mainly catabolic, leading to decreased food intake and weight loss. One other striking evidence of differential regulation between hypothalamus and peripheral tissues is the fuel gauge AMPK, whose activity is decreased by leptin in the hypothalamus but increased in skeletal muscle leading to a coordinate control of fuel expenditure and food intake [43]. More recently, a protein with a crucial function in the maintenance of glucose homeostasis, the cyclic AMP-response element binding protein (CREB) regulated protein co-activator 2 (CRTC2) has been described as displaying a differential regulation by the nutritional status in liver and hypothalamus [44]. At the fed state CTRC2 is cytoplasmic in hepatocytes and nuclear in hypothalamic neurons where it will differentially regulate hypothalamic and hepatic CRE gene expression [44]. Taken all together these different observations on the peripheral vs central effect of the nutritional status are logical if we consider the ventromedial hypothalamus as part of the feedback loop in charge not only of the maintenance of the energy stores but also of euglycemia, a sort of check point balancing the peripheral effects of the nutritional status. Having an opposite regulation of COUP-TFII expression between these different organs could then participate in this global homeostasis. In this paper, we described the unique and restricted localization of COUP-TFII expression to a subpopulation of VMH neurons expressing the transcription factor SF1. Actually, these VMH neurons expressing COUP-TFII are well character- ized for their involvement in controlling energy homeostasis. Indeed, SF1 gene knockout mice displayed an abnormal VMH development leading to obesity [23]. This demonstrates, outside of SF1 function in neuronal development, the involvement of such neurons in the control of food intake and fuel expenditure [23]. These neurons are known to control food intake through the production of BDNF, a neurotrophin whose expression in the VMH is increased in response to the activation of MC3/4-R [24,32,33]. These COUP-TFII positive neurons also express the MC4-R. Since mutations in the human MC4-R or deletion in mouse models have been associated with obesity, this receptor has received much attention as a potential drug target to treat this pathology [45,46,47,48]. Interestingly, most of this attention has been brought on its involvement in the control of food intake but it has been clearly shown that the melanocortin pathway play a global role in energy homeostasis. It stimulates oxygen con- sumption, inhibits basal insulin release and glucose tolerance, and this impaired insulin intolerance occurs prior to the onset of hyperphagia and obesity [49]. These data suggest that the central melanocortin system regulates not only energy intake, but also energy partitioning, as indicated by effects on insulin release and peripheral insulin responsiveness. As much as we know of the natural agonist and antagonist of this receptor, aMSH and AgRP, there is a lack of identified downstream effectors of this receptor. Indeed, so far, very few targets have been clearly identified. In the VMH and the dorsomedial hypothalamus, the BDNF gene expression is clearly modulated by MC4-R activation and several lines of evidence have also identified the Single Minded 1 as a downstream target of MC4-R in the paraventricullar hypothalamus [50,51]. Identifying downstream target genes, such as COUP-TFII, could be the first step in understanding the molecular mechanisms underlying the mela- nocortin-mediated regulation of energy homeostasis. Undoubt- edly, mice models with a specific invalidation of its expression restricted to the VMH neurons will be an invaluable tool in deciphering the proper implication of COUP-TFII as a central component of the hypothalamic integration of energy status and energy balance regulation. Neuronal COUP-TFII Regulation In conclusion, COUP-TFII expression is regulated in the VMH by insulin levels aside of circulating glucose and this suggests a possible role as a modulator of the counter regulation engaged by the VMH in response to neuroglucopenia or hypoglycemia. Materials and Methods Animals C57bl/6J male mice (Charles River, France) were used in this study. They were adapted to the facility environment for 2 weeks prior to the different studies. Animals were housed in colony cages with a 12-h light/12-h dark cycle in a temperature-controlled environment and fed ad libitum with a standard laboratory chow diet: composition 65% carbohydrate, 11% lipids, and 24% proteins (SAFE, France). All animal protocols were undertaken according to the Guidelines for Care and Use of Experimental Animals. Animal experimentation permit number 75-739 (Certi- ficat d’autorisation d’expe´rimenter sur animaux verte´bre´s num- ber 75-739) given by the veterinarian services board (Dr. Claudine Crochet, D.V.M). Animal care facility agreement number A-75-14-02. Streptozotocin injections Ten weeks old C57bl/6J male mice were used in these experiments. Streptozotocin (STZ) is dissolved extemporaneously in citrate buffer at pH 4.5 and administered daily at 180 mg/kg body weight (Sigma, St. Louis, MO, USA) for two days. Control mice received two citrate buffer injections. Food and water were provided ad libitum during the remaining 7 days of the experiment. After the injection, mice were checked daily for plasma glucose concentration via the tail vein with a blood glucometer (Roche diagnostic, Meylan, France). A subgroup of STZ treated mice received an intraperitoneal injection of insulin (1.5 U/kg) (Actrapid, Novo Nordisk) 2 hours prior to the sacrifice. Euglycemic-hyperinsulinemic (eGHI) clamped mice Ten weeks old C57bl/6J male mice were used in the eGHI experiments. Mice were catheterized at least 2 days before the experiment. They were anesthetized with a mixture of ketamine (100 mg/kg of body weight) and xylazine (10 mg/kg of body weight). The right jugular vein was catheterized with a silastic catheter for infusion. The free end of the catheter was tunneled under the skin to the back of the neck and passed through a piece of Tygon tubing, glued together, and secured to the skin [7]. Lines were flushed daily with 50 ml of 0.9% NaCl containing 5 mg/ml ampicillin and 20 IU/ml heparin. After surgery, animals were housed individually and weighed daily. After a 5 h fast on the day of experiment, awaken animals were placed unrestrained into their cage during the perfusion. Two groups of mice were studied: a saline-infused (control group) and glucose infused/insulin-infused (euglycemic-hyperinsulinemic [eGHI]) group. In eGHI mice, hyperinsulinemia was induced by a 3-h insulin infusion at a constant rate of 0.6 IU/kg/h (Actrapid; Novo-Nordisk) and euglycemia was maintained by a simultaneous glucose infusion at a flow rate adjusted to the basal glycemia, around 100 mg/dl. Blood glucose levels were determined from tail blood samples at time zero and then every 15 min. Steady state was reached when glucose measurements were constant for at least 20 min at a fixed glucose infusion rate and was achieved within 30 to 45 min. Plasma insulin concentrations were measured with a radioimmu- noassay kit (Insik-5 kit; Diasorin). At the end of the infusion, mice were killed by pentobarbital injection, and the hypothalamus frozen in liquid nitrogen and kept at 280uC until analysis. PLoS ONE | www.plosone.org 12 October 2010 | Volume 5 | Issue 10 | e13464