Impaired miR449a-induced downregulation of Crhr1 expression in low-birth-weight rats.

Low birth weight (LBW) is related to increased incidence of common cardiovascular and metabolic disorders, and psychopathologies later in life. Recent studies have suggested that maternal malnutrition affects fetal hypothalamic-pituitary-adrenal (HPA) axis programing although the mechanism is unknown. We demonstrated that LBW offspring delivered from malnourished dams showed prolonged elevated plasma corticosterone concentrations when compared with those of normal-birth-weight (NBW) offspring and impaired downregulation of corticotropin-releasing factor receptor type 1 (CRF-R1, Crhr1) in the anterior pituitary in restraint. Restraint increased expression of miR449a, which we had previously demonstrated to be involved in Crhr1 downregulation, in the anterior pituitary and serum exosomal miR449a contents through glucocorticoids in NBW offspring, but not in LBW offspring. Although plasma corticosterone concentrations were higher at 2000 h than at 0800 h in both LBW and NBW offspring, they were significantly higher in LBW offspring than in NBW offspring at 2000 and 0200 h. There were no significant diurnal changes in miR449a expression levels in the anterior pituitary of either NBW or LBW offspring, but the expression was significantly lower in LBW offspring than in NBW offspring at 1400, 2000, and 0200 h. The expression levels of GAS5, which inhibits glucocorticoid receptor (GR) binding to glucocorticoid-responsive element, in the anterior pituitary of LBW offspring were elevated when compared with those of NBW offspring. The downregulation of GR found in NBW offspring did not occur in restrained LBW offspring. These results indicate that impaired miR449a expression, probably induced by increased GAS5 expression, causes dysregulation of Crhr1 expression in the anterior pituitary, resulting in prolonged HPA axis activation in restrained LBW offspring.

Estrogens downregulate urocortin 2 expression in rat uterus.

Urocortin 2 (Ucn2) is a member of the corticotropin-releasing factor peptide family and is expressed by various tissues, including reproductive tissues such as the uterus, ovary, and placenta. However, the regulatory mechanisms of Ucn2 expression and the physiological significance of Ucn2 in these tissues remain unclear. We previously showed that passive immunization of immature female rats by i.p. injection of anti-Ucn2 IgG induces earlier onset of puberty. Therefore, this study was designed to clarify the site and regulatory mechanisms of Ucn2 expression in the uterus. Expression levels of Ucn2 mRNA in the uterus were higher in immature (2- and 4-week-old) and aged (17-month-old) rats than in mature (9-week-old) rats in the proestrus phase. In 9-week-old rats, mRNA expression levels and contents in the uterus were lower in the proestrus phase than in the diestrus phase, while plasma Ucn2 concentrations did not differ between the two phases. Ucn2-like immunoreactivitiy was detected in the endometrial gland epithelial cells of the uterus. S.c. injection of estradiol benzoate or an estrogen receptor α (ERα) agonist significantly reduced mRNA expression levels and contents of Ucn2 in the uterus when compared with vehicle-injected ovariectomized rats. By contrast, estradiol benzoate increased Ucn2 mRNA expression levels in the lung. Thus, estrogens downregulate Ucn2 expression in the uterus in a tissue-specific manner, and Ucn2 may play a role in the regulatory mechanisms of maturation of the uterus through ERα and estrous cycle.

miR-449a contributes to glucocorticoid-induced CRF-R1 downregulation in the pituitary during stress.

