Impaired Musculoskeletal Response to Age and Exercise in PPAR / Diabetic Mice

Fragility fractures are recognized complication of diabetes, but yet the underlying mechanisms remain poorly understood. This is particularly pronounced in type 2 diabetes in which the propensity to fall is increased but bone mass is not necessarily low. Thus, whether factors implicated in the development of insulin resistance and diabetes directly impact on the musculoskeletal system remains to be investigated. PPAR / mice have reduced metabolic activity and are glucose intolerant. We examined changes in bone and muscle in PPAR / mice and investigated both the mechanism behind those changes with age as well as their response to exercise. Compared with their wild type, PPAR / mice had an accelerated and parallel decline in both muscle and bone strength with age. These changes were accompanied by increased myostatin expression, low bone formation, and increased resorption. In addition, mesenchymal cells from PPAR / had a reduced proliferation capacity and appeared to differentiate into more of an adipogenic phenotype. Concomitantly we observed an increased expression of PPAR , characteristic of adipocytes. The anabolic responses of muscle and bone to exercise were also diminished in PPAR / mice. The periosteal bone formation response to direct bone compression was, however, maintained, indicating that PPAR controls periosteal bone formation through muscle contraction and/or metabolism. Taken together, these data indicate that PPAR deficiency leads to glucose intolerance, decreased muscle function, and reduced bone strength. On a molecular level, PPAR appears to regulate myostatin and PPAR expression in muscle and bone, thereby providing potential new targets to reverse bone fragility in patients with metabolic disturbances. (Endocrinology 155: 4686–4696, 2014)

S everal cohort studies and meta-analyses have demon- strated that people suffering from type 2 diabetes mellitus (T2DM) have a higher risk of fracture despite their higher-than-average bone mineral density (BMD) (1-3).The surprisingly higher incidence of fracture in T2DM may be explained by both a decrease in bone quality and an increased propensity to fall.Recent investigations in humans have indicated there is a negative alteration of the trabecular and cortical microarchitecture as well as the bone material properties, ie, components of bone quality, in patients with T2DM (4,5).There are several potential mechanisms for reduced bone quality in diabetic patients, including reduced bone formation, increased bone resorption, collagen modifications by advanced glycation end products, and lipid accumulation in the bone marrow (6).In addition, there are a number of mechanisms by which diabetes may contribute to falls, including peripheral neuropathy and decreased muscle strength (7,8).Interestingly, all T2DM features such as insulin resistance and reduced muscle strength have been associated with low levels of physical activity (9), suggesting that exercise training itself may be an efficient intervention in the management of T2DM.Several rodent models have been developed and/or are currently being investigated to help elucidate the pathophysiology of bone fragility in diabetes; however, most of these models have inherent limitations so that the molecular mechanisms that may link alterations in glucose and bone metabolism to muscle and bone alterations remain unclear (10).In particular, the molecular mechanisms for the potentially impaired musculoskeletal response to exercise have not been investigated.
Transcription factors of the peroxisome proliferatoractivated receptor (PPAR) family (PPAR␣, PPAR␤, PPAR␥) are known to play an important role in energy balance.PPAR␥ and more recently PPAR␤ have been shown to be implicated in metabolic dysregulation.More precisely, a decrease in PPAR␤ expression results in decreased glycolysis and lipogenesis in the liver while increasing fat infiltration in skeletal muscle (11)(12)(13).Thus, PPAR␤ Ϫ/Ϫ mice are metabolically less active and develop glucose intolerance (11).Conversely, activation of PPAR␤ genetically or pharmacologically promotes muscle oxidative capability by increasing the number of mitochondria formed in the cell, up-regulating myoglobulin, and increasing the proportion of type I (oxidative or slow twitch) muscle fibers (15)(16)(17).Hence, sufficient PPAR␤ expression positively modulates the capacity to perform physical activity, improves insulin sensitivity, and leads to reduced adiposity.It is not surprising therefore that a PPAR␤ agonist has been used in an animal model to successfully treat insulin resistance (18).
In addition to their lower muscle mass, reduced power, and resistance (15,16), PPAR␤ Ϫ/Ϫ mice have recently been reported to have increased bone turnover and decreased bone mass (20).This observation implicates PPAR␤ in the pathogenesis of bone fragility observed in diabetes.This work showed that in the absence of PPAR␤, Wnt-␤-catenin signaling was altered and associated with lower osteoprotegerin, higher osteoclast number, and osteopenia.However, the function of PPAR␤ on bone formation, particularly bone modeling, and its role in the bone response to exercise is yet to be elucidated.Here we characterize alterations in the biomechanical responses of bones from PPAR␤ Ϫ/Ϫ mice and identify new molecular targets, namely PPAR␥ and myostatin, that may contribute to the impairment of the muscle and bone functions in diabetes.

