Energetic response to repeated restraint stress in rapidly growing mice

Kevin D. Laugero and Gary P. Mobergdagger

Stress Research Unit, Department of Animal Science, University of California, Davis, California 95616


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There is a cost of stress that may result in the loss of normal biological function (e.g., growth). Repeated, and even single, applications of stressors have been shown to induce negative energy balance in rodents. However, here we addressed whether this energetic response changes during multiple stress exposure and whether there is complete recovery subsequent to the cessation of stress exposure. These questions were addressed in growing C57Bl/6 mice (31 day) by determining at different times the energetic and endocrine responses after the exposure to restraint (R) stress for 4 h applied once (R1), repeatedly over 3 days (R3), or repeatedly over 7 days (R7). Compared with control values, R elevated (P < 0.05) plasma corticosterone and reduced plasma insulin-like growth factor I on all days of exposure to the stressor. Seven days, but not 1 or 3 days of R, decreased the net growth (126%, P < 0.05) and deposition of fat (71%, P < 0.05) and lean (60%, P < 0.05) energy over the 7 days. Only R7 depressed the 7-day metabolizable energy intake (P < 0.05), and R7, but not R1 or R3, increased the overall energy expenditure (10%, P < 0.05). Our results demonstrate that repeated episodes of stress are energetically costly to the rapidly growing animal, but compensatory mechanisms mitigate this cost of repeated stress exposure and permit complete recovery of energy balance after the cessation of stress application.

growth; energy partitioning; feed intake; corticosterone


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STRESS CAUSES A SHIFT in biological function, a shift that comes at a biological cost to the individual (47). During stress, changes in metabolism help support the biological defenses an individual uses to maintain homeostasis (13, 21, 48, 57). These metabolic alterations during stress often result in the mobilization of energy away from energy-sensitive functions, such as growth in adolescents and maintenance of body weight in adults (12, 21, 57). As a result, stress can cause negative energy balance. For example, stress characteristically results in depressed body weight and food intake in rodents (16, 27, 36).

Previous reports have demonstrated that the stress-induced depression in body weight is maintained over time, even after the exposure to stress has ceased (27, 52). Although repeated stress (3 daily episodes) was reported to reduce food intake during and subsequent to the last day of stress exposure, one episode only of the stressor depressed food consumption on the day of stress. When the three daily episodes of restraint were applied to young (3-wk-old) rats, reductions in food intake and especially body weight were not as marked or as persistent as those observed in adult rats exposed to the repeated stressor. Results from a study by Marty et al. (42) suggest that chronic, intermittent restraint and immobilization maintain their inhibitory effects on growth and food intake in adult rats over the entire 4-wk experiment.

These findings, and previous results from studies conducted in our laboratory (39), raise two interesting questions about rapidly growing mice: 1) is the energetic response altered over the course of daily exposure to restraint stress? and 2) is there recovery of energy and body weight after the stress-induced depression of growth? To address these questions, we evaluated the energetic response to repeated restraint stress and poststress recovery in young, postweanling mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Male C57Bl/6 mice (B&K Universal, Fremont, CA) were individually housed in hanging wire mesh cages in a room maintained at 23 ± 2°C on a 14:10-h lighting schedule. Before each experiment, mice were acclimated for 1 wk, and all experiments began when mice were 31 days of age. Each experiment lasted 7 days, beginning at 0700 on day 1 and ending at 0700 on day 8. Each day at 0700, body weight was recorded, feed was removed, and feed intake (corrected for spillage) was measured. All mice were then returned to their home cage.

On day 1 of each experiment, all mice were weighed and the treated group was exposed to 4 h of restraint stress by placing each mouse into a well-ventilated restraint tube. During the 4-h restraint period, both experimental and control (nonrestrained) mice had no access to food or water. The restraint tube was a well-ventilated 50-ml plastic centrifuge tube. The tube was tied to the bottom of the cage with Silastic tubing, which was not accessible to the mouse. Several hole punches were evenly distributed throughout the length of each tube, and a bottom section of each tube was removed to allow free drainage of urine and feces. Once the tube had been fastened to the cage, the mouse was placed in the tube and a paper towel plug was used to prevent the mouse from escaping. Mice could move freely back and forth but could not turn around while in the tube. After the 4 h of restraint, mice were removed from the tube, and the tube was removed from the cage.

Immediately after the 4-h restraint period, both restrained and control mice were fed a semipurified test diet (PMI AIN-76A, PMI Feeds, St. Louis, MO) having a guaranteed analysis of 18.4% protein, 5.0% fat, 65.0% carbohydrate, and 5.0% fiber. With the exception of the 4-h restraint period, all mice had ad libitum access to feed and water. All experiments were approved by the University of California, Davis Animal Care and Use Committee.

