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Regular Article |
a Hormone Laboratory,
b Section of Pediatric Endocrinology, Hospital de Cruces, Barakaldo, 48903 País Vasco, Spain
c Department of Biochemistry and Molecular Biology, Universidad del País Vasco, 48903 País Vasco, Spain
d Department of Physiology, Universidad de Santiago de Compostela, 15705 Santiago de Compostela, Spain
ABSTRACT
Leptin is a polypeptide hormone originally thought to be produced exclusively by adipocytes. However, both leptin mRNA and leptin protein were identified in human placental trophoblast cells, suggesting a potential role in human pregnancy. In the present report, we examined the regulation of leptin mRNA levels and secretion by cAMP, glucocorticoids, and insulin in term human placental tissue. Placentae were obtained immediately after delivery from mothers with uncomplicated pregnancies. Leptin concentrations were measured by ELISA in the cultured media of trophoblast maintained in monolayer culture for 24, 48, and 72 h. Likewise leptin mRNA levels in these cultured human trophoblast cells were determined by reverse transcription-polymerase chain reaction. Treatment with forskolin and (Bu)2 cAMP led to a time- and dose-dependent increase in leptin release, significant after 48 and 72 h. Moreover, incubation with forskolin for 48 h also clearly increased leptin mRNA concentration. Leptin secretion and mRNA levels were also assessed after treatment with insulin or dexamethasone. We found a time- and dose-dependent increase in leptin release, significant after 48 and 72 h. Leptin mRNA levels were also increased after these treatments. All this supports a stimulatory role of cAMP pathway, insulin and dexamethasone in the leptin mRNA levels, and leptin release in trophoblast cells in vitro.
cAMP, insulin, leptin, trophoblast
INTRODUCTION
Pregnancy is associated with an increased adipose tissue mass. Increased lipid accumulation in fatty tissue during early pregnancy provides additional mobilizable fuel that can be drawn upon later in pregnancy and during lactation. Leptin, the obese (ob) gene product, is a newly discovered hormone that is mainly involved in humans in the regulation of body weight and reproduction [1]. Recent reports have demonstrated that leptin levels are elevated in serum during human and rodent gestations [2, 3]. Although increased adiposity during pregnancy might (as in obesity) be expected to underlie the hyperleptinemia of pregnancy, leptin levels are elevated to an extent that cannot be attributed to the increased basal metabolic index (BMI) [2, 4], suggesting that during this state there is an additional source of leptin. In fact, during gestation leptin mRNA and protein have been demonstrated in placentae from a wide range of species, despite different forms of placentation [2, 5–7]. Immunohistochemical studies have also shown the presence of leptin in the human placenta, specifically in the cytoplasm of syncytiotrophoblast cells [5]. Leptin is not found in the mesenchyme of the villi core or in blood vessels, suggesting that leptin is not taken up from the circulation but rather is synthesized and secreted locally. Leptin has also been detected in a cultured human choriocarcinoma cell line (BeWo) and is augmented during the course of forskolin (FK)-induced differentiation of cytotrophoblasts into syncytiotrophoblasts [2]. These observations are consistent with the detection of elevated plasma leptin levels in patients with gestational trophoblastic neoplasms, hydatiform moles, or choriocarcinoma [2].
Leptin levels in the plasma of pregnant women start to increase during the first trimester of gestation and are remarkably elevated during the second and third trimesters [2]. These latter values are comparable to those found in obese humans [2]. Within 24 h of delivery, maternal plasma levels decline to normal values [2].
Constitutive leptin production by the placenta might be viewed as a signal to the fetus of placental competence for nutrient transfer. Nevertheless, emerging data suggest that placental leptin production is regulated and that there are differences between the regulation of human placental leptin transcription and human leptin of adipose origin. In fact the human leptin gene in the placenta has an enhancer located 1.9 kilobases upstream that is activated by a novel, placental-specific transcription factor [8]. This clearly suggests the existence of independent regulators of leptin expression in adipose tissue and the placenta during pregnancy.
