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					Exercise and Adrenal Function                                                                           18

                                         Journal of Exercise Physiologyonline
                                                 Volume 11 Number 1 February 2008

 Managing Editor                              Systems Physiology: Immune Function
  Tommy Boone, Ph.D.
  Jon K. Linderman, Ph.D.
 Review Board
                                ACUTE TREADMILL RUNNING EXERCISE IN RATS
  Todd Astorino, Ph.D.
  Tommy Boone, Ph.D.            CLAUDIO GOBATTO1, MARIA MELLO1
  Lance Dalleck, Ph.D.
  Dan Drury, DPE.               1
  Hermann Engals, Ph.D.          Department of Physical Education/ UNESP/ Sao Paulo State University,
  Eric Goulet, Ph.D.            Rio Claro, Sao Paulo, Brazil.
  Robert Gotshall, Ph.D.
  Melissa Knight-Maloney,                                      ABSTRACT
  Len Kravitz, Ph.D.
  James Laskin, Ph.D.
                                Contarteze RVL, Manchado-Gobatto FB, Gobatto CA, Mello MAR.
  Derek Marks, Ph.D.            Different stress biomarkers sensitivity during acute treadmill running
  Cristine Mermier, Ph.D.       exercise in rats. JEPonline 2008;11(1):18-27. The level of stress during
  Daryl Parker, Ph.D.           acute or chronic exercise is important since higher levels of stress may
  Robert Robergs, Ph.D.         impair homeostasis. The adrenal gland is an essential stress-responsive
  Brent Ruby, Ph.D.
  Jason Siegler, Ph.D.
                                organ involved in the hypothalamic-pituitary-adrenal axis. The aim of the
  Greg Tardie, Ph.D.            study was to analyze the sensitivity of different stress biomarkers of the
  Chantal Vella, Ph.D.          adrenal gland during acute treadmill running at different intensities.
  Lesley White, Ph.D.           Adult rats performed three 25 min running tests at velocities of 15, 20
  Ben Zhou, Ph.D.               and 25 m/min, for determination of maximum lactate steady state
                                (MLSS). After obtaining individual MLSS animals were assigned to two
  Official Research Journal     groups: M, sacrificed after 25 minutes of exercise at MLSS, and AM,
 of The American Society of     sacrificed after exercise at 25% above MLSS. For comparison, a control
    Exercise Physiologists      group C was sacrificed at rest. Blood corticosterone concentrations, as
            (ASEP)              well, adrenal gland cholesterol and ascorbic acid concentrations were
      ISSN 1097-9751
                                used as biomarkers. Serum corticosterone concentrations were higher
                                after exercise in both M (1802,74±700,42) and AM (2027,96±724,94)
                                groups when compared C group (467,11±262,12), but were not different
                                as a function of exercise intensity. No difference in adrenal ascorbic acid
                                (M=2,37±0,66; AM=2,11±0,50 and C=2,54±0,53) and cholesterol
                                (M=1,04±0,12; AM=0,91±0,31 and C=1,15±0,40) levels were observed
                                when the three groups were compared. Serum corticosterone
                                concentrations showed to be sensitive to acute treadmill exercise
                                intensity. On the other hand, ascorbic acid and cholesterol
                                concentrations in adrenal were biomarkers not adequate to evaluate
                                exercise stress in rats.

                                Key Words: Ascorbic Acid, Cholesterol, Adrenal Gland, Corticosterone

  Exercise and Adrenal Function                                                                      19

Studies using animal models search to mimic the stress conditions that human beings are submitted
to, aiming at analyzing systemic, cellular and molecular alterations due to physical exercise practice
(1,2). However, physical exercise protocols designed for animals must simulate adequately situations
human beings are exposed to.

