The intestine, the major site of macronutrient breakdown and nutrient absorption, is extensively innervated by vagal afferents with peak density in the duodenum and lower density in the ileum and distal intestine (Jagger et al., 1997). Intestinal vagal afferents that terminate within muscle layers are directly responsive to mechanical stimuli (Clarke and Davison, 1978), similar to those innervating muscle layers of the stomach. Mucosal vagal afferents, however, are likely to respond directly to chemical signals from primary sense cells within the intestinal mucosa (Figure 3). In support of a paracrine action of enteroendocrine cells, subsets of intestinal vagal afferents are known to express receptors for a number of key hormones released by gut epithelial cells, including those released in response to luminal nutrients (Kakei et al., 2002; Raybould et al., 2003; Nakagawa et al., 2004; Vahl et al., 2007; Bucinskaite et al., 2009); a number of these important signal mediators are discussed below.
Intestinal vagal afferents are responsive to a wide array of luminal stimuli, including hyperosmotic and hypo-osmotic solutions, acids and bases, and breakdown products of carbohydrates, proteins and fats (Clarke and Davison, 1978; Mei, 1978; Mei and Garnier, 1986; Lal et al., 2001). Intestinal perfusion with water, for example, has been shown to specifically activate subsets of intestinal vagal afferents in cats and rats (Mei and Garnier, 1986; Zhu et al., 2001). Infusions of hyperosmotic solutions exceeding 500 mosM also directly activate nodose ganglion neurons in rats, and significantly inhibit food intake in pigs (Houpt et al., 1983; Zhu et al., 2001). This osmolarity is within the normal postprandial range of ingesta within the intestine and indicates that non-nutritive characteristics of ingesta contribute to the intestinal satiety signal, and signal largely via vagal pathways. Intestinal nutrients exert powerful effects on food intake and satiety via vagal pathways. Subdiaphragmatic vagotomy or abdominal afferent denervation using the neurotoxin capsaicin largely or completely blocks the inhibition of food intake following intestinal infusion of carbohydrates or fatty acids (Yox and Ritter, 1988; Walls et al., 1995). Correspondingly, expression of the immediate early gene product, c-fos, in vagal brainstem nuclei, closely follows intraduodenal infusion of these nutrients (Phifer and Berthoud, 1998). Together, these data highlight the critical role of intestinal vagus signals in satiety, and the rationale of targeting the vagus in managing obesity. There are also many examples of intestinal nutrient infusion slowing gastric emptying via vagal reflex pathways in animals and humans (Wilkinson and Johnston, 1973; Roze et al., 1977; Raybould and Holzer, 1992; Schwartz et al., 1993). In healthy humans, this rate is slowed to 1–3 kcal·min−1, a rate matched to the absorptive capacity of the small intestine (Brener et al., 1983; Raybould, 1998). However, it is important to appreciate that while slowing of gastric emptying may interact with intestinal satiety signalling, the suppression of food intake by intestinal signals per se does not require gastric emptying to be slowed.
Much research effort has been focussed on intestinal L-cells due to their ability to broadly detect digestion products of carbohydrates, fats and proteins, and in response, secrete the incretin hormone GLP-1. L-cells are distributed throughout the GI tract, with greatest density in the distal intestine (ileum, colon); yet not insignificant numbers are present in the proximal small intestine in most species (Bryant et al., 1983; Eissele et al., 1992). These cells use alternative post-translational processing of proglucagon to produce the bioactive peptides GLP-1, GLP-2 and oxyntomodulin, although most attention has focussed on the actions of GLP-1 (Holst, 2007). GLP-1 is released rapidly in proportion to caloric load and to the length of small intestine exposed to nutrient, and acts to increase satiation, stimulate insulin release, suppress glucagon secretion and slow gastric emptying (Schirra and Goke, 2005; Horowitz and Nauck, 2006; Little et al., 2006). In addition, complex carbohydrates that reach the distal intestine may be fermented to short-chain fatty acids, which can also interact with L-cells via other nutrient sensors (Holst, 2007; Reimann, 2010). While these distal L-cell mechanisms can powerfully influence GI motility and food intake (as the ileal and colonic brake mechanisms), they may not be significantly recruited under normal GI transit due to effective upper gut absorption; they are also less likely to signal via a vagal pathway.
