Mapping brain c-Fos immunoreactivity after insulin-induced voluntary lard intake: insulin- and lard-associated patterns.

In addition to the inhibitory role of central insulin on food intake, insulin also acts to promote lard intake. We investigated the neural pathways involved in this facet of insulin action. Insulin or saline was infused into either the superior mesenteric or right external jugular veins of streptozotocin-diabetic rodents with elevated steady-state circulating corticosterone concentrations. After postsurgical recovery, rats were offered the choice of chow or lard to eat. Irrespective of the site of venous infusion, insulin increased lard and decreased chow intake. After 4 days, lard was removed for 8 h. On return for 1 h, only insulin infused into the superior mesenteric vein resulted in lard intake. This facilitated distinction between the effects of circulating insulin concentrations (similar in the two insulin-infused groups) and lard ingestion on the patterns of c-Fos(+) cells in the brain, termed insulin- and lard-associated patterns, respectively. Insulin-associated changes in c-Fos(+) cell numbers were evident in the arcuate nucleus, bed nucleus of the stria terminalis and substantia nigra pars compacta, concomitant with elevated leptin levels and reduced chow intake. Lard-associated changes in c-Fos(+) cell numbers were observed in the nucleus of the tractus solitarius, lateral parabrachial nucleus, central nucleus of the amygdala, ventral tegmental area, nucleus accumbens shell and the prefrontal cortex, and were associated with lower levels of triglycerides and free fatty acids. The anterior paraventricular thalamic nucleus exhibited both patterns. These data collectively fit into a framework for food intake and reward and provide targets for pharmacological manipulation to influence the choice of food intake.

Hypothalamic-pituitary-adrenal dysfunction in Apoe(-/-) mice: possible role in behavioral and metabolic alterations.

Several neurological diseases are frequently accompanied by dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis regulates the secretion of glucocorticoids (GCs), which play important roles in diverse brain functions, including cognition, emotion, and feeding. Under physiological conditions, GCs are adaptive and beneficial; however, prolonged elevations in GC levels may contribute to neurodegeneration and brain dysfunction. In the current study, we demonstrate that apolipoprotein E (apoE) deficiency results in age-dependent dysregulation of the HPA axis through a mechanism affecting primarily the adrenal gland. Apoe(-/-) mice, which develop neurodegenerative alterations as they age, had an age-dependent increase in basal adrenal corticosterone content and abnormally increased plasma corticosterone levels after restraint stress, whereas their plasma and pituitary adrenocorticotropin levels were either unchanged or lower than those in controls. HPA axis dysregulation was associated with behavioral and metabolic alterations. When anxiety levels were assessed in the elevated plus maze, Apoe(-/-) mice showed more anxiety than wild-type controls. Apoe(-/-) mice also showed reduced activity in the open field. Finally, Apoe(-/-) mice showed age-dependent increases in food and water intake, stomach and body weights, and decreases in brown and white adipose tissues. These results support a key role for apoE in the tonic inhibition of steroidogenesis and HPA axis activity and have important implications for the behavioral analysis of Apoe(-/-) mice.

Chronic cold in adrenalectomized, corticosterone (B)-treated rats: facilitated corticotropin responses to acute restraint emerge as B increases.

Small elevations in corticosterone (B) administered exogenously exert potent inhibitory effects on both basal and stress-induced ACTH secretion. However, under conditions of chronic stress with chronic elevations in B, the hypothalamo-pituitary-adrenal system appears to balance the negative feedback signal of B with central neural facilitation so that the system remains fully responsive to acute stressors. In these studies, we tested whether: 1) circulating B concentrations affect responses to acute restraint in rats exposed to 5 days at 5-7 C (cold), compared with room temperature (control); and 2) facilitated ACTH secretion can be explained by increased CRF or vasopressin messenger RNA (mRNA) levels in the hypothalamic parvocellular paraventricular nuclei (PVN). Rats were adrenalectomized and supplied with B in doses that fixed plasma B at constant levels between approximately 2 and 20 microg/dl; rats were placed in cold or remained as controls. Increasing concentrations of fixed B decreased basal ACTH similarly in both groups. By contrast, as B levels increased, ACTH responses to restraint also increased in cold vs. control rats. Semiquantitative analysis of CRF mRNA by in situ hybridization revealed decreases of similar magnitude in both groups with increasing fixed B. Vasopressin mRNA levels also decreased with increasing fixed B in both groups, but with slightly less sensitivity to inhibition by B in cold exposed rats. Taken together, the decreases in mRNA for these major ACTH neuropeptide secretogogues in the parvocellular PVN are unlikely to explain facilitated ACTH responses in chronically stressed rats. We conclude that a brain site is stimulated by B that is proximal to the PVN; feedforward, positive effects of B are thus implicated in mediation of prior stress-induced facilitation of acute hypothalamo-pituitary-adrenal responses to stress.

