TY - JOUR
AU - Rubin,, Janet
AB - Abstract The contribution of marrow adipose tissue (MAT) to skeletal fragility is poorly understood. Peroxisome proliferator-activated receptor (PPAR)γ agonists, associated with increased fractures in diabetic patients, increase MAT. Here, we asked whether exercise could limit the MAT accrual and increase bone formation in the setting of PPARγ agonist treatment. Eight-week-old female C57BL/6 mice were treated with 20-mg/kg·d rosiglitazone (Rosi) and compared with control (CTL) animals. Exercise groups ran 12 km/d when provided access to running wheels (CTL exercise [CTL-E], Rosi-E). After 6 weeks, femoral MAT (volume of lipid binder osmium) and tibial bone morphology were assessed by microcomputer tomography. Rosi was associated with 40% higher femur MAT volume compared with CTL (P < .0001). Exercise suppressed MAT volume by half in CTL-E mice compared with CTL (P < .01) and 19% in Rosi-E compared with Rosi (P < .0001). Rosi treatment increased fat markers perilipin and fatty acid synthase mRNA by 4-fold (P < .01). Exercise was associated with increased uncoupling protein 1 mRNA expression in both CTL-E and Rosi-E groups (P < .05), suggestive of increased brown fat. Rosi increased cortical porosity (P < .0001) but did not significantly impact trabecular or cortical bone quantity. Importantly, exercise induction of trabecular bone volume was not prevented by Rosi (CTL-E 21% > CTL, P < .05; Rosi-E 26% > Rosi, P < .01). In summary, despite the Rosi induction of MAT extending well into the femoral diaphysis, exercise was able to significantly suppress MAT volume and induce bone formation. Our results suggest that the impact of PPARγ agonists on bone and marrow health can be partially mitigated by exercise. Despite its association with low bone density, hematologic disorders, and antidiabetic therapies (1–6), marrow adipose tissue (MAT) remains poorly understood. We have shown that MAT responds robustly to both diet and exercise intervention: MAT increases significantly in response to high-fat diet (HFD) and is suppressed by daily exercise (7). Exercise-induced bone formation correlated with suppression of MAT, such that exercise effects might be due to either calorie expenditure from this depot, or from mechanical biasing of mesenchymal stem cell (MSC) lineage away from fat and toward bone. Exercise is universally recognized as a means of suppressing obesity-associated white adipose tissue depots as well as enhancing bone density (8–12). Skeletal loading stimulates bone formation (7, 8, 13–15), whereas inactivity is permissive to decreased bone density, increased bone resorption and fracture risk (16–19). Importantly, under systemic challenges such as a HFD, which leads to rapid acquisition of MAT, exercise not only suppressed MAT acquisition but also stimulated bone formation (7). Thus, exercise effectively reduces MAT and induces bone anabolism despite dietary induction of MAT accumulation. We here focus on peroxisome proliferator-activated receptor (PPAR)γ agonists, which have been shown to increase MAT in rodents and to have adverse effects on bone quantity and fracture risk in humans (20–23). PPARγ agonist effects on bone health have been attributed to preferential biasing of MSC into the adipocyte, as opposed to osteoblast, lineage (24). In mice, reduction of MSC adipogenesis in the PPARγ hypomorph is associated with increased bone (24) and increased MSC adipogenesis is accompanied by decreased bone (25). In mice and in humans, there is evidence that, despite preservation of bone volume in obesity, increased MAT is associated with decreased bone quality (26, 27). As such, it has been suggested that increased marrow fat may arise at the expense of MSC progenitors entering the osteoblast lineage (14, 28). In vitro data from our lab suggest that in the face of enhanced PPARγ activation, physical stimulation can prevent adipogenesis (29), but this has not been tested in vivo. Experiments were thus designed to consider whether MAT, due to direct stimulation of MSC by the PPARγ agonist rosiglitazone (Rosi), could be influenced by exercise and whether PPARγ agonism would interfere with exercise-induced bone acquisition. Presented data will show that Rosi increased volumetric lipid accumulation in mouse femurs. Importantly, MAT acquisition in Rosi-treated mice was significantly suppressed by exercise. We will also demonstrate