Changes in production and lifestyle have gradually increased the proportion of obese people worldwide, especially in developed regions. Obesity is caused by the imbalance of energy intake and consumption and is mainly characterized by overweight and massive fat accumulation. The World Health Organization (WHO) has set body mass index≥ 30 kg/m² as the standard for judging obesity. About 13% of people suffer from obesity globally. If the recent trend continues, then 57.8% of the of the adult population will be overweight or obese by 2030 (Blüher, 2019; Caballero, 2019). Obesity cannot only greatly increase the risk of chronic metabolic diseases such as diabetes and hypertension but also is closely related to the health status of bones (Apovian, 2016).

Osteoporosis (OP) is characterized by decreased bone mineral density (BMD), low bone mass, and increased risk of fracture. The imbalance of bone homeostasis is the most fundamental cause of OP (Felsenberg and Boonen, 2005). The effect of obesity on bone is complex and could be benign. High body weight acts on bones, and it causes strong mechanical stimulation signals felt by bone cells, such as osteoblasts and osteocytes, and increase bone mass. In addition, the increase in estrogen level in obese people can directly promote the activity of osteoblasts, inhibit the activity of osteoclasts, and protect bones (Rinonapoli et al., 2021). However, scholars found that the positive effect of mechanical load on bone mass due to weight gain could not offset the adverse effect of obesity on bone mass. Obesity changes the bone microenvironment, inhibits the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, promotes adipogenic differentiation, and increases bone fat. The high inflammatory state caused by obesity and the secretion of adipokines also have a negative effect on bone (Forte et al., 2023; Piñar-Gutierrez et al., 2022; Shapses and Sukumar, 2012).

Regular physical exercises are conducive to physical health, and weight-bearing exercises are crucial to bone metabolism and homeostasis. Mechanical stress generated by exercise and the secretion of related factors can promote bone formation, reduce bone resorption, improve bone mass, and thus reduce the risk of falls (Qiu et al., 2023; Styner et al., 2014). Exercise can alleviate the acquisition of bone marrow adipose tissue, and promote the formation of bone mass in obese mice (Chubak et al., 2006). Although proper exercise can promote bone mass, the molecular mechanism of aerobic exercise-induced bone remodeling in obese individuals has not been elucidated. In the present study, we used high-fat diet (HFD) to build a mouse obese model and identified their bone phenotype. The obese male mice underwent a 10-week treadmill exercise to explore the effects of aerobic exercise on the bones and identify the related mechanisms. Results will provide exercise reference for children and adolescents with bone mass loss due to obesity.

The main findings of this study can be summarized as follows. Obesity induced by HFD inhibits bone formation, promotes bone resorption, and leads to decreased bone mass in obese male mice. The effects of HFD-induced obesity on the bones are mainly associated with the inhibition of the WNT signaling pathway and the dysregulation of adipokine production. Aerobic exercise can, to some extent, alleviate the inhibition of WNT pathway transmission caused by obesity, promote bone formation, and inhibit bone resorption.

Male mice were selected to avoid the significant interference of the estrogen cycle on bone metabolism. Bone metabolism, especially bone formation, is strongly regulated by estrogen, and the estrogen level in female mice fluctuates regularly with the estrous cycle (usually 4–5 days) (Almeida et al., 2017; Xiong and O'Brien, 2012). The periodic fluctuations of such endogenous hormones are themselves a powerful and difficult to control variable, which can significantly affect the baseline status and response of osteoblast activity, osteoclast formation, and key signaling pathways such as Wnt/β-catenin (Melville et al., 2014). Therefore, in the initial exploratory study of mechanisms, prioritizing the use of male animals with a relatively stable physiological hormone environment is an effective strategy to control variables and ensure that we can clearly observe the core causal chain between exercise and bone (Beery and Zucker, 2011).

Obesity not only increases the risk of chronic metabolic diseases, such as diabetes and hypertension, but is also closely related to skeletal health (Klein-Nulend et al., 1995). Mechanical load is one of the factors contributing to increased bone mass. Therefore, the increased body weight caused by obesity was thought to apply additional force to the body, thereby increasing bone mass to some extent and reducing the occurrence of OP (Gower and Casazza, 2013; Guo et al., 2021). However, in recent years, an increasing number of studies have confirmed that obesity leads to fat accumulation and trabecular bone loss (Kleinert et al., 2018). The mechanisms involved include changes in bone cell metabolism, increased oxidative stress and inflammation, and alterations in bone-regulating hormones. Nevertheless, the mechanisms by which exercise improves obesity-induced bone loss require further investigation. Therefore, we designed this study to examine changes in bone mass in obese male mice due to exercise and explore the related bone turnover mechanism.