The present study was designed in order to elucidate the regulation of leptin mRNA levels and leptin secretion in human placental trophoblastic cells maintained in monolayer culture. We evaluated the effect of the protein kinase A (PKA) pathway, glucocorticoids, and insulin (INS). Many hormones and proteins act on cells by activating second messenger pathways. Among them the cAMP-dependent pathway causes changes in cellular activity through PKA. By phosphorylating cytoplasmic and nuclear proteins this kinase apparently coordinates cellular processes, including the biosynthesis and release of peptides and hormones. The activation of PKA pathway is widely known to mediate the actions of numerous hormones in the placenta [9]. Therefore it is possible that this signalling pathway could be involved in leptin regulation. Our rationale for dissecting the effects of INS and glucocorticoids on leptin production in the placenta is based on the suggestion that the hyperleptinemia of pregnancy could have an important function in modulating maternal nutrient partitioning in order to optimize the provision of nutrients for fetal growth [10]. Thus, through modulation of maternal INS secretion and hepatic metabolism, leptin integrates maternal nutrient storage to the nutrient requirement of the fetus [10]. All this made INS and glucocorticoids good hormone candidates to regulate leptin expression and secretion in the placenta. On the other hand, the rise of glucocorticoids and INS in maternal serum at the end of gestation [11, 12], the expression of glucocorticoid and INS receptors in the placenta [13–17], and the existence of glucocorticoid- and INS-dependent regulation of a number of placental products [14, 18–21] are well documented.
MATERIALS AND METHODS
Cell Culture
Placentae were obtained immediately after delivery from mothers with uncomplicated pregnancies. Culture of trophoblastic cells was performed as previously described [22]. Briefly, several cotyledons were removed, rinsed with 0.9% NaCl at room temperature, dissected free of membranes and vessels, and minced until approximately 15 g of tissue were collected. The placental tissue was digested at 37°C in Ca2+-Mg2+-free Earle balanced salt solution (EBSS) with collagenase (0.25%), DNase (0.01%), and dispase (0.1%). After digestion, cells were washed with EBSS and centrifuged. The excess of erythrocytes was lysed by adding 10 ml of 0.83% freshly made ammonium chloride-PBS solution (pH 7.2) and kept in an ice bath for 10 min. After this time, 1 ml fetal bovine serum (FBS) was added onto the bottom of the tube carefully. The pellet was centrifuged at 100 x g, washed once, and resuspended in Dulbecco modified Eagle medium containing 25 mM Hepes and 25 nM glucose (DMEM-H-G) supplemented with 0.5% fetal calf serum (FCS), 100 IU/ml penicillin, and 2.5 µg/ml fungizone. Cells were plated in microwells and were incubated in a humidified 5% CO2 atmosphere at 37°C. Forskolin, (Bu)2 cAMP, dexamethasone (DEX), INS (Sigma Chemical Co., St. Louis, MO), or vehicle was added to the cells at the time of plating. The doses of FK and (Bu)2 cAMP used in this work correspond to the doses used by most authors [23, 24]. In relation to DEX and INS, the concentrations used here correspond to the blood levels observed in pregnant women [11, 12]. Culture medium was removed after 24, 48, and 72 h and stored at -20°C until used for leptin analysis. Cells were used for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis as described below.
Leptin Assay
An ELISA kit for leptin was purchased from R&D systems (Minneapolis, MN). The limit of sensitivity was 7.8 pg/ml. The intra-assay and interassay coefficients of variation were <10% (n = 16) for the three concentrations of leptin used (64.5, 146, and 407 pg/ml).
