Camille Tagliaferri, Amélie Dhaussy and Alain Huertas are employed by the commercial company Lesieur. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: CT MJD PL AD AH SW YW VC. Performed the experiments: CT MJD PL SG SM. Analyzed the data: CT SG MJA YW VC. Contributed reagents/materials/analysis tools: SG MJA AD AH VC. Wrote the paper: CT VC.
As the Mediterranean diet (and particularly olive oil) has been associated with bone health, we investigated the impact of extra virgin oil as a source of polyphenols on bone metabolism. In that purpose sham-operated (SH) or ovariectomized (OVX) mice were subjected to refined or virgin olive oil. Two supplementary OVX groups were given either refined or virgin olive oil fortified with vitamin D3, to assess the possible synergistic effects with another liposoluble nutrient. After 30 days of exposure, bone mineral density and gene expression were evaluated. Consistent with previous data, ovariectomy was associated with increased bone turnover and led to impaired bone mass and micro-architecture. The expression of oxidative stress markers were enhanced as well. Virgin olive oil fortified with vitamin D3 prevented such changes in terms of both bone remodeling and bone mineral density. The expression of inflammation and oxidative stress mRNA was also lower in this group. Overall, our data suggest a protective impact of virgin olive oil as a source of polyphenols in addition to vitamin D3 on bone metabolism through improvement of oxidative stress and inflammation.
Osteoporosis, defined as “a systemic skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fracture”
As vitamin D is lipophilic, supplementation of a lipid matrix such as olive oil is feasible and might be of public interest in the management of bone health. This is why we investigated the effect of the consumption of olive oil enriched or not with vitamin D, in the ovariectomized mice as a model of post-menopausal osteoporosis. Moreover, because the bone sparing effect of the polyphenols from olive oil has already been highlighted, possible synergy between those micronutrients and vitamin D was studied and compared to the impact of a refined olive oil (depleted in polyphenols) fortified with vitamin D or not.
All the experimental procedures were approved by the institution’s animal welfare committee (Comité d’Ethique en Matière d’Expérimentation Animale Auvergne: CEMEAA) and were conducted in accordance with the European’s guidelines for the care and use of laboratory animals (2010- 63UE). All efforts were made to minimize animal suffering.
Six weeks old female C57BL/6J mice were obtained from Janvier (Le Genest St Isle, France) and acclimated for 2 weeks under standard laboratory conditions. They were housed individually, on a 12-h light/dark cycle, in the animal facilities of the Human Nutrition Unit at INRA Research Center (Agreement no. C6334514), with free access to both food and water. After the acclimatization period, the rodents were randomly divided into six groups (n = 12/group), although respecting similar average body weight and composition (QMR EchoMRI-900TM, Houston, USA) in each experimental group. At 8 weeks of age, 4 groups of mice were bilaterally ovariectomized (OVX) and 2 batches were sham-operated (SH) under ketamine/xylazine anesthesia, before beginning consumption of the experimental diets (Scientific Animal Food and Engineering (SAFE), Augy, France). Mice were fed a standard diet modified from the AIN-93G powdered diet. 10% of the food was given as either refined olive oil (RO) (RO-SH and RO-OVX groups) or virgin olive oil (VO) (VO-SH and VO-OVX groups). The polyphenol composition of the virgin and refined olive oil is detailed in
Virgin olive oil | Refined olive oil | |||
Phenolic group | Phenolic compounds | Quantity (mg/kg) | Standard deviation | Quantity (mg/kg) |
Simple phenols | ||||
Tyrosol | 14.61 | 0.17 | n.d | |
Hydroxytyrosol | 14.15 | 0.01 | n.d | |
Secoiridoid derivatives | ||||
Oleuropein-aglycone di-aldehyde | 195.63 | 0.14 | n.d | |
Oleuropein-aglycone mono-aldehyde | 93.55 | 0.29 | n.d | |
Ligstroside-aglycone di-aldehyde | 199.22 | 0.26 | n.d | |
Lignan | ||||
Pinoresinol | 65.73 | 0.09 | n.d | |
Flavonoids | ||||
Apigenin | 1.16 | 0.02 | n.d | |
Luteolin | 3.21 | 0.02 | n.d | |
Total | 587.26 |
n.d., not detected.