Experiment 1a: Effect of repeated behavioral stress on growth and energetics. To evaluate the cost of repeated behavioral stress, we quantified growth and energetics of growing mice repeatedly exposed (R, n = 6) once/day for 7 days, or not exposed (Con, n = 8) to restraint stress. A comparative slaughter experimental design was employed to quantify any changes in 7-day protein (lean) and fat tissue energy. On the 1st day of experimentation and before the initiation of treatment, an initial group of mice (31 day) was decapitated and dissected into eviscerated carcass (C), gastrointestinal tract (contents removed) + liver (G), and remaining viscera (V). These components were analyzed for water (freeze-drying to constant weight), fat (difference in weight of dried component before and after ether-acetone extraction), and protein (Kjeldahl N × 6.25) content. Carcass lean energy (CLE), GI/Liver lean energy (GLE), and viscera lean energy (VLE) were determined by multiplying the dry gram protein content by the energy content of protein (assumed to be 5.4 kcal/g). Carcass fat energy (CFE), GI/Liver fat energy (GFE), and viscera fat energy (VFE) were determined by multiplying the dry gram content by the energy content of fat (assumed to be 9.0 kcal/g). Regression equations were generated from these data to express C, G, and V water, FE (fat energy), and LE (lean energy) as linear functions of body weight at age 31 days (Table 1). These regression equations were used to predict the initial state (i.e., FE, LE) of experimental mice.

                              
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Table 1.   Linear regression equations from experiments 1 and 2 expressing energy as a function of initial body wt

On the final day of experimentation, experimental mice were decapitated, dissected, and analyzed as described for 31-day-old mice. Changes in C, G, and V water, fat energy (FEDelta ), and lean energy (LEDelta ) were determined by the difference between the final (measured) and the initial (predicted from regression equations) values. Total body change in lean and fat energy content was taken as the sum of C, G, and V energy changes. Change in total (lean + fat) body energy (BEDelta ) was taken as the sum of LEDelta and FEDelta . The 7-day metabolizable energy intake (MEI) was calculated as the gram intake multiplied by the metabolizable energy content of the diet (3.79 kcal/g at maintenance, PMI Feeds). For mice gaining protein, 1.4 kcal/g protein gain was added to the value of total MEI. Energy expenditure (heat energy) was estimated by taking the difference between MEI and BEDelta .

Experiment 1b: Effect of repeated behavioral stress on corticosterone and insulin-like growth factor I. To obtain sufficient blood for hormone analyses and to avoid any stress associated with blood collection, it was necessary to kill mice by decapitation (within 30 s of removal from their home cage). Therefore, this experiment paralleled experiment 1a to examine the effects of repeated behavioral stress on circulating corticosterone and insulin-like growth factor I (IGF-I) over the 7-day experimental period. In experiment 1b, mice were randomly assigned to 1 of 8 day/treatment combinations, and blood was collected 4 h after the initiation of restraint on days 1, 3, 6, and 7. On each day of blood collection, four mice from each treatment group were decapitated. Restraint was initiated as described for experiment 1a, and at the selected times, a predetermined group of mice was decapitated for the collection of trunk blood into heparinized (15 U/ml blood) tubes kept on ice until plasma was separated by centrifugation at 1,000 g at 4°C for 30 min. After centrifugation, plasma was collected and stored at -70°C until assayed for corticosterone and IGF-I.

Experiment 2a: Growth and energetics of poststress recovery. In this experiment, we examined our young mice to determine if they were capable of recovering from the stress-induced inhibition of growth. To address this question, we first compared the effect of 1 (n = 9), 3 (n = 10), or 7 (n = 11) daily episodes of restraint stress only on growth over 7 days. Controls (n = 10) were not exposed to restraint. Subsequently, we replicated this experiment in a parallel study to quantify the effects of these three stressors on energy partitioning among energy expenditure and lean and fat tissues, as described in experiment 1a. With the exception of R7 (n = 6), each of the Con, R1, and R3 treatment groups represented an n of 5 in this parallel study of energy partitioning.

Experiment 2b: Effect of 1, 3, or 7 episodes of behavioral stress on corticosterone and IGF-I. To address the possibility of a prolonged or carryover effect of 1, 3, or 7 daily episodes of restraint on circulating corticosterone and IGF-I, we examined the plasma concentration of these hormones at 4, 28, 76, and 168 h after the initiation (0700, day 1) of restraint. At the selected times, mice from each treatment group were decapitated, and their trunk blood was collected for the analyses of corticosterone and IGF-I. On each of the selected times, five or six animals from each treatment group were decapitated for blood collection.

Hormone assays. Plasma corticosterone concentrations were determined by RIA with a corticosterone 125I system (ICN, Costa Mesa, CA). Samples were run in duplicate, and the intra- and interassay coefficients of variation were determined as 3.1 and 8.1%, respectively. Plasma IGF-I was extracted using an acid-ethanol procedure with cryoprecipitation (10). Human recombinant IGF-I (R&D Systems, Minneapolis, MN) was iodinated and purified as described by Hodgkinson et al. (29). IGF-I concentrations were determined by a nonequilibrium RIA (10). The polyclonal IGF-I antiserum (UB3-189), kindly provided by Drs. J. J. Van Wyk and L. Underwood, was obtained through the NIDDK National Hormone and Pituitary Program. Samples were run in duplicate, and the intra- and interassay coefficients of variation were determined as 4.2 and 8.2%, respectively.