Ribonucleic Acid Isolation and RT-PCR
Total RNA was isolated from trophoblasts using the method described by Chirgwin et al. [25]. Reverse transcription-PCR analysis for leptin mRNA was performed as already described [5]. Briefly, cDNAs were synthesized from 1 µg of total RNA using 200 units of Moloney murine leukemia virus reverse transcriptase (Gibco BRL, Gaithersburg, MD), and random primers (1 µM; Promega Co., Madison, WI). The reaction was carried out at 37°C for 50 min, 42°C for 15 min, and 95°C for 5 min in 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, 1 mM dNTPs (Gibco BRL), 10 mM dithiothreitol, and 30 U of ribonuclease inhibitor (RNase OUT; Gibco BRL). The PCR was performed with the following primers: upstream primer, 5'-ATGCATTGGGGAACCCTGTGCGG-3'; downstream primer, 5'-TGAGGTCCAGCTGCCACAGCATG-3'. The amplification of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an internal control. The primers designated for this purpose were the following: upstream 5'-CAGTCCCAGGGTCGTGATTA-3' and downstream 5'-AGCAAGTCTTTCAGTCCTGTC-3'. Polymerase chain reaction was performed using 0.2 mM dNTPs, 1.25 U of Taq-DNA polymerase (Gibco BRL), 5 µl of 10x PCR buffer (Gibco BRL; 18.6 mM Tris-HCl, 45.9 mM KCl, 3 mM MgCl2) in a total volume of 50 µl. The temperature and times used were 35 cycles at 94°C for 1 min for denaturation, 60°C for 1 min for annealing, 72°C for 1 min for polymerization, with a final elongation cycle at 72°C for 10 min. The PCR product was run in a 2% agarose gel stained with ethidium bromide. Amplified products were quantitated by densitometry using a digital imaging system (Molecular Analyst; Bio-Rad, Hercules, CA). The results were expressed in relation to HPRT amplification data.
Data Expression and Statistical Analysis
Each independent experiment was repeated at least three times. Data were expressed as a percentage of control. The results were analyzed by ANOVA followed by posthoc Bonferroni test. A P < 0.05 was considered as a criterion of significance. In order to analyze the kinetics of amplification by PCR of leptin and HPRT cDNA we used linear regression analysis, and the correlation coefficients (r) were calculated.
RESULTS
Basal Leptin Concentrations in Culture Media of Cultured Human Trophoblast
Leptin-like inmunoreactivity was detected in the culture media of human trophoblast maintained in monolayer cultured for 24, 48, or 72 h (Fig. 1).
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Effect of cAMP on Leptin Secretion
Treatment with FK (10-5 M) led to a time-dependent increase in leptin release, the effect being significant after 48 and 72 h (% control: 233.5 ± 77.7%, P < 0.005, and 316 ± 21.1%, P < 0.001, respectively), while shorter incubations (24 h) did not produce any significant effect (Fig. 2A). Also, the addition of FK at doses 10-7 M, 10-6 M, and 10-5 M for 72 h resulted in a dose-dependent increase of leptin secretion. The maximum effect was raised at a concentration of 10–6 M (% control: 469.9 ± 29.7%, P < 0.005) (Fig. 3A).
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In order to increase the intracellular levels of cAMP we also used (Bu)2 cAMP (10-5 M, 10-4 M, and 10-3 M). A significant dose- and time-dependent increase of leptin secretion was observed after these treatments (Figs. 2 and 3B).
Effect of DEX on Leptin Secretion
As can be observed in Figure 2C, leptin release was increased in a time-dependent manner after treatment with DEX (10-6 M). The maximum effect was seen after 72 h of incubation (% control: 903.7 ± 798%, P < 0.001). This stimulatory effect was dose dependent, as shown in Figure 3C, with a maximum effect at 10-6 M (% control: 1420.8 ± 695.2%).
Effect of INS on Leptin Secretion
The effect of INS on leptin secretion is shown in Figures 2 and 3D. After the cells were incubated with INS (10-7 M) for 24, 48, and 72 h, a time-dependent increase of leptin levels was observed. A significant stimulatory effect of leptin release was only obtained after 72 h of incubation (% control: 178.9 ± 45%, P < 0.001), while no significant changes were seen when the treatment spanned for 24 or 48 h (Fig. 2D).
As occurred before different concentrations of INS, 10-10 M to 10-7 M for 72 h elicited an increase in leptin levels in a dose-dependent manner, with a maximal response at a concentration of INS of 10-7 M (% control: 178.9 ± 45%, P < 0.001), while lower concentrations did not significantly increase leptin release (Fig. 3D).
