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In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

ABSTRACT: Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

SUBMITTER: Albers-Wolthers CHJ

PROVIDER: S-EPMC5484485 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Albers-Wolthers C H J CHJ de Gier J J Walen M M van Kooten P J S PJS Lambalk C B CB Leegwater P A J PAJ Roelen B A J BAJ Schaefers-Okkens A C AC Rutten V P M G VPMG Millar R P M RPM Kooistra H S HS

PloS one 20170626 6

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and ...[more]

PMID: 28650956

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Project description:The kisspeptin/Gpr54 signalling pathway plays a critical role in reproduction by stimulating the secretion of gonadotrophin-releasing hormone (GnRH), yet mice carrying mutations in Kiss1 (which encodes kisspeptin) or Gpr54 exhibit partial sexual maturation. For example, a proportion of female Kiss1(-/-) and Gpr54(-/-) mice exhibit vaginal oestrus, and some male Kiss1(-/-) and Gpr54(-/-) mice exhibit spermatogenesis. To characterise this partial sexual maturation, we examined the vaginal cytology of female Kiss1(-/-) and Gpr54(-/-) mice over time. Almost all mutant mice eventually enter oestrus, and then spontaneously transition from oestrus to dioestrus and back to oestrus again. These transitions are not associated with ovulation, and the frequency of these transitions increases with age. The oestrus exhibited by female Kiss1(-/-) and Gpr54(-/-) mice was disrupted by the administration of the competitive GnRH antagonist acyline, which also resulted in lower uterine weights and, in Kiss1(-/-) mice, lower serum follicle-stimulating hormone (FSH) and luteinising hormone (LH) concentrations. Similarly, male Kiss1(-/-) and Gpr54(-/-) mice treated with acyline had smaller testicular sizes and an absence of mature sperm. In addition to examining intact Kiss1(-/-) and Gpr54(-/-) mice, we also assessed the effects of acyline on gonadotrophin concentrations in gonadectomised mice. Gonadectomy resulted in a significant increase in serum FSH concentrations in male Gpr54(-/-) and Kiss1(-/-) mice. Acyline administration to gonadectomised Kiss1(-/-) and Gpr54(-/-) male mice lowered serum FSH and LH concentrations significantly. By contrast to males, gonadectomy did not result in significant gonadotrophin changes in female Kiss1(-/-) and Gpr54(-/-) mice, but acyline administration was followed by a decrease in LH concentrations. These results demonstrate that, although kisspeptin signalling is critical for the high levels of GnRH activity required for normal sexual maturation and for ovulation, Kiss1(-/-) and Gpr54(-/-) mice retain some degree of GnRH activity. This GnRH activity is sufficient to produce significant effects on vaginal cytology and uterine weights in female mice and on spermatogenesis and testicular weights in male mice.

Project description:GPR54, Kisspeptin-1 receptor (KISS1R), a member of rhodopsin family, plays a critical role in puberty development and has been proposed to be involved in regulation of energy metabolism. This study aims to explore the function of GPR54 in adipogenesis, lipid metabolism, and obesity in addition to its effect through hormones. Results showed that when fed a high-fat diet, the weight growth of castrated or ovariectomized Gpr54-/- mice was significantly slower than that of WT control, together with a lower triglyceride concentration. The ratio of white adipose tissue was lower, and average size of adipocytes was smaller in Gpr54-/- mice. Meanwhile, there were less adipose tissue macrophages (ATMs), especially pro-inflammatory macrophages. Expression of inflammatory related genes also indicated that inflammatory response caused by obesity was not as drastic in Gpr54-/- mice as in WT mice. Liver triglyceride in Gpr54-/- mice was reduced, especially in female mice. On the other hand, oil drop formation was accelerated when hepatocytes were stimulated by kisspeptin-10 (Kp-10). Primary mesenchymal stem cells (MSCs) of Gpr54-/- mice were less likely to differentiate into adipocytes. When stimulated by Kp-10, 3T3-L1 cell differentiation into adipocytes was accelerated and triglyceride synthesis was significantly promoted. These data indicated that GPR54 could affect obesity development by promoting adipocyte differentiation and triglyceride accumulation. To further elucidate the mechanism, genes related to lipid metabolism were analyzed. The expression of genes involved in lipid synthesis including PPARγ, ACC1, ADIPO, and FAS was significantly changed in Gpr54-/- mice. Among them PPARγ which also participate in adipocyte differentiation displayed a marked reduction. Moreover, phosphorylation of ERK, which involved in GPR54 signaling, was significantly decreased in Gpr54-/- mice, suggesting that GPR54 may promote lipid synthesis and obesity development by activating MAP kinase pathway. Therefore, in addition to the involvement in hormone regulation, our study demonstrated that GPR54 directly participates in obesity development by promoting adipocyte differentiation and fat accumulation. This provided evidence of involvement of GPR54 in lipid metabolism, and revealed new potentials for the identification and development of novel drug targets for metabolic diseases.

