Figure 4. Reduced satiation to exogenous and endogenous CCK in DIO rats. A) CCK feeding study (described in methods) in Sprague Dawley rats after 8 weeks on respective diets. CCK8S Mechanism of Onset of Hyperphagia in Obese Rats (0.22 nmol/kg; i.p.) significantly inhibited food intake compared to vehicle in LF and DR, but not DIO rats. N = 6. B) CCK8S (2.19 nmol/kg; i.p.) significantly inhibited food intake in all rats. N = 6. C) Protocol for leptin feeding study in which endogenous CCK was upregulated D) Endogenous CCK reduced food intake in LF fed and DR rats at 4, 6 and 8 weeks. Endogenous CCK reduced food intake in DIO rats at 4 weeks but not at 6 and 8 weeks. N = 6; data expressed as mean 6 SEM. Significant differences were represented as ** for p,0.01. doi:10.1371/journal.pone.0032967.g004 increased as previously described (Fig. 5A) [12]. In contrast, Y2 expression was constitutively low in VAN from DIO rats in the fed and fasted states (Fig. 5A). Similarly, in LF and DR rats, MCH1R and CB1 expression was high in VAN from fasted rats and decreased upon feeding (Fig. 5B–C); however, in DIO rats these proteins were constitutively expressed even in the fed state (Fig. 5B–C). Leptin resistance prevent CCK signaling in VAN Leptin-induced phosphorylation of the transcription factor STAT3 is required for EGR-1 expression and EGR-1 is involved in mediating gene expression [23]. We have previously shown that leptin resistance (as defined by an absence of phosphorylation of STAT3 in response to leptin) is present in VAN of DIO rats after 8 weeks of chronic high fat feeding [4]. We hypothesized, therefore, that the absence of pSTAT3 in leptin-resistant VAN of DIO rats would result in low EGR-1 abundance. To test this, we measured EGR-1 levels by western blot in the nodose ganglia of leptin- treated rats following 8 weeks on the respective diets. EGR-1 expression was high in VAN of both LF and DR rats in response to leptin (i.p.; 4.94 nmol/kg), but was absent in VAN from DIO rats (Fig. 6A–B). Leptin and CCK interact at the level of EGR1 in VAN. To study these interactions at the cellular level, we cultured VAN of DR and DIO rats and determined the effect of leptin-resistance on CCK-induced EGR-1 translocation to the nucleus. In cultured VAN from DR rats (Fig. 6C), CCK8S alone induced maximal EGR-1 activation at 10 nM but had little effect at lower doses. Leptin alone (10 ng/ml) had no effect on EGR-1 nuclear translocation but significantly increased the number of nuclear EGR-1-positive neurons in the presence of low doses of CCK (0.01–1 mM) (Fig. 6C). The dose-dependent response to CCK alone in VAN of DIO rats (Fig. 6D) was identical to that of VAN from DR (Fig. 6C) or LF rats [23]. However, the ability of leptin to act synergistically with CCK in VAN of DIO rats is completely abolished (Fig. 6D). Discussion We have previously shown that VAN from diet-induced obese rats are leptin-resistant; this leptin resistance occurs only in rats that express the obese phenotype and not in rats that remain lean when ingesting a HF diet. Here, we wanted to determine the consequences of impaired leptin signaling in VAN by studying EGR-1 signaling in VAN, changes in VAN neurochemical phenotype in response to feeding, and functional responses on control of food intake. The data show that in Zucker rats, the lack of leptin signaling is associated with a complete lack of phenotypic change of VAN normally induced by feeding and this is associated with an absence of CCK-induced inhibition of food intake in these rats. Moreover, in DIO rats, the lack of leptin signaling in VAN, results in a lack of phenotypic change in VAN in response to feeding, a decrease in CCK-induced nuclear EGR-1 and a lack of endogenous CCK-induced inhibition of food intake. Taken PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32967