Our own work has shown that a subset of proximal intestine L-cells in both mice and humans express GPCRs for sweet taste (Sutherland et al., 2007; Young et al., 2010). Sweet taste receptors are under dynamic luminal and metabolic control, and link the luminal presence of a broad range of sweet tastants, including sweeteners, to the intestinal release of GLP-1 (for review, see Young, 2011). In L-cell-based systems, and recently in human intestine, it has been shown that blockade of sweet taste receptors reduces glucose-stimulated GLP-1 [and peptide YY (PYY)] release, while mice that are deficient in sweet taste molecule expression show dysregulated glucose-stimulated GLP-1 release (Jang et al., 2007; Margolskee et al., 2007; Gerspach et al., 2011). Sweet taste receptors provide one example, among others, of sensors expressed on L-cells, which may transduce luminal carbohydrate signals (Tolhurst et al., 2009). A similarly diverse list of candidate GPCRs in the intestine respond to breakdown products of amino and fatty acids leading to the release of GLP; these have been reviewed by others [see Table 1 (Little and Feinle-Bisset, 2011b; Reimann, 2010)].
The functional importance of GLP-1 in food intake control is now well established – genetic variation around the GLP-1 allele strongly correlates with total food volume in mice strains (Kumar et al., 2008), while mice deficient in GLP-1 receptors are partially resistant to HFD-induced obesity (Hansotia et al., 2007). Evidence indicates that effects of gut-released GLP-1 on food intake are mediated largely via a vagal pathway arising from the proximal intestine (Hayes et al., 2011). Subdiaphragmatic vagotomy completely blocks the satiating effect of intraperitoneal GLP-1 infusion (targeting intestinal vagal afferents) in rats, indicating that these effects are dependent on vagal afferent activation (Punjabi et al., 2011). Indeed, GLP-1 receptors have been localized on abdominal vagal afferents, on nodose ganglion neurons and in brainstem neurons (notably within the dorsal vagal complex) (Drucker and Asa, 1988; Goke et al., 1995; Imeryuz et al., 1997; Merchenthaler et al., 1999; Kakei et al., 2002; Nakagawa et al., 2004). While GLP-1 has also been shown to excite vagal afferents directly, effects on satiety and gastric emptying were long considered to involve endocrine actions at central sites within brainstem and hypothalamic nuclei (Imeryuz et al., 1997; Kakei et al., 2002; Nagell et al., 2006; Nakade et al., 2006; Bucinskaite et al., 2009; Holmes et al., 2009). However, the relative importance of central site of action for gut-released GLP-1 is uncertain as GLP-1 undergoes rapid degradation within minutes at the site of peripheral release and within the circulation and liver by the ubiquitous enzyme dipeptidyl peptidase-IV (DPP-IV), with less than 10% reaching systemic circulation in intact form (Deacon et al., 1996; Holst, 2007). Moreover, circulating levels of active GLP-1 do not significantly rise after regular chow meals in non-anaesthetized rats (Punjabi et al., 2011), suggesting that the normal signalling mode of gut-derived GLP-1 is predominantly paracrine (vagal), rather than endocrine. It should be noted, however, that GLP-1 is also produced centrally within autonomic neurons of the ventrolateral and caudal nuclei of the solitary tract, which project to various hypothalamic nuclei, and direct activation of GLP-1 receptors within the caudal brainstem is capable of reducing food intake (Merchenthaler et al., 1999; Vrang et al., 2007; Hayes et al., 2008; Llewellyn-Smith et al., 2011). However, blockade of central GLP-1 receptors does not block the anorexia induced by intraperitoneal GLP-1 in rats, whereas peripheral GLP-1 receptor blockade led to increased light phase food intake in rats (Williams et al., 2009), arguing against a prominent role of central GLP-1 in intestinal vagal afferent-mediated satiety. In summary, it appears that gut-derived GLP-1 is the primary satiety signal triggered upon food intake, and that vagal processing is sufficient to exert this effect.
It is also well established that the peripheral administration of GLP-1 or GLP-1 analogues (e.g. exenatide, liraglutide) dose dependently reduce food intake leading to weight loss in animal models of obesity and in lean and obese humans (for review, see Hayes et al., 2010). In rats, this reduced food intake with liraglutide is due to combined actions at GLP-1 receptors on peripheral vagal afferents of the intestine (or hepatic portal region) and centrally (Kanoski et al., 2011). Peripheral GLP-1 also directs energy balance on both a short- and long-term basis via interaction with leptin, which is hypothesized to increase intestinal vagal afferent sensitivity to GLP-1, as occurs for another anorexigenic peptide, CCK. Thus, a satiating effect of GLP-1 in fasted mice is revealed by co-administration with leptin, and GLP-1 is ineffective in reducing food intake in leptin receptor-deficient mice. Both CNS and peripheral sites of action appear plausible for this action (for review, see Barrera et al., 2011).
