Physiological adaptations during endurance training below anaerobic

Eur J Appl Physiol (2013) 113:1859–1870 DOI 10.1007/s00421-013-2616-9

ORIGINAL ARTICLE

Physiological adaptations during endurance training below anaerobic threshold in rats Gustavo Gomes de Araujo • Marcelo Papoti Maria Andreía Delbin • Angelina Zanesco • Claudio Alexandre Gobatto

•

Received: 19 April 2012 / Accepted: 12 February 2013 / Published online: 2 March 2013 ! Springer-Verlag Berlin Heidelberg 2013

Abstract To assess the effects of continuous exercise training at intensities corresponding to 80 and 90 % of the lactate minimum test (LM), we evaluated antioxidant activity, hormone concentration, biochemical analyses and aerobic and anaerobic performance, as well as glycogen stores, during 12 weeks of swimming training in rats. Onehundred rats were separated into three groups: control (CG, n = 40), exercise at 80 (EG80, n = 30) and 90 % (EG90, n = 30) of LM. The training lasted 12 weeks, with sessions of 60 min/day, 6 days/week. The intensity was based at 80 and 90 % of the LM. The volume did not differ between training groups (X_ of EG80 = 52 ± 4 min; X_ of EG90 = 56 ± 2 min). The glycogen concentration (mg/100 mg) in the gastrocnemius increased after the training in EG80 (0.788 ± 0.118) and EG90 (0.795 ± 0.157) in comparison to the control (0.390 ± 0.132). The glycogen stores in the soleus enhanced after the training in EG90 (0.677 ± 0.230) in comparison to the control (0.343 ± 0.142). The aerobic performance increased by 43 and 34 % for EG80 and EG90, Communicated by William J. Kraemer. G. G. de Araujo ! C. A. Gobatto (&) Laboratory of Sports Applied Physiology, School of Applied Sciences, Campinas State University (UNICAMP), Pedro Zaccaria, 1300, Santa Luiza, Limeira, SP 13484-350, Brazil e-mail: [email protected]; [email protected] G. G. de Araujo Sports Science Research Group, Federal University of Alagoas (UFAL), Maceio´, Brazil M. Papoti School of Physical Education and Sport of Ribeirao Preto, University of Sao Paulo (USP), Ribeirao Preto, Brazil M. A. Delbin ! A. Zanesco Sao Paulo State University (UNESP), Sao Paulo, Brazil

respectively, in relation to baseline. The antioxidant enzymes remain unchanged during the training. Creatine kinase (U/L) increased after 8 weeks in both groups (EG80 = 427.2 ± 97.4; EG90 = 641.1 ± 90.2) in relation to the control (246.9 ± 66.8), and corticosterone (ng/mL) increased after 12 weeks in EG90 (539 ± 54) in comparison to the control (362 ± 44). The continuous exercise at 80 and 90 % of the LM has a marked aerobic impact on endurance performance without significantly biomarkers changes compared to control. Keywords Training ! Performance ! Biomarkers ! Endurance ! Rats Introduction A significant amount of research involving physical stress has been conducted in laboratory rats, for the purpose of increasing the understanding of the physiological adaptations that occur in response to exercise following various drug treatments, diets and pathologies (Clavel et al. 2002; Lambertucci et al. 2007; Cambri et al. 2010, 2011). However, exercise intervention in experimental physiology is not always simple because of several factors (Booth et al. 2010). Program length, session duration, frequency and intensity variables are difficult to systematize, and they make the prescription of exercise complex (de Araujo et al. 2012). Despite the significant number of studies involving training models in human beings (Issurin 2010), the transposition of these protocols for experimental physiology is inappropriate and as a consequence, misconceptions have been found around exercise adaptations (Booth et al. 2010). Thus, the metabolic effects of exercise on these rodents are frequently questioned due to a lack of information about the

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application of these variables during chronic physical stress (Booth et al. 2010). In an attempt to improve the prescription of exercise and its adaptations, studies have been made involving the determination of transition zone from aerobic and anaerobic metabolisms in rats (Pilis et al. 1993; Gobatto et al. 2001; Carvalho et al. 2005; Manchado et al. 2006; de Araujo et al. 2007; Contarteze et al. 2008). It is known that the anaerobic threshold is an important method to assess individually the endurance performance and to prescribe training intensities (Faude et al. 2009; de Araujo et al. 2012). Although there has been increasing interest in the scientific basis of intensity determination using this protocol, the necessary degree of physiological specificity is not present, as the intensity zone boundaries are not clearly anchored in underlying physiological events (Seiler and Kjerland 2006). The chronic exercise at or above anaerobic threshold has been considered an important method to enhance physiological adaptations related to ventricular enlargement, oxygen uptake, mitochondrial biogenesis and oxidative enzymes, thereby improving endurance performance (Faude et al. 2009; Bocalini et al. 2010; Cambri et al. 2011). On the other hand, the biomarker responses derived from chronic exercise below anaerobic threshold are not yet well established in rats and human beings (Booth et al. 2010; Seiler and Kjerland 2006). Some studies reported that acute exercises at or below the anaerobic threshold are associated with a blood lactate steady-state and reduced excretion of stress biomarkers when compared to workloads above the anaerobic threshold (Urhausen et al. 1994; Contarteze et al. 2008). Therefore, it is unclear whether these intensities applied for a prolonged period are sufficient to enhance the aerobic performance without significant changes in stress biomarkers. Studying the biomarker responses at these intensities is important either to experimental researches that use the exercise as a therapeutic intervention or to human studies that still need to establish the reference values on physiological responses below anaerobic threshold. This approach in laboratory rats may be an interesting strategy to avoid individual and external interferences often observed in humans (i.e., training status, nutritional condition, psychological factors, nationality, temperature, humidity, economic factors, sponsorship, calendar and other) and as a consequence provide a precise characterization of biomarkers adaptations (i.e., glycogen concentration in different tissues, enzymes, antioxidants enzymes, oxidative attack, metabolites and hormones) due to: (1) controlled environment; (2) possibility of invasive analysis and (3) internal control (i.e., same strain and age). In humans/ athletes, Seiler and Kjerland (2006) reported that the

