The Journal of Nutrition. First published ahead of print November 20, 2013 as doi: 10.3945/jn.113.183186.

The Journal of Nutrition

Nutritional Immunology

L-Arginine Supplementation Prevents Increases in

Intestinal Permeability and Bacterial Translocation
in Male Swiss Mice Subjected to Physical Exercise
under Environmental Heat Stress13
Katia Anunciacao Costa,4 Anne Danieli Nascimento Soares,4 Samuel Penna Wanner,5,6
Rosana das Gracas Carvalho dos Santos,4 Simone Odlia Antunes Fernandes,7
Flaviano dos Santos Martins,8 Jacques Robert Nicoli,8 Candido Celso Coimbra,5
and Valbert Nascimento Cardoso7*
4
Department of Foods, Faculty of Pharmacy, 5Department of Physiology and Biophysics, Institute of Biological Sciences, 6Department of
Physical Education, School of Physical Education, Physiotherapy, and Occupational Therapy, 7Department of Clinical Analysis and
Toxicology, Faculty of Pharmacy, and 8Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas
Gerais, Belo Horizonte, Minas Gerais, Brazil

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Abstract
Dietary supplementation with L-arginine has been shown to improve the intestinal barrier in many experimental models.
This study therefore investigated the effects of arginine supplementation on the intestinal permeability and bacterial
translocation (BT) induced by prolonged physical exercise under heat stress. Under anesthesia, male Swiss mice (5 wk old)
were implanted with an abdominal sensor to record their core body temperature (Tcore). After recovering from surgery, the
mice were divided into 3 groups: a non-supplemented group that was fed the standard diet formulated by the American
Institute of Nutrition (AIN-93G; control), a non-supplemented group that was fed the AIN-93G diet and subjected to
exertional hyperthermia (H-NS), and a group supplemented with L-arginine at 2% and subjected to exertional hyperthermia
(H-Arg). After 7 d of treatment, the H-NS and H-Arg mice were forced to run on a treadmill (60 min, 8 m/min) in a warm
environment (34C). The control mice remained at 24C. Thirty minutes before the exercise or control trials, the mice
received a diethylenetriamine pentaacetic acid (DTPA) solution labeled with technetium-99m (99mTc-DTPA) or 99mTc-Escherichia
coli by gavage to assess intestinal permeability and BT, respectively. The H-NS mice terminated the exercise with Tcore values of
;40C, and, 4 hours later, presented a 12-fold increase in the blood uptake of 99mTc-DTPA and higher bacterial contents in the
blood and liver than the control mice. Although supplementation with arginine did not change the exercise-induced increase in
Tcore, it prevented the increases in intestinal permeability and BT caused by exertional hyperthermia. Our results indicate that
dietary L-arginine supplementation preserves the integrity of the intestinal epithelium during exercise under heat stress, acting
through mechanisms that are independent of Tcore regulation. J. Nutr. doi: 10.3945/jn.113.183186.

Introduction
Several studies have demonstrated that immunonutrients, such
as L-arginine, reduce morbidity due to infections, prevent
damage to the intestinal mucosa, and aid in the recovery of
the intestinal barrier after specific damage (13). Arginine is a
1
Supported by Coordenacao de Aperfeicoamento de Pessoal de Nvel Superior protein synthesis, in intestinal healing, and in the control of
(CAPES/Brazil), Fundacao de Amparo a Pesquisa do Estado de Minas Gerais
(FAPEMIG), Conselho Nacional de Pesquisa (CNPq/Brazil), and Pro-Reitoria de
Pesquisa da Universidade Federal de Minas Gerais (PRPq/UFMG).
2
Author disclosures: K. A. Costa, A. D. N. Soares, S. P. Wanner, R. d. G. C. d.
Santos, S. O. A. Fernandes, F. d. S. Martins, J. R. Nicoli, C. C. Coimbra, and
V. N. Cardoso, no conflicts of interest.
3
Supplemental Tables 1 and 2 are available from the Online Supporting
Material link in the online posting of the article and from the same link in the
online table of contents at http://jn.nutrition.org.
* To whom correspondence should be addressed. E-mail: valbertcardoso@
yahoo.com.br.
conditionally essential amino acid because its nutritional
requirement increases under conditions of physiologic stress to
the organism, including trauma and sepsis (46). Arginine is
used by multiple metabolic pathways, including the polyamine
and NO synthesis pathways. These metabolites play important
roles in cellular signaling in enterocytes, in the stimulation of
blood flow and immune function (7,8).
