Exercise Training but not Curcumin Supplementation Decreases Immune Cell Infiltration in the Pancreatic Islets of a Genetically Susceptible Model of Type 1 Diabetes
Oharomari et al. Sports Medicine - Open
Exercise Training but not Curcumin Supplementation Decreases Immune Cell Infiltration in the Pancreatic Islets of a Genetically Susceptible Model of Type 1 Diabetes
Leandro Kansuke Oharomari 0 2
Camila de Moraes 1
Anderson Marliere Navarro 0 2
0 Department of Food and Nutrition, Pharmaceutical Sciences College , Araraquara, Sa?o Paulo , Brazil
1 School of Physical Education and Sport of Ribeira?o Preto, University of Sa?o Paulo , Ribeira?o Preto, Sa?o Paulo , Brazil
2 Department of Food and Nutrition, Pharmaceutical Sciences College , Araraquara, Sa?o Paulo , Brazil
Background: The main mechanism involved in the pathogenesis of autoimmunity is an uncontrolled inflammatory response against self-antigens. Therefore, anti-inflammatory factors, such as the intake of bioactive compounds and a physically active lifestyle, may decrease or cease the development of autoimmune diseases. Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic ? cell destruction. The non-obese diabetic (NOD) mouse is a model of spontaneous T1D and is the model most similar to human disease. Methods: To determine the effects of exercise training and curcumin supplementation on T1D progression, 48 NOD mice, 5 weeks old, were randomly divided into four groups: control, curcumin supplementation, trained, and trained plus curcumin. Every 2 weeks, blood glucose was measured using a glucometer. At the end of 20 weeks, a histopathological procedure was used to assess immune cells infiltration into pancreatic ? cells (insulitis). Results: Moderate intensity exercise training has the potential to protect pancreatic ? cells against an immune response in vivo. However, curcumin supplementation failed to attenuate insulitis in NOD mice. Conclusions: These data provide evidence that exercise training can mitigate T1D development in genetically susceptible mice.
Autoimmunity; Type 1 diabetes; Insulitis; Exercise training; Bioactive compound; Curcumin
Intense inflammatory response is a main feature of
autoimmune diseases. Type 1 diabetes (T1D), for instance,
occurs due to a chronic inflammatory response with enough
severity to destroy most of the pancreatic ? cells . The
incidence of some autoimmune diseases, including T1D,
has been increasing worldwide over the past decades [2?5],
and environmental factors are the most accepted
explanation for this phenomenon. For example, vitamin D status
 and the hygiene hypothesis  are being investigated.
Currently, physical inactivity and processed food intake
during childhood appear to be linked to the development
of chronic [8?10] and inflammatory disease  in early
stages of life. Furthermore, the Overload Hypothesis
suggests an association between a modern lifestyle and beta
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cell overload, which could make these cells more vulnerable
to an autoimmune response .
Intervention using functional foods with
antiinflammatory properties could decrease or halt the
autoimmune response, leading to a delay or even prevention
of autoimmunity. Dietary patterns characterized by high
bioactive compounds, such as a Mediterranean diet,
promote an ?anti-inflammatory environment? in humans
. Among several compounds, curcumin, a polyphenol
found in the rhizome of Curcuma longa, is one of the
most studied bioactive compounds due to its
antiinflammatory properties [14?16]. Additionally, studies
have shown that physical exercise also promotes
antiinflammatory responses through several mechanisms,
such as increasing antioxidant activity , releasing
myokines and hormones , modulating immune cell
metabolism , and decreasing inflammation signaling .
Currently, the non-obese diabetic (NOD) mouse, a
model of spontaneous T1D, is the model that is most
similar to human T1D . Over the last 30 years, it has
been used in research to improve the science of
autoimmunity . As in humans, T1D occurs in NOD mice
due to immune cell infiltration into pancreatic islets
(insulitis) and subsequent destruction of ? cells.
Additionally, it has been demonstrated by ample literature that
TNF-?, a pro-inflammatory cytokine, has an important
role in the pathogenesis of T1D in NOD mice [23?27].
Some studies have tested bioactive compounds in
NOD mice. Supplementation with cocoa flavonoids or
green tea catechin reduced diabetes incidence in this
animal model [28, 29]. However, no studies have tested
the potential of exercise training to prevent T1D in
NOD mice. Therefore, this study aimed to investigate
the effects of exercise training and curcumin
supplementation on T1D progression in NOD mice.
Forty-eight female NOD mice, 5 weeks old, were
randomly divided into four groups (n = 12): control (C),
curcumin supplementation (CUR), trained (T), and
trained + curcumin supplementation (TC). The
experimental protocol lasted 20 weeks. Animals were housed
collectively (four animals per cage) and kept in
conventional conditions (22?24 ?C and 12-h light-dark cycle)
with unlimited access to water and food, which was a
pelletized AIN-93G diet . Food intake was monitored
three times a week. Body weight was measured weekly.
