Combination treatment with pioglitazone and fenofibrate attenuates pioglitazone-mediated acceleration of bone loss in ovariectomized rats

in Journal of Endocrinology
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  • 1 Charles River Laboratories, F. Hoffmann-La Roche AG, Roche Diagnostics GmbH, 22022 Transcanadienne, Senneville, Montréal, Québec, Canada H9X 3R3

Peroxisome proliferator-activated receptor (PPAR) γ agonists, such as pioglitazone (Pio), improve glycemia and lipid profile but are associated with bone loss and fracture risk. Data regarding bone effects of PPARα agonists (including fenofibrate (Feno)) are limited, although animal studies suggest that Feno may increase bone mass. This study investigated the effects of a 13-week oral combination treatment with Pio (10 mg/kg per day)+Feno (25 mg/kg per day) on body composition and bone mass parameters compared with Pio or Feno alone in adult ovariectomized (OVX) rats, with a 4-week bone depletion period, followed by a 6-week treatment-free period. Treatment of OVX rats with Pio+Feno resulted in ∼50% lower fat mass gain compared with Pio treatment alone. Combination treatment with Pio+Feno partially prevented Pio-induced loss of bone mineral content (∼45%) and bone mineral density (BMD; ∼60%) at the lumbar spine. Similar effects of treatments were observed at the femur, most notably at sites rich in trabecular bone. At the proximal tibial metaphysis, concomitant treatment with Pio+Feno prevented Pio exacerbation of ovariectomy-induced loss of trabecular bone, resulting in BMD values in the Pio+Feno group comparable to OVX controls. Discontinuation of Pio or Feno treatment of OVX rats was associated with partial reversal of effects on bone loss or bone mass gain, respectively, while values in the Pio+Feno group remained comparable to OVX controls. These data suggest that concurrent/dual agonism of PPARγ and PPARα may reduce the negative effects of PPARγ agonism on bone mass.

Abstract

Peroxisome proliferator-activated receptor (PPAR) γ agonists, such as pioglitazone (Pio), improve glycemia and lipid profile but are associated with bone loss and fracture risk. Data regarding bone effects of PPARα agonists (including fenofibrate (Feno)) are limited, although animal studies suggest that Feno may increase bone mass. This study investigated the effects of a 13-week oral combination treatment with Pio (10 mg/kg per day)+Feno (25 mg/kg per day) on body composition and bone mass parameters compared with Pio or Feno alone in adult ovariectomized (OVX) rats, with a 4-week bone depletion period, followed by a 6-week treatment-free period. Treatment of OVX rats with Pio+Feno resulted in ∼50% lower fat mass gain compared with Pio treatment alone. Combination treatment with Pio+Feno partially prevented Pio-induced loss of bone mineral content (∼45%) and bone mineral density (BMD; ∼60%) at the lumbar spine. Similar effects of treatments were observed at the femur, most notably at sites rich in trabecular bone. At the proximal tibial metaphysis, concomitant treatment with Pio+Feno prevented Pio exacerbation of ovariectomy-induced loss of trabecular bone, resulting in BMD values in the Pio+Feno group comparable to OVX controls. Discontinuation of Pio or Feno treatment of OVX rats was associated with partial reversal of effects on bone loss or bone mass gain, respectively, while values in the Pio+Feno group remained comparable to OVX controls. These data suggest that concurrent/dual agonism of PPARγ and PPARα may reduce the negative effects of PPARγ agonism on bone mass.

Introduction

Bone is a highly specialized and dynamic tissue that undergoes constant remodeling by balancing bone formation and resorption (Clarke 2008, Eriksen 2010), processes that are regulated by osteoblasts and osteoclasts respectively (Clarke 2008, Eriksen 2010). Osteoblastogenesis, osteoclastogenesis, and activity of the resultant cell types are controlled by a variety of hormonal and humoral factors such as estrogen (Manolagas et al. 2002), thyroid hormones (Eriksen 2010), growth factors (Eriksen 2010), and cytokines (Clarke 2008, Eriksen 2010), the actions of which are mediated via specific receptors, including nuclear receptors (Boyce et al. 2009, Eriksen 2010).

