FERNANDA KLEIN MARCONDES,
ET AL .
ORIGINAL / ORIGINAL
Relação entre Exercício Físico de Alta
Intensidade e o Lactato Sangüíneo, em ratos
Relationship between High Intensity Exercise
and Blood Lactate in Rats
ANA PAULA TANNO
Farmacêutica e mestre em
odontologia –
Departamento de Ciências
Fisiológicas (FAM /SP )
TATIANA S OUSA CUNHA
Fisioterapeuta e mestre em
odontologia –
Departamento de Ciências Fisiológicas ( FOP /Unicamp/ SP )
MARIA JOSÉ COSTA SAMPAIO
MOURA
Biomédica, mestre e doutora em
ciências – Centro de Ciências da
Vida ( PUCC amp/ SP )
FERNANDA KLEIN MARCONDES *
Bióloga, mestre em ciências
biológicas e doutora em ciências –
Departamento de Ciências
Fisiológicas ( FOP /Unicamp/ SP )
* Correspondências: Av. Limeira,
901, 13414-903, Dep. de Ciências
Fisiológicas, Piracicaba/ SP
[email protected]
Saúde em Revista
Exercício Físico e Lactato em Ratos
R ESUMO Apesar de existirem muitos estudos sobre as adaptações
desencadeadas pelo treinamento físico aeróbio em animais de laboratório, pouco se sabe a respeito dos efeitos promovidos pelo treinamento
resistido de alta intensidade (AI). O objetivo deste estudo foi verificar
se o exercício físico resistido de AI promove elevação da concentração
sangüínea de lactato (L) acima do máximo estado estável do metabólito
(5,5 mmol/L). Ratos machos foram submetidos a uma sessão de salto
com peso na água (quatro séries/10 repetições por série/30 segundos de
intervalo entre as séries), com sobrecarga equivalente a 50% do peso
corporal. Amostras sangüíneas foram coletadas antes e durante o exercício, e a concentração de L foi determinada. Registrou-se o tempo
gasto para realização das repetições durante cada série. No repouso, a
concentração sangüínea de L foi 1,44 ± 0,10 mmol/L. Séries repetidas
do exercício resultaram em aumento significativo (p < 0,01) da concentração sangüínea de L: após a 1.ª série = 3,00 ± 0,21, 2.ª série = 5,12
± 0,30, 3.ª série = 6,52 ± 0,11 e 4.ª série = 8,64 ± 0,23 (mmol/L). Não
houve diferença significativa do tempo gasto para a realização das repetições. Esses resultados confirmam o caráter anaeróbio do protocolo
de treinamento resistido de AI, podendo ser um modelo para estudos
experimentais sobre capacidade anaeróbia e contribuindo na compreensão das adaptações que ocorreriam em humanos.
Palavras-chave ÁCIDO LÁCTICO – EXERCÍCIO – LEVANTAMENTO DE PESO.
ABSTRACT Despite the large amount of studies about the effects obtained
in response to the use of aerobic protocols in lab animals, little is
known about high-resistance training programs. The aim of this study
was to verify if high-resistance protocol promotes rise in blood lactate
(L) concentration higher than the maximal lactate steady state (5.5
mmol/L). Male rats were submitted to one session of weight lifting in
water (4 sets of jumps/10 repetitions/30 seconds rest between each set),
carrying an overload of 50% of body weight. Blood samples were
collected before and during the exercise protocol, and blood L
concentration was determined. The time spent to perform the repetitions
during each set was recorded. In state of rest, blood L concentration
was 1.44 ± 0.10 mmol/L. Repeated sets of exercise resulted in
significant (p < 0.01) enhancement of blood L concentration: after 1st
set = 3.00 ± 0.21, 2nd set = 5.12 ± 0.30, 3rd set = 6.52 ± 0.11 and 4th
set = 8.64 ± 0.23 (mmol/L). There was no statistical difference on the
times spent to perform the repetitions during each set. These data
confirm the anaerobic character of the high-resistance exercise protocol
used. Thus, it could be a helpful model for use in experimental studies
about anaerobic capacity and ways to improve it, as the first step to
understanding the adaptations that might occur in humans.
