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Vol. 52, Issue 1, 35-62, March 2000
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
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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.
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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.
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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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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).
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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
2-receptor) and 
-aminobutyric acid (GABA),
and feeding-inhibitory serotonin and dopamine (Table
1). Neuropeptides are important components of the feeding-regulatory systems.
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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).
TABLE 2
Hypothalamic stimulators of food intake and their effect on body weight
regulation
; 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 
-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
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, -endorphin, dynorphin, and
enkephalins, and their receptors, µ-opioid receptor, 
-opioid
receptor, and 
-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).
-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 -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 -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). -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,
-endorphin in particular may represent an important interconnected
orexigenic signal (Kalra et al., 1999a). -endorphin may situate
downstream from NPY, galanin, and GABA because all three molecules
stimulate -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.
-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
-endorphin (Rubinstein et al., 1996). The single-copy POMC gene
encodes the large precursor, which yields not only the opioid,
-endorphin, but also the nonopioid peptides, adrenocorticotropic hormone (ACTH) and -melanocyte-stimulating hormone (-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).
-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 -endorphin.
Normal and larger litter size (but no difference in body weight) are
reported in the µ- and -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 -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 -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).
,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
-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 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
2-adrenergic receptors in the PVN of the
hypothalamus induces feeding whereas the perifornical region contains
-adrenoceptors that inhibit feeding (Leibowitz, 1989, 1992). The
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 2-adrenergic receptors include
three distinct subtypes 2A,
2B, and 2C, among
which 2A and 2C
subtypes are expressed in the CNS (MacDonald et al., 1997). Mice
deficient in each of the 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 -MSH have been implicated in the regulation of feeding
behaviors (Table 3). -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, -MSH (Harrold et al., 1999).
TABLE 3
Hypothalamic inhibitors of food intake and their effect on body weight
regulation
). 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 -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 -endorphin and
anorexigenic -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).
,
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 -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).
). 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; Gibbs, 1985; Taylor and Garcia, 1985; Merali et al., 1993). Central administration of bombesin-receptor antagonists blocked the
satiety effect of bombesin and also enhanced food intake in satiated
rats (Flynn, 1993; Merali et al., 1993). Attempts to identify the
relevant neural circuits, using brain microinjections and lesioning,
have revealed that certain hypothalamic and hindbrain structures, such
as the PVN and the nucleus tractus solitarius (NTS), are particularly
sensitive to the feeding suppressant effects of bombesin (Kyrkouli et
al., 1987; Flynn, 1992). Meal-related fluctuations in the release of
bombesin (GRP)-like peptides were reported in the PVN of rats
(Plamondon and Merali, 1994). Bombesin might mediate its
feeding-suppressant effects through an interaction with CRF because CRF
antagonists attenuated the satiety effects of bombesin administered
centrally or peripherally (Plamondon and Merali, 1997). Animal studies
also suggested that at least some of the effects of bombesin-like
peptides on food intake are mediated through endogenous cholecystokinin
(CCK) release, although in humans, GRP can act independently to reduce
food intake (Gutzwiller et al., 1994). Because the effects of
systemically administered bombesin are abolished by total neural
disconnection of the gut from the brain and are attenuated by central
pretreatment of bombesin antiserum or antagonists, the satiety effects
of bombesin may be neurally communicated to the brain where bombesin
receptors participate (Plamondon and Merali, 1997).
; Wada et al., 1991;
Fathi et al., 1993). The GRP receptor binds GRP with higher affinity
than neuromedin B, and the neuromedin B receptor binds neuromedin B
with higher affinity than GRP (von Schrenck et al., 1990). In contrast,
bombesin receptor subtype-3 binds neither GRP nor neuromedin B with an
affinity higher than in the micromolar range, suggesting that the
natural ligand for this receptor remains to be determined. The GRP-and
neuromedin B-receptors are widely expressed in the CNS and
gastrointestinal tract, whereas bombesin receptor subtype-3 shows
limited expression in the CNS, notably hypothalamic nuclei such as the
PVN, ARC, and DMH (Wada et al., 1991, 1992; Fathi et al., 1993). The
GRP receptor is also expressed in the hypothalamus such as in the PVN,
and GRP is more potent than neuromedin B in inhibiting feeding. Although neuromedin B receptor is not highly expressed in these regions, it may participate in the control of food intake in the caudal
hindbrain (Ladenheim et al., 1997). It was thus suggested that both GRP
and neuromedin B receptors mediate bombesin-induced satiety response,
although the exact contribution of each of the receptor subtypes is
difficult to determine without highly specific and selective
agonists/antagonists available for functional studies.
Recently, as a complementary approach, mice were created that have a
disrupted gene for each of these bombesin receptor subtypes. GRP
receptor-deficient mice grew normally and had normal body weight
despite the lack of suppressive effect of bombesin on glucose intake
(Hampton et al., 1998). A compensatory mechanism was suggested in these
mice which had an increased sensitivity to CCK, another peptide
involved in regulating individual meal size (see Section. B. 5.). It was indicated that bombesin-induced appetite suppression is mediated through the GRP receptor. The mice also showed
hyperactivity during the dark period and abnormal social behavior (Wada
et al., 1997). Neuromedin B receptor-deficient mice were responsive to the feeding-suppressive effect of GRP and were not obese, and had
neither increased locomotor activity nor pronounced social responses
(Ohki-Hamazaki et al., 1999), suggesting that the function of
neuromedin B receptor and GRF receptor are distinct despite the
colocalization of these receptors in brain areas such as isocortex, hippocampus, amygdala, hypothalamus, and brainstem (Wada et al., 1997).
