Fluvastatin improves osteoporosis in fructose-fed insulin resistant model rats through blockade of the classical mevalonate pathway and antioxidant action.

Feeding rats with a high-fructose diet induced insulin resistance, leading to hypertension or metabolic disorders. Although hypertension is known to accelerate osteoporosis, it is not obvious whether insulin resistance would accelerate osteoporosis. In this study, we evaluated whether osteoporosis might accelerate in fructose-fed rats (FFR), and examined the effect of fluvastatin through a blockade of the mevalonate pathway and an antioxidant action. Stimulation of recombinant receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) expressed by osteoblasts/ stromal cells and macrophage-colony stimulating factor (M-CSF) significantly increased TRAP-positive multinuclear osteoclasts and pit formation, accompanied by an increase in reactive oxygen species as assessed by dichlorodihydrofluorescein (DCF) staining. Interestingly, it was completely abolished by treatment with fluvastatin, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), but not pravastatin. These actions of fluvastatin were partially abolished by co-treatment with geranylgeranylpyrophosphate (GGPP), but not farnesylpyrophosphate (FPP). In the estrogen-deficient model by ovariectomy, FFR exhibited a decrease in bone mineral density, activation of osteoclasts, and an increase in urinary deoxypyridinoline. Importantly, the treatment of fluvastatin, but not pravastatin, attenuated FFR-induced osteoporosis. The present study demonstrates that fructose fed to rats induced insulin resistance and accelerated osteoporosis, while fluvastatin, but not pravastatin, significantly attenuated osteoclast differentiation and activation through a blockade of the classical mevalonate pathway and an antioxidant action, leading to prevention of osteoporosis.

Eicosapentaenoic acid supplementation for chronic hepatitis C patients during combination therapy of pegylated interferon alpha-2b and ribavirin.

Eicosapentaenoic acid (EPA) (1.8 g/day) was administered to 12 chronic hepatitis C patients receiving combination therapy of pegylated interferon (PEG-IFN) alpha-2b and ribavirin for 48 weeks (EPA group). Twelve patients were not administered EPA (control group). All patients also received vitamin E and C (300, 600 mg/day, respectively) during the therapy. Serum alanine aminotransferase improved to a normal level in 8 of 12 patients from the EPA group and 6 of 12 patients from the control group after 12 weeks. Lymphocyte counts decreased significantly after 8 weeks in the control group, but not the EPA group. T-helper (Th) 1 decreased after 4 weeks in the control group, but not in the EPA group (two-way ANOVA; P < 0.05). Th1/Th2 ratios were elevated in 9 of 12 patients in the EPA group, and 3 out of 12 in the control group (P < 0.05) after 8 weeks. After 12 weeks, the arachidonic acid/EPA molar ratio of erythrocyte membrane phospholipid correlated negatively with the leukocyte count (n = 24, r = -0.439, P < 0.05) and the neutrophil count (n = 24, r = -0.671, P < 0.02). The hemoglobin level improved after 48 weeks compared with 24 weeks in only the EPA group. These findings suggest that EPA supplementation may be useful in therapy for chronic hepatitis C.

Erratum: Low levels of linoleic acid and α-linolenic acid and high levels of arachidonic acid in plasma phospholipids are associated with hypertension.

[This corrects the article DOI: 10.3892/br.2017.1015.].

Low levels of linoleic acid and α-linolenic acid and high levels of arachidonic acid in plasma phospholipids are associated with hypertension.

Dietary fat is an important determinant in the development and progression of high blood pressure (BP), a major risk factor for cardiovascular diseases and mortality. The aim of the present study was to determine the association between plasma phospholipid fatty acids and hypertension in Japanese men. The plasma level of linoleic acid (LA) in the subjects with hypertension (systolic BP ≥140 mmHg and/or diastolic BP ≥90 mmHg) was identified to be significantly higher than that in the healthy controls. Following adjustment for age, body mass index, physical activity, smoking status, alcohol consumption, salt intake, and serum levels of glucose and hemoglobin A1c, higher plasma levels of LA and α-linolenic acid (ALA), and lower levels of arachidonic acid (AA) were significantly associated with a lower prevalence of hypertension. The odds ratio (OR) for the highest quartile (Q4) versus the lowest quartile (Q1) of LA was 0.17 (P=0.003), the OR for Q4 versus Q1 of ALA was 0.26 (P=0.042) and the OR for Q4 versus Q1 of AA was 2.04 (P=0.047). These results indicate that elevated levels of LA and ALA, and reduced levels of AA in the plasma prevent hypertension.

