Investigating the effect of HFD since very young age on bones, we found the discrepancy of bone response to HFD-induced obesity. After 10 week HFD, the fat mass of HFD mice increased nearly 2.5 folds, yet the whole body BMD and BMC did not significantly increase corresponding to the fat mass gaining. It is observed lumber BMC, BMD, bone area decreased and femoral BMC, BMD and bone area increased.
The present study showed that HFD could no doubt cause overweight and obesity in female mice, the absolute lean mass did not differ between groups, but the lean percentage was significant lower in HFD mice. Hence, HFD-resulted obesity mainly caused by the increasing of body fat mass, which determined bone status in this study. After adjustment for fat mass, the total body BMD and BMC even tended to be lower than NFD mice, suggesting that fat mass is poorly contributes to bone mineralization. This is consistent with human study which concluded that lean mass has stronger effect on BMD than fat mass in young women[14]. At young ages, growing bone is actively acquiring bone mineral[15]. Healthy diet and sufficient physical activity are associated with leanness[16-17]. Conversely, obesity is highly related to poor diet and/or inactivity, thus, fat mass has not sufficient effect on bone density.
Mechanical loading, together with other biochemistry factors, promote the increase in BMC and bone size to compensate for the weight gain, which has been showed in our femoral measurements[18-21]. When evaluating the bone status in children and adolescence, it is recommended to use size-corrected bone BMC[22-23]. We adjusted femur BMD, BMC for body weight or fat mass, and these measurements in HFD mice were lower than NFD mice, although the difference was not significant, which might due to the small sample size. Several studies have proposed that extra weight from fat mass does not independently related to bone geometry. In overweight children and adolescents, proximal femur bone geometry is appropriately adapt to lean mass[24]. Dynamic force (muscle force), rather than static loads (body weight), causes a larger dynamic strain on bone and leads to the geometric adaptation[25-26]. Another study also reported that in young women, lean tissue mass has a greater effect on bone density than fat mass per kilogram of tissue mass[14]. Taking the less effect of fat mass on bone geometry into consideration, this result indicated that the femoral response to higher mechanical loading from the excess fat mass might not be sufficient to compensate for the surplus load.
This is the first time to report that HFD since young age could reduce absolute spine BMC and bone area in female mice. It has been reported in human study that obese girl had smaller vertebral area than girls of healthy weight after adjustment for the body size[5]. Regarding present study, high saturated fat diet seemed to exert more detrimental effects on growing vertebral bone beyond the negative influence of adiposity. To date, information concerning how HFD from young age affect vertebral mineral accrual is sparse. Some earlier reports suggest that obese children have limited ability to augment their spinal areal bone mineral density[27-28]. Yet, it is difficult to elucidate the true effect of fat mass on bone because of the complicated relationship[29]. One thing should be noted that the tempo of growth in bone size, mass and density is region-specific (girls)[30]. In terms of this, unlike in adulthood, the effect of exposure to a risk factor during growth depends on the maturational level and growth rate of the region exposed, and before puberty, limbs grow more rapidly than axial bones. The lumber spine is mainly composed of trabecular bone and has the different sensitivity to mechanical stress from cortical bone. In this study, the loss of numbers of trabeculae was observed, indicating the damage of trabecular architecture. Therefore, vertebral bone might be more sensitive to the deleterious effect of HFD despite the beneficial bone accommodation to the concomitant obesity.
The discrepancy of high bone mass and increased risk of fracture in obese children suggests that higher bone mass does not mean the higher bone quality[31]. As to bone quality, there is no current consensus about its definition. The authors prefer to the one proposed by Hernandez[32], that is, bone quality refers to the influence of factors that affect fracture but are not accounted for by bone mass or quantity. To analyze bone quality, one approach is to calculate the ratio of strength to density for each individual specimen. Bones with higher ratio are more biomechanically efficient. We examined the bone biomechanical properties of left femur and it turned out that the larger bone had a tendency to be stronger which is consistent with the study of obese male rats, but in present study, it did not attain statistical significance. When calculating the ratio of femur strength to femur bone density, higher ratio was seen in HFD mice, although the difference was not significant. This result could be explained as that HFD mouse have stronger weight-bearing bones because of greater load together with advanced maturity and bone ages, but this improvement of bone quality might be not enough relative to the weight gain because we did not see the significant difference of biomechanical properties between groups. In terms of appendicular bone status, how long can the biomechanical improvement maintain, how the peak bone value will be influenced if not changing the HFD, remain to be further studied. This reminds us another study with the results that prepubertal obese children had higher BMD, but a low BMD value was found after puberty[33].
In order to further understand the skeletal metabolism, we measured the specific bone metabolic markers, that is, bone resorption marker-pyridinoline (PYD) and bone formation marker-osteocalcin. We noted that both bone formation and resorption markers in HFD mice were lower than NFD mice, indicating a decreased bone turnover in HFD group. There are many factors influence the concentration of bone metabolic markers, such as age, pubertal stage, growth velocity, mineral accrual, hormonal regulation, nutritional status, etc[12]. In this study, nutritional status, prematurity and hormonal profiles are the disturbance to illustrate the impact of HFD on bone turnover, and there are fewer literatures to be referred about the bone turnover in young mice. However, human prospective study indicated that, bone markers increase rapidly during early puberty when growth velocity is highest and start to decrease after the growth spurt until reach values observed in adults during postpubertal period[34-35]. Bone markers are positively correlated with longitudinal growth, and the longer the pubertal increase of bone markers lasts, the higher pubertal growth increase, as well the higher bone mineral accrual[36]. According to this, our results indicate that the lower bone markers in HFD mice suggesting the lower growth velocity and subsequent less accumulation of bone mineral.
Our study has some limitations. We initially designed to use both male and female mice and analyze influence of HFD on bone development in different genders. But more male mice died during the experiment, resulting in less number far from enough for statistical analysis. In present study, only data of female mice were analyzed. The small sample size and relatively bigger standard deviation may influence the statistical analysis and diminish the significance. We did not measure serum endocrine factors, such as leptin, insulin, growth hormone or estrogen, which have been already identified to affect bone status. Further studies are needed to unveil the potential mechanisms of HFD-related bone phenotype. It has been reported that in adult mice, HFD can inhibit the intestinal absorption of calcium and consequently deleteriously affects bone mineralization, and the adverse effect is more apparent in cancellous bone than in cortical bone[37-38]. In addition, adiposity of bone marrow may cause the imbalance of differentiation from stem cells to adipocytes or osteoblasts[39]. As to the growing bone, the underline mechanisms might be more complicated. Although the results are not quite consistent with our experiment, a recent study also concluded that high fat diet intake during the growing period has deleterious effects on bone metabolism[40].
In conclusion, we studied the effect of HFD on bone growth. Our results showed femoral BMC, BMD and bone area increased in adaption to the excess body weight caused by HFD. However, these bone responses seem to be insufficient to compensate for the excess load regarding the body weight and fat mass. What is more conspicuous is the negative effect on lumbar spine growth. To conclude, our preliminary data suggest that the vertebral bone is more sensitive to HFD-induced bone loss compared to the long bones in young, growing animals. Considering that childhood and adolescence are crucial periods to acquire nearly half of the peak bone mass, HFD in young age might be one reason for the increasing prevalence of osteoporosis during adult and aging.
