Figure 5.

Leptin stimulates EGR1 promoter activity in vagal afferent neurons via STAT3, and ghrelin inhibits STAT3 translocation to the nucleus. A, Cultured vagal afferent neurons transfected with EGR1-luc do not exhibit increased luciferase activity in response to treatment with 10 nm CCK8s (16 h); however, 10 and 30 ng/ml leptin dose-dependently increased luciferase activity (*, P < 0.05 compared with control), and CCK8s had no further effect. Preincubation (30 min) with 10 nm ghrelin inhibited leptin-induced EGR1-luc activity (**, P < 0.05 compared with leptin alone). B, The PKC inhibitor Ro32-0432 and the inhibitor of Erk1/2 activation U0126 had no effect on leptin-induced (30 ng/ml) expression of EGR1-luc, whereas the STAT3 inhibitor AG490 prevented the action of leptin (*, P < 0.001 compared with response to leptin alone). C–H, Vagal afferent neurons cultured for 72 h and stained with antibody to STAT3 (C–E) and DAPI (F–H) in respective fields. Nuclear STAT3 staining was undetectable after transfer to serum-free medium for 2 h (C and F). Nuclear STAT3 was readily detectable after treatment with 30 ng/ml leptin in serum-free medium (D and G), whereas ghrelin (10 nm) in the presence of 30 ng/ml leptin inhibited nuclear localization of STAT3 (F and H). Representative images from six independent experiments are shown. Scale bar, 50 μm.