• n-3 fatty acids;
  • osteoporosis;
  • osteoclasts;
  • NF-κB;


  1. Top of page
  2. Abstract
  7. Acknowledgements

The mechanisms of action of dietary fish oil (FO) on osteoporosis are not fully understood. This study showed FO decreased bone loss in ovariectomized mice because of inhibition of osteoclastogenesis. This finding supports a beneficial effect of FO on the attenuation of osteoporosis.

Introduction: Consumption of fish or n-3 fatty acids protects against cardiovascular and autoimmune disorders. Beneficial effects on bone mineral density have also been reported in rats and humans, but the precise mechanisms involved have not been described.

Methods: Sham and ovariectomized (OVX) mice were fed diets containing either 5% corn oil (CO) or 5% fish oil (FO). Bone mineral density was analyzed by DXA. The serum lipid profile was analyzed by gas chromatography. Receptor activator of NF-κB ligand (RANKL) expression and cytokine production in activated T-cells were analyzed by flow cytometry and ELISA, respectively. Osteoclasts were generated by culturing bone marrow (BM) cells with 1,25(OH)2D3. NF-κB activation in BM macrophages was measured by an electrophoretic mobility shift assay.

Results and Conclusion: Plasma lipid C16:1n6, C20:5n3, and C22:6n3 were significantly increased and C20:4n6 and C18:2n6 decreased in FO-fed mice. Significantly increased bone mineral density loss (20% in distal left femur and 22.6% in lumbar vertebrae) was observed in OVX mice fed CO, whereas FO-fed mice showed only 10% and no change, respectively. Bone mineral density loss was correlated with increased RANKL expression in activated CD4+ T-cells from CO-fed OVX mice, but there was no change in FO-fed mice. Selected n-3 fatty acids (docosahexaenoic acid [DHA] and eicosapentaenoic acid [EPA]) added in vitro caused a significant decrease in TRACP activity and TRACP+ multinuclear cell formation from BM cells compared with selected n-6 fatty acids (linoleic acid [LA] and arachidonic acid [AA]). DHA and EPA also inhibited BM macrophage NF-κB activation induced by RANKL in vitro. TNF-α, interleukin (IL)-2, and interferon (IFN)-γ concentrations from both sham and OVX FO-fed mice were decreased in the culture medium of splenocytes, and interleukin-6 was decreased in sham-operated FO-fed mice. In conclusion, inhibition of osteoclast generation and activation may be one of the mechanisms by which dietary n-3 fatty acids reduce bone loss in OVX mice.


  1. Top of page
  2. Abstract
  7. Acknowledgements

It is well established that postmenopausal osteoporosis represents a major health and economic burden in our fast growing elderly population. Estrogen deficiency caused by either natural or surgical menopause predisposes women to develop osteoporosis. Current therapies for treating osteoporosis include estrogen and/or hormone replacement therapies (ERT and/or HRT), bisphosphonates, calcitonin, and raloxifene, as well as calcium and vitamin D3 supplementation. These therapies are indeed effective in preventing bone loss caused by menopause, but some are accompanied by adverse side effects, such as uterine bleeding, carcinogenesis, and cardiovascular disease.(1–7) Therefore, diet therapy and lifestyle changes that minimize bone loss in postmenopausal women would be very helpful to decrease the necessity for drug therapy to prevent osteoporosis.

Recently, there has been increasing evidence that in addition to lack of calcium and vitamin D3, deficiency of certain fatty acids in the diet may also contribute to bone loss.(8–10) Interestingly, essential fatty acid-deficient animals were found to develop severe osteoporosis coupled with increased renal and arterial calcification.(9) Although dietary supplementation of calcium, γ-linolenic acid (GLA, n-6), and eicosapentaenoic acid (EPA, n-3) in humans has been reported to decrease bone turnover and increase bone mineral density (BMD) in the femur and lumbar bones,(11–13) these findings have not been confirmed nor has the mechanism involved in the prevention of bone loss by n-3 fatty acids been determined. Studies on animals treated with different fatty acids have reported an influence on bone metabolism, including an increase in periosteal bone formation.(14,15) One of the possible mechanisms by which fish oil (FO) exerts its protective action against bone loss is decreased urinary calcium loss,(16) but the detailed cellular mechanisms of the beneficial effects of dietary FO on prevention of bone loss are not yet fully understood. In particular, the precise role of n-3 fatty acid intake in the prevention of bone loss has not been fully studied as yet in an ovariectomized animal model.