The hypothalamic-pituitary-adrenal axis is controlled by the feedback of glucocorticoids on the hypothalamus and pituitary. Stress increases CRF, ACTH, and glucocorticoid secretion. The expression of not only CRF mRNA in the hypothalamus and proopiomelanocortin mRNA in corticotrophs, but also CRF type 1 receptor (CRF-R1) mRNA and protein on corticotrophs are downregulated through glucocorticoids. However, the mechanisms underlying the glucocorticoid-induced CRF-R1 downregulation are not fully understood. Short RNA molecules, called microRNAs (miRNAs), are posttranscriptional regulators that usually induce translational repression or gene silencing via binding to complementary sequences within target mRNAs. We hypothesized that glucocorticoids may induce the expression of miRNAs in the pituitary, which are involved in glucocorticoid-induced downregulation of CRF-R1. We found 3 miRNAs with sequences predicted to bind to the CRF-R1 3' untranslated region (3'-UTR) by database search. Expression of 1 of these miRNAs (miR-449a) was significantly higher in the anterior pituitary of restrained rats than in that of unrestrained control rats. Expression of miR-449a was evident in many anterior pituitary cells, including corticotrophs. Although overexpression of miR-449a decreased CRF-R1 mRNA and CRF-R1 protein expression, knockdown of miR-449a attenuated dexamethasone-induced suppression of CRF-R1 mRNA and CRF-R1 protein expression in the monolayer-cultured pituitary cells. Notably, luciferase activity was significantly lower in cells cotransfected with a luciferase vector containing the CRF-R1 3'-UTR and a miR-449a vector. miR-449a expression was significantly increased by dexamethasone. Adrenalectomy attenuated restraint-induced increase in miR-449a expression in the pituitary. These results indicated that miR-449a plays an important role in stress-induced, glucocorticoid-mediated downregulation of CRF-R1 expression.

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.

Vagus nerve stimulation induced long-lasting enhancement of synaptic transmission and decreased granule cell discharge in the hippocampal dentate gyrus of urethane-anesthetized rats.

Vagus nerve stimulation (VNS) ameliorates deficits of hippocampal functions, such as contextual learning and memory, probably through direct modulation of neuronal activity. Previous studies showed that VNS enhanced excitatory synaptic transmission in the hippocampal CA3 area via activation of ß-adrenergic receptors. However, effects of VNS on excitatory synaptic transmission and action potential (AP) discharge of granule cells (GCs) in the dentate gyrus have not been studied. Urethane-anesthetized rats were used to investigate whether VNS influences synaptic transmission efficacy at perforant path-GC synapses and population spike discharge in the dentate gyrus. Intermittent burst stimulation of the left vagus nerve (30Hz for 30s at an inter-train interval of 5min for 1h) significantly enhanced the perforant path-GC synaptic transmission efficacy in the dentate gyrus for at least 2h, indicating that VNS can induce a long-lasting enhancement of synaptic transmission in this area, similar to the situation observed in the CA3 area. In contrast, a 60-min period of VNS significantly reduced population spike amplitude (a parameter reflecting synchronized AP discharge of GCs) for a given excitatory postsynaptic potential. These findings suggest that acute VNS enhances the excitatory synaptic transmission and reduces synchronized AP discharge of GCs in the dentate gyrus. It is likely that enhancement of excitatory synaptic transmission and reduction of GC excitability contribute VNS treatment efficacy for learning deficits and intractable epilepsy, respectively.

Therapeutic potential of ghrelin in restricting-type anorexia nervosa.

Anorexia nervosa (AN) is an eating disorder characterized by a decrease in caloric intake and malnutrition. It is associated with a variety of medical morbidities as well as significant mortality. Nutritional support is of paramount importance to prevent impaired quality of life later in life in affected patients. Some patients with restricting-type AN who are fully motivated to gain body weight cannot increase their food intake because of malnutrition-induced gastrointestinal dysfunction. Chronicity of AN prevents participation in social activities and leads to increased medical expenses. Therefore, there is a pressing need for effective appetite-stimulating therapies for patients with AN. Ghrelin is the only orexigenic hormone that can be given intravenously. Intravenous infusion of ghrelin is reported to increase food intake and body weight in healthy subjects as well as in patients with poor nutritional status. Here, we introduce the results of a pilot study that investigated the effects of ghrelin on appetite, energy intake, and nutritional parameters in five patients with restricting-type AN, who are fully motivated to gain body weight but could not increase their food intake because of malnutrition-induced gastrointestinal dysfunction.

Increased expression of miR-325-3p by urocortin 2 and its involvement in stress-induced suppression of LH secretion in rat pituitary.

