Ikuyo Tsukamoto

Insulin-dependent diabetes mellitus decreases osteoblastogenesis associated with the inhibition of Wnt signaling through increased expression of Sost and Dkk1 and inhibition of Akt activation.

Insulin-dependent diabetes mellitus (IDDM) is known to be associated with an increased risk of osteopenia. However, the cellular and molecular mechanisms for IDDM-induced alterations of the bone are not well understood. The effects of IDDM on bone metabolism were investigated using rats rendered diabetic by an injection of streptozotocin (STZ). After 4 weeks, the diabetic rats exhibited bone loss, low levels of osteocalcin, insulin-like growth factor-I (IGF-I) and bone alkaline phosphatase (ALP) activity with normal levels of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K activity, and urinary excretion of deoxypyridinoline (Dpd). Histological analysis showed a decrease in the number of osteoblasts with a normal number of osteoclasts in the metaphysis of the proximal tibia. The decreased expression of ALP, osteoclacin and collagen mRNA was associated with a decrease in the expression of runt-related transcription factor 2 (Runx2), Osterix and distal-less homeobox 5 (Dlx5) and an unaltered expression of bone morphogenic protein-2 (BMP2). The protein levels of Runx2, phosphorylated glycogen synthase kinase 3β (GSK3β), active β-catenin and β-catenin decreased. The activation of Akt was inhibited. The mRNA and protein levels of sclerosteosis (Sost) and Dickkopf 1 (Dkk1), inhibitors of Wnt signaling, increased. The mRNA expression of IGF-I and the IGF-I receptor (IGF-IR) was suppressed. These changes observed in the bone of diabetic rats were reversed by treatment with insulin, but not by normalization of the circulating IGF-I levels by treatment with IGF-I. These results suggest that insulin-deficiency in IDDM decreases osteoblastogenesis associated with inhibition of Wnt signaling through the increased expression of Sost and Dkk1 and the inhibition of Akt activation.

Curcumin suppresses increased bone resorption by inhibiting osteoclastogenesis in rats with streptozotocin-induced diabetes.

Curcumin is a potent inhibitor of the transcription factor activator protein-1 which plays an essential role in osteoclastogenesis. However, the effects of curcumin on bone metabolism have not been clarified in vivo. We reported herein the inhibitory effects of curcumin on the stimulated osteoclastic activity in insulin-dependent diabetes mellitus using rats with streptozotocin-induced diabetes. A dietary supplement of curcumin reversed the increase in levels of activity and mRNA of tartrate-resistant acid phosphatase (TRAP) and cathepsin K to control values. A histochemical analysis showed that the increase in TRAP-positive cells in the distal femur of the diabetic rats was reduced to the control level by the supplement. These results suggested that curcumin reduced diabetes-stimulated bone resorptive activity and the number of osteoclasts. When bone marrow cells were cultured with macrophage colony stimulating factor and receptor activator NF-kappaB ligand (RANKL), the increased activity to form TRAP-positive multinucleated cells and the increased levels of mRNA and protein of c-fos and c-jun in the cultured cells from diabetic rats decreased to control levels in the curcumin-supplemented rats. Similarly, the increased expression of c-fos and c-jun in the distal femur of the diabetic rats was significantly reduced by the supplement. These results suggested that curcumin suppressed the increased bone resorptive activity through the prevention of osteoclastogenesis associated with inhibition of the expression of c-fos and c-jun in the diabetic rats.

α-Adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy

The increases in activity of hepatic thymidylate synthetase and of thymidine kinase, which catalyze the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which had been given alpha-adrenoceptor antagonists (phenoxybenzamine and phentolamine) or adrenergic neuron blockers (guanethidine and reserpine). These suppressions were not observed with a beta-adrenoceptor antagonist (propranolol), or an anticholinergic agent (atropine methyl nitrate). The rise in the activity of the thymidylate-synthesizing enzymes was closely correlated with the increase in the DNA content of the liver. It is concluded that catecholamine regulates the increase in the activity of thymidylate synthetase and thymidine kinase, which are key enzymes in DNA synthesis in regenerating liver. It is also suggested that sympathetic nerves play an important role in liver regeneration.

