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.

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.

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.

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.

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.