Corticosterone-dependent metabolic and neuroendocrine abnormalities in obese Zucker rats in relation to feeding

The obese Zucker (fa/fa) rat is characterized by hyperphagia, hyperinsulinemia, an increase in fat deposition, and a hyperactivity in the hypothalamic-pituitary-adrenal (HPA) axis. The HPA axis in fa/fa rats is hypersensitive to stressful experimental conditions. Food deprivation even leads to a stress reaction in obese fa/fa rats. The present study was conducted to investigate the role of corticosterone in obese rats on the basal, fasting, and postprandial metabolic rate as well as on the central expression of the thyrotropin-releasing hormone (TRH) in these conditions. In addition, the study was aimed at clarifying whether the high levels of corticosterone in obese rats are responsible for the induction of the stress reaction to food deprivation in these animals. The present results demonstrate that whole body fat oxidation and postprandial metabolic responses in obese Zucker rats were improved by adrenalectomy (ADX). At the level of the central nervous system, ADX reversed a decrease in TRH mRNA expression in the paraventricular hypothalamus (PVH) detected in fasting animals. Considering all feeding conditions, the obese rats demonstrated lower TRH mRNA levels compared with lean animals. ADX resulted in an enhanced postprandial activation of the parvocellular PVH. In contrast, the magnocellular part of the PVH was less responsive to refeeding in ADX animals. Finally, ADX failed to prevent the stress response of obese rats to food deprivation. The present results provide evidence that the removal of adrenals resolve some of the metabolic defects encountered in obese Zucker rats. They also demonstrate that not all the abnormalities of the obese Zucker rats are attributable to the hyperactivity of the HPA axis.     

Physiological regulation of hypothalamic IL-1beta gene expression by leptin and glucocorticoids: implications for energy homeostasis

Interleukin-1beta (IL-1beta) is synthesized in a variety of tissues, including the hypothalamus, where it is implicated in the control of food intake. The current studies were undertaken to investigate whether hypothalamic IL-1beta gene expression is subject to physiological regulation by leptin and glucocorticoids (GCs), key hormones involved in energy homeostasis. Adrenalectomy (ADX) increased hypothalamic IL-1beta mRNA levels twofold, measured by real-time PCR (P < 0.05 vs. sham-operated controls), and this effect was blocked by subcutaneous infusion of a physiological dose of corticosterone. Conversely, hypothalamic IL-1beta mRNA levels were reduced by 30% in fa/fa (Zucker) rats, a model of genetic obesity caused by leptin receptor mutation (P = 0.01 vs. lean littermates), and the effect of ADX to increase hypothalamic IL-1beta mRNA levels in fa/fa rats (P = 0.02) is similar to that seen in normal animals. Moreover, fasting for 48 h (which lowers leptin and raises corticosterone levels) reduced hypothalamic IL-1beta mRNA levels by 30% (P = 0.02), and this decrease was fully reversed by refeeding for 12 h. Thus leptin and GCs exert opposing effects on hypothalamic IL-1beta gene expression, and corticosterone plays a physiological role to limit expression of this cytokine in both the presence and absence of intact leptin signaling. Consistent with this hypothesis, systemic leptin administration to normal rats (2 mg/kg ip) increased hypothalamic IL-1beta mRNA levels twofold (P < 0.05 vs. vehicle), an effect similar to that of ADX. These data support a model in which expression of hypothalamic IL-1beta is subject to opposing physiological regulation by corticosterone and leptin.     

Hypothalamic oxytocin neurons modulate hypophagic effect induced by adrenalectomy

