Short-Term Increased Physical Activity During Early Life Affects High-Fat Diet-Induced Bone Loss in Young Adult Mice

Abstract

Mechanical stresses associated with physical activity (PA) have beneficial effects on increasing BMD and improving bone quality. However, a high-fat diet (HFD) and obesity tend to have negative effects on bone, by increasing bone marrow adiposity leading to increased excretion of proinflammatory cytokines, which activate RANKL-induced bone resorption. In the current study, whether short-term increased PA via access to voluntary wheel running during early life has persistent and protective effects on HFD-induced bone resorption was investigated. Sixty 4-week-old male C57BL6/J mice were divided into two groups postweaning: without or with PA (access to voluntary running wheel 7-8 km/day) for 4 weeks. After 4 weeks with or without PA, mice were further subdivided into control diet or HFD groups for 8 weeks, and then all animals were switched back to control diet for an additional 4 weeks. Mice from the HFD groups were significantly heavier and obese; however, after 4 weeks of additional control diet their body weights returned to levels of mice on continuous control diet. Using μ-CT and confirmed by pQCT of tibias and spines ex vivo, it was determined that bone volume and trabecular BMD were significantly increased with PA in control diet animals compared with sedentary animals without access to wheels, and such anabolic effects of PA on bone were sustained after ceasing PA in adult mice. Eight weeks of a HFD deteriorated bone development in mice. Unexpectedly, early-life PA did not prevent persistent effects of HFD on deteriorating bone quality; in fact, it exacerbated a HFD-induced inflammation, osteoclastogenesis, and trabecular bone loss in adult mice. In accordance with these data, signal transduction studies revealed that a HFD-induced Ezh2, DNA methyltransferase 3a, and nuclear factor of activated T-cells 1 expression were amplified in nonadherent hematopoietic cells. In conclusion, short-term increased PA in early life is capable of increasing bone mass; however, it alters the HFD-induced bone marrow hematopoietic cell-differentiation program to exacerbate increased bone resorption if PA is halted. © 2021 Arkansas Children's Nutrition Center. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: BONE RESORPTION; EXERCISE; HIGH‐FAT DIET; WHEEL RUNNING.

Fig 1

Study characteristics. (A) Average running distance by mice assigned to physical activity. (B) Experimental process and animal euthanization time point and groups. (C) Specially designed individual mouse cage with an attached monitoring device, where mice can voluntary access running wheels. (D) Average running time for mice assigned to physical activity. (E) Average body weights of each group of mice from the experimental start point at 4 weeks of age to the end at 20 weeks of age. (F) Average fat and lean mass of each group of mice at second euthanization time point at 16 weeks of age. (G) Average fat and lean mass of each group of mice at third euthanization time point at 20 weeks of age. ***p < 0.001 by two‐way ANOVA with a HFD and early‐life PA as the main factors and their interactions. AVG, average; Con, control; HFD, high‐fat diet; PA, physical activity; sac'd, sacrificing day.

Fig 2

Early‐life increased physical activity (PA) via access to voluntary running wheel improves bone development. (A) Representative μCT images of the proximal tibia from one representative sample from each group of mice. Upper panel shows sagittal view and lower panel shows transverse view;, white lines and dots indicate trabecular or cortical bone tissues. (B–I) μCT measures of eight parameters from trabecular tibias from control and PA of 8‐month‐old mouse groups. Data are expressed as mean ± SD (n = 6 per group). *p < 0.05 by t test between control and PA group. (J) Representative images of transverse views of quantitative pQCT analysis of one slice of the proximal tibia from one sample from each group of mice. Color changes from black to white indicate bone density from low to high. BS/TV, bone surface density; BS/BV, bone surface/volume ratio; BV/TV, bone volume/total tissue volume; i.S, intersection surface; SMI, structure model index; SSI, stress–strain index; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.

Fig 3

Early‐life–increased physical activity (PA) via access to voluntary running wheel increases bone formation and resorption, as well as inflammation. (A–D) Real‐time PCR for alkaline phosphatase (ALP), osteocalcin (OC), Runx2, and β‐catenin mRNA expression in total RNA isolated from spine L3 vertebrae of either control or PA groups. (E–H) Real‐time PCR for matrix metallopeptidase 9 (MMP9), nuclear factor of activated T‐cells 1 (NFATc1), nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB), and cathepsin K mRNA expression in total RNA isolated from spine L3 vertebrae of either control or PA groups. Relative mRNA expression was normalized by GAPDH expression, and row data were presented without removing outliers. *p < 0.05 by t test between control and PA group. (I) Antibody array analysis showing increased inflammatory factor expression in PA groups in total proteins isolated from spine L3 vertebrae compared with those from control, the heat map analysis for comparison of all factors. p Value was by t test between control and PA groups.