Effect of cAMP, DEX, and INS on Leptin mRNA Levels
Reverse transcription-PCR of cultured trophoblastic cell RNA yielded a band of the expected size: 487 base pairs. The identity of the amplimer was confirmed by restriction enzyme cleavage (data not shown). In order to assure that the PCR reaction was performed in a linear range, samples were amplified for 30, 32, 35, 37, 40, and 42 cycles. As shown in Figure 4, the reaction was linear under the conditions used in this study.
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Treatment with FK (10-5 M) or DEX (10-6 M) for 48 h clearly increased leptin mRNA levels (about threefold). Likewise leptin mRNA content increased after incubation with INS (10-7 M) for 72 h (Fig. 5).
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DISCUSSION
Apart from its actions on body weight regulation and metabolism, leptin has significant physiological roles in humans concerning various aspects of reproduction [1]. These actions are believed to underlie nutritional features of reproduction that depend on the adequacy of energy storage. But leptin is also produced and secreted by the placenta [3, 5]. Although these data point to the fact that leptin is a novel trophoblast-derived hormone in humans, with a possible functional significance during pregnancy, its regulation and physiology is largely unknown.
We presented in this paper that leptin mRNA levels and leptin release are stimulated by PKA activation, DEX, and INS in term human placental tissue cultured in monolayer.
The PKA pathway plays a central role in biological signalling of various hormones in the placenta, such as epinephrine, prostanoids, and hCG [9]. In agreement with previous data [26], we found that PKA activation, obtained by increasing intracellular cAMP levels using both FK, an activator of adenyl cyclase, and (Bu)2 cAMP, an analogue of cAMP, leads to increased leptin secretion. Furthermore, our data suggest that this effect is mediated by an increase in leptin-gene transcription because leptin mRNA levels were also increased. Interestingly, our data are in clear contrast to what has been demonstrated in adipose tissue. In explant cultures of mature adipocytes, PKA suppresses leptin secretion and leptin mRNA levels [27]. Nevertheless, this discrepancy in the regulation of both leptin-producing tissues is not surprising, as the human leptin gene has a placenta-specific enhancer [8, 28], and a placenta-specific nuclear binding protein that is involved in leptin expression has been reported [28], indicating that the regulation of leptin production in placental trophoblasts is likely different from that in adipocytes.
Because of their long-observed effects on feeding behavior in animals, glucocorticoids were almost the first hormones tested for their potential effects on leptin gene expression [29]. It has been shown that an induction of leptin mRNA levels in adipose tissue after treatment with various glucocorticoids including DEX [30], and several glucocorticoid response element consensus binding sites have been found in the proximal promoter of the human leptin gene [31], indicating a direct transcriptional action of this hormone on the leptin gene. Because maternal free cortisol levels increase during gestation, glucocorticoid receptors have been identified in placental tissue, and also, glucocorticoid-dependent regulation of a number of placental products has been previously described, including hCG, corticotropin-releasing hormone, and prostaglandins [14, 18–20], we considered it interesting to evaluate the possible effects of these hormones on placental leptin levels. In this paper we also reported a stimulatory effect of DEX on leptin mRNA and leptin release in trophoblast cells in culture. Taking into account that pregnancy is a model of hypercortisolemia, it is possible that the elevated leptin release during gestation is mediated by cortisol [32]. In any event our data show that leptin responsiveness to glucocorticoids is similar in adipocytes and placental cells.