Glucocorticoids have major effects on food intake, as demonstrated by the decrease of food intake following adrenalectomy (ADX); however, the mechanisms leading to these effects are not well understood. Oxytocin (OT) has been shown to reduce food intake. We evaluated the effects of glucocorticoids on OT neuron activation and OT mRNA expression in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei induced by feeding. We also evaluated the effect of pretreatment with OT-receptor antagonist ([d(CH2)5,Tyr(Me)2,Orn8]-vasotocin, OVT) on food intake in ADX rats. Fos/OT neurons in the posterior parvocellular subdivision of the PVN were increased after refeeding, with a higher number in the ADX group, compared with sham and ADX+corticosterone (B) groups, with no difference in the medial parvocellular and magnocellular subdivisions of the PVN. ADX increased OT mRNA expression in the PVN both in fasting and refeeding condition, compared with sham and ADX+B groups. In the SON, refeeding increased the number of Fos/OT neurons, with a higher number in the ADX+B group. In fasted condition, OT mRNA expression in the SON was increased in ADX and ADX+B, compared with sham group. Pretreatment with OVT reversed the ADX-induced hypophagia, with no difference between sham and ADX+B animals. The present results show that glucocorticoid withdrawal induces a higher activation of PVN OT neurons in response to feeding, and an increase of OT mRNA expression in the PVN and OT-receptor antagonist reverses the anorexigenic effect induced by ADX. These data indicate that PVN OT neurons might mediate the hypophagic effect induced by adrenalectomy.     

Hypothalamic oxytocin neurons modulate hypophagic effect induced by adrenalectomy

Glucocorticoids have major effects on food intake, as demonstrated by the decrease of food intake following adrenalectomy (ADX); however, the mechanisms leading to these effects are not well understood. Oxytocin (OT) has been shown to reduce food intake. We evaluated the effects of glucocorticoids on OT neuron activation and OT mRNA expression in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei induced by feeding. We also evaluated the effect of pretreatment with OT-receptor antagonist ([d(CH2)5,Tyr(Me)2,Orn8]-vasotocin, OVT) on food intake in ADX rats. Fos/OT neurons in the posterior parvocellular subdivision of the PVN were increased after refeeding, with a higher number in the ADX group, compared with sham and ADX+corticosterone (B) groups, with no difference in the medial parvocellular and magnocellular subdivisions of the PVN. ADX increased OT mRNA expression in the PVN both in fasting and refeeding condition, compared with sham and ADX+B groups. In the SON, refeeding increased the number of Fos/OT neurons, with a higher number in the ADX+B group. In fasted condition, OT mRNA expression in the SON was increased in ADX and ADX+B, compared with sham group. Pretreatment with OVT reversed the ADX-induced hypophagia, with no difference between sham and ADX+B animals. The present results show that glucocorticoid withdrawal induces a higher activation of PVN OT neurons in response to feeding, and an increase of OT mRNA expression in the PVN and OT-receptor antagonist reverses the anorexigenic effect induced by ADX. These data indicate that PVN OT neurons might mediate the hypophagic effect induced by adrenalectomy.     

Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14 d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.     

Corticotrophin-releasing factor mediates hypophagia after adrenalectomy, increasing meal-related satiety responses

Adrenalectomy-induced hypophagia is associated with increased satiety-related responses, which involve neuronal activation of the nucleus of the solitary tract (NTS). Besides its effects on the pituitary–adrenal axis, corticotrophin-releasing factor (CRF) has been shown to play an important role in feeding behaviour, as it possesses anorexigenic effects. We evaluated feeding-induced CRF mRNA expression in the paraventricular nucleus (PVN) and the effects of pretreatment with CRF₂ receptor antagonist (Antisauvagine-30, AS30) on food intake and activation of NTS neurons in response to feeding in adrenalectomised (ADX) rats. Compared to the sham group, ADX increased CRF mRNA levels in the PVN of fasted animals, which was further augmented by refeeding. AS30 treatment did not affect food intake in the sham and ADX + corticosterone (B) groups; however, it reversed hypophagia in the ADX group. In vehicle-pretreated animals, refeeding increased the number of Fos and Fos/TH-immunoreactive neurons in the NTS in the sham, ADX and ADX + B groups, with the highest number of neurons in the ADX animals. Similarly to its effect on food intake, pretreatment with AS30 in the ADX group also reversed the increased activation of NTS neurons induced by refeeding while having no effect in the sham and ADX + B animals. The present results show that adrenalectomy induces an increase in CRF mRNA expression in the PVN potentiated by feeding and that CRF₂ receptor antagonist abolishes the anorexigenic effect and the increased activation of NTS induced by feeding in the ADX animals. These data indicate that increased activity of PVN CRF neurons modulates brainstem satiety-related responses, contributing to hypophagia after adrenalectomy.     