Bacterial translocation (BT)9 is associated with several
different critical conditions, including surgery, shock, ischemia,
9
Abbreviations used: AIN-93G, American Institute of Nutrition diet; BT, bacterial
translocation; C-NS, control and non-supplemented; cpm, counts per minute;
DTPA, diethylenetriamine pentaacetic acid; H-Arg, exertional hyperthermia and
arginine-supplemented; H-NS, exertional hyperthermia and non-supplemented;
MBq, Megabecquerel; MLN, mesenteric lymph node; sIgA, secretory immunoglobulin
A; 99m Tc, technetium-99m; Tcore, core body temperature.

2014 American Society for Nutrition.

Manuscript received July 31, 2013. Initial review completed August 29, 2013. Revision accepted November 1, 2013. 1 of 6
doi: 10.3945/jn.113.183186.
Copyright (C) 2013 by the American Society for Nutrition
and intestinal obstruction (9,10). Under these conditions, the
balance of the immune system function is lost, and the intestinal
mucosal barrier is damaged, making patients susceptible to
microbial invasion and the development of sepsis and multiple
organ failure (11). In addition to the pathologic conditions
enumerated above, severe hyperthermia, typically defined as a
core body temperature (T core) above 40C, also affects intestinal
function and may contribute to the occurrence of a disseminated
systemic inflammatory response. When associated with central
nervous system disturbances and multiple organ system failure,
severe hyperthermia is usually defined as heat stroke (12). Heat
stroke is a life-threatening illness characterized by myriad
inflammation, coagulation, and tissue abnormalities whose
severity and time course of progression vary widely between
individuals (13).
Prolonged dynamic exercise, particularly when performed
in hot environments, shifts the blood flow from the splanchnic
and renal vascular beds to the muscular and skin vascular beds
(14). This blood flow redistribution is important to ensure
the adequate supply of energetic substrates and oxygen to the
contracting muscles and for convective heat loss through the
blood vessels of the skin. However, depending on the amount of
environmental heat stress and the exercise intensity, splanchnic
vasoconstriction can result in local ischemia, which can enhance
both nitrosative and oxidative stress (15). This higher level of
metabolic stress, which is associated with the cytotoxic effects of
severe exertional hyperthermia, may disrupt the integrity of the
intestinal barrier, allowing the passage of bacteria from the
intestine to the systemic circulation (1618).
Supplementation with dietary L-arginine is a common prac-
tice among athletes and people who exercise regularly. There is
evidence that increasing the bioavailability of NO through
dietary supplementation with either L-arginine or nitrate allows
greater tolerance to physical exertion (19,20). Moreover, dietary
supplementation with arginine attenuates the cardiac oxidative
stress and inflammatory responses induced by exhausting exercise
(21). Given that treatment with arginine preserves the intestinal
barrier and reduces the BT induced by different experimental
models (2225), we hypothesized that dietary supplementation
with this amino acid would attenuate BT in mice subjected to
prolonged treadmill running under hot conditions, thereby decreas-
ing the risks associated with physical exertion under extreme
environmental conditions. In this context, the present study
investigated the effect of dietary arginine on intestinal perme-
ability and BT in mice subjected to forced treadmill-running
exercise under environmental heat stress.
Materials and Methods
Animals and diets. Male Swiss mice (4 wk old) weighing 1820 g were
provided by the animal care center at the Faculty of Pharmacy (Universidade
Federal de Minas Gerais) and were used in all experiments. The mice were
housed in individual cages under controlled light (05001900 h) and
temperature (24.0 6 2.0C) conditions with water and unpurified diet
provided ad libitum. The experiments were approved by the local Ethics
Committee for Animal Experimentation (protocol number 006/2011) and
complied with the Guide for the Care and Use of Laboratory Animals
published by the Institute of Laboratory Animal Resources.