Increased cathepsin K and tartrate-resistant acid phosphatase expression in bone of streptozotocin-induced diabetic rats.

The effect of insulin-dependent diabetes mellitus (IDDM) on bone metabolism was evaluated using the streptozotocin (STZ)-induced diabetic rat 1 week after the induction of diabetes. The urinary excretion of cross-linked N-telopeptides of type I collagen (NTx) and deoxypyridinoline (Dpd) in diabetic rats increased to 3.6-fold and 1.2-fold the control level, respectively. The amount of hydroxyproline and calcium in the distal femur of diabetic rats significantly decreased to 76% and 90% of the control, respectively. The levels of serum osteocalcin and alkaline phosphatase (ALP) activity in the distal femur of the diabetic rats were significantly reduced to about 40% and 70% of the control levels, respectively. The decrease in the expression osteocalcin was observed in distal femur of the diabetic rats, although the level of ALP mRNA was unchanged. The activity and the mRNA level of tartrate-resistant acid phosphatase (TRAP) increased to 1.5- and 2.3-fold the control level, respectively, in distal femur of the diabetic rats. The activity, protein, and mRNA levels of cathepsin K of diabetic rats also elevated to about 2-, 2.3-, and 2-fold the control levels, respectively. These results suggest that IDDM contributes to bone loss through changes in gene expression of TRAP and cathepsin K in osteoclasts as well as osteocalcin in osteoblasts resulting in increased bone resorptive activity and decreased bone formation.

Arsenite induces apoptosis in hepatocytes through an enhancement of the activation of Jun N-terminal kinase and p38 mitogen-activated protein kinase caused by partial hepatectomy.

To investigate the effects of arsenite on cell proliferation and the signal transduction in hapatocytes in vivo, rats received a single injection of sodium arsenite immediately after partial hepatectomy. Characteristic DNA fragmentation was observed at 4h after the arsenite-injection in partially hepatectomized liver, while it was not detected either in the control (partial hepatectomy only) or arsenite-injected normal (without partial hepatectomy) liver. The effect of the arsenite-injection on the activation of extracellular signal-regulated kinase (ERK) was not observed in the normal or the partially hepatectomized liver. The activity of p38 mitogen-activated protein kinase (MAPK) markedly increased after 15min to 2h after the arsenite-injection in partially hepatectomized liver while no or a less increase was observed in the arsenite-injected normal or the control, respectively. The Jun N-terminal kinase (JNK) was activated to a maximal level, about six-fold the maximum of the control, at 15min after the injection with partial hepatectomy. The arsenite-injection markedly increased the phosphorylated forms of c-Jun and ATF-2 and the protein levels of c-Jun, p53 and p21(WAF1/CIP1) in the partially hepatectomized liver. These results suggested that arsenite induced apoptosis in the hepatocytes in vivo, through the enhancement of the activation of JNK and p38 MAPK caused by partial hepatectomy and the p53-dependent p21(WAF1/CIP1) protein expression.

Manganese-induced apoptosis in hepatocytes after partial hepatectomy.

To investigate the apoptosis induced by manganese (Mn) in hepatocytes in vivo, rats received a single injection of manganese chloride immediately after partial hepatectomy. Characteristic DNA fragmentation was observed at 4 h after partial hepatectomy with Mn-injection. The activation of caspase-3 by Mn-injection was detected as early as 30 min and peaked at 1 h after partial hepatectomy. The activity of Jun N-terminal kinase (JNK) increased to a maximal level, which was about 10-fold the maximal level of the control, at 15 min after partial hepatectomy and this increase was maintained for 4 h in Mn-injected rats, while a transient increase was observed at 1 h in the control. No effect of the Mn-injection on the activation of p38 mitogen-activated protein kinase (MAPK) was observed. Western blot analysis revealed that the injection of Mn markedly increased c-Jun and phosphorylated c-Jun protein levels at 1 h after partial hepatectomy. An increase in p53 was also observed at 30 min after the Mn-injection and followed by the upregulation of p21(WAF1/CIP1) protein expression at 2 h after partial hepatectomy. These results suggested that the activation of JNK and the upregulation of c-Jun, p53 and p21(WAF1/CIP1) were involved in the apoptosis of hepatocytes induced by partial hepatectomy with manganese.