Animals
PPAR␤ Ϫ/Ϫ mice were generated from a mixed background (Sv129/C57BL/6) and have been previously described (21).Mice were housed five per cage, maintained under standard nonbarrier conditions, and had access to water and soft diet ad libitum (Harlan Teklad 2019, SDS).The mice were maintained at 22°C with a12-hour light, 12-hour dark cycle.A first group of mice was used to describe the role of PPAR␤ on bone and muscle phenotype in growing, young adult, adult, and old mice from 1 to 18 months old (n ϭ 8 female mice/group); a second group of 12-week-old male mice were used for the treadmill exercise (n ϭ 8 male mice/group), and a supplemental group of male mice aged 14 weeks were used for the axial compression studies (n ϭ 6 male mice/group).To measure the dynamic indices of bone formation, mice received sc injections of calcein 9 and 2 days before euthanasia.
The femurs, tibiae, and vertebrae were excised and cleared of fat and connective tissues.Bone and muscle tissue that were to be used for RNA analysis were freshly extracted and frozen on liquid nitrogen.Bones for histomorphometry analysis were immediately fixed in 10% formaldehyde for 48 hours at ϩ4°C.Other bones were placed in damp cloth and frozen at Ϫ20°C for subsequent microarchitectural and biomechanical tests.All animal procedures were designed in accordance with the Swiss Federal Act on Animal Protection, approved by the University of Geneva School of Medicine Ethical Committee and the State of Geneva Veterinarian Office.

Glucose tolerance tests
Glucose tolerance tests were performed after a 6-hour fast by measuring blood glucose on glucose strips and an Accu-check glucometer (Roche) at baseline and at 15, 30, 60, 90, and 120 minutes after an ip injection of glucose at 2 g/kg body weight.Blood was obtained after an incision at the base of the tail.

Handgrip test and locomotor activity
In vivo hindlimb grip strength was measured using an automated grip strength (Bioseb; In Vivo Research Instruments) as previously described (22).Each mouse was tested five times with a 40-second rest interval between tests.The average peak tension and the best attempts were used as a gauge of muscle strength.Two locomotor tests were performed using a treadmill to evaluate both endurance and aerobic maximum speed.For the evaluation of endurance, mice from each group were placed on the treadmill, submitted to moderate exercise at a speed of 14 m/min, and were left to run for as long as they could (mice were considered fatigued when they touched the back of the treadmill five times).The collected end point data were the distance and the time run by each mouse.For the evaluation of aerobic maximum speed, exercise workloads were selected to gradually progress in increments from moderate to maximal intensity.The exercise began with 5 minutes of exercise at a speed of 8 m/min, and speed was thereafter increased by 2 m/min every 2 minutes.The maximal speed was presented as the aerobic maximum speed.

Exercise training
Twelve-week-old male mice were randomly allocated into sedentary and exercise-trained groups for each genotype: PPAR␤ Ϫ/Ϫ and PPAR␤ ϩ/ϩ mice.The mice were trained 5 days per week for 6 weeks.The exercise protocol was determined and adapted to the maximal capacity of PPAR␤ Ϫ/Ϫ mice to perform an exercise on a treadmill: at a speed of 16 m/min, for 30 minutes, with an inclination of 5%.During the first acclimatization week, the treadmill speed and the duration of each running session were gradually increased from 8 m/min for 10 minutes to 16 m/min for 30 minutes.For the last 5 weeks, the running sessions consisted of 16 m/min for 30 minutes with a treadmill inclination of 8°, corresponding to moderate exercise at this specific age (16).

