This study investigated the effects and mechanisms of two types of exercise on T2DM bone resorption metabolism. We further interrogated the role of Sirt1/NF-κB signaling pathway in mediating bone resorption effects in T2DM mice. Our data demonstrate that two types of exercise inhibits the bone resorption in T2DM mice and the effect is mediated by Sirt1/NF-κB. Interestingly, downhill running conferred the best effect.
Advanced increase of glycation end products and abnormal expression of insulin-like growth factor due to high glucose and insulin deficiency for T2DM lead to abnormal enhancement of bone resorption, decrease of BMD and osteoporosis . Studies have proved that in T2DM patients, the cortical bone thickness and the number of bone trabecular are reduced significantly, and the morphological structure of bone is compromised seriously [23–25]. In this study, the morphological and structural degeneration of bone tissue was observed by both HE staining and Safranin O-Fast Green staining, indicating bone mass loss in T2DM mice resulting in significant degeneration in microstructure of bone tissue. This was closely related to the increased number of OCs and multinuclear OCs produced by differentiation in this study, for the secreted StrACP increased significantly with the number of OCs and multinuclear OCs, indicating that in T2DM mice, the bone resorption capacity was significantly enhanced, resulting in the secretion of organic acids on the bone surface enhancing bone erosion capacity, which caused the degradation of bone microstructure [26,27,12]. This process may be regulated by key pathways or factors. As a key factor for energy metabolism, Sirt1 also regulates bone metabolism. When Sirt1 is knocked out, the OC differentiation in mice will be inhibited and the bone mass lost significantly [28,29], which is closely related to the activation of NF-κB pathway by Sirt1 knockout causing up-regulation of IL-6 and downstream c-fos and NFATc1 genes and promotion of OC differentiation, fusion and bone resorption [30,31]. Although studies have confirmed that Sirt1 and NF-κB pathways regulate T2DM to promote OC differentiation and bone resorption respectively, the Sirt1 mediated NF-κB pathway for regulating T2DM to inhibit OC differentiation and bone resorption is still to be revealed. In this study, the mRNA and protein expressions for Sirt1 were down-regulated significantly and for NF-κB, IL-6, c-fos and NFATc1 up-regulated significantly in the bone of T2DM mice, suggesting that down-regulation of Sirt1 expression activated the NF-κB pathway in T2DM mice. When this pathway was activated, its target gene NFATc1 promoted OC differentiation, fusion and bone resorption capacity, and thus, the activity of StrACP, a serum biochemical marker for bone resorption, was enhanced significantly, resulting in further bone loss and degeneration of bone tissue microstructure . This may explain the increased number of OCs produced by differentiation, the enhanced StrACP activity, the degeneration of bone microstructure and the decrease of bone mass in this study. In T2DM mice, abnormal energy metabolism in OCs results in decreased NAD+/NADH ratio and inhibits Sirt1 expression through peroxisome proliferator- activated receptor γ coactivator 1α (PGC-1α) . After activated, the C-terminal domain of forkhead box O1 (FOXO1) binds to Sirt1 to form the FoxO3A-Sirt1 complex group, which down-regulates the Sirt1 expression together with v-ATPase V0subunit d2 (Atp6v0d2) and DC-STAMP in OCs, thus activating NF-κB and its downstream target genes like IL-6, c-fos, NFATc1, etc., and thus, the OC differentiation and bone resorption are promoted.