that exercise had differing effects on lipogenesis and thermogenesis in control (CTL) and Rosi groups. Our results suggest that effects of exercise to cause bone formation are preserved even in the face of direct stimulation of marrow adipogenesis by PPARγ agonist. Materials and Methods Animals and diet The University of North Carolina Institutional Animal Care & Use Committee approved the use and care of animals. Eight-week-old female C57BL/6 mice (n = 20) were randomly assigned to 1 of 2 diets for a period of 6 weeks: 1) regular diet (CTL) (item 5058; PicoLab Mouse Diet 20) or 2) a Rosi diet comprised of Rosi (item 71740; Cayman Chemical) incorporated into the regular diet at 0.02 g/kg of diet to achieve 3 mg/kg of body weight per day, consistent with previous studies as a commonly applied dose (30–32). Mice were fed ad libitum, the grams of food consumed were weighed, and calories per gram calculated. Female C57BL/6 mice were used because this gender and strain has been shown to be highly motivated to exercise daily when provided with access to running wheels (7). Exercise intervention Mice in either diet group were allocated to an exercise intervention for 6 weeks as previously described (7, 33). The groups were as follows: 1) regular diet (CTL); 2) CTL exercise (CTL-E); 3) Rosi; and 4) Rosi-E. There was n = 5 mice/group, and an additional experiment was combined in order to yield n = 10/group for mRNA data (see Figure 4). Change to Rosi and/or access to the exercise wheel were commenced simultaneously. Both CTL and running mice were individually housed for the duration of the running experiments to track individual running patterns monitored using a Mity 8 Cyclocomputer (model CC-MT400). CTL mice were housed similarly (one mouse/cage) without wheel access. This experiment was repeated and tibial mRNA results from 2 separate experiments was combined to achieve n = 10 per group. Rosi and exercise effect on white and brown fat markers Figure 4. Open in new tabDownload slide Whole tibias were harvested and mRNA isolated from C57BL/6 mice after 6 weeks of running ± Rosi. Relative expression is shown by real-time PCR normalized to GAPDH. Results are expressed as mean ± SEM (n = 10/group).). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Figure 4. Open in new tabDownload slide Whole tibias were harvested and mRNA isolated from C57BL/6 mice after 6 weeks of running ± Rosi. Relative expression is shown by real-time PCR normalized to GAPDH. Results are expressed as mean ± SEM (n = 10/group).). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Real-time PCR Total RNA from murine tibia or perigonadal fat pad was isolated, and 1 μg was reverse transcribed and analyzed via real-time PCR as previously described (34). Ten microliters of cDNA from each experimental condition were pooled and diluted 1:10 to 1:10 000 to generate a 5-point standard curve. A nontemplate CTL was added to each PCR. Standards and samples were run in duplicate. PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplicons. Specimen harvest and preparation Running wheels were maintained in the cage until the time of harvest. Animals were weighed and euthanized by CO2 inhalation followed by decapitation. Perigonadal fat pads were collected. Right and left femora and tibiae were harvested, and soft tissue was removed. Tibiae were prepared for microcomputer tomography (μCT) imaging by fixation with 10% neutral-buffered formalin at 4°C overnight (35). Femur specimens were prepared for osmium stain by fixation with 10% neutral-buffered formalin at 4°C overnight, followed by decalcification with 14% EDTA for 14 days (7). Quantification/imaging of MAT Our method for volumetric assessment of osmium staining of lipid in mouse bones has been previously described (7, 36). Briefly, femurs were fixed, then decalcified, and subsequently incubated with 1% osmium tetroxide/2.5% potassium dichromate for 48 hours. μCT imaging was performed at 10-μm isotropic resolution. Quantitative, volumetric image analysis (7) of the lipid binder, osmium was performed via standard density (Hounsfield unit [HU]) weighted volumetric measurements. Only locations with a minimal density of 2000 HU were incorporated in the analysis (7). Adipocyte number correlates with MAT quantified by osmium tetroxide-μCT imaging (7, 37). Bone microarchitecture The bone microarchitecture parameters of the proximal tibial metaphysis