The mice were surgically implanted with abdominal temperature
sensors, allowed to recover, and then divided into 3 groups: 1) mice fed
the standard American Institute of Nutrition diet (AIN-93G) and not
subjected to exertional hyperthermia [control/non-supplemented (C-NS)],
2) mice fed the standard AIN-93G diet and subjected to forced exercise
in the heat [hyperthermia/non-supplemented (H-NS)], and 3) mice fed
the AIN-93G diet supplemented with arginine at 2% of the total caloric
2 of 6 Costa et al.
content and subjected to forced exercise in the heat [hyperthermia/
supplemented (H-Arg)] (24,25). The standard AIN-93G diet was formu-
lated for growth, pregnancy, and lactation of rodents by the American
Institute of Nutrition and has been used extensively (26). The standard
and arginine-supplemented diets were isocaloric and isoproteic (Supple-
mental Table 1). In the supplemented diet, a portion of the casein was
replaced with L-arginine (Sigma-Aldrich).
The mice were fed the standard or supplemented diet for 7 d. Food
intake and body mass were measured once per day, and during the last
5 d of the supplementation period the mice were also familiarized with
running on a treadmill. On the day after completing the dietary monitoring
and the familiarization sessions, the mice were subjected to the experi-
mental trials.
Implantation of the abdominal temperature sensor. A telemetry
transmitter (G2 E-Mitter series; Mini Mitter) was implanted in the
abdominal cavity of each mouse to record the temperature. The mice
were weighed and anesthetized with ketamine (80 mg/kg body mass, i.p.)
and xylazine (8 mg/kg body mass, i.p.). A small incision was made in the
linea alba of the abdominal muscle, and the peritoneal cavity was exposed.
The sensor was inserted and sutured to the left lateral abdominal wall. The
abdominal muscle and skin were then sutured in layers (27,28). After a 5-d
recovery period, the mice were subjected to the running familiarization
protocol. This recovery period was sufficiently long for the mice to recover
and regain their presurgical body mass.
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Familiarization with the running exercise on a treadmill. The
familiarization protocol consisted of running on a treadmill for small
rodents (Modular Treadmill; Columbus Instruments) for 5 consecutive
days. Mice were encouraged to run by light electrical stimulation (0.5 mA)
provided by a grid located at the rear end of the treadmill belt. The
familiarization protocol has been described in detail elsewhere (28).
After the familiarization protocol, the mice were able to run for 5 min at
a constant speed of 8 m/min at a 5% incline with minimal exposure to
the electrical stimulation.
Experimental trials. On the day of the experimental trials, all mice
were weighed and allowed to rest for at least 2 h in the experimental
room. The mice from the hyperthermic groups were then transferred
from their home cages to the motor-driven treadmill. The mice in the
C-NS group were kept in their home cages at a room temperature of
24C, instead of being transferred to the treadmill.
Forced treadmill running at a constant speed of 8 m/min (5% inclination)
for 60 min in the heat (34C) was used to produce hyperthermia. Mice
subjected to this protocol, achieved T core values of ;40C (28), with no
interruption of the exertion before the predetermined exercise period had
finished. To heat the environment inside the chamber, an electric heater
(model AB1100; Britania) was positioned at the same level 45 cm distant
from the fan and turned on at 1200 W (29). The ambient temperature was
measured by using a thermocouple (YSI-400A; Yellow Springs Instruments)
that was placed inside the acrylic chamber that contained the treadmill. The
thermocouple was positioned on the ceiling halfway between the fan and the
electric grid.
Abdominal temperature was measured by telemetry every 30 s and used
as the T core value. The radio waves emitted by the abdominal sensors during
the experiments were captured by a receiving plate positioned on the side of
the treadmill. The frequency of the radio waves was converted into a
temperature value by using a data acquisition system (Vital View; Mini
Mitter). Abdominal temperature was measured in all mice used in the present
study, regardless of whether they had been assigned to experiments for
measuring the intestinal permeability, BT, or immunoglobulin concentrations.
Intestinal permeability. Intestinal permeability was assessed on the
basis of the diffusion of diethylenetriamine pentaacetic acid (DTPA)
solution labeled with technetium-99m (99mTc) administered by gavage.
DTPA is a macromolecule that rarely crosses the gastrointestinal barrier.
However, when intestinal permeability is increased as a result of damage
to the mucosa, DTPA permeation through the intestine (paracellular
pathway) is permitted, and greater concentrations of this macromolecule
can be measured in the bloodstream and urine (30).
