Obesity caused by an HFD will dilate adipose tissue and lead to chronic low-grade systemic inflammation. Ectopic adipose cells accumulate in the bone marrow cavity, impair bone regeneration and lead to osteoporosis (Halade et al., 2010). Bone marrow is the only tissue where adipocytes and bone cells directly interact (Horowitz and Tommasini, 2018). Studies have found that osteoporosis or bone mass loss often coexists with lipid metabolism disorders (Tian and Yu, 2015). When the number and volume of adipocytes in the bone marrow continue to increase, the microenvironment in the bone marrow cavity changes, causing the bone marrow lipid metabolism disorder, the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts decreases, the formation and function activation of osteoclasts increase, and the total amount of excessive bone loss, eventually leading to osteoporosis (Hu et al., 2018). Studies have shown that obesity is often accompanied by abnormal secretion of adipokines (Fasshauer and Blüher, 2015). Abnormal production of adipokines and activation of some pro-inflammatory signaling pathways in obese patients can induce accelerated inflammation. Inflammation activates various immune cells to produce many free radicals, aggravating oxidative stress, and oxidative stress (OS) also plays a crucial role in the pathogenesis of osteoporosis (Saltiel and Olefsky, 2017; Geng et al., 2019).

AMP-activated protein kinase (AMPK) is the regulatory switch of energy metabolism in organisms, and it plays a crucial role in lipid metabolism regulation (Hardie, 2015). Several studies have shown that it promotes the oxidation and decomposition of fatty acids, inhibiting lipid synthesis and other processes from inhibiting lipid accumulation (Kim et al., 2016; Garcia et al., 2019). In addition, AMPK can be activated under oxidative stress. The activated AMPK can produce ATP by promoting insulin sensitivity, fatty acid oxidation, and mitochondrial biosynthesis, thus reducing oxidative stress damage (Herzig and Shaw, 2018). Previous studies have reported that activating the AMPK signaling pathway can induce mitochondrial autophagy of bone marrow mesenchymal stem cells to inhibit intracellular oxidative stress and improve osteoporosis (Chen et al., 2021).

FA is a water-soluble vitamin widely found in fresh fruits, vegetables, and meat. FA is highly safe, preventing DNA damage, reducing oxidative stress and cell apoptosis. It has no significant side effects in the long-term use of small doses (Field and Stover, 2018). Studies have shown that FA is an effective free radical scavenger, which can inhibit lipid peroxidation and reduce the production of malonic acid (MDA) (Li et al., 2021). FA deficiency can cause an increase in homocysteine and a decrease in methionine, which further increases liver lipid synthesis and induces lipid accumulation in liver cells (Rizki et al., 2006). In addition, studies have shown that low FA levels can significantly reduce trabecular bone thickness and trabecular bone number, and there may be a direct relationship between FA and bone mineral density (Holstein et al., 2009). These results suggest that FA may prevent bone loss, but FA’s exact effect and mechanism on bone metabolism remain unclear.

Therefore, our current study aims to explore whether FA could affect bone health and related mechanisms of HFD-fed mice by regulating lipid metabolism and oxidative stress by using the AMPK signaling pathway as the entry point to provide valuable information-theoretical evidence for nutritional intervention strategies of HFD-induced osteoporosis.

FA has been widely used in the treatment of megaloblastic anemia and the prevention of neural tube defects in neonates due to its extensive pharmacological activity and reliable safety. In this study, we constructed an HFD-induced osteoporosis model in C57BL/6 J mice to explore the effect of FA on HFD-induced osteoporosis and its molecular mechanism. Our results show that FA reduced the metabolic disorders induced by an HFD, including insulin resistance, hyperlipidemia and systemic inflammation, and reduced the level of oxidative stress. More importantly, we found that FA significantly improved bone mineral density and bone microstructure, and activated the AMPK signaling pathway, promoting the expression of lipid oxidation-related enzymes and antioxidant enzymes. In conclusion, we believe that FA can reduce osteoporosis induced by an HFD, which may be related to the regulation of lipid metabolism and oxidative stress levels by FA through the activation of the AMPK signaling pathway (Figure 7).

With the increasing availability of cooking oils and animal foods, it is predicted that 20% of adults worldwide will be obese by 2030 (Kelly et al., 2008). Several studies have shown that HFD is related to the development of obesity, and under the circumstances of obesity and osteoporosis are closely related to diseases (Shapses and Sukumar, 2012; Fabiani et al., 2019). Therefore, this study constructed an osteoporosis model by feeding mice with a high-fat diet. The model of osteoporosis induced by a high-fat diet refers to the influence of bone metabolism by ingesting a large amount of fat, thereby inducing the occurrence of osteoporosis. Obesity and osteoporosis have common characteristics, such as genetic predisposition, common progenitor cells. Both adipocytes and osteoblasts are derived from bone marrow mesenchymal stem cells. A long-term high-fat diet promotes adipogenesis and inhibits bone formation. In this study, the HFD significantly increased the weight of the mice, while FA did not affect weight gain (Figures 1A,B) or food intake (Figure 1C), but it could reduce body fat percentage (Figure 1D). It is suggested that the effect of FA on HFD-induced osteoporosis may not be through affecting the body weight but body fat percentage of high-fat-fed mice.