Project description:GPR54 is a G-protein-coupled receptor, which binds kisspeptins and is widely expressed throughout the brain. Kisspeptin-GPR54 signaling has been implicated in the regulation of pubertal and adulthood gonadotropin-releasing hormone (GnRH) secretion, and mutations or deletions of GPR54 cause hypogonadotropic hypogonadism in humans and mice. Other reproductive roles for kisspeptin-GPR54 signaling, including the regulation of developmental GnRH secretion or sexual behavior in adults, have not yet been explored. Using adult wild-type (WT) and GPR54 knock-out (KO) mice, we first tested whether kisspeptin-GPR54 signaling is necessary for male and female sexual behaviors. We found that hormone-replaced gonadectomized GPR54 KO males and females displayed appropriate gender-specific adult sexual behaviors. Next, we examined whether GPR54 signaling is required for proper display of olfactory-mediated partner preference behavior. Testosterone-treated WT males preferred stimulus females rather than males, whereas similarly treated WT females and GPR54 KO males showed no preference for either sex. Because olfactory preference is sexually dimorphic and organized during development by androgens, we assessed whether GPR54 signaling is essential for sexual differentiation of other sexually dimorphic traits. Interestingly, adult testosterone-treated GPR54 KO males displayed "female-like" numbers of tyrosine hydroxylase-immunoreactive and Kiss1 mRNA-containing neurons in the anteroventral periventricular nucleus and likewise possessed fewer motoneurons in the spino-bulbocavernosus nucleus than did WT males. Our findings indicate that kisspeptin-GPR54 signaling is not required for male or female copulatory behavior, provided there is appropriate adulthood hormone replacement. However, GPR54 is necessary for proper male-like development of several sexually dimorphic traits, likely by regulating GnRH-mediated androgen secretion during "critical windows" in perinatal development.

Project description:BackgroundExcessive gestational weight gain (GWG), which is associated with adverse long-term effects on the health of the offspring, has become a major clinical problem. Accumulating evidence indicates that the ovary kisspeptin/GPR54 system directly participates in a series of physiological activities. We used a model of high-fat diet (HFD) during gestational to investigate offspring's ovarian function and whether kisspeptin/GPR54 system is involved.MethodsAfter introducing the male and confirmation of mating by checking a vaginal sperm plug, female rats were randomized into two groups: control diet called NCD group and high-fat diet called HFD group. After birth, all rats were changed into a control diet and litter size was adjusted to 12 pups per litter. Ovaries were collected for assessment at postnatal day (PND) 4 and PND 30. The timing of vaginal opening was recorded, and the estrous cyclicity was monitored for 2 consecutive weeks immediately. Primary granulosa cells and ovaries which were taken from PND 4 were collected for determination of the direct effect of kisspeptin-10 (kp-10) in vitro.ResultsNeonatal rats exposed to HFD during gestation had a lower number of secondary follicles in the ovary. The expression of follicle-stimulating hormone receptor (FSHR) and kisspeptin was not altered. At prepuberty, the number of antral follicles and preovulatory follicles was elevated with decreased type III follicles in the HFD group. While the expression of ovulation-related genes was decreased, the expression levels of follicular growth-related genes and steroidogenesis synthesis related genes were elevated. A significant increase in kiss1 mRNA and kisspeptin protein was detected without changes in kiss1r mRNA and GPR54. Maternal high-fat diet during gestation resulted in a significant advanced puberty onset and an irregular estrous cycle in offspring rats. In addition, the administration of kp-10 produced an increase in viability of primary granulosa cells and enlarged the size of oocytes.ConclusionsHFD exposure during maternal gestation had a long-term effect on reproductive function in the offspring and the increased ovarian kisspeptin/GPR54 system might be involved.

