Endocannabinoids in the Regulation of Appetite and Body Weight T

Review 297

Endocannabinoids in the regulation of appetite and body weight T. C. Kirkham

The discovery of cannabinoid receptors, together with ingesta. Moreover, there is strong evidence of an the development of selective cannabinoid receptor endocannabinoid role in energy metabolism and fuel antagonists, has encouraged a resurgence of cannabinoid storage. Recent developments point to potential clinical pharmacology. With the identification of endogenous benefits of cannabinoid receptor antagonists in the agonists, such as anandamide, scientists have sought management of obesity, and of agonists in the treatment of to uncover the biological role of endocannabinoid other disorders of eating and body weight regulation. systems; initially guided by the long-established actions of Behavioural Pharmacology 16:297–313 c 2005 Lippincott cannabis and exogenous cannabinoids such as D9-tetra- Williams & Wilkins. hydrocannabinol (THC). In particular, considerable research has examined endocannabinoid involvement in Behavioural Pharmacology 2005, 16:297–313 appetite, eating behaviour and body weight regulation. Keywords: eating, feeding, obesity, incentive, reward, cannabis, rimonabant, It is now confirmed that endocannabinoids, acting at anorexia brain CB1 cannabinoid receptors, stimulate appetite and ingestive behaviours, partly through interactions with School of Psychology, University of Liverpool, Liverpool, England. more established orexigenic and anorexigenic signals. Sponsorship: The author’s research is supported by the United Kingdom Key structures such as the nucleus accumbens and Biotechnology and Behavioural Sciences Research Council. hypothalamic nuclei are sensitive sites for the hyperphagic actions of these substances, and endocannabinoid activity Correspondence and rtequests for reprints to Professor T.C. Kirkham, School in these regions varies in relation to nutritional status of Psychology, University of Liverpool, Eleanor Rathbone Building, Bedford Street South Liverpool, L69 7ZA, England. and feeding expression. Behavioural studies indicate e-mail: [email protected] that endocannabinoids increase eating motivation by enhancing the incentive salience and hedonic evaluation of Received 13 June 2005 Accepted 26 July 2005

Cannabinoids, endocannabinoids and Two main cannabinoid receptors subtypes have been cannabinoid receptors identified. The CB1 receptor is widely distributed within The appetite-stimulating action of the cannabis plant the central nervous system and many peripheral tissues (Cannabis sativa) and its extracts has been documented for Devane et al., 1988; Matsuda et al.,1990.TheCB2 receptor many centuries (Abel, 1975; Kirkham and Williams, was until recently considered not to be significantly 2001a). This effect appears to be largely attributable to expressed in the central nervous system, being localized D9-tetrahydrocannabinol (THC), one of a large family of primarily in immune cells (Munro et al.,1993;Mackie, related ‘cannabinoid’ molecules first characterized in the 2005); however, there is new evidence for its expression 1960s (Gaoni and Mechoulam, 1964; Mechoulam et al., within brain neurons and glia (Onaivi et al.,2005).Bothof 1970). The physiological basis for the efficacy of plant- these are cell membrane receptors belonging to the G- derived cannabinoids in people has now been explained protein-coupled family of receptors (GPCR; Howlett, by the discovery within mammalian systems of cannabi- 2005). There is also growing evidence to support at least noid receptors and their endogenous ligands, the one other, non-CB1/non-CB2 cannabinoid receptor sub- ‘endocannabinoids’ (Pertwee, 2005a). The psychoactive type, but genes encoding other receptors have not been effects of the exogenous cannabinoids are thus explained identified (Fride et al.,2003;Begget al.,2005;Pertwee, by their ability to mimic the neural actions of the 2005a). It is generally accepted that the predominant endogenous agonists. It is increasingly apparent that the behavioural effects of cannabinoids are mediated by brain changes in eating motivation associated with cannabis CB1 cannabinoid receptors. However, these receptors are intoxication, or the administration of THC, reflect a also expressed in gastrointestinal, adipose and hepatic crucial role for these endocannabinoid systems in the tissues, so linking the endocannabinoids to important normal processes governing appetite, ingestive behaviour, peripheral processes related to energy storage and energy metabolism and body weight. This review will metabolism that may also affect appetite (Cota et al., examine primarily the evidence supporting endocannabi- 2003; Osei-Hyiamen et al., 2005). The availability of noid involvement in the behavioural and motivational selective CB1 receptor antagonists has been the prime aspects of these processes. mover in driving research in this area.

