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The Journal of Nutrition Vol. 128 No. 9 September 1998,
pp. 1415-1420
Department of Physiology, The University of Texas Health Science Center, San Antonio, TX 78284-7756
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ABSTRACT |
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Abstract
Introduction
Methods
Results
Discussion
References
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Chronic food restriction (FR) of rats and mice results in moderate hyperadrenocorticism, which may play a role in activating cellular mechanisms that retard aging. Previously, we reported that the FR-induced hyperadrenocorticism is not due to an activated hypothalamo-pituitary unit. Therefore, we investigated in a series of experiments whether adrenal responsiveness to adrenocorticotropic hormone (ACTH), in vitro and in vivo, is enhanced by FR. Three mo-old male Fischer 344 rats were either given free access to food (AL rats) or restricted to 60% of food consumed by AL rats (FR rats) from 6 wk of age. They were killed by decapitation in the morning (AM) and afternoon (PM) when endogenously circulating corticosterone levels are at their nadir and peak, respectively. In vitro, adrenal glands from FR rats (1.5 mo FR) produced more corticosterone per mg at all doses of ACTH than those from AL rats in both the AM and PM (diet main effect, P < 0.001). FR (1.5 to 2.5 mo) also enhanced adrenal responsiveness to physiologic (diet main effect, P < 0.05) and superphysiologic (diet main effect, P < 0.001) levels of ACTH administered in vivo to dexamethasone-treated rats. ACTH-receptor (ACTH-R) mRNA, normalized to adrenal mass or to total RNA, was not influenced by FR (1.5 mo). However, adrenal ACTH-R mRNA, as well as adrenal mass, per unit body weight was greater in FR than in AL rats (diet main effect, P < 0.001). These results indicate that enhanced adrenocortical responsiveness to ACTH plays a major role in the hyperadrenocortical state of chronically FR rats.
KEY WORDS: male Fischer 344 rats · food restriction · adrenal sensitivity · corticosterone
Corticosterone is unique among hormones affected by chronic food restriction (FR)4 in that its plasma concentration is elevated rather than reduced (Amario et al. 1987 Because of the potential importance of the hyperadrenocortical state to the effects of FR, we have undertaken studies to determine the mechanisms responsible for the elevated corticosterone in FR rats. Our previous study (Han et al. 1995 Animals and dietary procedures.
Male Fischer 344 rats were obtained at 4 wk of age from Charles River Laboratories (Kingston, NY) and housed singly in plastic cages (25.4 cm × 24.13 cm × 20.32 cm) with wire-mesh floors suspended on a Hazleton-Enviro Rack System (Hazleton Systems, Aberdeen, MD) in a barrier facility (Yu et al. 1985 In vitro adrenal response to ACTH.
Experiment 1.
The adrenal glands of 3-mo-old AL and FR rats decapitated within 10 s of cage disturbance were removed at 0830 and 1530 h, when levels of corticosterone were basal and maximal, respectively. The adrenals were defatted, weighed and divided into four approximately equal segments. The adrenal segments were placed in a 3-mm culture dish, rinsed once with 4°C Krebs-bicarbonate medium (CaCl2·2H2O, 2.5 mmol/L; MgSO4, 1.2 mmol/L; KCl, 4.7 mmol/L; KH2PO4, 1.2 mmol/L; NaCl, 0.1 mol/L; NaHCO3, 25 mmol/L; and glucose, 11 mmol/L) and washed four times with 37°C Krebs-bicarbonate medium containing O2 (95%) and CO2 (5%) for 1 min each. Adrenal glands were incubated at 37°C in 3 mL of Krebs-bicarbonate medium containing ascorbic acid (0.2 mmol/L) and 0, 33, 66 or 99 nmol/L rat ACTH1-39 (Peninsula Laboratories, Belmont, CA) with 95% O2:5% CO2 (v/v) gases for 1 h. Media were collected and stored at Experiment 2.
Experiment 2 was conducted in the same way as Experiment 1 except 0, 11, 22 or 33 nmol/L rat ACTH1-39 (Peninsula Laboratories) were used.
In vivo adrenal response to ACTH.
Superphysiologic levels of ACTH (Experiment 3).
Before the ACTH injection (2.5 h), 3- to 4-mo-old AL and FR rats were injected subcutaneously with dexamethasone-21-phosphate (C22H28FO8PNa2; 0.97 µmol/kg body weight, Sigma, St. Louis, MO) to suppress the endogenous ACTH release (Dijkstra et al. 1996 Physiologic level of ACTH (Experiment 4).