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Eur J Appl Physiol (2013) 113:1859–1870

training intensity is polarized away from the moderately hard intensity range represented by the anaerobic threshold. Therefore, understanding the organic responses at intensities below the anaerobic threshold can also be useful for athletes and coaches, since approximately 75 % of the training sessions has been performed below the anaerobic threshold (Seiler and Kjerland 2006). Thus, the purpose of this study was to characterize and compare the physiological adaptations in rats during 12 weeks of continuous training at intensities equivalent to 80 and 90 % of anaerobic threshold evaluated by lactate minimum test (LM). Specifically, aerobic and anaerobic performances, biochemical (creatine kinase, glucose, urea, creatinine), hormonal responses (corticosterone and testosterone), glycogen stores (soleus and gastrocnemius) and antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) were evaluated each 4 weeks of training. It was hypothesized that performance and physiological adaptations are dependent on the characteristics of particular training programs and that better adaptations in biomarkers and aerobic performance would be observed during training at 90 % of the anaerobic threshold than at 80 % of the anaerobic threshold due to the proximity of anaerobic threshold intensity.

Methods Animals All experiments involving animals were performed in accordance with the principles of laboratory animal care (NIH publication No. 86-23, revised 1985) and specific national laws (e.g., the current version of the German Law on the Protection of Animals). One-hundred male Rattus norvegicus albinus (Wistar) rats, 60 days old, were selected for this study. Rats that were healthy, with pelage in good condition, were used for the experiments. Rats were maintained in collective cages (5 rats/cage, 350 cm2/animal, 18 cm of height). The cages were changed 4 days/week during the experimental period. The animals received water and commercial chow (23.5 % protein, 6.5 % fat, 70 % carbohydrate, Purina 5008, St. Louis, MO, USA) ad libitum and were housed at 22 ± 2 "C with an inverted 12:12-h light–dark cycle (18:00–06:00 hours lights on). The luminosity intensity for the light period in the vivarium and the laboratory room was kept between 50 and 60 lux and 300 and 400 lux, respectively. The rats were kept in an experimental animal room with free access to food and water, which were supplied every day after the training sessions (13:00– 16:00 hours).


Adaptation to water The purpose of adaptation to the water was to reduce water stress without promoting physiological adaptations to physical training. The adaptation to the water environment consisted of 5-min (31 ± 1 "C) exposure daily, in cylindrical tanks (80-cm diameter 9 120-cm depth) subdivided into 4 PVC cylindrical compartments of 30-cm diameter 9 120-cm depth for individual swimming. For the first week of the adaptation period, the rats were placed in water that had a depth of 20 cm; for the second week of the adaptation period and during training periods, the water depth was approximately 100 cm. The use of tanks deeper than 100 cm with a smooth surface and individual compartments makes it impossible for the animals to rest at the tank bottom, forcing them to swim continuously (de Araujo et al. 2007). For the swimming exercise, the desired intensity of effort was obtained by attaching loads of lead to each animal’s back that were heavier than their body weights. This model of workload requires a swim to water surface and is considered a natural ability of rats (Gobatto et al. 2001). In addition, this model reduces the differences in buoyancy among individuals and standardizes the workload based on their body weights (Dawson and Horvath 1970). Groups The animals were separated into three groups. 1.

2.

3.

Control group (CG, n = 40) ? adaptation to the deep water (5 min, 2 days/week, without additional workload) during the experimental period. Ten rats were euthanized before the experimental period (baseline) and after 4, 8 and 12 weeks. Exercise group at 80 % of the anaerobic threshold (EG80, n = 30) ? training protocol lasted 12 weeks with exercise sessions up to 60 min/day, 6 days/week. The intensity of the training was based on 80 % of the anaerobic threshold for each individual rat as measured by the LM. Groups of ten rats each were euthanized after 4, 8 and 12 weeks. Exercise group at 90 % of the anaerobic threshold (EG90, n = 30) ? training protocol lasted 12 weeks with exercise sessions up to 60 min/day, 6 days/week. The intensity of the training was based on 90 % of the anaerobic threshold for each individual rat as measured by the LM. Groups of ten rats each were euthanized after 4, 8 and 12 weeks.