Studies on the physiological effects of exercise in rats demonstrated that the application of forced
endurance exercises causes an adverse situation to the organism in which the stress system may be
activated with large magnitude (3,4) In a number of stress situations such as exercise, fracture,
hypoglycemia and cold, glucocorticoids (in man basically cortisol, and corticosterone in most rodents)
contribute for the mobilization of energetic substrates with the objective of recovering injured tissues
and promoting organic homeostasis (1,2).However, long term exposition to high glucocorticoid
concentrations may unchain undesirable responses both for man and animal (5,6).

In the skeletal muscle, corticosterone acts directly promoting protein degradation especially in the
glycolytic fibers-rich white muscles (7,8,2). The treatment of L6 myotube culture with dexamethasone
(synthetic glucocorticoid) resulted in protein degradation through the nuclear cofactor p300 (9).
However, the most known muscular proteolysis mechanism induced by glucocorticoids is the
ubiquitin-proteasome system (10,11). The quick mobilization of aminoacids from muscular supplies
makes them available both as energy source and for the synthesis of other compounds (12,13,14).
Such situation, although useful in the homeostasis preservation, causes the weakening of the muscle
function and body protein unbalance (15). Therefore, acute/chronic forced exercises performed by
animal models may cause elevation of the corticosterone activity and alter the expected exercise
responses. Such situation shows the relevance of identifying the stress level of the animal during

The adrenal glands play a key role in the stress reactions, once they are involved both in the
hypothalamus-pituitary-adrenal axis (HPA) and the sympatho-adrenomedullary system (16,17,18).
The serum corticosterone concentration, final hormone of the HPA axis, is considered an important
stress biomarker (19,5). Knowing that the corticosterone synthesis by the adrenal cortex is derived
from the cholesterol molecule through an oxidation-reduction process, with the participation of the
ascorbic acid, another stress biomarker, which may be used, is the depletion of the cholesterol and
ascorbic acid concentrations in the adrenal glands (20,21,22).

It is already known in human beings that the exercise intensity changes the stress conditions, among
other aspects, and this condition results in distinct physiological responses in relation to the
acute/chronic effort. However, few studies involving different effort intensities in animals and their
specific stress responses are found in the literature (23).

The maximal lactate steady state protocol (MLSS) is considered the “gold standard” method for
determination of aerobic/ anaerobic metabolism transition (23,24). Our group initially described a
protocol for the individual MLSS determination in swimming rats, through exercises with continuous
loads (25). Later, using the same principle of exercise with continuous loads, Manchado et al. (26)
found individual MLSS of sedentary rats during treadmill running exercise.

Considering the importance of evaluating the stress level of animals submitted to exercise and the
possible effects of the different intensities in these responses, the aim of the present study was to
analyze which biomarker (serum corticosterone concentration or cholesterol and ascorbic acid
concentrations in the adrenal glands) is the most sensitive to measure the stress level of rats during
acute treadmill running exercise at different intensities. The intensities used in the present study
  Exercise and Adrenal Function                                                                       20

were: MLSS, interpreted as the maximal intensity with aerobic predominance and 25% above MLSS,
considered characteristically as anaerobic.

Sixty days old male Wistar rats (Rattus Norvegicus albinus) (n=23) at the beginning of the experiment
were used. The animals, provenient from the São Paulo State University UNESP Central Bioterium –
Botucatu/SP, were kept at the Bioterium of the Biodynamic Laboratory – Department of Physical
Education – UNESP – Rio Claro, in collective cages (5 rats per cage, all form the same experimental
group) at temperature of 25±1ºC and 12/12 hours light/dark photoperiod, with free access to water
and food (standard chow for rodents – Purina). All experiments involving animals were in agreement
with the European Convention for the Protection of Vertebrate Animals used for Experimental and
other Scientific Purposes (Council of Europe n. 123, Strasburg, 1985).

