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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 56:B391-B397 (2001)
© 2001 The Gerontological Society of America

Food Restriction Enhances Endogenous and Corticotropin-Induced Plasma Elevations of Free but Not Total Corticosterone Throughout Life in Rats

Eun-Soo Hana, Ted R. Evansa, Jian Hua Shua, Shuko Leeb and James F. Nelsona

a Department of Physiology, The University of Texas Health Science Center, San Antonio
b Aging Research and Education Center, The University of Texas Health Science Center, San Antonio

Eun-Soo Han, Department of Physiology, The University of Texas Health Science Center, San Antonio, TX 78229-3900 E-mail: han{at}uthscsa.edu.

Decison Editor: John Faulkner, PhD


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Chronic food restriction (FR), which retards many aging processes, enhances the endogenous diurnal peak of plasma total corticosterone (B) in young rats. Although the FR-dependent enhancement of total B disappears in aged rats, increased levels of the bioavailable fraction, free B, appear to be maintained. In young rats, we previously found that the FR-induced increase in the diurnal peak of total B is associated with increased adrenal response to corticotropin, also know as adrenocorticotropic hormone (ACTH). Here we show that the FR-enhanced adrenal response of total B to ACTH disappears with age but that the enhanced response of free B is maintained. We measured the endogenous diurnal peak and the response to ACTH of total and free B in 10-, 16-, and 22-month-old ad-libitum fed and FR male Fischer 344 rats in the afternoon, when plasma B peaks. At 10 and 16 months, FR rats showed enhanced total plasma B responses to ACTH relative to ad-libitum fed rats, but not at 22 months. By contrast, the response of free B to ACTH was enhanced by FR at all ages. The effect of FR on patterns of endogenous total and free diurnal B in these three age groups paralleled the ACTH-response data. The enhanced adrenocortical response of FR rats to ACTH does not reflect an increased expression of ACTH-receptor (ACTH-R) mRNA, because ACTH-R mRNA/µg adrenal RNA and ACTH-R mRNA/mg adrenal weight did not differ between ad-libitum fed and FR rats at any age.

CHRONIC food restriction (FR) potentiates the diurnal elevation of plasma corticosterone (B) in rats (1)(2) and mice (3). Afternoon plasma total B levels are higher in young male Fischer 344 FR rats than in ad-libitum (AL) fed rats, but this FR-induced difference in total B levels disappears with age (2). However, the finding that plasma levels of corticosterone-binding globulin decline with age in FR rats but not in AL rats provided evidence, based on mathematical calculation, that plasma free B remains elevated in aging FR rats (2).

Chronic FR retards many aging processes and extends life span. The elevation of B by FR, especially if the restriction is sustained throughout life, is of potential importance to understanding how FR extends life span. Clearly, chronic exposure to high levels of glucocorticoids and conditions of chronic stress-induced hypercorticism are deleterious (4)(5). However, the observation that moderate stress or intermittent exposure to elevated glucocorticoids is sometimes associated with extended life span (6)(7)(8) raises the possibility that moderate hyperadrenocorticism is compatible with or may even contribute to retarded aging (9)(10)(11).

The biosynthesis and secretion of B result in part from a linear activation of the hypothalamo-pituitary-adrenal axis. Hypothalamic release of corticotropin releasing hormone (CRH) stimulates the synthesis of proopiomelanocoritin (POMC) mRNA, the precursor of corticotropin, also known as adrenocorticotropic hormone or ACTH (12)(13)(14), and the release of ACTH from the corticotropes of anterior pituitary. Plasma ACTH binds adrenocortical ACTH-receptors (ACTH-R) and activates biosynthesis and release of B. The activity of the pituitary-adrenocortical axis exhibits a circadian pattern (15), reaching a peak around the time of onset of activity. Thus rats have their circadian peak at or shortly before the onset of darkness (16). The circadian rhythm of B in the plasma of rats is in phase with and driven by that of ACTH (17)(18). However, the circadian increase in B may also be in part driven by an increased adrenal response to ACTH (19)(20). Thus, the increased B in FR rats could be due to either increased ACTH, increased adrenal response to ACTH, or both. An increased adrenal response to ACTH could be due to an increased affinity of ACTH-R to ACTH, an increased number of ACTH-R, or an increased postreceptor activation of B biosynthetic pathways. It could also reflect an increased adrenocortical mass relative to body weight and blood volume with no change in the aforementioned parameters at the cellular level.