Feeding mice with a diet containing 60% fat resulted in a significant increase in body weight, elevated serum levels of TG, CHOL, and INS, and decreased glucose tolerance and insulin resistance (Gonnelli et al., 2014; Thyfault and Rector, 2020). Exercise can increase the body’s energy consumption, thereby reducing body weight and lowering fasting insulin levels (Nielson et al., 2011). After feeding 5-week-old C57BL/6 mice with HFD containing 60% fat for 12 weeks, the body weight consistently remained higher than that of control mice, and the obese male mice exhibited decreased glucose tolerance and insulin resistance. After 10 weeks of aerobic treadmill exercise, the body weight and fat mass of the mice significantly decreased. However, in the results of Figure 2B, the inter-group difference failed to reach the significant level observed in Figure 1H. We noticed that in the last week of the experiment, the mice underwent short-term fasting and injections for GTT and ITT. These necessary experimental procedures inevitably caused acute stress to the mice and might have temporarily affected their feeding behavior and energy metabolism. In addition, Supplementary Figure S1 of ours includes the weekly body weight measured during the exercise period for reference. The GTT AUC values of the four groups of mice showed that glucose clearance rates increased after aerobic exercise. Additionally, exercise improved serum CHOL and TG levels in obese male mice. The RT-PCR detection revealed that exercise reduced the expression of obesity-related fat genes in obese male mice. Hence, aerobic treadmill exercise, to some extent, alleviated abnormal lipid metabolism in obese male mice induced by HFD.

To control for the potential impact of circadian rhythms on exercise benefits, all exercise interventions in this study were strictly standardized to be conducted within the same time window (ZT 14). Evidence indicates that the physiological responses of organisms to exercise, including changes in energy metabolism and skeletal adaptability, are regulated by the intrinsic circadian clock (Sc et al., 2015; Wolff and Esser, 2012). Recent studies have clearly indicated that exercising at different stages of the animal’s active phase (ZT12-24) exerts distinctly different effects on bone and metabolism (Boudenot et al., 2021; Yu et al., 2024) For instance, exercise performed in the early stage of the active phase may primarily promote anabolism, whereas exercise conducted during the resting phase could disrupt normal rhythms and induce a stress response (Sato et al., 2019). Therefore, standardizing the exercise time is crucial for obtaining consistent and reproducible experimental results. In this study, treadmill training was conducted in male mice during the early active phase (ZT14). This choice aimed to mimic their natural activity peak and standardize the exercise intervention as a variable as much as possible, thereby ensuring that the observed effects of promoting bone formation and activating the Wnt pathway can be clearly attributed to exercise itself, rather than confounding effects caused by differences in exercise timing.

Under normal circumstances, the amount of old bone absorbed by osteoclasts is consistent with the amount of new bones generated by osteoblasts. This balance maintains the continuous renewal of bone tissue and its mechanical integrity throughout life, playing an important role in maintaining the homeostasis of trace elements, such as calcium and phosphorus, in the body. However, under certain physiological or pathological conditions, the activity of osteoblasts and osteoclasts may change accordingly. When the two processes of bone metabolism are no longer balanced, bone tissue will exhibit corresponding changes, such as physiological growth, development, and aging or have pathological conditions, such as OP, osteomalacia, bone metastases, and others. Obese males often exhibit significant decreases in BMD, BV/TV, Tb.N, and Tb.Th (Kang et al., 2019; Kang et al., 2017). However, after exercise intervention, the adverse effects of obesity on bones can be mitigated. Kang et al. (Kang et al., 2017) subjected obese rats to 8 weeks of swimming training, and the micro-CT results showed significant increases in BMD, BV/TV, and Tb.N in the femur and tibia of obese rats after swimming. The results consistently demonstrate that exercise can alleviate bone loss caused by obesity regardless of treadmill running, voluntary wheel running, swimming, or ladder climbing resistance exercise (Styner et al., 2014; Kang et al., 2019; Shi et al., 2022; Yan et al., 2021). Our micro-CT, Masson staining, and bone fluorescence results demonstrate that obesity decreases bone mass and inhibits the rate of femoral bone formation in mice. However, aerobic treadmill exercise can increase the trend of bone formation in obese male mice. Additionally, the HE staining results of the femur in each group of mice show similar findings, that is, obese male mice have widened trabecular spaces and reduced trabecular bone. After aerobic exercise, the trabecular bone in obese male mice significantly increases. Furthermore, the serum bone formation marker ALP and the expression of osteogenic genes OPN and ALP mRNA as well as the OCN protein in the bone are significantly reduced. ALP is a classic early-stage marker of osteogenic differentiation. The increase in its activity indicates the differentiation commitment of progenitor cells toward the osteoblast lineage, reflecting the initial stage of bone matrix maturation (Nakamura et al., 2020). In contrast, OPN is regarded as a middle-to-late-stage marker of the osteogenic process. By analyzing the expression of OPN, we aim to evaluate the differentiation progress and functional maturation status of osteoblasts (Chen et al., 2014). However, 10 weeks of aerobic treadmill exercise can promote the expression of osteogenesis-related genes and proteins. After extracting BMSCs, mechanical stimulation was simulated in vitro. The RT-PCR results were consistent with the above findings, that is, in vitro stretching of obese mouse BMSCs could promote the expression of osteogenesis-related genes. The above results indicate that after treadmill exercise, bone mass increases, suggesting changes in bone metabolism levels, although the difference was not significant. The reasons may involve issues, such as exercise selection, sample and parameter choices, etc. This also suggests that we should consider these factors in our future research to further explore the relationship between exercise and the prevention of bone metabolism diseases caused by obesity.