The intensity of training (EG80 and EG90) was calculated weekly and based on the LM according to the equations below. The training volume (min) was timed daily

1861

(Eq. 1) to calculate the total average (Eq. 2). The training load was the product of volume (min) and intensity (relative to the LM or absolute to the LM). The training load, in arbitrary units (AU), was determined according to the methods of de Araujo et al. 2012 (Eq. 3): X Volume ðminÞ ¼ daily minutes=6 days ð1Þ X_ ¼ Volumeweek 1 þ Volumeweek 2 þ Volumeweek 3 þ ! ! ! þ Volumeweek 12 =12

ð2Þ

Training load ðAUÞ ¼ Volume ðminÞ & LM intensity

ð3Þ

Lactate minimum test To determine the aerobic and anaerobic performances during the experimental period, we utilized the LM in accordance with the methods of de Araujo et al. (2007). The LM was applied in all groups after adaptation to the water (baseline) and after 4, 8 and 12 weeks of the experimental period. In addition to assessing the aerobic and anaerobic indexes, the test was periodically applied to update the prescription of individualized training intensity. Blood and tissues analyses Blood and tissues were collected after adaptation to the water (baseline) and 24 h after the last session in weeks 4, 8 and 12 of the experimental period. The antioxidant enzyme levels, hormone concentrations, metabolite levels and glycogen stores analyses are described below. At the end of the adaptation period (baseline values) and after 4, 8 and 12 weeks of the experimental period (following 24 h of rest), the animals were euthanized with 20 % chloral hydrate (0.3 mL/100 g-1 animal weight) for blood collection and tissue excision (soleus and white gastrocnemius). Blood was collected via cardiac puncture after thoracotomy into EDTA tubes (plasma) or dry tubes (serum) in accordance with the analyses. The plasma and serum were separated into aliquots using commercial kits. After the blood was collected, the soleus and the white gastrocnemius muscles were carefully dissected and then placed on filter paper to remove excess fat and connective tissues. The glycogen concentration of the tissue samples was immediately analyzed according to the methods of Dubois et al. (1956). Muscle (200–250 mg) samples were immediately digested in 0.5 mL of KOH 1 N (30 %) for 20 min. After this period, 20 lL of Na2SO4 was added for glycogen precipitation using 2.5 mL of ethanol (5 min of centrifugation). The colorimetric assay method was performed using 20 lL of phenol (80 %) and 2.0 mL of sulfuric acid. After 15 min of boiling, the absorbance was determined at 490 nm (Dubois et al. 1956).

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Metabolites For the lactate concentration measurement, the blood samples (25 lL) were collected from the tail and placed in microtubes (1.5 mL) containing 400 lL of 4 % trichloroacetic acid, which were then stored at 8 "C. The samples were centrifuged for 3 min, and 100 lL of plasma was placed into fresh tubes containing 500 lL of the following reagent: glycine/EDTA, hydrazine hydrate 88 % (pH 8.85), lactate dehydrogenase and b-nicotinamide adenine dinucleotide. The homogenized sample and reagent were incubated at 37 "C for 20 min, and absorbance was determined at 340 nm. To determine CK-NAC (N-acetyl-L-cysteine) activity, 50 lL of serum (stored at 2–8 "C during 1 week) was mixed with the kit reagent mix: imidazol (100 mmol/L), creatine kinase (30 mmol/L), ADP (2 mmol/L), glucose (20 mmol/L), NADP (2 mmol/L), hexokinase (2,500 U/L), glucose 6P (2,000 U/L), Mg (10 mmol/L) and AMP (5 mmol/L). The CK-NAC absorbance was determined at 340 nm (25 "C: U/L = D absorbance/min 9 3,333) 4, 5 and 6 min after the sample and reagent were mixed. The serum samples for urea analysis were stored for 2 months at -20 "C. Reagent preparation was based on standard reagent, urease, buffer (pH = 6.9, EDTA and NTP), sodium hydroxide and sodium hypochlorite. The homogenized sample (10 lL) and reagent (1 mL) were incubated at 37 "C for 5 min, and absorbance was determined at 600 nm (mg/dL = sample 9.60/standard value). Creatinine concentration was analyzed with a kinetic method, using 100 lL of sample (stored at 2–8 "C for 1 week) mixed with 1 mL of the following kit reagent: picric acid (60 mmol/L) and buffer. The creatinine absorbance was determined at 340 nm after 30 and 90 s (spectrophotometer analysis at 37 "C): mg/dL = D absorbance 9 standard factor (standard sample/D absorbance of standard sample). Testosterone and corticosterone For corticosterone and testosterone concentrations (ng/dL) measurement, 50 lL of the serum sample was added to polypropylene tubes containing 125I (105 mL). The radioactivity was measured in a gamma counter (1 min) following the instructions provided with the commercial Kit-Coat-A-Count from Diagnostic Products Corporation— DPC#. Antioxidant enzymes and sulfhydryl groups To measure the sulfhydryl groups, plasma (50 lL) was mixed in 1 mL of Tris–EDTA buffer for the first spectro-