 The mice from the C-NS, H-NS, and H-Arg groups (n = 18 for each TABLE 1 Body mass gain and daily food, calorie, nitrogen, and
group) received 0.1 mL of 13 MBq 99mTc-DTPA by gavage 30 min before arginine intakes of C-NS, H-NS, and H-Arg mice1
being subjected to the experimental trials (i.e., the 60 min of rest at temperate
conditions or physical exercise in the heat). At 1, 4, or 6 h after the trials, Experimental groups
groups of 6 mice were anesthetized, and 400 mL of blood was collected via
C-NS H-NS H-Arg P
cardiac puncture and placed in appropriate tubes for radioactivity quantifi-
cation. The percentage of the administered dose present in the blood was 2
Body mass gain , g 6.9 6 0.2 7.2 6 0.2 7.5 6 0.3 0.09
calculated by using the following equation: % dose = (cpm in blood/cpm of Food intake, g/d 6.7 6 0.2 6.5 6 0.2 6.9 6 0.2 0.30
standard) 3 100; where cpm represents the counts of radioactivity per minute.
Caloric intake, kcal/d 17.6 6 0.5 17.3 6 0.4 18.3 6 0.4 0.30
Nitrogen intake, g/d 0.15 6 0.00 0.15 6 0.00 0.16 6 0.00 0.30
BT. Three more groups of mice (n = 8 each) were used for the BT study.
Arginine intake, g/d 0.03 6 0.00a 0.03 6 0.00a 0.13 6 0.00b ,0.001
Thirty minutes before being subjected to the experimental trials, all of
the mice received 0.1 mL (1.8 MBq) of 99mTc-Escherichia coli 1
Values are means 6 SEMs, n = 32. Labeled means in a row without a common
containing 108 CFU by gavage. The procedures for radiolabeling the letter differ, P , 0.05. C-NS, control and non-supplemented; H-Arg, exertional
E. coli were performed as described by Diniz et al. (31). The percentage hyperthermia and arginine-supplemented; H-NS, exertional hyperthermia and non-
of 99mTc incorporated into the bacterial cells was determined by using supplemented.
2
the following equation: % labeling = (cpm of precipitate/cpm of During the 7 d of treatment with control or supplemented diets.
precipitate + cpm of supernatant) 3 100.
The mice were anesthetized and killed by decapitation at 4 h after the (measured as the T core index) of 37.47 6 0.12C during the
experimental trials. Blood, mesenteric lymph node (MLN), liver, spleen, 60 min of recording. Running at 34C induced a sharp and marked
lungs, and brain samples were collected, weighed, and placed in tubes for increase in T core. The temperature in the H-NS group was higher
radioactivity quantification. The samples were counted in an automatic than that in the C-NS group from minute 2 until the end of the
g counter (1480 model, Wallac Wizard; Perkin Elmer). The results are exercise period under heat stress (39.62 6 0.09C vs. 37.47 6
expressed as cpm/g of tissue or cpm/mL (for the blood only).
0.12C at minute 60; P < 0.001). The exertional hyperthermia in

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Immunoglobulin analysis. After being killed, the small intestines of other
the H-Arg group was not different from that in the H-NS group
mice that belonged to the same 3 groups (C-NS, H-NS, and H-Arg; n = 6 for throughout the exercise period under environmental heat stress (H-
each group) were collected, and the intestinal contents were withdrawn, NS vs. H-Arg: 39.62 6 0.09C vs. 39.41 6 0.09). Both the H-NS
weighed, and resuspended in PBS. The secretory immunoglobulin A (sIgA) and H-Arg mice reached T core values close to 40C within 15 min
concentration in the intestinal fluid was evaluated by ELISA using a goat anti- after the exercise had started; thereafter, T core values remained
mouse IgA (Sigma Chemical Co.) and a horseradish peroxidaseconjugated elevated until the end of the exercise period (Fig. 1).
goat anti-mouse IgA (Sigma), as previously described by Martins et al. (32).
The measurements were performed in duplicate. The sIgA is an important Intestinal permeability. The mice from the H-NS group
component of the intestinal immunity system that reduces the number of exhibited higher intestinal permeability than the mice from the
epithelium-adherent bacteria and limits BT (33).
