Asuka Mano-Otagiri

Gender differences in corticotropin and corticosterone secretion and corticotropin-releasing factor mRNA expression in the paraventricular nucleus of the hypothalamus and the central nucleus of the amygdala in response to footshock stress or psychological stress in rats.

Anorexia nervosa is mostly seen in adolescent females, although the gender-differentiation mechanism is unclear. Corticotropin-releasing factor (CRF), a key peptide for stress responses such as inhibition of food intake, increases in arousal and locomotor activity, and gonadal dysfunction, is thought to be involved in the pathophysiology of anorexia nervosa. CRF in the paraventricular nucleus of the hypothalamus (PVN) and CRF in the central nucleus of the amygdala (CeA) are involved in the regulation of stress responses, and gender differences in CRF mRNA expression in these regions in response to various stressors are controversial. We therefore examined CRF gene expression in the PVN and CeA as well as corticotropin (ACTH) and corticosterone secretion in response to a 60-min period of electric footshock (FS) or psychological stress (PS) induced by a communication box in both male and female rats in proestrus or diestrus in an effort to elucidate the mechanism underlying the gender difference in the activity of the hypothalamic-pituitary-adrenal (HPA) axis and the mechanism underlying the remarkable prevalence of anorexia nervosa in females. Female rats in proestrus showed higher basal plasma ACTH and CRF mRNA expression levels in the PVN and CeA than males. Females more rapidly showed higher plasma ACTH and corticosterone levels and a higher CRF mRNA expression level in the PVN in response to FS than males. Although females in both proestrus and diestrus showed significant increases in plasma ACTH and corticosterone and CRF mRNA expression in the PVN in response to PS, no significant responses of the HPA axis to PS were found in males. FS significantly increased CRF mRNA expression in the CeA in both females and males, with significantly higher peaks in females in proestrus than in males, while PS significantly increased CRF mRNA expression in the CeA only in males. These results suggest that gender affects differentially the function of the stress-related regions such as the PVN and CeA. The finding that CRF gene expression in the PVN responds to PS only in females may be a clue to elucidation of the neurobiological mechanism underlying the gender-differential prevalence of anorexia nervosa.

Ghrelin suppresses noradrenaline release in the brown adipose tissue of rats

To clarify the role of ghrelin in the regulatory mechanism of energy metabolism, we analyzed the effects of centrally and peripherally administered ghrelin on noradrenaline release in the brown adipose tissue (BAT) of rats using a microdialysis system. I.c.v. administration of ghrelin at a dose of 500 pmol suppressed noradrenaline release in BAT, and microinjection of ghrelin (50 pmol) into the paraventricular nucleus (PVN) or arcuate nucleus (ARC) of the hypothalamus also suppressed noradrenaline release in BAT. In addition, i.v. administered ghrelin (30 nmol) suppressed noradrenaline release in BAT, and this suppression was blocked by a vagotomy. Neither i.c.v. nor i.v. administration of des-acyl ghrelin, which does not bind to GH secretagogue receptor type 1a (GHS-R1a), affected noradrenaline release in BAT. These results indicate that ghrelin increases energy storage by suppressing the activity of the sympathetic nerve innervating BAT. It seems that the PVN and ARC, which express GHS-R1a, are the sites of action of ghrelin in the brain and that the action of peripheral ghrelin on the sympathetic nerve activity innervating BAT is mediated by the vagal nerve, which also expresses GHS-R1a.

Genetic suppression of ghrelin receptors activates brown adipocyte function and decreases fat storage in rats.

To clarify the role of ghrelin and its receptor (GHS-R) in the regulatory mechanism of energy metabolism, we analyzed transgenic (Tg) rats expressing an antisense GHS-R mRNA under the control of the tyrosine hydroxylase (TH) promoter. Tg rats showed lower visceral fat weight and higher O(2) consumption, CO(2) production, rectal temperature, dark-period locomotor activity, brown adipose tissue (BAT) weight and uncoupling protein 1 expression compared with wild-type (WT) rats on a standard diet. A high-fat diet for 14days significantly increased body weight, visceral fat weight, and the sizes of white and brown adipocytes in WT rats but not in Tg rats compared with the corresponding standard-diet groups. Antisense GHS-R mRNA was expressed and GHS-R expression was reduced in TH-expressing cells of the vagal nodose ganglion in Tg rats. Ghrelin administered intravenously suppressed noradrenaline release in the BAT of WT rats, but not in Tg rats. These results suggest that ghrelin/GHS-R plays an important role in energy storage by modifying BAT function and locomotor activity. As our previous study showed that peripheral ghrelin-induced noradrenaline release suppression in BAT is blocked by vagotomy, the present findings also suggest that vagal afferents transmit the peripheral ghrelin signal to the sympathetic nervous system innervating BAT.

