Gastrointestinal mechanisms of satiation for food

Transcription

1 Physiology & Behavior 81 (2004) Gastrointestinal mechanisms of satiation for food Robert C. Ritter Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, and Programs in Neuroscience, College of Veterinary Medicine, Washington State University, Pullman, WA , USA Abstract Satiation for food comprises the physiological processes that result in the termination of eating. Satiation is evoked by physical and chemical qualities of ingested food, which trigger afferent signals to the brain from multiple sites in the GI tract, including the stomach, the proximal small intestine, the distal small intestine and the colon. The physiological nature of each signal s contribution to satiation and overall control of food intake is likely to vary, depending on the level of the GI tract from which the signal arises. This article is a critical, though non-exhaustive, review of our current understanding of the mechanisms and adaptive value of satiation signals from the stomach and intestine. D 2004 Elsevier Inc. All rights reserved. Keywords: Gastrointestinal mechanism; Satiation; Food 1. Introduction The conviction that the gastrointestinal tract controls appetite for food is rooted in antiquity. It probably predates literary and artistic references to hunger and satiation that appear early in written historical records and persists in popular culture today. The potent and persistent association between food intake and the gastrointestinal tract derives from the fact that the gastrointestinal tract is highly innervated by sensory neurons and refers robust sensations, which are directly related to physical and chemical stimulation by ingested food. Some of the earliest experimental attempts to link the gastrointestinal tract with control of food intake were undertaken by W.B. Cannon and his student Washburn (1912). Cannon and Washburn recorded and correlated the strength of gastric contractions with conscious sensations that Cannon termed hunger pangs. While Cannon s efforts focused on the role of the gastrointestinal tract in the sensation of hunger, the desire to seek and eat food, he was acutely aware of the process of satiation. In his monograph, The wisdom of the body [229], he concluded: Cooperating with hunger and thirst in a way not yet clearly defined is the sensation of having had enough. Protection of the organism against being overstocked with food and water is thus obtained. The feeling of address: britter@vetmed.wsu.edu (R.C. Ritter). satiation is little understood, but it is important and deserves further attention. Couched as it is in his discourse on the role of the gastrointestinal tract in the sensation of hunger, three messages can be drawn from Cannon s statement. First, satiation derives, at least in part, from gastrointestinal signals. Second, satiation serves a protective function. And, third the mechanisms by which satiation occurs are incompletely understood. The situation has changed markedly in the last two decades, during which time satiation has received quite a lot of attention. Much of this attention has been focused on the role of the gastrointestinal tract in providing signals that control the process of satiation, a process which manifests itself in meal termination. In his use of the term feeling of satiation Cannon seems to imply that satiation involves conscious awareness of feedback signals, or at least an affective response to them. Indeed, studies of human subjects commonly assess the sensation of fullness using an analogue rating scale. However, there is no direct evidence that a conscious awareness of gastrointestinal feedback signals is necessary for satiation. In fact, gastrointestinal signals can control meal size in rats even in the absence of forebrain structures, which seem essential for sensory-perceptual function and affect. For example, rats that have been decerebrated at the collicular level exhibit satiation [90,92], and even display taste reactivity patterns that are associated with positive and negative gustatory properties of foods [91]. Nevertheless, few would argue that other components of conscious sensation or affect are pre /$ see front matter D 2004 Elsevier Inc. All rights reserved. doi: /j.physbeh

2 250 R.C. Ritter / Physiology & Behavior 81 (2004) served in the decerebrate preparation. Thus, gastrointestinal feedback to the caudal brainstem is sufficient for satiation. However, the participation of forebrain structures associated with feelings and conscious perception in satiation remains to be systematically investigated. The most compelling experimental evidence for GI involvement in satiation is that the removal of ingesta from the gastrointestinal tract during the ingestion of a meal increases food intake. In the mid- to late 1800s, human case reports indicated that people with gastric or intestinal fistulas remained hungry when most of the food they ate drained from the upper GI tract [30]. In 1895, Shumova- Simonovskia and Pavlov reported that dogs with experimental esophageal fistulas ate continuously, suggesting that stimuli for satiation were absent or attenuated [159]. More recent experiments, using chronically implanted esophageal [146] or gastric [81] cannulas, have enabled better control of the nutritional condition of experimental subjects, while providing temporal and kinetic information that convincingly demonstrates inhibition of food intake by the gastrointestinal tract. For example, animals in good nutritional condition, with chronically implanted gastric cannulas, eat continuously on test days when food is allowed to drain from the cannula. However, the same animals terminate ingestion rapidly when the cannula is closed, allowing ingesta to fill the stomach and empty to the small intestine [54], suggesting that the stomach and/or intestine provide signals that contribute to satiation for food. In this review, I intend to focus first on the locations from which the gastrointestinal sensory signals that contribute to satiation arise and on the physical/chemical nature of these signals. I also will discuss the potential mechanisms by which the signals are communicated to the central nervous system. Finally, I will conclude with recent results suggesting that sensitivity to intestinal signals that lead to satiation adapt in response to dietary conditions, and how this adaptation may be consistent with a protective function of satiation, vis-a-vi the gastrointestinal tract. The review only considers processes originating from the stomach and the small intestine and does not deal with potentially important signals that might arise pregastrically or from the large intestine or liver. Finally, I have attempted a fairly broad coverage, and no single area is exhaustively covered. With regard to the mechanisms and humoral mediators of satiation, ignorance far exceeds knowledge. In this area, I have taken the liberty of speculating on what seem to me promising leads. 2. Gastric satiation signals A variety of experimental results indicate that the stomach provides some of the inhibitory signals that participate in satiation. The stomach receives extensive sensory innervation from the vagus nerve [168] and spinal afferents via the splanchnic innervation [152]. In the rat topographical examination of anterogradely labeled vagal afferent, endings suggest that the stomach is the most heavily innervated visceral organ [232] and a prime candidate for GI monitoring of ingestion Extrinsic sensory innervation of the stomach The structure of the gastric vagal sensory innervation has been studied extensively by Berthoud, Powley and their colleagues [16]. Using anterograde tracers and confocal microscopy, they have described gastric vagal sensory endings with several distinct morphologies. Intraganglionic laminar endings (IGLEs) constitute vagal sensory nerve endings that are closely intercalated with neurons of the myenteric ganglia [155,186], while intramuscular arrays (IMAs) are extraganglionic endings imbedded in the smooth muscle of gastrointestinal organs including the stomach [16]. Based on their distribution and structure, Berthoud and Powley [16] have suggested that IGLEs may be in series tension receptors. In other words, they suggest that IGLEs may serve a function similar to Golgi tendon organs in somatic muscles. IMAs, on the other hand, might be organized and distributed to detect stretch or length [167], in a manner reminiscent of the muscle spindles of somatic muscle. Recently, Zagorodnyuk and Brookes [247] and Zagorodnyuk et al. [248] have applied anterograde tracers to discrete electrophysiologically identified vagal fibers innervating the guinea pig stomach. They reported discreet receptive fields for vagal fibers that were activated by stretching the wall of the stomach with von Frey hairs. Each receptive field was closely associated with a cluster of anterogradely labeled IGLEs, whereas randomly marked areas were not. They found no association between the receptive fields of activated fibers and IMAs. Thus, it appears that the IGLEs represent at least one form of mechanosensitive vagal ending in the stomach. It is important to point out that these experiments do not provide confirmation that IGLEs are selectively sensitive to tension, as opposed to stretch. Nor do they support or refute the stretch sensitivity of IMAs. Moreover, the fact that most electrophysiological experiments have not examined or cannot distinguish gastric stretch receptors from afferents that respond as in series tension receptors suggests that important characterization of gastric afferents that monitor length or volume has largely been overlooked. These distinctions may be of cardinal importance with regard to the role of specific gastric afferents in the control of food intake because, at least, some stimuli that reduce food intake, such as cholecystokinin (CCK), are associated with decreased gastric tension, a fact that appears to fly in the face of the notion that increased tension in the gastric wall contributes to satiation. The mechanisms by which mechanical deformation activates vagal afferents have not been worked out. However, reports utilizing cultured primary sensory neurons suggest that stretch-sensitive Ca ++ channels are expressed by some vagal [214] and spinal sensory neurons [172]. In this regard, it is interesting that Zagorodnyuk et al. [248] found that