The peroxisome proliferator-activated receptors (PPARs) are a group of ligand-activated nuclear receptors that play a key role in the regulation of lipid and carbohydrate metabolism, as well as inflammation, immunomodulation, and cellular differentiation (Karpe & Ehrenborg 2009, Lalloyer & Staels 2010). PPAR agonists are a diverse group of compounds and, although often grouped together, exhibit different selectivity/agonistic effects on PPAR subtypes. Thiazolidinediones (TZDs) work predominantly through the activation of PPARγ and have been shown to improve insulin sensitivity and glucose homeostasis in type 2 diabetes mellitus (Spiegelman 1998, Lalloyer & Staels 2010), while fibrates activate PPARα, with the primary effect of improving plasma lipids (Sierra et al. 2007, Lalloyer & Staels 2010). Accumulating evidence from animal studies as well as clinical trials indicates that activation of PPARγ results in loss of bone mass and/or strength (Kahn et al. 2006, Glintborg et al. 2008, Schwartz 2008) via effects on bone formation and resorption (Sottile et al. 2004, Grey et al. 2007), underlying the increased risk of fractures observed clinically with these agents (Kahn et al. 2006, Glintborg et al. 2008). The role of PPARα (and the third member of the PPAR family, PPARδ) in regulating bone health is much less understood although, recently, fibrates were reported to increase bone mass and strength in normal rats (Syversen et al. 2009) and in an animal model of osteoporosis, the ovariectomized (OVX) rat (Stunes et al. 2011).

Combined (dual) activation of PPARα and PPARγ has been pursued for over a decade due to the expectation of better control of cardiovascular risk factors vs solo activation of one of the PPARs, as both diabetic dyslipidemia and glucose control/insulin sensitivity are targeted (Lalloyer & Staels 2010). Several combined (dual) PPARα/γ agonists have reached phase III trials – muraglitazar (Kendall et al. 2006), tesaglitazar (Bays et al. 2007), and aleglitazar (Henry et al. 2009) – and although the development of muraglitazar and tesaglitazar was terminated because of safety concerns, the therapeutic potential of PPARα/γ agonists remains of high clinical interest. To our knowledge, the effect of combined activation of PPARγ and PPARα on bone biology has not been investigated previously.

The objective of this study was to investigate the effects of combination treatment with an agonist of PPARα (fenofibrate (Feno)) and PPARγ (pioglitazone (Pio)) on bone mass, bone density, and markers of bone turnover compared with the effect of Pio and Feno alone in OVX rats. In addition, the effect of discontinuation of treatment was investigated to evaluate the reversibility of observed effects.

Materials and Methods

Animals

The study was conducted in accordance with Standard Operating Procedures of Charles River Laboratories, Montreal, and F. Hoffmann-La Roche AG and the protocol was approved by the Institutional Animal Care and Use Committee. Animals were appropriately housed and experimental procedures were performed in accordance with guidelines of the Association for the Assessment and Accreditation of Laboratory and Animal Care and appropriate federal, state, or local guidelines.

At the beginning of treatment, animals were ∼7 months of age and ranged in weight from 318 to 454 g. Animals had free access to purified water and standard laboratory diet (PMI Certified rodent 5002, PMI Nutrition International, Inc., Saint Paul, MN, USA) throughout the study, except where indicated. The overall average temperature and relative humidity during the study were 21.9 °C and 50% respectively.

After a minimum acclimation period of 3 weeks, animals underwent surgical procedure (sham operation or ovariectomy). Prior to surgery, baseline bone densitometry and body weight measurements were obtained from all animals. Using a randomization procedure stratified according to body weight, animals were assigned to one of the following treatment groups: 1) sham vehicle control, 2) OVX vehicle control, 3) OVX Pio (10 mg/kg per day), 4) OVX Feno (25 mg/kg per day), or 5) OVX Pio (10 mg/kg per day)+Feno (25 mg/kg per day).

