Food intake regulating-neuropeptides are expressed and regulated through pregnancy and following food restriction in rat placenta
© Caminos et al; licensee BioMed Central Ltd. 2008
Received: 04 December 2007
Accepted: 02 April 2008
Published: 02 April 2008
Neuropeptide Y (NPY), agouti related peptide (AgRP), cocaine and amphetamine-regulated transcript (CART) and melanocortins, the products of the proopiomelanocortin (POMC), are hypothalamic peptides involved in feeding regulation and energy homeostasis. Recent evidence has demonstrated their expression in rat and human placenta.
In the current study, we have investigated the expression of those neuropeptides in the rat placenta by real-time PCR using a model of maternal food restriction.
Our results showed that placental-derived neuropeptides were regulated through pregnancy and following food restriction.
These data could indicate that placental-derived neuropeptides represent a local regulatory circuit that may fine-tune control of energy balance during pregnancy.
The regulation of body weight is carried out by a complex inter-organ circuit connecting the periphery and the brain, where neurons in the hypothalamus and brainstem exert potent effects on feeding and energy expenditure [1, 2]. Short-acting and long-term body-weight regulating signals mainly originate from: 1) the adipose tissue, such as leptin, and interleukin 6 (IL-6); 2) the pancreas, like insulin or amylin; 3) the gastrointestinal tract such as ghrelin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY), cholecystokinin (CCK) and neuropeptide W (NPW); 4) the sensory vagus nerve . These neural and humoral signals interact at the brain level with widely-located target receptors related with the nutritional state, metabolism and reproduction [2, 3].
Hypothalamic neuropeptide expression and regulation are stimulated by inputs from peripherally-derived hormonal and nutrient-related signals essential for the central control of energy homeostasis in mammals. Neuropeptide Y (NPY), melanocortins (POMC-derived products), agouti related peptide (AgRP), cocaine and amphetamine regulated transcript (CART), melanin concentrating hormone (MCH) and orexins, have been characterized in both rodent models and humans, by having either direct or indirect effects in the feedback loop of body weight regulation and reproduction [4, 5].
NPY and AgRP are two anabolic neuropeptides that promote weight gain by reducing energy expenditure and stimulating food intake. They are coexpressed in arcuate nucleus (ARC) neurons, which are inhibited by insulin and leptin [4, 6]. In contrast, CART and POMC, also coexpressed in the ARC neurons, are stimulated by inputs from insulin and leptin and these catabolic neuronal pathways act to reduce feeding and increase energy expenditure [4, 6].
Besides their role in food intake control, hypothalamic neuropeptides have also been identified in female reproductive tissues, such as the placenta and appear to have an important role in the physiology of pregnancy [7–14]. It is interesting to note that the placenta may play an important role not only in reproduction but is also an important site for translating inputs from diverse hormonal signals that powerfully influence energy balance within the fetus [15, 16]. The aim of this study was to evaluate the ontogenic mRNA expression pattern of NPY, AgRP, POMC, and CART throughout pregnancy in the rat placenta. In addition, we have determined the effects of chronic food restriction on their expression levels.
Pregnant female rats of the Sprague Dawley strain aged between 10–12 weeks (bred in the Animalario General USC; Santiago de Compostela, Spain), were housed in a temperature-regulated room with a 12 h light/12 h dark cycle, with tap water and standard rat chow ad libitum or a food-restricted diet (see below). At the end of the study period, animals were sacrificed, hypothalami and placentas were removed from each mother, and snap-frozen in liquid nitrogen for RNA extraction and real time semi-quantitative RT-PCR. All experiments and procedures in this study were carried out according to a protocol approved by the Ethics Committee of the University of Santiago de Compostela in accordance with the European Union normative for the care and use of experimental animals.
