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Vol. 52, Issue 1, 35-62, March 2000

Transgenic Approach to the Study of Body Weight Regulation

Akio Inui1

Second Department of Internal Medicine, Kobe University School of Medicine, Kobe, Japan

I. Introduction
II. Leptin and Body Weight Regulation
III. Neuropeptidergic Cascade Downstream of Leptin Signaling
IV. Key Components in Body Weight Regulation and Implications of Transgenic Animal Models
    A. Hypothalamic Stimulators of Food Intake
        1. NPY.
        2. MCH.
        3. Orexin.
        4. Galanin.
        5. Opioid Peptides.
        6. AGRP.
        7. Other Orexigenic Signals.
    B. Hypothalamic Inhibitors of Food Intake
        1. MC.
        2. CRF and Urocortin.
        3. GLP-1.
        4. Bombesin.
        5. CCK.
        6. Serotonin.
        7. Cytokine.
        8. Other Anorexigenic Signals.
    C. Regulators of Thermogenesis: Sympathetic Nervous System (SNS)-UCP Axis
    D. Other Regulators
V. Advanced Gene Targeting
VI. Conclusions
Acknowledgments
References


    Abstract
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Energy homeostasis is accomplished through a highly integrated and redundant neurohumoral system. Recently, novel molecular mediators and regulatory pathways for feeding and body weight regulation have been identified in the brain and the periphery. Because of the multitude and complexity of disturbances in energy intake, expenditure, and partitioning that are associated with obesity, it has been difficult to determine which abnormalities are causative versus less important phenomena that are consequences of the altered neuroendocrine and metabolic milieu. Transgenic technology has provided new opportunities to modify the complex body weight-regulating system and to assess the relative importance of the individual components. Observations of mutant mice have shed new light on the understanding of energy homeostasis equation. Once created, transgenic animal models may be useful in assessing the efficacy or determining the mode of action of potential new therapeutic agents. However, the interpretation of targeted mutation is sometimes not straightforward in unraveling the physiology because of the redundancy and compensation of the regulatory machinery, as well as the inherent problems of manipulation of the gene. Modifying the synthesis of a particular gene at all sites and developmental stages may be a relatively crude way of investigating its functions. Advanced gene-targeting strategies aimed at specific alterations (on and off) of a gene product at desired tissues and times could lead to a better understanding of the system.


    I. Introduction
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Most naturally existing single-gene mutations resulting in obesity in rodents have been cloned in recent years (Spiegelman and Flier, 1996; Chua and Leibel, 1997; Levine and Billington, 1998; York and Hansen, 1998). These mutations include yellow (Agouti, Ay), obese (ob/ob), diabetes (db/db), fat, tubby, and Zucker Fatty (fa/fa), which have been extensively studied in an effort to understand the physiological and biochemical basis for their obese phenotype. Obesity research has especially gathered momentum since the characterization of the obese (ob)2 gene and its product leptin (Friedman and Halaas, 1998).

Energy homeostasis is accomplished through a highly integrated and redundant neurohumoral system that minimizes the impact of short-term fluctuations in energy balance on fat mass (Bray and York, 1979, 1998; Rohner-Jeanrenaud, 1995; Kalra, 1997; Elmquist et al., 1998,1999; Flier and Maratos-Flier, 1998; Friedman and Halaas, 1998; Woods et al., 1998; Kalra et al., 1999a; Inui, 1999a). Recently, novel molecular mediators and regulatory pathways for feeding and body weight regulation have been identified in the brain (Elmquist et al., 1998,1999; Flier and Maratos-Flier, 1998; Woods et al., 1998; Kalra et al., 1999a; Inui, 1999a). These have generated great interest in the genetic framework of body weight regulation and the derangement of the tight control of energy homeostasis leading to obesity or anorexia/cachexia.

Transgenic technology, which permits the introduction of genes into the germ line of mice, and homologous recombinant gene knockout, which allows elimination of endogenous gene expression, are powerful tools for exploring the complex pathogenesis of obesity. The genetic manipulations have provided new models relevant to the study of each of the elements, as well as suggesting several illuminating, although sometimes confusing, insights into the underlying mechanisms (Levine and Billington, 1998; York and Hansen, 1998). The purpose of this review is to present recent advances in the understanding of body weight regulation, with a particular emphasis on transgenic animal models.


    II. Leptin and Body Weight Regulation
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The identification of the ob gene (Zhang et al., 1994) and the discovery that its encoded protein, leptin, is an adipocyte-derived hormone that is essential for normal regulation of body weight have greatly altered the field of metabolic physiology (Spiegelman and Flier, 1996; Flier, 1998). Leptin reduces appetite and increases energy expenditure when injected peripherally or i.c.v., and evidently elicits these effects via the central nervous system (CNS; Elmquist et al., 1998, 1999; Flier and Maratos-Flier, 1998; Friedman and Halaas, 1998; Sawchenko, 1998; Inui, 1999a). Leptin enters the brain by an active saturable system (Halaas et al., 1995; Banks et al., 1996) and acts through or in concert with several neuropeptides, monoamines, and other transmitter substances that affect food intake in the brain-gut axis (Figs. 1 and 2).



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  		Fig. 1.   A simplified cascade model for the interaction of leptin with hypothalamic neuropeptidergic effector molecules. Leptin acts as part of a feedback loop to maintain constant stores of fat (see also Fig. 2). A loss of body fat leads to a decrease in leptin, which activates feeding-stimulatory molecules in the hypothalamus, such as NPY. Conversely, an increase in body fat leads to an increase in leptin, which activates feeding-inhibitory molecules such as MC. NPY is the key component of the interconnected orexigenic network and its secretion and action are regulated by anorexigenic neuropeptides. In gray are the molecules of which the impact on body weight have been shown with either acute administration studies or chronic transgenic studies. MCH and orexin may situate downstream of NPY signaling. Other suggested interplays include those between leptinright-arrowTRH, TRHright-arrowNPY, GLP-1right-arrowCRF, alpha -MSHright-arrowMCH, and galaninright-arrowopioid, and also those in the opposite directions to the ones described in the figure, such as NPYright-arrowCRF and NPYright-arrowMC. These might have implications for the regulation of feeding and body weight, as well as for the activation of the hypothalamic-pituitary-adrenal axis. +, stimulatory input; -, inhibitory input



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  		Fig. 2.   Candidate molecules that control energy intake, expenditure and/or partitioning. The feeding-inhibitory molecules stimulate energy expenditure via the SNS-UCP axis, whereas the opposite applies to the feeding-stimulatory molecules. An increase in sympathetic activity is associated with an increase in the levels of UCP-1 mRNA in BAT, which produces thermogenesis, leading to reduced storage of fat. Genetic manipulation identified previously unknown regulators of energy homeostasis, including intracellular adhesion molecule-1, leukocyte integrin alpha Mbeta 2, metallothionein, and transcription factor Nhlh2, although fuller characterizations of these obese phenotypes need to be performed. beta 1 R, beta 1 adrenergic receptor; beta 3 R, beta 3-adrenergic receptor; LPL, lipoprotein lipase. Recently hypothalamic histamine was demonstrated to be involved in leptin signaling pathway (Yoshimatsu et al., 1999).

