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Biological Trace Element Research
ISSN 0163-4984
Biol Trace Elem Res
DOI 10.1007/s12011-018-1542-4
Effect of Duration of Exposure to Fluoride
and Type of Diet on Lipid Parameters and
De Novo Lipogenesis
Aline Dionizio, Heloisa Aparecida
Barbosa Silva Pereira, Tamara Teodoro
Araujo, Isabela Tomazini Sabino-Arias,
Mileni Silva Fernandes, et al.
1 23
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Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid
Parameters and De Novo Lipogenesis
Aline Dionizio
1
&Heloisa Aparecida Barbosa Silva Pereira
1
&Tamara Teodoro Araujo
1
&Isabela Tomazini Sabino-Arias
1
&
Mileni Silva Fernandes
1
&Karina Aparecida Oliveira
1
&Fabielle Sales Raymundo
1
&Tânia Mary Cestari
1
&
Fernando Neves Nogueira
2
&Rui Albuquerque Carvalho
3
&Marília Afonso Rabelo Buzalaf
1
Received: 14 June 2018 / Accepted: 4 October 2018
#Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
The effect of duration of chronic treatment with fluoride (F, 50 mg/L as NaF) on the lipid profile, lipid droplets and triglycerides
(TG) in liver was evaluated in mice with nonalcoholic fatty liver disease (NAFLD) previously induced by hyperlipidic diet and in
animals fed normocaloric diet. In addition, the effect of F administered for a short period (20 days) was evaluated on de novo
lipogenesis, by nuclear magnetic resonance. GRP78, Apo-E, and sterol regulatory element-binding protein (SREBP) were
quantified by Western blotting. Our data indicate that F interferes in lipid metabolism and lipid droplets, having a different action
depending on the exposure time and type of diet administered. F improved lipid parameters and reduced steatosis only when
administered for a short period of time (up to 20 days) to animals fed normocaloric diet. However, when NAFLD was already
installed, lipid parameters were only slightly improved at 20 days of treatment, but no effect was observed on the degree of
steatosis. In addition, lipid profile was in general impaired when the animals were treated with F for 30 days, regardless of the
diet. Moreover, F did not alter de novo lipogenesis in animals with installed NAFLD. Furthermore, hyperlipidic diet increased F
accumulation in the body. GRP78 increased, while Apo-E and SREBP decreased in the F-treated groups. Our results provide new
insights on how F affects lipid metabolism depending on the available energy source.
Keywords Fluoride .High-fat diet .Nonalcoholic fatty liver disease .Lipogenesis .NMR
Introduction
Fluoride (F) is an element naturally found in water. In some
sites the concentration is above the recommended limits,
which can cause deleterious effects among which the most
known is fluorosis that can be dental or skeletal [1,2].
Experimental studies where F is chronically administered to
animals in high doses have reported alterations involving dif-
ferent tissues such as liver, kidney, muscle, and heart and
affecting distinct proteins and enzymes involved in various
molecular processes such as oxidative stress [3–11].
Liver is an important target of xenobiotics like F, since it is the
main organ responsible for detoxifying the body and has a high
metabolic activity, besides neutralizing and eliminating toxic sub-
stances [12,13]. In addition, liver is responsible for lipid metab-
olism. Problems in the homeostasis of lipids, arising from an
instability in external lipid absorption or biosynthesis of internal
lipids, result in the generation of lipid inclusions in the liver [14].
Recent studies of our research group showed that male
Wistar rats fed hypercaloric diet and treated with water contain-
ing 50 mg/L F presented changes in the proteomic profile in
liver [5] and had reduction in plasma triglycerides and lipid
droplets in the liver [15]. These lipid droplets are a histological
spectrum of nonalcoholic fatty liver disease (NAFLD), which
includes several forms of macrovesicular steatosis in the form of
small and large drops, with or without portal triad inflammation,
followed by steatohepatitis, characterized by steatosis,
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12011-018-1542-4) contains supplementary
material, which is available to authorized users.
