Neuronal COUP-TFII Regulation We therefore investigated the effect of insulin or of a MC3/4-R agonist on hypothalamic COUP-TFII gene expression in a hypothalamic mouse cell line, the GT1-7 cells, known to express the insulin receptor, the COUP-TFII protein (data not shown) as well as the MC4-R receptor [29].First, the cells were cultured for 18 hours with 100 nM insulin. Under these conditions, we observed a strong repression of COUP-TFII mRNA levels by insulin in cells directly exposed to insulin (figure 6A). On the other hand, when the cells are cultured for 18 hoursin the presence of increasing amounts of the MC4-R agonist, the melanotan II (MTII), a aMSH analog, a concentration of 1 nM MTII is sufficient to cause a significant increase of COUP-TFII mRNA Figure 6. COUP-TFII gene expression in response to the MC4-R activation in GT1-7 cell line. GT1-7 cells were cultured for 18 hours in the presence of either (A) insulin (100 nM) or increasing amount of (B) MTII or (C) DiBcAMP. Results are expressed as a fold increase over the value obtained without MTII (A) or without DiBcAMP (B). (D) Transient transfection of a minimal COUP-TFII promoter in response to DiBcAMP. Data are expressed as the mean 6 SEM. (n = 4). *P#0.05 **P#0.01 when compared to cells cultured in the absence of MTII or DiBcAMP. doi:10.1371/journal.pone.0013464.g006 PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13464