n-3 polyunsaturated fatty acids stimulate osteoclastogenesis through PPARγ-mediated enhancement of c-Fos expression, and suppress osteoclastogenesis through PPARγ-dependent inhibition of NFkB activation.

n-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been reported to suppress osteoclastogenesis in vivo. In this study, the effect of PUFAs on receptor for activation of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis was examined using bone marrow-derived monocytes/macrophage precursor cells (BMMs) or bone marrow cells (BMCs) in vitro. EPA and DHA stimulated the osteoclastic differentiation of BMMs, but n-6 PUFAs, linoleic acid and arachidonic acid had no effect. The stimulation of osteoclastogenesis of BMMs by EPA and DHA was associated with enhancement of the gene expressions of c-Fos, tartrate-resistant acid phosphatase, cathepsin K and peroxisome proliferator-activated receptor-γ (PPARγ) and the protein levels of c-Fos, PPARγ and nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent-1 (NFATc1). The PPARγ agonists, rosiglitazone and GW1929, also stimulated the osteoclastogenesis of BMMs. The PPARγ antagonists, T0070907 and GW9662, inhibited the stimulations of osteoclastogenesis and c-Fos expression by EPA or DHA. However, EPA and DHA inhibited the osteoclastogenesis in BMCs including BMMs and mesenchymal stem cells (MSCs). This inhibition was associated with suppression of the expression of RANKL and nuclear factor-κB (NFκB)-regulating genes, cyclooxygenase 2, TNFα and IL-6 in BMCs and MSCs. The agonists and antagonists of PPARγ showed that the inhibitions of NFκB transcriptional activity and osteoclastogenesis by EPA and DHA were PPARγ-dependent. These results suggest that EPA and DHA directly act on BMMs and stimulate osteoclastogenesis through enhancing c-Fos expression mediated by PPARγ but suppress osteoclastogenesis through the PPARγ-dependent inhibition of NFκB activation of MSCs in BMCs.

Increase in osteoclastogenesis in an obese Otsuka Long-Evans Tokushima fatty rat model.

In the present study, the effects of obesity on bone metabolism were investigated using a hyperphagic and obese rat model, the Otsuka Long­Evans Tokushima fatty (OLETF) rat, which exhibits normal glycemic control at 8 weeks of age. Body weight, food intake, fat mass, markers of bone resorption, the activities of tartrate­resistant acid phosphatase (TRAP) and cathepsin K, the number of osteoclasts in the proximal tibia, and the serum C­terminal crosslinking telopeptide level were higher in OLETF rats than those in control rats (Long­Evans Tokushima Otsuka; LETO). However, no differences in markers of bone formation, alkaline phosphatase activity, the number of osteoblasts in the proximal tibia or the serum osteocalcin level were observed. mRNA and protein levels of c­fms, receptor for activation of nuclear factor­κB (RANK), RANK ligand (RANKL), TRAP and cathepsin K were significantly increased in OLETF rats, although those levels of macrophage colony­stimulating factor (M­CSF) and osteoprotegerin (OPG) were similar to those in LETO rats. The level of serum tumor necrosis factor α (TNFα), and that of TNFα mRNA in bone, increased in association with the activation of NFκB. Furthermore, a frequency analysis and a colony formation assay respectively showed that the number of osteoclast precursors and the number of colony­forming cells induced by M­CSF each increased in OLETF rats compared with the control group. These results suggested that hyperphagia­induced obesity with normal glycemic control induces the upregulation of osteoclastogenesis that is associated with an increase in the expression of c­fms, RANK and RANKL, which is induced by TNFα, via the activation of NFκB.

Apoptosis induced by 5-(N,N-hexamethylene)-amiloride in regenerating liver after partial hepatectomy.

