ABSTRACT
Fluoride (F) has been employed worldwide to control dental caries. More recently, it has been suggested that the consumption of low doses of F in the drinking water may reduce blood glucose levels, introducing a new perspective for the use of F for the management of blood glucose. However, the exact mechanism by which F affects blood glucose levels remains largely unexplored. Given that the small gut plays a pivotal role in glucose homeostasis, the aim of this present study was to investigate the proteomic changes induced by low doses of F in the ileum of female non-obese-diabetic (NOD) mice. Forty-two female NOD mice were divided into two groups based on the F concentration in their for 14 weeks: 0 (control) or 10 mg/L. At the end of the experimental period, the ileum was collected for proteomic and Western Blotting analyses. Proteomic analysis indicated an increase in isoforms of actin, gastrotropin (confirmed by Western Blotting), several H2B histones and enzymes involved in antioxidant processes, as well as a decrease in enzymes essential for energy metabolism. In summary, our data indicates 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.
Keywords: ileum, diabetes, fluoride, glucose homeostasis, epigenetic
INTRODUCTION
Fluoride (F) has been widely employed worldwide to control dental caries, primarily through drinking water and toothpaste (Bratthall et al. 1996; Buzalaf and Whitford 2011; Iheozor-Ejiofor et al. 2015). However, excessive ingestion may cause various alterations in the organism, including oxidative stress (Pereira et al. 2018; Pereira et al. 2016; Pereira et al. 2013), perturbations in the lipid metabolism (Dionizio et al. 2018a), changes in intracellular homeostasis and cell cycle, leading to apoptosis (Barbier et al. 2010),and morphological and proteomic alterations in the duodenum (Melo et al. 2017), jejunum (Dionizio et al. 2018b) and ileum (Dionizio et al. 2021).
The gastrointestinal tract (GIT) is the main route of exposure to F. Approximately 25% of the ingested F is absorbed in the stomach as hydrofluoric acid, (Whitford and Pashley 1984), and around 75% is absorbed in the small intestine in its ionic form (F-), independent of pH (Nopakun and Messer 1990; Nopakun et al. 1989). Consequently, gastrointestinal symptoms like nausea, vomiting, abdominal pain, and diarrhea are the first signs of F toxicity (Whitford 2011).
More recently, it has been suggested that consumption of low doses of F in drinking water, similar to those added to artificially fluoridated water, may increase insulin sensitivity or reduce blood glucose levels (Lima Leite et al. 2014; Lobo et al. 2015; Malvezzi et al. 2019; Trevizol et al. 2020; Trevizol et al. 2023), which is very relevant from the perspective of public health, since water fluoridation is recognized among the top ten public health achievements of the last century (Centers for Disease and Prevention 1999). However, the exact mechanisms by which F influences glucose homeostasis remain unclear.
The small gut is central to glucose homeostasis, as the jejunum senses nutrients and regulates hepatic glucose production, and the entire small gut secretes both glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic peptide (GIP), enhancing insulin secretion (Kamvissi-Lorenz et al. 2017). GLP-1 enhances insulin secretion, accounting for around 70% of the insulin secretion in the presence of high levels of glucose (Miyawaki et al. 2002).
In this study, NOD mice, a validated model for evaluating agents and protocols that prevent or reverse type 1 diabetes (T1D) (Mullen 2017), were used to assess the proteomic changes induced by low doses of F in the ileum. This aims to provide insights into the mechanisms by which F affects glucose homeostasis.
MATERIAL AND METHODS
Animals, fluoride treatment and ileum collection
The experimental protocol was approved by the Animals Ethics Committee of Bauru School of Dentistry (CEUA-Proc. 013/2017). Forty-two female NOD mice were randomly divided into two groups based on the fluoride concentration (as sodium fluoride) in the drinking water administeredad libitum for 14 weeks: 0 (control) or 10 mgF/L (as sodium fluoride). The drinking was water administered ad libitum to the animals for 14 weeks (Trevizol et al. 2020). This F concentration aims to mimic the consumption of water from the public supply by humans, since the metabolism of F by rodents is 5-10 times faster compared to humans (Dunipace et al. 1995). Female NOD mice were selected because they develop insulitis and diabetes to a higher degree than their male counterparts. The treatment period of 14 weeks was chosen because insulitis starts at 2-4 and 5-7 weeks in females and males, respectively, and the development of hyperglycemia is observed between 12 and 30 weeks (Mullen 2017). During the experimental period, the animals received low F (~ 2 mg/Kg) and normocaloric diet ad libitum . The animals were euthanized at the conclusion of the experimental period (exposure to CO2 followed by decapitation). The ileum was collected following the procedure described in a previous publication (Dionizio et al. 2021) for proteomic and western blotting analyses (n=12 animals per group). Plasma fluoride, plasma glucose and plasma insulin were assayed and the results are reported in a previous publication (Trevizol et al. 2020).
