Tag Archives: animal

AMPK Activators: Lipoic Acid, Metformin, EGCG, Berberine, Resveratrol Can Inhibit Bone Resorption in Mice

Abstract

AMP kinase acts as a negative regulator of RANKL in the differentiation of osteoclasts.

AMP-activated protein kinase (AMPK) has been reported to stimulate differentiation and proliferation of osteoblasts, but the role of AMPK in the physiology of osteoclasts has not been investigated.
Osteoclasts were differentiated from mouse BMMϕs. TRAP-positive multinucleated cells were considered to be osteoclasts using TRAP staining, and resorption area was determined by incubation of cells on dentine discs. Signaling pathways were investigated using Western blotting and RT-PCR.
RANKL induced phosphorylation/activation of AMPK-α in BMMϕs and stimulated formation of TRAP-positive multinucleated cells. Pharmacological inhibition of AMPK with compound C and siRNA-mediated knockdown of AMPK-α1, the predominant α-subunit isoform in BMMϕs, increased RANKL-induced formation of TRAP-positive multinucleated cells and bone resorption via activation of the downstream signaling elements p38, JNK, NF-κB, Akt, CREB, c-Fos, and NFATc1. STO-609, an inhibitor of CaMKK, completely blocked the RANKL-induced activation of AMPK-α, but KN-93, an inhibitor of CaMK, did not. siRNA-mediated TAK1 knockdown also blocked RANKL-induced activation of AMPK-α. The AMPK activators metformin, (-)-epigallocatechin-3-gallate, berberine, resveratrol, and α-lipoic acid dose-dependently suppressed formation of TRAP-positive multinucleated cells and bone resorption.
AMPK negatively regulates RANKL, possibly by acting through CaMKK and TAK1. Thus, the development of AMPK activators may be a useful strategy for inhibiting the resorption of bone that is stimulated under RANKL-activated conditions.

Lee YS, Kim YS, Lee SY, Kim GH…
Bone Nov 2010
PMID: 20696287

Lipoic Acid Prevents Steroid-Induced Bone Death in Rabbits

Abstract

Lipoic acid prevents steroid-induced osteonecrosis in rabbits.

The objective of this study was to investigate in vivo effects of lipoic acid (LA) in preventing steroid-induced osteonecrosis and the possible pathway in a rabbit model. Sixty rabbits were divided into 2 groups: rabbits were intraperitoneally injected with LA aqueous solution at 36 mg/kg of body weight per day for 4 weeks in Group A and rabbits were injected with physiologic saline (PS) as a control in Group B. At 2 weeks after starting treatment, they were intramuscularly injected once with 20 mg/kg of methylprednisolone acetate (MPSL). The femora were histopathologically examined for the presence of osteonecrosis. The plasma levels of total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), glutathione (GSH), endothelin (ET) and malondialdehyde (MDA) were assayed at 2 weeks after the injection of MPSL. The incidence of osteonecrosis was significantly higher in Group B (73.1%) than in Group A (20.8%). The GSH level was higher in Group A than in Group B after the LA injection. The plasma MDA and ET levels were lower in Group A than in Group B at 2 weeks after the MPSL administration. Lipoic acid can prevent the development of steroid-induced osteonecrosis in rabbits. Inhibited oxidative stress and amendment of vascular endothelial dysfunction is a possible mechanism for this effect.

Lu BB, Li KH
Rheumatol. Int. Jun 2012
PMID: 21431293

Lipoic Acid Increases Bone Mass and May Reduce Resorption in Mice

Abstract

Lipoic acid increases the expression of genes involved in bone formation in mice fed a high-fat diet.

Antioxidant lipoic acid (LA) has been reported to have a potential prophylactic effect on bone loss induced by high-fat diet (HFD). The aim of this work was to examine the hypothesis that LA decreases bone resorption-related gene expression and increases bone formation-related gene expression in HFD-fed mice, preventing a shift in the bone metabolism balance toward resorption. Male C57BL/6 mice were fed a normal diet, HFD, or HFD plus 0.1% LA for 12 weeks. The bone metabolism-related genes differentially expressed between mice fed HFD and those fed HFD supplemented with LA were identified through complementary DNA microarray. The supplemental LA significantly increased bone mineral density and bone antioxidant capacity in mice fed HFD (P < .05). Compared with the HFD-fed mice, LA induced the decreased expression of genes associated with bone resorption, such as Mmp9 (1.9-fold) and Ctsk (2.3-fold), and increased those genes associated with bone formation, such as Col1a1 (1.3-fold) and Alp1 (1.5-fold). Furthermore, LA upregulated many genes involved in the Igf signaling pathway, such as Igf-1 (increased 1.7-fold), and downregulated genes involved in the p53 apoptotic pathway, such as p53 (decreased 2.3-fold), thus attenuating the HFD-induced inhibition of bone formation. Lipoic acid induced upregulation of Il12a (2.1-fold) and downregulation of Tgfbr1 (4.3-fold) and Il17a (11.3-fold), which may reduce bone resorption. In summary, LA supplementation during HFD could affect bone density, altering gene expression.

