Neuronal COUP-TFII Regulation Figure 8. Regulation of hepatic and pancreatic COUP-TFII mRNA levels by activation of the melanocortin pathway. (A–D) RT-qPCR analysis of mRNA from livers (A–B) or pancreas (C–D) of ad libitum fed mice receiving a ICV injection of CSF (%), or 18 h fasting mice receiving a ICV injection of CSF (grey bar), or 18 h fasting mice with a ICV injection of MTII (&). (A) Hepatic PEPCK mRNA levels (B) Hepatic COUP-TFII mRNA levels. (C) Pancreatic COUP-TFII mRNA levels (D) Pancreatic Neuro D mRNA levels. Data are expressed as mean 6 SEM (n = 5) *P#0.05 when compared to control fed mice receiving a CSF injection. 1 P#0.05 when compared to control fasting mice receiving a CSF injection. doi:10.1371/journal.pone.0013464.g008 expressing this receptor will be activated throughout the hypothal- amus. Using a hypothalamic cell line expressing the MC4-R receptor, we have shown that its direct activation induces COUP- TFII mRNA levels in these cells, so part of the MTII effect on COUP-TFII gene expression in vivo is mediated through a direct activation of the melanocortin receptors expressed on the VMH neurons. MC3/4-R belongs to the family of G protein-coupled seven-transmembrane-domain receptors and the binding of its agonist induces an increased cAMP production [30]. A COUP-TFII gene promoter analysis had identified cAMP response element localized in the first 300 bp from the transcription start site [31]. In our hypothalamic cell line it did drive also the cAMP dependant activation of the COUP-TFII gene. So in vivo, an activation of the MC3/4-R could affect COUP-TFII gene expression directly through a transcriptional mechanism regardless of the peripheral effect of MTII on the other nuclei. Our previous studies had shown that insulin was down- regulating COUP-TFII gene expression in liver and pancreas. In regards to COUP-TFII functions in those tissues, i.e controlling the glucose dependant insulin secretion by the pancreatic beta cells and gluconeogenesis by the hepatocytes, this coordinated down- regulation is therefore contributing to glucose homeostasis [7]. In the hypothalamus, we observed an up regulation of its expression in fed mice, i.e. when peripheral insulinemia is at his highest. This apparent opposite effect of insulin on COUP-TFII gene expression is reminiscent of the overall observation regarding peripheral vs central insulin effects on energy homeostasis. In peripheral tissues, insulin is rightly considered as the anabolic hormone: insulin injections induce hypoglycemia with a temporary increase in food intake as a consequence and it will promote fuel storage in peripheral organs, mainly in liver and adipose tissues. On the other hand, the central effect of insulin on the hypothalamus is PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13464