Hypothalamic serotonin in control of eating behavior, meal size, and body weight

Hypothalamic Serotonin in Control of Eating Behavior, Meal Size, and Body Weight Sarah F. Leibowitz and Jesline T. Alexander Serotonin (5-HT) has been implicated in the control of eating behavior and body weight. Stimulants of this monoamine reduce food intake and weight gain and increase energy expenditure, both in animals and in humans. This article reviews evidence that supports a role for hypothalamic serotonergic receptor mechanisms in the mediation of these effects. A variety of studies in rodents indicate that, at low doses, 5-HT or drugs that enhance the release of this neurotransmitter preferentially inhibit the ingestion of carbohydrate, more than fat or protein. This phenomenon is mediated, in part, by 5-HT receptors located in various medial hypothalamic nuclei. A negative feedback loop exists between the consumption of this macronutrient and the turnover of 5-HT in the hypothalamus. That is, carbohydrate ingestion enhances the synthesis and release of hypothalamic 5-HT, which in turn serves to control the size of carbohydrate-rich meals. A model is described that proposes the involvement of circulating hormones and glucose in this feedback process. These hormones, including insulin, corticosterone, and the adipose tissue-derived hormone, leptin, have impact on serotonergic function as well as satiety. This model further suggests that 5-HT exerts its strongest effect on appetite at the start of the natural feeding cycle, when carbohydrate is normally preferred. Clinical studies provide evidence that is consistent with the proposed model and that implicates 5-HT in disturbances of eating and body weight disorders. Biol Psychiatry 1998;44: 851– 864 © 1998 Society of Biological Psychiatry Key Words: Serotonin, hypothalamus, eating behavior, insulin, leptin, eating disorders

Introduction

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tudies indicate that serotonin (5-HT) has a suppressive effect on food intake and body weight (Blundell 1984, 1986; Curzon 1990; Leibowitz et al 1988; Simansky 1996). This effect of serotonergic stimulation has been demonstrated with both peripheral and central injections

From the Rockefeller University, New York, New York. Address reprint requests to Dr. Sarah F. Leibowitz, The Rockefeller University, 1230 York Avenue, New York, NY 10021. Received January 9, 1998; revised April 13; accepted April 17, 1998.

© 1998 Society of Biological Psychiatry

of serotonergic agonists. Moreover, the opposite effect, an enhancement of food consumption, has been observed with receptor antagonists and other drugs which reduce 5-HT activity. This report will focus on brain mechanisms involved in this effect of 5-HT. For a review of 5-HT’s role in the periphery, the reader is referred to a recent review by Simansky (1996).

Serotonin and Its Receptor Subtypes in Medial Hypothalamic Nuclei Hypothalamic microinjection of serotonergic agents into chronically brain-cannulated rats produces a potent and selective effect on feeding patterns and food choice, at much smaller doses (1–10 nmol) than those used in intraventricular and peripheral injection studies (Leibowitz et al 1988). This phenomenon is observed in different nuclei of the medial hypothalamus, most particularly the paraventricular (PVN), ventromedial (VMN), and suprachiasmatic (SCN) nuclei, and to a more variable extent, the dorsomedial nucleus (DMN) (Figure 1). These nuclei are essential for normal control of nutrient intake, and there is some evidence to suggest that the integrity of the hypothalamus, although not necessarily one specific nucleus (Fletcher et al 1993), is required for normal responsiveness to serotonergic drugs (Blundell 1984; Leibowitz et al 1988, 1990; Sclafani and Aravich 1983; Shor-Posner et al 1986; Stanley et al 1985; Weiss et al 1986). The suppressive effect on food intake can be seen with exogenous 5-HT, as well as with agents that enhance synaptic availability of endogenous 5-HT. These include d-norfenfluramine and fluoxetine, which enhance the release or block synaptic uptake of endogenous 5-HT (Leibowitz et al 1988; Shor-Posner et al 1986; Weiss et al 1986, 1990, 1991). In addition to reducing food intake, serotonergic stimulation of the PVN has been shown to enhance energy metabolism (Sakaguchi and Bray 1989) and specifically lipid oxidation, while reducing carbohydrate oxidation (Boschmann et al 1996). It also increases circulating levels of glucose and corticosterone (CORT) (Scheurink et al 1993). In humans, the serotonin reuptake inhibitor fluoxetine is found to increase energy expenditure (Bross and Hoffer 1995). 0006-3223/98/$19.00 PII S0006-3223(98)00186-3

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Figure 1. Schematic diagrams of coronal sections of the rat brain showing location of sites in individual subjects that exhibited a significant (circle) or insignificant (triangle) feeding-inhibition response to 5-HT injection. POM, medial preoptic area; SCN, suprachiasmatic nucleus; PVN, paraventricular nucleus; DMN, dorsomedial nucleus; VMN, ventromedial nucleus; PFH, lateral perifornical hypothalamus; PH, posterior hypothalamus. Reproduced with permission from Leibowitz et al (1990).

