It has been previously shown that a 50 mM glucose stimulus directly into the 3V generates a neuronal response mediated by changes in the expression of anorexigenic and orexigenic neuropeptides28. Here, we tested if this glucose-triggered response was maintained in rats following GK knockdown. At 48 h after transduction, rats were subjected to a 24-h fasting period. Control and GK knockdown rats were next i.c.v. injected with 50 mM D-glucose (or saline). At 2 h after the glucose challenge, the expression of feeding-regulatory neuropeptides was analyzed by qRT-PCR (Fig. 5A–D). After glucose injection, control rats (Ad-shβGal-EGFP) showed a 50% and 70% decrease in the orexigenic peptides, NPY and AgRP, respectively (Fig. 5A,B, respectively) with respect to saline treatment. In contrast, a 70% and 200% increase in the anorexigenic peptides, POMC and CART, respectively, was observed in glucose-injected control rats compared to saline-injected control rats (Fig. 5C,D, respectively). This decrease in orexigenic neuropeptides and increase in anorexigenic neuropeptides is the expected normal response to glucose29. However, when saline and glucose treatment responses were compared in GK knockdown animals, no statistically significant differences were detected in orexigenic neuropeptides (Fig. 5A,B). Regarding anorexigenic neuropeptides, CART expression showed no difference between saline-injected and glucose-injected GK knockdown animals (Fig. 5D). However, POMC expression was decreased in glucose-injected GK knockdown animals compared to saline-injected GK knockdown animals (Fig. 5C). Saline-injected animals (control and GK-inhibited) showed significant differences in anorexigenic neuropeptides, which suggest that GK could be participating in the normal response to starvation. These results clearly indicate that GK knockdown animals lost the glucose-regulated expression of feeding-regulating neuropeptides.Figure 5Expression of neuropeptides involved in feeding regulation. (A–D) Control (Ad-shβGal-EGFP-injected animals) and AdshGK-injected animals (Ad-shGK) were subjected to a 24-h fasting period. After that, saline or D-glucose was injected into the 3 V. After 2 h, NPY (A), AgRP (B), POMC (C) and CART (D) mRNA levels were assessed via qRT-PCR. Data are relative to cyclophilin (media ± SEM; n = 6) and processed by the ΔΔCT method. Statistics: Non-parametric ANOVA; *p < 0.05.