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Choline uptake into the presynaptic terminal of cholinergic neurons is mediated by the high-affinity choline transporter and is essential for acetylcholine synthesis. In a previous study, we reported that P2X(2) purinoceptors are selectively expressed in OFF-cholinergic amacrine cells of the mouse retina. Under specific conditions, P2X(2) purinoceptors acquire permeability to large cations, such as N-methyl-D-glucamine, and therefore potentially could act as a noncanonical pathway for choline entry into neurons. We tested this hypothesis in OFF-cholinergic amacrine cells of the mouse retina. ATP-induced choline currents were observed in OFF-cholinergic amacrine cells, but not in ON-cholinergic amacrine cells, in mouse retinal slice preparations. High-affinity choline transporters are expressed at higher levels in ON-cholinergic amacrine cells than in OFF-cholinergic amacrine cells. In dissociated preparations of cholinergic amacrine cells, ATP-activated cation currents arose from permeation of extracellular choline. We also examined the pharmacological properties of choline currents. Pharmacologically, alpha,beta-methylene ATP did not produce a cation current, whereas ATP gamma S and benzoyl-benzoyl-ATP (BzATP) activated choline currents. However, the amplitude of the choline current activated by BzATP was very small. The choline current activated by ATP was strongly inhibited by pyridoxalphosphate-6-azophenyl- 2', 4'-sulfonic acid. Accordingly, P2X(2) purinoceptors expressed in HEK-293T cells were permeable to choline and similarly functioned as a choline uptake pathway. Our physiological and pharmacological findings support the hypothesis that P2 purinoceptors, including P2X(2) purinoceptors, function as a novel choline transport pathway and may provide a new regulatory mechanism for cholinergic signaling transmission at synapses in OFF-cholinergic amacrine cells of the mouse retina.
NEW & NOTEWORTHY Choline transport across the membrane is exerted by both the high-affinity and low-affinity choline transporters. We found that choline can permeate P2 purinergic receptors, including P2X(2) purinoceptors, in cholinergic neurons of the retina. Our findings show the presence of a novel choline transport pathway in cholinergic neurons. Our findings also indicate that the permeability of P2X(2) purinergic receptors to choline observed in the heterologous expression system may have a physiological relevance in vivo.

DOI： 10.1152/jn.00506.2016

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Diethylstilbestrol (DES), a strong estrogenic compound, is well-known to affect the reproductive system. In this study, we investigated the effects of DES administration on gonadotropin levels and ovarian steroidogenesis in prepubertal rats. DES treatment acutely reduced serum LH levels, followed by a reduction in the expression of various steroidogenesis-related genes in theca cells. Serum FSH levels were almost unaffected by DES-treatment, even though Cyp19a1 expression was markedly reduced. Serum progesterone, testosterone and estradiol levels were also declined at this time. LH levels recovered from 12 h after DES-treatment and gradually increased until 96 h with a reduction of ERα expression observed in the pituitary. Steroidogenesis-related genes were also up-regulated during this time, except for Cyp17a1 and Cyp19a1. Consistent with observed gene expression pattern, serum testosterone and estradiol concentrations were maintained at lower levels, even though progesterone levels recovered. DES-treatment induced the inducible nitric oxide synthase (iNOS) in granulosa cells, and a nitric oxide generator markedly repressed Cyp19a1 expression in cultured granulosa cells. These results indicate that DES inhibits thecal androgen production via suppression of pituitary LH secretion and ovarian Cyp17a1 expression. In addition, DES represses Cyp19a1 expression by inducing iNOS gene expression for continuous inhibition of estrogen production in granulosa cells.

DOI： 10.1038/s41598-017-08780-7

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Cardiomyocytes possess a non-neuronal cardiac cholinergic system (NNCCS) regulated by a positive feedback system; however, its other regulatory mechanisms remain to be elucidated, which include the epigenetic control or regulation by the female sex steroid, estrogen. Here, the NNCCS was shown to possess a circadian rhythm; its activity was upregulated in the light-off phase via histone acetyltransferase (HAT) activity and downregulated in the light-on phase. Disrupting the circadian rhythm altered the physiological choline acetyltransferase (ChAT) expression pattern. The NNCCS circadian rhythm may be regulated by miR-345, independently of HAT, causing decreased cardiac ChAT expression. Murine cardiac ChAT expression and ACh contents were increased more in female hearts than in male hearts. This upregulation was downregulated by treatment with the estrogen receptor antagonist tamoxifen, and in contrast, estrogen reciprocally regulated cardiac miR-345 expression. These results suggest that the NNCCS is regulated by the circadian rhythm and is affected by sexual dimorphism.