0955-8810 c 2005 Lippincott Williams & Wilkins

The endocannabinoids are eicosanoid compounds, humans may be summarized briefly in relation to a few synthesized from arachidonic acid. The first of these key studies. substances to be identified was arachidonoylethanolamine, now known as anandamide (Devane et al., 1992). For example, in an early study, Hollister (1971) examined Subsequently, 2-arachidonoylglycerol (2-AG) was isolated acute THC effects on the consumption of chocolate milk (Mechoulam et al., 1995; Sugiura et al., 1995), followed by shakes and found significantly increased intake, elevated the identification of the putative endocannabinoids hunger ratings and enhanced food appreciation. Over noladin ether (2-arachidonyl glyceryl ether), N-arachido- several years Foltin, Haney and colleagues have con- noyldopamine (NADA), and O-arachidonoylethanolamine ducted the most in-depth analyses, using a residential (virodhamine) (Hanus et al., 2001; Porter et al., 2002). laboratory setting (e.g. Foltin et al., 1986, 1988; Haney These substances are agonists at CB1 receptors, while et al., 1999, 2005; Hart et al., 2002). Early studies virodhamine is apparently a partial agonist that also has demonstrated substantial increases in daily caloric intake antagonist activity at the CB1 receptor (Porter et al., 2002; after cannabis smoke inhalation, primarily through an Howlett, 2004; Pertwee, 2005a). The neuromodulatory increase in the frequency and consumption of sweet actions of the endocannabinoids involve novel mechan- snack foods. It is likely, however, that these apparently isms that, as with the discovery of the endogenous opioid selective actions may be partly attributed to the peptides, are likely to revolutionize thinking about neural prevailing test conditions. For example, increased food regulation. Thus, endocannabinoids are thought to act intake was predominantly observed during social access primarily as retrograde signals: released by postsynaptic periods, suggesting a degree of social facilitation. By neurons, they bind to presynaptic heteroceptors to contrast, we have found that oromucosal application of modulate the release of inhibitory and excitatory THC will increase intake, but this effect is not restricted neurotransmitters through multiple GPCR-linked effec- to sweet foods, as the drug also significantly increased the tor mechanisms (Schlicker and Kathmann, 2001; Howlett size of savoury set meals. Moreover, these effects were et al., 2004; Vaughan and Christie, 2005). The metabolic obtained when volunteers were tested individually, pathways of anandamide and 2-AG have now been suggesting that cannabinoids can have broad effects on characterized in great detail, and we can anticipate that appetite that are not limited to specific flavours, food these pathways – together with endocannabinoid uptake types or the context in which food is available (Townson mechanisms – will complement receptors as targets for and Kirkham, unpublished observations). the pharmacological analysis of the physiological functions of these substances (Fowler et al., 2001; Di It is probable, although so far untested, that these actions Marzo et al., 2005). are CB1 receptor-mediated, since the broader psychological actions of cannabis in people are reversed by the Cannabinoids and human appetite selective CB1 antagonist rimonabant (SR141716; Huestis The established behavioural actions of cannabis and the et al., 2001). Finally, in addition to studies in healthy exogenous cannabinoids invited speculation that the individuals, a small number of clinical trials have been newly discovered endocannabinoids might be of impor- conducted to assess the possible benefits of cannabinoids tance to appetite processes. To date only anandamide and in the treatment of conditions involving wasting and 2-AG have been investigated to any significant extent appetite loss, such as cancer cachexia and AIDS. Although with respect to their actions on feeding behaviour, but it these trials were hampered by the nature of the seriously is clear that endocannabinoids do play a significant role in ill populations in which they were tested, beneficial behavioural and metabolic aspects of energy regulation. effects were uncovered. Notably, treatment with THC (generally as the synthetic form, dronabinol) leads to improvement in both appetite ratings and actual con- Despite recent discoveries, much of our theorizing about sumption levels; while weight loss is attenuated – with cannabinoid function is still based on historical accounts weight gain in some instances (Regelson et al., 1976; of the actions of cannabis and its derivatives in people. Plasse et al., 1991; Beal et al., 1995). Consequently, THC, Unfortunately, political considerations have made it in the form of dronabinol, has been approved for the notoriously difficult to conduct the necessary scientific amelioration of appetite and weight loss (and for its anti- studies in humans (Robson, 2005), and a great deal emetic action; Kirkham, 2004). remains to be determined about the precise psychological and physiological actions of cannabinoids that underlie their hyperphagic effects. The limited literature in Cannabinoids and feeding in animal models relation to cannabinoids and human appetite has been Since the 1970s THC has been shown to stimulate reviewed extensively elsewhere (Kirkham and Williams, feeding in a variety of animal models, after systemic or 2001a, c, 2004; Kirkham, 2004), and in this review I shall central administration (Brown et al., 1977; Anderson- focus on findings from the animal literature. However, Baker et al., 1979; Williams et al., 1998), although several the principal effects of exogenous cannabinoids in laboratories initially reported hypophagic actions through

the use of very high, sedative doses. With the discovery of antagonist rimonabant (Rinaldi-Carmona et al., 1994) and the endocannabinoids, the pace of this aspect of feeding its sister compounds have largely driven research in this research has accelerated. In this regard it is important field. Indeed, a role for endocannabinoids in feeding was that the hyperphagic action of THC has been shown to largely posited on the basis of the intake suppressant be mediated by CB1 cannabinoid receptors: THC- actions of rimonabant, which were reported long before induced feeding is reversed by the selective CB1 any actions of the endogenous CB1 agonists on feeding antagonist rimonabant, but not the CB2-selective antago- had been demonstrated. An anorectic action of rimona- nist SR144258 (Williams and Kirkham, 2002a). The bant was first reported by Arnone and colleagues at Sanofi hyperphagic effect of THC in rats is also remarkably in 1997. The ability of the drug to suppress ingestion was potent, causing animals to overconsume even when not restricted to food, as sucrose and ethanol drinking replete (Williams et al., 1998). Interestingly, low doses were also reliably affected (although water drinking in of D8-THC, have been reported to have significantly thirsty animals was unaffected). Consequently, it was greater hyperphagic potency than D9-THC, and to exert argued that the ability of the antagonist to exert an fewer cannabimimetic side-effects (Avraham et al., 2004). anorectic action was evidence for tonic endocannabinoid Such data indicate the importance of exploring the activity in feeding-related systems (Arnone et al., 1997). behavioural actions of other, largely un-investigated An apparently greater effect of rimonabant on sweet food, phytocannabinoids. together with the effects on alcohol, led to the notion that the drug exerted its effects through reward pathways The hyperphagic actions of THC have been replicated to modify the appetitive value of ingesta (Simiand et al., following administration of the endocannabinoids anan- 1998). Reliable anorectic actions of rimonabant, or its damide and 2-AG. These substances increase food intake analogues (e.g. AM281, AM251, O-3259 and O-3257), in rodents following systemic or central injection and have since been reported (typically at doses above 3 mg/ kg, i.p. or p.o.). Intake suppression has been observed their actions are CB1 receptor-mediated, being blocked following systemic or central administration in satiated or by rimonabant but not the CB2 antagonist SR144528 (Williams and Kirkham, 1999; Hao et al., 2000; Jamshidi food-deprived animals, and after acute or chronic treat- and Taylor, 2001; Go´mez et al., 2002; Kirkham et al., 2002). ments (Columbo et al., 1998; Shearman et al., 2003; Importantly, anandamide and 2-AG will promote feeding Werner and Koch, 2003; Chen et al., 2004; Rutkowska, when administered into hypothalamic nuclei and into the 2004; Wiley et al., 2005). Sanofi have recently reported shell of the nucleus accumbens (Jamshidi and Taylor, the ability of a new CB1 antagonist, SR147778, to dose- 2001; Kirkham and Williams, 2001c; Kirkham et al., 2002). dependently reduce food intake in fasted or non-deprived These regions are firmly associated with eating motiva- rats, and to reduce sucrose consumption in rats and mice tion, and their sensitivity to the hyperphagic actions of (Rinaldi-Carmona et al., 2004). A number of studies have anandamide and 2-AG strongly supports a key role for also investigated the effects of CB1 blockade in genetic endocannabinoids in the control of eating. models of obesity. Thus rimonabant and AM251 have been shown to exert reliable effects on food intake and body weight in obese fa/fa rats, and ob/ob and agouti Recently, a third putative endogenous agonist, noladin ether, has been shown to increase food intake after yellow A(y) mice, often with greater effects than in lean littermates (e.g. Vickers et al., 2003; Zhou and Shearman, systemic treatment in food-restricted mice (Avraham et 2004). al., 2005). This action is again CB1-mediated, being reversed by rimonabant, and is obtained with very low doses (0.001 mg/kg, i.p.). Although there has been some The ability of CB1 antagonists to suppress food intake debate about whether noladin ether is naturally present soon led to assessment of their potential as anti-obesity in the brain (Oka et al., 2003), its relative stability treatments. Consequently, there has been considerable compared to anandamide or 2-AG (which are metabolized interest in the chronic effects of these drugs on intake rapidly) may yet provide a useful tool for probing and body weight. In an early study, Colombo et al. (1998) endocannabinoid function in appetite. Clearly, more work demonstrated that daily administration of rimonabant is required to assess the feeding actions of each suppressed intake of lab chow and induced persistent endogenous cannabinoid: given the topicality of this area weight loss in rats. Although the anorectic action of the of research, there are surprisingly few studies addressing drug dissipated after 5 days, suppression of body-weight the behavioural actions of these agonists. gain was evident across the full course of a 14-day experiment. More recently, Vickers et al. (2003) demonstrated that sub-chronic oral treatment with rimonabant Anorectic actions of cannabinoid receptor dose-dependently decreased food (chow) intake and antagonists body weight gain in both lean and genetically obese Complementing the feeding actions of CB1 agonists is Zucker (fa/fa) rats. Once again, anorectic effects were no the ability of CB1 receptor blockade to suppress eating in longer evident after 4 days in lean animals, but reduction laboratory species. Experiments with the prototypic CB1 of body weight gain was maintained over 28 days. These