Procedures were the same as for the previous experiment, except for the addition of a morning sampling time, the amount of ACTH injected and the times sampled after injection. Experiments were done at 0800 or 1300 h, when levels of corticosterone were around basal or peak, respectively. Blood samples (140-280 µL) were collected just before the ACTH (0.27 nmol/kg body weight) or vehicle injection (t = 0) and at 5, 15, 30, 60 and 120 min thereafter. The dose of ACTH was chosen to yield circulating levels that were within the physiologic range. The plasma was immediately prepared and stored at Adrenal collection and RNA preparation.
For measurement of adrenal ACTH-R mRNA, 3-mo-old rats (5 AL and 5 FR rats at 0430, 0930, 1330, 1530, 1730 and 2130 h, n = 60) were weighed and killed by decapitation within 10 s of disturbance of their cage. Adrenal glands were dissected, weighed, quickly frozen in liquid nitrogen and stored at Slot blot analysis of ACTH-R mRNA.
Duplicates of each adrenal RNA sample (10 µg) were adjusted to 80 µL with DEPC-treated H2O, diluted with 100 µL deionized formamide (BRL, Gaithersburg, MD), heated for 5 min at 65°C, chilled on ice and diluted with 20 µL 20× SSC (3 mol/L NaCl, 0.3 mol/L trisodium citrate, pH 7.0). An 80-µL aliquot from each duplicate was applied to GeneScreen (NEN, Boston, MA) presoaked in 20× SSC, using a Schleicher and Schuell (Keene, NH) slot blot minifold. 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. A 32P-labeled cRNA probe complementary to mouse ACTH-R cDNA was synthesized from pBluescript II KS containing a 200-bp EcoRI-SalI fragment from the transmembrane domain of the mouse ACTH-R gene obtained from Dr. Roger Cone (Mountjoy et al. 1994 Radioimmunoassay of ACTH and corticosterone.
The measurement of corticosterone was performed with an 125I corticosterone kit for rats and mice (ICN Biochemicals, Carson, CA), and the measurement of ACTH was performed with an 125I ACTH kit for humans (INSTAR, Stillwater, MN) as previously described (Han et al. 1995 Statistical analysis.
Data are expressed as means and SEM and were analyzed by one-way, two-way or three-way ANOVA (Dunn and Clark 1987 In vitro adrenal responsiveness to ACTH in FR and AL rats.
In vitro adrenal responsiveness to ACTH was tested in 3-mo-old AL and FR rats. In Experiment 1 (Fig. 1), ACTH enhanced the amounts of corticosterone secreted in the media at all doses (ACTH dose main effect, P < 0.001); however, the responses to 33, 66 and 99 nmol/L ACTH did not differ. We concluded that adrenal response was maximal at 33 nmol/L ACTH. Therefore, we repeated the experiment with lower doses of ACTH. The responses were less than those in Experiment 1. There was an ACTH dose main effect (P < 0.001). Within the range of lower doses, however, the magnitude of the response again did not differ among doses (Fig. 1).
In vivo adrenal responsiveness to ACTH in FR and AL rats.
Superphysiologic levels of ACTH (Experiment 3).
In vivo adrenal responsiveness to superphysiologic levels of ACTH was tested with 3- to 4-mo-old AL and FR rats (Fig. 2). There was an ACTH dose main effect (P < 0.001) for plasma levels of ACTH. There was no significant difference in plasma ACTH levels between the FR and AL groups. Plasma corticosterone levels were elevated by ACTH at all doses in relation to baseline values (Tukey-Kramer test, P < 0.001). Moreover, the response of FR rats to ACTH was nearly twice that of AL rats (diet main effect, P < 0.001).
Physiologic levels of ACTH (Experiment 4).
Because plasma levels of ACTH in Experiment 3 exceeded those typically seen in vivo, we repeated the experiment with a separate group of rats and used a lower dose of ACTH (0.27 nmol ACTH/kg body weight). The results for the vehicle-injected rats in the AM and PM were pooled because there was no effect of time of day on either ACTH or corticosterone levels (Fig. 3). Although there also were no differences between AM and PM samples in rats injected with ACTH, the results are graphed separately to underscore this similarity.
Adrenal ACTH-R mRNA.