In vivo axial compression
Fourteen-week-old male mice were subjected to axial compression stimulation on 3 alternate days per week for 2 weeks.The loading apparatus used had been specifically adapted for mice tibiae as previously described (23).Mice were anesthetized by isoflurane 2%, and the tibiae were placed on the stimulation machine between the moving pad on the proximal side (the knee) and the fixed pad on the distal side (the foot).The left tibia of each mouse was subjected to dynamic axial stimulation, using the following parameters: peak load ϭ 12 N; peak strain (midshaft cortex) ϭ 1500 ⑀; pulse period (trapeze shaped pulse) ϭ 0.1 second; rest time between pulses ϭ 10 seconds; and full cycle frequency (pulse ϩ rest) ϭ 0.1 Hz.A total of 40 cycles (ϳ7 min) were applied per session.The nonstimulated right tibia served as an internal control.

In vivo measurement of body composition and BMD
Body composition and total body, femoral, and spinal BMD (grams per square centimeter) were measured in vivo by dualenergy X-ray absorptiometry (PIXImus2; GE Lunar) at 12 and 18 weeks of age in the exercise experiment; at 13 and 16 weeks of age in the axial compression experiment; and at 1, 4, 12, and 18 months for the aging experiment.Lean limb mass was evaluated by positioning the region of interest perpendicular to the vertebral column and with the corner of the region of interest aligned to the anterior margin of the hip.

Ex vivo measurement of morphology and microarchitecture
Microcomputed tomography (UCT40; Scanco Medical AG) was used to assess the trabecular microarchitecture in the excised fifth lumber spine body and distal femur and cortical bone geometry at the midshaft femoral diaphysis as previously described (24).Trabecular and cortical bone regions were evaluated using an isotropic voxel resolution of 12 m.For the vertebral trabecular region, we evaluated 250 transverse computed tomography slices between the cranial and caudal end plates, excluding 100 m near each endplate.For the femoral and tibial trabecular regions, to eliminate the primary spongiosa, we analyzed 100 slices from the 50 slices under the distal growth plate.Femoral cortical geometry was assessed using 50 continuous computed tomography slides (600 m) located at the femoral midshaft.For the trabecular bone regions, we assessed the bone volume fraction (BV/TV, percentage), trabecular thickness (micrometers), trabecular number (millimeters Ϫ1 ), trabecular connectivity density (millimeters Ϫ3 ), and structural model index.For the cortical bone at the femoral and tibial midshaft, we measured the cortical tissue volume (CtTV; cubic millimeters), cortical bone volume (CtBV; cubic millimeters), the marrow volume (cubic millimeters), and the average cortical width (micrometers).To evaluate bone marrow adiposity tibias, we processed protocol with osmium staining as described (25).After labeling of lipids by osmium tetroxide, the bones were imaged using energy of 45 keV.

Testing of mechanical resistance
The night before the mechanical testing, bones were thawed slowly at 7°C and then maintained at room temperature.A threepoint bending test was performed on the femur as follows: bones were placed on the material testing machine on two supports separated by a distance of 9.9 mm, and the load was applied to the midpoint of the shaft.The mechanical resistance to failure was tested using a servocontrolled electromechanical system (Instron 1114; Instron Corp) with the actuator displaced at 2 mm/ min.Outcomes measured were ultimate force, stiffness, and energy as described previously (26).

Histomorphometry
After 48 hours of fixation, bones were dehydrated in absolute acetone and embedded in methyl-methacrylate (Merck), and 8-m-thick transversal sections of the midshaft were cut with a Leica Polycut E microtome (Leica Corp Microsystems AG) and mounted unstained for the evaluation of fluorescence.Five-micrometer-thick sagittal sections were stained with modified Goldner's trichrome, and histomorphometric measurements were performed on the secondary spongiosa of the proximal tibia metaphysis and on the endocortical and periosteal bone surfaces in the middle of the tibia using a Leica Q image analyzer (Leica Corp) at ϫ40 magnification.All parameters were calculated and expressed according to standard formulas and nomenclatures (27): mineral apposition rate (micrometers per day), mineralizing surface per bone surface (percentage), bone formation rate (cubic micrometers per square micrometer per day).Osteoclast surface per bone surface and numbers were evaluated only at 4 and 18 months of age.

