Cell culture The mouse GT1-7 hypothalamic cell line [52] was used between passage 31 and 37. The cells were grown in 5% CO2- 95% air at 37uC in Dubelco modified Eagle medium (DMEM) containing Glutamax 1x supplemented with 10% (vol/vol) of heat inactivated fetal calf serum and 100 U/ml penicillin-streptomycin. For melanotan II (MTII) (Phoenix pharmaceuticals, Burlingame, CA, USA) or Dibutyryl-cAMP (DiBcAMP) (Sigma, St. Louis, MO, USA) dose response experiments, cells were seeded in 12 well plates at a density of 150,000 cells/well. Twenty four hours after plating, cells were cultured in presence of MTII or DiBcAMP for 16 h prior to the RNA extraction. Plasmids, transfection and reporter gene assay The reporter plasmid -328 (Apa I) to +873 (BglII) relative to the transcription initiation site was described in Perilhou et al. (-328/ luc) [53]. Cells were plated at density 150,000/well in GT1-7 medium. Transient transfections were carried out with Lipofecta- mine 2000 reagent (Invitrogen, Cergy, France), 800 ng of reporter plasmid and 100 ng of internal control. The following day the cells were treated with DiBcAMP for 6 hours and cell extracts were assayed for reporter enzyme activities, using the dual luciferase kit (Promega, Charbonnieres, France) as described previously [53]. Isolation of total mRNA and quantitative analysis by real time RT-PCR Total RNAs were extracted and purified from hypothalamus, cultured GT1-7 cells and liver using the RNA-Plus reagent (Q- BIOgene, Montreuil, France) according to the manufacturer instructions. For RNA extraction from flash frozen pancreas, the tissues were powdered in liquid nitrogen using a steel mortar and pestle. A frozen minute amount of powdered pancreas was then immediately homogenized in a chaotropic buffer and RNA were extracted using the RNAeasy kit (Qiagen, Courtaboeuf, France) according to the manufacturer instructions. Reverse transcription was performed with 1 mg of total RNA using the Superscript II reverse transcriptase (Invitrogen, Cergy, France). Real time PCR was performed using 6.25 ng of reverse-transcribed RNA subsequently mixed with Fast Start DNA Master Sybr Green (Roche, Meylan, France), 2 mM MgCl2 and various set of gene specific forward and reverse primers in a final volume of 10 ml. The relative amount of each mRNA was calculated using the 2nd derivative Ct method. Quantification was obtained after normal- ization to cyclophilin mRNA and inter-assay variations using an RT-calibrator [54]. Forward and reverse primers used for the specific amplification of mouse cDNA fragments were: COUP-TFII: 59-CGC TCC TTG CCG CTG CT-39 and 59-AAG AGC TTT CCG AAC CGT GTT-39, Cyclophilin: 59-TTG CCA TTC CTG GAC CCA AA-39 and 59-ATG GCA CTG GTG GCA AGT CC-39 Neuro D1: 59-GCC CAG CTT AAT GCC ATC TTT-39 and 59- CAA AAG GGCTGC CTT CTG TAA-39 Brain-derived neurotrophic factor (BDNF): 59-CAG GGG CAT AGA CAA AAG -39and 59-CTT CCC CTT TTA ATG GTC-39, Pro- opiomelanocortin (POMC): 59-ATG CCG AGA TTC TGC TAC AGT CG-39and 59-TC ATC TCC GTT GCC AGG AAA CAC -39, Agouti related protein (AgRP): 59-TT TGG GAG AGC GTG TAA CTG AA-39and 59-CT CCA TAT CGT GGT CTG AAA CTG-39, Phosphoenolpyruvate carboxykinase (PEPCK): 59 GTG CTG GAG TGG ATG TTC GG 39and 59 CTG GCT GAT TCT CTG TTT CAG G 39, Neuropeptide Y(NPY): 59 TTC TGT GCT TTC CTT CAT 39 and 59 GTG TTT GGG CAT TCT G 39. Neuronal COUP-TFII Regulation Western blot analysis and ECL detection Nuclear extracts were prepared from fresh tissues as described previously by Bossard et al. [55]. Western blot were done using 30 mg of nuclear extracts and