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Marked Regulatory Shifts in Gonadal, Adrenal, and Metabolic System Responses to Repeated Restraint Stress Occur within a 3-Week Period in Pubertal Male Rats

Department of Physiology, University of California, San Francisco, California 94143-0444

Address all correspondence and requests for reprints to: Dr. Mary F. Dallman, Department of Physiology, Box 0444, University of California, San Francisco, California 94143-0444. E-mail: . dallman{at}itsa.ucsf.edu

	Abstract

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We compared testosterone (T), corticotropin (ACTH), corticosterone (B), and leptin responses to three daily 3-h bouts of restraint and blood sampling as well as energy balance of male rats in early (40 d of age) and late (60 d of age) puberty. Rats either remained intact or were adrenalectomized and replaced with B clamped at basal mean values (ADX+B). Hormones, weight gain, food intake, and fat depot weight were measured during or after the days of stress. The major effects of restraint on T, ACTH, and energetic responses were age dependent, but clamped B affected the effects of restraint seen in intact rats at each age. T secretion was inhibited in 40-d-old and was stimulated in 60-d-old rats after restraint. ACTH responses were high, but diminished with repetition of stress in intact, but not ADX+B, 40-d-old rats. ACTH responses were lower, but constant across days, in both intact and ADX+B 60-d-old rats. Younger rats gained weight during the period of stress, whereas older rats stopped gaining weight. We conclude that the central regulation of stress responses shifts markedly between early and late puberty, although stress-induced B responses are important at both ages. In early puberty, priority is placed on maintaining normal ponderal growth, whereas in late puberty, priority is placed on maintaining reproductive capability.

	Introduction

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STUDIES OF THE effects of stress-induced activity in the hypothalamic-pituitary-adrenal (HPA) axis on sex hormone secretion generally have shown that reproductive function is inhibited by stress-induced activation of both corticotropin-releasing factor (CRF) (1, 2) and glucocorticoids. However, this relationship is reciprocal, because the hypothalamic-pituitary-gonadal (HPG) axis also exerts organizational and activational effects on basal and stress-induced HPA activity (reviewed in Ref. 3). The stage of development, reproductive state, metabolic state and social status determine not only the activity in the HPA and HPG axes, but also the mechanisms that mediate their interactions.

Although chronic stress generally suppresses testosterone (T) secretion in both male rodents and primates, including man (3), we and others have shown that different types of stressors do not inhibit T in rapidly growing, male rats entering puberty (40 d of age) as they do in young male rats leaving puberty (60 d of age) (4). Moreover, manipulation of T concentrations at the two ages showed that the younger rats required a very narrow range of T concentrations to survive cold stress. The difference in energy balance between 40- and 60-d-old rats in the cold was striking, and it appeared that 40-d-old rats, entering puberty, put their resources into ponderal growth, whereas 60-d-old rats, leaving puberty, put their resources into maintenance of reproductive capability when both groups were stressed (4).

Because the stressor of chronic cold causes major demands on energy balance, we wanted to test the generality of the different responses in younger and older rats using a stressor that presumably places fewer metabolic demands on the animal. In this study we used 3-h daily bouts of restraint on 3 consecutive days to ask both whether there are age-dependent differences in responses, and whether these differences occur in intact rats as well as those that are adrenalectomized (ADX) with corticosterone (B) replacement (ADX+B). We chose this stress paradigm for two main reasons. First, restraint has been considered by many as a more psychological type of stressor than cold (5). Second, its effects on energy balance have been previously studied in considerable detail (6, 7, 8).

We measured basal and stress-induced concentrations of T, ACTH, B, and leptin at different times during the 3 h of restraint across each of the 3 d of restraint to determine the effects of age on responsivity of the hormones. Leptin was measured because it is clearly involved in the regulation of energy balance and activity in the HPA and HPG axes (9, 10, 11). Changes in body weight (BW), food intake (FI), and white adipose tissue (WAT) depot weights were also measured as indexes of energy balance.

	Materials and Methods

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Animals
Male Sprague Dawley rats delivered from Bantin and Kingman (Gilroy, CA) were used. The rats were in early puberty, 40–41 d old (190 g BW), or late puberty, 60–61 d old (265 g BW), at the time of initiating restraint stress. Rats were singly housed in hanging wire cages and were maintained under standard conditions of light (lights on, 0600–1800 h) and temperature (22 ± 2 C) for a minimum of 3 d before the experiments, to allow adaptation to the new environment. Animals were offered Purina rodent chow (Diet 5008, Ralston Purina Co., St. Louis, MO) and tap water (or 0.5% saline after ADX) ad libitum. Protocols were approved by the committee on animal research, University of California (San Francisco, CA). All experimental procedures were performed between 0900–1300 h to minimize any circadian influence.