effects were greater in obese Zucker rats than in lean attenuate eating and facilitate weight loss with an controls, with daily food intake initially reduced by as interrupted dosing regime suggests that tolerance to much as 40% and persistent weight loss evident over the the effects of CB1 antagonists may not be an obstacle to first 2 weeks of treatment. Moreover, the weakening of the long-term application of these drugs in the treatment the anorectic action of the drug was considerably delayed of obesity. in obese animals, although their intake subsequently remained lower than in vehicle-treated controls. Even The mechanism underlying the rapid loss of CB1 after significant anorectic effects had waned, the rate of antagonist anorexia with repeated treatment is not weight gain remained significantly suppressed in antago- understood, nor well-explored. This loss of effect, often nist-treated rats. Withdrawal of rimonabant on day 28 described as tolerance, has been reported with respect to resulted in rebound hyperphagia and significant weight other effects of these compounds (e.g. Carai et al., 2004). gain (Vickers et al., 2003). The differential change in efficacy on food intake and body weight is presumably unrelated to metabolic The effects of chronic CB1 blockade has also been tolerance, but may reflect behavioural tolerance in addressed in mice made obese through the provision of a response to persistent intake suppression (see below), high-fat diet. This diet-induced obesity is a good model or pharmacodynamic differences between brain and the for the most common form of human obesity and its peripheral cannabinoid systems, which may be more consequences, including visceral obesity and diabetes. directly linked to weight change (discussed in a later Ravinet-Trillou et al. (2003) reported that daily rimona- section). Rubino et al. (2000) proposed that desensitiza- bant administration reduced food intake by almost 50% tion to the initial behavioural effects of chronic rimona- during the first week of a 5-week study. This initial bant treatment may involve, variously: uncoupling of the anorectic effect gradually diminished, but intake re- CB1 receptor from its transduction system; homeostatic mained suppressed compared to vehicle-treated controls elevation of endocannabinoid synthesis; or tolerance to throughout the whole test period. Overall, body weights increased endocannabinoid levels resulting from disin- were reduced substantially after 1 week and stabilized at hibition of their normally negative control over specific that lower level until the end of the experiment. Carcass neural pathways. analysis showed that rimonabant significantly reduced adipose stores, halving the proportion of body fat seen in It is notable that one group has reported that a single controls fed the same high-fat diet, while preserving lean dose of AM251 can lead to intake suppression for up to 6 mass. A follow-up study confirmed these effects and days post-treatment (Chambers et al., 2004), despite the demonstrated that the drug-induced changes in intake reported half-life in mouse brain for this compound being and body composition were dose-dependent. Addition- of the order of several hours (Gatley et al., 1996). ally, elevated plasma levels of insulin, leptin and insulin McLaughlin et al. (2003) have also reported that the resistance that accompany the development of obesity, motivational effects of rimonabant and AM251 have a were substantially reduced by antagonist treatment. long duration, with significant suppression of food- motivated behaviour evident for up to 22 h after Hildebrandt et al. (2003) confirmed the general effec- administration. Although such a persistent effect has to tiveness of chronic CB1 receptor blockade in dietary be separated clearly from non-specific actions (such as obesity using the rimonabant analogue, AM251. The drug the induction of conditioned taste aversion; see below), dose-dependently suppressed intake, initially by as much the advantages to therapeutic regimes of intermittent, as 60% of control levels. Significant, dose-related weight long-lasting antagonist treatments and the avoidance of loss was evident after 3 days of treatment and was tolerance are obvious. maintained over an initial 2-week period of daily oral administration; with significant intake suppression evident until day 12. Rebound hyperphagia was apparent CB1 receptor knockout mice –/– during a subsequent drug-free inter-treatment phase, but The availability of CB1 receptor knockout mice (CB1 ) body weight gain was negligible until after 4–5 drug-free has provided important supporting evidence for endo- days. Reliable anorexia and weight loss were reinstated cannabinoid involvement in appetite regulation. Ravinet- when AM251 treatment was given for a further 2 weeks – Trillou et al. (2003) characterized the phenotype of these again with very marked initial intake suppression. In this animals when fed either standard chow or a high-fat diet. –/– second drug stage, attenuation of the intake-suppressing When maintained on chow, CB1 mice were leaner and +/+ actions of the drug was evident somewhat earlier, but slightly hypophagic compared to the wild-type (CB1 ) –/– weight loss was maintained with marked reductions in animals. When fed the palatable, high-fat diet, CB1 adiposity. Plasma leptin and cholesterol levels were also mice did not display the hyperphagia characteristic of significantly reduced at the highest dose, with an the wild-type mice and did not develop obesity. Addi- –/– additional tendency for plasma insulin to be reduced tionally, although CB1 mice did display a preference (Hildebrandt et al., 2003). The ability of AM251 to for the high-fat diet, this was acquired more slowly than