Relationships among adrenal weight, corticosterone, and stimulated adrenocorticotropin levels in rats.

Based on results of previous studies, we tested the hypothesis that increased adrenal weight in the absence of increased corticosterone secretion would inhibit the magnitude of the ACTH response to ether vapor. Adrenal hypertrophy was induced by treatment of rats for 3 days with aminoglutethimide, cyanoketone, or metyrapone, three agents that act to inhibit enzymes required for corticosterone synthesis. On the fourth day, resting plasma ACTH and corticosterone levels were normal; however, the logarithm of the ACTH response to ether was directly related to total adrenal weight. This result did not support the hypothesis. Unilateral adrenalectomy and treatment with cyanoketone resulted in total adrenal weight equivalent to that of control rats bearing two adrenals. Resting ACTH levels were normal, but stimulated ACTH was significantly greater in these rats than in controls or in rats with two adrenals, suggesting that there is an interaction between the effects of unilateral adrenalectomy and adrenal enzyme inhibition. As anticipated, adrenal hypertrophy with increased corticosterone production caused by ACTH infusion resulted in a significant negative relationship between resting and stimulated ACTH levels and adrenal weight. We conclude that when adrenals are enlarged by means that prevent excessive corticosterone secretion, there is a mechanism associated with the increase in adrenal weight that correlates directly with the magnitude of stimulated ACTH secretion. We have reexamined the results of our previous results in the light of these experiments.

Drug-induced adrenal hypertrophy provides evidence for reset in the adrenocortical system.

These studies were performed to determine how bilateral adrenal hypertrophy persists despite normal resting ACTH levels in male rats for weeks after a 3-day course of treatment with cyanoketone (CK). This drug blocks corticosterone synthesis by binding to the enzyme 3 beta-ol-dehydrogenase-delta 5, delta 4-isomerase (EC 1.1.1.51). Although plasma ACTH levels were significantly elevated at all times during the course of treatment with CK, ACTH and corticosterone levels of CK-treated rats were normal thereafter compared to those in control rats killed in the morning. Despite these normal ACTH levels, adrenal weight remained elevated (by 100%) for at least 14 days. We determined that increased ACTH secretion was required to initiate and maintain the adrenal growth response to CK. Rats pretreated with hypophysectomy or dexamethasone (1 mg/kg) did not respond with increases in adrenal weight after CK treatment. Sustained elevations in ACTH during CK treatment were required for increased adrenal weight, because rats treated with dexamethasone 3 h after CK injection did not have enlarged adrenals at 24 h. Increased adrenal weight was not sustained after removal of supranormal levels of ACTH achieved by infusion, suggesting that after adrenal hypertrophy is achieved by ACTH, elevated ACTH levels are also required to maintain adrenal enlargement. Finally, ACTH and corticosterone levels were measured in the evening (at the peak of the diurnal rhythm in the adrenocortical system of rats) in CK-treated rats. Evening ACTH levels were significantly elevated in CK-treated rats compared to those in controls 7, 10, and 14 days after the onset of 3 days of treatment with CK; however, corticosterone levels were normal. The effects of CK on the adrenocortical system were exerted via the adrenal, since adrenalectomy normalized the amplitude of the diurnal rhythm of ACTH in CK-treated rats compared to that in adrenalectomized controls. We conclude 1) that adrenal hypertrophy after CK is maintained by increased ACTH secretion which occurs daily in the evening; and 2) that the results provide evidence for a daily reset of the corticosteroid feedback sensor in the adrenocortical system. This conclusion arises from the findings that there is a normal rhythm in corticosterone levels in CK-treated rats and that morning ACTH levels are normal although evening ACTH levels are significantly elevated.

Roles of type I and II corticosteroid receptors in regulation of basal activity in the hypothalamo-pituitary-adrenal axis during the diurnal trough and the peak: evidence for a nonadditive effect of combined receptor occupation.