A specific role for medial hypothalamic serotonergic receptors in the control of feeding is supported by the finding that hypothalamic administration of general 5-HT

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antagonists effectively and dose-dependently blocks the feeding-inhibitory effect of 5-HT in the PVN (Leibowitz et al 1988; Weiss et al 1986). Pharmacologic evidence has suggested that central 5-HT1, as opposed to 5-HT2 or 5-HT3, receptor subtypes mediate the feeding-suppressive action of serotonergic stimulation in the medial hypothalamus (Curzon 1990; Curzon et al 1997; Garattini 1995). This is supported by the evidence that peripheral or hypothalamic injections of serotonergic or b-adrenergic antagonists with relatively high affinity for 5-HT1 receptors, but not selective antagonists of 5-HT2 or 5-HT3 receptors, significantly attenuate the feeding-suppressive action of serotonergic agonists injected peripherally or into the PVN (Curzon 1990; Dourish et al 1989; Hammer et al 1990; Kennett et al 1987; Leibowitz et al 1988; Massi and Marini 1987; Neill and Cooper 1989; Simansky 1996). These serotonergic receptor antagonists on their own may additionally potentiate feeding behavior (Coscina et al 1994; Currie and Coscina 1996; Neill and Cooper 1989), presumably by blocking the action of the endogenous 5-HT at its postsynaptic 5-HT1 receptors. Further differentiation of the 5-HT1 receptor indicates that, in the rat, the 5-HT1B and possibly the 5-HT1C subtypes are specifically involved in 5-HT-induced hypophagia, in contrast to the 5-HT1A receptor, which may mediate the opposite response, hyperphagia (Curzon 1990; Dourish et al 1986; Kennett and Curzon 1988a; Hutson et al 1988; Weiss et al 1986). In the rat, receptor binding assays in micropunched tissue (Leibowitz and Jhanwar-Uniyal 1989) and autoradiographic analyses of brain sections (Pazos and Palacios 1985) have identified a dense concentration of 5-HT1B and 5-HT1C receptor sites in the hypothalamus, in contrast to a relatively low concentration of 5-HT1A receptors. The evidence indicates, further, that the highest density of 5-HT1B receptor sites can be found in medial hypothalamic nuclei, in particular, the PVN, VMN, and SCN (Leibowitz and Jhanwar-Uniyal 1989), precisely where endogenous 5-HT is believed to act in modulating nutrient intake in the rat (Leibowitz et al 1990; Weiss et al 1986, 1990, 1991). While studies in humans, similar to rats, have revealed a suppressive effect of serotonergic agonists on eating behavior (Hill and Blundell 1986; Silverstone and Goodall 1986; Wurtman and Wurtman 1984), the failure to detect 5-HT1B receptors in human brain (Peroutka 1988) suggests that 5-HT1C or possibly 5-HT1D receptors, which exhibit similar characteristics to 5-HT1B receptors in rodents, may be involved in mediating the modulatory effects of serotonergic stimulation. A recent investigation with mutant mice supports the proposal that the 5-HT2C receptor subtype, previously termed 5-HT1C, is involved in eating and body weight regulation. In addition to evidence obtained in pharmaco-

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Figure 2. Percent change in macronutrient intake relative to vehicle control baseline after PVN injection of 5-HT, d-norfenfluramine, and fluoxetine, *p , .05. Results are obtained from data presented in Weiss et al 1986, 1990, 1991.

logic studies (Lawton and Blundell 1993), it has been demonstrated that mice lacking functional 5-HT2C receptors become obese as adults (Dourish et al 1997; Tecott et al 1997). These mice exhibit hyperphagia and consume larger meals, indicating a deficit in satiety. These traits precede the increase in body weight (Tecott et al 1997), suggesting the primary importance of eating behavior and meal size in generating the obesity syndrome. This mutant is also found to be less responsive to the feeding-suppressive effect of fenfluramine, reflecting the disturbance in serotonergic function (Dourish et al 1997). In another mutant mouse, anx, the anorexic behavior of this animal at a young age is suggested to be attributed, in part, to an overactive 5-HT innervation of the medial hypothalamic nuclei (Son et al 1994). It is clear that there are other sites, besides the medial hypothalamus, where 5-HT acts in the control of feeding behavior and metabolism. For example, diets with an amino acid imbalance cause primary anorexia and conditioned taste aversion. This effect, which is blocked by 5-HT antagonists, is mediated in part through actions in the piriform cortex (Hammer et al 1990). Serotonin also contributes to postingestive onset of satiety through brain stem structures (Li et al 1994) as well as through systems in the periphery (Simansky 1996; Simansky et al 1992).