Our previous studies suggested that the enhanced adrenocortical state of 3-month-old FR rats does not involve a linear activation of the hypothalamo-pituitary-adrenal axis (21), but that an increased adrenal response of FR rats to ACTH is most likely responsible for this state (22). The aim of our present study was twofold. First, we sought to test the hypothesis that the diurnal elevation of free B is sustained in FR rats throughout life, using a direct measurement of the free fraction of B in the plasma. Second, we sought to determine the basis for the age-related loss of the FR enhancement of total B. We hypothesized that the loss of difference in the total B levels between aged AL fed rats and FR rats reflected a loss of difference in the response of total B to ACTH, whereas the sustained differences in free B reflected a sustained elevated response of free B to ACTH. We also measured adrenal ACTH-R mRNA of young and aging AL fed rats and FR rats to determine whether the amount of the ACTH-R message paralleled the differential response during aging to ACTH.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals and Dietary Procedures
Male Fischer 344 rats were obtained at 4 weeks of age from Charles River Laboratories (Kingston, NY) and housed singly in plastic cages (10 in. x 9.5 in. x 8 in., or 25 cm x 24 cm x 20 cm) with wire mesh floors suspended on a Hazleton-Enviro Rack System (Hazleton Systems, Aberdeen, MD) in a barrier facility (23). Animals were kept on a cycle of 12 hours of darkness and 12 hours of light (lights on from 4 AM to 4 PM except in the ACTH-R mRNA measurement, in which lights were on from 5:30 AM to 5:30 PM). The presence of murine viral antibodies (Sendai, Reo-3, GP-VII, PVM, KRU, H-1, SDA, LCM, and Adeno) and mycoplasma antibodies was monitored quarterly with serum samples from sentinel animals by Microbiological Associates (Rockville, MD). All tests for pathogenic organisms were negative. All procedures and experiments involving use of rats were approved by the Institutional Animal Care and Use Committee and are consistent with the National Institutes of Health (NIH) Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Education, the Guide for the Care and Use of Laboratory Animals, and the Animal Welfare Act.

For the first 2 weeks (i.e., until 6 weeks of age) all rats were fed ad libitum a standard semisynthetic diet (21). At six weeks, approximately half of the rats (group AL) were allowed to continue to eat this diet ad libitum until death or experiments. The other half (group FR) were restricted to 60% of the mean food intake of group AL until death or experiments. Food intake by AL rats was measured twice a week and the amount ingested per day calculated. FR rats were provided with food 1 hour before the start of the dark phase of the light cycle.

Plasma Collection, and Total and Free Corticosterone Measurements
For measurement of plasma total and free corticosterone, plasma was collected at 7:30 AM and 1:30 PM from 9-, 15-, and 21-month-old AL and FR rats. The sampling times were chosen because we observed that, at 7:30, total B levels for both AL and FR rats were basal and, at 1:30, FR rats had the maximum elevated total B levels compared with AL rats, with the light cycle applied (lights on from 4 AM to 4 PM). Sixty rats (10 AL and 10 FR per each age) were tail bled (each rat was bled once at each time point; bleedings were 3 or 4 days apart) without anesthesia. Blood collection was completed within 2 minutes of initial disturbance of the animal to ensure that plasma levels of B were obtained from nonstressed animals (2). Plasma samples were stored at -70°C. Plasma concentrations of total B were measured by radioimmunoassay (RIA; see RIA of ACTH and corticosterone). The percentage of free B in undiluted plasma was determined directly by centrifugal ultrafiltration dialysis, a previously described procedure (24) with some modifications. Corticosterone, [1,2,6,7-3H (N)] (71.70 Ci/mmol) and glucose D-[14C (U)] (298.00 mCi/mmol) were obtained from New England Nuclear Corporation (Boston, MA) and kept at -20°C.