Abnormally enhanced bone resorption in T2DM degrades the morphology of bone tissue and decreases the bone mass. For the bone, an organ sensitive to mechanical stimulation, the mechanical load generated by exercise on T2DM bone is transformed into biological signals to inhibit OC differentiation and bone resorption capacity as well as bone resorption, thus improving the microstructure of bone tissue and bone mass . Studies have verified that in T2DM mice, the abnormal increase of bone resorption results in significant degeneration of bone phenotypes like distal tibial and femoral cancellous bones as morphometric indicators, while 8-week downhill running may improve the indicators in T2DM significantly [13,35,36]. High-intensity intermittent exercise is an important method for improving bone resorption, but there are still few reports on the comparison between the exercise modes for improving bone resorption in T2DM. In this study, both 8-week high-intensity intermittent exercise and downhill running significantly improved the morphological structure of tibial and femoral cancellous bones in T2DM mice, but compared with high-intensity intermittent exercise, downhill running had a more significant effect on the morphological structure of tibia and femur. This might be closely related to the great mechanical stimulation of bone in T2DM mice by running downhill, which may improve the microstructure of bone tissue significantly . In this study, both exercise modes improved the morphological structure of distal tibial cancellous bone in the mice significantly, but the downhill running had a more significant effect. The results were consistent with Moon's study, which reckons that the effect of downhill running on the bone of T2DM mice results in more significant improvement in the morphological structure of bone tissue, supporting the positive effect of downhill running on bone . An analysis of relevant mechanism has shown that in T2DM mice, downhill running inhibits OC differentiation and fusion as well as bone resorption capacity and secretion of StrACP (a biochemical marker for bone resorption) significantly in T2DM mice . In this study, the total number of OCs and the number of ≥ 3 nuclei OCs produced by BMM differentiation in T2DM mice were decreased significantly under both exercise modes. In addition, a comparison between the two modes showed that the total number of OCs and the number of ≥ 3 nuclei OCs decreased more significantly in the downhill running group. StrACP secretion was inhibited by the decrease in OCs and relevant bone resorption capacity. Moreover, exercise inhibited the decline in bone erosion of OCs in T2DM mice and improved the microstructure of bone tissue, especially for cancellous bone . A study by Chen Xianghe et al.  has also verified that exercise inhibited OC differentiation, fusion and bone formation in T2DM mice, the mechanism is closely related to the inhibition of CN/NFAT pathway in the bone by exercise, and downhill running has a more significant effect for its direct mechanical stimulation on the bone.
The process for exercise inhibiting OC differentiation and bone resorption in T2DM is regulated by signaling pathways or key proteins like OPG/RANKL/RANK, G protein coupled receptor 48 (GPR48) and p38MAPK [41,42]. For the Sirt1/NF-κB pathway, an important signal pathway that regulates T2DM for promoting OC differentiation and bone resorption, relevant effects have been verified in this study, and furthermore, Sirt1 and NF-κB pathway play important regulatory roles in exercise inhibition of OC differentiation and bone resorption respectively [43–45]. However, there are few studies concerning this pathway regulating exercise for inhibiting OC differentiation and bone resorption in T2DM patients. In this study, after 8-weeks of intervention by exercise, the expression of Sirt1 mRNA and protein was up-regulated in group TDG, mRNA expressions for NF-κB and NFATc1 and protein expressions for c-fos and NFATc1 down-regulated, but for the mRNA expressions for IL-6 and c-fos and protein expressions for NF-κB and IL-6, the effects were not significant, while the expression of Sirt1 mRNA and protein in group TDP was up-regulated significantly and for NF-κB, IL-6, c-fos and NFATc1 down-regulated significantly. Although high-intensity intermittent exercise up-regulated the expressions for Sirt1 and down-regulated the expressions for c-fos and NFATc1 significantly, the expressions for NF-κB were not significantly changed. Relevant studies have shown that NF-κB and IL-6 play their role at a translation level, suggesting that Sirt1 also mediates other pathways to regulate bone resorption in T2DM mice through target genes c-fos and NFATc1. However, downhill running can inhibit NF-κB pathway through up-regulating Sirt1 expressions in the bone of T2DM mice. This has explained the changes in OC differentiation, cancellous bone microstructure and BMD under the two exercise modes in this study. Compared with group TDG, the mRNA and protein expressions for Sirt1 of group TDP had up-regulated significantly, mRNA expressions for Il-6 and NFATc1 had down-regulated significantly, and mRNA expressions for NF-κB and c-fos had down-regulated but not significantly. However, the protein expressions for NF-κB, IL-6, c-fos and NFATc1 were down-regulated significantly. Overall compared with high-intensity intermittent exercise, downhill running can significantly up-regulate Sirt1 expressions and inhibit NF-κB pathway in T2DM mice, for the direct effect of downhill running on the bone of T2DM mice up-regulates expressions for Sirt1, thus inhibiting the expressions for IκBα and preventing it from binding to NF-κB subunit P65 through nucleation, and thus, NF-κBp65 is deacetylated and the expressions for IL-6 and target genes c-fos and NFATc1 are down-regulated . Downhill running may also promote the expressions of Wnt1 inducible signaling pathway protein 1 (WISP1) in the bone of T2DM mice significantly, thus up-regulating Sirt1 and its NF-κB pathway . Furthermore, this is related to up-regulation of AMPK expressions in the bone for T2DM by downhill running . AMPK can transcriptionally activate Nicotinamide Phosphoribosyl transferase (Nampt), thus increasing the NAD+/NADH ratio, and activating Sirt1 and downstream NF-κB pathway . Downhill running can activate Sirt1/NF-κB pathway in the bone of T2DM mice significantly, which has also explained the OC differentiation and bone resorption capacity decreased and bone microstructure and BMD significantly improved after inhibition of bone resorption in this study in terms of molecular mechanism.