Serotonin’s Impact on Eating Patterns and Macronutrient Ingestion Studies of meal patterns, using computer-automated procedures, demonstrate that medial hypothalamic 5-HT has a

specific role in controlling the temporal aspects of feeding. Hypothalamic as well as peripheral administration of serotonergic agonists affects feeding patterns by producing a significant decrease in the size and duration of individual meals, in association with a reduced rate of eating (Blundell 1984, 1986; Leibowitz et al 1988; ShorPosner et al 1986). Since the latency to meal onset and the frequency of meals taken are not affected, it is proposed that endogenous 5-HT may influence primarily the termination rather than the initiation of eating. Through hypothalamic administration of 5-HT, as well as studies employing systemic injection of serotonergic agents, evidence has accumulated to indicate a role for 5-HT in the modulation of the animals’ appetite for specific foods (Blundell 1984, 1986; Li and Anderson 1984; Shor-Posner et al 1986; Weiss et al 1990, 1991; Wurtman and Wurtman 1977). This role has been proposed to involve the control of carbohydrate and protein intake or perhaps the ratio of these two macronutrients, with serotonergic stimulation reducing the proportion of carbohydrate in the diet. This phenomenon was initially demonstrated in a two-diet self-selection paradigm, first with peripherally administered fenfluramine and fluoxetine (Wurtman and Wurtman 1977) and then with PVN injection of 5-HT or norfenfluramine (Leibowitz et al 1990; Shor-Posner et al 1986; Weiss et al 1990). The opposite pattern has been detected with a reduction in brain 5-HT after intraventricular injection of a serotonergic neurotoxin (Li and Anderson 1984). Subsequent studies have examined this phenomenon in freely feeding animals offered separate sources of pure

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Figure 3. Responsiveness of seven hypothalamic sites to 5-HT injection (2.5 nmol), indicated by a percent change in feeding scores relative to vehicle control baseline. Measurements of protein, carbohydrate, and fat were taken 1 hour after injection. See legend to Figure 1 for abbreviations. *p , .05, **p , .01, and ***p , .001. Reproduced with permission from Leibowitz et al (1990).

macronutrient diets (Leibowitz et al 1988; Weiss et al 1990, 1991). These studies reveal that injections of 5-HT, or the serotonergic agonists d-norfenfluramine and fluoxetine, directly into the medial hypothalamus preferentially and dose-dependently suppress carbohydrate consumption, while having little or no effect on or possibly enhancing the ingestion of protein or fat (Figure 2). This effect, similarly seen with central injection of 5-HT in mice (Currie 1993), can be detected in several medial hypothalamic nuclei, although the PVN and VMN are most responsive (Figure 3). It is predominantly observed at low doses of the 5-HT agonists, whereas higher doses or chronic stimulation with 5-HT agonists may additionally reduce fat intake (Kanarek and Dushkin 1988; Weiss et al 1990, 1991). A meal pattern analysis of this preferential effect on nutrient intake demonstrates that 5-HT in the PVN and VMN is active in terminating specifically carbohydrate-rich meals, while reducing the rate of carbohydrate eating and enhancing the satiating value of this nutrient (Kennett and Curzon 1988b). This is consistent with other evidence obtained with electrolytic brain lesions, distinguishing these two nuclei as having an essential role in controlling satiety for carbohydrate (Sclafani and Aravich 1983; Shor-Posner et al 1985). When peripherally administered, the serotonergic agents d-fenfluramine, fluoxetine, and quipazine also suppress carbohydrate intake; however, depending upon the drug dose and nutritional state of the animal, they may be less selective in their effect on macronutrient choice (Blundell 1986; Kanarek and Dushkin 1988; Leibowitz et al 1988; Shor-Posner et al 1986; Weiss et al 1986, 1990, 1991). Particularly at higher doses, they cause a reduction in fat as well as carbohydrate ingestion, while having lesser impact on or sometimes enhancing protein consumption. With peripheral injection of 5-HT antagonists, the

converse pattern is obtained, namely, a strong enhancement in feeding attributed primarily to a robust increase in carbohydrate and fat as opposed to protein ingestion (Dourish et al 1989; Leibowitz et al 1988; Shor-Posner et al 1986). A meal pattern study with peripherally administered metergoline demonstrates a specific effect of this antagonist in potentiating the carbohydrate concentration of a meal, specifically by reducing the satiating impact of this macronutrient (Leibowitz et al 1993). This 5-HT antagonist also stimulates feeding when injected directly into the PVN (Coscina et al 1994; Currie and Coscina 1996).

Feedback Loop: Impact of Carbohydrate Ingestion on Hypothalamic Serotonin The evidence favors the possibility that hypothalamic 5-HT, at least at low-to-moderate levels of stimulation, has a greater effect in reducing carbohydrate ingestion as compared to protein or fat ingestion. What is the significance of this relationship between 5-HT and dietary carbohydrate? A link between this monoamine and a specific macronutrient is substantiated by results showing a feedback effect of carbohydrate ingestion itself on 5-HT in the hypothalamus and brain stem. Animals consuming a high-carbohydrate diet, compared to a low-carbohydrate/ high-protein diet, show increased level of circulating tryptophan (TRP), the 5-HT amino acid precursor (Fernstrom et al 1975; Noach 1994; Wurtman and Wurtman 1995), and an increased level of 5-HT content in whole hypothalamus (Schweiger et al 1989; Thibault 1994). This diet promotes uptake of TRP into the brain and its subsequent conversion to 5-HT and also reduces concentrations of other amino acids that compete with TRP for transport into the brain. Insulin, which is released after a

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Figure 5. Feedback loop relating medial hypothalamic 5-HT, circulating hormones, and glucose in the control of carbohydraterich meal size.