Batches of 3H-corticosterone were purified weekly with a Waters 600 E Multisolvent Delivery System (Waters Chromatography Division, Millipore Corp., Bedford, MA), connected with an Alltima C18/5 micron column (length, 250 mm; i.d. 4.6, mm; Alltech Association, Inc., Deerfield, IL). Acetonitrile/filter (0.2 µm) sterilized double-distilled H2O (1:1) was used as elution solvent. The two fractions with the highest radioactivity in the B peak, which together accounted for approximately 45% of the total injected radioactivity, were pooled and used in the assays. Purified 3H-corticosterone (75,000 cpm) was transferred into a 1.5-ml microcentrifuge tube together with 14C-glucose (3,000 cpm) and dried briefly in a SpeedVac (Savant Instruments, Inc., Farmingdale, NY). All procedures were done in a warm room at 37°C; the plasma sample (25 µl) was added to the tube, mixed briefly by aspiration, and allowed to equilibrate at 37°C for 30 minutes. After incubation, 2 µl was transferred to a scintillation vial with 10 ml of Liquiscint (National Diagnostics, Atlanta, Georgia) for measurement of the isotope ratio in plasma. Twenty microliters of the same incubation was transferred into an Ultrafree-MC filter unit with a 5000 nominal molecular weight limit, low-binding regenerated cellulose membrane (Millipore Corp.), which had been incubated at 37°C for 30 minutes. The filter unit was centrifuged at 3000 g for 10 minutes at 37°C. Two microliters of filtrate were collected for measurement of isotope ratio in filtrate. 3H and 14C radioactivities were counted simultaneously on a Beckman LS 7500 Liquid System (Beckman Instruments, Inc., Fullerton, CA). After correction for background counts and for 3H and 14C energy overlap in the liquid scintillation counting system, the percentage of free steroid was calculated by the following formula:

The percentage of free B was multiplied by the total B concentration in the sample, as determined by RIA, to provide the free B concentration.

Adrenal Response to ACTH
Rats used for this experiment were the same as those used for total and free B measurements 1 month earlier. Both 3 and 2 hours before the ACTH injection, 10-, 16-, and 22-month-old AL and FR rats were subcutaneously injected with dexamethasone-21-phosphate (500 µg/kg body weight each time; Sigma, St. Louis, MO) to suppress the endogenous ACTH release (25). Two hours after the second dexamethasone injection, an incision placed 1–1.5 in. (2.5–3.8 cm) in from the tail tip was made with a razor blade and a sample of blood was removed (140 µl) in heparinized capillary tubes without anesthesia and followed by a subcutaneous injection of 1.25 µg/kg body weight rat ACTH1-39 (Peninsula Laboratories Inc., Belmont, CA) or vehicle control (pyrogen-free saline with 18 µg/ml ascorbic acid and 0.1 mg/ml bovine serum albumin) at 1:30 PM when the FR rats showed the maximum levels of total B. Additional blood samples (140–280 µl) were collected at 5, 15, 30, 60, and 120 minutes after injection. Plasma was immediately prepared by centrifugation. The plasma was stored at -70°C until assayed to determine plasma levels of total B and ACTH by RIA, and free B by centrifugal ultrafiltration dialysis as described in previous section. ACTH solutions were prepared in pyrogen-free saline (0.9% NaCl; Sigma) containing 18 µg/ml ascorbic acid and 0.1 mg/ml high grade bovine serum albumin, pH 4.3 (25), and they were stored at -20°C. Dexamethasone-21-phosphate was dissolved in pyrogen-free saline (25) and stored at room temperature with protection against light.

