GLP-1 increases Kiss-1 mRNA expression in kisspeptin-expressing neuronal cells.

Feeding-related metabolic factors exert regulatory influences on the hypothalamic-pituitary-gonadal axis. Glucagon-like peptide-1 (GLP-1) is an anorexigenic hormone synthesized from the ileum in response to food intake. The purpose of this study was to examine the direct effect of GLP-1 on hypothalamic kisspeptin and gonadotropin-releasing hormone (GnRH) expression using the rat clonal hypothalamic cell line rHypoE-8. GLP-1 significantly increased Kiss-1 mRNA expression in rHypoE-8 cells up to 1.94 ± 0.22-fold. This effect of GLP-1 on Kiss-1 gene expression was also observed in GT1-7 GnRH-producing neurons and in primary cultures of fetal rat brain. GLP-1 increased cAMP-mediated signaling, as determined by cAMP response element activity assays, but failed to activate extracellular signal-regulated kinase pathways. Another anorexigenic factor, leptin, similarly increased Kiss-1 mRNA levels up to 1.34 ± 0.08-fold in rHypoE-8 cells. However, combined treatment with GLP-1 and leptin failed to potentiate their individual effects on Kiss-1 mRNA expression. Gnrh mRNA expression was not significantly increased by GLP-1 stimulation in rHypoE-8, but kisspeptin significantly stimulated the expression of Gnrh mRNA in these cells. Our current observations suggest that the anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in these hypothalamic cell lines and in neuronal cells of fetal rat brain and affects the expression of Gnrh mRNA.

Expression of gonadotropin-inhibitory hormone receptors in mouse pituitary gonadotroph LßT2 cells and hypothalamic gonadotropin-releasing hormone-producing GT1-7 cells.

Gonadotropin-inhibitory hormone (GnIH) was first identified in quail as a novel neurohormone that acts directly on the anterior pituitary to inhibit gonadotropin release. GnIH inhibits not only gonadotropin release from the pituitary gland but also inhibits the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. In this study, we examined how GnIH receptors were regulated in pituitary gonadotroph cells and GnRH-producing neurons in the hypothalamus. In the mouse pituitary gonadotroph cell line LßT2, GnRH increased expression of the GnIH receptor, G-protein coupled receptor 74 (GPR74). GnRH also stimulated the expression of GPR74 and GPR147 in primary cultures of rat anterior pituitary cells. In addition, when GnRH was administered to LßT2 cells in a pulsatile manner, low frequency GnRH pulse stimulation stimulated GPR74 and GPR147 expression more than did high frequency GnRH pulses. In the mouse hypothalamic GnRH-producing cell line GT1-7, hypothalamic kisspeptin did not significantly increase the expression of GnIH receptors. However, the intermittent administration of kisspeptin to GT1-7 cells significantly increased GPR74 and GPR147 mRNA expression. The overexpression of either constitutively active MEK kinase (MEKK) or protein kinase A (PKA) in LßT2 cells increased the expression of GPR74 mRNA. Conversely, in GT1-7 cells, although the overexpression of either MEKK or PKA failed to stimulate GnIH receptor expression, the combined overexpression of both kinases together increased GPR74 and GPR147 mRNA levels. Our current observations suggest that two central controllers of reproductive function, GnRH and kisspeptin, stimulate the expression of GnIH receptors in pituitary gonadotroph cells and hypothalamic GnRH neurons.

Kisspeptin induces expression of gonadotropin-releasing hormone receptor in GnRH-producing GT1-7 cells overexpressing G protein-coupled receptor 54.

Kisspeptin signaling through its receptor is crucial for many reproductive functions. However, the molecular mechanisms and biomedical significance of the regulation of GnRH neurons by kisspeptin have not been adequately elucidated. In the present study, we found that kisspeptin increases GnRH receptor (GnRHR) expression in a GnRH-producing cell line (GT1-7). Because cellular activity of G protein-coupled receptor 54 (GPR54) and GnRHR was limited in GT1-7 cells, we overexpressed these receptors to clarify receptor function. Using luciferase reporter constructs, the activity of both the serum response element (Sre) promoter, a target for extracellular signal-regulated kinase (ERK), and the cyclic AMP (cAMP) response element (Cre) promoter were increased by kisspeptin. Although GnRH increased Sre promoter activity, the Cre promoter was not significantly activated by GnRH. Kisspeptin, but not GnRH, increased cAMP accumulation in these cells. Kisspeptin also increased the transcriptional activity of GnRHR; however, the effect of GnRH on the GnRHR promoter was limited and not significant. Transfection of GT1-7 cells with constitutively active MEK kinase (MEKK) and protein kinase A (PKA) increased GnRHR expression. In addition, GnRHR expression was further increased by co-overexpression of MEKK and PKA. The Cre promoter, but not the Sre promoter, was also further activated by co-overexpression of MEKK and PKA. GnRH significantly increased the activity of the GnRHR promoter in the presence of cAMP. The present findings suggest that kisspeptin is a potent stimulator of GnRHR expression in GnRH-producing neurons in association with ERK and the cAMP/PKA pathways.