*Marília Afonso Rabelo Buzalaf
mbuzalaf@fob.usp.br
1
Present address: Department of Biological Sciences, Bauru School of
Dentistry, University of São Paulo, Alameda Octávio Pinheiro
Brisolla, 9-75, Bauru, SP 17012-901, Brazil
2
Department of Biomaterials and Oral Biology, School of Dentistry,
University of São Paulo, Avenida Professor Lineu Prestes 2227, São
Paulo, SP 05508-000, Brazil
3
Department of Life Sciences, Faculty of Science and Technology,
University of Coimbra, Calçada Martim de Freitas, Edifício São
Bento, 3000-456 Coimbra, Portugal
Biological Trace Element Research
https://doi.org/10.1007/s12011-018-1542-4
Author's personal copy
inflammation and cellular injury, or nonalcoholic steatohepatitis
[16–19]. This alteration is inwardly related with disturbances in
metabolism, particularly those involving oxidative stress and,
consequently, lipid peroxidation [20,21], which, interestingly,
are also affected by F [15,22–24]. A recent study revealed an
increase in 78 kDa glucose-regulated protein (GRP78—marker
of oxidative stress) and a decrease in apolipoprotein-E (Apo-
E—involved in transport and delivery of lipids to the body) in
the liver of rats treated with 50 mg/L F and hypercaloric diet
[15]. This was possibly related to the reduction in lipid droplets
in the liver of animals treated with F and hypercaloric diet [5,
15], since increase in GRP78 inhibits endoplasmic reticulum
stress, thus reducing hepatic steatosis [25]. In addition, decrease
in Apo-E induced by F reduces the availability of lipoproteins to
deliver fat to liver, which also reduces steatosis [26]. The study
by Pereira et al. [5] was the first one to report beneficial effects
(reduction in lipid droplets in the liver due to alterations in the
expression of proteins involved in steatosis) of high dose of F in
the liver of animals fed hypercaloric diet. In that study, the
animals were treated with F for 60 days. It would be interesting
to see if these beneficial effects also occur when the animals are
treated with F for shorter periods, since the effects of F have
been reported to be time-dependent. In addition, it is important
to evaluate if these effects also occur when the animals are fed a
normocaloric diet, since some studies report increase in total
cholesterol, triglycerides (TG), very low-density lipoprotein
(VLDL), or low-density lipoprotein (LDL) when animals are
exposed to F and fed a normocaloric diet [27–29].
Thus, our primary aim was to evaluate the effect of dura-
tion of chronic treatment with F on the lipid profile, lipid
droplets, and TG in liver, in mice with NAFLD previously
induced by hyperlipidic diet and in animals fed normocaloric
diet. In order to elucidate the mechanisms involved in the
alterations induced by F in animals with installed NAFLD or
not, de novo lipogenesis (LDN) was evaluated using nuclear
magnetic resonance (NMR) and GRP78; Apo-E and sterol
regulatory element-binding protein (SREBP) were evaluated
by Western blotting.
Materials and Methods
All experimental protocols were approved by the Ethics
Committee for Animal Experiments of Bauru Dental School,
University of São Paulo (protocol: 001/2014; 008/2015).
The first study, involving different durations of exposure to
F, was conducted using Swiss male mice (26–44 g) that were
obtained from São Paulo State University (UNESP, Bauru).
The animals were housed in the Central Vivarium of Bauru
School of Dentistry, University of São Paulo. After an adapta-
tion period of 7 days, the animals were randomly distributed
into two groups (n= 60/group). One of them received for
30 days an in-house prepared high-fat diet (hyperlipidic semi-
purified unbalanced diet with 5350 kcal/kg) [30,31] for induc-
tion of hepatic steatosis (hyperlipidic). The other group re-
ceived, for the same period, normocaloric diet (Presence®,
Purina, 3028 kcal/kg [32] (normocaloric)). After this period,
for each type of diet, the animals were divided into two groups
(n= 30/group), based on the type of drinking water to which the
animals had free access: deionized water or water containing
50 mg/L F (as sodium fluoride). In both cases, the diet initially
administered was maintained. Furthermore, treatment with F
was done during three distinct periods: 10, 20, or 30 days, for
each condition (Fig. 1(A)). At the end of the study, the animals
were weighed and then received an intramuscular injection of
anesthetic and muscle relaxant (ketamine chlorhydrate and
xylazine chlorhydrate, respectively). Blood was collected, and
plasma was obtained and frozen at −20 °C until the analysis of
TG, HDL, total cholesterol, LDL, VLDL, and F. Liver was also
collected. The right lobe was used for histopathological analy-
sis, while the remaining was employed for TG, F, and Western
blotting analysis.
The second study, involving LDN and Western blotting
analyses, weanling Swiss male mice (body weight ranging
between 18 and 27 g) were obtained from Multidisciplinary
Center for Biological Research in the Area of Science in
Laboratory Animals (CEMIB)-UNICAMP. They were
housed in the Central Vivarium of Bauru School of
Dentistry, University of São Paulo, and randomly distributed
into two groups (n= 12/group) that received, for 30 days,
high-fat diet (hyperlipidic semi-purified unbalanced diet with
5350 kcal/kg) [30,32] for induction of NAFLD or
normocaloric diet (Presence®, 3.028 kcal/kg) [32]. All the
animals were housed in standard cages (three per cage) with
chow and water ad libitum. During this period, all animals had
free access to deionized water. After this period, for each type
of diet, the animals were further divided into two groups (n=
6/group), based on the type of drinking water to which they
had free access for 20 days: deionized water or water contain-
ing 50 mg/L F (as sodium fluoride) (Fig. 1(B)). In both cases,
the diet initially administered was maintained. Three days
before euthanasia, the animals received by gastro gavage a
volume of
2
H
2
O in order to enrich body water in deuterium
around 3% (assuming 75% of body water). For the remainder
of the study, 3%
2
H
2
O was added to the drinking water to keep
the body water enrichment throughout the study. At the end of
20 days, the animals were weighed and then received an in-
tramuscular injection of anesthetic and muscle relaxant (keta-
mine chlorhydrate and xylazine chlorhydrate, respectively).
Liver was also analyzed by NMR.