Tissue Collection and RNA Preparation
For measurement of ACTH-R mRNA, adrenals were collected from 6-, 12-, 18-, and 24-month-old AL and FR rats. Sixty rats per age (30 AL and 30 FR; 5 AL and 5 FR rats each at 4:30 AM, 9:30 AM, 1:30 PM, 3:30 PM, 5:30 PM, and 9:30 PM) were weighed and then decapitated within 10 seconds of dis-turbance of their cage. Adrenals were dissected, weighed, quickly frozen in liquid nitrogen, and stored at -70°C. Total RNA was extracted separately from the adrenals of each animal as previously described (26). The RNA yield of each sample was determined spectrophotometrically, assuming 1 OD260 unit = 40 µg/ml. Samples were stored in diethylpyrocarbonate (DEPC)-treated water at -70°C. The quality of the RNA extracted from each sample was monitored by 1.0% agarose formaldehyde gel electrophoresis. All samples had 260:280 ratios of ~2 and exhibited discrete 28S and 18S bands.

Slot Blot Analysis for ACTH-Receptor mRNA in Total Adrenal RNA
Duplicates of each adrenal RNA sample (10 µg) were brought to 80 µl with DEPC-treated H2O diluted with 100 µl deionized formamide (BRL, Gaithersburg, MD), heated for 5 minutes at 65°C, chilled on ice, and diluted with 20 µl 20 x standard sodium citrate (SSC). An 80-µl aliquot from each duplicate was applied to GeneScreen (NEN, Boston, MA) presoaked in 20 x SSC, using a Schleicher and Schuell (Keene, NH) slot-blot minifold. As a way to construct a standard curve of the hybridization signal for samples, serial dilutions from a pool of rat adrenal RNA ranging from 1 to 10 µg were applied in duplicate to the membranes. 32P-labeled cRNA probe complementary to mouse ACTH-R cDNA was synthesized from pBluescript II KS± containing a 200 base pair EcoRI-SalI fragment from transmembrane domain of the mouse ACTH-R gene obtained from Dr. Roger Cone (27). The riboprobe was synthesized with T7 RNA polymerase (Promega, Madison, WI), following reaction conditions specified by the vendor (labeled to approximately 1 x 108 cpm/µg input DNA with 32P-CTP). Northern blot analysis (26) showed that the probe is specific for adrenal RNA (only a single band was detected in adrenal RNA) and no specific band was detected in RNA from other tissues tested as negative controls (i.e., liver, testis; data are not shown). Hybridization was performed as previously described (28).

Signal quantitation was performed with a storage phosphorimaging system (Molecular Dynamics, Sunnyvale, CA), and the signal intensities of test samples were compared with those of the standard curve. Standards used to generate this curve were processed in parallel with the test samples. The standard curves were linear (r2 > .98).

Radioimmunoassay of ACTH and Total Corticosterone
The measurement of B was performed with 125I cortico-sterone kit for rats and mice (IKCN Biochemicals, Carson, CA), and the measurement of ACTH was performed with a 125I-ACTH kit for humans (INSTAR, Stillwater, MN) as previously described (21).

Statistical Analysis
Data are expressed as means and standard error of the mean. Results from the total and free B measurements were analyzed by a three-way (age, dietary group, and time of sampling) analysis of variance (ANOVA) (29) with repeated measures on one factor (time of sampling). Results from the adrenal response to the ACTH test were analyzed by a four-way (age, dietary group, time of sampling, and ACTH dose) ANOVA (29) with repeated measures on one factor (time of sampling). Data from the measurements of ACTH-R mRNA per microgram of RNA, ACTH-R mRNA per milligram of adrenal weight, ACTH-R mRNA per gram of body weight, and adrenal weight, body weight, and adrenal weight per body weight were analyzed by a three-way (age, dietary group, and time of sampling) ANOVA. The Box-Cox transformation (30) was used to meet the assumptions of normality of ANOVA. Selected mean differences between dietary groups at each age were evaluated by the t test (31). Differences with a value of p < .05 were considered significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
FR Elevates the Endogenous Peak of Free but not Total B Throughout Life
Morning (7:30 AM) and afternoon (1:30 PM) plasma total and free corticosterone concentrations in 9-, 15-, and 21-month-old AL and FR rats are shown in Fig. 1. In both AL and FR rats, plasma total and free B levels rose markedly between 7:30 AM and 1:30 PM (p < .0005; ANOVA). The levels of total and free B also increased with age (p < .01 and p < .05, respectively; ANOVA). There was also a significant interaction of dietary treatment and time of sampling (p < .05 for total B and p < .005 for free B; ANOVA). This reflects the observation that FR, as shown previously, mainly enhanced the afternoon elevation of B. When the rats were 9 months of age, the afternoon plasma total and free B concentrations of FR rats were significantly higher than those of AL rats (p < .05 for total B and p < .01 for free B; t test). However, for total B, this afternoon difference disappeared in 15- and 21-month-old rats. In contrast, afternoon free B levels in FR rats were significantly higher than those in AL rats at all ages (p < .01 for 9-month-old rats and p < .05 for 15- and 21-month-old rats; t test). There were no differences in morning values for plasma total and free B between AL and FR rats except for 15-month-old rats for total B (p < .05; t test).