Blood glucose was measured every 14 days using a drop of
tail blood and a glucometer (FreeStyle Lite?Abbott). Mice
were considered diabetic when their blood glucose level
was higher than 250 mg/dL. Diabetic animals that lost
more than 25% of their body weight were killed before
20 weeks. All procedures were reviewed and approved by
the Ethics Committee on Animal Use in Research
(CETEA-FMRP, protocol no. 190/2014) in compliance
with the Ethical Principles in Animal Research adopted by
the National Council for the Control of Animal
Experimentation (CONCEA) and following the guidelines for
the care and use of laboratory animals .
Sample size was calculated based on the primary
outcome in this study, which was immune cell infiltration
into pancreatic islets (insulitis). As insulitis is a nominal
qualitative variable, the sample size was determined
using the following equation:
Considering that each section of pancreas has
approximately 10 islets and that in control groups,
approximately 60% have some level of insulitis even in the
absence of T1D , sample size was established to
reach statistical significance when the applied
interventions reduced the number of islets with some level of
insulitis at least by half (30%).
The AIN-93 diet was prepared twice a week, protected
from light, and kept frozen. The food offered to the CUR
and TC groups had curcumin added (500 mg/kg).
Curcumin was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Considering the daily food intake of the mice, the
curcumin dose reached approximately 65 mg/kg per day.
Exercise Training Protocol
All mice from the T and TC groups performed a maximal
incremental running test at the beginning of the
experimental protocol. This test began at 10 m/min and increased by
1.7 m/min every 2 min until exhaustion (determined when
the animal touched the bottom of the bay five times within
1 min). The speed at which exhaustion occurred was
considered 100%. Adjustment of exercise load was done every
4 weeks by repeating the same incremental test. According
to the animals? performance in the incremental test, they
were divided into three running groups (best, middle, and
lower) to guarantee that exercise intensity was maintained
and to respect the differences in mouse performance.
Exercise training was carried out for 20 weeks,
5 days a week. All exercise sessions were performed
in the morning. The exercise session consisted of a
5minute warm-up followed by 60 min of running at
training intensity and a 2-min cool down. Training
intensity varied during week sessions. On Monday,
Wednesday, and Friday, the mice ran at 60% of their
maximal speed reached in the incremental test; on
Tuesday and Thursday, mice had recovery sessions
running at 30% of the maximal speed.
Cytokine and Insulin Concentration
Following overnight fasting, the animals were euthanized
by decapitation 48 h after the last exercise session,
without previous anesthetic. The serum obtained was used
to determine glucose concentration by the colorimetric
method (Labtest, Lago Santa, Brazil), and frozen samples
(?70 ?C) were used to measure insulin, TNF-?, and IL-6
using Luminex xMAP? technology with a Milliplex? kit
(Millipore Corporation) analyzed on MAGPIX?; the
standard curve of all analytes had R2 = 1.
A homeostatic model assessment of insulin resistance
(HOMA-IR) was calculated to estimate insulin resistance
as previously described .
Pancreas was fixed in formalin (10%), embedded in
paraffin, and stained with hematoxylin-eosin. Images were
taken on an optical microscope (Olympus BX61VS).
Three sections of each pancreas were analyzed. Islets
were counted as previously described  and labeled
as: No Insulitis (absence of immune cells), Pre-Insulitis
(few immune cells around the islet), and Insulitis (>25%
of cells infiltrated and destruction of islet architecture).
Counts were performed manually by two independent
researchers (blinded). Insulitis extension was calculated
as previously described :
?NONo Insulitis? ? ?NO Pre Insulitis 2? ? ?NOInsulitis 3?
N OTotal Islets
Data are presented as the means ? standard deviation.
Analysis of variance (one-way ANOVA) followed by a
Tukey post hoc test was performed, except for HOMA-IR
and IL-6, which did not have a normal distribution and
were analyzed using a Kruskal-Wallis test. Weekly blood
glucose was analyzed using a repeated measure ANOVA.
A log-rank test was used to compare diabetes incidence. A
Chi-square test was used to compare insulitis distribution.
The level of significance adopted was p < 0.05.
Although the daily food intake was approximately 10%
greater in the trained group than in the control group,
body weight gain was approximately 41% lower in the
trained mice after 20 weeks. Only four animals
developed T1D, two each in groups C and CUR. Insulin levels
tended to be lower in CUR and T, but the difference
only reached a statistical level of significance in the TC
group. HOMA-IR was not modified by curcumin or
exercise training (Table 1). Blood glucose was similar
among groups during the 20 weeks, as seen in Fig. 1.
Despite the low incidence of T1D, insulitis was seen
in all groups but in different levels. The trained and
trained + curcumin groups had 50% less immune cell
infiltration in pancreatic islets than the sedentary
groups (Fig. 2a). In addition, insulitis extension was
also reduced with exercise training (Fig. 2b).
Regarding cytokine levels, IL-6 and TNF-? were not
modified by curcumin or exercise training (Fig. 3).
To the best of our knowledge, this study is the first to
show that exercise training has the potential to protect
pancreatic ? cells against an immune response in vivo.