Negative feedback regulation of basal activity in the hypothalamo-pituitary-adrenal (HPA) axis requires less corticosterone (B) at the trough (morning) than at the peak (evening) of the diurnal rhythm. It has been hypothesized that in the morning in rats, occupation of the high affinity, type I corticosteroid receptors is sufficient to inhibit adrenalectomy (ADX)-induced increases in plasma ACTH secretion, whereas in the evening, regulation occurs through the occupation of the lower affinity type II corticosteroid receptors. To examine this hypothesis, the sensitivity of ACTH to inhibition by two different doses of B or of dexamethasone (DEX) were compared in ADX rats killed in the morning or the evening (B has a higher affinity for type I receptors in vitro and in vivo; in vivo, DEX has a higher affinity for type II receptors). The requirement for greater concentrations of corticosteroids to inhibit ACTH secretion in the evening was verified. The effect of these treatments on the number of neurons immunoreactive for vasopressin (AVP) and on the expression of AVP messenger RNA (mRNA) in the parvocellular portion of the paraventricular nuclei was also examined. In the morning, plasma concentrations of B equivalent to the IC50 for the reduction of plasma ACTH in the morning reduced the amount of AVP mRNA, but not immunoreactive AVP cell number as compared with ADX rats. DEX reduced plasma ACTH in the morning but did not prevent high levels of expression of AVP mRNA or protein. AVP mRNA was more sensitive to B in the morning than in the evening. Antagonist to the type I receptor (spironolactone) given chronically to ADX rats treated with B increased plasma ACTH secretion at both times of day, even though the plasma B concentrations suggested occupancy of a large proportion of the type II receptors. To test the hypothesis that an interaction between the type I and II receptor is necessary for the control of HPA activity at the peak of the diurnal rhythm, ADX rats were given B or DEX, alone or in combination. DEX reduced evening plasma ACTH only in the presence of very low concentrations of B, suggesting that for full potency, type II receptor occupation requires type I receptor occupation. In summary, these results demonstrate that occupation of type I corticosteroid receptors is capable of controlling basal activity in the HPA axis in the morning and that in the evening, type I receptor occupation potentiates the inhibition of plasma ACTH by occupation of type II receptors.

Regulation of activity in the hypothalamo-pituitary-adrenal axis is integral to a larger hypothalamic system that determines caloric flow.

We have previously reported that there are diurnal rhythms in the magnitude of ACTH responses to stressors and in the sensitivity of stress-induced ACTH responses to facilitation induced by prior stress and to corticosterone (B) feedback induced by exogenous B. In all cases ACTH was more responsive in the morning than in the evening in nocturnally feeding rats. We have also shown in adrenalectomized rats that an overnight fast reduces ACTH responses to restraint in the morning compared with rats fed ad libitum, and we have shown that calorie-containing gavage during the fast increases the amplitude of ACTH responses to restraint in fasted rats. Therefore, this diurnal rhythm is not associated with B feedback and is associated with calories. In these studies we asked whether young, male intact rats that were deprived of food overnight had: 1) hypothalamo-pituitary-adrenal (HPA) axis responses during the fasting period; 2) altered basal activity in the HPA axis; 3) altered responsivity of ACTH to restraint; and 4) altered sensitivity of restraint-induced ACTH responses to facilitation or B feedback. Our results show that food deprivation: 1) induces marked ACTH and B responses during the fast that mirrors the pattern of food intake in fed rats, with an approximately 3-h lag; 2) results in essentially no change in basal ACTH in the morning; 3) reduces ACTH responsivity to stress in the morning; and 4) reduces ACTH responsivity to prior stress-induced facilitation and exogenous B-induced feedback. We conclude that: 1) the HPA axis serves as a default pathway to feeding when food is not available; 2) the diurnal rhythms in restraint-induced ACTH secretion are determined by food intake; and 3) the HPA axis is integral to a larger hypothalamic system that mediates energy flow.

Reset of feedback in the adrenocortical system: an apparent shift in sensitivity of adrenocorticotropin to inhibition by corticosterone between morning and evening.

There is evidence in man and rats that higher circulating levels of glucocorticoids are required to normalize basal unstimulated ACTH levels at the peak of the circadian rhythm than at the trough. To explore this phenomenon, we tested the inhibitory effect of constant levels of corticosterone on plasma ACTH in the morning (AM) and evening (PM) in young male rats implanted with fused pellets of corticosterone-cholesterol at the time of adrenalectomy (ADX+B) and studied 5 days later. There was a marked shift of the plasma corticosterone-ACTH inhibition curve to the right between AM and PM, demonstrating that the efficacy of corticosterone feedback inhibition of ACTH is less in the PM. Comparison of plasma ACTH and corticosterone levels during 24 h in sham-adrenalectomized rats (SHAM-ADX), adrenalectomized rats (ADX), and ADX+B revealed constantly low ACTH in SHAM-ADX, constantly high ACTH in ADX, and biphasic ACTH levels in ADX+B. Corticosterone levels were biphasic in SHAM-ADX and were constant in the other two groups. These results again showed a shift in corticosterone feedback efficacy as a function of the time of day and also suggested that basal ACTH secretion is maintained in the low normal range in intact rats because of the marked diurnal rhythm in corticosterone. The sensitivity of the pituitary ACTH response to exogenous CRF did not change between AM and PM in either intact or ADX+B showing that the shift in feedback sensitivity to corticosterone does not reside in the pituitary. The response of the entire adrenocortical system to histamine stress was shown to be equivalent in both the AM and PM, suggesting that feedback sensitivity of the entire system to corticosterone does not change as a function of the time of day. We conclude from these results that there is an apparent diurnal change in ACTH sensitivity to corticosterone feedback that can be defined operationally as reset. We believe that the site of feedback being tested shifts solely from the pituitary in the AM (at the nadir of the rhythm) to the brain and the pituitary in the PM (at the peak of the rhythm). The lack of the normally high transients of corticosterone that occur in SHAM-ADX rats results in increased brain drive of the pituitary in ADX+B.