Keywords LACTIC ACID – EXERCISE – WEIGHT LIFTING.
23
FERNANDA KLEIN MARCONDES,
INTRODUCTION
It is becoming increasingly clear that a
person’s health and well-being are improved
by physical activity. In this context, physical
activity should be viewed as providing
stimuli that stress many systems of the body
to various degrees and thus promote very
specific and varied adaptations according to
the type, intensity, and duration of the exercise performed.1
Numerous studies have addressed
substrate metabolism during exercise, but the
exact relation between exercise intensity and
substrate mobilization from different endogenous stores has not been fully elucidated.2 It
is known that the use of each energetic
substrate is related to the intensity and duration of the exercise performed. For obvious
reasons, a significant number of these studies
have been conducted on laboratory animals
and almost all of them used exercise
protocols of low and moderate intensity,
which have already been characterized.3
Despite the large amount of information
about the effects obtained in response to the
use of aerobic protocols in rats, little is
known about high-resistance training programs that are associated with increased body
weight, lean body mass, muscle cross-sectional area and glycogen storage capacity.4, 5
The use of swimming rats as a model of high
intensity exercise presents advantages over
the others, since swimming is a natural ability
of the rat, thus avoiding selection of animals,
which is necessary in experimental protocols
using treadmill running.6 In this case, incremental exercise is obtained by adding progressively heavier loads in relation to body
weight, attached to the animal’s chest or tail.
The major limitation of swimming studies is
the lack of knowing the effort intensity of the
rat’s performance.
A tool, widely used for determining effort intensity during exercise is the blood lactate concentration. In human subjects blood
24
ET AL .
lactate concentration increases exponentially
with exercise intensity. The break point in
the blood lactate vs workload curve during
incremental exercise has been termed the
blood lactate threshold or anaerobic threshold
(AT); 7 although anaerobioses as the sole
cause of accelerated lactate production has
been questioned.8, 9 In rats this procedure is
limited by the small amount of information
available concerning lactate kinetics during
exercise. Only few studies dealing with lactate threshold determinations in rats have
been reported in the literature, most of them
using treadmill running as an ergometer.10, 11
According to Gobatto et al.12 the maximal lactate steady state, for sedentary rats
submitted to acute swimming exercise, occurs at blood lactate concentrations of 5.5
mmol/L. On the other hand, Pilis et al. 11
found an anaerobic threshold at blood lactate
concentration of approximately 4 mmol/L in
rats during a treadmill exercise test. Also in
humans, these differences are probably found
because of the type of exercise used (running
or swimming) to determine the value of
blood lactate at AT.
Thus, this study was designed to verify
if the protocol of jumping up and down into
the water carrying a load (high-resistance exercise protocol)4, 5 promotes rise in blood lactate to upon the maximal lactate steady state
(5.5 mmol/L established by Gobatto et al.12).
Understanding the kinetics of lactate using
the protocol of weigh lifting in the water,
which seems to be less stressful than the others, and proving its anaerobic characteristic,
it could be used in further studies on the role
of energetic substrates, like glycogen, in this
type of exercise.
METHODS
Animals: the experiments were approved
by the institutional Committee for Ethics in
Animal Research (Unicamp/COBEA – protocol number 391-1) and were conducted in
SAÚDE REV., Piracicaba, 8 (20): 23-29, 2006
FERNANDA KLEIN MARCONDES,
conformance with the policy statement of the
American College of Sports Medicine on
research with experimental animals. Male
Wistar rats, 60 days old, weighing 250-300g
were used for this study. Animals were
housed in a collective cage (three or four rats
per cage), in a temperature-controlled room
(22°C ± 2°C) with lights on from 6 am to 6
pm. Animals received commercial chow for
rodents and filtered water ad libitum.