However, curcumin supplementation failed to attenuate
insulitis in NOD mice.
Curcumin is a polyphenolic compound that exhibits low
bioavailability . The route of administration and the
dose of curcumin used in this study were established
based on previous studies that aimed to simulate a rich
polyphenol diet [36?39]. Although chronic oral curcumin
(500 ppm in diet) reached detectable concentrations in
plasma (0.035 ?g/ml) and in brain tissue (0.469 ?g/ml) in
a dose-dependent study , our study indicates that this
concentration was not enough to prevent an immune
response against pancreatic ? cells. However, intraperitoneal
curcumin supplementation (25 mg/kg body weight)
inhibited leucocyte infiltration in accelerated murine models of
T1D . These data suggest that the concentration of
curcumin required to modulate immune function could
not be reached using dietary strategies.
The low incidence of T1D observed was expected
because animals were fed a gluten-free diet and were not
Table 1 Food intake, weight gain, T1D incidence, insulin level, and HOMA-IR after the 20-week experimental protocol
Data are the mean ? standard deviation
aDifferent from control
Fig. 1 Blood glucose during the 20-week experimental protocol from
control (C), curcumin (CUR), trained (T), and trained + curcumin (TC).
NOD mice were 5 weeks old at the beginning. Data are
mean ? standard deviation
in a germ-free environment [41, 42]. However, NOD
mice present ~60% immune cell infiltration in islets even
in the absence of T1D , which is consistent with our
findings. Exercise training lowered this rate to 30%.
The effect of exercise training in diminishing body
weight gain is well documented and was observed in the
present study, showing the importance of physical exercise
for caloric balance. Lower body weight gain could be one
mechanism that explains the reduction of insulitis in the
T and TC groups. Since obesity induces chronic
inflammation  and the Overload Hypothesis proposes that
environment factors, such as obesity, increase T1D risk
, this line of thought is strengthened.
Another possible mechanism to explain the effects of
exercise training is through dendritic cells modulation. It is
well known that dendritic cells modulate both innate and
adaptive immune responses, and the role of these cells on
the development of T1D was recently demonstrated .
Several studies have shown that exercise training decreases
the number of dendritic cells or diminishes their response
[45?48]. Thus, exercise could mitigate autoimmunity by
shaping dendritic cell activation.
Regarding cytokine signaling, IL-6 has been recognized
as one of the myokines produced during exercise training
. In 2010, exercise training was documented as an
anti-inflammatory approach, which is able to prevent type
2 diabetes, cardiovascular diseases, cancer, and dementia
. Although no significant differences were seen in
cytokine levels, the CUR, T, and TC groups exhibited an
IL-6 concentration more than twofold higher than the C
group (p = 0.07). In an ex vivo study, the pancreas of
trained animals had fewer apoptosis biomarkers than
sedentary animals. When an IL-6 blocker was added to the
trained animals? pancreas, the apoptosis biomarkers rose
to sedentary levels. The authors concluded that the benefit
of exercise training on pancreatic ? cell survival is through
the IL-6 pathway . However, the results of the present
study cannot reinforce the role of IL-6 in preventing
insulitis because the CUR group had the same insulitis
markers as the C group, which suggests that the effect of
exercise training is due to other mechanisms.
Fig. 2 Quantitative/representative analysis of insulitis. Insulitis distribution (a), insulitis extension (b), and representative image used to label insulitis
level (c). Control (C), curcumin (CUR), trained (T), and trained + curcumin (TC).*Different from control; #different from curcumin
Fig. 3 Cytokine levels after the 20-week experimental protocol from
control (C), curcumin (CUR), trained (T), trained plus curcumin (TC)
In conclusion, moderate intensity exercise training has the
potential to protect pancreatic ? cells against an immune
response in NOD mice. The limitation of the present study
is a lack of mechanisms that establish a causal effect, as well
as the lack of an evaluation of other inflammatory markers,
and both pancreatic and blood oxidative stress, epinephrine
levels, and the characterization of infiltrating immune cells.
Those measurements are an important area of future
research. Therefore, additional and prospective studies are
needed to uncover the mechanisms that explain the link
between exercise training and autoimmunity.
AIN: American Institute of Nutrition; ANOVA: Analysis of variance; C: Control;
CONCEA: Conselho Nacional de Controle de Exeperimenta??o Animal;
CUR: Curcumin; FMRP: Faculdade de Medicina de Ribeir?o Preto;
HOMAIR: Homeostatic model assessment of insulin resistance; IL: Interleukin;
NOD: Non-obese diabetic; T: Trained; T1D: Type 1 diabetes; TC: Trained +
curcumin; TNF: Tumor necrosis factor
LKO conceived, designed the study, performed the experiments, analyzed the
data, and wrote the paper. CDM and AMN conceived and designed the study,
contributed to discussions, and edited the manuscript. All authors reviewed the
manuscript. All authors read and approved the final manuscript.
All applicable international, national, and/or institutional guidelines for the
care and use of animals were followed. This article does not contain any
studies with human participants performed by any of the authors.
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