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Bombesin
Chapter 142
Bombesin Ellen E. Ladenheim
ABSTRACT Bombesin (BN), a tetradecapeptide originally isolated from anuran skin, belongs to a large family of structurally related amphibian and mammalian peptides. Since their discovery, BN and two mammalian BN-like peptides, gastrin-releasing peptide (GRP), and neuromedin B (NMB) have been shown to exhibit diverse behavioral and physiological actions in a number of species. Receptor subtypes for GRP and NMB and a third receptor subtype, BRS-3, have been identified. BN-like peptides have been shown to suppress food intake after peripheral or central administration and have been proposed as putative satiety signals. Specific agonists and antagonists have been instrumental in determining the individual contributions of BN receptor subtypes to food intake and energy homeostasis. Studies in knockout mice have provided an additional means to evaluate their physiological function. Recent studies have shown that BN-like peptides and receptors participate in long-term effects on food intake and body weight.
BACKGROUND Bombesin (BN) is a tetradecapeptide isolated in 1970 by Erspamer et al. from the skin of the European frog Bombina bombina. It is part of a large family of amphibian and mammalian peptides that are biologically active in mammals (see Bombesin-like Peptides in Amphibian/Skin Peptides section of this book). Since their discovery, BN-like peptides have been shown to affect many gastrointestinal (GI) processes including gastrin release, gastric acid secretion, pancreatic enzyme secretion, gallbladder contraction, and intestinal motor activity. Aside from their well-known effects on gastrointestinal function, BN-like peptides also participate in a number of important neuroregulatory processes such as thermoregulation, glucose homeostasis, circadian rhythms, and food intake.10 Although BN itself is not present in mammals, several peptides that are structurally homologous to BN have been detected in mammalian viscera and central nervous system (CNS). These peptides include several forms of gastrinreleasing peptide (GRP1–27, GRP1–29, and GRP18–27, also known as neuromedin C) and several forms of neuromedin B (NMB1–32, NMB2–32, and NMB23–32). Bombesin and GRP 1064
share an identical carboxyl terminal heptapeptide sequence, the minimal fragment necessary for their biological activity. NMB contains a portion of these amino acid residues but more closely resembles the amphibian peptides, ranatensin, and litorin.24 The distribution of BN/GRP-like immunoreactivity in the gastrointestinal tract is mainly confined to nerve cell bodies in the myenteric ganglia and in nerve fibers of these ganglia. Similarly, NMB-like immunoreactivity is found throughout nerve fibers in the gastrointestinal tract, as well as the pancreas, spinal cord, and pituitary gland. In the CNS, immunohistochemical mapping of GRP and NMB, as well as mRNA studies, have shown distinct and discrete distribution of the two peptides in rat brain. GRP mRNA is highly expressed in the isocortex, hippocampal formation, anterior olfactory nucleus, and several hypothalamic nuclei. NMB mRNA expression is most prominent in the olfactory bulb, dentate gyrus, and dorsal root ganglion.7 Because of structural similarities between BN and GRP it was initially believed that GRP was the mammalian counterpart to BN and thus all BN binding sites in the periphery and brain represented GRP receptors. Early work evaluating the effect of BN-like peptides on smooth muscle contraction found that they displayed varying degrees of potency suggesting the existence of multiple receptor subtypes. Subsequent studies by von Schrenck et al.10 identified and characterized two pharmacologically distinct BN receptor subtypes in pancreatic acinar cells and esophageal muscularis mucosa. BN binding sites in the pancreas displayed a high affinity for GRP and BN and a low affinity for NMB, whereas esophageal BN binding sites had a high affinity for NMB and BN and a low affinity for GRP. Because of these pharmacological characteristics esophageal BN receptors were termed “NMB-preferring” or BB1 and pancreatic BN receptors were termed “GRP-preferring” or BB2. BN has a high and equal affinity for NMB-R and GRP-R, and thus, its activity may be attributed to binding to either or both receptor subtypes. These receptors were later cloned, and an additional mammalian BN receptor subtype, BN receptor subtype-3 (BRS-3 or BB3) was identified through homology screening and found to Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00142-1 Copyright © 2013 Elsevier Inc. All rights reserved.
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share approximately 50% structural identity with GRP-R and NMB-R. Unlike GRP-R and NMB-R, BRS-3 has a low affinity for all naturally occurring BN-like peptides. To date, no endogenous ligand for BRS-3 has been identified and it is termed an “orphan” receptor.10 Receptors for BN-like peptides have been identified in a variety of tissues in the periphery and central nervous system. Using an approach similar to that used to characterize peripheral BN receptor subtypes, initial studies evaluated the relative ability of BN, GRP, and NMB to inhibit binding of radioactively labeled BN in rat brain. These studies revealed two distinct binding patterns consistent with those reported in peripheral tissues. Subsequent autoradiographic mapping studies determined that the predominant BN receptor subtype in the rat brain is NMB-R with the highest densities in the anterior olfactory nucleus, nucleus accumbens, and thalamus. The distribution of GRP-R is more limited with the highest densities in several hypothalamic nuclei, basal ganglia and central nucleus of the amygdala.16 The results of the binding experiments were in general agreement with in situ hybridization studies examining expression patterns of GRP-R and NMB-R mRNA. Localization of BRS-3 mRNA was initially reported to be much more limited with the highest expression levels in the arcuate (Arc), dorsomedial (DMH), ventromedial (VMH), and paraventricular (PVN) nuclei of the hypothalamus. A later study using an antibody directed toward BRS-3 found a much wider distribution in the CNS with particularly strong immunoreactivity in the cerebral cortex, hippocampal formation, hypothalamus, and thalamus.7 In the periphery, BN binding sites in the stomach were similarly differentiated where the majority of binding was GRP-R. In gastric regions, BN binding sites were localized to the circular muscle layer with the highest density in the gastric fundus. In primates, the highest levels of NMB-R mRNA were detected in the testis and stomach, whereas in rodent peripheral tissue, significant NMB-R expression was found in esophagus, intestine, testis, and uterus.7 Immunohistochemical evaluation of gastrointestinal BRS-3 distribution demonstrated BRS-3-like immunoreactivity at all levels of the gastrointestinal tract primarily in neurons of the myenteric and submucosal ganglia and in fibers in the longitudinal and circular muscle layers.29 In general, the distinct distributions of BN-like peptides and receptors support the view that they function as separate regulatory agents in the periphery and CNS.