Pancreatic acinar cell amylase gene expression: selective effects of adrenalectomy and corticosterone replacement.

To examine the role of glucocorticoids in the regulation of the acinar pancreas, adult male rats were adrenalectomized (Adx) and replaced with no corticosterone (B), normal B, or high B. Plasma B concentration, body weight gain, and thymus weight were used as independent measures of treatment efficacy. Compared to controls, Adx animals had a 75 +/- 0.5% (n = 30) reduction in pancreatic amylase content; a 50% decrease occurred within 1 day and the maximal 75% decrease was observed after 5 days. In Adx animals, amylase content was normalized by normal B replacement and was increased to 235 +/- 39% (n = 30) of control by high B replacement. Furthermore, in all Adx rats, pancreatic content of amylase and plasma B concentration was significantly correlated (r = 0.81, n = 30). The effect of adrenalectomy was selective for amylase; contents of ribonuclease, chymotrypsin, and elastase were not altered. However, the effects of high B replacement were not selective, and increased the content of all digestive enzymes. To determine whether the changes in enzyme content were associated with changes in messenger RNA (mRNA), pancreatic RNA was probed with 32P-labeled complementary DNAs for amylase, ribonuclease, and chymotrypsin. After adrenalectomy and B replacement there was a significant correlation only between amylase mRNA (r = 0.87, n = 13) and plasma B concentration. These data indicate that physiological levels of B have a selective effect on pancreatic amylase gene expression. In contrast, high levels of B have the separate, nonselective effect of increasing the content of all digestive enzymes without increasing corresponding mRNA levels.

Pituitary-adrenocortical responses to persistent noxious stimuli in the awake rat: endogenous corticosterone does not reduce nociception in the formalin test.

Although glucocorticoids inhibit inflammation and are used to treat painful inflammatory rheumatic diseases, the contribution, if any, of endogenous pituitary-adrenocortical activity to the control of pain remains unclear. We report that injection of dilute formalin into the hindpaw not only evokes inflammation and pain-related behavior, but it also increases ACTH and corticosterone to a greater extent than restraint and saline injection alone. This difference was particularly robust during the final periods of pain-related behavior in the formalin test, when the ACTH and corticosterone (B) levels in the restraint/saline control group had returned to normal. These results indicate that formalin-evoked increases in ACTH and B reflect nociceptive input, rather than the stress associated with handling. To test the hypothesis that the formalin-induced increase in corticosterone reduces pain and inflammation, we next evaluated the effect of adrenalectomy (to prevent activation of glucocorticoid receptors) or high-dose dexamethasone (to saturate glucocorticoid receptors) on nociceptive processing in the formalin test. Neither adrenalectomy nor dexamethasone changed behavioral or cardiovascular nociceptive responses. Furthermore, the increases in blood pressure and heart rate produced by formalin may not be mediated by adrenomedullary catecholamine release. In addition, we conclude that the nociceptive component of the formalin stimulus is sufficient to activate the pituitary-adrenocortical system in the awake rat, but that the resulting release of corticosterone does not feed back and reduce nociceptive processing.

Constant corticosterone replacement normalizes basal adrenocorticotropin (ACTH) but permits sustained ACTH hypersecretion after stress in adrenalectomized rats.