Adrenal ACTH-R mRNA was measured to determine if it was associated with the enhanced adrenal responsiveness to ACTH in FR rats (Fig. 4). Adrenal glands were collected at six different time points but data were pooled because there was no effect of time of day on ACTH-R mRNA levels in either AL or FR rats. When ACTH-R mRNA was expressed per microgram total adrenal RNA or per milligram adrenal gland, there was no significant difference between the AL and FR groups (Figs. 4A, B). However, when total content of adrenal ACTH-R mRNA was normalized to body weight, FR rats had >50% more ACTH-R mRNA/g body weight than AL rats (diet main effect, P < 0.001). This outcome reflects the fact that adrenal mass is not reduced proportionately to body weight in FR rats. Although absolute weight of the adrenal gland is reduced in FR rats by ~15% (Table 1; diet main effect, P < 0.05), body weight is reduced ~30% (diet main effect, P < 0.001). As a consequence, adrenal weight normalized to body weight in FR rats is ~30% greater than that in AL rats (diet main effect, P < 0.001).
This paper reports two major findings. First, adrenals from chronically FR rats exhibited enhanced release of corticosterone after in vivo and in vitro exposure to exogenous ACTH. Second, there was no elevation in adrenal ACTH-R mRNA when normalized to micrograms total RNA in FR rats. However, the amount of adrenal ACTH-R mRNA normalized to body mass was >50% greater in FR than in AL rats. As discussed below, this could contribute to the heightened corticosterone response of FR rats to ACTH. These findings support the hypothesis that the elevated plasma levels of corticosterone observed in chronically FR rats are related to changes in the adrenal gland. They do not, however, provide evidence explaining the origin of those changes.
INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References
, Klebanov et al. 1995, Sabationo et al. 1991, Stewart et al. 1988). The hyperadrenocorticism of FR rodents is qualitatively different from that associated with chronic stress or a Cushingoid state. It is not associated with any of the pathologic sequelae that characterize these syndromes, and it does not involve continuous or near-continuous exposure to elevated plasma levels of glucocorticoid. FR-induced hyperadrenocorticism is confined to the period or periods of the day preceding feeding, and peak levels are less than those observed after restraint stress (Masoro et al. 1995, Sabatino et al. 1991). This hyperadrenocortical state is not associated with impaired immune function (Weindruch and Walford 1988, Yu 1990), muscle atrophy (Yu et al. 1982) or accelerated hippocampal neuronal loss (O'Steen et al. 1990), all of which can be induced by chronic stress (Sapolsky 1992) or Cushingoid syndromes (Krieger 1982). There is no evidence that this level of hyperadrenocorticism is deleterious, and some evidence suggests that it may be involved in the retardation of aging by FR (Leakey et al. 1994, Nelson 1992, Schwartz and Pashko 1994). For example, the resistance of FR mice to one type of chemically induced carcinogenesis requires adrenal input (Pashko and Schwartz 1992). FR is also associated with attenuated inflammatory response (Klebanov et al. 1995, Lakshmi et al. 1992), a well-known characteristic of enhanced glucocorticoid action (Munck et al. 1984).
) suggested that the hyperadrenocortical state of FR rats does not involve an activation of the hypothalamo-pituitary component of the axis, because adrenocorticotropic hormone (ACTH) levels are not elevated in FR rats. Therefore, we hypothesized that the basis for the hyperadrenocorticism in FR rats resides in the adrenal cortex, possibly the consequence of an enhanced responsiveness of the adrenal to ACTH. This study addressed this question by investigating the effect of FR on adrenal sensitivity to ACTH in vitro and in vivo. After finding enhanced adrenal sensitivity to ACTH, we measured adrenal ACTH-receptor (ACTH-R) mRNA to determine whether greater expression of ACTH-R message is associated with the enhanced adrenal sensitivity of FR rats.
MATERIALS AND METHODS
Abstract
Introduction
Methods
Results
Discussion
References
). Animals were kept on a 12-h light:dark cycle (lights on at 0530 h for the in vitro study and ACTH-R mRNA measurement, and at 0400 h for the in vivo study). 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 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.