3 R.C. Ritter / Physiology & Behavior 81 (2004) vagal-afferent discharge in response to poking the gastric wall was blocked by gadolinium, which is thought to block stretch-activated calcium channels. Gastric sensory innervation is not limited exclusively to the detection of stretch or tension. Stroking of the gastric mucosa activates some vagal afferent fibers [ ]. In addition, the application of strong acid or base to the gastric mucosa also activates vagal afferents [36]. On the other hand, Ozaki and Gebhardt [156] reported that splanchnic sensory fibers innervating the stomach did not respond to the intragastric application of oleate or glucose. Finally, some gastric vagal-afferent fibers appear to be thermosensitive [65]. Little is known regarding the anatomy of these afferents. However, vagal afferent fibers have been observed in the gastric submucosa, and it seems probable that these fibers may be responsive to physical and/or chemical mucosal stimulation by ingesta Pyloric cuff experiments and modalities for gastric satiation Gastric participation in the process of satiation has been subject to extensive experimental scrutiny. Sham-feeding preparations, in which ingested food is removed from the stomach during ingestion, constitute some of the earliest experimental evidence associating the GI tract with satiation [53,54]. Indeed, there are scores of published reports demonstrating that hungry animals eat significantly longer and consume much more food when the ingesta is allowed to drain from the stomach via an open gastric cannula (sham feeding). While sham-feeding experiments suggest gastrointestinal control of food intake, they obviously do not specifically implicate the stomach as the sole source of signals that terminate feeding because opening the cannula not only prevents the stomach from filling, it also prevents or reduces the entry of ingesta to the small intestine. Likewise, loading the stomach via a gastric cannula or catheter not only fills the stomach, but also may increase the stimulation of the small intestine as the stomach empties. The issue of gastric emptying and intestinal stimulation is especially significant during a meal because Kaplan et al. [109] have shown that gastric emptying occurs at a much more rapid rate during filling (eating) than it does after filling has stopped. In fact, as much as 40% of a liquid meal leaves the stomach prior to meal termination. In other words, it appears that gastric and intestinal stimulations during a meal are virtually simultaneous. Therefore, gastric preloads actually load the intestine nearly as much as they do the stomach, and the stimulation of the stomach and intestine is virtually simultaneous. By reversibly occluding the pylorus, using a surgically implanted noose or inflatable cuff [57,166,171,201,242], it is possible to prevent the emptying of material from the stomach to the intestine. When rats are eating very large meals [55] or receiving substantial gastric preloads with the pylorus occluded [63,166], they eat less than they do with the cuff open. In other words, when the ingesta is prevented from leaving the stomach, a large meal may distend the stomach more rapidly and terminate eating sooner. However, during the ingestion of smaller, more physiological meals, rats generally eat similar amounts, regardless of whether the pylorus is occluded or patent [57,171,201]. These results suggest that signals from the stomach are sufficient to terminate ingestion, without contribution from postgastric stimuli. However, when gastric volume is measured during such manipulations, the stomachs of rats with occluded pylori contain much more ingesta at meal end than those with patent pylori [201]. In other words, gastric stimulation alone is sufficient to terminate meals, but the amount of gastric stimulation required is greater than occurs when ingesta is allowed to enter the small intestine. Thus, under normal conditions, meal termination likely results from a combination of gastric and postgastric signals. This conclusion is strengthened by the observation that rats eat less when the pylorus is occluded after the rats are allowed to eat for 20 min with the pylorus open than they do when not permitted to have the preclosure feeding period [57]. This observation contrasts with the often-replicated observation that when no pylorus-open prefeeding is allowed, rats eat the same amount with the pylorus occluded as they do when it is not occluded. Taken together, these observations suggest that postgastric stimuli may add to or enhance the stomach s contribution to the process of satiation. Electrophysiological experiments in rats [198] and results from studies in humans, in which reports of gastrointestinal sensation and feelings of hunger and fullness were obtained, are consistent with this conclusion ([68,69]; see below). Satiation produced by food in the stomach depends on mechanical rather than chemical stimulation by constituents of the meal. Phillips and Powley [166] observed that when gastric emptying is prevented by closing a pyloric cuff, loads of isotonic saline ( ml) produced volumerelated reductions of intake equivalent to comparable loads of liquid diet. In addition, they found that varying nutrient concentration, osmotic concentration or ph, also had no effect on the reduction of intake, independent of the volume infused. In a more recent study, Eisen et al. [63] reported that only large (12 ml) gastric loads significantly reduced intake by rats with the pylorus occluded. However, like Phillips and Powley [166], they found that intragastric infusions of nonnutritive saline were as effective for reducing 30-min intake as were infusions of liquid diet in pylorusoccluded rats. Eisen et al. [63] also examined the effect of their intragastric loads on 3-min food intake and on licking parameters. They found that saline and liquid diet loads reduced 3-min intake to the same extent. Interestingly, however, they observed that liquid diet preloads reduced lick rates more than did saline preloads. Thus, while the gastric signals that reduce food intake appear to be mechanical in nature, the Eisen et al. [63] results suggest that gastric chemosensation might influence the dynamics of food intake.

4 252 R.C. Ritter / Physiology & Behavior 81 (2004) Neural afferents for gastric satiation Vagal and spinal afferents and gastric satiation Surprisingly, the nature of the afferents that communicate gastric satiety signals from the gut to the brain remains obscure. Schwartz et al. [199] combined left vagal afferent rhizotomy with contralateral subdiaphragmatic vagotomy, producing animals that were devoid of vagal sensory innervation below the diaphragm. The animals exhibited increased meal size compared with sham-operated rats. However, subdiaphragmatic vagal deafferentation failed to attenuate reduction of food intake by intragastric preloads of liquid diet. It is important to remember that spinal sensory neurons supply innervation to the abdominal viscera including the stomach. Therefore, the Schwartz et al. results suggest that the reduction of food intake by intragastric preloads might be mediated, in part, by spinal afferents. Ritter and Ladenheim [183] found that rats pretreated with capsaicin to destroy small unmyelinated sensory neurons in both vagal and spinal sensory nerves did not differ from control rats with regard to the reduction of food intake by intragastric preloads. Systemic capsaicin treatment destroys a majority of both the spinal and vagal sensory innervation to the viscera, as this innervation is comprised largely of small unmyelinated C fibers. Thus, response to gastric preloads does not depend on capsaicin-sensitive vagal or spinal afferents. Capsaicin, however, does not destroy all spinal or vagal afferents. Most somatic afferents that carry information on stretch or pressure are resistant to capsaicin neurotoxicity [105], and it is clear that some visceral afferents also are insensitive to capsaicin. Indeed, Berthoud et al. [15] have observed that gastric IGLEs largely survive capsaicin treatment, although intestinal vagal sensory afferents are almost completely eliminated by capsaicin ([15,104]; Fig. 4). These observations are consistent with electrophysiological findings (Fig. 1), which demonstrated that responses to gastric distension by neurons in the dorsal vagal complex of the hindbrain are similar in capsaicin treated and control rats [184]. Therefore, it is possible that either spinal- or vagal-capsaicin-resistant afferents are sufficient to mediate the effects of intragastric preloads on reduction of food intake. Results reported for preweanling rat pups by Lorenz et al. [129] are consistent with this conclusion. They found that vagotomy increased milk consumption and attenuated the reduction of food intake by gastric preloads in 6- to 12-day old rats with closed pyloric cuffs [128]. They also found that vagotomy increased oral intake, and cervical spinal transection, combined with vagotomy, increased intake further over vagotomy alone [129]. Thus, it seems that there is experimental evidence for control of meal size by vagal and nonvagal sensory neurons. However, the specific participation of the gastric innervation in this control remains uncertain because in all results reported so far, the surgical treatments used eliminated vagal sensory innervation to all of the abdominal viscera, not just innervation of the stomach Primary afferent neurotransmitters and gastric satiation The sensory neurotransmitters involved in the response to gastric stimuli are as enigmatic as the peripheral neural substrates themselves. Neurons of the nodose and spinal sensory ganglia express a variety of putative neurotransmitters. Substances proposed as vagal sensory neurotransmitters and neuromodulators, based on immunohistochemical studies or the presence of transcripts, include glutamate [3,122,188], nitric oxide [71], acetylcholine [158], calcito- Fig. 1. Extracellular recordings from hindbrain neurons firing in response to gastric distension or near coeliac artery infusion of cholecystokinin octapeptide (CCK). Left column: records from vehicle-treated rat. This hindbrain neuron was typical of 19 units that reduced their firing rate during gastric distension. Note that cholecystokinin octapeptide (CCK-8; 7 pmol) totally inhibited firing by this neuron for 39 s. Stimulus bars under gastric distensions represent the stimulus period from 30 to 34 s. Right column: records from a capsaicin-treated rat. This hindbrain unit was typical of 15 neurons that reduced their firing rates during gastric distension. Note, however, that neither 7 pmol nor 70 pmol (data not shown) of CCK-8 altered the firing rate of this neuron. Since hindbrain neurons recorded in this study were separated by several synapses from the primary vagal afferents activated by gastric distension or CCK, these data do not indicate that the same afferent neurons respond to both distension and CCK. Indeed, the fact that capsaicin reduces responses to CCK, but not distension, suggests that gastric distension- and CCK-responsive afferents may constitute distinct populations of vagal-afferent neurons that converge on higher order neurons in the dorsal vagal complex of the hindbrain. Data traces excerpted from Ritter et al. [184].

