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a Department of Physiology and
b Department of Medicine, Faculty of Medicine, University of Santiago de Compostela, 15705 Santiago de Compostela, Spain
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Leptin, the obese (ob) gene product, is a newly discovered hormone that is involved in the regulation of body weight by suppressing appetite and stimulating energy expenditure [2]. Most studies have identified the hypothalamus as the major site of leptin action [3, 4]. The effects of leptin are mediated by specific receptors (Ob-R), which belong to the cytokine receptor superfamily with a single membrane-spanning domain [5]. Several alternatively spliced isoforms of Ob-R have been reported, which share extracellular and transmembrane domains, but they differ in intracellular C-terminal tails [5–7]: a long isoform (Ob-Rb), and several shorter subtypes (Ob-Ra, Ob-Rc, Ob-Re, and Ob-Rf). The long isoform (Ob-Rb), with demonstrable signalling ability, is predominantly expressed in the hypothalamus [8]. Mice deficient in this receptor isoform (db/db) exhibit a similar obese phenotype to the ob/ob mice, indicating that the Ob-Rb mediates the weight-reducing effects of leptin [6, 9]. The function of the short isoforms is not completely understood. Nevertheless, several roles have already been assigned to them: antagonists of Ob-Rb, transporters of leptin through the blood-brain barrier [5, 10–12], and a circulating binding protein for Ob-Re [6]. In any case, the action of leptin would finally depend on the regulation of the expression of the different Ob-R isoforms in the target tissue.
Recent reports have demonstrated that leptin levels are elevated in serum during human and rodent gestation [13–18]. Whether this hyperleptinemia is due to an increase in the production of leptin in the adipose tissue and/or placenta, or to an increase in the levels of a leptin-binding protein in serum (Ob-Re isoform), is still a matter of debate [14–16, 19, 20]. In any event, the paradoxically high levels of leptin during pregnancy indicate the existence of a physiological state of leptin resistance in the hypothalamus that might explain the increased food intake observed in this situation.
During rodent lactation, serum leptin concentration falls to levels found in nonpregnant animals [13, 17] or below [14]. The mechanisms responsible for the massive hyperphagia present in this state are still not totally understood.
In the present study, we compared the gestational profile of leptin mRNA expression in both placenta and adipose tissue during the gestation in the rat in order to evaluate a possible role of these tissues in the hyperleptinemia found in pregnancy. Our main goal was to gain further insight into the mechanisms responsible for the hyperphagia observed during pregnancy and lactation by evaluating the differential regulation of the expression of the Ob-R subtypes in the hypothalamus in these physiological states. For this purpose, we determined the hypothalamic mRNA levels of the different leptin receptor isoforms in nonpregnant, pregnant, and lactating rats.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague-Dawley rats were housed under controlled lighting (12L:12D) and temperature, with free access to food and water. On the day of proestrus, 3 females were housed with a male, and the day when sperm were observed in the vaginal smear was designated Day 0 of pregnancy. At least six animals were used for each experimental group. All in vivo experiments were conducted in accordance with the Guiding Principles for the Care and Use of Research Animals promulgated by the Society for the Study of Reproduction and the Ethic Committee of the University of Santiago de Compostela (Spain).
Sample Collection
Placental and white adipose tissue was obtained from rats at gestational Days 14 (D14), 16 (D16), 18 (D18), and 20 (D20). Hypothalami were obtained from nonpregnant female rats, rats at D18, and lactating rats at neonatal Day 7 (L7) and Day 14 (L14). To isolate the hypothalamus, animals were decapitated, and their brains were rapidly removed. The hypothalamus, defined by the posterior margin of the optic chiasm and the anterior margin of the mamillary bodies to the depth of approximately 2 mm, was dissected out.
Tissue samples were frozen immediately on dry ice and maintained at -80°C until processed.
Determination of Leptin by RIA
Animals were killed by decapitation, and trunk blood was collected for subsequent measurement. Serum was stored at -20°C until assayed. Serum leptin levels were measured using a kit for rat leptin (Linco Research, St. Charles, MO), as already described [21].
Quantification of Leptin mRNA Levels by Reverse Transcription (RT)-Polymerase Chain Reaction (PCR)
Total RNA was isolated by the guanidinium-isothiocyanate-phenol-chloroform extraction method [22] and was quantified by optical density at 260 nm.
Leptin mRNA levels in placenta and adipose tissue were determined by semiquantitative RT-PCR as described previously by ourselves and others [23, 24].