Maternal chronic food-restriction
A maternal chronic food-restriction model was used throughout pregnancy to study the effect of long-term undernutrition on placental neuropeptide mRNA expression as previously described . Briefly, virgin rats were mated on the day of proestrus and the day on which spermatozoa were present in vaginal smear, was designated gestational day 1. The pregnant rats were divided into two dietary groups: pregnant rats fed ad libitum and food-restricted group of pregnant rats fed 30% of ad libitum intake. Food-restricted animals were fed every day at 18:00 hours. Rats were sacrificed at gestational days 12, 16 and 21. We used 9 hypothalami and placentas per experimental group, extracted from 9 different mothers. All the samples were analyzed individually and samples were not pooled.
RNA Isolation and real-time RT-PCR
Primers used for real-time RT-PCR
Primer Sequence (5'-3')
Product size (bp)
Statistical analyzes were performed using GraphPad Instat (version 3.05). All group values were expressed as mean ± SEM. Differences between groups were analyzed by using one-way analysis of variance (ANOVA) or Student's t-test. P < 0.05 was considered significant.
NPY mRNA expression is down-regulated by food restriction in the rat placenta
AgRP mRNA expression is up-regulated by food restriction in the rat placenta
POMC mRNA expression shows a biphasic response to food restriction during pregnancy in rat placenta
CART mRNA expression is down-regulated by food restriction in rat placenta
Although the expression of feeding neuropeptides in placenta is well established, little is known about the regulation of their expression during pregnancy and by nutritional status. In this study, we investigated the placental mRNA expression levels of NPY, AgRP, CART and POMC throughout pregnancy (from day 12 to day 21) in fed and food-restricted rats.
Fetal development is critically determined by the availability and flux of nutrients and oxygen across the placenta during pregnancy. The placenta tissue is an active endocrine organ involved in the control of not only metabolism and energy balance, but also of other relevant body functions including reproduction [23, 24]. Previous studies have demonstrated the placental expression of hormones, such as ghrelin and leptin [25–27], as well as neuropeptides such as NPY, AgRP, CART and POMC [7–11] are involved in the regulation of energy homeostasis. However, despite these data, both the physiogical relevance and the regulation of these molecules in placental tissues remain unclear.
In the hypothalamus, food restriction induces opposite and compensatory changes in neuropeptide expression. Thus, orexigenic neuropeptides such as AgRP and NPY are up-regulated by decreased food availability whilst anorexigenic neuropeptides such as CART and POMC are downregulated under the same conditions. Interestingly, AgRP, CART and POMC follow the same expression pattern in placental tissues but NPY is down-regulated in food-restricted rats. The reason for this discrepancy is unclear, but it could be related to an altered hormonal milieu after food restriction. In this sense, recent data from our group demonstrates that pregnancy hormones, such as prolactin, play a major role on hypothalamic NPY expression . Whether this interaction is present in placental tissues will merit further investigation. In addition, these data support the fact that contrary to the pattern in the ARC, AgRP and NPY are probably coexpressed by different cell populations in placental tissue and may be modulated by different signals.
Whatever the case, the discrepancies in NPY expression pattern suggest that an anabolic role for placental NPY in states of increased energy demand is not clear and this possibility merits further investigation. The data from AgRP, CART and POMC suggest that their expression may be under the same, or at least, a similar transcriptional control to that in the hypothalamus. Whether placental neuropeptide protein levels correlate with mRNA expression, as in the hypothalamus, will also merit further investigation.
In summary, we demonstrate that central signals involved in energy homeostasis in the hypothalamus are also expressed and modulated by nutritional status in the rat placenta. These neuropeptides display a specific ontogenic expression pattern which change over the entire gestational range. They are also affected by chronic food restriction. Altogether, these data suggest that neuropeptides could play a role in the homeostatic response to energy availability in rat placenta.
We are very grateful to Dr. Chris Lelliott (AstraZeneca R&D; Mölndal, Sweden) for his comments and criticisms. This work has been supported by grants from Xunta de Galicia (ML: GRC2006/66), Fondo Investigationes Sanitarias (ML: PI061700), Ministerio de Educacion y Ciencia (CD: BFU2005), Mútua Madrileña (CD and ML) and European Union (CD: LSHM-CT-2003-503041).
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