Inhibitory effect of ghrelin on food intake is mediated by the corticotropin-releasing factor system in neonatal chicks

It is known that, in rats, central and peripheral ghrelin increases food intake mainly through activation of neuropeptide Y (NPY) neurons. In contrast, intracerebroventricular (ICV) injection of ghrelin inhibits food intake in neonatal chicks. We examined the mechanism governing this inhibitory effect in chicks. The ICV injection of ghrelin or corticotropin-releasing factor (CRF), which also inhibits feeding and causes hyperactivity in chicks. Thus, we examined the interaction of ghrelin with CRF and the hypothalamo-pituitary-adrenal (HPA) axis. The ICV injection of ghrelin increased plasma corticosterone levels in a dose-dependent or a time-dependent manner. Co-injection of a CRF receptor antagonist, astressin, attenuated ghrelin-induced plasma corticosterone increase and anorexia. In addition, we also investigated the effect of ghrelin on NPY-induced food intake and on expression of hypothalamic NPY mRNA. Co-injection of ghrelin with NPY inhibited NPY-induced increase in food intake, and the ICV injection of ghrelin did not change NPY mRNA expression. These results indicate that central ghrelin does not interact with NPY as seen in rodents, but instead inhibits food intake by interacting with the endogenous CRF and its receptor.     

Corticotropin releasing factor stimulates growth hormone secretion in neonatal rats

Corticotropin releasing factor (CRF) both stimulates ACTH secretion from the pituitary and inhibits secretion of growth hormone (GH) in adult rats through actions in the CNS. The purpose of the present study was to evaluate these pituitary and central actions of CRF in neonatal rats, in which the hypothalamo- pituitary adrenal (HPA) axis is relatively hypo-functional. The results of this study show that central or peripheral administration of CRF evokes a marked dose-related rise in serum corticosterone in 6-day old rats. The same doses of CRF stimulate, rather than inhibit GH secretion. These results suggest that CRF has unique central actions early in ontogeny.     

ICV CRF and isolation stress differentially enhance plasma corticosterone concentrations in layer- and meat-type neonatal chicks

The present study compared the plasma corticosterone concentrations between meat- and layer-type neonatal chicks (Gallus gallus) (1) exposed to isolation-induced stress or (2) injected intracerebroventricularly (ICV) with corticotropin-releasing factor (CRF). Both types of neonatal chicks housed in groups were individually introduced to an open field arena and locomotion and distress-induced vocalizations were monitored for 10 min. The responses of the two strains were remarkably different, with meat-type chicks being less active than layer-type chicks. Distress-induced vocalizations were drastically decreased over time in meat-type chicks while they remained high in layer-type chicks throughout the test. Plasma corticosterone concentrations measured at the end of the test were significantly higher in layer-type chicks than in meat-type ones. Plasma corticosterone concentrations measured 10 min after the ICV injection of CRF were significantly higher in layer- than meat-type chicks. These results indicate that meat-type chicks have either a greater capability to acclimatize to novel environments, or a blunted HPA axis compared with layer-type chicks.     

Dietary eicosapentaenoic acid supplementation accentuates hepatic triglyceride accumulation in mice with impaired fatty acid oxidation capacity