according to Perilhou et al. [7]. Immunohistological studies Two-month-old C57Bl/6J male mice were decapitated and their brains were removed, frozen at 235uC for 2 minutes in isopentane and stored at 280uC. Frozen brains were mounted and placed in a cryostat and 7 mm sections were cut in the coronal plane. One section out of 20 was stained by the Kluver Berrera method which by staining the myelin in a bluish hue and the cells structures in violet allows the visualization of the different brain structures [56]. For COUP-TF immunostaining, sections were fixed in 1% paraformaldehyde for 20 min at 4uC then permeabilized with 0.1% triton in 1X Phosphate buffer saline (PBS) for 10 min at room temperature. The sections were incubated with a mouse IgG blocking agent (Mouse-on-Mouse, Vector, USA), then incubated overnight at 4uC with mouse monoclonal primary antibodies: COUP-TFII/NR2F2 (PP- H7147-00, dilution 1:50) or the COUP-TFI/NR2F1 (PP- H8132-00, dilution 1:100). The following day, the sections were washed and revealed using an anti-mouse antibody coupled with a peroxidase labeling enhancement system (Envision DAB-HRP, Dakocytomation). On the same slide, a serial section was treated without the primary antibody as a control for secondary antibody specificity. For immunofluorescence double staining, the slides were incubated overnight at 4uC with mixed primary antibodies directed against SF1 (rabbit anti SF1 polyclonal antibody, Ab3436, AbCam, dilution 1:100) and against COUP-TFII (PP- H7147-00, dilution 1:50), or against COUP-TFII and MC4-R (goat anti MC4-R polyclonal antibody, R-19, Santa Cruz, dilution 1:100), then with mixed secondary antibodies: FITC conjugated anti-mouse and Texas Red conjugated anti rabbit for SF1/ COUP-TFII double staining or Alexa 568 conjugated anti-mouse and Alexa 488 conjugated anti-goat for MC4-R/COUP-TFII double staining, at room temperature for 1 h. The slides were mounted in DAPI (4-6-diamidino-2-phenylindole) containing Vectashield (Vector Laboratories, Philadelphia, PA, USA) for the nuclei counterstaining and stored at 4uC until observation. The different specificity controls were done on the same slides: a section was incubated with only one of the primary antibody then incubated with the mixed secondary antibodies. A serial section is also treated without any primary antibody, as a control for the secondary antibodies specificity. Intracerebroventricular (ICV) injection Twelve weeks old C57bl/6J male mice were anaesthetized with isofluran (2.5%). After the initial anaesthesia, they were connected at the snout to a device providing isofluran at 1.5% for the surgery duration. As soon the animals were anaesthetized, they received an injection of xylazine and xylocaine (10 mg/kg of body weight). A chronic guide cannula (26 ga, Plastic One Products, Roanoke, VA) was implanted into the right lateral brain ventricle to coordinates X = 21 mm, Y = 20.4 mm, Z = 22.5 mm from the Bregma using a Kopf stereotaxic instrument (David Kopf Instruments, Tujunga, CA, USA). The 26 stainless steel gauge guide cannula (Plastic One Products, Roanoke, VA, USA) was maintained in place by dental cement anchored to one stainless steel jewelry screw fixed to the skull. A 28 gauge dummy cannula was inserted to prevent clogging of the guide cannula. After the surgery, the animals were housed individually with direct bedding for 10 days during which they were handled (about 5 min daily) and accustomed to the placement of the ICV injector. Paracet- PLoS ONE | www.plosone.org 13 October 2010 | Volume 5 | Issue 10 | e13464