To characterize further the effects of providing a constant corticosterone signal after bilateral adrenalectomy, we have compared the effects of bilateral adrenalectomy with no replacement (ADX) and with replacement with a corticosterone pellet implanted sc at surgery (B-PELLET) to those of sham-adrenalectomy (SHAM) on pituitary and plasma ACTH concentrations during the first 3 postoperative days. In ADX rats, plasma ACTH concentrations were elevated at all times compared to those in the SHAM group; pituitary ACTH content decreased during the first 12 h, then increased and was not different from that in the SHAM group thereafter. Replacement of corticosterone at the time of adrenal surgery in B-PELLET rats resulted in no differences in pituitary and plasma ACTH concentrations from SHAM values, suggesting that immediate steroid replacement prevents the major adrenalectomy-induced changes in central regulatory components governing basal activity of the adrenocortical system. Although B-PELLET rats had normal basal morning ACTH concentrations 5 days after surgery, they exhibited augmented and sustained ACTH responses to five different ACTH-releasing stimuli (injection, restraint, chlorpromazine, and, under pentobarbital anesthesia, morphine or sham adrenalectomy). The circulating corticosterone concentrations were maintained at relatively constant, low levels (3-6 micrograms/dl). Because these concentrations appear to restore basal morning ACTH concentrations to normal, but do not restore the ACTH response to stress to normal, we conclude that a different corticosterone signal is required to normalize stress-induced ACTH responses.

Circadian variations in plasma corticosterone permit normal termination of adrenocorticotropin responses to stress.

We previously reported that adrenalectomized rats given constant corticosterone via a sc pellet (B-PELLET) hypersecrete ACTH in response to stress. Although lacking a feedback signal, B-PELLET rats do not secrete ACTH indefinitely after stress; plasma ACTH levels in these animals returned to those in sham-operated (SHAM) rats within 1-4 h after 2-min restraint. To distinguish between the requirement for circadian or stress-induced increases in corticosterone, we compared changes in ACTH and corticosterone levels after stress in SHAM and B-PELLET rats with those in cyanoketone-treated rats (CK) and adrenalectomized rats given corticosterone in their drinking fluid (B-WATER). B-WATER rats exhibited sustained increases in plasma corticosterone after lights-off, correlating with the nocturnal feeding period. Morning plasma corticosterone levels in B-WATER rats were constant and even lower than those in B-PELLET rats; however, B-WATER rats did not differ from SHAM rats in their ACTH response to ip injection. CK rats, which have an approximately normal circadian corticosterone rhythm but do not have significant corticosterone responses to acute stimuli, also exhibited plasma ACTH levels similar to those of SHAM rats at all times after 5-min restraint. Compared with SHAM and B-WATER rats in the same experiment, B-PELLET rats tended to hypersecrete ACTH 60 min after 5 min of restraint, but only had significantly elevated plasma ACTH relative to both groups 45 min after 10 min of restraint. We conclude that circadian, rather than stress-induced, increases in corticosterone may be sufficient for normal termination of ACTH responses to stress.

Pharmacological evidence that the inhibition of diurnal adrenocorticotropin secretion by corticosteroids is mediated via type I corticosterone-preferring receptors.

These studies were performed to determine pharmacologically the corticosteroid receptor type that mediates the effects of corticosterone (B) on ACTH secretion in adrenalectomized rats. We have compared the effects of treating young male rats at the time of adrenalectomy and throughout the next 5 days with B, dexamethasone (DEX), or aldosterone (ALDO) in doses that elevated plasma levels to concentrations in the range between 0.2-30 nM. Plasma ACTH, corticosteroid-binding globulin (CBG), and thymus weight were measured in the morning or evening, and these steroid-sensitive end points were related to the circulating concentrations of B (total B - CBG-bound B), total DEX, and total ALDO. For the inhibition of ACTH the rank order of potency of the three steroids was B greater than DEX greater than or equal to ALDO in the morning (estimated IC50, 0.7 +/- 0.1, 2.3 +/- 0.5, and 4.9 +/- 1.6 nM for B, DEX, and ALDO, respectively). There was a significant shift to the right in steroid efficacy between morning and evening (estimated IC50 in the evening, 3.9 +/- 0.2 and 9.3 +/- 0.8 nM for B and DEX; ALDO at the concentrations achieved was ineffective). The rightward shift in efficacy may result from the circadian increase in drive to ACTH secretion. The rank order of potency for B and DEX on ACTH and the agreement between the steady state IC50 values achieved for these steroids and the Kd values determined for B and DEX with type I receptors in vitro strongly suggest that feedback control of basal diurnal ACTH by corticosteroids is mediated by association with type I, B-preferring receptors. By contrast, DEX was 3 times more potent than B on CBG (estimated IC50, 1.5 and 4.5 nM, respectively) and tended to be more effective on thymus weight, suggesting that the effects of corticosteroids on these peripheral targets are mediated by association of the steroids with type II glucocorticoid receptors. ALDO coinfused with DEX or B did not alter the inhibitory effects of these on ACTH, suggesting that ALDO does not interfere with these type I, B-preferring receptors in vivo. Because there is little if any evidence for type I corticosteroid receptors in the hypothalamus, these results strongly suggest that the majority of corticosteroid feedback inhibition of basal morning and evening ACTH secretion is mediated transynaptically by the activity of extra-hypothalamic neurons.

