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).