ADX Rats Research Articles

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb.

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH(2)O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA (P < 0.04) and protein (P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA (P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium (P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats (P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.

Repeated Immobilization Stress Increases Uncoupling Protein 1 Expression and Activity in Wistar Rats.

Repeat immobilization-stressed rats are leaner and have improved cold tolerance due to enhancement of brown adipose tissue (BAT) thermogenesis. This process likely involves stress-induced sympathetic nervous system activation and adrenocortical hormone release, which dynamically enhances and suppresses uncoupling protein 1 (UCP1) function, respectively. To investigate whether repeated immobilization influences UCP1 thermogenic properties, we assessed UCP1 mRNA, protein expression, and activity (GDP binding) in BAT from immobilization-naive or repeatedly immobilized rats (3 h daily for 4 weeks) and sham operated or adrenalectomized (ADX) rats. UCP1 properties were assessed before (basal) and after exposure to 3 h of acute immobilization. Basal levels of GDP binding and UCP1 expression was significantly increased (140 and 140%) in the repeated immobilized group. Acute immobilization increased GDP binding in both naive (180%) and repeated immobilized groups (220%) without changing UCP1 expression. In ADX rats, basal GDP binding and UCP1 gene expression significantly increased (140 and 110%), and acute immobilization induced further increase. These data demonstrate that repeated immobilization resulted in enhanced UCP1 function, suggesting that enhanced BAT thermogenesis contributes to lower body weight gain through excess energy loss and an improved ability to maintain body temperature during cold exposure.

Corticosterone infused intracerebroventricularly inhibits energy storage and stimulates the hypothalamo-pituitary axis in adrenalectomized rats drinking sucrose.

When allowed to drink sucrose, bilaterally adrenalectomized (ADX) rats exhibit normal weight gain, food intake, sympathetic neural activity, and ACTH compared with sham-ADX rats. Furthermore, ADX rats drinking sucrose have normal corticotropin-releasing factor (CRF) mRNA throughout brain. In ADX rats without sucrose, all of these variables are abnormal. Systemic corticosterone (B) replacement also restores these variables in ADX rats to normal. To test whether B acts centrally, we infused B or saline intracerebroventricularly into ADX rats under basal conditions and after repeated restraint. Rats were exposed to no stress or 3 h/d restraint for 3 d. Body weights and food and fluid intakes were measured. Brains were analyzed using immunocytochemistry against glucocorticoid receptors (GR) and CRF. Intracerebroventricular B blocked the positive effects of sucrose on metabolism, increased basal ACTH concentrations, and augmented ACTH responses to restraint on d 3. B-infused rats exhibited nuclear GR staining in perirhinal cortex, hippocampus, and hypothalamic paraventricular nuclei, showing that infused B spreads effectively. CRF staining in the paraventricular nucleus of the hypothalamus was higher in B- than in saline-infused rats. We conclude that under basal conditions B acts systemically, but not in the brain, to restore metabolism and neuropeptides after adrenalectomy. By contrast, tonic GR occupancy in brain initiates metabolic and ACTH responses characteristic of stress.

Selective aldosterone blockade prevents angiotensin II/salt-induced vascular inflammation in the rat heart.