The adrenocortical system responds slowly to removal of corticosterone in the absence of concurrent stress.

After removal of corticosteroid feedback by surgical or pharmacological adrenalectomy, plasma ACTH increases more rapidly than can be explained by changes in receptor-mediated gene expression. In aminoglutethimide-treated rats, plasma ACTH increased only at doses much higher than those inhibiting plasma corticosterone, suggesting that adrenal enzyme blockers may themselves be stressful. To determine the adrenocortical system response to stressless corticosterone removal, adrenalectomized rats maintained for 5 days on corticosterone in the drinking water were switched to steroid-free fluid (-B) or again given steroid (+B); additional rats were adrenalectomized (ADX). Plasma ACTH did not differ between -B and +B rats until 18-24 h after steroid removal, regardless of whether steroid was withdrawn at the circadian maximum or minimum. Plasma ACTH was similar between -B and ADX rats 0.5-14 days after corticosterone removal, although morning plasma ACTH was more stable in -B rats at 4-7 days. Evening plasma ACTH increased significantly after day 3 in ADX and -B rats. Unlike ADX rats, -B rats did not exhibit pituitary ACTH depletion at 12 and 24 h, but both -B and ADX groups had significantly elevated pituitary ACTH by 6.5 days. We conclude that 1) rapid increases in ACTH secretion after surgical or pharmacological adrenalectomy result from interaction between stress and loss of corticosteroid feedback; 2) no immediate interaction occurs between loss of feedback and circadian stimuli; and 3) the effects of steroid withdrawal may require at least 3 days to be stably expressed.

Adrenalectomy in the neonate: adult-like adrenocortical system responses to both removal and replacement of corticosterone.

Neonatal rats exhibit a period of diminished responsiveness to stress between days 3-10 of life, which has been shown to be associated with an increased sensitivity to corticosterone (B) inhibitory feedback. In this study we further investigated B feedback potency on regulation of ACTH by examining 1) the time course of changes in pituitary ACTH secretion and content, plasma B and B-binding globulin (CBG) concentrations, and thymus weight after adrenalectomy (ADX) performed on 5-day-old pups, with or without sc 5% B pellet replacement, and 2) the time required for acute (B injection) and the B dose required for constant (B pellet) inhibition of ACTH secretion in 10-day-old ADX neonates. As in adult rats, ADX in neonates caused an immediate (3 h) large increase (13-fold) in plasma ACTH levels compared to that in sham-operated rats, followed by a decrease by 12 and 24 h after surgery and a further and sustained increase during the next 4 days. Pituitary ACTH stores were diminished in ADX rats by 3, 12, and 24 h and increased thereafter. Five percent B pellet replacement abolished ADX-induced changes in plasma and pituitary ACTH until days 4-5, when plasma ACTH was slowly released from B inhibition (circulating B values were similar to ADX values). By day 10 of life, inhibition of plasma ACTH by calculated free B showed an IC50 of 1.09 nM. Plasma CBG concentrations exhibited a clear developmental pattern in sham-operated rats, being lower on days 6-8 than earlier or later. Typical ADX-induced increases in CBG levels were observed from day 3 on after surgery, at the same time as a transient decrease in CBG levels occurred in ADX plus 5% B rats. On day 10 of age, inhibition of CBG by calculated free B demonstrated an IC50 of 1.5 nM. Although no enlargement of the thymus was observed after neonatal ADX, thymus weight was significantly diminished by 12 h after B replacement and in a dose-related manner at 5 days with B pellets containing 5-25% B. The thymus contained mostly type II glucocorticoid receptors, which did not up-regulate 3 h or 5 days after ADX. Acute sc injection of B (10-34 micrograms/g BW) in 10-day-old rats inhibited ADX-induced ACTH secretion within 30 min, and the estimated half-time for the inhibition was 40 min. By 2 h after B injection, plasma ACTH levels were comparable to those in sham-operated animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Plasma adrenocorticotropin is more sensitive than transcortin production or thymus weight to inhibition by corticosterone in rats.