Leptin concords well with the postulate of a lipostatic system for weight control, which was proposed to explain the relative stability of weight over time in many animal species as well as their capacity to respond well to short-term fluctuations in energy balance to restore body weight to previous levels. Leptin is an afferent signal from the periphery to the brain in a homeostatic feedback loop that regulates adipose tissue mass (Figs. 1 and 2; Schwartz et al., 1992; Bray and York, 1998; Flier, 1998; Friedman and Halaas, 1998). The level of leptin is positively correlated with body fat mass, and dynamic changes in plasma leptin concentrations in either direction activate the efferent energy regulation pathways. Rising levels of leptin signal the brain that excess energy is being stored, and this signal brings about adaptations of decreased appetite and increased energy expenditure that resist obesity. Transgenic overexpression of leptin in the liver by using the human serum amyloid P component promoter has resulted in markedly decreased food intake and body weight gain with the complete disappearance of white adipose tissue and brown adipose tissue (BAT; Ogawa et al., 1999). Conversely, a loss of body fat leads to a decrease in leptin, and the physiological response is to increase appetite and decrease energy expenditure, both of which induce a positive energy balance and weight gain. Ob/ob mice, homozygous for a spontaneous mutation on the ob gene, failed to produce leptin and exhibited hyperphagia and obesity. Mutations in leptin receptors seen in db/db mice and fa/fa rats, resulted in an obese phenotype identical with that of ob/ob mice (Friedman and Halaas, 1998).

Shortly after the discovery of leptin it was also found that, with the exception of the ob/ob mice, obese rodents exhibit increased levels of serum leptin (Maffei et al., 1995; Frederich et al., 1995). The concept that obese rodents and humans are resistant to their endogenous leptin began to emerge. Recent studies have suggested that leptin resistance, also referred to as reduced leptin sensitivity, may play a significant role in the development of obesity (Strader et al., 1998). It may result not only from a structural aberration in the leptin receptor, but also from defective transport of leptin across the blood-brain barrier (BBB) and/or defects localized downstream in the signal transduction pathway of leptin (Caro et al., 1996; Bjorbaek et al., 1998). The observation of central leptin responsiveness in obese rodents in the face of peripheral leptin resistance may suggest a role for the reduced efficacy of leptin transport to the CNS (Van Heek et al., 1997).


    III. Neuropeptidergic Cascade Downstream of Leptin Signaling
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There is now a growing recognition that expression of appetite is chemically coded in the hypothalamus (Bray and York, 1979, 1998; Rohner-Jeanrenaud, 1995; Elmquist et al., 1998, 1999; Sawchenko, 1998; Woods et al., 1998; Inui, 1999a; Kalra et al., 1999a). Classic studies described syndromes of ravenous overeating and obesity as a consequence of lesions centered in the ventromedial nucleus (VMH; Hetherington and Ranson, 1940) and of a failure to eat and drink after damage to the lateral hypothalamus (LH; Anand and Brobeck, 1951), the dual-center model (Stellar, 1954). It is now known that other hypothalamic sites such as the paraventricular nucleus (PVN) and dorsomedial nucelus (DMH) also contain neural mechanisms that affect feeding behavior (Hetherington and Ranson, 1940; Stellar, 1954; Gold, 1973; Swanson and Sawchenko, 1983; Bernardis and Bellinger, 1996; Fig. 2). There are terminal fields of neurons from the arcuate nucleus (ARC), which is located at the base of the hypothalamus and contains orexigenic (feeding-stimulatory) and anorexigenic (feeding-inhibitory) neurotransmitters and neuromodulators. The biologically active, long form of the leptin receptor is produced in various hypothalamic sites including ARC, VMH, DMH, PVN, and LH (Mercer et al., 1996; Schwartz et al., 1996; Friedman and Halaas, 1998).

Figure 1 shows a simplified model for the interaction of leptin with hypothalamic neuropeptidergic effector molecules within a regulatory feedback loop. The model is based on the findings obtained mostly from acute administration studies, and it emphasizes the feeding drive systems that would underlie both obesity and hypothalamic response to starvation. Neuropeptide Y (NPY) is the most potent orexigenic peptide activated by the fall of leptin, and consists of an interconnected orexigenic network that includes galanin, opioid peptides, melanin-concentrating hormone (MCH), orexin, and agouti-related protein (AGRP; Morley, 1987; Inui et al., 1991; Qu et al., 1996; Flier and Maratos-Flier, 1998; Sakurai et al., 1998; Woods et al., 1998; Inui, 1999a,b; Kalra et al., 1999a). Most of these peptides are up-regulated in ob/ob mice, and their expressions are increased through fasting in wild-type mice and are inhibited by leptin administration (Inui, 1999a). Other effector molecules functioning in this homeostatic loop are the anorexigenic neuropeptides such as corticotropin-releasing factor (CRF), melanocortin (MC), glucagon-like peptide-17-36 amide (GLP-1), neurotensin, and cocaine- and amphetamine-regulated transcript (CART), the expression of which is down-regulated in ob/ob mice and stimulated by leptin (Schwartz et al., 1995; Flier and Maratos-Flier, 1998; Woods et al., 1998; Inui, 1999a; Kalra et al., 1999a). The administration of the receptor antagonists of these peptides effectively blocks the reduction of food intake and body weight induced by leptin (Inui, 1999a). An imbalance in the operation of either orexigenic or anorexigenic pathways is thought to perturb the regulatory microenvironment, leading to hyperphagia and abnormal weight gain (Kalra et al., 1999a). It has yet to be determined by which mechanisms the orexigenic network escapes the inhibitory influences of leptin and anorexigenic signals.

The orexigenic and anorexigenic substances decrease and increase sympathetic nervous activity, respectively, thereby regulating energy expenditure and body fat stores (Fig. 2). This is achieved by modulating thermogenesis in BAT and possibly in other sites such as white adipose tissue and muscle, through induction of the mitochondrial uncoupling protein UCP-1 and the newly identified UCP-2 and UCP-3 (Rohner-Jeanrenaud, 1995; Collins et al., 1996; Spiegelman and Flier, 1996; Fleury et al., 1997; Gong et al., 1997; Bray and York, 1998). The reciprocal relationship between food intake and sympathetic activity has been shown to be robust among various neurotransmitter substances (Bray, 1993; Bray and York, 1998). Other neurotransmitters that affect food intake and energy expenditure include feeding-stimulatory norepinephrine (via alpha 2-receptor) and gamma -aminobutyric acid (GABA), and feeding-inhibitory serotonin and dopamine (Table 1). Neuropeptides are important components of the feeding-regulatory systems.