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  		Figure 1. Plasma total and free corticosterone (B) concentrations in ad-libitum-fed (AL) and food-restricted (FR) 9-, 15-, and 21-month-old male Fischer 344 rats. (Data are expressed as the mean ± SE; n = 10 per diet/time point.) When the rats were 9 months of age, afternoon plasma total and free B concentrations of FR rats were significantly higher than those of AL rats (p < .05 for total B and p < .01 for free B; t test), but when they were 15 and 21 months of age, only afternoon free B levels in FR rats were significantly higher than those in AL rats (p < .05; t test).

 
FR Enhances the Response of Free but Not Total B to ACTH Throughout Life
In vivo adrenal response to ACTH (1.25 µg ACTH/kg of body weight) was tested in the same rats at 10, 16, and 22 months (1 month after measuring endogenous B levels; Fig. 2). We only tested adrenal response to ACTH in the afternoon, when plasma B is at its peak, and the differences between FR and AL rats are greater. The dose of ACTH was chosen to give peak levels of plasma B near those occurring during the spontaneous diurnal elevation of B in rats.



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  		Figure 2. In vivo adrenal response to adrenocorticotropic hormone (ACTH) in 10-, 16-, and 22-month-old ad-libitum-fed (AL) and food restricted (FR) rats. (Data are expressed as the means ± SE; n = 6 per diet/time point.) Graphs show ACTH, total corticosterone, and free corticosterone (B) measurements after injection of 1.25 µg ACTH/kg of body weight. When the rats were 10 and 16 months of age, peak levels of total and free B were significantly higher in FR rats compared with AL rats (p < .05 except for free B at 10 months, which is p < .0001; t test), but when they were 22 months of age, only the peak level of free B was significantly higher in FR rats compared with AL rats (p < .05; t test).

 
There were no significant differences in the levels of ACTH in different age groups or the two dietary groups following injection of ACTH. There was an overall significant difference in the levels of total B in the two dietary groups (p < .05; ANOVA) with ACTH injection. When the rats were 10 and 16 months of age, peak levels of total B were significantly higher in FR compared with AL rats (p < .05; t test). However, when they were 22 months of age, the difference disappeared between two dietary groups. Peak levels of free B, however, were maintained at higher levels in FR rats at all three ages (Fig. 2, p < .0001 for 10-month-olds, and p < .05 for 16- and 22-month-olds; t test).

Table 1 shows the relationship between the effects of FR on endogenous and ACTH-stimulated total and free B across the life span. Both the relative increase, expressed as percent above the AL values, and the statistical significance of the increase of total and free B in FR animals were strikingly related across the two experimental conditions.