Experiments
Effects of age on responses to repeated restraint in intact rats.
Rats of both ages were exposed to 3 h of restraint stress (0900–1200 h) on 3 consecutive days. This restraint protocol is similar to that previously reported (6, 7, 8); however, control rats were not pair-fed during the 3-d period. Some of the rats of each age were exposed to repeated restraint plus tail blood sampling, whereas the others served as nonmanipulated controls in which only FI and BW was measured. All rats were decapitated under basal conditions on the morning of d 4. There were three control rats and five restrained rats per group at 40 d of age and four control rats and six restrained rats per group at 60 d of age.

Immediately before restraint and within 2 min of the time they were taken from the home cage, a blood sample (control, time zero) was collected from a cut made over a lateral tail vein. These samples were considered to reflect basal conditions. Rats were immediately placed in restraint tubes, and blood samples were collected 30, 90, and 180 min later by gently dislodging the clot that had formed over the initial cut. Samples of 300 µl were collected into EDTA-coated capillary tubes, kept on ice, and centrifuged in the cold. Aliquots of plasma were stored at -20 C until hormone concentrations were assayed. After the 180-min sample was collected in restraint, rats were returned to their home cages. During the period of restraint, control rats were untouched and had free access to food and water.

Effects of age and restraint stress in ADX+B rats.
On experimental d 0, under isoflurane (Abbott Laboratories, North Chicago, IL) anesthesia, all rats were bilaterally ADX by the dorsal approach and replaced with one pellet of fused B/cholesterol weighing 100 mg. Forty- and 60-d-old rats were replaced with 25% B/75% (w/w) cholesterol pellets and with 35% B/65% (w/w) cholesterol pellets, respectively. These percentages of B were designed to provide the rats of each BW with plasma B concentrations in the normal basal daily range (5 µg/dl) (12). After surgery all rats were supplied with fresh food and 0.5% NaCl to drink and were monitored for at least 4 h for full recovery from anesthesia before being returned to their home cages. On each of the 3 d after surgery, these rats experienced the same restraint paradigm and manipulations as intact rats in the first experiment. All rats were decapitated under basal conditions on the morning of d 4. There were eight control and four restrained rats per group at 40 d of age; there were six control and five restrained rats per group at 60 d of age.

Measures
Body weight and food intake was recorded daily in the morning. All rats were killed on the morning of d 4 by decapitation within 10 sec after they had been taken from their cages. Basal hormone concentrations were determined. Mesenteric and sc WAT as well as adrenals (if present) and thymuses were dissected, cleaned, and weighed.

Plasma hormones
All plasma hormone concentrations were measured by RIA. Plasma B and T kits were obtained from ICN (ICN Biomedicals, Inc., Costa Mesa, CA). The T assay has a sensitivity of 0.1–0.14 ng/ml and an intraassay coefficient of variation of 3.5%. The leptin kit was obtained from Linco Research, Inc. (St. Charles, MO). The ACTH RIA used a specific antiserum generously donated by Dr. William Engeland (University of Minnesota, Minneapolis, MN) at a final dilution of 1:120,000 and [¹²⁵I]ACTH as a trace (INCSTAR Corp., Stillwater, MN).

Statistics
Organs and fat depot weights within experiments were analyzed by two-way ANOVA with age and stress as the main factors. Across experiments these were analyzed by two-way ANOVA within stress condition with age and adrenal status as main factors. Within experiments, BW, FI, caloric efficiency (CE), and hormone levels (within day or control, time zero, across the 3 d of restraint) were analyzed by ANOVA for repeated measures with age and stress as main factors. Basal hormone concentrations on the fourth day of restraint were analyzed separately, because we did not want to compare values collected from tail nick blood to blood obtained by decapitation. For food intake in 60-d-old intact rats, initial values before the period of stress differed between restraint and control groups. One-way ANOVA with stress as a main factor and time as a repeated measure was used to test whether restraint had a significant effect on food intake in this group. Within experimental days, the total area under the curve (AUC) using hormone levels from 0–180 min was calculated. The AUC corresponding to the stress response after 30, 90, and 180 min of restraint after being normalized against time zero values was also calculated. Across experiments, data were analyzed with three-way ANOVA for repeated measures, using age and adrenal status as main factors and days as repeated measure. Unpaired t tests were used to assess the effect of age, adrenal status, or stress within experimental day and paired t tests were used to assess the effect of either time or day. Significance was assumed at P 0.05; when 0.05 < P < 0.1, it was noted and considered a trend. All data are presented as the mean ±SEM.

