Increased marrow adipogenesis does not contribute to age‐dependent appendicular bone loss in female mice

Abstract Marrow adipocytes and osteoblasts differentiate from common mesenchymal progenitors in a mutually exclusive manner, and diversion of these progenitors toward adipocytes in old age has been proposed to account for the decline in osteoblasts and the development of involutional osteoporosis. This idea has been supported by evidence that thiazolidinedione (TZD)‐induced activation of PPARγ, the transcription factor required for adipocyte differentiation, increases marrow fat and causes bone loss. We functionally tested this hypothesis using C57BL/6J mice with conditional deletion of PPARγ from early mesenchymal progenitors targeted by the Prx1‐Cre transgene. Using a longitudinal littermate‐controlled study design, we observed that PPARγ is indispensable for TZD‐induced increase in marrow adipocytes in 6‐month‐old male mice, and age‐associated increase in marrow adipocytes in 22‐month‐old female mice. In contrast, PPARγ is dispensable for the loss of cortical and trabecular bone caused by TZD or old age. Instead, PPARγ restrains age‐dependent development of cortical porosity. These findings do not support the long‐standing hypothesis that increased marrow adipocyte differentiation contributes to bone loss in old age but reveal a novel role of mesenchymal cell PPARγ in the maintenance of cortical integrity.

the distal growth plate. Cortical dimensions were determined at the diaphysis (18 slices, midpoint of the bone length as determined in scout view), and the metaphysis, starting 8-10 slices away from the growth plate so as to avoid the growth plate, and proceeding proximally for 151 slices, to obtain cross-sectional images drawn to exclude trabecular elements. In some experiments (as indicated), only the proximal third (50 slices) of the metaphysis was used for analysis. Cortical bone was measured at a threshold of 200 mg/cm 3 . Trabecular analyses were performed on contours of the cross-sectional images drawn to exclude cortical bone and were measured at a threshold of 220 mg/cm 3 . Trabecular analysis of the femoral head was made beginning at the first slice exhibiting trabecular bone, and proceeding approximately 90 slices towards the proximal growth plate near the neck. Tibiae were scanned from the proximal end to the distal tibiofibular joint; the latter 10 slices were used for measurement of cortical indices. Trabecular bone was evaluated using 100 slices from the proximal end to the tibiofibular joint. Trabecular architecture was determined using sphere filling distance-transformation indices without assumptions about the bone shape as a rod or plate.
Analysis of cortical integrity were performed using a single slice midway between the upper and lower limits of the metaphysis. Slices were scored in a blind fashion by three different observers, as follows: 1, no porosity and intact endosteum; 2, porosity with intact endosteum; 3, porosity with loss of the endosteal boundary; 4, extensive porosity with loss of the endosteal boundary. For femoral porosity measurements, slices were analyzed from a point immediately distal to the third trochanter to a point immediately adjacent to the primary spongiosa. In some experiments, only the distal third of the metaphysis was analyzed as described above. After defining endosteal and periosteal boundaries, an additional image processing script ("peel-iter = 2") was used to eliminate false voids caused by imperfect wrap of the contours to the bone surface.
Images were binarized with a threshold of 365 mg/cm 3 . Cortical bone volume and void volume were determined with the "cl_image" script and used to calculate porosity. To avoid inclusion of osteocyte lacunae and canalicular space, void volumes < 31,104 μm 3 (18 voxels) were excluded in the determination of porosity.
The fourth lumbar vertebra (L5) was scanned from the rostral growth plate to the caudal growth plate to obtain 233 slices. BV/TV in the vertebra was determined using 100 slices (1.2 mm) of the anterior (ventral) vertebral body immediately inferior (caudal) to the superior (cranial) growth plate. Trabecular bone analyses were performed on contours of cross-sectional images, drawn to exclude cortical bone, as described for femoral trabecular bone. Cortical bone thickness was determined on the ventral cortical wall using contours of cross-sectional images, drawn to exclude trabecular bone, as described for femoral cortical bone.
Knees fixed in 4% paraformaldehyde were scanned with the MicroCT40 system using an energy of 70 kVp and a 300ms integration time (isotropic voxel size = 12µm), at a threshold of 200 mg/cm 3 ; 500 slices were collected, starting from the proximal end of patella, including the entire tibiofemoral joint, and ending in the tibial metaphysis.

Enumeration of Osx1+ osteoblast progenitors
Bone marrow cells were obtained femora and tibiae, and then depleted of CD45+ hematopoietic cells using a biotin-conjugated rat antibody specific for mouse CD45 (eBioscience, San Diego, CA, USA; 14-0451, 1:100), and three round of treatment with anti-rat IgG Dynabeads (Invitrogen, Grand Island, NY, USA) at a bead:cell ratio of approximately 4:1. Osx1-TdRFP+ cells were sorted in an Aria II cell sorter (BD Bioscience, San Jose, CA, USA) using the PE-A fluorochrome gate, as we have previously described (Kim et al., 2017).  Table 5.

Glucose tolerance test
Glucose (2 g/kg body weight) was injected i.p. between 9 and 11 AM to 22-mo-old control and PPAR ∆Prx1 mice that had been fasted for 16 h. Blood was obtained from the tail vein measured immediately prior to glucose administration, and 15, 30, 60, 90, and 120 min afterwards. Blood glucose was determined using a glucometer (Bayer Healthcare, Contour Next).