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  		Table 1. Percent Increase of Total and Free B in FR Compared With AL Rats

 
FR Does Not Increase ACTH-R mRNA
Adrenal ACTH-R mRNA was measured to determine whether ACTH-R mRNA increased in FR rats in concert with the increased adrenal response to ACTH. Fig. 3 shows the levels of ACTH-R mRNA, normalized per microgram of adrenal RNA (A), per milligram of adrenal weight (B), and per gram body weight (C) in 6-, 12-, 18-, and 24-month-old AL and FR rats. These were a different group of rats than those used for the B measurements. Although tissues were collected and studied at six different time points throughout the day, only mean values of all time points are shown, because there were no significant interaction effects of time and diet on the variables measured. ACTH-R mRNA levels did not differ between the AL and FR groups at any ages, whether expressed per microgram of total RNA or per milligram of adrenal weight. However, FR rats had higher levels of ACTH-R mRNA/g of body weight compared with AL rats (Fig. 3; p < .0005; ANOVA); these differences were significant at 6 and 18 months but disappeared at 24 months. As the animals grew older, the level of ACTH-R mRNA per microgram of RNA and per milligram of adrenal weight decreased in both AL and FR rats. However, when normalized to body weight, ACTH-R mRNA did not decrease significantly with age.



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  		Figure 3. Effect of food restriction on adrenocorticotropic hormone-receptor (ACTH-R) mRNA in adrenal glands of 6-, 12-, 18-, and 24-month-old rats. (Data are expressed as the means ± SE.) ACTH-R mRNA levels did not differ between the ad-libitum-fed (AL) and food restricted (FR) groups at any ages, whether expressed per microgram of total RNA or per milligram of adrenal weight. However, FR rats had higher levels of ACTH-R mRNA/g of body weight compared with AL rats (p < .0005; analysis of variance). A, ACTH-R mRNA/µg of adrenal RNA (n = 23–26 per age/diet group); B, ACTH-R mRNA/mg of adrenal weight (n = 20–25 per age/diet group); C, ACTH-R mRNA/g of body weight (n = 20–25 per age/diet group).

 
Adrenal weights were significantly lower in FR rats than in AL rats (Fig. 4; p < .0005; ANOVA) and increased with age (p < .0005; ANOVA). Although FR rats weighed less than AL rats (Fig. 4), the decrease in adrenal weight of FR rats did not scale to the decrease in body weight. As a consequence, adrenal weight, like ACTH-R mRNA, normalized to body weight was greater in FR rats than in AL rats at 6, 12, and 18 months of age (Fig. 4; p < .05; t test). At 24 months, these increases in FR rats were no longer present. The disappearance of the difference in total B level differences at old ages between AL and FR rats may be related in part to the disappearance of the difference in the adrenal to body weight ratios at old age.



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  		Figure 4. Effect of food restriction on adrenal weight, body weight, and their ratio for 6-, 12-, 18-, and 24-month-old rats. (Data are expressed as the means ± SE.) Adrenal and body weights were significantly lower in food restricted (FR) rats than in ad-libitum-fed (AL) rats (p < .0005; analysis of variance); however, the decrease in adrenal weight of FR rats did not scale to the decrease in body weight. As a consequence, adrenal weight normalized to body weight was greater in FR rats than in AL rats (p < .0005; analysis of variance). A, adrenal weight (n = 23–25 per age/diet group); B, body weight (n = 23–26 per age/diet group); C, adrenal weight/body weight ratio (n = 23–25 per age/diet group).

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Corticosterone (B) is unique among hormones affected by chronic FR. Instead of showing the suppressed activity characteristic of most hormonal responses to FR (9), B levels in plasma are moderately elevated in FR rats and mice, especially during the diurnal peak (1)(2)(3)(32). This enhanced adrenocortical state does not, however, reflect increased hypothalamic-pituitary activity—anterior pituitary and plasma levels of ACTH are reduced in FR rats compared with AL rats (21). The elevated B of FR rats primarily reflects an enhanced response of the adrenal gland to ACTH and not an increase in plasma levels of ACTH (22).

The results of this study confirm and extend these earlier findings, most of which were limited to young rats. The results show that the enhanced response of total B to ACTH in FR rats extends to 18 months of age, and, for free B, extends to 22 months. Thus, in F344 rats, chronic FR increases the sensitivity of the adrenal gland to ACTH for much of the adult life span. This observation buttresses the earlier evidence, based on measurements of plasma B levels, that the FR animal is in a moderately elevated state of adrenocortical activity throughout much of its life span. However, evidence based on plasma levels of B alone can always be questioned because of the possibility that the process of obtaining samples reveals a differential response to the stress of sampling between FR and AL animals. However, finding that the adrenal gland has enhanced responsiveness to ACTH and that circulating and pituitary ACTH is lower, not higher as might be expected in a stressed animal, provides stronger evidence that the adrenal axis is altered in FR animals. Moreover, the increased B of FR has physiological significance (i.e., negative feedback suppression of B on ACTH) (22).