It is known that dietary sources of long-chain n-3 fatty acids are essential for maintaining optimum health, because mammals cannot synthesize fatty acids with double bonds past the Δ9 position. Thus, dietary intake of essential fatty acids has far-reaching consequences on membrane composition in all cells in the body and particularly on the production of inflammatory cytokines by immune cells such as macrophages and T-cells. Epidemiological studies have documented low rates of cardiovascular disease along with improvements in triglyceride and high-density lipoprotein (HDL) levels, blood pressure, indices of inflammation, and other cardiovascular risk factors with even moderate fish consumption.(17–19) We and others have described the protective role of n-3 fatty acids with or without restricting calories on autoimmune renal diseases in animal models.(20–23) We further observed that inhibition of renal disease in B/W mice was associated with decreased expression of proinflammatory cytokines (interleukin [IL]-1, IL-6, and TNF-α).(24–26) n-3 fatty acids also have suppressive effects on lymphocyte proliferation.(27) Because IL-1, IL-6, and TNF-α are closely linked to osteoclastogenesis and T-cells play a critical role in the pathogenesis of osteoporosis,(28) we hypothesize that dietary n-3 fatty acids can prevent bone loss induced by ovariectomy through regulating T-cell function and cytokine production, thereby inhibiting activation and maturation of osteoclasts. In this study, we examined the effects of dietary fatty acids on bone mass in OVX mice and compared the effects of selected n-3 and n-6 fatty acids on osteoclastogenesis in vitro.


  1. Top of page
  2. Abstract
  7. Acknowledgements


RANKL antibody was from R&D Systems (Minneapolis, MN, USA). All other antibodies were from BD Pharmingen (San Diego, CA, USA). Diet components, including corn oil (CO) and FO (menhaden oil), were from ICN Biomedicals (Aurora, OH, USA). RPMI 1640 medium was from Cellgro (Herndon, VA, USA). Fetal calf serum was from Hyclone (Logan, UT, USA). All other media components were from GIBCO (Invitrogen Corp., Carlsbad, CA, USA). 1,25(OH)2D3 was from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Fatty acids were from Matreya, Inc. (State College, PA, USA). The BCA kit for protein determination was from Pierce Chemical Co. (Rockford, IL, USA). TRACP solution was No. 387 from Sigma Chemical Co. (St Louis, MO, USA). [32P]-ATP was from Perkin Elmer-NEN (Boston, MA, USA). Oligonucleotides for NF-κB and AP-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). T4 polynucleotide kinase and Sephadex G-25 M columns were from Promega (Madison, WI, USA). Poly [d(I-C)] was from Roche Diagnostics (Indianapolis, IN, USA). All other chemicals were highest grade available from Sigma Chemical Co.


Eight-week-old female Balb/c mice were obtained from Jackson Laboratories and housed, five mice per cage, in a temperature-controlled room at 24°C on a 12:12 h dark-light cycle in the laboratory animal care facility at the University of Texas Health Science Center at San Antonio. The animals were fed American Institute of Nutrition (AIN)-76 based semipurified diets differing only in the fat provided (Table 1).(26) Diets contained either 5% CO or 5% FO + 0.5% CO to prevent essential fatty acid deficiency. The fatty acid composition of the oils used in the diets is given in Table 2. CO contains 0.8 g/kg α-tocopherol, and 0.2 g/kg was added. FO was supplemented with 1 g/kg α-tocopherol to match the concentration in the CO. To further protect these oils from oxidation, 0.2 g/kg of tert-butyl hydroquinone (TBHQ) was added to both CO and FO as previously described.(21) After 2 months of feeding, mice were either sham-operated or ovariectomized (OVX). Sixteen weeks after surgery, mice were subjected to DXA to determine bone density and body composition. One week later, they were killed, bones were fixed for histological analysis, and splenocytes were isolated. All procedures were conducted in compliance with the guiding principles in the “Care and Use of Animals” published by the American Journal of Physiology and were approved by the Institutional Animal Care and Use Committee.