C-NS group 4 h after the end of the experimental trials (Fig. 2);
Statistical analysis. All variables were tested for normality by using the
however, no differences were observed between these 2 groups
Shapiro-Wilk test and for homogeneity of variance by using the Levene at 1 and 6 h after the end of the trials. Mice fed arginine and
test. The normally distributed data are expressed as means 6 SEMs. The subjected to exertional hyperthermia had reduced intestinal
abdominal temperature curves were compared between the experimental permeability compared with mice from the H-NS group 4 h after
groups and time points by using 2-factor ANOVA, with repeated the exercise. Moreover, the intestinal permeability of the H-Arg
measures only for the time factor. Increases in body mass; daily intakes of group was not different from that of the C-NS group at any of
unpurified diet, calories, and nitrogen; intestinal permeability data; and the time points studied (Fig. 2).
sIgA concentrations were compared between the experimental groups by
using 1-factor ANOVA. To select the most appropriate post hoc test, the BT. In the next step, the effects of exertional hyperthermia and
CV for each variable was calculated. When the CV was <15%, Tukeys
arginine supplementation on BT were investigated at 4 h after
test was chosen, and when the CV was 15%, Duncans test was used
(34). The correlation between the thermoregulatory variables and
the end of the experimental trials (i.e., the time point at which
intestinal permeability was assessed by using the Pearson coefficient.
The BT data were expressed as medians because they were not
normally distributed. The BT was compared between the experimental
groups by using the nonparametric Kruskal-Wallis ANOVA, followed by
Dunns post hoc test. The correlation between the thermoregulatory
variables and BT was assessed by using the Spearman coefficient.
The level of significance was set at P < 0.05. All analyses were performed
by using the program Sigma Plot version 11.0 (Systat Software, Inc.).

Results
Body mass gain and food intake. Body mass gain during the
7 d of treatment with control or arginine supplemented diets and
the daily food, caloric, and nitrogen intakes were similar among
all groups studied. The arginine intake of the mice in the H-Arg
group was ;3 times higher than that of the non-supplemented
groups (Table 1). FIGURE 1 Abdominal temperature of C-NS, H-NS, and H-Arg mice
during the experimental trials. Values are means 6 SEMs, n = 32. *Different
Abdominal temperature during the experimental trials. from C-NS mice, P , 0.001. C-NS, control and non-supplemented; H-Arg,
The mice that were allowed to move freely in their home cages at exertional hyperthermia and arginine-supplemented; H-NS, exertional hyper-
24C (C-NS group) had an average abdominal temperature thermia and non-supplemented.

Arginine, bacterial translocation, and exercise 3 of 6


FIGURE 2 Intestinal permeability determined as the blood concen-
tration of 99mTc-DTPA for C-NS, H-NS, and H-Arg mice 1, 4, and 6 h
after the end of the experimental trials. Values are means 6 SEMs,
n = 18. *Different from C-NS and H-Arg, P , 0.05. % dose = [(cpm in
blood 3 100)/cpm of administered dose]. cpm, counts of radioactivity
per minute; C-NS, control and non-supplemented; DTPA, diethylenetri-
amine pentaacetic acid; H-Arg, exertional hyperthermia and arginine-
supplemented; H-NS, exertional hyperthermia and non-supplemented;
99m
Tc, technetium-99m.
hyperthermia had significantly increased intestinal permeabil-
ity). No differences were observed in the amounts of 99mTc-E.
coli in the MLN, spleen, lungs, or brain between the 3 groups
(Table 2). In contrast, BT to the blood and liver was higher in the
H-NS group than in the C-NS and H-Arg groups, indicating that
dietary supplementation with arginine prevented exertional
hyperthermia-induced BT to the blood and liver.
sIgA secretion in the intestinal fluid. The intestinal fluid sIgA
concentration was lower in the C-NS group (0.70 6 0.14 mg/g)
than in the H-NS (1.16 6 0.12 mg/g) and H-Arg (1.38 6 0.08
mg/g) groups (P < 0.01), which did not differ from one another.
Correlation analyses. Correlation analyses were performed
to determine whether the effects of arginine supplementation
on intestinal permeability and BT were associated with changes in
thermoregulation. The intestinal permeability and BT variables of
the mice in the H-NS and H-Arg groups were not significantly
associated with any of the thermoregulatory variables calculated
(Supplemental Table 2).
Discussion
In the present study, the effects of dietary supplementation with
arginine on the intestinal barrier and BT were investigated in
mice subjected to prolonged physical exercise under heat stress.
We observed that exercise performed at 34C increased the T core
to ;40C and induced a greater degree of intestinal permeability
and BT to the blood and liver than was observed in mice allowed
to move freely at temperate conditions. Supplementation with
arginine prevented the increases in both intestinal permeability
and BT induced by prolonged running under heat stress but did
not affect the magnitude of exercise-induced hyperthermia.
