Our study provides the first evidence that administration of the PPARα agonists fenofibrate and Wyeth 14643 to a certain level can maintain bone quality at sham levels in an ovariectomized rat model. Furthermore, this study differentiates the PPARα agonists from the PPARγ agonists regarding skeletal effects, since the PPARγ agonist pioglitazone exerts the opposite effect by further boosting ovariectomy-induced bone loss in rats.
Ovariectomized control rats had significantly lower gain in femoral BMC and BMD associated with reduced biomechanical strength parameters compared to sham-operated controls. The relatively young age of the rats may explain why the differences found between the SHAM and OVX controls were less pronounced than expected. According to Kahrode et al, 2008 [46
], rats from 2-15 months are applied in the rat OVX model, and while use of older animals is attractive due to steady bone turnover rate, the use of young adult rats can also provide consistent, reproducible and interpretable results. Ovariectomy of skeletally immature rats results in achievement of a lower peak bone mass (total bone mass present at the end of the skeletal maturation, which for rats are considered to occur between 47-61 weeks of age [47
In our study, fenofibrate had a partly preventive effect on bone in this rat model of osteoporosis, as femoral and whole body BMC and BMD were maintained at the same levels as for sham-operated rats, and this group also exhibited significantly higher femoral BMC and BMD than ovariectomized controls. Fenofibrate also prevented deterioration of the bone architecture to a certain level, as reflected in maintenance of trabecular BV, trabecular thickness Tb.Th, and TV. We have previously shown that fenofibrate increased femoral BMD and reduced bone medullary area in intact female rats [12
] suggesting an inhibition of endosteal bone resorption. Chan et al.
found that fibrates directly inhibit human osteoclast activity and function [22
], and Okamoto et al.
demonstrated that fenofibrate suppresses osteoclast differentiation by inhibiting NFκB-signaling [48
]. We have, however, not been able to confirm a direct effect of fenofibrate on human osteoclasts [13
]. This is consistent with the findings of Still et al
., who showed that administration of bezafibrate to rats caused no change in either the number of osteoclasts or the serum levels of resorption markers [9
In contrast to the ovariectomized control group, rats given fenofibrate and Wyeth 14643 had similar bending moment and energy absorption as the sham group at the femoral neck, indicating maintained mechanical strength in spite of the ovariectomy.
Furthermore, administration of fenofibrate and Wyeth 14643 resulted in maintenance of femoral BMC at the same level as the sham control group.
We have previously shown that fenofibrate stimulates osteoprotegerin (OPG) release from the mouse preosteoblast cell line MC3T3-E1 [13
], and it has also been found to enhance plasma OPG in humans [49
]. These findings suggest an antiresorptive effect of fenofibrate.
In a previous study, we demonstrated that fenofibrate elevated plasma osteocalcin levels in intact female rats [13
]. In the present study, however, the plasma osteocalcin levels were similar in the ovariectomized group and the fenofibrate group, and both groups had elevated osteocalcin levels compared to the sham-operated group. It is therefore difficult to differentiate if the elevated level of plasma osteocalcin level in the fenofibrate group is due to increased bone formation or increased bone turnover associated with ovariectomy.
We and others have previously shown that activation of PPARα increases osteoblast gene expression of differentiation markers [13
], increases ALP activity, induces osteoblastic maturation and matrix calcification [50
] in MC3T3-E1 cells. In the present study we found that fenofibrate significantly enhanced the number of ALP-, calcium- and collagen-positive colonies in bone marrow cells from rats.
Still et al.
showed that bezafibrate and linoleic acid increased BMD and periosteal bone formation in male rats [9
]. Since the most pronounced effect was found for linoleic acid, which preferentially is a PPARδ agonist, they concluded that the effects were mediated through PPARδ activation. Wyeth 14643 and fenofibrate possess high and equal affinity to PPARα [51
], and we demonstrate that administration of these PPARα agonists to some level maintain bone mass and biomechanical at sham levels.