and mid diaphysis were quantified ex vivo using high-resolution (resolution = 8 μm; E = 55 kVa; I = 145 μA) x-ray μCT (μCT40; Scanco Medical) as previously described (7, 38). Intracortical porosities were quantified as previously described (39, 40). Briefly, the interface of trabecular and cortical surfaces was manually contoured. In order to resolve the 2 compartments, the natural contour was preserved at the endosteum, where the cortical surface was delineated from trabecular struts based on variations in discrete density values (mgHA/ccm). Separation of these compartments was followed by use of an automated algorithm, which was then applied to quantify bone content in each. Trabecular bone was subtracted from total area of evaluated region of bone, thus determining cortical bone. Fixed density thresholds were used to separately quantify cortical and trabecular bones across all groups. Intracortical porosities were calculated by subtracting the cortical bone volume from the total cortical bone volume, thus resolving porosities within the cortical compartment. Statistics Before undertaking the experiment, we calculated that having 6 mice in each group would provide at least 80% power. Prestudy power, calculated under certain specified assumptions, represents the prestudy probability that we will obtain a significant result at the conclusion of the study. As such, the prestudy power calculation is not directly relevant for interpreting our observed results, for which we did obtain a significant comparison for n = 5. Statistical significance was evaluated by two-way ANOVA with correction for multiple comparisons via a Tukey post hoc test (GraphPad Prism 6.05). Exercise intervention and drug intervention (Rosi) were used as the analysis variables. Results Mice treated with Rosi exercise similarly to CTLs Food consumption and running exercise (measured by daily running distance) did not differ between Rosi-E and CTL-E. CTL and Rosi mice consumed 151.8 ± 1.98 and 149.0 ± 13.4 g of food for the 6-week experiment, respectively (Figure 1A). Daily calorie intake for CTL and Rosi mice was 569.4 ± 7.44 and 558.8 ± 50.24 kcal (Figure 1B). Exercise did not significantly alter daily calorie intake, but there was a trend for increased daily food and calorie intake in CTL-E mice compared with CTL (Figure 1, A and B). Body weight did not differ significantly between the groups at the beginning of the experiment (CTL 18.76 ± 0.23, CTL-E 18.48 ± 0.55, Rosi 18.42 ± 0.35, Rosi-E 19.00 ± 0.32) (Figure 1D). At the end of the 6-week experiment, there was a trend for greater weight in the Rosi group (CTL 21.74 ± 0.41, CTL-E 21.58 ± 0.53, Rosi 22.50 ± 0.46, Rosi-E 22.38 ± 0.70) (CTL vs Rosi, P = ns; Figure 1D). Mice treated with Rosi exercise similarly to CTLs Figure 1. Open in new tabDownload slide Eight-week-old C57BL/6 mice had access to running wheels at the start of the experiment. A, Food intake per group for the duration of the experiment (6 wk). B, Kilocalories consumed. C, Daily running distance. D, Body weight. Results are expressed as mean ± SEM. Figure 1. Open in new tabDownload slide Eight-week-old C57BL/6 mice had access to running wheels at the start of the experiment. A, Food intake per group for the duration of the experiment (6 wk). B, Kilocalories consumed. C, Daily running distance. D, Body weight. Results are expressed as mean ± SEM. Voluntary access to running wheels was provided to both CTL-E and Rosi-E at the time of pharmacologic intervention, a resource used by both groups through the course of the study (Figure 1C). CTL-E and Rosi-E mice ran an average of 11.9 ± 0.99 and 12.18 ± 0.95 km/d, respectively (P = not significant [ns]) (Figure 1C), with activity logs demonstrating the use of the wheels for an average of 6 h/d, with no difference between groups. Exercise attenuated MAT despite PPARγ agonist MAT was quantified in the femur using volumetric μCT imaging of the lipid-binder, osmium (7). Visualization was performed by superimposing and averaging the images of each femur (n = 5 per group) and colored labeling of osmium according to HU density (Figure 2, A–C). Exercise suppresses MAT accumulation despite PPARγ agonist treatment Figure 2. Open in new tabDownload slide Visualization of osmium stain by μCT in (A) sagittal, (B) coronal, and (C) axial planes. Quantification of osmium as a measure of MAT in (D) whole femur, (E) diaphysis, (F) metaphysis, and (G) epiphysis. H, 3D representation of regions. Results are expressed as mean ± SD (n = 5/group). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Figure 2. Open in new tabDownload slide Visualization of osmium stain by μCT in (A) sagittal, (B) coronal, and (C) axial planes. Quantification of osmium as a measure of MAT in (D) whole femur, (E) diaphysis, (F) metaphysis, and (G) epiphysis. H, 3D representation of regions. Results are expressed as mean ± SD (n = 5/group). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Rosi treatment was associated with a marked increase in MAT acquisition in the whole femur as well as specific anatomic regions of the femur; whole femur MAT (in mm3 osmium/femoral volume) was 11% in CTL, as compared with 52% in Rosi (P < .0001). More specifically, MAT was 9% in the distal epiphysis of CTL as compared with 62% in Rosi-treated mice (P < .0001). MAT was 18% in the metaphyseal region of CTL as compared with 47% in Rosi (P < .0001), and 9% in diaphyseal shaft of CTL as compared with 51% in Rosi (P < .0001). In CTL-E, daily running for 6 weeks resulted in significantly lower volumetric MAT of 6.9%, as compared with 11.5% to CTL (P < .01). A similar running regimen also effectively suppressed MAT in Rosi-E, with total MAT of 51% in Rosi falling to 42% in Rosi-E (P < .0001). Running exercise decreased the extension of adipose tissue into the diaphysis (Figure 2) and decreased osmium signal through the bone in both CTL mice and those treated with Rosi. Running increased trabecular bone quantity in CTL-E and Rosi-E mice Bone microarchitecture parameters were measured in the proximal tibial metaphysis and diaphysis to assess response to both Rosi and exercise (Figure 3). Although Rosi increased cortical porosity in both Rosi and Rosi-E groups relative to CTL and CTL-E (P < .0001 and P < .01, respectively) (Figure 3C), Rosi did not significantly compromise bone volume/total volume (BV/TV), trabecular number, trabecular thickness (Tb.Th), or trabecular spacing (Figure 3D) in comparison with CTL. Exercise increased BV/TV in CTL-E by 21% relative to CTL, reinforcing the potent effect of 6-week running on trabecular bone enhancement (P < .05; Figure 3D) (7). Notably, we found that in the setting of Rosi treatment, exercise increased trabecular BV/TV to an even greater extent than in the CTL mice, a percent difference of 26% (Rosi-E vs Rosi) (P < .01; Figure 3D). In parallel, there was a significant increase in Tb.Th in Rosi-E mice compared with Rosi (P < .01) (Figure 3D). Neither running exercise nor Rosi altered cortical parameters other than porosity (Figure 3E). Exercise increases trabecular bone quantity Figure 3. Open in new tabDownload slide Trabecular and cortical bone microarchitecture measured via μCT. Representative μCT 3D reconstructions for trabecular (A) and cortical (B) bone. C, Cortical porosity. D, Trabecular bone parameters measured by μCT include volume/total volume (BV/TV), trabecular number (Tb.N), Tb.Th, and trabecular spacing (Tb.Sp). E, Cortical bone parameters measured by μCT, including cortical bone area (Ct.Ar), cortical total area (Tt.Ar), Ct.Ar/Tt.Ar, and cortical thickness (Ct.Th). Results are expressed as mean ± SD (n = 5/group).). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Figure 3. Open in new tabDownload slide Trabecular and cortical bone microarchitecture measured via μCT. Representative μCT 3D reconstructions for trabecular (A) and cortical (B) bone. C, Cortical porosity. D, Trabecular bone parameters measured by μCT include volume/total volume (BV/TV), trabecular number (Tb.N), Tb.Th, and trabecular spacing (Tb.Sp). E, Cortical bone parameters measured by μCT, including cortical bone area (Ct.Ar), cortical total area (Tt.Ar), Ct.Ar/Tt.Ar, and cortical thickness (Ct.Th). Results are expressed as mean ± SD (n = 5/group).). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Running and Rosi effect on MAT gene expression Next, we wished to understand whether Rosi and exercise regulate fat formation pathways. We analyzed total tibia mRNA expression of fat and lipogenesis markers. Fatty acid synthase (FASN) and perilipin were significantly increased after Rosi treatment as compared with CTL (Figure 4), consistent with the large increase in total bone fat. Exercise had no measurable effect on gene expression associated with MSC adipogenesis and lipid droplet formation in either group (FASN and perilipin) (Figure 4). In CTL mice, exercise was associated with trend for higher FASN compared with that measured in the tibia mRNA of sedentary mice. However, this did not reach significance. Two-way ANOVA revealed a significant effect of exercise on uncoupling protein (UCP)1 mRNA (overall P value for exercise effect < .05) in mouse tibia (Figure 4). Rosi with or without exercise did not affect UCP1 mRNA. The expression of type II iodothyronine deiodinase (Dio2), another brown fat marker, was not significantly affected by Rosi or exercise. We compared effects of Rosi and running exercise on MAT to concurrent effects on gonadal fat depots. In the 6 weeks of study, neither variable altered fat pad FASN. Rosi significantly increased fat pad UCP1 expression (P < .05) and this effect was inhibited by exercise (Figure 5). An effect of PPARγ agonist to increase UCP1 has been demonstrated previously in white adipose tissue (41). These differences between MAT and perigonadal fat depots reinforce that not all fat is created equal. Fat pad lipogenic and thermogenic response Figure 5. Open in new tabDownload slide Perigonadal fat pads were harvested and mRNA isolated from C57BL/6 mice after 6 weeks of running ± Rosi. Relative expression is shown by real-time PCR normalized to GAPDH. Results are expressed as mean ± SEM (n = 5/group). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Figure 5. Open in new tabDownload slide Perigonadal fat pads were harvested and mRNA isolated from C57BL/6 mice after 6 weeks of running ± Rosi. Relative expression is shown by real-time PCR normalized to GAPDH. Results are expressed as mean ± SEM (n = 5/group). #, significant effect due to Rosi by two-way ANOVA; †, significant effect due to exercise by two-way ANOVA. Statistical significance for between-group comparisons is noted as follows: *, P < .05; **, P < .01; ***, P < .001; and ****, P < .0001. Discussion The physiologic function of marrow fat is unknown, and its effect on health, specifically bone health, remains unclear. PPARγ agonists have been shown to increase marrow fat and may be associated with decreased bone quality (4, 22). We undertook a study to assess qualitative as well as volumetric quantitative changes in marrow fat due to PPARγ agonist, and to ask whether PPARγ-induced marrow fat interfered with exercise-stimulated bone density. We demonstrated that in the setting of PPARγ agonist, marrow fat was remarkably increased, out of proportion to body weight, suggesting that MSC might be more sensitive to the effects of Rosi than other fat depots. Importantly, exercise induction of bone formation proceeded despite the presence of a strong stimulus for marrow adipogenesis. Our study is the first to qualitatively visualize and to volumetrically quantify marrow fat in the setting of PPARγ agonist; this quantification was performed in the load-bearing, appendicular skeleton, within distinct regions: diaphysis, metaphysis and epiphysis. Six weeks of Rosi treatment significantly increased MAT throughout the femur. Not only was MAT increased in the metaphysis, similar to increases seen on a HFD (7), but it was increased in the entire femur, extending well into the diaphysis, where it was not observed after 6 weeks of high-fat feeding (7). It is interesting to note that accumulation of MAT in Rosi-treated mice occurred without a difference in body weight in growing animals. This suggests that the PPARγ agonist specifically induced MSC adipogenesis in the bone marrow compartment. It is unclear why the bone marrow MSC might be so sensitive to PPARγ lineage biasing compared MSC located in other fat depots. It may be that marrow MSC are more “available” during a time of skeletal apposition and can be easily subverted by the strong effects of a PPARγ agonist; it would be important to examine this question in older animals with less marrow MSC reserve or a longer course of treatment. Alternatively, the MSC population variances that exist between fat depots may be responsible for the expansion of adipocytes in one compartment vs another. The mRNA profile of MAT was analyzed after Rosi treatment. A significant increase in tibial mRNA defining adipose phenotype, perilipin and FASN, followed Rosi treatment as would be consistent with the known effect of PPARγ to increase MSC adipogenesis (29, 42). We also noted that exercise increased UCP1 in bone mRNA by two-way ANOVA. UCP1 