Glucose-dependent insulinotropic peptide (GIP)
GIP-secreting K-cells are located within the proximal small intestine, and are responsive to digestion products of carbohydrates and fat, as well as certain amino acids (Baggio and Drucker, 2007). GIP is rapidly released postprandially in proportion to calorific load (Pilichiewicz et al., 2007a), and acts to stimulate insulin release and promote lipid storage in adipocytes, the latter an emerging role that may link overnutrition to obesity (Yip et al., 1998; Baggio and Drucker, 2007; Kim et al., 2007). There is recent interest in GIP in the obesity setting, given that GIP knockout mice are resistant to diet-induced obesity (Miyawaki et al., 2002). Indeed, it has been proposed that part of the benefits of Roux-en-Y gastric bypass in obesity management may be due to surgical removal of intestinal K-cells (for review, see Paschetta et al., 2011). However, in contrast to GLP-1, exogenous GIP does not alter the rate of gastric emptying in response to intestinal carbohydrates, and correspondingly, vagal afferents in rodents lack receptors and functional responses to GIP (Nishizawa et al., 1996; Meier et al., 2004; Nakagawa et al., 2004). Moreover, while the density of intestinal K-cells may increase in obesity, GIP has no direct effects on hunger, desire to eat, satiety or prospective consumption in humans (Cho and Kieffer, 2011; Edholm et al., 2011). It should be noted that K-cells co-secrete the peptide xenin, which exerts satiating effects at a peripheral and/or central site of action, the latter which may occur independent of known hypothalamic satiety centres (Leckstrom et al., 2009; Taylor et al., 2011). It remains to be established whether a peripheral vagal pathway is involved in this action of xenin.
PYY is produced within L-cells that co-secrete GLP-1 in both the proximal and distal intestine, and is released in response to fatty acids, carbohydrates and to a lesser extent, amino acids (for review, see Holst, 2007). PYY release is in proportion to caloric load and acts to both increase satiation and slow gastric emptying (Batterham et al., 2006; Pilichiewicz et al., 2007b; Helou et al., 2008). Two endogenous forms mediate these effects, with PYY3–36– the predominant circulating form – produced following cleavage of PYY1–36 in circulation by the ubiquitous enzyme DPP-IV (Karra et al., 2009). Along with other members of this peptide family (neuropeptide Y, pancreatic polypeptide), PYY peptides interact with specific GPCRs Y1, 2, 4, 5 and 6 with differential specificity; PYY1–36 binds to all Y-class receptors, while PYY3–36 shows high affinity for Y2 receptors, and lower affinity for Y1 and Y5 receptors. Intraperitoneal administration of PYY3–36 in rodents was shown to exert a dose-dependent anorectic effect on food intake (Batterham et al., 2002), which could be completely blocked by subdiaphragmatic vagotomy (Abbott et al., 2005). Indeed, Y2 receptors have been identified on both intestinal vagal afferents and within the hypothalamus (arcuate nucleus) indicating that anorectic effects of Y2 receptor activation may be achieved via paracrine activation of intestinal vagal afferents, via direct central activation by circulating PYY3–36, or by both pathways (Zhang et al., 1997; Fetissov et al., 2004; Koda et al., 2005). Like GLP-1, PYY has complex effects on food intake upon central administration in rodents. Direct administration to the hypothalamic arcuate nucleus inhibits food intake via a Y2 receptor-dependent manner, while i.c.v. administration exerts orexigenic effects in the hypothalamic paraventricular nucleus via a Y1 and Y5 receptor-dependent manner (for review, see Cummings and Overduin, 2007). Together, these findings highlight the complex role of Y2 receptors in both vagal and central pathways regulating satiety.
Vagal expression of Y2 receptors is known to be modified by feeding status in rats, and in association with prevailing levels of the gut hormone CCK. Thus, Y2 receptor levels are low in fasted mice and high in fed mice, or in fasted mice infused with CCK (Burdyga et al., 2008). This action of CCK is mediated via release of the vagal afferent neuronal peptide derived from cocaine- and amphetamine-regulated transcript (CART), which acts in an autocrine manner to increase Y2 receptor levels (De Lartigue et al., 2011; reviewed in Dockray and Burdyga, 2011). Pharmacological blockade of Y2 receptors in rats has been shown to abolish feeding inhibition by PYY3–36, while germline knockout mice deficient in Y2 receptors are hyperphagic and develop obesity (Batterham et al., 2002; Scott et al., 2005); similar findings have been revealed in a PYY-specific knockout mouse (Naveilhan et al., 1999). Thus, there is ample evidence to suggest that vagal Y2 receptors are an important satiety-signalling mechanism.