Compensation for neuromedin B receptor deficiency by overexpression of
GRP receptor or bombesin receptor subtype-3 was not observed. Bombesin
receptor subtype-3-deficient mice developed obesity, which is
characterized by a reduced metabolic rate (oxygen consumption), hyperphagia, and increased feed efficiency without defective
temperature regulation and physical activity (Ohki-Hamazaki et al.,
1997). Hypertension, increased plasma levels of leptin, and impaired glucose and lipid metabolism were associated with the obesity of these
animals. Because bombesin shows only low affinity for bombesin receptor
subtype-3, it would be warranted to identify the natural ligand for
this receptor and its role in the appetite regulation.
5. CCK.
CCK is a peptide hormone secreted from gut endocrine
cells and a neurotransmitter in the brain, which can reduce food intake (McLaughlin and Baile, 1981; Baile et al., 1986; Morley, 1987; Gibbs
and Smith, 1992: Crawley and Corwin, 1994; Smith and Gibbs, 1994;
Schick et al., 1994). CCK exists in multiple molecular forms in the
circulation and the brain, ranging from 58 to 39, 33, 22, 8, and 4 amino acids in length, although neither CCK-33 nor CCK-39 has been
isolated from brain. The COOH-terminal octapeptide CCK-8 is well
conserved between species and is the smallest form that retains the
full range of biological activities (Inui et al., 1989; Hirosue et al.,
1993). CCK stimulates receptors in the pyrolic sphincter and the
afferent limb of the abdominal vagus nerve, which in turn relays a
message to the brainstem and the PVN of the hypothalamus where CCK mRNA
is also present (Morley, 1987; Ingram et al., 1989; Gibbs and Smith,
1992; Smith and Gibbs, 1994). CCK is also released from hypothalamic
sites after intragastric nutrients and has a definite neuronal
influence on food intake in the CNS (Baile and Della-Fera, 1985; Schick
et al., 1986, 1987). CCK reduces and CCK antagonists increase feeding
after administration into the cerebral ventricle (Baile et al., 1986;
Inui et al., 1987, 1989; Hirosue et al., 1993). Mapping of
CCK-sensitive brain sites in the rat revealed that active sites lie not
only in the LH, but also in the medial pons and lateral medulla in the
vicinity of the NTS, where vagal afferent fibers terminate (Schick et
al., 1994).
; Lotti and Chang, 1989;
Crawley and Corwin, 1994). Although CCK-B receptor is a major receptor
in the brain, CCK-A receptor mRNA is also present in localized areas of
the brain including the PVN of the hypothalamus (Honda et al., 1993). A
large body of evidence indicates that the CCK-A receptor-specific
antagonist L-364,718 (MK-329, devazepide) blocks the inhibition of food
intake by exogenously administered CCK-8, and administration of
L-364,718 to mice, rats, pigs, or monkeys increases food intake (Moran
et al., 1992, 1993; Hirosue et al., 1993; Crawley and Corwin, 1994).
Administration of the CCK-A antagonist into the PVN decreased the
satiating effect of peripheral CCK-8, suggesting that CCK released from
central neurons could participate in the processing of the information produced by peripheral CCK-8 (Smith and Gibbs, 1994). However, the
CCK-B receptor antagonist, L-365,260, also stimulated food intake and
delayed the postprandial satiety sequence in partially satiated rats
(Dourish et al., 1989). L-365,260 increased food intake after injection
into the LH of rats, with limited duration of action (Schick et al.,
1994). These results indicate that under certain conditions the CCK-B
receptor may have a role in regulating feeding. The inability of CCK to
reduce food intake over an extended period has been interpreted to
indicate that it is a meal-related signal and does not have a role in
the regulation of body adiposity.
Recently, it was demonstrated that exogenous CCK-8 rapidly mobilizes
gastric leptin with a concomitant increase in plasma leptin
concentrations (Bado et al., 1998). Evidence was provided for a
synergistic interaction between leptin and CCK leading to short-term
reduction in food intake (Barrachina et al., 1997). The food-reducing
effect of leptin injected with CCK was specific and not associated with
alterations in gastric emptying or locomotor behavior. The leptin-CCK
interaction was blocked by systemic capsaicin and L-364,718 but not by
L-365,260, suggesting an involvement of CCK-A receptors in the vagal
afferents. It was further suggested that CCK can contribute to the
long-term control of feeding and body weight when central leptin levels
are elevated (Matson and Ritter, 1999). The availability of mice
lacking either CCK-A or CCK-B/gastrin receptors provided an alternative
approach to further address these issues. It was clearly shown that
CCK-A, but not CCK-B, receptors are involved in mediating the satiety
effect of exogenous CCK (Kopin et al., 1999). Administration i.p. of CCK-8 failed to decrease food intake in mice lacking CCK-A receptors, whereas CCK-8 decreased food intake by up to 90% in both wild-type and
CCK-B receptor-deficient mice. However, CCK-A receptor-deficient mice
showed normal day- and night-food intake and body weight that is
comparable to the corresponding age- and sex-matched wild-type controls
(Kopin et al., 1999). The similarity in weights persisted throughout
the rapid growth phase and extended well into adulthood. CCK-B receptor
deficient mice also showed normal body weight but have a remarkable
atrophy of the gastric mucosa demonstrating its importance in
maintaining the normal cellular composition and function of the gastric
mucosa (Nagata et al., 1996; Langhans et al., 1997). The loss of a
functional CCK-B receptor did not affect the expression of CCK-A
receptor mRNA in brain areas such as the cerebral cortex (Nagata et
al., 1996). The role of CCK-A receptor in the control of satiety and
body weight should be further examined in knockout mice because
congenital mutation in the CCK-A receptor gene may be associated with
obesity in humans and rats (Miller et al., 1995; Moran et al., 1998).
