The effects of RGD on pre- and post-prandial POMC and NPY expression in the arcuate nucleus and NTS were examined. In the arcuate nucleus, POMC levels rose predictably following food intake for both control and RGD animals. POMC expression levels were low in the NTS, and no significant differences were detected in either group following food intake. Decreased plasma leptin levels, and trends towards increased baseline arcuate and NTS NPY mRNA expression were observed in animals exposed to RGD. This marginal elevation in baseline NPY following RGD may contribute to the hyperphagia that occurs in BN, BED, and certain types of obesity.
Following food intake, NPY expression declined to normal levels in RGD rats. This would imply that RGD leads to a change in feelings of hunger, but not satiety. It is perplexing that, despite this trend towards elevated NPY, animals do not show any differences in food intake patterns at the time of sacrifice, as compared to control animals. One possibility is that, following RGD, sensitivity to NPY is decreased. Though we have demonstrated no effect of RGD on POMC expression, it is possible that the relative decrease in NPY is leading to less inhibition of CART or MC4r activity, resulting in a greater feeling of satiety. One would expect a longer inter-meal interval if this were the case. As our measurements of food intake lacked the temporal resolution required to test this, further investigation is needed.
RGD animals had lower leptin levels, as compared to controls, but this reduction was unrelated to daily food intakes or body weights. It is difficult to ascertain whether the trend towards decreased leptin resulted from alterations in gastric or peripheral leptin. Since animals weighed the same and did not differ in cumulative daily food intakes, it is unlikely that they had increased adiposity; however, the possibility remains that RGD, or the altered food intake patterns resulting from RGD, led to an alteration in fat mass accumulation. Future studies will investigate this phenomenon via nuclear magnetic resonance to determine if and when overall adiposity is altered in RGD, and fat pads will be weighed upon sacrifice to determine any regional differences in fat accumulation.
If distension contributes to gastric leptin secretion, overstimulation of mechanoreceptors via RGD could lead to a desensitization of the secretory granules, and an elevated threshold for leptin secretion. Gastric leptin exerts at least some of its effects via the vagus nerve, as evidenced by a reduction in food intake in sham-operated but not vagotomized rats following a physiological dose of leptin [45
]; it is likely that mechanoreceptors in the gut are one of its targets. If this is the case, it is possible that irregularities present in binge eating disorders, such as the higher threshold for fullness and increased gastric capacity, are due, at least in part, to alterations in gastric leptin. Given the want for knowledge pertaining to gastric leptin, more research is warranted to characterize this important hormone. Irrespective of its source, the RGD-induced leptin suppression despite similar body weights is consistent with the human literature. While it would be premature to suggest a causal role for RGD on plasma leptin levels, a relationship between the two should not be ruled out.
Regardless of treatment group, all rats exhibited similar growth patterns, and consumed equal amounts of food throughout the experimental period. Though unsurprising, given the transient nature of the inflations, this is in contrast to previous findings in rats and humans that have been given permanent, indwelling gastric balloons, in which total daily food intake levels and body weights were reduced for a period of time following implantation [13
]. The periodicity of feeding, however, was altered during the first two weeks of RGD, with experimental rats consuming their food at least an hour later than the control group. Many others have observed this phenomenon [47
]. In the human literature, individuals reported an increased feeling of fullness after balloon distension, as well as a decreased desire to eat. This effect was transient, and both human subjects and RGD rats consumed equivalent daily calories as controls [50
]. Two possible explanations for this delay in meal-initiation are that the mechanoreceptors in the GI tract continue to signal that distension is occurring for a period of time beyond inflation, or that the signal that the stomach is no longer distended has not been relayed to the NTS or hypothalamic nuclei. A third possibility is that the mechanical distension of the stomach increased the efficacy of the chemoreceptors in the GI tract, thereby increasing the effect of even a small amount of food. Hormonal involvement is also likely, as both CCK [51
] and leptin [52
] are known to amplify the afferent signal resulting from gastric distension.