Statistical analyses. All data were analyzed using least squares ANOVA procedures (SAS/STAT User's Guide, 1990). Comparisons between treatment groups for 7-day MEI, changes in body weight, lean energy (LEDelta ), fat energy (FEDelta ), body energy (BEDelta ), and energy expenditure were analyzed in a statistical model that included the effect of treatment. For hormone data, differences between treatment groups were analyzed in a model that included the effects of treatment, day of collection, and their interaction. Where indicated, as a means of adjusting energetic responses to a common MEI, analysis of covariance was employed. Data from experiments evaluating daily body weight, daily body weight gain, daily relative body weight gain [body wt gain (g)/body wt (g) or body wt (g.75)], feed intake, daily relative feed intake [feed intake/body wt (g) or body wt (g.75)], and daily feed conversion [body wt gain (g)/feed intake (g)] were evaluated using a repeated-measures model (22). If a significant treatment × day interaction was present in the repeated-measures analysis, data were stratified by day and differences between treatment groups were analyzed using least squares procedures (SAS/STAT User's Guide, 1990) in a statistical model that included the effect of treatment. For all analyses, differences in means were determined with the PDIFF option in PROC GLM (SAS/STAT User's Guide, 1990), and a level of P < 0.05 was considered statistically significant. A P < 0.10 was considered to indicate tendencies.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experiment 1a: Effect of repeated behavioral stress on growth and energetics. Although the initial body weights did not differ before the initiation of treatment (P > 0.10), repeated restraint stress significantly (P < 0.0002) depressed final body weight and total body weight gain over the 7 days of repeated restraint (Fig. 1A). The repeated behavioral stressor also reduced (P < 0.0005) daily body weight, daily body weight gain (Fig. 1B), and daily feed intake (Fig. 1C). However, the effects of repeated restraint on daily body weight and daily body weight gain were dependent on the day (Pstress × day interaction < 0.0001). Relative body weight gain and relative feed intake were also reduced by repeated restraint stress (P < 0.0001). Although repeated restraint stress suppressed absolute feed intake on all 7 days, this stressor reduced absolute and relative body weight gain and relative feed intake only on days 1-4. To determine whether the stress-induced reduction in daily body weight was strictly due to differences in feed intake between stressed and nonstressed mice, daily feed conversion was calculated. Repeated restraint stress depressed (P < 0.0001) daily feed conversion on only the first 2 days (Fig. 1D).


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Fig. 1.   Experiment 1a: Effect of repeated behavioral stress on daily body weight (A), body weight gain (B), feed intake (C), and feed efficiency (gain/feed) (D). Values are least-squares means ± SE. C, control (n = 8, ); R7, repeated restraint (n = 6, ).

Repeated behavioral stress reduced (P < 0.0002) lean and fat energy deposition (Fig. 2, A and B), MEI (Fig. 3A), and total energy expenditure (Fig. 3B) over the 7-day experiment. However, stress can increase basal expenditure of energy (1, 24, 51, 59), and because energy expenditure depends on MEI (which was also reduced by the repeated stressor), it seemed reasonable that the reduction in MEI offset a stress-induced increase in basal energy expenditure in the present experiment. To address this possibility, we also examined energy expenditure after adjusting for differences in MEI between repeatedly stressed and nonstressed mice. When the effect of MEI was removed, energy expenditure was increased (P < 0.009) by the repeated stressor (Fig. 3C).


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Fig. 2.   Experiment 1a: Effect of repeated behavioral stress on change in total body (A) and eviscerated carcass (B) lean energy (LE) and fat energy (FE) over 7 days. Values are least-squares means ± SE. C, control (n = 8); R7, repeated restraint over 7 days (n = 6). * Significantly different from C (P < 0.05).



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Fig. 3.   Experiment 1a: Effect of repeated behavioral stress on metabolizable energy intake (MEI, A), heat energy (B), and heat energy adjusted to a common MEI (C). Values are least-squares means ± SE. C, control (n = 8); R7, repeated restraint (n = 6). * Significantly different from C (P < 0.05).

Experiment 1b: Effect of repeated behavioral stress on corticosterone and IGF-I. On each day of determination, restraint stress increased (P < 0.0001) circulating corticosterone (Fig. 4A) and reduced (P < 0.0001) circulating IGF-I (Fig. 4B).


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Fig. 4.   Experiment 1b: Effect of repeated behavioral stress on plasma corticosterone (A) and insulin-like growth factor I (IGF-I, B) 4 h after initiation of stressor. Values are least-squares means ± SE. C, control (n = 4); R7, repeated restraint (n = 4). * Significantly different from C (P < 0.05).

Experiment 2a: Growth and energetics of poststress recovery. Initial body weights did not differ (P > 0.10) between treatment groups. One, three, or seven episodes of restraint stress initially depressed body weight (P < 0.0001), body weight gain (P < 0.0001), and feed intake (P < 0.0001) in all animals (Fig. 5). R1, R3, and R7 equally depressed each of these parameters from day 1 to day 2 (P > 0.1). Likewise, R3 and R7 depressed body weight, weight gain, and feed intake to the same degree over the first 3 days (P > 0.1). As in experiment 1, only those mice that were exposed to 7 days of repeated restraint stress had depressed final body weight (P < 0.0001) and total body weight gain (P < 0.0001) over the 7-day experiment. However, body weight was restored to control levels by day 3 in animals exposed to one episode of restraint and by day 7 in mice exposed to three episodes of the stressor. Mice exposed to one or three daily episodes of restraint stress appeared to respond with enhanced feed intake, feed conversion, and growth after the last episode of restraint. To answer this question, the growth and feed intake data were analyzed from day 2 to day 8 for those animals that experienced one episode of restraint and from day 4 to day 8 for mice exposed to three episodes of restraint. From day 2 to day 8, separate analyses were made between control mice and mice exposed to a single episode of restraint and between mice exposed to one or seven episodes of restraint. From day 4 to day 8, we compared the data, in two separate analyses, between control mice and mice exposed to three episodes of restraint and between mice exposed to three or seven episodes of restraint.


