The mechanism(s) for how physically active organisms are resistant to many damaging effects of acute stressor exposure is unknown. Cellular induction of heat-shock proteins (e.g., HSP72) is one successful strategy used by the cell to survive the damaging effects of stress. It is possible, therefore, that the stress-buffering effect of physical activity may be due to an improved HSP72 response to stress. Thus the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Adult male Fischer 344 rats were housed with either a mobile running wheel (Active) or a locked, immobile wheel [sedentary (Sed)] for 8 wk before stressor exposure. Rats were exposed to either inescapable tail-shock stress (IS; 100 1.6-mA tail shocks, 5-s duration, 60-s intertrial interval), exhaustive exercise stress (EXS; treadmill running to exhaustion), or no stress (controls). Blood, brain, and peripheral tissues were collected 2 h after stressor termination. The kinetics of HSP72 induction after IS was determined in cultured mesenteric lymph node cells. Activation of the stress response was verified by measuring serum corticosterone (RIA). Tissue and cellular HSP72 content were measured using HSP72 ELISA in cell lysates. Both Active and Sed rats had elevated levels of serum corticosterone after stress. In contrast, Active but not Sed rats exposed to IS and/or EXS had elevated HSP72 in dorsal vagal complex, frontal cortex, hippocampus, pituitary, adrenal, liver, spleen, mesenteric lymph nodes, and heart. In addition, Active rats exposed to IS demonstrated a faster induction of lymphocyte HSP72 compared with Sed rats. Thus Active rats responded to stress with both greater and faster HSP72 responses compared with Sed rats. These results indicate that previous physical activity potentiates HSP72 expression after a wide range of stressors. Facilitated induction of HSP72 may contribute to the increased stress resistance previously reported in physically active organisms.
- acute stress
- heat-shock proteins
physically active organisms are protected from many of the damaging effects of stressor exposure. For example, it has been previously reported that physical activity can prevent the negative effect of stress on behavioral depression (9, 35), anxiety (11), immune function (12, 15, 23), and splenic apoptosis (2). The mechanism(s) for the stress-buffering effect of physical activity remains unclear. One feature of the stress response that could be altered by physical activity status and can contribute to stress resistance that has not yet been thoroughly investigated is the cellular stress response.
The generation of the stress response is under tight regulatory control and is designed to facilitate an organism's chance of survival during challenge. This is accomplished at both the system (e.g., muscular, cardiovascular, respiratory, and neuroendocrine) and single-cellular levels. One way cells can resist damage and/or death induced by stress is to synthesize a highly conserved set of intracellular proteins, termed heat-shock proteins (HSPs) (25). A variety of stressors are known to induce HSPs, and these proteins confer protection and/or tolerance against stress-induced oxidative, heat, and cytokine insult (24, 36). The 70-kDa HSP (HSP70) family of proteins includes a constitutive 73-kDa protein (HSC73) and a highly stress-inducible 72-kDa protein (HSP72) (37). HSP72 is essential for cellular recovery after stress as well as survival and maintenance of normal cellular function. Furthermore, HSP72 prevents protein aggregation and also refolds damaged proteins. Importantly, expression of high levels of HSPs has been associated with an increased ability of cells to withstand challenges that would otherwise lead to cell injury and/or death (36). It would be reasonable to speculate, therefore, that one potential adaptation produced by physical activity that could contribute to stress resistance would be improvements in the cellular HSP response.
In fact, there is some evidence to support this idea. Fehrenbach et al. (13) demonstrated that in vitro heat shock of human peripheral blood leukocytes significantly stimulated HSP27 and HSP70 mRNA, with physically active individuals exhibiting the greatest increases. Additionally, Gonazalez et al. (17) demonstrated that HSP72 was expressed at higher levels in the skeletal muscles of treadmill-trained rats compared with sedentary animals after an exhaustive exercise challenge. Although these studies lend support to the hypothesis that habitual physical activity might better prepare an organism to deal with subsequent stressor exposure at the cellular level, they are limited for a variety of reasons. First, HSPs are ubiquitously induced after stress (29, 36, 39, 45), yet these studies only investigated peripheral blood lymphocytes and skeletal muscle. Furthermore, whole organism stressors affect multiple tissues (36), and physical activity selectively changes features of the stress response such that some systems demonstrate adaptations while others do not (9, 10, 15, 27, 35, 47); thus it is important to examine more than a single tissue or system during an experiment. Last, in light of the facts that previous preconditioning (i.e., exposure to stress) results in a larger HSP response to a subsequent stressor (25) and that treadmill training has been reported to be chronically stressful to rats (34), the results from the previously mentioned study using treadmill training are difficult to interpret.