5 R.C. Ritter / Physiology & Behavior 81 (2004) nin-gene-related peptide (CGRP; [208]), SP, galanin, CART [26] and others [59,251]. Extracellular recording from neurons in the NTS or dorsal motor nucleus during gastric stimulation support the participation of CGRP, acetylcholine and glutamate in the central processing of signals from the stomach [158]. Furthermore, injections of a plethora of peptides and other neuroactive substances into the dorsal vagal complex reduce food intake. However, there are no reports that unequivocally link specific vagal afferent neurotransmitters to control of food intake by gastric sensory stimuli. Partosoedarso and Blackshaw [158] have reported that antagonists to CGRP, M1 acetylcholine receptors and non- NMDA-type glutamate receptors attenuate the responses of the neurons of the ferret dorsal motor nucleus to gastric stimulation. In this study, the blockade of the M1 receptor selectively attenuated responses to gastric mechanoreception, suggesting that cholinergic synapses are involved in the hindbrain integration of gastric mechanoreceptive signals. However, currently available data do not support the participation of central cholinergic transmission in control of food intake by the stomach. For example Rauhofer et al. [170] have reported that meal size in rats eating with closed pyloric cuffs is not altered by atropine, suggesting that muscarinic neurotransmission is unnecessary for the control of meal size by the stomach. In addition Covasa et al. [47] found that neither systemic atropine nor infusion of atropine into the fourth cerebral ventricle increased the 30-min food intake following 16-h food deprivation. A variety of anatomical and functional data suggest that glutamate is a transmitter of some vagal sensory neurons [2,3,125,188]. In this regard, it is interesting that several groups have reported that the blockade of NMDA-type glutamate receptors increases meal size [29,223,249]. Furthermore, Treece et al. [224] demonstrated that increased food intake following systemic NMDA-receptor antagonist injection was abolished in rats with lesions of the dorsal vagal complex. In separate experiments, they also found that nanoinjection of NMDA-type antagonist into the dorsal vagal complex markedly increased meal size (Fig. 2; [223]). Covasa et al. [48] reported that NMDA receptor blockade increased gastric emptying, suggesting that gastric modulation might account for increased feeding produced by the NMDA antagonist (Fig. 3). However, they also found that NMDA receptor blockade did not increase meal size in food-deprived rats allowed to eat while their pylorus was occluded [48]. Therefore, a direct role for glutamate, acting at NMDA-type receptors in the gastric control of meal size, remains unclear and in need of further investigation. The potential role of enteric neurotransmitters in communication of gastric mechanical stimulation to vagal afferent fibers also has not been investigated. However, the anatomical association between IGLEs and enteric neurons would seem ideal for this sort of communication. Mechanical distension activates specific subpopulations of myenteric neurons in the guinea pig small intestine [182] and in myenteric neurons of the rat stomach [58]. How- Fig. 2. Increased intake of liquid food (15% sucrose) following nanoinjection of the NMDA channel blocker, MK-801, into the dorsal vagal complex. (A) Average intake of sucrose following injection of 30 nl MK-801 or saline vehicle into the dorsal vagal complex, medial to the solitary tract. (B) Average intake of sucrose following injection of 30 nl MK-801 or saline vehicle into hindbrain sites ventral or lateral to the medial portions of the dorsal vagal complex. (C) Schematic representation of cannula placements at which MK-801 injection evoked increased sucrose intake (E) and sites at which no increase in intake was evoked (). Taken from Treece et al. [223]. Injections sites are mapped onto plates from the stereotaxic atlas of Paxinos and Watson [160].

6 254 R.C. Ritter / Physiology & Behavior 81 (2004) coeliac artery reduces food intake at doses that are not effective when injected intravenously [111], suggesting that receptors in the upper gastrointestinal tract mediate bombesin s participation in satiation. On the other hand, participation of bombesin-like peptides in satiation might not be mediated entirely by neural afferents. Ladenheim and Ritter [116] found that lesions of the dorsal vagal complex attenuate reductions of food intake by both central and peripheral bombesin administration. Furthermore, a subsequent report by Ladenheim et al. [118] indicates that the blockade of hindbrain GRP receptors attenuates the reduction of food intake by peripherally administered, as well as central, GRP. The participation of centrally located bombesin receptors in the control of food intake is further supported by reports that fourth ventricle injection of either a GRP receptor antagonist [211] or a neuromedin B receptor antagonist [117] increases food intake in rats. These results suggest that endogenous bombesin-like peptides could provide an endocrine afferent that contributes to meal termination. In this regard, it also is interesting that mice lacking GRP receptors eat larger meals and gain more weight than wildtype mice do [114]. Fig. 3. Effect of intraperitoneal administration of 0.9% NaCl or MK-801 (100 Ag/kg) on volume emptied from the stomach over 10 min following a 5-ml intragastric load of either 0.9% NaCl or 15% sucrose. Data represents means F S.E.M. * Significant differences compared with intraperitoneal NaCl ( P <.01). From Covasa et al. [48]. ever, we do not know whether the activated enteric neurons have an afferent relationship to vagal sensory fibers or whether transmitters released by activated myenteric neurons activate vagal afferent fibers Paracrine and endocrine substances and gastric satiation Bombesin-related peptides The stomach, like other alimentary organs, produces a variety of signaling substances that might participate in satiation. For example, bombesin, an amphibian analogue of mammalian gastrin releasing peptide (GRP) and neuromedin B, reduce food intake when injected systemically or into the forebrain or hindbrain cerebral ventricles [79,116]. Bombesin-related peptides are localized in intrinsic neurons of the stomach [98,144] and in the nucleus of the solitary tract, where primary vagal afferents terminate [130]. Reduction of food intake by systemic bombesin or bombesin-like peptides is attenuated by removing both vagal and spinal afferent innervation from the upper GI tract [212]. In addition, systemic capsaicin treatment attenuates reduction of food intake by peripheral bombesin injection [115]. These data are fundamentally consistent with evidence indicating that both vagal and spinal afferents contribute to the reduction of food intake by gastric stimulation. Furthermore, near arterial infusion of bombesin via the Ghrelin Recently, ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, has been localized in the gastric mucosa and proximal intestinal mucosa [113]. Peripheral injection of ghrelin increases food intake in rats and humans [239,240]. Furthermore, ghrelin reduces vagal afferent discharge, and surgical vagotomy or treatment of the abdominal vagus with capsaicin reportedly eliminates increased food intake following systemic ghrelin injection [52]. Systemic administration of a ghrelin receptor antagonist is reported to decrease food intake in both lean and obese mice [5], suggesting that endogenous ghrelin might participate in the physiological control of food intake. In addition, intracerebral injection of antighrelin antisera has been reported to reduce food intake in rats [8], lending support to the hypothesis that central ghrelin receptors also participate in the control of food intake. For the time being, these results must be cautiously interpreted, as the specificity of reduction of food intake by these antighrelin treatments has not been fully explored. Finally, although plasma ghrelin levels rise prior to a meal, when the stomach may be empty, the relationship between gastric fullness and plasma ghrelin levels may not always be straightforward. For example, Murakami et al. [149] have reported that plasma ghrelin concentrations exhibit a bimodal pattern of elevations, with ghrelin levels peaking at the end of the light period when the stomach is nearly empty, as well as at the end of the dark period, when it is nearly full. Nevertheless, the possibility that the reduction of plasma ghrelin during a meal might participate in reduction of food intake by gastric stimulation is interesting but, as yet, incompletely explored.

7 R.C. Ritter / Physiology & Behavior 81 (2004) Gastric leptin The fat cell hormone, leptin, has recently become a candidate substance for gastric signaling. The signaling form of the leptin receptor is expressed by vagal afferent neurons [28,31]. In addition, Peters et al. [164] have reported that exogenous leptin increases phosphorylation of STAT3 in the nodose ganglia, suggesting that circulating leptin may modulate vagal afferent signaling via a transcriptional mechanism. Plasma leptin concentrations in the systemic circulation are not closely correlated with meals, and most circulating leptin appears to come from white adipose tissue. However, leptin is produced by cells of the gastric mucosa [7,35,145,206] and may be released during feeding. The gastric production and secretion of leptin raises the possibility that leptin could also act in a paracrine fashion to acutely activate vagal afferents innervating the gastric wall. While there are currently no data to directly support this hypothesis, it is interesting that Peters et al. [164] have found that leptin induces a rapid increase in cytosolic Ca ++ in cultured vagal sensory neurons. This increase in Ca ++ depends on the opening of voltage-sensitive Ca ++ channels, suggesting that leptin can rapidly activate vagal-afferent neurons. Peters et al. [164] also found that many nodose neurons that respond to leptin also respond to CCK. Indeed, leptin enhanced the activation of cultured vagal sensory neurons by CCK. These results are consistent with extracellular recording results from Wang et al. [234], indicating synergy between leptin and CCK in the activation of some vagalafferent fibers. Furthermore, these results are consistent with reports that leptin and CCK act synergistically to reduce food intake [11,66,133,233]. Whether paracrine gastric leptin participates in satiation remains to be directly tested. However, the fact that leptin can produce rapid changes in membrane conductance and influx of extracellular calcium is consistent with effects on the terminals of gastric sensory neurons. While the stomach may sense the volume of ingesta arriving from a meal, the bulk of evidence does not support a direct role for this organ in monitoring the chemical constituents or caloric value of ingesta. However, intestinal nutrient stimulation markedly inhibits gastric emptying in proportion to the caloric concentration of gastric contents entering the duodenum [136]. Hence, it is reasonable that the chemical stimulation of the intestine may amplify gastric mechanical signals and thereby enable gastric signals to participate in the monitoring of nutrient content of meals [73]. Furthermore, the fact that the stomach secretes substances such as ghrelin and leptin raises the possibility that the stomach may supply humoral satiety signals, the salience of which could be modulated by metabolic or nutritional status. Therefore, the involvement of gastric mechanoreception in the behavioral response to nutrient contents of meals and variations of metabolic state remains a potentially rich field for investigation. 3. Satiation signals from the intestine Most enzymatic digestion and absorption of macronutrients occurs in the proximal small intestine. Consequently, the small intestine comprises the final opportunity for monitoring ingesta prior to absorption and assimilation. The intestinal mucosa and submucosa are extensively innervated by vagal afferents [13], which communicate with higher order neurons in the hindbrain and forebrain. In addition, the small intestine secretes a cadre of peptides that may serve as humoral afferents, informing the brain either directly or via activation of peripheral neural afferents. These features of the small intestine and its innervation suggest the capability to control food intake in response to the quantity and quality of ingesta during an ongoing meal Extrinsic sensory innervation of the small intestine The small intestine is innervated by extrinsic sensory fibers from both the spinal dorsal root ganglia and the nodose ganglia of the vagi. The density of vagal innervation is highest in the proximal duodenum and it is least dense in the ileum ([13,104]; Fig. 4). The small intestine, Fig. 4. Distribution of anterogradely labeled vagal intraganglionic laminar nerve endings in the rat small intestine. Intact rats and capsaicin-treated rats were given intranodose ganglion injections of either vehicle or horse radish peroxidase conjugated to wheat germ agglutinin. After 72 h, rats were perfused and intestinal whole-mounts were prepared for histochemical localization of anterogradely labeled nerve endings using tetramethylbenzadine. The figure depicts the average number of IGLEs/ cm 2 at four intestinal levels for vehicle- (n=3) and capsaicin-treated (n=3) rats. D1 and D2, duodenum; Jej, jejunum. Error bars indicate standard errors of the means. Note that IGLEs are most numerous in the duodenum, less numerous in the jejunum and sparse in the ileum. Also, note that IGLEs are greatly reduced in the intestine of capsaicin-treated rats, indicating that nearly all intestinal IGLEs are endings of capsaicinsensitive vagal afferents. From Jagger et al. [104].