                              
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  		TABLE 1
Hormones, neurotransmitters, and neuropeptides that affect food intake

The feeding-stimulatory molecules include norepinephrine, gamma -aminobutyric acid, and seven classes of neuropeptides, whereas the feeding-inhibitory molecules include serotonin, dopamine, and a long list of brain-gut peptides.


    IV. Key Components in Body Weight Regulation and Implications of Transgenic Animal Models
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A. Hypothalamic Stimulators of Food Intake

1. NPY. NPY, a 36-amino acid peptide, is one of the most abundant and widely distributed neurotransmitters in the mammalian brain (Tatemoto, 1982; Sahu and Kalra, 1993; Billington et al., 1994; Leibowitz, 1995; Kalra, 1997; King and Williams, 1998; Inui, 1999b; Kalra et al., 1999a; Table 2). The ARC is the major site of expression for NPY within neurons in the hypothalamus that project to PVN, DMH, LH, and other hypothalamic sites. Although NPY can produce diverse effects on behavior and other functions, its most noticeable effect is the stimulation of feeding after central administration (Sahu and Kalra, 1993; Billington et al., 1994; Kalra, 1997; King and Williams, 1998; Inui, 1999b; Kalra et al., 1999a). The feeding-stimulatory effect of NPY is approximately 500 times more potent on a molar basis than norepinephrine (King and Williams, 1998). Multiple injections of NPY into the PVN or cerebral ventricle result in obesity, indicating that NPY is capable of overriding powerful inhibitory signals on food intake and body adiposity (Stanley et al., 1986; Stanley, 1993). NPY produces a shift to positive energy balance by increasing food intake, by decreasing energy expenditure primarily with a reduction in thermogenesis in BAT (Egawa et al., 1991), and by facilitating fat deposition in white adipose tissue partly through increased insulin activity (Zarjevski et al., 1993).


                              
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  		TABLE 2
Hypothalamic stimulators of food intake and their effect on body weight regulation

NPY synthesis in the ARC and its release into the PVN, the most abundant projection, are regulated by afferent signals such as leptin, insulin (both inhibitory), and glucocorticoids (stimulatory; Schwartz et al., 1992; Sahu and Kalra, 1993; Stanley, 1993; Leibowitz, 1995; Kalra, 1997; King and Williams, 1998; Inui, 1999b; Kalra et al., 1999a). NPY synthesis and secretion are all up-regulated in models of energy deficiency or increased metabolic demand such as starvation, insulin-dependent diabetes mellitus, lactation and physical exercise (Sahu and Kalra, 1993; Stanley, 1993; Kalra, 1997; Turton et al., 1997; Inui, 1999b; Kalra et al., 1999a). The NPY neurons that are activated by fasting are the neurons that express the long form of the leptin receptor (Baskin et al., 1999). A primary physiological role of the ARC NPY neurons may thus be to restore normal energy balance and body fat stores under conditions of energy deficit, the signals of which are falling leptin and/or insulin occurring in these conditions.

It is also known that hyperphagia and obesity in several genetic and experimental models are associated with augmentation of NPYergic signaling. Genetically obese ob/ob mice, db/db mice, and fa/fa rats exhibit increased preproNPY mRNA in the ARC and increased NPY levels and release in the PVN (Sanacora et al., 1990; Wilding et al., 1993; Dryden et al., 1995). Augmented NPY gene expression in the ARC and increased NPY levels as well as release in the PVN are observed in streptozotocin-induced diabetic rats, which may precede the onset of hyperphagia (Williams et al., 1988, 1989; Sahu et al., 1990, 1992, 1997). Arcuate NPY mRNA expression may also be abnormally high and not responsive to energy restriction before phenotypic expression of obesity in diet-induced obese rats (Levin and Dunn-Meynell, 1997; Levin, 1999). In this model, once obesity is fully expressed, NPY levels are depressed but become responsive to energy restriction to defend the higher body weight. Lesions in the VMH are associated with hyperphagia and body weight gain in rodents and humans (Hetherington and Ranson, 1940; Bray and York, 1979). However, preproNPY mRNA levels in the ARC and NPY levels as well as release from the PVN were unexpectedly suppressed in hyperphagic VMH-lesioned rats (Kalra, 1997; Dube et al., 1999). Obesities induced by neurotoxins such as colchicine, gold thioglucose, and monosodium glutamate also exhibit suppressed hypothalamic NPY gene expression and NPY levels or release in the PVN (Bergen and Mobbs, 1996; Stricker-Krongrad et al., 1996; Jain et al., 1998). However, feeding in response to NPY was increased and hyperphagia was suppressed by blockade of NPY or NPY receptors (Dube et al., 1995; Bergen and Mobbs, 1996; Stricker-Krongrad et al., 1996; Kalra et al., 1997, 1998). Both low and high abundance of NPY can be associated with hyperphagia and obesity (Kalra et al., 1999a). Increased NPY receptor abundance and sensitivity, and up-regulation of NPY in novel hypothalamic sites such as DMH may underlie the hyperphagia (Kalra et al., 1997, 1998, 1999a). Furthermore, increased induction and responsiveness of other orexigenic signals such as galanin were reported in colchicine-treated rats (Kalra et al., 1999a; Pu et al., 1999a). Therefore, diminution of leptin feedback or leptin resistance contributes to the hyperphagia and obesity through modifications of the NPY orexigenic network.

In view of the above evidence indicating a key role for NPY in energy homeostasis, it was surprising that mice in which the NPY gene had been deleted by homologous recombination were phenotypically normal except for an increase in susceptibility to seizures (Erickson et al., 1996a; Palmiter et al., 1998). These NPY-deficient mice were sensitive to the anorectic effect of leptin, indicating that leptin acts through pathways other than those involving NPY. Subsequent experiments with mice with a deficiency in both leptin and NPY demonstrated that the double mutant mice are halfway between normal lean and ob/ob mice in terms of body weight and fat mass, indicating that NPY is required for full expression of the ob phenotype (Erickson et al., 1996b). The absence of NPY, however, did not attenuate the development of obesity induced by a high-fat diet, chemical lesions of the hypothalamus, impaired BAT function due to a diphtheria toxin transgene or the lethal agouti mutation (Ay; Hollopeter et al., 1998a). The responses of NPY-deficient mice to anorectic and orexigenic substances including NPY, CRF, dexfenfluramine (an enhancer of serotonergic transmission), and MT (a melanocortin-4 receptor agonist) were unaltered although the initial response to galanin might be lost (Hollopeter et al., 1998b). It remains to be determined whether NPY is involved in hyperphagia and obesity only under extreme conditions such as in ob/ob mice (Hollopeter et al., 1998a) or whether the normal phenotype is due to compensation by other orexigenic signals that replace NPY and maintain seemingly normal feeding and body weight regulation.

