Diet-Stimulated Marrow Adiposity Fails to Worsen Early, Age-Related Bone Loss

Abstract

Introduction: Longitudinal effect of diet-induced obesity on bone is uncertain. Prior work showed both no effect and a decrement in bone density or quality when obesity begins prior to skeletal maturity. We aimed to quantify long-term effects of obesity on bone and bone marrow adipose tissue (BMAT) in adulthood.

Methods: Skeletally mature, female C57BL/6 mice (n = 70) aged 12 weeks were randomly allocated to low-fat diet (LFD; 10% kcal fat; n = 30) or high-fat diet (HFD; 60% kcal fat; n = 30), with analyses at 12, 15, 18, and 24 weeks (n = 10/group). Tibial microarchitecture was analyzed by µCT, and volumetric BMAT was quantified via 9.4T MRI/advanced image analysis. Histomorphometry of adipocytes and osteoclasts, and qPCR were performed.

Results: Body weight and visceral white adipose tissue accumulated in response to HFD started in adulthood. Trabecular bone parameters declined with advancing experimental age. BV/TV declined 22% in LFD (p = 0.0001) and 17% in HFD (p = 0.0022) by 24 weeks. HFD failed to appreciably alter BV/TV and had negligible impact on other microarchitecture parameters. Both dietary intervention and age accounted for variance in BMAT, with regional differences: distal femoral BMAT was more responsive to diet, while proximal femoral BMAT was more attenuated by age. BMAT increased 60% in the distal metaphysis in HFD at 18 and 24 weeks (p = 0.0011). BMAT in the proximal femoral diaphysis, unchanged by diet, decreased 45% due to age (p = 0.0002). Marrow adipocyte size via histomorphometry supported MRI quantification. Osteoclast number did not differ between groups. Tibial qPCR showed attenuation of some adipose, metabolism, and bone genes. A regulator of fatty acid β-oxidation, cytochrome C (CYCS), was 500% more abundant in HFD bone (p < 0.0001; diet effect). CYCS also increased due to age, but to a lesser extent. HFD mildly increased OCN, TRAP, and SOST.

Conclusions: Long-term high fat feeding after skeletal maturity, despite upregulation of visceral adiposity, body weight, and BMAT, failed to attenuate bone microarchitecture. In adulthood, we found aging to be a more potent regulator of microarchitecture than diet-induced obesity.

Keywords: Advanced image analysis (MRI-CT); Aging; Obesity; Osteoporosis; Translational research.

Fig. 1.

Body weight and white adipose tissue accumulate in response to HFD, initiated in adulthood. Female 12-week-old B6 mice were allocated to LFD (n = 40 LFD) or HFD (n = 30 HFD) interventions for a 12-week experimental period. A schematic demonstrating the experimental timeline (a) is provided. The weekly experimental (b) as well as harvest (c) weights are plotted. Arrow in (b) denotes start of experimental intervention. Harvest fat pad weight (d) and fat pad as a % of body weight (e) are shown. Data in (b) were analyzed via mixed effects with multiplicity corrected p values via Bonferroni. Data (c–e) analyzed via two-way ANOVA with Turkey’s post hoc; p values for main effects (age, diet) shown below plots; data as individual animal data points. Box plot horizontal lines represent medians ± IQR. Group differences shown if p < 0.05 as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****p < 0.0001.

Fig. 2.

Trabecular bone parameters decline with experimental age, with lesser attenuation of the cortical compartment. Female 12-week-old skeletally mature B6 mice were allocated to LFD or HFD intervention for 12 weeks (n = 10/group). a Trabecular bone microarchitecture was assessed at the proximal tibial metaphysis via micro-CT. b Quantitative measures of cortical bone at the mid-tibial diaphysis via μCT. c Biomechanical measures of the cortical mid-diaphysis. Data as individual animal data points. Box plot horizontal lines represent medians ± IQR; analysis via two-way ANOVA with Tukey’s post hoc; p values for main effects (age, diet) are noted below plots, and between-group comparisons added if p < 0.05 as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****p < 0.0001. Measures of resistance to bending pMOI of Ixx- or Iyy-axes. Tb.BMDa, apparent trabecular bone mineral density; Tb.BV/TV, trabecular bone volume fraction; Tb.N, trabecular number per unit length; Tb.Th, trabecular thickness via direct 3D methods; Tb.Sp, trabecular separation, or the mean distance between trabecular using direct 3D methods; Tb.Conn.D, trabecular connectivity density; Tb.SMI, trabecular structural model index; Conn.D, trabecular connectivity density; Ct.Ar/Tt.Ar, cortical bone volume fraction; Tt.Ar, total area; Ct.Ar, cortical area; Ct.BMDa, apparent cortical bone mineral density; Ct.Th, cortical thickness; pMOI, polar moment of inertia; Ixx, inertia around the X-axis; Iyy, inertia around the Y-axis.

Fig. 3.

Dietary intervention and aging associated with increased quantity of BMAT via 9.4 Tesla MRI during the experimental period, with the largest effect of diet localized to the distal femoral compartment. Skeletally mature female 12-week-old B6 mice allocated to LFD or HFD for a 12-week period. a Visualization of BMAT by MRI. Each image is an average of each of the individual images superimposed on each other per experimental group; top panel, sagittal cut; bottom panel, coronal cut. Data as individual animal data points. Box plot horizontal lines represent medians ± IQR expressed as % relative to 12-week LFD. Volumetric analyses on the whole femur (b) as well as pre-specified anatomical regions in the distal femur (c) and proximal femur (d) were analyzed via two-way ANOVA with Tukey’s post hoc; p values for main effects (age, diet) noted below plots; between-group differences shown if *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Fig. 4.

Bone marrow adipocyte size is increased in HFD initiated in adulthood. Female 12-week-old C57BL/6 mice allocated to baseline 12-week-old LFD or HFD interventions for a 12-week experimental period. a, b Bone marrow adipocyte area quantified via ImageJ. b The Gaussian distribution using a non-linear least squares regression to visualize 95% CI (horizontal lines) of adipocyte size per group, including the means. c Representative distal femoral sections stained via methyl green and probed via immunohistochemistry for PLIN1 via brightfield microscopy at indicated magnification. d Adipocyte number per mm² via ImageJ. e Osteoclast number per bone surface. d and e are shown as individual animal data with bars indicating median. a shows individual measurements per animal (M1, mouse 1; M2, mouse 2; M3, mouse 3). Two-way ANOVA p values for main effects (age, diet) below (d and e) in italics; between-group differences shown as follows: *, p < 0.05; **, p < 0.01.

Fig. 5.

Diet and experimental age effect on adipose, metabolism, bone, and inflammatory markers in the tibia. Female 12-week-old C57BL/6 mice allocated to baseline 12-week-old control group (CTL) as well as LFD or HFD interventions for a 12-week experimental period. Target gene mRNA % relative to GAPDH was quantified via qPCR (n = 3–4) in murine tibia for adipose/lipid droplet, metabolism, bone turnover, and inflammatory markers. Data as individual animal data points with bars indicating median ± range. Analysis via 2-way ANOVA with Tukey’s post hoc; p values for main effects (age, diet) shown above each plot.