We studied the role of aldosterone (aldo) in myocardial injury in a model of angiotensin (Ang) II-hypertension. Wistar rats were given 1% NaCl (salt) to drink and randomized into one of the following groups (n = 10; treatment, 21 d): 1) vehicle control (VEH); 2) Ang II infusion (25 ng/min, sc); 3) Ang II infusion plus the selective aldo blocker, eplerenone (epl, 100 mg/kg.d, orally); 4) Ang II infusion in adrenalectomized (ADX) rats; and 5) Ang II infusion in ADX rats with aldo treatment (20 micro g/kg.d, sc). ADX rats received also dexamethasone (12 micro g/kg.d, sc). Systolic blood pressure increased with time in all treatment groups except the VEH group (VEH, 136 +/- 6; Ang II/NaCl, 203 +/- 12; Ang II/NaCl/epl, 196 +/- 10; Ang II/NaCl/ADX, 181 +/- 7; Ang II/NaCl/ADX/aldo, 236 +/- 8 mm Hg). Despite similar levels of hypertension, epl and ADX attenuated the increase in heart weight/body weight induced by Ang II. Histological examination of the hearts evidenced myocardial and vascular injury in the Ang II/salt (7 of 10 hearts with damage, P < 0.05 vs. VEH) and Ang II/salt/ADX/aldo groups (10 of 10 hearts with damage, P < 0.05). Injury included arterial fibrinoid necrosis, perivascular inflammation (primarily macrophages), and focal infarctions. Vascular lesions were associated with expression of the inflammatory mediators cyclooxygenase 2 (COX-2) and osteopontin in the media of coronary arteries. Myocardial injury, COX-2, and osteopontin expression were markedly attenuated by epl treatment (1 of 10 hearts with damage, P < 0.05 vs. Ang II/salt) and adrenalectomy (2 of 10 hearts with damage, P < 0.05 vs. Ang II/salt). Our data indicate that aldo plays a major role in Ang II-induced vascular inflammation in the heart and implicate COX-2 and osteopontin as potential mediators of the damage.

Effects of adrenalectomy on AGRP, POMC, NPY and CART gene expression in the basal hypothalamus of fed and fasted rats

Glucocorticoids regulate body energy balance through both peripheral and central mechanisms. In order to understand the central mechanisms that mediate these effects of glucocorticoids we studied the effects of adrenalectomy (ADX) and food deprivation on the expression of four neuropeptide genes (measured by S1 nuclease protection assay) in the medial basal hypothalamus (MBH), which are known to regulate energy balance: pro-opiomelanocortin (POMC), agouti-related peptide (AGRP), neuropeptide Y (NPY), and cocaine and amphetamine regulated transcript (CART). Adult male rats were ADX or sham operated (SHAM), and studied 1–2 weeks later. In the first study effects of ADX and corticosterone replacement on POMC and AGRP expression were determined. ADX decreased POMC and AGRP gene expression in the MBH by 27 and 38%, respectively, compared to SHAM rats. Corticosterone treatment increased the expression of POMC by 87% and AGRP by 45% in ADX rats. The second study was designed to determine if glucocorticoids are necessary for the fasting induced changes in POMC, AGRP, NPY and CART in the MBH. ADX caused a 20–30% decrease in the expression of all four neuropeptide genes in the MBH. As expected, fasting suppressed POMC and CART expression and increased AGRP and NPY expression. The fasting-induced increases in AGRP and NPY persisted after ADX but no further significant decreases in POMC or CART were noted after fasting in ADX rats. Plasma leptin and insulin declined significantly after ADX and increased with corticosterone replacement; both leptin and insulin declined further in fasted, ADX animals. In conclusion, ADX decreases both anorexigenic, POMC and CART, and orexigenic, AGRP and NPY, neuropeptide gene expression in the MBH. AGRP and NPY decrease after ADX despite the fall in plasma leptin and insulin concentrations which in other situations would increase these neuropeptides. Furthermore, glucocorticoids are not required for fasting-induced upregulation of AGRP and NPY expression.

Adrenalectomy enhances the insulin sensitivity of muscle protein synthesis.