NPY transgenic animals were also created by using a novel CNS neuron-specific expression vector of human Thy-1 gene fragment linked to mouse NPY cDNA (Inui et al., 1998) or additional copies of the NPY structural gene introduced into the mouse (Thiele et al., 1998) or the rat (Michalkiewicz et al., 1999). NPY-overexpressing mice showed anxiety-like behavior via CRF neuronal system (Inui et al., 1998) or decreased ethanol consumption with increased sensitivity to ethanol-induced sedation (Thiele et al., 1998). Ethanol consumption and resistance appeared to be inversely related to NPY levels (Thiele et al., 1998). However, they exhibited seemingly normal food intake and body weight regulation, although the transgene expression may be minimal in the hypothalamus of these animals. Transgenic rats also showed only gender-dependent increases or decreases in food intake and body weight despite the hypertension and enhanced cardiovascular responsiveness to adrenergic stimulation and stress (Michalkiewicz et al., 1999).

NPY activates at least six G protein-coupled receptor subtypes, Y1 to Y6, all of which have been cloned except for the Y3 receptor (Balasubramaniam, 1997; Blomqvist and Herzog, 1997; Gehlert, 1998; Michel et al., 1998). NPY analogs exhibit varying degrees of affinity and specificity for these Y receptors, as well as potency in stimulating feeding (Inui, 1999b). Recent studies on NPY have focused on the Y receptors, because new anti-obesity drugs may emerge from pharmacological characterization of the Y receptors and their antagonists. The Y5 receptor has been isolated as the receptor that has pharmacological properties most closely matching a proposed feeding receptor (Gerald et al., 1996; Schaffhauser et al., 1997; Criscione et al., 1998). However, recent progress in the development of nonpeptide ligands suggests that NPY receptors other than the Y5 may mediate the appetite-stimulating effect of NPY. The potent anorectic effects of Y1 receptor-specific antagonists in various rodent models of obesity support the initial suggestion that the Y1 receptor is involved in appetite regulation (Stanley, 1993; Kanatani et al., 1996, 1998; Lopez-Valpuesta et al., 1996; Ishihara et al., 1998; Wieland et al., 1998). It is also postulated that another Y receptor closely related to Y1 and Y5 may have a role in mediating feeding induced by NPY (Inui, 1999b; Kalra et al., 1999a).

Recently, gene knockout experiments were performed on the Y1 and Y5 receptors (Marsh et al., 1998; Pedrazzani et al., 1998; Kanatani et al., 1999). Both types of mutant mice fed and grew normally, although they developed unexpected late-onset obesity due to hyperphagia in Y5-deficient mice or lowered metabolic rate associated with reduced locomotor activity in Y1-deficient mice. However, the Y1 receptor-deficient mice exhibited a markedly reduced feeding response to fasting, as well as a slightly reduced daily food intake and NPY-stimulated feeding. The Y5 agonist (Peptide YY(PYY)3-36)-induced feeding response, as well as fasting-induced corticosterone response (Pralong et al., 1999) were reduced in this mouse model. Y1-receptor deficiency also led to impaired insulin secretion to glucose and changes in UCP expression such as up-regulation of UCP-1 in BAT and down-regulation of UCP-2 in white adipose tissue (Kushi et al., 1998). The Y5 receptor deficient mice exhibited normal responses to fasting and leptin, but the feeding response to PYY3-36 was markedly reduced and the residual response was eliminated by the simultaneous administration of the Y1 receptor antagonist. Although shifts in the orexigenic network may be involved in the late-onset obesity in Y1 and Y5 receptor mutant mice, these observations indicate that both Y1 and Y5 receptors are involved in the regulation of feeding, but Y1 receptor may play a more prominent role in mediating feeding induced by NPY. The Y5 receptor appears not to be required for the development of the ob/ob obesity syndrome, because mice deficient for both leptin and the Y5 receptor were indistinguishable from littermate ob/ob mice in body tempeature, body weight, food intake, and adiposity (Marsh et al., 1998). The Y2 receptor is a predominant form of NPY receptors in the brain that is thought to be presynaptic and to suppress release of transmitters such as norepinephrine and NPY in the hypothalamus (Broberger et al., 1997; Blomqvist and Herzog, 1997; Michel et al., 1998; Gehlert, 1998). The Y2 receptor subtype may have either a minor or no role in the regulation of feeding (Stanley, 1993). The Y2 receptor agonist has no effect on feeding after i.c.v. administration, although it might reduce nocturnal food intake, especially the carbohydrate component of the meal, after injection into the PVN (Leibowitz and Alexander, 1991; Inui, 1999b). Very recently, the Y2 receptor subtype has been inactivated. The mutant mice developed mild obesity caused by hyperphagia and displayed an attenuated feeding response to leptin but a normal response to fasting (Patrik, 1999). The observations indicate the importance of the Y2 receptor in feeding and body weight regulation and show that it is an essential mediator of the leptin response. The Y2 receptor was also found to be important in the basal control of heart rate (Patrik, 1999). The identification of NPY feeding receptors and their complimentary and/or overlapping functions is obviously of great importance to the molecular basis of the hypothalamic regulation of feeding and body weight. The gene knockout experiment on the respective Y receptors singly or in combination could be particularly useful for addressing this.

2. MCH. A population of neurons in the LH and zona incerta produce a cyclic 19-amino acid peptide, MCH, which was initially discovered in salmon pituitaries as a regulator of skin color change (Vaughan et al., 1989; Nahon, 1994). MCH potentiated nocturnal feeding after central administration, and MCH gene expression was stimulated by fasting and augmented in ob/ob mice (Qu et al., 1996; Rossi et al., 1997). However, MCH-induced feeding was small and of short duration relative to NPY, and chronic administration had no effect on daily (cumulative) food intake and body weight (Rossi et al., 1997). It was even reported that MCH had a potent anorectic effect after administration into the cerebral ventricle or the zona incerta-LH area, which was highly dependent on the light/dark cycle (Presse et al., 1996). However, ablation of the gene led to a thin phenotype associated with reduced food intake and an inappropriately increased metabolic rate, indicating a role of MCH on the energy homeostasis equation (Shimada et al., 1998). MCH-deficient mice showed reduced amounts of leptin and pro-opiomelanocortin (POMC) mRNA in the ARC. Because deletion of a single gene encoding an orexigenic peptide can result in leanness despite the interconnected orexigenic network, MCH may act downstream of leptin and NPY signaling cascade, as might be expected from the immunohistochemical demonstration of projection from NPY neurons in the ARC to MCH neurons in the LH (Broberger et al., 1998a; Elias et al., 1998).

