Effect of a HF diet on body weight and food intake Sprague Dawley rats carry a polygenic susceptibility to high fat diet-induced obesity; individuals become either diet-induced obese (DIO) or remain lean (diet-induced obese resistant DR) when ingesting high fat diet [27]. We have recently demonstrated that chronic (8 week) ingestion of a high fat diet leads to the development of leptin resistance in VAN of DIO, but not in DR or low fat (LF) fed control rats, and that this occurs in the absence of measurable leptin resistance in the arcuate nucleus of the hypothalamus [4]. Hence this rat model was used to determine whether leptin signaling in VAN is required for CCK-signaling and its subsequent anorexigenic effects. A total of 24 Sprague Dawley rats were fed a HF diet for 8 weeks. Rats with the highest body weight at 8 weeks were assigned to the DIO group (n = 12), the other rats were designated DR (n = 12). After 8 weeks, DIO rats had significantly higher body weight compared to either LF fed controls rats or DR rats (p,0.01, Fig. 3A); there was no significant difference between DR and LF controls (p.0.05). DIO rats had a significantly higher body weight gain after 4 weeks on a HF diet compared to both the DR and LF controls (p,0.01; p,0.001, respectively; Fig. 3B). In the first week on a HF diet, both DIO and DR groups had a higher caloric intake than LF fed controls (p,0.001). Following acclimation to the HF diet in the first week, DR rats had equal caloric intake to the LF fed controls. DIO rats had a consistently higher caloric intake than either LF and DR rats but this only reached significance after 5 weeks (p,0.001, p,0.01, respectively, Fig. 3C). Leptin resistance in VAN decreases CCK-induced inhibition of food intake Administration of CCK8S (i.p., 0.22 nmol/kg) after 12 hr fasting reduced food intake by 27% in LF and DR rats compared to saline (Fig. 4A; p,0.01) but had no effect in DIO rats (Fig. 4A; p.0.05). However, a higher dose of CCK8S (i.p., 2.19 nmol/kg) significantly reduced food intake in DIO as well as LF and DR rats compared to saline (Fig. 4B; p,0.01). To determine whether leptin resistance in VAN of DIO rats would inhibit the satiating effects of endogenous CCK, we used a previously described feeding protocol in which a small preload meal was used to release endogenous CCK [23]. In this protocol we found that exogenous leptin had no effect on food intake in fasted rats. After a preload meal (which increases endogenous CCK), we observed that exogenous leptin inhibited food intake, and these effects were abolished by a CCK1R antagonist, lorglumide [23]. Therefore, we concluded that leptin potentiates the satiating effects of endogenous CCK. Here we show using this same protocol (Fig. 4C) that after 4 weeks on the respective diets, leptin significantly potentiated CCK- induced inhibition of food intake in LF, DR, and DIO rats (LF, p,0.01; DR, p,0.01; DIO, p,0.01; Fig. 4D). After 6 weeks on a HF diet, DIO rats receiving leptin (i.p.; 4.94 nmol/kg) no longer significantly inhibited food intake even in the presence of endogenous release of CCK by a preload meal (LF: p,0.01; DR: p,0.01; DIO: p.0.05; Fig. 4D). After 8 weeks leptin had no effect on food intake in DIO rats (LF: p,0.01; DR: p,0.01; DIO: p.0.05; Fig. 4D). In contrast, LF fed and DR rats after 6 and 8 weeks on LF or HF diets, respectively, had reduced feeding in response to exogenous leptin (i.p..; 4.94 nmol/kg) compared to vehicle following the release of endogenous CCK induced by the preload meal (p,0.01; Fig. 4D). Leptin resistance in VAN decreases CCK-induced alteration in neurochemical phenotype Under fasting conditions, VAN of LF and DR rats expressed low Y2 abundance while in the fed state Y2 abundance is Mechanism of Onset of Hyperphagia in Obese Rats Figure 3. Effect of a high-fat (HF) diet on body weight and food intake A) Body weight of rats ingesting either a LF diet or HF diet (45% kcal) for 8 weeks. There was a significant increase in body weight in diet-induced obesity (DIO) rats compared with diet-induced obesity-resistant (DR) and LF animals. B) The increase in body weight of LF, DR, DIO rats expressed as a percent of weight gain from initial weight. DIO rats became significantly heavier than LF fed animals after 4 weeks of HF diet, and after 4 weeks of a HF diet compared to the DR rats. C) Average daily food intake (kcal) per week over the 8 weeks on the diets. In the first week, all rats on the HF diet had a significantly higher energy intake than LF fed rats but this initial hyperphagia only lasted for one week after which there was no significant difference in caloric intake between the groups. At week 5, the DIO group had a significantly higher energy intake than LF rats and DR rats. N = 12 per group; data expressed as mean 6 SEM. Significant differences were represented as a,b,c between groups. doi:10.1371/journal.pone.0032967.g003 PLoS ONE | www.plosone.org 4 March 2012 | Volume 7 | Issue 3 | e32967