After confirming that adrenalectomy per se does not affect skeletal muscle protein synthesis rates, we examined whether endogenously produced glucocorticoids modulate the effect of physiological insulin concentrations on protein synthesis in overnight-fasted rats 4 days after either a bilateral adrenalectomy (ADX), ADX with dexamethasone treatment (ADX + DEX), or a sham operation (Sham; n = 6 each). Rats received a 3-h euglycemic insulin clamp (3 mU. min(-1). kg(-1)). Rectus muscle protein synthesis was measured at the end of the clamp, and the phosphorylation states of protein kinase B (Akt), eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), and ribosomal protein S6 kinase (p70(S6K)) were quantitated before and after the insulin clamp. The basal phosphorylation states of Akt, 4E-BP1, and p70(S6K) were similar between ADX and Sham rats. Insulin significantly enhanced the phosphorylation of Akt (P < 0.03), 4E-BP1 (P = 0.003), and p70(S6K) (P < 0.002) in ADX but not in Sham rats. Protein synthesis was significantly greater after insulin infusion in ADX than in Sham rats (P = 0.01). Glucocorticoid replacement blunted the effect of insulin on Akt, 4E-BP1, and p70(S6K) phosphorylation and protein synthesis. In conclusion, glucocorticoid deficiency enhances the insulin sensitivity of muscle protein synthesis, which is mediated by increased phosphorylation of translation initiation-regulatory proteins.

Endogenous glucocorticoids modulate experimental anti-glomerular basement membrane glomerulonephritis.

The influence of endogenous glucocorticoids (GC) on glomerular injury was studied in a rat model of heterologous anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Sprague-Dawley rats underwent adrenalectomy (ADX) or sham-operation 3 days prior to i.v. administration of both nephritogenic (100 microgram/g) and subnephritogenic (50 microgram/g) doses of sheep anti-rat GBM globulin. Administration of a subnephritogenic dose of anti-GBM globulin resulted in GN in adrenalectomized animals only. Similarly, ADX performed prior to administration of anti-GBM in the nephritogenic dose range resulted in exacerbation of GN compared with sham-operated animals (24 h protein excretion: 190.8 +/- 32.8 versus 42.5 +/- 2.6 mg/24 h; P < 0.005). In ADX animals receiving subnephritogenic doses of anti-GBM injury was manifested by abnormal proteinuria (62.7 +/- 5.8 mg/24 h), accumulation of neutrophils which peaked at 6 h (7.2 +/- 1.37 neutrophils per glomerular cross-section (neut/gcs)) and macrophage accumulation in glomeruli at 24 h (6.8 +/- 1.2 macrophages/gcs). Sham-adrenalectomized animals given the same dose of anti-GBM globulin developed minimal or no glomerular injury: urinary protein excretion (8.7 +/- 1.5 mg/24 h, P < 0.001); neutrophils (0.2 +/- 0.04 neutrophils/gcs, P < 0.001); macrophages (1.2 +/- 0.5 macrophages/gcs, P < 0.001). The increased cellular recruitment to glomeruli in adrenalectomized animals was associated with glomerular endothelial P-selectin expression. P-selectin expression was not detected in sham-operated rats after anti-GBM injection. Complement deposition in glomeruli was minimal in both groups. Physiologic GC replacement of ADX rats receiving subnephritogenic-dose anti-GBM reversed the observed susceptibility to GN development, with urinary protein excretion (7.8 +/- 1.12, P < 0.005) and no detectable P-selectin expression or leucocyte accumulation in glomeruli. These results suggest that endogenous GC modulate heterologous anti-GBM nephritis in rats and that this may be attributable, in part, to regulation of P-selectin expression.

Increased somatostatin mRNA expression in periventricular nucleus of rat hypothalamus during hypoxia.