Daily oral dosing by gavage of vehicle or test substance(s) commenced in treatment groups 1–5 at the end of the bone depletion period (4 weeks after surgery). Animals were treated at approximately the same time each day for 13 weeks. Ten of the animals in groups 1–5 were killed at week 13, while treatment was discontinued in the remaining ten animals from each of these groups for 6 weeks (i.e. end of the study at week 19).

Body weight and food consumption were measured weekly, starting from the last week of the acclimation period and extending through the treatment and treatment-free periods. In addition, body weight was measured at randomization and on the day of termination (as an overnight fasted body weight).

Laboratory analysis

At the end of the bone depletion period, blood was collected from overnight fasted animals at weeks 5 or 6 and 12 or 13 of the treatment period and at week 19 (i.e. end of the treatment-free period). Plasma was analyzed for assessment of triglyceride (TG), insulin, and biomarkers of bone turnover (serum osteocalcin (bone formation) and urinary C-telopeptide of type I collagen (CTx; bone resorption)). TG was measured by an enzymatic colorimetric assay (GPO-PAP, Roche Diagnostics GmbH), while insulin and osteocalcin were measured using rat RIA kits (LINCO Research, Billerica, MA, USA and Biomedical Technologies, Inc., Stoughton, MA, USA respectively). CTx was assessed with a rat ELISA (Immunodiagnostic Systems, Tyne and Wear, UK). Adiponectin was measured by a colorimetric ELISA assay (B-Bridge International, Inc., Cupertino, CA, USA).

Bone densitometry measurements

Bone densitometry was evaluated by dual-energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT). Animals were anesthetized using isoflurane and were maintained under the effect during the scans. Scans were acquired once prior to surgery, at the end of the bone depletion period, during weeks 5/6 and 12/13 of the treatment period, and at the end of the treatment-free period at week 19.

Dual-energy X-ray absorptiometry

DXA was used to measure area (cm2), bone mineral content (BMC (g)), and bone mineral density (BMD (g/cm2)) using a Hologic Discovery A densitometer (Hologic, Inc., Bedford, MA, USA) with small animal hi-res software version 12.3. The percent coefficient of variation for rat BMD at the spine and femur was 1.1–1.5%. In addition, lean mass and fat mass were reported from whole-body DXA scans. Bone densitometry measurements of the whole body, lumbar spine (L1–L4), and whole and regionalized right femur were acquired from all animals. The initial scans acquired from each animal were compared with follow-up scans to ensure appropriate and consistent positioning of scan sites.

Peripheral quantitative computed tomography

In vivo pQCT scans were performed using an XCT Research SA+ bone scanner with software version 5.50D (Stratec Medizintechnik, Pforzheim, Germany). Bone densitometry measurements were performed on the right proximal tibia of all animals. One slice each was obtained in the metaphysis and in the diaphysis, acquired at 14 and 50%, respectively, of the total bone length distal to the reference line set to the tibial proximal end. For follow-up scans, positioning and placement of CT scan lines were verified using the scout scan and compared with the initial scout scan. The percent coefficient of variation for pQCT parameters, including trabecular BMD and cortical BMD, was 0.4–0.8%.

Statistical analyses

All statistical procedures were pre-specified in the study protocol. Data are presented as group means and s.e.m., unless otherwise stated. The homogeneity of group variances was evaluated using Levene's test at the 0.05 significance level. If differences between group variances were not found to be significant (P>0.05), then a parametric one-way ANOVA was performed. When significant differences among the means were indicated by the overall ANOVA F test (P≤0.05), the t-test on least squares means was used to perform all combinations of two-by-two group mean comparisons, except for the sham vehicle control group, which was compared only with OVX vehicle control. If Levene's test indicated heterogeneous group variances (P≤0.05), then the non-parametric Kruskal–Wallis test was used to compare all considered groups. When the Kruskal–Wallis test was significant (P≤0.05), the Wilcoxon rank-sum test was used to perform the pairwise group comparisons of interest as above.