Figure 4. Top, left: Serotonin fiber density in the median eminence in rats consuming a high-fat (HFD), control (MFD), or high-carbohydrate (HCD) diet. Top, right: Serotonin cell density in the dorsal and median raphe nuclei in rats on a HCD versus HFD. Bottom: Photomicrographs of 5-HT cell density in the dorsal and median raphe nuclei in rats on a HFD (left) or HCD (right). *p , .05. V, ventricle.

high-carbohydrate meal (Thibault 1994), stimulates the absorption of these large neutral amino acids (LNAA), thereby increasing the ratio of TRP:LNAA. This feedback mechanism may be disturbed in conditions of decreased insulin sensitivity, e.g., obesity, resulting in an overconsumption of carbohydrates (Fernstrom et al 1975; Noach 1994; Weltzin et al 1994). Studies in this laboratory, using immunohistochemistry, demonstrate a strong, site-specific effect of a high-carbohydrate diet on 5-HT-synthesizing neurons in the brain stem as well as 5-HT-containing fibers in the hypothalamus. In animals consuming a high-carbohydrate (65%) diet, compared to those on a high-fat diet with 15% carbohydrate, a marked increase in 5-HT is evident in the dorsal and median raphe nuclei (Figure 4) and also in the median eminence (ME), which receives a dense 5-HT innervation from the raphe nuclei (Chang and Leibowitz unpublished results). These results place the hypothalamic 5-HT system within a negative feedback loop controlling eating behavior. In this model (Figure 5), the ingestion of the macronutrient, carbohydrate, stimulates the production of the

monoamine, which then performs the function of terminating the ingestion of this nutrient and producing satiety. With excess serotonergic stimulation of the medial hypothalamus, anorexia results, e.g., in the anx mouse (Son et al 1994) or in cachexia (Holden and Pakula 1996; Meguid et al 1996). On the other hand, with a deficiency of 5-HT function, e.g., with lesions or receptor antagonists of the 5-HT system, hyperphagia and weight gain become evident (Lambert et al 1993; Myers et al 1995; Paez et al 1993).

Serotonin and a Single Meal As described above, the evidence predominantly indicates that the major effect of hypothalamic 5-HT is on eating behavior and particularly meal size, and this effect in turn has impact on body weight. Therefore, to investigate further this relationship between 5-HT and carbohydrate intake, one needs to focus on a single meal of carbohydrate and the role of 5-HT in controlling the size of this meal. In a preliminary study (Orosco et al 1997), a high-carbohydrate meal, compared to a high-fat meal, is found to cause an increase in release of 5-HT in the PVN, a site where 5-HT is believed to act in suppressing carbohydrate ingestion (Blundell 1986; Leibowitz et al 1988, 1990). This finding, consistent with studies in humans showing increased TRP:LNAA ratio after a high-carbohydrate meal (Lyons and Truswell 1988), indicates that the activity of the hypothalamic 5-HT system shifts significantly within a brief time period relevant to a single meal. By focusing on this brief interval, a better understanding of the control mechanisms related to 5-HT may be achieved. What are the properties of a high-carbohydrate meal that cause this change in brain 5-HT? Experiments from this laboratory, utilizing a chronic blood collection procedure in freely moving rats (Leibowitz and Yun unpublished results), demonstrate that a high-carbohydrate meal, compared to a low-carbohydrate/high-fat meal, is

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associated with high circulating levels of insulin, glucose, and the adrenal steroid, CORT. This pattern, consistent with evidence obtained through analyses of trunk blood (Boivin and Deshaies 1995; Heseltine et al 1990), can be observed during the first 90-min period after presentation of the macronutrient diets at the onset of the natural feeding cycle. Can these changes in circulating hormones and glucose help us to understand the increase in 5-HT observed in relation to carbohydrate ingestion? That they may contribute to the phenomenon, as indicated by the model in Figure 5, is suggested by evidence that CORT and insulin can directly affect the production of 5-HT. For example, in adrenalectomized (ADX) rats, CORT is shown to stimulate 5-HT metabolism in the brain (Chalmers et al 1993; Jhanwar-Uniyal et al 1987). In a study of discrete hypothalamic areas, this steroid enhances levels of 5-HT and its major metabolite, 5-hydroxyindoleacetic acid (5-HIAA), specifically in the PVN (Jhanwar-Uniyal et al 1987). Moreover, acute stress, through an increase in circulating CORT, increases 5-HT turnover while enhancing forebrain 5-HT2C receptor density (Holmes et al 1995; Vahabzadeh and Fillenz 1994). The serotonergic system is also responsive to insulin, which is released by the ingestion of carbohydrate. Insulin administration enhances 5-HT turnover and release in vitro (Dunbar et al 1995; Vahabzadeh et al 1995). Streptozotocin diabetes, in contrast, is associated with a decrease in 5-HT turnover rate (Martin et al 1995), which may contribute to the hyperphagia characteristic of diabetic animals (Bellush and Reid 1994). In addition to insulin, blood glucose levels also rise rapidly in response to a carbohydrate-rich meal. An increase in 5-HT levels in the brain has been reported after glucose administration in intact rats (Vahabzadeh et al 1995), and the injection of glucose together with insulin reverses the stimulatory effect of insulin on 5-HT expression (Vahabzadeh et al 1995).

