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Chronic sulforaphane oral treatment accentuates blood glucose impairment

and may affect GLUT3 expression in the cerebral cortex and hypothalamus of
rats fed with a highly palatable...

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DOI: 10.1039/c3fo60039d · Source: PubMed

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Chronic sulforaphane oral treatment accentuates blood

glucose impairment and may affect GLUT3 expression in
Cite this: Food Funct., 2013, 4, 1271
the cerebral cortex and hypothalamus of rats fed with a
highly palatable diet
C. G. Souza,*ab B. P. Riboldi,a F. Hansen,a J. D. Moreira,a D. G. Souza,a A. M. de Assis,a
L. M. Brum,c M. L. S. Perrya and D. O. Souzaa

Received 27th January 2013
Accepted 24th May 2013
DOI: 10.1039/c3fo60039d
www.rsc.org/foodfunction
Obesity and insulin resistance are the key factors underlying the etiology of major health problems such as
hypertension, diabetes and stroke. These important health issues lead researchers to investigate new
approaches to prevent and treat obesity and insulin resistance. Good candidates are the phytochemical
compounds that have been extensively studied in the field. Therefore, the aim of this study was to test
whether sulforaphane (SFN, 1 mg kg
1, 4 months treatment), a potent inducer of antioxidant enzymes
present in cruciferous vegetables, had some beneficial effects on obesity and insulin resistance induced
by a highly palatable (HP) diet in male Wistar rats. Glucose tolerance, serum and hepatic lipid levels,
lipid profile, ALT, AST, urea and creatinine, GLUT1 and GLUT3 levels in the cerebral cortex, hippocampus
and hypothalamus were analyzed. Glucose tolerance was lower in the HP diet groups, especially in the
HP group treated with SFN. Except for the liver triacylglycerols, no differences were found in serum
lipids, hepatic and kidney markers of the HP diet groups. Although expression of GLUT1 was similar
between groups for all three brain structures analyzed, expression of GLUT3 in the cortex and
hypothalamus had a tendency to decrease in the HP diet group treated with SFN. In conclusion, SFN at
the specific dose was able to accentuate glucose intolerance and may affect GLUT3 expression in the
cerebral cortex and hypothalamus.

1 Introduction
Obesity and insulin resistance are key factors in the etiology of
major diseases like hypertension, diabetes, cardiovascular
disease, stroke and even some types of cancer.1–3 The link
between excessive body weight and insulin dysfunction in the
above mentioned disease is the increase of body fat that leads to
an increased release of free fatty acids into the blood, which
accumulates in other tissues.4,5 This set of alterations culmi-
nates in hyperglycemia and increased oxidative stress resulting
in health problems.6 It is important to remember that one of the
main causes of obesity and insulin resistance in the modern
world is the elevated consumption of food with a high content
of sugar and fat.7
a
Department of Biochemistry, ICBS, Universidade Federal do Rio Grande do Sul-
UFRGS, Rua Ramiro Barcelos, 2600 anexo, CEP 90035003, Porto Alegre, RS, Brazil.
E-mail: carolguerini@hotmail.com; Fax: +55 51 33085540; Tel: +55 51 33085559
b
Medical School, Internal Medicine Department, Universidade Federal do Rio Grande
do Sul - UFRGS, Brazil
c
Cardiology Institute of Rio Grande do Sul, FUC, Porto Alegre, RS, Brazil
Results from our group demonstrated that animals fed with
a highly palatable diet (enriched with sucrose) for four months,
used as a model to induce obesity, had increased oxidation of
proteins in the cerebral frontal cortex and anxiety-like behavior,
elevated body fat, and high levels of glucose and hepatic tri-
acylglycerols.8 Although the mechanisms behind these changes
are unknown, it is clear that alterations in glucose metabolism
are implicated.
So, studies with phytochemical compounds that can prevent
or attenuate metabolic derangements have been extensively
developed with several positive results.9–11 It has been recently
demonstrated that sulforaphane (SFN), a potent antioxidant
that acts as a phase 2 enzyme inducer present in cruciferous
vegetables such as broccoli and cauliower,12 prevents strep-
tozotocin-induced diabetes.13 Therefore, the aim of this work
was to study the effect of oral sulforaphane administration, at a
concentration similar to that obtained by the ingestion of 150 g
of fresh broccoli,14 on obesity and insulin resistance induced by
a highly palatable (HP) diet. To address this issue we analyzed
peripheral and central parameters of the glucose metabolism
in a dietary model to induce obesity with a highly palatable
diet.