For densitometry data (DXA and pQCT), individual treatment period results were adjusted to the related pre-surgery and end of bone depletion periods by computing the relative difference (i.e. the percentage change from pre-surgery and baseline respectively). DXA lean mass, DXA fat mass, pQCT muscle area, and pQCT fat area were also adjusted to the most recent body weight relative to each scanning occasion. These derived variables were submitted to the analysis described above. The original measurements (unadjusted data) were submitted to the above statistical analysis only for the pre-treatment period. A modified Bonferroni correction was applied.

Results

For ease of visualization, data are shown from pre-surgery to the end-of-treatment period (i.e. treatment groups 1–5, n=10 per group), while the effect of discontinuation of treatments is described within the text, except for body weight and BMD at the femur.

Body weight

Mean body weight gains were statistically significantly higher for OVX controls compared with sham controls up to week 8 and comparable for the remainder of the study period (Fig. 1A). Pio-treated animals showed trends toward a greater increase in weight gain compared with OVX controls, while the Feno-treated group showed trends toward a decreased rate of weight gain. These differences, however, did not attain statistical significance. Mean body weights and body weight gains in the Pio+Feno-treated animals were comparable to OVX controls during the treatment (Fig. 1A) and treatment-free periods.

Figure 1
Figure 1

(A) Group mean body weights, (B) insulin, and (C) triglyceride levels at pre-treatment and during the treatment period. Body weight chart also shows the effect of ovariectomy post-randomization and the impact of discontinuation of treatment. Insulin and triglyceride data are mean±s.e.m. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0356

Plasma insulin and TG levels

OVX increased insulin (Fig. 1B), TG (Fig. 1C), and total cholesterol levels (data not shown). Compared with OVX controls, there was a decrease in plasma insulin levels in Pio-treated animals and Pio+Feno-treated animals at weeks 5 and 12 (Fig. 1B). There were also decreases in plasma TG in Pio-treated animals, in Feno-treated animals, and in Pio+Feno-treated animals at weeks 5 and 12 (Fig. 1C). Adiponectin levels were also significantly increased by treatment with Pio or Pio+Feno during the treatment period (at week 12/13, OVX controls: 8.6±0.8 μg/ml, Pio: 22.0±2.2 μg/ml, and Pio+Feno 17.7±1.8 μg/ml; P<0.01 for both vs OVX controls). The significant reductions in insulin and TG observed with Pio were maintained after discontinuation of treatment but were lost in other treatment groups (Supplementary Table 1, see section on supplementary data given at the end of this article).

Biochemical markers of bone turnover

Compared with the sham group, OVX increased plasma levels of osteocalcin by 60% (Fig. 2A) and CTx by 151% (Fig. 2B). A characteristic age-related decline in both osteocalcin and CTx was also noted, but there was no effect of any treatment on these markers of bone turnover in OVX rats (Fig. 2A and B).

Figure 2
Figure 2

Mean levels of (A) osteocalcin (bone formation marker) and (B) CTx (bone resorption marker) at the end of the bone depletion period and during treatment. Data are mean±s.e.m. *P ′≤0.05 vs OVX; P ′= adjusted P value (modified from Bonferroni adjustment).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0356

Body composition

At the whole-body level, DXA values (relative to body weight) showed a loss of muscle mass (26.5%) and gain of fat mass (64.5%) in OVX rats compared with the sham group (Fig. 3A and B). Pio resulted in further gains in fat mass and a slightly greater loss of lean mass, while Feno partially prevented the OVX-induced fat gain, with no notable effect on muscle mass. Feno partially prevented (by ∼50%) Pio-induced fat gains, with no effect on muscle mass. During the treatment-free period, the rate of loss of lean mass and gain in fat mass was slower for Pio-treated animals compared with OVX, suggesting reversibility of the Pio effect.