8 256 R.C. Ritter / Physiology & Behavior 81 (2004) like the stomach, contains both IGLEs and IMAs. Vagalafferent fibers also course through the submucosa and extend into the lamina propria of the intestinal villi [13], through which absorbed nutrients and enteric hormones pass on their way to the capillary circulation and lymphatics. While vagal afferent fibers of the lamina propria may come close to the abluminal surfaces of enterocytes and enteroendocrine cells of the intestine, synapse-like contacts have not been observed between epithelial and identified vagal-afferent terminals [14]. Moreover, nerve fibers and terminals do not extend from the lamina propria into the basolateral spaces of the mucosa, nor do they make direct contact with the intestinal luminal contents. Spinal-afferent fibers form dense networks around blood vessels in the submucosal layer of the small intestine. Many of these afferent fibers are immunoreactive for CGRP and/or substance P (SP) [82,83,213]. Both CGRP and SP are found in fibers that extend into the villus lamina propria [103]. As with vagal afferents, there is no evidence for any direct contact of spinal afferents with luminal contents or specialized contacts between spinal afferents and cells of the intestinal mucosa. Most, if not all, spinal- and vagal-afferent fibers innervating the small intestine are sensitive to the neurotoxin, capsaicin [104]. CGRP immunoreactivity is nearly absent in the submucosal plexus of capsaicin-treated rats [217]. Likewise, capsaicin treatment results in almost complete disappearance of vagal IGLEs and IMAs from the small intestine ([104]; Fig. 4). Therefore, in contrast with the stomach innervation [15], which includes capsaicin-insensitive vagal afferents, most intestinal sensory fibers are destroyed by systemic capsaicin treatment. Electrophysiological recordings of fibers teased from the vagal trunks or from perivascular nerve bundles in the mesentery of the small intestine indicate that the extrinsic sensory innervation of the small intestine responds to a variety of stimuli. For example, vagal afferents have been found to respond to mechanical stimulation of the intestinal mucosa, hyperosmotic solutions, acids and bases [36]. At least some vagal afferent fibers appear to respond selectively to the intestinal infusion of carbohydrate [138] or fatty acids [119,139,169]. Therefore, the extrinsic innervation appears to be capable of providing the brain with signals that are specific to individual macronutrient classes. However, it is uncertain whether vagal afferents can respond directly to nutrients in the extracellular space or whether responses to nutrients are mediated by substances released from the intestinal mucosa or enteric neurons. Vagal-afferent fibers from the intestine respond to a variety of peptides and amines such as CCK [177,196] and serotonin (5-HT) [250]. It is likely that vagal responses to paracrine and endocrine substances like these modulate or mediate vagal-afferent activation by luminal nutrients. In fact, studies with cultured vagal sensory neurons indicate Fig. 5. An example of concentration-response characteristics of calcium responses induced by CCK-8 in an isolated nodose neuron. Traces represent the calcium signal from single nodose neurons. The bars over the traces represent periods when test substances were added to the perfusate. (A) Calcium response to 5-HT (serotonin) by vagal-afferent neuron that does not respond to CCK-8, either prior to or after exposure to 5-HT. (B) Dose-related calcium response to CCK-8 in a vagal-afferent neuron. Note that a more detailed report and analysis of CCK sensitivity of vagal afferent neurons in culture can be found in Simasko et al. [203] and in Simasko and Ritter [202]. that the nodose ganglia contain neurons that are sensitive to a variety of neuroactive peptide and nonpeptide substances, including CCK (Fig. 5; [203]), 5-HT [163], leptin [162,164], GABA, etc. [6,124] and glucagon-like peptide 1 (GLP-1) [107]. The diversity of vagal sensitivities could provide coding for a wide variety of specific stimuli Sensory modalities for intestinal satiation The intestinal infusion of liquid diet inhibits food intake in a variety of mammals including monkeys [80], rats [176] and humans [34,134,237]. Although reductions of food intake following intestinal infusion may continue for hours after the infusion has ended, the reduction of food intake begins within seconds or minutes of the start of infusion, suggesting that, at least, the early effects of infusion are not due to the postabsorptive or systemic metabolic effects of infused nutrients [80,244]. Indeed, experiments with infusions of specific macronutrients (see below) further support the hypothesis that reduction of food intake by macronutrients is mediated by signals arising from the intestine itself. Intestinal infusion of nutrients or hyperosmotic nonnutrient solutions inhibit gastric emptying [37,77,137,195]. It is possible that the inhibition of gastric emptying may participate in the control of food intake by intestinal nutrients. However, many experiments have demonstrated that intestinal infusions inhibit food intake during sham

9 R.C. Ritter / Physiology & Behavior 81 (2004) feeding, when drainage of ingesta via an open gastric cannula obviates gastric filling effects on food intake (see, e.g., Refs. [67,80,127,244,246]). Therefore, while it is probable that intestinal signals and gastric emptying can interact to control food intake, the inhibition of gastric emptying is not necessary for control of food intake by intestinal stimulation Osmotic stimuli and intestinal satiation Ingesta entering the intestine presents a complex assortment of colligative and chemical signals to the intestinal sensory systems. Material emptying during a meal may be hypertonic and can contain a variety of macronutrient constituents, each of which might influence food intake by a distinct mechanism. Houpt et al. [99] demonstrated that intestinal infusion of hyperosmotic solutions inhibited food intake in pigs. They also measured the osmotic pressure of the duodenal contents after eating and showed that infusions with osmotic concentrations within the physiological range were effective for the reduction of food intake. Tracy and Ritter (unpublished observations) also found that nonnutritive infusions inhibited sham feeding in rats. The threshold for the inhibition of intake at an infusion rate that was comparable to the within-meal gastric emptying rate was approximately 700 mosm, which was similar with the concentrations that Houpt [99] found effective in swine. The results of these experiments suggest that nonnutritive characteristics of ingesta may contribute to the process of satiation at the intestinal level. Macronutrient components of intestinal infusates appear to reduce food intake via mechanisms that are independent of their osmotic concentration. For example, the intestinal infusion of oligosaccharides or glucose reduces real or sham feeding substantially, even when their osmotic concentration is isotonic or even hypotonic [244]. Likewise, micellar solutions or long-chain fatty acids [246] or triglyceride emulsions [84] reduce food intake despite the fact that they contribute negligibly to the osmotic pressure of an isotonic intestinal infusion Fats as intestinal satiation signals Both real and sham feeding are reduced by intestinal infusions of all three macronutrient groups carbohydrates, fats and amino acids. However, the chemical forms in which nutrients are infused have a significant effect on their ability to reduce food intake. For example, infusion of the lipolytic digestion products of triolein (oleic acid and 2-monoglyceride) reduce food intake more than the unhydrolyzed triglyceride does [87]. Duodenal infusion of a long-chain triglyceride reduces food intake in rats. However, intestinal infusion of glycerol has little or no effect on food intake (Ritter and Brenner, unpublished observations). These results suggest that the intestine monitors absorbable lipolytic products, fatty acids and 2- monoglyceride, rather than unhydrolyzed fat. The fact that the inhibition of pancreatic lipase by orlistat (Hoffman- LaRoche, Basal, Switzerland) attenuates the reduction of food intake by intestinal triglyceride infusion is consistent with this hypothesis [142]. The chemical characteristics of fatty acids themselves also affect their ability to reduce food intake during intestinal infusion. For example, Yox and Ritter [244] found that while oleic acid (C18) profoundly inhibited food intake in sham-feeding rats, equicaloric infusions of octenoic acid (C8) did not significantly reduce intake (Fig. 6). Similar results have been reported by McCaffery et al. [135]. The fact that longer chain fatty acids are more effective for the inhibition of food intake than shorter chain fatty acids may have something to tell us about the mechanisms by which they reduce food intake. First, fatty acids longer than 14 carbons are in micellar solution in the intestinal lumen. They are incorporated into triglycerides after absorption into intestinal epithelial cells and packaged in lipoprotein envelopes (chylomicrons), prior to being secreted into the lymphatic drainage of the intestine [165,226], which enters the systemic circulation at the thoracic duct. In contrast to long-chain fatty acids, shortand medium-chain fatty acids have greater solubility in intestinal luminal contents, even without micellization. Unlike long-chain fatty acids, short- and medium-chain acids are not incorporated into chylomicrons for secretion into the lymphatic drainage. Rather, they are absorbed directly into the hepatic portal venous drainage of the small intestine. Hence, available data suggest that only fatty acids that are absorbed via the chylomicron pathway produce intestinal satiation signals. The importance of this absorption pathway for fatty acid-induced satiation seems to be supported by the observation that Pluronic L-81, a detergent that prevents fat absorption by inhibiting chylomicron formation [227], attenuates the reduction of food intake following an 8-h infusion of fat into the intestine [142,190]. It is not clear, however, that the effects of these prolonged fat infusions actually exercise the same mechanisms for reducing food intake as are activated by the briefer infusions reported by others because the reduction of food intake by these prolonged infusions do not depend on vagal sensory neurons. Nevertheless, the apparent involvement of chylomicron formation in the reduction of food intake by fat has led to the hypothesis that a chylomicron component, apolipoprotein A-IV (apo A-IV), may be the signal for fat-induced satiation. However, while apo A-IV itself has significant effects on food intake, it seems unlikely that it accounts for fat-induced satiation in its entirety (see below) Carbohydrate as an intestinal satiation signal Many investigators have reported that intestinal infusions of sugars reduce food intake. Unfortunately, in most experiments, high molar concentrations have been used, such that the role of osmotic stimuli cannot be clearly separated from specific chemical stimulation [241]. Using