Urocortin 2 (Ucn2) is a member of the corticotropin releasing factor (CRF) peptide family, which binds to CRF type 2 receptor. We previously reported on expression of Ucn2 in proopiomelanocortin cells of rat pituitary and its inhibitory action on LH secretion. We also demonstrated that Ucn2 is involved in the mechanism underlying immobilization-induced suppression of LH secretion; the details remain unclear. Here, we found that Ucn2 increased the expression of miR-325-3p, one of three microRNAs with predicted sequence for binding to LH ß-subunit 3'-untranslated region (3'-UTR) in monolayer cultured rat anterior pituitary cells, and that miR-325-3p was expressed in LH cells of the anterior pituitary. Immobilization also increased miR-325-3p expression in the anterior pituitary, and its increase was blocked by pretreatment with anti-Ucn2 IgG. Overexpression of miR-325-3p in cultured pituitary cells significantly suppressed intracellular contents and secretion of LH, while miR-325-3p knockdown blocked Ucn2-induced suppression of intracellular contents and secretion of LH. Coexpression of miR-325-3p with LH ß-subunit 3'-UTR-fused luciferase vector significantly suppressed luciferase activity compared with that of mock transfectants. These results suggest that miR-325-3p is involved in immobilization-induced suppression of LH translation and secretion and that Ucn2 plays a role in the increase in miR-325-3p expression.

Microinjection of different doses of corticotropin-releasing factor into the medial prefrontal cortex produces effects opposing anxiety-related behavior in rats.

Corticotropin-releasing factor (CRF) in the medial prefrontal cortex (mPFC) is suggested to play an important role in mediating fear, anxiety, and depression. The results of the studies of the actions of CRF in the mPFC regarding anxiety-related behavior, however, seem contradictory. In one study, microinjection of CRF into the mPFC produced an increase in anxiety-related behavior on the elevated plus maze, whereas in another study CRF produced an anxiolytic-like effect. To test whether the different doses of CRF used in these experiments are responsible for the differing results, we examined the dose-dependent effects of CRF (0.015, 0.05, 0.15, 0.5, and 1.0 µg/0.5 µL/site) microinjected into the bilateral mPFC of male Wistar rats on anxiety-related behavior in the elevated plus maze. We found that microinjection of 0.05 µg CRF significantly decreased the number of open-arm entries, whereas 1.0 µg CRF significantly increased the time spent on the open arms. The results indicate that CRF has effects opposing anxiety-related behavior in the elevated plus maze: anxiety-related behavior at a lower dose and an anxiolytic-like effect at a higher dose.

Corticotropin-releasing factor (CRF) receptor subtypes in mediating neuronal activation of brain areas involved in responses to intracerebroventricular CRF and stress in rats.

Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF(1)) and CRF2 receptor (CRF(2)). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF(1) and CRF(2), are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF(1)- and CRF(2)-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF(1) and/or CRF(2). However, the manner of involvement for CRF(1) and CRF(2) in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF(1&2)-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.

Involvement of CRF2 receptor in the brain regions in restraint-induced anorexia.

We have reported that corticotropin-releasing factor (CRF) receptor subtypes, CRF1 and CRF2, are involved in stress-induced anorexia. To clarify in which brain regions the CRF receptor is involved in mediating stress-induced anorexia, we examined the effect of microinjecting CRF1-selective or CRF2-selective antagonist into the lateral septum or the bed nucleus of the stria terminalis (BNST), which are implicated in regulating stress response. The results demonstrated that injecting antisauvagine-30 into the lateral septum or the BNST significantly attenuated restraint-induced anorexia, whereas injecting antalarmin into these regions did not affect anorexia. These results suggest that the CRF2 receptor in the lateral septum and the BNST is involved in the stress-induced inhibitory mechanism of feeding behavior.

The effects of ghrelin/GHSs on AVP mRNA expression and release in cultured hypothalamic cells in rats.