). At 6 wks, half of the rats (group AL) were allowed to continue to eat this diet ad libitum and the other half (group FR) were restricted to 60% of the mean food intake of the AL group until killing. Rats were killed by decapitation for adrenal collections for ACTH-R mRNA measurements (3 mo of age, 1.5 mo FR) or initiation of in vivo (3-4 mo of age, 1.5-2.5 mo FR) or in vitro (3 mo of age, 1.5 mo FR) ACTH sensitivity. Vitamins and minerals were not restricted for FR rats. Food intake of AL rats was measured twice a week and the amount ingested per day calculated. FR rats were provided with food 1 h before the start of the dark phase of the light cycle. Rats in both groups gained weight during the studies, although FR rats gained less weight than AL rats (body weights of 5 wk-old rats were ~70 g). FR rats are healthy, more active than AL rats, and live longer and show fewer diseases than AL rats (Masoro et al. 1980, Yu 1994).
20°C for corticosterone measurement by RIA. A pilot study using adrenal glands from young AL rats indicated that the method and range of ACTH concentrations produced saturated corticosterone response isotherms for ACTH (unpublished observations).
). Two hours later, an incision ~2.5-3.5 cm from the tail tip was made with a razor blade for sampling blood. A sample of blood was removed (140-280 µL) in heparinized capillary tubes for basal hormone determination just before a subcutaneous injection of 1.1, 2.2 or 3.3 nmol/kg body weight rat ACTH1-39 (Peninsula Laboratories) at 1300 h (t = 0). Additional blood samples (140-280 µL) were collected at 5, 15, 30 and 60 min after ACTH injection. Plasma was immediately prepared and stored at 70°C. In this experiment, we tested adrenal responsiveness to ACTH only in the afternoon, when plasma corticosterone levels are at their peak (1300-1400 h). ACTH solutions were prepared in pyrogen-free saline (154 mmol/L NaCl, Sigma) containing 0.1 mmol/L ascorbic and 0.1 g/L high grade bovine serum albumin (BSA) (pH 4.3) (Dijkstra et al. 1996) and stored at 20°C. Dexamethasone-21-phosphate was dissolved in pyrogen-free saline (Dijkstra et al. 1996) and stored at room temperature with protection against light.
70°C for ACTH and corticosterone measurements by RIA.
70°C. Total RNA was extracted separately from each adrenal gland as previously described (Sambrook et al. 1989). The RNA yield of each sample was determined spectrophotometrically, assuming that 1 OD260 unit = 40 mg/L. Samples were stored in diethylpyrocarbonate (DEPC)-treated water at 70°C. The quality of each RNA sample was monitored by 10 g/L agarose formaldehyde gel electrophoresis. All samples had A260:A280 ratios of ~2 and exhibited discrete 28S and 18S bands.
). The riboprobe was synthesized with T7 RNA polymerase (Promega, Madison, WI) according to reaction conditions specified by the vendor with 32P-CTP (29.6 TBq/mmol, NEN). Northern blot analysis (Sambrook et al. 1989) showed that the probe was specific for adrenal RNA. Only a single band was detected in adrenal gland, and no specific band was detected in tissues that were tested as negative controls (liver, testis) (data not shown). Hybridization was performed as previously described (Nelson et al. 1988).
).
). When the distribution of values was not normal, the Box-Cox transformation (Box and Cox 1964) was used to meet the assumption of normality of ANOVA. Main effects, (ACTH doses, time of sampling and dietary treatment) and their interactions were assessed. The Tukey-Kramer test (Kirk 1995) for multiple comparison of mean differences was used to make comparisons among diet groups and ACTH doses. Differences among sampling times were evaluated by t test (Hines and Montgomery 1972). Differences with a P-value <0.05 were considered significant.
RESULTS
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (29K):
[in a new window]
Fig 1.
In vitro adrenal responsiveness to adrenocorticotropic hormone (ACTH) in 3-mo-old male Fischer 344 rats with free access to diet (AL) or food restricted (FR). Values are means ± SEM. Experiment 1 shows corticosterone measurements for AM and PM samples with 0, 33, 66 and 99 nmol/L ACTH (n = 5 adrenal glands per group). *Significantly different from AL (Tukey-Kramer test, P < 0.05). Experiment 2 shows corticosterone measurements for AM and PM samples with 0, 11, 22 and 33 nmol/L ACTH (n = 7 adrenal glands per group). Although there was a main effect of group (P < 0.001), there was no significant difference between AL and FR rats at each dose of ACTH.
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Fig 2.
In vivo adrenal responsiveness to superphysiologic levels of ACTH in 3- to 4-mo-old male Fischer 344 rats with free access to diet (AL) or food restricted (FR). Values are means ± SEM (n = 3 rats per group). Graphs show ACTH and corticosterone measurements with 1.1 nmol (upper panel), 2.2 nmol (middle panel), and 3.3 nmol (lower panel) ACTH/kg body weight injections. There was a significant ACTH dose effect (P < 0.001) on plasma ACTH, but effects in FR and AL rats did not differ. There was a main effect of group (P < 0.001) on plasma corticosterone, but there were no significant differences between AL and FR rats at any given time.
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Fig 3.
In vivo adrenal responsiveness to physiologic levels of ACTH in 3- to 4-mo-old male Fischer 344 rats with free access to diet (AL) or food restricted (FR). Values are means ± SEM. Graphs show ACTH and corticosterone measurements with AM and PM samples for vehicle (upper panel, n = 4 rats per group), AM samples for 0.27 nmol (middle panel, n = 5 rats per group), and PM samples for 0.27 nmol (lower panel, n = 5 rats per group) ACTH/kg body weight injections. Plasma ACTH did not differ in AL and FR rats. Although there was a main effect of group (P < 0.05) on plasma corticosterone, there were no significant differences between AL and FR rats at each bleeding time.
11 mol/L and 3.28 × 1011 mol/L, significantly greater than baseline values (t test, P < 0.05) but much less than those after the higher doses of ACTH used in Experiment 3. The plasma levels of ACTH in AL and FR rats did not differ, and there was a robust elevation of plasma corticosterone after injection of ACTH (ACTH dose main effect, P < 0.001). Moreover, the elevation of corticosterone in FR rats was greater than that in AL rats (diet main effect, P < 0.05). This elevation in corticosterone did not differ between AM and PM samples in either diet group.
View larger version (25K):
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Fig 4.
Adrenocorticotropic hormone receptor (ACTH-R) mRNA measurements in 3-mo-old male Fischer 344 rats with free access to diet (AL) or food restricted (FR). Values are means ± SEM (n = 29 rats per group for panel A, 16 rats for group AL and 21 rats for group FR for panel B, and 27 rats for group AL and 28 rats for group FR for panel C. The numbers vary because some of samples measurements were not available.) *Significantly different from AL (ANOVA, P < 0.05).
View this table:
Table 1.
Adrenal weight, body weight and their ratio in 3-mo-old male Fischer 344 rats with free access to diet (AL) or
food restricted (FR)1
DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References
, Stewart et al. 1988). We have observed that the difference of total corticosterone levels between AL and FR rats at diurnal peak is higher after 1.5-2.5 mo of FR than after a longer period of FR. At older ages, the difference between AL and FR rats in the level of total corticosterone diminishes, although FR rats maintain the higher plasma levels of free corticosterone, which is the biologically active form. This is associated with, and probably the consequence of reduced plasma concentration of corticosterone-binding globulin in FR compared with AL rats. Therefore, we used 1.5- to 2.5-mo FR periods in these experiments.
). Previously, we investigated whether FR alters ACTH biosynthesis and secretion in 3-mo-old AL and FR rats, having hypothesized that elevated ACTH was the cause of the hyperadrenocortical state of FR (Han et al. 1995). Surprisingly, anterior pituitary and plasma ACTH levels were lower in FR than in AL rats, suggesting that factors other than elevated ACTH may account for FR-induced hyperadrenocorticism.
reported corticosterone responsiveness to exogenous ACTH was found to be ~2.5 times greater in the evening (at lights off) than in the morning (at lights on) in rats. More recently, Dijkstra et al. (1996) reported that there was no AM/PM difference in corticosterone responses when ACTH was given in vehicle of pH 4.3-7, but that they could replicate the AM/PM difference using the strongly acidic vehicle (pH 1-1.9) used by Dallman and colleagues (1978). We used the less acidic vehicle of Dijkstra et al. (1996) and confirmed the absence of AM/PM differences in AL rats. This lack of an AM/PM difference with the less acidic vehicle was also observed in FR rats. Our results thus confirm and extend the recent finding that in vivo adrenal responsiveness to ACTH does not appear to vary diurnally.
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FOOTNOTES |
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Manuscript received 14 November 1997. Initial reviews completed 17 January 1998. Revision accepted 6 May 1998.
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ACKNOWLEDGMENTS |
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We thank Roger Cone for providing the cDNA probe necessary for our experiment; 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; M. S. Shuko Lee for help on statistical analyses of the data; and Kim Kennedy and Nancy Markum for preparation of the manuscript.
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LITERATURE CITED |
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Abstract
Introduction
Methods
Results
Discussion
References
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