RNA extraction and quantitative PCR
The whole tibia was excised, and both tibial extremities were cut to remove the bone marrow from the diaphysis by flushing with cold PBS.Muscle gastrocnemius was frozen by dipping them on liquid nitrogen.Muscle and tibial diaphysis plus extremities were immediately pulverized to a fine powder and homogenized in peqGold Trifast (peQLab Biotechnologie GmbH) using FastPrep system apparatus (QBiogene).Total RNA was extracted and then purified on minicolumns (RNeasy minikit; QIAGEN) in combination with a deoxyribonuclease treatment (ribonuclease free deoxyribonuclease set; QIAGEN) to avoid DNA contamination.Single-stranded cDNA templates for quantitative real-time PCR analyses were carried out using Super-Script III reverse transcriptase (Invitrogen AG) following the manufacturer's instructions.Quantitative RT-PCR was performed using predesigned TaqMan gene expression assays (references in Supplemental Material).Relative quantities were calculated with the formula, relative quantity ϭ E Ϫ cycle threshold using an efficiency (E) of 2 by default.For each gene the mean quantity was calculated from triplicates for each sample, and this quantity was normalized to the similarly measured mean quantity of the glyceraldehyde-3-phosphate dehydrogenase normalization gene.Finally, normalized quantities were averaged for three to four animals.

Bone marrow mesenchymal stem cell enriched culture
Mesenchymal stem cells (MSCs) were isolated from the femurs of PPAR␤ Ϫ/Ϫ and PPAR␤ ϩ/ϩ adult mice.For this purpose, after removing soft tissue and epiphysis, bone marrow was flushed with DMEM medium.Marrow was then submitted to a 15-min enzymatic digestion.Collagenase activity was neutralized and marrow was then resuspended in ␣MEM (Gibco) supplemented with 10% fetal calf serum (Amimed), antibiotics (penicillin 100 U/mL, streptomycin 100 g/mL; Gibco), glutamine (200 mM; Gibco), amino acids (Amimed), and amphotericin B (0.25 g/mL; Amimed).The cells were plated at 10 6 /cm 3 cells in a T75 flask and incubated at 37°C with 5% CO 2 , and the media were changed every 3 days until the cells reached 80% confluence.Outcomes were the quantification of MSC PPAR␤ Ϫ/Ϫ and PPAR␤ ϩ/ϩ differentiation into adipocyte and osteoblast populations.For adipogenesis, when MSCs reached 100% of confluence, adipogenesic media (DMEM with 10% fetal bovine serum, 1 m dexamethasone, 0.5 mm isobutylmethylxanthine, 10 g/mL insulin, and 100 m indomethacin) were added.After 3 days of the adipogenic medium, cells were returned to a medium with DMEM, l0% fetal calf serum, l m dexamethasone, and 5 pg/mL insulin that was refreshed twice per week.After 14 days in culture, adipocytes were visualized by oil red staining and quantified at 540 nm on a spectrophotometer.For osteoblastogenesis, when the MSCs reached 100% of confluence, osteogenic media (DMEM with 10% fetal bovine serum, 10% 100 mM ␤-glycerophosphate, 10 Ϫ8 M dexamethasone, 5 mg ascorbic ac- id-2 phosphate) were added and replaced twice per week.After 14 days in culture, calcium depositions were visualized by alizarin red S staining and quantified at 405 nm on a spectrophotometer.

Collection of serum
Blood from all mice was obtained by a submandibular collection at 1 and 18 months of age.After centrifugation, serum was removed and stored at Ϫ80°C until analysis.Serum carboxy-terminal collagen cross-links and osteocalcin were measured according to the manufacturer's instructions (SBA Sciences and Biomedical Technologies Inc).Myostatin was measured in serum by an ELISA with a kit from United States Biological Corp) following the manufacturer's instructions.

Data analysis
We first tested the effects of exercise or loading within groups (PPAR␤ Ϫ/Ϫ and PPAR␤ ϩ/ϩ ) by paired or unpaired t tests.In the mechanical loading experiments, we compared stimulated and nonstimulated tibia in the same animal using a paired t test.For the exercise investigation, we compared sedentary vs trained animals using unpaired t tests.To compare the effect of genotype and the response to loading (mechanical loading and exercise), we used a two-way ANOVA.As appropriate, post hoc testing was performed using Fisher's protected least squares difference.The P value of interaction between the genotype and loading (mechanical stimulation or exercise) was mentioned only when it was found to be significant.We then tested the effects of repeated measures within groups (for the effects of aging) by a one-way repeated-measures ANOVA with the genotype used as a factor.Differences were considered significant at P Յ .05.Data are presented as mean Ϯ SEM.