+/+ in CB1 littermates. Knockout mice also show et al. (2005) reported that AM251 dose-dependently reduced consumption of sucrose compared to the wild reduced consumption of a novel, flavoured solution with type (Poncelet et al., 2003). Additionally, Di Marzo et al. which it had been paired 4 days previously. Additionally, –/– (2001) have shown that CB1 animals display a the antagonist induced aversive gaping responses to reduced hyperphagic response to fasting, eating less than saccharine in taste reactivity tests. Such effects are wild-type littermates. clearly of concern in trying to interpret the anorectic actions of CB1 antagonists (and particularly in relation to their possible application in human obesity). Non-specific effects of antagonists Although antagonist-anorexia has been widely replicated, Aversive or illness-inducing effects of the antagonists may there has been some debate about the mode and still be independent of any ability to specifically suppress specificity of action of these drugs on eating motivation. appetite, since the endocannabinoids are separately For example, rimonabant and its analogues have inverse associated with the complex mechanisms that influence agonist properties in some assays, suggesting that nausea and vomiting (Darmani et al., 2003, 2005; suppression of food intake may reflect this ‘inverse Kirkham, 2004). Notably, cannabinoid CB receptors are cannabimimetic’ action rather than competitive blockade 1 widely expressed in the brain stem dorsal vagal complex of endocannabinoid tone (Pertwee, 2005b). Arguably, associated with triggering emetic responses. And, using these compounds are neutral antagonists at low doses and vomiting species, it has been demonstrated that rimona- inverse agonists at higher concentrations. It is certainly bant can induce vomiting at high systemic doses the case that relatively high systemic doses of these drugs ( > 10 mg/kg, i.p.; Darmani, 2001). Such effects might are required to reliably inhibit feeding (typically > 3 mg/kg). be anticipated given the accepted medicinal use of However, behaviourally silent rimonabant doses effec- dronabinol and nabilone for the suppression of nausea and tively attenuate the feeding actions of exogenously emesis, and the demonstration in animals that ananda- administered endocannabinoids, which would tend to mide and other CB agonists will suppress vomiting and support the active role of CB agonists in driving food 1 1 conditioned rejection responses to flavours associated intake. A recent suggestion is that rimonabant-like drugs with illness (Limebeer and Parker, 1999; Parker et al., may act as neutral antagonists to displace agonists from 2003). It should be noted that, conversely, 2-AG has been one site on the CB receptor, but exert inverse agonist 1 shown to have emetic actions (Darmani, 2002). More effects by acting at a separate allosteric site, so analyses are required to resolve these issues, but we modulating the constitutive activity of the receptor presume that, in most instances, cannabinoid-induced (Pertwee, 2005b). The new availability of potent neutral feeding does not involve the suppression of nausea. CB1 antagonists such as NESS 0327, will help resolve these issues (Ruiu et al., 2003). An additional concern may Ultimately, of course, confirmation of the role of be that at high doses rimonabant can bind to CB2 receptors (Pertwee, 2005b). However, the inability of the endocannabinoids in the stimulation of eating is not dependent on the demonstration of behaviourally specific selective CB2 antagonist SR144528 to block cannabinoid- induced feeding (Williams and Kirkham, 2002a), or to intake suppression by CB1 antagonists. None the less, suppress feeding in its own right (Wiley et al., 2005) rimonabant and its analogues have helped confirm the selectivity of agonist actions at CB1 receptors by block- supports the key involvement of CB1 receptors in the behavioural actions of rimonabant. ing their hyperphagic effects at doses that are much lower than those required to produce overt behavioural effects. Another important issue in relation to the mechanisms whereby antagonists suppress food intake relates to the possibility of induction of non-specific malaise, or of Endocannabinoids and eating motivation behaviours that are incompatible with the expression of Having established that agonist and antagonist inter- eating. At doses that overlap those found to suppress food actions at CB1 receptors can respectively stimulate or intake, CB1 antagonists have been reported to have suppress feeding, we are left the task of ascertaining the anxiogenic activity, and to promote behaviours such as underlying mechanisms through which these changes are wet-dog and head shakes, forepaw fluttering and facial effected. Again, we are faced with a relative paucity of rubbing that might interfere with feeding (Navarro et al., data – particularly of detailed psychometric analyses of 1997; Rubino et al., 2000). It should be noted that these agonist and antagonist effects in humans. Animal data are effects are not universally reported in feeding studies also generally restricted to measures of changing food (although, of course, they may not be routinely assessed). intake levels, which of themselves may not impart any However, there is some evidence for the possibility that clear indication of motivational specificity. Nevertheless, antagonists may induce conditioned taste aversion (CTA) we shall summarize the available findings here and argue at anorectic doses, and that this effect may partially for a dual role of central endocannabinoids in the account for intake suppression. For example, McLaughlin initiation and maintenance of eating.

A common theme in hypotheses about how cannabinoids CB1 receptor agonists, while antagonist treatments affect eating motivation reflects the kinds of effects attenuate responding (Gallate and McGregor, 1999; described earlier: an increased sensitivity to the sensory Gallate et al., 1999). In line with the antagonist studies properties of foods and apparently preferential effects on are reports that CB1 knockout mice have reduced preferred, highly palatable foods. As already noted, the sensitivity to the motivating properties of food. Thus, –/– earliest studies of the effects of rimonabant on feeding CB1 animals show lower levels of responding for were specifically couched in terms of reward processes. sucrose in operant situations, exhibiting lower break Arnone et al. (1997) compared the effects of repeated oral points than wild-type mice (Sanchis-Segura et al., 2004). treatments in rats fed either normal, bland maintenance Interestingly, antagonist effects on operant responding diets or more palatable supplements (45 mg sucrose are evident with bland as well as palatable foods pellets). After 1 week of daily administration, rimonabant (Freedland et al., 2000; Pe´rio et al., 2001), and rimonabant was reported to suppress intake of the more palatable has proved equi-anorectic when tested with foods of food selectively, with intake of chow being unaffected. differing macronutrient content (Verty et al., 2004a). This However, on closer inspection the data reveal that suggests that endocannabinoids modulate appetitive rimonabant had suppressed chow intake in the initial processes per se, to provide a general gain in the incentive stages of testing, but this effect disappeared over several value of food. Moreover, our more detailed observational days (a phenomenon subsequently reported by other analyses and monitoring of meal patterns in freely fed rats groups). Recalling our earlier discussion, it is possible that treated with cannabinoids reveal that stimulation of CB1 aversive effects contributed to the initial suppression of receptors directly increases the salience of food, irrespec- both normal and palatable food intake. In this study tive of need or energetic status. Thus, exogenous and animals had only restricted access to food, suggesting that endogenous cannabinoids can induce feeding almost as behavioural tolerance developed in response to cumula- soon as chow becomes available; even when animals are tive caloric deficit, through which animals overcame tested under conditions in which motivation to eat is deleterious effects of the drug to restore consumption of normally minimal – for example, after pre-satiation by the more familiar chow. However, the authors specifically overconsumption of a highly palatable food (Kirkham ruled out any action of rimonabant to promote neophobic et al., 2002; Williams and Kirkham, 2002b). Typically, in responses to the sweet food. A more clear-cut demonstra- our experiments, elevated food intake after cannabinoid tion of selective suppression of palatable foods was treatment primarily involves the advance of meal onset, subsequently reported by Simiand et al. (1998), who rather than a marked increase in meal duration or meal reported that in marmosets (Callithrix jacchus) low doses of size. Crucially, once initiated, the subsequent pattern of rimonabant reduced intake of a cane sugar mix without cannabinoid-induced feeding behaviour is identical to suppressing intake of standard pellets. that of untreated rats feeding spontaneously under home cage conditions (Kirkham and Williams, 2001a; Williams These early findings were interpreted generally in terms and Kirkham, 2002b). The latter finding indicates that of antagonist actions on orosensory reward processes, or cannabinoids provoke feeding through adjustments to on palatability responses to food. There is now strong natural feeding processes rather than, for example, by evidence to support specific involvement of endocanna- inducing stereotyped behaviour (Wiley et al., 2005). binoids in both the appetitive and consummatory aspects Overall, the ability of agonists to increase responding of eating motivation (Freedland et al., 2001; Thornton- for food and specifically to reduce eating latency implies Jones et al., 2005). Indeed, direct evidence in support that stimulation of CB1 receptors directly activates eating of endocannabinoids in general reward processes has motivation. recently been obtained through the demonstration that anandamide is self-administered by squirrel monkeys Of the various brain loci linked to feeding, the shell (Saimiri sciureus) (Justinova et al., 2005). We shall briefly subregion of the nucleus accumbens (AcbSh) has discuss the evidence that endocannabinoids are linked to particularly strong associations with appetitive processes. the processes that instigate food seeking and meal Neural activity here is believed to signal incentive initiation, as well as those that actively maintain ingestion salience and to facilitate the generation of motor patterns during meals. The data indicate that endocannabinoids orienting an animal toward potentially rewarding stimuli contribute both to incentive processes, and to hedonic – such as food in a hungry animal (Kelley, 1999). evaluation of food during eating – what Berridge (2000) Importantly, the AcbSh contains a relatively high density has respectively described as ‘wanting’ and ‘liking’ of CB1 receptors (Herkenham et al., 1991; Breivogel and processes. Childers, 1998) and is a particularly sensitive site for the induction of endocannabinoid-induced feeding. Bilateral Early evidence for a role of cannabinoids in incentive administration of 2-AG into this region induces sub- processes came from studies examining drug effects on stantial short-term, rimonabant-reversible hyperphagia operant responding. It was found that rats will work (Kirkham et al., 2002). In line with the notion that harder to obtain palatable ingesta after administration of cannabinoids increase the incentive value of food,

