In this study it was seen that the ligature-induced periodontitis reproduced the main findings of the periodontitis in humans [26]. DM potentiated alveolar bone loss as seen macroscopically and confirmed by micro-CT analysis. DM reduced the trabecular thickness and bone volume; increased osteoclast counts and reduced the number of osteoblasts in alveolar bone. It was seen higher inflammatory infiltrate in the periodontium of diabetic rats. Taking together, these findings confirm the noxious effect of hyperglycemia on bone tissue [27]. The treatment with ATV, in turn, was not able to reverse neither bone loss nor inflammation caused by diabetes. Interestingly, ATV increased by 20% blood glucose levels in diabetic animals, suggesting that its use can be associated to high risk for diabetes.
ATV has well-known pleiotropic effects, such as anti-inflammatory and bone anabolic properties. Its local and systemic use has already been demonstrated to be beneficial for periodontitis [8, 9, 28]. In diabetic patients, the local delivery of ATV was found to be effective to reduce bone resorption [29–31]. However, the systemic use of ATV in diabetic individuals was associated to higher risk for DM [11]. This study investigated the effect of ATV, administered systemically, in diabetic rats submitted to periodontitis and from the best of our knowledge this is the first time that this investigation is performed.
The results of this studies revealed that ATV increased glycemia in diabetic animals. Numerous mechanisms have been proposed for statin-associated diabetes. It was reported that ATV reduce translocation into the cell membrane of glucose transporter 4 (GLUT4), a facilitated transporter for peripheral insulin-mediated glucose influx thereby inducing insulin resistance [32]. ATV also can contribute to an increased risk of diabetes by inhibiting the phosphorylation events or G-protein activation needed to initiate insulin signal transduction [33]. In addition, ATV has been shown to inhibit adipocyte differentiation. Undifferentiated adipocytes do not secrete insulin-sensitizing hormone. The excess of these cells may increase insulin resistance [32]. Moreover, it is known that increased intracellular calcium concentration opens the calcium channels to trigger insulin secretion, the altered function or levels of calcium channels may therefore influence glucose homeostasis. Studies have shown that ATV, may impair calcium channels [32]. Other mechanisms have investigated the role of MicroRNAs on insulin levels and sensitivity, and ATV has been shown to induce the hepatic expression of microRNA-33a, which could lead to decreased insulin secretion [34]. Taking together, these mechanism can explain why ATV can contribute to hyperglycemia, as seen in this study, increasing therefore the risk for diabetes.
This study also showed that ATV could not reverse or mitigate bone loss induced by DM and EP. High glucose levels, seen in ATV group, has toxic effects on several cell and tissues, including bone. Poor glycemic control increases the relative risk of hip fracture [35], with reduction of bone mineral density [36, 37] accompanied to low bone turnover [38]. In alveolar bone, one study corroborated our findings, showing that simvastatin was not able to protect bone in animals with DM and submitted to EP [39]. The potential mechanism by which hyperglycemia induces bone loss is based on changes in bone formation and resorption [3].
Our results showed that in diabetic animals with periodontitis treated with ATV there was a significant reduction on osteoblasts number. Hyperglycemia reduces the expression of transcription factors that regulate osteoblast differentiation, such as Runx2 [40]. High glucose levels decrease osteoblast migratory potential and chemotaxis leading to an inefficient movement of these cells to the erosion site resulting in uneven mineralization and bone fragility [41]. Hyperglycemia can also reduce alkaline phosphatase activity and mineralized matrix formation and promote osteoblast apoptosis [42].
This study revealed that ATV treatment in diabetic rats submitted to periodontitis enhanced the number of osteoclasts. High glucose levels promote direct osteoclast differentiation and activation via upregulation of macrophage-colony stimulating factor, receptor activator of nuclear factor kappa-B ligand (RANKL) and vascular endothelial growth factor [43, 44]. In addition, cathepsin K expression, a marker of osteoclast activity, is elevated in hyperglycemic conditions [43].
Moreover, the treatment with ATV did not modulate inflammation caused by ligature-induced periodontitis in diabetic rats. A higher degree of inflammation and a more persistent inflammatory response following periodontitis are reported in rats with DM [45]. High glucose levels stimulate greater production of chemokines that induce neutrophil recruitment as well production of reactive oxygen species (ROS). Macrophages under hyperglycemic condition increase the production of interleukin-1 and tumor necrosis factor (TNF). Dendritic cells are also impacted by diabetes, increasing the generation of Th1 or Th17 lymphocytes or reducing the formation of regulatory T cells. It is well known that the exacerbation of inflammatory response has a negative effect on bone metabolism, contributing to bone loss.
Despite the interesting findings, this study has its own limitations. Ligature-induced periodontitis can mimic the most important findings of human periodontitis but does not reproduce all pathogenic mechanism of the diseases as in humans [46]. The study also lacks deeper evaluation of molecular and biological mechanisms of bone resorption in diabetic animals treated with ATV. Finally, the effect of ATV should be also evaluated in other models of DM, to confirm our data.
In summary these findings suggest that ATV contribute to the unbalance the glycemic level induced by diabetes. The deleterious effect of ATV in diabetic animals submitted to periodontitis is marked by increase on bone resorption and inflammation with reduction of bone formation. However, more studies are needed to confirm these findings.