Body weight, lean mass, muscle strength, and BMD
First, we confirmed that PPAR␤ Ϫ/Ϫ mice were glucose intolerant at 4 and 18 months and found that this alteration was maintained, but not exacerbated, as they age (Supplemental Figure 1A).We found the same trend at the age of 12 months but the difference was not statistically significant.

Bone microarchitecture, turnover, and biomechanical properties
Age-related changes in trabecular microarchitecture followed a similar pattern in the distal femur and caudal vertebrae, although in the femur the loss of BV/TV occurred earlier and was more dramatic (Figure 2A).At the vertebrae, the decline in BV/TV was higher in PPAR␤ Ϫ/Ϫ at 12 and 18 months of age (Figure 2B and Supplemental Table 1), whereas trends were observed only at the distal femur.In the same region, ie, the metaphysis of the distal femur, old PPAR␤ Ϫ/Ϫ mice exhibit higher osmium staining, indicating more bone marrow adiposity (ϩ217% of adipocyte volume per tissue volume vs PPAR␤ ϩ/ϩ , P Ͻ .05)(Figure 2C).In the midshaft femur, cortical tissue volume and bone volume stabilized at the age of 12 months.At 18 months of age, PPAR␤ Ϫ/Ϫ mice exhibited lower cortical tissue volume and bone volume compared with PPAR␤ ϩ/ϩ mice (Figure 2, D and E) together with increased endocortical porosity (Figure 2F).These changes were associated with higher osteoclast number and surface in PPAR␤ Ϫ/Ϫ vs PPAR␤ ϩ/ϩ mice (Supplemental Table 1), whereas the bone-forming indices were lower, particularly at the periosteal surfaces at a latter age (Figure 2, G-J).Consistent with bone histomorphometry, at 18 months of age, bone resorption (carboxy-terminal collagen cross-links) and bone formation (osteocalcin) markers were higher and lower, respectively, in PPAR␤ Ϫ/Ϫ than PPAR␤ ϩ/ϩ mice (Supplemental Table 2), indicating that bone turnover and coupling is altered in the absence of PPAR␤.In addition, we found higher circulating levels of myostatin in PPAR␤ Ϫ/Ϫ mice compared with PPAR␤ ϩ/ϩ mice.
As evaluated by three-point bending, ultimate force and stiffness were lower in PPAR␤ Ϫ/Ϫ mice compared with PPAR␤ ϩ/ϩ mice at 4, 12, and 18 months (Figure 2, K and L).At 18 months of age, the energy to fracture was also significantly lower in PPAR␤ Ϫ/Ϫ compared with PPAR␤ ϩ/ϩ mice (Figure 2M).

Altered osteoblast differentiation in PPAR␤deficient cells
To clarify the molecular mechanisms by which PPAR␤ deficiency alters the bone forming activity, we first examined bone mRNA expression levels in the old mice (18 mo of age).Expression levels of genes associated with osteoblast and osteocyte differentiation, as well as Foxo1 and Hmox, were significantly altered in PPAR␤ Ϫ/Ϫ mice (Figure 3A).

Response to physical activity and loading
We first evaluated whether moderate physical activity can restore the musculoskeletal phenotype of PPAR␤ Ϫ/Ϫ mice, particularly muscle force and bone formation at periosteum surfaces.
Having established that PPAR␤ modulates the muscle and bone response to moderate physical activity, we next asked whether the lack of skeletal response in PPAR␤ Ϫ/Ϫ was secondary to the muscle dysfunction and/or could be explained by an intrinsic failure of the bone mechanotransduction.To this aim, mice were subjected to direct axial compression of the tibia in vivo for 2 weeks.Contrasting with exercise, axial compression of the tibia significantly increased BMD and CtBV in both PPAR␤ ϩ/ϩ and PPAR␤ Ϫ/Ϫ mice (Figure 6, A-C).
Loading also stimulated periosteal bone formation similarly in PPAR␤ ϩ/ϩ and PPAR␤ Ϫ/Ϫ mice (Figure 6, D and F).However, at the endocortical  surface, loading increased the bone-forming indices only in PPAR␤ ϩ/ϩ mice (Figure 6, E and F).Taken together, these data indicate that PPAR␤ deficiency alters the periosteal modeling response induced by muscle activity but not by direct mechanical loading, whereas alterations of endosteal bone remodeling occur independently of muscle activity.