	Results

Top Abstract Introduction Materials and Methods Results References

 
Responsivity in the HPG axis: T as a function of age and B responses
Intact rats (Fig. 1, top panels).
Basal T concentrations (time zero) differed as a function of age and time across the 3 experimental days (age, P = 0.034; time, P = 0.041; interaction, P = NS). Initial T concentrations increased with days of restraint; however, the effect was not significant in the 40-d-old rats, and the analysis was dominated by the marked increase in basal T in the 60-d-old rats. On d 4, 60-d-old, but not 40-d-old, previously restrained rats sampled under basal conditions still had higher basal T concentrations than those collected by tail nick sampling on d 3 (T at 40 d, 2.3 ± 0.6 and 1.7 ± 0.6 ng/ml; 60 d, 1.5 ± 0.1 and 5.8 ± 1.0, control and prior restraint, , respectively; mean ±SEM).

View larger version (25K): [in this window] [in a new window]   Figure 1. T is inhibited by restraint in 40-d-old, but not 60-d-old, intact rats and is stimulated in ADX+B at 60 d. Plasma T during restraint in intact (top panels) and ADX+B (bottom panels) rats is shown. Within these figures, the total AUC from 0–180 min of restraint is shown in the insets. In all cases results are expressed as the mean ± SEM (n = 4–6 rats/group). , Significant effect of age at time zero (by t test, P < 0.05). *, Significant effect of age on total AUC within day (by unpaired t test, P < 0.05). A and B, Bars labeled with different capital letters indicate significant effects of day of exposure to restraint in 60-d-old rats (by paired t test, P < 0.05).

The AUC for total T within each day of restraint did not change in either group across days of restraint.

ADX+B rats (Fig. 1, bottom panels).
There was no significant effect with time or an interaction between repeated restraint and days. On d 4, basal T concentrations in 40- and 60-d-old rats did not differ (T at 40 d, 1.5 ± 0.2 and 2.0 ± 0.5 ng/ml; 60 d, 2.1 ± 0.6 and 1.4 ± 0.4 ng/ml, control and prior restraint, respectively; mean ± SEM).

Plasma T concentrations did not change during the 3 h of restraint in 40-d-old rats, whereas they increased in 60-d-old rats (d 1: age, P = 0.02; time, P = 0.001, age x time, P < 0.0001; d 2: age, P = 0.01; time, P = 0.032; age x time, P = NS; d 3: age, P < 0.001; time, P = 0.001; age x time, P = 0.001). Without a B response to restraint, T was stimulated by the stress in 60-d-old rats.

The overall AUC for T did not change across days in 40-d-old rats. The overall AUC for T was increased in 60-d-old rats on the third day of restraint compared with the first 2 d.

Responsivity in the HPA axis: ACTH and B as a function of age and B responses
ACTH in intact rats (Fig. 2, top panels).
Basal ACTH (time zero) did not differ between ages. There were no significant differences among basal ACTH concentrations across d 1–3 between the groups of rats. On d 4 basal ACTH concentrations did not differ among the four groups.

View larger version (27K): [in this window] [in a new window]   Figure 2. ACTH responses habituate during consecutive exposures to restraint in 40-d-old, but not in 60-d-old, rats. Plasma ACTH (top panels) and B (bottom panels) during restraint. Within these figures, the total AUC from 0–180 min of restraint is represented as bars in the insets. *, Significant effect of age on total AUC within day (by unpaired t test, P < 0.05). Bars labeled with different lowercase letters indicate significant effects of day of exposure to restraint in 40-d-old rats, whereas capital letters are used for 60-d-old rats (by paired t test, P < 0.05; when the letter is italicized, 0.06 > P > 0.05; n = 4–6 rats/group).

The total AUC for ACTH reflected the decrement in response across days in the 40-d-old rats, and there was no difference across days of restraint at 60 d of age.

ACTH in ADX+B rats (Fig. 3, top panels).
Basal ACTH (time zero) was maintained by the B pellets in the range of intact rats and did not differ between 40- and 60-d-old rats across days. On d 4 basal ACTH concentrations were elevated in the previously restrained 60-d-old rats only (interaction of age x stress, P = 0.03).

View larger version (28K): [in this window] [in a new window]   Figure 3. The lack of B secretion during consecutive exposures to restraint in ADX+B rats leads to enhanced ACTH response in 40-d-old, but not 60-d-old, rats. For details, see Fig. 2. *, Significant effects of age on total AUC within day (by unpaired t test, P < 0.05). #, 0.06 > P > 0.05. n = 4–5 rats/group.