Reduced mitochondrial fatty acid (FA) β-oxidation can cause accumulation of triglyceride in liver, while intake of eicosapentaenoic acid (EPA) has been recommended as a promising novel therapy to decrease hepatic triglyceride content. However, reduced mitochondrial FA β-oxidation also facilitates accumulation of EPA. To investigate the interplay between EPA administration, mitochondrial activity and hepatic triglyceride accumulation, we investigated the effects of EPA administration to carnitine-deficient mice with impaired mitochondrial FA β-oxidation. C57BL/6J mice received a high-fat diet supplemented or not with 3% EPA in the presence or absence of 500 mg mildronate/kg/day for 10 days. Liver mitochondrial and peroxisomal oxidation, lipid classes and FA composition were determined. Histological staining was performed and mRNA level of genes related to lipid metabolism and inflammation in liver and adipose tissue was determined. Levels of pro-inflammatory eicosanoids and cytokines were measured in plasma. The results showed that mildronate treatment decreased hepatic carnitine concentration and mitochondrial FA β-oxidation and induced severe triglyceride accumulation accompanied by elevated systemic inflammation. Surprisingly, inclusion of EPA in the diet exacerbated the mildronate-induced triglyceride accumulation. This was accompanied by a considerable increase of EPA accumulation while decreased total n-3/n-6 ratio in liver. However, inclusion of EPA in the diet attenuated the mildronate-induced mRNA expression of inflammatory genes in adipose tissue. Taken together, dietary supplementation with EPA exacerbated the triglyceride accumulation induced by impaired mitochondrial FA β-oxidation. Thus, further thorough evaluation of the potential risk of EPA supplementation as a therapy for NAFLD associated with impaired mitochondrial FA oxidation is warranted.     

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF₁ receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF₁ receptor binding affinities (15 and 10 nM) and functional activities in reporter gene assay (15 and 9.5 nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10 mg/kg inhibited [¹²⁵I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [¹²⁵I]-CRF binding, indicating that only Compound A inhibited central [¹²⁵I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF₁ receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.     

Central corticotropin-releasing factor (CRF) may attenuate somatic pain sensitivity through involvement of glucocorticoids

Corticotropin-releasing factor (CRF) is an important regulator of physiological functions and behavior in stress. Analgesia is one of the characteristics of stress reaction and CRF is involved in providing stress-induced analgesia, however, the underlying mechanisms remain to be determined. Exogenous CRF mimics stress effects on pain sensitivity and causes analgesic effect. The present study was performed to investigate the participation of endogenous glucocorticoids in analgesic effects induced by central administration of CRF in anesthetized rats. The participation of glucocorticoids was studied by pharmacological suppression of the hypothalamic-pituitary-adrenocortical (HPA) axis as well as an occupation of glucocorticoid receptors by its antagonist RU 38486. Since CRF administration causes the release of β-endorphin from the pituitary, the opioid antagonist naltrexone was used to determine the contribution of opioid-dependent mechanism to CRF-induced analgesia. An electrical current threshold test was applied for measurement of somatic pain sensitivity in anesthetized rats. Intracerebroventricular administration of CRF (2 μg/rat) caused analgesic effects (an increase of pain thresholds) and an increase in plasma corticosterone levels. Pretreatment with naltrexone did not change analgesic effects of central CRF as well as corticosterone levels in blood plasma. However, pharmacological suppression of the HPA axis leading to an inability of corticosterone release in response to CRF resulted in an elimination of CRF-induced analgesic effects. Pretreatment with RU 38486 also resulted in an elimination of CRF-induced effects. The data suggest that CRF-induced analgesic effects may be mediated by nonopioid mechanism associated with endogenous glucocorticoids released in response to central CRF administration.     

Chronic ethanol exposure potentiates the locomotor-activating effects of corticotropin-releasing factor (CRF) in rats

The effects of chronic exposure (21 days) to ethanol vapors on locomotor response to intracerebroventricular (i.c.v.) administration of corticotropin releasing factor (CRF) was investigated in male Wistar rats. Responses to CRF were tested during chronic exposure, 1 1/2 hours following removal of ethanol vapors, and two weeks after withdrawal of ethanol. A greater sensitivity to the locomotor-activating effects of CRF was found in ethanol-treated rats as compared to their controls during ethanol exposure (P less than 0.001) and 90 min following removal of ethanol vapors (P less than 0.001) but not two weeks following withdrawal. These results support clinical findings of a reversible activation in the hypothalamic-pituitary-adrenal (HPA) axis in alcoholism. In addition, it appears that chronic exposure to ethanol can also modify central neuronal systems specifically responsive to the locomotor activating effects of CRF.     