Ghrelin, the endogenous ligand for growth hormone secretagogues (GHSs) receptor (GHS-R), increases adrenocorticotropin (ACTH) and cortisol (corticosterone) as well as GH secretion in humans and animals. However, the site of GHSs action to induce ACTH secretion is not fully understood. To clarify the mechanisms of the action of ghrelin/GHSs on ACTH secretion, we analyzed the effects of KP-102 and ghrelin on the mRNA expression and release of corticotropin releasing factor (CRF) and arginine vasopressin (AVP), ACTH secretagogues, in monolayer-cultured hypothalamic cells of rats. Incubation of cells with KP-102 for 4h and 8h and with ghrelin for 4h significantly increased AVP mRNA expression and release without changing CRF mRNA expression. CRF levels in culture media were undetectable. Suppression of GHS-R expression by siRNA blocked ghrelin- and KP-102-induced AVP mRNA expression and release. NPY significantly increased AVP mRNA expression and release. Furthermore, treatment of cells with anti-NPY IgG blocked KP-102-induced AVP mRNA expression and release. We previously reported that KP-102 significantly increases NPY mRNA expression in cultured hypothalamic cells. Taken together, these results suggest that ACTH secretion by ghrelin/GHSs is induced mainly through hypothalamic AVP, and that NPY mediates the action of ghrelin/GHSs.

Role of urocortin 2 secreted by the pituitary in the stress-induced suppression of luteinizing hormone secretion in rats.

We have previously shown that urocortin 2 (Ucn 2), a member of the corticotropin-releasing factor (CRF) peptide family that binds to CRF type 2 receptor, is expressed in proopiomelanocortin (POMC) cells of rat pituitary and that its secretion and expression are increased by CRF in both the anterior and intermediate lobes and suppressed by glucocorticoids in the anterior lobe. We have also shown that Ucn 2 secreted by POMC cells acts on gonadotrophs expressing CRF type 2 receptors and inhibits the expression and secretion of gonadotropins. In the present study, we examined whether pituitary Ucn 2 is involved in stress-induced inhibition of gonadotropin secretion. A 90-min period of immobilization stress increased POMC mRNA expression without influencing Ucn 2 mRNA expression and suppressed luteinizing hormone (LH) ß-subunit mRNA expression in the anterior lobe and plasma LH levels, while it increased both POMC and Ucn 2 mRNA expression in the intermediate lobe of the pituitary. Pretreatment with anti-CRF IgG blocked immobilization-induced increases in plasma ACTH and corticosterone and in POMC mRNA expression in both pituitary lobes and Ucn 2 mRNA expression in the intermediate pituitary. It also blocked immobilization-induced suppression of plasma LH and LH ß-subunit mRNA expression. Pretreatment with anti-Ucn 2 IgG blocked immobilization-induced suppression of plasma LH and LH ß-subunit expression without affecting immobilization-induced ACTH and corticosterone release and POMC or Ucn 2 mRNA expression. These results suggest that CRF suppresses the secretion and expression of LH probably through pituitary Ucn 2 in stress.

Levetiracetam suppresses development of spontaneous EEG seizures and aberrant neurogenesis following kainate-induced status epilepticus.

Electroencephalographic (EEG) seizures and behavioral convulsions begin to appear spontaneously a few weeks after chemoconvulsant-induced status epilepticus (SE) and thereafter become more intense. This indicates the progressive development of a long-lasting epileptic focus. In addition, chemoconvulsant-induced SE increases neuronal proliferation in the dentate subgranular zone (SGZ) and ectopic migration of newborn neurons into the dentate hilus of adult animals. These seizure-induced newborn neurons, especially ectopic granule cells in the dentate hilus, are believed to facilitate the development of epileptic foci in animal models of temporal lobe epilepsy. In the present study, we examined the effects of a novel antiepileptic drug, levetiracetam, on the appearance of spontaneous EEG seizures and on the generation of newborn neurons, especially of ectopic granule cells in the dentate hilus, following kainate-induced SE. Levetiracetam treatment for 25 days, initiated 24 hours after induction of kainate-induced SE, significantly decreased the mean duration of spontaneous EEG seizures 58 days later. Levetiracetam treatment also prevented an SE-induced increase in the number of ectopic granule cells observed 58 days after kainate administration by suppressing neuronal proliferation in the dentate SGZ and abnormal migration of newborn neurons from the dentate SGZ to the hilus. These results are in accord with a previous report that an antimitotic agent that reduced the number of newborn neurons significantly decreased the frequency of spontaneous convulsions 1 month after pilocarpine-induced SE. This evidence from the kainate model of temporal lobe epilepsy suggests that levetiracetam may exert antiepileptogenic effects through the suppression of seizure-induced neurogenesis.