The total AUC for ACTH increased with days of restraint in the 40-d-old, but not the 60-d-old, rats.

B in intact rats (Fig. 2, bottom panels).
There were no differences between ages in basal (time zero) plasma B concentrations across days in 40- and 60-d-old rats. There were no significant effects of age or prior stress on basal plasma B concentrations on d 4.

As expected, plasma B concentrations increased in response to restraint stress on each day in both age groups. Overall B responses to restraint were similar at both ages (age, d 1, 2 and 3, P = NS), although there were trends for age x stress interactions on all days. Similar to the effect observed in ACTH, 40-d-old rats showed lower overall B concentrations on d 3 compared with d 1 (P = 0.016). Sixty-day-old rats did not show differences among the overall B values across the 3 experimental days. Only after 90 min of restraint on d 2 did 60-d-old rats have decreased B concentrations compared with d 1 (P = 0.013).

The total AUC for B did not differ across days in the 40-d-old rats, but was reduced with repetition in the 60-d-old rats.

B in ADX+B rats (Fig. 3, bottom panels).
B replacement in ADX rats resulted in similar basal plasma B concentrations across days at both ages. There were no significant effects of age or prior stress on basal plasma B concentrations on d 4.

As expected, because the source was exogenous, overall plasma B at both ages was not significantly affected by restraint.

The total AUC for B increased slightly with days of restraint in both groups of rats, probably because of a restraint-induced shift in blood flow away from liver, thus reducing the clearance rate of the exogenous steroid.

Leptin
Intact rats (Fig. 4, top panels).
Overall, basal (time zero) leptin concentrations were lower in the less fat 40-d-old than in 60-d-old rats, independently of adrenal status (P < 0.001), but they were not different across days at either age. On d 4, basal leptin concentrations were not measured.

View larger version (27K): [in this window] [in a new window]   Figure 4. Repeated restraint induced minor effects on plasma leptin concentrations independently of age and adrenal condition. For details, see Fig. 1.

The total AUC for leptin did not change across days in the 40-d-old rats, but decreased significantly between d 1 and d 2–3 in the 60-d-old rats.

ADX+B rats (Fig. 4, bottom panels).
Basal (time zero) leptin concentrations were lower in 40- than 60-d-old rats. Leptin was lower on d 3 than d 2 in 40-d-old rats. There were no significant changes in basal leptin with repeated restraint in 60-d-old rats. On d 4 basal leptin concentrations did not differ between the rats of the two ages.

On d 1 a progressive decrease in leptin levels was observed from 30–180 min in 40-d-old rats. In 60-d-old rats, leptin at 180 min was lower than at time zero. There were no significant effects of restraint on leptin concentrations at either age on d 3.

The total AUC for leptin was lower in 40- than 60-d-old rats, but there were no significant differences in leptin AUC across days of restraint.

Comparisons of the hormonal responses (AUC above baseline, time zero) to repeated restraint in younger and older, intact and ADX+B rats (Fig. 5)
T responses (Fig. 5, first row).
There were significant main effects of age and adrenal status on the AUC for T after the onset of restraint (all P < 0.01). There were essentially no T responses to restraint in the younger rats, whereas there were positive T responses to restraint in the older rats, particularly in the older rats that were ADX+B. The interaction between adrenal status and days was also significant (P < 0.01), with a tendency for a three-way interaction (P < 0.06). These interactions reflect the fact that on the third day of restraint intact rats had lower T responses to restraint, whereas the ADX rats with clamped B responded positively.

View larger version (21K): [in this window] [in a new window]   Figure 5. Responses of T and ACTH to restraint are affected by B responses. All data represent the response (30, 90, and 180 min) to restraint after subtraction of the basal (time zero) values to produce daily response AUCs. Top row, T response decreases in intact and increases in ADX+B rats. A similar pattern, but different amplitude, of the responses is observed in 40-d-old (left panels) and 60-d-old (right panels) rats. Second row, ADX+B increases ACTH responses to restraint across days in 40-d-old, but not 60-d-old, rats. Third row, Forty-day-old intact rats adapt their B responses to restraint. Bottom row, Plasma leptin is not affected by either adrenal condition or age. In all cases results are expressed as the mean ± SEM (n = 4–6 rats/group). *, Significant effects of ADX+B on AUC within day and age (by unpaired t test, P < 0.05). Within age and adrenal status, different lowercase letters (40 d) or capital letters (60 d) indicate significant differences among days (P < 0.05, by paired t test).