Proteomics and bioinformatics analyses
The proteomic analysis was performed exactly as previously described (Dionizio et al., 2018). Briefly, the ileum was homogenized, proteins were extracted (lysis buffer A - 7 M urea, 2 M thiourea, 40 mM DTT, all in AMBIC solution) and the samples were quantified using the Bradford method (Bradford 1976). Samples were then reduced (5 mM DTT; BioRad, cat# 161-0611) and alkylated (10 mM IAA; GE, cat# RPN 6302V). Digestion was performed by the addition of 2% (w/w) trypsin overnight (Promega, cat #V5280). To stop the digestion, 5% TFA was added. Supernatant was purified using C 18 Spin columns (Pierce, cat #89870) and then resuspended in a solution containing 3% ACN and 0.1% formic acid.
The Xevo G2 (Waters) mass spectrometer coupled to the nanoACQUITY (Waters) system for the peptide analysis and Protein Lynx Global Server (PLGS) version 3.03 software was used to process and search for continuous LC-MSE data, as previously described (Dionizio et al. 2020; Trevizol et al. 2020). Peptides were identified using the software’s ion counting algorithm and a search on the updated Mus musculus database (UniProtKB). The PLGS detected difference in expression between the groups, used t-test, with p<0,05. The software CYTOSCAPE® (Java®) was employed to construct networks of molecular interaction between the identified proteins, supported by ClusterMarker® and ClueGO applications.
The mass spectrometry proteomics data have been deposited at the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Perez-Riverol et al. 2019) with the dataset identifier PXD031865.
Western blotting analysis
The Western blotting was conducted as previously described (Dionizio et al. 2021; Yan et al. 2012). Ileum protein extracts were obtained by lysing homogenized tissue in lysis buffer A, supplemented with protease inhibitors (Roche Diagnostics, Mannheim, Germany). Protein samples (40 μg) were resolved in 10% Tris-HCl polyacrylamide gels and subsequently transferred to a Polyvinylidene difluoride (PVDF) membrane. Membranes were probed with commercially available Gastrotropin (1:500 dilution) (Abcam, Cambridge, MA, USA) and β-Actinin (1:200 dilution), followed by HRP-conjugated anti-rabbit antibody (1:10000) for Gastrotropin andβ-Actinin (1:1000 dilution) (Cell Signaling, Danvers, MA, USA) and ECL Plus detection reagents (GE Biosciences, Piscataway, NJ, USA). The relative Gastrotropin andβ-Actinin band densities were determined by densitometrical analysis using the Image Studio Lite software from LI-COR Corporate Offices-US (Lincoln, Nebraska USA). In each case, density values of bands were corrected by subtraction of the background values. The results were expressed as the ratio of Gastrotropin to β-Actinin . For Western blotting data, the software GraphPad Prism (version 7.0 for Windows, La Jolla, CA, USA) was used. Data were analyzed by Kolmogorov–Smirnov test for data homogeneity and Mann-Whitney Test with p = 0.0037.
RESULTS
Proteomic analysis
In the proteomic analysis of the ileum, a total of 129 proteins with different expression were identified. The number of up- and down-regulated proteins identified in the 10 mgF/L group compared to the control group (0 mgF/L) was 15 and 114, respectively (Table 1). Among the up-regulated proteins, Actins , Gastrotropin and several H2B histones were found. Conversely, among the down-regulated proteins, were H2A , H3 and H4histones, in addition to enzymes involved in energy metabolism, such asAlpha-enolase, Malate dehydrogenase, Pyruvate Kinase PKM, Aldehyde dehydrogenase , as well as several antioxidant enzymes, including isoforms of Glutathione-S-Transferase and Peroxirredoxin(Table 1).
Table 1. Differently expressed proteins from the ileum of 20-week-old female NOD mice, treated for 14 weeks with water containing 10 mg/L fluoride (as NaF) or not (control).