Experimental groups
The animals were distributed into 3 groups, according to sacrifice condition as follows:
    Maximal Lactate Steady State in Treadmill Running (M): group composed of seven rats
      that were selected, adapted to treadmill running and submitted to the maximal lactate steady
      state test, which were sacrificed shortly after 25-minutes of continuous exercise at maximal
      lactate steady state intensity.
    Above the Maximal Lactate Steady State Intensity in Treadmill Running (AM): group
      composed of six rats that were selected, adapted to treadmill running and submitted to the
      maximal lactate steady state test, which were sacrificed shortly after exhaustive exercise at
      intensity 25% above the maximal lactate steady state.
    Control (C): group composed of ten rats that were sacrificed in rest.

Selection of the running rats and adaptation to treadmill exercise
The selection of running rats occurred for 10 consecutive days with animals running for 5 minutes at
velocity of 15 m/min. The animals that ran for 9 or 10 days were selected for the experiment. Two
days later, the selected animals were adapted to the treadmill exercise. This adaptation consisted of
daily running performed 5 days/week during 3 weeks, with duration of 1-5 min and velocity 5-10
m/min, in a graded adaptation process. The adaptation was aimed at reducing the ergometer-induced
stress without promoting physiological alterations related to physical training, since short duration and
slow velocity were utilized.

Maximal lactate steady state test (MLSS) in treadmill running
After adapted to exercise, all running animals were submitted to 3 exercise tests at velocities of 15,
20 and 25 m/min. The tests were conducted with 48 hours of interval between them with velocities
sequence randomly distributed. Each test consisted of 25 minutes of continuous running at velocity
previously established or until exhaustion. Blood was collected, through a small incision at the tip of
the tail, in rest and each 5 minutes of exercise during the test. One single incision at the beginning of
the test was sufficient to complete all blood draws. The blood collection was performed in 30s in order
to avoid that the removal of the animal from the treadmill for a long period of time caused additional
stress, thus interfering on the exercise response. The lacticemia level during the test was considered
as stable when no difference above 1 mmol/L was observed from 10 to 25 minutes of exercise, as
previously stated for human beings (24,27) and rodents (28).
     Exercise and Adrenal Function                                                                                     21

Analysis of the blood lactate
The blood samples (25 μL) collected from the animals during tests were placed in eppendorf tubes
(1.5 mL) containing 50 μL of 1% sodium fluoride which were stored in freezer for later lactate
concentration analyze in YSI model 1500 SPORT lactate analyzer.

Sacrifice of animals
Animals were sacrificed by decapitation at rest or shortly after 25 minutes of acute or exhaustive
exercise (depending on the group) for blood collection, for determination of corticosterone (Coat-A-
Count Kit from Diagnostic Products Corporation – DPC). The adrenal glands were removed for the
immediate ascorbic acid (left adrenal gland) and cholesterol (right adrenal gland) assessment.
Ascorbic acid was measured using the dichlorophenolindophenol reactive (29). For cholesterol
measurement, the tissue was first treated with KOH 30% in a boiling water bath for 30 min, followed
by addition of a saturated Na2SO4 solution. Ethanol 70% was then added and the mixture was
centrifuged at 2000 rpm for 5 min. The supernatant was saved for cholesterol determination by
colorimetric method (30).

Statistical analysis
The statistical procedures included the One-way Analysis of Variance (ANOVA) for independent
samples followed by the post-hoc Student-Newman-Keuls test wherever suitable. The significance
level adopted was of (P<0.05).


Figure 1 shows the results corresponding to tests performed to identify the maximal lactate steady
state (MLSS) during treadmill running exercises. Based on the results from the individual tests, it was
possible calculating the mean blood lactate values during the tests for all animals evaluated.

During treadmill running exercise at velocities of 15 and 20 m/min, stabilization of the blood lactate
concentration values was observed from 10 to 25 minutes of exercise, with mean values of 3.2±0.1
and 3.8±0.1 mmol/L, respectively. At velocity of 25 m/min, a progressive increase on the blood lactate
concentration was observed. At this velocity, the animals did not complete the test due to exhaustion,
reached after 15 minutes of exercise. Therefore, MLSS was obtained at velocity of 20 m/min.