Discussion
Our results establish several important new findings regarding the role of PPAR␤ in the pathogenesis of bone fragility in diabetes.First, in the absence of PPAR␤, there is an accelerated and parallel decline of muscle strength   The metabolic syndrome and T2DM are characterized by a compromised ability of insulin to control glucose disposal in muscle, increased hepatic glucose production, and overt postprandial hyperglycemia.In accordance with the literature, our study confirms that PPAR␤ Ϫ/Ϫ mice are metabolically less active and glucose intolerant from young adulthood to older age (11).In our study the glucose intolerance was not significant at 12 months of age, but we still observed the same trend as at an earlier age.This was probably due to an artifact of glucose measurement or mice not completely fasted because PPAR␤ Ϫ/Ϫ mice are again intolerant at 18 months of age.We specifically provide three lines of evidence indicating that PPAR␤ is also implicated in the pathogenesis of bone fragility that could be observed in metabolic syndrome.
First, PPAR␤ deficiency not only alters the proportion of type II/type I fibers and muscle oxidative function (28) but also plays a major role in the maintenance of muscle mass and strength during aging.These two latter factors are now recognized as significant determinants of bone fragility and frailty (29,30).Most prominently, PPAR␤ deficiency increased myostatin levels both locally and systemically, which can negatively impact on both muscle development and osteoblastic function (31)(32)(33).Indeed, in vitro, PPAR␤ activation induces myogenesis by inhibiting myostatin expression (34).Interestingly, type 2 diabetic patients have an up-regulated plasma myostatin associated with increased body mass index, higher fasting plasma glucose, and blood insulin sensitivity (35,36).Myostatin and its receptors, type IIA and IIB activin Rc, have become prominent targets for the development of therapeutic inhibitors (37), with the potential for use in muscle dysfunction associated with T2DM and sarcopenic patients (now in phase II-III clinical trials, see http://clinicaltrials.gov/ct2/results?termϭmyostatin&SearchϭSearch).Thus, increased myostatin expression in PPAR␤-deficient mice may at least partly explain their poor muscle strength.
Second, in bone, PPAR␤ has been shown to regulate Opg expression and osteoclastogenesis through the ␤-catenin signaling pathway (20).We also observed decreased Ctnnb and Opg expression in PPAR␤-deficient cells and an increase in osteoclast number and bone resorption markers in PPAR␤ Ϫ/Ϫ mice.In accordance, we observed the appearance of cortical porosity in PPAR␤ Ϫ/Ϫ mice.Furthermore, our data demonstrate that PPAR␤ in bone cells plays a role in the regulation of osteoblast differentiation and function with lower expression of Collagen 1a, Runx2, and Osterix.
We also show an alteration of osteoblastic oxidative stress in the absence of PPAR␤, as previously demonstrated in myoblasts, endothelial cells, and the ROS17/2.8 osteoblastic cell line (38 -40).In vivo our results indicate that bone formation indices are negatively altered at the endocortical surfaces at early time points and later on at the periosteum.Consistent with this, PPAR␤ Ϫ/Ϫ exhibits a lower BMD and cortical structure, ie, CtTV and CtBV in adult and old mice.With regard to the biomechanical properties, the femurs of the knockout mice exhibit lower ultimate force, stiffness, and energy to fracture.Interestingly, young adult PPAR␤ Ϫ/Ϫ mice exhibit a degradation of biomechanical properties without any significant changes in BMD and microarchitecture, suggesting an alteration of bone material properties.Previous rodent models suggest that a dysregulation of glucose metabolism leads to an accumulation of reactive oxygen species such as advanced glycation end-products and superoxide dismutase, which interact with bone matrix collagen and therefore change the mechanical properties of the bone (10).This has been demonstrated in Zucker rats and yellow Kuo Kondo mice (41); however, all these models exhibit a low BMD.Therefore, PPAR␤ Ϫ/Ϫ mice are unique in that they display similar key metabolic and skeletal characteristics as in human T2DM patients, namely lower mechanical properties and higher cortical porosity in the absence of low BMD (5).