Extraction of phenolic compounds was carried out using one milliliter of syringic acid (0,015 mg/ml in methanol and used as internal standard) which was added at 2.0 g of olive oil before being evaporated under nitrogen. Then 6 mL of a methanol/water (80/20) mixture was added and vigorously mixed during 5 min. To finish, the extraction mixture was centrifuged at 5000 rpm during 25 min. The phenolic compounds were then quantified using an HPLC system (Agilent 1200 series) equipped with a diode array detector. Separation was performed on a column Waters Spherisorb ODS-2 (C18; 5 µm; 4.6 mm×250 mm) using a ternary gradient elution (water and 0.2% of phosphoric acid; methanol and acetonitrile). The detection was made at 280 nm and the injection volume was 20 µL.
Micro-architecture was investigated using X-ray radiation micro-CT (Viva CT 40, Scanco Medical, Brüttisellen, Switzerland). Scans were performed on the dried femurs at 55 keV with a 10-µm cubic resolution. The secondary spongiosa and associated cortical bone were scanned within the distal metaphasis. Trabecular bone volume (BV/TV, %), trabecular number (Tb.N,/mm), trabecular thickness (Tb.Th, µm), trabecular spacing (Tb.Sp, µm), the degree of anisotropy (DA), and the structural model index (SMI) were analyzed. Cortical thickness and porosity, bone surface and area as well as medullary area were determined as well. Moreover, BMD of the primary spongiosa was determined using an eXplore CT 120 scanner (GE Healthcare, Fairfield, CT). Acquisitions were performed with X-ray tube settings at 100 kV and 50 mA. BMD (mg/cc) was estimated as the mean converted gray-scale level within the region of interest of cortical and trabecular bone.
Serum PINP (N-terminal propeptide of type I procollagen) levels, a specific and sensitive marker of bone formation, was measured using a mouse competitive enzyme immunoassay (EIA) assay (Immunodiagnostic Systems EURL, Paris, France), according to the manufacturer’s protocol The sensitivity was 0.7 ng/mL. The intra- and inter-assay precisions were 6.4 and 9.2%, respectively.
Serum CTX-1 (collagen type 1 cross-linked C-telopeptide, a bone resorption marker) was determined using a mouse-specific enzyme-linked immunosorbent assay (ELISA) (Immunodiagnostic Systems EURL, Paris, France), according to the manufacturer’s protocol. The detection limit was 2.0 ng/mL. The intra- and inter-assay variations were 5.6 and 10.5%, respectively.
Total RNA from powdered femurs was extracted using TRIzol reagent according to the protocol provided by the manufacturer (Invitrogen Life technology, Carlsbad, CA). After validating the RNA quality, high capacity cDNA reverse transcription kit (Applied Biosystems Life technology, Carlsbad, CA) was used to convert RNA into cDNA. Taqman Low Density Array (TLDA) (Applied Biosystems Life technology, Carlsbad, CA) was performed on reverse transcription products, using a 7900 HT Fast Real-Time PCR system (7900HT Fast Real-Time PCR system.). Gene expression was calculated relative to that of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the comparative threshold cycle method (2−ΔΔCT).
Sequence references are: Gapdh;Gm12070;Gm10481-Mm03302249_g1, Trap (Acp5)-Mm00475698_m1, Alpl-Mm01187117_m1, Ocn (Bglap;Bglap-rs1;Bglap2)-Mm03413826_mH, Ccl2-Mm00441243_g1, Col1a1-Mm00801666_g1, Comp-Mm00489490_m1, Ctsk-Mm00484036_m1, Esr1-Mm00433147_m1, Il1b-Mm99999061_mH, Il6-Mm99999064_m1, Itgb3-Mm00443980_m1, Lrp5-Mm00493179_m1, Mmp2-Mm01253621_m1, Nos2-Mm00440488_m1, Pparg-Mm01184322_m1, Sfrp1-Mm03053883_s1, Sost-Mm03024247_g1, osterix (Sp7)-Mm00504574_m1, osteopontin (Spp1)-Mm00436767_m1, Tlr2-Mm00442346_m1, Tlr4-Mm00445273_m1, Rank (Tnfrsf11a)-Mm00437135_m1, Opg (Tnfrsf11b)-Mm01205928_m1, Rankl (Tnfsf11)-Mm00441908_m1.
