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Effect of WAY 161503 on MCKOs 1 The Effect of WAY 161503 on Food Intake in Mice with Disrupted Melanocortin Receptors Kaihan Fakhar, Kim Robertson, Neil Rowland University of Florida
Effect of WAY 161503 on MCKOs 2 Abstract Previous studies have investigated the anorectic effects of selective agonists on the 5 HT2c receptors that are known to be involved in appetite regulation. Research has also shown that melanocortin receptors 3 and 4, thought to be downstream from the serotonin rec eptors, are an integral component of this pathway because their disruption results in distinguishable phenotypes. To further understand the receptors involved, we tested the effects of a known serotonergic agonist, WAY 161503, on four different melanocorti n genotypes: WT, MC3R / MC4R / and the double receptor knock out (DKO). At a WAY 161503 dosage of 8 mg/kg all genotypes ate under 16% of their baseline except for the MC3R / which only reduced food intake by 49%. Brain Fos ir induced by WAY 161503 wa s only significant in the paraventricular nucleus of the double knockout mice. The data suggests that the melanocortin 3 receptor is directly involved in the feeding behavior pathway downstream from the 5 HT2c receptor.
Effect of WAY 161503 on MCKOs 3 The Effect of WAY 161503 on Food Intake in Mice with Disrupted Melanocortin Receptors Whether people can control their obesity is a highly controversial subject; a major finding in this area of research has been the link between serotonin and eating behavior. Evidence shows that increased levels of serotonin are associated with a decline in eating (Latham & Blundell, 1979). This has led investigators to target serotonin receptors and establish a link between their activation and a suppression of appetite ( Blundell, 1992). Downstream from t hese serotonin receptors are melanocortin receptors which seem to regulate energy homeostasis (Heisler et al., 2003). Determining the effect of serotonin agonists on different genotypes of melanocortin receptor knockout mice could help to better understand their interaction, potentially leading to appetite suppressant drugs without harmful side effects. Serotonin Agonists There are fourteen known serotonin (5 HT) receptor subtypes, four of which have been shown to be involved in food intake. These four inc lude 5 HT1a, 5 HT1b, 5 HT2a, and 5 HT2c (Kitchener & Dourish, 1994). 5 HT1a receptors are different from the others because they exist primarily as somatodendritic autoreceptors in the raphe nucleus (Vickers, Bickerdike, & Dourish, 2001). When these recept ors are activated, the result is a decrease in postsynaptic 5 HT receptor activation which will not produce appetite suppression. The other three receptors seem to increase serotonin release when activated, inducing hypophagia (Vickers, Bickerdike, & Douri sh, 2001). However, use of agonists at the 5 HT2a receptor by Kitchener & Dourish resulted in a number of other behavioral effects besides hypophagia, such as nausea and sedation, which may be alternative explanations for the absence of eating behavior (Ki tchener & Dourish, 1994). Genetic knockouts of the 5 HT1b receptor resulted in significant weight gain compared to wild types, signifying its role in feeding behavior (Bouwknecht, van der Gugten, Hijzen, Maes, Hen, & Olivier, 2001). The 5 HT2c receptor see ms to be the most directly involved in feeding behavior, specifically those in the paraventricular nucleus (PVN). 5 HT2c genetic knock outs exhibit an increase in number and duration of feeding sessions (Nonogaki et al., 1998).