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Fig. 5.   Experiment 2a: Effect of 1 episode (R1), 3 episodes (R3), or 7 episodes (R7) of behavioral stress on daily body weight (A), body weight gain (B), feed intake (C), and feed conversion (gain/feed) (D). Values are least-squares means ± SE. C, control (n = 10); R1 (n = 9); R3 (n = 10); R7 (n = 11).

Compared with seven daily episodes of restraint stress and controls, one episode of the stressor altered (P < 0.0001) daily body weight, daily body weight gain, daily feed intake, and daily feed conversion from day 2 to day 8. However, these effects of a single episode of restraint on body weight, body weight gain, daily feed intake, and daily feed conversion depended on the day (Pstress × day interaction < 0.0001). Exposure to one restraint episode increased body weight gain only on day 2 (P < 0.0011) and day 3 (P < 0.0358). Only on day 3 was the relative feed intake of mice exposed to a single episode of stress higher (P < 0.007) than the relative feed intake of nonrestrained mice. However, one episode of restraint increased (P < 0.005) the relative feed intake above that of mice exposed to seven episodes of the stressor on days 2-4. One episode of restraint increased (P < 0.0033) the feed conversion on day 2, relative to controls, and on days 2 and 3 compared with mice repeatedly restrained over 7 days (P < 0.0004).

From day 4 to day 8, exposure to three daily episodes of behavioral stress altered (P < 0.0001) daily body weight, daily body weight gain, and daily feed conversion, and with the exception of daily body weight (Pstress × day interaction < 0.0001), three daily episodes of the stressor increased daily body weight gain and daily feed conversion on each of the 4 days after the 3 days of stress exposure (Pstress × day interaction > 0.10). Relative to nonrestraint, three restraint episodes increased (P < 0.003) relative feed intake on days 4-6. Compared with seven restraint episodes, three episodes of the stressor increased (P < 0.003) relative feed intake on days 4-6.

Subsequent to this experiment, which compared the effect of one, three, or seven daily episodes of restraint stress only on growth, a parallel study examined the energetic response to one, three, or seven daily episodes of the stressor. Only seven daily episodes of repeated restraint stress altered (P < 0.05) energy partitioned into lean and fat tissues over the 7 days (Fig. 6). Compared with nonrestraint and one or three episodes of restraint, seven daily episodes of the stressor depressed (P < 0.05) the deposition of energy into total and carcass lean and fat tissues. Relative to controls and a single restraint episode, three and seven episodes of the stressor suppressed (P < 0.05) MEI. However, the MEI of mice exposed to seven daily episodes of behavioral stress was even lower (P < 0.05) than the MEI of mice exposed to three stress episodes. Overall (7-day) energy expenditure was not affected by restraint (P > 0.10), but when heat energy was adjusted for differences in MEI, only mice repeatedly exposed to seven daily episodes of restraint had a higher (P < 0.05) overall energy expenditure than nonstressed mice and mice exposed to one or three daily restraint episodes.


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Fig. 6.   Experiment 2a: Effect of 1 episode (R1), 3 episodes (R3), or 7 episodes (R7) of behavioral stress on total and eviscerated carcass LE change (A), total and eviscerated carcass FE change (B), heat energy adjusted to a common MEI (C), and MEI (D). Values are least-squares means ± SE. C, control (n = 5); R1 (n = 5); R3 (n = 5); R7 (n = 6). Groups with different letters are significantly different (P < 0.05).

Experiment 2b: Effect of one, three, or seven daily episodes of behavioral stress on corticosterone and IGF-I. The behavioral stressor elevated (P < 0.0001) circulating corticosterone on each of the designated sampling days, when sampling occurred 4 h after the initiation of the stressor (Fig. 7). The corticosterone concentrations of mice experiencing a single episode of restraint stress on day 1 did not differ from control levels on days 2, 4, and 8. Likewise, the plasma concentrations of corticosterone in mice exposed to only three episodes of the stressor did not differ from control concentrations on days 4 and 8.


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Fig. 7.   Experiment 2b: Effect of 1 episode (R1, A), 3 episodes (R3, B), or 7 episodes (R7, C) of behavioral stress on plasma corticosterone 4 h after initiation of the stressor on days 1, 2, and 4. On day 8, blood was collected 24 h after initiation of the stressor on day 7. Values are least-squares means ± SE. C, control (n = 5); R1 (n = 5); R3 (n = 5); R7 (n = 6). * Significantly different from C (P < 0.05).

Each daily episode of restraint stress decreased (P < 0.0001) circulating IGF-I 4 h after the initiation of the stressor (Fig. 8). Twenty-four hours after the initiation of the stressor on day 7, IGF-I tended (P < 0.10) to be lower in mice exposed to seven daily episodes of restraint. On days 2, 4, and 8, there were no differences in circulating IGF-I concentrations between controls and mice exposed to a single restraint episode. Mice exposed to three episodes of the stressor had comparable IGF-I concentrations to controls on days 4 and 8.