Therefore, the purpose of the current study was to determine whether prior voluntary freewheel running facilitates the stress-induced induction of HSP72 in central (brain), peripheral, and immune tissues. Voluntary freewheel running was chosen because this type of physical activity does not activate the stress response (33, 37). The following tissues were examined: brain (hypothalamus, hippocampus, dorsal vagal complex, prefrontal cortex), peripheral tissues (pituitary, adrenal, heart, liver, triceps), and immune tissues (spleen and mesenteric lymph nodes). These tissues were chosen to extend the previous observations and because they are known to be stress responsive. The triceps muscle was selected because previous studies have indicated that this particular muscle shows physiological adaptations to freewheel running (41), and it is unknown if the HSP72 response to stress is modulated by voluntary freewheel running. In addition to examining multiple stress-responsive tissues, the generality of the effect across stressors was tested by assessing the effect of exposure to two different stressors, i.e., inescapable tail-shock stress (IS) and treadmill running to exhaustion [exhaustive exercise stress (EXS)]. Finally, to begin to investigate how physical activity changes HSP72 responses, examination of the rate of HSP72 induction after stress was tested in lymphocytes from physically active and sedentary rats. We hypothesize that habitually physically active rats will have both greater and faster HSP72 responses to stress, suggesting that physical activity may contribute to stress resistance at the cellular level.
Adult male viral-free Fischer (F344) rats (200–300 g, 8–9 wk old at start of experiments; Harlan Labs) were used in all experiments (5–6 animals/experimental group). Preliminary data revealed that five to six animals per experimental group were sufficient to achieve statistical significance. The animal colonies were maintained in a pathogen-free barrier facility with a 12:12-h light-dark cycle (lights on 0600–01800). Colony room temperature was maintained at 22–23°C. Rats were given at least 1 wk to habituate to the colonies before experimentation. All rats were handled and weighed each day for at least 3 days before each study began. Care and use of the animals were in accordance with protocols approved by the University of Colorado Institutional Animal Care and Use Committee and fully conform to the “Guiding Principles for Research Involving Animals and Human Beings” of the American Physiological Society (1).
Rats were individually caged in Nalgene Plexiglas cages (45 × 25.2 × 14.697 cm) with a stainless steel open running wheel attached (1.081 wheel circumference). Physically active rats (Active) had a mobile running wheel and ran for 8 wk before stressor exposure. Sedentary controls (Sed) were housed in the same environment except that the running wheel was locked and remained immobile. Voluntary freewheel running was the chosen modality because, in contrast to forced treadmill training, voluntary freewheel running does not produce negative adaptations that are indicative of chronic stress in rats (34, 35). The caging environment meets National Institutes of Health floor space standards for a single rodent. Total daily running distances were monitored hourly by computer with the VitalView Automated Data-Acquisition System (Bend, OR). Rats were weighed weekly.
Animals either remained undisturbed in their home cages as controls (Active-No Stress, n = 6, 16 wk old, 334.5 ± 3 g; Sed-No Stress, n = 6, 16 wk old, 347.5 ± 2 g the day of experimentation) or were exposed to IS (Active-IS, n = 6, 16 wk old, 335.3 ± 3 g; Sed-IS, n = 6, 16 wk old, 346.2 ± 2 g the day of experimentation). IS rats were transported to an adjacent room and placed in Plexiglas restraining tubes (15 × 7 cm). Contact beams were placed on the tail of the rat, and 100 1.6-mA tail shocks (5-s duration, variable intertrial interval ∼60 s; range 30–90 s) were administered. The total IS session lasted ∼100 min.