Impact of high altitude on the hepatic fatty acid oxidation and synthesis in rats

High altitude (HA) affects energy metabolism. The impact of acute and chronic HA acclimatization on the major metabolic pathways is still controversial. In this study, we aimed to unveil the impact of HA on the key enzymes involved in the fatty acid (FA) metabolism in liver. Rats were exposed to an altitude of 4300 m for 30 days and the expressions of two key proteins involved in FA β-oxidation (carnitine palmitoyl transferase I, CPT-I; and peroxisome proliferator-activated receptor alpha, PPARα), two proteins involved in FA synthesis (acetyl CoA carboxylase-1, ACC-1; and AMP-activated protein kinase, AMPK), as well as the total ketone body in the liver and the plasma FFAs were examined. Rats without HA exposure were used as controls. We observed that the acute exposure of rats to HA (3 days) led to a significant increase in the expressions of CPT-I and PPARα and in the total hepatic ketone body. Longer exposure (15 days) caused a marked decrease in the expression of CPT-I and PPARα. By 30 days after HA exposure, the expression levels of CPT-I and PPARα returned to the control level. The hepatic ACC-1 level showed a significant increase in rats exposed to HA for 1 and 3 days. In contrast, the hepatic level of AMPK showed a significant reduction throughout the experimental period. Plasma FFA concentrations did not show any significant changes following HA exposure. Thus, increased hepatic FA oxidation and synthesis in the early phase of HA exposure may be among the important mechanisms for the rats to respond to the hypoxic stress in order to acclimatize themselves to the stressful environments.     

Expression of hypothalamic neuropeptides and the desensitization of pituitary-adrenal axis and hypophagia in the endotoxin tolerance

Repeated exposure to lipopolysaccharide (LPS) induces desensitization of hypothalamus-pituitary-adrenal axis (HPA) responses and hypophagia. We investigated the interplay between the neural circuitries involved in the control of food intake and HPA axis activity following single or repeated LPS injections. Male Wistar rats received a single or repeated i.p. injection of LPS (100 microg/kg) for 6 days and were subdivided into four groups: 6 saline, 5 saline+1 LPS, 5 LPS+1 saline and 6 LPS. Animals with a single exposure to LPS showed increased plasma levels of ACTH, CORT, PRL, TNF-alpha and also CRF mRNA in the paraventricular nucleus of the hypothalamus. These animals exhibited a reduced food intake and body weight associated with an increase of CART expression in the arcuate nucleus (ARC). Leptin plasma levels were not altered. On the other hand, repeated LPS administration did not alter ACTH, CORT, PRL and TNF-alpha, but it reduced leptin level, compared to single LPS or saline treatment. Furthermore, repeated LPS administration did not increase CRF or CART mRNA expression. Food intake and weight gain after repeated LPS injections were not different from saline-treated animals. There was no change in NPY and POMC mRNA expression in the ARC after single or repeated injections of LPS. In conclusion, desensitization induced by repeated exposure to LPS involves the blockade of HPA axis activation and anorexigenic response, which are both associated with an unresponsiveness of TNF-alpha production and CRF and CART expression in the hypothalamus.     

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.     

Chronic leptin administration in developing rats reduces stress responsiveness partly through changes in maternal behavior