6. Serotonin.
Serotonin (5-HT) originates from the midbrain
dorsal raphe nucleus and projects to the hypothalamus, including the
PVN and the VMH. It is an important modulator of many developmental,
behavioral, and physiological processes, including sleep, appetite,
temperature regulation, pain perception, and motor activity (Vanhoutte
et al., 1993). Intense interest has been focused on understanding the
role of serotonin in clinical conditions such as depression, alcoholism, and drug abuse, as well as obsessive-compulsive disorder and anxiety disorders (Leibowitz, 1992; Wurtman and Wurtman, 1995; Ramamoorthy and Blakely, 1999). Serotonin and its agonists inhibit food
intake when administered either peripherally or centrally in freely
feeding or food-deprived animals (Leibowitz, 1989; Sahu and Kalra,
1993; Blundell et al., 1995; Simansky, 1996; Curzon et al., 1997;
Heinrichs et al., 1998). Stimulants of this monoamine reduce weight
gain and increase energy expenditure in both animals and humans.
Cannula mapping and lesion studies indicate that this effect is
anatomically localized to the medial hypothalamus, specifically the
PVN, VMH, and suprachiasmatic nuclei (Leibowitz, 1989). The serotonergic system may have a selective effect on macronutrient intake; serotonin in the PVN dose-dependently suppresses carbohydrate intake (Leibowitz, 1989; Thibault and Booth, 1999). However, several studies call into question the idea. Serotonergic drugs such as dexfenfluramine and fluoxetine can inhibit fat intake, indicating that
a selective effect on carbohydrate intake may not be the most prominent
feeding response to serotonergic drugs (Blundell et al., 1995; Heisler
et al., 1997; Smith et al., 1998). Based on the studies in which
relatively selective agonists and/or antagonists for serotonin receptor
subtypes were used, it was suggested that serotonin-induced satiety was
mediated by postsynaptic 5-HT1B receptor sites
(Leibowitz, 1989; Samanin and Garattini, 1996). Functional
interrelationships between serotonin and CRF, CCK, or NPY were also
suggested, which were thought to be through
5-HT2A, 5-HT2C, or
5-HT2A(2C) receptors (Samanin and Garattini,
1996; Curzon et al., 1997). However, serotonin interacts with a large number of receptors, probably up to 20 different ones (Hoyer et al.,
1994; Brunner and Hen, 1997). Thus, it is difficult to assign specific
functions to specific receptors by this pharmacological approach alone.
There are actually no selective ligands for a number of receptor
subtypes such as the 5-HT1D,
5-HT1E, 5-HT1F, 5-HT5, 5-HT6, and
5-HT7 that have been identified by molecular biological techniques (Brunner and Hen, 1997). Some of these receptors may possess a high affinity for ligands that were thought to be specific to other receptors.
; Nonogaki et al., 1998). Initial
experiments showed that mutant mice had elevated body weight (and
epilepsy) and were resistant to the anorectic effect of
m-chlorophenylpiperazine, an agonist at
5-HT1B and 5-HT2C
receptors, indicating that 5-HT2C receptors
contribute substantially to the serotonergic regulation of body weight.
5-HT2C receptors are known to be expressed in the
PVN, LH, ARC, and DMH (Wright et al., 1995; Nonogaki et al., 1998). The
obesity syndrome in the mutant mice was distinguished by its
developmental pattern of physiological changes, with hyperphagia preceding the development of obesity and reduced sensitivity to leptin
and insulin (Nonogaki et al., 1998). The mice thus differ from other
rodent models of obesity such as ob/ob and db/db
mice, which are characterized primarily by metabolic dysregulation. The
5-HT2C receptor-deficient mice were more
sensitive to a high-fat diet that induced obesity, and at an older age
they developed insulin resistance and hyperglycemia. These results
indicate a dissociation of serotonin and leptin in the regulation of
feeding and indicate that a perturbation of brain serotonin systems can predispose to type 2 diabetes. In contrast, mice lacking the
5-HT1B receptor developed normally and had normal
food intake although they exhibited motor impulsivity, enhanced
aggressive behavior, elevated alcohol consumption, and increased
sensitivity to cocaine (Saudou et al., 1994; Crabbe et al., 1996;
Brunner and Hen, 1997). Serotonin transporter plays a key role in the
regulation of serotonergic neurotransmission by inactivating serotonin
after its release into the synaptic cleft. It is the primary target for
widely used serotonin reuptake-inhibiting antidepressant drugs
(Ramamoorthy and Blakely, 1999). Mice lacking serotonin transporter
exhibited normal body weight although adaptive changes in serotonin
synthesis, turnover, or metabolism were evidenced by the substantial
depletion of neuronal serotonin and 5-hydroxyindoleacetic acid in the
brain regions (Bengel et al., 1998).
7. Cytokine.
Cytokines are protein molecules possessing a
spectrum of biological activities that are produced by the lymphocyte
and/or the monocyte/macrophage during various disease states such as infection and cancer (Plata-Salaman, 1991; Sternberg, 1997). Several proinflammatory cytokines, most notably tumor necrosis factor-alpha (TNF-), interleukin (IL)-1, and IL-6 induce anorexia and body weight
loss (Plata-Salaman, 1996; Inui, 1999c). A large body of evidence
suggests that the anorexia is mediated by central neural mechanisms and
that the primary targets of action reside in the hypothalamus, the area
with highest densities of receptors for most cytokines (Hopkins and
Rothwell, 1995). TNF- and IL-1 produce anorexia in rats at doses
estimated to yield pathophysiological concentrations in the
cerebrospinal fluid of animal models or patients with wasting disorders
(Sonti et al., 1996a). These cytokines are released into the
circulation and are transported to the CNS through the BBB and the
circumventricular organs, leaky areas in the BBB (Licinio and Wong,
1997). Peripheral cytokines can exert their effects on the brain via
neural pathways or second messengers such as nitric oxide and
prostanoids in the brain vasculature. Cytokines are also generated
within the brain, including the hypothalamus, in response to microbial
and inflammatory products or peripherally released cytokines. Cytokines
such as IL-1 antagonize the NPY feeding system directly or indirectly
via stimulation of leptin secretion (Grunfeld et al., 1996; Sonti et
al., 1996b; Gayle et al., 1997; Sarraf et al., 1997). Conversely,
leptin can increase hypothalamic levels of immunoreactive IL-1
(Luheshi et al., 1999). The leptin receptor is most closely related to
glycoprotein 130, which is a common signal transducer among receptors
for members of the IL-6 subfamily (Friedman, 1998; Bessesen and
Faggioni, 1998). Therefore, excessive leptin or leptin-like signaling
resulting from the elaboration of inflammatory cytokines in the
overlapping and probably redundant cytokine network may lead to
anorexia and unopposed weight loss in wasting illness (Inui, 1999c,d).
