Endocrine Abstracts

Hypothalamic MC4R receptors are G_alphas-coupled GPCR’s that mediate the satiety response to αMSH. MC4R expression predominates in the PVN/VMH regions of the hypothalamus, regulating satiety gene expression (e.g. Bdnf). Human MC4R mutations often result in compromised G_alphas-cAMP-PKA signalling and obesity onset. However, downstream signalling pathways have yet to be elucidated. We have investigated the potential for αMSH to stimulate phosphorylation of multiple MAPK family proteins in a range of immortalised hypothalamic cell lines (N29, N39, N40, N42 and GT1–7) by Western blotting and immunocytochemistry. Initial studies revealed 100 nM alphaMSH stimulated ERK phosphorylation within 15 min in all cell lines apart from N29 cells, suggesting the presence of MC3R or MC4R receptors. MC4R expression was confirmed by RT-PCR. Subsequent studies were performed in N39 cells due to low background levels of phosphoERK. Time-course studies showed alphaMSH stimulated phosphoERK, phosphoJNK and phospho-p38MAPK levels after 5 min and for up to 30 min. A concentration of 100 pM alphaMSH was sufficient to cause ERK phosphorylation in dose-response studies. Phosphorylation of ERK following alphaMSH treatment was dependent on MEK activity (as determined by blockade with UO126) and partially required PKA activity (H-89 pre-treatment). However, JNK phosphorylation was sensitive to pharmacological blockade of MEK, PKA, PKC and calcium entry, suggesting divergent signalling pathways are activated by the MC4R in N39 cells. Fluorescent immunocytochemistry for phosphoERK localisation revealed enhanced nuclear localisation from 30 min to 90 min post-alphaMSH treatment. To examine the effect of physiological antagonists of the MC4R, GT1-7 cells were co-treated with 100 nM AGRP and 100 nM alphaMSH that resulted in diminished ERK phosphorylation. Collectively, these data suggest alphaMSH can cause activation of three MAPK pathways in hypothalamic cell lines via multiple signalling intermediates. The biological consequences of activating these pathways in N39 cells remains to be established.