Neuronal COUP-TFII Regulation Figure 7. Regulation of hypothalamic COUP-TFII mRNA levels in vivo by activation of the melanocortin pathway. (A-C) RT-qPCR analysis of mRNA levels from hypothalamus 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) BDNF mRNA levels (B) Neuro D mRNA levels (C) COUP-TFII mRNA levels. Data are expressed as mean 6 SEM (n = 5) *P#0.05 **P #0.01 when compared to control fed mice receiving a CSF injection. 1 P#0.05 when compared to control fasting mice receiving a CSF injection. (D) Glucose and plasma insulin concentrations from the different mouse models. doi:10.1371/journal.pone.0013464.g007 the melanocortin system. Changes in the expression of these neuropeptides in the ARH are regarded as a key mechanism mediating the insulin effect in the central nervous system. In keeping with this mechanism, we observed that in our nutritional and hormonal, eGHI and STZ mouse models, variations of circulating plasma insulin are closely associated with variation of the hypothalamic COUP-TFII mRNA levels, paralleling the modulation of POMC gene expression and therefore of the melanocortin receptor agonist (aMSH) production. Our experi- ments on a hypothalamic cell line with insulin showed that this hormone was able to repress COUP-TFII mRNA expression when applied directly on the cells expressing COUP-TFII as it was observed on a pancreatic beta cell line or on cultured hepatocytes [7]. In vivo, the observed induction of COUP-TFII gene expression by the fed state could be therefore mediated indirectly by insulin through the conjunction of decreasing amount of AgRP and increasing amount of POMC leading downstream to an activation of the MC3/4-R. MC4-R and MC3-R have been shown to be widely distributed throughout different hypothalamic regions, with a higher expression in the ARH, the VMH, the dorso medial hypothalamus, the paraventricular nucleus and the median eminence [27,28]. There- fore in our in vivo experiments with a central ventricular injection of a MC3/4-R agonist, the MTII, a complex network of neurons PLoS ONE | www.plosone.org 10 October 2010 | Volume 5 | Issue 10 | e13464