Figure 3
Figure 3

Analysis of (A) lean mass and (B) fat mass by DXA at whole-body level and (C) muscle area and (D) fat area by pQCT at the proximal tibia. Data are mean±s.e.m. and show percentage change values adjusted to body weight. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0356

These results were generally corroborated by pQCT analysis of soft tissue at the tibial diaphysis site, where OVX induced an accelerated loss of muscle area (14%) while increasing the fat area (67%) (Fig. 3C and D). Using values adjusted to body weight, treatment of OVX rats with Pio significantly increased fat area (40%), with a slight loss of muscle area (8%), compared with OVX vehicle controls. Feno partially prevented OVX- and Pio-induced fat gains, as the mean fat area was slightly lower for animals treated with Pio+Feno relative to Pio. In contrast, Feno (alone or in combination with Pio) had no effect on muscle mass (Fig. 3C). During the treatment-free period, loss of fat was noted for both Pio‐ and Pio+Feno-treated animals, with mean values comparable to OVX vehicle controls (Supplementary Table 2, see section on supplementary data given at the end of this article). No effect was noted for Feno-treated animals (Supplementary Table 2).

Bone densitometry

Dual-energy X-ray absorptiometry

Whole body

DXA analysis revealed that OVX prevented BMD gains compared with sham controls over the study period (Fig. 4A). Treatment of OVX rats with Pio resulted in further reduction of whole-body bone mass (BMC and BMD; BMC data are shown in Supplementary Table 3, see section on supplementary data given at the end of this article). In contrast, treatment of OVX rats with Feno partially (∼50%) restored OVX-induced reduction in bone mass, as evidenced by generally higher BMC and BMD values; however, values remained lower compared with sham controls at the end of the treatment period (Fig. 4A, BMC data are shown in Supplementary Table 3). At the whole-body level, combination of Pio+Feno did not have a notable effect on Pio-induced bone loss in OVX rats. Over the treatment-free period, a slight loss and a slight gain of bone mass were noted for Feno- and Pio-treated animals, respectively, indicating partial reversibility of the effect of these compounds on bone (Supplementary Table 3).

Figure 4
Figure 4

BMD analysis by DXA at (A) whole body, (B) lumbar spine, (C) global femur in the treatment groups, and (D) global femur during treatment and follow-up treatment-free period. Data depicted are mean±s.e.m. and show percentage change from pre-surgery levels. BMD, bone mineral density. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0356

Lumbar spine

At the lumbar spine, DXA analysis showed a marked loss of bone (24% BMC and 19% BMD; BMC data are shown in Supplementary Table 3) in OVX rats from the end of the bone depletion period and throughout the study (Fig. 4B). Treatment with Pio resulted in further loss of bone mass (6% BMC and 6% BMD), whereas Feno reduced OVX-induced bone loss (41% prevention of BMD loss at week 12), as demonstrated by the higher BMD values compared with OVX controls. At this site, Feno partially (∼60%) prevented Pio-induced bone loss in Pio+Feno-treated OVX rats. During the treatment-free period, trends for reversal of effects of Pio and Feno were noted.

Femur

At the femur, most notably at sites rich in trabecular bone (distal and proximal ends (data not shown)), a significant loss of bone mass (15% for both BMC and BMD; P≤0.05) was noted in OVX vehicle controls compared with sham vehicle controls over the study period. Treatment of OVX rats with Pio resulted in further loss of bone mass (3% BMC and 3% BMD), whereas Feno attenuated OVX-induced bone loss (36% prevention of BMD loss at week 12), as indicated by the higher BMD values compared with OVX controls (Fig. 4C). Combination treatment of OVX rats with Feno+Pio indicated that Feno partially prevented Pio-induced bone loss. The results obtained over the treatment-free period in the femur were generally similar to the lumbar spine, with reversal of bone loss in Pio-treated animals and a slight reduction in BMD in Feno-treated animals (Fig. 4D).