We reported that hypoxia inhibited the growth hormone (GH) and induced somatostatin (SS) release from the hypothalamic median eminence (ME) of rats. This study is designed to examine the SS mRNA alterations in the periventricular nucleus (PeN) of the hypothalamus in rats and the possible involvement of glucocorticoid (GC) during hypoxia. Rats were exposed to hypoxia in a simulated hypobaric chamber. SS mRNA levels in the PeN were tested by in situ hybridization. Hypoxia of 5-km altitude (10.8% O(2)) for 2, 5 and 24 h increased the SS mRNA expression by 34.72%, 50.31% and 95.05% (p<0.05), respectively. Severe hypoxia of 7-km altitude (8.2% O(2)) enhanced the SS expression by 79.08% (p<0.01), 74.90% (p<0.01) and 71.40% (p<0.05), respectively. Prolonged hypoxia (5 km for 5 days) exposure augmented a 2.5-fold SS mRNA (p<0.001). One week post adrenalectomy (ADX), SS mRNA level was significantly increased. During hypoxia, 5 km for 5 h, SS mRNA in ADX rats was not further increased. An increased SS mRNA was showed by pretreatment with low dose of dexamethasone (DEX) (125 microg/kg, i.p.) to ADX animals but this increase was depressed by a high dose of DEX (500 microg/kg, i.p.). The data suggested that (1) hypoxia stimulated the expression of SS mRNA in the PeN of rat hypothalamus. (2) Increased circulating GC levels might play a role in upregulating the SS mRNA in the rat PeN during hypoxia.

Inhibition of neuronal nitric oxide synthase increases adrenalectomy-induced granule cell death in the rat dentate gyrus

Recent studies have shown that the expression of neuronal nitric oxide synthase (NOS) mRNA is increased after adrenalectomy (ADX). However, the role of increased NO production after ADX in the dentate gyrus is unknown. In this study, the relationship between NO inhibition and apoptosis in the dentate gyrus after ADX was examined. 7-Nitroindazole (7-NI; 30 mg/kg, i.p.), a selective inhibitor of neuronal NOS, was injected 1 day before ADX and subsequently once every 24 h. Then 4 days after ADX, dentate granule cell death was evaluated using silver impregnation and Nissl staining methods. Inhibition of neuronal NOS by 7-NI increased the number of dying granule cells ∼4-fold in the dentate gyrus of the ADX rats, compared to vehicle-injected ADX controls. These results suggest that increased NO production after ADX may play an endogenous neuroprotective role in the dentate gyrus.

Centrally regulated blood pressure response to vasoactive peptides is modulated by corticosterone.

To investigate the role of brain glucocorticoid (GR) and mineralocorticoid receptors (MR) in centrally evoked blood pressure responses, the effects of intracerebroventricular (i.c.v.) administration of angiotensin II and vasopressin were studied in adrenalectomized rats with and without corticosterone or aldosterone replacement. Five groups were examined: (i) Adrenalectomy (ADX); (ii) ADX + a subcutaneously implanted 20-mg corticosterone pellet (low corticosterone); (iii) ADX + 100 mg corticosterone pellet (high corticosterone); (iv) ADX + 6 microg/24 h aldosterone via Alzet minipump (Aldo); and (v) Sham adrenalectomy (Sham). Pressor responses to 150 ng angiotensin II and 50 ng vasopressin i.c.v. were determined in freely moving rats using biotelemetry. The results show that, compared to sham rats, ADX rats showed significantly reduced pressor responses. This reduction of the pressor response to angiotensin II could be reversed and even further enhanced by replacement of the ADX rats with high corticosterone concentrations. In contrast, with aldosterone, a depressor type response was observed. Corticosterone replacement could not restore the pressor response to vasopressin. We conclude that the pressor response to centrally administered vasoactive substances is substantially attenuated by removal of the adrenals and that, in the case of angiotensin II, this is due to the lack of high concentrations of circulating corticosterone occupying both MR and GR. However, predominant MR occupancy appears to play an opposite role and attenuates the angiotensin II-induced pressor response.

Do sex steroids regulate glutamine synthesis with age?