Table Table 1. Diet Ingredients (g/kg)
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Table Table 2. Fatty Acid Composition of Oils (% of Total)
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Fatty acid analysis

Blood was collected by retro-orbital bleeding under anesthesia and centrifuged to obtain plasma. Plasma total lipids were extracted by the method of Bligh and Dyer(29) using chloroform-methanol (1:2) and chloroform-methanol-water (1:2:0.8). Extractions were performed in an atmosphere of nitrogen, and butylated hydroxy toluene (BHT) was added to prevent oxidation during processing. The organic phase containing the total lipid extracts was evaporated under a stream of nitrogen and the residue methylated according to the method of Kates.(30) Fatty acid methyl esters were separated and quantified by gas-liquid chromatography (GLC) using a Hewlett-Packard 5890A series II gas chromatograph, equipped with a DB225MS capillary column (J&W Scientific, Folsom, CA, USA) and a flame ionization detector (FID). The injector and detector port temperatures were 225°C and 250°C, respectively. The oven temperature was maintained at 170°C for 1 minute and then increased to 215°C at the rate of 5°C/min. Helium was used as the carrier gas. The running time of each sample was approximately 36 minutes. The fatty acid methyl esters were identified by comparison of retention times with fatty acid methyl ester standard (68A) from Nu Chek (Elysian, MN, USA). Quantification was done by an integrator (Hewlett-Packard 3396 series II) attached to the GLC machine. Oil samples were treated similarly beginning at the methylation step.

BMD analysis

Bone mineral content (BMC) and BMD were measured using a Lunar PIXImus (General Electric) mouse bone densitometer, and data analysis was done with PIXImus software.(31–33) During the measurements, the animals were anesthetized and were lying in a prone position with posterior legs maintained in external rotation with tape. The hip, knee, and ankle articulations were in 90° flexion. The measurements were made at the distal end of the left femur (DLF; knee joint), including cancellous (trabecular) bone, in a region of the bone shaft next to the end of the bone (CLF), including cortical bone, and in lumbar vertebrae (L1-L4).

Flow cytometric analysis of splenocyte subsets and RANKL expression on T-cells

Single cell suspensions of spleen were prepared as described previously.(34) The Fc receptor on the splenocytes was blocked with anti-CD16/CD32. Aliquots of 105 cells were incubated with phycoerythrin (PE)-labeled anti-CD4, fluorescein isothiocyanate (FITC)-labeled anti-CD8 or PE-labeled anti-CD19. For RANKL staining, 105 cells were incubated with FITC-labeled anti-CD4 and anti-CD8 monoclonal antibodies and biotinylated anti-RANKL antibody. After washing twice, cells were further incubated with streptavidin-conjugated PE. The samples were analyzed with a FACScan (Becton Dickinson) flow cytometer using Cell Quest software.

Histological analysis of lumbar vertebrae

The vertebrae were fixed in formaldehyde and then sectioned and stained with H&E. The bone structure was assessed under a light microscope.

Cytokine concentration

Cytokines released into the culture medium were measured in splenocytes cultured with and without Con-A activation (2.5 μg/ml, 48 h) by standard ELISA techniques as described previously.(35) In brief, microtiter plates were coated with purified anti-cytokine antibodies overnight at 4°C and washed. Samples were added followed by incubation at room temperature. The plates were washed, and biotinylated anti-cytokine antibodies were added. After incubation and washing, streptavidin-alkaline phosphatase (AKP) conjugate was added. The plates were again incubated and washed, and the chromogen substrate was added. The plates were incubated at room temperature to achieve the desired maximum absorbance and read at 410 nM in an ELISA reader.

Osteoclastogenesis in vitro

Bone marrow cells were isolated from whole bone marrow of 4- to 6-week-old mice and cultured in tissue culture dishes in α-MEM containing 10% FBS, at 37°C in 5% CO2. After culturing for 2 h, the adherent cells were discarded. Nonadherent cells were plated at 1 × 106/well in 48-well tissue culture plates in the presence of 10−8 M 1,25(OH)2D3 and cultured for 7 days. One-half of the media and 1,25(OH)2D3 were changed on days 2 and 4. The fatty acids EPA, docosahexaenoic acid (DHA), linoleic acid (LA), arachidonic acid (AA), a combination of EPA and DHA, or a combination of LA and AA in ethanol were added on day 4.

TRACP staining

After 7 days in culture, adherent cells were fixed, and TRACP staining was performed using TRACP solution 387 from Sigma Chemical Co. according to the manufacturers instructions. TRACP+ cells appeared dark red. TRACP+ multinucleated cells with three or more nuclei were considered osteoclastic cells. For the cell-free TRACP assay, cells were lysed in lysis buffer (0.2% NP-40 in distilled water), and TRACP solution was added. TRACP activity was determined by measuring absorbance at 405 nm on a microplate reader (BioRad Laboratories, Hercules, CA, USA).