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photometric (FS) analysis (412 nm). Then, 20 lL of 5.5diotibis-2-nitrobenzoic (DTNB) 10 mM was added and diluted in methanol, and a second spectrophotometric (SS) analysis was performed after 15 min. A sample containing DTNB and buffer Tris–EDTA was analyzed in a spectrophotometer as a blank. The total amount of sulfhydryl groups was calculated using molar absorbance = 13,600/ cm-1 M-1 (SS – FS - B 9 1.57 mM). Catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity were measured using commercial kits. The plasma aliquots for the CAT, SOD and GPx analyses were stored for 20 days at -20 "C. SOD activity was determined using an EIA Cayman Chemical Assay Kit# by the xanthin and xanthin oxidase method (absorbance was 460 nm). GPx activity was determined using an EIA Cayman Chemical Assay Kit#. In the presence of glutathione reductase and NADPH, the oxidized glutathione was immediately converted to the reduced form with a concomitant oxidation of NADPH to NAD?. The absorbance was measured every 5 min at 340 nm. CAT activity was measured using an EIA Cayman Chemical Assay Kit#. The sample was mixed in the kit reagents (Buffer, formaldehyde standard, KOH, methanol, hydrogen peroxide and potassium periodate), and the absorbance was measured at 540 nm. Statistical analyses All the dependent variables were subjected to the normality test using the Shapiro–Wilk W test. All analyses were conducted with a statistical software package (Statistica#, version 7.0, Tulsa, OK, USA), and data are presented as the mean ± standard deviation (SD). An analysis of variance (ANOVA) was used to examine changes over time (0, 4, 8 and 12 weeks) within the lactate minimum variables (aerobic and anaerobic performances), glycogen stores (soleus and gastrocnemius) and biochemical analyses (lactate, creatine kinase, urea, creatinine, glutathione peroxidase, catalase, superoxide dismutase, testosterone and corticosterone) among the groups (CG, EG80 and EG90). When a significant interaction effect was found, a Newman Keuls post hoc test was used to identify where the difference existed among groups. For non-parametric samples, the Kruskal–Wallis method was used, followed by Dunn’s method. The effect size and Cohen’s d (Cohen 1988) were calculated for all the variables among the periods (baseline, 4, 8 and 12 weeks) for CG, EG80 and EG90. The thresholds for small, moderate, and large effects were 0.20, 0.50, and 0.80, respectively. Effect sizes (ES) were determined by the formula: (mean1 - mean2)/pooled SD. The significance level was set a priori a B 0.05.


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Results When compared to baseline (0 week), the body weights (g) of the groups were significant at 4 (CG: P = 0.00, effect size = 1.98; EG80: P = 0.00, effect size = 1.67; EG90: P = 0.00, effect size = 2.01), 8 (CG: P = 0.00, effect size = 4.23; EG80: P = 0.00, effect size = 3.33; EG90: P = 0.00, effect size = 2.99) and 12 (CG: P = 0.00, effect size = 3.62; EG80: P = 0.00, effect size = 5.85; EG90: P = 0.00, effect size = 4.61) weeks. In addition, after 8 weeks, the body weights in the CG were higher than those of EG80 (P = 0.04, effect size = 0.44) and EG90 (P = 0.00, effect size = 1.23) at the same age (Table 1). Figure 1a shows that in EG80, the training volume was reduced at 7 weeks in comparison to 2 weeks (P = 0.00, effect size = 0.75). No differences were found between the other weeks and between the other groups (X_ of EG80 = 52 ± 4 min; X_ of EG90 = 56 ± 2 min, P = 0.59, effect size = 0.44) (Fig. 1a). The training load calculated by absolute intensity (intensity corresponding to the LM in grams) in EG90 was 6, 15 and 8 % higher in comparison to the training load in EG80 during weeks 0–4, weeks 5–8 and weeks 9–12, respectively (Fig. 1b). No differences were found between the groups in training load calculated by relative intensity (% of the LM) during weeks 0–4, weeks 5–8 and weeks 9–12. However, the training load (% of the LM) in EG90 during weeks 5–8 and weeks 9–12 was 16 and 11 % higher than the training load in EG80 during those same periods (Fig. 1c). As shown in Fig. 2, aerobic capacity (% of the LM) was reduced by 19 and 13 % in CG after 12 weeks (P = 0.00, effect size = 1.51) and 4 weeks (P = 0.01, effect size = 1.18), respectively, in comparison to baseline. For EG80, the LM (% of BW) was 15 % lower (P = 0.00, effect size = 1.49) after 12 weeks compared to baseline and 15 % higher than the CG after 8 weeks (P = 0.00, effect

Table 1 Body weight (g) before experimental period (baseline), after 4, 8 and 12 weeks in control (CG) and exercise groups (EG80 and EG90) CG

EG80

EG90

Baseline (0 week)