intra-accumbens 2-AG also significantly advances the As the differential actions of dopamine receptor-selective onset of feeding without markedly affecting other meal drugs indicate, the precise relationship between endo- parameters. Moreover, the rapidity of onset and magni- cannabinoids and dopamine activity in these systems is tude of 2-AG hyperphagia injected into the AcbSh are still uncertain (for a more comprehensive discussion see greater than the effects of anandamide or noladin ether Lupica et al., 2004). However, it is likely that these seen after peripheral administration or injection into interactions may involve coordination of the activity of other brain sites (Williams and Kirkham, 1999; Jamshidi ventral tegmental dopamine neurons. Specifically, it has and Taylor, 2001; Avraham et al., 2005). Importantly, and been proposed that stimulation of accumbens CB1 in accordance with the proposed action of CB1 agonists to receptors may suppress glutaminergic activity, with promote feeding by increasing the ‘wanting’ of food, we consequent inhibition of the GABAergic medium have also shown that acute food deprivation provokes spiny neurons that normally constrain the firing significant increases in anandamide and 2-AG levels of VTA dopamine neurons (van der Stelt and Di Marzo, within the limbic forebrain (which incorporates the 2003). Inhibitory prefrontal cortex-VTA afferents also AcbSh). Interestingly, there is indirect evidence from appear to undergo similar regulation by endocannabi- antagonist experiments for elevated endocannabinoid noids. The finding that VTA dopamine neurons activity during fasting. Specifically, we found that the themselves release 2-AG, which then acts to fine-tune anorectic action of rimonabant was significantly enhanced their activity, strengthens the potentially key role for in food-deprived rats compared to non-deprived animals these neuromodulators in the attribution of incentive (Kirkham and Williams, 2001a). salience and reward anticipation. Endocannabinoids, therefore, may be essential for the orientation to Mesolimbic dopamine neurons, arising in the ventral motivationally significant stimuli, and the elicitation of tegmental area (VTA) and projecting to the nucleus appropriate behavioural responses, such as food seeking accumbens, are regarded as being key to incentive (Melis et al., 2004; Lupica and Riegel, 2005). It should be processes and food wanting (Berridge, 2000, 2004). noted that serotonin (5-HT) has been linked to reward These pathways play an important role in the generation processing (Higgins and Fletcher, 2003). However few of emotional arousal and behavioural activation in data are available on possible endocannabinoid–5-HT response to stimuli that predict reward (Ikemoto and interactions in this regard. Current information is Panksepp, 1999; Spanagel and Weiss, 1999). Food stimuli confined to the reported failure of the indirect seroto- cause dopamine release in the nucleus accumbens after nergic agonist dexfenfluramine to affect THC-induced deprivation, or if the food is novel or palatable. feeding (Rowland et al., 2001; Williams and Kirkham, Importantly, there is growing support for an influence of 2002a). endocannabinoids on mesolimbic dopaminergic activity (Hermann et al., 2002; van der Stelt and Di Marzo, 2003; The hypothalamus is also considered to play a key role Lupica et al., 2004). For example, doses of THC within in integrating the multiple chemical and behavioural the hyperphagic range stimulate dopamine release in the components of feeding and weight regulation. Moreover, nucleus accumbens (Gardner, 2005; Gardner and Vorel, the accumbens and hypothalamus are functionally linked 1998; Malone and Taylor, 1999). Verty et al. (2004b) have components of feeding regulatory pathways (Stratford reported that THC hyperphagia is attenuated by a and Kelley, 1999; Stratford, 2005). It is therefore behaviourally silent dose of the D1 antagonist significant that not only will cannabinoid administration SCH23390, suggesting that mesolimbic dopamine signal- into hypothalamic nuclei induce eating, but that hypo- ling may be crucial to the appetitive actions of thalamic endocannabinoid activity apparently changes cannabinoids. with nutritional status and the expression of eating behaviour. For example, levels of 2-AG (but not anan- However, dopamine–endocannabinoid interactions are damide) are increased in the hypothalamus after likely to be complex, since SCH23390 has been shown 24-h food deprivation in rats (Kirkham et al., 2002) and to increase anandamide levels in limbic forebrain, while mice (Hanus et al., 2003). Interestingly, 2-AG levels the D2 antagonist reticlopride elevates 2-AG levels (Patel decline as animals eat, falling to control levels as animals and Hillard, 2003). Rimonabant can also prevent the satiate (Kirkham et al., 2002). These changes are wholly enhancement of food incentive value induced by compatible with the behavioural actions of cannabinoids dopamine agonists. Thus, the D3 receptor-preferring (i.e. an increased urgency to feed, but only marginal agonist quinelorane reinstates responding for food changes in meal size), and indicate the key involvement unexpectedly delivered during extinction, and this action of hypothalamic endocannabinoids in food wanting or is blocked by low doses of rimonabant (Duarte et al., craving (Kirkham and Williams, 2001c; Williams and 2004). By contrast, rimonabant had no effect on the Kirkham, 2002b). Accordingly, the decline of 2-AG levels response priming effect of food alone, indicating that CB1 during feeding would also indicate a minor, or indirect, receptors subserve the ability of dopamine to enhance role for this agent in the maintenance of feeding once it the motivational valence of food stimuli. has been initiated.