Behavioral and cardiac responses after intracerebroventricular corticotropin-releasing hormone (CRH) administration: role of adrenal cortical hormones.

Intracerebroventricularly (icv) administered corticotropin-releasing hormone (CRH) produces a dose-dependent increase in heart rate in association with behavioral activation. The present study was designed to investigate whether these CRH-induced responses are dependent on adrenal function. The effects of adrenalectomy (ADX) and subsequent corticosterone replacement were studied. Administration icv of 300 ng of CRH failed to produce behavioral activation and tachycardia in ADX rats. Corticosterone replacement restored the CRH-induced behavioral response to preoperative levels, whereas the CRH-induced tachycardia was partially restored. This latter result may be related to the fact that the baseline heart rate of ADX animals appeared to be significantly higher than that of corticosterone-treated ADX animals. It is concluded that circulating adrenal corticosterone in ADX rats is involved in the expression of the behavioral and cardiac effect of central CRH.     

Adrenalectomy enhances endotoxemia-induced hypophagia: higher activation of corticotrophin-releasing-factor and proopiomelanocortin hypothalamic neurons

Inflammatory and infectious processes evoke neuroendocrine and behavioral changes known as acute-phase response that includes activation of the hypothalamo-pituitary-adrenal (HPA) axis and reduction of food intake. Besides its action as the most important ACTH secretagogue, corticotrophin-releasing factor (CRF), synthesized in the paraventricular nucleus (PVN), is also involved in the control of food intake. Alpha-melanocyte stimulating hormone (α-MSH) in the arcuate nucleus also plays a role in the energy homeostasis, possessing anorexigenic effects. To investigate the participation of neuropeptides involved in the regulation of food intake during endotoxemia, we administrated lipopolysaccharide (LPS) in sham-operated and adrenalectomized (ADX) male Wistar rats to evaluate food intake, hormone responses and Fos-CRF and Fos-α-MSH immunoreactivity in the PVN and arcuate nucleus, as well as CRF and POMC mRNA expression in these hypothalamic nuclei. In sham-operated rats, treatment with LPS (100 µg/kg) showed lower food intake, higher plasma ACTH and corticosterone levels, as well as an increase in Fos-CRF double labeled neurons and CRF mRNA expression in the PVN, with no changes in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to saline treated rats. After LPS treatment, ADX rats showed further increase in plasma ACTH levels, marked decrease of food intake, higher Fos-CRF immunoreactive neurons in the PVN and CRF mRNA expression, as well as an increase in Fos-α-MSH immunoreactivity and POMC mRNA expression in the arcuate nucleus, compared to sham-operated rats treated with LPS. In conclusion, the present data indicate that the marked hypophagia during endotoxemia following ADX is associated with an increased activation of CRF and POMC neurons in the hypothalamus and an increased mRNA expression of these neuropeptides.     

The role of estradiol in adrenal insufficiency and its interaction with corticosterone on hydromineral balance

Estradiol (E2) plays an important role in controlling the homeostasis of body fluids. Several studies have reported the involvement of the hypothalamic pituitary adrenal axis (HPA) in the homeostatic control of hydromineral balance and the influence of estrogens on the modulation of this system. Nevertheless, until now, the physiological relevance of HPA axis activity on the hydromineral balance in females has not yet been fully elucidated. Therefore, the objective of the present study was to evaluate the effects of E2 (20 μg/animal) pretreatment on neuroendocrine and hydroelectrolyte changes induced by adrenalectomy (ADX) with or without glucocorticoid hormone replacement (corticosterone, CORT; 10 mg/kg) in ovariectomized rats (OVX). The results show that sodium appetite, natriuresis and the elevated plasma angiotensin II (ANG II) concentration induced by ADX were attenuated by E2 pretreatment. Additionally, a reduction of AT1 mRNA expression in the subfornical organ (SFO) and an increase in plasma atrial natriuretic peptide (ANP) concentrations by E2 pretreatment were observed. E2 pretreatment reversed the reduction in water intake induced by ADX in ADX CORT-replaced rats. Moreover, E2 pretreatment attenuated corticotropin releasing factor (CRF) mRNA expression in the paraventricular nucleus (PVN) induced by ADX. In contrast, E2 pretreatment increased CRF mRNA expression in the PVN in ADX CORT-replaced rats. Taken together, these results suggest that E2 has an important role in the modulation of behavioral and neuroendocrine responses involved in the maintenance of body fluid homeostasis in ADX rats with or without glucocorticoid replacement therapy.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.     