due to targeted disruption of the gene does not
prevent the development of obesity induced by a high fat or high calory
diet, or gold thioglucose injection except for a small decrease in
adiposity (Uysal et al., 1997; Ventre et al., 1997). Gold thioglucose
causes chemical ablation of the VMH. However, a significant improvement
in insulin sensitivity was observed in mice lacking TNF- or type 1 (p55) and type 2 (p75) receptors for TNF-, confirming previous
implications of TNF- as a mediator of insulin resistance in obesity.
Normal circadian variations in food intake and body weight gain were
reported in the TNF double receptor knockout mice (Leon et al., 1997).
IL-1 plays a central role in local inflammatory and systemic responses after infection. The IL-1 family of proteins consists of at least three
different gene products: IL-1, IL-1, and an IL-1 receptor antagonist (Plata-Salaman, 1991, 1996). Mice lacking IL-1 or IL-1
receptor type were normal in size, weight, or gross appearance of
organs (Zheng et al., 1995; Labow et al., 1997). However, the IL-1
mutant mice resisted anorexia and fever development when challenged
with turpentine, which causes localized inflammation and tissue injury
(Zheng et al., 1995). IL-1 receptor antagonist overexpressing mice were
created under the control of the glial fibrillary acidic protein
(CNS-specific expression) or under the control of the endogenous
promoter (general overexpression). Neither of these mutant mice showed
any developmental or weight abnormalities (Hirsch et al., 1996;
Lundkvist et al., 1999). However, IL-1 receptor antagonist null mice
had reduced body weight, suggesting a physiological role of the IL-1
receptor antagonist in normal energy homeostasis (Hirsch et al., 1996).
The involvement of IL-6 in the generation of fever and anorexia has
also been actively studied (Plata-Salaman, 1991, 1996). Mice lacking
IL-6 consumed the same quantity of food and water and had similar body
weight as control mice under normal conditions (Kopf et al., 1994;
Kozak et al., 1997). However, they were also resistant to fever,
anorexia, and body weight loss induced by turpentine, but not by
influenza and lipopolysaccharide, which both induce TNF- (Kozak et
al., 1997). IL-6-deficient mice also showed a higher degree of
aggressive behavior, whereas IL-6-overexpressing mice created by the
neuron-specific enolase promoter showed a tendency to be more involved
in affiliative-type social interactions (Alleva et al., 1998).
8. Other Anorexigenic Signals.
Thyrotropin-releasing hormone
(TRH) was originally isolated from the hypothalamus. It regulates
thyrotropin secretion from the anterior pituitary and possibly feeding
behavior (Morley, 1987). TRH reduced both feeding and drinking after
i.c.v. administration in rats (Morley, 1987; Gibbs and Smith,
1992). The TRH metabolite, cyclohistidyl proline diketopiperazine
(Cyclo-His-Pro), produced a more potent and long-lasting inhibition of
food intake leading to weight loss. Recent studies suggest that NPY may
inhibit the activity of PVN TRH neurons, which may be a necessary
component to evoke the feeding response (Diano et al., 1998). TRH is
expressed in the PVN at reduced levels during fasting and is restored
to normal by leptin administration (Légradi et al., 1997).
TRH-deficient mice showed obvious hypothyroidism and transient growth
retardation with reduced body weight gain, which was restored by
T4 replacement (Yamada et al., 1997). The mice
also showed a marked decrease in insulin secretion leading to
hyperglycemia, implicating a pathophysiological role of TRH in
pancreatic islets.
). Neurotensin is produced in the ARC, PVN, and DMH of
the hypothalamus and its microinjection into the PVN decreases food
intake (Stanley et al., 1983; Alexander et al., 1989; Kalra et al.,
1999a). Leptin increases neurotensin gene expression in the
hypothalamus (Sahu, 1998) and the reduced expression and/or content of
neurotensin is associated with the development of obesity in
ob/ob mice and fa/fa rats (Beck et al., 1989;
Wilding et al., 1993). Neurotensin inhibits the orexigenic effect of
MCH, but not that of NPY, after i.c.v. coadministration (Tritos et al.,
1998a). A hypothalamic neuropeptide CART (cocaine- and
amphetamine-regulated transcript) was recently suggested to be an
endogenous satiety factor (Kristensen et al., 1998; Kuhar and Dall
Vechia, 1999). The expression of CART mRNA in the ARC is
decreased in food-deprived animals and is almost absent in
ob/ob mice, although in the latter it is restored by leptin
treatment. CART peptide is a potent inhibitor of feeding and markedly
inhibits NPY- induced feeding after i.c.v. administration (Kristensen
et al., 1998; Lambert et al., 1998; Kuhar and Dall Vechia, 1999).
Administration of antiserum against CART increases nocturnal feeding.
These results suggest that neurotensin and CART are anorexigenic
signals in the brain, although genetic evidence is lacking at present.