Xiao Y, Cui J, Shi Y, Le G
Nutr Res Apr 2011
PMID: 21530805

Lipoic Acid + Vitamin C Inhibit Bone Resorption in Rats

Abstract

Therapeutic effects of alpha lipoic acid and vitamin C on alveolar bone resorption after experimental periodontitis in rats: a biochemical, histochemical, and stereologic study.

Alpha lipoic acid (ALA) and vitamin C (Vit-C) are very important and powerful antioxidants that have been used for the treatment of many diseases. The present study aims to investigate the role of ALA and Vit-C substances in the treatment of alveolar bone resorption in periodontal diseases.
Thirty-six male Wistar albino rats were randomly divided into four groups as follows: 1) control rats; 2) rats with experimental periodontitis (PED); 3) rats with PED treated with ALA (ALA); and 4) rats with PED treated with ALA+Vit-C (ALA+Vit-C). PED was simulated by placing ligatures around the neck of teeth for 5 weeks. After ligature removal, the PED group was given a single intragastric dose of 1 mL saline, and the ALA and ALA+Vit-C groups were treated with an intragastric dose of 50 mg/kg ALA and ALA+Vit-C for 15 days, respectively. Levels of serum bone alkaline phosphatase (B-ALP) and myeloperoxidase (MPO) activity in gingival tissues were analyzed. To evaluate the osteoclast activation, expression of activated receptor activator nuclear factor-kappa B ligand (RANKL) and bone density index (BDI) were determined stereologically in the bone sections obtained from the mandibles of the rats.
The results showed statistically significant differences between the PED group and groups treated with antioxidant according to B-ALP, MPO, RANKL, and BDI values (P <0.05). ALA and ALA+Vit-C treatments showed beneficial effects on the mesial/distal periodontal bone support at the ligature-induced periodontitis tooth areas.
This study shows that ALA and Vit-C treatment provides therapeutic effects on inhibition of alveolar bone resorption and periodontal tissue destruction.

Akman S, Canakci V, Kara A, Tozoglu U…
J. Periodontol. May 2013
PMID: 22702517

Review: Depression and Bone Mass

Abstract

Depression and bone mass.

Although it has been repeatedly suggested that low bone mineral density (BMD) is disproportionately prevalent among patients with depressive disorders, so far depression has not been officially acknowledged as a risk factor for osteoporosis. In a recent meta-analysis comparing depressed with nondepressed individuals we report that BMD is lower in depressed than nondepressed subjects. The association between depression and BMD is stronger in women than men, and in premenopausal than postmenopausal women. Only women psychiatrically diagnosed for major depression display significantly low BMD; women diagnosed by self-rating questionnaires do not. Using a mouse model for depression, we demonstrate a causal relationship between depressive-like behavior and bone loss. The depression-induced bone loss is associated with increases in skeletal norepinephrine and serum corticosterone levels. Bone loss, but not the depressive behavior, could be prevented by a beta-blocker. Hence, depression appears as a significant risk factor for low BMD, causing bone loss through stimulation of the sympathetic nervous system.

Bab IA, Yirmiya R
Ann. N. Y. Acad. Sci. Mar 2010
PMID: 20392233

Lipoic Acid Inhibits Resorption from Adrenaline

Abstract

β-Adrenergic signaling stimulates osteoclastogenesis via reactive oxygen species.