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2 Results
The animals' body weight did not change aer four months of
diet and treatment. However, the adipose tissue represented by
retroperitoneal and epididymal fat pads, was two fold higher in
HP and HP + SFN groups than the SC group (Table 1, P > 0.05
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and P < 0.05, respectively). Although the liver TAG, a marker of

insulin resistance, was elevated in HP and HP + SFN groups
(Table 1, P < 0.05), the liver weight, liver cholesterol, serum TAG,
total cholesterol and HDL cholesterol were similar for all groups
(Table 2, P > 0.05).
In order to evaluate the chronic effects of sulforaphane in the
liver and kidney function ALT, AST, urea and creatinine were
measured and the levels of these markers in the blood were
similar for all groups, suggesting the absence of the hepato- and
nephrotoxicity effect by the chronic use of SFN (Table 2, P > 0.05).
On the other hand, glucose intolerance induced by the HP
diet was accentuated in the HP + SFN group when compared
with SC and HP groups. Fasting glycemia at time 0 was equal in
all groups (P > 0.05) (Fig. 1A). However, aer glucose overload,
the glycemic levels in HP and HP + SFN groups were higher than
the SC group at 30 minutes, and the level for the HP + SFN group
was higher than the HP group (P < 0.05). This difference
between the two HP groups disappeared aer 60 minutes of
testing, and both remaining HP + SFN and HP groups were Fig. 1 (A) Glucose Tolerance Test (GTT) and (B) Area Under the Curve (AUC) of all
more intolerant to glucose than the SC group (P < 0.05). At the groups (n ¼ 7). For GTT, blood glucose was measured before (0) and 30, 60 and
end of the test the glycemic levels were similar for all groups, 120 minutes after glucose injection (2 mg g
1 body weight). Data are expressed
as mean  standard deviation of groups. Statistical analysis performed with
with a slight increase for both HP groups compared with the SC
Repeated Measures Anova followed by Bonferroni's test for GTT and One Way
group (P > 0.05). The difference in glycemic levels during the Anova followed by Duncan's post-hoc test for AUC. a, b and c ¼ different letters
test can be appreciated by the area under the curve (AUC), mean P < 0.05 among groups.
with statistical differences only for SC and HP groups (Fig. 1B,
P < 0.05).

We also measured the expression of glucose transporters in

Table 1 Body composition and lipid content in the liver of SC, HP and HP + SFN
groups (n ¼ 7). Data are expressed as mean  standard deviation. Statistical the cerebral cortex, hippoccampus and hypothalamus. In the
analysis performed with One Way Anova followed by Duncan's post-hoc test. a,b ¼ cerebral cortex we found no difference in GLUT1 expression
different letters mean P < 0.05 among groups (Fig. 2A, P > 0.05). However, the expression of GLUT3 tended to
be lower in the HP + SFN (Fig. 2B, P ¼ 0.05) when compared
SC (n ¼ 7) HP (n ¼ 7) HP + SF (n ¼ 7)
with HP and SC groups. Also, we were able to observe a
Final body weight (g) 328  17 343  26 339  15 decrease of GLUT3 levels with the increased blood glucose
Adipose tissue (RP + EP) (g) 8  3a 16  4b 15  3b levels for all groups (negative correlation between GLUT3
Liver weight (g) 10  1 10  1 91 levels and AUC; r ¼
0.4, P ¼ 0.05, data not shown). These data
Liver TAG (mg%) 0.9  0.3a 1.5  0.3b 1.4  0.4b suggest a possible inuence of glycemic levels on GLUT3
Liver cholesterol (mg%) 1.7  0.4 1.7  0.4 1.4  0.2
expression in the cortex. No correlation was observed between
GLUT1 expression in the cerebral cortex and AUC values. Like
Table 2 Serum biochemical parameters of SC, HP and HP + SFN groups (n ¼ 7). the GLUT1 and GLUT3 expression in the hippocampus (Fig. 3A
Data are expressed as mean  standard deviation or median (min–max). Statis- and B, P > 0.05), the GLUT1 expression in the hypothalamus
tical analysis performed with One Way Anova followed by Duncan's post-hoc test was similar in all groups (Fig. 4A, P > 0.05). However, the
or the Kruskall–Wallis test. No differences were found among groups
expression of hypothalamic GLUT3 in the HP + SFN group
SC (n ¼ 7) HP (n ¼ 7) HP + SF (n ¼ 7)
(Fig. 4B, P ¼ 0.06) had a tendency to decrease when compared
with HP and SC groups. Similar to what we observed in the
Serum TAG (mg dL
1) 92 (43–222) 99 (52–176) 115 (34–269) cerebral cortex, here was a tendency of GLUT3 levels to
Total cholesterol (mg dL
1) 62  6 64  8 57  11 decrease in this structure with the increased blood glucose
HDL cholesterol (mg dL
1) 34  2 38  1 38  1
levels for all groups (negative correlation between GLUT3
ALT (IU L
1) 3 (2–5) 3 (2–6) 4 (2–6)
AST (IU L
1) 8 (4–13) 8 (4–17) 13 (4–27) levels and AUC; r ¼
0.5, P ¼ 0.06, data not shown). Again, no
Urea (mg dL
1) 54  8 46  9 49  8 correlation was found between hypothalamic GLUT1 levels
Creatinine (mg dL
1) 0.4  0.1 0.4  0.1 0.4  0.1 and AUC values.