PERIPHERAL EFFECTS OF BN-LIKE PEPTIDES ON FOOD INTAKE A role for BN as a putative satiety signal stems from experiments showing that exogenous administration of BN dose dependently suppresses food intake in a variety of experimental paradigms, and in a number of animal species, including humans. The inhibition of food intake produced
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by peripherally administered BN is followed by a normal postprandial sequence of behaviors that are characteristic of meal ingestion. The reduction of food intake by BN is not accompanied by behavioral signs of toxicity, such as sickness or malaise, nor are there nonspecific effects on water intake. The decrease in meal size produced by BN is consistent with a behavioral profile that indicates an increase in post-ingestive feedback signaling. Studies in human subjects have demonstrated that intravenous administration of BN reduced intake of a test meal without producing significant side effects.6 Although an increase in meal-related plasma BN-like immunoreactivity has been reported, the general consensus is that BN’s actions on food intake are mediated through neurocrine or paracrine mechanisms. Supporting this view are studies demonstrating an increase in BN-like immunoreactivity in the antral region of the stomach in response to meal intake, coupled with the localization of BN-like immunoreactivity in gut neurons.23 BN has an equal and high affinity for both GRP and NMB receptors. The discovery that BN interacted equally with GRP and NMB receptors presented the possibility that BN was mimicking the actions of GRP and NMB but that both GRP and NMB produced their effects on food intake at distinct receptor populations. A comparison of feeding suppression produced by peripheral administration of BN, GRP1–27, GRP18–27 and NMB in rats showed that, at intermediate doses, the rank order of potency is BN > GRP1–27 = GRP18–27 > NMB. However, at the lowest and highest doses, BN, GRP1–27, and GRP18–27 are equivalent. The biphasic nature of the dose response curve for GRP1–27 and GRP18–27 suggested that these peptides may be interacting with GRP-R at low doses and with both GRP-R and NMB-R at high doses. NMB is consistently less potent than BN in reducing food intake, suggesting that it is interacting only with NMB-R. A more recent study examining the satiety effects of GRP1–29 in rats (the predominant form in this species) found equivalent potency to GRP1–27. It is believed that the increased potency of BN to reduce food intake, compared with its mammalian counterparts, partially results from a relative resistance to enzymatic degradation. Supporting this idea are studies showing that acetylation of GRP18–27, which improves resistance to degradation, increased its ability to reduce feeding, making it equivalent to that of BN.21 The notion that the effects of BN on food intake may result from the combined action at both receptor subtypes is also supported by studies showing that peripheral administration of equimolar doses of GRP and NMB reduces food intake and elicits a microstructural licking pattern equivalent to that of BN.31 Moreover, when a dose of NMB that produces a maximal suppression of food intake is given in combination with a submaximal dose of GRP, the reduction in food intake is significantly enhanced beyond suppression
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produced by either peptide administered alone.21 Additional studies show that a specific NMB-R receptor antagonist blocks feeding inhibition by peripherally administered NMB, but not by GRP, suggesting that NMB and GRP reduce food intake by acting at distinct receptor populations.20 Although several studies found that peripherally administered GRP-R antagonists prevent the suppression of food intake by peripherally administered BN, attempts to increase food intake using GRP-R antagonists, or to block feeding suppression produced by intragastric loads, have been inconsistent.4,23 The reason for this is unresolved; however, it is possible that the antagonists have limited potency or bioavailability and are unable to access sites that are activated by endogenously released BN-like peptides.
CENTRAL EFFECTS OF BN-LIKE PEPTIDES ON FOOD INTAKE The presence of BN-like immunoreactivity and BN binding sites in brain regions implicated in controlling food intake suggested that central BN-like peptides may also play a role in satiety. Several early studies showed that BN administered into the lateral cerebral ventricles (LV) in rats produces a potent suppression of food intake.12 However, the reduction in food intake is accompanied by a decrease in water intake and an increase in grooming and locomotion, suggesting a nonspecific effect of BN on feeding that is dissimilar to the actions of peripherally administered BN. Subsequently, studies were undertaken to examine the specific brain sites where BN may produce its anorexic effects. Early studies using site specific injections of high doses of BN into various hypothalamic nuclei including the PVN, DMH, VMH, and lateral hypothalamus (LH) significantly reduced food intake, as did injections into extrahypothalamic areas such as the amygdala and periaqueductal gray. However, in all instances, feeding suppression was accompanied by increased grooming activity suggesting that the reduction in food intake may be secondary to competing behaviors.13 Other experiments using smaller BN doses found that direct injections into the LH and amygdala specifically reduce food intake without producing competing behavioral effects.6,33 Initial studies focused primarily on forebrain mediation of BN satiety; however the presence of binding sites for BNlike peptides and BN-like immunoreactive nerve terminals and cell bodies in hindbrain regions known to participate in ingestive control (e.g., nucleus of the solitary tract (NTS)) suggest that this region may be responsive to BN’s feeding effects. Several investigators found that injections of BN into the fourth cerebral ventricle (4V), in close proximity to the NTS, are highly effective in reducing food intake in rats4 The doses required to suppress feeding after 4V administration are up to 100 times lower than those that elicit reduced
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food intake after LV administration. Further, this action is specific for food and does not produce a conditioned taste aversion or elicit competing behaviors such as grooming and locomotor activity that are associated with injection at other brain sites. Studies evaluating the effect of direct microinjections of BN into the NTS found this brain region to be particularly sensitive for feeding inhibition by BN.23 Although several studies have examined the feeding effects of centrally administered BN receptor agonists, a systematic comparison across peptides has not been conducted. It is assumed that the order of potency is BN>GRP>NMB based on studies examining each peptide individually or comparing one peptide against BN using a limited range of doses. Although the majority of BN binding sites in the vicinity of the 4V are NMB-R, studies using specific GRP-R antagonists have identified the contribution of hindbrain GRP-R to the feeding inhibitory actions of BN. Two reports showed that pretreatment with selective GRP-R antagonists facilitates feeding in sated rats, supporting a role for endogenously released GRP in reducing food intake.5,23 Injections of GRP1–27 and GRP18–27 into the central amygdala have also been reported to transiently decrease food intake without producing changes in body temperature or stereotypic behaviors.3 Relatively little attention has been given to the role of central NMB-R in food intake. Agonist studies have not been particularly informative because central NMB exhibits very poor potency to inhibit feeding. However a role for hindbrain NMB-R in ingestive control was determined in studies using a specific NMB-R antagonist.19 As shown with GRP-R antagonists, 4V administration of an NMB-R antagonist alone also increases food intake and blocks the effects of centrally administered BN. In addition, 4V administration of GRP-R and NMB-R antisense oligonucleotides prevents the suppression of food intake by centrally administered BN, supporting a role for both receptor subtypes in mediating BN’s central feeding effects.23
NEURAL PATHWAYS MEDIATING THE EFFECTS OF BN-LIKE PEPTIDES Several studies have been conducted to determine the site of action and neural substrates underlying the satiety actions of peripheral and central BN-like peptides. Early work evaluating the effect of adrenalectomy, hypophysectomy, vagotomy, and lesions of the ventromedial and dorsomedial hypothalamus reported that none of these surgical manipulations were effective in preventing BN’s ability to suppress food intake.6 By contrast, Stuckey et al. examined the effect of individual or combined lesions of the afferent innervation of the GI tract and found that, while vagotomy alone fails to reduce BN satiety, combined bilateral subdiaphragmatic vagotomy, spinal cord transaction (T6), and dorsal rhizotomy (T1–T6) significantly attenuate the ability of peripheral
