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The distribution of serotonin within the hypothalamus and its role in the pathogenesis of obesity.

Brensha Williams Fayetteville State University Scientific Communications April 7, 2010 Instructor: Dr. James Raynor

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Abstract Human obesity represents a significant peril across the globe. By obtaining a better understanding of the neuro-chemical mechanisms involved in the regulation of food intake, it may be possible to develop more effective treatments for obesity. The brain monoaminergic system plays an important role in eating behavior. Because obesity is associated with variations in serotonergic concentrations, there is still uncertainty as to whether or not this abnormality is solely responsible for the pathogenesis of obesity. It is hypothesized that serotonergic abnormalities within certain areas of the hypothalamus contribute to the persistence of obesity and its participation in food intake and control. The diet-induced model of obesity demonstrates that abnormal hypothalamic serotonin turnover levels, and its close association with dopamine concentration levels, are involved in the regulation of food intake and control. When SpragueDawley rats are placed on a high fat and calorie diet, over half develop diet-induced obesity (DIO), while the rest are diet-resistant (DR). When the rats are fed a low-fat diet, DIO and DRprone rats weigh the same, but DIO rats obtain numerous abnormalities of neural transmission. After the onset of obesity, these abnormalities are conflictingly normalized. This normalization may contribute to the persistence of obesity. Future studies on abnormal hypothalamic neurotransmission focus on the normalization of the monoamine system through gene therapy.

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1. Introduction With more than 1 billion adults overweight, obesity has become an epidemic globally. Although obesity has not been deemed a disease, it represents a significant peril across the globe. Obesity predisposes patients to the development of chronic diseases, including coronary heart disease, certain types of cancers, and diabetes. Its consequences drastically increase mortality rates and healthcare costs. Obesity represents a combination of factors including genetic, social, and environmental. Past and present research findings have shown that abnormal levels of serotonergic neurotransmission are closely related with concentrations of the neurotransmitter dopamine. (Laviano and Meguid, 2003; 89). Decreased levels of the neurotransmitter serotonin affect mood and behavior, which may lead to obesity. Currently, drug treatments for obesity interfere with the monoamine system and it is difficult to develop targeted drug treatments for obesity due to limited knowledge of interactions in the monoamine system. (Ramos, Meguid, Campos, and Coelho, 2005). These drug treatments increase the levels of serotonin in the brain, while decreasing dopamine levels. The underlying mechanisms of obesity are poorly understood and by better understanding the neurochemical mechanisms involved in the regulation of overeating, it may be possible to develop better drug treatments to decrease appetite and mitigate obesity. (Laviano and Meguid, 2003; 89). In recent studies, the ventromedial nucleus of the hypothalamus (VMN) and lateral hypothalamic area (LHA) of the brain, link both neurotransmitters serotonin and dopamine and their actions on different areas of the hypothalamus. Both VMN and LHA variations in neurotransmission levels have shown the relationship between meal size and number. (Laviano et al., 2007). It is hypothesized that serotonergic abnormalities within certain areas of the hypothalamus contribute to the persistence of obesity and its participation in food intake and

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control. The diet-induced obesity model is used to simulate obesity in animals; therefore, this model can be used as an effective tool to prove or disprove this hypothesis. 1. Materials and Methods 1.1 Diet-Induced Obesity Model With the diet-induced model, three month old male Sprague-Dawley rats were individually habituated at a temperature of 23 -24 on 12-hour light and dark periods. The rats were fed

Purina rat chow and given unlimited access to food and water. After the first three experiments, 24-30 animals were either classified as Diet-Induced Obesity (DIO) or Diet-Resistant (DR). In the first experiment, chow fed DIO and DR prone animals were capitulated after 1 hour during the onset of the light phase (lights on at 600 hours). In experiment two, chow-fed DIO and DR prone rats were capitulated at 1 hour before the dark phase (lights off at 1800 hours). In experiment three, food was removed 1 hour after the light phase of the DIO and DR prone animals. The DIO and DR prone animals were sacrificed 48 hours after that. An additional experiment was performed on DIO and DR rats after abnormalities of serotonergic turnover were found. In experiment four, 24 rats were placed on a High Energy (HE) diet for a 14 week period. The HE diet consisted of 8% corn oil, 44% sweetened condensed milk, and 48% Purina rat chow. After two weeks on the diet, the heaviest gainers were categorized into the DIO group and the lowest weight gainers were assigned to the DR group. Eight of the intermediate weight gainers were put back on a low fat diet control. The rats were weighed every 2 weeks for a 14 week period and then sacrificed 1 hour before the dark cycle. (Hussanain and Levin, 2001). 2.1 Accumulation and analysis of 24-hr urine Norepinephrine turnover levels