Adaptation to the water: all rats were
adapted to the water before the experiment
began. The adaptation consisted of sessions
of weight lifting (once a day for five days) in
water at 30°C ± 2°C 13 , with incremental
number of sets (two to four) and repetitions
(five to ten) and 30 seconds rest between
each set, carrying a load of 50% body weight
strapped to the chest. The sessions were performed between 1-2 pm and on the last day
of the adaptation period, the animals were
performing four sets of ten repetitions (lifts).
The purpose of the adaptation was to reduce
the stress without, however, promoting
physical adaptation.
Exercise-test protocol: before the exercise-test protocol started, a blood sample was
collected for determining lactate in the state
of rest. Then the rats, carrying an overload of
50% of body weight strapped to the chest,
ET AL .
were individually submitted to one session of
weight lifting (fig. 1). The session of weight
lifting consisted of four sets of jumps (ten
repetitions per set), with 30 seconds rest between each set. 4, 5, 14 A jump repetition was
counted only if the rat put its snout out of the
water. Using a stopwatch, the time spent to
perform ten repetitions during each set was
recorded. During the rest period, new blood
samples were taken for lactate measurements.
Blood samples and lactate analysis:
blood samples (25 µL) were collected from a
cut at the tail tip before and during the highresistance exercise protocol, and deposited in
Eppendorf tubs (1.5 µL capacity) containing
50 µL sodium fluoride (1%). To avoid blood
dilution with residual water on the animal’s
tail, the rats were quickly dried with a towel
immediately before blood collection. The lactate concentrations were determined using a
lactate analyzer (YSI model 1500 SPORT).15
Statistical analysis: data are presented
as mean ± S.E.M. Analysis of differences
regarding blood lactate concentration and
the time spent to do ten repetitions during
each set was performed using a repeated
measures analysis of variance (one-way
Anova). When necessary, the Tukey-Kramer
post-hoc comparison test was used. The statistical significance was set at p < 0.01.
Figure 1. Illustration of the exercise protocol (jumping up and down in water or weigh lifting in water).
The dimensions of the plastic tank used to perform the training are shown in the figure.
Saúde em Revista
Exercício Físico e Lactato em Ratos
25
FERNANDA KLEIN MARCONDES,
RESULTS
In figure 2, the data about blood lactate
concentration in state of rest and after each
one of four sets of high-intensity exercise in
water are presented. In the state of rest, the
blood lactate concentration of the sedentary
rats was 1.44 ± 0.10 mmol/L. Repeated sets
of high-intensity exercise resulted in significant enhancement of blood lactate concentration, as follows: after first set = 3.00 ± 0.21
mmol/L, second set = 5.12 ± 0.30 mmol/L,
third set = 6.52 ± 0.11 mmol/L and after the
fourth set = 8.64 ± 0.23 mmol/L (p < 0.01).
The data about the time spent to perform
each set of ten jump repetitions are presented
in Table 1. There were no statistical differences on the times spent during each set.
Figure 2. Blood lactate concentrations (mmol/L)
during high-resistance exercise protocol (weight
lifting in water), carrying an overload of 50% body
weight (n = 7). Results are mean ± S.E.M. Different
letters mean significant differences (p < 0.01).
DISCUSSION
Many studies have been carried out to
design experimental models for inducing
muscle hypertrophy in laboratory animals
using treadmill, swimming, and other training
apparatus simulating human weigh lifting.4, 5,
16
However, it is clear that without a systematic approach to the investigation of the phe26
ET AL .
Table 1. Time spent (s) to perform ten jump
repetitions during each one of four sets of the
high-resistance exercise protocol (weight lifting in
water), carrying an overload of 50% body weight.
Sets
N
Time (s)a
First
7
22 ± 1.32
Second
7
20.83 ± 1.41
Third
7
22.33 ± 1.42
Fourth
7
26.24 ± 3.75
Mean ± S.E.M
a
nomenon, there is a lack of control and manipulation of the independent variables,
which makes its difficult to test the validity
of these models. 17 Another problem is the
stress caused by these high intensity exercise
protocols since some of them use electrical
stimuli (foot shock) to force the animals to
perform the exercise.
