Ghrelin stimulates corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) play a central role in regulating the stress response. In response to stress, CRF and AVP neurons in the hypothalamic paraventricular nucleus secrete the peptides to stimulate the release of adrenocorticotropic hormone from the anterior pituitary. Ghrelin, an endogenous ligand of the growth hormone-releasing peptide receptors (GHSR), has been shown to stimulate the release of CRF and AVP by rat hypothalamic explants. However, little is known about the ability of the ghrelin signaling pathways to activate the CRF and AVP genes in the hypothalamus. In the present study, we examined the direct effect of ghrelin on CRF and AVP gene expression in hypothalamic 4B cells, which show the characteristics of the hypothalamic parvocellular paraventricular nucleus neurons. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. Ghrelin stimulated the promoter activities and mRNA levels for both CRF and AVP. The involvement of a protein kinase pathway was examined using inhibitors. Protein kinase A and phospholipase C pathways were shown to be involved in ghrelin-induced increases in both CRF and AVP promoter activities. GHSR type 1a (GHSR1a) mRNA levels were also increased by ghrelin, and these ghrelin-induced levels were suppressed by a GHSR1a antagonist. Thus, ghrelin-dependent pathways are involved in the regulation of CRF and AVP gene expression in the hypothalamus: ghrelin, an orexigenic hormone, stimulates CRF, an anorexigenic/anxiogenic factor in the hypothalamus, resulting in hypothalamic-pituitary-adrenal axis activation to stimulate the release of glucocorticoids.

Regulation of corticotropin-releasing factor and urocortin 2/3 mRNA by leptin in hypothalamic N39 cells.

Corticotropin-releasing factor (CRF) activates the pituitary-adrenal axis during stress, and shows anorectic effects via CRF type 1 receptors in the hypothalamus. Both urocortin (Ucn) 2 and Ucn3 also act as anorectic neuropeptides via CRF type 2 receptors. Leptin, a product of the obesity gene secreted mainly from adipose tissue, reduces food intake and increases energy expenditure. A possible interaction between leptin and CRF/Ucns has been suggested, as leptin can regulate expression and activation of CRF and Ucns in the hypothalamus. This study aimed to explore the possible function of leptin in the hypothalamus, and its effects in regulating CRF and Ucns. The study identified mRNA expression of the leptin receptor (Ob-R) and its subtypes, CRF, and Ucn2/3 in mouse hypothalamic N39 cells. Leptin stimulated signal transducer and activators of transcription type 3 (STAT3) phosphorylation, directly increased the mRNA levels of both CRF and Ucn2/3 in hypothalamic cells, and increased Ob-Rb mRNA levels. A Janus kinase inhibitor inhibited the leptin-mediated increase in STAT3 phosphorylation, and then the increases in CRF and Ucn2/3 mRNA levels. Leptin may contribute to a stress response or anorectic effect via the regulation of CRF and Ucn2/3 in the hypothalamus.

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells.

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.

Action of glucagon-like peptide 1 and glucose levels on corticotropin-releasing factor and vasopressin gene expression in rat hypothalamic 4B cells.

Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) are the two major regulatory peptides in the hypothalamic-pituitary-adrenal (HPA) axis. Glucagon-like peptide-1 (GLP-1), an important regulator of metabolism or energy homeostasis, is implicated in the regulation of the HPA axis in response to stress and may act directly on CRF and AVP neurons. To elucidate the direct regulation of CRF and AVP genes by GLP-1 in the hypothalamus, we examined the effect of GLP-1 in hypothalamic 4B cells, which show the characteristics of hypothalamic paraventricular nucleus neurons. The mRNA of GLP-1 receptor was detected in 4B cells by RT-PCR. GLP-1 significantly stimulated both CRF and AVP mRNA levels. Cells were transfected with CRF or AVP promoter to examine the activity of each promoter. GLP-1 directly stimulated the activities of both CRF and AVP promoters in hypothalamic 4B cells. Basal promoter activities of both CRF and AVP were increased in higher glucose medium. In addition, CRF and AVP promoter activities were increased by GLP-1 in standard or low glucose medium but not in higher glucose medium. An equimolar concentration of metabolically inactive l-glucose failed to mimic the effect of d-glucose, indicating that the event was caused by changes in glucose levels and not by hyperosmolality. Together, these data suggest that GLP-1 would contribute to stress responses through activation of CRF and AVP genes in the hypothalamic cells. Hyperglycemia may be one of the stressors enhancing the syntheses of CRF and AVP in the hypothalamus.

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells.

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.