Specific and sensitive determination of lipid hydroperoxides with chemical derivatization into 1-naphthyldiphenylphosphine oxide and high-performance liquid chromatography

Abstract A highly sensitive and specific high-performance liquid chromatographic (HPLC) method to determine the hydroperoxide level in biological samples involving the oxidation of 1-naphthyldiphenylphosphine into its oxide was developed. The detection limit of the method applied to the determination of cumene hydroperoxide was 1 pmol and the yield of the phosphine oxide based on the hydroperoxide in the concentration range from 0.2 μM to 1 mM was 85–100%. The linearity between the amount of the hydroperoxide and the HPLC peak height was excellent in a wide range (1 pmol to 5 nmol). The recoveries of cumene hydroperoxide from saline and homogenates of mouse brain, heart, kidney and liver were 88.5 ± 10, 83.3 ± 13, 75.3 ± 1.0, 46.3 ± 13.4 and 18.7 ± 10%, respectively. The recovery of exogenously added lecithin hydroperoxide from mouse liver homogenate was 52.4 ± 9.8%, while that from saline was 63.6 ± 5.4%. The peroxide levels in the brain, heart, kidney and liver of 5-week-old mice were determined to be 474 ± 170, 426 ± 183, 655 ± 121 and 239 ± 31 pmol/mg protein, respectively.

Retinoic acid repressed the expression of c-fos and c-jun and induced apoptosis in regenerating rat liver after partial hepatectomy

Retinoic acid (RA), which was injected within 4 h after partial hepatectomy (PH), inhibited DNA synthesis in regenerating liver. The inhibition was accompanied by apoptosis, evidenced by in situ end labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was obvious at 4 h and reached a maximum at 8 h after injection. Northern blot analysis revealed that RA repressed the expression of c-fos and c-jun at 15 and 30 min with the up-regulation of retinoic acid receptor gamma (RARgamma) and RARbeta at 2 h after PH. The transglutaminase II mRNA level and activity were increased by RA injection at 4 h and 8 h after PH, respectively. The mRNA levels of thymidylate synthase and thymidine kinase, which are rate determining enzymes of DNA synthesis, decreased in RA injected rats. No change was seen in the expression of p53 and p21WAF1/CIP1 which have been suggested to participate in the apoptosis process. These results suggest that RA exerts the antiproliferative activity only on the early stage of liver regeneration accompanied by the repression of c-fos and c-jun expression and induction of apoptosis.

Quercetin inhibited DNA synthesis and induced apoptosis associated with increase in c-fos mRNA level and the upregulation of p21WAF1CIP1 mRNA and protein expression during liver regeneration after partial hepatectomy

Quercetin, a widely distributed bioflavonoid, inhibited DNA synthesis in regenerating liver after partial hepatectomy. This inhibition was accompanied by apoptosis, evidenced by in situ end-labeling and gel electrophoresis of DNA fragmentation. Characteristic DNA fragmentation was detected as early as 2 h after injection. Northern blot analysis revealed that quercetin induced the increases in c-fos and p21WAF1CIP1 mRNA levels within 2 h. The expression of p21 protein was also enhanced, while p53 mRNA and protein levels were not affected by quercetin. These results suggest that quercetin-induced apoptosis is associated with the increase in c-fos mRNA level and the upregulation of p21 mRNA and protein expression, probably in a p53-independent pathway.

Effect of colchicine and vincristine on DNA synthesis in regenerating rat liver

The increases in the activities of hepatic thymidylate synthetase and thymidine kinase were significantly suppressed at 24 h after 70% partial hepatectomy in rats which had been administered a microtubule disrupter, colchicine or vincristine. The decrease of these enzymic activities was accompanied by a reduction of DNA content in 24 h regenerating liver. The immunoblotting assay showed that the depression of the thymidylate synthetase activity by the injection of colchicine or vincristine was due to the decrease of the enzyme protein. These results indicate that colchicine and vincristine inhibit the DNA synthesis during liver regeneration by inhibiting the induction of the key enzyme in DNA synthesis.