3. Orexin. The orexins are a recently identified class of neuropeptides that also were described as hypocretins (Sakurai et al., 1998; De Lecea et al., 1998). Orexin A and orexin B are 33- and 28- amino acid peptides, respectively, sharing 46% identity. Both peptides are coded by the same gene, and are localized in neurons in the dorsal and lateral hypothalamic areas and perifornical hypothalamus. Administration i.c.v. of orexin A and orexin B stimulated feeding in a dose-related fashion, with orexin A significantly more effective than orexin B, possibly through activation of both orexin A and orexin B receptors (Sakurai et al., 1998). Microinjection studies indicated that orexins act in the limited areas in the hypothalamus such as the LH, perifornical hypothalamus, and PVN, despite broad distribution of orexin fibers in hypothalamic and extrahypothalamic sites (Edwards et al., 1999; Kalra et al., 1999a; Sweet et al., 1999). However, orexin was found to be less effective than NPY in stimulating feeding (Edwards et al., 1999; Kalra et al., 1999a). Orexin may be more likely involved in the control of energy metabolism than of food intake (Lubkin and Stricker-Krongrad, 1998). Although fasting up-regulated orexin gene expression in the hypothalamus, down-regulation of the gene expression was observed in the ob/ob and db/db mice (Yamamoto et al., 1999). However, this may be due, in part, to the accompanying hyperglycemia in the animals because leptin acutely inhibits orexin gene expression. Very recently, orexin-overexpressing or -deficient mice were created, and both types of the mutant mice showed decreased body weight (T. Sakurai and M. Yanagisawa, personal communication). Orexin-overexpressing mice have reduced body weight despite increased food intake due to an inappropriately increased metabolic rate, and orexin-deficient mice have slightly reduced body weight despite markedly reduced food intake due to a decreased metabolic rate. The gene knockout experiment, together with established synaptic contacts between NPY neurons in the ARC and orexin neurons in the LH, suggests that orexin may also function as a downstream effector molecule of NPY signaling (Broberger et al., 1998a; Elias et al., 1998; Horvath et al., 1999).

4. Galanin. Galanin is a 29-amino acid peptide that is distributed in discrete subpopulations in the ARC, DMH, and PVN of the hypothalamus (Leibowitz, 1989, 1995). Galanin stimulates feeding in rats after injection into the cerebral ventricle, as well as into the PVN, LH, VMH, and central nucleus of the amygdala (Kyrkouli et al., 1990a; Schick et al., 1993; Corwin et al., 1993). Like MCH and orexin, galanin-induced feeding is less remarkable than that of NPY, and continuous galanin infusion was ineffective in inducing sustained hyperphagia and obesity (Smith et al., 1994). A close anatomical and functional relationship exists between neurons producing galanin and other orexigenic signals (Horvath et al., 1996). NPY neurons are in direct contact with galanin neurons in the ARC and PVN, and galanin may partly mediate NPY-induced feeding (Kalra et al., 1999a). Involvement of beta -endorphin and norepinephrine (NE) in galanin-induced feeding was also suggested immunohistochemically, as well as from the attenuated feeding response to galanin by pretreatment with naloxone, an opioid receptor antagonist and rauwolscine, an alpha 2-adrenergic receptor antagonist, respectively (Kyrkouli et al., 1990b; Dube et al., 1994). Although the NPY system is closely associated with carbohydrate ingestion and use, channeling nutrients toward the synthesis of fat, galanin may function primarily in controlling fat ingestion and enhancing fat deposition through a reduction in energy expenditure (Leibowitz, 1995). Galanin may be active during the middle period of the natural feeding cycle, and a high-fat diet can enhance galanin production in the PVN, which was closely linked to body adiposity (Akabayashi et al., 1994; Leibowitz et al., 1998). Galanin may also be involved in hyperphagia seen in VMH regioned animals (Pu et al., 1999a; Kalra et al., 1999b). However, it needs to be further clarified how galanin constitutes an important orexigenic signal in natural feeding as well as hyperphagia in genetically obese rodents (Beck et al., 1993; Corwin et al., 1995). It was recently reported that galanin-deficient mice have markedly reduced synthesis and secretion of prolactin in the hypothalamus but they grow normally and have unaltered NPY and GLP-1 content in the hypothalamus (Wynick et al., 1998).

5. Opioid Peptides. The opioid system is composed of three families of biologically active peptides, beta -endorphin, dynorphin, and enkephalins, and their receptors, µ-opioid receptor, kappa -opioid receptor, and delta -opioid receptor, respectively (Levine and Billington, 1989, 1997; Mansour et al., 1995; Kalra et al., 1999a; Kieffer, 1999). Novel µ-selective endomorphins have also been identified in the brain (Zadina et al., 1997). One of the many functions of opioid peptides in the brain is involvement in mediation of the hunger component in the control of food intake (Baile et al., 1986). Opioid peptides may potentiate fat as well as protein ingestion (Leibowitz, 1992). beta -Endorphin, derived from precursor POMC, and dynorphin from prodynorphin, stimulate feeding after central administration (Baile et al., 1986; Morley, 1987; Inui et al., 1991; Lambert et al., 1993a; Kalra, 1997; Kalra et al., 1999a). POMC neurons are localized in the ARC and innervate the PVN, VMH, and other areas of the hypothalamus, where microinjection of beta -endorphin and opiate agonists that bind to the µ-opioid receptors stimulate feeding (Baile et al., 1986; Kalra et al., 1999a). Dynorphin-producing neurons are also found in various regions of the hypothalamus, including the ARC and PVN. The opioid receptor antagonists, especially the µ- and kappa -antagonists, decreased feeding in animals and humans (Morley, 1987; Cole et al., 1995). Antagonists such as naloxone and naltrexone decreased body weight during chronic administration, and were more potent in decreasing food intake and weight gain in obese than in lean rodents (Baile et al., 1986). beta -Endorphin reduced sympathetic nerve activity in BAT, suggesting a potential role for opioids in thermogenesis (Egawa et al., 1993). Although the opioid-evoked feeding is modest, beta -endorphin in particular may represent an important interconnected orexigenic signal (Kalra et al., 1999a). beta -endorphin may situate downstream from NPY, galanin, and GABA because all three molecules stimulate beta -endorphin release in the hypothalamus, and opioid antagonists such as naloxone inhibit feeding stimulated by any one of the three (Morley, 1987; Lambert et al., 1993b, 1994; Dube et al., 1994; Kalra, 1997; Kalra et al., 1999a). However, in contrast to NPY, POMC gene expression appears to be decreased in rats with diabetes or experiencing an energy deficit (Levine and Billington, 1997; Kalra et al., 1999a). Opioid peptides may provide the palatability and rewarding aspects of feeding rather than those for energy needs.

beta -Endorphin-deficient mice were created by introducing a point mutation into the POMC gene that translates to a truncated prohormone lacking the entire COOH-terminal amino acid region encoding beta -endorphin (Rubinstein et al., 1996). The single-copy POMC gene encodes the large precursor, which yields not only the opioid, beta -endorphin, but also the nonopioid peptides, adrenocorticotropic hormone (ACTH) and alpha -melanocyte-stimulating hormone (alpha -MSH). The homozygous mice had normal birth weights and growth and development into adulthood. However, after puberty, the mice attained 10-15% greater body weight than wild-type mice. No significant changes in CRF mRNA in the PVN were reported. Mice that lack µ-receptors have been generated by several laboratories, and the main biological actions of morphine were abolished in the mutant mice, including analgesia, reward and physical dependence (Matthes et al., 1996; Kieffer, 1999). kappa -Opioid deficient mice were reported to have a modified nociceptive threshold in response to visceral pain (Simonin et al., 1998). However, only limited information is available on feeding and body weight regulation in these mutant mice as in the mice lacking beta -endorphin. Normal and larger litter size (but no difference in body weight) are reported in the µ- and kappa -receptor deficient mice, respectively. No apparent compensatory changes in the expression of the opioid peptides or the remaining opioid receptor subtypes have been noted (Kieffer, 1999). Research is warranted on the role of each opioid receptor in palatability (Levine and Billington, 1997), as well as the inactivation of all components of the opioid system.

