however, in obese Zucker rats, Y2 expression was constitutively low in VAN and feeding failed to inhibit MCH1R and CB1 expression in VAN. Therefore in the absence of leptin signaling, VAN are unable to alter their neurochemical phenotype in response to endogenous CCK after a meal. Similarly, VAN of DIO rats did not respond to feeding, exhibiting elevated Y2 abundance, as well as reduced MCH1R and CB1 expression compared to LF or DR rats. DIO rats develop leptin resistance in VAN, prior to measurable leptin resistance in the ARC, therefore we conclude that hypothalamic leptin resistance is not required for CCK induced signaling and satiation. We hypothesize instead that reduced VAN sensitivity to CCK, as a result of leptin resistance in these neurons, is responsible for the altered expression of these receptors and would have important functional consequences on vagally mediated regulation of food intake. Changes in receptor expression on VAN of DIO animals suggest that the sensitivity of VAN to the gastrointestinal hormones that bind these receptors will be altered. Reduced Y2 expression in VAN of obese animals would prevent signaling of the anorexigenic hormone PYY3–36 via VAN in response to a meal. PYY3–36 is an anorectic peptide released from L cells in response to a meal which inhibits pancreatic enzyme secretion [35], influences gastric emptying [36] and inhibits food intake [37]. Subdiaphragmatic vagotomy attenuates the effects of PYY3–36 on food intake in rats [38,39] suggesting that Y2 receptor downreg- ulation in VAN of obese animals would prevent the satiating effects of PYY3–36. Furthermore constitutively high MCH1R and CB1 expression in VAN of obese animals suggests that anandamide released from the gut and MCH from VAN would prolong orexigenic signaling. Thus altered receptor expression could increase the orexigenic tone of the vagus, potentially resulting in hyperphagia. DIO rats develop hyperphagia between weeks 4 and 5 following chronic HF feeding. In the first 4 weeks, DIO and DR rats consume the same amount of food, but by week 5 the DIO rats consume 10 kcal per day more than the DR rats (Fig. 3C). Interestingly we provide evidence that functional leptin resistance develops between weeks 4 and 6 of a HF diet, which coincides with the onset of hyperphagia in these rats (Fig. 4D). There is evidence in the literature to suggest that altered CCK sensitivity could result in hyperphagia. Both knockout mice and OLEFT (Otsuka Long Evans Tokushima Fatty) rats that lack the CCK1R eat larger meals than their respective controls. Crucially OLETF (Otsuka Long Evans Tokushima Fatty) rats, which lack the CCK-1 receptor gene, have increased average meal size, with reduced meal frequency that is insufficient to compensate for the increase in meal size, resulting in hyperphagia [40]. Interestingly, pair feeding OLETF rats with intact controls (LETO rats) prevented the development of obesity [41]. This suggests that reduced sensitivity to CCK results in hyperphagia and could directly lead to the development of obesity. While mice lacking the CCK1R eat larger and longer meals both in response to chow or a high fat diet compared to wild type mice [42], they have statistically insignificant increases in total daily food intake and maintain normal body weight. The reason for the difference in response between knockout mice and OLEFT rats to the absence of CCK1R is not clear. However, it should be noted that the knockout mice are on a 129S genetic background, which is known to confer resistance to obesity [43]. Mice with this genetic background have been reported to have low feeding efficiency (small weight gain per calorie consumed), and high basal energy expenditure [44]. Another important difference is that OLEFT and LETO rats express the NPY gene in the dorsal motor Mechanism of Onset of Hyperphagia in Obese Rats nucleus of the hypothalamus (DMH) while wild-type and CCK1 receptor2/2 mice do not. Crucially, DMH NPY gene expression is dysregulated in OLEFT rats so that in response to a high-fat diet, NPY expression is significantly reduced in LETO rats but not in OLETF rats [45]. In conclusion, we demonstrate that the onset of leptin resistance in VAN of DIO rats results in the reduced sensitivity of VAN to CCK. This leads to altered expression of receptors in VAN known to play a role in the regulation of food intake. We hypothesize that these changes result in increased orexigenic signaling and reduced anorexic sensing of hormones from the gut resulting in the development of hyperphagia. Materials and Methods Ethics Statement All experiments with animals reported complied with relevant national and international guidelines. All experimental procedures involving rats complied with an animal studies protocol (#15202) approved by the UC Davis Institutional Animal Care and Use Committee (IACUC) and PHS animal welfare assurance to UC Davis (#A3433-01). Rats Male Sprague Dawley rats (initial weight 180 g; Harlan San Diego, n = 36) were housed at 22uC under a 12 h light (6am– 6pm), 12 h dark (6pm–6am) cycle with ad libitum access to food and water. Rats were kept on chow (13% kcal/g fat; Purina 5001) or high fat (HF) diet (45% kcal/g fat; Research Diets D12451) for 8 weeks. Body weight and food intake were measured twice a week. Feeding studies were performed between weeks 4 and 8. After week 8 on respective diets, rats were fasted overnight, and were injected the following morning (9–10 am) either leptin (4.94 nmol/kg, i.p.) or saline (400 ml, i.p.). After 1 h rats were deeply anesthetized with a mixture of sodium phenytoin and pentobarbital sodium (0.2 ml/100 g i.p., Beutha- nasia-D Special C-III; Shering, Kenilworth, NJ) and tissue was collected. Male Zucker rats (12 lean and 12 obese; 6 weeks old; Charles River) were fed chow for three weeks before tissue collection, during which time all feeding studies were performed. Feeding studies Rats were fasted for 12 h starting at 9am. CCK feeding study: CCK (0.22 nmol/kg or 2.19 nmol/kg; i.p.) or saline (400 ml, i.p.) were administered and food intake was recorded every 20 min for 2 h. The low dose of CCK was chosen based on work by Savastano and Covasa [10] demonstrating that DIO rats were insensitive to this dose of CCK. We selected a 10 times higher dose to show that DIO rats had reduced sensitivity to CCK rather than an inability to respond. Leptin feeding study: Leptin (4.94 nmol/ kg; i.p.) or saline (400 ml; i.p.) were administered. Leptin dose was selected based on previously published work in our laboratory [4,23]. Animals were allowed to consume 10 kcal of chow for 40 min; food intake was then recorded every 20 minutes for 2 h as previously described [4]. All rats within each group (LF, DR and DIO) were paired according to body weight; one rat in each pair received either vehicle or treatment. Peptides and drugs Leptin (rat) was obtained from Sigma (St. Louis, MO). Cholecystokinin, CCK (octapeptide, sulfated) was purchased from Bachem (Torrance, CA). PLoS ONE | www.plosone.org 8 March 2012 | Volume 7 | Issue 3 | e32967