Serotonin–Carbohydrate Feedback Loop in the Control of Meal Size What is the significance, relative to meal size, of these endocrine and neurochemical events, including increased insulin, glucose, and CORT in the blood and enhanced 5-HT synthesis in the hypothalamus? The functional importance of this feedback loop may be found in the natural characteristics of a high-carbohydrate meal. Meal pattern analyses demonstrate that a high-carbohydrate meal is distinctive in terms of its size and frequency (Shor-Posner et al 1991, 1994). A carbohydrate-rich meal (approximately 8 kcals) is generally smaller, by 10 –30%, compared to a high-fat meal (10.5 kcals), and it occurs at more frequent intervals, with 12.0 vs. 10.4 meals, respec-

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Figure 6. Representative meal patterns of high-carbohydrate eater (top) and high-fat eater (bottom) across the 12-hour feeding cycle. See text for description of meal patterns.

tively, occurring during the natural feeding cycle. In recent analyses of this 12-hour cycle (Alexander and Leibowitz unpublished results), the carbohydrate-rich meals are found to be most frequent during the first 2 hours, whereas the high-fat meals occur in the middle (hours 3–5) and end (hours 10 –11) of the cycle. This general pattern can be seen in animals that show a natural preference for carbohydrate. In these carbohydratepreferring rats, meal size is significantly smaller than that observed in fat-preferring animals, and the intermeal interval is significantly shorter and the meals more frequent (Figure 6). This smaller size of a carbohydraterich meal may be due, in part, to the fact that carbohydrate in some situations is more satiating than fat, in both animals (Shor-Posner et al 1991, 1994) and obese humans (Johnstone et al 1996; Lawton et al 1995; Rolls and Hammer 1995). These traits of a high-carbohydrate meal may be attributed to the neurochemical and endocrine events unique to this meal (Figure 5). Considering the inhibitory effect of 5-HT on carbohydrate ingestion as described above, it is likely that this monoamine, which is released by carbohydrate ingestion from raphe neurons innervating the medial hypothalamus (Figure 4), serves to terminate the meal and produce a state of satiety. Insulin itself may also be involved in this effect, perhaps through its stimulatory effect on 5-HT synthesis. This hormone, released by a carbohydrate-rich meal, can act directly within the medial hypothalamus to inhibit feeding (McGowan et al 1990; Plata-Salaman and Oomura 1986) and reverse the hyperphagia observed in diabetic animals (Tang et al 1997; Woods et al 1996). A similar action may be attributed to glucose itself, which inhibits feeding behavior while

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enhancing satiety (Heseltine et al 1990; Kurata et al 1986; McHugh and Moran 1986; Tordoff and Friedman 1986).

Diurnal Rhythm of Hypothalamic Serotonergic System Experiments indicate that a rhythm of serotonergic activity, in relation to feeding behavior, exists across the light/dark cycle, as reflected by temporal shifts in responsiveness to medial hypothalamic 5-HT1 receptor stimulation and in the release and utilization of endogenous 5-HT. The time of strongest serotonergic activity exists at the beginning of the active feeding period, just at the transition between light and dark (Leibowitz et al 1988; Shor-Posner et al 1986; Weiss et al 1991). At the onset of the dark cycle, the active period for the freely feeding rat, serotonergic stimulation of the PVN, VMN, or SCN is most effective in suppressing food intake and, in particular, carbohydrate ingestion (Figures 1–3) (Currie and Coscina 1996; Leibowitz et al 1990; Weiss et al 1990). Protein and fat ingestion are unaffected or even enhanced by 5-HT at this time, and at other points in the dark cycle, the serotonergic agonists cause no change in either total food intake or macronutrient choice. Moreover, carbohydrate ingestion is most effective at the onset of the feeding period in enhancing TRP concentrations and 5-HT synthesis in the brain (Fernstrom and Fernstrom 1995). The proposal that hypothalamic serotonergic control of feeding is expressed phasically primarily at the beginning of the natural feeding cycle is supported by various biochemical studies that have revealed that endogenous 5-HT activity in the medial hypothalamus rises or peaks specifically at this time. This change at dark onset has been revealed in the PVN, VMN, and SCN with measurements of either synaptic 5-HT content, synthesis, metabolism or uptake (Faradji et al 1983; Hery et al 1982; Martin 1991; Mason 1986; Meyer and Quay 1976; Stanley et al 1989). At this time, carbohydrate-rich meals are found to predominate under natural feeding conditions (Figure 6) (Shor-Posner et al 1991; Tempel et al 1989). Moreover, a single hypothalamic injection of 5-HT reduces the size and carbohydrate content of only the first three meals of this nocturnal feeding cycle (Leibowitz et al 1993). A similar effect is observed with the serotonergic compounds fluoxetine and d-norfenfluramine, which through microdialysis studies are found to enhance the synaptic release of endogenous 5-HT in the PVN (Paez and Leibowitz 1993). Thus, it is proposed that 5-HT in the medial hypothalamus plays a specific role in terminating these initial carbohydrate meals. The monoamine may act by stimulating PVN or VMN “satiety” neurons known to control intake of this macronutrient (Sclafani and Aravich

