Descripción: Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling moleculeforthe central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 μM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 μm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway. Copyright © 2009.

Descripción: Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway. © 2012 Maymó et al.

Descripción: The aims of this study were to investigate the negative action of leptin on some intraovarian ovulatory mediators during the ovulatory process and to assess whether leptin is able to alter the expression of its ovarian receptors. Immature rats primed with gonadotrophins were used to induce ovulation. Serum leptin concentration was diminished 4 h after human chorionic gonadotrophin (hCG) administration, whereas the ovarian expression of leptin receptors, measured by western blot, was increased by the gonadotrophin treatment. Serum progesterone level, ovulation rate and ovarian prostaglandin E (PGE) content were reduced in rats primed with equine chorionic gonadotrophin (eCG)/hCG and treated with acute doses of leptin (five doses of 5 μg each). These inhibitory effects were confirmed by in vitro studies, where the presence of leptin reduced the concentrations of progesterone, PGE and nitrites in the media of both ovarian explants and preovulatory follicle cultures. We also investigated whether these negative effects were mediated by changes in the expression of the ovarian leptin receptors. Since leptin treatment did not alter the expression of ovarian leptin receptor, the inhibitory effect of leptin on the ovulatory process may not be mediated by changes in the expression of its receptors at ovarian level, at least at the concentrations assayed. In summary, the ovulatory process was significantly inhibited in response to an acute treatment with leptin, and this effect may be due, at least in part, to the direct or indirect impairment of some ovarian factors, such as prostaglandins and nitric oxide. © 2006 Society for Reproduction and Fertility.

Descripción: Testicular macrophages as well as endothelial cells, which are intimately associated with Leydig cells, constitute a potential source of paracrine nitric oxide (NO) in the testis. In the present study, we investigated the effect of NO donors on MA-10 murine Leydig tumor cell line and rat Leydig cell steroidogenesis. We show that NO donors inhibit human CG- induced steroidogenesis in both type of cells. We also studied NO mechanism of action. Contrary to what is observed in many other systems, NO inhibitory effect on Leydig cell steroidogenesis is not mediated by cyclic GMP (cGMP) because NO fails to increase cGMP production, and cGMP analogs do not reproduce NO effect. NO does not modify the production of cAMP, the main second messenger that mediates gonadotropin action. When we studied NO effect over the steroidogenic pathway in MA-10 cells, we found that NO was inhibiting the conversion of cholesterol to pregnenolone. Taken together these results show an inhibitory effect of NO donors on Leydig cell steroidogenesis, and suggest that NO can be directly inhibiting cholesterol side-chain cleavage enzyme (cytochrome P450(scc)) as it does with other heme proteins, including different cytochromes P459.

Descripción: To investigate the expression of leptin receptors (Ob-R) in the rat hypothalamus-pituitary-ovarian axis, immature rats were treated with eCG/ hCG and Ob-R expression was evaluated by western blot analysis. The Ob-R expression increased 24 h after eCG administration in all the tissues assayed. In the hypothalamus, these levels immediately decreased to those obtained without treatment. In the pituitary, the Ob-R expression continued to be elevated 48 h after eCG administration, whereas the hCG injection did not modify these levels. Similar results were obtained with the ovarian long isoform. To assess the effect of leptin on its receptors, Ob-R was assessed in hypothalamus, pituitary and ovarian explants cultured in the presence or absence of leptin (0.3-500 ng/ml). In the hypothalamus, we found a biphasic effect: the Ob-R expression was either reduced or increased at low or high concentrations of leptin respectively. LH-releasing hormone secretion increased at 1 ng/ml. In the pituitary, Ob-R increased at 10 or 30 ng/ml of leptin for the long and short isoforms respectively. Leptin also induced an increase in LH release at 30 ng/ml. In the ovarian culture, the presence of leptin produced an increase in Ob-R expression at different ranges of concentrations and a dose-dependent biphasic effect on the progesterone, production. In conclusion, all these results clearly suggest that leptin is able to modulate the expression of its own receptors in the reproductive axis in a differential way. Moreover, the positive or negative effect that leptin exerts on the ovulatory process may be dependent on this regulation. © 2008 Society for Endocrinology.