineralization by means of a warfarin-administration model. Thirty-six 4-week-old male F344 rats received warfarin (warfarin group) or distilled water (control group), and were fixed after 4, 8 and 12 weeks with an aldehyde solution. Tibiae and femora were employed for histochemical analyses of alkaline phosphatase, osteocalcin and tartrate-resistant acid phosphatase, and for bone histomorphometry and electron microscopy. After 4, 8 and 12 weeks, there were no marked histochemical and histomorphometrical differences between control and warfarin groups. However, osteocalcin immunoreactivity was markedly reduced in the warfarin-administered bone. Mineralized nodules and globular assembly of crystalline particles were seen in the control osteoid. Alternatively, warfarin administration resulted in crystalline particles being dispersed throughout the osteoid without forming mineralized nodules. Immunoelectron microscopy unveiled lower osteocalcin content in the warfarin-administered osteoid, which featured scattered crystalline particles, whereas osteocalcin was abundant on the normally mineralized nodules in the control osteoid. In summary, Gla proteins appear to play a pivotal role in the assembly of mineralized nodules.

Comparison of intestinal absorption of vitamin K2 (menaquinone) homologues and their effects on blood coagulation in rats with hypoprothrombinaemia

To examine the physiological activities of vitamin K2 (menaquinone, MK) homologues with different numbers of isoprene units, MK with 1-14 isoprene units and menadione (MK-0) were administered to rats with hypoprothrombinaemia, and the absorption, concentration in liver and ameliorating effect of these MK on hypoprothrombinaemia were compared. Hypoprothrombinaemia was induced by giving a vitamin K-deficient diet and warfarin (0.06 mg/kg body weight) for 8 days. Before MK treatment, the MK were undetectable in plasma and liver. At 6 hr after oral MK administration (0.1 mg/kg): MK was not detected in the plasma in rats treated with MK with 1, 2, 3 or more than 12 isoprene units; the MK level in the liver was increased but blood coagulation activity was not improved in rats treated with MK with 0, 9, 10 or 11 isoprene units; the MK level in the liver was increased and hypoprothrombinaemia was slightly improved in rats treated with MK with 7 or 8 isoprene units; and the MK level in the liver was increased and hypoprothrombinaemia was markedly improved in rats treated with MK with 4, 5 or 6 isoprene units. Almost identical results were observed 3 hr after intravenous injection of MK with 4, 5 or 6 isoprene units (10 nmol/kg). These findings suggest that the number of isoprene units of MK is an important factor in its absorption and incorporation into the liver and that the ameliorating effect of MK on hypoprothrombinaemia does not parallel their concentrations in the liver.

Overexpression of granulocyte colony-stimulating factor induces severe osteopenia in developing mice that is partially prevented by a diet containing vitamin K2 (menatetrenone).

Mice transgenic for granulocyte colony-stimulating factor (G-CSF) exhibit severe osteopenia with an increase of osteoclast number and acceleration of bone resorption in adult mice. To examine the effect of G-CSF overexpression on developing bone, bone mineral density levels were examined from 4 weeks through 36 weeks after birth. Peak bone mass was observed at around 24 weeks of age irrespective of G-CSF expression. Apparent osteopenia was observed as early as 4 weeks of age without detectable developmental retardation in bone length and skeletal structure. Morphological examination confirmed a reduction of cancellous bone and cortical bone at this early stage of life, indicating that overexpression of G-CSF results in apparent osteopenia in developing mice, similar to that in adult animals. The effect of vitamin K2 (menatetrenone) (MK4) on bone phenotypes during development was then examined. Mice were fed chow containing either 0.05 mg MK-4 per 100 g or 20.0 mg MK-4 per 100 g for 12 weeks as the control and experimental diets, respectively. This treatment did not change bone length, irrespective of the type of mouse or diet. Peripheral quantitative computed tomography (pQCT) revealed an increase of in CT value bone of MK4-treated mice. Taken together, these results indicate that overexpression of G-CSF induces an apparent reduction of bone mass and results in osteopenia in developing mice. The bone reduction was partially restored by feeding the mice MK4, suggesting a choice for treatment on the osteopenia induced by G-CSF.

Comparison of inhibitory effects of warfarin on γ-carboxylation between bone and liver in rats

The purposes of this study were to clarify that warfarin (WF, vitamin K antagonist) levels that inhibit γ-carboxylation are different in liver and bone (experiment 1), and to investigate whether the plasma osteocalcin (OC) level reflects bone OC levels (experiments 2 and 3). Four-week-old male rats were treated with 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 mg/l of WF solution as drinking water for 4 weeks. Blood coagulation activity, an index of γ-carboxylation of prothrombin in the liver, was significantly decreased in rats receiving 0.8 mg/l or larger doses of WF. A significant decrease of plasma γ-carboxylated OC (GlaOC), an index of γ-carboxylation of OC in bone, was shown in rats receiving 0.2 mg/l or larger doses. Significantly lower OC levels in the femoral diaphysis and metaphysis were shown in the 0.2 mg/l and 0.4 mg/l groups. However, femoral bone mineral density (BMD) values in the WF-treated groups were almost the same as those in the intact group. In experiment 2, we evaluated changes in bone OC levels 4 weeks after discontinuing an 8-week WF treatment. Four-week-old male rats received 0.8 mg/l WF as drinking water for 8 or 12 weeks. Recovery of the OC level after discontinuing the WF treatment was shown in the femoral metaphysis, but not in the diaphysis. In experiment 3, 0.3 mg/kg WF was administrated to 25-week-old male rats three times a week for 8, 12, or 16 weeks. In aged rats, decreased bone OC was shown in the femoral metaphysis, but not in the diaphysis. From these findings, it is suggested that the effects of WF on γ-carboxylation are likely to appear in bone at lower doses than in the liver, that the bone OC level does not always correspond directly to plasma GlaOC, and that the bone OC level is not directly linked with BMD.