Animals either remained undisturbed in their home cages as controls (Active-No Stress, n = 5, 17 wk old, 338.8 ± 3 g; Sed-No Stress, n = 5, 17 wk old, 343.9 ± 2 g the day of experimentation) or were exposed to a bout of EXS (Active-EXS, n = 5, 17 wk old, 338.0 ± 3 g; Sed-EXS, n = 5, 17 wk old, 345.8 ± 3 g the day of experimentation). EXS rats were transported to an adjacent room and placed on a treadmill and forced to run until they reached volitional exhaustion. The exhaustive exercise protocol consisted of running on a motorized treadmill on a 10% grade, at a speed of 17.5 m/min during the initial 5 m of the test and 20 m/min thereafter. A shock grid at the back of the treadmill provided a mild but aversive foot shock (1.6 mA) if the pace of the rat slowed below treadmill rate. Very few shocks were administered during the bout of exercise and occurred within the first few minutes and final few minutes of exercise. Volitional exhaustion was defined as the inability to continue to run on the treadmill as observed by a failure to run after 10 consecutive foot shocks. The total duration of treadmill testing lasted between 20 and 95 min, depending on prior physical activity status.
Blood and tissue collection.
Two hours after IS, EXS, or No Stress, animals were killed by decapitation. Preliminary studies indicated that 2 h after the termination of stress was the optimal time point for HSP72 induction in the majority of tissues (data not shown). Trunk blood was collected for later measurement of serum corticosterone in 50-ml conical tubes. Tubes were stored on ice and immediately spun in a refrigerated centrifuge. Aliquots of serum were taken and stored at −20°C until time of assay. The pituitary and brain were quickly removed after decapitation. Brains were dissected on a frosted glass plate placed on top of crushed ice. Brain structures, which included hypothalamus, hippocampus, prefrontal cortex, and dorsal vagal complex, along with the pituitary, were placed in microfuge tubes and quickly frozen in liquid nitrogen. Left and right adrenal, liver, spleen (cut in half), mesenteric lymph nodes, right ventricle of the heart, and left triceps were aseptically dissected and then placed in microfuge tubes and flash frozen in liquid nitrogen. Tissues were removed from liquid nitrogen and stored at −70°C until time of tissue processing.
Brain tissue processing.
Each tissue was added to 0.25–1.0 ml of cold Iscove's culture medium containing 5% fetal calf serum and a cocktail enzyme inhibitor (in mM: 100 amino-n-caproic acid, 10 EDTA, 5 benzamidine-HCl, and 0.2 phenylmethylsulfonyl fluoride). Total protein was mechanically dissociated from tissue using an ultrasonic cell disrupter (Heat Systems, Farmingdale, NY). Sonication consisted of 10 s of cell disruption at setting 10. Sonicated samples were centrifuged at 35,000 g for 10 min at 4°C. Supernatants were removed and stored at −20°C until time of assay.
Peripheral tissue processing.
Each tissue was placed in homogenizing buffer (HSP70 Extraction Reagent, StressGen Biotechnologies, Victoria, British Columbia, Canada, and 1 protease inhibitor cocktail tablet, Boehringer Mannheim, Mannheim, Germany, per 50 ml of Extraction Reagent). For each ∼0.5 ml piece of tissue, 1 ml of homogenizing buffer was used. Tissues were then dissociated using sterile, modified, glass tissue homogenizers, and cells were lysed by exposure to extraction reagent. The cell lysates were transferred to microfuge tubes and centrifuged at 30,000 g for 10 min at 4°C. Supernatants were removed and stored at −20°C until time of assay.
Cell isolation, processing, and culture procedure for HSP72 kinetics analyses.
To investigate the kinetics of HSP72 induction after stress, mesenteric lymph nodes were dissected from rats exposed to IS or No Stress 30 min after stressor termination. Mesenteric lymph nodes were aseptically dissected and then placed in dissection medium [15 ml of Iscove's medium (GIBCO, Grand Island, NY) with 1% penicillin-streptomycin (GIBCO)] over ice. Tissues were dissociated using sterile, modified, glass tissue homogenizers, and cells were counted using a Coulter particle counter. Cells were then resuspended in culture medium [Iscove's medium (GIBCO) containing 10% FBS (GIBCO) with 1% penicillin-streptomycin (GIBCO) and 1% l-glutamine (GIBCO)] at 6.25 × 106cells/ml. One-hundred twenty-five microliters of each suspension was plated into a well of a sterile 96-well round bottom culture plate (Falcon) and placed in culture at 37°C, 95% air-5% CO2. Cells were then removed from culture at 2.5, 3.5, and 5.5 h after the termination of IS, placed in microfuge tubes, and centrifuged at 30,000g for 10 min at 4°C. Pellets were resuspended with 150 μl of lysis buffer (HSP70 Extraction Reagent) and centrifuged again. Supernatants were removed and stored at −20°C until time of assay. HSP72 was measured from cell lysates using ELISA (StressGen Biotechnologies).
