Hypercholesterolemia promotes an osteoporotic phenotype

Abstract

A role for hypercholesterolemia in the development of osteoporosis has been suggested in published reports. However, few studies contain direct evidence of a role for maintenance of cholesterol homeostasis in bone health. Using isocaloric high-fat/high-cholesterol and low-fat/no-cholesterol diets in a 4-month feeding study combined with micro computed tomography analysis, we demonstrated in two different mouse strains that mice with hypercholesterolemia lose cortical and trabecular bone in the femurs and vertebrae (bone mineral density was decreased on average by ≈90 mg/mL in the cortical vertebrae in one strain) and cortical bone in the calvariae (bone mineral density was decreased on average by ≈60 mg/mL in one strain). Mechanical testing of the femurs demonstrated that loss of bone in the mice with hypercholesterolemia caused changes in the mechanical properties of the bone including loss of failure load (failure load was decreased by ≈10 N in one strain) and energy to failure. Serologic and histomorphologic analyses suggested that hypercholesterolemia promotes osteoclastogenesis. These studies support a role for hypercholesterolemia in the development of osteoporosis and provide a model with which to test intervention strategies to reduce the effects of hypercholesterolemia on bone health.

Figure 1

Effects of diet on weight and blood chemistry. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were weighed (A), and serum cholesterol (B), triglyceride (C), and testosterone (D) concentrations were determined. A: Body mass. Data are plotted as mean ± SD versus diet group. There is no statistically significant difference between the 2 groups in either strain. B–D: Serologic measures. Mice were bled via cardiac puncture, and the serum was subjected to various tests. B: Serum cholesterol concentration was measured in the collected serum via Infinity colorimetric assay. Data are plotted as mean ± SD cholesterol concentration versus group. C: Triglyceride concentration was measured as described in Materials and Methods. Data are plotted as triglyceride concentration versus group. D: Testosterone concentration was measured as described in Materials and Methods. Data are plotted as mean ± SE testosterone concentration versus diet group. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005.

Figure 2

Effects of diet on femoral cortical bone. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and their femors were removed, cleaned, and analyzed using microCT. Measurements included tissue mineral density (TMD) (A), thickness (B), cortical surface area (CSA)(C), moment of inertia (Iyy) (D), and bone mineral density (BMD). Data are given as mean ± SD versus group. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005.

Figure 3

Effects of diet on femoral trabecular bone. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and their femors were removed, cleaned, and analyzed using microCT. Measurements included bone volume fraction (BVF) [bone volume/total volume (BV/TV)] (A), trabecular spacing (distance between trabeculae) (B), number of trabeculae (C), trabecular thickness (D), and tissue mineral density (TMD) (E). Data are given as mean ± SD versus group. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.005.

Figure 4

Effects of diet on the calvariae. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and their calvariae were removed, cleaned, and analyzed using microCT. Measurements included bone mineral density (BMD) (A), thickness (B), tissue mineral density (TMD) (C), bone mineral content (BMC) (D), and tissue mineral content (TMC) (E). Data are given as mean ± SD versus group. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, ***P ≤ 0.005.

Figure 5

Effects of diet on the trabecular bone of vertebrae. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and L4 was removed, cleaned, and analyzed using microCT. Measurements included number of trabeculae (A), trabecular spacing distance between trabeculae (B), trabecular thickness (C), and bone volume fraction (BVF) [bone volume/total volume (BV/TV)] (D). Data are given as mean ± SD versus group. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, ***P ≤ 0.005.

Figure 6

Effects of diet on the cortical bone of the vertebrae. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and L4 was removed, cleaned, and analyzed using MicroCT. Measurements included bone mineral content (BMC) (A), bone mineral density (BMD) (B), tissue mineral content (TMC) (C), and tissue mineral density (TMD) (D). Data are given as mean ± SD versus group. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, ***P ≤ 0.005.

Figure 7

Mechanical properties of femors. Mice (14 to 16 per group) fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and their femors were removed, cleaned, and analyzed via mechanical testing. Whole-bone mechanical properties of the femors to failure were performed in 4-point bending using a servohydraulic testing machine. Load-displacement curves were then analyzed for whole-bone yield load, ultimate load, stiffness, failure energy, and displacement ratio (ultimate displacement:yield displacement), and other measures using MATLAB software. The difference between “start” (centroid coordinate position) and “end” positions was used to calculate stiffness (A), ultimate load (B), failure load (C), and energy (D). Data are given as mean ± SD. In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. *P ≤ 0.05, ***P ≤ 0.005.

Figure 8

Bone loss in mice with hypercholesterolemia is linked to increased osteoclastic activity and osteoclasts. Mice fed an isocaloric high-fat/high-cholesterol diet (black bars) or a low-fat/no-cholesterol diet (white bars) for 4 months were sacrificed, and serum pyridinoline (PYD) concentration in the murine blood was determined via enzyme-linked immunosorbent assay (A), the femors were analyzed at histology, and the osteoclasts were counted (B). A: Data are plotted as mean ± SD PYD concentration versus group (n = 8 or 9 mice per group). B: Five-micrometer sections of femors were cut and stained using the Acid Phosphatase, Leukocyte (TRAP) kit, and the total number of osteoclasts in 5 ×20 fields were counted. Data are given as mean ± SD number of osteoclasts versus group (n = 7 bones per group). In all cases, the Student's t-test was used to calculate significance. Data were considered significant at P < 0.05. C: Histomorphometric images demonstrate osteoclasts (arrows) in trabecular regions of the femors in C57BL/6 mice. These are representative images of the ×20 fields used in the osteoclast quantification at ×40 magnification. Left: Most panels are enlargements of original ×60 images demonstrating osteoclasts used for quantification with characteristic TRAP staining (light pink) and multiple nuclei (purple). *P ≤ 0.05.