Leptin and insulin regulate transcription and alter the excitability of various arcuate nucleus (ARC) neuronal subpopulations. One subtype is the orexigenic neuropeptide Y (NPY) expressing neurons, which are inhibited by leptin and insulin. Obesity is associated with CNS leptin and insulin resistance, which may be due to defective signalling within target neurons. Leptin and insulin stimulate various signalling pathways in ARC neurons, including the phosphoinositide-3 kinase (PI3K) pathway, and affect actin dynamics, in a PI3K-dependent manner, to alter the electrical activity of certain neurons (Mirshamsi et al. 2004). However, these actions have not been investigated within defined ARC neuronal subtypes. The NPY-expressing neuronal cell line (N29/4) was cultured as described (Belsham et al. 2004). For immunoblotting, cells were treated with leptin or insulin for various times in the absence and presence of the PI3K inhibitors LY294002 or wortmannin or the F-actin stabilising agent, jasplakinolide. Levels of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3) were determined by staining with the pleckstrin homology domain of the general receptor for phosphoinositides-1 protein fused to GFP, and F-actin examined as described by Mirshamsi et al. (2004). Leptin (10 nM) and insulin (1 nM) increased levels of phosphorylated mitogen activated protein kinase (p-MAPK), protein kinase B (p-PKB) and its downstream target glycogen synthase kinase 3 (p-GSK3) (n = 8), and phosphorylation of the latter two proteins was dependent on PI3K activity (n = 4). Stimulation of p-PKB and p-GSK3 levels by insulin was sustained (up to 60 minutes examined; n = 8). In contrast leptin induced a transient increase in p-PBK and p-GSK3, lasting less than 15 minutes (n = 8). Insulin induced a sustained increase in PIP3 (up to 60 minutes; n = 3) whereas the leptin increase in PIP3 was transient (< 15 minutes; n = 3). F-actin staining revealed that insulin (10 nM) did not alter F-actin levels (n = 4) whereas leptin (10 nM) caused rapid (within 15 minutes) F-actin disruption (n = 6). Leptin-driven increased p-PKB and p-GSK3 remained transient after stabilisation of F-actin with jasplakinolide (n = 4). Therefore, the change in F-actin is not responsible for the transient nature of the leptin PI3K response. The differences in leptin and insulin signalling, via the PI3K pathway, observed in this hypothalamic cell line, may be relevant to the mechanism by which leptin and insulin induce differential actions on excitability of ARC neurons (Choudhury et al. 2005).