levels in the dose-response study (figure 6B). The MC4-R is a Ga s-coupled receptor known to stimulate cAMP production following agonist stimulation [30]. Indeed, we observed a dose dependant accumulation of COUP-TFII mRNA in response to increasing amount of the cAMP analog DiB-cAMP (figure 6C). Previous studies from Soosaar et al. [31] had identified a functional cAMP response element (CRE) binding site in the promoter region of the mouse COUP-TFII gene. Following this observation, we asked whether cAMP had a direct transcriptional effect on the COUP-TFII gene promoter in an hypothalamic cell line. To demonstrate the direct implication of cAMP in the control of the COUP-TFII gene promoter, the GT1-7 cells were transiently transfected with a reporter plasmid carrying the luciferase gene controlled by the 328 bp 59 regulatory region. We observed that the cAMP treatment induces in these neuronal cells the -328/luc COUP-TFII construct (figure 6D). All the experiments performed in this hypothalamic cell line showed that COUP-TFII gene expression was increased by an activation of the MC4-R signaling pathway. This led us to hypothesize that, in vivo, the fed state induced COUP-TFII expression through a local activation of the melanocortin pathway in the VMH. In vivo MC3/4-R activation induces COUP-TFII mRNA levels in the hypothalamus Therefore we tested the effects of intracerebroventricular (ICV) injections of the MC3/4-R agonist MTII on COUP-TFII mRNA levels in the hypothalamus. In our ICV experiments, mice were subdivided into three groups: one group fed ad libitum on a regular laboratory chow received an ICV injection of cerebrospinal fluid (CSF). A second group fasted for 18 hours prior to the experiment received an ICV injection of CSF, the last group fasted for 18 hours received a single MTII injection. Mice were sacrificed 4 hours after the injections. As an activation of the hypothalamic MC3/4-Rs is known to induce the expression of the brain derived neurotrophic factor (BDNF) expression, a neurotrophin involved in energy homeosta- sis [24,32,33], we analyzed its expression in our different groups of mice. We observed higher BDNF mRNA levels in the fasting MTII injected mice than in the control fasting mice (figure 7A). Under these experimental conditions, a higher expression of COUP-TFII mRNA in the fed mice than in the fasted mice was observed, therefore the ICV per se did not disturb the previously observed COUP-TFII mRNA regulation by the nutritional status (figure 7C). On the other hand, the MTII-injected fasting mice displayed significantly higher COUP-TFII mRNA levels than the control fasting mice injected with CSF (figure 7C). Therefore, a central activation of the melanocortin pathway in the fasting mice induces COUP-TFII mRNA. In fasting mice, when insulinemia and glycemia are low, an activation of the melanocortin pathway leads to an activation of COUP-TFII expression in the ventromedial hypothalamus. A central activation of the melanocortin pathway represses COUP-TFII mRNA levels in the liver and in the pancreas Previous data from our group had clearly shown that COUP- TFII mRNA levels are inhibited in the liver and in the pancreas of fed mice and by insulin in cultured hepatocytes and in a pancreatic beta cell line [7]. As an activation of the melanocortin pathway in the hypothalamus is known to affect gene expression through an activation of the autonomous nervous system, leading to variations in the phosphorylation state of the insulin signaling pathway in Neuronal COUP-TFII Regulation peripheral organs [34][35]we asked whether a central activation of the melanocortin pathway could also contribute to the regulation of hepatic and pancreatic gene expression. In our experiments, a central MTII injection failed to lower insulin levels in fasting mice, as insulinemia was already physiologically at its lowest (figure 7D). The ICV MTII injection leads to a slight but significant increase of blood glucose concentrations (figure 7D) as previously described [36]. First, we analyzed the expression of an hepatic gene known to be modulated by a central induction of the melanocortin pathway, the phosphenolpyruvate carboxykinase gene (PEPCK), encoding an enzyme involved in hepatic gluconeogenesis. As expected, the fed state was characterized by lower hepatic PEPCK mRNA levels compared to the fasted state (figure 8A) and the central injection of MTII in fasting mice was followed by a strong inhibition of PEPCK mRNA expression, even lower than at the fed state (figure 8A). When looking at the hepatic COUP-TFII mRNA expression, it was higher in the control fasting mice compared to the control fed mice as expected (figure 8B). However, an ICV MTII injection in fasting mice strongly inhibited COUP-TFII mRNA expression in the liver (figure 8B). We also analyzed in these mouse models the pancreatic COUP- TFII expression and observed a higher expression in the control fasting mice than in the control fed mice as previously described (figure 8C) [7]. On the other hand, the MTII injection in fasting mice significantly inhibits COUP-TFII mRNA expression in the pancreas (figure 8C). The MTII injection did not alter the neuro D mRNA expression, a control gene whose expression is restricted to the pancreatic beta cells (figure 8D). In fasting mice, a direct activation of the melanocortin pathways in the central nervous system reproduced the hypothalamic, hepatic and pancreatic gene COUP-TFII expression profiles observed in fed mice. Discussion The hypothalamus is a complex structure constituted of anatomically distinct nuclei that work in a coordinate fashion to regulate metabolic functions. Among them, the ventromedian hypothalamus assumes several functions involved in sexual and defensive behaviors. In addition, experiments with lesions of the medial hypothalamus allowed the characterization of the ventro- medial nucleus as the satiety center, its destruction resulting in hyperphagia and obesity [37]. More recent studies have also implicated neurons of the VMH as critical for courterregulatory responses to hypoglycemia, since sympathetic response to glucopenia are blocked by lesions of the VMH or by local infusion of glucose in the VMH [38]. In this paper, we report a COUP-TFII gene expression restricted to a subpopulation of neurons in the ventromedial hypothalamus expressing the SF1 protein and described as expressing the melanocortin receptor MC4-R [22,23,24,25]. We then suggested that the fed state induction of COUP-TFII gene expression in the VMH could be indirect, through an activation of the melanocortin pathway by insulin. Indeed, even as the insulin receptors are present in neurons from the different nuclei, the highest expression is mainly described in the arcuate nuclei (ARH) [39,40]. Moreover, binding assays with radiolabeled insulin had shown a stronger binding in the ARH compared to the VMH. These observations led to the idea that the prominent site of insulin action are the neurons of the ARH over the other hypothalamic nuclei [41]. Upon binding to its receptor in the ARH, insulin triggers a repression of the AgRP and NPY gene expression and an increase in POMC gene expression [26,42]. The POMC gene product is aMSH which is with AgRP part of PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13464