The effects of a specific inhibitor of the Na+/H+ exchanger, 5-(N,N-hexamethylene)-amiloride (HMA), on liver regeneration after partial hepatectomy were investigated. A single injection of HMA inhibited DNA synthesis and caused apoptosis in regenerating liver. Characteristic DNA fragmentation was observed at 4 h after partial hepatectomy with HMA-injection. The activity of Jun N-terminal kinase (JNK) increased to a maximal level at 15 min after partial hepatectomy in HMA-injected rats, while it was not detected until 30 min in the control. Western blot analysis revealed that the injection of HMA markedly increased c-Jun and phosphorylated c-Jun protein levels at 30 min after partial hepatectomy. An increase in p53 was also observed at 30 min after the HMA-injection and was followed by the upregulation of p21WAF1/CIP1 protein expression at 1 h after partial hepatectomy. These results suggested that HMA induced apoptosis accompanied by the activation of JNK and the upregulation of c-Jun, p53 and p21WAF1/CIP1 expression at an early stage of liver regeneration.

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.

Jun N-terminal kinase activation and upregulation of p53 and p21(WAF1/CIP1) in selenite-induced apoptosis of regenerating liver.

To investigate apoptosis induced by selenite in hepatocytes in vivo, rats received a single injection of sodium selenite immediately after partial hepatectomy. Characteristic DNA fragmentation in gel electrophoresis and in situ end-labeling and the increase in caspase-3 activity were observed at 4 h after partial hepatectomy with selenite injection. The activation of Jun N-terminal kinase (JNK) was observed as early as 15 min and increased to about 10-fold the maximal level of the control at 1 and 2 h after partial hepatectomy in selenite-injected rats, while a transient increase was observed at 1 h in the control. Western blot analysis revealed that the c-Jun and the phosphorylated c-Jun protein markedly increased after 30 min and reached a maximal level at 1 and 2 h after partial hepatectomy with selenite injection, although c-Jun and a faint band of the phosphorylated c-Jun were observed after 1 h in the control. The levels of c-jun mRNA and c-Fos protein and mRNA in selenite-injected rats also increased more than in the control. The rise in the p53 protein level after partial hepatectomy with selenite injection was followed by the upregulation of p21(WAF1/CIP1) mRNA and protein expression. These results suggested that selenite induced apoptosis accompanied by the activation of caspase-3 and JNK and the upregulation of c-jun, c-fos, p53 and p21(WAF1/CIP1) at the early stage of liver regeneration.

Fish oil suppresses bone resorption by inhibiting osteoclastogenesis through decreased expression of M-CSF, PU.1, MITF and RANK in ovariectomized rats.

Previous studies have identified a positive correlation between the intake of n­3 fatty acids and bone mineral density in postmenopausal women. The aim of the present study was to determine the effects of fish oil on bone metabolism and to investigate the underlying mechanism using ovariectomized rats. Ovariectomized or sham­operated (sham) female rats were fed AIN­76A­based diets containing 5% corn or fish oil for 2 weeks. Fish oil was found to decrease the plasma levels of arachidonic and linoleic acids, but increased the levels of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids. Fish oil reversed the increased activity and number of osteoclasts, and decreased calcium (Ca) and hydroxyproline (Hyp) content of the proximal tibia to sham values without affecting the activity or number of osteoblasts. In addition, fish oil suppressed increases in the mRNA and protein levels of macrophage colony­stimulating factor (M­CSF), PU.1, microphthalmia-associated transcription factor (MITF), receptor for activation of NFκB (RANK) and RANK ligand (RANKL) and serum levels of tumor necrosis factor α (TNFα), interleukin­6 (IL-6) and prostaglandin E2 (PGE2). Fish oil was also found to suppress NFκB activation induced by ovariectomy. These results indicate that increases in plasma n­3 fatty acid levels by fish oil led to the suppression of NFκB activation and subsequent downregulation of TNFα, followed by suppression of M­CSF and RANKL. Dietary fish oil suppressed ovariectomy­stimulated osteoclastogenesis by inhibiting the expression of M­CSF, PU.1, MITF and RANK in the early stages of osteoclastogenesis, upstream of RANKL signaling.

Zinc supplementation inhibits the increase in osteoclastogenesis and decrease in osteoblastogenesis in streptozotocin-induced diabetic rats.