Effect of WAY 161503 on MCKOs 4 One difficulty encountered w hen classifying the functions of these receptors using a receptor blockade (rather than genetic knockouts) is finding a selective agonist or antagonist. Many of the drugs bind to a few receptors but with different affinities. The administration of 5 HT2c a gonists, such as d fenfluramine, has been shown to decrease fat intake in humans (Heisler et al. 2002). These findings make this receptor the most appropriate target for studies looking to investigate feeding behavior without disrupting other behavior. Man y agonists at the 5 HT2c receptor also seem to activate other 5 HT receptors that have undesirable side effects, so a very selective agonist is necessary (Kitchener & Dourish, 1994). Kitchener and Dourish found that some agonists activating the 5 HT2c rece ptor also activate the 5 HT2a receptor which may be responsible for hallucinogenic behaviors. Although there are a couple of selective agonists at the 5 HT2c receptor, WAY 161503 has been the most studied and has been shown to only activate that particular receptor (Rosenzweig Lipson et al., 2006). Melanocortin Repectors Melanocortin receptors (MCR) themselves do not have specific 5 HT binding sites, but manipulating them has significant effects on obesity. Pro opiomelanocortin (POMC) neurons produce alpha (Garfield et al., 2009). There are four known melanocortin receptors, melanocortin 1 through 4. Mice with these receptors genetically knocked out have been ver y useful in understanding the MCRs functions (Garfield et al., 2009). MC3RKOs exhibit a phenotype that is bigger and leaner than wild type mice, while MC4RKOs appear fatter than both wild type as well as MC3RKO mice (Skibicka & Grill, 2009). Most interesti ng is MC3 + 4RKO (DKO) mice which are more obese than the other genotypes. The reason for this is not understood and implies that there may be an intricate connection between these receptors (cite). It seems that a variety of confounding variables such as age, sex, and the way in which obesity is measured may at least partially explain inconsistent findings on the subject ( Bouwknecht et al., 2001).
Effect of WAY 161503 on MCKOs 5 weight depending on the receptor they bind to. Agonists of both the MC3R and MC4R decrease food intake (Skibicka & Grill, 2009), implicating them as the primary melanocortin receptors regarding feeding behavior. Research has also shown a connection between the 5 HT and mel anocortin pathways. Heisler et al. (2002) found that 5 HT2CRs are expressed on many POMC neurons and that this system converges on melanocortin receptors in the arcuate nucleus. They showed conclusively that the 5 HT2CR action on POMC neurons is upstream f rom the melanocortin receptors by administering a MC3/4R agonist in 5 HT2cRKO and wild type mice at a young enough age that the body weights were not significantly different. The mice given the agonist experienced a significant reduction in eating behavior but the difference was constant for both genotypes. POMC mRNA expression was also not significantly different between genotypes. Figure 1. Hypothesized Pathway from 5 HT Neuron to Melanocortin Receptor. 5 HT drugs require functional melanocortin pathwa ys to exert their effects on food intake as illustrated by Figure 1(Heisler et al., 2003) (pg. 172). There are a few models of the possible connection between 5 HT and melanocortin receptors, and Figure 1 is one of the most common hypotheses. 5 HT
Effect of WAY 161503 on MCKOs 6 drug ind uced hypophagia is attenuated by genetic blockade of downstream melanocortin receptors (Heisler et al., 2003), and if the signal initiated by the 5 HT neuron is not eventually received by a functional melanocortin receptor, the signal is lost. 5 HT2c recep tor agonists were experimentally shown to promote hypophagia via downstream activation of melanocortin 4 receptors (Lam et al., 2008). This information establishes a role for both serotonin and melanocortin in regulating feeding behavior. Evidence shows t hat the 5 HT2c receptor seems to have the largest role among the serotonin receptors and the same goes for melanocortin 3 and 4 among the melanocortin receptors (Garfield et al., 2009). The interaction between the two types of receptors is complicated, but it appears that the melanocortin receptors are downstream from the 5 HT2c receptors (Heisler et al., 2002). The link may be in POMC neurons where an increase in serotonin action causes a decrease in appetite. It is plausible that this action can be modele d by use of a 5 neuron and subsequent melanocortin activation. Using genetic knock outs, more specific functions can be examined. 5 HT2c receptor knock out mice eat more often and their meals are longer i n duration, ultimately resulting in obesity (Tecott, Sun, Akana, Strack, Lowenstein, Dallman et al., 1995). Melanocortin receptor 3 and 4 knock out mice also turn obese, although to different degrees (Chen, Marsh, Trumbauer, Frazier, E.G., Guan, X., Yu, H. et al, 2000). The evidence of a connection among the pathways makes it interesting to investigate the effect of a 5 HT2c agonist on the different melanocortin genotypes that have shown varying levels of obesity and feeding behaviors. We investigate the effect of the selective 5 HT2c agonist, WAY 161503, on food intake in adult mice with melanocortin 3 and/or 4 receptor knocked out. We also measure Fos ir to estimate brain activation following the administration of WAY 161503. Materials and Methods Anima ls and Housing