About 1 µg of total RNA was reverse-transcribed into cDNA using 200 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (Gibco/BRL, Gaithersburg, MD) and random primers (Promega, 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 a total volume of 30 µl containing 20 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 1 mM dNTPs (Promega), 10 mM dithiothreitol (DTT), and 30 U of ribonuclease inhibitor (RNase OUT; Gibco/BRL).
PCR was carried out using 3 µl of cDNA template and 25 pmol of forward (5'-TCACCCCATTCTGAGTTTTGTC-3') and reverse (5'-CGCCATCCAGGCTCTCT-3') primers; PCR was performed using 0.2 mM dNTPs (Promega), 1.25 U of Taq polymerase (Gibco/BRL), 5 µl of 10-strength PCR buffer (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 37 cycles at 94°C for 1 min for denaturation, 60°C for 1 min for annealing, and 72°C for 1 min for polymerization, with a final elongation cycle at 72°C for 10 min.
Amplifications were carried out in a thermal cycler (Eppendorf, Hamburg, Germany). The amplified products were resolved in 2% agarose gels stained with ethidium bromide and visualized in a digital imaging system (Molecular Analyst; Bio-Rad, Cambridge, MA). The size of the PCR product was 203 base pairs (bp). ß-Actin amplification was used as an internal control. The primers designated for this purpose were the following: forward 5'-TACAACCTCCTTGCAGCTCC-3' and reverse 5'-GGATCTTCATGAGGTAGTCAG-3'. The product size was 603 bp.
Quantification of Alternatively Spliced Isoforms of Leptin Receptor mRNA Levels
The expression of the five leptin receptor isoforms, designated as Ob-Ra, Ob-Rb, Ob-Rc, Ob-Re, and Ob-Rf, in the hypothalamus of nonpregnant, pregnant, and lactating rats were assessed by RT-PCR using 20- to 22-mer specific primers (34 cycles, annealing at 54°C) as follows:
Ob-Ra: forward, 5'-CCTATCGAGAAATATCAGTTTA-3', and reverse, 5'-TCAAAGAGTGTCCGCTCTCT-3'; Ob-Rb: forward, 5'-CCTTGAAACATTTGAGCATCTTT-3' and reverse, 5'-CGATGCACTGGCTGACAGAA-3'; Ob-Rc: forward, 5'-ATTGTACCGGTAATTATTTCCT-3', and reverse, 5'-CTGCAACCTTAGATATCTTGG-3'; Ob-Re: forward, 5'-GCAGAATCAGCACACACTGTT-3', and reverse, 5'-GTAAAAGCACAGTACACATACC-3'; Ob-Rf: forward, 5'-AGAGGATATATAGTGGATGCCG-3', and reverse, 5'-CACAAATGAGCCATCTTCAAACC-3'.
The amplification of hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an internal control. The primers designated for this purpose were the following: forward, 5'-CAGTCCCAGGGTCGTGATTA-3', and reverse, 5'-AGCAAGTCTTTCAGTCCTGTC-3'; the size of the single generated HPRT product was 201 bp.
Specificity of the Products Amplified by RT-PCR
We found leptin mRNA in the adipose tissue and placenta, as described by others [20]. Figures 3 and 4 show the amplified product of the expected size (203 bp). The mRNAs encoding Ob-Ra, Ob-Rb, Ob-Rc, Ob-Re, and Ob-Rf were detected in the hypothalamus, as already reported [10, 24]. The specificity of the signals was further demonstrated by restriction analysis of the fragments. Furthermore, RT-PCR without reverse transcriptase was also performed as a negative control (data not shown).
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In order to assure that the PCR reaction was performed in a linear range, samples were amplified for 15, 20, 25, 30, 35, and 40 cycles. A shown in Figure 1 and in reference [24], the reaction was linear under these conditions for each pair of primers used in this study.
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Statistical Analysis
All data were expressed as mean ± SEM. The statistical significance of the differences observed in the parameters evaluated between experimental groups was assessed using ANOVA and followed by the Student-Neuman-Keuls method for multiple comparisons. A P < 0.05 was considered as criterion of significance.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Serum leptin levels significantly increased as pregnancy progressed in relation to those of nonpregnant rats (control). During lactation, leptin levels were restored to control values (Fig. 2).
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Leptin mRNA Levels in Adipose Tissue and in Placenta Increased During Pregnancy
Our data demonstrated an increase in leptin mRNA levels in rat adipose tissue and placenta as pregnancy advanced (Figs. 3 and 4).