298.4 ± 36.6

292.2 ± 33.7

286.1 ± 33.4

4 weeks

372.7 ± 38.4a

350.7 ± 36.2a

356.8 ± 37.1a

8 weeks

a

12 weeks

429.8 ± 25.5

a

474.8 ± 60.9

a,b

391.3 ± 36.9#,a

a,b

460.6 ± 42.3a,b,c

405.2 ± 34.2 454.3 ± 21.7

#

Significantly different in relation to CG

a

Significantly different in relation to 0 week

b

Significantly different in relation to 4 weeks

c

Significantly different in relation to 8 weeks

size = 1.02). The LM (% of LM) was reduced by 14 % after week 12 in EG90 compared to week 4 (P = 0.00, effect size = 1.22). In addition, the aerobic capacity as determined by percentage of the LM in EG90 was 12 % higher than in the CG after 4 weeks (P = 0.01, effect size = 0.81) (Fig. 2). However, the LM, as determined by absolute intensity, showed an inverse curve in comparison to the relative values. Whereas the relative intensity decreased during the experimental period, the absolute intensity increased significantly after 12 weeks in relation to baseline in all groups (Fig. 2). After 12 weeks, the absolute LM in the CG, EG80 and EG90 was increased by 18, 31 and 26 %, respectively, in comparison to baseline. Exceptionally, the absolute intensity in EG80 was increased significantly after 12 weeks when compared to 8 weeks (P = 0.04, effect size = 0.78) (Fig. 2). The anaerobic performance (Tlim 13 %) decreased in EG90 and the CG after 4 (EG90: P = 0.00, effect size = 2.00; CG: P = 0.00, effect size = 1.33), 8 (EG90: P = 0.00, effect size = 1.64; CG: P = 0.00, effect size = 2.11) and 12 weeks (EG90: P = 0.00, effect size = 1.18; CG: P = 0.00, effect size = 1.61) in comparison to baseline. The anaerobic index decreased in EG80 after 8 (P = 0.01, effect size = 1.71) and 12 weeks (P = 0.01, effect size = 1.87) in relation to week 0 (Fig. 3), and even after 4 weeks, a decreasing trend was seen (P = 0.05, effect size = 0.56). As shown in Fig. 3, there are reductions of 55 and 66 % at the end of training in EG80 and EG90, respectively, compared to baseline. Figure 4 shows that the glycogen concentrations in the gastrocnemius and soleus were significantly higher after training in both groups (EG80 and EG90) compared to the CG. The glycogen stores in the soleus increased by 51 % (P = 0.01, effect size = 1.78) in EG90 in comparison to the CG after 12 weeks (Fig. 4b). However, the glycogen concentration in EG80 showed a large effect size (1.31) after 12 weeks in relation to the CG. Glycogen concentration in the gastrocnemius increased approximately 102 % in both groups (EG80: P = 0.00, effect size = 3.15; EG90: P = 0.00, effect size = 2.79) after the exercise program, in comparison to the CG (Fig. 4a). As shown in Table 2, was not found significant change in testosterone, urea, creatinine and peak lactate during the experimental period. However, the concentration of corticosterone increased after 12 weeks in EG90 compared to EG80 (P = 0.03, effect size = 1.13) and the CG (P = 0.02, effect size = 1.19) (Table 2). The antioxidant enzymes (SOD, GPx and CAT) unchanged during the experimental period, but the oxidative attack as measured by levels of sulfhydryl groups was higher after 8 (EG80: effect size = 1.23; EG90: effect size = 1.89) and 12 weeks (EG80: effect size = 1.18; EG90: effect size = 2.09) in both groups compared to the CG (Table 2). The CK

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1864 65

60

Volume (min)

Fig. 1 Volume (min), absolute training load (AU) and relative training load (AU) in EG80 and EG90. Training volume during 12 weeks in EG80 and EG90 (a). The absolute load was the product between intensity of LM (g) and training volume (b). The relative load was the product between intensity of LM (%) and training volume (c). Hash indicates significantly different in relation to 7 week of EG80


55

#

50

45

Training load calclulated by relative intensity (UA)

Training Load calculated by absolute intensity (UA)

40

1000 900 800 700 600 500 400 300 200 100 350 300 250 200 150 100

1

2

3

4

5

6 7 Week EG80

concentration was 54 % higher in EG80 (effect size = 0.48) and 131 % higher in EG90 (effect size = 0.93) compared to CG after 8 weeks (Table 2).

Discussion To the best of our knowledge, this is the first study to investigate the effects of chronic exercise below anaerobic threshold on volume, biomarkers and aerobic/anaerobic performance in rats. The findings of this investigation

123

8

9

10

11

12

EG90

demonstrated that chronic exercise on these rodents can be applied to understand precisely the relation among intensity, duration and frequency of training (Booth et al. 2010; de Araujo et al. 2012). By physical training applied in rats, it was possible to obtain some advantages concerning the physiological alterations in relation to human beings due to possibility of controlled experimental environment (i.e., diet, time of manipulation, temperature and sleep) and amplified number and frequency of biochemical/invasive analysis. Thus, chronic exercise in laboratory rats is important to characterize the metabolic effects and provide

Eur J Appl Physiol (2013) 113:1859–1870 C

5.00 A

4.50

18 16

B

#

4.00

#

14

AB

12

3.50

10 8

3.00 0

4

8

12

0

4

CG

8

12

0

EG80

4

8

12

EG90

Load (% of BW)

Load (g)

190 170 150

Tlim13% (s)

Fig. 3 Values of Tlim13 % (s) timed after exhaustion for hyperlactatemia induction during 12 weeks in CG, EG80 and EG90. A Significantly different in relation to baseline. B Significantly different in relation to 4 weeks

20

*

*

A

Load (g)

Load (% of BW)