ACTH responses (Fig. 5, second row).

B responses (Fig. 5, third row).
Age and time did not differ; the major effects of adrenal status and interactions (all P < 0.02) dominated the analysis.

Leptin responses (Fig. 5, fourth row).
There were no significant main effects or interactions of the leptin responses to restraint. Although leptin tended to be inhibited during restraint (AUCs are on the whole negative), the negative response appeared to drift up to no response with succeeding exposures to restraint.

Responsivity in metabolic variables as a function of age and B responses
BW in intact rats (Fig. 6, top panels).
BW increased with time (P < 0.000), and the interaction of age x time was significant (P = 0.04). This reflects the fact that although the ponderal growth rate in controls was similar at the two ages, the daily increase in BW tended to be greater in stressed 40- than 60-d-old rats. Repeated restraint blocked BW gain in 60-d-old rats. The increase in BW was also affected in 40-d-old rats after the first exposure to restraint (d 1–2), but it recovered after the second exposure. By d 4, 60-d-old stressed rats tended to have lower BW than their controls (P < 0.1), but restraint did not affect final BW in the 40-d-old group.

View larger version (29K): [in this window] [in a new window]   Figure 6. Restraint decreases BW gain in 60-d-old, but not in 40-d-old, rats independently of adrenal status. Absolute BW for intact (top row) and ADX+B (bottom row) rats are shown for 40-d-old (left column) and 60-d-old (right column) rats. Results are expressed as the mean ± SEM (n = 3–8 rats/group). *, Significant effects of restraint within day and age (by unpaired t test, P < 0.05).

BW in ADX+B rats (Fig. 6, bottom panels).

FI in intact rats (Fig. 7, top panels).
Initial FI before stress (d 0–1) was similar at both ages. FI was 3–4 g/d greater in the younger than the older rats when it was normalized to BW (P < 0.001), and BW x time was significant (P < 0.001), indicating variability of FI with time, particularly in the rats exposed to restraint. Restrained 40-d-old rats decreased FI on d 1–2 and 3–4 compared with prestress levels, but were not significantly different from the control. Sixty-day-old rats decreased FI throughout the period of restraint (P < 0.05); however, there was no significant difference between the control and restrained groups at this age because of the considerably different intake during d 0–1.

View larger version (26K): [in this window] [in a new window]   Figure 7. Restraint decreases FI at both ages and in both adrenal conditions. ADX+B decreases FI further in 40 d-old, but not in 60-d-old, rats. Absolute FI in intact (top row) and ADX+B (bottom row) groups is shown for 40-d-old (left column) and 60-d-old (right column) rats. Results are expressed as the mean ± SEM (n = 3–8 rats/group). * and #, Effects of restraint within day and age (*, P < 0.05; #, 0.06 > P > 0.05; by unpaired t tests). Different lowercase letters (40 d) or capital letters (60 d) indicate significant differences compared with prestress FI levels (P < 0.05, by paired t test).

FI in ADX+B rats (Fig. 7, bottom panels).

Comparison of ADX+B to intact rats on FI within age showed that surgery on d 0 reduced FI on d 0–1 at both ages. During the period of restraint, 40-d-old ADX+B rats maintained lower levels of FI than intact rats. There were no differences in FI between ADX+B and intact 60-d-old rats during the stress period.

CE in intact rats (Fig. 8, top panels).
CE (grams of BW gained/calories ingested) on d 0 was significantly greater in 40- than in 60-d-old rats (P = 0.015). There were significant effects of age (P = 0.005), restraint stress (P = 0.0011), and interaction between time and stress (P = 0.006). Restraint stress decreased CE on d 1 and 2, but not on d 3 in both groups. However, it should be noted that mean CE remained positive across all days after restraint in the 40-d-old rats, whereas it was negative or 0 on all days after restraint in the 60-d-old group. CE was significantly greater in 40-d-old than in 60-d-old rats on d 2 and 3.

View larger version (32K): [in this window] [in a new window]   Figure 8. Restraint decreases CE in intact rats at both ages, whereas in ADX+B rats, the effects of restraint on CE are age and time dependent. CE in intact (top row) and ADX+B (bottom row) rats is shown for 40-d-old (left column) and 60-d-old (right column) rats. Results are expressed as the mean ± SEM (n = 3–8 rats/group). * and #, Effects of restraint within day and age (*, P < 0.05; #, 0.06 > P > 0.05; by unpaired t test). Within the stress group, different bold lowercase letters (40 d) or capital letters (60 d) indicate significant differences (P < 0.05, by paired t test). Plain letters (neither bold nor italic) are used for control groups. When a letter is italicized, 0.06 > P > 0.05.