Zinc (Zn) has been shown to stimulate bone formation and inhibit osteoclastic bone resorption and osteoclastogenesis. However, the effects of Zn on bone metabolism in diabetic animals remain to be clarified in vivo. Here, the effects of Zn supplementation on bone metabolism, including osteoclastogenesis and osteoblastogenesis, were investigated using streptozotocine (STZ)-induced diabetic rats. Zn-supplemented water (7.5 mg/L) was given for 1 week to diabetic rats injected with STZ (30 mg/kg body weight) 1 week earlier. The Zn supplement prevented a decrease in the activity and mRNA of alkaline phosphatase (ALP), osteocalcin mRNA, and hydroxyproline and calcium levels, and an increase in the activity and mRNA of tartrate-resistant acid phosphatase (TRAP) and cathepsin K in the proximal tibia of diabetic rats. Histological analysis revealed that the Zn supplement inhibited the diabetes-induced increase and decrease in the number of osteoclasts and osteoblasts, respectively, in the metaphysis of the proximal tibia. The increase in mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP, and cathepsin K and decrease in the expression of Runx2, Dlx5, osterix, ALP, osteocalcin, and collagen were prevented by the supplement. The decrease in ß-catenin, phosphorylated GSK3ß, phosphorylated Akt, insulin-like growth factor 1 (IGF-1), and IGF-1 receptor (IGF-1R) protein levels in diabetic rats was also inhibited, although Zn did not affect the diabetes-increased gene and protein expression of Sost and Dkk1. These results suggested that Zn prevented the diabetes-induced increase in osteoclastogenesis and decrease in osteoblastogenesis by inhibiting RANK expression and stimulating IGF-1/IGF-1R/Akt/GSK3ß/ß-catenin signaling, respectively.

A crucial role for reactive oxygen species in macrophage colony-stimulating factor-induced RANK expression in osteoclastic differentiation.

Macrophage colony-stimulating factor (M-CSF) is essential for differentiation from hematopoietic precursor cells into osteoclasts. M-CSF transiently increased the intracellular level of reactive oxygen species (ROS) through an NADPH oxidase (Nox) and induced the expression of receptor for activation of nuclear factor-κB (RANK) in early-stage osteoclast precursor cells (c-fms+RANK-). Blocking of the activity of Nox with diphenylene iodonium inhibited ROS production, activation of extracellular signal-regulated kinase (ERK), and the expression of RANK, PU.1 and MITF. The suppression of Nox2, but not Nox1, expression by RNA interference inhibited ROS production and RANK expression. These results suggested that ROS produced in response to M-CSF via a process mediated by Nox2 acted as an intracellular signaling mediator for RANK expression through the activation of ERK and the expression of PU.1 and MITF in early-stage osteoclast precursor cells.

Ethanol increases osteoclastogenesis associated with the increased expression of RANK, PU.1 and MITF in vitro and in vivo.

Ethanol has been known to induce osteopenia. However, the cellular and molecular mechanisms responsible for its effect have not been well characterized. This study investigated the effects of ethanol on bone metabolism and osteoclastogenesis using rats fed an ethanol-containing liquid diet (35% of calories from ethanol) for 3 weeks. Ethanol increased the activities of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K, without affecting the levels of serum osteocalcin or bone alkaline phosphatase activity. Histological analysis showed an increased number of osteoclasts in the proximal tibia, but no significant change in the number of osteoblasts. The mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP and cathepsin K were significantly increased, although those of macrophage colony-stimulating factor and c-fms were unaltered. The mRNA and protein levels of PU.1 and microphthalmia-associated trascription factor (MITF) also increased. Further, the osteoclastic differentiation of bone marrow-derived macrophage/monocyte precursor cells (BMMs) in vitro was stimulated by ethanol. The increased osteoclastogenesis of BMMs was associated with increased levels of RANK, PU.1 and MITF expression, activated extracellular signal-regulated kinase (ERK), and reactive oxygen species (ROS). Higher lipid peroxide levels and lower glutathione levels were also observed in the serum of the ethanol-fed rats. These results suggested that ethanol promoted osteoclastogenesis by increasing RANK expression through increases in the production of ROS, activation of ERK and expression of PU.1 and MITF.

Vitamin C-deficiency stimulates osteoclastogenesis with an increase in RANK expression.