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Fig. 8.   Experiment 2b: Effect of 1 episode (R1, A), 3 episodes (R3, B), or 7 episodes (R7, C) of behavioral stress on plasma IGF-I 4 h after initiation of stressor on days 1, 2, and 4. On day 8, blood was collected 24 h after initiation of stressor on day 7. Values are least-squares means ± SE. C, control (n = 5); R1 (n = 5); R3 (n = 5); R7 (n = 6). * Significantly different from C (P < 0.05). ** Tended to be different from C (P < 0.07).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We found that repeated exposure to an acute episode of restraint stress induced a cost that significantly suppressed normal growth in the growing mouse. This cost to normal growth resulted, at least in part, from altered deposition of energy into lean and fat tissues. Although the overall effect of the repeated stressor was inhibitory, the energetic response to daily restraint was not constant over the 7-day study. Interestingly, although there was an attenuated energetic response to repeated restraint, there were comparable and repeated increases in circulating corticosterone and decreases in IGF-I each time mice were exposed to the stressor. These results support the view that stress can lead to a shift of energy from growth to those functions that prepare the individual for survival (12, 21, 25, 53, 58). Furthermore, our results demonstrate that there is a shift in the energetic response to repeated stress from energy mobilization to energy preservation in young, rapidly growing mice.

Although the body weights of mice exposed to one or three episodes of restraint were initially depressed, the body weights returned to control levels because of a rapid enhancement of growth after the last exposure to stress. In fact, mice exposed to only one or three episodes of the stressor failed to show any net impairment of lean and fat energy deposition, which was in contrast to the animals exposed to seven daily episodes of stress. We previously found (40) that a single episode of restraint significantly reduced the energy deposited into lean and fat tissue 24 h after the initiation of restraint. Therefore, those results and our present work suggest that 1 or 3 days of restraint stress initially impaired energy deposition, but during the poststress period, this energy was replenished by increased rates of energy deposition into both lean and fat tissues. As a result, the mice overcame the inhibitory effects of stress on growth by a period of compensatory growth that enabled them to fully recover energy and normal body weight.

Although other investigators (27, 52) have demonstrated a sustained reduction of body weight after 3 days of restraint stress, this very interesting finding was in adult rats. The drive for growth and the attainment of a mature body weight may have been so great in our mice that compensatory mechanisms to ensure such a body weight, after stress, are intact in such young animals. In contrast, repeated exposure to behavioral stress on all 7 days prevented any compensatory growth, recovery of energy, or the recovery of normal body weight by the final day after seven daily episodes of stress. It is quite possible that, given a recovery period after the 7 days of repeated stress, these mice would also have recovered. However, the longer an individual is incapable of reaching normal stature, the greater that individual is at risk for developing pathologies related to abnormal growth. For example, psychosocial short stature resulting from chronic stress is related to delayed puberty, eating disorders, and/or vulnerability to disease (58).

Although determining the mechanisms that lead to altered energy deposition and growth was not the primary focus of this study, we did evaluate factors known to influence these functions. Feed and metabolizable energy consumption were significantly reduced by repeated behavioral stress, and this response is consistent with the reduced feed or energy intake that results from other types of stress (18, 33, 34, 37). On the other hand, feeding was enhanced during the recovery period after either one or three episodes of stress. Growth depends on nutrient and energy availability, as dictated by intake and its partitioning among tissues (25). Therefore, it is possible that, in this study, the stress-induced reduction in feed and energy intake was responsible for impaired growth, whereas increased intake may have stimulated catch-up growth seen in recovering mice. However, the depressed feed conversion in mice repeatedly exposed to seven episodes of behavioral stress and the enhanced feed conversion in recovering mice indicate that some factor(s) other than altered feed intake affected growth. However, the total effect of the repeated stress was not completely independent from this altered feed consumption, as suggested by the treatment × day interaction effect on daily feed conversion in repeatedly stressed mice and mice recovering from one episode of restraint. Daily feed conversion in mice recovering from three episodes of stress was, however, significantly enhanced on each of the 4 days after the last day of stress, suggesting that mechanisms controlling the efficiency of energy deposition were necessary for rapid recovery of a normal body weight.

Changes in energy expenditure may also explain the altered growth and energy deposition in our repeatedly stressed and recovering mice. In this study, both MEI and total heat energy production were reduced by the repeated exposure to stress. However, this does not preclude the possibility that stress increased basal heat energy production (1, 20, 24, 51, 59). In fact, when differences in MEI between stressed and control mice were removed, only repeated exposure to seven episodes of restraint caused significantly higher net energy expenditure. Thus, although the total energy expenditure may have been suppressed by the significant reduction in MEI (7, 8, 17, 20, 35), the examination of energy expenditure, independent from the effects of MEI, suggests that mice repeatedly exposed to stress had an elevated basal energy expenditure. This stress-induced increase in basal energy expenditure probably caused the overall expenditure to be higher than one would expect from an equivalent reduction of MEI in nonstressed mice. Therefore, the stress-induced elevation in basal expenditure of energy is a cost that increases the quantity of energy partitioned into heat as opposed to growth, and may, in part, have accounted for the impaired growth in the repeatedly stressed mice.