CCK-secreting I-cells are located largely within the proximal small intestine and are more responsive to digestion products of fatty and amino acids than to carbohydrates (Rehfeld, 1978; Cummings and Overduin, 2007). It is well established that CCK release occurs in proportion to caloric load, but independent of the length of small intestine exposed (Liddle et al., 1985; Little et al., 2006). Release of CCK by fatty acids is critically dependent on acyl chain length, with only fatty acids ≥12 carbon atoms able to trigger release, and subsequent suppression of food intake (Hunt and Knox, 1968; McLaughlin et al., 1999; Feltrin et al., 2004). Short-chain fatty acids, in contrast, engage specific nutrient detectors in the distal intestine, which may influence satiety via non-vagal pathways as part of the ileal or colonic brake mechanisms (for review, see Kaemmerer et al., 2010). Activation of intestinal vagal afferents by fat involves the packaging of fatty acids within enterocytes into chylomicrons, followed by the production and basolateral release of apolipoprotein A-IV; apolipoprotein A-IV then triggers CCK release, activating CCK1 receptors on adjacent vagal afferent endings (Glatzle et al., 2003; Whited et al., 2006; Lo et al., 2007). An alternate signal pathway for CCK-dependent activation of vagal afferents has also been proposed for oleoylethanolamine, a lipid amide that also triggers intestinal release of apolipoprotein A-IV in a manner dependent on the activation of peroxisome proliferator-activated receptor alpha (Fu et al., 2003). However, much less is known of how CCK is released by proteins to activate intestinal vagal afferents. Evidence so far has revealed that the proton-coupled oligopeptide transporter PepT1 (peptide transporter 1) is indirectly involved via a CCK-dependent pathway (Darcel et al., 2005; Matsumura et al., 2005; Liou et al., 2011).
There is abundant evidence from our laboratory, and others, that intestinal vagal afferents express the CCK1 receptor, and are responsive to CCK (Cottrell and Iggo, 1984a; Moran et al., 1987; Blackshaw and Grundy, 1990; Broberger et al., 2001; Lal et al., 2001; Partosoedarso et al., 2001). Exogenous administration of CCK potently suppresses food intake and slows gastric emptying in rats and humans – effects blocked by vagotomy in rats (Smith et al., 1985; Schwartz et al., 1993; Sullivan et al., 2007; Brennan et al., 2008). In a similar manner, pharmacological blockade of CCK1 receptors dose dependently inhibits food intake and gastric emptying effects of exogenous and endogenous CCK in rats and humans (Fried et al., 1991; Moran et al., 1992; 1994; Yox et al., 1992; Beglinger et al., 2001). Genetic polymorphisms around the CCK (and leptin) allele in humans have also been shown to associate with specific eating patterns and meal size (de Krom et al., 2007), supporting a specific role for CCK in human satiety signalling. Despite strong evidence of a peripheral site of action, a central site for CCK satiety has been shown, based on expression and function of CCK receptors in hypothalamic satiety centres, and evidence that blood–brain barrier permeant CCK1 receptor antagonists modify food intake in vagotomized rats (Schick et al., 1990; Honda et al., 1993; Reidelberger et al., 2004). A recent and elegant magnetic resonance study in humans, however, has revealed that activation of brain regions upon intestinal exposure to intralipid was consistent with activation via a vagal pathway, and could be abolished by pharmacological blockade of CCK1 receptors (Lassman et al., 2010). Satiation in humans in response to long-chain fatty acids, and gastric emptying of hexose sugars are also reported to be blocked by the CCK1 receptor antagonist loxiglumide (Matzinger et al., 2000; Little et al., 2010), highlighting the importance of CCK satiety signals in response to a range of dietary macronutrients. Levels of CCK1 receptors in vagal afferents do not show significant nutritional plasticity in health, but serve as triggers to modulate levels of other anorexic and orexigenic receptors with feed status (for review, see (Dockray and Burdyga, 2011). CCK activation of vagal afferents and satiety signalling, though, is inhibited by orexigenic hormones including ghrelin, orexin A and the endogenous cannabinoid receptor agonist, anandamide (Burdyga et al., 2006a; 2010; 2006b; De Lartigue et al., 2011). In contrast, CCK activation of a subset of CCK1 receptor expressing vagal afferents is likely to be potentiated by anorectic actions of melaninocortin-4 receptor agonists (such as α-melanocyte-stimulating hormone) acting via a central presynaptic mechanism (Wan et al., 2008; Gautron et al., 2010), as well as by leptin.