Hypothalamic Ob-Rb mRNA Levels Were Specifically Reduced During Gestation
Ob-Rb mRNA levels were significantly lower in pregnant (gestational Day 18) than in nonpregnant rats. Interestingly, no changes were found in the content of the mRNAs encoding the short forms of the leptin receptor (Ob-Ra, Ob-Rc, Ob-Re, and Ob-Rf) (Figs. 5 and 6).
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Hypothalamic Ob-Re and Ob-Rf mRNAs Were Increased During Lactation
During lactation, Ob-Rb mRNA levels were restored to values found in the nonpregnant rats. In contrast, Ob-Re significantly increased during the first week of lactation, and Ob-Rf mRNA levels were highest during the second week. The rest of the short subtypes (Ob-Ra and Ob-Rc) were not significantly altered (Figs. 5 and 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the precise mechanisms by which leptin acts are still unknown, it has been shown that it binds to leptin receptors mainly in the hypothalamus to reduce food intake and increase energy expenditure [2, 8]. It is thought that these effects of leptin are mediated by the long isoform of the leptin receptor (Ob-Rb), which contains the full intracellular domain. The function of the other shorter subtypes is not completely understood. Ob-Ra, which is predominant in all peripheral tissues, is capable of leptin-mediated signalling but much less effectively than the full-length receptor [26]. It has been suggested that this isoform, together with the other short subtypes, could function as specific transport systems for leptin, as they are present in high amounts in the choroid plexus and in brain microvessels [5, 6, 10, 11]. On the other hand, Ob-Re, which is spliced in front of the transmembrane domain, might be a soluble binding protein for leptin [6]. The other short isoforms could behave as functional antagonists by sequestering leptin and preventing its binding to the Ob-Rb subtype [12]. In order to establish whether the leptin-resistant state of pregnancy could be mediated by changes in the pattern of expression of the long and short forms of the leptin receptor, we determined the levels of expression of the different isoforms in the hypothalamus. We found a significant reduction of the mRNA encoding the fully active form of the leptin receptor (Ob-Rb) in the hypothalamus of pregnant rats in comparison to nonpregnant animals, but we found no changes in the short forms. This specific down-regulation of the Ob-Rb at the hypothalamic level would explain, at least partially, the state of leptin resistance during pregnancy. However, our data do not allow us to identify whether these changes are specific to some subsets of hypothalamic neurons or whether they are a generalized phenomenon within the hypothalamus.
During lactation, serum leptin concentration returned to the levels found in nonpregnant rats. The energy demand in this state is very high and is met primarily by increased food intake, although there is some mobilization of reserves, especially adipose tissue lipids [27]. The factors responsible for the massive increase in appetite during lactation remain poorly understood. It has been shown by others [13, 17], and by ourselves in the present report, that serum leptin levels are similar in lactating and in nonpregnant animals. Furthermore we did not observe any change in the hypothalamic expression levels of the long form of the leptin receptor (Ob-Rb), but very interestingly, the levels of the mRNAs of the short forms Ob-Re and Ob-Rf were found to be elevated during lactation. As previously stated, it is likely that Ob-Re encodes a soluble binding protein. In most peptide hormone/binding protein systems, such as growth hormone, with which leptin exhibits striking similarities [28], the bound form of the hormone is unable to bind and activate its receptor. In such a case, the binding protein would act as an inhibitor of the hormone action. Assuming that the leptin-binding protein would inhibit leptin signalling, the higher levels of Ob-Re mRNA, together with a higher expression of a short biologically inactive form (Ob-Rf) in the hypothalamus of rats during lactation, would cause leptin resistance that could also contribute to the hyperphagia observed in this state.
In summary, we have shown that pregnant rats exhibited a marked hyperleptinemia associated with an increase in leptin mRNA levels in both adipose tissue and placenta. Furthermore, our data suggest that the hyperphagia observed during pregnancy could be due to a specific decrease of the long fully active form of the leptin receptor (Ob-Rb) at the hypothalamic level. On the other hand, an increase in the levels of the soluble binding protein of the leptin receptor Ob-Re together with an increased expression of one of the short, probably inactive forms of the receptor (Ob-Rf) could be responsible for the increased food intake present during lactation. In conclusion, these data indicate the existence of different regulatory mechanisms on leptin receptor gene expression during pregnancy and lactation that should allow a greater understanding of the adaptive responses that take place in these physiological settings.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Xunta de Galicia and Pedro Barrié de la Maza Foundation. 
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: October 29, 1999.
Received: May 27, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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