Whereas increased basal energy expenditure may have inhibited normal energy deposition in mice exposed to seven episodes of restraint, mice recovering from stress may have incurred a greater capacity for energy deposition because of lowered basal energy expenditure over the period of recovery. Although our measure of energy expenditure reflects the net sum of all 7 days, the energy expended over the specific period of recovery may have been reduced. A reduced expenditure of energy has been shown to occur in animals experiencing compensatory growth after a period of nutritional stress (19). Because, for a given intake, a lower basal energy expenditure can lead to a greater efficiciency of energy deposition (4), the enhanced feed conversion in our recovering mice may reflect a depressed energy expenditure during the recovery period.

Finally, the hormonal status of an individual dictates energy partitioning, and thus growth capacity (21, 25, 53). Thus stress-induced alterations in hormones known to affect this partitioning may affect growth. We measured circulating concentrations of corticosterone and IGF-I in the present study. IGF-I is an important growth factor (41, 50, 55), and its circulating levels are positively correlated with body size and growth (5, 6, 45, 46). Therefore, repeated inhibition of this growth factor may have contributed to the impaired growth in mice exposed repeatedly to the behavioral stress. Additionally, it has been reported that elevated circulating corticosterone, as seen in this study, has catabolic effects on both protein and fat tissue (30) and inhibits growth and feed efficiency (15, 16, 56). Therefore, the repeated increases in circulating corticosterone in mice exposed repeatedly to restraint stress may have contributed to the alterations in energy partitioning and growth in these behaviorally stressed mice.

It remains uncertain whether changes in the growth hormone/IGF-I and adrenal-cortical systems play a role in stimulating compensatory growth. Our results showed that circulating concentrations of corticosterone and IGF-I in mice exposed to restraint only on day 1 were similar to the concentrations of experimental controls on day 2, and mice exposed to restraint on days 1-3 had circulating concentrations of corticosterone and IGF-I similar to those in experimental controls when the levels of these hormones were determined on day 4. However, our morning determination of corticosterone and IGF-I is not necessarily indicative of the animal's hormonal status at any given time throughout the period of recovery, and thus we cannot exclude these hormones from those factors regulating the enhanced growth of our recovering mice. Furthermore, other factors, such as the receptors and binding proteins for corticosterone and IGF-I, may alter the impact of these circulating hormones on growth-related processes.

We demonstrated that repeated episodes of stress are energetically costly to the rapidly growing animal. However, the results suggest that there are counterregulatory mechanisms that become engaged to attenuate the detrimental effects of repeated stress on growth. Furthermore, there exist poststress mechanisms that induce rapid recovery of energy in young mice. Thus there appear to be mechanisms that serve to preserve excessive energy loss during repeated stress and to enhance poststress recovery of body weight in the young, rapidly growing animal.


    ACKNOWLEDGEMENTS

We thank Dr. Chris Calvert for expert advice on growth and energetics and guidance in many areas of the experimentation. Our thanks go to Dr. Anita M. Oberbauer for help with the IGF-I assay and to Dr. Edward J. DePeters and Scott Taylor for technical help with nutritional analyses.


    FOOTNOTES

dagger Deceased 13 August 1999.

Address for reprint requests and other correspondence: K. D. Laugero, Department of Physiology, University of California, San Francisco, CA 94143-0444 (E-mail: laugero{at}itsa.ucsf.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 11 May 1999; accepted in final form 22 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Benson, BN, Calvert CC, Roura E, and Klasing KC. Dietary energy source and density modulate the expression of immunological stress in chicks. J Nutr 123: 1714-1723, 1993[ISI][Medline].

2.   Berdanier, CD, and Shubeck D. Interaction of glucocorticoid and insulin in the response of rats to starvation-refeeding. J Nutr 109: 1766-1771, 1979[ISI][Medline].

3.   Berdanier, CD, Wurdeman R, and Tobin RB. Further studies on the role of adrenal hormones in the responses of rats to meal feeding. J Nutr 106: 1791-1800, 1976[ISI][Medline].

4.   Bernier, JF, Calvert CC, Famula TR, and Baldwin RL. Maintenance energy requirement and net energetic efficiency in mice with a major gene for rapid postweaning gain. J Nutr 116: 419-428, 1986[ISI][Medline].

5.   Blair, HT, McCutcheon SN, Mackenzie DDS, Gluckman PD, and Ormsby JE. Variation in plasma concentration of insulin-like growth factor-I and its covariation with liveweight in mice. Aust J Biol Sci 40: 287-293, 1987[ISI][Medline].

6.   Blair, HT, McCutcheon SN, Mackenzie DDS, Ormsby JE, Siddiqui RA, Breier BH, and Gluckman PD. Genetic selection for insulin-like growth factor-I in growing mice is associated with altered growth. Endocrinology 123: 1690-1692, 1988[Abstract].

7.   Blaxter, KL. Methods of measuring the energy metabolism of animals and interpretation of results obtained. Fed Proc 30: 1436-1443, 1971[ISI][Medline].

8.   Blaxter, KL. Energy Metabolism in Animals and Man. Cambridge, UK: Cambridge University Press, 1989.

9.   Bouillon, DJ, and Berdanier CD. Role of glucocorticoid in adaptive hyperlipogenesis in the rat. J Nutr 10: 286-297, 1980.