Measurement of proteins.
Serum corticosterone was measured using a commercially available RIA kit (ICN Pharmaceuticals, Costa Mesa, CA). HSP72 was measured from tissue lysates using a commercially available ELISA for rat HSP72 (StressGen Biotechnologies). Both the intra-assay and interassay coefficients of variations are <10%. Validation of HSP72 changes in rodents using a quantitative ELISA has been verified using Western blot analyses and reported to be more sensitive and more quantifiable than the Western blot technique (33). All samples were assayed at optimal dilutions and according to manufacturer's instructions. Tissue total protein was measured using the Bradford assay as described previously (16). The concentration of HSP72 protein per unit of total protein (pg/μg) is presented.
One-factor ANOVA was performed to analyze rat body weight data. A two (Active vs. SED) × three (baseline, IS, EXS) ANOVA was used to test the effect of stress on serum corticosterone. Two-factor repeated-measures ANOVA was used to examine the effects of stress and physical activity on HSP72 in cell culture samples over time. To preserve statistical power while having unequal group sizes, one-way ANOVAs were performed for analysis of HSP72 after either IS or EXS stress in Active and Sed rats. Post hoc pairwise comparison analyses were conducted using the Fisher protected least significant difference test (F-PLSD); α was set at 0.05. Data are presented as group means ± SE.
Freewheel running distance and body weight.
Rats (Active) in the current studies voluntarily ran in their wheels an average of 3.185 ± 0.32 km/wk and gained slightly less body weight than their Sed counterparts [Active mean = 336.9 ± 3.3 g; Sed mean = 345.0 ± 4.6 g,F(1,113) = 1.991, P = 0.16].
Time to exhaustion.
As would be expected, rats (Active) that lived with a running wheel for 8 wk before treadmill testing ran significantly longer than their Sed counterparts [Active mean = 62.2 ± 13.3 min; Sed mean = 20.4 ± 0.9 min, F(1,8) = 9.8,P = 0.01].
Effects of IS and EXS stress on serum corticosterone.
To verify that both IS and EXS induced a reliable stress response, serum corticosterone was measured from trunk blood samples taken from rats that were not exposed to stress (No Stress) and rats that were killed 2 h after stressor termination (Fig.1). A two × three ANOVA revealed no main effect of physical activity (P = 0.9) but a statistically significant effect of stress condition [F(2,29) = 18.8, P = 0.0001] and a significant stress condition × physical activity status interaction [F(2, 29) = 4.8,P = 0.01].
Post hoc analyses revealed that rats exposed to either IS or EXS stress had elevated stress hormones for at least 2 h after stressor termination compared with nonstressed controls (Active-No Stress vs. Active-IS, F-PLSD, P < 0.01; Active-No Stress vs. Active-EXS, F-PLSD, P < 0.01; Sed-No Stress vs. Sed-IS, F-PLSD, P < 0.01; Sed-No Stress vs. Sed-EXS, F-PLSD, P < 0.01). Two hours after stressor termination, Active rats had higher serum corticosterone after IS stress (Active-IS vs. Sed-IS, F-PLSD, P = 0.02) and a trend for a lower serum corticosterone after EXS stress (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.06).
Effects of IS on HSP72 induction in brain.
The effect of IS on HSP72 levels in brain is shown in Fig.2, A–D. IS significantly induced HSP72 response only in Active and not Sed rats for most brain areas tested. ANOVA revealed significant differences between groups in dorsal vagal complex [F(3,19) = 4.481,P = 0.015].
