In this study, we innovatively compared the differences in bone metabolism between pathological obesity (in mice) induced by a high-fat diet and healthy obesity (in ground squirrels) induced by natural fattening before hibernation. We measured body weight, adipose tissue wet weight, bone microstructure, bone mechanical properties, and protein expression levels related to bone formation, bone resorption and Wnt signaling. The results showed that obese mice (high-fat diet) showed no obvious alterations or abnormalities in bone microstructure as compared with controls, but bone strength increased and the expression levels of proteins related to bone formation and bone loss increased and maintained a dynamic balance. By contrast, bone formation was enhanced in pre-hibernation ground squirrels, which was manifested as an enhancement of bone microstructure, improvement of bone strength, increased expression levels of bone formation-related proteins (RunX2 and ALP), increased expression levels of bone resorption-related proteins, and enhanced Wnt signaling.
The body weight and adipose tissue wet weight of OB mice and PRE ground squirrels were significantly increased compared with CON mice and SA ground squirrels, respectively (Table 2 and Table 3). After being fed a high-fat diet, the body weight of mice in the OB group increased significantly by 10.6% compared with the CON group. However, after natural fattening, the body weight of ground squirrels in the PRE group increased much more with a significant 62.5% increase over the SA group (Table 2). This indicated that the degree of obesity in pre-hibernating ground squirrels was much greater than in mice. At the same time, our data showed that after fattening, the change in adipose tissue of mice occurred in perirenal and mesenteric adipose, whereas in ground squirrels, adipose accumulation was mainly in mesenteric and subcutaneous depots with lesser accumulation of perirenal adipose. There is so little subcutaneous adipose in mice that it cannot be easily separated by surgical procedures. In fact, we can’t deny that subcutaneous adipose is always present and may be extracted by other methods. Previous studies have shown an inverse relationship between visceral fat and bone density. Therefore, compared with mice, the characteristics of less visceral fat exhibited by ground squirrels may be related to the increase in bone density.
There was no significant change in the microstructure of the femur or tibia in the OB group, but the bone mineral parameters BMC of mice showed a significant increase, TMD and TMC showed an increasing trend (P = 0.095 and 0.070, respectively) in femur after fattening (Table 4). This indicated that the bone microstructure of mice was in a balanced state, but bone minerals had a tendency to increase in femur. By contrast, ground squirrels showed some substantial differences between SA and PRE states. The BS/BV and Tb.N parameters of the PRE group were significantly increased by 36.4% and 28.6%, respectively, whereas Tb.Th, Tb.Sp, Ma.Ar, TMC and BMC were all strongly reduced by 31.6%, 24.3%, 88.6%, 82.8% and 81.9%, respectively, indicating that the bone formation of the femur in the PRE group was enhanced (Table 4). The microstructural changes of the ground squirrel tibia was similar to those of the femur. The Tb.N showed an increasing trend (P = 0.064), Tb.Sp was significantly decreased (-33.0%, P < 0.05), and BV/TV and BMD were significantly increased (40.4% and 20.1%, respectively, P < 0.05), which indicated that the bone formation was enhanced in the tibia (Table 5). In conclusion, there was no tissue specificity in bone formation on the femur and tibia between the mice and ground squirrels. This is consistent with another study, which shows that obese Wistar rats induced by high-fat diet have no difference in bone formation between femur and tibia. Compared with the mice, the bone mass in the PRE group increased in both femur and tibia of ground squirrels, which showed that healthy obesity was not harmful to their bones. However, interestingly, the bone minerals of the femurs in the two models showed opposite changes, an increase in the OB mice and a decrease in the PRE ground squirrels. The mineral density is related to the mechanical properties of bones and therefore, we postulated that these two types of obesity have opposite effects on the mechanical properties of bones. Hence, we next determined the mechanical properties of bones from the two obesity models.
For this study, we recorded changes in the mechanical properties of the bones of mice fed the high-fat diet for 3 months and of ground squirrels fattened before hibernation. Using a three-point bending test, the stiffness of the femur in the OB group was significantly increased by 32.7%, but the mechanical properties of the tibia did not change significantly (Table 6). This indicated that the increased bending resistance of the OB mice was mainly manifested in the femur, thereby reducing the risk of fracture. This may be an adaptation to the higher load caused by weight gain. Compared with the SA ground squirrels, the bones of the PRE group also showed different mechanical properties. The ultimate bearing capacity, stiffness and ultimate bending energy of the femur in the PRE group did not change significantly compared with SA group (Table 6), which indicated that the mechanical properties of the femur did not change during the approach to the hibernation season. However, the ultimate bearing capacity and stiffness of the tibia in the PRE group showed an increasing trend (P = 0.051 and 0.095, respectively, Table 6), which suggests that an increase in bending resistance in the PRE group was mainly manifested in the tibia, which could also reduce the risk of fracture. The changes of bone microstructure and mechanical characteristics were related to changes in bone remodeling function. Therefore, we examined the expression levels of key proteins that regulate bone formation, bone resorption, and Wnt signaling pathways.