Maternal adrenalectomy affects development of adrenal medulla

This work investigates the effects of maternal adrenalectomy (ADX) on the development of the adrenal medulla. Adrenal catecholamines (AC) were measured at postnatal day (PN) 1, 8, 12 and 22 in rat offspring of ADX dams and in pups of control dams. The pups of ADX rats showed a reduction in AC concentrations in the adrenal medulla at PN 1, 12 and 22, although these were higher than in the pups of sham dams at PN 8. Further, in the pups of control mothers, there was an increase in ACs during the first two weeks of life whereas pups of ADX mothers only showed increases in noradrenaline, dopamine and adrenaline levels at day 8. These results suggest that maternal absence of corticosterone affects the medulla catecholamine content during development. These data support the idea that a maternal glucocorticoids are involved in the differentiation or/and maturation of the adrenal medulla.     

Time course of vasopressin and oxytocin secretion after stress in adrenalectomized rats.

To characterize the participation of vasopressin (AVP) and oxytocin (OT) in hypothalamus-pituitary-adrenal regulation after adrenalectomy (ADX), we evaluated corticosterone, ACTH, AVP and OT plasma concentrations and AVP and OT content of the paraventricular nucleus (PVN) at different periods (3 h, 1, 3, 7 and 14 days) in sham or ADX rats under basal conditions and after immobilization stress. ADX animals showed undetectable corticosterone levels, while sham animals showed a marked increase in corticosterone and ACTH 3 h after surgery, then lowering to basal control levels. ADX rats showed high basal ACTH levels with a triphasic response without changes after immobilization. After three hours, the ADX group showed higher OT levels than the sham group. OT was increased after immobilization stress in sham and ADX groups. AVP plasma levels did not change throughout the basal or stress studies in either group. There was a decrease in hypothalamic AVP content 1 and 3 days after ADX under basal and stress conditions. Plasma osmolality showed a significant decrease in the ADX group at 3, 7, and 14 days. In conclusion, there are different pituitary-adrenal axis set points after removal of the glucocorticoid negative feedback. The role of vasopressinergic and oxytocinergic neurons in the ACTH secretion after ADX or immobilization stress appears to differ. Magnocellular AVP is unlikely to contribute to ACTH secretion in response to ADX or immobilization stress. On the other hand, OT is elicited by immobilization stress and might contribute to the ACTH secretion during short-term ADX.     

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.     

Effect of the exercise-induced increase in glucocorticoids on endurance in the rat

To investigate the effect of the increase in glucocorticoids during exercise on endurance, rats were either sham operated (SO) or adrenalectomized. All adrenalectomized rats were given a subcutaneously implanted corticosterone pellet at the time of adrenalectomy. Adrenalectomized rats were injected with corticosterone (ADX Cort) or corn oil (ADX) 5 min before exercise. Rats were killed at rest or after running on a treadmill (21 m/min, 15% grade) until exhaustion. SO rats ran 138 +/- 6 min compared with 114 +/- 9 min for ADX Cort and 89 +/- 8 min for ADX. All differences in run times were significant (P less than 0.05). Corticosterone levels were similar in exhausted SO and ADX Cort groups. ADX exhausted rats had corticosterone levels similar to resting values in SO and ADX rats. Inhibition of the rise in glucocorticoids during exercise had no effect on liver glycogen, liver adenosine 3',5'-cyclic monophosphate, plasma insulin, blood glucose, lactate, glycerol, or 3-hydroxybutyrate, plasma norepinephrine, or red quadriceps and soleus glycogen. Plasma free fatty acids were significantly depressed at exhaustion in ADX rats compared with SO. These data show that glucocorticoids exert effects within the time frame of a prolonged exercise bout and play a role in increasing endurance.     