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At 1500 hours, the rats were placed in metabolic cages for 24-hours with unlimited access to food and water. Urine was collected and placed in a solution of ice-cold 2 N HCl that contained 2% sodium metabisulfite with 10 pg/ temperature of -85 of dihydroxybenzylamine. The samples were stored at a

and the pH was adjusted between 2 and 3, and then extracted for high

performance liquid chromatography analysis. (Hussanain and Levin, 2001). 2.2 Brain procedure The rats were weighed, and then capitulated by decapitation. After the brains were removed, they were frozen immediately for 30 minutes, and then equilibrated at -12 in a cryostat. To

dissect the ventromedial (VMN) and dorsomedial hypothalamus (DMN), the brains were micropunched. The lateral nuclei in the VMN and DMN were punched using the blunt end of a 21gauge needle. Tissue punches were placed in 50 of .01 M sodium acetate/0.1 M citric acid

buffer with a pH of 3.0, and then homogenized for 10 seconds using an ultrasonic cell disruptor set at 70% power. Last, the homogenate was placed in a centrifuge for 10 minutes at 4 15,300 g. (Hussanain and Levin, 2001). 2.3 High Performance Liquid Chromatography and electrochemical analysis The samples were examined using a Coulochem II HPLC and Model 5011 electrochemical detector with a 3 M particle size for a Model MD-150 column. The mobile phase consisted of 7.5 mM sodium phosphate, monohydrate, and 1.7 mM octanesulfonic acid, 100 triethanolamine, 25 1 at

Na2 EDTA, and 10% acetonitrile. Phosphoric acid was used and the pH

was adjusted to 3.0. (Hussanain and Levin, 2001). 2.4 Data analysis

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Data was accessed using a Systat 8.0 statistical software using codes for phenotype, condition, and main area. After differences were located between samples, each part of the brain was tested by the software by using codes for phenotype and condition. Any significant phenotype differences were determined by implementing the Scheffe test. (Hussanain and Levin, 2001). 2. Results The diet-induced model of obesity is another method used to simulate obesity in animals. When researchers placed three week old Sprague-Dawley rats on a diet high in fat and calories, about two-thirds developed diet-induced obesity (DIO). (Laviano and Meguid, 2003). The remaining pups were diet-resistant. In another trial, DIO and diet-resistant prone rats were fed a low-fat diet. Both groups weighed the same, but the DIO prone rats possessed a high number of abnormalities of neural function, many of which are normalized when they become obese after chronic exposure to a high-fat diet. (Laviano and Meguid, 2003). A recent study reporting these abnormalities of the DIO prone rats revealed alterations in brain serotonin turnover, leading researchers to believe that this neurochemical anatomy predisposes DIO prone rats to become obese when dietary fat and caloric density are increased. (Laviano and Meguid, 2003). Serotonin turnover was expressed in experiments 1 and 2 under the feeding conditions on a low fat diet. The animals that were sacrificed at the light phase showed no significant change in serotonin turnover. However, in experiment 3, DIO-prone and DR-prone rats revealed a reduction in VMN serotonin turnover. This reduction of serotonin turnover was significantly more pronounced in DIO-prone rats that DR-prone rats. (Hussanain and Levin, 2001). In experiment 4, the DIO and DR rats were fed the HE diet for 14 weeks and restricted to a low-fat chow. At the end of the 14 week time frame, the DIO rats gained 50% more weight than the DR group. During this time,

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both groups contained comparable levels of low serotonin turnover in all brain areas. The graph below represents the changes in serotonin turnover in response to a 48-hour fast in DR-prone and DIO-prone rats:

Figure 1. Serotonin turnover after 14 weeks on the HE diet. Hassanain, M. and Levin, B. Dysregulation of hypothalamic serotonin turnover in diet-induced obese rats. (2002). Brain Research 929. 175-180. Abbreviations: Free-feeding (FF), Food-Deprived (FD).