In both studies, the temperature and humidity in the
climate-controlled room, which had a 12-h light/dark cycle,
were 23 ± 1 °C and 40–80%, respectively. These diets had a
low F content (< 2 mg/kg). The administration of drinking
water containing 50 mg/L F to mice leads to plasma F levels
correspondent to those found in humans consuming water
Dionizio et al.
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containing ~ 10 mg/L F [31], which occurs in areas of endem-
ic fluorosis [33].
F Analysis
Liver and plasma were determined after overnight
hexamethyldisiloxane (HMDS)-facilitated diffusion [1,34]
using the ion-specific electrode (Orion Research, Model
9409) and a miniature calomel electrode (Accumet #13-620-
79) both coupled to a potentiometer (Orion Research, Model
EA 940) (n= 10) as previously described [15].
Histological Analysis
Histological Processing and Analysis
The right liver lobules (n= 10) were fixed in 10% formalin in
phosphate buffer for a week and processed for histology (par-
affin embedded). Semi-serial sections were performed at 5 μm
using a microtome (Microm, model HM 340 E, Germany).
Sections were included in slides and stained with hematoxylin
and eosin (HE) using routine histological protocols [35]. An
Olympus Upright BX43 binocular microscope was used
(Olympus Corporation Microscopes, Tokyo, Japan) to exam-
ine and evaluate the histological sections, together with a pho-
tographic camera Olympus SC30 (Olympus Corporation
Microscopes, Tokyo, Japan) and the Olympus CellSens 1.14
software (Olympus Corporation Microscopes, Tokyo, Japan).
Morphometric Analysis
It was performed blindly, using a Zeiss microscope in 40X
magnification with reticular integration (10mm × 10mm).
Tenfieldswereselectedbysystematicsampling[15,35]in
each lobule (n= 3). Nonalcoholic steatosis was classified ac-
cording to the percentage of lipid droplets, as follows: normal
(≤12.9%), mild (13.0–37.9%), moderate (38.0–61.3%), or
Fig. 1 Experimental design of the first study (A) and second study (B)
Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis
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severe (≥61.4%) (adapted from Brunt, Tiniakos [17], with
modifications Pereira et al. [15]).
Morphological Analysis
The morphological analysis comprised evaluation of steatosis
per graded score. For this, three blind examiners performed
the evaluations and was used a Zeiss microscope in 10X mag-
nification. The qualitative classification of liver lipid content
was based in the amount (0 to 5) and size (macro- or
microdroplets), as follows: none (0), few microdroplets (1),
moderate amount of microdroplets and few macrodroplets (2),
many microdroplets and moderate amount of macrodroplets
(3), microdroplets and agglomerate macrodroplets (4), and
many macrodroplets throughout the tissue (5) [15].
This analysis needs calibration evaluated by kappa (k)co-
efficient. For this, 33% of the samples were evaluated and this
was repeated after 15 days for the purpose of intra-examiner
calibration. The kcoefficients obtained were > 0.8.
Lipid Profile Analysis
TG quantitation in plasma and liver was determined using a
commercial kit (Doles, Belo Horizonte, MG, Brazil). For this,
200 mg of liver tissue from each animal was homogenized in
0.5 mL 0.1% TritonX-100, sonicated for 45 s and centrifuged
at 4000 rpm for 10 min, obtaining the supernatant for analysis.
HDL and total cholesterol analysis in plasma were quanti-
fied by commercial kit (Doles, Belo Horizonte, MG, Brazil).
Determinations of LDL and VLDL were derived using the
Friedewald formula [36].
Extraction of Liver Lipids
We used the method of Folch et al. [37]withsomemodifica-
tions proposed by Christie [38]. The remaining liver was ini-
tially weighed and added to 5 mL of methanol per gram of
liver. The homogenization was performed for about 5 min
(LHNH Uniscience®) under vigorous stirring. After this step,
10 mL of chloroform per gram tissue was added and kept
under stirring for 3 min. The mixture was centrifuged, the
supernatant separated, and the solid residue subjected to new
extraction by addition of 6 mL of 2:1 (v/v) chloroform-
methanol solution, followed by vigorous homogenization for
5 min. The lipid present in chloroform was obtained by drying
(without heating) and preserved for further analysis by NMR.
Analysis of De Novo Lipogenesis by NMR
The de novo lipogenesis by NMR analysis was conducted in
the Department of Life Sciences at the Faculty of Science and
Technology, University of Coimbra, Portugal. The lipid sam-
ples were dissolved in 525 μL chloroform not deuterated
(CHCl
3
)togetherwith25μL of standard deuterated and not
deuterated pyrazine (internal standard) in CHCl
3
and 50 μL
hexafluorobenzene (C
6
F
6
) to capture fluorine lock. For each
sample, proton and deuterium spectra were obtained. These
spectra allowed the quantification of the total amount of lipids
isolated from each liver and also the percentage of deuteration
of the same lipid for the purpose of quantifying the de novo
lipogenesis process. The spectra were acquired on a Bruker
500 MHz NMR spectrometer using a SE-
2
H-X probe (
1
H)
5 mm (type: PH SEX 500S2
2
H-H-F-05 Z). For protons, the
acquisition of a single transient was enough but for deuterium,
accumulation of multiple transients had to be performed to
obtain the ratio signal/noise suitable for quantitative analysis.