Comparison of pituitary-adrenal responsiveness between insulin tolerance test and growth hormone-releasing peptide-2 test: a pilot study.

Insulin tolerance test (ITT) is the gold standard for assessing the hypothalamic-pituitary-adrenal (HPA) function. GH-releasing peptide (GHRP)-2, which has a strong GH-stimulating activity, is useful for diagnosing GH deficiency as well as ITT. Additionally, GHRP-2 is also known to activate HPA axis. There have been no comparative studies of pituitary-adrenal responsiveness between GHRP-2 test and ITT in patients with hypothalamic/pituitary disease. To assess whether GHRP-2 test could be an alternative to ITT for diagnosing HPA axis failure, both ITT and GHRP-2 test were performed in 15 patients suspected of hypopituitarism. A 100mug dose of GHRP-2 was administered intravenously and plasma ACTH and serum cortisol concentrations were measured. In ITT, a peak cortisol value over 18mug/dl is considered normal. Nine patients were diagnosed as HPA axis failure by ITT. Their median peak cortisol in GHRP-2 test was 11.4mug/ml. In 6 patients diagnosed as normal HPA axis status by ITT, their median peak cortisol in response to GHRP-2 test was 21.4mug/dl, significantly higher (p=0.0032) than seen in patients diagnosed as HPA axis failure. There was a strong correlation between the peak cortisol in GHRP-2 test and ITT (r=0.817; p<0.0001). When the cut-off value for the peak cortisol in GHRP-2 test was set to 13-14mug/dl for diagnosing HPA axis failure, the specificity and sensitivity were 100% and 88.9%, respectively. Although further studies that include normal subjects are needed, these preliminary results suggest the possibility that GHRP-2 test may be an alternative to ITT for assessing HPA axis function.

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.

Ghrelin increases hunger and food intake in patients with restricting-type anorexia nervosa: a pilot study.

Ghrelin increases hunger sensation and food intake in various patients with appetite loss. Anorexia nervosa (AN) begins with psychological stress-induced anorexia and some patients cannot increase their food intake partly because of malnutrition-induced gastrointestinal dysfunction. The effects of ghrelin on appetite, food intake and nutritional parameters in anorexia nervosa (AN) patients were examined. Five female restricting- type AN patients (age: 14-35 y; body mass index: 10.2-14.6 kg/m(2)) had persistently complained of gastrointestinal symptoms and failed to increase body weight. They were hospitalized for 26 days (6 days' pretreatment, 14 days' ghrelin-treatment, and 6 days' post-treatment) and received an intravenous infusion of 3 microg/kg ghrelin twice a day. Ghrelin infusion improved epigastric discomfort or constipation in 4 patients, whose hunger scores evaluated by visual analogue scale questionnaires also increased significantly after ghrelin infusion. Daily energy intake during ghrelin infusion increased by 12-36 % compared with the pre-treatment period. Serum levels of total protein and triglyceride as nutritional parameters significantly increased after ghrelin treatment. There were no serious adverse effects including psychological symptoms. We found that ghrelin decreases gastrointestinal symptoms and increases hunger sensation and daily energy intake without serious adverse events in AN patients. Although the present study had major limitations of the lack of a randomized, placebo-controlled group, non-blindness of the investigators and the small number of patients recruited, it would contribute to further investigations for therapeutic potential of ghrelin in AN patients.

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.

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.