The rats were weighed at baseline and weekly throughout the study, and femoral and whole body DXA scans were performed in duplicates at baseline, and after two and four months.
Bone Regulates Browning and Energy Metabolism Through Mature Osteoblast/Osteocyte PPARγ Expression
Animals were anesthetized with 2. Blood samples were collected by cardiac puncture during final anaesthesia. The livers were dissected and weighed. The coefficient of variation CV was 2. The proximal femurs, including the femoral head and the metaphysis of the dissected formalin-fixed femurs were scanned in a SkyScan microtomograph SkyScan, Antwerp, Belgium , with a voxelsize of Femoral head and metaphysis data sets were analyzed separately.
Cortcial bone and trabecular bone were separated using in-house developed software. For each cross-section in the 3D dataset, a virtual mask of the total bone was created and used to identify the bone marrow regions in the original image. The marrow regions were expanded into a mask of the total marrow cavity using a close operation. Bone inside the total marrow cavity is considered trabecular bone, the remainder is regarded as cortex.
Cortical bone volume Ct. The shafts were fractured The proximal femur was fixed in a clamp, the cam of the rotating wheel engaged the femoral condyles, and a fulcrum positioned All tests were done at a loading rate of 0. Ultimate bending moment M was calculated as the ultimate load before failure multiplied by the moment arm by which the load was applied Newton Meter, Nm.
Energy absorption and stiffness were read directly or calculated from the computer recordings as previously described [ 43 ].
The detection limit was 3. Detection limits were 3. Briefly, tibiae and femurs were removed and all soft tissue was removed. The proximal ends were cut off, and the bones briefly centrifuged g, 10 s. The bone marrow pellets from each animal were pooled and resuspended in culture medium and passed through a gauge needle to achieve a single cell suspension.
Mineralizing fibroblast-colony-forming unit mCFU-f cultures assays were performed essentially as previously described by Scutt et al. Experiments were performed with cells from three female rats in three individual experiments, and with at least three parallel wells for each concentration of the drugs. The media were changed after 5 days, and thereafter every second day for 21 days. Wells were stained for alkaline phosphatase ALP by addition of 0. Calcium-positive colonies were stained with 1.
Collagen-positive colonies were stained with 1. All measurements were performed in duplicates or triplets. All data were tested for normality with the Shapiro-Wilk normality test. Normally distributed parameters were tested with two-tailed unpaired Student's t-test, or one-way ANOVA with Bonferroni's post test, while parameters that were not normally distributed were tested with Mann-Whitney's two tailed test, or Kruskal Wallis test with Dunn's post test. Significance was assumed at p -values lower than 0.
Correlations between ultimate bending moment in the femoral neck and shaft and whole body and femoral BMD were analyzed with a two-way Spearman's Rank correlation test.
Core level regulatory network of osteoblast as molecular mechanism for osteoporosis and treatment
There were no differences in body weight, lean or fat mass between the groups seven days after surgery, when administration of test substances was initiated. The body weight increased in all groups from baseline until the end of the study Figure 1A. There was no difference in body weight between the four OVX groups during the study, but they all had significantly higher body weight than the SHAM group after two and four months Figure 1A. SHAM; 2. OVX; 2. We did not detect any liver pathology by gross visual inspection, however livers were not further examined, as this was not within the scope of this study.
There were no differences in whole body or femoral BMC or BMD between the groups seven days after surgery at the beginning of administration. All ovariectomized groups had a significantly higher whole body BMC than the SHAM group after two and four months, but there was no significant difference in whole body BMC between the ovariectomized groups at any time Figure 2A.
V, Ct. Furthermore, Tb. Th in the femoral head and the Ct. However, Ct. Th in the femoral head compared to the other groups Tables 1 and 2. In conclusion, ovariectomized rats given fenofibrate, and to some extent rats given Wyeth , partly maintained bone architectural structures at sham levels in the femur, while ovariectomized rats given pioglitazone had further deterioration of femoral bone architecture. The OVX controls had significantly lower bending moment and energy absorption in the femoral neck compared to SHAM, while the PIO OVX group had significantly lower bending moment and energy absorption than all the other groups except OVX controls in the femoral neck, and all the other groups for the femoral neck Table 3.
There was no significant difference between the groups regarding stiffness, neither in the femoral neck nor shaft Table 3. Plasma leptin levels were significantly higher in the OVX 6. Plasma adiponectin levels were significantly increased in all ovariectomized groups OVX; Also, pioglitazone led to adipocyte forming colonies, with clearly visual formation of lipid droplets data not shown. Effects of fenofibrate and pioglitazone on osteoblast differentiation.
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, [ 46 ], rats from 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 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.
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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 , 13 ] suggesting an inhibition of endosteal bone resorption. Chan et al. 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.
In contrast to the ovariectomized control group, rats given fenofibrate and Wyeth had similar bending moment and energy absorption as the sham group at the femoral neck, indicating maintained mechanical strength in spite of the ovariectomy.
Ciglitazone—a human PPARγ agonist—disrupts dorsoventral patterning in zebrafish [PeerJ]
Furthermore, administration of fenofibrate and Wyeth 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.
Osteoclasts: differentiation and function
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. 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. Similar findings were recently demonstrated in humans [ 30 — 33 , 52 , 53 ].
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Pioglitazone is still used for treatment of diabetes mellitus 2, while rosiglitazone has been withdrawn due to adverse effects [ 54 ].