Figure 3- Representative expression of proteinsGastrotropin and constitutive β-Actinin in samples of individual animals (n = 6) from each group. Densitometric analysis was performed for 6 animals per group. Densitometry was analyzed using the software Image Studio Lite. Bars indicate SD. n = 6
DISCUSSION
The results of plasma F concentration, glucose and plasma insulin are reported in a previous publication (Trevizol et al. 2020). Low-dose F exposure significantly elevated plasma F levels, lowered blood glucose by 20%, and did not impact insulinemia. These data are extremely relevant from a public health point of view, since water fluoridation, a worldwide recognized measure for the prevention of dental caries (Buzalaf 2018), could also aid in controlling hyperglycemia. However, the potential mechanisms underlying the effects of F on glycemic are yet to be elucidated. A feasible explanation could be the well-documented effects of F in diminishing energy metabolism (Araujo et al. 2019; Dionizio et al. 2021; Dionizio et al. 2018b; Khan et al. 2018; Malvezzi et al. 2019; Trevizol et al. 2020; Trevizol et al. 2023). Exposure to F has been linked to changes in enzymes involved in energy metabolism and the antioxidant system, as observed in the liver of NOD mice (Malvezzi et al. 2019; Trevizol et al. 2020) and various intestinal segments of rats (Dionizio et al. 2021; Dionizio et al. 2018b; Melo et al. 2017).
In the current study, we generally noted a reduction in several enzymes involved in energy metabolism, such as Alpha-enolase, Malate dehydrogenase, Pyruvate Kinase PKM, Aldehyde dehydrogenase, observed both in the duodenum (unpublished data) and in the ileum of F-treated mice. A decrease in glycolytic enzymes following exposure to low doses of F has been similarly documented in other studies with rats (Araujo et al. 2019) and NOD mice (Malvezzi et al. 2019; Trevizol et al. 2020). Additionally, there was also a reduction in proteins related to lipid metabolism, such as Fatty acid-binding proteins, which play a role in intracellular lipid transport (Consortium 2023). Among these proteins is Gastrotropin , also known asFatty acid-binding protein 6 , an important transport protein involved in the enterohepatic circulation of bile salts. Predominantly expressed in the ileum, it participates in the absorption of vitamin B12 and binds to bile acids, crucial for the effective digestion and absorption of dietary fats (Davis and Attie 2008; Grober et al. 1999; Iiizumi et al. 2007; Landrier et al. 2006; Marvin-Guy et al. 2005; Thompson et al. 2017).
In this study, Gastrotropin levels increased in the ileum of NOD mice after F treatment, contrasting with findings in the ileum of rats treated with a similar concentration of F in the drinking water, where this protein was reduced (Dionizio et al. 2021). However, in the study by Dionizio et al. (2021), the mice were not diabetic, which might explain the different expression patterns observed. Modifications in receptors and proteins related to bile acids (BAs) may be crucial in regulating BAs, lipids, and glucose homeostasis, as well as managing inflammatory responses, barrier function, and preventing bacterial translocation in the intestinal tract (Ding et al. 2015; Gillard et al. 2022). Furthermore, other FABP family proteins such as FABP1 and FAPB2, are often associated with type 2 diabetes pathology, where alterations can lead to changes in lipid metabolism and disruptions in proteins related to fatty acids, lipid transport and intestinal absorption (Sianipar et al. 2022; Tsai et al. 2020).
It is recognized that genes involved in antioxidant defense are less expressed in spontaneously diabetic lymphopenic rats (BB rats), a factor implicated implicated in β-cell damage and the development of diabetes in these animals (Bogdani et al. 2013). In our study, we noted a reduction in peroxirredoxin and isoforms ofGlutathione S-transferase in the ileum. In the liver of NOD mice treated with low doses of F, these enzymes were also diminished (Malvezzi et al. 2019; Trevizol et al. 2020). The observed increase in these antioxidant enzymes in the current study indicated a potential counterregulatory mechanism of detoxification in the ileum of NOD mice.
Environmental factors can cause epigenetic changes that regulate gene expression and influence immune cell function. Epigenetics provides multiple molecular mechanisms to elucidate the environmental effects implicated in the development of autoimmune diabetes. The three primary epigenetic modifications aree DNA methylation, histone modification, and microRNA alteration (Xie et al. 2014). In the ileum, we noted significant changes in histone expression, histone-modifying proteins, and numerous other proteins involved in transcription and translation, such as ribonucleoproteins. This epigenetic influence of F on histone modification was similarly observed in the islets of female NOD mice (Trevizol et al. 2023). Additionally, dysregulation in histone acetylation, governed by histone deacetylases (HDACs) and histone acetyltransferases (HATs), is implicated in diabetes pathogenesis (Gray and De Meyts 2005). Histone acetylation has been demonstrated to preserve insulin secretion under cytokine insult and prevent NF-κB-induced apoptosis in β-cell lines and islets from newborn mice (Larsen et al. 2007). In our previous study (Trevizol et al. 2023), we observed an increase in inflammatory infiltrate in the group exposed to 10 mgF/L in their drinking water. Additionally, proteomic analysis of the islets revealed substantial increases in histones H3.1 and H3.2 (about 100-fold), as well as in histone H4 (more than 60-fold), alongside a 6-fold rise in histone acetyltransferase KAT6B and histone acetyltransferase KAT8, exclusively found with F treatment. The proportion of histone H3 acetylation was also assessed, showing more than a 2-fold increase in the F-exposed group compared to the control, though the difference was not significant. The acetylation of H3K14 was analyzed as it is a target of KAT6B and KAT8.
During histone acetylation, an acetyl functional group is transferred from acetyl Co-A to the histone (Gruber et al. 2019). In the current study, Krebs’ cycle enzymes, like malate dehydrogenase and citrate synthase, were diminished in the ileum. These observations suggest a decrease in Krebs’ cycle flow, potentially reducing acetyl Co-A levels. Furthermore, the reduction of enzymes in the glycolytic pathway, also noted in our findings, contributes to the lowered acetyl Co-A levels (Wellen et al. 2009). Therefore, the substantial alterations in the expression of histones, histone-modifying proteins and various proteins involved in transcription and translation may represent effort to sustain translation amidst reduced acetyl Co-A availability.
We consider these findings particularly intriguing, as recent research has shown F-induced hypermethylation in crucial genes linked to various toxicity effects (Ma et al. 2020; Meng et al. 2021). Indeed, the concept of F-induced epigenetic changes has only recently emerged in scientific literature and warrants further investigation. In summary, our data indicate an adaptive response of the organism to preserve protein synthesis in the ileum, despite significant alterations in energy metabolism typically induced by F, therefore highlighting the safety of controlled fluoridation in water supplies.