Leptin and insulin act on energy homeostasis through hypothalamic systems, notably two populations of arcuate nucleus (ARC) neurones, the pro-opiomelanocortin (POMC) and neuropeptide Y (NPY) containing neurones. The anorectic actions of both hormones increase POMC and decrease NPY mRNA levels (Niswender & Schwartz, 2003). However, obesity engenders CNS leptin and insulin resistance, which may be due, at least in part, to defective signalling within the ARC. It is therefore important to determine the intracellular signalling mechanisms they utilise in ARC neurones. Leptin drives the janus kinase 2 (JAK2)- signal transducer and activator of transcription 3 (STAT3) pathway to alter transcription and activates phosphoinositide-3 kinase (PI3K) and mitogen activated protein kinase (MAPK) pathways (Niswender & Schwartz, 2003). Although insulin activates PI3K and MAPK in ARC neurones, effects on the JAK2-STAT3 pathway have not been reported.Male Sprague-Dawley rats (4-6 weeks) were killed humanely, hypothalamic slices (400 µm) obtained and equilibrated in aCSF at 22°C for 20 mins, then at 35°C for 1 hr. Isolated ARC wedges were incubated in 0.1 or 1 nM insulin ± 10 µM LY294002 or 10 nM wortmannin for up to 20 minutes, prior to lysis, SDS-PAGE and immunoblotting, whereas whole hypothalamic slices were treated with 0.1 nM insulin for up to 20 minutes, fixed and permeabilised for immunohistochemistry.As expected, insulin caused a rapid ( 1 minute) increase (n =7) in the levels of phosphorylated MAPK (p-MAPK), protein kinase B (p-PKB) and glycogen synthase kinase-3 (p-GSK3), the latter two acting as a sensitive assay for PI3K activity. Insulin also induced rapid tyrosine phosphorylation of STAT3 (p-STAT3) in the ARC immunoblots (n = 7). Furthermore, inhibition of PI3K with wortmannin (n =3) or LY294002 (n = 3) reduced p-PKB, p-GSK3, p-MAPK and p-STAT3 levels, indicating that PI3K activity is central to all three signalling paths. Intact slice studies established that insulin increased STAT3 phosphorylation in various subpopulations of ARC neurones. Dual fluorescence immunohistochemistry, with an anti-ACTH antibody showed that some of the p-STAT3 positive neurones were likely to be POMC-containing. Thus, insulin stimulates phosphorylation of proteins in the PI3K, MAPK and JAK- STAT pathways within the ARC, recapitulating the actions of leptin. Further work is required to determine the nature of the non-POMC, p-STAT3 positive ARC neurones and the exact signalling mechanism controlling STAT3 phosphorylation.