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1983; Shor-Posner et al 1985) and by SCN neurons that determine the circadian rhythms of physiological systems (Faradji et al 1983; Hery et al 1982; Meyer and Quay 1976). The hypothalamic–pituitary–adrenal axis is also most active at this time of the diurnal cycle. Corticosterone peaks in its release before the start of the natural feeding cycle, and through glucocorticoid receptors, enhances the ingestion of carbohydrate (Akabayashi et al 1994; Kreiger 1979; Tempel and Leibowitz 1994). This steroid may potentiate 5-HT production to terminate the carbohydrate meal. Corticosterone, which is actually released by serotonergic stimulation possibly acting through 5-HT2C receptors in the PVN (Feldman et al 1987; Fuller 1992), may be needed to counteract the hypoglycemic action of insulin. This effect of insulin is most likely to occur at a time, e.g., at the beginning of the natural feeding cycle, when glycogen stores are low and insulin sensitivity is high (Armstrong 1980; Tempel and Leibowitz 1994). In the process of terminating carbohydrate-rich meals characteristic of the early active period, 5-HT may interact antagonistically with norepinephrine (NE) and its a2noradrenergic receptors in the PVN (Currie and Coscina 1996; Currie et al 1994; Paez and Leibowitz 1993). Evidence suggests that NE in the PVN is most functional at the onset of the natural feeding cycle, when it acts in synergy with CORT to stimulate carbohydrate feeding. Its action at this time is specifically antagonized by administration of 5-HT agonists.

Leptin: Adipose Tissue Hormone Impacting on Eating and Body Weight The recent discovery of the fat-derived hormone, leptin, has substantiated the concept of a lipostatic factor, one that governs energy balance through a negative feedback loop originating in adipose tissue and acting on hypothalamic neurons. This hormone was discovered through the identification of the mutation responsible for producing obesity in the ob/ob mouse (Zhang et al 1994). Homozygosity for the ob (obese) gene results in reduced energy expenditure and brown adipose tissue activity, hyperphagia, and hyperinsulinemia, which produce obesity in the fa/fa Zucker rat. Leptin is expressed only in fat and is secreted into the circulation. This hormone may act as a satiety factor, leading to a decrease in food intake and reduced body weight after injection into ob/ob mice (Campfield et al 1996; Schwartz and Seeley 1997). There is also evidence that leptin affects overall energy expenditure, increasing both metabolic rate and body temperature (Campfield et al 1996; Mantzoros et al 1997; Salbe et al 1997).

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Leptin binding sites, which correspond to various classes of leptin receptor, have been identified in the hypothalamus, predominantly the VMN and arcuate nucleus (ARC), and also the choroid plexus (Hakansson et al 1998; Mercer et al 1996; Wilding et al 1997). Leptin enters the brain via a saturable, active transport system that is independent of the insulin transporter (Banks et al 1996). This system effectively transports leptin across the blood– brain barrier in the ME, where specialized transport occurs between the circulation and central nervous system. Binding sites in the choroid plexus may also represent leptin transporters in these regions (Malik and Young 1996; Mercer et al 1996). A single gene, which has homology to the cytokine receptors, appears to encode the various leptin receptor isoforms in the brain (Wilding et al 1997). There are various factors that determine leptin synthesis and release. The average daily circulating concentrations of leptin are closely related to body fat mass (Frederich et al 1995a), with leptin expression rising in various models of obesity (Frederich et al 1995b; Funahashi et al 1995; Maffei et al 1995; Murakami and Shima 1995; Trayhurn et al 1995; Zhang et al 1994). In addition, leptin secretion from fat is determined by hormonal status. Insulin has a modest stimulatory effect on ob gene expression (Trayhurn and Rayner 1996), and there is some evidence to suggest that glucose uptake may also affect leptin production (Havel 1997; Mizuno et al 1996). Glucocorticoids can also enhance leptin synthesis and secretion in rats (Slieker et al 1996).