Peripheral quantitative computed tomography

In vivo pQCT at the proximal tibial metaphysis showed significant reduction of bone mass in OVX control rats (Fig. 5B and C), most of which was attributed to the trabecular bone compartment (62% BMC and 61% BMD). Treatment with Pio further increased bone loss (9% for both trabecular BMC and BMD), whereas Feno prevented or slightly restored OVX-induced bone loss (Fig. 5A). Concomitant treatment with Feno prevented Pio-induced bone loss in OVX rats (∼80% for trabecular BMD), resulting in BMD levels comparable to OVX controls. At the end of the treatment-free period, in vivo pQCT values were similar to values at the end of treatment, showing no evidence of reversal of the effects at the tibial metaphysis. In contrast to the effects observed at the tibial metaphysis, no clear effects of treatment were observed on BMD at the tibial diaphysis (Fig. 5D) (cortical bone site).

Figure 5
Figure 5

(A) Total, (B) trabecular, (C) cortical/subcortical, and (D) cortical BMD analysis by pQCT in the proximal tibial metaphysis (A–C) or diaphysis (D). Data depicted are mean±s.e.m. and show percentage change from pre-surgery levels. BMD, bone mineral density. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

Citation: Journal of Endocrinology 212, 2; 10.1530/JOE-11-0356

Discussion

Clinical use of PPARγ agonists such as Pio or rosiglitazone is associated with an increased risk of fractures (Kahn et al. 2006, Schwartz 2006, Glintborg et al. 2008). In vivo preclinical studies in models of osteoporosis, such as the OVX rat, have documented that TZD PPARγ agonists exacerbate OVX-induced bone loss (Sottile et al. 2004, Kumar 2009, Stunes et al. 2011). The underlying mechanisms of PPARγ-induced bone effects are not fully understood although, collectively, clinical and preclinical data suggest impacts on both bone formation and osteoblast activity, as well as bone resorption and osteoclastogenesis (Schwartz 2008). Whether the deleterious bone effects of PPARγ agonists can be reversed by co-treatment strategies, or by discontinuation of treatment, has not been investigated systematically, although a preliminary non-clinical study showed that the bone antiresorptive agent alendronate could reduce rosiglitazone-induced loss of bone (Kumar et al. 2009).

This study provides the first evidence that co-treatment with a PPARγ and PPARα agonist reduces the deleterious effects of PPARγ agonism on BMC and/or BMD in mature OVX rats. Treatment with Feno partially prevented Pio-induced bone loss, in particular at sites rich in trabecular bone, with BMC and BMD values remaining generally lower compared with OVX controls, suggesting that administration of Feno counteracted some of the negative Pio bone effects. As Feno and Pio are selective PPARα and PPARγ agonists, respectively, the data are consistent with the compounds acting as functional, rather than pharmacological, antagonists in bone. Furthermore, they suggest that dual agonists of PPARα/γ may exhibit lower or no deleterious bone effects in comparison with PPARγ agonists.

Dual agonists of PPARα/γ have long been pursued for their potential to provide better control of cardiovascular risk factors vs solo activation of PPARα or PPARγ, as both dyslipidemia and glucose control/insulin sensitivity are targeted; however, their effect on bone has not been reported. The results of this study may thus have potential clinical safety implications for dual PPARα/γ agonists, as they show that concurrent/dual agonism of PPARγ and PPARα may minimize the negative effect of PPARγ agonism on bone mass/density.

During the preparation of this manuscript, Stunes et al. (2011) reported that Pio augmented OVX-induced loss of BMD, while Feno (and another PPARα agonist, pirinixic acid) maintained BMD at sham levels, determined by DXA and micro-computed tomography at the femur. While the data from the Pio and Feno groups of our study are broadly in line with those reported by Stunes et al., a number of important methodological differences should be highlighted. First, we used mature (7-month-old) OVX rats, which have a lower bone turnover rate compared with young adult rats (3-month-old), minimizing the effect of growth on bone. Secondly, we selected a longer bone depletion period (4 weeks vs 1 week) to ensure that bone loss had been established prior to initiation of treatment. Indeed, the peak effect on bone mass, bone turnover markers, and body weight was noted 4 weeks following surgery, with only a slight progression of the response for the remaining study period. Thirdly, the doses of Pio and Feno employed in our study were lower (10 and 25 mg/kg per day vs 35 and 90 mg/kg per day for Pio and Feno respectively). These lower doses were chosen to minimize possible off-target effects of the compounds while providing adequate pharmacological activity, as evidenced by changes observed in several pharmacodynamic indicators, i.e. decreases in plasma TG in all treated groups, decreases in insulin in Pio and Pio+Feno groups, and increases in adiponectin in Pio and Pio+Feno groups. Fourthly, we studied several bone sites, including the femur, lumbar spine, and tibia, and also included treatment discontinuation to assess the reversibility of treatment effects to increase the reliability of the conclusions. Finally, this manuscript reports the effects of combination treatment with both a PPARα and a PPARγ agonist, providing data to support the predicted beneficial effects of dual agonism in comparison with the deleterious effect of PPARγ agonism alone.