In Figure 1, both DIO and DR rats had comparably low serotonin turnover levels in all brain areas during the light phase compared to their values prior to diet exposure. 3. Discussion Both free feeding DIO and DR rats revealed statistically low serotonin turnover among the several hypothalamus areas of the brain tested. The results suggest that DIO-prone rats possess numerous abnormalities of hypothalamic serotonergic metabolism. The DIO model of obesity demonstrates that changes in diet modulate gene expression. Furthermore, the DIO

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model data points to the critical role of genetics in the determination of obesity predisposal. It appears that changes in the composition of the diet might greatly impact hypothalamic neurotransmission caused by variations in gene expression in obese-prone animals. (Laviano and Meguid, 2003). Once obesity develops, abnormalities of several neurotransmitters in the hypothalamus are normalized, contributing to the persistence of obesity. Construing this evidence into human obesity, it can be assumed that abnormalities of brain serotonin levels are involved in the pathogenesis of obesity. However, obesity is an accumulation of a variety of factors, making it unlikely that abnormal neurotransmission is the only factor involved in the pathogenesis of obesity. In addition to its role in influencing food intake, hypothalamic serotonin seems to impact energy expenditure. (Laviano and Meguid, 2003). In a study conducted by Ohliger-Frerking et al., dorsal raphe nucleus serotonergic neurons were projected to the ventromedial nucleus (VMN) of the hypothalamus to influence feeding. The study revealed that the neurons from obese Zucker rats exhibited a larger depolarization and firing rate in response to phenylephrine than the cells from lean Zucker rats. (Laviano and Meguid, 2003). This suggested that dorsal raphe nucleus serotonergic neurons from obese rats have enhanced adrenergic activity. Furthermore, serotonergic neurotransmission through various receptors reduce food intake, therefore, promoting weight loss. To date, a number of experimental studies have indicated that abnormal hypothalamic serotonergic activities contribute to obesity. Additionally, abnormal levels of the

neurotransmitter, dopamine, in conjunction with serotonin contribute to body weight gain. When

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considered together, the modulation of hypothalamic serotonin and dopamine levels may result in reduced food intake and control, thus leading to sustained weight loss. This data suggests that hypothalamic neurotransmission represents a common ground on which various appetite-related messengers communicate. Because of the abnormalities existing in the signaling pathways of the obese rat, interrupted hypothalamic monoaminergic neurotransmission is an indication suggesting its significance in the persistence of obesity. Future studies on abnormal hypothalamic neurotransmission focus on the normalization of the monoamine system through gene therapy. Currently, drug treatments for obesity work by suppressing appetite related messengers in the brain. However, it must by articulated that the side effects of such medications may limit their use. (Laviano and Meguid, 2003). The development of sibutramine raises new enthusiasm because its effects are not limited by severe side effects.

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References: 1. Hassanain, M., & Levin, B. (2002). Dysregulation of hypothalamic serotonin turnover in diet induced obese rats. Brain Research 929. 175-180. 2. Laviano, A., & Meguid, M. (2003). Serotonin and Obesity. Current Medicinal Chemistry - Central Nervous System Agents, 3(2), 89-100. doi:NO_DOI. 3. Ramos, E., Meguid, M., Campos, A., & Coelho, J. (2005). Neuropeptide Y, melanocytestimulating hormone, and monoamines in food intake regulation. Nutrition, 21(2), 269-279. doi:10.1016/j.nut.2004.06.021. 4. Coscina, D., & Magder R. (1984). Effects of serotonin-depleting midbrain lesions on the defense of hypothalamic obesity. Physiology & Behavior, Volume 33, Issue 4, October 1984, 575-579 5. Coscina, D., McArthur, R., Stancer, H., & Godse, D. (1978). Association of altered brain norepinephrine and serotonin with the obesity induced by goldthioglucose in mice. Pharmacology Biochemistry and Behavior, Volume 9, Issue 1, July 1978, 123128 6. Huang, X.F., Zavitsanou, K., Huang, X., Yu, Y., Wang, H., Chen, F., Lawrence, A., & Deng, C. (2006). Dopamine transporter and D2 receptor binding densities in mice prone or resistant to chronic high fat diet-induced obesity. Behavioural Brain Research, Volume 175, Issue 2, 15 December 2006, 415-419. 7. Laviano, A., Molfino, A., Preziosa, I., Muscaritoli, M., Cascino, A., & Fanelli, F. (2007). Brain serotonin and the control of food intake under physiological and pathological conditions. Nutritional Therapy and Metabolism, Volume 25, Issue 2, 49-55.

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