In adult rodents, leptin has been shown to significantly alter the activity of several neuroendocrine functions, including the activity of the hypothalamic-pituitary-adrenal (HPA) axis. Leptin is generally believed to be inhibitory to HPA activity in adults. Developing rat pups have high circulating levels of leptin, which begs the question of leptin's physiological role in controlling basal and stress-induced adrenocortical activity in neonatal rats. In this study, we treated rat pups daily from days 2-9 (or 6-10) of life with either vehicle or leptin (1 or 3 mg/kg body wt, ip) and determined the effects on body weight gain, fat pad deposits, and HPA activity in 10-day-old pups. We measured hypothalamic CRF mRNA levels in vehicle- and leptin-treated pups by in situ hybridization and determined plasma ACTH, corticosterone, and leptin concentrations under basal conditions or following exposure to a 3-min ether stress. Because leptin activates sympathetic activity and energy expenditure in adults and possibly also in rat pups, and because litter temperature is an important determinant of maternal behavior, we also investigated whether chronic leptin administration would modify aspects of maternal care that are important for the maintenance of HPA function. Chronic leptin treatment increased circulating levels of leptin and had significant dose-related metabolic effects, including reduced body weight gain and fat pad weight in 10-day-old pups. Basal expression of CRF mRNA in the PVN or secretion of ACTH and corticosterone was not modified by leptin treatment. In contrast, chronically elevated leptin concentrations during the neonatal period significantly lowered CRF expression in the PVN 60 min after stress and reduced the duration of the ACTH response to stress in pups, suggesting that glucocorticoid feedback on the HPA axis might be altered by this treatment. In addition, mothers caring for pups injected with leptin displayed longer bouts of anogenital licking of pups than mothers of vehicle-treated rats. Given that this particular type of pup stimulation has been shown to influence stress responsiveness, it is possible that the maternal response modulates the effects of exogenous leptin treatment. In conclusion, our results demonstrate that the leptin signal is functional during the early developmental period and that leptin can modulate the hormonal response to stress in young rats either by a direct effect on the HPA axis or indirectly through changing some aspects of maternal behavior.     

Repeated citalopram treatment but not stress exposure attenuates hypothalamic-pituitary-adrenocortical axis response to acute citalopram injection

Many experimental, clinical and epidemiological studies have shown a direct connection between exposure to stress or adverse life events and disease, but little is known about the effect of stress on the action of drugs. The aim of this study was to test the hypothesis that previous exposure to stress changes the action of the antidepressant drug citalopram (10 mg/kg, i.p.) on hypothalamic-pituitary-adrenocortical (HPA) axis function, gene expression of selected neuropeptides and serotonin reuptake. Three different stress models were used, which included immobilization, restraint and unpredictable stress stimuli. Samples of plasma for hormone measurement were taken from conscious cannulated animals. Changes in corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) gene expression in the paraventricular nucleus of the hypothalamus and the anterior pituitary, respectively, and the ability of citalopram to inhibit serotonin reuptake were investigated. The exposure to three different stress models did not influence citalopram action on individual parameters of HPA axis and on serotonin reuptake. On the other hand, repeated administration of the drug led to significant attenuation of ACTH and CRH mRNA responses. The present results allow to suggest that the stressors used did not influence serotonergic neurotransmission to the extent that would modify HPA axis response to citalopram challenge. Activation of HPA axis by acute citalopram treatment was found to be accompanied by increased CRH gene expression in the hypothalamus. Repeated administration of the drug led to the development of tolerance to activation of central and peripheral components of HPA axis, but not to serotonin reuptake inhibition.     

Immediate early gene expression associated with induction of brooding behavior in Japanese quail

Certain species can be induced to foster infant or neonatal animals through the process of sensitization. We induced brooding behavior in adult Japanese quail through repeated exposure to foster chicks across five 20-min trials. Brooding behavior was characterized by a bird allowing chicks to approach and remain underneath its wings while assuming a distinctive stationary crouching posture, preening, and feather fluffing. Birds who did not show brooding behavior actively avoided chicks. Among the birds that brooded chicks, females brooded chicks for longer durations compared to males. Brooding females continued a regular daily egg laying pattern; males showed no significant changes in testosterone levels after exposure to chicks. In a second experiment, we measured expression of two immediate early gene (IEG) protein products, ZENK and Fos, to identify the brain regions activated or inhibited by brooding behavior in females. ZENK and Fos expression in brooding or sensitized females (SF) were compared with expression in nonmaternal females with chicks (NMF) and with females without chicks and with blocks as control objects (BL). There was a reduced density of ZENK-like immunoreactive (ZENK-lir) cells in the medial preoptic nucleus (POM) in NMF birds. In SF birds, the density of Fos-like immunoreactive (Fos-lir) cells was elevated in the bed nucleus stria terminalis, medial portion (BSTm), and ectostriatum (E). These experiments begin to define the neural circuitry underlying brooding behavior in Japanese quail, and establish a model for future studies of the neural mechanisms of avian parental behavior.     