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Fig. 2 Western blotting analysis of glucose transporters (A) GLUT-1 and (B)
GLUT-3 in the cerebral cortices of SC (white bars), HP (black bars) and HP + SFN
groups (filled bars) (n ¼ 4). Data are expressed as mean  standard deviation
(n ¼ 4 animals per group). At the top of the figure are representative images of
the immunocontent of transporters. b-Actin was used as a protein loading
control. Statistical analysis performed with One Way Anova followed by Duncan's
post-hoc test.
3 Discussion
This was the rst study that demonstrated the effects of chronic
oral administration of SFN in a model of obesity and insulin
resistance induced by the HP diet. Surprisingly we found an
increased glycemia in HP diet + SFN treated animals, since the
dose of SFN used (1 mg kg
1) was based on the amount of SFN
present in 150 g of fresh broccoli,14 a portion commonly
ingested in a meal.
As demonstrated by previous studies from our group15,16 the
HP diet induced obesity and insulin resistance, based on
increased levels of adipose tissue, glucose intolerance and
hepatic triacylglycerols when compared with SC, even without
changes in the serum lipid prole. SFN had no role in pre-
venting or ameliorating any alteration promoted by the HP diet,
and did not produce a toxic effect in the liver or kidney func-
tionality, suggested by the unaltered levels of ALT, AST, urea
and creatinine. On the other hand, the increased glycemic levels
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Fig. 3 Western blotting analysis of glucose transporters (A) GLUT-1 and (B)
GLUT-3 in hippocampi of SC (white bars), HP (black bars) and HP + SFN groups
(filled bars) (n ¼ 4). Data are expressed as mean  standard deviation (n ¼ 4
animals per group). At the top of the figure are representative images of the
immunocontent of transporters. b-Actin was used as a protein loading control.
Statistical analysis performed with One Way Anova followed by Duncan's post-
hoc test.
in GTT at 30 minutes in HP diet + SFN-treated animals indicated
higher glucose intolerance than the HP group. This hypothesis
is reinforced by the tendency of GLUT3 decreased expression in
the cortex and hypothalamus of HP diet + SFN-treated animals,
which was not observed in animals receiving only the HP diet.
GLUT1 and GLUT3 are glucose transporters present in the
central nervous system (CNS). While GLUT1 is involved in
glucose crossing through the endothelial cells of the blood–
brain barrier (BBB), GLUT3 transports glucose into the neural
cells.17 Nevertheless, whether hyperglycemia increases or
decreases GLUT-1 and GLUT-3 expression in the brain is still
under debate. Zhang and colleagues18 observed an increase of
GLUT1 and GLUT3 mRNA levels in diabetic rats with or without
brain ischemia and reperfusion. Likewise, Peeyush et al., Rea-
gan et al. and Sherin et al.19–21 described an increased GLUT3
gene expression in the brainstem, hippocampus and cortex of
diabetic rats that received different types of treatments. On the
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Fig. 4 Western blotting analysis of glucose transporters (A) GLUT-1 and (B)
GLUT-3 in the hypothalamus of CT (white bars), HP (black bars) and HP + SFN
groups (filled bars) (n ¼ 4). Data are expressed as mean  standard deviation ( n ¼
4 animals group). At the top of the figure are representative images of the
immunocontent of transporters. b-Actin was used as a protein loading control.
Statistical analysis performed with One Way Anova followed by Duncan's post-
hoc test.
other hand, the decrease in GLUT1 expression in the hypo-
thalamus,22 thalamus, cerebellum and hippocampus, mediated
by high glucose levels has been described.23 Similar to our
ndings, Hou et al.24 demonstrated decreased GLUT1 and
GLUT3 expression in the brain of diabetic rats, with transporter
levels being negatively correlated with blood glucose. However,
it is important to mention that all studies cited above measured
mostly GLUT1 and GLUT3 mRNA that may not always be
translated into the protein content. Different from glucose
transporter 4 (GLUT4), GLUT1 and GLUT3 are not regulated by
insulin but rather by blood glucose levels. It seems that the
hyperglycemia downregulates brain glucose transporters in an
attempt to protect the brain against damage caused by an inux
of glucose into the cells. The critical point is when blood glucose
levels reduce and cells fail to correct this downregulation,
resulting in a relative insufficiency of glucose in the brain,