The mice from all groups received isocaloric and isoproteic
diets (Supplemental Table 1), and they exhibited similar food intakes
and body mass gains (Table 1). The mice in the supplemented group
received arginine at 2% of the total caloric value and consumed ;3
times more arginine than the C-NS and H-NS groups. Taken
together, these data indicate that the experimental outcomes
observed in the supplemented group can be attributed to the
4 of 6 Costa et al.
effects of arginine alone and not to differences in protein or
calorie levels.
Increased intestinal permeability was observed in the mice
forced to exercise under heat stress at 4 h after the running
protocol; however, 2 h later, the gastrointestinal function of the
H-NS mice had recovered, and the intestinal permeability of
these mice was similar to that of the C-NS mice at this time
point (Fig. 2). These results suggest that the increase in intestinal
permeability induced by physical exercise (under the conditions
of the current investigation) is short-lived compared with the
intestinal permeability observed in other experimental models,
such as intestinal obstruction, in which intestinal permeability
gradually increases with time, with maximal values observed 18
h after the obstruction procedure (3537).
Dysfunctions in the intestinal barrier, with consequent
increases in permeability, have been suggested to be predisposing
factors for BT (38). One of the proposed mechanisms underlying
the greater permeation of molecules from the gut is the release of
circulating and local proinflammatory cytokines, which pro-
mote the contraction of the epithelial cell cytoskeleton, thus
opening the paracellular space and allowing BT (39). Because
increased intestinal permeability is associated with BT, our next
step was to evaluate the bacterial biodistribution in mice subjected
Downloaded from jn.nutrition.org by guest on January 28, 2017
to physical exercise under heat stress. The hyperthermia induced
by prolonged exercise at 34C increased BT to the blood and
liver at 4 h after the running protocol (Table 2); this time point
corresponds to the time at which the increased intestinal perme-
ability was observed. Our results are consistent with the results
of experiments in humans that found exercise-induced increases
in intestinal permeability (17,40) and in plasma LPS concentra-
tions (41,42).
The physiologic stress model used in the current investigation
likely favored the direct passage of bacteria through the portal
circulation. According to Wiest and Ratch (43), there are multiple
potential routes by which a microorganism can translocate from
the gut to extraintestinal sites. BT may occur through direct
migration across the intestinal wall, via the MLNs, or via the
portal circulation. In contrast to our findings, other studies have
detected bacteria at similar time points in the MLN and other
organs or body compartments, such as the spleen, liver, peritoneal
cavity, lungs, and blood (25,37). Therefore, it is likely that the
bacterial dissemination and the route of BT vary according to the
type and magnitude of the inflammatory stimulus (44).
The increases in BT observed in the H-NS mice most likely
occurred in response to the reduced splanchnic blood flow and
TABLE 2 Bacterial translocation, as determined by the
biodistribution of 99mTc- Escherichia coli in the organs and blood
of C-NS, H-NS, and H-Arg mice1
Experimental groups
C-NS H-NS H-Arg P
Blood, cpm/mL 3 10 3
0.26 (0.230.29)a
0.63 (0.511.03)b
0.38 (0.190.49)a
0.01
Liver, cpm/g 3 103 0.96 (0.811.26)a 2.61 (2.255.52)b 1.36 (0.941.44)a 0.01
MLN, cpm/g 3 103 1.45 (0.155.16) 1.86 (0.252.23) 0.56 (0.310.90) 0.35
Spleen, cpm/g 3 103 0.29 (0.160.48) 0.35 (0.230.52) 0.28 (0.240.43) 0.90
Lung, cpm/g 3 103 0.59 (0.271.18) 0.56 (0.271.24) 0.55 (0.330.89) 0.99
Brain, cpm/g 3 103 0.10 (0.090.23) 0.24 (0.121.09) 0.12 (0.070.23) 0.25
1
Values are medians (firstthird quartiles), n = 8. Labeled medians in a row without a
common letter differ, P , 0.05. C-NS, control and non-supplemented; cpm, counts per
minute; H-Arg, exertional hyperthermia and arginine-supplemented; H-NS, exertional
hyperthermia and non-supplemented; MLN, mesenteric lymph node; 99mTc, techne-
tium-99m.
