Mechanism of Onset of Hyperphagia in Obese Rats Figure 6. EGR-1 expression in VAN of DIO rats. A) Protein expression of EGR-1 in VANs of LF, DR and DIO rats treated with leptin. B) Densitometry analysis of A showing that EGR-1 levels in VAN are significantly reduced in DIO rats compared to LF and DR rats. N = 3. C) and D) Quantification of the number of EGR-1 immunopositive neurons in cultured VAN from DR rats (C) and DIO rats (D) fed a high fat diet for 8 weeks. C) Leptin reduced the concentration of CCK required to induce EGR-1 translocation in cultured VAN. D) Leptin had no effect on CCK induced EGR-1 translocation in cultured VAN. N = 6. Data expressed as mean 6 SEM. Significant differences were represented as a,b,c between groups. doi:10.1371/journal.pone.0032967.g006 Leptin acts synergistically with CCK and can modulate CCK signaling in VAN [23,24,28] and leptin has also been found to potentiate the inhibitory action of CCK on food intake [23,24]. Zucker rats are completely insensitive to leptin as a result of a single amino acid substitution of a glutamine for a proline in the leptin receptor gene (Ob-R) [25,29]. Zucker rats homozygous for the fa gene are morbidly obese and characterized by fat cell hypertrophy and hyperplasia [30], increased adipose tissue, lipoprotein lipase activity [31], hyperinsulinemia, hypertriglycer- idemia, and hyperphagia [32] compared to their lean heterozy- gous counterparts. Unsurprisingly, we found that exogenous administration of leptin in these rats failed to phosphorylate STAT3 in both the arcuate nucleus and the nodose ganglia, while lean Zucker rats responded normally to leptin. We demonstrate that loss of leptin signaling in Zucker rats inhibits CCK induced signaling in VAN and at a dose that inhibits food intake in lean Zucker rats, CCK failed to inhibit food intake in obese Zucker rats. Under normal physiological conditions leptin and CCK interact at the level of EGR1 in VAN. Leptin upregulates expression of EGR1 in these neurons, while CCK activates EGR1. Inhibition of EGR1 abolishes leptin potentiation of CCK-induced protein synthesis in VAN [23]. Here we demonstrate that altered leptin signaling, as a result of VAN resistance to leptin following prolonged ingestion of a HF diet, prevents high EGR1 expression in VAN. To study the interactions between leptin and CCK at the cellular level, we used cultured VAN from DR and DIO rats. Interestingly the dose-dependent translocation of EGR1 to the nucleus in response to CCK alone in VAN of DIO rats was identical to that of VAN from DR rats, demonstrating that the sensitivity of VAN from DIO rats to CCK alone is not changed (Fig. 6D). However, the ability of leptin to act synergistically with CCK in the DIO rats is completely abolished. Therefore the response to CCK is unaltered, but rather the potentiating effect of leptin on CCK is lost in VAN of DIO rats, suggesting that leptin activation of VAN is required for optimal CCK signaling. Similarly, the satiating effects of CCK in DIO rats are not completely abolished, instead higher concentrations of CCK are required in these rats to inhibit food intake. Administration of a low dose of CCK (0.22 nmol/kg) after a 12 hr fast inhibited food intake in LF and DR rats but had no effect in DIO rats. However, a 10-fold higher dose of CCK produced a significant reduction in food intake in both DIO and DR rats. We propose that loss of the potentiating effects of leptin in DIO rats is responsible for the reduced sensitivity VAN to CCK signaling and subsequently that higher concentrations of CCK are required for satiation. Feeding in the presence of a CCK1 receptor antagonist completely abolishes feeding induced Y2 expression and inhibition of MCH1R and CB1, and injection of CCK in fasted animals increases Y2 and inhibits MCH1R and CB1 [12–15,33,34] _ENREF_13. Hence, under normal physiological conditions, CCK is required for the regulation of the neurochemical phenotype in VAN [13]. Here we found that lean Zucker rats had high Y2 and low MCH1R and CB1 abundance in VAN; PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e32967