Effect of glucocorticoid on liver regeneration after partial hepatectomy in the rat.

The increase in activities of hepatic thymidylate synthetase (EC 2.1.1.45) and thymidine kinase (EC 2.7.1.21), which catalyse the formation of thymidylate through the de novo and salvage pathways, respectively, were significantly suppressed during liver regeneration in rats which were given glucocorticoids (hydrocortisone and dexamethasone) or indomethacin. These drugs also prevented the augment of hepatic DNA content in 24 h regenerating liver.

Methotrexate-induced apoptosis in hepatocytes after partial hepatectomy.

To investigate apoptosis induced by methotrexate in hepatocytes in vivo, rats received a single injection of methotrexate immediately after partial hepatectomy and apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and gel electrophoresis of DNA. Characteristic DNA fragmentation was obvious at 2 h and peaked at 4 h after partial hepatectomy with methotrexate injection. TUNEL-positive staining was observed in nuclei and nuclear fragments of hepatocytes in the methotrexate-injected liver (partial hepatectomy with methotrexate), with negligible background staining in the control (partial hepatectomy only) and in the methotrexate-injected normal (normal with methotrexate) rat liver. The involvement of the c-Jun N-terminal kinase (JNK) activator protein 1 (AP-1) pathway and p53 in apoptosis was also examined. The activity of JNK increased at 15 min and peaked at 1 h after partial hepatectomy. This increase was repressed by methotrexate injection. Western blot analysis showed that the levels of c-Fos and c-Jun protein expression, which increased at 1 h after partial hepatectomy, were also reduced by methotrexate. The levels of p53 protein were markedly increased after partial hepatectomy with methotrexate injection. The increase in p53 protein was followed by an up-regulation of p21(WAF1/CIP1) protein at 2 h after partial hepatectomy. These results suggested that the inhibition of the JNK-AP-1 pathway and concurrent up-regulation of p53 and p21(WAF1/CIP1) were involved in hepatocyte apoptosis induced by partial hepatectomy with methotrexate.

Impaired Expression of Noncollagenous Bone Matrix Protein mRNAs During Fracture Healing in Ascorbic Acid–Deficient Rats

In scorbutic patients, fractures are slow to heal because of impaired collagen synthesis. To investigate the influence of impaired collagen synthesis on the differentiation and proliferation of osteogenic and chondrogenic cells, we examined the expression of genes encoding bone matrix proteins, including osteonectin (ON), osteopontin (OPN), osteocalcin (OC), and matrix Gla protein (MGP), as differentiation markers for osteogenic and chondrogenic cells during fracture healing in Osteogenic Disorder Shionogi (ODS) rats, which have a hereditary defect in the ability to synthesize ascorbic acid (Asc). In ODS rats without Asc supplementation, intramembranous ossification was completely inhibited. Although a few fibroblast‐like cells expressing ON mRNA were observed, no OPN mRNA‐expressing cells were detected. During endochondral ossification, a small amount of metachromatic staining cartilage appeared at the fracture site, but there was no provisional calcification zone in the cartilage. Chondrocytes expressed ON and MGP mRNAs, but not OPN mRNA. When Asc was given to these rats, callus formation was soon detected around the fracture site, while OPN mRNA was expressed by differentiated osteoblasts and hypertrophic chondrocytes. Our data indicate that impaired collagen synthesis due to Asc deficiency inhibited the increase of ON and MGP mRNA‐expressing cells as well as the appearance of OPN mRNA‐expressing cells. Since OPN is considered to play an important role in normal and pathological mineralization, lack of OPN mRNA expression accompanying impaired collagen synthesis may have a role in defective mineralization and delayed fracture healing in scurvy.