10 258 R.C. Ritter / Physiology & Behavior 81 (2004) Fig. 6. (A) Percent suppression of 30-min intake of 15% sucrose following intraintestinal infusions of oleic or octanoic acid. Intraintestinal infusions of oleic, but not octanoic, acid significantly reduced 30-min sham intake of sucrose in vehicle-treated rats, suggesting that the satiation effects of fatty acid depend on chain length. Reduction of intake in capsaicin-treated rats was significantly less than that which occurred in vehicle-treated rats, suggesting that capsaicinsensitive fibers mediate the satiation effects of long-chain fatty acid anions such as oleate. (B) Percent suppression of 30-min sham sucrose intake in response to intraintestinal infusions of casein hydrolysate or L-phenylalanine. Intraintestinal casein hydrolysate infusions produced a small, but significant, reduction of sham feeding in both vehicle- and capsaicin-treated rats. Moreover, there was no difference in suppression produced in either group of rats. In contrast, intraintestinal infusions of a 3% L-phenylalanine solution produced a strong suppression of sham feeding in vehicle-treated rats. L-phenylalanine-induced suppression of sham ingestion was significantly attenuated in capsaicin-treated rats. From Yox and Ritter [244]. 180 mm infusions of glucose, maltose and maltotriose, we have observed that the reduction of food intake by equimolar concentrations of these sugars is greatest for maltotriose and least for glucose [180]. It should be noted that 180 mm maltotriose is three times as concentrated calorically as 180 mm glucose is. However, even in the unlikely event that all of the glucose in the maltose or maltotriose were liberated instantaneously during infusion, the osmotic concentration of the infusates would be 360 or 540 mosm, which in our hands, are below the threshold for osmotic effects on feeding. Although the intestinal infusion of oligosaccharides potently reduces food intake, we have found that administration of the amyloglucosidase inhibitor, acarbose, attenuates reduction of food intake by intestinal maltotriose (Fig. 7). Acarbose also attenuates induction of Fosimmunoreactivity in the NTS following the intestinal infusion of maltotriose (Fig. 8; [181]), suggesting that the inhibition of oligosaccharide hydrolysis attenuates vagal-afferent activation by maltotriose infusion. Thus, it appears that the liberation of monosaccharide, glucose, is necessary for the reduction of food intake by oligosaccharides such as maltotriose. It remains possible that other monosaccharides can contribute to satiation. However, Walls et al. [230] reported that infusions of 3% glucose significantly reduced real feeding, but 3% fructose infusion had no effect. These data suggest that at low concentrations, glucose plays a special role in satiation by intestinal carbohydrate. The mechanism by which intestinal glucose is detected is not known. In this regard, it is interesting that Meyer et al. [142] have reported that duodenal infusions of monosaccharides, such as xylose, 3-O-methyl glucose and alphamethyl glucose, all reduce food intake when infused into the intestine. All three of these monosaccharides are substrates for the sodium-linked glucose transporter (SGLT-1). While this observation might suggest that glucose transport is necessary for the reduction of intake by intestinal infusion, intravenous glucose is notoriously ineffective for reduction of short-term food intake. Furthermore, our previously published results [180] indicate that satiation by intestinal oligosaccharides is directly related to glucose in the lumen and inversely related to blood glucose. Finally, we recently demonstrated that inhibition of SGLT-1, by intestinal phlorizin infusion, fails to attenuate reduction of food intake and vagal-afferent activation by maltotriose or glucose, even though the transport of glucose to the blood is almost completely blocked [181]. Thus, it appears that the reduction of food intake by intestinally infused carbohydrates requires hydrolysis to glucose, but that transport of glucose

11 R.C. Ritter / Physiology & Behavior 81 (2004) Fig. 7. (A) The elevation of blood glucose following the intestinal infusion of maltotriose in 18-h fasted rats is nearly abolished by the coadministration of the amyloglucosidase inhibitor, acarbose. (B) Intestinal maltotriose infusion significantly reduced solid food intake. The reduction of intake was reversed by acarbose. These data suggest that the reduction of food intake by oligosaccharides depends upon their hydrolysis to monosaccharides. from the intestine to the blood is not necessary. In other words, the stimulus for glucose-induced satiation appears to arise through an action of glucose on the luminal side of the intestinal mucosa. If glucose absorption is not required for the activation of the neural afferents that mediate satiation, then, that task must fall to an endocrine or paracrine secretion of the intestinal mucosa. However, the identity of the product(s) that activate vagal afferent satiation signaling is uncertain (see below) Amino acids and proteins as intestinal satiation signals Vagal afferents innervating the small intestine respond to amino acids and hydrolyzed protein. For example, Jeaningros [106] reported that intestinal infusion of a variety of amino acids triggers increased discharge by vagal afferent fibers in the cat. Furthermore, there appeared to be some selectivity of individual fibers for specific amino acids. Schwartz and Moran [197] have reported that intestinal infusion of casein hydrolysate, which contains a mixture of peptides and amino acids, also increased vagal afferent discharge. Likewise, Eastwood et al. [61] demonstrated that a subset of vagal fibers innervating the small intestine included responders to casein hydrolysate. Intestinal infusion of partially hydrolyzed protein and amino acids both are reported to decrease food intake. For example, Gibbs et al. [78] and Yox and Ritter [244] reported that isotonic infusions of L- but not D-phenylalanine reduced real and sham feeding in rats (Fig. 6B). Meyer et al. [142] compared the effects of intestinal infusion of several amino acids on the reduction of food intake, but observed significant reductions only when either phenylalanine or tryptophan were infused. Other amino acids were essentially without effect. Similarly, Yox and Ritter [244] reported that the infusion of casein hydrolysate produced only a slight reduction of food intake, compared with equicaloric infusions of L-phenylalanine, oleate and maltose (Fig. 6B). Therefore, it is likely that substrates that mediate reduction of food intake by amino acids are rather narrowly tuned to respond to only certain ones. However, it may be that sensory mechanisms distinct from those that respond to individual amino acids mediate the reduction of food intake by unhydrolyzed or partially hydrolyzed protein. In this regard, it is noteworthy that the reduction of food intake by intestinal infusion of partially hydrolyzed protein is attenuated by CCK-A receptor antagonists [44], whereas reduction of food intake by the amino acid L-phenylalanine is not attenuated by CCK receptor antagonists ([22,25,243]; see Table 1) Receptive area for intestinal satiation signals The efficacy of nutrient infusions at different levels of the small intestine has been examined by several investigators. Meyer et al. [143] reported that the reduction of food intake by nutrient infusions depends on the total length of the intestine that is contacted by the infusion. We found that