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BN to reduce food intake.6 A subsequent study found that rhizotomy and spinal cord section, in the absence of vagotomy, also attenuate BN’s feeding effects. More recently, it was shown that vagotomy combined with splanchnic afferent and efferent denervation of the gut lessen the effects of GRP1–29 on meal size in rats.35 To more clearly define the pathways through which BNlike peptides exert their satiety actions, studies were undertaken to evaluate the effects of capsaicin on the suppression of food intake produced by BN. Capsaicin, a derivative of red pepper, is a neurotoxin that selectively damages a subset of primary sensory fibers that include both vagal and spinal neurons. Adult rats treated systemically with capsaicin exhibit a significant attenuation in the feeding inhibitory response to peripheral BN administration.18 A subsequent report showed that neonatal capsaicin treatment, which produces a more complete deafferentation, completely abolishes peripheral BN’s feeding effects in adult rats without affecting the inhibition of food intake produced by third ventricular (3V) administration.23 Because of the distinct roles of BN-like peptides to reduce food intake, additional studies were conducted in adult rats treated neonatally with capsaicin to examine whether different neural substrates underlie the feeding suppression by BN, GRP, and NMB.17 The suppression of food intake by peripheral administration of BN and NMB is abolished or attenuated by capsaicin treatment, whereas the effects of GRP remained intact except at the highest dose. These results suggest that BN-like peptides use distinct neural pathways to reduce feeding. The presence of gastric BN binding sites and changes in gastrointestinal BN-like immunoreactivity in response to meal intake led to the proposal that BN may produce its effects on food intake through a gastric mechanism, although evidence to support this has been equivocal. In support of a gastric site of action for BN, it was shown that local celiac artery infusions of BN are much more effective in suppressing food intake than either intraperitoneal or superior mesenteric artery infusions.11 However, another experiment that compared the feeding effects of various doses of BN with the gastric inhibitory effects of BN, concluded that BN’s satiety actions are dissociated from its effects on gastric emptying, as is the potency of BN to reduce food intake compared with its relative affinity for gastric receptors.9 These results suggest that BN’s effects on food intake are not mediated by a gastric site or via a gastric inhibitory mechanism. Although BN-like peptides have been shown to excite mechanosensitive gastric branch vagal afferents, electrophysiological studies have determined that this is an indirect action secondary to its stimulation of gastric motor effects, consistent with the lack of BN binding sites in the vagus nerve. The neuronal pathways through which peripheral and central BN-like peptides exert their effects on food intake have been also been examined by evaluating patterns of
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c-fos immunoreactivity, a marker of neuronal activation. Peripheral injection of BN induces c-fos in the area postrema (AP), medial NTS, parvocellular PVN, and to a lesser extent in central nucleus of amygdala, parabrachial nucleus, supraoptic, and magnocellular PVN. A comparison between peripheral and central (4V) BN administration found that both routes of administration show strong fos-LI in the PVN. Fos-like immunoreactivity was also detected in the medial NTS, central nucleus of amygdala, and bed nucleus of the stria terminalis, though the regional distribution differs depending on the route of administration. Differences were also found in the AP where peripheral, but not central, administration induced c-fos activation.1,30 A more recent study compared the effects systemic administration of different forms of GRP on c-fos activation in the gastrointestinal tract and hindbrain. Myenteric neurons in the stomach and neurons in the area postrema are activated by GRP18–27, GRP1–27, and GRP1–29. In the duodenum, both GRP1–27 and GRP1–29 increase c-fos activation in the myenteric plexus, whereas only GRP1–29 increases fos-LI in the submucosal plexus.34 The necessity of central BN-like peptide receptors to the peripheral effects of BN-like peptides has also been examined. Merali et al. (1988) first reported that 3V infusion of BN antisera, or a GRP-R antagonist, blocks the suppression of food intake by peripheral BN administration. Other studies have shown that ablation of the AP/NTS complex or blockade of hindbrain GRP receptors with 4V GRP-R antagonists attenuate the ability of peripherally administered BN or GRP to suppress feeding.23 Converging behavioral and anatomical evidence indicates that the NTS is a critical region for the feeding effects of BN. This region receives sensory input from the gastrointestinal tract via vagal and spinal neurons. Within the NTS, BN-ir is located in the medial subnucleus, an area that is associated with nerve terminals that synapse on cell bodies or dendrites of vagal motoneurons. Compelling evidence for the importance of the caudal hindbrain to BN’s feeding effects is provided in experiments showing that either peripheral or 4V injections of BN reliably suppress sucrose intake in chronic decerebrate rats, a surgical preparation that severs communication between the hindbrain and forebrain. These results support the idea that local neural circuitry residing in the caudal hindbrain, independent of forebrain systems, is sufficient to mediate the effects of BN-like peptides on ingestive behavior.4 Although the necessity of central neural substrates, particularly in the NTS, play a critical role in BN’s effects on food intake there are still questions remaining concerning how the signal is transmitted. Whether peripheral administration of BN-like peptides causes a central release of GRP (or NMB) or whether they gain direct access to critical hindbrain receptors has not been fully determined.
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CONTRIBUTION OF BN RECEPTOR SUBTYPES TO FOOD INTAKE USING GENETICALLY MODIFIED MICE The development of mice with targeted deletions for each BN receptor subtype has provided a complementary means to evaluate the individual roles of BN receptor subtypes to ingestive control and other physiological effects of BN-like peptides.26 The first of these to be developed are mice lacking the GRP-R. In studies to evaluate the overall contribution of GRP-R to food intake, GRP-R KO mice fail to suppress intake after exogenously administered of either BN or GRP, suggesting that GRP-R alone mediates the satiety effect of BN-like peptides in mice. An analysis of 24-h meal patterns reveals that GRP-R KO mice exhibit an increase in meal size consistent with a deficit in satiety signaling, but 24-h food consumption is not different from control mice because of a reduction in the total number of meals consumed. A long-term analysis of body weight found that body weights between GRP-R KO mice and WT controls are comparable in early life although a small but significant elevation in body weight occurs in aged GRP-R KO mice. Additional studies show GRP-R deficient mice display increased nighttime locomotor activity, loss of GRP-induced hypothermia, and increased social investigatory behaviors. Metabolic disturbances, including decreased secretion of glucagon-like peptide 1 and abnormalities in insulin release and glucose tolerance, are also present in GRP-R KO mice. Unlike GRP-R KO mice, previous studies in mice lacking NMB-R (NMB-R KO) found no differences in food intake and body weight between NMB-R KO and WT control mice. However, a subsequent study showed that although female NMB-R KO mice fed a chow diet show no differences in body weight, when presented with a high fat diet they are partially protected against body weight gain and adipose accumulation and exhibit normal glucose tolerance compared with that of WT littermates.28 Recent attention has been directed toward the role of BRS-3 in food intake and energy homeostasis. Progress in determining the physiological function of BRS-3 was hindered by the lack of an identifiable natural ligand or a suitable synthetic ligand. To assess the function of BRS-3, Ohki-Hamazaki and colleagues generated mice with a targeted deletion of BRS-3 (BRS-3 KO) and found that they develop obesity accompanied by hypertension, impairment of glucose tolerance, and insulin resistance. In addition, BRS-3 KO mice exhibit reduced metabolic rate, increased feeding efficiency and hyperphagia, and showed alterations in glucose transport.26 Additional behavioral and physiological studies were conducted to identify factors contributing to increased food consumption and body weight in BRS-3 KO mice.15 A primary deficit leading to the obese phenotype of BRS-3 deficient
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mice is an uncompensated increase in meal size resulting in an overall increase in total food intake. Although the increase in body weight could be overcome by pair-feeding to the caloric intake of WT controls, they still exhibit significantly elevated leptin concentrations and body fat distribution, confirming a metabolic dysregulation in BRS-3 KO mice. Despite the finding that BRS-3 KO mice increase their meal size, they exhibit a normal response to peripherally administered peptides (e.g., cholecystokinin and BN) that participate in meal-related controls of food intake. A study evaluating the effects of centrally administered peptides reported that mice lacking BRS-3 are leptin resistant, hyperresponsive to the orexigenic effects of exogenously administered melanin concentrating hormone (MCH), and have elevated levels of MCH mRNA in the lateral hypothalamus. Studies have also suggested that hyper-responsiveness to taste stimuli may contribute to overeating in BRS-3 deficient mice particularly when exposed to highly palatable foods.26 BRS-3 KO mice exhibit a stronger taste preference for saccharin solution and a stronger aversion to quinine solution compared with that of WT littermates. Using a conditioned taste aversion paradigm, BRS-3 KO mice display a stronger aversion to both saccharin and sodium chloride solutions after intraperitoneal lithium chloride administration. A preliminary study reported exaggerated hyperphagia and weight gain in BRS-3 KO mice maintained on a high fat diet, a characteristic that is consistent with other mouse models of obesity.