Late pregnancy is associated with a reduced threshold for glucose-stimulated INS secretion and enhanced secretory response to glucose [33]. Enhanced INS secretion during late pregnancy could compensate for maternal peripheral INS resistance and thereby permit maternal fuel storage when the nutrient supply is abundant [34]. We demonstrated a stimulatory role of INS on placental leptin levels. In support of our data it has been shown that there is a three- to fivefold increase in leptin expression in placentae from INS-treated diabetic women [35]. This is interesting as the placenta contains large numbers of INS receptors and responds acutely to hyperinsulinemia with increased glucose uptake and phosphorylation [36]. All this emphasizes a role for INS in regulating placental metabolic processes and fits with the idea that INS is an important modulator of leptin gene expression, even though its role in leptin release in adipose tissue is still a matter of debate. Thus, although in rodent adipocytes insulin acts on leptin secretion in a stimulatory way, in humans, INS does not have the acute effect of raising leptin concentration in vivo, not when administered during a clamp test and not when released endogenously after meals. Only a chronically high INS level clearly raises leptin concentration [1]. There is some evidence that leptin may inhibit glucose-stimulated INS secretion [33], suggesting the existence of a negative feedback system between leptin and INS [37]. It is not known to what extent the hyperleptinemia of pregnancy could contribute to the development of INS resistance in maternal tissues in late pregnancy. However, it has been reported that leptin increases fatty acid partitioning toward oxidation from esterification [38] and inhibits basal and INS-stimulated glycogen synthesis in skeletal muscle [39]. A potential role for the high levels of leptin during pregnancy therefore could be related to terminate INS-dependent energy storage (as glycogen or lipid) after the attainment of adequate reserves, thereby directing nutrients in excess of the maternal requirement toward the fetus [10].
Although there is little doubt that peptide factors produced by the human placenta are fundamental for inhibition and maintenance of pregnancy, the physiological relevance of placental leptin expression and secretion is yet unclear. On a theoretical basis placenta-derived leptin could be involved in 1) the local modulation of hormone release, 2) the regulation of placental cell growth and differentiation, and 3) playing a homeostatic role in either the mother or the fetus after being secreted into the maternal and fetal circulation. Data regarding the first two possibilities are still scanty [40], and no firm conclusions can be drawn. On the other hand, although a definitive proof is still lacking, it is likely that the placenta is a clear source of leptin in the human maternal circulation. Thus, although at early stages of pregnancy leptin levels in the maternal serum are highly correlated with weight, BMI, and skinfold thickness, during late gestation this correlation does not occur, suggesting that during the third trimester of pregnancy the regulation of leptin levels by the adipocytes differ from that in nonpregnant women, and/or that another source of leptin, such as the placenta, is present [2, 5]. Moreover, a rapid decline in maternal circulating leptin levels is observed when placental function vanishes as occurs after parturition and after a spontaneous abortion [41]. Finally, the placental pathology is associated with variations in leptin levels [2].
In conclusion, the present data validate the use of term placenta in vitro to model determinants of leptin expression in this tissue, as these cells not only actively secrete leptin in culture but also demonstrate a range of responses to treatments that parallels the reported in vivo situations. These should lead to a greater insight into different aspects related to placental endocrinology and a variety of gestational disorders.
ACKNOWLEDGMENTS
We especially acknowledge all the registered nurses in the delivery room for their collaboration.
FOOTNOTES
First decision: 12 December 2000.
1 This work was supported by Fondo de Investigación Sanitaria (FISS 99/0678), Spanish Ministry of Health, and Lilly, S.A. (Spain). 
2 Correspondence: Rosa María Señarís, Department of Physiology, Faculty
of Medicine, C/ San Francisco s/n, 15705 Santiago de Compostela, Spain.
FAX: 34 981 574145; fsrsr{at}usc.es
Accepted: April 19, 2001.
Received: October 27, 2000.
REFERENCES
This article has been cited by other articles:
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 J. Zhao, T. H. Kunz, N. Tumba, L. Clamon Schulz, C. Li, M. Reeves, and E. P. Widmaier Comparative analysis of expression and secretion of placental leptin in mammals Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2003; 285(2): R438 - R446. [Abstract] [Full Text] [PDF]
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P. Cameo, P. Bischof, and J. C. Calvo Effect of Leptin on Progesterone, Human Chorionic Gonadotropin, and Interleukin-6 Secretion by Human Term Trophoblast Cells in Culture Biol Reprod, February 1, 2003; 68(2): 472 - 477. [Abstract] [Full Text] [PDF]
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