Electrophoretic mobility shift assay

Nuclear extracts were prepared from bone marrow-derived macrophages as described previously,(35) and protein concentration was determined using a standard BCA kit. EMSA was performed as described(35) using a DNA probe (5′-AGTTGAGGGGACTTTCCCAGG C-3′) containing the NF-κB binding site (italics). For specific and nonspecific binding analysis, 100 ng of unlabeled double-stranded NF-κB and unlabeled double-stranded AP-1 oligonucleotide were added before adding labeled probes.

Statistical analysis

The basic hypothesis concerns the main effect of ovariectomy on bone density and related parameters and how it is influenced by the dietary source of oil. This results in a two-factor ANOVA with two levels of diet (CO or FO) as one factor and two levels of surgery (sham or ovariectomy) as the other factor. Student's t-test was used to determine individual differences between pairs of groups or Newman-Kuel's test was used to compare all groups with one another.


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  2. Abstract
  7. Acknowledgements

Effects of dietary fatty acids on plasma fatty acid content

The fatty acid content of plasma from mice on different diets is shown in Table 3. LA (C18:2, n-6) and AA (C20:4, n-6) content was significantly decreased, and palmitoleic (C16:1), EPA (C20:5, n-3), and DHA (C22:6, n-3) content was significantly increased in plasma from FO-fed mice. Ovariectomy significantly increased AA content in plasma from CO-fed mice and DHA content in plasma from FO-fed mice.

Table Table 3. Effect of Diet on Plasma Fatty Acid Composition
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Effects of varying dietary fatty acids on the decrease in uterine weight in ovariectomized mice

Uterine weights were significantly lower in both OVX groups (CO: −72.7 ± 3.4%; FO: −58.3 ± 3.2%; p < 0.05) compared with sham-operated groups, indicating that the mice were estrogen deficient. Although significantly lower than in the FO-fed sham-operated group, the uterine weight in FO-fed OVX mice was about 40% higher than that in CO-fed OVX mice, suggesting that an estrogen-like activity in the FO may prevent bone loss. A histological study indicated that the atrophy of uterine epithelial cells in OVX mice was partially prevented by feeding FO (data not shown).

Effects of different dietary fatty acids on BMD and BMC

As shown in Table 4, the BMD in the distal left femur was significantly (p < 0.05) lower in both groups of OVX mice than that in sham-operated mice. DXA scans of the distal end of the femur, the proximal end of the tibia, and the lumbar region of the spine of the CO-fed mice showed the most bone loss in the OVX animals. Comparing OVX mice fed with CO (low in n-3 fatty acids) and FO (high in n-3 fatty acids), the bone loss was significantly (p < 0.05) higher in the CO-fed mice. Confirming previous results of others,(9,36,37) we too have observed that FO is quite effective in maintaining higher BMD in OVX mice compared with CO.

Table Table 4. Effect of Dietary Fatty Acids on BMD (g/cm2) From DXA
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Effects of dietary fatty acids on splenocyte subset distribution

To determine the effect of FO on lymphocytes, spleen lymphocyte subsets were also measured, and the data are shown in Table 5. A significant decrease in the CD4+ T-cell subset, with no change in CD8+ cells and an increase in CD19+ B-cells, was found in CO-fed OVX mice. However, there was no marked difference in B-cells found between sham-operated and OVX mice in the FO-fed group.

Table Table 5. Effect of Dietary Fatty Acids on Splenocyte Subset Distribution
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Effects of ovariectomy and dietary fatty acids on RANKL expression on activated CD4+ T-cells

The analysis of RANKL in activated spleen cells showed very minimal RANKL staining on activated CD8+ T-cells, whereas the proportion of RANKL+CD4+ T-cells was significantly higher in CO-fed OVX mice than in CO-fed sham-operated mice. In FO-fed mice, the proportion of RANKL+CD4+ T-cells was unchanged by OVX (Fig. 1).

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Figure FIG. 1.. Effect of dietary fat and ovariectomy on RANKL expression on splenic CD4 cells. Female Balb/c mice were fed diets containing either 5% CO or 5% FO for 2 months and then were either sham-operated or ovariectomized (OVX). Four months after surgery, mice were killed, and splenocytes were isolated and analyzed by flow cytometry using FITC-labeled anti-CD4 mAb and PE-labeled anti-RANKL mAb. Means of columns not labeled with the same letter were significantly different by Newman-Kuel's test at p < 0.05.

