In order to remain consistent with today’s society and the fast paced life, the average person must learn how to live a life of small conveniences. The comfort of not having to leave the car to get a full course meal for only a few dollars at McDonald’s is overwhelming to the majority of Americans. As a result, people slowly and subtly overeat and gorge their bodies with the most unhealthy and unfortunately deadly substances necessary to produce large amounts of food in short periods of time. Statistics show that one in two Americans is overweight, and nearly one in five is obese. According to Atlantic Monthly correspondent, Ellen Ruppel Shell, obesity is one of many potentially terminal disorders including heart disease, diabetes, and hypertension¹. Scientists continue to fight the obesity epidemic in labs attempting to understand what leads people to overeat and thus become obese. It has been recognized that specific regions, hormones, and nerves in the brain play a considerable role in obesity, food regulation, and feeding behavior. Using rats as testing subjects, due to the similar anatomy of the rat brain to the human brain, scientists have been able to identify particular neural pathways that play a significant role in feeding regulation⁷.

Among many other regions of the brain, the dorsomedial hypothalamus (DMH) has been identified as a region that contributes greatly to eating behavior. The region, located in the posterior of the brain (Figures A1), is known to influence cardiovascular and anxiety responses as well as to control triglycerides and fatty acid levels². However, most distinctly the DMH regulates feeding behavior³. Melanocortin-4 receptor (MC4-R) in different regions of the brain, among them the DMH, plays critical roles in regulating feeding behavior. Previous studies have proven that genetic disruption of the MC4-R causes obesity⁵⁴. Questions still remain about which sites are crucial for MC4-R action.

The MC4-R’s natural ligand, a -melanocyte-stimulating hormone (a -MSH), has been shown to be an endogenous, agonist of the MC4-R which plays a restorative, inhibitory role in feeding and energy storage⁵. As an agonist the ligand binds to its MC4-R to stimulate a mode of communication for the hormone and receptor. Other studies have suggested that a -MSH can modify such MC4-R activity as appetite-regulation⁶.

A synthetic antagonist of the MC3/MC4 receptors was formed to test how feeding and energy storage would be affected by the elimination of the MC4 receptor. SHU 9119, a combination of amino acids, is an antagonist which interrupts the MC4 receptor neural pathways (Figure A2)⁷. Because obesity results as a consequence of the disruption of the MC4-R, SHU 9119 should cause overeating. The drug stimulates feeding because the body will no longer recognize when it is satiated due to the lack of regulation by the MC4 receptor. The drug distracts the path between MC4-R’s ligand, a -MSH, and itself preventing it from doing its job of regulating feeding and energy storage⁸.

In this study, rats were treated with SHU 9119 through injection in the DMH region to observe the effect of feedings given the following variables. The rats were provided the option of their typical diet of plain water/plain chow and a preferred diet of 10% sucrose water/plain chow. SHU 9119 was administered in a dosage of 50 picomoles per .5 microliters. The purpose of the study was to show the types of changes that would occur in the feeding behavior patterns of rats due to the variables provided.

It is recognized that the rats would normally consume more of the preferred diet than of the normal diet because the rats are attracted to its taste. It was expected that the SHU 9119 would encounter MC4 receptors in the dorsomedial hypothalamus sending a signal that the rat is still hungry. The rats were expected to consume more of each diet when treated because they would not recognize that they are satiated.

Fourteen male Sprague-Dawley rats (Figure A3), weighing 225-250 g, were individually housed in conventional hanging cages (Figure A4) in a temperature-controlled room (21° -22° C). The rats used in the study underwent a cannulation surgical procedure performed prior to this project for another experiment. The rats were anesthetized with Nembutal (40 mg/kg) and fitted with 28-gauge stainless steel guide cannula in the DMH (Figure A5). The cannula was implanted using the following coordinates:0.5 mm lateral, 2.5 mm posterior to bregma, and 7.3 mm below the skull surface, as determined by The Rat Brain Atlas by Paxinos and Watson (1986). The injector extended 1 mm beyond the tip of the guide cannula. For all cannulations, the incisor bar was set at 3.3 mm below the ear bars. At least 7 days of recovery were allowed after surgery before experimental trials were conducted.

The study extended over a 5-6 week period. First, a baseline study was performed over a 5-day period to provide a standard measurement of consumption as well as to familiarize the rats with the diet options without the influence of a treatment. All fourteen rats were exposed to the typical diet of plain Tek Lad Rodent chow and plain, room temperature tap water for a 24-hour period. Consumption was measured at 24-hours after the initial presentation of food. The rats then had a 24-hour period undisturbed on their normal diet. After the 24-hour period of rest, the fourteen rats were given the plain chow and preferred liquid of 10% sucrose water. Again, the consumption was measured at 24-hours after the initial feeding.

On the first day of the experiment, the rats were divided into four groups with four different combinations of treatment and diet. Rats received their first treatment on the first day of week two. All rats were injected with either 50 picomoles of SHU 9119 or 50 picomoles of saline in the DMH administered in a volume of 0.5 microliters over 30 seconds. The group of rats that received saline stood as the control for comparison for that day. At two hours, four hours, and twenty-four hours after initial feeding and injection, food intake and fluid intake were measured and recorded. Over the remaining weeks, each rat, acting as its own control, received the four combinations of diet and treatment (Figure A6).

