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 together, the data suggest that leptin resistance in VAN may account for the well known decrease in lipid- and CCK-induced satiation and hyperphagia following chronic HF feeding. Inaccurate monitoring of nutrients in the intestinal lumen may lead to a lack of satiety and contribute to diet-induced hyperphagia and obesity.
Leptin acts synergistically with CCK and can modulate CCK signaling in VAN 
and leptin has also been found to potentiate the inhibitory action of CCK on food intake 
. 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) 
. Zucker rats homozygous for the fa
gene are morbidly obese and characterized by fat cell hypertrophy and hyperplasia 
, increased adipose tissue, lipoprotein lipase activity 
, hyperinsulinemia, hypertriglyceridemia, and hyperphagia 
compared to their lean heterozygous 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 
. 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 (). 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 
_ENREF_13. Hence, under normal physiological conditions, CCK is required for the regulation of the neurochemical phenotype in VAN 
. Here we found that lean Zucker rats had high Y2 and low MCH1R and CB1 abundance in VAN; 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 
, influences gastric emptying 
and inhibits food intake 
. Subdiaphragmatic vagotomy attenuates the effects of PYY3–36
on food intake in rats 
suggesting that Y2 receptor downregulation 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 (). 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 ().
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 
. Interestingly, pair feeding OLETF rats with intact controls (LETO rats) prevented the development of obesity 
. This suggests that reduced sensitivity to CCK results in hyperphagia and could directly lead to the development of obesity.
While mice lacking the CCK1
R eat larger and longer meals both in response to chow or a high fat diet compared to wild type mice 
, 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 CCK1
R 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 
. Mice with this genetic background have been reported to have low feeding efficiency (small weight gain per calorie consumed), and high basal energy expenditure 
. Another important difference is that OLEFT and LETO rats express the NPY gene in the dorsal motor nucleus of the hypothalamus (DMH) while wild-type and CCK1 receptor−/−
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 
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.