Typical parameters of acquisition included a pulse reading
radio frequency of 45° and a time of interpulse repetition of 5 s
to allow relaxation all deuterium both of the lipid sample and
of the internal reference (pyrazine). This internal standard,
whose amount is known, allowed to quantify absolutely both
the total amount of lipid (proton spectrum) as the percentage
of de novo lipogenesis, by assessing the percentage of incor-
poration of deuterium from body water into the units of acetyl-
CoA used in the de novo synthesis. Processing of the spectra
was done using the NUTSproTM software and deconvolution
of the resonances necessary for the quantitative analysis was
performed using a subroutine of the same software, which
allows a successive iteration between calculated and experi-
mental spectrum, reducing at each step the difference and
allowing quantitation of the areas of each resonance and eval-
uation of multiple spectral parameters, namely widths at half
height [39–41].
Western Blotting Analysis
The Western blotting was performed as previously described
[42]. Liver protein extracts were obtained by lysing homoge-
nized tissue in Ripa buffer (0.5% sodium deoxycholate, 0.1%
SDS, 1% NP-40) 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 commer-
cially available rabbit polyclonal anti-Apo-E (1:500 dilution)
(Abcam, Cambridge, MA, USA), anti-GRP-78 (1:500)
(Abcam, Cambridge, MA, USA), anti-SREBP-1c (1:500)
(Abcam, Cambridge, MA, USA), and anti-α-tubulin
(1:2000) (Abcam, Cambridge, MA, USA), followed by
HRP-conjugated anti-rabbit antibody (1:10000) for GRP-78,
Apo-E, and α-tubulin and for SREBP-1c anti-mouse antibody
(1:10000) and ECL Plus detection reagents (GE Biosciences,
Piscataway, NJ, USA). Relative Apo-E, GRP-78, α-tubulin,
and SREBP band densities were determined by
densitometrical analysis using the Image Studio Lite software
from LI-COR Corporate Offices-US (Lincoln, NE, USA). In
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all instances, density values of bands were corrected by sub-
traction of the background values. The results were expressed
as the ratio of GRP-78, Apo-E, and SREBP-1 to that of α-
tubulin.
Statistical Analysis
The software Statistica (version 10.0 for Windows, StatSoft,
Inc., Tulsa, USA, 2011) was used. After checking normality
and homogeneity, data were evaluated by two-way ANOVA
(experimental time and F concentration as criteria) and
Tukey’s test, for each type of diet separately. For Western
blotting data, the software GraphPad Prism (version 7.0 for
Windows, La Jolla, CA, USA) was used. Data were evaluated
by unpaired ttest. The level of significance, in all cases, was
set to 5%.
Results
For the animals fed normocaloric diet, body weights tended to
be more homogeneous, despite the animals treated with F for
30 days had significantly higher body weight when compared
with animals treated with F or not for 10 days and animals
treated with F for 20 days (Fig. 2(A)). On the other hand, for
the hyperlipidic diet, treatment with F for 10 and 20 days
significantly increased and reduced the body weight,
Fig. 2 Mean body weight of mice fed normocaloric (A) or hyperlipidic
(B) diets that received drinking water containing 0 mg/L or 50 mg/L
fluoride for 10, 20, or 30 days. For each type of diet, distinct lower case
letters indicate significant differences among the groups (two-way
ANOVA and Tukey’s test, p< 0.05). Bars indicate SD. n=10
Fig. 3 Mean fluoride concentrations in plasma (A, B) and liver (C, D) of
mice fed normocaloric or hyperlipidic diets that received drinking water
containing 0 mg/L or 50 mg/L fluoride for 10, 20, or 30 days. (A) Plasma
concentration, normocaloric diet. For each type of diet, distinct lower case
letters indicate significant differences among the groups (two-way
ANOVA and Tukey’s test, p< 0.05). Bars indicate SD. n=10
Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis
Author's personal copy
respectively, while at 30 days, F did not interfere in the body
weight (Fig. 2(B)).
F administration significantly increased plasma F concen-
trations for both types of diet (Fig. 3(A, B)). Animals fed
normocaloric diet had a significant decrease in plasma F con-
centration along time (Fig. 3(A)), while those fed hyperlipidic
diet treated for 20 and 30 days had significantly higher plasma
F concentrations when compared to those treated for 10 days,
but without significant difference between 20 and 30 days
(Fig. 3(B)).
In addition, F administration also significantly increased
liver F concentrations for both types of diet (Fig. 3(C, D)).
For the normocaloric diet, groups treated with F presented a
reduction in liver F concentrations along time. However, the
difference was significant only between animals treated for
10 days when compared with those treated for 30 days (Fig.
3(C)). On the other hand, liver F concentrations were signifi-
cantly higher at 30 days, when compared with 10 and 20 days,
for animals fed hyperlipidic diet (Fig. 3(D)).
Histological analysis (Fig. 4) revealed that animals fed with
normocaloric diet had a low amount of small lipid droplets
(Fig. 4(A)), while those fed with hyperlipidic diet presented a
high amount of intermediate and big lipid droplets (Fig. 4(B)).