BN-LIKE PEPTIDES/RECEPTORS AND ENERGY HOMEOSTASIS Although a major focus for determining a role for GRP and NMB in ingestive control has been to evaluate their function as short-term or meal-related satiety signals there is also evidence to suggest that they interact with energy regulatory pathways and may contribute to long-term energy balance. Previous studies show that peptides that play a critical role in energy homeostasis work in concert with gastrointestinal signaling peptides to reduce food intake. Leptin, the protein product of the ob gene, is synthesized and released by adipocytes and circulates at levels proportional to body fat. Although many of the hypothalamic signaling pathways involved in leptin’s actions have been identified, the specific mechanisms mediating their effects on food intake are not clear. Several studies show that the effect of leptin on food intake is characterized by a reduction in meal size without affecting meal frequency, a behavioral profile shared by satiety peptides. A study evaluating the interaction of leptin with BN found that when a subthreshold dose of leptin administered into the 3V is combined with a peripheral injection of BN, there is a significant potentiation in the reduction of food intake. Consistent with the behavioral results, c-fos activation in the NTS is increased
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by combined dosages of leptin and BN beyond the individual response to either peptide. These results support the idea that the effects of leptin on food intake may be mediated, in part, by modulating meal-related satiety signals such as BN.25 In the brain, the preponderance of GRP and GRP receptors are found in several hypothalamic nuclei that play important roles in energy homeostasis. GRP mRNA is localized in the hypothalamic PVN in a region that receives input from neurons in the Arc. Previous studies have shown that direct bilateral PVN infusion of BN (presumably acting on GRP-R) results in a decrease in food intake and a robust increase in blood glucose, free fatty acids, and corticosterone,8 suggesting that BN binding sites in the PVN may participate in regulating circulating metabolic fuels and the hypothalamic–pituitary–adrenal (HPA) axis. Other studies show that food ingestion evokes a rapid elevation of GRP levels in various hypothalamic sites, including the PVN and Arc.23 A follow-up study determined how these changes correlate with release during different phases of ingestion and found that during food consumption there is a substantial decrease in the release of BN-like peptides in the PVN which is then followed by an increase in release during the postprandial phase. These results suggest that the decline in release during the prandial phase is conducive to meal initiation, whereas the elevation in release provides a signal for meal termination. A more recent study found that challenges that represent different nutritional states alter expression of GRP mRNA expression in the PVN.14 Food deprivation significantly decreases GRP mRNA expression in the PVN, whereas activation of the melanocortin system with the melanocortin 4 receptor (MC4-R) agonist MTII increases levels of PVN GRP mRNA and prevents the reduction in GRP mRNA induced by fasting. Further, this population of neurons respond to MTII injection, as seen by c-fos activation, and co-expresses MC4-R. These results show that food deprivation and stimulation of the melanocortin system produce opposing changes in GRP gene expression in the PVN, suggesting that GRP-containing neurons in the PVN may be part of the hypothalamic signaling pathway controlling food intake and energy balance. Electrophysiological studies have evaluated the effects of GRP and NPY on identified Arc NPY cells.32 The results indicate that nanomolar amounts of both the peptides produce strong direct excitatory effects on NPY neurons, surpassing the actions of other energy regulatory peptides such as ghrelin and orexin. Similar excitation is also seen in Arc POMC neurons. Because these two sets of neuronal populations are generally activated in opposition to one another it is surmised that BN-like peptides may function to initiate broad activation, rather than excitation or inhibition of specific cellular populations in the Arc.
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Several studies have also identified NMB as an important contributor to energy balance regulation. NMB and NMB-R are significant regulators of the HPA axis through modulation of thyrotropin secretion.27 Hoggard et al. have reported that NMB mRNA is present in human and rodent adipose tissue and that the expression levels can be changed through alterations in energy balance and leptin signaling. Neuromedin B has also been proposed as a strong candidate for gene linking eating behaviors to the susceptibility to obesity in humans.2 The Quebec Family Study, a large prospective study designed to identify the genetics of obesity and related diseases, found that a missense mutation within exon 2 of the NMB gene was significantly associated with eating behaviors and obesity, resulting in twice as much body fat gain over a six-year period compared with those without the mutation. Subjects that are homozygous for the mutation are twice as likely to exhibit high levels of disinhibition and susceptibility to hunger and to exhibit positive associations for sweet craving. As mentioned above, the physiological role for BRS-3 has been examined primarily by evaluating the consequences of BRS-3 deletion in genetically engineered mice. A recent advance in studying the role of BRS-3 in energy homeostasis has been the development of specific agonist and antagonist ligands for BRS-3.22 Experiments using a specific BRS-3 antagonist, show that chronic LV infusion results in increased food intake, body weight, and adiposity. Conversely, acute BRS-3 agonist administration reduces food intake and increases fasting metabolic rate. Chronic delivery is effective in lowering food intake and fat mass in diet-induced obese mice. Additional studies examined the interaction of BRS-3 activation with other central neural pathways that are important in energy homeostasis.22 Results obtained in a variety of KO mice (i.e., Npy(−/−), Mc4r(−/−), Cnr1(−/−) and Lepr(db/db)) indicate that BRS-3 agonists are still capable of reducing food intake in these mice and thus activation of BRS-3 seems to be independent of these pathways. BRS-3 also plays a role in body temperature regulation, but this seems to be secondary to its effects on energy metabolism. In general, results from experiments in BRS-3 KO mice and with BRS-3 agonist and antagonist compounds have led to the conclusion that BRS-3 plays a more significant role in long-term or metabolic controls of energy balance than either GRP-R or NMB-R.
ACKNOWLEDGMENT The author was supported by Grant DK-019302 from the National Institute of Diabetes, Digestive and Kidney Diseases.
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REFERENCES 1. Bonaz B, De Giorgio R, Tache Y. Peripheral bombesin induces c-fos protein in the rat brain. Brain Res 1993;600:353–7. 2. Bouchard L, Drapeau V, Provencher V, Lemieux S, Chagnon Y, Rice T, et al. Neuromedin beta: a strong candidate gene linking eating behaviors and susceptibility to obesity. Am J Clin Nutr 2004;80:1478–86. 3. Fekete EM, Bagi EE, Toth K, Lenard L. Neuromedin C microinjected into the amygdala inhibits feeding. Brain Res Bull 2007;71:386–92. 4. Flynn FW. Bombesin-like peptides in the regulation of ingestive behavior. Ann N Y Acad Sci 1994;739:120–34. 5. Flynn FW. Fourth ventricular injection of selective bombesin receptor antagonists facilitates feeding in rats. Am J Physiol 1993;264:R218–21. 6. Gibbs J, Smith GP. The actions of bombesin-like peptides on food intake. Ann N Y Acad Sci 1988;547:210–6. 7. Gonzalez N, Moody TW, Igarashi H, Ito T, Jensen RT. Bombesinrelated peptides and their receptors: recent advances in their role in physiology and disease states. Curr Opin Endocrinol Diabetes Obes 2008;15:58–64. 8. Gunion MW, Tache Y. Bombesin microinfusion into the paraventricular nucleus suppresses gastric acid secretion in the rat. Brain Res 1987;422:118–28. 9. Hostetler AM, McHugh PR, Moran TH. Bombesin affects feeding independent of a gastric mechanism or site of action. Am J Physiol 1989;257:R1219–24. 10. Jensen RT, Battey JF, Spindel ER, Benya RV. International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states. Pharmacol Rev 2008;60:1–42. 11. Kirkham TC, Gibbs J, Smith GP. Satiating effect of bombesin is mediated by receptors perfused by celiac artery. Am J Physiol 1991;261:R614–8. 12. Kulkosky PJ, Gibbs J, Smith GP. Feeding suppression and grooming repeatedly elicited by intraventricular bombesin. Brain Res 1982;242:194–6. 13. Kyrkouli SE, Stanley BG, Leibowitz SF. Bombesin-induced anorexia: sites of action in the rat brain. Peptides 1987;8:237–41. 14. Ladenheim EE, Behles RR, Bi S, Moran TH. Gastrin-releasing peptide messenger ribonucleic acid expression in the hypothalamic paraventricular nucleus is altered by melanocortin receptor stimulation and food deprivation. Endocrinology 2009;150:672–8. 15. Ladenheim EE, Hamilton NL, Behles RR, Bi S, Hampton LL, Battey JF, et al. Factors contributing to obesity in bombesin receptor subtype3-deficient mice. Endocrinology 2008;149:971–8. 16. Ladenheim EE, Jensen RT, Mantey SA, Moran TH. Distinct distributions of two bombesin receptor subtypes in the rat central nervous system. Brain Res 1992;593:168–78. 17. Ladenheim EE, Knipp S. Capsaicin treatment differentially affects feeding suppression by bombesin-like peptides. Physiol Behav 2007;91:36–41. 18. Ladenheim EE, Ritter RC. Capsaicin attenuates bombesin-induced suppression of food intake. Am J Physiol 1991;260:R263–6.