It should be noted that in contrast to the effects of acute observed in drug-free animals when the palatability of the deprivation, Hanus et al. (2003) found that hypothalamic test solution is increased (Higgs et al., 2003). Conversely, 2-AG decreased after chronic (12 days) food restriction. rimonabant alters drinking in a way that is consistent with These discrepancies were hypothesized to reflect differ- a reduction in the palatability of the sucrose solution. ent adaptive strategies in response to acute or chronic Direct comparison of the effects of changing palatability food deprivation. In the short term, energy needs and CB1 agonists or antagonists on ingestive behaviour resulting from environmentally imposed fasting, such as reinforces these conclusions. Thus, we found that in might arise from behavioural adaptations to an animal’s simple drinking tests, the effect of THC on ingestion of a particular ecological niche, could involve periodic upreg- 20% sucrose solution directly mimics the behavioural ulation of endocannabinoids to instigate food seeking. alterations following presentation of a more palatable, Conversely, during periods of famine, it may aid survival 30% sucrose solution: increasing the duration (but not to conserve energy by reducing the motivation to engage frequency) of drinking bouts. In rats freely drinking 30% in foraging – perhaps by reducing the conscious sucrose, rimonabant exerts an opposite action (Rogers et –/– experience of hunger (Hanus et al., 2003). It is the case al., unpublished observations). Additionally, CB1 mice that circadian rhythms in brain endocannabinoids have are less responsive to sweet taste, consistently drinking been demonstrated in the rat, which is primarily a less of a range of sucrose solutions than the wild type nocturnal feeder. Specifically, Valenti et al. (2004) found (Sanchis-Segura et al., 2004). Moreover, these differences that anandamide levels in the nucleus accumbens, pre- were abolished when sucrose solutions were adulterated frontal cortex, striatum and hippocampus were signifi- with bitter quinine, indicating that they arise from cantly higher in the dark phase than during daytime. The differences in the rewarding consequences of palatable story is complicated somewhat by the finding that 2-AG ingesta rather than from any sensory impairment. –/– levels in these regions displayed the converse pattern, Confirming our antagonist data, CB1 mice drinking being higher during the light phase. These differences, sucrose display reduced bout size but normal bout and the differential regional variation in anandamide and frequency. These findings thus support the hypothesis 2-AG noted in relation to fasting and feeding, emphasize that endocannabinoid activity contributes significantly to the limitations of current knowledge about the specific the hedonic evaluation of ingesta, and that CB1 stimula- role of individual endocannabinoids, and the urgent need tion or blockade/deletion can respectively render food to establish more precisely the neuroanatomical, motiva- more or less pleasurable. tional and contextual specificity of the behavioural actions of CB1 agonists. Consistent with this notion is the fact that key components of the neural mechanisms underlying food

We should also note here that CB1 receptors located in palatability lie within the AcbSh and, as already noted, 2- feeding-relevant hindbrain areas, such as the dorsal motor AG administered into this site produces a profound nucleus of the vagus (DMV) and the nucleus tractus hyperphagic response (Kirkham et al., 2002). Fasting, solitarius (NTS), may also be subject to cannabinoid which would be expected to elevate the reward value of regulation (Derbenev et al., 2004). Thus, the cannabinoid food, also increases levels of anandamide and 2-AG in the receptor agonist CP55,940 has been reported to enhance limbic forebrain (Kirkham et al., 2002). Moreover, Harrold milk intake with greater potency when administered into et al. (2002) have shown that accumbens CB1 receptors the fourth ventricle than when injected into the third are down-regulated in rats that overconsume palatable ventricle (Miller et al., 2004). food supplements. This latter effect is consistent with increased activation of these receptors by endocannabi- The data discussed above link endocannabinoids to noids, and again suggests they mediate the hedonic appetitive processes in feeding. However, their role may evaluation of palatable foods. be extended to involvement in food ‘liking’. Such a role is evident in anecdotal reports of cannabis users (Tart, There are, of course, some contradictory aspects of our 1970), and recent animal studies have provided support data that do not fully support this account. For instance, for a specific interaction of endocannabinoids with food we noted the lack of effect of intra-accumbens 2-AG on palatability. As we have seen, CB1 receptor blockade was meal duration (while eating latency was reduced): reported to preferentially attenuate the intake of enhancement of palatability would be anticipated to palatable, sweet foods (Arnone et al., 1997; Simiand extend meal duration. Possibly, the use of a relatively et al., 1998) and reduce operant responding for sweet food bland test food (chow) limited our ability to detect such (Perio et al., 2001); while CB1 knockout mice consume effects in these experiments. Additionally, the precise less sucrose than wild types (Poncelet et al., 2003). We location of drug infusion within the AcbSh may be critical have examined the actions of CB1 receptor ligands on the to the exact behavioural profile of drug-induced changes microstructure of sucrose drinking and found that to ingestive behaviour (Reynolds and Berridge, 2002). alterations to licking behaviour induced by exogenous However, in support of the palatability/accumbens and endogenous CB1 agonists are reminiscent of those hypothesis are recent data from Berridge’s group. Using