In addition PPAR␤ Ϫ/Ϫ mice were characterized by a higher fat infiltration in the bone marrow, which can also influence bone quality.This fat accumulation was associated with an up-regulation of PPAR␥ expression.In vitro, we confirm a higher transdifferentiation activity of PPAR␤ Ϫ/Ϫ MSCs into adipocytes, paralleled by an overexpression of PPAR␥.Activation of the PPAR␥2 isoform leads to MSC differentiation toward the adipocyte lineage at the expense of the osteoblast (42).Importantly, there has been increasing evidence that the inverse correlation between bone and fat phenotypes also exists in osteoporosis (42) and T2DM patients (43).
Third, exercise was unable to restore muscle and/or bone mass and strength in PPAR␤ Ϫ/Ϫ mice, contrasting with preclinical and clinical studies showing an improvement of muscle function by exercise through an increase in glycogen synthesis and a decrease in lipid accumulation in rodent models of type 2 diabetes (44,45) and T2DM subjects (46,47).In contrast, direct mechanical loading of the tibiae restored periosteal bone formation but not the endocortical response.We hypothesize that the lack of periosteal bone response to exercise in PPAR␤ Ϫ/Ϫ may be attributed to lower muscle contraction in these mice because muscle stimulation is not involved in the bone response to direct mechanical loading.This explanation is consistent with reports in the literature describing lower muscle function correlating with reduced PPAR␤ expression (48,49), with the lower grip strength observed in the PPAR␤ Ϫ/Ϫ mice and with the known effects of muscle contraction on bone structure (50).Alternatively, the lack of PPAR␤ in muscle may have altered the expression of a bone growth factor such as Igf-1, bone morphogenetic protein (Bmp), and/or other myokines, known to be involved in the musculoskeletal system in response to exercise (51,52).In our model, Igf-1 is not significantly affected; however, Bmps and Mstn are expressed, respectively, less and more in PPAR␤ Ϫ/Ϫ mice compared with their wild-type littermates.Thus, further studies aimed at investigating whether the use of a myostatin inhibitor or BMP treatment can restore the bone response to exercise of PPAR␤ Ϫ/Ϫ mice are warranted.
To potentially explain the absence of bone formation at the endocortical surfaces in loaded PPAR␤ Ϫ/Ϫ mice, two major hypotheses are suggested.First, the absence of PPAR␤ may alter the structure of the bone matrix, particularly at the endocortical surface, and therefore impair its mechanotransduction properties, as we previously reported for the periosteal surfaces in periostin-deficient mice (26).Second, bone marrow stem cells are capable of sensing exogenous mechanical signals and shift MSC differentiation toward osteoblastogenesis and away from adipogenesis (53,14).These alterations have been related to decreased Runx2 and increased PPAR␥ expression, respectively (19), two transcriptions factors modulated by PPAR␤.Therefore, the absence of a bone response to axial compression at the endocortical compartment could be attributed to the default of MSCs to differentiate into the osteoblastic lineage.
In conclusion, the absence of PPAR␤ leads to three major systemic changes, namely glucose intolerance, decreased muscle strength, and exercise capacity, together with low bone formation and response to exercise.At the molecular level, PPAR␤ appears to regulate myostatin and PPAR␥ levels in muscle and bone, thereby providing potential new mechanisms to explain bone fragility related to metabolic syndromes and potentially identifying new molecular targets for the treatment of muscle and bone fragility in diabetes.

Figure 2 .
Figure 2. Effect of aging in the absence of PPAR␤ on bone microarchitecture, bone formation indices, and strength.A, Trabecular BV/TV of femur.B, BV/TV of the vertebral body.C, Upper panel, Illustration of three-dimensional trabecular structure of the distal femur in mice aged 18 months.Middle panel, Two-dimensional illustration of adipocytes stained at the distal femur by osmium and visualized by microcomputed tomography.Lower panel, Three-dimensional reconstruction of adipocyte volume present in the marrow space.CtTV (D) and CtBV (E) at the midshaft femur are shown.F, Two-dimensional reconstruction of the cortical midshaft illustrating the lower CtBV in part by increasing the endocortical porosity.G-J, Bone formation indices at endocortical (Ec) and periosteal (Ps) surfaces.MAR, mineral apposition rate; MPm/BPm, mineralization perimeter on bone perimeter.K-M, Biomechanical properties of the cortical femur obtained by three-point bending tests.*, P Ͻ .05;**, P Ͻ .01;***, P Ͻ .001significant difference vs PPAR␤ Ϫ/Ϫ mice.Continuous lines, PPAR␤ ϩ/ϩ ; hatched lines, PPAR␤ Ϫ/Ϫ show means (Ϯ SEM).