DOI： 10.1007/s12265-017-9750-4

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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 V 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 mu g/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.

DOI： 10.1111/jne.12440

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We previously developed cardiac ventricle-specific choline acetyltransferase (ChAT) gene-overexpressing transgenic mice (ChAT tgm), i.e. an in vivo model of the cardiac non-neuronal acetylcholine (NNA) system or non-neuronal cardiac cholinergic system (NNCCS). By using this murine model, we determined that this system was responsible for characteristics of resistance to ischaemia, or hypoxia, via the modulation of cellular energy metabolism and angiogenesis. In line with our previous study, neuronal ChAT-immunoreactivity in the ChAT tgm brains was not altered from that in the wild-type (WT) mice brains; in contrast, the ChAT tgm hearts were the organs with the highest expression of the ChAT transgene. ChAT tgm showed specific traits in a central nervous system (CNS) phenotype, including decreased response to restraint stress, less depressive-like and anxiety-like behaviours and anti-convulsive effects, all of which may benefit the heart. These phenotypes, induced by the activation of cardiac NNCCS, were dependent on the vagus nerve, because vagus nerve stimulation (VS) in WT mice also evoked phenotypes similar to those of ChAT tgm, which display higher vagus nerve discharge frequency; in contrast, lateral vagotomy attenuated these traits in ChAT tgm to levels observed in WT mice. Furthermore, ChAT tgm induced several biomarkers of VS responsible for anti-convulsive and anti-depressive-like effects. These results suggest that the augmentation of the NNCCS transduces an effective and beneficial signal to the afferent pathway, which mimics VS. Therefore, the present study supports our hypothesis that activation of the NNCCS modifies CNS to a more stress-resistant state through vagus nerve activity.

DOI： 10.1042/CS20160277

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We previously reported that satellite cells possess the ability to produce angiogenic factors, including fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF) in vivo. However, whether C2C12 cells possess a non-neuronal cholinergic system (NNCS) or non-neuronal ACh (NNA) remains to be studied; therefore, we investigated the system using C2C12 cells and its regulatory mechanisms. C2C12 cells synthesized ACh, the level of which was comparable with that of cardiomyocytes, and the synthesis was augmented by the acetylcholinesterase inhibitor galantamine. The ChAT promoter activity was upregulated by nicotine or galantamine, partly through nicotinic receptors for both agents as well as through a non-nicotinic receptor pathway for galantamine. Further, VEGF secretion by C2C12 cells was also increased by nicotine or galantamine through nicotinic receptors as well as partly through non-nicotinic pathways in the case of galantamine. These results suggest that C2C12 cells are equipped with NNCS or NNA, which is positively regulated through nicotinic or non-nicotinic pathways, particularly in the case of galantamine. These results provide a novel concept that myogenic cells expressing NNA can be a therapeutic target for regulating angiogenic factor synthesis.

DOI： 10.1016/j.intimp.2015.04.057

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Ischemic preconditioning (IPC) renders the targeted organ resistant to prolonged ischemic insults, leading to organoprotection. Among several means to achieve IPC, we reported that remote ischemic preconditioning (RIPC) activates the non-neuronal cardiac cholinergic system (NNCCS) to accelerate de novo ACh synthesis in cardiomyocytes. In the current study, we aimed to optimize a specific protocol to most efficiently activate NNCCS using RIPC. In this study, we elucidated that the protocol with 3 min of ischemia repeated three times increased cardiac ChAT expression (139.2 ± 0.4%; P < 0.05) as well as ACh (14.2 ± 2.0× 10(-8) M; P< 0.05) and ATP content (2.13 ± 0.19 μmol/g tissue; P < 0.05) in the heart. Moreover, in the specific protocol, several characteristic responses against energy starvation and for obtaining adequate energy were observed; therefore, it is suggested that RIPC evokes a robust response by the heart to activate NNCCS through the modification of energy metabolism.