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Effects of dietary fatty acids on bone structure changes with ovariectomy in vertebrae of Balb/c mice

To compare bone loss in the lumber spine, vertebrae were fixed in formalin and prepared for histological examination. A sample photomicrograph is presented in Fig. 2; it is quite clear that trabecular bone loss is much higher in the vertebrae of CO-fed mice with OVX, whereas the vertebrae of FO-fed OVX mice appear to be near normal.

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Figure FIG. 2.. Histology of lumbar vertebrae. Female Balb/c mice were fed diets containing either 5% CO or 5% FO for 2 months and then were either sham-operated or ovariectomized (OVX). Four months after surgery, mice were killed, and vertebrae were fixed in formaldehyde and sectioned and stained with H&E. The bone structure was photographed under a light microscope. It seems that there was a significant trabecular bone loss in the CO/OVX mice, whereas the FO/OVX section seems near normal compared with sham-operated animals.

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Effects of fatty acids on RANKL-induced NF-κB activation of bone marrow-derived macrophages

Electrophoretic mobility shift assays showed that 40 μM DHA and EPA alone or in combination inhibited NF-κB activation induced by RANKL in bone marrow cell-derived macrophages (BMM) in vitro (Fig. 3). In contrast, there was no change with LA or AA, indicating a selective suppression of NF-κB activation by n-3 fatty acids.

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Figure FIG. 3.. The n-3 fatty acids, DHA and EPA, inhibited RANKL-induced NF-κB activation in bone marrow-derived macrophages. BMMs were generated by culturing bone marrow cells from Balb/c mice with GM-CSF (10 ng/ml) for 3 days and then were purified and cultured with n-3 (DHA, EPA) and n-6 (LA, AA) fatty acids for 48 h. Total cell nuclear extracts were prepared, and NF-κB DNA binding activity was determined by EMSA. To assess the specificity of binding, 50× excess of cold NF-κB or AP-1 (irrelevant probe) was added to the reaction. One representative experiment is shown.

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Effects of fatty acids on the osteoclastogenesis and TRACP activity

To elucidate the effects of selected n-3 and n-6 fatty acids on osteoclastogenesis, bone marrow cells were cultured in the presence of 1,25-(OH)2D3, and TRACP activity was examined (Fig. 4). Compared with vitamin D3 alone, the lowest TRACP activity was observed in wells containing DHA, EPA, and DHA and EPA combined, and it was shown to inhibit in a dose-dependent fashion. In contrast, there were no differences among the control, LA, AA, or the combination of LA and AA groups. Multinucleation and TRACP staining are markers of osteoclast maturation. It is clear that both EPA and DHA, alone or in combination, caused a significant decrease in osteoclast maturation compared with the n-6 fatty acids LA and AA (Fig. 5). To our knowledge, this is the first report of n-3 fatty acids inhibiting osteoclastogenesis in vitro. GLA and di-homogamma linolenic acid (DGLA), n-6 fatty acids reported to have beneficial effects on bone density, were not tested in these studies.

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Figure FIG. 4.. Effect of fatty acids on TRACP enzyme activity in bone marrow cells cultured for 7 days with 1,25(OH)2D3. DHA or EPA alone or in combination inhibited bone marrow-derived macrophage TRACP activity in a dose-dependent manner. *Significantly different from medium alone by Dunnet's t-test, p < 0.05.

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Figure FIG. 5.. The effects of n-3 and n-6 fatty acids on osteoclast generation in vitro. Osteoclasts were generated as described as in the Materials and Methods section. It is shown that n-3 fatty acids DHA and EPA alone or in combination selectively inhibited osteoclast differentiation in vitro. Results are from one representative experiment.

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Effect of dietary fatty acids on cytokine concentrations

In the splenocyte culture medium, TNF-α concentrations were decreased by FO feeding in both sham and OVX mice, IL-6 was decreased by FO feeding in sham-operated mice but not OVX mice, IL-2 was decreased by FO feeding only in OVX mice, and IFN-γ was decreased by FO feeding in both sham and OVX mice (Table 6).