6. AGRP. AGRP is a recently discovered 132-amino acid peptide that has generated intense interest because a growing body of evidence indicates it has a major role in the regulation of feeding and body weight (Ollmann et al., 1997; Shutter et al., 1997; Wilson et al., 1999). AGRP was identified by virtue of its sequence similarity to the product of the Agouti coat color gene, a paracrine-signaling molecule produced normally in the skin that inhibits the effect of alpha -MSH, a pigment factor, on MC-1 receptor (Bultman et al., 1992; Lu et al., 1994; Leibel et al., 1997). Instead of being expressed only at a certain time during hair growth, Agouti is constitutely expressed throughout the body of yellow Agouti (Ay) mice, and this ectopic Agouti expression gives rise to pleiotropic effects including yellow coat color, obesity, insulin resistance, hyperglycemia, and increased body length. The dominant obesity syndrome was produced by expressing wild-type Agouti cDNA under the control of a ubiquitous promoter such as beta -actin in transgenic mice (Klebig et al., 1995; Ollmann et al., 1997). Because mice homozygous for null mutations of Agouti do not display abnormalities of weight regulation (Wilson et al., 1999) and because ubiquitous overexpression of AGRP in transgenic mice recapitulates the increased body weight gain and body length phenotype, obesity and diabetes caused by ectopic Agouti expression occurring naturally in the yellow mice or by transgenic technology are likely explained by the ability of Agouti to mimic AGRP (Graham et al., 1997; Ollmann et al., 1997).

AGRP is expressed only in the ARC of the hypothalamus in the brain, and all of the AGRP-producing neurons are NPY-positive and project to various hypothalamic (such as PVN and DMH) and extrahypothalamic sites (Broberger et al., 1998a,b; Elias et al., 1998; Hahn et al., 1998; Haskell-Luevano et al., 1999). Like NPY, expression of AGRP is up-regulated in leptin deficiency due to fasting or mutation (Ollmann et al., 1997; Shutter et al., 1997; Hahn et al., 1998; Wilson et al., 1999). It is yet to be determined whether NPY and AGRP are in the same or parallel pathways for appetite regulation. AGRP is a potent and selective antagonist of MC-3 and MC-4 receptors (Yang et al., 1999), the melanocortin receptors implicated in control of energy balance (see Section B. 1.). The inhibition of melanocortin receptors may thus lead to the obese phenotype that is associated with hyperphagia, decreased thermogenesis, and increased caloric efficiency (Fan et al., 1997; Miltenberger et al., 1997).

Humans also have an agouti gene that is normally expressed in adipose tissue, unlike in the mouse. To model human agouti expression, transgenic mice were generated that express murine agouti at high levels in adipose tissue under the regulatory control of the adipocyte lipid-binding protein (aP2) promoter (Mynatt et al., 1997). The aP2-agouti transgenic mice are not obese or diabetic, but combined insulin treatment promotes obesity, an implication for human obesity.

7. Other Orexigenic Signals. GABA, a predominant inhibitory transmitter in the CNS, can stimulate feeding (Morley, 1987; Kalra et al., 1999a). Central administration of the GABAA receptor agonist muscimol either i.c.v. or by microinjection into the PVN and other sites in the brain stimulated feeding, a response blocked by the specific GABAA receptor antagonist, bicuculline (Morley et al., 1981; Tsujii and Bray, 1991; Stratford and Kelley, 1997). GABA is coexpressed in an NPY-producing subpopulation of neurons in the ARC and is reported to have anatomical and functional relationships with other orexigenic signals such as galanin and beta -endorphin (Blasquez et al., 1994; Horvath et al., 1997). These results suggest that GABA is a component in the interconnected orexigenic network (Pu et al., 1999b; Kalra et al., 1999a). Mice devoid of GABAA receptor beta 3 subunit that is an essential component of the receptor, developed epilepsy, hypersensitive behavior, cleft palate, and a high incidence of neonatal motility (Homanics et al., 1997). The mutant mice that survived were runts until weaning but achieved normal body size by adulthood. GABA is synthesized by two isoforms of glutamic acid decarboxylase, GAD-65 and GAD-67. GAD-67 deficient mice exhibited a perinatal lethal phenotype, although GAD-65 deficient mice exhibited increased anxiety-like behaviors with normal glucose tolerance and body weight (Asada et al., 1997; Condie et al., 1997; Kash et al., 1997, 1999). Another genetic approach is needed to examine the role of GABA in appetite regulation independent from its effect on normal development

It has been shown that activation of alpha 2-adrenergic receptors in the PVN of the hypothalamus induces feeding whereas the perifornical region contains beta -adrenoceptors that inhibit feeding (Leibowitz, 1989, 1992). The alpha 2-adrenergic system is a selective system for carbohydrate intake and is particularly active at the onset of the animals' active cycle. Infusions of NE into the VMH, but not the PVN, produce a sustained hyperphagia, reduced sympathetic activity, increased insulin, and obesity over a 20-day period (Shimazu et al., 1986). The alpha 2-adrenergic receptors include three distinct subtypes alpha 2A, alpha 2B, and alpha 2C, among which alpha 2A and alpha 2C subtypes are expressed in the CNS (MacDonald et al., 1997). Mice deficient in each of the alpha 2-adrenergic receptor subtypes were generated, which were viable and appeared grossly normal, although detailed analysis on feeding and body weight regulation has not been reported (Link et al., 1996; MacMillan et al., 1996; MacDonald et al., 1997).

Growth hormone-releasing hormone (GHRH) has been shown to increase feeding after central administration (Vaccarino et al., 1985; Morley, 1987; Inui et al., 1991). This action was shared by the new class of penta- or hexapeptides of growth hormone-releasing peptides (Okada et al., 1996). The feeding response to GHRH may follow as an inverted U-shaped dose-response curve with higher doses inhibiting feeding. Mouse-metallothionein-human GHRH transgenic mice were developed previously (Hammer et al., 1985). Overproduction of GHRH was observed in several tissues, including the pituitary, pancreas, and arcuate nucleus of the hypothalamus. The mice exhibited a dramatic increase in somatotrope function and an accelerated rate of growth, providing an animal model of acromegaly.

VGF is a secreted polypeptide of unknown function that is synthesized by neurons and is abundant in the hypothalamus (Levi et al., 1985; Snyder and Salton, 1998). Mice lacking VGF displayed dramatically decreased body weight and body fat, the major defect of which is due to excess energy expenditure and not to decreased food intake (Hahm et al., 1999). The mice had increased oxygen consumption at rest and increased locomotor activity despite normal sympathetic tone and somewhat reduced levels of thyroid hormone, suggesting that VGF may play a novel role in energy expenditure regulation.