Fig 4

Short‐term early‐life–increased physical activity (PA) does not prevent later life high‐fat diet (HFD)–induced bone loss. (A) Representative μCT images of the proximal tibia from one sample from each group of mice. Upper panel shows sagittal view and lower panel shows transverse view; white lines and dots indicate trabecular or cortical bone tissues. (B–G) μCT measures of six parameters from trabecular tibias from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. Data are expressed as mean ± SD (n = 6 per group), analyzed by two‐way ANOVA with a HFD and early‐life PA as the main factors, and their interactions were tested. Additionally, *p < 0.05, **p < 0.01, ***p < 0.001 by Tukey's multiple comparison. (H) Representative images of transverse views of quantitative pQCT analysis of one slice of the proximal tibia from one sample from each group of mice. Color changes from black to white indicate bone density from low to high. BS/TV, bone surface density; BV/TV, bone volume/total tissue volume; HF, high fat; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness; SMI, structure model index; SSI, stress–strain index.

Fig 5

Short‐term early‐life–increased physical activity (PA) via access to voluntary running wheel does not ameliorate high‐fat diet (HFD) bone resorption, it exacerbated HFD‐induced inflammation in bone. (A–D) Real‐time PCR for nuclear factor of activated T‐cells 1 (NFATc1), cathepsin K, osteopontin (OPN), and β‐catenin mRNA expression in total RNA isolated from spine L3 vertebrae from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐month‐old mouse groups. Data were expressed as mean ± SD (n = 6 per group). p Value was analyzed by two‐way ANOVA with a HFD and early‐life PA as the main factors and their interactions. Additionally, *p < 0.05 by t test compared with no PA/control group. (E) Antibody array analysis showing increased inflammatory factor expression in PA/control, no PA/HF, and PA/HF of 16‐month‐old mouse groups in total proteins isolated from spine L3 vertebrae compared with those from no PA/control group, the heat map analysis for comparison of all factors. Data are expressed as mean ± SD. p Value was determined by two‐way ANOVA with a HFD and early‐life PA as the main factors, and their interactions. HF, high fat.

Fig 6

Early‐life–increased high‐fat diet (HFD) does not prevent HFD‐induced epigenetic regulation of osteoclastogenesis. (A) Bone marrow cells were isolated from femur of all second euthanized mice from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups, and suspended for 48 h. Nonadherent bone marrow cells were recultured in the presence of 30 ng/ml RANKL for 5 days. Attached stromal cells were kept in culture until confluence as colony‐forming unit fibroblasts for isolation of RNA. Pictures showing osteoclast morphology from one represented well of cell cultures of all four groups after TRAPase staining. (B) Osteoclast numbers per well from cultures of nonadherent bone marrow cells from all four groups of no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. (C–F) Real‐time PCR for Ezh2, DNA methyltransferase 3a (DNMT3a), interferon regulatory factor 8 (IRF8), and nuclear factor of activated T‐cells 1 (NFATc1) mRNA expression in total RNA isolated from nonadherent hematopoietic bone marrow cells from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. (G–J) Real‐time PCR for alkaline phosphatase (ALP), osteocalcin (OC), collagen 1 (col 1), and osteopontin (OPN) mRNA expression in total RNA isolated from attached stromal cells as colony‐forming unit fibroblasts from no PA/control, PA/control, no PA/HF, and PA/HF of 16‐week‐old mouse groups. Data were expressed as mean ± SD (n = 6 per group). p Value was analyzed by two‐way ANOVA with a HFD and early‐life PA as the main factors and their interactions. Additionally, * p < 0.05 by t test compared with no PA/control to other groups. HF, high fat.

Fig 7

Short‐term early‐life–increased PA exacerbates HFD‐induced persistent effects on bone loss. (A) Representative μCT images of the proximal tibia from one sample from each group of mice. Upper panel shows sagittal view and lower panel shows transverse view; white lines and dots indicate trabecular or cortical bone tissues. (B–G) μCT measures of six parameters from trabecular tibias from no PA/con/con, PA/con/con, no PA/HF/con, and PA/HF/con of 20‐week‐old mouse groups. All data were expressed as mean ± SD (n = 6 per group), p value was analyzed by two‐way ANOVA with a HFD and early‐life PA as the main factors, and their interactions. Additionally, * p < 0.05 by t test compared with no PA/control group.Data were expressed as mean ± SD (n = 6 per group. p Value was analyzed by two‐way ANOVA with a HFD and early life PA as the main factors and their interactions. Additionally, *p < 0.05, **p < 0.01, ***p < 0.001 by Tukey's multiple comparison. (H) Representative images of transverse views of quantitative pQCT analysis of one slice of the proximal tibia from one sample from each group of mice. Color changes from black to white indicate bone density from low to high. BS/TV, bone surface density; BV/TV, bone volume/total tissue volume; Con, control; HF, high fat; SMI, structure model index; SSI, stress–strain index; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness.