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Department of Physiology and Program in Neuroscience, 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.
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Introduction |
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Because ingestion of sucrose by ADX rats appears to take the place of B under basal conditions (3, 7), this suggests that the steroid may act to restore ADX rats to normal through its systemic effects on metabolism. However, it is also possible that B acts directly at the brain through a pathway that parallels or intersects with the one(s) acted on by the metabolic effects of sucrose. One study suggests that this is not so. The efficacy of systemic and intracerebroventricular (icv) B replacement was tested on metabolism in ADX rats, and the researchers found that although systemic B was restorative, icv B caused weight loss (8). Thus, there is some direct evidence that B acts systemically, not centrally, to restore metabolic deficits in ADX rats.
However, that finding does not explain where B acts to restore CRF and ACTH to normal in ADX rats. We and others have generally assumed that corticosteroids act on a brain site to restore ADX mammals to the sham-ADX basal state (4, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). However, direct evidence for this assumption is not strong. An effect of B that occurs several days after implantation of the steroid in brain (21) may be indirect and a function of steroid-induced changes in energy balance.
We asked here whether B acts directly on a pathway in brain that is parallel or identical to that excited by the effects of sucrose drinking that restores ADX rats to normal. We measured feeding and metabolic and hormonal variables, and assessed brain glucocorticoid receptor (GR) and neuropeptide responses to icv B or icv saline in ADX rats drinking sucrose. The results clearly show that although B infusion does occupy GR in the brain, it blocks the normal restorative effects of sucrose on basal metabolism and activity in the hypothalamo-pituitary-adrenal axis. However, there are also distinct effects of icv B infusion on metabolism and changes in ACTH induced by repeated restraint. The results suggest that constant GR occupancy in the brain results in a state change that is appropriate for responding to constant stress.
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Materials and Methods |
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110 ng B hemisuccinate/24 h). Immediately after the intracranial surgery, all rats were bilaterally ADX by the dorsal approach. After surgery and for the next 2 d, all rats were given 25 µg/ml B in 0.2% ethanol-0.5% NaCl to drink. This was to ensure that all rats survived the major combined surgeries of icv cannula implantation and subsequent adrenalectomy. Plasma B concentrations were less than 1 µg/dl in all of the ADX rats included in the study and did not differ among the groups (not shown).
Experiment 1: basal
In experiment 1, B-NaCl drinking fluid was removed on d 0, the onset of the experiment; all rats were provided with 0.5% saline, and half of the rats were provided with an extra bottle of 30% sucrose (wt/vol) to drink. During the next 4 d, body weight and saline, sucrose, and food intake were measured daily. Caloric intake was determined as the sum of chow intake (3.31 kcal/g) and sucrose intake (1.33 kcal/ml solution). Rats (six or seven rats per group) were decapitated under basal conditions within 4 h of lights on. Thymus glands and white adipose tissue depots were collected and weighed. Five milliliters of trunk blood were collected on 0.1 ml 0.3 N EDTA in chilled plastic tubes for subsequent measurement of metabolic (free fatty acids, triglycerides, and glucose) and hormonal (ACTH, B, insulin, and leptin) variables in plasma. Plasma concentrations of metabolites and hormones were determined in duplicate by colorimetric assays and RIAs as reported previously (3).
Experiment 2: stress
In experiment 2, rats (six or seven rats per group) surgically prepared in the same way as those in experiment 1 were given 25 µg/ml B in 0.2% ethanol-0.5% NaCl to drink for 2 d after surgery. This bottle was removed on d 0, when all rats were given sucrose as well as 0.5% saline to drink throughout the experiment. On d 0, 1, and 2, body weight was measured, and all rats were tube-restrained for 3 h (0800–1100 h). Blood was collected daily from a scalpel nick into a tail vein just before (0 min), and blood was also collected after 90 and 180 min of restraint as previously described (22). Separated plasma was frozen in aliquots for subsequent analysis of ACTH. Saline, sucrose, and food intakes were also measured daily. Caloric intake was determined as described for experiment 1. On d 2 (final episode of restraint), rats were decapitated at 180 min for collection of trunk blood and brains. Upon removal, brains were immediately placed into ice-cold 4% paraformaldehyde for 2 wk and then were transferred into a fresh 4% paraformaldehyde/30% sucrose solution for 2 additional d. Three brains from each group were sectioned at 30 µm on a sliding microtome, and sections were stored at 4 C in microwell plates filled with Tris-buffered saline (TBS; pH 7.5) until immunocytochemical analysis. Thymus glands and white adipose tissue depots were collected and weighed.
Immunocytochemical analysis
The analyses were conducted to determine whether our B infusion procedure activated GR and to semiquantify any effects of icv B on CRF expression in the paraventricular nucleus of the hypothalamus (PVN) and median eminence (ME). Expression of these proteins in the PVN and ME is important to understanding the regulation of activity in the HPA axis, and both are responsive to glucocorticoids. For examination of CRF and GR expression, adjacent sections from a multiple series were transferred to 24-well plates, rinsed 3 times each for 20 min at room temperature in TBS, and then rinsed in 10%methanol/3%H2O2 for 20 min. Subsequently, sections were rinsed 3 times in TBS and then exposed overnight (4 C) to a primary antibody specific for either CRF (r70, rabbit antirat, 1:4000; gift from W. Vale, The Salk Institute, La Jolla, CA) or GR (rabbit antirat, 1:2000; Affinity BioReagents, Inc., Golden, CO). The primary antibody was diluted in TBS/0.5% Triton X-100 (vol/vol)/0.2% gelatin (wt/vol). After incubation with the primary antibody, brain sections were rinsed again 3 times for 20 min each time at room temperature in TBS and subsequently incubated for 60 min at room temperature with a biotinylated secondary IgG antibody (Vector Laboratories, Inc., Burlingame, CA) diluted 1:400 in TBS/0.5% Triton X-100/0.2% gelatin (goat antirabbit). After this reaction, sections were rinsed as described above and then incubated for 1 h with avidin-biotin-peroxidase Elite complex (ABC Elite, Vector Laboratories, Inc.) at room temperature, followed by three 20-min rinses in TBS at room temperature. To visualize CRF or GR immunoreactivity, we used a 0.025% 3,3'-diaminobenzidine/0.01% H2O2 incubation solution that yields a red reaction product (5- to 7-min reaction). Sections were washed in TBS, mounted, and coverslipped using an aqueous medium at room temperature. Images of each of multiple brain regions were captured using a digital camera (Leica Corp., Rockleigh, NJ) and Spot 3.3 software system (Diagnostic Instruments, Inc., Sterling Heights, MI), and images were analyzed at a common anatomical level across all animals. GR activation was assumed by qualitative visualization of nuclear localization of GR immunoreactivity. Relative CRF and immunoreactivity were determined by analyzing the specific staining intensity by threshold subtraction followed by densitometry, using ImageJ 1.26t (W. Rasband, NIH, Bethesda, MD).
Statistical analysis
Data were analyzed by two-way ANOVA and ANOVA, adjusted for repeated measures [icv, drink (experiment 1), and time]. Post hoc tests determined individual points of significance. Two-tailed P 
0.05 was taken as significant; P 0.10 was considered to be a significant trend.
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Results |
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).). ADX rats drinking sucrose and saline with icv saline infusions ate less chow than the saline-drinking rats with icv saline infusions (Fig. 1B), but ingested more total calories and gained more body weight than any other group (Fig. 1, C and D). Chow intake, caloric intake, and body weight gain were not affected by the icv infusion in ADX rats drinking only saline (Fig. 1, B–D). In marked contrast, ADX rats infused with icv B and drinking sucrose decreased their chow intake below any other group (Fig. 1B) and therefore ingested fewer total calories and gained less body weight than the icv saline-infused rats drinking sucrose (Fig. 1, C and D). Thus, B infused into brain did not have effects similar to drinking sucrose on energy balance in ADX rats. In addition, icv B infusion blocked the metabolic efficacy of sucrose drinking. The total body weight gain over the 4 d of the experiment was significantly greater in the icv saline-infused, sucrose-drinking group than in any other group (Fig. 1E). This is primarily because of the increased caloric intake in this group. However, in addition to decreased food intake, icv B inhibited the sucrose-induced increase in caloric efficiency [caloric efficiency (weight gained/Cal ingested): icv saline, saline, 4.3 ± 0.86; icv saline, sucrose, 7.10 ± 1.06; icv B, saline, 3.29 ± 2.26; icv B, sucrose, 0.34 ± 2.09; P 0.05]. Mesenteric white adipose tissue weights were decreased by icv B and were increased by sucrose drinking, with no interaction between these variables (Fig. 1F).
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). 0.05), but there was no significant effect of icv infusion or interaction. Plasma glucose concentrations were not affected by either icv infusion or sucrose drinking. However, triglyceride concentrations were increased and nonesterified fatty acids were reduced in sucrose-drinking ADX rats compared with those drinking only saline. Intracerebroventricular B infusion tended (P 0.10) to reduce triglyceride concentrations.