Glutamine synthetase, a key enzyme in the production of glutamine, is known to be induced by glucocorticoids and preserved in skeletal muscle during aging, but the effect of other steroids, such as sex steroids (progesterone, estradiol), is unknown in vivo. The aim of this study was to determine whether progesterone or estradiol plays a role in the regulation of glutamine synthetase (GS) with aging. The effects of glucocorticoids and sex steroids on muscle GS activity and mRNA expression were measured in adult (6-8 mo; n = 7 in each group) and aged (26 mo; n = 10 in each group) female Wistar rats after adrenalectomy (ADX), ovariectomy (OV), or both (ADXOV) and were compared with those in sham-operated (Sham) control rats. In tibialis anterior muscle, ADX noticeably decreased both GS activity and expression irrespective of age (50-60%; P < 0.05), whereas OV had no effect at either age. Progesterone and estradiol replacement had no effect on the recovery of muscle GS response in either ADX or OV rats, regardless of age. In contrast, heart GS activity was decreased by ADX in aged animals only. These results suggest that the reproductive endocrine status of female rats does not affect muscle GS activity either in muscle or in heart, in young or aged animals, and that the heart GS response to steroids may be differently regulated in aged rats.

Involvement of corticosterone in cardiovascular responses to an open-field novelty stressor in freely moving rats

The aim of the present study was to investigate the modulatory action of different concentrations of circulating corticosterone occupying either predominantly mineralocorticoid receptors (MR) or both MR and glucocorticoid receptors (GR) in control of cardiovascular responses to a novelty stressor. Six groups of rats were instrumented with radiotelemetry transmitters: sham-operated controls, adrenalectomised (ADX) controls, ADX with chronic implantation of a 20-mg corticosterone pellet, ADX with chronic implantation of a 100-mg corticosterone pellet, ADX receiving acute bolus injection of 0.25 mg/kg of corticosterone, and ADX with both implantation of a 20-mg corticosterone pellet and bolus treatment. Exposure to the novelty of an open field caused an increase in blood pressure, heart rate, body temperature and exploratory locomotor activity. The pressor response was dose-dependently increased in ADX rats implanted with a corticosterone pellet. Bolus injection of corticosterone at 10 min prior to novelty had no effect. The tachycardia was reduced in ADX rats compared to sham-operated controls, and this effect was restored by implantation of a 20-mg, but not 100-mg, corticosterone pellet. Bolus injection of corticosterone facilitated the return of heart rate towards baseline levels. The increase in body temperature was reduced in ADX rats, a deficit that was normalised by implantation of either corticosterone dose but not by acute bolus treatment. Locomotor activity was not different between the groups except for a slightly more rapid decline of locomotor activity in both groups treated with a bolus injection of corticosterone. These data show an important role of putative brain MR in maintaining adequate cardiovascular and behavioural responsiveness to a mild psychological stressor, while additional acute or chronic occupation of GR has further differential and sometimes opposing effects.

Comparable Effects of Adrenalectomy and C Fiber Depletion on Delayed-Type Hypersensitivity in Rats

Objective: To determine the influence of the hypothalamic-pituitary-adrenal (HPA) axis and sensory C fibers on cell-mediated immunity, the time course of allergic contact dermatitis (ACD) was examined in normal, adrenalectomized (ADX) and capsaicin (CAP)-treated rats. Methods: Five days after sensitization with 2,4-dinitro-1-fluorobenzene (DNFB) onto the back, rats were challenged with DNFB on the pinna. ADX or CAP treatment was performed a week before the sensitization. Results: Ear swelling in normal rats reached a peak 24 h after the challenge and subsided gradually to about half of this peak 72 h afterwards. Plasma ACTH levels increased about 9 times more than those of nonsensitized rats 72 h after the challenge. ADX rats, in which the plasma corticosterone level was significantly lower than that in sham-operated animals, exhibited more rapid development of ACD, which reached a peak 12 h after the challenge. However, the swelling returned to about half of the maximum within 48 h. In CAP-treated rats, ACD reached a peak 6 h after the challenge and recovered quickly as in ADX rats. Plasma ACTH levels in CAP- treated rats were significantly lower than those in vehicle-treated rats. Conclusions: The rapid development of and recovery from ACD in ADX and CAP-treated rats, both of which showed suppression of the HPA axis, indicated a biphasic action of the HPA axis on ACD, i.e. inhibitory in the early phase but augmentative in the late phase of ACD.