B. Hypothalamic Inhibitors of Food Intake

1. MC. The MC system involves peptides that are processed from the polypeptide precursor POMC, which is produced by neurons in the ARC of the hypothalamus and the nucleus of the tractus solitarius (Adan and Gispen, 1997). Several of the peptide products of the POMC gene such as alpha -MSH have been implicated in the regulation of feeding behaviors (Table 3). alpha -MSH and the MC mimetics inhibit feeding in rats, mice, and agouti obese mice, an effect that is counteracted by MC antagonist (Fan et al., 1997). To date, five MC receptors have been characterized, of which MC-3 and MC-4 receptors are expressed in the hypothalamus of the brain (Adan and Gispen, 1997). A highly selective MC-4 receptor antagonist augments feeding in satiated animals and long-term blockade increases food intake and body weight gain leading to obesity (Kask et al., 1998a,b; Skuladottir et al., 1999). Altered energy balance causes selective changes in MC-4, but not MC-3, receptor binding in hypothalamic regions such as VMH, DMH, and ARC (Harrold et al., 1999). Some of the POMC neurons express functional long form of the leptin receptor, and both POMC mRNA levels and plasma leptin levels decrease after fasting and in the ob/ob mouse (Schwartz et al., 1997; Cheung et al., 1997; Mizuno et al., 1998). Up-regulation of MC-4 receptor binding was observed in such leptin-deficient food-restricted rats as well as leptin-resistant fa/fa Zucker rats, whereas down-regulation of the receptor binding was observed in diet-induced obese rats, probably reflecting changes in the release of endogenous ligand, alpha -MSH (Harrold et al., 1999).


                              
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  		TABLE 3
Hypothalamic inhibitors of food intake and their effect on body weight regulation

Targeted disruption of the MC-4 receptor produced many of the hallmark features of the yellow obese syndrome without producing yellow fur (Huszar et al., 1997). In this model, the magnitude of hyperphagia and weight gain were significantly higher than that observed in agouti transgenic mice or the yellow mutant mice of the same genetic background, suggesting complete versus partial antagonism of the receptor (Miltenberger et al., 1997). MC-4 receptor-deficient mice did not respond to the anorectic actions of the cyclic MC agonist MT, a potent MC-3 and MC-4 receptor agonist, indicating that alpha -MSH inhibits feeding primarily by activating MC-4 receptor (Marsh et al., 1999). Obese MC-4 receptor-deficient mice were resistant to both peripherally and centrally administered leptin, although young nonobese mice showed a blunted response to feeding-inhibitory actions of leptin. It was also suggested from double-mutant studies that obesity that results from leptin deficiency is not caused by altered MC signaling because Ay was observed to have an additive effect on weight gain in adrenalectomized ob/ob animals (Boston et al., 1997). These data demonstrate that melanocortin signaling transduced by MC-4 receptor is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. The MC-4 receptor-deficient mice responded normally to the anorexigenic signals such as ciliary neurotrophic factor and urocortin, as well as to the orexigenic signals such as NPY and PYY, indicating that these neuromodulators may act independently or downstream of MC-4 receptor signaling (Marsh et al., 1999). However, enhanced response to CRF was observed in these mice, which may suggest compensatory changes in the absence of the MC-4 receptor signaling. Taken together, these results indicate the importance of MC signaling in the energy homeostasis equation, the regulation of which could be mediated by changes in agonist and/or antagonist levels, that is, a tonic restraint on feeding by MC through MC-4 receptor and removal of this restraint by AGRP leading to hyperphagia and obesity. NPY may be a downstream target of melanocortin signaling because aberrant expression of NPY in the DMH of the hypothalamus is observed in the models of the agouti obesity syndrome (Kesterson et al., 1997) and MC-4 receptor antagonist-induced feeding appears to be mediated by NPY Y1 receptor (Kask et al.,1998b). This may be in keeping with the notion that the drive for food is chemically coded through the release of orexigenic signals (Morley, 1987; Kalra et al., 1999a; Inui, 1999a). The functional interrelationships between orexigenic beta -endorphin and anorexigenic alpha -MSH needs to be clarified.

Very recently, mahogany protein was identified. It is expressed in various regions of the body, including the VMH of the hypothalamus (Gunn et al., 1999; Nagle et al., 1999). The mahogany protein is thought to be involved in the agouti pathway to compensate for agouti overexpression. This protein may act at or upstream of melanocortin receptors and may be involved in suppression of diet-induced obesity, the most common form of human obesity (Nagle et al., 1999).

2. CRF and Urocortin. CRF is a 41-amino acid mammalian neurohormone that is best known as the major physiological regulator of pituitary ACTH secretion and, in addition, stimulates complimentary stress-related endocrine, autonomic, and behavioral responses (Vale et al., 1981; Owens and Nemeroff, 1991; Turnbull and Rivier, 1997). There is considerable evidence indicating that CRF is an endogenous inhibitor of food intake. Injection of CRF into the brain, specifically into the PVN of the hypothalamus, a major site of CRF expression, decreases spontaneous feeding or fasting-induced feeding (Morley, 1987; Schwartz et al., 1995; Levine and Billington, 1997; Heinrichs et al., 1998). CRF decreases feeding stimulated by GABA agonist (muscimol), norepinephrine, dynorphin, and NPY (Levine et al., 1983). Chronic administration of CRF causes sustained anorexia and progressive body weight loss (Schwartz et al., 1995). Central pharmacological blockade with CRF antagonists or antisense oligonucleotide, immunoneutralization, or immunotoxin targeting of CRF in the hypothalamus enhances basal and NPY-stimulating feeding, suggesting that CRF may tonically restrain the actions of orexigenic signals (Heinrichs et al., 1991; Menzaghi et al., 1993; Hulsey et al., 1995). Both CRF and NPY may exert local site-specific effects on feeding behavior within the PVN relative to the extrahypothalamic site that constitutes a sensitive substrate for nonappetite behavioral actions of these peptides (Heinrichs et al., 1998). Central CRF blockade also inhibits anorexia evoked by stress such as physical restraint or by interleukin (IL)-1, suggesting that CRF may be directly related to stress-related changes in feeding (Krahn et al., 1986; Uehara et al., 1989). CRF mediates its actions through interaction with two distinct receptor subtypes, CRF-1 and CRF-2, which have been cloned and characterized (Chalmers et al., 1996; Turnbull and Rivier, 1997). CRF-2 receptor is primarily involved in the feeding-suppressive and thermogenic response to CRF and CRF-related peptides (Martinez et al., 1998; Smagin et al., 1998). Urocortin is a 40-amino acid peptide that is a potent activator of CRF-2 rather than CRF-1 receptors (Vaughan et al., 1995; Spina et al., 1996). Urocortin reduces food intake and promotes weight loss at doses that do not activate the stress response (Spina et al., 1996; Asakawa et al., 1999). This makes urocortin all the more likely to be a regulator of energy homeostasis, although its role in appetite-regulating pathways needs to be determined (Inui, 1999a; Kalra et al., 1999a).