is localized in the mitochondrial inner membrane of mammalian brown adipose tissue and is therefore a specific marker for brown adipose tissue (1). This increase with exercise may indicate a brown phenotype within MAT adipocytes. However, this requires further confirmation. Dio2, a second marker of brown adipose tissue, was not significantly affected by Rosi or running exercise. In contrast, fat pad perilipin was unaffected by either measure, consistent with other studies (41). Interestingly, exercise increase of UCP1 may be consistent with increases in irisin (43), although irisin's role in exercise physiology remains unclear (44). In humans, MAT quantified by MR Spectroscopy in PPARγ-treated patients was shown to be decreased in the spine (45) whereas it was increased in another study which considered both spine and femur sites (4). Differences may be due to specific PPARγ agonist choice, sample size or underlying population selection. Despite the reduced use of PPARγ agonists for diabetes mellitus due to concern regarding heart failure and fractures (46), a new class of antidiabetic agents being studied for clinical use, fibroblast growth factor 21 mimetics, appear to enhance PPARγ effects at multiple sites (47). Fibroblast growth factor 21 mimetics exhibit similar metabolic and bone effects to PPARγ agonists, underlining the clinical importance of understanding the effects of this pathway on MAT (48, 49). In our study, 6 weeks of Rosi was associated with significantly increased intracortical porosity in the bones of young animals. This type of microarchitectural change is associated with reduced bone quality (50). Despite an increase in intracortical porosity, Rosi did not significantly alter trabecular or cortical bone quantity during the treatment period (Figure 3). It is certainly possible that longer treatment would produce declines. Lecka-Czernik and coworkers (22) suggested that PPARγ agonists have minimal effects on bone in young mice only causing significant negative changes in the skeletons of older animals. Accrued negative effects of PPARγ-induced MAT on bone are not well understood, because they may be direct (biasing of MSC pool) (22, 29) and indirect (effects of local fat on hematopoietic niche function) (35). Further, PPARγ-associated bone loss may arise from stimulation of bone resorption as PPARγ can induce osteoclastogenesis (51). Published studies (22, 52) have examined the effects of Rosi via dynamic histomorphometry and showed that in young animals, Rosi decreased bone formation rate implicating biasing of bone marrow MSC away from the osteoblast lineage. Other studies noted significant reductions in bone quality with PPARγ agonist therapy, for instance, the lower ultimate bending moment, deflection, and energy absorption found in femoral shafts of rats treated with pioglitazone (53). Rosi has been shown to significantly decrease compressive strength of rat L4 vertebra (54). Because increased intracortical porosity exponentially reduces bone strength (55–57), our data showing that Rosi increases intracortical porosity likely indicate that bone strength is reduced. Conversely, it is established that running exercise improves biomechanical properties of bone; it would be interesting to see whether exercise can mitigate the effect of PPARγ agonist to increase bone fragility, something we hope to answer in the future. Significantly, running exercise, which prevents MAT accumulation in both CTL and HFD-fed mice (7), effectively suppressed MAT volume in the setting of Rosi treatment while stimulating bone acquisition. Mechanical input has the ability to prevent differentiation along the adipogenic lineage (28), even in the presence of thiozolidinedione biasing (29). Mechanical force promotes cytoskeletal complexity (58, 59)and through mTORC2/Akt/GSK3β signaling activate βcatenin (60), signals associated with biasing away from adipogenesis and toward osteogenesis (61). Because exercise subjects the marrow to mechanical signals, CTL of MSC lineage is likely involved in the inverse relationship of marrow fat and bone during running. Also, it is tempting to speculate that MAT might represent a physiologic local energy depot analogous to the deposition of fat in exercising muscle (62) to provide necessary calories to fuel skeletal anabolism (7, 63). This is possibly linked to an effect on de novo lipogenesis, reflected in that FASN which