favoring neurological damage.24
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Paper
In this study we did not investigate the mechanism underlying
the hyperglycemia in the presence of SFN, and even not being one
of our goals, it still is a limitation of our data. However, a recent
work of Fu et al.25 showed that prolonged SFN exposure decrease
insulin secretion by attenuation of glucose-stimulated reactive
oxygen species (ROS) production, which is essential to glucose-
stimulated insulin secretion (GSIS) in pancreatic b-cells. Besides
its well-known deleterious effects when excessively produced, at
physiological levels ROS work as intracellular second messen-
gers, therefore ROS derived from normal glucose metabolism act
as a necessary intermediate signal to mediate GSIS in pancreatic
b-cells. Fu and colleagues25 demonstrated that acute SFN expo-
sure stimulates basal insulin secretion while prolonged exposure
persistently increases the antioxidant capacity, leading only to a
fractional increase in basal insulin secretion. This means that
SFN may inhibit postprandial GSIS when insulin is needed for
maintaining blood glucose levels, and we believe this is a
reasonable explanation for the effects on GTT found in our SFN +
HP diet model of treatment. To the best of our understanding,
the present results were greatly inuenced by the SFN dose used
and chronic time of treatment.
Different from the present results, Song et al.13 demonstrated
that SFN in a lower dose (40 mg kg
1, ip) and in a very short
period of treatment (3 days) was able to prevent streptozotocin-
induced diabetes by decreasing reactive oxygen species
production and suppressing the NF-kB pathway in the pancreas,
preserving b-cells and insulin production. In fact, another work
from our group26 based on the paper of Song et al.13 corrobo-
rates their ndings using different doses of SFN (0.1, 0.25 and
0.5 mg kg
1), lower than that we used in this study (1 mg kg
1),
but we have to consider the changes in the diabetes induction
model and SFN intake that was shorter and not concomitant
with the hyperglycemic state.26 To complement that, recent
results of our group (Souza et al., unpublished data) also using
streptozotocin to induce diabetes showed that a treatment with
0.5 SFN mg kg
1 i.p., for 21 days aer diabetes installation, did
not promote any changes in fasting glycemia but was able to
ameliorate insulin responsiveness in diabetic animals, without
nephron or hepatotoxicity in diabetics and non-diabetics/non-
insulin resistant animals. But, again we have to consider that
the diabetes induction model and SFN dose and time of treat-
ment were different from the present one.
It is also important to emphasize that the SFN used in our
protocol was an isolated compound, absorbed directly, without
intestinal enzymatic mechanisms, which is a different process
compared to SFN contained in cruciferous vegetables. It is
known that vegetables can have antinutritional factors that
affect absorption of some nutrients. That could explain why
isolated SFN had a hyperglycaemic effect different from that in
fresh broccoli when taken orally. Regardless, it is important to
remember that the benet or toxicity of antioxidants usually is
dose-dependent, which is well-explained by the concept of
hormesis, dened as a biphasic dose-response of cells or
organisms to an exogenous or endogenous factor (chemical
agents, dietary ingestion, oxidative stress, etc.) in which the
factor induces stimulatory or benecial effects at low doses and
inhibitory or adverse effects at high doses.27
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4 Experimental
4.1 Reagents
R,S-Sulforaphane was purchased from LKT Laboratories (St.
Paul, MN). Diagnostic kits were obtained from Labtest (Lagoa
Santa, MG, Brazil).
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4.2 Animals and diets
Twenty one 60 day old male Wistar rats were obtained from the
Central Animal House of our Department and were maintained
under a standard dark–light cycle (lights on between 7:00 a.m.
and 7:00 p.m.), at a room temperature of 22  2  C. Animal care
followed the official governmental guidelines in compliance
with the Federation of Brazilian Societies for Experimental
Biology and was approved by the Ethics Committee of the
Federal University of Rio Grande do Sul, Brazil.
Rats were divided into three groups: (1) the control group
(SC, n ¼ 7), which received standard laboratory rat chow (50%
carbohydrate, from starch, 22% protein and 4% fat); (2) the