12 260 R.C. Ritter / Physiology & Behavior 81 (2004) when nutrients are infused into the distal ileum, little or no reduction of food intake occurs (Huff and Ritter, unpublished observations). However, when comparable infusions of isotonic nutrient solutions are made into the duodenum, 30-min food intake is reduced by 40% or more. Infusion into the midjejunum reduces intake significantly, but less than duodenal infusion. These results, while consistent with those of Meyer et al. [143] also suggest that the majority of satiation signals arising from low-nutrient concentrations come from proximal small intestine. This interpretation is also consistent with the fact that vagal innervation of the small intestine is denser in the duodenum than in the distal small intestine [15,104]. However, these results do not rule out control of food intake by distal intestinal signals, especially under conditions where nutrient load or intestinal motility may result in escape of nutrients from their primary site of digestion and absorption in the duodenum and jejunum Neural afferents for intestinal satiation Fig. 8. Intestinal infusion of maltotriose induces increased Fos-immunoreactivity in the nucleus of the solitary tract (NTS; top panel). Note that Fos-immunoreactivity following saline vehicle infusion was virtually absent in this experiment (data not shown). Bottom panel shows that acarbose abolishes maltotriose-induced Fos-immunoreactivity in the rat dorsal hindbrain. Most of the reduction of food intake observed following intestinal nutrient infusion is mediated by vagal sensory neurons. Yox et al. [246] found that reductions of sham feeding by maltose or oleate were virtually abolished in rats that had been subjected to total subdiaphragmatic vagotomy. They also reported that the reduction of intake by L- phenylalanine was attenuated, but not abolished, in vagotomized rats. In another study, Yox et al. [245] also found that systemic [244] or fourth ventricle injection of capsaicin abolished or attenuated sham feeding by intestinal oleate, L- phenylalanine or maltose (Fig. 6). Taken together, these results indicate that in the reduction of food intake by intestinal carbohydrate, some amino acids and fat are mediated by capsaicin-sensitive vagal afferents. The role of the afferent component of the vagus is supported in subsequent reports by Walls et al. [230,231]. They sectioned the vagal afferent roots unilaterally as they emerged from the medulla and sectioned the contralateral vagal trunk below the diaphragm. The result of this procedure is complete vagal deafferentation of the abdominal viscera, with partial deeferentation due to the unilateral subdiaphragmatic vagotomy. Rats prepared in this manner did not reduce their food intake in response to intestinal infusion of Table 1 Sham intake of sucrose following intraperitoneal administration of the CCK receptor antagonist, devazepide (MK-329), prior to intraintestinal nutrient infusion. From Brenner and Ritter [25] Devazepide Sucrose (Ag/kg) Oleate (0.08 kcal/ml) Maltotriose L-Phenylalanine (0.13 kcal/ml) (0.345 kcal/ml) (0.13 kcal/ml) F 3.15 (12) F 3.80 (10) F 3.35 (8) F 4.36 (8) F (8) F 6.31 (9) F 3.67 (6) F 7.23 (6) F 5.88 * (9) F 4.84 (6) F 8.33 (7) F 4.91 * (8) F 7.24 (6) F 5.9 (6) F 4.21 (10) F 6.12 * (7) F 6.02 * (6) F 4.91 * (4) F 5.42 * (5) F 6.31 * (6) F 8.18 (8) F 4.75 * (3) F 7.34 * (6) F 5.42 * (5) F 4.37 (7) Saline/saline F 3.72 (12) F 3.4 (10) F 3.28 (6) F 3.4 (8) Devazepide was administered intraperitoneally 5 min prior to the intraintestinal nutrient infusion (10 ml/10 min). Values are mean 30-min sham intakes in milliliters F S.E.M. beginning 5 min after the start of intraintestinal infusion. Saline/saline represents the baseline condition saline injection coupled with saline infusion. Numbers in (parenthesis) indicate n for each experiment. * P <.01, significantly different from intraintestinal nutrient infusion alone.

13 R.C. Ritter / Physiology & Behavior 81 (2004) carbohydrate, fat or amino acid. Walls et al. [230,231] also reported that a section of the coeliac branch of the vagus was sufficient to abolish the reduction of food intake by infusions of glucose, maltose, L-phenylalanine and 1.4% oleate. They hypothesized that coeliac vagotomy was effective because the coeliac branch appears to be the main source of vagal innervation to the duodenum. While this finding is in general agreement with the prior report of Yox et al. [246], the fact that coeliac vagotomy abolished the reduction of food intake by L-phenylalanine in the Walls et al. [230,231] study is inconsistent with the report of Yox et al. [246] that vagotomy or capsaicin treatment did not completely eliminate reduction of food intake by this nutrient. One possible interpretation for this apparent discrepancy is that, at least, some of L-phenylalanine s effect is mediated by capsaicin-insensitive spinal afferents that join the vagus below the level of the truncal vagal cuts of Yox et al. [246]. However, at this time, the existence or participation of such afferents in satiation has not been experimentally assessed. While it is not known whether satiation induced by intestinal nutrient infusion depends upon afferent innervation of the intestinal mucosa or the myenteric plexus, the results of Tamura and Ritter [216] suggest that innervation of the mucosa may be most important. They found that rats exhibited transient insensitivity to reduction of food intake by intraintestinal oleate infusion [216] for 24 h following the irrigation of the intestinal lumen with capsaicin. Reduced sensitivity to intestinal oleate infusion was correlated with the depletion of calcitonin gene-related peptide from nerve fibers and terminals in the submucosal plexus, but not with the myenteric plexus [217]. These results suggest that the reduction of food intake by intestinal fat depends on capsaicin-sensitive vagal-afferent fibers that innervate the upper small intestinal mucosa Paracrine and endocrine mediators of intestinal satiation In rats, most meals are less than 20 min in length. During real feeding of a liquid diet, the rate of ingestion begins to decrease during the first few minutes and steadily declines until the animal stops eating between 10 and 20 min after the start of the meal [54]. These behavioral kinetics indicate that signals responsible for meal termination begin to develop during the early moments of the meal. Like mealinduced satiation, reduction of food intake during intestinal nutrient infusion begins within minutes or seconds of the start of infusion, suggesting that satiation signals originate while food is entering the intestine. As discussed above, the reduction of food intake by intestinal nutrient infusion depends on small capsaicin-sensitive vagal sensory neurons that innervate the intestine. However, the terminals of these neurons do not contact the luminal contents. These facts lead to the hypothesis that intestinal nutrients trigger the secretion of neuroactive substances from the intestinal epithelium and that these substances activate vagal sensory neurons, resulting in reduction of food intake. The intestinal epithelium secretes a variety of substances during digestion and absorption. These include the wellknown gastrointestinal peptide hormones, biogenic amines, various cytokines, growth factors, lipid membrane components and apolipoprotein components of chylomicrons [20]. Members of each of these classes of substance and others have been implicated in the control of food intake, and it is plausible that satiation in response to a mixed nutrient meal involves several mucosally derived substances acting in concert to generate satiation. The substances discussed in the following paragraphs are amongst those that could contribute to satiation following nutrient stimulation of the intestinal mucosa Cholecystokinin (CCK) CCK is secreted by a population of mucosal endocrine cells (I cells) in the orad small intestine. Plasma CCK concentrations rise following intestinal infusions of longchain fatty acids and unhydrolyzed or partially hydrolyzed proteins [22,126]. In rats, the reduction of food intake by exogenous CCK has been documented repeatedly in rats [4,81], monkeys [67,78] and humans [112,209,210]. Reduction of food intake by exogenous CCK is attenuated by the destruction of capsaicin-sensitive vagal-afferent neurons [131,183,205,207]. Thus, vagal sensory neurons are necessary for reduction of food intake by both exogenous CCK and intestinal nutrients [230,231,244]. CCK exerts its physiological actions via two G-proteincoupled receptors, the CCK-A (CCK-1) or CCK-B (CCK-2) receptor [154,235]. The injection of exogenous CCK reduces both real and sham feeding by acting at peripheral CCK-A receptors [21,147]. Binding sites for CCK receptors are transported in vagal sensory neurons [148]. Furthermore, recent in situ hybridization studies indicate that between 30% and 40% of vagal sensory neurons transcribe CCK-A receptor mrna [27]. Likewise, calcium imaging of dissociated nodose ganglion neurons [203] indicates that about this same proportion of vagal sensory neurons respond to exogenous CCK via CCK-A receptor activation. Finally, extracellular recording from vagal-afferent fibers indicates that activation of vagal afferents by some intestinal stimuli is attenuated by CCK-A receptor antagonists [61,119]. All of these data support a role for CCK in vagally mediated reduction of food intake by intestinal nutrients. Reductions of food intake by intestinal nutrient infusions are attenuated or abolished by CCK-A receptor antagonists in humans [134] and rats [25,88,175,243]. In the rat Yox et al. [243] and Brenner et al. [25] found that CCK-A antagonists virtually abolished the reduction of food intake by intestinal infusions of oleate, maltose or maltotriose in sham-feeding rats (Table 1; [25,243]). Subsequently, Matzinger et al. [134] demonstrated that CCK-A receptor antagonism also attenuates satiation by intestinal fat infusions in humans. Others have reported that CCK-A antag-