Effect of WAY 161503 on MCKOs 7 Male and female mice with genotypes of wild type, MC3R / MC4R / and double knock out (DKO) of C57BL/6 background were used. Body weights ranged from 19 55g and varied largely based on the genotype. Animals were housed in individual polyc arbonate cages containing Sani Chips bedding (Harlan, Madison WI) and a translucent igloo (Bio Serv, Frenchtown NJ). The vivarium was maintained at a temperature of 232 C and a 12:12 light:dark cycle was used (lights on 07:00 19:00). The experiment was c onducted in the first half of the light phase, and animals were given ad libitum access to tap water and Purina 5001 pellets. The use of animals in this project was approved by the Institutional Animal Care and Use Committee at the University of Florida. T esting Diet For 3 weeks mice were acclimated to eating fruit Serv, Frenchtown, NJ) for 30 minutes a day. Crunchies are 190mg spherical fruit flavored, dessert like, pellets that contain the same macronutrient c omposition as chow. Previous work in this lab has shown that mice eat up to half their daily food intake from this treat (is there a source available for this information?). Following this acquisition period, the behavioral experiments use d a t. The Crunchies were given once per day for 30 minutes and the intake recorded. All mice were given 10 brightly colored Crunchies a day, 3 lime flavored, 3 orange flavored, and 4 grape flavored. Intake was recorded as the number of pellets consumed in the time allotted. Since all mice were presented with the same treats, the average intakes could be compared across genotypes. O n these baseline days, no drug treatment was given and baseline intake was a three day average. On three different test days, at le ast one week apart, mice were injected subcutaneously with either vehicle or one of the two dosages of WAY 161503 fifteen minutes prior to presentation of the treats so that a dose effect curve could be established. Intakes were recorded as percentage of b aseline Crunchies consumed. Previous work in this lab has established the 50% inhibitory dose in wild type mice to be about 6 mg/kg. Each mouse received each dosage in random order so that the data could be combined for a total of N=72 (6 mice of each geno type
Effect of WAY 161503 on MCKOs 8 X 3 different treatments). These results were analyzed statistically, using 1 way ANOVA with dose of drug and genotype as the main factors, for genotype or genotype x dose effects. Drugs WAY 161503 (Tocris Bioscience, Ellisville MO), which was given 1 5 minutes prior to Crunchie presentation on test days, was dissolved in saline and given at dosages per body weight of 4 and 8 mg/kg. At 8 mg/kg the solution was warmed to prevent precipitation. Fos analysis In a second experiment, at least 1 week after co mpletion of the behavioral studies, the same mice were treated with either WAY 161503 at a dosage of 8 mg/kg or vehicle and were anesthetized (Sleepaway, Fort Dodge) one hour later;WAY 161503 has a biologically effective period of about one hour (Rosenzwei g Lipson et al., 2006) The mice were perfused with saline followed by fixative (paraformaldehyde), the brains removed, and sliced into 100 micron sections including the paraventricular nucleus, area postrema, and nucleus of the solitary tract. The section s were then reacted with a primary Fos antibody c fos (4) (rabbit polyclonal, Santa Cruz Biotechnology, Santa Cruz, CA), then with secondary antibody (goat anti rabbit IgG, Zymed Laboratories, Carlsbad, CA), and a colorizing reaction (peroxidase, Vector La boratories, Burlingame, CA), mounted on slides, and examined under a light microscope, as described in Li & Rowland (1996). The number of Fos containing cells were counted in the aforementioned brain areas. Results Table 1 shows the difference in average body weight that reflects the phenotypic differences among the different genotypes of mice. There were six mice per genotype and the differences in baseline Crunchie intake are also shown. Converting the Crunchie int ake to percentage of body weight consumed