The FR-based elevation of endogenous total B levels disappears as rats age (2). The current results indicate that the enhanced response of total B to exogenous ACTH also disappears as rats age. These results suggest that an age-related change in adrenal sensitivity underlies the loss of the enhanced diurnal elevation of total B in FR rats. As FR rats age, their enhanced adrenal responsiveness to ACTH by FR disappears. The basis for this change in sensitivity is not clear. We examined ACTH-R mRNA levels and found no evidence for a differential in receptor message levels that could explain either the enhanced response or its loss during aging in the FR animals. The finding that adrenal mass, normalized to body weight, is greater in FR than in AL rats when they are young, but is no longer greater in old rats, provides one explanation for the differential response's disappearance with age. However, it should be noted that in young rats, the in vitro response of adrenal tissue to ACTH was enhanced by FR even when normalized to tissue mass (22). Thus, there may be alterations in the postreceptor pathway through which the FR-induced changes and ultimate loss of enhanced adrenal sensitivity are also mediated.

Although the FR enhancement of the endogenous diurnal peak of total B disappears with age, Sabatino and colleagues (2) hypothesized that free B would remain elevated in old FR rats, based on their finding that the plasma concentration of corticosterone-binding globulin declines with age in FR rats but not in AL rats. A decline in corticosterone-binding globulin, which has high affinity for B and, along with albumin, sequesters over 95% of circulating B, would theoretically increase the free fraction of B in the circulation. Our direct measurement of the free fraction of plasma B supports their hypothesis. When the rats were 22 months of age, when total plasma B concentrations no longer differed between those that were AL and those that were FR rats, the difference remained for free B. Because free B is believed to be the better index of biologically active B in the circulation than total B, this result indicates that FR rats remain exposed to a moderate hyperadrenocorticism through much of their life. Although we and others have argued that a moderate hyperadrenocorticism could contribute to the antiaging actions of FR by enhancement of the same protective mechanisms that are activated by stress-induced elevations of B (3)(9)(10)(11), direct testing of this hypothesis remains to be done. It is also possible that the elevations of B have little to do with the antiaging actions of FR and may reflect a hormonal metabolic adjustment to a more exaggerated circadian pattern of exogenous caloric availability.

Conclusions
In conclusion, this study has provided additional evidence to indicate that the FR state in rats is associated with a moderate hyperadrenocorticism that persists through much of the adult life span. It has established that this persistence is most strongly evidenced in the elevation of the free fraction of plasma B, which is biologically the most significant fraction. Finally, the study has shown that increased adrenal sensitivity to ACTH is associated with the moderate hyperadrenocorticism of FR through middle age. When this increased sensitivity wanes at later ages, free B remains elevated in FR animals. The persistence of elevated free B into old age is remarkable, considering that different mechanisms appear to maintain this state during the adult life span, and supports the argument that maintaining an enhanced adrenocortical state is important to the physiological requirements of the FR animal.


   Acknowledgments
 
This work was supported by grants from the NIH (AG 00746-03) and the Aging Research and Education Centers at the University of Texas Health Science Center at San Antonio to Dr. Eun-Soo Han and by NIH Grant AG 14674 to Dr. James F. Nelson.

We thank Dr. Roger Cone for providing us with the cDNA probe necessary for our experiments; Dr. Helen Bertrand for excellent supervision of the barrier facility and the feeding regimens; Anthony Rodarte, Jose Arguillo, Joseph Hogue, and Esteban Arredondo for excellent care of the animals; and Ms. Kim Kennedy for manuscript preparation.

Received August 7, 2000

Accepted April 9, 2001


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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