As expected, ovariectomy resulted in increases in body weight gain and fat mass accumulation, accompanied by a lack of gain in bone mass and/or loss of bone mass (particularly at sites rich in trabecular bone), consistent with marked increases in bone turnover markers (CTx and osteocalcin), compared with sham controls. Treatment with Pio exacerbated the OVX-induced loss of bone mass, as shown by DXA and in vivo pQCT. In contrast, treatment with Feno reduced OVX-induced bone loss. BMC and BMD values were generally higher in Feno-treated animals than OVX controls but remained lower compared with sham controls. In contrast to the clear effect of OVX on CTx and osteocalcin, none of the treatments (Pio, Feno, or Pio+Feno) exerted any effect on these parameters in this study. Although both CTx and osteocalcin are used to monitor bone turnover (resorption and formation), the literature, particularly non-clinical reports, is not entirely consistent. For example, Feno increased osteocalcin in intact female rats (Syversen et al. 2009) but had no effect in OVX rats (Stunes et al. 2011), despite clear bone protective effects in both studies. This may be due to the elevated bone turnover levels in OVX rats, which can mask additional effects of treatment with PPARα agonists. Furthermore, measurements of markers of bone turnover are made at single time points and therefore subtle effects of treatments may not be reflected in levels of bone markers, despite the fact that changes are occurring in bone structure, as evidenced by BMC/BMD analysis. In addition, in vitro studies indicate the presence of both PPARα and PPARγ in preosteoblast cells (Jackson & Demer 2000, Syversen et al. 2009) and a role for PPARα in osteoblast differentiation and osteoclastogenesis and bone resorption has been suggested (Chan et al. 2007, Syversen et al. 2009): Feno stimulated mRNA expression of several osteoblast differentiation markers (e.g. sialoprotein and collagen I) in MC3T3-E1 cells (Syversen et al. 2009) and inhibited osteoclast formation (Chan et al. 2007). In contrast, PPARγ activation by high doses of troglitazone and ciglitazone decreased maturation of MC3T3-E1 cells (Jackson & Demer 2000) and rosiglitazone has been shown to decrease the expression of several osteoblast markers in vitro (Hasegawa et al. 2008). The latter is consistent with our finding that Pio exacerbated OVX-induced loss of bone and is in accordance with several other studies showing that TZD PPARγ agonists suppress osteogenesis (Akune et al. 2004) and/or reduce alkaline phosphatase activity (Hasegawa et al. 2008). Thus, PPARγ may exert direct inhibitory effects on osteogenesis/osteoblast function in addition to its well-established effects through stimulation of adipogenesis from marrow progenitor cells that can reciprocally give rise to adipocytes or osteoblasts (Akune et al. 2004). Related to this, we observed that treatment with Feno had a greater effect in counteracting Pio-induced bone loss compared with Pio-induced fat gain, i.e. the net effect of combining Pio+Feno was to suppress the Pio effect on both bone and fat.

The partial protective effect of Pio+Feno co-treatment on bone loss compared with Pio was apparent across a number of bone sites, including lumbar spine, tibia, and femur. In general, the effect of all treatments on bone mass at the tibial diaphysis was marginal. At the lumbar spine and tibial metaphysis, combined treatment with Pio+Feno prevented Pio-induced bone loss by up to 60 and 80%, respectively, while an ∼36% prevention of bone loss was observed at the femur.