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19. Ladenheim EE, Taylor JE, Coy DH, Carrigan TS, Wohn A, Moran TH. Caudal hindbrain neuromedin B-preferring receptors participate in the control of food intake. Am J Physiol 1997;272:R433–7. 20. Ladenheim EE, Taylor JE, Coy DH, Moran TH. Blockade of feeding inhibition by neuromedin B using a selective receptor antagonist. Eur J Pharmacol 1994;271:R7–9. 21. Ladenheim EE, Wirth KE, Moran TH. Receptor subtype mediation of feeding suppression by bombesin-like peptides. Pharmacol Biochem Behav 1996;54:705–11. 22. Majumdar ID, Weber HC. Biology and pharmacology of bombesin receptor subtype-3. Curr Opin Endocrinol Diabetes Obes 2012;19:3–7. 23. Merali Z, McIntosh J, Anisman H. Role of bombesin-related peptides in the control of food intake. Neuropeptides 1999;33:376–86. 24. Minamino N, Kangawa K, Matsuo H. Neuromedin B and neuromedin Ca. Two mammalian bombesin-like peptides identified in porcine spinal cord and brain. Ann N Y Acad Sci 1988;547:373–90. 25. Moran TH, Aja S, Ladenheim EE. Leptin modulation of peripheral controls of meal size. Physiol Behav 2006;89:511–6. 26. Ohki-Hamazaki H, Iwabuchi M, Maekawa F. Development and function of bombesin-like peptides and their receptors. Int J Dev Biol 2005;49:293–300. 27. Oliveira KJ, Cabanelas A, Veiga MA, Paula GS, Ortiga-Carvalho TM, Wada E, et al. Impaired serum thyrotropin response to hypothyroidism in mice with disruption of neuromedin B receptor. Regul Pept 2008;146:213–7. 28. Paula GS, Souza LL, Cabanelas A, Bloise FF, Mello-Coelho V, Wada E, et al. Female mice target deleted for the neuromedin B receptor have partial resistance to diet-induced obesity. J Physiol 2010;588:1635–45. 29. Porcher C, Juhem A, Peinnequin A, Bonaz B. Bombesin receptor subtype-3 is expressed by the enteric nervous system and by interstitial cells of Cajal in the rat gastrointestinal tract. Cell Tissue Res 2005;320:21–31. 30. Rowland NE, Crews EC, Gentry RM. Comparison of Fos induced in rat brain by GLP-1 and amylin. Regul Pept 1997;71:171–4. 31. Stratford TR, Gibbs J, Smith GP. Simultaneous administration of neuromedin B-10 and gastrin-releasing peptide(1–27) reproduces the satiating and microstructural effects of bombesin. Peptides 1996;17:107–10. 32. van den Pol AN, Yao Y, Fu LY, Foo K, Huang H, Coppari R, et al. Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong Renilla green fluorescent protein in NPY neurons. J Neurosci 2009;29:4622–39. 33. Vigh J, Lenard L, Fekete E, Hernadi I. Bombesin injection into the central amygdala influences feeding behavior in the rat. Peptides 1999;20:437–44. 34. Washington MC, Sayegh AI. Gastrin releasing peptides increase Foslike immunoreactivity in the enteric nervous system and the dorsal vagal complex. Peptides 2011;32:1600–5. 35. Wright SA, Washington MC, Garcia C, Sayegh AI. Gastrin releasing peptide-29 requires vagal and splanchnic neurons to evoke satiation and satiety. Peptides 2012;33:125–31.
Bombesin Ellen E. Ladenheim
ABSTRACT Bombesin (BN), a tetradecapeptide originally isolated from anuran skin, belongs to a large family of structurally related amphibian and mammalian peptides. Since their discovery, BN and two mammalian BN-like peptides, gastrin-releasing peptide (GRP), and neuromedin B (NMB) have been shown to exhibit diverse behavioral and physiological actions in a number of species. Receptor subtypes for GRP and NMB and a third receptor subtype, BRS-3, have been identified. BN-like peptides have been shown to suppress food intake after peripheral or central administration and have been proposed as putative satiety signals. Specific agonists and antagonists have been instrumental in determining the individual contributions of BN receptor subtypes to food intake and energy homeostasis. Studies in knockout mice have provided an additional means to evaluate their physiological function. Recent studies have shown that BN-like peptides and receptors participate in long-term effects on food intake and body weight.
BACKGROUND Bombesin (BN) is a tetradecapeptide isolated in 1970 by Erspamer et al. from the skin of the European frog Bombina bombina. It is part of a large family of amphibian and mammalian peptides that are biologically active in mammals (see Bombesin-like Peptides in Amphibian/Skin Peptides section of this book). Since their discovery, BN-like peptides have been shown to affect many gastrointestinal (GI) processes including gastrin release, gastric acid secretion, pancreatic enzyme secretion, gallbladder contraction, and intestinal motor activity. Aside from their well-known effects on gastrointestinal function, BN-like peptides also participate in a number of important neuroregulatory processes such as thermoregulation, glucose homeostasis, circadian rhythms, and food intake.10 Although BN itself is not present in mammals, several peptides that are structurally homologous to BN have been detected in mammalian viscera and central nervous system (CNS). These peptides include several forms of gastrinreleasing peptide (GRP1–27, GRP1–29, and GRP18–27, also known as neuromedin C) and several forms of neuromedin B (NMB1–32, NMB2–32, and NMB23–32). Bombesin and GRP 1064
share an identical carboxyl terminal heptapeptide sequence, the minimal fragment necessary for their biological activity. NMB contains a portion of these amino acid residues but more closely resembles the amphibian peptides, ranatensin, and litorin.24 The distribution of BN/GRP-like immunoreactivity in the gastrointestinal tract is mainly confined to nerve cell bodies in the myenteric ganglia and in nerve fibers of these ganglia. Similarly, NMB-like immunoreactivity is found throughout nerve fibers in the gastrointestinal tract, as well as the pancreas, spinal cord, and pituitary gland. In the CNS, immunohistochemical mapping of GRP and NMB, as well as mRNA studies, have shown distinct and discrete distribution of the two peptides in rat brain. GRP mRNA is highly expressed in the isocortex, hippocampal formation, anterior olfactory nucleus, and several hypothalamic nuclei. NMB mRNA expression is most prominent in the olfactory bulb, dentate gyrus, and dorsal root ganglion.7 Because of structural similarities between BN and GRP it was initially believed that GRP was the mammalian counterpart to BN and thus all BN binding sites in the periphery and brain represented GRP receptors. Early work evaluating the effect of BN-like peptides on smooth muscle contraction found that they displayed varying degrees of potency suggesting the existence of multiple receptor subtypes. Subsequent studies by von Schrenck et al.10 identified and characterized two pharmacologically distinct BN receptor subtypes in pancreatic acinar cells and esophageal muscularis mucosa. BN binding sites in the pancreas displayed a high affinity for GRP and BN and a low affinity for NMB, whereas esophageal BN binding sites had a high affinity for NMB and BN and a low affinity for GRP. Because of these pharmacological characteristics esophageal BN receptors were termed “NMB-preferring” or BB1 and pancreatic BN receptors were termed “GRP-preferring” or BB2. BN has a high and equal affinity for NMB-R and GRP-R, and thus, its activity may be attributed to binding to either or both receptor subtypes. These receptors were later cloned, and an additional mammalian BN receptor subtype, BN receptor subtype-3 (BRS-3 or BB3) was identified through homology screening and found to Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00142-1 Copyright © 2013 Elsevier Inc. All rights reserved.