Dopamine neural circuits have been studied intensively because of their
involvement in movement control, drug abuse, and cognition. However,
the role of dopamine in the central control of feeding behavior is
complex and still controversial. Both dopamine-dependent anorexia and
overeating are reported and may depend on behavioral processes that
indirectly affect feeding (Samanin and Garattini, 1996). Any change of
dopaminergic function could result in marked changes of arousal and
motor behavior that make it particularly difficult to study the role of
this amine in feeding. However, several neuropeptides such as galanin,
CCK, and bombesin stimulate dopamine release in the nucleus accumbens
(Schwartz et al., 1988; Hoebel et al., 1994, 1996). Galanin injected
into the PVN evokes both feeding behavior and dopamine release in the
nucleus accumbens, suggesting that dopamine is involved in reinforcing
ongoing behavior, including the tendency to choose a fat-rich meal
(Hoebel et al., 1994). Conditioned taste stimuli excites or inhibits
dopamine release in the accumbens depending on the meaning for the
animal (Hoebel et al., 1996). It is likely that different aspects of feeding such as satiety and reward are regulated by dopamine in different brain regions. Recent studies suggest that the perifornical area of the LH may be a brain region in which dopamine suppresses feeding (Leibowitz and Rossakis, 1979; Parada et al., 1988; Leibowitz, 1989). Dopamine agonists appear to affect a motivational component of
feeding because appropriate doses injected in this region reduced feeding without having an effect on motor behavior (Parada et al.,
1988). Dopamine D1 receptor may be involved in
this action (Samanin and Garattini, 1996) although recent molecular
cloning has identified five dopamine receptor subtypes in the CNS
(Civelli et al., 1993). Mice unable to synthesize dopamine specifically in dopaminergic neurons were created by inactivating the tyrosine hydroxylase (TH) gene then by restoring TH function in noradrenergic cells by using noradrenergic-specific dopamine--hydroxylase promoter (Zhou and Palmiter, 1995). This dopamine deficiency was not lethal to
the mice, although they became hypoactive and stopped feeding a few
weeks after birth. With the administration of
L-dihydroxyphenylalanine, the product of TH, the mice
became active and consumed more food, leading to nearly normal growth.
Relatively mild feeding deficits were also observed in the mice lacking
dopamine D1 or D2
receptors, and they grew to 70% or 85% of normal body weight,
respectively (Drago et al., 1994; Xu et al., 1994; Baik et al., 1995).
However, they also showed decreased rearing behavior (Drago et al.,
1994), locomotor hyperactivity (Xu et al., 1994) or Parkinsons-like
locomotor impairment (Baik et al., 1995). The phenotypic changes of
D5 receptor-deficient mice, if generated, may be
interesting because D5 receptor is largely
confined to the limbic system and hypothalamus and is practically
absent in the striatum and nucleus accumbens (Tiberi et al., 1991;
Samanin and Garattini, 1996).
Pancreatic polypeptide (PP) is a 36-amino acid hormone produced within
the pancreatic islets and the exocrine pancreas, and consists of PP
family of regulatory peptides with NPY and PYY (Taylor, 1989; Gehlert,
1998). Previous studies demonstrated that PP administration reduces
food intake, body weight, or weight gain in dogs, genetically obese
mice, and patients with Prader-Willi syndrome who have hyperphagia and
PP deficiencies (McLaughlin and Baile, 1981; Sun et al., 1985; Berntson
et al., 1993). However, a reduction of food intake by PP is modest, and
other studies failed to observe it or observed it only after high
pharmacological doses (Billington et al., 1983; Taylor and Garcia,
1985). Transgenic overproduction of PP, by using the cytomegalovirus
immediate early enhancer-chicken -actin hybrid promoter, developed
lean mice that had specifically reduced food intake and fat mass in
body composition analysis (Ueno et al., 1999). Enterostatin is the amino-terminal pentapeptide released from pancreatic procolipase by
tryptic hydrolysis after pancreatic secretion has been stimulated by
the presence of fat in the duodenum (York and Lin, 1996;
Erlanson-Albertsson and York, 1997). Gastric procolipase is also
activated to release colipase and enterostatin (Winzell et al., 1998).
After peripheral or i.c.v. administration, enterostatin selectively
inhibits dietary fat intake. The peripheral effects appear to be
dependent on the hepatic vagal afferents through which signals activate
specific brain nuclei, including the PVN and NTS (York and Lin, 1996). Enterostatin inhibits the -opioid system and it chronically reduces body weight and body fat (Erlanson-Albertsson and York, 1997). Enterostatin may be a determinant of fat appetite and the
susceptibility to develop obesity on high-fat diets, although
transgenic models for this peptide have not yet been reported.
C. Regulators of Thermogenesis: Sympathetic Nervous System (SNS)-UCP Axis
Activation of the SNS and the consequent releases of NE in adipose
tissue leads to weight loss (Strader et al., 1998). Activation of
-adrenergic receptor in white adipose tissue stimulates lipolysis, which depletes fat stores and releases fatty acids. Activation of
-adrenoceptors in BAT increases energy expenditure via
thermogenesis. BAT is a specialized form of adipose tissue that
functions as a thermogenic organ in rodents (Himms-Hagen, 1992). The
production of heat after SNS stimulation results in the production of
uncoupling protein (UCP-1 or thermogenin). Oxidation of fuels by
mitochondria produces a proton gradient across the inner mitochondrial
membrane. It is the energy produced by this gradient that is used by
the mitochondria to convert ADP to ATP. UCP-1 allows this proton
gradient to be dissipated independent of phosphorylation and thereby
uncouples fuel oxidation from ATP generation. BAT plays an important
role in overall energy balance in rodents, especially in cold
environments and after fluid ingestion. Recently, a number of other
uncoupling proteins have been identified (Fleury et al., 1997; Gong et
al., 1997; Millet et al., 1997). UCP-2 is expressed in many tissues, including white adipose tissue and skeletal muscle, the major sites
considered for thermogenesis in humans. It is up-regulated in white
adipose tissue in response to fat feeding (Fleury et al., 1997). A
third uncoupling protein homolog, UCP-3, is preferentially and
abundantly expressed in skeletal muscle in humans and rodents (Gong et
al., 1997; Millet et al., 1997).