Post hoc analyses revealed significant effects of IS on HSP72 in Active rats in the dorsal vagal complex (Active-No Stress vs. Active-IS, F-PLSD, P = 0.02) and the frontal cortex (Active-No Stress vs. Active-IS, F-PLSD, P = 0.05). In all brain areas tested, there were no significant post hoc differences in HSP72 induction between Active-IS and Sed-IS, and physical activity alone did not effect HSP72 induction.
Effects of IS on HSP72 induction in peripheral stress-responsive tissue.
The effect of IS on HSP72 in peripheral, stress-related tissues is shown in Fig. 3, A–D. ANOVA (1 × 4) revealed significant differences between the groups in the pituitary [F(3,19) = 3.176,P = 0.047], adrenal [F(3,19) = 7.013, P = 0.0023], and liver [F(3,18) = 3.745,P = 0.029].
Post hoc analyses indicated significant elevations in HSP72 in both physically active (Active-No Stress vs. Active-IS, F-PLSD,P < 0.05) and sedentary rats (Sed-No Stress vs. Sed-IS, F-PLSD, P < 0.05) for every tissue examined. The effect of IS on HSP72 was greater in physically active vs. sedentary rats in the pituitary (Active-IS vs. Sed-IS, F-PLSD,P < 0.05) and adrenal (Active-IS vs. Sed-IS, F-PLSD, P = 0.05). Physical activity alone did not effect HSP72 induction in any tissue.
Effects of IS on HSP72 induction in skeletal muscle tissue.
It appears the voluntary freewheel running is insufficient to induced HSP72 adaptations in the triceps muscle. The data also suggest that at the 2-h time point, IS does not induce triceps HSP72. No difference was found for any group in the triceps muscle (Active-No Stress = 8.3 ± 3.1 pg/μg; Active-IS = 10.3 ± 3.1 pg/μg; Sed-No Stress = 4.0 ± 0.5 pg/μg; Sed-IS = 7.6 ± 1.4 pg/μg).
Effects of IS on HSP72 induction in immune tissues.
The effect of IS on HSP72 in immune tissues is shown in Fig.4, A and B. ANOVA (1 × 4) revealed significant differences between the groups in spleen [F(3,19) = 5.173,P = 0.008] and mesenteric lymph nodes [F(3,19) = 4.297, P = 0.017].
Post hoc analyses indicated significant elevations in HSP72 in both physically active (Active-No Stress vs. Active-IS, F-PLSD,P < 0.05) and sedentary rats (Sed-No Stress vs. Sed-IS, F-PLSD, P < 0.05) in the spleen but not the mesenteric lymph nodes. In this tissue, IS did not elevate HSP72 in sedentary rats. IS elevated HSP72 more in physically active than sedentary rats (Active-IS vs. Sed-IS, F-PLSD, P < 0.05). Physical activity alone did not effect HSP72 induction in any tissue.
Effects of EXS on HSP72 induction in brain.
The effect of EXS on HSP in brain is shown in Fig.5, A–D. EXS significantly induced HSP72 response only in Active and not in Sed rats for most brain areas tested. ANOVA (1 × 4) revealed a significant difference between groups in hippocampus [F(3,16) = 5.162, P = 0.01], cortex [F(3,16) = 6.858,P = 0.003], and dorsal vagal complex [F(3,16) = 6.919, P = 0.0034].
Post hoc analyses revealed significant effects of EXS on HSP72 in physically active rats in the hippocampus (Active-No Stress vs. Active-EXS, F-PLSD, P = 0.02), the frontal cortex (Active-No Stress vs. Active-EXS, F-PLSD, P = 0.01), and dorsal vagal complex (Active-No Stress vs. Active-EXS, F-PLSD,P = 0.006). EXS did not significantly increase HSP72 in sedentary rats in any brain region tested (Sed-No Stress vs. Sed-IS, F-PLSD, P > 0.10). The effect of EXS on HSP72 was statistically greater in physically active vs. sedentary rats in hippocampus (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.003), prefrontal cortex (Active-EXS vs. Sed-EXS, F-PLSD,P = 0.0005), and dorsal vagal complex (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.0086). Physical activity alone did not effect HSP72 induction.
Effects of EXS on HSP72 induction in peripheral stress-related tissues.