Fig. 2 Absolute (g) and relative Load (% of BW) correspondent to LM in CG, EG80 and EG90 obtained in baseline period, 4, 8 and 12 weeks. A Significantly different in relation to baseline. B Significantly different in relation to 4 weeks. C Significantly different in relation to 8 weeks. Asterisk indicates significantly different in relation to CG. Hash indicates significantly different in relation to 4, 8 and 12 weeks

1865

130 110 90

A

A

70

AB

A

AB

A A

AB

50 30 0

4

8 CG

comparative evidence with human beings in relation to training design below anaerobic threshold, physiological adaptations and performance (Gobatto et al. 2001; de Araujo et al. 2012; Pereira et al. 2012). The main finding of the present study is that aerobic training at an intensity equivalent to 80 and 90 % of the anaerobic threshold increased muscle glycogen stores and aerobic capacity but did not cause significant biochemical and metabolic changes. EG90 did not show significant increases in aerobic performance compared to EG80 after the experimental period, contradicting our hypothesis. In addition, the training program at 90 % of the anaerobic threshold did not cause greater changes in biomarkers than EG80. However, the corticosterone concentration increased after 12 weeks at 90 % of the anaerobic threshold in comparison to the CG and EG80. Although the LM (%) remained unchanged after the training program in comparison to the CG, the aerobic performance determined by the absolute method (g) showed increases after 4, 8 and 12 weeks in EG90 and EG80 in relation to baseline. Body weight The gain or loss of body weight during a training program is related to the health of rats (Hohl et al. 2009). The reduction of body weight is one of the symptoms related to

12

0

4

8

12

EG80

0

4

8

12

EG90

a training overload and an inappropriate recovery period (Armstrong and VanHeest 2002; Pereira et al. 2012). However, a training intensity below the anaerobic threshold increased the body weight at the end of 12 weeks, similar to the CG group. These data suggest that the increase in body weight in EG80 and EG90 represents an anabolic condition during long periods of training (Pereira et al. 2012). Aerobic and anaerobic performance In the current study, we used the LM to predict the intensity of effort equivalent to the anaerobic threshold, i.e., that corresponds to the maximal lactate steady state (MLSS), which is considered the gold-standard test in the determination of aerobic capacity (de Araujo et al. 2007). The LM test enabled the determination of individualized aerobic and anaerobic parameters without interfering with the training sessions, as it was applied during a single session (Tegtbur et al. 1993; Voltarelli et al. 2002; de Araujo et al. 2007). Thus, it can be concluded that the LM test is a practical and responsive protocol to evaluate, prescribe and monitor the training in rats (de Araujo et al. 2012). The aerobic performance in the control group was reduced, which may be related to the long periods that the rats were maintained in cages, leading to a sedentary

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A

1.20

Glycogen Concentration (mg/100mg)

Fig. 4 Glycogen stores in gastrocnemius (a) and soleus (b) in baseline, 4, 8 and 12 weeks of experimental period in CG, EG80 and EG90. A Significantly different in relation to baseline (0 week). B Significantly different in relation to 4 weeks. C Significantly different in relation to 8 weeks. Asterisk indicates significantly different in relation to CG


ABC

1.00

ABC

*

*

0.80 0.60 0.40 0.20 0.00 0

4

8

12

Week CG

EG90

1.20 BC

1.00

(mg/100mg)

Glycogen Concentration

B

EG80

0.80

*

BC

0.60 0.40 0.20 0.00 0

4

8

12

Week CG

lifestyle and reduced spontaneous activity (Bocalini et al. 2010; de Araujo et al. 2012). The LM measured by absolute intensity (g) indicated that the aerobic performance was higher after the experimental period in EG80 and EG90 compared to at baseline, as well as at 4 and 8 weeks. Although the intra-groups differences after 12 weeks were insignificant, the absolute LM increased by 31, 26 and 18 % in EG80, EG90 and the CG, respectively, compared to baseline (Gobatto et al. 2001; Clavel et al. 2002). When the relative intensity of LM (%) was analyzed, EG80 and EG90 attenuated the aerobic loss in comparison to the control group. In these circumstances, the endurance training at 80 and 90 % of the anaerobic threshold can be considered beneficial toward developing aerobic capacity, because (Clavel et al. 2002; Habouzit et al. 2009) the LM (%) after 4, 8 and 12 weeks in the CG showed natural aerobic damage in comparison to the baseline values (de Araujo et al. 2012). However, the LM during the endurance training either attenuated the aerobic reduction by relative LM or increased the aerobic capacity by absolute LM. The natural growth of rats limits the interpretations of aerobic adaptations using the swimming model (Dawson and Horvath 1970; Reis et al. 2011). We suggested that relative intensity as measured by LM (%) could be applied to

123

EG80

EG90

correct pronounced differences in intra-group body weights (de Araujo et al. 2007). Therefore, the absolute method may be more useful to indicate overload adaptations in these conditions rather than relative intensity in rats (Reis et al. 2011). The reduction in the anaerobic performance after training (EG80 and EG90) was expected due to high volume and low intensity, leading to the reduction of the following anaerobic characteristics: type II muscle fibers and glycolytic enzymes (Carrow et al. 1967; Costill et al. 1988, 1991; Fitts et al. 1989). However, the glycogen stores increased after the training program, but this was not converted into greater anaerobic performance than that at baseline. The long period of endurance training stimulated glycogen synthesis but did not allow for efficient glycogenolysis in order to promote hyperlactatemia (Costill et al. 1991; Nakatani et al. 1997; Vandenberghe et al. 1999; Hargreaves 2004). In part, the inefficient glycogenolysis may be demonstrated by the lack of elevation in blood lactate levels following the hyperlactatemia induction phase in the LM (Nakatani et al. 1997; Vandenberghe et al. 1999; Hargreaves 2004). However, specific analyses (i.e., glycogen synthase a-b, fosforilase a-b and lactate dehydrogenase) are necessary to verify this phenomenon.