Relationship between Serotonin and Leptin in Controlling Eating Behavior The above results demonstrate a pattern for leptin similar to that seen for 5-HT, in terms of their impact on food intake and energy expenditure as well as their responsiveness to circulating hormones or glucose. To date, there appear to be no investigations of a direct interaction between 5-HT, leptin, or their respective receptor subtypes. There is some evidence, however, to suggest that these two substances may function in parallel circuits, under similar conditions, to control eating and possibly meal size. In the hypothalamus, 5-HT and ob receptors are generally concentrated in the same nuclei, namely, the VMN, ARC, PVN, and ME (Mengod et al 1990; Pazos and Palacios 1985; Wilding et al 1997). Stimulation of these receptors by the respective agonists reduces food intake. Leptin facilitates the firing of neurons in the PVN and VMN (Shiraishi et al 1997), where lesions produce carbohydrate hyperphagia and obesity (Shor-Posner et al

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1985). Moreover, both 5-HT and leptin inhibit the production of hypothalamic peptides that normally enhance food intake (Dryden et al 1996; Stephens et al 1995; Wilding et al 1997; Leibowitz et al 1996). This is seen with neuropeptide Y, a potent stimulant of feeding behavior (Tempel and Leibowitz 1994; Wilding et al 1997; Stanley et al 1985; Wang et al 1998). Injection of leptin also reduces the expression of hypothalamic galanin, which stimulates feeding (Leibowitz et al 1998; Sahu 1998), and it enhances peptides that normally suppress feeding (Costa et al 1997; Schwartz et al 1996). Further, both leptin and 5-HT appear to synergize with another peptide, cholecystokinin (CCK), which inhibits feeding behavior. This peptide, released from the gut during a meal, provides appropriate postingestive signals for terminating the meal (Gibbs et al 1993; Crawley and Corwin 1994), acting through CCK-A receptors possibly located on PVN neurons (Dourish 1992). Cholecystokinin may come from serotonergic neurons as a cotransmitter, having longer-lasting effects on feeding than the monoamine alone. It may also be released in the hypothalamus in response to vagal inputs that are relayed from the stomach to the hypothalamus. The proposal that 5-HT and CCK work together is supported by the finding that 5-HT receptor blockers antagonize CCK’s anorectic effect (Crawley and Corwin 1994; Smith and Gibbs 1994). Behavioral analyses reveal that leptin, similar to 5-HT, produces satiety when injected directly into the medial hypothalamus (Satoh et al 1997). In rats on macronutrient diets, leptin reduces the ingestion of carbohydrate in addition to fat, while having little impact on protein intake (Bedrin et al 1996). Moreover, consumption of a carbohydrate-rich meal increases circulating levels of leptin (Leibowitz and Yun unpublished results), similar to its effect on hypothalamic 5-HT (see above). This is in contrast to a fat-rich meal, which has little impact on leptin (Schrauwen et al 1997; Leibowitz and Yun unpublished results) as well as on 5-HT. These similarities between 5-HT and leptin support a role for both substances in the initiation of satiety (Figure 5). As described above, this effect is likely to be strongest for carbohydrate-rich meals, which increase leptin levels and hypothalamic 5-HT production, are more responsive to serotonergic agonists at low doses, and are generally smaller in size compared to fat-rich meals. The actions of these substances, however, may also be exhibited with meals rich in both carbohydrate and fat. Whether 5-HT and leptin directly interact in this process is unknown. This possibility may be suggested by the finding that ob mice, which have a deficiency of leptin, are also less responsive to the suppressive effects of serotonergic stimulation on carbohydrate ingestion (Currie 1993).

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Clinical Studies of Brain Serotonin and Serotonergic Drugs in Eating and Body Weight Disorders: Gender Differences These findings in animals may have implications for understanding human brain mechanisms of appetite control, which exhibit some similarities in their responsiveness to serotonergic stimulation. As in animal studies, administration of serotonergic agents in humans suppresses eating behavior, possibly by reducing the rate of eating as well as the size of meals through the intensification of satiety (Hill and Blundell 1986; Silverstone and Goodall 1986; Wurtman and Wurtman 1984). In some studies (Wurtman and Wurtman 1995), they cause a preferential reduction in the consumption of carbohydrate, as well as a decrease in the “craving” for snacks rich in this macronutrient. Moreover, administration of agents that antagonize serotonergic activity are generally found to increase calorie intake and hunger ratings in human subjects and possibly enhance intake of “sweet highcarbohydrate” foods (Silverstone and Goodall 1986). In addition to carbohydrate, dietary fat may also be important in these effects of the serotonergic drugs on appetite (Blundell and Lawton 1995). Clinical studies have also revealed disturbances in the activity of the serotonergic system in patients with eating disorders (Brewerton 1995; Jimerson et al 1990; Kaye 1997). Of particular interest are bulimic patients, who appear to have a disorder in the production of satiety. These patients are found to overconsume carbohydrate, or carbohydrate combined with fat, in all but their largest meals (Weltzin et al 1994; Wurtman and Wurtman 1995). They also exhibit a deficiency of brain 5-HT activity, decreased postsynaptic serotonergic receptor responsiveness, and a tendency toward smaller postmeal increases in the plasma ratio of TRP:LNAA, which may determine brain 5-HT synthesis (Fernstrom and Fernstrom 1995; Jimerson et al 1990; Kaye 1997; Wurtman and Wurtman 1995). Bulimic patients are reported to have reduced prolactin levels in basal conditions as well as after stimulation with 5-HT agonists (Heninger et al 1984; Kaye 1997; Weltzin et al 1994). Disturbances in brain 5-HT have also been seen in patients with anorexia nervosa. In particular, after long-term weight restoration, anorexic subjects exhibit a pattern indicative of enhanced 5-HT turnover, opposite to that detected in bulimic patients (Kaye 1997). Some studies with serotonergic compounds, such as, fenfluramine and fluoxetine, have reported positive results in the treatment of patients with eating disorders (Bever and Perry 1997; Jimerson et al 1990; Kaye 1997; Toornvliet et al 1996). These agents, which enhance endogenous serotonergic activity substantially, decrease binge eating