The effects of vitamin C (VC) on osteoclastogenesis were studied in vivo using ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rats and in vitro using bone marrow-derived monocyte/macrophage cells (BMMs). The results confirmed previous findings of increases in the number of osteoclasts and in bone resorption at 2 and 3 weeks, but not 1 week, of VC-deficiency in ODS rats. The mRNA and protein levels of receptor activator nuclear factor kappaB (RANK) ligand and osteoprotegerin, and the mRNA level of macrophage-colony stimulating factor (M-CSF) in the proximal tibia of VC-deficient rats did not differ from those in VC-supplemented control rats. However, the mRNA levels of RANK, c-fos and c-jun were significantly increased at as early as 1 week of VC-deficiency. These results suggested that VC-deficiency stimulated osteoclastogenesis by increasing RANK expression. The osteoclastic differentiation of BMMs was suppressed in the presence of VC. The suppressed osteoclastogenesis was associated with decreased levels of RANK, c-fos and c-jun. The pretreatment of BMMs with VC or PD 98059, a specific inhibitor of extracellular signal regulated kinase (ERK)-activating MEK1, decreased the expression of RANK induced by M-CSF. VC inhibited the M-CSF-induced activation of ERK. These results suggested that VC-deficiency increased osteoclastogenesis by increasing RANK expression mediated through the activation of ERK.

Administration of zinc inhibits osteoclastogenesis through the suppression of RANK expression in bone.

Zinc (Zn) has been known to inhibit osteoclastic bone resorption and stimulate osteoblastic bone formation. However, the mechanisms responsible for these effects have not been well characterized in vivo. Here, the effects of a dietary administration of Zn on osteoclastogenesis and osteoblastogenesis were investigated in Zn-adequate rats. The administration of Zn decreased the activities of bone tartrate-resistant acid phosphatase (TRAP) and cathepsin K, without affecting the serum osteocalcin level. Histological analysis showed a decrease in the number of osteoclasts with a normal number of osteoblasts in the metaphysis of the proximal tibia. The mRNA levels of receptor for activation of NF-κB (RANK), c-fos, c-jun, TRAP and cathepsin K were significantly decreased, although those of RANK ligand, macrophage colony-stimulating factor and c-fms were unaltered. The gene expression of bone morphogenic protein-2, Runx2, Dlx5, osterix, alkaline phosphatase, osteocalcin and collagen was not affected. The level of the RANK protein decreased, while the levels of the Runx2 and ß-catenin proteins were unchanged. Further, the osteoclastic differentiation of precursor cells in vitro was suppressed. The suppressed osteoclastogenesis was associated with decreased levels of reactive oxygen species, extracellular signal-regulated kinase (ERK) activation and RANK expression. A lower lipid peroxide level and a higher glutathione level were also observed. These results suggested that Zn-administration did not affect osteoblastogenesis but decreased osteoclastogenesis by inhibiting RANK expression through suppression of the production of reactive oxygen species and ERK activation in Zn-adequate rats.

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 3ß (GSK3ß), active ß-catenin and ß-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.

Zinc deficiency decreases osteoblasts and osteoclasts associated with the reduced expression of Runx2 and RANK.

The effects of Zinc(Zn)-deficiency on the function and differentiation of osteoblasts and osteoclasts were investigated in vivo using rats, which were fed a Zn-adequate (control) or Zn-free diet (ZD) or pair-fed a Zn-adequate diet (PF) for 3 weeks. Levels of Zn, insulin, insulin-like growth factor I (IGF-I), and osteoclacin in serum and the activities and numbers of osteoblasts and osteoclasts in bone decreased in ZD rats compared with the control and PF rats. The frequency analyses showed that the precursors of osteoblasts and osteoclasts decreased in bone marrow of ZD, but not PF, rats. The expression of receptor for activation of NF-κB (RANK) decreased with the Zn-deficiency, although RANK ligand, osteoprotegerin, macrophage colony-stimulating factor, and c-fms levels were unaltered. The protein level of a transcription factor MITF, but not PU.1, decreased. The expression of Runx2 decreased associated with the decrease in ß-catenin protein and the suppression of glycogen synthase kinase 3ß (GSK3ß) inhibition and Akt activation. The gene expression of the insulin receptor, IGF-I and the IGF-I receptor was decreased with a reduced level of transcription factor SP-1. These results suggested that a deficiency of Zn decreased osteoclastogenesis associated with the reduced expression of RANK through a decrease in MITF protein, and osteoblastogenesis associated with the reduced expression of Runx2 through the inhibition of Wnt/ß-catenin signaling via the suppression of GSK3ß inhibition and Akt activation preceded by the reduced level of SP-1 protein.