It has been already demonstrated that the
high-resistance exercise protocol used in this
study14 is efficient for inducing skeletal muscle hypertrophy in rats.18 Resistance training is
a physiological challenge that can promote
hypertrophic responses in both human and
animal models19 since hypertrophic growth of
skeletal muscle is stimulated by short bursts of
muscle activity against high resistance. Muscle hypertrophy is induced throughout a process of satellite cell activation, proliferation,
chemotaxis, and fusion to existing myofibers.
Although there are still a number of questions
with regard to the role of the satellite cell in
muscle remodeling, the primary physiological
consequence of the hypertrophic response is
to produce a muscle with a greater capacity
for peak force generation.20
In addition to the hypertrophic character
of the protocol used in this work, the data
now confirm its anaerobic nature, based on
the value of the blood lactate concentration
SAÚDE REV., Piracicaba, 8 (20): 23-29, 2006
FERNANDA KLEIN MARCONDES,
after the sets of jumps. The rats did not show
difficulty in performing the protocol and it
produced hyperlactemia. As can be seen, the
blood lactate concentration enhanced as the
sets were performed and after the third set of
jumps, it was higher than that of the anaerobic threshold established by Gobatto et al.12
Irrespective of the characteristics of the
exercise performed, energy is required to
produce work (in this case, to produce the
contractile activity). Carbohydrate, fat, and
protein, obtained either directly from daily
meals or from endogenous stores in the body,
provide the substrates that fuel the chemical
reactions that in turn are catalyzed by enzymes and cofactors. In these reactions, the
chemical energy of substrates is converted to
an energetic compound that cells can harness, namely adenosine triphosphate (ATP).21
Then, the ATP hydrolysis to adenosine diphosphate (ADP) and inorganic phosphate
(Pi) provides the energy required for contractile activity. ATP can be re-synthesized
anaerobically from phosphocreatine (PCr),
through the creatine kinase reaction, or from
glycolitic process in the sarcoplasma. 22 The
glycolitic process represents the initial stage
of glucose and glycogen metabolism and
leads to pyruvate or lactate production. 23
Moreover, ATP can also be re-synthesized
aerobically, within the mitochondria, by
chemical reactions that consume oxygen
(oxidative phosphorylation).24
It is known that intense exercises of
short duration, like that used in this work,
are heavily dependent on energy from
anaerobic sources. Many studies have demonstrated that glycogen in liver and skeletal
muscles is important to maintain physical
performance during exercise, 25 mainly if its
duration is between approximately eight and
thirty five seconds. Thus, this work demonstrated that the mean period of time spent to
perform each set of ten jumps in water was
not longer than thirty seconds. It also emphasizes that energy production during this
Saúde em Revista
Exercício Físico e Lactato em Ratos
ET AL .
type of exercise is primarily dependent on
the anaerobic pathway.
During the type of exercise used in this
study, there is a large breakdown of glycogen,
and it may reappear as hexose phosphates or
as lactate (it is important to remember that in
the presence of oxygen it can also be fully
oxidized). The increasing demand for
glycolitic energy production promotes a decrease in intracellular pH during progressive
work26 because the lactate production exceeds
the bicarbonate buffering capacity, and part of
lactate produced may be transferred from
muscle to blood.27 Thus, it explains the elevation in blood lactate concentration as the sets
of jumps were performed. The extent to which
lactate increases in the blood can be altered by
fitness or with training.28
It is important to mention that some
authors make reservation about the use of
lactate concentration as a direct index of lactate production by muscles, because muscles
as well as others tissues and organs are also
consumers of lactate by oxidative metabolism
for their energetic needs. 29 However, it remains a widely used tool in the exercise
physiology laboratory for understanding the
characteristics of the exercise performed.
