Caloric restriction leads to high marrow adiposity and low bone mass in growing mice

Abstract

The effects of caloric restriction (CR) on the skeleton are well studied in adult rodents and include lower cortical bone mass but higher trabecular bone volume. Much less is known about how CR affects bone mass in young, rapidly growing animals. This is an important problem because low caloric intake during skeletal acquisition in humans, as in anorexia nervosa, is associated with low bone mass, increased fracture risk, and osteoporosis in adulthood. To explore this question, we tested the effect of caloric restriction on bone mass and microarchitecture during rapid skeletal growth in young mice. At 3 weeks of age, we weaned male C57Bl/6J mice onto 30% caloric restriction (10% kcal/fat) or normal diet (10% kcal/fat). Outcomes at 6 (n = 4/group) and 12 weeks of age (n = 8/group) included body mass, femur length, serum leptin and insulin-like growth factor 1 (IGF-1) values, whole-body bone mineral density (WBBMD, g/cm(2)), cortical and trabecular bone architecture at the midshaft and distal femur, bone formation and cellularity, and marrow fat measurement. Compared with the normal diet, CR mice had 52% and 88% lower serum leptin and 33% and 39% lower serum IGF-1 at 6 and 12 weeks of age (p < .05 for all). CR mice were smaller, with lower bone mineral density, trabecular, and cortical bone properties. Bone-formation indices were lower, whereas bone-resorption indices were higher (p < .01 for all) in CR versus normal diet mice. Despite having lower percent of body fat, bone marrow adiposity was elevated dramatically in CR versus normal diet mice (p < .05). Thus we conclude that caloric restriction in young, growing mice is associated with impaired skeletal acquisition, low leptin and IGF-1 levels, and high marrow adiposity. These results support the hypothesis that caloric restriction during rapid skeletal growth is deleterious to cortical and trabecular bone mass and architecture, in contrast to potential skeletal benefits of CR in aging animals.

Figure 1

Body mass, bone length, and serum hormone levels at 6 and 12 weeks of age. (A) Body mass (g; n = 8 to 12/group). (B) Femur length (mm; n = 8 to 12/group). (C) Serum leptin (ng/mL; n = 4 to 5/group). (D) Serum IGF‐1 (ng/mL; n = 4 to 6/group). Significant differences (p < .05) as shown.

Figure 2

Whole‐body bone densitometry by DXA at 6 and 12 weeks of age. (A) Whole‐body bone mineral density (WBBMD, g/cm²). (B) Whole‐body bone mineral content (WBBMC, g). (C) Total‐body fat (%) (n = 8 to 12/group). Brackets indicate significant differences (p < .05) for unscaled (solid line) and body mass–adjusted (dashed line) values.

Figure 3

Caloric restriction decreased both midshaft cortical and distal femur trabecular bone properties.

Figure 4

Dynamic histomorphometry showed a substantial reduction in bone mineralization in 30% CR mice (B) versus normal diet controls (A) at 12 weeks of age. Calcein labels administered 9 and 2 days before euthanization indicated more doubly labeled surface and greater interlabel distance (yellow lines) in normal diet mice (A) compared with CR mice (B).

Figure 5

Distal femur marrow fat increased with CR versus normal diet mice. (A) Normal diet (12 weeks of age). (B) CR (12 weeks of age; adipocytes shown by arrows). (C) Adipocyte number per total area via histomorphometry at 6 (left) and 12 weeks (right) of age. Significant differences (p < .05) as shown. (D) Bone images with color intensity coding for fat content showed increased distal femur marrow fat at 6 weeks of age in 30% CR mice (right) versus normal diet controls (left).