In order to quantify these lipid inclusions, morphometric
(Fig. 5(A, B)) and morphological analyses (Fig. 5(C, D)) were
applied. The morphometric analysis revealed that for the ani-
mals treated with the normocaloric diet, administration of F
for 20 days significantly reduced the percentage of lipid drop-
lets when compared to all the other groups (Fig. 5(A)). The
percentage of lipid droplets was higher for the animals fed
with hyperlipidic diet, but a tendency for reduction was found
for the animals treated with F for 20 days (Fig. 5(B)).
Regarding the morphological analysis (Fig. 5(C, D)), the
average scores found for the normocaloric diet were much
lower than those found for the animals fed hyperlipidic diet.
In addition, they were significantly lower for the animals treat-
ed for 20 days, regardless of the F administration, in compar-
ison to the other periods (Fig. 5(C)). For hyperlipidic diet, data
were more homogeneous, without noticeable differences
among the groups, despite a tendency for reduction was also
found at 20 days, but only for the animals treated with F (Fig.
5(D)).
In order to investigate the effect of F in lipid metabolism in
animals with NAFLD and normal animals, as a function of the
type of diet and duration of treatment, lipid parameters were
evaluated in plasma and TG in the liver (Figs. 6and 7). For the
animals fed normocaloric diet, treatment with F for 10 days
significantly reduced liver TG, plasma TG, and VLDL in
comparison to the animals that received deionized water
(Fig. 6(A–C)). When the animals were treated for 20 days, a
significant decrease was observed in liver TG, total cholester-
ol, and LDL (Fig. 6(A, D, F)) in the animals treated with F
when compared to those receiving deionized water. However,
treatment with F for 30 days significantly increased total cho-
lesterol and HDL (Fig. 6(D, E)) in comparison to the animals
receiving deionized water.
For the groups that received the hyperlipidic diet, treatment
with F for 10 days did not provoke any alterations in the lipid
parameters. However, treatment with F for 20 days signifi-
cantly reduced total cholesterol and LDL (Fig. 7(D, F)), but
treatment for 30 days significantly reduced HDL (Fig. 7(E)),
in comparison to the animals receiving deionized water. When
the animals fed hyperlipidic diet were compared to those re-
ceiving normocaloric diet and deionized water, for each time
period, treatment for 10 days reduced plasma TG and in-
creased LDL, while treatment for 20 days reduced total cho-
lesterol and LDL. Treatment for 30 days, however, increased
total cholesterol, LDL, HDL, and liver TG (three-way
ANOVA, data not shown).
Fig. 4 HE staining of liver of mice fed normocaloric (A) or hyperlipidic
(B) diets that received drinking water containing 0 mg/L or 50 mg/L
fluoride for 10, 20, or 30 days. The lipid droplets are represented by
dashed circles (diameter ≤3μm), yellow arrows (diameter > 3 μm), or
black arrow (diameter around 20 μm). The bar corresponds to 100 μm
Dionizio et al.
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The calculations of de novo lipogenesis were performed
using the NMR spectra. For the analysis of
1
H, the group
calculated is the methyl group (CH
3
) and for the analysis of
2
H, the methyl group labeled with deuterium was used
(C
2
H
1
H
2
)(SupplementaryFig.A1). Animals fed hyperlipidic
diet had less de novo lipogenesis when compared with those
fed normocaloric diet. The treatment with F did not alter de
novo lipogenesis in animals with NAFLD. For the animals fed
normocaloric diet, there was a trend for reduction in de novo
lipogenesis upon treatment with F (Fig. 8).
To investigate the mechanism by which F alters the lipid
profile, key proteins related to lipid metabolism were identi-
fied and quantified by Western blotting. GRP-78 is related to
de novo lipogenesis, Apo-E is involved in lipid transport and
delivery to the liver [43], and SREBP participates in the acti-
vation of TG synthesis [25]. Regardless of the type of diet,
GRP-78 was significantly increased, while Apo-E and
SREBP were significantly decreased upon exposure to F
(Fig. 9(A, B)).
Discussion
Recently, the effect of F in lipid metabolism has been reported
in many studies [5,15,27–29,44]. However, only a few
studies evaluated how this effect is affected by the type of diet
and duration of treatment with F [5,15]. In our study, in
general, F had a beneficial effect on lipid parameters, which
could suggest a preventive action of F against lipid disorders.
To the best of our knowledge, this is the first study that eval-
uated the effect of F on lipid metabolism and on de novo
lipogenesis in animals with installed NAFLD. This evaluation
is important in order to see if a therapeutic effect of F could
also be envisioned in these cases. Regarding the dose of F
evaluated, lower doses of F, such as 10–25 mg/L, would have
been be more realistic. However, we chose to treat the animals
with 50 mg/L F because this concentration has been reported
to cause more alterations in lipid metabolism in comparison to
lower doses [5,15]. Considering that rodents metabolize F
five times faster than humans [31], this dose corresponds to
~ 10 mg/L F in the drinking water for humans. It is a high
concentration, but it can be found naturally in the water in
areas of endemic fluorosis worldwide [33].