A misconception that continues to be
widespread is the idea that lactate is the
major cause of both fatigue during exercise
and post exercise muscle soreness. In fact,
lactate has little direct effect on either, even
during short bouts of exercise of relatively
high intensity. Rather than lactate causing
fatigue per se, it is the accumulation of H +
ions during glycolysis that contributes to fatigue. High concentrations of H + lower the
blood pH which adversely influences energy
production and muscle contraction.10 Despite
there being no significant differences in the
time spent to perform the exercise between
the four sets, it can be seen that the time
spent on the last set of jumps was longer
than the others, indicating that the occurrence
of this phenomenon, in response to this pro27
FERNANDA KLEIN MARCONDES,
tocol, might start at this point. Probably if
there were a fifth set of jumps, the time spent
might be longer than the previous one, since
there was only thirty seconds between each
set to re-establish the glycogen stores.
Successful subjects in anaerobic types
of sports may therefore have a larger anaerobic capacity and be able to release energy at
a higher rate. It has already been shown that
performances in these kinds of sports are
improved by training, suggesting that anaerobic capacity is trainable. 30 We know that
ET AL .
there are metabolic differences between humans and rats and that quantitative differences in some physiological parameters,
would be found. But we must emphasize that
the general principles that regulate some of
them are valid for both species. Therefore,
after the confirmation of the anaerobic character of this protocol, one believes it could
be a helpful model for use in experimental
studies about anaerobic capacity and ways to
improve it, as the first step to understanding
the adaptations that could occur in humans.
REFERÊNCIAS BIBLIOGRÁFICAS
1. Coyle EF. Physical activity as a metabolic stressor. Am J Clin Nutr 2000;72(2 Suppl):512-20.
2. Romijn JA, Coyle EF, Sidossis LS, Gastaldelli A, Horowitz JF, Endert E et al. Regulation of endogenous
fat and carbohydrate metabolism in relation to exercise intensity and duration. Am J Physiol set/1993;265(3
Pt 1):380-91.
3. Antonutto G, Di Prampero PE. The concept of lactate threshold. A short review. J Sports Med Phys Fitness
mar/1995;35(1):6-12.
4. Cunha TS, Tanno AP, Moura MJCS, Marcondes FK. Influence of high-intensity exercise training and
anabolic androgenic steroid treatment on rat tissue glycogen content. Life Sci jun/2005;77(9):1.030-43.
5. Cunha TS, Moura MJCS, Bernardes CF, Tanno AP, Marcondes FK. Vascular sensitivity to phenylefrine in
rats submitted to anaerobic training and nandrolone treatment. Hypertension jul/2005;46(2):1.010-5.
6. Gobatto CA, Sibuya CY, Azevedo JRM, Luciano E, Kokubun E, Mello MAR. Characterization of exercise
intensity and of physical training effects using a swimming model for Wistar rats. Motriz 2001;7(1):57-62.
7. Mader A, Heck H. A theory of the metabolic origin of “anaerobic threshold”. Int J Sports Med jun/
1986;7(Suppl 1):45-65.
8. Brooks GA. Intra and extra-cellular lactate shuttles. Med Sci Sports Exerc abr/2000;32(4):790-9.
9. Myers J, Ashley E. Dangerous curves. A perspective on exercise, lactate, and the anaerobic threshold. Chest
mar/1997;111(3):787-95.
10. Langfort J, Zarzeczny R, Pilis W, Kaciuba-Uscilko H, Nazar K, Porta S. Effect of sustained
hyperadrenalinemia on exercise performance and lactate threshold in rats. Comp Biochem Physiol A Physiol
maio/1996;114(1):51-5.
11. Pilis W, Zarzeczny R, Langfort J, Kaciuba-Uscilko H, Nazar K, Wojtyna J. Anaerobic threshold in rats.
Comp Biochem Physiol out/1993;106(2):285-9.
12. Gobatto CA, Mello MAR, Sibuya CY, Azevedo JRM, Santos LA, Kokubun E. Maximal lactate steaty state
in rats submitted to swimming exercise. Comp Biochem Physiol – Part A 2001;130:21-7.
13. Harri M, Kuusela P. Is swimming exercise or cold exposure for rats? Acta Physiol Scand fev/
1986;126(2):189-97.
14. Voltarelli FA, Gobatto CA, Mello MAR. Determination of anaerobic threshold in rats using the lactate
minimum test. Braz J Med Biol Res nov/2002;35(11):1.389-94.