                             MLSS=20 m/min
 Lactate (mmol/L)

                                                                                            15 m/min
                    4                                                                       20 m/min
                    2                                                                       25 m/min

                         0      5       10       15       20          25         30
                                             Time (min)

Figure 1. Blood lactate concentration (mean ± SD) during test to identify the maximal lactate steady state (MLSS) of rats
in treadmill running exercise. To do this, rats were submitted to 3 running sessions with 25 minutes of duration (n=07) at
velocities of 15; 20 and 25 m/min. Blood samples were collected from the tip tail of rats each 5 minutes of exercise for
lactate dosage. MLSS was defined as the highest velocity at which blood lactate concentration did not varied by more
than 1 mmol/L between minutes 10 and 25 of the constant exercise.
             Exercise and Adrenal Function                                                                               22

Results presented in Figure 2 correspond to the serum corticosterone concentrations after exercise.
The serum corticosterone concentrations of the animals submitted to treadmill running (M and AM)
presented higher values when compared to values found for the control group (C) (P<0.05). No
difference was found for the groups M and AM (P<0.05). The statistical power was 0.9245.
                                                                    Data corresponding to ascorbic acid and cholesterol
 Corticosterone (ng/mL)

                                               *     *              concentrations in the adrenal gland of animals after
                                                                    sacrifice are found in figure 3. No differences in
                                                                    adrenal ascorbic acid and cholesterol levels were
                                                                    observed when the three groups were compared
                                                                    (P<0.05). The statistical power was 0.8130 for
                                                                    ascorbic acid and 0.8726 for cholesterol.
                                               M     AM         C
  Figure 2. Serum corticosterone concentrations
  (mean ± SD) of animals at the end of the      The exercise intensity employed in researches
  experiment in rest and after exercise session at
                                                involving human beings modulates the adrenal glands
  intensities corresponding to the maximal lactate
                                               activity, where high intensities promote higher serum
  steady state (MLSS) and 25% higher in treadmill
  running exercises. M = maximal lactate steadycortisol concentrations and consequent biochemical
  state; AM = above maximal; 25% higher than   alterations on the adrenal glands such as the depletion
  MLSS and C = control. *, P<0.05 difference inof the cholesterol and ascorbic acid concentrations
  relation to the control group.
                                               (31,17). However, few studies involving the
                                               determination precise of effort intensities in animals
and their specific stress responses can be found. Therefore, the present study was aimed at
analyzing different adrenal gland activity biomarkers with the objective of verifying their sensitivity in
measuring alterations of the stress levels of rats submitted to acute treadmill running exercises at
precise intensities: equivalent and 25% above the MLSS.

The procedure considered “gold standard” for the identification of the aerobic/anaerobic transition
zone during exercise is the Maximal Lactate Steady State (MLSS). The MLSS represents the highest
intensity at which it is possible observing blood lactate stabilization during endurance exercise, due to
the balance between production and clearance from blood lactate (32,27,26).
                                         3,5                                                       2
                                                                         Cholesterol (mg/100mg)

                Ascorbic acid (μg/mg)


                                          2                                                       1,2

                                         1,5                                                      0,8
                                          0                                                        0
                                               M    AM      C                                           M    AM      C
                                                   groups                                                   groups

Figure 3. Ascorbic acid (µg/mg) and cholesterol (mg/100mg) in adrenal gland of animals at the end of the experiment in
rest and after exercise session at intensities corresponding to the maximal lactate steady state (MLSS) and 25% higher in
treadmill running exercises. M = maximal lactate steady state; AM = above maximal; 25% higher than MLSS and C =
control. No significant differences were seen.

The mean individual MLSS of running rats was obtained at 20m/mim and blood lactate concentration
of 3.8±0.1 mmol/L. These results were quite similar to those obtained by Manchado et al. (26)
  Exercise and Adrenal Function                                                                         23

(3.9±0.3 mmol/L), who used similar protocol to that employed in the present study to determine the
MLSS of sedentary rats during treadmill running exercise.