10.   Breier, BH, Gallaher BW, and Gluckman PD. Radioimmunoassay for insulin-like growth factor-I: solutions to some potential problems and pitfalls. J Endocrinol 128: 347-357, 1991[Abstract].

11.   Carstens, GE, Johnson DE, and Ellenberger MA. Energy metabolism and composition of gain in beef steers exhibiting normal and compensatory growth. In: Energy Metabolism of Farm Animals. Wageningen, The Netherlands: Pudoc, 1989, p. 131.

12.   Chrousos, GP, and Gold PW. The concepts of stress and stress system disorders. Overview of physical and behavioral homeostasis. JAMA 267: 1244-1252, 1992[Abstract].

13.   Chrousos, GP, and Gold P. Stress: basic mechanisms and clinical implications. Ann NY Acad Sci 771: xv-viii, 1995[Medline].

14.   Cunningham, DL. Cage type and density effects on performance and economic factors of caged layers. Poultry Sci 61: 1944-1949, 1982[ISI].

15.   Dallman, MF, Akana SF, Strack AM, Hanson SE, and Sebastian RJ. The neural network that regulates energy balance is responsive to glucocorticoids and insulin and also regulates HPA axis responsivity at a site proximal to CRF neurons. Ann NY Acad Sci 771: 730-742, 1995[Abstract].

16.   Dallman, MF, Strack AM, Akana SF, Bradbury MJ, Hanson ES, Scribner KA, and Smith M. Feast and famine: critical role of glucocorticoids with insulin in daily energy flow. Front Neuroendocrinol 14: 303-347, 1993[ISI][Medline].

17.   DeBoer, JO, Roovers LA, van Raaij JMA, and Hautvast JG. Adaptation of energy metabolism of overweight women to low energy intake, studied with whole body calorimeters. Am J Clin Nutr 44: 585, 1986[Abstract].

18.   Dess, NK. Suppression of feeding and body weight by inescapable shock: modulation by quinine adulteration, stress reinstatement, and controllability. Physiol Behav 45: 975-983, 1989[ISI][Medline].

19.   Dulloo, AG, and Girardier L. Adaptive changes in energy expenditure during refeeding following low-calorie intake: evidence for a specific metabolic component favoring fat storage. Am J Clin Nut 52: 415-420, 1990[Abstract].

20.   Edens, NK, Gil KM, and Elwyn KH. The effects of varying energy and nitrogen intake on nitrogen balance, body composition, and metabolic rate. Clin Chest Med 7: 3-17, 1986[ISI][Medline].

21.   Elsasser, TH, Kahl S, Steele NC, and Rumsey TS. Nutritional modulation of somatotropic axis-cytokine relationships in cattle: a brief review. Comp. Biochem. Physiol. 116A: 209-221, 1997[ISI].

22.   Fox, DG, Preston RL, Dockerty TR, and Klosterman EW. Protein and energy utilization during compensatory growth in beef cattle. J Anim Sci 34: 310, 1972[ISI].

23.   Gill, JL, and Hafs HD. Analysis of repeated measurements of animals. Anim Sci 33: 331-336, 1971.

24.   Groenink, L, Compaan J, van der Gugten J, Zethof T, van der Heyden J, and Olivier B. Stress-induced hyperthermia in mice. Ann NY Acad Sci 771: 252-256, 1995[ISI][Medline].

25.   Hammond, J. Physiological limits to intensive production of animals. Br Agr Bull 4: 222-225, 1951.

27.   Harris, RB, Zhou J, Youngblood BD, Rybkin II, Smagin GN, and Ryan DH. Effect of repeated stress on body weight and body composition of rats fed low- and high-fat diets. Am J Physiol Regulatory Integrative Comp Physiol 275: R1928-R1938, 1998[Abstract/Free Full Text].

28.   Hemsworth, PH, Barnett JL, and Hansen C. The influence of handling by humans on the behavior, growth, and corticosteroids in the juvenile female pig. Horm Behav 15: 396-403, 1981[ISI][Medline].

29.   Hodgkinson, SC, Moore L, Napier JR, Davie SR, Bass JJ, and Gluckman PD. Characterization of IGFBPs in ovine tissue fluids. J Endocrinol 120: 429-438, 1989[Abstract].

30.   Kaplan, NM. The adrenal glands. In: Textbook of Endocrine Physiology, edited by Griffin JE, and Ojeda SR. New York: Oxford University Press, 1992, p. 247-275.

31.   Kaul, L, and Berdanier DD. Effect of pancreatectomy or adrenalectomy on the responses of rats to meal feeding. J Nutr 105: 1176-1185, 1975[ISI][Medline].

32.  Ketelslegers JM, Maiter D, Maes M, Underwood LE, and Thissen JP. Nutritional regulation of insulin-like growth factor-I. Metabolism 44: 10, and Suppl 4: 50-57, 1995.

33.   Klasing, KC. Nutritional aspects of leukocytic cytokines. J Nutr 118: 1436-1446, 1988[ISI][Medline].

34.   Klasing, KC, Laurin DE, Ping RK, and Fry MD. Immunologically mediated growth depression in chicks: influence of feed intake, corticosteone, and interleukin-1. J Nutr 117: 1629-1637, 1987[ISI][Medline].