Bone metabolism is maintained by the dynamic balance of bone formation and bone resorption. RunX2 is the main driving factor of bone formation and promotes the differentiation and maturation of osteoblasts. The expression level of RunX2 in the OB mice did not change significantly as compared to CON group (Fig. 2B), which was different a previous study that showed a significant decrease in RunX2 mRNA levels in 4-week-old male rats fed with a high-fat diet for 22 weeks. By contrast, the expression level of RunX2 in PRE ground squirrels was significantly up-regulated compared with SA group, indicating that bone formation in the PRE group was enhanced. The differential expression of RunX2 may be the reason for the different changes in bone microstructure between the PRE ground squirrels and the OB mice. OCN plays an important role in regulating calcium metabolism of the bone, mainly promoting bone mineralization. In this study, the expression level of OCN in OB mice was up-regulated, whereas the expression level of OCN in PRE ground squirrels was down-regulated (Fig. 2C). This indicates that the OB mice had increased bone mineralization ability, whereas the PRE ground squirrels could be decreasing bone mineralization activity as the hibernation season approaches. In addition, OCN not only plays a role in bone formation, but also affects energy regulation and, hence, the different changes in the expression of OCN in OB mice and PRE ground squirrels may also contribute to differential regulation of energy metabolism. This idea requires further experimental. ALP protein is one of the phenotypic markers of osteoblasts and can directly reflect the activity or function of osteoblasts. The expression level of ALP in both the OB mice and the PRE ground squirrels was significantly increased (Fig. 2D), which indicated that the osteoblasts in both groups were in good activity and function. This is consistent with a previous study on 6-week-old male C57BL/6 mice fed a high-fat diet for 14 weeks, the results showing that the expression level of ALP in obese mice was significantly increased. Studies have shown that ALP can promote the absorption of calcium ions by bones. We speculate that due to insufficient obesity in the high-fat OB model in the present study, only a 10.6% weight gain was achieved compared with the controls. In order to adapt to the higher load caused by moderate obesity, the bones significantly increased the expression level of ALP, thereby promoting the absorption of calcium salts by the bones, increasing the bone minerals, and enhancing the mechanical strength of the bones. This was also in line with the increasing trend of TMD, TMC and BMC obtained by Micro CT in this study. Hence, we propose that the difference in the expression level of RunX2 was a main reason for the difference in bone formation between the two models. Compared with the OB group, the bone formation in the PRE group was at a higher level.
In terms of bone resorption, the expression levels of RANKL, Cathepsin K and MMP9 increased significantly in both the OB mice and the PRE ground squirrels (Fig. 3). The enhancement of bone resorption in obese mice was consistent with previous studies[34, 61, 62]. We speculated that the reason why bone loss did not occur in the OB mice was that both bone formation and bone resorption were up-regulated to achieve a dynamic balance of high expression. Although there was no bone loss in the OB mice, the high expression levels of bone resorption proteins may be a potential risk for bone loss in mice. Studies have shown that bone loss occurred in mice when they were extremely obese. The expression levels of bone resorption proteins were significantly increased in the PRE group of ground squirrels, but the bone substance was also increased, which may be caused by greater bone formation than bone resorption.
In addition, Wnt signaling also plays an important role in the regulation of bone remodeling. In the present study, the expression levels of P-β-catenin and GSK-3β in OB mice were significantly increased (38.8% and 20.2%, respectively, Fig. 4), and Wnt signaling was weakened, which could lead to an increase of bone resorption and a decrease of bone formation, which is consistent with a study that showed that obesity inhibited the Wnt signaling pathway. Different responses were seen in ground squirrels, where the expression level of GSK-3β in the PRE group was significantly increased (1.4-fold, Fig. 4C), but the expression level of P-β-catenin did not change (Fig. 4B), which indicated that the Wnt signal was strengthened and bone formation was enhanced before hibernation. The differential expression of Wnt signals in the two models was also the cause of bone changes. Activation of typical Wnt signaling by inhibiting GSK-3β has been shown to increase bone mass, which may involve many mechanisms. However, although GSK-3β inhibitors can promote osteogenesis, we should note that the activity of GSK-3β is not only manifested in osteogenesis, but is also related to other intracellular biological processes, which has raised concerns about possible side effects of long-term treatment with these inhibitors in humans. In addition, over-inhibition of GSK-3β has the risk of tumorigenicity.
When comparing the two models, we found that weight gain will cause a significant increase in the expression of bone resorption proteins in both the OB mice and the PRE ground squirrels. The bone substance of the mice did not change significantly, which may be caused by an unchanged expression level of RunX2 and the significant increases in the expression levels of OCN and ALP. Although body weight of the OB mice only increased by 10.6% compared with the control group, the weight gain also brought a great risk of bone loss to the OB mice, which was manifested as a significant up-regulation of bone resorption proteins and weakened Wnt signaling. Different from mice, ground squirrels showed different regulatory mechanisms at work. Although the expression levels of bone resorption proteins in the PRE group also increased significantly, the protein expression levels of RunX2, ALP and GSK-3β increased significantly, resulting in greater bone formation than bone resorption and a net increase in bone mass. This mechanism, which is different from pathological obesity, suggests that ground squirrels fattened before hibernation can be studied as an anti-obesity bone loss model.