The effect of EXS on HSP in peripheral stress-related tissues is shown in Fig. 6, A–D. ANOVA revealed significant differences between the groups in pituitary [F(3,19) = 3.176, P = 0.047], adrenal [F(3,16) = 24.55,P = 0.0001], heart [F(3,16) = 28.07,P = 0.0001], and liver [F(3,15) = 3.416, P = 0.04].
Post hoc analyses indicated significant elevations in HSP72 in both physically active (Active-No Stress vs. Active-IS, F-PLSD,P < 0.05) and sedentary rats (Sed-No Stress vs. Sed-IS, F-PLSD, P < 0.05) in the adrenal, heart, and liver. EXS increased pituitary HSP72 in physically active rats (Active-No Stress vs. Active-EXS, F-PLSD, P = 0.0006) but not sedentary rats. The effect of EXS on HSP72 was reliably greater in physically active vs. sedentary rats in the pituitary (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.004), adrenal (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.0001), and heart (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.0002). Physical activity alone did not have an effect on HSP72 in any tissue except the heart (Active-No Stress vs. Sed-No Stress, F-PLSD, P = 0.05).
Effects of EXS on HSP72 induction in skeletal muscle tissue.
No difference was found for any group in the triceps muscle after voluntary freewheel running or 2 h after EXS [Active-No Stress = 5.2 ± 0.6 pg/μg; Active-EXS = 5.2 ± 1.6 pg/μg; Sed-No Stress = 3.1 ± 0.6 pg/μg; Sed-EXS = 4.2 ± 1.1 pg/μg; data not shown].
Effects of EXS on HSP72 induction in immune tissues.
The effect of EXS on HSP72 in immune tissues is shown in Fig.7, A and B. ANOVA (1 × 4) revealed significant differences between the groups in spleen [F(3,16) = 8.477,P = 0.0013] and mesenteric lymph nodes [F(3,16) = 5.567, P = 0.0082].
Post hoc analyses indicated significant elevations in HSP72 in both physically active (Active-No Stress vs. Active-IS, F-PLSD,P < 0.05) and sedentary rats (Sed-No Stress vs. Sed-IS, F-PLSD, P < 0.05) in the spleen. In the mesenteric lymph nodes, EXS elevated HSP72 in active rats (Active-No Stress vs. Active-IS, F-PLSD, P < 0.01) but not sedentary rats. The effect of EXS in the mesenteric lymph node was statistically greater in active vs. sedentary stressed rats (Active-EXS vs. Sed-EXS, F-PLSD, P = 0.010). Physical activity alone had no impact on HSP72 levels.
IS-HSP72 kinetics analyses in lymph node cell cultures.
Rats were exposed to IS, and mesenteric lymph node cells were put into culture 30 min after stressor termination. Cells were removed from culture at 2.5, 3.5, and 5.5 h after IS termination, and HSP72 was measured in cell lysates. As shown in Fig.8, IS induced HSP72 in lymph node cells faster in active vs. sedentary rats. A two × two repeated-measures (time) ANOVA revealed significant main effects of group [IS vs. No Stress, F(1,15) = 9.4,P = 0.01], activity status [Active vs. Sed,F(1,15) = 38.6, P = 0.0007], and time [F(2,30) = 5.2,P = 0.01]. There was also a significant group × activity status interaction [F(1,15) = 9.9, P = 0.0065].
Post hoc analyses revealed a significant effect of IS on HSP72 in active rats 2.5 h (Active-No Stress vs. Active-IS, F-PLSD,P = 0.03), 3.5 h (Active-No Stress vs. Active-IS, F-PLSD, P = 0.004), and 5.5 h (Active-No Stress vs. Active-IS, F-PLSD, P = 0.004) after IS termination. IS significantly increased HSP72 in sedentary rats at only the 3.5-h time point (Sed-No Stress vs. Sed-IS, F-PLSD, P = 0.04). The effect of IS on HSP72 induction was statistically greater in physically active vs. sedentary rats 2.5 h (Active-IS vs. Sed-IS, F-PLSD, P = 0.02) and 5.5 h (Active-IS vs. Sed-IS, F-PLSD, P = 0.03) after IS termination. These data suggest that physically active rats induce HSP72 faster after stressor exposure than do sedentary rats. Physical activity alone did not effect HSP72 induction.