14 262 R.C. Ritter / Physiology & Behavior 81 (2004) onists attenuate the reduction of food intake by peptone and protein infusions as well [44,153]. Yox et al. [243] also found that neither CCK-A (Table 1) nor CCK-B antagonists attenuated reduction of food intake by L-Phe [25,243]. Thus, it is clear that some, but not all, intestinal nutrients reduce food intake by CCK-A receptor dependent mechanisms. CCK is produced by neurons in the brain as well as by enteroendocrine cells [1,96,97,194]. Furthermore, most CCK antagonists penetrate the blood brain barrier [102,238]. Therefore, the attenuation of nutrient-induced reduction of food intake by CCK-A antagonists could be due to peripheral and/or central actions of the receptor antagonists. Initially, most investigators assumed that CCK, released by nutrient stimulation and circulating in the plasma, was responsible for reducing food intake. However, Brenner et al. [22] found that while maltose and oleate reduced food intake substantially, only oleate detectably elevated plasma CCK (Fig. 9). Intestinal maltose infusion did not. Furthermore, they found that intestinal infusion of unhydrolyzed casein, at a concentration calorically equivalent to oleate or maltose, substantially elevated plasma CCK, but did not significantly reduce sham feeding. These results produced a conundrum that still exists. Apparently CCK-A receptors are necessary for the reduction of food intake by some nutrients, which do not stimulate release of endocrine CCK. On the other hand, triggering secretion of endocrine CCK is not, in and of itself, sufficient to reduce feeding. This conundrum led to two explanatory hypotheses, which are not mutually exclusive. First, it may be that brain CCK-A receptors are important for the control of food intake by intestinal nutrients. Second, there might be sources of peripheral CCK secreted in small quantities, at high concentrations, close to vagal afferent nerve fibers. Such paracrine CCK secretion might mediate the satiation response to intestinal nutrients that do not elevate plasma CCK concentrations. Injection of exogenous CCK into a variety of brain sites reduces food intake [17,56,89,192,193]. Furthermore, several reports indicate that very small doses of CCK-A receptor antagonist injected into the hypothalamus increase food intake [18,62]. While these data support a role for brain CCK in control of food intake, the importance of brain CCK receptors in control of food intake by intestinal nutrients remains to be determined. In fact, Brenner and Ritter [24] were unable to attenuate the reduction of food intake by intestinal oleate, even when the central dose of CCK-A antagonist they used exceeded that which was effective when injected peripherally (Table 2). More recently, Reidelberger et al. [175] reported that the reduction of food intake by intestinal lipids and carbohydrates is attenuated, although not abolished, by injections of a CCK-A antagonist that does not cross the blood brain barrier. Likewise, Brenner and Ritter [21] demonstrated that a peptide CCK receptor antagonist, which presumably is excluded from the brain, increases food intake when injected in the absence of exogenous CCK. Fig. 9. Suppression of sham feeding and peak plasma CCK concentration after intraintestinal nutrient infusion. (A) Mean plasma CCK concentration in the plasma samples collected 10 min after the start of intraintestinal infusion of oleate, maltose, L-phenylalanine or casein. (B) Mean percent suppression of 30-min sham intake of sucrose after the intraintestinal infusion of oleate, maltose, L-phenylalanine or casein. Note that maltose infusion reduced food intake but did not elevate plasma CCK, while casein infusion elevated CCK but did not significantly reduce food intake. From Brenner et al. [22]. Intraarterial infusion experiments have demonstrated that exogenous CCK infused near [32,51] or directly into [50] the arterial supply of the upper small intestine reduces food intake at doses below those required by intravenous infusion. These near-arterial infusions support the role of peripheral CCK receptors in control of food intake. Specifically, they suggest that the vagal afferents that mediate the reduction of food intake by exogenous CCK reside on terminals innervating the upper gastrointestinal tract. Furthermore, Cox [49] demonstrated that the infusion of a CCK-A receptor antagonist directly into the pancreaticoduodenal artery increases meal size at doses that were ineffective when injected intravenously. This result is consistent with the hypothesis that endogenous CCK, which is released from the upper small intestine,

15 R.C. Ritter / Physiology & Behavior 81 (2004) Table 2 Effect of intraperitoneal or intracerebroventricular CCK receptor antagonists on the suppression of sucrose sham feeding by intraintestinal oleate. From Brenner and Ritter [24] Intraperitoneal devazepide (13) [75 Ag/rat] Lateral intracerebroventricular devazepide (8) [75 or 150 Ag/rat] Lateral intracerebroventricular L (5) [150 Ag/rat] Fourth intracerebroventricular devazepide (6) [300 Ag/rat] Oleate + vehicle F F F F 5.55 Antag + oleate F 4.56 * F F F 4.67 Vehicle F vehicle F 3.64 * F 4.97 * F 3.43 * F 6.17 * CCK receptor antagonists or vehicles were administered 5 min prior to intraintestinal oleate or vehicle infusion (10 ml/10 min). Five minutes after the beginning of intestinal infusions, 15% sucrose was introduced. Values are mean 30 min sham intake in milliliters F S.E.M. beginning 5 min after the start of intraintestinal infusion. Numbers in parenthesis indicate n for each experiment. * P V.01, significantly different from intraintestinal oleate infusion/vehicle. acts on vagal terminals innervating this part of the GI trait. Taken together, the results of these studies strongly support the participation of peripheral, gastrointestinal, CCK receptors in reduction of food intake by nutrients, but do not rule out a role for central CCK-A receptors in other aspects of control of food intake. The results also are consistent with, but not conclusive for, a proposed paracrine mechanism of CCK action in the control of food intake. At this time, there are no data to directly support or refute the existence of paracrine CCK secretion or a role for such secretion in reduction of food intake by intestinal nutrients. Most studies of CCK s effects on food intake have been conducted using synthetic CCK-8, injected intraperitoneally. This approach may mimic paracrine exposure of vagal endings by locally released CCK, which might not appear in the systemic. Greenberg and Smith [85] and Greenberg et al. [86] have demonstrated that CCK-8 is ineffective when injected into the hepatic portal circulation. Because gut peptides enter the systemic circulation only after running the gauntlet of hepatic portal catabolism, these data suggest that CCK-8 released from the gut would have to act locally and not in an endocrine fashion. Indeed, Reeve et al. [174] recently reported that CCK-58 is the only endocrine form of CCK in the rat. Unfortunately, we do not know whether smaller forms of CCK may be released, but not detected in the systemic circulation. Experiments at the cellular level also yield circumstantial evidence for paracrine CCK action. In some cells, the CCK- A receptor exhibits both high- and low-affinity binding sites or states [157,215]. High-affinity sites have a Kd for CCK in the pm range, while low-affinity sites have a Kd in the nm range. Presumably, the low-affinity sites mediate effects of CCK at higher concentrations, whereas high-affinity sites are adapted to respond to lower endocrine CCK concentrations. Pharmacological studies indicate that exogenous CCK reduces food intake by acting at the low-affinity sites of the CCK-A receptor [236]. Our recent examinations of CCK responsiveness in cultured vagal sensory neurons suggest that all vagal afferent responses to CCK are mediated by the low-affinity CCK-A receptor site ([203]; but see also Ref. [120]). These data suggest that vagal sensory neurons that mediate responses to CCK may well be best adapted for detecting paracrine, rather than endocrine, CCK secretion Apolipoprotein A-IV (apo A-IV) Apo A-IV is a lipoprotein component of chylomicrons, the synthesis of which varies directly with fat absorption [165]. Fujimoto et al. [76] demonstrated that exogenous apo A-IV reduces food intake in rats. Furthermore, the infusion of chylous lymph from rats that had received intestinal fat infusions also reduces food intake, while the lymph from rats not infused with fat has no effect. The detergent, pluronic L-81, inhibits chylomicron synthesis and attenuates reduction of food intake by intestinal fat [142,190], suggesting that a chylomicron component such as apo A-IV may contribute to the reduction of food intake by intestinal fat. Thus, the characteristics of apo A-IV seem appropriate to signal the reduction of food intake by intestinal fat. One potential problem with a role for apo A-IV in the reduction of food intake by fat is that this substance seems to act in the brain to reduce food intake [225], whereas the reduction of food intake by intestinal fat is mediated by vagal sensory neurons [246]. There are, however, two possible mechanisms by which vagal sensory fibers might play a role in the reduction of food intake by apo A-IV. First, the increase in synthesis of apo A-IV associated with fat absorption is attenuated in rats that either are vagotomized or treated with capsaicin [108]. At this point, it is not clear that this lesioninduced reduction in apo A-IV synthesis results in its reduced secretion. However, it is conceivable that vagal fibers enhance apo A-IV secretion in response to fat, but that this substance then acts at a nonvagal site, e.g., in the brain. An alternative possibility is that apo A-IV could trigger the secretion of some other substance that does act on the vagus to reduce food intake. In this regard, it is interesting that the blockade of chylomicron formation with pluronic L-81 [227] is reported to block the elevation of plasma CCK in response to intestinal fat infusion [173]. Hence, it is possible that CCK secretion, stimulated by apo A-IV and acting on the vagus, could mediate all or part of the satiety effects of apo A-IV.