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). 0.05) increased by icv B and tended (P 0.10) to be decreased by sucrose drinking (Fig. 2).
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). 0.05). This decrease in caloric intake was due to the inhibitory effects of icv B on sucrose intake (P 0.05) on both days and of chow intake on the first day of restraint (icv x day, P 0.05). With repeated restraint, icv B depressed central mesenteric fat depot weight (P 0.05), and although body weight gain was not significantly different between the two groups, icv B tended (P 0.10) to exacerbate total body weight loss per unit calorie ingested (caloric efficiency) over the 2 d of repeated restraint (icv saline, -0.50 ± 0.90; icv B, -3.52 ± 1.45).
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7).). On d 0, when B in the drinking saline had just been removed, both groups of rats responded similarly to restraint (time, P 0.05; no interaction). By contrast, on d 1, although initial ACTH concentrations were higher in both groups than on d 0, the icv B-infused rats had no elevation of ACTH during restraint, whereas the icv saline-infused rats did respond to the stressor. Moreover, at 90 min of restraint on d 2, there was a significant icv infusion x day interaction (P 0.05); further inspection demonstrated that icv B significantly (P 0.05) enhanced stress-induced increases in plasma ACTH at 90 min of restraint only on d 2 (Fig. 4).
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0.10; Fig. 5
). CRF immunoreactivity in the ME was not different between the two groups; however, the staining was heavy in both, and further antibody dilution might have shown a difference between the groups (Fig. 5).
B infusion into a lateral ventricle did activate GR in broad areas of brain, as shown by the nuclear localization of GR immunoreactivity in cells of the hippocampus (Fig. 6), PVN (Fig. 7), and perirhinal cortex (Fig. 8), suggesting that the steroid reached GRs in the brain widely. Saline-infused animals primarily exhibited GR-positive staining in the cell cytosol (compare left and right panels in Figs. 68).
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Discussion |
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However, approximately 100 ng/d B, icv, blocked the positive metabolic effects of sucrose under basal and stress conditions and also affected CRF and ACTH responses to repeated stress. Thus, B acts directly on the brain to modulate activity in the HPA axis and energy storage when brain GR are occupied. This action of B may be due to its activation of central CRF neurons, which are known to reduce food intake, increase sympathetic nervous system output, and consequently raise the metabolic rate and result in a general catabolic state (23). Together, our results strongly suggest separate glucocorticoid feedback axes that are concentration dependent (see Fig. 9).
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GR occupancy in the brain that was induced by the constant infusion of B probably mediated at least three of the biological effects we observed. 1) Facilitated ACTH secretion in response to repeated restraint. Facilitation requires that glucocorticoid concentrations be in the stress range (27, 28). 2) Stimulation of basal ACTH concentrations. This may have resulted from an action of B on CRF synthesis in the central amygdala (CeA) (29), which has been shown to occur when systemic B concentrations are in the stress range (30), and after chronic stress (31). Similarly, B implants that activate GRs in the CeA reduce mesenteric white adipose tissue weight specifically in conjunction with chronic stress (32). 3) Finally, the action of icv B on the reduction of chow intake in rats drinking sucrose has been shown to occur when plasma B concentrations are in the stress range, but not in the normal range (1, 33). This suggests that brain GR occupancy is required for this inhibitory effect of B to occur.
Figure 9 schematizes our results and provides a model for the following discussion. Intact rats are shown under basal and chronic stress conditions at the top of Fig. 9; ADX rats given sucrose to drink with either icv saline or icv B are shown at the bottom. The essence of the schemes is that there are two afferent feedback signals to brain: one (to date unidentified) (7) transmits information about the metabolic state, and the other (B) transmits information about the state of adrenal activity. There are two efferent limbs, both driven by CRF. One drives premotor sympathetic neurons that act to increase metabolic rate and mobilize substrate from muscle and fat. The other drives B secretion that inhibits muscle and fat accretion and stimulates hepatic gluconeogenesis. Under basal, well nourished conditions (or after ADX, sucrose, or icv saline), there is moderate feedback from the metabolic limb of the afferent system and little or no feedback from the B limb. With chronic stress (or after ADX, sucrose, or icv B) the strengths of the two feedback signals reverse, and a major effect of this is to stimulate CRF synthesis and secretion from the amygdala and PVN, resulting in strong stimulation of sympathetic outflow.