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share approximately 50% structural identity with GRP-R and NMB-R. Unlike GRP-R and NMB-R, BRS-3 has a low affinity for all naturally occurring BN-like peptides. To date, no endogenous ligand for BRS-3 has been identified and it is termed an “orphan” receptor.10 Receptors for BN-like peptides have been identified in a variety of tissues in the periphery and central nervous system. Using an approach similar to that used to characterize peripheral BN receptor subtypes, initial studies evaluated the relative ability of BN, GRP, and NMB to inhibit binding of radioactively labeled BN in rat brain. These studies revealed two distinct binding patterns consistent with those reported in peripheral tissues. Subsequent autoradiographic mapping studies determined that the predominant BN receptor subtype in the rat brain is NMB-R with the highest densities in the anterior olfactory nucleus, nucleus accumbens, and thalamus. The distribution of GRP-R is more limited with the highest densities in several hypothalamic nuclei, basal ganglia and central nucleus of the amygdala.16 The results of the binding experiments were in general agreement with in situ hybridization studies examining expression patterns of GRP-R and NMB-R mRNA. Localization of BRS-3 mRNA was initially reported to be much more limited with the highest expression levels in the arcuate (Arc), dorsomedial (DMH), ventromedial (VMH), and paraventricular (PVN) nuclei of the hypothalamus. A later study using an antibody directed toward BRS-3 found a much wider distribution in the CNS with particularly strong immunoreactivity in the cerebral cortex, hippocampal formation, hypothalamus, and thalamus.7 In the periphery, BN binding sites in the stomach were similarly differentiated where the majority of binding was GRP-R. In gastric regions, BN binding sites were localized to the circular muscle layer with the highest density in the gastric fundus. In primates, the highest levels of NMB-R mRNA were detected in the testis and stomach, whereas in rodent peripheral tissue, significant NMB-R expression was found in esophagus, intestine, testis, and uterus.7 Immunohistochemical evaluation of gastrointestinal BRS-3 distribution demonstrated BRS-3-like immunoreactivity at all levels of the gastrointestinal tract primarily in neurons of the myenteric and submucosal ganglia and in fibers in the longitudinal and circular muscle layers.29 In general, the distinct distributions of BN-like peptides and receptors support the view that they function as separate regulatory agents in the periphery and CNS.
PERIPHERAL EFFECTS OF BN-LIKE PEPTIDES ON FOOD INTAKE A role for BN as a putative satiety signal stems from experiments showing that exogenous administration of BN dose dependently suppresses food intake in a variety of experimental paradigms, and in a number of animal species, including humans. The inhibition of food intake produced
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by peripherally administered BN is followed by a normal postprandial sequence of behaviors that are characteristic of meal ingestion. The reduction of food intake by BN is not accompanied by behavioral signs of toxicity, such as sickness or malaise, nor are there nonspecific effects on water intake. The decrease in meal size produced by BN is consistent with a behavioral profile that indicates an increase in post-ingestive feedback signaling. Studies in human subjects have demonstrated that intravenous administration of BN reduced intake of a test meal without producing significant side effects.6 Although an increase in meal-related plasma BN-like immunoreactivity has been reported, the general consensus is that BN’s actions on food intake are mediated through neurocrine or paracrine mechanisms. Supporting this view are studies demonstrating an increase in BN-like immunoreactivity in the antral region of the stomach in response to meal intake, coupled with the localization of BN-like immunoreactivity in gut neurons.23 BN has an equal and high affinity for both GRP and NMB receptors. The discovery that BN interacted equally with GRP and NMB receptors presented the possibility that BN was mimicking the actions of GRP and NMB but that both GRP and NMB produced their effects on food intake at distinct receptor populations. A comparison of feeding suppression produced by peripheral administration of BN, GRP1–27, GRP18–27 and NMB in rats showed that, at intermediate doses, the rank order of potency is BN > GRP1–27 = GRP18–27 > NMB. However, at the lowest and highest doses, BN, GRP1–27, and GRP18–27 are equivalent. The biphasic nature of the dose response curve for GRP1–27 and GRP18–27 suggested that these peptides may be interacting with GRP-R at low doses and with both GRP-R and NMB-R at high doses. NMB is consistently less potent than BN in reducing food intake, suggesting that it is interacting only with NMB-R. A more recent study examining the satiety effects of GRP1–29 in rats (the predominant form in this species) found equivalent potency to GRP1–27. It is believed that the increased potency of BN to reduce food intake, compared with its mammalian counterparts, partially results from a relative resistance to enzymatic degradation. Supporting this idea are studies showing that acetylation of GRP18–27, which improves resistance to degradation, increased its ability to reduce feeding, making it equivalent to that of BN.21 The notion that the effects of BN on food intake may result from the combined action at both receptor subtypes is also supported by studies showing that peripheral administration of equimolar doses of GRP and NMB reduces food intake and elicits a microstructural licking pattern equivalent to that of BN.31 Moreover, when a dose of NMB that produces a maximal suppression of food intake is given in combination with a submaximal dose of GRP, the reduction in food intake is significantly enhanced beyond suppression
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produced by either peptide administered alone.21 Additional studies show that a specific NMB-R receptor antagonist blocks feeding inhibition by peripherally administered NMB, but not by GRP, suggesting that NMB and GRP reduce food intake by acting at distinct receptor populations.20 Although several studies found that peripherally administered GRP-R antagonists prevent the suppression of food intake by peripherally administered BN, attempts to increase food intake using GRP-R antagonists, or to block feeding suppression produced by intragastric loads, have been inconsistent.4,23 The reason for this is unresolved; however, it is possible that the antagonists have limited potency or bioavailability and are unable to access sites that are activated by endogenously released BN-like peptides.
CENTRAL EFFECTS OF BN-LIKE PEPTIDES ON FOOD INTAKE The presence of BN-like immunoreactivity and BN binding sites in brain regions implicated in controlling food intake suggested that central BN-like peptides may also play a role in satiety. Several early studies showed that BN administered into the lateral cerebral ventricles (LV) in rats produces a potent suppression of food intake.12 However, the reduction in food intake is accompanied by a decrease in water intake and an increase in grooming and locomotion, suggesting a nonspecific effect of BN on feeding that is dissimilar to the actions of peripherally administered BN. Subsequently, studies were undertaken to examine the specific brain sites where BN may produce its anorexic effects. Early studies using site specific injections of high doses of BN into various hypothalamic nuclei including the PVN, DMH, VMH, and lateral hypothalamus (LH) significantly reduced food intake, as did injections into extrahypothalamic areas such as the amygdala and periaqueductal gray. However, in all instances, feeding suppression was accompanied by increased grooming activity suggesting that the reduction in food intake may be secondary to competing behaviors.13 Other experiments using smaller BN doses found that direct injections into the LH and amygdala specifically reduce food intake without producing competing behavioral effects.6,33 Initial studies focused primarily on forebrain mediation of BN satiety; however the presence of binding sites for BNlike peptides and BN-like immunoreactive nerve terminals and cell bodies in hindbrain regions known to participate in ingestive control (e.g., nucleus of the solitary tract (NTS)) suggest that this region may be responsive to BN’s feeding effects. Several investigators found that injections of BN into the fourth cerebral ventricle (4V), in close proximity to the NTS, are highly effective in reducing food intake in rats4 The doses required to suppress feeding after 4V administration are up to 100 times lower than those that elicit reduced