Sympathetic signaling regulates bone resorption through receptor activator of nuclear factor-κB ligand (RANKL) expression via the β-adrenergic receptor (β-AR) on osteoblasts. Reactive oxygen species (ROS) are known as one type of osteoclast regulatory molecule. Here we show that an antioxidant, α-lipoic acid (α-LA), treatment prevent the β-adrenergic signaling-induced bone loss by suppressing osteoclastogenesis, and sympathetic signaling directly regulates osteoclastogenesis through β2-AR expressed on osteoclasts via intracellular ROS generation. In an in vitro study, the β-AR agonist isoprenaline increased intracellular ROS generation in osteoclasts prepared from bone marrow macrophages (BBMs) and RAW 264.7 cells. Isoprenaline enhanced osteoclastogenesis through β2-AR expressed on BMMs and RAW 264.7 cells. The antioxidant α-LA inhibited isoprenaline-enhanced osteoclastogenesis. Isoprenaline increased the expression of osteoclast-related genes such as nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1, tartrate-resistant acid phosphatase, and cathepsin K on osteoclasts. α-LA also inhibited isoprenaline-induced increases of these gene expressions. These in vitro results led to the hypothesis that β-adrenergic signaling directly stimulates osteoclastogenesis via ROS generation. In an in vivo study, isoprenaline treatment alone caused oxidative damage in local bone and reduced bone mass because of an increase in bone resorption, and, in α-LA-treated mice, isoprenaline did not increase tibial osteoclast number even though the RANKL-to-osteoprotegerin ratio increased. These in vitro and in vivo results indicate that β-adrenergic signaling, at least in part, directly stimulates osteoclastogenesis through β2-AR on osteoclasts via ROS generation.

Kondo H, Takeuchi S, Togari A
Am. J. Physiol. Endocrinol. Metab. Mar 2013
PMID: 23169789

Lipoic Acid Prevents the Bone Inhibition and Resorption from a High Fat Diet and Dyslipidemia in Mice

Abstract

Dyslipidemic high-fat diet affects adversely bone metabolism in mice associated with impaired antioxidant capacity.

The present study examined impacts of dyslipidemic high-fat diet on the bone antioxidant system and bone metabolism in growing mice. Furthermore, the relationship was studied between them.
Male C57BL/6 mice (4 wk old) were fed with normal diet, high-fat diet (HFD), or HFD supplemented with 0.1% antioxidant lipoic acid (LA). After 13-wk feeding, the markers of plasma lipids status, bone metabolism in plasma and in urine, and femora oxidative stress were measured. To provide molecular evidence for abnormal bone metabolism affected by HFD, bone cell-specific mRNA levels were tested by real-time quantitative polymerase chain reaction. Moreover, insulin-like growth factor I and tumor necrosis factor-alpha in plasma and their mRNA levels in femur were measured.
The feeding dyslipidemic HFD induced both inhibitory bone formation reactions and enhancement of bone resorption reactions, accompanied by impaired bone antioxidant system, low levels of insulin-like growth factor I in plasma and in bone, and high levels of tumor necrosis factor-alpha in plasma but not in bone. In contrast, these alternatives were prevented completely or partially in mice fed LA supplement. Further, plasma propeptide of І collagen C-propeptide as a marker of bone formation was positively correlated with both total antioxidant capacity (r=0.683, P<0.001) and reduced glutathione/oxidized glutathione ratio (r=0.565, P<0.003) of bone. Cross-linked N-telopeptides of bone type І collagen as a marker of bone resorption was negatively correlated with both total antioxidant capacity (r=-0.753, P<0.001) and glutathione/oxidized glutathione ratio (r=-0.786, P<0.001).
Dyslipidemia induces impaired bone antioxidant system. Oxidative stress could be an important mediator of hyperlipidemia-induced bone loss.

Xiao Y, Cui J, Li YX, Shi YH…
Nutrition Feb 2011
PMID: 20392601

Lipoic Acid Protects from Radiation-Induced Bone Loss in Mice

Abstract

Oxidative stress and gamma radiation-induced cancellous bone loss with musculoskeletal disuse.

Exposure of astronauts in space to radiation during weightlessness may contribute to subsequent bone loss. Gamma irradiation of postpubertal mice rapidly increases the number of bone-resorbing osteoclasts and causes bone loss in cancellous tissue; similar changes occur in skeletal diseases associated with oxidative stress. Therefore, we hypothesized that increased oxidative stress mediates radiation-induced bone loss and that musculoskeletal disuse changes the sensitivity of cancellous tissue to radiation exposure. Musculoskeletal disuse by hindlimb unloading (1 or 2 wk) or total body gamma irradiation (1 or 2 Gy of (137)Cs) of 4-mo-old, male C57BL/6 mice each decreased cancellous bone volume fraction in the proximal tibiae and lumbar vertebrae. The extent of radiation-induced acute cancellous bone loss in tibiae and lumbar vertebrae was similar in normally loaded and hindlimb-unloaded mice. Similarly, osteoclast surface in the tibiae increased 46% as a result of irradiation, 47% as a result of hindlimb unloading, and 64% as a result of irradiation + hindlimb unloading compared with normally loaded mice. Irradiation, but not hindlimb unloading, reduced viability and increased apoptosis of marrow cells and caused oxidative damage to lipids within mineralized tissue. Irradiation also stimulated generation of reactive oxygen species in marrow cells. Furthermore, injection of alpha-lipoic acid, an antioxidant, mitigated the acute bone loss caused by irradiation. Together, these results showed that disuse and gamma irradiation, alone or in combination, caused a similar degree of acute cancellous bone loss and shared a common cellular mechanism of increased bone resorption. Furthermore, irradiation, but not disuse, may increase the number of osteoclasts and the extent of acute bone loss via increased reactive oxygen species production and ensuing oxidative damage, implying different molecular mechanisms. The finding that alpha-lipoic acid protected cancellous tissue from the detrimental effects of irradiation has potential relevance to astronauts and radiotherapy patients.