A further aspect of this study was an assessment of the effect of a 6-week treatment-free period, which followed the 13-week treatment. In vivo, the net effect of combining Pio and Feno was essential to cancel the opposing effects on fat and bone. During the treatment-free period, the loss of the negative effects of Pio and positive effects on Feno on bone mass and fat mass resulted in all groups trending toward OVX vehicle controls.

Conclusions

Data from this study show that Pio exacerbated bone loss in the mature OVX rat model, predominantly affecting sites rich in trabecular bone. These effects were generally reversible after discontinuation of treatment. In contrast to Pio, Feno increased bone mass and, importantly, co-treatment with Pio+Feno partially prevented Pio-induced loss of bone. These data suggest that concurrent/dual agonism of PPARγ and PPARα may minimize the negative effect of PPARγ agonism on bone mass in rats. While the translation of these data to humans remains to be established, they raise the possibility that treatment with a dual PPARα/γ agonist may be an effective therapeutic regimen to manage diabetes cardiovascular risk and minimize negative effects on bone mass in patients at risk.

Supplementary data

This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-11-0356.

Declaration of interest

A B, E S, and M W are employees of F Hoffmann La-Roche, Switzerland. F B is employed by Roche Diagnostics GmbH, Germany. S Y S, R S, and M A are employees of Charles River Laboratories, Canada.

Funding

This study was performed at Charles River Laboratories and was funded by F. Hoffmann-La Roche AG, Switzerland.

Author contribution statement

A B, E S, M W, F B, and S Y S were involved in the study design, data, and statistical analysis; interpretation of results and discussion; and writing and critical assessment of the manuscript. R S was involved in data acquisition, interpretation of results and discussion, and writing and critical assessment of the manuscript. M A was involved in study conduct and critical assessment of the manuscript.

Acknowledgements

The authors thank A-E Salman, A Vandjour, W Riboulet, C Wohlgensinger, V Griesser, and A Wallier for providing technical assistance for biochemical assays (F. Hoffmann-La Roche AG, Switzerland) and the imaging technical team for scanning assessments (Charles River Laboratories, Canada). Editorial support for the development of this manuscript was provided by Moh Tadayyon (MediTech Media, UK), funded by F. Hoffmann-La Roche Ltd.

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      Society for Endocrinology

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    (A) Group mean body weights, (B) insulin, and (C) triglyceride levels at pre-treatment and during the treatment period. Body weight chart also shows the effect of ovariectomy post-randomization and the impact of discontinuation of treatment. Insulin and triglyceride data are mean±s.e.m. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

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    Mean levels of (A) osteocalcin (bone formation marker) and (B) CTx (bone resorption marker) at the end of the bone depletion period and during treatment. Data are mean±s.e.m. *P ′≤0.05 vs OVX; P ′= adjusted P value (modified from Bonferroni adjustment).

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    Analysis of (A) lean mass and (B) fat mass by DXA at whole-body level and (C) muscle area and (D) fat area by pQCT at the proximal tibia. Data are mean±s.e.m. and show percentage change values adjusted to body weight. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

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    BMD analysis by DXA at (A) whole body, (B) lumbar spine, (C) global femur in the treatment groups, and (D) global femur during treatment and follow-up treatment-free period. Data depicted are mean±s.e.m. and show percentage change from pre-surgery levels. BMD, bone mineral density. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).

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    (A) Total, (B) trabecular, (C) cortical/subcortical, and (D) cortical BMD analysis by pQCT in the proximal tibial metaphysis (A–C) or diaphysis (D). Data depicted are mean±s.e.m. and show percentage change from pre-surgery levels. BMD, bone mineral density. *P ′≤0.05 vs OVX; P ′≤0.05 vs pioglitazone; P ′≤0.05 vs fenofibrate; P ′= adjusted P value (modified from Bonferroni adjustment).