Data were evaluated by a repeated measure one factor ANOVA and means were compared using Scheffe’s F-test. All data are expressed as mean + Standard Error of Means (S.E.M.).

This study provided the opportunity to observe changes in feeding behavior when rats’ MC4 neural receptor was blocked by SHU 9119. The results indicate that SHU 9119 significantly stimulated feeding on the chow/tap water diet as was hypothesized. However, the rats did not consume more than usual on the chow/10% sucrose diet when injected with SHU 9119.

SHU 9119 is supposed to contact and interrupt the feeding regulatory MC4 receptor pathways¹⁰. Consequently, the rats, according to previous studies, should not recognize when they are satiated and therefore they should eat significantly more when treated with the SHU 9119 than they would if they were not treated. As Figure B1 shows, the baseline study revealed that the rats consumed more on the preferred diet of chow/10% sucrose than they did on the typical diet of chow/tap water probably due to its favorable flavor. All comparisons were made at 24 hours because as Figures B2 and B3 show, there was no significant change in feeding observed before 24 hours on either treatment.

Figure B4 demonstrates that SHU 9119 did significantly stimulate feeding on the typical diet, but did not significantly increase consumption on the preferred diet. On the typical diet of chow/tap water, the consumption level increased significantly, approximately 100 kilocalories. However, on the preferred diet, SHU 9119 may have had an adverse effect on the rats because the graph shows that they consumed less when treated with SHU 9119 than they did when they were not treated. The increase in consumption on the typical diet confirms that SHU 9119 did encounter MC4 receptors. However, the fact that there was no increase in consumption under the influence of SHU 9119 on the chow/10% sucrose diet suggests that the treatment is not effective when rats are on a preferred diet.

A possible explanation that the rats’ consumption did not increase on the preferred diet with the injection of SHU 9119 as predicted it would because the sucrose solution was diluted to 10%. It is possible that while the taste of the sucrose solution was pleasurable to the rats, the dilution may have forced them to work too hard to gross the net amount of kilocalories needed. It has also feasible that the rats may have needed more consistency in diet. On the typical diet, the rats received kilocalories from eating chow only. Although they drank the plain tap water, for various reasons, they did not acquire any kilocalories from the liquid. On the preferred diet the rats received kilocalories from both the chow and the sucrose solution. However, because the rats enjoyed the taste of the sucrose solution they drank much more than they ate. It is possible that the rats may have simply become tired of drinking. There is also reason to believe that the rats may have reached their maximum point of consumption because as Figure B4 shows the amount of the preferred diet consumed was fairly consistent on both treatments. Considering that possibility, it was concluded that SHU 9119 could not increase consumption beyond the animal’s normal maximum capacity.

Further research was performed through grants at the Veteran Affairs Medical Center to address and eliminate the factors which may have contributed to the possibility of the rats becoming tired of consuming liquid, the inconsistency in kilocalorie availability, and the rats eating beyond maximum capacity. In the study conducted sucrose pellets were provided with plain tap water as the preferred diet along with chow and plain tap water as the normal diet. The new diet option would eliminate the inconsistency in diet. The rats would only receive kilocalories from the solid portion of the diet. The sucrose pellets would eliminate the concern of the rats working too hard to acquire the amount of kilocalories sought out as well as the concern of the rats having an imbalance of liquids to solids.

As more studies are performed it is expected that a greater understanding of obesity will be acquired. Understanding the roles as well as the significance of such neural receptor pathways as the MC4-R could lead researchers to a new and more thorough understanding of feeding regulation and consequently weight control. With the continual success of obesity studies and research, obesity will no longer be an epidemic, but rather reduced to merely a rare, genetic disorder.

From this study it has been concluded that SHU 9119 is effective as a stimulator for consumption when administered with a normal diet. However, according to the results of this study, SHU 9119 is not effective when administered with a preferred, abnormal diet. It has been concluded that SHU 9119 conditionally effective as it only increased consumption on one of the two diets. The SHU 9119 did encounter MC4 receptors in the dorosmedial hypothalamus as hypothesized, however, do to various conditions- such as the rats’ boredom on the preferred diet the rats only consumed more than usual on the typical diet. The rats’ increased feeding on the typical diet confirms that the rats were not able to recognize that they were satiated proving the effectiveness and ability of SHU 9119, an MC4-R antagonist, to stimulate consumption.

(Wei 1997)

Figure B1: Total consumption of chow/10% sucrose water diet and chow/tap water diet. Values are means + S.E.M. The asterisk denotes a significant difference as compared to chow/tap water diet (p=.001)

Figure B3: Total consumption in kilocalories of chow/10% sucrose water and chow/tap water diets with SHU 9119 treatment at 2, 4, and 24 hour intervals. Values are means + S.E.M. Shows most significant consumption occurred at 24 hours with SHU 9119.

Figure B4: Total kilocalorie intake at 24 hour interval of chow/10% sucrose water diet and chow/tap water diet with treatments. Values are means + S.E.M. The asterisk denotes significant difference in kilocalorie intake compared to saline treatment on chow/tap water diet. Shows significant increase in kilocalorie intake with SHU 9119 treatment on chow/tap water diet (p=.001). Also shows insignificant increase in kilocalorie intake with SHU 9119 on chow/10% sucrose diet.