Project description:Kisspeptin-10 (KP-10), a potent vasoconstrictor and inhibitor of angiogenesis, and its receptor, GPR54, have currently received much attention in relation to pre-eclampsia. However, it still remains unknown whether KP-10 could affect atherogenesis. We evaluated the effects of KP-10 on human umbilical vein endothelial cells, human monocyte-derived macrophages, human aortic smooth muscle cells in vitro, and atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice in vivo. KP-10 significantly increased the adhesion of human monocytes to human umbilical vein endothelial cells, which was significantly inhibited by pretreatment with P234, a GPR54 antagonist. KP-10 stimulated mRNA expression of tumor necrosis factor-α, interleukin-6, monocyte chemotactic protein-1, intercellular adhesion molecule-1, vascular adhesion molecule-1, and E-selectin in human umbilical vein endothelial cells. KP-10 significantly enhanced oxidized low-density lipoprotein-induced foam cell formation associated with upregulation of CD36 and acyl-CoA:cholesterol acyltransferase-1 in human monocyte-derived macrophages. In human aortic smooth muscle cells, KP-10 significantly suppressed angiotensin II-induced migration and proliferation, but enhanced apoptosis and activities of matrix metalloproteinase (MMP)-2 and MMP-9 by upregulation of extracellular signal-regulated kinase 1 and 2, p38, Bcl-2-associated X protein, and caspase-3. Four-week-infusion of KP-10 into ApoE-/- mice significantly accelerated the development of aortic atherosclerotic lesions with increased monocyte/macrophage infiltration and vascular inflammation as well as decreased intraplaque vascular smooth muscle cells contents. Proatherosclerotic effects of endogenous and exogenous KP-10 were completely canceled by P234 infusion in ApoE-/- mice. Our results suggest that KP-10 may contribute to accelerate the progression and instability of atheromatous plaques, leading to plaque rupture. The GPR54 antagonist may be useful for prevention and treatment of atherosclerosis. Thus, the KP-10/GPR54 system may serve as a novel therapeutic target for atherosclerotic diseases.

Project description:BackgroundKisspeptins and their G-protein coupled receptor, GPR54 are required for GnRH release and have been associated with anti-metastatic tumour cell behaviour in model systems. The latter might suggest that their overexpression would be associated with a better prognosis in cancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) could also impact tumour behaviour in a negative manner. Here, for the first time, we associate the immunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a large cohort of ovarian carcinomas.MethodsImmunohistochemical analysis for kisspeptin and GPR54 was performed on a tissue microarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcome data. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and +2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive and designated as 1, while all other cases were considered negative and designated 0. All statistical analysis was conducted using two-sided tests and a p value equal to or less than 0.05 was considered significant.ResultsKisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariable disease specific survival (p = 0.0023, p = 0.0092), as well as in overall survival (p = 0.0006, p = 0.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determined by multivariable disease specific (p = 0.0046) and overall survival analysis (p = 0.0170), while GPR54 is an independent marker for overall survival only (p = 0.0303). Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102).ConclusionKisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.

Dataset Information

In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Publications

In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

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