31.9 ± 8.2

551 ± 31

SOD (U/mL)

Sulfhydryl Groups (lM)

289.7 ± 58.1

358.5 ± 95.8

51.5 ± 18.8

0.84 ± 0.30

6.89 ± 0.89

523 ± 107

30.0 ± 3.3

5.8 ± 2.5

323 ± 54

373 ± 55

2.65 ± 1.15

EG80 (n = 10)

367.4 ± 65.3

61.2 ± 17.6

0.83 ± 0.26

7.20 ± 0.89

640 ± 117

29.9 ± 5.0

8.5 ± 2.4

314 ± 45

428 ± 73

2.41 ± 0.56

EG90 (n = 10)

246.9 ± 66.8

55.5 ± 14.2

0.63 ± 0.13

6.29 ± 1.4

356 ± 100

33.0 ± 2.4

5.1 ± 1.9

321 ± 47

315 ± 52

3.04 ± 0.82

CG (n = 10)

8 weeks

D,

427.2 ± 97.4 *

60.5 ± 11.0

0.69 ± 0.16

6.06 ± 0.98

548 ± 225*

34.9 ± 4.0

9.0 ± 5.0

339 ± 46

324 ± 80

3.22 ± 1.40

EG80 (n = 10)

641.1 ± 90.2

60.1 ± 20.2

0.62 ± 0.11

7.05 ± 0.99

A,B,D,

651 ± 183*

32.9 ± 2.8

7.6 ± 5.7

332 ± 93

370 ± 54

3.56 ± 0.94

EG90 (n = 10)

* 215.9 ± 39.0

53.1 ± 5.9

0.68 ± 0.11

7.52 ± 0.60

449 ± 39

35.6 ± 5.1

5.9 ± 1.3

351 ± 56

362 ± 44

2.63 ± 0.62

CG (n = 10)

12 weeks

173.1 ± 17.1

51.8 ± 9.1

0.71 ± 0.09

7.00 ± 0.74

609 ± 222*

34.6 ± 5.7

8.6 ± 3.7

421 ± 138

372 ± 44

2.10 ± 0.60

EG80 (n = 10)

337.8 ± 43.7

52.3 ± 9.2

0.75 ± 0.12

6.75 ± 0.74

637 ± 161*

34.4 ± 4.1

6.5 ± 2.3

393 ± 62

539 ± 54*,#

1.88 ± 0.65

EG90 (n = 10)

* Significantly different in relation to CG; # significantly different in relation to EG80; A significantly different in relation to baseline (0 week); B significantly different in relation to 4 week; D significantly different in relation to 12 week

The antioxidant activity (SOD, CAT, GPx) and sulfhydryl groups during 12 weeks of experimental period

277.4 ± 45.1

Creatine kinase (U/L)

52.6 ± 13.0

0.64 ± 0.11

0.71 ± 0.13

50.4 ± 4.75

7.17 ± 1.05

485 ± 142

36.0 ± 4.3

5.85 ± 0.99

Urea (mg/dL)

Peak lactate (mmol/L) Creatinine (mg/ dL)

Metabolites

4.1 ± 1.0

5.4 ± 1.8

304 ± 25

298 ± 35

GPx (U/mL)

CAT (U/mL)

3.79 ± 1.16 335 ± 48

2.82 ± 0.72

CG (n = 10)

CG (n = 10)

370 ± 55

Corticosterone (ng/mL) Antioxidant

Testosterone (ng/ mL)

Hormones

4 weeks

Baseline

Table 2 Serum concentrations testosterone, corticosterone, creatinine, urea, creatine kinase and peak lactate in baseline (0 week) and after 4, 8 and 12 weeks of training in CG, EG80 and EG90