It has been generally assumed that glucocorticoids act directly on the brain to modulate energy balance and activity in the HPA axis under basal conditions in ADX rats. Various brain lesions have been shown to alter basal activity in the HPA axis (34, 35, 36, 37), and the results have been interpreted as interference by the lesions with a B feedback site. However, finding a specific site in brain at which B inhibits basal HPA activity has been generally unsuccessful (21, 38, 39, 40, 41, 42). This is not true for corticosteroid feedback on HPA activity under stressful conditions. Implants of B in amygdala, preoptic area, and prefrontal cortex have been shown to inhibit stress-induced ACTH secretion (32, 42, 43, 44).
Under basal conditions, high circulating B stimulates insulin secretion and relative fat deposition in ADX rats (2, 45, 46). Systemic glucocorticoids are also required for normal feeding behavior, and high doses can stimulate feeding (1, 2, 45–47). Intracerebroventicular infusion of dexamethasone, a potent synthetic glucocorticoid, in non-ADX rats stimulates feeding and body weight gain, but in the PVN alone it does not stimulate the normal carbohydrate feeding response in non-ADX rats that it does in ADX rats (48, 49). When corticosteroids were infused icv into basal ADX rats, there were, if anything, negative effects on feeding (8). However, these effects are state dependent. For example, under stress, when circulating insulin concentrations are likely to be depressed, body weight and fat depot loss occur (6). Again, the site and mechanism through which glucocorticoids act to modulate feeding behavior and fat deposition are unknown.
There is considerable evidence that the effects of B on metabolism and ACTH secretion are state dependent. Systemic replacement of basal ADX rats with stress concentrations of circulating B inhibits body weight gain, although it causes obesity (1, 2, 50). Replacement with stress levels of circulating B in ADX rats inhibits basal activity in the HPA axis (50, 51), but in previously stressed rats, high B allows facilitated ACTH responses to novel stress (27, 28). Furthermore, high systemic replacement concentrations of B also increase central fat deposition in unstressed, but not diabetic, stressed rats (47, 50). B implants over the CeA in combination with clamped low systemic mean daily concentrations of B depress fat depots, but only when stress is applied simultaneously (32). The latter is clearly a GR-mediated effect exerted in brain and was observed in this study as well. However, high concentrations of steroid also act in the periphery to modulate anabolic and catabolic processes. Glucocorticoids increase hepatic gluconeogenesis, gathering substrate from peripheral muscle and fat stores (52).
ADX reduces CRF in the CeA, and local implants (29), high systemic replacement (30), and stress (31) increase CRF in the CeA. CRF from the CeA is secreted in response to both stress and feeding, and this neuropeptide may mediate many of the feeding, autonomic, and metabolic responses to stress (53, 54). Therefore, exposure of the brain to a GR-occupying concentrations of B in the absence of high peripheral B alters neural systems (e.g. CRF) that together inhibit some energy-related functions, such as caloric intake and fat deposition. The state of the organism (e.g. stress, refeeding, or basal) is likely to dictate the net effect of B in the brain on the physiology of the animal.
In summary, it appears that to achieve feedback on energy balance and in the HPA axis under basal conditions, the peripheral actions of B are required. When B is elevated to chronically occupy GR in the brain, without systemic elevations in B, it provides the central nervous system with a stress signal that overrides the systemic effects of sucrose on metabolism through activating neural systems that suppress energy balance and turn on the HPA axis. We have previously shown that pathways in the brain, including the paraventricular thalamus and basomedial, basolateral, and central nuclei of the amygdala, are specifically sensitive to acute stress superimposed on chronic stress (55, 56, 57, 58, 59). When the results of the present studies are added to the above, they reinforce the idea that there is a central chronic stress network. Once triggered by GR occupancy, this network (perhaps mediated by increased CRF synthesis and secretion) results in decreased feeding, increased sympathetic outflow, facilitated ACTH responses, and altered behaviors characteristic of chronically stressed individuals (60, 61).
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Acknowledgments |
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Footnotes |
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This work was supported by Grant DK-59735 (to K.D.L.) and in part by Grant DK-28172 (to F.G.).
Abbreviations: ADX, Adrenalectomized; B, corticosterone; CeA, central nucleus of amygdala; CRF, corticotropin-releasing factor; CSF, cerebrospinal fluid; GR, glucocorticoid receptor; icv, intracerebroventricularly; ME, median eminence; MR, mineralocorticoid receptor; PVN, paraventricular nucleus of the hypothalamus; TBS, Tris-buffered saline.
Received June 12, 2002.
Accepted for publication August 7, 2002.
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and 
, ADX rats given sucrose as well as saline to drink; 
and 
, ADX rats given only saline to drink; 
, significant (P 
); E, relative mesenteric white adipose tissue (mWAT) weight. 