Data are expressed as the mean ± SEM. Homoscedasticity was checked by Levene’s test and the Grubbs test was used to identify outliers using XLStat (Addinsoft, Paris, France). Statistical analysis was performed by one-way analysis of variance (ANOVA) using XLStat. When a significant effect was detected, a post hoc Tukey test was applied to locate pairwise differences between conditions. A p value less than or equal to 0.05 was considered to be statistically significant.
The quality of the experiment was checked, notably castration efficiency was confirmed by uterine atrophy (p<0.0001) in the OVX rodents (RO-OVX: 13.0±1.4 mg; RO-OVX-VD3∶18.4±2.4 mg; VO-OVX: 12.0±1.3 mg
Dietary consumption was evaluated twice during the experiment: 11 days (J11) then 23 days (J23) after the surgery. While the consumption did not statistically differ between the groups at J11 mice (3.13±0.07), surprisingly at J23, the SH mice ate significantly more than the OVX (RO-OVX: 3.29±0.12 g; RO-OVX-VD3∶3.11±0.14 g; VO-OVX: 3.22±0.11 g; VO-OVX-VD3∶3.09±0.15 g vs RO-SH: 4.05±0.22 g; VO-SH: 3.64±0.15 g; p<0.001; ANOVA).
During the experimental period, mean body weight, as well as lean and fat mass, increased in all the experimental groups, which is a good indicator of health. At the end of the investigation, the OVX mice exhibited a higher body weight (p<0.0001) than the SH animals (RO-OVX: 21.18±0.29 g
Body weight and composition were assessed at the end of the experiment. Values are means ± SEM. *p<0.05 vs RO-SH, ‡p<0.05 vs VO-SH based on ANOVA analysis with Tukey’s post hoc test. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy. Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3.
Spleen weight was evaluated as it is a primary indicator of inflammation. As expected, a splenomegaly was observed in OVX mice (RO-OVX: 71.4±3.2 mg; RO-OVX-VD3∶79.6±2.6 mg; VO-OVX: 73.2±2.7 mg; VO-OVX-VD3∶76.1±4.4 mg vs RO-SH: 57.7±2.2 mg; VO-SH: 51.4±1.7 mg; p<0.0001; ANOVA)). This parameter was not modified by any of the diet as all the OVX animals exhibited same values.
As expected in such an animal model, BMD of the 3 compartments of interest (i.e. cortical bone, primary and secondary spongiosa) was impaired in all the OVX groups (p<0.0001; p<0.0001; p = 0.0002, respectively; ANOVA) (
Femoral cortical, primary and secondary spongiosa bone mineral density (A) and serum levels of CTX1 and PINP (B) in mice fed with olive oil enriched or not with vitamin D3.Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice received refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed. *p<0.05 vs RO-SH, ‡p<0.05 vs VO-SH, $p<0.05 vs VO-OVX, £p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; BMD, bone mineral density; CTX-1, collagen type 1 cross-linked C-telopeptide; PINP, N-terminal propeptide of type I procollagen.
RO-SH | VO-SH | RO-OVX | VO-OVX | RO-OVX-VD3 | VO-OVX-VD3 | ANOVA | |
1.57±0.09 | 1.67±0.06 | 1.76±0.05 | 1.83±0.08 | 1.83±0.07 | 1.67±0.06 | NS | |
0.85±0.02 | 0.86±0.01 | 0.80±0.01‡ | 0.79±0.01*‡ | 0.79±0.01*‡ | 0.79±0.01*‡ | p<0.0001 | |
142±5 | 140±3 | 136±3 | 131±4 | 126±4 | 138±4 | NS | |
7.54±0.34 | 7.92±0.25 | 8.60±0.35 | 8.98±0.49 | 8.76±0.51 | 7.60±0.25 | NS | |
0.84±0.02 | 0.87±0.01 | 0.85±0.01 | 0.84±0.01 | 0.84±0.01 | 0.83±0.01 | NS | |
2.80±0.10 | 2.79±0.11 | 2.98±0.06 | 3.02±0.07 | 2.99±0.05 | 2.92±0.07 | NS | |
8.32±0.84 | 7.87±0.85 | 4.88±0.38*‡ | 4.89±0.48*‡ | 5.26±0.44*‡ | 5.31±0.61*‡ | p<0.001 | |
2.33±0.17 | 2.21±0.18 | 1.43±0.10*‡ | 1.45±0.12*‡ | 1.51±0.11*‡ | 1.47±0.14*‡ | p<0.0001 | |
35.3±1.2 | 36.9±1.2 | 34.1±0.8 | 33.3±0.9 | 34.7±0.8 | 35.6±0.8 | NS | |
405±33 | 444±42 | 692±46*‡ | 708±70*‡ | 667±58*‡ | 691±64*‡ | p<0.001 | |
1.49±0.05 | 1.43±0.02 | 1.51±0.04 | 1.47±0.02 | 1.49±0.02 | 1.52±0.02 | NS |
Values are means ± SEM. *p<0.05 vs RO-SH, ‡p<0.05 vs VO-SH based on ANOVA analysis with Tukey’s post hoc test. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy, SMI, structural model index; BV/TV, trabecular bone volume; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp, trabecular spacing; DA, degree of anisotropy. Following sham operation or ovariectomy, mice received refined or virgin olive oil for 4 weeks. Two additional groups of ovariectomized mice received refined or virgin olive oil enriched with vitamin D3.