After attainment of MLSS, the animals were submitted to a 25-minutes exercise session at MLSS
intensity or to exhaustive exercise session at intensity 25% higher than MLSS, in the attempt of
verifying if slight alterations in the exercise intensity cause distinct stress-associated physiological

Since there are limitations in the studies with human beings, there has been a proliferation of
genetically altered rat models of human metabolic diseases over the past few years, increasing
interest in how closely rat physiology resembles that of the human being. Although it has already
been established in literature that the increase in the glucocorticoids concentration in humans during
exercise is proportional to its intensity (33,34,35), few studies with animal models are aimed at
identifying precisely alterations in intensity of acute exercises (23) and its effects on blood
corticosterone concentration. Moreover, no studies associating the sensitivity of different stress
biomarkers during acute treadmill running exercises performed by rats at distinct intensities can be
found until the present moment in the literature.

The performance of treadmill running exercise at both intensities (M and AM) led to significant
increases in serum corticosterone concentrations when compared to animals in rest. These results
were similar to those reported by Kawashima et al. (36) and Huang et al. (37), in studies on the acute
effect of the treadmill running exercise in rats. It is worth emphasizing that in both studies, the precise
identification of the effort intensity individually performed by the animal during exercise was not

Glucocorticoids cause fast mobilization of aminoacids from their cellular stores, becoming available
both for energetic sources and widely for glucose synthesis, essential for tissues (2). The most known
action of glucocorticoids is its capacity of stimulating the liver gluconeogenisis, usually elevating 6 to
10 times the gluconeogenisis rate. Jungas et al. (38) showed that daily supply of amino acids
provided in the diet cannot be totally oxidized to CO2 in the liver because such a process would
provide far more ATP than the liver could utilize. Instead, most amino acids are oxidatively converted
to glucose and stored as glycogen. Studies conducted with sheep fetuses and rats treated with
corticosterone infusion showed that the blood glucose concentration increased supported by 14C-
Lactate substrate. Concurrently with the decrease of the 14C-Lactate and corticosterone
concentrations, a decrease on the blood glucose levels was observed (39,40). Corticosterone usually
promotes gluconeogenisis by direct acting on the phosphoenolpyruvate carboxykinase enzyme
activity (PEPCK) or through the increased sensitivity of the adrenaline and glucagon hormones
responsible for the hepatic gluconeogenisis (41). Thus, glucocorticoids provide high supply of
energetic substrates for adverse situations.

On the other hand, the systematic mobilization of aminoacids may cause protein unbalance. A study
using prednisolone treatment (synthetic glucocorticoid) conducted in human beings, showed that the
glucocorticoid caused increase in free aminoacids concentration in arterial blood (42).

Cholesterol and ascorbic acid concentrations in the adrenal glands of the animal after acute treadmill
running exercise were also evaluated. No differences between the groups studied, M, AM and
control, were found. A small amount of cholesterol is used by the adrenal glands to synthesize the
cortex steroid hormones. The cholesterol used in the steroid synthesis may be synthesized by the
cortex cells from acetate; however, most of them are originated from the plasma low-density
lipoproteins (LDL).(43) Hepatic lipase (HL), a liver-expressed lipolytic enzyme, hydrolyzes
  Exercise and Adrenal Function                                                                             24

triglycerides and phospholipids in lipoproteins and promotes cholesterol delivery through receptor-
mediated whole particle (LDLR) and selective cholesterol uptake. HL activity also occurs in the
adrenal glands, which utilize lipoprotein cholesterol to synthesize glucocorticoids in response to
pituitary ACTH. It is likely that the role of adrenal HL is to facilitate delivery of exogenous cholesterol
for glucocorticoid synthesis. Dichek HL et al. (44) in study of corticosterone response to eight daily
ACTH injections in HL-deficient (hl-/-), LDLR-deficient (Ldlr-/-), and HL- and LDLR-doubly deficient
(Ldlr-/- hl-/-) mice, showed on day 5, plasma corticosterone levels were reduced by 57, 70, and 73%
and on day 8 by 76, 59, and 63% in hl-/-, Ldlr-/-, and Ldlr-/- hl-/- mice, respectively. These results
demonstrate that plasma cholesterol might directly interferer in corticosterone concentrations.