The purpose of the present investigation was to test the hypothesis that habitually physically active rats have both greater and faster HSP72 responses to stress, suggesting that physical activity may contribute to stress resistance at the cellular level. The results of the current studies support this hypothesis. Physically active rats exposed to either IS or EXS stress responded with a greater induction of HSP72 in response to stress than did sedentary rats. Physically active stressed rats had increased HSP72 in nearly every tissue tested. In contrast, sedentary stressed rats had increased HSP72 only in pituitary, adrenal, liver, and spleen, and the increase was smaller than in the physically active-stressed rats. Moreover, physically active rats exposed to IS stress induced HSP72 in lymphocytes at a faster rate than did sedentary rats. Thus the impact of stress on HSP72 induction was potentiated in physically active rats. This study is the first comprehensive examination of the effects of voluntary freewheel running on stress-induced HSP72 expression in multiple stress-responsive tissues. The results are consistent with previous work conducted on skeletal muscle (17) and peripheral blood leukocytes (13) demonstrating that physical activity status can modulate the HSP response to stress.
One implication of the current data is that the greater increase in HSP72 could protect cells in both the brain and periphery from stress-induced damage. It has been previously reported that physical activity can prevent many of the deleterious consequences of stress (2, 9, 11, 12, 15, 23, 35). A greater and faster induction of HSP72 after stress, therefore, could be an important cellular mechanism for the stress-buffering effects of freewheel running. Indeed, there is evidence in the literature to suggest that elevated HSPs can promote cell survival after stressor exposure in brain, peripheral, and immune tissues. Fink et al. (14) were among the first to demonstrate that HSP72 alone protects neurons in the brain after various neuronal insults, and others have subsequently demonstrated the HSP72 upregulation in brain tissues protects those tissues from subsequent lethal damage (30, 48). High levels of HSP72 in peripheral tissues have also been reported to prevent 1) pancreatic cell injury after a supramaximally stimulating dose of cholecystokinin (3), 2) tissue damage in the liver after heat stress (19),3) myocardial injury after ischemia/reperfusion (31), and 4) endotoxin-driven apoptosis in rat cells (6). Furthermore, underexpression of HSP72 increases susceptibility to hypoxia and reoxygenation injury (38). The immune system benefits from HSP induction as well. For example, HSP72 has been found to confer protection to human peripheral blood monocytes from bacteria-induced apoptosis (18), protect tumor cells from TNF-mediated monocyte cytotoxicity (21), protect monocytes from their own cytotoxicity (22), and provide thermotolerance for murine T cells (7). Thus it is reasonable to suggest that an increased induction of HSPs after stress would be a beneficial and adaptive response of the physically active organism.
It is unclear how physical activity modulates HSP72 induction, but a variety of mechanisms are tenable. It has been determined that trained athletes demonstrate higher leukocyte and skeletal muscle HSP72 mRNA expression at rest than do sedentary individuals (13, 28). Thus it has been proposed that trained cells provide high HSP72 transcript levels for immediate translation when necessary (13) (e.g., after stressor exposure). It is also possible that temporal dynamics of HSP72 induction change in a physically active rat. This is in fact the case in aged eukaryotic organisms. Hall et al. (19) demonstrated that aged rats have a reduced ability to generate HSPs in response to thermal challenge, such that aging delays the induction of HSPs. The altered HSP70 response with aging was attributed to a dysfunction in transcriptional regulation. It is, therefore, feasible to speculate that physical activity changes the pattern of HSP expression by exhibiting faster or longer-lasting HSPs due to improved transcriptional regulation. Furthermore, it has been reported that physical activity can increase the efficiency of translation (46), enhance mRNA stability (20), and inhibit protein degradation (44). These adaptations could potentially result in changes in the pattern of HSP expression in physically active organisms; however, they have yet to be observed regarding HSPs and deserve further investigation.