DOI： 10.1016/j.intimp.2015.06.004

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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.

DOI： 10.1210/me.2012-1357

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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.

DOI： 10.1016/j.neulet.2013.02.006

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Nemoto T, Mano A, Shibasaki T. Increased expression of miR-325-3p by urocortin 2 and its involvement in stress-induced suppression of LH secretion in rat pituitary. Am J Physiol Endocrinol Metab 302: E781-E787, 2012. First published January 17, 2012; doi: 10.1152/ajpendo.00616.2011.-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 beta-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 beta-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.

DOI： 10.1152/ajpendo.00616.2011

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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 4 h and 8 h and with ghrelin for 4 h 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. (C) 2011 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.peptides.2011.04.007

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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.

DOI： 10.1016/j.regpep.2009.11.010

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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.

DOI： 10.1677/JOE-08-0374

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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.

DOI： 10.1016/j.neulet.2009.03.054

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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.

DOI： 10.1016/j.psyneuen.2008.09.003

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GH secretagogue (GHS)/ghrelin stimulates GH secretion by binding mainly to its receptor (GHS-R) on GHRH neurons in the arcuate nucleus (Arc) of the hypothalamus. GHRH, somatostatin, and neuropeptide Y (NPY) in the hypothalamus are involved in the regulatory mechanism of GH secretion. We previously created transgenic (Tg) rats whose GHS-R expression is reduced in the Arc, showing lower body weight and shorter nose-tail length. GH secretion is decreased in female Tg rats. To clarify how GHS-R affects GHRH expression in the Arc, we compared the numbers of GHS-R-positive, GHRH, and NPY neurons between Tg and wild-type rats. Immunohistochemical analysis showed that the numbers of GHS-R-positive neurons, GHRH neurons, and GHS-R-positive GHRH neurons were reduced in Tg rats, whereas the numbers of NPY neurons and GHS-R-positive NPY neurons did not differ between the two groups. The numbers of Fos-positive neurons and Fos-positive GHRH neurons in response to KP-102 were decreased in Tg rats. Competitive RT-PCR analysis of GHRH mRNA expression in the cultured hypothalamic neurons showed that KP-102 increased NPY mRNA expression level and that NPY decreased GHRH mRNA expression level. KP-102 increased GHRH mRNA expression level in the presence of anti-NPY IgG. GH increased somatostatin mRNA expression. Furthermore, GH and somatostatin decreased GHRH mRNA expression, whereas KP-102 showed no significant effect on somatostatin mRNA expression. These results suggest that GHS-R is involved in the up-regulation of GHRH and NPY expression and that NPY, somatostatin, and GH suppress GHRH expression. It is also suggested that the reduction of GHRH neurons of Tg rats is induced by a decrease in GHS-R expression.

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Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.

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RATIONALE: Stress-induced inhibition of food intake is reportedly blocked by a selective corticotropin-releasing factor (CRF) type 1 receptor (CRF1) antagonist, suggesting the involvement of CRF1 in the inhibitory mechanism. CRF1 and CRF2 are considered to function in the inhibition of food intake by CRF-related peptides with different time courses. OBJECTIVES: This study was designed to clarify whether CRF2 is also involved in stress-induced inhibition of food intake and to examine the relation of CRF1to CRF2 in the inhibitory mechanism. METHODS: Antisauvagine-30 (AS-30), a selective CRF2 antagonist, and/or CRA1000, a selective CRF1 antagonist, were pre-administered intracerebroventricularly and intraperitoneally, respectively, to male Wistar rats deprived of food for 24 h before the animals were exposed to a 1-h period of stressors and food intake in 1 h after stress exposure was examined. The effect of both antagonists on locomotor activity was also examined. RESULTS: Pre-administration of 5-30 microg of AS-30 attenuated inhibition of food intake induced by restraint, electric footshock or emotional stress using a communication box. CRA1000 also attenuated the restraint-induced inhibition of food intake at doses of 5 and 10 mg/kg body weight. The reversal of restraint-induced inhibition of food intake by co-administration of AS-30 and CRA1000 was not larger than that by AS-30 or CRA1000 alone. Both antagonists did not affect locomotor activity. CONCLUSIONS: These results suggest that not only CRF1, but also CRF2, are involved in stress-induced inhibition of food intake, and that both subtypes of CRF receptor function probably in series in 1 h after stress exposure.