CCK acts predominantly as a short-term satiety signal, as long-term administration in animals and humans does not alter body weight, as reduced meal size is effectively offset by increased meal frequency. In fact, the satiating actions of intraperitoneal CCK administration are lost as early as 24 h in rodents (Crawley and Beinfeld, 1983; West et al., 1984). However, anorectic effects of CCK are augmented by the long-term satiety hormone leptin, when CCK responses are potentiated at the level of vagal afferents, the brainstem and hypothalamus (for review, see Strader and Woods, 2005; Dockray and Burdyga, 2011). Accordingly, the adiposity of an individual, which is tightly linked to circulating leptin levels, is likely to sculpt responsiveness to short-term CCK signals. For example, anorectic effects of CCK would likely be decreased in both lean individuals and in leptin-resistant states, such as obesity. It is also important to note that rats deficient in CCK1 receptor expression are obese and hyperphagic, while mice lacking CCK1 receptors are lean, but become obese following a HFD (Schwartz et al., 1999; Bi et al., 2007); variable effects of a HFD have been reported in CCK knockout mice (Lacourse et al., 1999; Lo et al., 2011). Together, these data indicate that CCK exerts acute anorectic effects on meal size and timing, but exerts these actions predominantly via peripheral vagal actions to control food intake.
Serotonin-secreting enterochromaffin (EC) cells are a prominent enteroendocrine cell type distributed throughout the GI tract. EC cells release 5-HT in response to a wide array of chemical and mechanical cues, including acids, bases, carbohydrates, long- and short-chain fatty acids, as well as following distension or exposure to high-osmolarity stimuli (for reviews, see Grundy, 2006; 2008; Blackshaw et al., 2007; Bertrand, 2009; Raybould, 2009; Bertrand and Bertrand, 2010). Vagal afferents in rats and humans are known to express the 5-HT3 receptor, while in rats, both 5-HT3A and 5-HT3B subtypes are expressed and may form homomeric or heteromeric receptors each with different agonist sensitivity (Glatzle et al., 2002; Morales and Wang, 2002; Lang and Grafe, 2007). Following GI release, 5-HT exerts paracrine effects via activation of 5-HT receptors present on enteric nerves and vagal nerve endings within the mucosa; circulating 5-HT levels are kept low due to specific uptake into platelets. A number of other bioactive compounds have been identified in EC cell populations, including chromogranin A, melatonin, ATP, GABA, uroguanylin and dynorphin although less is known of their actions in the GI tract (see Bertrand and Bertrand, 2010).
While parenteral 5-HT administration has been shown to reduce meal size and duration in rats, vagotomy appears to increase this effect (Fletcher and Burton, 1985; 1986). Additionally, outcomes of studies using 5-HT3 receptor antagonists to influence satiety in animals and humans have generally been inconsistent, raising questions for a peripheral role of 5-HT in food intake regulation (for review, see Aja, 2006). Indeed, most attention has been focussed on central sites of satiety actions of 5-HT acting on 5-HT1 and 5-HT2 receptor subtypes (for review, see Atkinson, 2008). Despite this, evidence in rodents has indicated a role of peripheral 5-HT3 receptors in vagal pathways activated by carbohydrate, fatty acids and hyperosmolar stimuli leading to suppression of food intake, slowing of gastric emptying and pancreatic secretion (Li et al., 2000; Zhu et al., 2001; Raybould et al., 2003; Savastano et al., 2005; 2007; Wu et al., 2005). It has also been suggested that similar populations of intestinal vagal afferents activated by CCK in rats are responsive to 5-HT (Li et al., 2004); this could potentiate satiety signal arising in this common pool of vagal afferents, particularly in the presence of mixed meal components. A similar mechanism has been shown for gastric vagal afferent mechanosensitivity, which is augmented by both CCK and 5-HT and has been suggested is the primary peripheral satiety action of peripheral 5-HT (Bozkurt et al., 1999; Daughters et al., 2001; Hayes et al., 2006). Additional experiments are required to ascertain the precise role of peripheral GI release of 5-HT and vagal 5-HT3 receptors in food intake regulation, although this contribution is likely to be less than other direct vagal mediators of satiety.