As far as biomarkers of bone metabolism are concerned, no differences were observed between the groups consuming RO (
A TLDA analysis allowed assessing the potential impact of the diets on a number of genes, in order to understand the involved mechanisms. Forty eight genes were investigated, therefore only the genes whose expression was different between the groups are presented. The RO-SH group was used as a reference.
Consistent with the bone biomarkers data, bone turnover genes were up-regulated in animals under estrogen deprivation. Indeed, genes representative of bone formation (osteocalcin (OCN; p = 0.0001), osteopontin (OPN; p = 0.025), type I collagen (Col1a1; p = 0.007; ANOVA without the vitamin D3 groups)) (
Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ‡p<0.05 vs VO-SH, #p<0.05 vs RO-OVX, $p<0.05 vs VO-OVX, £p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; ALP, alkaline phosphatase; OCN, osteocalcin; OPN, osteopontin; Col1a1, type I collagen; Lrp5, low density lipoprotein receptor-related protein 5; Sfrp1, secreted frizzled related sequence protein 1; Sost1, sclerostin; Esr1, estrogen receptor 1.
Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ‡p<0.05 vs VO-SH, #p<0.05 vs RO-OVX, $p<0.05 vs VO-OVX, £p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; TRAP, tartrate-resistant acid phosphatase; Ctsk, catepsin K; Itg-β3, β3-integrin; MMP-2, matrix metalloproteinase 2; RANK, Receptor activator of nuclear factor-kappaB (NF-kappaB); RANKL, RANK ligand; OPG, osteoprotegerin.
Following sham operation or ovariectomy, the mice received refined or virgin olive oil for 4 weeks: RO-SH, VO-SH, RO-OVX, VO-OVX. Two additional groups of ovariectomized mice were given refined or virgin olive oil enriched with vitamin D3: RO-OVX-VD3 and VO-OVX-VD3. Values are means ± SEM. ANOVA with Tukey’s post hoc test were performed on the 6 groups (symbols above histograms), on the 4 groups without vitamin D3 (symbols above the solid line) and on the 4 OVX groups (symbols above the dotted line). *p<0.05 vs RO-SH, ‡p<0.05 vs VO-SH, #p<0.05 vs RO-OVX, $p<0.05 vs VO-OVX, £p<0.05 vs RO-OVX-VD3. RO, refined olive oil; VO, virgin olive oil; VD3, vitamin D3; SH, sham operation; OVX, ovariectomy; IL-1β, interleukin-1β; IL-6, interleukin-6; CCl2, chemokine (C-C motif) ligand 2; Tlr2, toll like receptor 2; Tlr4, toll like receptor 4; Nos2, nitric oxide synthase.