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In adipocytes, peroxisome proliferator-activated receptor (PPAR)-gamma activates adipocyte differentiation and glucocorticoid (GC) stimulates the expression of PPAR-gamma mRNA. The local tissue concentrations of GC, in turn, are modulated by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). To clarify the change of energy metabolism in condition of reduced energy intake, we investigated whether food restriction alters the adipocyte size and levels of PPAR-gamma, GC receptor (GR), and 11beta-HSD1 mRNA expression in the white adipose tissues of normal rats. Male Wistar rats weighing 340 g were housed under free feeding or 20% reduction of food intake for 2 or 14 days. We found that 2-day food restriction did not cause any change in the mean size or number of adipocytes in the omentum, while 14-day food restriction decreased the size and increased the number of adipocytes. In addition, the levels of PPAR-gamma2, GR, and 11beta-HSD1 mRNA expression in the omentum were lower in the food-restricted rats after 2 days, while they did not differ after 14 days. Also, after both 2 and 14 days, plasma concentrations of free fatty acid (FFA) were higher in the food-restricted rats than in control rats. Finally, plasma concentrations of adrenocorticotropin (ACTH) and corticosterone were the same in the both groups after 2 days, although they were higher in the food-restricted rats after 14 days. These results suggest that adipocyte differentiation in the omentum of food-restricted rats is attenuated after 2 days but recovers after 14 days, resulting in an increase in the number of small adipocytes. It is also likely that lipolysis induced during the 14-day period of food restriction decreased the size of adipocytes. Further, food restriction may affect the efficiency of local GC effects by altering GR and 11beta-HSD1 mRNA expression. Also, higher levels of plasma GC and recovery of GR and 11beta-HSD1 mRNA expression may contribute to the recovery of the levels of PPAR-gamma2 mRNA expression in the omentum and result in the recovery of adipocyte differentiation.

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Growth hormone secretagogues (GHSs) stimulate GH secretion and food intake. GHS receptor (GHS-R) mRNA has been identified mainly in the arcuate nucleus (Arc) and ventromedial nucleus of the hypothalamus and in the pituitary. Ghrelin, an endogenous ligand for GHS-R, has recently been purified from rat stomach. Although ghrelin is also expressed in the hypothalamus, the physiological significance of the ghrelin/GHS-R system is still unknown. We have created transgenic (Tg) rats expressing an antisense GHS-R mRNA under the control of the promoter for tyrosine hydroxylase (TH), thus selectively attenuating GHS-R protein expression in the Arc. Tg rats had lower body weight and less adipose tissue than did control rats. Daily food intake was reduced, and the stimulatory effect of GHS treatment on feeding was abolished in Tg rats. GH secretion and plasma insulin-like growth factor-I levels were reduced in female Tg rats. These results suggest that GHS-R in the Arc is involved in the regulation of GH secretion, food intake, and adiposity.

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DOI： 10.1016/s1388-2457(02)00019-6

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Root conduction time (RCT), defined as the time difference between M-wave latency by cervical magnetic stimulation (CMS) and the total peripheral motor conduction time calculated from the shortest F-wave latency, was investigated in patients with inflammatory demyelinating neuropathies (IDP) and amyotrophic lateral sclerosis (ALS). The minimal threshold for CMS also was studied. In the IDP patients, conduction in the proximal motor root segment was considered abnormal in 52% by the RCT and in 47% by the minimal threshold for CMS, whereas both were normal in 85% of the ALS patients. These findings suggest that the RCT and minimal threshold for CMS might be additional parameters for evaluating motor nerve conduction in IDP.