According to Pignatelli (31), in experimental studies using rats, the cholesterol content in the adrenal
gland is directly associated to the duration, intensity and type of training the animals were submitted
to. In the conditions of the present study, the acute exercise was not able of changing the cholesterol
concentrations in the adrenal gland. Probably, the high levels of serum corticosterone observed in
exercised animals were supported by the cholesterol from the plasma low-density lipoproteins (LDL).
These results were similar to those found by Kucharczyk & Ziemlanski (43), who did not find any
effect of the exercise load on the cholesterol concentration in the adrenal gland of wistar rats fed a
standard rodent diet while in a group of rats receiving 30% of calories from rapeseed oil, the trained
rats accumulated more rapidly cholesterol in the adrenals than the untrained rats.
In relation to the ascorbic acid concentration of the exercised animals, our results are not in
agreement with those found by Umegaki et al. (45), who found reduced ascorbic acid concentration in
the adrenal gland of rats submitted to treadmill running exercise.

The ascorbic acid and the dehydroascorbic acid are found in high amounts in organs composed of
tissues presenting intense metabolic activity such as adrenal, gonads, thyroid and pituitary glands,
once the dehydroascorbic acid may act in the cell reations involved with oxygen transportation (20).
However, its participation in the steriogenesis is still unclear.When the metabolic responses to
treadmill running exercise at both intensities (M and AM) are analyzed, no differences in
corticosterone and ascorbic acid and cholesterol in adrenal gland levels were observed.

According to Guszkowska (46), the activity of the hypothalamic-pituitary-adrenal axis during acute
exercise depends on intensity, producing higher activity at higher intensities. When human beings
perform physical exercises with O2 consumption higher than 60% of the VO2 max, the ACTH and
cortisol secretion are proportional to the exercise intensity (47). However, although corticosterone
seemed to be sensitive to the treadmill running exercise, an increase of 25% on the MLSS intensity
was not sufficient to cause additional alterations on the serum concentrations of untrained rats.


In short, it was possible identifying the effort intensity (velocity) in which the lactate concentrations
were stable with the protocol employed here, indicating that this protocol was suitable for the
individual MLSS identification in rats.

Among the adrenal activity indices analyzed, only the serum corticosterone concentrations responded
to the treadmill running exercise. Serum corticosterone concentrations seem to be more sensitive
biomarker than adrenal ascorbic acid and cholesterol concentrations to infer on the stress level of rats
submitted to treadmill running exercise. Moreover, the elevation of the exercise intensity 25% above
MLSS does not seem to impose additional stress to the animal as indicated by biomarkers utilized.
  Exercise and Adrenal Function                                                                 25

Further studies are required such as timecourse analysis of the corticosterone secretion throughout
the exercise, to corroborate this conclusion.


This study was supported by CAPES, CNPq e FAPESP. Grateful acknowledgment to Clarice Yoshio
Sibuya, Eduardo Custódio and José Roberto Rodrigues da Silva, who provided essential help to the

Address for correspondence: MELLO, M.A.R. PhD., Department or private address, Department of
Physical Education, UNESP, São Paulo State University, Rio Claro, Sao Paulo, Brazil. CEP: 13.506-
900, Phone:+55 19 3526-4320; Fax:+55 19 3526-0009; E-mail:


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  Exercise and Adrenal Function                                                                     26

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  Exercise and Adrenal Function                                                                   27

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