15. Simões HG, Grubert-Campbell CS, Kokubun E, Denadai BS, Baldissera V. Blood glucose responses in
humans mirror lactate responses for individual anaerobic threshold and for lactate minimum in track tests.
Eur J Appl Physiol Occup Physiol jun/1999;80(1):34-40.
16. Duncan ND, Williams DA, Lynch GS. 1998 Adaptations in rat skeletal muscle following long-term
resistance exercise training. Eur J Appl Physiol Occup Physiol mar/1998;77(4):372-8.
17. Docherty D, Sporer B. A proposed model for examining the interference phenomenon between concurrent
aerobic and strength training. Sports Med dez/2000;30(6):385-94.
28
SAÚDE REV., Piracicaba, 8 (20): 23-29, 2006
FERNANDA KLEIN MARCONDES,
ET AL .
18. Rosante MC, Robert-Pires CM, Marquetti RC, Baldissera V, Perez SEA, Ninomyia M. Anabolic androgenic
steroids do not induce hypertrophy in rats submitted to weight lifting training. Proceedings of XVIII FeSBE
Anual Meeting; 27-30/ago/2003; Curitiba/PR. São Paulo: FeSBE/USP; 2003. p. 7.
19. Rosenblatt JD, Yong D, Parry DJ. Satellite cell activity is required for hypertrophy of overloaded adult rat
muscle. Muscle Nerve jun/1994;17(6):608-13.
20. Hawke TJ, Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol ago/
2001;91(2):534-51.
21. Hargreaves M. Skeletal muscle metabolism during exercise in humans. Clin Exp Pharmacol Physiol mar/
2000;27(3):225-8.
22. Gastin PB. Energy system interaction and relative contribution during maximal exercise. Sports Med
2001;31(10):725-41.
23. Green HJ. How important is endogenous muscle glycogen to fatigue in prolonged exercise? Can J Physiol
Pharmacol fev/1991;69(2):290-7.
24. Bottinelli R, Reggiani C. Human skeletal muscle fibres: molecular and functional diversity. Prog Biophys
Mol Biol 2000;73(2-4):195-262.
25. Murakami T, Shimomura Y, Fujitsuka N, Sokabe M, Okamura K, Sakamoto S. Enlargement glycogen store
in rat liver and muscle by fructose-diet intake and exercise training. J Appl Physiol mar/1997;82(3):772-5.
26. Iwanaga K, Sakurai M, Minami T, Kato Y, Sairyo K, Kikuchi Y. Is the intracellular pH threshold an
anaerobic threshold from the view point of intracellular events? A brief review. Appl Human Sci mar/
1996;15(2):59-65.
27. Medbo JI. Glycogen breakdown and lactate accumulation during high-intensity cycling. Acta Physiol Scand
set/1993;149(1):85-9.
28. Dubouchaud H, Butterfield GE, Wolfel EE, Bergman BC, Brooks GA. Endurance training, expression, and
physiology of LDH, MCT1, and MCT4 in human skeletal muscle. Am J Physiol Endocrinol Metab abr/
2000;278(4):571-9.
29. Brooks GA. Current concepts in lactate exchange. Med Sci Sports Exerc ago/1991;23(8):895-906.
30. Medbo JI, Burgers S. Effect of training on the anaerobic capacity. Med Sci Sports Exerc ago/1990;22(4):501-7.
Submetido: 24/mar./2006
Aprovado: 15/ago./2006
Agradecimentos
As autoras agradecem aos professores doutores Sérgio Eduardo de Andrade Perez, Cássio
M. Robert-Pires e Vilmar Baldissera pelo
uso do laboratório para as dosagens de
lactato, ao senhor José Carlos Lopes pelo auxílio técnico, a César Acconci pela elaboração da ilustração do protocolo de exercício
físico, à Margery Galbraith pela correção do
texto em inglês e à FAPESP, FAEPEX/Unicamp e
CAPES pelo financiamento.
Saúde em Revista
Exercício Físico e Lactato em Ratos
29