Fig. 3.

NPY decreases PC2 and Egr-1. A: Egr-1 protein in the ARC of control and NPY icv-infused rats (control n = 5, NPY n = 4; t-test, P = 0.0297). B–G: experiments were performed using the hypothalamic POMC-positive N43-5 cell line. B: N43-5 cells were transfected with PC2 promoter-luciferase construct along with pRL-renilla SV40. Twenty-four hours later, cells were treated with vehicle control (V) or cAMP activators forskolin (FK10 μM), MTII (50 μM), or 8-Br-cAMP (100 μM) for 1.5 h and then lysed. Values are shown normalized to renilla (n = 3/group ANOVA, P < 0.05). Results were repeated in 2 independent experiments (n = 3/group/experiment). C: PC2 protein levels with and without overexpression of Egr-1 by cDNA transfection (n = 5/group, t-test, P < 0.0001). Results were repeated in 2 independent experiments (n = 3/group/experiment). D: Egr-1 protein levels with and without NPY (100 μM; n = 6/group, t-test, P < 0.0337). Results were repeated in an independent experiment (n = 3/group). E: PC2 promoter activity (normalized to renilla) in cells treated with 100 μM NPY for 0, 30, 60, and 90 min (n = 6/group, ANOVA, P = 0.0004). Results were repeated in an independent experiment (n = 3/group). F: PC2 protein levels at 0, 30, 60, and 90 min post-NPY treatment (100 μM) in N43-5 cells transfected with PC2, NPY-Y1 receptor (NPY-Y1R), and Egr-1 (n = 4/group, ANOVA, P = 0.0042). Results were repeated in an independent experiment (n = 3/group). G: PC2 protein levels at 0, 30, 60, and 90 min post-NPY treatment (100 μM) in N43-5 cells transfected with PC2 and NPY-Y1R but not Egr-1 (n = 4/group, Kruskal-Wallis test, P = 0.067). Results were repeated in an independent experiment (n = 3/group). Tukey's test was used for all post hoc analyses. See text for definitions. Data are means ± SE. *P < 0.05, **P < 0.01 vs. control.