After adrenalectomy, ACTH, corticosterone-binding globulin (CBG), and thymus wet weight increase in rats as a consequence of the removal of corticosterone (B) and decrease again in response to replacement with glucocorticoids. We have studied the effect of replacing adrenalectomized male rats with a variety of different concentrations of B. Plasma concentrations of ACTH and CBG and thymus wet weights were related to the measured concentration of free ultrafilterable B in plasma. In other experiments, the clearance of [125I]CBG was determined in adrenalectomized rats with and without B replacement, and the time required for the changes in plasma CBG concentrations after removal and/or replacement of B in adrenalectomized rats was determined. Five to 7 days after adrenalectomy and institution of a relatively constant B replacement signal, plasma CBG concentrations were highly correlated with circulating B concentrations (r2 = 0.745; P less than 0.001). The effect of B was on the CBG production rate, since clearance did not change. Because CBG concentrations decrease as total B concentrations increase, there is an amplification of free B concentrations with increasing total B. The relationships of plasma ACTH and CBG and thymus wet weight to circulating free B levels showed that 50% inhibition of ACTH was achieved at a free B concentration of 0.8 +/- 0.05 nM, whereas 50% inhibition of CBG and thymus wet weight were achieved at free B concentrations of 4.6 +/- 0.9 and 4.4 +/- 0.6 nM, respectively. These inhibition values correlate well with the known Kd values for the high affinity type I B receptor (0.5 nM) and for the lower affinity type II glucocorticoid receptor (2.5-5 nM), respectively, suggesting that ACTH secretion, CBG production, and thymus wet weight are regulated by the association of B with these receptor types.

Streptozotocin-diabetic rats exhibit facilitated adrenocorticotropin responses to acute stress, but normal sensitivity to feedback by corticosteroids.

A variety of chronic stress paradigms have been shown to increase basal activity in the hypothalamic-pituitary-adrenocortical axis, resulting in hypercorticoidism. Despite this, chronically stressed rats typically exhibit facilitated ACTH responses to acute novel stress, suggesting that the activity of some central neural component(s) in the axis is facilitated by chronic stress. We have used the chronic stress of streptozotocin (STZ)-induced diabetes in rats to determine diurnal sensitivity of basal and stimulated ACTH secretion to exogenous corticosterone (B) feedback in vivo. Control and STZ-diabetic rats were adrenalectomized or adrenalectomized and implanted with a 30% or 50% B pellet at the time of vehicle/STZ injection. Rats were killed 5 days later, under basal conditions or after 6 min of restraint, in the morning or evening. We show that basal ACTH secretion in both the morning and evening was similarly suppressed by B in STZ-diabetic and control rats. However, stress-induced ACTH secretion was significantly greater in STZ-diabetic compared to control rats throughout the range 3-7 micrograms/dl B, when tested in the morning. Suppression of evening stress-induced ACTH secretion by B was also significantly different in STZ-diabetic rats; however, the IC50 values for the inhibition of ACTH by B did not differ. This result shows that in the evening after stress and under basal conditions in both the morning and evening, sensitivity to B feedback is normal in chronically stressed, STZ-diabetic rats. Despite the observed facilitation of morning stress-induced ACTH secretion in STZ-diabetic rats, there were no differences in hypothalamic CRF content between control and STZ-diabetic tissue. We conclude that 1) the facilitatory input to the paraventricular nucleus functions primarily at the time of the circadian trough to maintain or enhance acute stress responsiveness in chronically stressed, hypercorticoid rats; and 2) the sensitivity of ACTH to inhibition by B is normal in rats chronically stressed by STZ-induced diabetes.

Aldosterone and dexamethasone both stimulate energy acquisition whereas only the glucocorticoid alters energy storage.

Corticosteroids stimulate and insulin inhibits energy acquisition (food intake); conversely, corticosteroids inhibit and insulin stimulates energy storage (body weight gain). Thus, together these hormones mediate long-term energy balance. This study tested whether the stimulatory action of corticosteroids on food intake was mediated by association with high affinity mineralocorticoid receptors (MRs) or lower affinity glucocorticoid receptors (GRs). Young male rats were adrenalectomized (ADX) and given vehicle (control) or streptozotocin (diabetic); subgroups of rats were infused with vehicle, aldosterone (Aldo, an MR agonist in vivo), dexamethasone (Dex, a GR agonist in vivo), or Aldo&Dex for the 5 days after ADX. Sham-ADX rats were included. Food intake, body weight gain, and epididymal white adipose and interscapular brown adipose tissue stores were weighed. ADX decreased food intake by approximately 24%, and food intake was not increased by diabetes as it was in sham-ADX rats. In control ADX rats, Dex, but not Aldo, stimulated insulin, and food intake was not significantly affected by either hormone; together, Aldo and Dex restored insulin and food intake to sham-ADX rats. Food intake in diabetic ADX rats was significantly increased by each treatment (ADX < Aldo < Dex < Aldo&Dex = sham). Aldo increased body weight through an increase in fluid volume (estimated by decreased plasma protein concentration); however, fat stores were not different from ADX. Dex reduced body weight in control rats but maintained fat stores; in diabetic rats, body weight and fat stores were less than or similar to ADX. We conclude that: 1) corticosteroids, acting through association with both MRs and GRs, stimulate food intake; 2) insulin counteracts the GR-mediated stimulation of food intake in control rats; and 3) Dex and insulin, which is stimulated by Dex, selectively maintain or increase body fat stores, probably at the expense of protein stores.