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food intake after LV administration. Further, this action is specific for food and does not produce a conditioned taste aversion or elicit competing behaviors such as grooming and locomotor activity that are associated with injection at other brain sites. Studies evaluating the effect of direct microinjections of BN into the NTS found this brain region to be particularly sensitive for feeding inhibition by BN.23 Although several studies have examined the feeding effects of centrally administered BN receptor agonists, a systematic comparison across peptides has not been conducted. It is assumed that the order of potency is BN>GRP>NMB based on studies examining each peptide individually or comparing one peptide against BN using a limited range of doses. Although the majority of BN binding sites in the vicinity of the 4V are NMB-R, studies using specific GRP-R antagonists have identified the contribution of hindbrain GRP-R to the feeding inhibitory actions of BN. Two reports showed that pretreatment with selective GRP-R antagonists facilitates feeding in sated rats, supporting a role for endogenously released GRP in reducing food intake.5,23 Injections of GRP1–27 and GRP18–27 into the central amygdala have also been reported to transiently decrease food intake without producing changes in body temperature or stereotypic behaviors.3 Relatively little attention has been given to the role of central NMB-R in food intake. Agonist studies have not been particularly informative because central NMB exhibits very poor potency to inhibit feeding. However a role for hindbrain NMB-R in ingestive control was determined in studies using a specific NMB-R antagonist.19 As shown with GRP-R antagonists, 4V administration of an NMB-R antagonist alone also increases food intake and blocks the effects of centrally administered BN. In addition, 4V administration of GRP-R and NMB-R antisense oligonucleotides prevents the suppression of food intake by centrally administered BN, supporting a role for both receptor subtypes in mediating BN’s central feeding effects.23
NEURAL PATHWAYS MEDIATING THE EFFECTS OF BN-LIKE PEPTIDES Several studies have been conducted to determine the site of action and neural substrates underlying the satiety actions of peripheral and central BN-like peptides. Early work evaluating the effect of adrenalectomy, hypophysectomy, vagotomy, and lesions of the ventromedial and dorsomedial hypothalamus reported that none of these surgical manipulations were effective in preventing BN’s ability to suppress food intake.6 By contrast, Stuckey et al. examined the effect of individual or combined lesions of the afferent innervation of the GI tract and found that, while vagotomy alone fails to reduce BN satiety, combined bilateral subdiaphragmatic vagotomy, spinal cord transaction (T6), and dorsal rhizotomy (T1–T6) significantly attenuate the ability of peripheral
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BN to reduce food intake.6 A subsequent study found that rhizotomy and spinal cord section, in the absence of vagotomy, also attenuate BN’s feeding effects. More recently, it was shown that vagotomy combined with splanchnic afferent and efferent denervation of the gut lessen the effects of GRP1–29 on meal size in rats.35 To more clearly define the pathways through which BNlike peptides exert their satiety actions, studies were undertaken to evaluate the effects of capsaicin on the suppression of food intake produced by BN. Capsaicin, a derivative of red pepper, is a neurotoxin that selectively damages a subset of primary sensory fibers that include both vagal and spinal neurons. Adult rats treated systemically with capsaicin exhibit a significant attenuation in the feeding inhibitory response to peripheral BN administration.18 A subsequent report showed that neonatal capsaicin treatment, which produces a more complete deafferentation, completely abolishes peripheral BN’s feeding effects in adult rats without affecting the inhibition of food intake produced by third ventricular (3V) administration.23 Because of the distinct roles of BN-like peptides to reduce food intake, additional studies were conducted in adult rats treated neonatally with capsaicin to examine whether different neural substrates underlie the feeding suppression by BN, GRP, and NMB.17 The suppression of food intake by peripheral administration of BN and NMB is abolished or attenuated by capsaicin treatment, whereas the effects of GRP remained intact except at the highest dose. These results suggest that BN-like peptides use distinct neural pathways to reduce feeding. The presence of gastric BN binding sites and changes in gastrointestinal BN-like immunoreactivity in response to meal intake led to the proposal that BN may produce its effects on food intake through a gastric mechanism, although evidence to support this has been equivocal. In support of a gastric site of action for BN, it was shown that local celiac artery infusions of BN are much more effective in suppressing food intake than either intraperitoneal or superior mesenteric artery infusions.11 However, another experiment that compared the feeding effects of various doses of BN with the gastric inhibitory effects of BN, concluded that BN’s satiety actions are dissociated from its effects on gastric emptying, as is the potency of BN to reduce food intake compared with its relative affinity for gastric receptors.9 These results suggest that BN’s effects on food intake are not mediated by a gastric site or via a gastric inhibitory mechanism. Although BN-like peptides have been shown to excite mechanosensitive gastric branch vagal afferents, electrophysiological studies have determined that this is an indirect action secondary to its stimulation of gastric motor effects, consistent with the lack of BN binding sites in the vagus nerve. The neuronal pathways through which peripheral and central BN-like peptides exert their effects on food intake have been also been examined by evaluating patterns of
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c-fos immunoreactivity, a marker of neuronal activation. Peripheral injection of BN induces c-fos in the area postrema (AP), medial NTS, parvocellular PVN, and to a lesser extent in central nucleus of amygdala, parabrachial nucleus, supraoptic, and magnocellular PVN. A comparison between peripheral and central (4V) BN administration found that both routes of administration show strong fos-LI in the PVN. Fos-like immunoreactivity was also detected in the medial NTS, central nucleus of amygdala, and bed nucleus of the stria terminalis, though the regional distribution differs depending on the route of administration. Differences were also found in the AP where peripheral, but not central, administration induced c-fos activation.1,30 A more recent study compared the effects systemic administration of different forms of GRP on c-fos activation in the gastrointestinal tract and hindbrain. Myenteric neurons in the stomach and neurons in the area postrema are activated by GRP18–27, GRP1–27, and GRP1–29. In the duodenum, both GRP1–27 and GRP1–29 increase c-fos activation in the myenteric plexus, whereas only GRP1–29 increases fos-LI in the submucosal plexus.34 The necessity of central BN-like peptide receptors to the peripheral effects of BN-like peptides has also been examined. Merali et al. (1988) first reported that 3V infusion of BN antisera, or a GRP-R antagonist, blocks the suppression of food intake by peripheral BN administration. Other studies have shown that ablation of the AP/NTS complex or blockade of hindbrain GRP receptors with 4V GRP-R antagonists attenuate the ability of peripherally administered BN or GRP to suppress feeding.23 Converging behavioral and anatomical evidence indicates that the NTS is a critical region for the feeding effects of BN. This region receives sensory input from the gastrointestinal tract via vagal and spinal neurons. Within the NTS, BN-ir is located in the medial subnucleus, an area that is associated with nerve terminals that synapse on cell bodies or dendrites of vagal motoneurons. Compelling evidence for the importance of the caudal hindbrain to BN’s feeding effects is provided in experiments showing that either peripheral or 4V injections of BN reliably suppress sucrose intake in chronic decerebrate rats, a surgical preparation that severs communication between the hindbrain and forebrain. These results support the idea that local neural circuitry residing in the caudal hindbrain, independent of forebrain systems, is sufficient to mediate the effects of BN-like peptides on ingestive behavior.4 Although the necessity of central neural substrates, particularly in the NTS, play a critical role in BN’s effects on food intake there are still questions remaining concerning how the signal is transmitted. Whether peripheral administration of BN-like peptides causes a central release of GRP (or NMB) or whether they gain direct access to critical hindbrain receptors has not been fully determined.