Table Table 6. Effect of Diet and OVX on Cytokine Production by Splenocytes
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  1. Top of page
  2. Abstract
  7. Acknowledgements

In this study, bone density was measured in vivo by DXA in cancellous and cortical bone of femur, tibia, and lumbar spine, and bone histology was analyzed. The results clearly showed that dietary FO can prevent bone loss caused by estrogen deficiency, whereas CO seems to increase bone loss after OVX. While some n-6 fatty acids such as GLA and DGLA have been reported to have similar effects,(38,39) these fatty acids are not found in appreciable quantities in CO and were not tested separately in this study. Our results are consistent with others' earlier findings, showing that lowering the ratio of dietary n-6/n-3 fatty acids results in increased bone marrow cellularity(40) and bone strength.(41) Although loss of BMD in OVX mice was attenuated by n-3 fatty acids, we did not find any significant effect of n-3 fatty acids on BMD in sham-operated mice. In the present study, although the uterine weight in FO-fed OVX mice was much lower than in FO-fed sham-operated mice, it is significantly higher than that from CO-fed OVX animals, indicating a possible estrogen-like activity of FO as well, which needs further investigation.

The critical role of fatty acids in bone metabolism was confirmed by the observation that EPA-deficient animals developed severe osteoporosis coupled with increased renal and arterial calcification.(9) This finding is somewhat similar to the clinical picture seen in the elderly, in whom osteoporosis is associated with calcification of the arteries and kidneys.(9) It has been reported that the increased deposition of calcium in both kidneys and aorta was reduced by FO and EPA in male Sprague-Dawley rats.(42) A well-controlled human study also revealed that polyunsaturated fatty acids (PUFAs) such as GLA and EPA have beneficial effects on bone density in elderly people.(38) In this study, oral supplements of PUFAs were reported to be incorporated in increased amounts into the intestinal membrane vesicles, which in turn enhanced calcium transport.(16) This seems to be one of the possible mechanisms by which GLA and EPA are able to enhance BMD and prevent osteoporosis.

It is recognized that one of the mechanisms by which estrogen deficiency causes bone loss is stimulation of osteoclast differentiation,(43,44) a process induced by the stimulation of osteoclast progenitors by macrophage colony-stimulating factor (M-CSF) and RANKL simultaneously.(45,46) In normal physiological conditions, the differentiation of osteoclast progenitors into mature osteoclasts in bone marrow depends on the balance between OPGL-RANK signaling and the levels of biologically active OPG produced by stromal cells and osteoblasts.(47) However, during stimulation, additional bone marrow cells participate in regulating osteoclast formation by producing pro- and anti-osteoclastogenic cytokines. T-cells are the main cell type that modulates osteoclast formation by expression of RANKL,(28,48,49) osteoprotegerin,(50) and IFN-γ.(51) During inflammation and autoimmune arthritis, activated T-cell production of RANKL promotes bone resorption and bone loss, whereas release of IFN-γ limits T-cell-induced bone wasting. Recently it was shown that activated T-cells play an essential causal role, not only in inflammation-induced bone loss, but also in the bone wasting induced by estrogen deficiency. In fact, T-cells express estrogen receptors, and estrogen directly regulates T-cell function and cytokine production.(52–55) The results reported herein showed that after activation ex vivo, RANKL+/CD4+ cells in spleen were increased in CO-fed OVX mice but unchanged in FO-fed OVX mice compared with the respective sham-operated mice. This suggests that downregulation of RANKL expression on T-cells may be one of the mechanisms involved in the prevention of estrogen deficiency-induced bone loss by FO.

It is well established that osteoporosis associated with a decline in estrogen production in postmenopausal women results in increased production of several osteoclastogenic cytokines such as IL-1, IL-6, and TNF-α.(44,56) These cytokines induce the expression of COX-II in osteoblastic and stromal cells, resulting in an increase in the production of prostaglandin E (PGE-2).(57) TNF-α, together with PGE-2, enhances the expression of RANKL, not only in stromal cells,(58) but also in pre-B-cells, which then cooperatively stimulate osteoclast progenitors through RANK-RANKL interaction and promote the differentiation of these cells into mature osteoclasts. This process ultimately leads to osteoporosis.(59) We have also measured the cytokines TNF-α and IL-6 in this study and found that FO downregulates TNF-α and IL-6. In the past, the anti-inflammatory effect of FO and its fatty acids (e.g., EPA) on levels of mRNA for pro-inflammatory cytokines has been established in vivo(60) and also in osteoblasts in vitro.(61) We and others have also described the inhibition of pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) mRNA expression by n-3 fatty acids in immune cells as well as in kidney tissues from autoimmune disease animal models.(20,21,23,), 62–68 In addition, n-3 fatty acids can decrease the production of PGE-2 and perhaps downregulate cyclo-oxygenase-II (COX-II) activity(69) in local tissues. In addition, nitric oxide (NO) is postulated to play an important role in bone metabolism, and it is also known that both DHA and EPA enhance NO formation.(70,71) This may be another mechanism of the anti-osteoporotic action of n-3 fatty acids. We speculate that by modulating the dietary ratio of n-6/n-3 fatty acids, bone growth could be optimized during bone remodeling.