Constitutive activation of STAT-3 and downregulation of SOCS-3 expression induced by adrenalectomy.

Removal of adrenal steroids by adrenalectomy (ADX) slows or reverses the development of many forms of obesity in rodents, including those that are leptin or leptin receptor deficient. Obesity is associated with hyperleptinemia and leptin resistance. We hypothesized that glucocorticoids impair leptin receptor signaling and that removal thereof would activate the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) signaling pathway. The inhibitory effect of leptin (2.5 microg icv) on food intake was enhanced in ADX rats. A combination of ribonuclease protection assays, RT-PCR, Western blots, and mobility shift assays was used to evaluate the leptin signaling pathway in whole hypothalami from sham-operated, ADX and corticosterone-replaced ADX (ADX-R) Sprague-Dawley rats that were treated acutely with either saline vehicle or leptin intracerebroventricularly. ADX increased the expression of leptin receptor mRNA, increased STAT-3 mRNA and protein levels, induced constitutive STAT-3 phosphorylation and DNA binding activity, and also reduced suppressor of cytokine signaling-3 (SOCS-3) mRNA and protein levels. ADX and leptin treatment increased STAT-3 phosphorylation, but with no concomitant increase in DNA binding activity. Leptin and ADX decreased NPY mRNA expression, but their combination did not further decrease NPY mRNA. Corticosterone supplementation of ADX rats partially reversed many of these effects. In conclusion, ADX through activation of STAT-3 and inhibition of SOCS-3 activates the JAK-STAT signaling pathway. These effects most probably explain the ability to prevent the development of obesity by removal of adrenal steroids.

Sympathetic innervation of mammary glands mediates suckling-induced reflex inhibition of milk yield in rats

Previous work has shown that physiologic activation of the sympathetic system may inhibit milk yield (ME) in rats. Thus, adrenal catecholamines (CAs) are released by suckling, but it is not known whether such inhibition results also from reflex activation by the same stimulus of neural sympathetics upon the mammary gland. The present experiments were designed to determine whether suckling inhibits ME induced by oxytocin (OT) in the urethane-anesthetized lactating rat, and whether such inhibition results from adrenal and/or neurally released CAs. Rats were isolated (6 h) from their pups and then anesthetized. OT (0.8 mU every 2 min) was administered intravenously to the mothers during suckling. Rats were either chronically implanted with cannulae into the lateral cerebral ventricles (intracerebroventricularly), bilaterally adrenalectomized (ADX), hypophysectomized (HX), spinal cord transected (SCT: T3–T4), or had the nipple area (NA) locally anesthetized before suckling. MEs were low in control, sham, ADX and HX rats, but not in rats given the β-adrenergic blocker propranolol (PROP; intravenously or intracerebroventricularly injected), nor in SCT, NA or PROP-HX rats. As revealed by ductal resistance measurements as an indicator of ductal tone, suckling-induced inhibition of ME was due to ductal constriction within the mammary glands. These effects of suckling, however, could be prevented by prior activation of ductal mechanoreceptors. Together, these results indicate that suckling inhibits ME through the reflex activation of neurally mediated central β-adrenergic mechanisms, and that these effects, in turn, can be regulated by ductal mechanoreceptor activation.

Sucrose Ingestion Normalizes Central Expression of Corticotropin-Releasing-Factor Messenger Ribonucleic Acid and Energy Balance in Adrenalectomized Rats: A Glucocorticoid-Metabolic-Brain Axis?

Both CRF and norepinephrine (NE) inhibit food intake and stimulate ACTH secretion and sympathetic outflow. CRF also increases anxiety; NE increases attention and cortical arousal. Adrenalectomy (ADX) changes CRF and NE activity in brain, increases ACTH secretion and sympathetic outflow and reduces food intake and weight gain; all of these effects are corrected by administration of adrenal steroids. Unexpectedly, we recently found that ADX rats drinking sucrose, but not saccharin, also have normal caloric intake, metabolism, and ACTH. Here, we show that ADX (but not sham-ADX) rats prefer to consume significantly more sucrose than saccharin. Voluntary ingestion of sucrose restores CRF and dopamine-β-hydroxylase messenger RNA expression in brain, food intake, and caloric efficiency and fat deposition, circulating triglyceride, leptin, and insulin to normal. Our results suggest that the brains of ADX rats, cued by sucrose energy (but not by nonnutritive saccharin) maintain normal activity in systems that regulate neuroendocrine (hypothalamic-pituitary-adrenal), behavioral (feeding), and metabolic functions (fat deposition). We conclude that because sucrose ingestion, like glucocorticoid replacement, normalizes energetic and neuromodulatory effects of ADX, many of the actions of the steroids on the central nervous system under basal conditions may be indirect and mediated by signals that result from the metabolic effects of adrenal steroids.

Sucrose ingestion normalizes central expression of corticotropin-releasing-factor messenger ribonucleic acid and energy balance in adrenalectomized rats: a glucocorticoid-metabolic-brain axis?

Both CRF and norepinephrine (NE) inhibit food intake and stimulate ACTH secretion and sympathetic outflow. CRF also increases anxiety; NE increases attention and cortical arousal. Adrenalectomy (ADX) changes CRF and NE activity in brain, increases ACTH secretion and sympathetic outflow and reduces food intake and weight gain; all of these effects are corrected by administration of adrenal steroids. Unexpectedly, we recently found that ADX rats drinking sucrose, but not saccharin, also have normal caloric intake, metabolism, and ACTH. Here, we show that ADX (but not sham-ADX) rats prefer to consume significantly more sucrose than saccharin. Voluntary ingestion of sucrose restores CRF and dopamine-beta-hydroxylase messenger RNA expression in brain, food intake, and caloric efficiency and fat deposition, circulating triglyceride, leptin, and insulin to normal. Our results suggest that the brains of ADX rats, cued by sucrose energy (but not by nonnutritive saccharin) maintain normal activity in systems that regulate neuroendocrine (hypothalamic-pituitary-adrenal), behavioral (feeding), and metabolic functions (fat deposition). We conclude that because sucrose ingestion, like glucocorticoid replacement, normalizes energetic and neuromodulatory effects of ADX, many of the actions of the steroids on the central nervous system under basal conditions may be indirect and mediated by signals that result from the metabolic effects of adrenal steroids.

Effects of Prenatal Ethanol Exposure on Hypothalamic‐Pituitary‐Adrenal Regulation After Adrenalectomy and Corticosterone Replacement

Previous studies have demonstrated that rats prenatally exposed to ethanol (E) exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness, demonstrated by increased and/or prolonged elevations of adrenocorticotropin (ACTH) and/or corticosterone (CORT) after stress. This study investigated possible mechanisms of HPA hyperresponsiveness in E rats by manipulating CORT feedback regulation of HPA activity via adrenalectomy (ADX) with or without CORT replacement. Male Sprague-Dawley rat offspring from prenatal E, pair-fed (PF) and ad libitum-fed control (C) groups were tested at 90 to 120 days of age. Rats were either sham-operated or underwent ADX, with or without CORT replacement. CORT (25 microg/ml) was replaced via the drinking water to achieve basal plasma CORT levels and maintain a phasic CORT signal. Seven days after surgery, animals were decapitated at the diurnal peak either under basal conditions or after a 15-min restraint stress, and trunk blood was collected. After ADX, loss of the CORT feedback signal resulted in increased plasma ACTH in all groups compared with those in sham animals. In addition, under basal conditions, ADX E rats had significantly greater plasma ACTH levels than both PF and C rats. However, no differences were seen in ADX rats after stress. CORT replacement after ADX was partially effective in normalizing ACTH levels under both basal and stress conditions, with no differences among E, PF, and C animals. These results suggest that E males may exhibit enhanced stimulatory inputs to the hypothalamus, increased pituitary sensitivity to secretagogues, or both, which may be revealed after ADX. In contrast, E animals seem similar to controls in their ability to use an exogenous CORT signal to regulate HPA activity.

