There are many reports about ACTH and β-endorphin responses to exercise (4), and it also has been argued that exercise training may influence the physiological stress response to nonexercise challenges (i.e., there may be a cross-stressor adaptation) (15). For example, it has been shown that ACTH responses to nonexercise stressors such as immobilization (17) and foot shock (18) are augmented after treadmill exercise training. In order for these responses and adaptations to be better understood, it important to consider the precursor molecule proopiomelanocortin (POMC). POMC gives rise to β-endorphin, ACTH, α-melanocyte-stimulating hormone (α-MSH), and corticotropin-like intermediate lobe peptide (CLIP). POMC has emerged as a potential neuroregulator of energy balance (7,11). Moreover, POMC has been shown to play a role in the feedback component of the response of the hypothalamic-pituitary-adreno-cortical (HPAC) axis to acute and chronic stressors such as foot shock, prolonged swimming or restraint (14). Exercise is a complex, and the influence of POMC mRNA expression in brain areas other than the hypothalamus and pituitary has not, yet, been reported. Hence, the purpose of this investigation was to examine the effects of acute and chronic treadmill exercise on POMC mRNA expression not only in the hypothalamus but also in the frontal cortex and hippocampus, brain areas that are involved in the stress process—including memory and the processing of emotions such as fear.
MATERIALS AND METHODS
Animals and Exercise
Male Sprague-Dawley rats, weighing between 180 and 220 g and supplied by the Laboratory Animal Center of Beijing Medical University, were housed in groups of 4–5 animals per cage with free access to water and food under standard conditions (22°C, 40% humidity and a light/dark cycle, light between 06:00 and 18:00). This experiment closely followed the standard of National Administration Committee for Laboratory animals of China and American College of Sports Medicine policy.
Rats were forced to run on a motor-driven treadmill. Treadmill running was stimulated during the adaptation period through the use of sound (15∼30 db) and mild electric shock presented to the rats’ tails. The treadmill grade was 0°, and the speed and duration are described in Table 1.
Control group (A, N = 13).
Rats in this group did not engage in any form of exercise from the start of the experiment to the end.
Exercise control group (B, N = 12).
After 5 wk of adapting exercise, the rats run at a speed of 35 m·min−1 for 20 min·d−1 for 2 wk but were not run on the day they were sacrificed.
Experimental exercise group (N = 56).
After 5 wk of adapting exercise, the rats run at a speed of 35 m·min−1 for 25 min·d−1 for 2 wk. This group of rats did run on the day they were sacrificed.
On the day they were sacrificed, rats from the experimental exercise group were randomly divided into four subgroups, namely C, E, F, and G, which respectively became the preexercise control group, 0-min group, 30-min postexercise group, and 3-h postexercise group.
All rats were placed under deep anesthesia with 10% chloral hydrate (400 mg·kg−1, i.p.) perfused through the ascending aorta with approximately 100 mL of cold physiological saline. This was followed by 300 mL of cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were then removed and placed in the same fixative at 4°C for 6–10 h and followed by immersion in a 20% sucrose solution with a 0.1 M phosphate buffer (PB, pH 7.4) at 4°C for more than 24 h before sectioning. The brains were rapidly frozen with liquid nitrogen and sectioned horizontally (10 μm) on a freezing microtome (LEICA-CM 1900, Heidelberg, Germany) and thaw-mounted onto 2% 3-amino propyltriethoxysilane-coated microscope slides.
In Situ Hybridization
For in situ hybridization studies, tissue sections were treated as follows: each section was incubated with 0.3% methanol/hydrogen peroxide in dark place, at 37°C for 30 min. Subsequently the section was placed in room temperature 0.2 N HCl for 10 min and then transferred to a 100 μg·mL−1 protein kinase K for 20 min at 37°C, followed by fixing in 4% paraformaldehyde for 5 min at room temperature. After each of the above three steps, the slide preparation was washed in phosphate-buffered saline (PBS) (pH 7.4) 3 × 10 min per wash. The preparation was then washed in both cold 70% ethanol and 100% ethanol for 5 min each solution and then hybridized with the POMC Bio-cRNA probe, which had been labeled with random primer (RP kit, Promega Beijing, China; biotin labeling kit for PCR, Sino-American Biotechnology Company; POMC plasmid as a kind gift offered by Dr. Dominic J. Autelitano, Molecular Physiology Laboratory, Baker Medical Research Institute, Australia).
Each section was covered with 22-μL hybridization solution (10-L 50% formamide, 4-μL 20 × standard saline citrate (SSC), 2-μL × 20 Denhard’s solution, 1-μL salmon sperm DNA, 1-μL 1-M DTT, 2-μL 50% Dextra sulfate, and 2-μL POMC Bio-cRNA probe) and hybridized in humidified boxes containing 50% formamide overnight (24 h) at 42°C. After hybridization, the sections were washed in 2 SSC for 30 min and 1 SSC for 30 min. The incubation was followed by a soak in avidin-biotin-peroxidase complex (Vector Labs, Burlingame, CA, 1:100) at 37°C for 1 h, and washed in PBS (pH 7.4) for 10 min at room temperature, then followed by 0.05% DAB (diaminobenzidine)/0.01% hydrogen peroxide, and examined by microscope. Control slices were treated with the same procedure with omission of the cRNA probe but this had no effect on the staining.
Image Pattern Analysis
The quantity and comparative levels of POMC mRNA expression were determined by the positive signal intensities as scanned by a image pattern instrument (QTM 970, Cambridge, England), the amplified time was 40 × 2 × 20 (objective lens × transmission × circuit). Six or nine sections were randomly measured in each group and the values of integral optical density were used to indicate relative amounts of mRNA expression.