ADX+B (Fig. 8, bottom panels).

Tissue weights (Table 1)
Thymus.
Independently of adrenal manipulation, absolute thymus weights were similar at both ages and restraint stress did not affect them. Relative thymus weight (grams per 100 g BW) was lower in the older rats in both experiments (P < 0.0001); however, thymus weight did not differ between intact and ADX+B groups, showing adequate B replacement.

Subcutaneous fat.
In intact rats there was a significant effect of age (P = 0.04) and restraint (P = 0.01) on absolute sc fat weight. Forty-day-old rats had lower depot weights than 60-d-old rat and restraint decreased sc fat weights to a similar extent at both ages. Only the effect of restraint was significant on the relative weights of sc fat depots. In ADX+B rats there was a significant effect of age (P = 0.0002), sc fat was lighter in 40- than 60-d-old rats. However, restraint stress did not affect sc fat weight. No significant effects were observed in relative weights. Comparison of both experiments by t test showed that ADX+B did not affect either absolute or relative sc fat depot weights in 60-d-old rats. However, ADX+B decreased both absolute and relative sc fat depot weights in control 40-d-old rats.

Discussion
Effects of restraint on the HPG and HPA axes, and interactions between them
These studies reveal the importance of interactions among pubertal age and stress in regulation of the HPA and HPG axes in young male rats. Forty- and 60-d-old male rats display very different regulation of both the gonadal and adrenocortical system responses to restraint. Moreover, B concentrations exogenously maintained at daily mean unstressed concentrations (12) affect both axes. Forty-day-old intact, but not ADX+B, rats decrease T concentrations in response to stress, and basal concentrations do not change in either group. In 60-d-old intact rats, T responses to restraint tend to increase, and the absence of stress-induced B secretion leads to significant T responses during restraint. Basal T concentrations increase with days of restraint in 60-d-old intact rats. In rats entering puberty at 40 d, ACTH responses to repeated restraint habituate across days; this requires increased B, because ACTH responses increase across days in B-clamped ADX rats. By contrast, in intact 60-d-old rats, restraint causes ACTH responses of similar amplitude on each day, and this is unaffected by clamped B.

The HPG axis.
T responses to chronic cold in gonadally intact groups of ADX+B 40-d- and 60-d-old rats are age dependent. Thus, chronic cold significantly decreases plasma T in 60-d-old, but not 40-d-old, rats (4). Moreover, the ability of 40-d-old rats to cope with chronic cold stress is so highly sensitive to plasma T concentrations that a mean difference of ±0.5 ng/ml in plasma T determines the life or death of younger rats. By contrast, a wide range of T concentrations had little metabolic effect in 60-d-old rats. Therefore, we suggested that maintenance of plasma T within a narrow range in 40-d-old rats might be an adaptive regulatory strategy to maintain growth under cold conditions. Here, we show that plasma T in 40-d-old rats is very slightly modulated within and across the days of restraint, whereas marked changes in T are observed with time and days after restraint in 60-d-old rats. These results support the hypothesis that the maintenance of fairly constant plasma T levels in 40-d-old rats accounts, at least partially, for stability of energy balance.

Initial T concentrations increase with days of restraint in 60-d-old intact rats; with clamped B, initial concentrations do not change with time. This suggests that stress-induced elevations in B may affect HPG function positively. By d 3 of restraint, initial T concentrations were significantly increased in the intact 60-d-old rats, suggesting that restraint and the B responses to it increase basal activity in the HPG axis. By d 4, basal concentrations of T were still higher than on d 3 in intact rats. However, further studies are required to determine whether overall basal activity in the HPG axis is increased in response to restraint or whether the elevation in initial T concentrations on d 3 and 4 results from an acute anticipatory response of T to restraint.

The intact 40-d-old rats acutely decrease T concentrations during restraint and decrease it to a greater extent on d 3 than on d 1. We do not know whether the acute decrease in T is mediated by inhibition of hypothalamic function and/or by stress-induced changes directly on pituitary or gonadal function. However, because similar acute decreases did not occur in 40-d-old ADX+B rats, this effect appears to be at least partially dependent on stress-induced B secretion.