In the present study, one of the variables was the duration
of the treatment with F. In a previous study, it was observed
that treatment of rats with 50 mg/L F in the drinking water for
20 days reduced lipid droplets in the liver and also decreased
plasma TG, while no alterations were detected when the treat-
ment with F lasted 60 days [15]. For this reason, we decided to
evaluate the periods of 10, 20, and 30 days of treatment. This
was proven to be important, since considerable variation in the
results was observed depending on the duration of the treat-
ment. For LDN analysis, the treatment was conducted for
20 days because treatment of rats with 50 mg/L F in the
Fig. 5 Morphometric (A, B) and morphological (C, D) analyses of lipid
droplets in the liver of mice fed normocaloric or hyperlipidic diets that
received drinking watercontaining0 mg/L or 50 mg/L fluoride for 10, 20
or 30 days. (A) Mean percentage of lipid droplets for animals fed
normocaloric diet; (B) mean percentage of lipid droplets for animals fed
with hyperlipidic diet; (C) average of scores, based on morphological
analysis, for animals fed normocaloric diet; (D) average of scores, based
on morphological analysis, for animals fed hyperlipidic diet. For each
type of diet, distinct lower case letters indicate significant differences
among the groups (two-way ANOVA and Tukey’stest,p< 0.05). Bars
indicate SD. n=10
Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis
Author's personal copy
drinking water for the same period reduces lipid droplets in the
liver and improves the lipid profile, while treatments for 10,
30 (this study), or 60 [15] days do not provoke any alterations.
We found only slight differences in body weight and liver
weight (Fig. 2). In most of the studies, when administered at
the concentration employed in the present study, F does not
alter body weight [5,24]. However, in these studies, F was
administered for a longer time (usually more than 42 days). In
the present study, it was noticeable that F administration for
20 days in animals with installed NAFLD significantly re-
duced body weight (Fig. 2(B)).
One interesting finding of the present study was the fact
that when F was administered in conjunction with the
normocaloric diet, F concentrations in liver and plasma re-
duced along time, while they increased in the group with
installed NAFLD (Fig. 3). The main routes of F elimination
from plasma are urinary excretion or uptake in the skeleton
[2]. Thus, the reduction in plasma F concentrations along time
when the animals were fed the normocaloric diet might be due
to one of these events. Studies conducted in the 1950’sreport
an increase in the absorption of F by dietary fat [45,46], since
high levels of fat in the duodenum reduce the rate of gastric
emptying, which in turn increases the degree of gastric absorp-
tion of F [1], thus increasing plasma F levels [45,46].
Additional studies on this topic are necessary to provide the
basis for understanding these events, as well as to clarify why
plasma and tissue F concentrations increase along time with
consumption of a hyperlipidic diet. The increase in the reten-
tion of F in the organism when the hyperlipidic diet is con-
sumed is relevant, since this might increase F toxicity, thus
increasing the risk of occurrence of dental or skeletal fluorosis.
There is only one study that associated body mass index with
dental fluorosis in children, but no correlation was found [47].
However, it should be evaluated if children that ingest a diet
with high fat content have a higher risk to develop dental
fluorosis. On the other hand, a recent study reported that over-
weight and obesity are risk factors for skeletal fluorosis in
adults [48].
It is well known that F induces oxidative stress, which
could lead to an increase in steatosis [4,11,22–24,44,
Fig. 6 Means of the lipid profiles in plasma and liver of mice fed
normocaloric diet receiving 0 mg/L or 50 mg/L fluoride in the drinking
water for 10, 20, or 30 days. (A) Liver TG. (B) Plasma TG. (C) VLDL.
(D) Total cholesterol. (E) HDL. (F) LDL. For each variable, distinct lower
case letters indicate significant differences among the groups (two-way
ANOVA and Tukey’s test, p< 0.05). Bars indicate SD. n=10
Dionizio et al.
Author's personal copy
49–51]. However, in the present study, the animals treated
with F for 20 days and fed with normocaloric diet presented
a reduction of lipid droplets in the liver (Fig. 5(A)). In the
meantime, for the animals that had installed NAFLD, F did
not interfere in the degree of steatosis, which might be related
to a possible adaptation of the animals to the hyperlipidic diet
[52]. As expected, the severity of steatosis, as evaluated by
scores, was much higher in the animals fed hyperlipidic diet
[15,53].
Our results for the lipid profile show that F can exert a
distinct effect, depending on the duration of exposure and type
of diet consumed. For the animals fed normocaloric diet, a
significant improvement in lipid profile was seen at 10 and
20 days of treatment with F. However, animals treated for
30 days had increased total cholesterol levels, which was
due to increase in HDL levels (Fig. 6). In the case of the
hyperlipidic diet, however, a beneficial effect of F was only
observed at 20 days (Fig. 7), even when we compared the
fluoride-treated animals wen the animals receiving
normocaloric diet and fluoride-free water (that could be
regarded as Bcontrol^). In general, the studies available in
the literature report an impairment in lipid parameters upon
exposure to F in doses similar to the ones employed in the
present study [27–29,44]. However, the duration of the treat-
ment with F in these studies is longer, which is consistent with
the tendency of deleterious effect of F found for the period of
30 days in the present study. In other words, F seems to be able
to improve the lipid parameters only when administered in the
Fig. 7 Means of the lipid profiles in plasma and liver of mice fed
hyperlipidic diet receiving 0 mg/L or 50 mg/L fluoride in the drinking
water for 10, 20, or 30 days. (A) Liver TG. (B) Plasma TG. (C) VLDL.
