onto with a carbohydrate rich diet [7]. In fact, COUP-TFII gene expression is decreased by secreted insulin in response to glucose in pancreatic beta cells and by insulin and glucose in hepatocytes [7]. In situ hybridization data showed that COUP-TFII mRNA was also expressed in the hypothalamus [11,12]. As an actor of glucose homeostasis, whose expression is modulated by the nutritional status in several tissues and as it is expressed in the hypothalamus whose function is central to energy homeostasis, it was of interest to decipher the regulation of COUP- TFII expression by the nutritional status in the hypothalamus. In this paper, we report 1) a differential regulation of COUP- TFII mRNA levels by the nutritional status with an induction of its expression in the hypothalamus in the fed state 2) COUP-TFII protein expression in a subpopulation of ventromedial hypotha- lamic neurons 3) COUP-TFII expression in the hypothalamus and in a hypothalamic cell line being controlled by the melanocortin pathway leading to a direct COUP-TFII tran- scriptional activation. Results Hypothalamic COUP-TFII expression is induced by the fed state in adult mice Previous studies from our lab and from others had observed an expression of COUP-TFII mRNA in embryonic and adult hypothalamus [12,13,14]. From our previous studies on COUP- TFII mRNA regulation in liver and pancreas, we looked at the regulation of COUP-TFII mRNA levels in the hypothalamus of C57bl/6J adult mice according to their nutritional status. Mice were subjected to a 24 hours fast and compared with mice refed a regular chow diet for 6 hours, which causes a major switch leading to elevated plasma glucose (from 102610.2 mg/dl at the fasted state to 16569.5 mg/dl in the fed mice) and plasma insulin levels (from 0.660.2 at the fasted state to 1.960.3 in the fed mice). In the hypothalamus, the fed state is characterized by increased proopio- melanocortin (POMC) and decreased agouti related protein (AgRP) mRNA and neuropeptide Y (NPY) mRNA contents [15,16,17], as observed in our mice (figure 1A). Refed mice displayed significantly higher COUP-TFII mRNA levels (48%) in the hypothalamus than the fasted mice (figure 1B). In these experiments, we analyzed also COUP-TFII protein expression. We observed a significantly higher expression in the hypothalamus of the fed mice than in the hypothalamus of the fasted mice with the characteristic migration profile with 2 bands, the minor higher molecular weight band corresponding to a post translational modified COUP-TFII protein [18]. In these experiments, the ratio of the two bands was modified in a similar way between the fasted and the fed state (figure 1C). Taken altogether, these results suggested that the changes in hypothalamic COUP-TFII expression could be centrally regulated by signals of energy supplies such as blood glucose or plasma insulin levels. To confirm this hypothesis, we investigated the effects of glucose and/or insulin in the control of hypothalamic COUP-TFII mRNA levels. As insulin and glycemia are so tightly linked, in order to distinguish between the relative implication of glucose or insulin on hypothalamic COUP-TFII regulation, we used the euglyce- mic/hyperinsulinemic (eGHI) clamp technique to impose high circulating insulin while maintaining euglycemia with an intra- jugular glucose infusion (figure 2A). First, we analyzed POMC, AgRP and NPY mRNAs levels in these clamped mice (figure 2B) and observed a significant 3 fold increase in POMC mRNA levels and a 2.3 and 1.6 fold decrease respectively in AgRP and NPY mRNA levels in the eGHI mice compared to the control mice (figure 2B). In these experiments, COUP-TFII mRNA levels were Neuronal COUP-TFII Regulation significantly increased by a 2.2 fold in the eGHI clamped mice compared to the control mice (figure 2C). Thus, insulin per se was able to induce COUP-TFII mRNA levels in the hypothalamus independently of glucose. Conversely, we analyzed COUP-TFII mRNA levels in the hypothalamus of insulinopenic mice. We generated type 1 diabetic mouse models using streptozotocin injections. The control mice received citrate buffer injections (the streptozotocin vehicle). Once the hyperglycemia (470650 mg/dl) was stable (i.e. 8 days) in the STZ mice, the hypothalamic gene expression was analyzed (figure 3A). In parallel, a subgroup of STZ treated mice received an insulin injection (1.5 U/kg) two hours prior to the sacrifice. At the time of sacrifice, the glycemia of these insulin injected STZ mice had dropped down to 120627 mg/dl (figure 3A). In the STZ treated mice, POMC mRNA levels were drastically decreased by a 8 fold, whereas in the STZ subgroup receiving the insulin injection, POMC mRNA levels were reinduced by a 3.4 fold. Conversely, hypothalamic AgRP and NPY mRNA levels were respectively 3 and 10 fold higher in STZ treated mice than in the control citrate mice, however the insulin injection rapidly repressed their expressions (figure 3B). In these experiments, COUP-TFII mRNA levels dropped dramatically (80%) in insulinopenic mice compared to their control mate whereas exogenous insulin injection induced a rapid and significant increase in COUP-TFII mRNA levels within 2 hours compared to the STZ mice (figure 3B). Taken all together, these results showed that insulin is able to induce a rapid increase in hypothalamus COUP-TFII gene expression. These results are quite intriguing since our previous data had clearly demonstrated that COUP-TFII gene expression was decreased by insulin in hepatocytes and in pancreatic b cells and decreased in liver and pancreas of fed mice [7]. Therefore, we questioned how the fed state could have such an opposite effect on COUP-TFII mRNA expression in the hypothalamus compared to the liver and the pancreas. COUP-TFII expression is restricted to a subpopulation of VMH neurons The hypothalamus is a complex structure consisting of anatomically distinct nuclei that work in a coordinate fashion to regulate vegetative functions [19]. In order to decipher the mechanisms by which insulin is able to stimulate hypothalamic COUP-TFII gene expression, we needed to identify precisely the nuclei expressing this transcription factor. Therefore, we did a study of COUP-TFII protein expression profile by immunohisto- chemistry on cryostat sections of C57bl/6J fed mice brains (figure 4). After a Kluver Barrera staining, the hypothalamic nuclei were identified using a mouse brain atlas as well as morphological brain markers (figure 4A). Throughout all hypothalamic sections, we detected the COUP-TFII protein only in the ventromedial nuclei (VMH) with the very characteristic perinuclear profile (figure 4B–H). This protein expression profile is reminiscent of the COUP-TFII mRNA expression profiles observed by in situ hybridization studies (Allen Brain Atlas). These data confirmed the highly restricted expression of the protein. The secondary antibody specificity was confirmed on cryostat sections done without primary antibody incubation (figure 4C). Similar exper- iments using a primary monoclonal antibody directed against the COUP-TFI homologue, a closely related protein, were done and no expression was observed in the VMH while it was clearly expressed in cortical subpopulations on the same cryostat section, as previously described [20] (figure 4I–J). The VMH remains poorly defined in terms of cellular and molecular genetic markers as tool to identify cell subgroups. PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13464