Eur J Appl Physiol (2013) 113:1859–1870 1867

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1868

Training The training prescription based on 80 and 90 % of the anaerobic threshold was intended to provide an intensity that would stimulate aerobic gains while the animals endured 60 min of continuous effort. To perform long periods of continuous exercise, the intensity of effort should be near the zone of the anaerobic threshold, which enables maintenance over time without allowing blood lactate to accumulate (Gobatto et al. 2001; Seiler and Kjerland 2006). Continuous training is a classical method to develop endurance capacity and is characterized by constant intensity without recovery time (Carvalho et al. 2005). An intensity of 80 % of the anaerobic threshold corresponds approximately to the first lactate threshold and 90 % of the anaerobic threshold corresponds to between the first lactate threshold and the anaerobic threshold (second lactate threshold) (Faude et al. 2009). At exactly the anaerobic threshold intensity, the time to exhaustion is usually below 60 min (Billat et al. 2003), and the rats could likely not perform for this time period. The target volume of training was 60 min, but the total average times for EG80 and EG90 were 52 and 56 min, respectively. The volume in EG80 remained above the overall average until 6 weeks but decreased to the end of the training period. However, the volume in EG90 oscillated near the overall average over the 12 weeks. The longlasting exercise series, might lead to undesirable effects associated with incomplete recovery, such as muscle lesions and incomplete restoration of glycogen stores between exercise sessions (Halson and Jeukendrup 2004; Hohl et al. 2009). Thus, the decrease in volume may be a consequence of the spontaneous recovery required to support the next series. Although training at 80 and 90 % of the anaerobic threshold has shown positive biomarker adaptations, a segmented training or high-intensity training could induce more significant results (Garcıá-Pallare´s et al. 2010; de Araujo et al. 2012). Just as in human beings, it is necessary to develop a specific training program in rats with periods of recovery and variation between the volume and intensity to create different cellular and enzymatic adaptations (Issurin 2010; de Araujo et al. 2012). Series of chronic exercise without intensity variation and recovery periods can result in exceeding the exercise tolerance, promoting the disruption of homeostasis, immune suppression and muscle fatigue (Fry et al. 1992; Halson and Jeukendrup 2004). Biomarkers In the present study, the CK concentration indicated muscle damage after 8 weeks of training (Fry et al. 1992; Brancaccio et al. 2007). However, the CK curve was not

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accompanied by similar changes in antioxidant enzymes, hormonal concentrations and levels of other biochemical markers. After 12 weeks, the CK concentration decreased at the same time the volume decreased. Thus, the CK concentration either increased after the volume increased or decreased after the volume decreased. The CK concentration was shown to be a sensitive biomarker in training, but other studies are necessary to correlate the volume performed with the amount of muscle damage. Exercise stimulates the production of superoxide anion (O!' 2 ) in tissues and blood due to large increases in oxygen uptake (Ji 1999; Lambertucci et al. 2007). Concomitant shifts in the oxidant/antioxidant balance induced by chronic exercise may result in an increased antioxidant capacity (Ji 1999). In the present study, despite the evidence of oxidative attacks suggested by sulfhydryl group analysis after 8 and 12 weeks, this ROS formation was not sufficient to promote a significant endogenous antioxidant response. GPx, SOD and CAT activities measured in plasma remained unchanged in the groups during the experimental period. It is likely that the endurance training below the anaerobic threshold resulted in impaired mitochondrial activity compared to endurance training above the anaerobic threshold, which increases oxygen consumption and reactive oxygen species by approximately 10-folds to 20-folds (Ji 1999). Most studies with laboratory animals involving antioxidant analyses have prioritized tissue samples, because they provide specific information in addition to the feasibility of extraction (Powers et al. 1994; Lambertucci et al. 2007). However, the vast majority of the relevant human studies have used blood samples to measure the antioxidant adaptations due to the unavailability of biopsies (Nikolaidis and Jamurtas 2009). In addition, it is assumed that the plasma is a more accurate reflection of tissue redox status (Nikolaidis and Jamurtas 2009). Thus, we analyzed plasma antioxidant enzymes to allow for data comparison with and transposition to human beings. The blood produces significant amounts of ROS due to increases in temperature, decreases in blood pH, decreases in blood oxygen partial pressure and increases in the concentration of blood lactate (Nikolaidis and Jamurtas 2009). These physiological imbalances are associated with high intensity or intensities above the anaerobic threshold (Ji 1999). Studies have shown that during physical exercise at intensities below the anaerobic threshold, physiological conditions are held in a steady state. Despite the reduced organic stress at intensities below the anaerobic threshold, these intensities promote significant aerobic gains (Carvalho et al. 2005; Lambertucci et al. 2007). Testosterone is an anabolic hormone related to protein synthesis and the deposition of glycogen in muscles (Halson and Jeukendrup 2004). In contrast, cortisol is a


catabolic hormone that induces lipolysis, proteolysis and hepatic glycogenolysis (Meeusen et al. 2004). Testosterone and cortisol levels have been used to estimate the anabolic and catabolic phases during training programs (de Araujo et al. 2012). High corticosterone concentrations, which represent a stressful period of training, were observed after 12 weeks in EG90. However, the testosterone levels remained unchanged during the training in both groups (EG80 and EG90). Flynn et al. (1994) examined testosterone and cortisol concentrations throughout the season in competitive runners and swimmers and reported that large changes in volume are required to show significant changes in these parameters. Our results showed that corticosterone might be used as an important biomarker to indicate a catabolic phase during high-volume training. In summary, the endurance training in rats can be easily implemented in other studies involving experimental interventions in laboratory animals or in human beings, because enables a better understanding of the physiological, metabolic and performance alterations. Specifically, continuous aerobic training did not cause significant biomarker alterations, except for CK in both groups after 8 weeks and corticosterone concentrations in EG90 after 12 weeks. In addition, the training at 80 and 90 % of the anaerobic threshold during the 12 weeks induced glycogen super-compensation in the soleus and gastrocnemius muscles to increase the absolute aerobic performance after 12 weeks in comparison to baseline. However, the adaptations in aerobic capacity were similar for both exercise protocols at intensities below anaerobic threshold. Acknowledgments The authors thank FAPESP (04/01205-6; 06/ 58411-2) for the financial support.

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