TRACP staining and Masson’s trichrome staining results show that obesity induced by HFD tends to increase bone resorption indicators in mice, while aerobic treadmill exercise mitigates this trend. After aerobic exercise intervention, the expression of NFATC1 mRNA and the level of the ctsk protein in the tibia of obese male mice decreased significantly. Previous experimental results also showed that HFD upregulates bone resorption in mice, while treadmill exercise training can alleviate the expression of bone resorption (Cao and Picklo, 2015). Ca and P are essential minerals in the human body. They form the basic components of bones as phosphates and confer bones their appropriate strength. Only 1% of Ca is present in the blood. When the balance of Ca and P in the blood is disrupted, the body automatically mobilizes Ca and P stored in the skeletal system to regulate the balance (Blaine et al., 2015). This experiment found that blood Ca levels significantly increased in HFD mice and HFD + EX group mice. The elevated levels of CTX-1 in urine typically indicate increased rates of bone matrix degradation and bone resorption, which is common in diseases such as osteoporosis. In elderly individuals, a significant negative correlation was found between urinary CTX-1 concentration and the degree of obesity; obese patients had relatively lower levels of CTX-1 in their urine (Djafari et al., 2021). In this experiment, obesity significantly increased in urinary CTX-1 levels in mice. After extracting BMSCs, mechanical stimulation was simulated in vitro. The RT-PCR results were consistent with the above findings, namely, that in vitro stretching of obese male mice BMSCs could inhibit the expression of osteoclast-related genes. Hence, exercise can improve the function of osteoclasts induced by HFD in obese male mice and reduce bone resorption.

The classical WNT/β-catenin signaling pathway is one of the most conserved regulatory factors in evolution and plays a crucial role in regulating tissue development and maintaining homeostasis (Liu et al., 2022). Meanwhile, the Wnt/β-catenin signaling pathway is also a core regulator of bone metabolism. Studies have reported that the Wnt/β-catenin signaling pathway regulates the differentiation of MSCs into osteoclasts by inhibiting the adipogenic transcription factor C/EBPα and PPARγ, thereby playing a crucial role in the regulation of bone homeostasis. Additionally, the interaction between β-catenin and TCF (T-cell factor) proteins can upregulate the expression of OPG (Osteoprotegerin) in osteoclasts, which serves to block the differentiation of osteoclasts (Rodda and McMahon, 2006). In this study, RNA-seq, Western blot, and RT-PCR consistently confirmed that obesity induced by a high-fat diet significantly inhibited the Wnt/β-catenin signaling pathway in the tibia of mice, which was manifested by the downregulation of β-catenin and Ccn4. However, 10 weeks of aerobic treadmill exercise effectively reversed this inhibitory trend, as evidenced by the increased expression of β-catenin, Wif1, and Ccn4. We propose that the activation of skeletal Wnt signaling by exercise may be achieved through the synergistic effect of two aspects.