A wide variety of stressful stimuli induce an increase in HSP synthesis; however, the exact signal for HSP72 induction after IS and EXS is unknown. The earliest HSP investigators determined that a rise in a cell's temperature resulted in an increase in HSPs (40), and certainly exposure to IS and EXS would result in stress-induced hyperthermia (5, 8). For several reasons, however, it is unlikely that differences in stress-induced hyperthermia between active and sedentary stressed rats are the mechanisms for the potentiation of HSP72 reported after stress in physically active rats. First, the facilitated induction of HSP72 in physically active rats is tissue specific within the brain, peripheral, and immune tissues. Assuming one region of the brain or peripheral tissue would not be exposed to large differences in temperature, heat cannot be the sole signal responsible for HSP induction. Second, there is no evidence that the core body temperature elevations produced by IS would vary between physically active and sedentary rats. Finally, it has been demonstrated that increases in HSP70 during intense exercise challenge can be independent of body temperature (42). Thus differences in core body temperature after stress are not a likely mechanism for these effects; however, only direct measures of core body temperature will determine if this is an accurate conclusion.
There are many other factors that are released by IS and EXS that have been reported to induce HSP72. For example, both IS and EXS produce a large increase in sympathetic activation (32, 43), and it has been reported that α-adrenergic signaling induces the transcriptional upregulation of HSP70 (26). Physical activity training is known to modulate the sympathetic nervous system (SNS) (4), and it is believed that the SNS contributes to HSP induction (26); thus physically active rats might be better equipped to induce HSP72 after stress due to SNS adaptations. This possibility is currently under investigation.
An alternative explanation for the facilitated HSP72 response observed in active rats is that physically active rats experience the stress to a greater extent. However, there is a great deal of evidence to refute this idea. In fact, there is evidence to suggest that many features of the stress response are either decreased or not different in physically active compared with sedentary rats. For example, physically active rats compared with sedentary rats exposed to stress have decreased sympathetic output (15, 47), decreased activity in stress-reactive brain areas (9), and no change in hypothalamic-pituitary-adrenal axis activity (9, 35). Results from the present study confirm and extend these results. As expected, both sedentary and physically active rats exposed to IS and EXS had elevated serum corticosterone in the circulation 2 h after stressor termination. Physically active rats had a slight increase in corticosterone after IS and slight decrease in corticosterone after EXS. Taken together, these results are consistent with the observation that physical activity training does not change the corticosterone response to stress (8,14). Given that both IS and EXS produced similar changes in HSP72 in brain, peripheral, and immune tissues, modulation of the corticosterone response to stress or global changes in stress sensitivity are not likely mechanisms for the effect of physical activity on HSP72 induction.
Another potential mechanism by which the increased HSP72 induction demonstrated in active rats might be explained is differences in the duration of stressor exposure. As previously mentioned, during EXS active rats were able to run significantly longer than sedentary rats before reaching exhaustion. Thus the argument can be made that an increased duration of stressor exposure resulted in increased induction of HSP72. However, stressor duration cannot explain these findings because the duration of IS stress was equal between the groups, and yet physically active-IS rats still had increased HSP72 responses.
The results of the current studies do indeed suggest a generality of physical activity effects across stressors. Rats were exposed to two different stressors, either IS stress or EXS stress, and despite differences in the time, duration, and nature of the stressors, the HSP72 results were remarkably similar. After either stressor, physically active rats had a greater increase in HSP72 than sedentary rats in brain, peripheral, and immune tissues. These data suggest that the cellular adaptation produced by physical activity can be triggered by a variety of stressors or challenges.
It is of interest to consider the implication of these findings from a broader perspective. It is unclear which of the tested environments (sedentary vs. active) results in a normal physiological state. It would be easy to argue that, in fact, the sedentary condition is the abnormal physiological state and that the physically active condition is the normal physiological state. Given this perspective, our results would be equally demonstrative of the stress susceptibility of being sedentary. It is feasible to hypothesize that the lack of physical activity has hindered the expression of adaptive cellular mechanisms such as HSP induction that are in place to successfully cope with stress. Regardless of one's interpretation, the results of the current study clearly suggest important differences in stress physiology between physically active and sedentary organisms at the cellular level.
Address for reprint requests and other correspondence: M. Fleshner, Dept. of KAPH, Campus Box 354, Univ. of Colorado, Boulder, CO 80309-0354 (E-mail:).
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First published October 24, 2002;10.1152/ajpregu.00513.2002
- Copyright © 2003 the American Physiological Society