35.   Kleiber, M. The Fire of Life: An Introduction to Animal Energetics. New York: Krieger, 1975.

36.   Krahn, DD, Gosnell BA, Grce M, and Levine AS. CRF antagonist partially reverses CRF- and stress-induced effects on feeding. Brain Res Bull 17: 285-289, 1986[ISI][Medline].

37.   Krahn, DD, Gosnell BA, Levine AS, and Morley JE. Localization of the effects of corticotropin-releasing factor on feeding (Abstract). Proc Soc Neurosci 10: 302, 1984.

38.   Langley, LL, and Clarke RW. The reaction of the adrenal cortex to low atmospheric pressure. Yale J Biol Med 14: 529-546, 1942.

39.   Laugero, KD, and Moberg GP. Endocrine and energetic response to acute and repeated behavioral stress in the growing mouse (Abstract). FASEB J 12: 4, 1998.

40.  Laugero KD and Moberg GP. Effects of acute behavioral and immunological stress on growth and energetics in mice. Physiol Behav. In press.

41.   Lowe, WL. Biological actions of insulin-like growth factors. In: Insulin-Like Growth Factors: Molecular and Cellular Aspects, edited by LeRoith D. Boca Raton, FL: CRC, 1991, p. 49-85.

42.   Marty, O, Marty J, and Armario A. Effects of chronic stress on food intake of ras: influence of stressor intensity and duration of daily exposure. Physiol Behav 55: 747-753, 1994[ISI][Medline].

43.   McEwen, BS. Protective and damaging effects of stress mediators. Semin Med Beth Israel Deaconess Med Ctr 338: 171-179, 1998.

44.   McEwen, BS, and Stellar E. Stress and the individual: mechanisms leading to disease. Arch Intern Med 153: 2093-2101, 1993[Abstract].

45.   Merimee, TJ, Zapf J, and Froesch ER. Dwarfism in the pygmy. An isolated deficiency of insulin-like growth factor I. N Engl J Med 305: 965-968, 1981[Abstract].

46.   Merimee, TJ, Zapf J, Hewlett B, and Cavalli-Sforza LL. Insulin-like growth factors in pygmies. The role of puberty in determining final stature. N Engl J Med 49: 825, 1957.

47.   Moberg, GP. Biological response to stress: key to assessment of animal well-being? In: Animal Stress, edited by Moberg GP. Bethesda, MD: Am Physiol Soc, 1985, p. 27-49.

48.   Moberg, GP. Suffering from stress: an approach for evaluating the welfare of an animal. Acta Agric Scand A Anim Sci Suppl 27: 46-49, 1996.

49.   Perhach, JL, and Barry H, III. Stress response of rats to acute body or neck restraint. Physiol Behav 5: 443-448, 1970[ISI][Medline].

50.   Sara, VR, and Hall K. Insulin-like growth factors and their binding proteins. Physiol Rev 70: 591-613, 1990[Free Full Text].

51.   Shibata, H, and Nagasaka T. Contribution of nonshivering thermogenesis to stress-induced hyperthermia in rats. Jpn J Physiol 32: 991-995, 1982[ISI][Medline].

52.   Smagin, GN, Howell LA, Redmann S, Jr, Ryan DH, and Harris RB. Prevention of stress-induced weight loss by third ventricle CRF receptor antagonist. Am J Physiol Regulatory Integrative Comp Physiol 276: R1461-R1468, 1999[Abstract/Free Full Text].

53.   Steele, NC, and Elsasser TH. Regulation of somatomedin production, release and mechanism of action. In: Animal Growth Regulation. New York: Plenum, 1989, p. 295-316.

54.   Sterling, P, and Eyer J. Allostasis: a new paradigm to explain arousal pathology. In: Handbook of Life Stress, Cognition, and Health, edited by Fisher J, and Reason J. New York: Wiley, 1988.

55.   Stewart, CEH, and Rotwein P. Growth, differentiation, and survival: multiple physiological functions for insulin-like growth factors. Physiol Rev 76: 1005-1026, 1996[Abstract/Free Full Text].

56.   Strack, AM, Sebastian RJ, Schwartz MW, and Dallman MF. Glucocorticoids and insulin: reciprocal signals for energy balance. Am J Physiol Regulatory Integrative Comp Physiol 268: R142-R149, 1995[Abstract/Free Full Text].

57.   Stratakis, CA, and Chrousos GP. Neuroendocrinology and pathophysiology of the stress system. Ann NY Acad Sci 771: 1-18, 1995[Abstract].

58.   Stratakis, CA, Gold PW, and Chrousos GP. Neuroendocrinology of stress: implications for growth and development. Horm Res 43: 162-167, 1995[ISI][Medline].

59.   Weissman, C. The metabolic response to stress: an overview and update. Anesthesiology 73: 308-327, 1990[ISI][Medline].

60.   Williams, B, and Berdanier CD. Effects of diet composition and adrenalectomy on the lipogenic responses of rats to starvation-refeeding. J Nutr 112: 534-541, 1982[ISI][Medline].

61.   Yambayamba, ESK, Price MA, and Foxcroft GR. Hormonal status, metabolic changes, and resting metabolic rate in beef heifers undergoing compensatory growth. J Anim Sci 74: 57-69, 1996[Abstract/Free Full Text].


Am J Physiol Endocrinol Metab 279(1):E33-E43
0193-1849/00 $5.00 Copyright © 2000 the American Physiological Society




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