highly palatable diet group (HP, n ¼ 7), which received an
enriched sucrose diet (65% carbohydrates (34% from
condensed milk, 23% from starch and 8% from sucrose), 25%
protein, 10% fat derived from soybean oil); (3) the highly
palatable diet group which received the same enriched
sucrose diet + sulforaphane (HP + SFN, n ¼ 7). All animals had
free access to food and water during the four months of
treatment.
4.3 Oral sulforaphane treatment
All animals were gavaged daily in the early morning with either
deionized water (SC and HP groups) or sulforaphane (SFN) 1 mg
kg
1 diluted in deionized water (HP + SFN). The SFN solution
was prepared weekly, adjusted to animals' body weight, and
kept under refrigeration.
4.4 Glucose tolerance test (GTT)
A glucose tolerance test was performed one week before the
animals were sacriced. A 50% glucose solution was injected
into the animals (i.p, 2 mg g
1) aer 6 h of fasting.28 The blood
sample was taken by a small tail puncture immediately before
and 30, 60, and 120 minutes aer the injection. At each time,
glucose was measured with a glucose meter (AccuChek Active,
Roche Diagnostics, USA). The glucose levels were also evalu-
ated by the analysis of the area under the curve (AUC).
4.5 Blood and tissue preparation
At the end of the fourth month all groups were anesthetized
using sodium thiopental (40 mg kg
1), blood samples were
taken by cardiac puncture and immediately centrifuged at
5000  g for 10 minutes to obtain serum and evaluate
biochemical parameters. Liver, retroperitoneal and epididymal
fat pads were dissected and weighed. The cerebral cortex,
hippocampus and hypothalamus were dissected and a sample
of each was obtained to perform posterior western blotting
analysis.
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4.6 Biochemical parameters
Serum levels of total cholesterol, high density lipoprotein (HDL)
cholesterol, triacylglycerols (TAG), urea, creatinine and enzy-
matic activities of alanine aminotransferase (ALT) and aspartate
aminotransferase (AST) were measured using commercial kits
(Labtest- Lagoa Santa, MG, Brazil). Liver triacylglycerol (TAG)
was measured using a 100 mg liver sample, which was
homogenized in a 1 : 20 saline solution (0.9%). The assay was
performed with commercial kits (Roche Diagnostics, Indian-
apolis, USA) using an aliquot of this mixture. Liver cholesterol
was determined by the method of Bergmeyer.29
4.7 Western blotting analysis
Cerebral cortices, hippocampi and hypothalamus were dissected
out and immediately homogenized in a 25 mM HEPES solution
(pH 7.4) with 0.1% SDS and protease inhibitor cocktail (Sigma,
USA). Samples (20 mg protein per well) were placed in a 10% SDS-
PAGE mini-gel and transferred to a nitrocellulose membrane using
a Trans-Blot system (Bio-Rad, S~ ao Paulo/SP, Brazil). Membranes
were processed as follows: (1) blocking with 5% bovine serum
albumin (Sigma, USA) for 1 h; (2) incubation with primary anti-
body overnight: 1 : 200 rabbit anti-GLUT1 and rabbit anti-GLUT3
(Santa Cruz Biotechnology); (3) incubation with horseradish
peroxidase-conjugated secondary antibody for rabbit 1 : 2000 (GE
Healthcare UK Limited) overnight and (4) chemiluminescence
(ECL, Amersham Pharmacia Biotech, S~ ao Paulo/SP, Brazil) was
detected using X-ray lms (Kodak X-Omat, Rochester, NY, USA).
The same steps were repeated to incubate the membranes with
anti-b-actin primary antibody overnight 1 : 3000, followed by
incubation with horseradish peroxidase-conjugated secondary
antibody for mouse 1 : 2000. The lms were scanned and band
intensities were analyzed using Image J soware (developed at the
US National Institutes of Health and available on the website
http://rsb.info.nih.gov/nih-image/). In order to determine the
adequate amount of protein to be assayed, different protein
concentrations were used in the same gel for each antibody tested.
4.8 Statistical analysis
Data were analyzed with SPSS 17.0 soware. Glucose tolerance
was analyzed by Repeated Measures analyses of variance
(ANOVA) and post-hoc Bonferroni's test. Parametric variables
were tested by One-Way ANOVA and post-hoc Duncan's test and
non-parametric variables were tested by Kruskall Wallis's test
followed by Dunns' post-hoc test. Results are expressed as mean
 standard deviation or median (min–max).
Acknowledgements
This work is dedicated to life and memory of Marcos Luiz Santos
Perry, beloved friend, mentor and Professor of Biochemistry.
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