(D) Total cholesterol. (E) HDL. (F) LDL. For each variable, distinct lower
case letters indicate significant differences among the groups (two-way
ANOVA and Tukey’s test, p< 0.05). Bars indicate SD. n=10
Fig. 8 Average of percentage of de novo lipogenesis in mice fed
normocaloric or hyperlipidic diets that received 0 mg/L or 50 mg/L fluo-
ride in the drinking water for 20 days. Distinct lower case letters indicate
significant differences between the types of diet (two-way ANOVA and
Tukey ’stest,p<0.05).BarsindicateSD.n=6
Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis
Author's personal copy
short time (up to 20 days according to our model). One pos-
sibility for this dual effect of F depending on the duration of
the treatment might be related to changes in calcium metabo-
lism induced by F, due to the high affinity that these elements
have to one another [54]. It was recently shown that lipid
membranes have substantial calcium-binding capacity, with
potential implications in calcium signaling [55]. It is also
known that lipolysis is affected by calcium [56]. Additional
studies should be conducted in attempt to clarify this dual
effect of F on lipid parameters, especially what shifts its effect
from a beneficial to a harmful action.
The significantly lower de novo lipogenesis (Fig. 8)found
for the animals fed hyperlipidic diet was expected, because
there are many free fatty acids circulating in animals fed with
this diet. Thus, inhibitory mechanisms that prevent lipid syn-
thesis from non-lipid sources (de novo lipogenesis) will be
activated, as has been previously shown [57]. In addition,
experimental models used to induce obesity can have influ-
ence de novo lipogenesis. In other words, the nutritional com-
position of the diet interferes on de novo lipogenesis [41,58],
which might help to explain the differences found in the stud-
ies that use Bhigh-fat^or normocaloric diets [58]. Several
studies where animals had NAFLD revealed a decrease in
lipogenesis [58–61], in agreement with our results. This can
be related to the composition of the hyperlipidic diet used in
our study, which contains lard [58–61]. Moreover, the in-
crease in de novo lipogenesis found for the animals fed
normocaloric diet can be due to the high carbohydrate content
(43.7%) of this diet [58,62]. Regarding the influence of treat-
ment with F on de novo lipogenesis according to the type of
diet, those fed with normocaloric diet had a trend for reduction
in de novo lipogenesis upon treatment with F.
Based on these results and on the current literature, a mech-
anism can be proposed to try to explain how F interferes on
lipid metabolism and de novo lipogenesis, depending on the
type of diet consumed, when the animals are treated with
50 mg/L F in the drinking water for 20 days (Fig. 10). The
normocaloric diet used in this study contains 43.7% carbohy-
drates, while the hyperlipidic diet has 35% fat (31% from
animal source (39% saturated fat) and 4% from vegetal source
(soybean oil)) [32]. This causes an increase in the availability
of carbohydrates and lipids in the organism. Upon treatment
with normocaloric diet in the absence of F, the increase in the
ingestion of carbohydrates leads to an increase in acetyl-CoA,
which in turn increases pathways related to cholesterol, thus
increasing low-density lipoprotein (LDL) and de novo lipo-
genesis. Increase in these precursors might, in turn, increase
liver lipid droplets and triglycerides (TG) (Fig. 10(A)) [63].
When the animals are concomitantly treated with F, the in-
crease in carbohydrates from the diet increases acetyl-CoA,
as mentioned above, but F induces endoplasmic reticulum
oxidative stress, which increases GRP-78 and this, in turn,
reduces SREBP that deactivates de novo lipogenesis (Fig. 9)
[5,15,25]. This deactivation reduces the amount of available
TG, thus diminishing lipid accumulation in the liver (both as
lipid droplets and TG). F also affects the cholesterol pathway,
through the inhibition of Apo-E [5,15], which diminishes the
transport of cholesterol, thus reducing its accumulation in the
organism, as well as the LDL levels (Figs. 9and 10(B)). The
consumption of hyperlipidic diet, in the absence of treatment
Fig. 9 Representative expression
of proteins GRP-78, Apo-E,
SREBP, and of the constitutive
protein α-tubulin in samples of
individual animals (n=4) from
each group. Densitometric analy-
sis was performed for four ani-
mals per group. (A) Protein ex-
pression in the liver of mice in the
group fed normocaloric diet for
20 days; (B) protein expression in
the liver of mice fed hyperlipic
diet for 20 days. Densitometry
was analyzed using the software
Image Studio Lite. For each type
of diet, distinct letters denote sig-
nificant differences between ani-
mals treated with F or not (un-
paired ttest, p< 0.05) Bars indi-
cate SD. n=4
Dionizio et al.