taste reactivity tests, they found that intra-accumbens There is evidence elsewhere in the brain for interactions administration of anandamide specifically increases the between endocannabinoids and opioids; notably within number of positive ingestive responses to intra-oral the hypothalamus, and particularly the paraventricular infusions of sweet solutions, indicating that anandamide nucleus (PVN) in which orexigenic and anorexigenic can indeed enhance the hedonic impact of sweet taste pathways converge. Importantly, both CB1 and opioid (Mahler et al., 2004). Finally, it is important to emphasize receptors are expressed in the PVN, and it is a sensitive that the role of endocannabinoids in palatability re- site for the hyperphagic actions of both cannabinoid and sponses is modulatory, since CB1 antagonists or CB1 opioid receptor agonists. It is of interest then, that deletion in knockout animals does not wholly remove the feeding induced by injection of morphine into this site rewarding properties of ingesta (Sanchis-Segura et al., can be reversed by rimonabant (Verty et al., 2003). 2004). Although we have so far considered feeding motivation in Endocannabinoid interactions with terms of discrete wanting and liking components, it may orexigenic/anorexigenic systems be that the borders between these are less distinct than Given these associations between endocannabinoid currently conceptualized. Previously, we have argued that palatability, it is noteworthy that the endocannabinoids the synergy between endogenous cannabinoid and opioid may have important functional relationships with the systems made apparent when CB1 and opioid receptors endogenous opioid systems that mediate orosensory are simultaneously blocked is indicative of some priming reward (Cooper and Kirkham, 1993; Kelley et al., 2002; function of the endocannabinoids: endocannabinoid Bodnar, 2004). It is of interest in this regard that CB1 activity in the prelude to feeding may facilitate the knockout mice are not only unresponsive to cannabinoids, positive, opioidergic hedonic response to the orosensory but display a reduced sensitivity to the rewarding effects properties of food during ingestion (Kirkham and of opiate drugs (Ledent et al., 1999). In rats, the Williams, 2001b). In essence, the two systems might hyperphagic action of THC is reversed by the general co-operate to ensure that effort expended in obtaining opioid receptor antagonist, naloxone (Williams and food is optimized by rendering the food palatable and so Kirkham, 2002a). Importantly, the facilitatory effects of encouraging its actual consumption. To stand aside from aCB1 agonist on responding for palatable solutions were the data briefly, and raise a debating point: imagine for a reversed by both a CB1 antagonist and naloxone (Gallate few moments the subjective experience of eating one’s and McGregor, 1999). Moreover, low doses of rimonabant favourite food. Think how good it will taste, how and opioid antagonists that are behaviourally inactive pleasurable and satisfying it will be, how your mouth will when administered singly, combine synergistically to water, and the lip-smacking that will accompany the first produce a profound anorectic action when co-adminis- mouthful. I think it is fair to say that anticipation of food tered (Kirkham and Williams, 2001b; Rowland et al., 2001; can involve at least a trace of the pleasure that will Chen et al., 2004). Given the established ability of opioid ultimately be derived from the physical experience of antagonists to reduce the hedonic evaluation of foods and tasting and eating it. Is it possible that this subjective to reverse CB1 agonist-stimulated ingestion, the marked experience reflects the overlap of endocannabinoid and anorexia induced by combined CB1 and opioid receptor opioid function? blockade again strengthens the proposition that endocannabinoids also contribute to orosensory reward pro- Historically, the consequences of independent interven- cesses. tions in endocannabinoid or opioid processes have been operationally distinguishable. For example, cannabinoids In opposition to this hypothesis is a report that feeding primarily reduce eating latency without affecting meal induced by intra-accumbens morphine administration duration (i.e. actions on appetitive processes); while was not reversed by rimonabant (Verty et al., 2003). opioids typically do not alter eating latency but extend However, intra-accumbens administration of either mor- meal duration by enhancing palatability (i.e. actions on phine or anandamide increases the liking of sweet consummatory processes). A closer temporal relationship, solutions (Pecina and Berridge, 2000; Mahler et al., or merging, of direct, primary cannabinoid influences on 2004), and there is ultrastructural evidence that canna- appetitive motivation and their secondary facilitation of binoid–opioid interactions are mediated by activation opioid consummatory components may be revealed by of CB1 and m-opioid receptors within the same, or some recent work by Solinas and Goldberg (2005). They synaptically linked, reward-relevant neurons in the AcbSh reported that THC and morphine dose-dependently (Pickel et al., 2004). Moreover, systemic administration of increased break points for food reinforcement, while THC has been shown to stimulate b-endorphin release in rimonabant and naloxone dose-dependently decreased both the VTA and AcbSh (Solinas et al., 2004), and CB1 break points. Confirming our findings in free-feeding rats, agonist-induced DA release in the accumbens is blocked the effects of THC were blocked by naloxone. But by intra-VTA infusion of the m1-selective antagonist, more surprisingly, the effects of morphine were blocked naloxonazine (Tanda et al., 1997). by rimonabant. These data potentially demonstrate

reciprocal cannabinoid–opioid mediation of appetitive representative model of diet-induced obesity (DIO), behaviour. The authors concluded that ‘activation of both induced by overconsumption of palatable foods. Thus, endogenous cannabinoid and opioid systems appears to Harrold et al. (2002) found that although rats with DIO jointly facilitate motivational effects of foody and this were hyperleptinaemic, there was no correlation between facilitatory modulation appears to critically depend on plasma leptin levels and hypothalamic CB1 expression interactions between these two systems’ (Solinas and (while CB1 receptors were upregulated in the nucleus Goldberg, 2005). Given the extensive evidence for accumbens in these animals). cannabinoid–opioid interactions in many other aspects of neural function, more research into these interactions In the first report of the anorectic action of rimonabant, in relation to appetite is clearly warranted. Arnone et al. (1997) also reported that the drug could block the ability of neuropeptide Y (NPY) to increase As might be expected, functional interactions between intake of a palatable sucrose solution. Despite this endocannabinoids and systems implicated in feeding are finding, the considerable potency of NPY as an orexigen, not restricted to the opioids. Indeed, there is a growing and its key interactions with other hypothalamic neuro- body of evidence to link endocannabinoids with a wide peptides regulating feeding, subsequent analysis of range of other factors currently implicated in the regula- potentially important relationships between this peptide tion of appetite (Cota et al., 2003; Di Marzo and Matlas, and the endocannabinoids has been limited. Further 2005; Sharkey and Pittman, 2005). The following overview investigation of functional interactions is warranted how- is largely phenomenological: despite the promulgation of ever, since Poncelet et al. (2003) reported that rimonabant substantive models for the place of endocannabinoids in can prevent NPY hyperphagia, and that the ability of –/– appetite and energy homeostasis, it is a matter of some NPY to stimulate feeding is abolished in CB1 mice, concern that the number of review articles in this area now although rimonabant is as effective in reducing food seriously outweighs the volume of original research articles intake in NPY knockout mice as in the wild type (Di – not least in relation to the specific behavioural actions of Marzo et al., 2001). the endocannabinoids themselves. An important component of appetite regulation – through Given its pre-eminence in current accounts of energy the co-ordination and integration of neural, metabolic regulation, much emphasis has been given to the and hormonal signals, is the hypothalamic melanocortin demonstration of apparent interactions between the system. Verty et al. (2004c) reported that in rats sub- adipokine leptin and the endocannabinoids. Leptin, anorectic, i.c.v. doses of the melanocortin MCR4 receptor which originates in adipose tissue and affects a number agonist a-melanocyte stimulating hormone (a-MSH) and of appetite-related factors in the hypothalamus, has been rimonabant combined synergistically to suppress feeding. proposed to be a core component in the regulation of Feeding stimulated by the MCR4 antagonist JKC-363 was long-term food intake and weight control (Friedman, dose-dependently attenuated by rimonabant, whereas 2002). It is therefore of interest that functional relation- THC-induced eating was unaffected by a-MSH. These ships between cannabinoids and leptin are indicated by results were interpreted as indicating that CB receptors recent research, and that endocannabinoid synthesis may 1 are located downstream from melanocortin receptors, and be regulated by leptin. Thus, Di Marzo et al. (2001) that endocannabinoids exert an inhibitory action on the reported that exogenous leptin administration, which ability of the melanocortin system to inhibit feeding. exerts an anorectic action, suppresses hypothalamic endocannabinoid levels in normal rats; while genetically obese, chronically hyperphagic rats (fa/fa) and mice (ob/ The same group of researchers also reported functional ob) express elevated, leptin-reversible, hypothalamic interactions between endocannabinoids and another appe- anandamide or 2-AG levels. Unfortunately, this study tite-inhibiting peptide, oxytocin. Again, subthreshold provided no indication of the extent to which these doses of i.c.v. rimonabant and oxytocin produced supra- changes reflect altered leptin signalling alone, rather than additive suppression of feeding. Although THC hyper- being a consequence of differences in eating behaviour. phagia was unaffected by oxytocin, rimonabant dose- However, rimonabant has been shown to be more dependently attenuated feeding induced by the oxytocin effective in suppressing food intake in obese Zucker receptor antagonist tocinoic acid. These data indicate (fa/fa) rats, which may be partly due to a greater that endocannabinoids can negatively modulate the sensitivity to the drug as a consequence of the upregula- inhibitory actions of oxytocin (Verty et al., 2004d). –/– tion of central cannabinoids. Additionally, CB1 mice have an enhanced sensitivity to the intake-suppressing Very little is presently known about the interaction of the actions of intracerebroventricular (i.c.v.) leptin (Ravinet- endocannabinoids with other neuropeptides involved in Trillou et al., 2003). We should note that data contra- energy control and food intake. However, there are dicting a simple relationship between leptin and canna- indications from histological analyses suggesting that binoid function have been obtained using the more further research would be fruitful. For example, Cota et al.