Androgens Alter Corticotropin Releasing Hormone and Arginine Vasopressin mRNA Within Forebrain Sites Known to Regulate Activity in the Hypothalamic‐Pituitary‐Adrenal Axis

To reveal direct effects of androgens, independent of glucocorticoids, we studied the effects of gonadectomy (GDX) in adrenalectomized (ADX) rats with or without androgen replacement on corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) mRNA expression within various forebrain sites known to regulate the hypothalamic-pituitary-adrenal axis. These included the medial parvocellular portion of the paraventricular nucleus of the hypothalamus (mp PVN), the central and medial nuclei of the amygdala and bed nuclei of the stria terminalis (BNST). In the mp PVN, ADX stimulated both CRH and AVP mRNA expression. Combined ADX + GDX inhibited only AVP, and testosterone and dihydrotestosterone (DHT) restored AVP mRNA. In the central nucleus of the amygdala, ADX decreased CRH mRNA expression, and this response was unaffected by GDX +/- testosterone or DHT replacement. In the medial amygdala, AVP mRNA expression was decreased by ADX, abolished by ADX + GDX, and restored by androgen replacement. ADX had no effect on CRH and AVP mRNA expression in the BNST. GDX + ADX, however, reduced CRH mRNA expression only within the fusiform nuclei of the BNST and reduced the number of AVP-expressing neurones in the posterior BNST. Androgen replacement reversed both responses. In summary, in ADX rats, AVP, but not CRH mRNA expression in the amygdala and mp PVN, is sensitive to GDX +/- androgen replacement. Both CRH- and AVP-expressing neurones in the BNST respond to GDX and androgen replacement, but not to ADX alone. Because androgen receptors are not expressed by hypophysiotropic PVN neurones, we conclude that glucocorticoid-independent, androgenic influences on medial parvocellular AVP mRNA expression are mediated upstream from the PVN, and may involve AVP-related pathways in the medial amygdala, relayed to and through CRH- and AVP-expressing neurones of the BNST.

Induction of cyclooxygenase-2 in thick ascending limb cells by adrenalectomy.

Adrenalectomized (ADX) and sham-operated rats received either dexamethasone (DEX) or vehicle. Renal tissue was used for morphologic analysis, assessment of cyclooxygenase-2 (COX-2) protein expression and mRNA accumulation, and quantitation of COX-2 activity. In untreated or shamoperated rats, COX-2 protein was observed in a subset of tubular epithelial cells (<2%), which were located mainly in the cortex. All COX-2-positive cells also expressed Tamm-Horsfall glycoprotein, a highly selective marker for thick ascending limb (TAL) cells. After ADX, >30% of TAL cells expressed COX-2 in a manner consistent with recruitment of COX-2-positive TAL cells toward the medulla. Treatment of ADX rats with DEX reduced the number of COX-2-positive cells to that observed in sham-operated or intact rats. COX-2 mRNA accumulation was increased by ADX and partially attenuated by treatment with DEX. Western blot analysis of cortical microsomes revealed a substantial increase in COX-2 expression in ADX rats, compared with ADX/DEX-treated, sham-operated, or intact rats. The increase in COX-2 protein expression was associated with a twofold increase in prostaglandin E(2) formation by cortical microsomes obtained from ADX rats, compared with sham-operated rats. It is concluded that ADX induces expression of enzymatically active COX-2, such that expression occurs in the cortical TAL and proceeds in a defined pattern toward the outer medullary TAL. It is suggested that ADX induces expression of TAL cells that, in the basal state, do not express COX-2 protein.