The role of brown fat in obesity has been demonstrated in mice in which
expression of a transgene for diphtheria toxin under the control of a
BAT-specific UCP gene promoter was used to specifically ablate BAT
(Lowell et al., 1993). The mutant mice developed obesity due to
decreased thermogenesis and lowered body temperature (Klaus et al.,
1998). However, they were affected later by unexpected hyperphagia
despite extreme hyperleptinemia (Lowell et al., 1993; Melnyk and
Himms-Hagen, 1998; Mantzoros et al., 1998). The expression of NPY,
AGRP, and MCH were all decreased in the hypothalamus of the mutant mice
in contrast to leptin-deficient ob/ob mice, although aberrant expression of NPY was observed in the DMH of the hypothalamus (Tritos et al., 1998b). Such de novo expression of NPY was reported in
other models of obesity such as Agouti, MC-4-receptor knockout mice,
tubby mice, and after disruption of the VMH (Inui, 1999a,b; Kalra et
al., 1999a). The hyperphagia may suggest the existence of a link
between BAT function and the hypothalamic system for satiety (Mantzoros
et al., 1998; Melnyk and Himms-Hagen, 1998). This is consistent with
the theory that sympathetic efferent output tonically inhibits food
intake (Bray and York, 1998). The BAT-deficient mice also developed
profound insulin resistance and diabetes that were sensitive to diet.
Mice lacking UCP-1 in BAT were cold-sensitive, indicating a defect in
thermoregulation (Enerbäck et al., 1997). However, UCP-1
deficiency caused neither hyperphagia nor obesity in mice fed either a
standard or a high-fat diet. This may have been due to the induction of
UCP-2 in the brown fat of the mutant mice. It was reported that mice
overexpressing the UCP-1 gene from the aP2 gene promoter in BAT and
white adipose tissue exhibited a striking redistribution of regional
fat but no change in body weight. However, they were partially
resistant to obesity induced by genetic (yellow obese), dietary
(high-fat), or age related factors (Kopecky et al., 1995, 1996). The
potential role of glycerol-3-phosphate dehyrogenase (GPDH) in energy
expenditure via futile cycling was suggested by results obtained with
transgenic overexpression of GPDH gene, a gene normally expressed in
BAT and other tissues (Kozak et al., 1991). In this case, mice
developed BAT hypertrophy that was associated with reduced amounts of
white adipose tissue.
The 1-, 2-, and
3-adrenergic receptor subtypes are all
involved in control of lipolysis and UCP-1 activation in rodent adipose
tissue (Strader et al., 1998). 3-Adrenoceptor
is expressed primarily in adipose tissue, whereas
1-adrenoceptor and
2-adrenoceptor are more widely expressed.
Selective 3-adrenergic receptor agonists increased the metabolic rate, leading to weight loss and improvement in
glucose tolerance in obese rodents (Lipworth, 1996). Mice lacking the
3-adrenoceptor possess only a modest tendency
to become obese relative to normal controls (Susulic et al., 1995;
Revelli et al., 1997). However, there was a compensatory increase in
the level of 1- (but not
2-) adrenergic receptor in the white adipose tissue and BAT of the knockout mice, and activation of this receptor could have prevented excessive adiposity. Because of the differences in
sites of expression and pharmacology between human and rodent 3-adrenergic receptors, mice expressing human
but not murine 3-adrenergic receptor were
developed under the control of human gene regulatory elements (Ito et
al., 1998). These humanized animals are useful for the development of
sufficiently selective treatments for human obesity. The transgenic
mice overexpressing 1-adrenergic receptor in
the adipose tissue under the control of the aP2 gene promoter
were resistant to diet-induced obesity (Soloveva et al., 1997). The
increased lipolytic activity observed in these mutant mice suggests
that total - adrenergic receptor activity, rather than the
particular subtype, may determine overall lipolytic state. 1-Adrenergic receptor may also be involved in
the proliferation of brown fat cells (Soloveva et al., 1997).
Sympathetic stimulation of protein kinase A in BAT promotes
thermogenesis through UCP. Protein kinase A has two regulatory and two
catalytic subunits, and the RII regulatory subunit is abundant in
BAT and white adipose tissue and brain, with limited expression
elsewhere. Targeted disruption of the RII subunit, however, resulted
in lean mice with hyperphagic tendency (Cummings et al., 1996). The
mice were resistant to diet-induced obesity due to chronic activation
of BAT thermogenesis and elevated body temperature. Mutant BAT
exhibited a compensatory increase in RI, which almost entirely
replaced lost RII and elevated basal protein kinase A activity,
leading to increased UCP, excess energy expenditure, and leanness. The mice that cannot synthesize norepinephrine and epinephrine by inactivating the gene encoding dopamine -hydroxylase were
hyperphagic but did not become obese because of an elevated basal
metabolic rate (Thomas and Palmiter, 1997). These mice were
cold-intolerant because they had impaired peripheral vasoconstriction
and were unable to induce thermogenesis in BAT through UCP-1. The
unexpected increase in the basal metabolic rate was not due to
hyperthyroidism, compensation by UCP-1, or shivering. These results
indicate that BAT has an effect on the overall metabolic rate both
dependent on and independent of the SNS-UCP axis.
D. Other Regulators
Obesity may occur by preferential nutrient uptake into white
adipose tissue. This was demonstrated by the specific overexpression of
human glucose transporter-4 (GLUT4) in white adipose tissue by using
the aP2 promoter (Shepherd et al., 1993). GLUT4 is a major facilitative
glucose transporter isoform in skeletal muscle and adipose tissue
(Mueckler, 1994). The transgenic mice do not exhibit hyperphagia but
produce hyperplasia of adipocytes and increased fat mass, suggesting
that increased uptake of glucose into white adipose tissue is
sufficient to produce obesity. GLUT4-null mice had severely depleted
adipose tissue and growth retardation (Katz et al., 1995). Conversely,
the targeted overexpression of lipoprotein lipase (LPL) in skeletal
muscle by using the muscle creatine kinase promoter and enhancer
prevents diet-induced obesity by diverting lipoprotein-derived
triglyceride fatty acids away from storage in adipose tissue to
oxidation in muscle (Jensen et al., 1997). Acylation-stimulating
protein is generated by adipocytes in the postprandial period and acts
on adipose tissue to stimulate triglyceride synthesis and glucose
transport (Baldo et al., 1993; Sneiderman et al., 1997). The
significance of acylation-stimulating protein and other molecules
involved in the metabolic pathways needs to be examined in transgenic models.