Characterization of CRF, AVT, and ACTH cDNA and pituitary-adrenal axis function in Japanese quail divergently selected for tonic immobility

Higher corticosterone (CORT) responses to acute stress have previously been reported in quail selected for short (STI) duration of tonic immobility (TI) than for long TI (LTI), although behavioral studies indicated that LTI quail were more fearful. To investigate adrenal and pituitary function in these quail lines and their possible involvement in the differences in hypothalamic-pituitary-adrenal (HPA) axis reactivity, we measured CORT responses to adrenocorticotropin (1-24 ACTH), corticotropin-releasing factor (CRF), and arginine vasotocin (AVT) after characterizing the nucleotide acid sequences of these peptides in quail. Although maximum adrenal responses, assessed by ACTH challenge, were higher in STI quail, adrenal sensitivity was comparable for the two genotypes. It is therefore unlikely that differences in HPA axis reactivity involved the adrenal level. AVT and ACTH induced comparable CORT responses in both genotypes, whereas those induced by CRF were much lower. AVT is thus more potent than CRF in quail, but the respective maximum pituitary capacity of both genotypes to secrete ACTH was similar, and it is doubtful that the AVT pathway is involved in the difference in HPA axis reactivity between genotypes. On the other hand, the higher CORT responses induced by CRF in STI quail suggest that CRF might be involved in the differences in HPA axis reactivity between LTI and STI genotypes.     

Regulation and role of p21-activated kinase 3 by corticotropin-releasing factor in mouse pituitary

Corticotropin-releasing factor (CRF) is a major regulatory peptide in the hypothalamic-pituitary-adrenal (HPA) axis under stress conditions. In response to stress, CRF, produced in the hypothalamic paraventricular nucleus, releases adrenocorticotropic hormone (ACTH) from the anterior pituitary (AP). ACTH in turn stimulates the release of glucocorticoid from the adrenal glands. Glucocorticoid then inhibits hypothalamic production of CRF and pituitary production of ACTH. Mice lacking a functional gene for CRF (CRF KO) showed severe impairment of the HPA axis, indicating that CRF is required for its regulation. We applied oligonucleotide microarray analysis to the AP of CRF KO to identify gene expression induced by CRF. Twenty-four genes showed less than 60% expression in CRF KO compared with normal mice. Real-time PCR analysis revealed that p21-activated kinase 3 (Pak3), prohormone convertase type 1 (PC1), and CRF-binding protein (BP) mRNA expression levels were increased by CRF in AP cells. Both Pak3 and PC1 were also increased by dexamethasone in AP cells, while CRF-BP mRNA levels were reduced. Therefore, both Pak3 and PC1 mRNA levels would be regulated by both CRF and glucocorticoids. Pak3 knockdown inhibited CRF-induced cell viability in AtT-20 cells, suggesting the important role of Pak3 in the proliferation of corticotrophs.     

低氧暴露对大鼠下丘脑CRF分泌的影响

目的和方法：利用人工模拟低氧及放射免疫测定（RIA）法,观察不同低氧（5km,7km）暴露对大鼠下丘脑分泌促肾上腺皮质激素释放激素（C orticotropin-releasing factor,CRF）的作用和血浆皮质酮水平的变化。比较低氧暴露不同时间（2h,24h,5d,15d）后,大鼠下丘脑CRF分泌和血浆皮质酮的变化。结果：低氧暴露2h,24h后下丘脑的CRF分泌明显增加,血浆皮质酮水平显著升高,这种变化随着低氧程度的加深而增强,随着低氧暴露时间的延长（5d）上述变化减弱。至慢性低氧暴露15d后,下丘脑CRF分泌及血浆皮质酮水平与对照组相比已无显著差异,基本恢复到对照水平。结论：动物暴露于低氧环境后,下丘脑CRF分泌及血浆皮质酮水平变化为：低氧暴露2h,24h后CRF分泌和皮质酮分泌被激活;低氧暴露5d后CRF分泌和皮质酮分泌活动减弱,并开始恢复;至低氧暴露15d后,这种分泌活动基本恢复,进入恢复期。