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Publishing type：Research paper (scientific journal)   Publisher：American Physiological Society  

We attempted to clarify whether leptin and uncoupling protein 1 (UCP1) are involved in the action of nicotine on the energy balance. Male Wistar rats were infused subcutaneously with nicotine (12 mg · kg⁻¹ · day⁻¹) for 4 or 14 days. At the end of the 4-day period, the plasma concentrations of leptin of the nicotine-treated and pair-fed rats were lower than those of the freely fed rats, although the levels of leptin mRNA expression in various white adipose tissues did not differ among the three groups. At the end of the 14-day nicotine infusion period, plasma concentrations of leptin were higher, and leptin mRNA expression in the omentum and epididymal and retroperitoneal adipose tissues was stronger in the nicotine-treated rats than in the pair-fed and freely fed rats. UCP1 mRNA expression in the brown adipose tissue of nicotine-treated was stronger than that of the pair-fed rats. These results suggest that continuous nicotine infusion differentially affects the synthesis and secretion of leptin according to the duration of infusion and stimulates UCP1 mRNA expression, probably in a manner independent of leptin.

DOI： 10.1152/ajpendo.2001.280.6.e867

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Publishing type：Research paper (scientific journal)  

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Publishing type：Research paper (scientific journal)  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：間脳・下垂体・副腎系研究会  

副腎皮質刺激ホルモン放出因子(CRF)1とCRF2の非共通部分を多く含むアミノ酸配列のフラグメントを抗原としてCRF1の特異的抗体を作成し、下垂体におけるCRF1様免疫活性の局在を解明しようと試みた。CRF1フラグメントを添加した抗体を用いた免疫染色では、CRF1の免疫活性は完全に消失していたが、CRF2フラグメントを添加した抗体を用いた免疫染色では、CRF1の免疫活性は変わらなかった。前葉ではCRF1の免疫活性は点在していた。約40%のコルチコトロフにCRF1の発現が認められ、それに比べその他のホルモン産生細胞でのCRF1の発現は少なかった。中葉では免疫活性は前葉ほど強くはないが、殆ど全てのメラノトロフにCRF1の免疫活性が認められた。後葉では前中葉と比べると免疫活性は弱く、点状に存在し、核周囲には殆ど認められないことからCRF1の免疫活性は神経線維に存在すると思われた。

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(公財)成長科学協会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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J-GLOBAL

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(公財)成長科学協会  

ラットを用いて高脂肪摂取下でのエネルギー代謝調節機構における成長ホルモン(GH)とグレリン及びグレリン受容体(GHSR)の役割の詳細を調べた。8週齢のSDラットを標準食を与えた対照群と高脂肪食を2週間与えたHFD群に分けた。HFD群の14日間の体重増加及び摂取カロリーは対照群より有意に高かった。HFD群では対照群と比較して血中IGF-I値及びBATのUcp1発現量は増加傾向を示し、下垂体のGH発現量は有意に低値を示し、視床下部のGHSR及びグレリン発現量は2群間で有意差はみられなかった。グレリンを脳室内に投与後BAT内のNA分泌量を測定すると、対照群では投与から40分間NA分泌が約50%まで抑制され、HFD群では投与から20分間NA分泌が約70%まで抑制された。

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(公財)成長科学協会  

加齢に伴う体脂肪蓄積機序におけるGHとグレリンの関与様式をラットで検討した。副睾丸周囲と後腹膜の体重100g当たりの脂肪重量は週齢を経るごとに増加したが、腸間膜の体重100g当たりの脂肪重量は、20週において有意に増加した。20週齢の血中グルコース濃度は4週齢と比較して有意に高値を示した。血中インスリン濃度およびIGF1濃度は8週齢と20週齢で高値を示した。褐色脂肪組織のUCPI mRNA発現量は4週齢が最も多かった。下垂体におけるGH mRNA発現量では、20週齢は4週齢と比較して有意に低値を示した。視床下部におけるGHSR mRNA発現量は20週齢において4週齢と比較し有意に多かった。4週齢及び8週齢ラット脳室内へのグレリン投与は基礎分泌および生理食塩水投与群と比較してノルアドレナリンの有意な分泌抑制をもたらした。

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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J-GLOBAL