Effect of corticosterone on the prolactin response to psychological and physical stress in rats

Both corticosterone and prolactin (PRL) levels increase in response to stress. In these studies we examined the effect of corticosterone on the PRL response to both physical (footshock) and psychological (novel environment) stress. Three groups of rats were used: sham adrenalectomized (SHAM), adrenalectomized (ADX), and adrenalectomized with corticosterone replacement (ADX+CORT). The corticosterone-treated animals received 80 ug corticosterone/ml drinking water. Blood samples were drawn via an indwelling cannula and PRL values determined using radioimmunoassay. ADX rats showed a consistently greater PRL response to being placed on a platform above water (novel environment) or when receiving intermittant footshock than did ADX+CORT rats. The PRL response of the latter group was similar to that of the SHAM animals. These findings indicate that corticosterone levels of an animal can significantly attenuate the magnitude of the PRL response to both physical and psychological stress. These findings further emphasize that the PRL response to stress is dependent not only upon the immediate action of the stressor, but also the prior stress history of the animal.     

HPA-axis responses during experimental colitis in the rat

We investigated the responses of the hypothalamic-pituitary-adrenal (HPA) axis during experimental colitis induced by intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid in the rat. On days 3 and 7 after induction of colitis, the corticotropin-releasing hormone (CRH) mRNA level in the parvocellular paraventricular nucleus (pPVN) of the hypothalamus was reduced, the plasma ACTH level remained at the basal level, and the plasma corticosterone (Cort) level was high. Induction of colitis on day 3 after adrenalectomy with Cort pellet replacement (ADX + Cort) resulted in a marked increase in CRH mRNA on day 7 after induction of colitis compared with noncolitic ADX + Cort animals. Pair feeding to match the food intake of the colitic animals resulted in no significant change in CRH mRNA in the pPVN, plasma ACTH, and Cort compared with healthy control animals. These findings indicated that CRH mRNA expression in the pPVN was inhibited by glucocorticoid feedback during this experimental colitis, and the decrease in food intake during colitis was not simply responsible for the expression of CRH mRNA. It is inferred that the HPA axis including the CRH level in the pPVN is altered in patients with inflammatory bowel disease.     

Sleep deprivation can inhibit adult hippocampal neurogenesis independent of adrenal stress hormones

Sleep deprivation (SD) can suppress cell proliferation in the hippocampal dentate gyrus of adult male rodents, suggesting that sleep may contribute to hippocampal functions by promoting neurogenesis. However, suppression of cell proliferation in rats by the platform-over-water SD method has been attributed to elevated corticosterone (Cort), a potent inhibitor of cell proliferation and nonspecific correlate of this procedure. We report here results that do not support this conclusion. Intact and adrenalectomized (ADX) male rats were subjected to a 96-h SD using multiple- and single-platform methods. New cells were identified by immunoreactivity for 5-bromo-2'-deoxyuridine (BrdU) or Ki67 and new neurons by immunoreactivity for BrdU and doublecortin. EEG recordings confirmed a 95% deprivation of rapid eye movement (REM) sleep and a 40% decrease of non-REM sleep. Cell proliferation in the dentate gyrus was suppressed by up to 50% in sleep-deprived rats relative to apparatus control or home cage control rats. This effect was also observed in ADX rats receiving continuous low-dose Cort replacement via subcutaneous minipumps but not in ADX rats receiving Cort replacement via drinking water. In these latter rats, Cort intake via water was reduced by 60% during SD; upregulation of cell proliferation by reduced Cort intake may obscure inhibitory effects of sleep loss on cell proliferation. SD had no effect on the percentage of new cells expressing a neuronal phenotype. These results demonstrate that the Cort replacement method is critical for detecting an effect of SD on cell proliferation and support a significant role for sleep in adult neurogenesis.