calcium in culture [21]. However, leptin also plays a crucial role in modulating the sensitivity of these neurons to CCK. Leptin markedly enhances vagal afferent discharge [20], cytosolic calcium [21,22], and translocation of the early gene EGR-1 to the nucleus [23] in response to low doses of CCK. Leptin regulates EGR-1 levels while CCK activates EGR-1 in VAN. Inhibition of EGR-1 abolishes leptin-induced sensitization of VAN to CCK and reduces CCK-induced expression of CART in VAN [23]. Vagally- mediated changes in function, such as inhibition of food intake and gastric emptying in response to CCK, are also enhanced by leptin [23,24]. We have recently demonstrated that chronic ingestion of a high fat diet leads to the development of leptin resistance in VAN of diet- induced obese rats, compared to low fat fed control rats or rats resistant to the obesigenic effects of a high fat diet [4]. In the present study, we hypothesize that leptin resistance in VAN of DIO rats is responsible for the reduced sensitivity of these neurons to CCK, resulting in an altered neurochemical phenotype in VAN and hyperphagia. Firstly, Zucker rats which have a genetic deletion of the leptin receptor were used to determine the effect of altered leptin function on VAN. Secondly, Sprague-Dawley rats with a predisposition to diet-induced obesity (DIO), that develop leptin resistance in VAN when fed a HF diet [4], were used to determine changes in CCK-induced inhibition of food intake and signaling in VAN in response to long term ingestion of a high fat diet. Mechanism of Onset of Hyperphagia in Obese Rats Results Reduced sensitivity of VAN to CCK in obese Zucker rats Initial studies using Zucker rats demonstrated that leptin signaling is required for CCK-induced activation of VAN and its subsequent anorexigenic effects. Zucker rats are a genetic model of leptin receptor deficiency [25,26]. As previously reported, homozygous Zucker rats (LepRfa/LepRfa), weighed significantly more than lean Zucker rats when fed on chow (p,0.01; Fig. 1A). Exogenous leptin (4.94 nmol/kg; i.p.) phos- phorylated STAT3 in the nodose ganglia and arcuate nucleus of lean Zucker rats; however as expected, leptin had no effect on STAT3 phosphorylation in either arcuate or vagal afferent neurons from obese Zucker rats (Fig. 1B–E). To determine whether CCK-induced inhibition of food intake requires leptin signaling, food intake in fasted lean and obese Zucker rats was measured following administration of CCK8S (i.p.; 0.22 nmol/kg). In lean Zucker rats, CCK induced a 25% reduction in food intake compared to saline; CCK had no effect on food intake in obese Zucker rats (Fig. 1F). To determine whether leptin signaling is required in the regulation of the neurochemical phenotype of VAN, immunore- activity of Y2, MCH1R and CB1 was examined in the nodose ganglia of lean and obese Zucker rats in the presence or absence of Figure 1. Altered CCK induced satiety in obese Zucker rats. A) Obese Zucker rats weigh significantly more than lean Zucker rats (N = 12; t- test). B) Immunoblot of pSTAT3 in VAN of lean (top) and obese (bottom) Zucker rats treated with vehicle or leptin. C) Densitometry analysis of B showing that STAT3 is phosphorylated in response to leptin (i.p) in VAN of lean Zucker rats but not in obese Zucker rats. N = 4 D) Immunoblot of pSTAT3 in hypothalamus of lean and obese Zucker rats treated with leptin. E) Densitometry analysis of D showing that STAT3 is phosphorylated in response to leptin in the arcuate nucleus of the hypothalamus of lean Zucker rats, but not obese Zucker rats. (N = 4).F) CCK8S (i.p., 0.22 nmol/kg) significantly inhibited food intake in lean Zucker rats but not obese Zucker rats. N = 6. Data expressed as mean 6 SEM. Significant differences were represented as a,b,c between groups in one-way ANOVA. Significant differences were represented as * for p,0.05; ** for p,0.01; and *** for p,0.001 in Student’s t-test. doi:10.1371/journal.pone.0032967.g001 PLoS ONE | www.plosone.org 2 March 2012 | Volume 7 | Issue 3 | e32967