Author's personal copy
with F, increases the availability of lipids in the organism, thus
augmenting the levels of circulating lipids, as well as of ace-
tyl-CoA. This increases the transport of lipids to the tissues,
thus increasing circulating cholesterol and LDL as well as
lipid droplets in the liver. On the other hand, due to the in-
crease in the levels of circulating lipids, there is no need to
form TG and de novo lipogenesis is reduced (Fig. 11(A)).
When the animals are concomitantly treated with F, the great
availability of lipids from the diet reduces de novo lipogenesis
and increases lipid droplets in the liver. However, since F
inhibits Apo-E (Fig. 9)[5,15], the transport of cholesterol is
diminished, thus reducing cholesterol and LDL levels
(Fig. 11(B)) [5,15,63,64].
The data obtained in the present study in conjunction with
the available literature provide new insights on how F affects
lipid metabolism depending on the available energy source.
Taken together, our results show that F is able to improve lipid
parameters and reduce steatosis only when administered for a
short period of time (up to 20 days) to animals fed
normocaloric diet. However, when NAFLD is already
Fig. 10 Proposed mechanism of action of fluoride (F) on lipid metabo-
lism and de novo lipogenesis. The animals were fed normocaloric diet
(increasing acetyl-CoA due to the ingestion of carbohydrates) and treated
with 0 or 50 mg/L F in the drinking water for 20 days. (A) Normocaloric
diet and 0 mg/L F: increase in acetyl-CoA increases cholesterol pathways
(increase in total cholesterol and LDL) and de novo lipogenesis,
augmenting lipid droplets and TG in the liver. (B) Normocaloric diet
and 50 mg/L F: F causes endoplasmic reticulum oxidative stress, increas-
ing GPR78, which in turn reduces SREBP, reducing de novo lipogenesis
and available TG, which reduces lipid droplets and TG in the liver. F also
affects the cholesterol pathway, through inhibition of Apo-E, which re-
duces the transport and circulating levels of cholesterol, as well as of LDL
Effect of Duration of Exposure to Fluoride and Type of Diet on Lipid Parameters and De Novo Lipogenesis
Author's personal copy
installed, lipid parameters are only slightly improved at
20 days of treatment, but no effect can be seen on the degree
of steatosis (Supplementary Fig. A2). In addition, lipid profile
was in general impaired when the animals were treated with F
for 30 days, regardless of the diet. Moreover, it was observed
that the retention of F in the organism is higher when
hyperlipidic diet is consumed, which can have important im-
plications in F toxicity and should be better investigated.
In summary, when the diet contains a high fat content,
F does not alter de novo lipogenesis, but when the main
source of energy in the diet is derived from carbohy-
drates, F tends to reduce de novo lipogenesis. It should
be noted that the aim of this study was to better
understand the mechanisms by which F alters the lipid
metabolism. Despite beneficial effects observed when
the animals were treated with F up to 20 days, there
was impairment in the longer treatment period. For this
reason, our results do not support the consumption of F
in the long term as a preventive method against lipid
disorders. Additional studies should be conducted in or-
der to investigate if the beneficial effects are maintained
if the consumption of F is interrupted.
Acknowledgments We are grateful to Dr. Leandro Pereira de Moura for
the help with the preparation of the hyperlipidic diet and Dr. José Roberto
Bosqueiro for the help with obtaining the animals.
Fig. 11 Proposed mechanism of action of fluoride (F) on lipid metabo-
lism and de novo lipogenesis. The animals were fed hyperlipidic diets
(increasing acetyl-CoA due to the ingestion of lipids) and treated with 0 or
50 mg/L F in the drinking water for 20 days. (A) Hyperlipidic diet and
0 mg/L F: increase in circulating lipids and acetyl-CoA augments the
transport of lipids to the tissues and reduces the formation of TG through
de novo lipogenesis. (B) Hyperlipidic diet and 50 mg/L F: increase in
circulating lipids increases acetyl-CoA and reduces de novo lipogenesis,
besides increasing lipid droplets in the liver. F inhibits Apo-E, reducing
the transport and concentration of cholesterol, as well as of LDL
Dionizio et al.
Author's personal copy
Author Contributions Dionizio A, Pereira HABS, and Buzalaf MAR
conceived the experiments. Dionizio A, Pereira HABS, Nogueira FN,
and Carvalho, RA conducted the experiments. Dionizio A, Pereira
HABS, Araujo TT, Sabino-Arias IT. Fernandes MS. Oliveira KA.
Nogueira FN, Carvalho, RA, Raymundo FS, and Cestari TM participated
in the research experiments. Dionizio A, Pereira HABS, and Buzalaf
MAR drafted the article and analyzed and interpreted the results. All
authors reviewed and approved the manuscript.
Funding Information We thank Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) for the concession of a Master scholarship
to the first author (grant number 2013/25756-0 and 2015/10988-9).
Compliance with Ethical Standards
All experimental protocols were approved by the Ethics Committee for
Animal Experiments of Bauru Dental School, University of São Paulo
(protocol: 001/2014; 008/2015).
Conflict of Interest The authors declare that they have no conflict of
interest.
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