HFD has been reported to inhibit bone formation and enhance bone resorption (Tencerova et al., 2018; Roberts et al., 2021). Our study also proved that HFD could reduce bone mass and destroy bone microstructure in mice. The BV/TV of the HFD group was 46.7% lower than that of the Control group which was consistent with previous studies. The FA intervention can significantly improve the bone microstructure of mice on an HFD (Li et al., 2019; Lin et al., 2019). The specific manifestation is the increase in BV/TV, Tb. N and the connection density of trabecular bone after the intervention of FA, and the Tb. Th has no obvious change (Figures 2B,C), suggesting that the main effect of FA may be to increase the number of trabecular bone rather than thickening existing trabecular bone. Consistent with micro-CT analysis, the H&E staining of distal femur slices confirmed that the number and thickness of femur trabecular bone were reduced in the HFD compared with the control group, and FA attenuated these changes (Figure 2D). In addition, the number of osteoclasts was reduced and the number of adipocytes was increased in the HFD-fed mice after FA supplementation (Figures 2E,F). Meanwhile, FFA levels in bone homogenates were significantly reduced after FA supplementation (Figure 2G). Studies have shown that CTX-1 and PINP play an essential role in the pathogenesis of osteoporosis (Szulc, 2018). CTX-1 is an important osteoclast regulator, and the increase of CTX-1 can be used as a marker of the active function of osteoclast, which plays a vital role in osteoclast generation and osteoclast-mediated local bone loss (Eastell and Szulc, 2017). CTX-1 was significantly decreased after folic acid supplementation, but the effect on the number of osteoclasts was not obvious, suggesting that the effect of folic acid supplementation on osteoclasts may be more reflected in effect on their activity rather than the number. Type I collagen is the most abundant type of collagen in the human body and the only type of collagen in mineralized bone. The metabolites of its synthesis and decomposition can indirectly reflect the status of bone turnover. The content of PINP in serum reflects the ability of osteoblasts to synthesize collagen (Krege et al., 2014). The clinical study of Trovas GP (Trovas et al., 2002) showed that the bone resorption indexes NTX and CTX-1 decreased more significantly than the bone formation indexes BALP and PINP after the use of calcitonin, which can significantly reduce the bone turnover rate. Our results also show that FA reduced the serum CTX-1 and PINP levels of mice on an HFD (Figures 2H,I), and CTX-1 decreased more significantly than PINP, and the possible causes were analyzed: Generally, osteoporosis is caused by the increase of osteoclasts than osteoblasts, which breaks the balance between bone resorption and bone formation during normal bone reconstruction, resulting in osteoporosis. The bone formation index showed hyperfunction along with the increase of bone resorption index, which led to the increase of compensatory bone formation, which also led to the increase of bone formation index. However, the inhibition of this mechanism after short-term use of folic acid led to the decrease of bone metabolism index. It is possible that with the prolongation of folic acid supplementation, it will promote the proliferation of osteoblasts and increase bone formation indexes. Therefore, the results of this study cannot be considered contradictory at present.

Based on previous studies, we speculate that FA may affect the occurrence of osteoporosis by regulating lipid metabolism. Studies have shown that an HFD can cause glucose and lipid metabolism disorders in mice (Chen et al., 2019). The results of GTT and ITT show that the HFD group has impaired glucose regulation and reduced insulin hypoglycemic effect, indicating that HFD can induce glucose metabolism disorder and insulin resistance in mice. FA supplementation improved glucose regulation and insulin sensitivity in mice (Figures 3A,B). At the same time, FA can adjust the blood lipid level of HFD-fed mice (Figures 3C–F) and reduce the serum FFA content of mice (Figure 3G). Previous studies have shown that abnormal lipid metabolism and glucose metabolism are closely related to osteoporosis (Saoji et al., 2018; Veldhuis-Vlug and Rosen, 2018; Hu et al., 2019). Studies have found that long-term FA deficiency can lead to obesity, lipid metabolism disorders and glucose metabolism disorders, and appropriate FA supplementation can reduce the risk of dyslipidemia (Gargari et al., 2011; Dehkordi et al., 2016). Our results also show that FA intervention can reduce insulin resistance induced by an HFD and regulate lipid levels in mice, suggesting that FA has a positive effect on glycemic and lipid metabolism disorders. These beneficial effects are closely related to a reduction in lipid deposition in the body. We speculate that these changes may indicate that FA supplementation promotes lipid metabolism in the body, increasing additional lipid consumption.