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in bulimic patients, and this effect appears to occur independently of changes in symptoms of depression. Furthermore, a possible effectiveness of antidepressant agents that block 5-HT uptake in maintaining a healthy body weight in anorexics has also been described (Kaye 1997). Eating disorders occur predominantly in female subjects and generally develop during the adolescent years (Silverman 1983). Moreover, disturbances in their carbohydrate preferences, as well as the distribution of their carbohydrate diet across the 24-hour cycle, are common (Wurtman and Wurtman 1989). This is of interest in light of recent studies showing gender differences in the eating patterns of animals. For example, female rats exhibit a stronger preference for carbohydrate than male rats; they consume more calories of carbohydrate relative to protein or fat, and they show a diurnal shift compared to males, with a larger portion of their carbohydrate consumed during the inactive period (Leibowitz et al 1991). This preference pattern, similarly seen in humans (Drewnowski et al 1992), is evident sometime between weaning and puberty, with peak carbohydrate intake in female subjects occurring just prior to the onset of puberty. The possibility that this gender difference in appetite for carbohydrate may be linked to brain 5-HT (Wurtman and Wurtman 1989) is supported by the evidence that female rats exhibit a higher density of medial hypothalamic 5-HT1B as well as 5-HT1A receptors compared to males, as well as a greater responsiveness to serotonergic agents (Datla and Curzon 1997; Leibowitz and Jhanwar-Uniyal 1989). They also show faster 5-HT turnover rate and responsiveness to agents that modulate brain 5-HT synthesis (Haleem et al 1990), and their 5-HT system appears to be more perturbable and less adaptive to stress compared to males (Kennett et al 1986). The overexpression of 5-HT in the hypothalamus of different rodent populations is, in fact, associated with a state of anorexia (Holden and Pakula 1996; Meguid et al 1996; Son et al 1994). In humans, female subjects have been reported to have higher cerebrospinal fluid 5-HIAA levels than male subjects (Heninger et al 1984). Moreover, clinical evidence indicates that dieting alters the responsiveness of women, but not men, to the 5-HT precursor, TRP (Goodwin et al 1987). Investigation of the relationship of this neurochemical profile to gender differences in natural meal patterns and macronutrient preferences should help us to understand how the brain serotonergic system may contribute to the development of disturbances in appetite and eating patterns in humans. The involvement of leptin, which has been implicated in the onset of puberty (Cheung et al 1997) and is found to be at much higher levels in female subjects (Saad et al 1997), must also be given serious consideration.

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Obesity is a significant health problem because of its associated medical complications. Pharmacologic agents, e.g., dexfenfluramine and fluoxetine, which block 5-HT uptake have been examined as adjunctive therapy to standard weight management programs and found to have clinically meaningful benefits on weight loss (Bever and Perry 1997; Davis and Faulds 1996; Goldstein et al 1995). An additional agent, sibutramine, which blocks uptake of both 5-HT and norepinephrine (Jackson et al 1997), is also being considered for clinical use. The efficacy, specificity, and safety of these compounds are continually being investigated, not only in the management of obesity but also in the control of appetite in diabetics, binge eating, premenstrual overeating, and neuroleptic-induced weight gain. Selective agonists of the 5-HT2C receptor, which are involved in the anorexic effect (see above), may also prove effective in reducing eating behavior or body weight (Dourish 1995).

This research was supported by U.S. Public Health Service grant MH 43422. The authors wish to thank Dr. Guo-Quing Chang for contributing the photomicrograph (Figure 4). This work was presented at the Neuroscience Discussion Forum “A Decade of Serotonin Research” held at Amelia Island, Florida in November 1997. The conference was sponsored by the Society of Biological Psychiatry through an unrestricted educational grant provided by Eli Lilly and Company.

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