Intact 60-d-old rats increase plasma T after restraint on d 1 and 2, but not on d 3; over the 3 d, restraint does not stimulate total T concentrations significantly. However, 60-d-old rats with clamped B increased T during restraint on all days. In the 60-d-old rats, high, stress-induced circulating B concentrations appear to inhibit the T response to restraint while stimulating basal concentrations of T. Thus, constant B concentrations at the unstressed daily mean appear to sensitize gonadal responses of rats leaving puberty to repeated restraint.

The HPA axis.
The overall ACTH and B responses to restraint are greater in male rats entering puberty than in those leaving puberty. Intact 40- and 60-d-old rats have maximal stress-induced ACTH and B responses 30 min after the onset of restraint in the morning, as reported previously (13). Thereafter, from 90–180 min of restraint, hormone concentrations decrease, occasionally to initial values, even during continued restraint. By contrast, rats with clamped B do not decrease ACTH concentrations during the periods of repeated restraint. These results suggest strongly that independently of age, B secretion effectively inhibits ACTH secretion during restraint.

A clear habituation of ACTH responses to restraint occurs in 40-d-old, but not 60-d-old, rats within three repetitions of the stressor. Adult rats also characteristically habituate adrenocortical responses to restraint, but this appears to require more than three repetitions (reviewed in Ref. 14). In response to restraint, activation of the HPA axis requires occupancy of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) (15). Blockade of MR, but not GR, prevents habituation to repeated restraint (16). Furthermore, in the presence of B, CRF induces up-regulation of hippocampal MR (17). Thus, occupancy of both MR and GR, as well as an action of CRF are probably required for habituation of ACTH responses to restraint. From the present studies it appears that habituation of ACTH occurs more rapidly in 40-d-old than in 60-d-old rats, and that marked elevations in B, and thus high GR occupancy, are required to express habituation.

Interactions between the HPG and HPA axes: proposed mechanisms
In male rats T is intimately involved in stress responsivity. The overall effect of T on stress responsiveness in adult males is inhibitory. For instance, the number of Fos-expressing cells in the neuroendocrine paraventricular nucleus (PVN) is highly negatively correlated with circulating T concentrations after restraint stress (18), and there is a tight inhibitory relationship between circulating T concentrations and the amplitude of ACTH and B responses to restraint (19). Moreover, T stimulates arginine vasopressin (AVP) mRNA expression in PVN, amygdala, and bed nucleus of the stria terminalis (BNST) (20). In amygdala and BNST, the decrease in AVP mRNA expression that follows treatment with dexamethasone is due to the glucocorticoid-induced suppression of T and is not a direct action of the glucocorticoid (21). Thus, stress- or glucocorticoid-induced changes in T can modulate AVP in a variety of brain sites.

In adult male rats there is good evidence that chronic or repeated stressors increase AVP mRNA expression in the PVN CRF-expressing cells and AVP peptide content in the median eminence (22). Just as T appears to play a major role in the regulation of brain AVP mRNA and peptide, B has a similar role in the regulation of CRF mRNA expression (23). High B inhibits CRF mRNA expression in the PVN and peptide in the median eminence, but stimulates CRF mRNA in the amygdala and BNST (24, 25). Late pubertal-adult male rats exposed to 10 d of repeated restraint exhibit normal CRF, but high AVP, mRNA in the neuroendocrine PVN after the tenth bout of restraint, whereas these responses were opposite (high CRF, but low AVP, mRNA) in rats restrained only once (26). These effects of restraint are altered in ADX rats treated with basal or stress levels of B. Both CRF and AVP mRNAs are stimulated by repeated restraint in the presence of clamped basal B concentrations (26), suggesting that it was the high B that acted to inhibit CRF mRNA after repeated restraint in intact rats. By contrast, both CRF and AVP mRNA expression were decreased with clamped stress level B concentrations (26), suggesting that, as in the dexamethasone experiments (21), the constant high B inhibited T concentrations and, thus, vasopressin mRNA.

Based on our current findings together with those cited above, we speculate that a major difference in the regulation of the response to repeated restraint in 40- and 60-d-old rats is the stimulated T secretion observed in the older groups. The higher T concentrations would be predicted to cause increased AVP mRNA and peptide expression in PVN and median eminence, respectively, and increased AVP mRNA in the extended amygdala. Thus, suppression of CRF responsiveness in PVN in 60-d-old compared with 40-d-old rats would be expected by the overall inhibitory effect of T. This hypothesized action of T could result in consistent ACTH responses to restraint as observed in the older rats that secrete T in response to restraint. Inhibition of T responses to restraint in the younger rats suggests that they may be activating their HPA axis to a greater extent through stimulation of ACTH by CRF than by AVP after repeated restraint.