was minimally induced in the tibia by running exercise in CTL mice. Exercise has been shown to increase production of the secreted thermogenic protein Irisin from muscle, which in turn “browns” white adipose tissue (43, 64). In our experiment exercise significantly increased the expression of UCP1 in bone mRNA. Additionally, relative to total MAT, exercise, in the absence of Rosi, increased expression of brown fat markers as well. This suggests that exercise may brown MAT in the setting of exercise. Importantly, exercise increased bone quantity in the setting of Rosi's powerful adipose biasing of MSC. The trabecular bone gains noted with voluntary exercise in rodents correlate with human studies demonstrating improved bone health with exercise (7, 65). The suppression of MAT with exercise was of the same order of magnitude whether in the presence and absence of Rosi, leading us to speculate that MAT might supply some daily energy for the similar distances run in the CTL-E and Rosi-E groups. That the effect of exercise to build bone in the setting of Rosi treatment was perhaps even higher than that in CTL-E, raises the interesting possibility that increased MAT stores might provide additional fuel for skeletal growth. Analogously, it is thought that muscle triglyceride is the preferred fuel source for energy requirements of endurance exercise training (9, 10). Our data are reassuring in that not only can exercise induce bone growth in the face of a HFD (7) but that “pathologic” marrow fat due to PPARγ induction does not interfere with energy required for the anabolic tissue building of exercise. In summary, after 6 weeks of Rosi treatment, significant accumulation of lipid was noted in the marrow of mouse femurs accompanied by increased markers of lipogenesis and lipid droplet formation, consistent with adipocyte biasing of the MSC pool. Exercise suppressed this increase in volumetric marrow fat and concurrently stimulated trabecular bone formation. Our results suggest that marrow fat deposition, driven either by MSC biasing with PPARγ agonist or due to HFD, can supply energy requirements for exercise and anabolism. That exercise effectively induced bone even in the face of a potent pharmacologic stimulator of marrow fat underscores the importance of functional loading to skeletal structure. It is possible that exercise causes browning of MAT as UCP1 mRNA expression was increased by exercise. However, this finding requires further investigation. That exercise can suppress MAT volume and increase bone quantity even in the presence of Rosi underscores that exercise can be a powerful regulator of bone formation, biasing marrow stem cells toward the osteogenic lineage to fulfill an adaptive need for bone formation. As such, our data reinforce that exercise can be an effective treatment for negative effects on bone and fat due to both overeating and iatrogenic etiologies. Acknowledgments We thank Dr Mark Weaver for performing our power analysis and checking our statistical analysis methodology. We also thank Joseph Temple for assistance with data acquisition. This work was supported by National Institutes of Health Grants AR062097 and P30DK056350 (to M.S.), AR042360 and AR056655 (to J.R.), AR 43598 and EB 14351 (to C.R.), and DK092759 (to M.C.H.). Disclosure Summary: The authors have nothing to disclose. Abbreviations BV/TV bone volume/total volume μCT microcomputer tomography CTL control CTL-E CTL exercise FASN fatty acid synthase GAPDH glyceraldehyde-3-phosphate dehydrogenase HFD high-fat diet HU Hounsfield unit MAT marrow adipose tissue MSC mesenchymal stem cell ns not significant PPAR peroxisome proliferator-activated receptor Rosi rosiglitazone Tb.Th trabecular thickness UCP uncoupling protein. References 1. Bredella MA , Torriani M , Ghomi RH , et al. . Vertebral bone marrow fat is positively associated with visceral fat and inversely associated with IGF-1 in obese women . Obesity (Silver Spring) . 2011 ; 19 : 49 – 53 . Google Scholar Crossref Search ADS PubMed WorldCat 2. 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TI - Exercise Regulation of Marrow Fat in the Setting of PPARγ Agonist Treatment in Female C57BL/6 Mice
JF - Endocrinology
DO - 10.1210/en.2015-1213
DA - 2015-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/exercise-regulation-of-marrow-fat-in-the-setting-of-ppar-agonist-5dTF5PSruB
SP - 2753
VL - 156
IS - 8
DP - DeepDyve
ER -