Data are expressed as means ± standard deviations ( ± SD), and comparisons between groups were made using analysis of variance (ANOVA) followed with Duncan’s test. Significance was accepted at the P < 0.05 level.
Under microscopic observation, POMC mRNA expression appeared as golden particles in the cytoplasm. They were widely distributed in cortex, hippocampus, thalamus, and hypothalamus cells. POMC mRNA expression in the frontal cortex, hippocampal CA1, and hypothalamus was quantified using image pattern analysis, the results of which are shown in Table 2.
Changes in POMC mRNA expression in the frontal cortex: the integral optical density was markedly higher in the preexercise group than in either the normal control or exercise control group. Levels were significantly lower at 0 min, 30 min, and 3 h after the cessation of exercise as compared with the preexercise group (F = 29.7;df = 5,43;P < 0.01), but there were no significant differences at 0 min, 30 min, and 3 h after the cessation of exercise within the group (P > 0.05).
Changes in POMC mRNA expression in the hippocampal CA1: the integral optical density showed no marked difference preexercise among the three groups (P > 0.05). In the postexercise group, it was significantly lower at 0 min after the cessation of exercise as compared with that of the preexercise state, but levels significantly increased returning to preexercise levels at 30 min and 3 h.
Changes in POMC mRNA expression in the hypothalamus: in the preexercise state, the integral optical density in the exercise control group was noticeably higher than in the normal control group. It was also increased at 0 min, 30 min, and 3 h after the cessation of exercise than it was at preexercise state (F = 2.9;df = 5,42;P < 0.05 or P < 0.01) in comparison with the preexercise group, but there was no significant difference between groups at 0 min, 30 min, and 3 h (P > 0.05).
POMC is the precursor of ACTH, β-endorphin, and α-MSH and is primarily synthesized in the pituitary gland. Extrapituitary sites of POMC gene expression have been reported in various organs including such brain sites as the hypothalamus, nucleus amygdala, and cortex (5), the testes and ovaries (3,6,8,10), adrenal medulla (9), and gastrointestinal tract, lung, and spleen (1). It has also been shown to have a role in the immune system (2,13). POMC fibers are generally lightly distributed throughout the nucleus, but the area ventromedial to the anterior commissure region appears to contain the most dense POMC innervation, the origins of which are believed to lie in the POMC-producing cells in the nucleus arcuatus of the hypothalamus (12).
Exercise influences peripheral mechanisms involved in the regulation of energy balance. Although the central mechanisms are less well understood, exercise-induced changes in POMC gene expression may be involved. Kim et al. (11) reported that POMC expression in arcuate nucleus and α-MSH peptide levels in paraventricular nucleus are decreased in diabetes. Also, insulin may influence arcuate and pituitary POMC activity in neurons involved in the regulation of energy metabolism. Furthermore, Guan et al. (7) and colleagues reported that the altered hypothalamic POMC and/or neuropeptide Y functions may be important contributing factors for the development of obesity in mutant mice.
In our research, POMC positive cells were seen to be widely distributed in the cortex, hippocampus, thalamus, hypothalamus, and their corresponding nuclei, as determined using the in situ hybridization method. In response to chronic exercise stress, we found the integral optical density of POMC mRNA became markedly elevated in the frontal cortex, but there was no obvious change in hippocampal CA1 or hypothalamic levels. Immediate postexercise, the integral optical density of POMC mRNA was markedly decreased in the frontal cortex and hippocampal CA1 areas. This may be due to the fact that high-intensity acute exercise depresses POMC gene expression. Measurements taken 30 min and 3 h postexercise showed that the POMC mRNA levels in the frontal cortex still had not returned to preexercise levels. Levels measured in the hippocampal CA1 area, however, showed a return to preexercise levels even after 30 min. The POMC mRNA in the hypothalamus showed a continuous increase in levels postexercise, possibly due to the fact that high-intensity acute exercise stimulates hypothalamic POMC gene expression. The above results indicate POMC gene expression varies depending on different brain regions and that, in response to sustained high-intensity acute exercise, its expression levels and recovery rates also differ.
POMC mRNA in the frontal cortex was elevated in the preexercise group as compared with the exercise control and normal control groups, but the postexercise groups had expression levels similar to the nonexercise controls. The elevated levels shown by the preexercise group is possibly a result of anticipation of forced treadmill running or as a conditioned fear effect. Prior research has found that electric shock, and its controllability, has effects on brain neural function (16). Nonetheless, our prior research (4) found that high-intensity acute exercise has an inhibitory effect on the POMC mRNA expression levels in the frontal cortex; it may be considered that the elevated POMC gene expression in the preexercise group but lack of elevation at later time is an indication that the exercise stress-induced effect is transient. The decreased POMC levels in the exercise control group suggest that chronic exercise may lead to a tonic, compensatory decrease in POMC gene expression.
In conclusion, we have observed that the changes of POMC gene expression in different areas of the rat brain including the frontal cortex, hippocampal CA1, and hypothalamus as responding to chronic and high-intensity treadmill acute exercise were “bidirectional,” showing decreases in some areas and increases in others. Furthermore, the duration of the changes also varied depending on brain area. These results suggest that the altered central POMC mRNA may be related to such chronic and high-intensity acute exercise.
We wish to thank the cooperative unit of the China National Research Institute of Sports Science for animal housing, exercising, and surgery, Prof. Huang Qifu for photography, Prof. Zhang Zhengsheng for image pattern analysis, Dr. Dominic J. Autelitano for his kind gift of POMC plasmid, Ms. Liu Xiaonan for animal brain sectioning, and Ms. Brenda Hood for correcting the English. We also wish to thank Associate Editor Patrick J. O’Connor for his kind help and for carefully handling the manuscript.
This project was supported by a grant from the National Natural Science Foundation of China (NSFC, No. 39430140).
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