Exercise may safeguard the normal function of the Wnt pathway by improving the obesity-related systemic pathological environment. Obesity is a state of chronic inflammation, characterized by elevated levels of circulating inflammatory factors such as TNF-α and IL-6 (Hotamislig and il, 2006). These inflammatory factors are potent inhibitors of the Wnt pathway, as they can upregulate the expression of Wnt antagonists Dkk1 and sclerostin (Poole et al., 2005). Wannenes et al. (Wannenes et al., 2014).cultured osteoblasts by using serum from obese patients and found that the WNT pathway was inhibited, with its downstream target genes downregulated. When using palmitic acid (PA) to simulate high-fat-induced in vitro obesity state, the expression levels of WNT3a and β-catenin were significantly decreased in the PA group compared with those in the control group (Guo et al., 2022). This indicates that obesity itself directly inhibits the Wnt pathway. Therefore, we hypothesize that aerobic exercise, as an effective anti-inflammatory intervention (Pedersen and Saltin, 2015), may reduce the induction of Dkk1/sclerostin by lowering the inflammatory levels in the whole body and local bones, thereby indirectly relieving the inhibition on the Wnt pathway and enabling the recovery of β-catenin signaling. Although this study did not directly detect Sost, the observed change in the expression of Wif1 (another antagonist) in the exercise group suggests that the regulation of antagonists may be a core mechanism. Future studies that directly detect changes in the levels of inflammatory factors and Sost protein in serum and bone tissue after exercise will strongly validate this hypothesis.

The mechanical load exerted by exercise on bones may be a direct pathway. Bones are highly mechanically sensitive tissues, In clinical settings, strength training can upregulate the expression of WNT-related genes in the human body (Cardaci et al., 2020), and mechanical stimulation can directly inhibit the expression of Sost in osteocytes (Robling et al., 2008; Tu et al., 2012)while promoting the secretion of Wnt ligands by osteoblasts (Lawson et al., 2022). We detected changes in Ccn4 (WISP1). As a downstream target gene and secretory protein of the Wnt pathway, Ccn4 itself can enhance the differentiation and proliferation of osteoblasts and inhibit adipogenesis through autocrine/paracrine mechanisms (Zuo et al., 2010). Therefore, the upregulation of Ccn4 by exercise is not only a response to the activation of the Wnt pathway, but also likely constitutes a positive feedback loop itself, which further amplifies the osteogenic effect of exercise.In summary, under the state of obesity induced by a high-fat diet, factors such as chronic inflammation lead to the inhibition of the Wnt pathway and a reduction in bone formation. On one hand, aerobic treadmill exercise reduces the inhibition of the pathway through its systemic anti-inflammatory effect. On the other hand, it directly activates the pathway via local mechanical load. These two forces act together to reverse the obesity-induced inhibition of the Wnt/β-catenin signal, ultimately leading to favorable outcomes including enhanced osteoblast activity and suppressed osteoclast formation, thereby improving bone metabolic homeostasis. This mechanism not only deepens our understanding of the role of exercise in protecting bones, but also provides a solid theoretical basis for using exercise as a non-pharmacological strategy to treat obesity-related.

This study conducted an endpoint assessment immediately after the completion of the 10-week intervention, which allowed us to demonstrate that exercise ultimately promotes bone formation and inhibits bone resorption. However, bone remodeling is a dynamic, multi-stage continuous process. It is well-known that mechanical loading stimulation on bones first triggers a transient activation of bone resorption, followed by sustained bone formation, and there may be an anabolic window period throughout this process. Due to the lack of dynamic observations at intermediate time points during the intervention (e.g., Week 2, Week 4, Week 6) in this study, we were unable to depict detailed kinetic curves of exercise-induced changes in bone metabolism markers (such as P1NP and CTX-1) or the expression of key factors in the Wnt pathway. Therefore, we cannot determine at which specific stage of the intervention exercise exerts its strongest effect, nor can we completely rule out the possibility of a transient increase in bone resorption that may occur in the early stage. Future studies, by performing longitudinal analyses at multiple time points, will be able to more accurately reveal the temporal mechanism of aerobic exercise against obesity-related bone loss and provide key data for optimizing the duration and frequency of exercise interventions, and future studies that use CT to assess the dynamic changes of adipose tissue will be of great value for comprehensively interpreting the interaction between lipid metabolism and bone health during exercise response.

This study demonstrated the connection between obesity and bone turnover. Obesity induced by HFD leads to decreased bone formation, increased bone resorption, and increased adipogenesis. Aerobic exercise, however, mitigates changes in bone structure. Our findings suggest that the impaired bone mass accumulation caused by obesity may be due to the inhibition of the WNT signaling pathway, and exercise intervention may restore its activity. The results provide a foundation for further exploration of the mechanisms of obesity-induced impaired bone mass accumulation. However, the mitigating effect of exercise on obesity-induced bone loss was not immediate. Therefore, in subsequent studies, different forms of exercise will be investigated, focusing on enhancing the WNT signaling pathway as an effective intervention to prevent or improve bone loss in obese patients.

This study reveals that 10 weeks of aerobic exercise improves the imbalance of glycolipid metabolism and bone metabolism in high-fat obese male mice by activating the WNT pathway and regulating adipokines, inhibits bone resorption and promotes bone formation, providing a basis for exercise intervention for obese-induced bone loss.