Effect of WAY 161503 on MCKOs 9 in Crunchies at 190mg per Crunchie, WTs actually ate the most at ~5% and the DKOs ate the least at ~3%. Figure 1 shows the percentage of Crunchies that the mice ate after administration of WAY compared to their nor mal baseline intake. At the lower dosage, WAY (4 mg/kg) significantly decreased food intake only in the DKO and MC4R / genotypes. At the higher dosage, WAY (8 mg/kg) significantly reduced food intake in all genotypes, but to a much lesser extent in the MC 3R / genotype. The MC3R / genotype of mice ate approximately 50% of their baseline levels compared to ~10% in the DKO genotype, ~15% in the 4RKO genotype, and ~16% in the wild types. Fos analysis in the PVN, AP, and NTS only showed a significant differen ce of activity in the PVN between groups, which can be seen in Figure 2. The DKO mice showed the most activity in the PVN. Cellular activation was similar and low across genotypes in the AP. All genotypes showed more activity in the NTS than the AP, but th ere was no significant difference among genotypes. Discussion The results of this experiment showed that when the melanocortin 3 receptor is knocked out, the 5 HT2c agonist, WAY 161503, reduces feeding behavior to a smaller extent than in wild type, melano cortin 4 knock out, and double knock out mice. This suggests a more crucial role for the MC3R in the hypothesized pathway from 5 HT2c to MC in appetite regulation than the MC4R. It is important to consider other factors that may contribute to the food inta ke in these mice. Other studies, particularly Lam et al. (2008), reported that MC4R knock outs had no reduction in food intake while wild type mice significantly decreased food intake when administered with a similar 5 HT2c agonist, BVT.X. A potential expl anation for this is that the mice used in our experiment ranged from 2 6 months, while those in their experiment were much younger. It is possible that compensatory mechanisms adjust to regulate feeding behavior when these receptors are disrupted, and at a young age those mechanisms have not fully developed. The mice in the Lam et al. experiment were also floxed at the melanocortin 4 receptor. This
Effect of WAY 161503 on MCKOs 10 means that the gene for this receptor was transcriptionally blocked by loxp sites. This process localizes the knock out to a particular location. We did not use this localized knock out in our experiment. It is also possible that the 5 HT2c agonist BVT.X simply works through a different mechanism than WAY 161503. Another topic of interest is the fact that the MC3R knock outs showed a less significant reduction in food intake compared to other groups, while the DKO did not. If the MC3R is the crucial receptor, then it would make sense that both of these groups should exhibit similar feeding behaviors. This was not t he case. Perhaps the age range of mice could have been narrower, but the results imply something more significant is responsible. Future studies could seek to find a compensatory process in the knock out mice that is perhaps different between the MC3R knoc k outs and the DKOs, which may also explain Fos ir discrepancies discussed later. There is a potential limitation to this study due to the fact that all of the mice received every treatment. Each test day six mice received vehicle, six received WAY at 4 mg /kg, and six received WAY at 8 mg/kg. No mouse ever received the same treatment twice but, although unlikely, it is possible that the mice may have developed a slight tolerance to the drug that skewed results. Also, the experiment showed conclusively that WAY 161503 significantly reduced Crunchie intake in all genotypes of mice, but we cannot be certain that this effect translates to less palatable food, although Crunchies consist of the same macronutrient composition as normal chow. Measuring chow intake i s more difficult because the physical shape of the pellets make them difficult to count while the spherical and brightly colored Crunchies are easier to keep track of. It is an important consideration, though, and future studies could investigate the effec t on regular chow intake as well. The Fos ir investigation showed that the highest levels of brain activation due to WAY 161503 were generally in the PVN. In the PVN, the activation was significantly higher in the DKOs compared to all other genotypes but most interestingly, the MC3R knock outs. While it is plausible to predict a low
Effect of WAY 161503 on MCKOs 11 level of activation on all groups other than wild type due to disruption of the 5 HT2c to melanocortin pathway, this may be evidence of a compensatory mechanism that results in different basal firing rates of POMC neurons. It would further suggest that the mechanism is different between the DKO and the MC3R knock out (due to significantly different levels of fos ir between the two genotypes in the PVN shown in Figure 2), which c ould potentially explain the differences in feeding behavior. The results of this experiment better our understanding of the 5 HT2c to melanocortin pathway that regulates appetite. We have evidence that suggests it is the downstream MC3R, rather than the M C4R, that is critical to suppression of feeding behavior. Future studies should more closely control the age of mice, as well as look at an even higher dosage of WAY 161503. Since Fos activation was generally low across all genotypes in the areas observed, a higher dosage may show more distinguishable results.