The PIO OVX group exhibited enhanced bone loss and impaired bone architecture, reflected in a significant decrease in cortical volume and thickness in both femoral head and shaft, and an elevated total volume. In accordance with these findings, we also found reduced mechanical strength in this group. These results are in agreement with several earlier studies, all showing reduced bone formation and/or a decrease in bone mass, in rodents treated with glitazones, mainly rosiglitazone [24
]. Similar findings were recently demonstrated in humans [30
]. Pioglitazone is still used for treatment of diabetes mellitus 2, while rosiglitazone has been withdrawn due to adverse effects [54
]. We show that pioglitazone causes negative skeletal effects similar to those seen for rosiglitazone. Plasma levels of osteocalcin were significantly reduced in the PIO OVX group compared to all the other groups, suggesting that the negative effect of PPARγ agonists on the skeleton is caused by inhibition of bone formation, rather than elevated bone resorption. This is supported by the fact that plasma CTx was significantly lower in the rats receiving pioglitazone. Pioglitazone also reduced the number of ALP-, calcium- and collagen -positive colonies in vitro
in bone marrow cells from rats. This is in accordance with several other studies, showing that pioglitazone [55
] and the PPARγ agonist BRL-49653 [56
] stimulate adipogenesis, inhibit osteogenesis and reduce ALP activity in mesenchymal cells from mouse and rat bone marrow, respectively. We have previously studied the effect of pioglitazone on human osteoclast differentiation and activity [13
], but could not detect any effect with the doses used in our study (0.1-10 μM). Studies regarding the effects of PPARγ agonists on osteoclasts are contradictory; some studies report an inhibition of osteoclast differentiation and activity [20
], while others demonstrate a pro-osteoclastogenic effect of PPARγ [22
]. Since we could not demonstrate a direct effect of either fenofibrate or pioglitazone on osteoclasts [13
], we speculate whether the observed skeletal effects of PPAR agonists mainly are mediated through the regulation of mesenchymal cells within the bone marrow.
The doses of rosiglitazone used in previous rodent studies were 3.0-25 mg/kg/day, whereas we used a higher daily dose of pioglitazone (35 mg/kg). Rosiglitazone is a more potent PPARγ agonist than pioglitazone [4
], and several studies have estimated comparable glycemic effects of rosiglitazone 2-8 mg with pioglitazone 15-45 mg, respectively [59
Most studies of glitazones treatment in rodents [24
], also report enhanced bone marrow adiposity, supporting evidence from in vitro
studies in which PPARγ activation in bone cells was found to promote adipogenesis at the expense of osteoblastogenesis [14
]. We did not measure the fat content in bone marrow; the PIO OVX group, however, displayed significantly higher body fat mass and lower % lean mass than all the other groups, showing that PPARγ activation stimulates adipogenesis in general.
The FENO OVX group maintained % lean mass at sham levels in contrast to the ovariectomized controls, which is in accordance with our previous study where fenofibrate increased lean mass in intact female rats [13
PPARα activation is found to induce hepatomegaly in rodents [8
] and in accordance with this we found increased liver weights in the FENO and WY groups. This was not examined any further, as this was beyond the scope of this study.
The skeletal effects of PPAR agonists may also be indirect, for instance through regulation of adipokine production in adipose tissue. There is a connection between the amount of body fat and bone mass as recently reviewed by several authors [63
]. Adipose tissue produces several hormones assumed to be involved in the regulation of bone metabolism, such as adiponectin [66
] and leptin [68
]. However, there are conflicting results concerning the skeletal effects of adipokines [63
In our study we found significant differences in plasma leptin and adiponectin levels between the groups, especially the PIO OVX group exhibited higher levels than the others. In humans, circulating leptin concentrations correlate with body mass index (BMI) and the total amount of body fat [70
]. We found that the WY OVX and the FENO OVX groups had similar plasma leptin levels as SHAM, despite a significantly higher body weight and fat mass. Adiponectin is known to stimulate insulin sensitivity, and usually correlates negatively with increased fat mass [72
]. In spite of the large enhancement in fat mass, the PIO OVX group exhibited the highest level of plasma adiponectin among the groups. This is in accordance with previous studies demonstrating that glitazones increase circulating adiponectin levels in humans as well as rodents [75
]. It is difficult to elaborate whether these differences explain the various skeletal effects of PPARα and PPARγ agonists.