The diurnal rhythm in adrenocorticotropin responses to restraint in adrenalectomized rats is determined by caloric intake.

There is a diurnal rhythm in ACTH responses to stressors that peaks, in nocturnally feeding rats, at the time of lights on, in the morning (AM). To determine whether this rhythm is subordinate to the rhythm in food intake, we tested the effects of removing food during the night or the day on ACTH responses in the AM or evening (PM) to the stimulus of restraint in 5-day-adrenalectomized rats. An overnight fast reduced the ACTH response to restraint with tail blood sampling in the AM to the low magnitude observed in the PM in rats fed ad libitum; by contrast, a fast of equivalent duration imposed during the day had no effect on the ACTH response to the stressor in the PM. Short term fasts did not alter the normal AM-PM rhythm in basal ACTH levels. The fasts did, however, significantly decrease the pituitary ACTH concentration at both times of day, suggesting that lack of food had stimulated ACTH secretion during the preceding 14 h. Providing calories by either gavage or manipulation of food presentation increased ACTH responses to restraint in fasted adrenalectomized rats in both the AM and PM. Although four of four experiments showed that provision of calories to fasted rats resulted in increased ACTH responses to the stimulus of restraint, none of the manipulations of caloric intake fully restored ACTH responses in fasted rats to the high amplitude observed in ad libitum fed rats in the AM. We conclude that 1) unlike the circadian rhythm in basal activity in the hypothalamic-pituitary-adrenalocortical (HPA) system, the diurnal rhythm in ACTH responsiveness to stimuli is tightly coupled to the endogenous rhythm in energy intake; and 2) caloric deprivation per se appears to activate the HPA system at some time during the 14- to 17-h fast, but does not produce the normal facilitation in the AM response to acute restraint that is induced by chronic or prior stimulation of the HPA axis.

Feedback and facilitation in the adrenocortical system: unmasking facilitation by partial inhibition of the glucocorticoid response to prior stress.

Previously stressed animals remain responsive to subsequent stressors, despite secreting an adequate corticosteroid signal during the first stress which should act to damp the response to a second stress. We have previously postulated that stress acts to facilitate subsequent responses in the adrenocortical system, and that this facilitation is balanced by the corticosteroid feedback signal. To test this hypothesis directly, we treated young male rats with cyanoketone (CK) to partially block the adrenal capacity to synthesize corticosterone (B). Subsequently, groups of CK- or vehicle (VEH)-treated rats were exposed to the FIRST stress of 30-min restraint with small blood samples collected at 0, 15, and 30 min. The FIRST stress was given to subgroups of rats 12, 9, 6, or 3 h before lights off (12 h) or lights on (24 h). At 12 or 24 h, rats were again restrained with blood samples at 0 ("basal") and 30 min (SECOND stress). Control groups were stressed for the first time when the experimental groups received their SECOND stress. Plasma ACTH and B concentrations were measured. Although in the absence of stress, basal B concentrations were normal in CK-treated compared to VEH-treated rats throughout the day, the B response to the FIRST stress was reduced by 60% in the CK- compared to the VEH-treated group. When the FIRST stress was performed during the time of lights on, "basal" plasma ACTH was elevated in CK groups at 12 h (lights off) compared to levels in both previously stressed VEH groups and unstressed CK controls. There was no difference at this time of day in the magnitude of the ACTH response to the SECOND stress in CK rats compared to that in CK rats receiving their only stress (controls) or that in VEH-treated rats receiving the SECOND stress. When first stress was performed during the time of lights off, "basal" plasma ACTH at 24 h (lights on) in CK and VEH rats were not different compared to levels in their respective unstressed controls. The ACTH response to the SECOND stress at 24 h was elevated in all previously stressed CK groups compared to that in either CK control or VEH groups. At neither time of day were SECOND stress ACTH concentrations in VEH rats different from those in control VEH rats. At 12 h (lights off), but not at 24 h (lights on), "basal" ACTH was significantly elevated in VEH rats above the unstressed VEH control values.(ABSTRACT TRUNCATED AT 400 WORDS)