Effect of WAY 161503 on MCKOs 12 References Blundell, J.E. (1992). Serotonin and the biology of feeding. American Journal of Clinical Nutrition 55:155S 159S. Bouwknecht, J. A., van der Gugten, J., Hijzen, T. H., Maes, R. A A., Hen, R., & Olivier, B. (2001). Male and female 5 HT1B receptor knockout mice have higher body weights than wildtypes. Physiology & Behavior, 74 (4 5): 507 516. Chen, A.S., Marsh, D.J., Trumbauer, M.E., Frazier, E.G., Guan, X., Yu, H., et al. (2000). Inactivation of the mouse melanocortin 3 receptor results in increased fat mass and reduced lean body mass. Nature Genetics, 26:97 102. Garfield, A.S., Lam, D.D., Marston, O.J., Przydzial, M.J., Heisler, L.K. (2009). Role of central melanocortin1. pathways in energy homeostasis. Trends in Endocrinology & Metabolism 20(5):203 215. Heisler, L.K ., Cowley M.A ., Kishi T ., Tecott L.H ., Fan W ., Low M.J ., Smart J.L ., Rubinstein M ., Tatro J.B ., Zigman J.M ., Cone R.D ., Elmquist J.K (2003). Central serotonin and melanocortin pathways regulating energy homeostasis. New York Academy of Sciences 994:169 174. Heisler, L. K., Cowley, M. A., Tecott, L. H., Fan, W., Low, M. J., Smart, J. L., Rubinstein, M., Tatro, J. B., Marcus, J. N., Holstege, H., Lee, C. E., Cone, R. D., and Elmquist, J. K. (2002). Activation of central melanocortin pathways by fenfluramine. Science 297(5581):609 611. Kitchener, S. J. and Dourish, C. T. (1994). An examination of the behavioural specificity of hypophagia induced by 5 HT1b, 5 HT1c and 5 HT2 receptor agonists using the post prandial satiety sequence in rats. Psychopharmacology (Berl) 113(3 4):369 377.
Effect of WAY 161503 on MCKOs 13 L.K. (2008) Serotonin 5 HT2c Receptor Agonist Promotes Hypophagia via Downstream Activation of Melanocortin 4 Receptors. Endocrinology 149(3): 1323 1328 Latham, C.J., and Blundell, J.E. (1979). Serotonergic influences on food intake: Effect of 5 hydroxytryptophan on parameters of feeding behaviour in deprived and free feeding rats. Pharmacology, Biochemistry, and Behavior, 11(4): 431 437. Li, BH., Rowla nd, N.E. (1996). Effect of Chronic Dexfenfluramine on Fos in Rat Brain. Brain Research, 728: 188 192. Nonogaki, K., Strack, A. M., Dallman, M. F., Tecott, L. H. (1998). Leptin independent hyperphagia and type 2 diabetes in mice with a mutated serotonin 5 HT2c receptor gene. Nature Medicine 4:1152 1156. Rosenzweig Lipson, S., Zhang, J., Mazandarani, H., Harrison, B. L., Sabb, A., Sabalski, J., et al. (2006). Antiobesity like effects of the 5 HT2C receptor agonist WAY 161503. Brain Research 1073 1074 : 24 0 251. Skibicka, K. P. and Grill, H. J. (2009). Hypothalamic and hindbrain melanocortin receptors contribute to the feeding, thermogenic, and cardiovascular action of melanocortins. Endocrinology 150(12):5351 5361. Tecott, L.H., Sun, L.M., Akana, S.F., Strack, A.M., Lowenstein, D.H., Dallman, M.F., & Julius, D. (1995). Eating disorder and epilepsy in mice lacking 5 HT2c Receptors. Nature, 374:542 546. Vickers, S.P., Bickerdike, M.J., & Dourish, C.T. (2001). 5 HT2c Receptor Modulation and the Treatment
Effect of WAY 161503 on MCKOs 14 of Obesity. Diabetes, Obesity, and Metabolism 1(4), 207 214. Table 1. Average Body Weight and baseline Crunchie Intake for Each Genotype Genotype Average Body Weight (g) Baseline Crunchie Intake (number of crunchies) WT 28.0 7.6 MC3R / 25.2 5.5 MC4R / 48.2 8.3 DKO 38.7 5.9
Effect of WAY 161503 on MCKOs 15 Figure 1 Percentage of Baseline Crunchies Eaten after Various Dosages of WAY 161503 Administration 0 20 40 60 80 100 120 0 4 8 Percentage of Baseline Crunchies Eaten Dosage of WAY 161503 (mg/kg) WT MC3R-/MC4R-/DKO
Effect of WAY 161503 on MCKOs 16 Figure 2. FOS Activity in Mice Administered with WAY