Genetic manipulation can identify previously unknown regulators of
energy balance. The genes encoding receptors that mediate leukocyte
adhesion were demonstrated to be involved in the regulation of adipose
tissue mass. Mice deficient in intracellular adhesion molecule or its
counterreceptor, leukocyte integrin M2 (Mac-), developed
late-onset obesity without overeating (Dong et al., 1997). Leukocyte
functions may thus influence energy expenditure and/or lipid
metabolism. Metallothioneins comprise a family of highly conserved
metal-binding proteins that have a role in the detoxification of heavy
metals as well as other functions. The metallothionein-null mice became
obese at a young age and were hyperphagic in established obesity
(Beattie et al., 1998). The family of basic helix-loop-helix genes
comprises transcription factors involved in growth and development.
Mice deficient in Nhlh2, a transcription factor made in the
hypothalamus, displayed adult-onset obesity with impaired gonadal
growth associated with puberty (Good et al., 1997). Decreased
expression of POMC in the hypothalamus was observed in these mutant
mice. Growth hormone (GH) affects carbohydrate and lipid metabolism,
and GH-deficient human adults become obese and insulin resistant.
Transgenic rats with low serum GH concentrations and lack of pulsatile
secretion exhibit normal body growth, yet develop a type of obesity
with diabetes that is responsive to stimulation of pulsatile GH
secretion (Ikeda et al., 1998) The signal transducer and activator of
transcription, STAT5b, has been implicated in signal transduction
pathways for a number of cytokines and growth factors, including GH.
STAT5b-deficient mice showed dwarfism associated with later development
of obesity without substitution by the highly homologous STAT5a (Udy et
al., 1997). The transcription factor CCAAT/enhancer-binding protein (C/EBP) is enriched in liver and adipose tissue, and controls the
expression of a wide variety of genes coding for important metabolic
pathways, including gluconeogenesis and lipid synthesis. A subset of
the homozygous C/EBP-deficient pups die within 24 h after birth,
whereas the remainder are viable, but appear to have less adipose lipid
accumulation (Liu et al., 1999). Mice homozygous for deletion in the
genes for both C/EBP and C/EBP had a more severe reduction in
size of the white adipose tissue (Tanaka et al., 1995), and there was
no up-regulation of expression C/EBP, which is known to be required
for the normal development of white adipose tissue. Sterol regulatory
element-binding protein-1c is another transcription factor that has
been implicated in adipocyte differentiation. Overproduction of the
nuclear form of sterol regulatory element-binding protein-1c driven by
the aP2 promoter developed mice with markedly decreased fat tissue and
insulin resistance and diabetes mellitus (Shimomura et al., 1997,
1999). The nuclear receptors known as peroxisome proliferator-activated receptor (PPAR) family contains 3 distinct isotypes: , , and (Spiegelman and Flier, 1996). PPAR is expressed in the liver and
other tissues, and controls the expression of numerous genes related to
lipid metabolism. A striking metabolic defect was observed in
PPAR-null mice, characterized by a progressive, sexually dimorphic dyslipidemia with pronounced adiposity in females and steatosis in
males (Costet et al., 1998). The mice exhibited enhanced accumulation of lipid in the liver and defective responses to fasting such as severe
hypoglycemia (Kersten et al., 1999). CD36 (FAT) is a membrane protein
that may have a role in the muscle uptake of long-chain fatty acids.
Muscle targeted overexpression of CD36 by using the promoter of the
muscle creatine kinase gene was associated with enhanced muscle fatty
acid oxidation and lower body weight and fat mass (Ibrahimi et al.,
1999). Protein tyrosine phosphatase 1B (PTP-1B) has been implicated in
the negative regulation of insulin signaling. Disruption of the gene
yielded healthy mice that had increased insulin sensitivity and obesity
resistance on a high-fat diet (Elchebly et al., 1999). The disruption
of the leukocyte antigen-related PTP, which has also been suggested to
affect the insulin signaling cascade, produced mice with body weights
that were half those of control mice in some (Ren et al., 1998), but
not other (Schaapveld et al., 1997), studies.
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V. Advanced Gene Targeting |
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There are several reasons to achieve conditional, i.e.,
cell-type-specific and/or inducible, gene targeting beyond classical targeted mutagenesis of the mouse germline (Rajewsky et al., 1996). Germline mutations may result in lethality, in which case there is no
mouse to study gene function. Genes may exert their function at several
stages of ontogeny and in different cell types. It is difficult to
distinguish such spatiotemporal roles of the gene by classical gene
targeting. The phenotype of transgenic mouse models may be the
consequence of developmental abnormality or compensation by the related
gene family, rather than the physiological role of a targeted gene
product per se. Furthermore, obesity and associated physiological
derangements such as diabetes are complex traits, developing later in
life in most cases and involving interactions between genetic factors
and acquired factors such as diminished exercise. It may be preferable
to initiate a gene-targeting event in adult animals without mutation
during development.