Previously, a transgenic mouse model of CRF overexpression was developed that exhibited an increase in anxiogenic behavior as well as a change in female sexual receptivity (Stenzel-Poore et al., 1992, 1994). The mice had elevated levels of activity in the hypothalamic-pituitary-adrenal axis and became obese, resembling Cushing's syndrome in humans. CRF-deficient mice were also developed which revealed a fatal glucocorticoid requirement for lung maturation and a deficient, sexually dimorphic adrenal response to stress and impaired adrenal rhythmicity (Muglia et al., 1995, 1997). However, they did not exhibit increased food intake or body weight under basal conditions, nor did they display smaller decreases in feeding after adrenalectomy known to up-regulate CRF production and release (Jacobson, 1999). Corticosterone replacement completely blocked the adrenalectomy-induced decrease in feeding and body weight in all mice and frequently stimulated food intake in CRF-deficient mice (Jacobson, 1999). This was achieved at plasma corticosterone levels above the circadian peak, which probably occupied type 2 glucocorticoid receptors. There was no significant difference in the plasma levels of leptin and insulin between the control and the knockout mice, although the knockout mice tended to have lower insulin levels (Muglia et al., 1997). These results indicate that factors in addition to CRF are involved in controlling basal and glucocorticoid-associated effects on feeding. NPY could potentially mediate the effects of glucocorticoid on appetite in this model (Zakrzewska et al., 1999). CRF-1 receptor-deficient mice have been generated and their phenotype confirms the obligatory role of this receptor in stress-related reactions as shown by severely blunted adrenal response to stress and decreased anxiety levels (Smith et al., 1998; Timpl et al., 1998; Turnbull et al., 1999). However, resting ACTH secretion is maintained largely by arginine vasopressin-dependent mechanisms, and IL-6 may be involved in the pituitary-adrenal axis activation of inflammation in these mice, suggesting considerable plasticity in the mechanisms of important neuroendocrine response (Turnbull et al., 1999). A compensatory increase in the expression of CRF was detected within the PVN of the hypothalamus, but no detectable alteration was observed in the expression of CRF-2 receptor, CRF-binding protein, arginine vasopressin, or mineralocorticoid and glucocorticoid receptors (Smith et al., 1998; Timpl et al., 1998).

Although both CRF- and CRF-1 receptor-deficient mice appeared to have no gross disturbances in body weight regulation, the overexpression of CRF-binding protein confirmed the previous implication of CRF and urocortin in the regulation of feeding and body weight. The transgenic mice overexpressing CRF-binding protein in the pituitary under the control of the pituitary glycoprotein hormone alpha -subunit promoter and those overexpressing broadly in the body including the liver and brain under the control of mouse metallothionein-1 promoter, produced an altered circadian pattern of food intake and a sexually dimorphic body weight gain, respectively (Burrows et al., 1998; Lovejoy et al., 1998). These results are consistent with a previous report that increased availability of CRF/urocortin in the hypothalamus by the chronic administration of CRF 6-33, a high-affinity CRF-binding protein inhibitor, significantly decreased body weight in Zucker obese rats that normally have reduced CRF content in the hypothalamus, primarily by increasing sympathetic tone and energy expenditure (Heinrichs et al., 1996).

Almost all obesities depend on the presence of adrenal glucocorticoids and an overactivity of type II corticosteroid receptors (York and Hansen, 1998). All types of hyperphagia and obesity syndromes are reversed or prevented by adrenalectomy and can be readily restored by steroid replacement (Tempel and Leibowitz, 1994). Chronic excessive stimulation of type as well as type receptor in obesity results in increased fat storage and excessive food intake, with a strong preference for fats (Tempel and Leibowitz, 1994). The mice in which type II corticosteroid receptor antisense RNA construct was expressed primarily in neural tissue by using a human neurofilament gene promoter, paradoxically developed obesity despite clear evidence for reduced glucocorticoid receptor activity in the hypothalamus, cerebral cortex, and liver (Pépin et al., 1992). The type of obesity produced was associated with reduced food intake and oxygen consumption during the dark phase (thus an increased energetic efficiency; Richard et al., 1993).

3. GLP-1. GLP-1, is produced by differential post-translational processing of the proglucagon gene in the CNS and gut (Drucker, 1998). In the CNS, GLP-1 is predominantly synthesized in the brainstem, which projects to the hypothalamic sites such as the PVN and DMH (Shimizu et al., 1987; Kreymann et al., 1989; Larsen et al., 1997). These hypothalamic sites richly contain GLP-1 binding sites and GLP-1 receptor mRNA (Shughrue et al., 1996; Turton et al., 1996). It was recently reported that hypothalamic GLP-1 may be a physiological satiety factor (Turton et al., 1996). Administration i.c.v. of GLP-1 reduced food intake in fasted rats and hyperphagia in the obese Zucker rats (Tang-Christensen et al., 1996; Donahey et al., 1998). Repeated administration of GLP-1 reduced food intake and body weight without an apparent tachyphylaxis in response (Meeran et al., 1999). The GLP-1-receptor antagonist, exendin 9-39, stimulated feeding in satiated animals, and daily administration of exendin 9-39 augmented food intake and body weight gain. The anorectic effects of GLP-1 may be mediated through NPY signaling because GLP-1 inhibited and exendin 9-39 augmented NPY-induced feeding, respectively (Turton et al., 1996; Kalra et al., 1999a). The GLP-1 receptor antagonist also blocked the leptin-induced inhibition of food intake and body weight, indicating that the GLP-1 pathway may be one of the targets for the anorectic effects of leptin (Goldstone et al., 1997).

However, targeted disruption of the GLP-1 receptor gene in mice resulted in a phenotype with normal feeding and body weight, despite fasting hyperglycemia, abnormal glycemic excursions after glucose challenge, and reduced levels of glucose-stimulated insulin secretion (Scrocchi et al., 1996). Obesity fails to develop in these mice with aging or high-fat feeding (Scrocchi and Drucker, 1998). These results support the previous implications of an essential role of GLP-1 as an incretin that potentiates the release of insulin through glucose-dependent mechanisms, but do not support those in body weight regulation.

4. Bombesin. Bombesin is a tetradecapeptide originally purified from the skin of the European frog Bombina bombina (Taché and Brown, 1982; Spindel, 1986). The two known mammalian bombesin-like peptides are neuromedin B and gastrin-releasing peptide (GRP). In the CNS, these neuropeptides are thought to play a role in the regulation of feeding behavior, metabolism, and thermoregulation. Central administration of bombesin and bombesin-related peptides elicit suppression of food intake in a variety of species, although bombesin is more potent than either mammalian peptide (McLaughlin and Baile, 1981