Kondo H, Yumoto K, Alwood JS, Mojarrab R…
J. Appl. Physiol. Jan 2010
PMID: 19875718 | Free Full Text

Lipoic Acid Inhibits Osteoclasts and Bone Loss from Inflammation in Mouse Cells

Abstract

alpha-Lipoic acid inhibits inflammatory bone resorption by suppressing prostaglandin E2 synthesis.

alpha-Lipoic acid (LA) has been intensely investigated as a therapeutic agent for several pathological conditions, including diabetic polyneuropathy. In the present study, we examined the effects of LA on osteoclastic bone loss associated with inflammation. LA significantly inhibited IL-1-induced osteoclast formation in cocultures of mouse osteoblasts and bone marrow cells, but LA had only a marginal effect on osteoclastogenesis from bone marrow macrophages induced by receptor activator of NF-kappaB ligand (RANKL). LA inhibited both the sustained up-regulation of RANKL expression and the production of PGE2 induced by IL-1 in osteoblasts. In addition, treatment with either prostaglandin E2 (PGE2) or RANKL rescued IL-1-induced osteoclast formation inhibited by LA or NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, in cocultures. LA blocked IL-1-induced PGE2 production even in the presence of arachidonic acid, without affecting the expression of COX-2 and membrane-bound PGE2 synthase. Dihydrolipoic acid (the reduced form of LA), but not LA, attenuated recombinant COX-2 activity in vitro. LA also inhibited osteoclast formation and bone loss induced by IL-1 and LPS in mice. Our results suggest that the reduced form of LA inhibits COX-2 activity, PGE2 production, and sustained RANKL expression, thereby inhibiting osteoclast formation and bone loss in inflammatory conditions.

Ha H, Lee JH, Kim HN, Kim HM…
J. Immunol. Jan 2006
PMID: 16365401 | Free Full Text

Lipoic Acid Suppresses Osteoclasts

Abstract

Alpha-lipoic acid suppresses osteoclastogenesis despite increasing the receptor activator of nuclear factor kappaB ligand/osteoprotegerin ratio in human bone marrow stromal cells.

Growing evidence has shown a biochemical link between increased oxidative stress and reduced bone density. Although alpha-lipoic acid (alpha-LA) has been shown to act as a thiol antioxidant, its effect on bone cells has not been determined. Using proteomic analysis, we identified six differentially expressed proteins in the conditioned media of alpha-LA-treated human bone marrow stromal cell line (HS-5). One of these proteins, receptor activator of nuclear factor kappaB ligand (RANKL), was significantly up-regulated, as confirmed by immunoblotting with anti-RANKL antibody. ELISA showed that alpha-LA stimulated RANKL production in cellular extracts (membranous RANKL) about 5-fold and in conditioned medium (soluble RANKL) about 23-fold, but had no effect on osteoprotegerin (OPG) secretion. Despite increasing the RANKL/OPG ratio, alpha-LA showed a dose-dependent suppression of osteoclastogenesis, both in a coculture system of mouse bone marrow cells and osteoblasts and in a mouse bone marrow cell culture system, and reduced bone resorption in a dose-dependent manner. In addition, alpha-LA-induced soluble RANKL was not inhibited by matrix metalloprotease inhibitors, indicating that soluble RANKL is produced by alpha-LA without any posttranslational processing. In contrast, alpha-LA had no significant effect on the proliferation and differentiation of HS-5 cells. These results suggest that alpha-LA suppresses osteoclastogenesis by directly inhibiting RANKL-RANK mediated signals, not by mediating cellular RANKL production. In addition, our findings indicate that alpha-LA-induced soluble RANKL is not produced by shedding of membranous RANKL.

Koh JM, Lee YS, Byun CH, Chang EJ…
J. Endocrinol. Jun 2005
PMID: 15930166 | Free Full Text