Since FA can reduce lipid accumulation and improve bone microstructure in HFD-fed mice, we next try to explore the mechanism of FA. AMPK is a protein kinase that exists in the body, which can participate in the regulation of energy, glycolipid metabolism and mitochondrial homeostasis. AMPK is known as an energy regulator, and activation of AMPK is of great significance in treating obesity and obesity-related metabolic disorders (Carling, 2017; Garcia and Shaw, 2017). Many studies have shown that the activation of AMPK can control the overall lipid metabolism of cells by regulating ACC. ACC is a key enzyme that catalyzes lipid synthesis. Carnitine palmitoyl transferase 1 (CPT-1) is responsible for transporting fatty acids to mitochondria for oxidation, and ACC can convert acetyl-CoA to malonyl-CoA, an inhibitor of CPT-1. So the inactivation of ACC can increase fatty acid transport and subsequent oxidation, thereby reducing lipid synthesis (Vancura et al., 2018; Fadó et al., 2021). Our results show that the intervention of FA significantly increases the phosphorylation level of AMPK and ACC, and further up-regulates the expression of CPT1. This indicates that FA may activate AMPK, further promote the expression of CPT-1, and promote fatty acid oxidation to reduce lipid synthesis and lipid accumulation in the bone marrow cavity, thereby attenuating the osteoporosis induced by HFD.

Obesity is usually accompanied by chronic low-grade systemic inflammation, which leads to oxidative stress, and both inflammation and oxidative stress show harmful effects on the osteogenic differentiation of BMSCs (Napoli et al., 2017). The NF-κB signaling pathway mediated by TNF-α can regulate lipid metabolism, and IL-6 can promote systemic inflammation, leading to metabolic syndrome and abnormal lipid metabolism (Engin, 2017; Wang and He, 2018). In our study, FA significantly reduced the levels of TNF-α and IL-6 in the serum of mice, and reduced the inflammatory response (Figures 5A,B), suggesting that FA may regulate lipid metabolism by regulating TNF-α and IL-6. FA significantly reduced the expression of MDA. It increased the activity of the antioxidant enzyme SOD (Figures 5C,D), indicating that an HFD can induce lipid peroxidation in the body and reduce the activity of antioxidant enzymes in bone tissue, while FA supplementation improved the expression. Nrf2 is a transcription factor that protects cells and maintains bone metabolism from oxidative damage by regulating excessive antioxidant and cellular defense pathways (Dai et al., 2020). Under physiological conditions, Nrf2 exists in the cytoplasm and binds to kelch-like epichlorohydrin-related protein 1 (Keap1), and its activity is inhibited. When oxidative stress occurs in the body, phosphorylation of a series of protein kinases leads to the decoupling and translocation of Nrf2 and Keap1 into the nucleus, where Nrf2 binds with the antioxidant reaction element ARE to initiate the transcription and expression of AREs regulated antioxidant oxidase genes (Yamamoto et al., 2018). Several studies have shown that Nrf2 can be activated by AMPK (Lv et al., 2017; Duan et al., 2019). Nrf2 activation can activate downstream antioxidant enzymes, such as SOD, HO-1, NQO1, GCLM, etc., to play its antioxidant role and quickly and effectively remove excessive ROS produced in the body (Cuadrado et al., 2019). Our results showed that FA could up-regulate Nrf2 signaling pathway activity in the HFD-fed mice and promote the expression of downstream proteins HO-l, NQOl and GCLM, thereby activating antioxidant enzymes to mediate oxidative stress reduction (Figures 6A–C). These results suggest that FA may up-regulate the activity of antioxidant enzymes through the Nrf2 signaling pathway, protect cells from oxidative stress and inflammation-induced damage, and inhibit the activity of osteoclasts, thus contributing to the osteogenic differentiation of BMSCs, and thus reducing the high lipid-induced osteoporosis.

The present study has several limitations. First, the detailed mechanisms underlying the ability of FA to activate AMPK signalling have not been fully elucidated. Second, pharmacological parameters such as the optimal doses and treatment timing that are critical for clinical studies have not been well explored. Third, The potential mechanism of folic acid affecting skeletal health in mice was only preliminarily explored using animal models. Future work should target the molecular mechanisms underlying the protective effect of FA and explore its optimal administration in large animal models.

In summary, factors such as lipid metabolism disorders, inflammation, and oxidative stress damage together contribute to the formation and development of osteoporosis. This study shows that FA may have the effects of regulating metabolism, anti-inflammatory, and antioxidant. Therefore, FA may regulate lipid metabolism and oxidative stress in the HFD-fed mice by activating the AMPK signaling pathway, reducing osteoporosis induced by an HFD. Based on the positive effects of FA on the bone health of mice in this study, FA may provide a new idea for nutritional intervention strategies for osteoporosis caused by an HFD.

Osteoporosis has become a worldwide epidemic. Among people over the age of 50, the incidence is one-third for women and one-fifth for men. It is estimated that the number of patients with osteoporosis worldwide will exceed 1 billion in 2020. Studies have found that obesity caused by a HFD will expand adipose tissue and cause chronic low-grade systemic inflammation. Ectopic adipose cells accumulate in the bone marrow cavity, which may impair bone regeneration and lead to osteoporosis. We proposed that FA may have a preventive effect on HFD-induced osteoporosis. Our research shows that FA can attenuated bone loss and improved bone microarchitecture in the HFD-fed mice. After the intervention of FA, the body fat rate of obese mice was reduced, and related metabolic disorders such as insulin resistance, hyperlipidemia, and systemic inflammation were alleviated. Thus, FA may provide a new idea for nutritional intervention strategies for osteoporosis caused by a HFD.