Recent sophistication of molecular genetic techniques has made it
possible to circumvent such potential problems by spatiotemporally regulating a gene-targeting or transgene expression (Moller, 1994; Kühn et al., 1995; Gossen et al., 1995; Rajewsky et al., 1996, Son and Joh, 1997; Rohrer and Kobilka, 1998). The tissue-specific targeting is achieved by tissue-specific expression of cre
recombinase enzyme, which will mediate excision of the targeted gene
having lox P recognition sites. By this Cre/lox P
system, the gene of interest will remain and be functional in all
non-cre-expressing cells, but will be lost in
cre-expressing cells. A related technique is inducible gene
targeting, which enables the desired genetic change at any point in
development, merely by the addition of an inducer substance, such as
tetracycline binary system (Gossen et al., 1995). Although the
technique has not yet been applied to a component of the
feeding-regulatory cascade, Cre/lox P mediated, tissue-specific targeting was successfully achieved to reveal the role
of insulin receptor in muscle and pancreatic cells for fat
metabolism (Brüning et al., 1998), insulin secretion (Kulkarni et
al., 1999), and hippocampal
N-methyl-D-asparate receptor for
spatial memory (Tsien et al., 1996a,b). A caveat of both the tissue-specific and inducible system is the penetrance of the knockout,
which may affect experimental results (Rohrer and Kobilka, 1998). This
is because both systems rely on the cre expression and
targeted excision may not occur in all cells or with the same efficiency. Differences in phenotype for transgenic mice such as
D1 dopamine receptor knockouts (Drago et al.,
1994; Xu et al., 1994) may be due to mouse strain heterogeneity or
differences in the methods of gene disruption (Rohrer and Kobilka,
1998). Manipulation of the gene may modify the expression of other
genes located near the intended one and may thus confound the
interpretation of the phenotypic changes (Olson et al., 1996).
Regardless of the potential shortcomings, however, advanced gene
targeting will undoubtedly be instrumental in furthering our
understanding of body weight regulation.
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VI. Conclusions |
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The application of transgenic technology to create animal models
that address the pathogenesis of obesity is a recent development that
is now gaining rapid momentum and receiving deserved attention (Moller,
1994; Chua and Leibel, 1997; York and Hansen, 1998; Levine and
Billington, 1998). 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 methods have provided new opportunities to modify the
complex body weight regulating system and then to assess the relative
importance of the individual components (Figs. 1 and 2). Once created,
transgenic animal models are useful to assess the efficacy or determine
the mode of action of potential new therapeutic agents.
The complicated nature of appetite and energy homeostasis regulation
may be suggested from the essentially normal phenotype of NPY knockout
mice (Erickson et al., 1996a), despite the potent appetite-stimulating
and body weight-increasing effects of NPY observed in various animal
models and species (Woods et al., 1998; Inui, 1999a; Kalra et al.,
1999a). The functional discrepancy between pharmacological and genetic
manipulations needs to be clarified. However, it may include the
redundancy and plasticity of the regulatory machinery for the energy
homeostasis equation, a characteristic of regulated biological systems
(Levine and Billington, 1998; Kalra et al., 1999a). It was reported
that GRP receptor-deficient mice may have an increased sensitivity to
CCK, another peptide involved in regulating individual meal size
(Hampton et al., 1998). Other such examples are the UCP-1
(Enerbäck et al., 1997) and 3-adrenergic receptor
(Susulic et al., 1995) knockout mice in which UCP-2 and
1-adrenergic receptor may substitute, respectively. Therefore, in some cases the lack of any obvious effect of a targeted mutation may not necessarily mean that the gene product is not involved
in the regulation of energy homeostasis. Rather, modifying the
synthesis of a particular gene at all sites and developmental stages
may be a 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 are a recent elaboration which can obviously lead to a better understanding of the role the gene product plays in body weight regulation. In conjunction with conventional pharmacological manipulations, these powerful genetic tools will provide more sophisticated animal models and unprecedented insights into the underlying mechanisms of obesity, leading to new treatments.
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Acknowledgments |
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TopPreviousNextReferences
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I am indebted to Prof. Masato Kasuga and Prof. Shigeaki Baba (Kobe University) for many stimulating discussions. I sincerely thank Dr. Etsuro Itoh (Ube Research Laboratory) for cooperation. The work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.
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Footnotes |
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1 Address for correspondence: Dr. Akio Inui, Associate Professor, Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. E-mail: inui{at}med.kobe-u.ac.jp
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Abbreviations |
|---|
ACTH, adrenocorticotropic
hormone;
AGRP, Agouti-related protein;
-MSH, -melanocyte-stimulating hormone;
aP2, adipocyte lipid-binding
protein;
ARC, arcuate nucleus of the hypothalamus;
BAT, brown adipose
tissue;
BBB, blood-brain barrier;
CART, cocaine- and
amphetamine-regulated transcript;
CCK, cholecystokinin;
C/EBP, transcription factor CCAAT/enhancer-binding protein;
CNS, central
nervous system;
CRF, corticotropin-releasing factor;
DMH, dorsomedial
nucleus of the hypothalamus;
GABA, -aminobutyric acid;
GAD, glutamic
acid decarboxylase;
GH, growth hormone;
GHRH, growth hormone-releasing
hormone;
GLP-1, glucagon-like peptide-17-36 amide;
GLUT4, glucose transporter-4;
GPDH, glycerol-3-phosphate dehydrogenase;
GRP, gastrin-releasing peptide;
5-HT, 5-hydroxytryptamine serotonin;
IL, interleukin;
LH, lateral hypothalamus;
MC, melanocortin;
MCH, melanin-concentrating hormone;
mRNA, messenger ribonucleic acid;
NE, norepinephrine;
POMC, proopiomelanocortin;
NPY, neuropeptide Y;
NTS, nucleus tractus solitarius;
ob, obese;
PP, pancreatic polypeptide;
PPAR, peroxisome proliferator-activated receptor;
PTP, protein tyrosine
phosphate;
PVN, paraventricular nucleus of the hypothalamus;
PYY, peptide YY;
SREBP, sterol regulatory element-binding protein;
STAT, signal transducer and activator of transcription;
SNS, sympathetic
nervous system;
TH, tyrosine hydroxylase;
TNF-, tumor necrosis
factor-;
TRH, thyrotropin-releasing hormone;
UCP, uncoupling
protein;
VMH, ventromedial nucleus of the hypothalamus.
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