16 264 R.C. Ritter / Physiology & Behavior 81 (2004) Additional investigation of the interaction between apo A-IV and CCK is required to address this issue Peptide YY 3 36 (PYY 3 36 ) PYY 3 36 is secreted by enteroendocrine cells located primarily in the ileum and colon. Circulating immunoreactivity for the peptide is elevated soon after eating, and plasma levels are reported to rise following carbohydrate and protein meals in humans [161]. Peripheral injection of exogenous PYY 3 36 is reported to reduce food intake in rats and people [12] by acting at the Y2 receptor subtype. PYY 3 36 injection into the rat hypothalamus reduces food intake, suggesting that circulating PYY 3 36 might act directly on the brain to reduce food intake. Although most PYY 3 36 is secreted by the distal small intestine and colon, secretion from these structures apparently occurs prior to direct distal intestinal stimulation by digesta. These results suggest that PYY 3 36 may be released from the distal small intestine and colon as a reflex response to upper small intestinal stimulation. This suggestion is supported by the fact that removing parts of the cecum and proximal colon, or cutting the vagi, abolished the elevation of PYY 3 36 in the plasma following intraintestinal liquid diet infusion [75]. These observations suggest that PYY 3 36 might contribute to the satiation induced by intestinal stimulation. However, much more work is needed to determine the site(s) of action of peripheral PYY The fact that intrahypothalamic PYY 3 36 reduces food intake does not prove that the hypothalamus is the sole site of the hormone s action on feeding. Furthermore, although a selective nonpeptide Y2 receptor antagonist exists [60], there are, as yet, no reports relating the effects of peripheral or central Y2 receptor blockade on food intake or body weight. On the other hand, knockout of the Y2 receptor is reported to induce a mild obesity, which is dependent on increased food intake [151]. In a more recent study, conditional knockout of Y2 receptor expression in the hypothalamus resulted in increased food intake, but also in decreased body weight [189]. At this time, there are no published reports on the effect of vagotomy or capsaicin treatment on the response to exogenous PYY Given the importance of the vagus to the response to intestinal nutrients, definitive experiments to evaluate vagal participation are essential to establish the mode and site of action for this interesting peptide. At least, for now, the possibility that PYY 3 36 might participate in the reduction of food intake via a direct vagal sensory route of action remains open Glucagon-like peptide 1 (GLP-1) GLP-1 is a posttranslational product of the preproglucagon gene. GLP-1 is secreted from endocrine L cells in the small intestine, but also is synthesized by a small population of neurons in the brain [141]. Intestinal GLP-1-secreting cells occur predominantly in the distal small intestinal and colonic mucosa [64]. A great amount of attention has been focused on central GLP-1 effects on food intake [179,200,219,220]. However, peripheral administration of GLP-1 and GLP-1 analogs reduces food intake in humans and other animals [93,94,141,150,187,221]. Intestinal nutrient infusions trigger GLP-1 release from the ileum [123], and GLP-1 release correlated with the reduced feelings of hunger in humans [19,121]. GLP-1 receptors in the brain are responsible for reduction of food intake by intracerebral injections of GLP-1 [110,218,228]. Furthermore, central administration of GLP-1 receptor antagonists attenuate the reduction of food intake by injections of LiCl [178,200,219], suggesting that endogenous GLP-1 participates in the reduction of food intake by illness. However, a role for vagal afferents in the reduction of food intake by the peripheral peptide cannot be ruled out. In fact, GLP-1 does activate vagal-afferent neurons in culture [107], and vagal afferents are necessary for the inhibition of gastric emptying by this peptide [101]. Furthermore, there is some data to indicate that vagal-afferent innervation participates in the release of intestinal GLP-1 [185]. Therefore, GLP-1 remains a viable candidate as a mediator of satiation produced by intestinal nutrient stimulation Diet and intestinal satiation Fat digestion products in the small intestine strongly inhibit food intake and gastric emptying. However, the persistence of overeating and increased weight gain by rats and humans eating high-fat diets indicates that satiation signals from the intestine do not necessarily prevent excess fat intake. This observation is similar with those made concerning the control of food intake and body weight by the fat-cell hormone, leptin. Exogenous leptin reduces food intake by reducing meal size [70]. However, humans and other animals gain weight and become obese despite increased endogenous leptin secretion. In the case of leptin, it has been suggested that dietary or environmental conditions might result in reduced leptin sensitivity [72,95,191]. An acquired reduction in sensitivity to gastrointestinal signals might also explain their failure to universally prevent overeating and obesity. On the other hand, such an explanation begs the question of what the adaptive values of GI controls are in the first place. Over the past few years, Covasa et al. [38,42,45,46] have performed an extensive series of studies to determine the effect of high-fat diets on satiation by intestinal stimuli. The clear results are that rats maintained on high-fat diet are less sensitive to the reduction of food intake by intestinal fatty acid than are rats maintained on low-fat diets (Fig. 10). These results are consistent with earlier reports that humans fed with high-fat diets eat more and feel less satiated than those maintained on low fat do [74]. Diminished sensitivity to reduction of food intake by intestinal fat infusion is accompanied by decreased inhibition of gastric emptying following intestinal fat as well [43]. Interestingly, adaptation to high-fat diet also results in increased gastric emptying of fat in humans [33]. Finally, high-fat diet is associated with increased pancreatic lipase

17 R.C. Ritter / Physiology & Behavior 81 (2004) Fig. 10. Oleate-induced reduction in food intake in rats adapted to low-fat (LF) and isocaloric high-fat (HF) diet. Data are shown as percent reduction of 30-min food intake after 17-h food deprivation. Intestinal infusion of oleate caused significantly greater reduction of intake at all oleate doses (0.08, 0.06 and 0.03 kcal/ml) in low- than in high-fat diet-adapted rats. * Significantly different from LF ( P <.05). From Covasa and Ritter [46]. content and secretion [23]. Reports from other investigators also indicate that adaptation to high-fat diets results in increased ability to absorb fat digestion products [9,10,100,204]. Thus, with continued exposure to fat, the GI tract adapts such that it can more efficiently digest and absorb fat calories. Moreover, increased ability to process fat is accompanied by decreased satiation in response to fat digestion products in the upper small intestine. Thus, it appears that sensitivity to at least one intestinal satiation signal is reduced under the very conditions when it could be most helpful in opposing excessively positive energy balance. Reduction of food intake by intestinal infusion of fat or fatty acids is attenuated or abolished by CCK-A receptor antagonists [25,88,243]. Not surprisingly, therefore, rats maintained on high-fat diets also are less sensitive to the reduction of food intake and the inhibition of gastric emptying by exogenous CCK than are the low-fat-fed rats (Fig. 11; [38,45]; but see also Torregrossa and Smith [222], whose results were largely negative with regard to dietary fat effects on reduction of food intake by exogenous CCK). Thus, reduced sensitivity to satiation by intestinal fat may reflect reduced neuronal sensitivity to CCK. Indeed, an examination of neurons in the nucleus of the solitary tract, the nodose ganglia and the intrinsic enteric neurons of the small intestine revealed diminished expression of Fos in response to exogenous CCK or intestinal fatty acid in high-fat-fed rats (Fig. 12; [38 40]). These data suggest the possibility of a causal relationship between high-fat feeding and reduced neuronal sensitivity to CCK. Fat digestion products in the small intestine stimulate the secretion of CCK from intestinal I cells, and postprandial plasma CCK concentrations are higher in highthan in low-fat-fed animals. To determine whether the elevation of plasma CCK or some other consequence of fat ingestion might be responsible for reduced sensitivity to intestinal fat or CCK, rats were adapted to a low fat, Fig. 11. Reduced response to CCK in high-fat-fed rats. CCK-induced reduction of food intake in rats adapted to low-fat (LF) and high-fat (HF) diets. Data shown are percent reduction of 30-min food intake following 17-h food deprivation. CCK caused significantly greater reduction of intake at 0.5 and 0.25 Ag/kg dose in low- than in high-fat-adapted rats ( P <.01). From Covasa and Ritter [45].

18 266 R.C. Ritter / Physiology & Behavior 81 (2004) Fig. 12. Differences in the density of nuclear Fos-immunoreactivity in representative sections of dorsal hindbrain from rats adapted to a LF or a HF diet. Counts of immunoreactive nuclei indicated that 2 h after the intestinal infusion of 0.06 kcal/ml oleate, Fos-immunoreactivity was significantly greater in LF rats (A,AV) than in HF rats (B,BV; P <.05). Scale bar, 0.1 mm. From Covasa et al. [41]. high-protein diet that stimulates CCK secretion. When tested with exogenous CCK, both high-fat- and highprotein-fed rats reduced food intake less in response to exogenous CCK than rats fed the control low-fat diet [42]. Furthermore, the infusion of CCK by osmotic minipump also resulted in reduced sensitivity to satiation by acute doses of exogenous CCK [42]. Taken together, these results indicate that diets that are rich in nutrients that elevate circulating CCK concentrations result in reduced sensitivity to the satiation by intestinal fat and exogenous CCK (Fig. 13). One interpretation of these results is that the adaptive value of inhibition of food intake by intestinal fat is metering the delivery of fat for efficient digestion and Fig. 13. CCK-induced suppression of food intake in rats adapted to a low-fat (LF), high-fat (HF), and high-protein (HP) diets. HF and HP, but not LF, have been shown to elevate plasma CCK concentrations. Intraperitoneal administration of 125 or 250 ng/kg dose of CCK produced significantly greater reduction of intake in LF-adapted rats than in HF- or HP-adapted rats. * Significant difference from LF ( P <.05). From Covasa et al. [42].

19 R.C. Ritter / Physiology & Behavior 81 (2004) not limiting excessive caloric consumption in the interest of overall energy homeostasis. On the other hand, one may also argue that regardless of the physiological role of fatinduced intestinal satiation, the reduction of sensitivity to intestinal fat, and CCK, could contribute to overconsumption and development of obesity. 4. Summary and synthesis Current experimental results on control of food intake by signals from the gastrointestinal tract present an intriguing but complex picture. Studies on the effects of dietary adaptation to fat, as well as some other findings, suggest that satiation signals from the GI tract function to limit ingestion in the interest of efficient digestion. Afferent signals arise from the entire length of the GI tract, including the stomach, the proximal small intestine, the distal small intestine and the colon. The adaptive value of signals from various levels of the GI tract are likely to be different and distinct. Most signals from the stomach and proximal small intestine arise while a meal is in progress and when digestion and absorption are just beginning. These signals evoke responses that alter ongoing ingestion, and the rate at which nutrients are presented to the normal absorptive surface. Signals from the distal small intestine and the colon are likely to evoke responses to deal with incomplete digestion or absorption. These areas of the GI tract deal with the reclamation of fluid, electrolyte and digestive secretions, such as bile acids. The appearance of undigested nutrients here signals potential digestive pathology that might threaten hydration and electrolyte balance. The common feature of both upper and lower GI signals controlling food intake is that they probably exist, first and foremost, to promote efficient digestion and absorption and to protect the animal from the deleterious effects of excessive consumption and inefficient digestion. The primacy of GI controls for GI function, in no way, precludes their recruitment for participation in other physiological processes such as control of energy balance. The inhibition of food intake in response to GI stimulation also affords the first post-oral clue of increased energy availability. Recent experimental results suggest that GI signals are modulated by humoral and neural afferents reflective of energy storage or utilization [11,66,132,133,162,234] and, thereby, may adjust the caloric intake in the interest of non-gi-related physiological functions. Finally, the integration of GI sensory information does not end with the first central synapse of primary vagal afferents. 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