Nicotine suppresses energy storage through activation of sympathetic outflow to brown adipose tissue via corticotropin-releasing factor type 1 receptor

Nicotine is known to stimulate energy expenditure, although the precise mechanism is unclear. To clarify the involvement of corticotropin-releasing factor (CRF) in the mechanism by which nicotine increases energy expenditure, the effect of intraperitoneal injection of nicotine (0.1 or 0.5mg/kg) on the release of noradrenaline (NA), a stimulator of thermogenesis, in brown adipose tissue (BAT) important for energy expenditure was examined in rats. We also examined the effects of CRF receptor subtype antagonists on the nicotine-induced change in BAT NA release. Nicotine significantly increased BAT NA release at a dose of 0.5mg/kg, and the increase was completely blocked by antalarmin, a CRF type 1 receptor antagonist, but not by antisauvagine-30, a CRF type 2 receptor antagonist. These results suggest that nicotine increases energy expenditure by activating BAT function, and that CRF type 1 receptors are involved in the mechanism by which nicotine affects energy balance.

Effects of intracerebroventricular ghrelin on food intake and Fos expression in the arcuate nucleus of the hypothalamus in female rats vary with estrous cycle phase.

Intracerebroventricular (icv) injection of ghrelin increases food intake via activation of neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons, which express growth hormone secretagogue receptor type 1a (GHS-R1a), in the arcuate nucleus of the hypothalamus (Arc) in male rats. Conversely, elevation in endogenous estrogens or exogenous estrogens decreases food intake, but the precise mechanism mediating this estrogenic effect is unknown. We studied whether the effects of icv ghrelin on food intake and on the expression of Fos, a marker of neuron activation, vary with estrous cycle phase in female rats. Icv ghrelin (100pmol) significantly increased food intake after injection in diestrus, but it did not affect food intake in proestrus during light phase. Icv ghrelin increased the number of Fos-positive neurons in the Arc both in proestrus and diestrus; however, a significantly larger number of Fos-positive neurons appeared in diestrus than in proestrus. Real-time RT-PCR analysis showed no significant difference in GHS-R1a mRNA expression levels in the mediobasal hypothalamus between diestrus and proestrus. These results indicated that not only the orexigenic effect but also the Fos-inducing effect of icv ghrelin were influenced by the estrous cycle phase; and both effects were reduced in proestrus but not in diestrus. Most NPY/AgRP neurons seemed to be influenced indirectly by estrogens during proestrus because only a few of the NPY/AgRP neurons present in the Arc express ERα. The change in GHS-R1a expression levels in the hypothalamus during estrous cycle is not probably involved in the estrous cycle-induced changes in ghrelin action because there was no difference in GHS-R1a mRNA expression between diestrus and proestrus.

Distribution of Corticotrophin‐Releasing Factor Type 1 Receptor‐Like Immunoreactivity in the Rat Pituitary

Corticotrophin‐releasing factor (CRF) regulates the hypothalamic‐pituitary‐adrenal axis response to stress through its type 1 receptor (CRF1) in the corticotrophs of the anterior pituitary. Although CRF1 mRNA expression has been confirmed in the rat pituitary, the distribution pattern of CRF1 protein in the pituitary has not been reported. Therefore, we generated an antiserum against the amino acid fragment corresponding to the 177–188 sequence of the first extracellular loop of the rat CRF1. Using the antiserum, CRF1‐like immunoreactivity (CRF1‐LI) was detected in the anterior lobe cells of the rat pituitary where some of them expressed intense signals. CRF1‐LI also appeared in the intermediate lobe cells and on the fibre‐like elements of the posterior lobe of the pituitary. Dual immunofluorescence labelling showed that corticotrophs exhibited the highest percentage of CRF1 (male: 27.1 ± 3.0%, female: 18.0 ± 3.0%), followed by lactotrophs (male: 6.7 ± 3.0%, female: 12.1 ± 1.3%), gonadotrophs (male: 2.6 ± 1.0%, female: 7.5 ± 0.5%), thyrotrophs (male: 2.9 ± 0.1%, female: 5.3 ± 1.2%) and somatotrophs (male: 1.1 ± 0.3%, female: 1.2 ± 0.5%). The percentage of CRF1‐LI‐positive cells that were corticotrophs was significantly higher in male rats than in female rats, whereas CRF1‐LI‐positive lactotrophs and gonadotrophs were significantly higher in female rats than in male rats. Almost all of the melanotrophs were positive for CRF1 in the intermediate lobe (98.9 ± 0.2%). CRF1‐LI and the percentage of CRF1‐LI in corticotrophs were decreased in the anterior pituitary, and the distribution patterns were altered from a diffuse to punctate one by adrenalectomy; the changes were restored by treatment with dexamethasone (100 μg/kg bw). These results suggest that CRF1 is involved in the modulation of the functions of the pituitary; moreover, protein expression and the distribution patterns of CRF1 are regulated by glucocorticoids in the rat anterior pituitary.