Age-dependent effects of repeated restraint on energy balance and ponderal growth
BW and food intake.
Young male rats entering puberty (40 d old) continue to gain BW better during both cold (4, 27) and restraint, as shown here, than young rats leaving puberty (60 d old), provided that the rats have elevated B concentrations. At 40–45 d, intact rats or ADX rats provided with high circulating B concentrations do not decrease overall FI significantly under conditions of chronic cold or repeated restraint; FI by 60- to 65-d-old rats decreases and is unaffected by circulating B (27). Similarly, we previously found marked effects of circulating B on food intake and hypothalamic neuropeptide Y mRNA content in early pubertal male rats with diabetes (28). We do not know whether similar effects of B on food intake occur in older diabetic rats, although the neuropeptide Y mRNA content is increased in intact adult male diabetic rats (29). Here, the presence of functional adrenals alters the differential age-dependent metabolic responses to restraint, primarily through effects on FI and CE. Intact rats at 40 d of age do not significantly decrease FI during restraint, whereas at 60 d FI is decreased. Caloric efficiency decreases, particularly in the 60-d-old group. In the 40-d-old rats CE remained positive during the last days of restraint, whereas in the 60-d-old rats it was either negative or 0. Clamped B reduces food intake in 40-d-old rats and does not affect FI during restraint at 60 d of age. In younger (40-d-old), but not older (60-d-old), male rats, stress concentrations of B are critical for maintenance of FI with repeated restraint, as they are in cold (25). The older rats clearly alter FI using mechanisms other than circulating B concentrations. The effects of B on food intake in younger rats resemble those in mice (30, 31), suggesting that their higher surface/volume ratio may require maintenance of nearly normal food intake for survival.

Older (60-d-old) rats may modulate stress-induced reduction in food intake through the central anorexogenic effects of CRF, as these reductions in FI and BW are blocked by third ventricular injections of a CRF antagonist (7). Many repeated stressors do appear to excite the central CRF system, which may be responsible for the coordinated changes in several aspects of central regulation that occur after chronic stress in rats (32). Alternatively, the relatively high T concentrations in the older rats may be converted in part to estrogen through the action of aromatase (33, 34). Estrogens are well known to reduce food intake (35).

Fat depots.
Regulation of fat metabolism also clearly differs as a function of age. The younger rats exhibit fat loss in both central and peripheral fat depots after restraint, whereas only the measured peripheral fat stores are reduced in the older rats. This reduction in fat stores is almost certainly a B-mediated effect, as there is no reduction in fat stores at either age when animals are restrained under conditions of clamped B. We believe that the effects of B on fat mass may be mediated through an action of B on the brain regulation of sympathetic outflow. Increased sympathetic outflow to WAT would increase fat mobilization (36). B implants in the central nucleus of the amygdala not only increase CRF mRNA levels and anxiety behavior (37), but also decrease fat depot weight, specifically in chronically stressed rats (38). Because CRF is known to stimulate sympathetic outflow (39, 40) and CRF in amygdala is well sited for control of sympathetic outflow (41), the hypothesis is logical. In addition, T serves as an antiglucocorticoid (42) and may serve to inhibit to some extent the actions of stress-induced B secretion at GRs in 60-d-old rats.

There are minor changes in plasma leptin concentrations as a function of repeated restraint, but it appears that this hormone may not play a major role in either neuroendocrine or metabolic responses to the stressor in any group of rats.

Conclusion
Male rats entering puberty (40 d old) and leaving puberty (60 d old) have different regulation of behavioral, neuroendocrine, and metabolic responses to stress. The younger rats defend ponderal growth, whereas the older rats appear to defend reproductive capacity. The younger rats also emphasize different neuroendocrine axes for their hormonal responses to stress. Finally, it is common to find in the literature results from groups of male rats with weights spanning the range of 200–300 g; additionally, different rat strains grow to different final sizes and weights. The results of this study show that it is important to specify not only the weight range, but also the ages of male rats used in all studies.

	Acknowledgments

 

	Footnotes

 
This work was supported in part by Grant DK-28172.

Abbreviations: ADX, Adrenalectomized; ADX+B, adrenalectomized with corticosterone replacement; AUC, area under the curve; AVP, arginine vasopressin; B, corticosterone; BNST, bed nucleus of the stria terminalis; BW, body weight; CE, caloric efficiency; CRF, corticotropin-releasing factor; FI, food intake; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; MR, mineralocorticoid receptor; PVN, paraventricular nucleus; T, testosterone; WAT, white adipose tissue.

Received October 25, 2001.

Accepted for publication April 3, 2002.

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