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(公財)成長科学協会  

growth hormone secretagogue(GHS)がgrowth hormone-releasing factor(GRF)の発現調節に関与しているか検討するため,ラットから摘出した視床下部をGHS含有培養液で培養後,全RNAを抽出してRT-PCR法でGRF mRNA発現量を測定し対照ラットと比較した.結果,発現量は対照ラットよりも多く,GHSがGRF発現調節に促進的に関与している可能性が示唆された.次に,エネルギー調節に対するGHSRの関与を検討するため,GHSR発現抑制トランスジェニックラット(Tgラット)から褐色脂肪組織,白色脂肪組織(皮下,大網,後腹膜,副睾丸周囲)を採取し組織重量を計測するとともに,酸素消費量,二酸化炭素産生量を測定して呼吸商を算出し,対照ラットと比較した.褐色脂肪組織重量には有意差を認めなかった.白色脂肪組織では皮下,大網に有意差なく,後腹膜と副睾丸周囲の重量はTgラットが有意に軽かった.酸素消費量,二酸化炭素産生量はともにTgラットが高値を示し,呼吸商には有意差を認めなかった

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：間脳・下垂体・副腎系研究会  

anti-sauvagine-30(AS-30)を脳室内投与後に拘束,フットショック,心理ストレスを負荷し,ストレス負荷後1時間の摂食量を測定して,各々のストレスによる摂食抑制機序へのCRFR2の関与の有無を検討した.又,CRA1000及びAS-30の両投与の拘束による摂食抑制への影響を検討した.拘束,フットショック,心理ストレスなどのストレスによる摂食抑制をCRFR2選択的拮抗薬であるAS-30が阻止し,摂食量を回復させ,CRFR2がストレス時の摂食抑制機序に関与していることを示唆された.よって,拘束,フットショック,心理ストレスによる摂食抑制機序にはCRFR1のみならずCRFR2も関与していることが示唆された

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(公財)成長科学協会  

トランスジェニック(TG)ラットの視床下部におけるgrowth hormone secretagogue(GHS)受容体(GHSR),GH-releasing factor(GRF),ソマトスタチン(SRIF)ニューロンを定量し,GH分泌機序における視床下部GHSRの役割を解明した.TGラットの弓状核のGHS-R陽性ニューロン数はWTラットの約30%に減少していた.弓状核の外側部に認められるGRFニューロンは,TGラットでWTラットの57%に有意に減少し,両ラット群のGHSR陽性ニューロン数とGRFニューロン数との関係には正の相関が認められた.室周囲核でのSRIFニューロン数にはWTラットとTGラットとの間に有意差は認められなかった.弓状核でのSRIFニューロン数も室周囲核と同様にWTラットとTGラットとの間で有意差は認められなかった

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Language：Japanese   Publisher：(株)北隆館  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：日本ストレス学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(公財)成長科学協会  

GHSRのアンチセンス遺伝子の導入により,視床下部弓状核のGHSRが減少したトランスジェニックラットを用いて,その成長ホルモン分泌パターン,GHSの一つであるKP-102に対する成長ホルモンの反応パターンと視床下部GHSR発現ニューロンの変化とを解析し,成長ホルモン分泌調整におけるGHSRの役割について考察した.GHSRは視床下部弓状核のGRFニューロンとNPYニューロンに共存し,GHSRが摂食,脂肪量,GH分泌等の調節に関与していることが明らかになった

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：間脳・下垂体・副腎系研究会  

副腎皮質刺激ホルモン放出因子(CRF)ペプチドファミリーのウロコルチン(Ucn) IIおよびUcn III におけるUcn IIの免疫組織化学検索と摂食行動,運動活動量に与える両ペプチドの作用をマウスとラットを用いて検討した.免疫組織化学方法ではマウス・ラット共に視床下部室傍核に陽性細胞が多く認められ,視床下部弓状核において少量の陽性細胞と多数の陽性線維が認められた.Ucn IIは投与後6時間より9時間で,Ucn IIIは投与後4時間より8時間で摂食量がvihicleを投与したラットに比べ有意に減少した.ラットにおける両ペプチドの投与による摂食量の減少は見られなかった.ホームゲージにおける運動活動量は,CRF投与後4時間にわたり活動量の上昇が見られ,Ucn IIIのみを投与したラットでは活動量を低下させることが示された.また,CRFによる行動変化に対してUcn IIが拮抗して作用することが示唆された

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Language：Japanese   Publisher：(一社)日本神経学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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Language：Japanese   Publisher：(一社)日本内分泌学会  

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