As far as bone formation is concerned, osteoblast genes were not differently expressed in the SH rodents, whether olive oil was refined or not. In OVX animals, low density lipoprotein receptor-related protein 5 (Lrp5), a receptor by which the Wnt/β-catenin signal is transduced, was less expressed in those that were given the virgin oil (p = 0.001; ANOVA without the vitamin D3 groups) (
With regards to gene expression in the mice that have been given the refined oil, the only differences that were observed target OCN, secreted frizzled related sequence protein 1 (Sfrp1) and Esr1 genes (
In the case of the virgin oil, fortification with vitamin D was associated to a correction of the expression of Esr1 gene (similar values in both VO-SH and VO-OVX-VD3, while the expression was higher in VO-OVX than in VO-SH) (
Broad-based preventive strategies designed to lower the risk of osteoporosis need to be implemented. This is why the concept of a healthy diet providing adequate amounts of various micronutrients deserves mention. Actually, within Europe, conspicuous differences is encountered in the severity of osteoporosis, the lowest incidence being reported in the Mediterranean area
The ovariectomized rodent is, by far, the most widely used animal model for post-menopausal osteoporosis. Actually, characteristics of skeletal physiology in the murine model share similarities with those of early post-menopausal women, in many respects. These include: increase rate of bone turnover with resorption exceeding formation, greater loss of cancellous than cortical bone; and similar skeletal response to various stimuli.
In the present study, as expected and previously shown
Olive oil, whether it was refined (depleted in polyphenols) or not, was not able to prevent the increase in body weight observed in the OVX mice. This is consistent with previous data published by our team showing that neither olive oil nor oleuropein could modify this parameter in OVX rats
With regards to bone health, BMD was not significantly different in the OVX mice whether they were submitted to virgin or refined olive oil consumption. In other words, olive oil, when given alone, was not efficient to prevent OVX-induced osteopenia. This was consistent with the lack of significant difference in both CTX1 and OCN serum levels between those two groups. Consistently with our data, Puel et al.
Ovariectomy-induced bone loss was prevented (both at the cortical and trabecular levels) by the combination of virgin olive oil and vitamin D3, but not by virgin olive oil alone, or even by refined olive oil and vitamin D. Therefore, our data support the idea that the beneficial impact of the Mediterranean diet involves, at least in part, olive oil. This food combination could be part of a strategy to restrain the development of osteoporosis and such a beneficial effect could be explained by exacerbation of the polyphenol-bone sparing properties thanks to vitamin D. Even though the impact of olive oil could be attributed to its fatty acids
In the present case, the combination of virgin olive oil and vitamin D3 indeed slowed down the ovariectomy-accelerated bone turnover. Moreover, transcriptomic analyses showed that osteoblastic and osteoclastic markers were down-regulated in VO-OVX-VD3 mice compared to the other OVX groups (
The effect of ovariectomy is represented by dotted boxes, the arrow (before the gene name), showing the modification of gene expression. The effect of virgin olive oil and vitamin D3 is represented by the grey boxes, the arrow (after the gene name) showing the impact on gene expression. The arrows between the genes outline regulation pathways. ALP, alkaline phosphatase; CCl2, chemokine (C-C motif) ligand 2; Col1a1, type I collagen; Ctsk, catepsin K; Esr1, estrogen receptor 1; IL-1β, interleukin-1β; IL-6, interleukin-6; Itg-β3, β3-integrin; Lrp5, low density lipoprotein receptor-related protein 5; MMP-2, matrix metalloproteinase 2; Nos2, nitric oxide synthase; OCN, osteocalcin; OPN, osteopontin; Sost1, sclerostin; Tlr2, toll like receptor 2; Tlr4, toll like receptor 4; TRAP, tartrate-resistant acid phosphatase.
The bone sparing effect observed in the animals that were given the combination of virgin olive oil and vitamin D was concomitant with decreased oxidative stress and inflammation gene expressions. Bone metabolism is impaired in the presence of an excess of reactive oxygen species, which leads to bone loss
Regarding inflammation, virgin olive oil enriched with polyphenols has been shown to exhibit protective effects in two models of inflammation
In conclusion, our study shows that virgin olive oil fortified with vitamin D3 is able to counteract the bone loss induced by estrogen deprivation. Such a bone sparing effect could be explained by an improvement of both inflammation status and oxidative stress. Thus, although further data are required, virgin olive oil fortified with vitamin D might be a potential nutritional alternative for osteoporosis prevention.
The authors thank Mehdi Djelloul-Mazouz, Alexandre Teynie and Christophe Del’homme for providing everyday care to the animals. Fig. 6 was produced with the assistance of Servier Medical Art.