There have also been reports of a role of NF-κB in the pathogenesis of osteoporosis. Mice null for NF-κB1 and NF-κB2 developed osteopetrosis and contain very few osteoclasts compared with normal controls.(72) This indicated the essential role of the NF-κB signal pathway in osteoclast generation and activation.(73–75) We used bone marrow-derived macrophages, which can differentiate into osteoclasts in vitro when stimulated by soluble RANKL, to assess the effects of n-3 and n-6 fatty acids on RANKL-induced NF-κB activation. It is clear from the present data that EPA and DHA alone and in combination inhibited RANKL-induced NF-κB activation in contrast to the n-6 fatty acids, LA and AA, which had no effect. These are similar to previous results reported by Camandola et al.,(76) which showed that FO can inhibit lipopolysaccharide (LPS)-induced NF-κB activation in a macrophage cell line. Consistent with the NF-κB results, we also found that the n-3 fatty acids, DHA and EPA, alone or in combination, inhibited TRACP activity in primary bone marrow cells after culturing with vitamin D3 for 7 days, and significantly inhibited osteoclastogenesis in vitro as assessed by TRACP staining, whereas the n-6 fatty acids, LA and AA, had no effect on TRACP activity or osteoclastogenesis. To our knowledge, this is the first data showing inhibition of osteoclast formation by n-3 fatty acids, which supports the in vivo results that dietary FO attenuated OVX-induced RANKL expression on T-cells.

In this study, we also analyzed spleen lymphocyte subsets. The result is of equally great interest. A significant increase in B-cells and decrease in CD4+ T-cells was found in the CO-fed OVX group. However, much to our surprise, there was no difference found between sham-operated and OVX mice in the FO-fed group. Interestingly, it has been shown that OVX increases B-cells, particularly in the bone marrow, and may also promote osteoclastogenesis in the presence of T-cells.(77,78) OVX does not cause bone loss in the absence of T-cells in nude mice.(79) Thus, the relationship between bone loss and decreased T- and increased B-cells in the absence of estrogen is of much interest. Steroids, and particularly estrogens, are powerful regulators of bone mass. They are involved in the differentiation of the B-lymphocyte hematopoietic lineage. B-lymphocyte precursors declined dramatically in the bone marrow of pregnant or estrogen-treated mice. Reciprocally, the same populations increased in hypogonadal, ovariectomized mice.(78,80) A role of B-cells in the pathogenesis of OVX-induced bone loss has been postulated.(81,82) Onoe et al.(83) suggested a possible role for different cytokines and RANKL in the bone loss caused by increased B-lymphocyte numbers. Other investigators showed that osteoclasts and B-lymphocytes have a common precursor,(84) and osteoclasts can form from a highly purified B220+ bone marrow cell population when stimulated with M-CSF and RANKL. This indicated that the direct effect of estrogen withdrawal on the proposed osteoclast and B-lymphocyte precursors is an important event in the pathogenesis of bone loss that occurs after ovariectomy.(85) Therefore, the effect of FO on B-lymphocyte lineage development and on osteoclastogenesis through the B-cell lineage pathway merits further study.

In conclusion, the results of this study provide strong support for the contention that dietary FO can prevent OVX-induced bone loss. FO downregulates expression of RANKL on activated T-cells and inhibits NF-κB activation in osteoclast progenitors, thereby inhibiting osteoclastogenesis. This may be one of the primary mechanisms of the protective effects of FO on bone density. The long-term anti-inflammatory effects of dietary FO or concentrated purified FO supplements on bone loss along with the pro-inflammatory effects of n-6 fatty acid-containing lipids during aging need further clarification.


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  2. Abstract
  7. Acknowledgements

Work was supported in part by National Institutes of Health Grants AG14541 and AG13693.


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  2. Abstract
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