Interestingly, RGD rats resumed normal patterns of eating by weeks 3 and 4. This phenomenon is less well characterized, however it coincides with the cessation of weight loss in humans and animals with permanently inflated, indwelling gastric balloons [12
], as well as the increased fullness threshold, and higher tolerance for large balloon inflations in BN [26
]. Given that leptin potentiates gastric distension sensitive neurons in the NTS under normal circumstances [52
], it is possible that the leptin levels present in RGD animals are insufficient to amplify the distension-induced vagal afferent signal to threshold levels. This lack of modulation could eliminate the delay in meal initiation observed during the first two weeks of RGD. Studies of the change in neuroendocrine profiles and gene expression at time points when food intake patterns between RGD and control animals were discordant would provide insight into the cause of the meal pattern adaptation.
The vagus nerve is often associated with satiety mechanisms, including those derived from gastric distension. Selective and complete vagotomy led to a reduced or entirely eliminated ability to detect changes in gastric volume, and led to a transient increase in food intake for the first meal after surgery. Somewhat paradoxically, despite their inability to detect distension in the stomach; completely vagotomized animals consume fewer calories after 48 hours recovery [53
], and exhibit an increase in arcuate NPY expression, and a decrease in serum leptin, which were not observed following splanchectomy [54
]. While it is extremely unlikely that the balloon implantation surgery drastically damaged the vagus (as control animals who received the same apparatus were unaffected), nor is it likely that the moderate degree of distension inflicted upon these animals led to gross changes to vagal morphology, it is possible that RGD led to a temporary desensitization of mechanoreceptors, and therefore influenced food intake.
Another explanation for this adjustment in meal patterns is that RGD differentially affects subpopulations of mechanoreceptors. For example, intramuscular arrays, which are involved in the gastric accommodation reflex, may be more active in RGD animals, perhaps as an adaptation to the repeated strain incurred during the inflationary periods. Intraganglionic laminar endings, which are responsible for distension-related satiety signaling [55
], may exhibit a diminished response as a result of RGD, thereby elevating the threshold for fullness. Neural tracing studies may provide insight into both of these possibilities.
A fourth possibility is that RGD rats learn to dissociate gastric distension from energy consumption. The regulation of food intake is crucial for survival, and therefore remarkably plastic [56
]. Redundant systems are in place, and the manipulation of one often leads to a subsequent change in another. It is likely that the feeding-regulatory system has adapted, either via neuroendocrine feedback systems, or learning mechanisms, in order to utilize more reliable cues for caloric intake.
The goal of these experiments was to induce distension related to moderate overeating, rather than hyperdistension that could elicit feelings of malaise. Despite using a conservative volume to inflate the intra-gastric balloons, the possibility persists that the stomachs of RGD rats were larger or more compliant than those of control rats by the end of the four-week period. Because volumes used to inflate the balloon were based on the amount of a liquid diet consumed by a non-RGD rat it is possible that, despite increases over time, the inflationary volume ceased to provide adequate distension. In order to eliminate this possibility, future inflationary levels will be based on pressure, rather than volume. Furthermore, in order to better represent the extremely large quantities of food typically ingested during binge episodes, and therefore better correlate studies of RGD with disorders such as BN and BED, animals with implanted balloons that have been trained to binge-eat [58
] will be used to determine the inflationary volumes and levels of gastrointestinal pressure to be utilized later in the experiment. In addition, future studies will address possible effects of RGD on elements of gastric function, including gastric compliance and rate of emptying.
Since BN is a disorder that includes both binge-eating and compensatory behaviors, including self-induced vomiting, it is possible that the rat, which lacks the emetic reflex, may be an inappropriate model for such a disorder. Interestingly, we found a trend toward altered NPY expression in the caudal brainstem, a region associated with emesis in animals with that ability [59
]. NPY itself has been demonstrated to occasionally induce an emetic response [60
]; it is possible that NPY contributes to both binge-eating and subsequent purging. As other factors consistent with BN were observed in a non-vomiting animal, it is possible that gastric distension alters the relationship between food consumption and malaise-avoidance. Study of this relationship would be greatly enhanced by modifying the paradigm to work in an animal capable of vomiting, and would provide better insight into BN.
RGD, as implemented in the present set of experiments, induced alterations in food intake patterns, and influenced plasma leptin levels and NPY mRNA expression, without a noticeable impact on body weight, or total daily food intake. Since this paradigm uses a simple mechanism for manipulating the stomach, it avoids many of the confounding factors present in more naturalistic models of eating disorders and overconsumption of food, and may serve as a valuable complement to the human subjects research that is the current standard for the study of disorders in which binge-eating is a component.