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CONTRIBUTION OF BN RECEPTOR SUBTYPES TO FOOD INTAKE USING GENETICALLY MODIFIED MICE The development of mice with targeted deletions for each BN receptor subtype has provided a complementary means to evaluate the individual roles of BN receptor subtypes to ingestive control and other physiological effects of BN-like peptides.26 The first of these to be developed are mice lacking the GRP-R. In studies to evaluate the overall contribution of GRP-R to food intake, GRP-R KO mice fail to suppress intake after exogenously administered of either BN or GRP, suggesting that GRP-R alone mediates the satiety effect of BN-like peptides in mice. An analysis of 24-h meal patterns reveals that GRP-R KO mice exhibit an increase in meal size consistent with a deficit in satiety signaling, but 24-h food consumption is not different from control mice because of a reduction in the total number of meals consumed. A long-term analysis of body weight found that body weights between GRP-R KO mice and WT controls are comparable in early life although a small but significant elevation in body weight occurs in aged GRP-R KO mice. Additional studies show GRP-R deficient mice display increased nighttime locomotor activity, loss of GRP-induced hypothermia, and increased social investigatory behaviors. Metabolic disturbances, including decreased secretion of glucagon-like peptide 1 and abnormalities in insulin release and glucose tolerance, are also present in GRP-R KO mice. Unlike GRP-R KO mice, previous studies in mice lacking NMB-R (NMB-R KO) found no differences in food intake and body weight between NMB-R KO and WT control mice. However, a subsequent study showed that although female NMB-R KO mice fed a chow diet show no differences in body weight, when presented with a high fat diet they are partially protected against body weight gain and adipose accumulation and exhibit normal glucose tolerance compared with that of WT littermates.28 Recent attention has been directed toward the role of BRS-3 in food intake and energy homeostasis. Progress in determining the physiological function of BRS-3 was hindered by the lack of an identifiable natural ligand or a suitable synthetic ligand. To assess the function of BRS-3, Ohki-Hamazaki and colleagues generated mice with a targeted deletion of BRS-3 (BRS-3 KO) and found that they develop obesity accompanied by hypertension, impairment of glucose tolerance, and insulin resistance. In addition, BRS-3 KO mice exhibit reduced metabolic rate, increased feeding efficiency and hyperphagia, and showed alterations in glucose transport.26 Additional behavioral and physiological studies were conducted to identify factors contributing to increased food consumption and body weight in BRS-3 KO mice.15 A primary deficit leading to the obese phenotype of BRS-3 deficient
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mice is an uncompensated increase in meal size resulting in an overall increase in total food intake. Although the increase in body weight could be overcome by pair-feeding to the caloric intake of WT controls, they still exhibit significantly elevated leptin concentrations and body fat distribution, confirming a metabolic dysregulation in BRS-3 KO mice. Despite the finding that BRS-3 KO mice increase their meal size, they exhibit a normal response to peripherally administered peptides (e.g., cholecystokinin and BN) that participate in meal-related controls of food intake. A study evaluating the effects of centrally administered peptides reported that mice lacking BRS-3 are leptin resistant, hyperresponsive to the orexigenic effects of exogenously administered melanin concentrating hormone (MCH), and have elevated levels of MCH mRNA in the lateral hypothalamus. Studies have also suggested that hyper-responsiveness to taste stimuli may contribute to overeating in BRS-3 deficient mice particularly when exposed to highly palatable foods.26 BRS-3 KO mice exhibit a stronger taste preference for saccharin solution and a stronger aversion to quinine solution compared with that of WT littermates. Using a conditioned taste aversion paradigm, BRS-3 KO mice display a stronger aversion to both saccharin and sodium chloride solutions after intraperitoneal lithium chloride administration. A preliminary study reported exaggerated hyperphagia and weight gain in BRS-3 KO mice maintained on a high fat diet, a characteristic that is consistent with other mouse models of obesity.
BN-LIKE PEPTIDES/RECEPTORS AND ENERGY HOMEOSTASIS Although a major focus for determining a role for GRP and NMB in ingestive control has been to evaluate their function as short-term or meal-related satiety signals there is also evidence to suggest that they interact with energy regulatory pathways and may contribute to long-term energy balance. Previous studies show that peptides that play a critical role in energy homeostasis work in concert with gastrointestinal signaling peptides to reduce food intake. Leptin, the protein product of the ob gene, is synthesized and released by adipocytes and circulates at levels proportional to body fat. Although many of the hypothalamic signaling pathways involved in leptin’s actions have been identified, the specific mechanisms mediating their effects on food intake are not clear. Several studies show that the effect of leptin on food intake is characterized by a reduction in meal size without affecting meal frequency, a behavioral profile shared by satiety peptides. A study evaluating the interaction of leptin with BN found that when a subthreshold dose of leptin administered into the 3V is combined with a peripheral injection of BN, there is a significant potentiation in the reduction of food intake. Consistent with the behavioral results, c-fos activation in the NTS is increased
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by combined dosages of leptin and BN beyond the individual response to either peptide. These results support the idea that the effects of leptin on food intake may be mediated, in part, by modulating meal-related satiety signals such as BN.25 In the brain, the preponderance of GRP and GRP receptors are found in several hypothalamic nuclei that play important roles in energy homeostasis. GRP mRNA is localized in the hypothalamic PVN in a region that receives input from neurons in the Arc. Previous studies have shown that direct bilateral PVN infusion of BN (presumably acting on GRP-R) results in a decrease in food intake and a robust increase in blood glucose, free fatty acids, and corticosterone,8 suggesting that BN binding sites in the PVN may participate in regulating circulating metabolic fuels and the hypothalamic–pituitary–adrenal (HPA) axis. Other studies show that food ingestion evokes a rapid elevation of GRP levels in various hypothalamic sites, including the PVN and Arc.23 A follow-up study determined how these changes correlate with release during different phases of ingestion and found that during food consumption there is a substantial decrease in the release of BN-like peptides in the PVN which is then followed by an increase in release during the postprandial phase. These results suggest that the decline in release during the prandial phase is conducive to meal initiation, whereas the elevation in release provides a signal for meal termination. A more recent study found that challenges that represent different nutritional states alter expression of GRP mRNA expression in the PVN.14 Food deprivation significantly decreases GRP mRNA expression in the PVN, whereas activation of the melanocortin system with the melanocortin 4 receptor (MC4-R) agonist MTII increases levels of PVN GRP mRNA and prevents the reduction in GRP mRNA induced by fasting. Further, this population of neurons respond to MTII injection, as seen by c-fos activation, and co-expresses MC4-R. These results show that food deprivation and stimulation of the melanocortin system produce opposing changes in GRP gene expression in the PVN, suggesting that GRP-containing neurons in the PVN may be part of the hypothalamic signaling pathway controlling food intake and energy balance. Electrophysiological studies have evaluated the effects of GRP and NPY on identified Arc NPY cells.32 The results indicate that nanomolar amounts of both the peptides produce strong direct excitatory effects on NPY neurons, surpassing the actions of other energy regulatory peptides such as ghrelin and orexin. Similar excitation is also seen in Arc POMC neurons. Because these two sets of neuronal populations are generally activated in opposition to one another it is surmised that BN-like peptides may function to initiate broad activation, rather than excitation or inhibition of specific cellular populations in the Arc.
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Several studies have also identified NMB as an important contributor to energy balance regulation. NMB and NMB-R are significant regulators of the HPA axis through modulation of thyrotropin secretion.27 Hoggard et al. have reported that NMB mRNA is present in human and rodent adipose tissue and that the expression levels can be changed through alterations in energy balance and leptin signaling. Neuromedin B has also been proposed as a strong candidate for gene linking eating behaviors to the susceptibility to obesity in humans.2 The Quebec Family Study, a large prospective study designed to identify the genetics of obesity and related diseases, found that a missense mutation within exon 2 of the NMB gene was significantly associated with eating behaviors and obesity, resulting in twice as much body fat gain over a six-year period compared with those without the mutation. Subjects that are homozygous for the mutation are twice as likely to exhibit high levels of disinhibition and susceptibility to hunger and to exhibit positive associations for sweet craving. As mentioned above, the physiological role for BRS-3 has been examined primarily by evaluating the consequences of BRS-3 deletion in genetically engineered mice. A recent advance in studying the role of BRS-3 in energy homeostasis has been the development of specific agonist and antagonist ligands for BRS-3.22 Experiments using a specific BRS-3 antagonist, show that chronic LV infusion results in increased food intake, body weight, and adiposity. Conversely, acute BRS-3 agonist administration reduces food intake and increases fasting metabolic rate. Chronic delivery is effective in lowering food intake and fat mass in diet-induced obese mice. Additional studies examined the interaction of BRS-3 activation with other central neural pathways that are important in energy homeostasis.22 Results obtained in a variety of KO mice (i.e., Npy(−/−), Mc4r(−/−), Cnr1(−/−) and Lepr(db/db)) indicate that BRS-3 agonists are still capable of reducing food intake in these mice and thus activation of BRS-3 seems to be independent of these pathways. BRS-3 also plays a role in body temperature regulation, but this seems to be secondary to its effects on energy metabolism. In general, results from experiments in BRS-3 KO mice and with BRS-3 agonist and antagonist compounds have led to the conclusion that BRS-3 plays a more significant role in long-term or metabolic controls of energy balance than either GRP-R or NMB-R.
ACKNOWLEDGMENT The author was supported by Grant DK-019302 from the National Institute of Diabetes, Digestive and Kidney Diseases.
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