consequent tissue hypoxia, which may have damaged the
intestinal mucosal barrier and increased the intestinal permea-
bility (45). No increases in the number of the 99mTc-E. coli were
observed in the MLNs, spleens, lungs, or brains of the H-NS
mice, suggesting that the blood and liver macrophages could
phagocytose most of the translocated bacteria, decreasing the
circulating concentrations of 99mTc-E. coli and their diffusion to
other organs. These results are less dramatic than those produced
by our research group using an intestinal obstruction model. In
that model, greater bacterial spreading was observed, because the
bacteria reached all organs and body compartments investigated
(25,35,36). The lower extent of BTobserved after physical exercise
under heat stress suggests that the conditions used to induce hyper-
thermia (T core reached nonlethal levels, mice could drink water and
were immediately removed from heat stress after completing the 60-
min running protocol) likely did not promote a severe inflammatory
response that would be associated with high amounts of BT. Al-
though the magnitude of hyperthermia was not sufficiently severe to
induce sepsis, the present experimental model has high ecological
validity because it mimics the routines of athletes who undergo
prolonged exertion in warm climates, when Tcore values 40C are
often recorded (41,46).
Pretreatment with arginine prevented the exercise-mediated
increase in intestinal permeability (Fig. 2) and BT to the blood
and liver (Table 2). The values recorded in the H-Arg mice were
similar to those measured in the C-NS mice. We also observed
that dietary arginine supplementation did not affect the exercise-
induced increase in T core values (Fig. 1). These findings indicate
that the prevention of increased intestinal permeability and BT
in the supplemented group was not a consequence of attenuating
hyperthermia during exercise under heat stress. Moreover, in the
context of the previous evidence that central NO protects the
body against exaggerated hyperthermia during exercise (47), our
data suggest that dietary L-arginine does not increase the brain
bioavailability of NO to concentrations that would produce
thermoregulatory effects.
Previous studies have demonstrated the importance of argi-
nine in hypercatabolic, stress-associated states, including endo-
toxemia induced by LPS (2,24,25). Dietary supplementation
with arginine prevents BT by reducing histologic necrosis,
increasing villous height, and attenuating gut mucosal injury
(8,25). These protective cellular effects, which have been attrib-
uted to the actions of polyamines or NO, allow the maintenance of
high rates of protein synthesis and intestinal healing, modulate
the immune response, and prevent vascular stress (4850). There is
evidence that arginine, via NO synthesis and the consequent
production of intestinal sIgA, modulates the expression of Th1/
Th2 cytokines, preventing exaggerated inflammatory responses
(51,52). However, in the present study, the H-Arg group did not
exhibit significantly higher sIgA concentrations than the H-NS
group. Therefore, the arginine-mediated prevention of the in-
creases in intestinal permeability and BT was independent of
sIgA synthesis. Other mechanisms that are likely involved in
the preventive action of arginine but were not investigated here
include the inhibition of oxidative stress in enterocytes, improved
macrophage function, decreased expression of intestinal proin-
flammatory cytokines, and maintenance of tight junction struc-
ture (2,48). Moreover, changes in the composition of intestinal
microbiota may have also accounted for the protection associated
with arginine supplementation, because arginine has beneficial
effects on swine nutrition by regulating amino acid utilization and
metabolism in the small-intestinal microbiota (53).
In conclusion, supplementation with dietary arginine was an
effective intervention that preserved the intestinal barrier and
prevented BT during prolonged physical exercise under heat
stress. The arginine-mediated effects on intestinal permeability
and BT were not due to the attenuation of exertional hyperther-
mia or an increased secretion of intestinal IgA. Therefore, this
investigation provides preliminary evidence that dietary arginine
supplementation may represent an important intervention to
attenuate the intestinal dysfunction caused by exertional hyper-
thermia.
Acknowledgments
We thank Vanderli Pacheco da Silva for technical assistance.
K.A.C., C.C.C., and V.N.C. designed the research; K.A.C.,
A.D.N.S., S.P.W., R.d.G.C.d.S., and F.d.S.M. conducted the
research; K.A.C., S.P.W., S.O.A.F., J.R.N., C.C.C., and V.N.C.
analyzed the data; K.A.C., S.P.W., and V.N.C. wrote the
manuscript; V.N.C. had primary responsibility for final content.
All authors read and approved the final manuscript.
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