(2003) have shown co-localization in the PVN of the CB1 concentrations increasing in 24-h fasted rats (Go´mez receptor with the anorexigens cocaine amphetamine- et al., 2002). Moreover, the respective hyperphagic or regulated transcript (CART), corticotrophin-releasing anorectic actions of intraperitoneal anandamide and hormone (CRH) and orexigenic melanin-concentrating rimonabant were attenuated by capsaicin-induced de- hormone (MCH). There is also evidence for functional afferentiation of peripheral sensory nerves. These find- interactions between endocannabinoids and orexin A, an ings suggested that stimulation or blockade of peripheral orexigenic peptide that is expressed in the lateral CB1 receptors may influence central motivational pro- hypothalamus and which has been linked to the cesses, and have been interpreted as indicating a possible stimulation of feeding (Sakurai, 2005). More specifically, role for intestinal anandamide as a ‘hunger signal’. evidence was obtained for cross-talk between CB1 Unfortunately, this notion was originally posited on the receptors and the orexin OX1R receptor (Hilairet et al., basis that the authors could only obtain anandamide –/– 2003). Additionally, CB1 knockout mice show higher hyperphagia and rimonabant anorexia after systemic but levels of mRNA for the anorexigen CRH (Cota et al., not central administration, despite the fact that ananda- 2003), and CRH1 receptors are co-localized with CB1 in mide, 2-AG, THC and CB1 antagonists had already been several brain regions, including the hypothalamus (Her- reported by different groups to exert reliable effects on –/– mann and Lutz, 2005). The phenotype of CB1 intake via intracerebral or i.c.v. injection (Jamshidi and animals (characterized by hypophagia, reduced body Taylor, 2001; Koch and Matthews, 2001; Kirkham et al., weight and reduced fat mass compared to their wild- 2002). type littermates) may thus be interpreted as reflecting the pre-eminence of anorexigenic systems in these Nevertheless, subsequent study revealed that vagal animals, in the absence of counter-regulatory cannabinoid afferents originating in the stomach and duodenum, input. which express receptors (CCK1) for the putative satiety hormone cholecystokinin (CCK), also express CB1 Recently, evidence has been obtained for significant receptors. CB1 expression was increased by prolonged interactions between endocannabinoids and the orexi- food deprivation and rapidly reduced on re-feeding. genic peptide, and putative hunger signal, ghrelin. Interestingly, these respective effects were blocked by Ghrelin is a gut–brain peptide that peripherally is the CCK1 receptor antagonist, lorglumide, and mimicked released principally from gastric tissues, but which is also by CCK injection in fasted animals. It was concluded that synthesized within the hypothalamus (Kojima and CCK may naturally inhibit the ability of peripheral Kangawa, 2005). Circulating ghrelin levels are closely anandamide to stimulate feeding via vagal activity, and correlated with meal taking: rising in advance of meals so fasting may release vagal cannabinoid signals from CCK and declining rapidly post-prandially. Recently, we found inhibition. CB1 receptors are also expressed by CCK- that feeding stimulated by intrahypothalamic (PVN) containing neurons within the brain (McDonald and ghrelin injection is blocked by pre-treatment with Mascagni, 2001), although the implications of this rimonabant, suggesting that expression of ghrelin hyper- association to feeding have not been studied. Given the phagia is dependent on an intact endocannabinoid system contradictory data referred to above, more research is (Tucci et al., 2004). Interestingly, we have also shown that clearly required to assess the relative importance to behaviourally silent doses of ghrelin and THC will normal feeding regulation of gut-derived endocannabi- stimulate feeding when combined, indicating a synergis- noids, and to determine whether changing gut endocan- tic interaction between the two systems (Tucci and nabinoid levels reflect nutritional status directly, or are Kirkham, unpublished findings). Further support for related to their role in regulating gut motility and cannabinoid–ghrelin interactions comes from the report gastrointestinal enzyme secretion (Izzo et al., 2001; by Cani et al. (2004) that plasma levels of ghrelin are Coutts and Izzo, 2004). suppressed by systemic rimonabant treatment. Endocannabinoid regulation of fat Peripheral cannabinoids and feeding metabolism and adiposity stimulation Several findings raise the possibility of important In addition to the central nervous system, cannabinoids cannabinoid influences on adiposity/body weight that and CB1 receptors are also present in gastro-intestinal, may be distinct from their direct actions on appetite adipose and hepatic tissues, suggesting a variety of (Cota et al., 2003). Indeed, rimonabant and analogues possible influences of peripheral endocannabinoids on have been shown to reduce adiposity in diet-induced the regulation of appetite and body weight (Izzo et al., obese mice and genetically obese rodents, independently 2001; Cota et al., 2003; Osei-Hyiamen et al., 2005). A role of their primary anorectic actions (Ravinet-Trillou et al., for peripheral endocannabinoids in the control of feeding 2003; Vickers et al., 2003). Thus, chronic CB1 blockade was indicated by observations that anandamide is only transiently suppresses food intake, while weight synthesized within gut tissues, with small intestine loss persists for the duration of treatment. Moreover,

pair-feeding tests in which control animals receive the a lipogenic action: increasing fatty acid synthesis by same amount of food voluntarily consumed by antagonist- upregulating expression of the lipogenic gene transcrip- treated animals, showed that weight loss was greater with tion factor sterol response element-binding protein 1c rimonabant. Additionally, when deprived of food for 24 h, (SREBP-1c), and its target enzymes acetyl-coA carbox- rimonabant-treated obese mice lose more weight than ylase-1 (ACC1) and fatty acid synthase (FAS). Moreover, similarly deprived controls (Ravinet-Trillou et al., 2003). obesity induced by overconsumption of fat leads to elevated anandamide levels in the liver (through reduc-

As we have noted, CB1 knockout mice are resistant to tion of its enzymatic breakdown), upregulation of hepatic diet-induced obesity, and do not exhibit the insulin resis- CB1 receptors, and increased hepatic fatty acid synthesis tance normally occurring in high-fat-fed mice (Ravinet- (Osei-Hyiamen et al., 2005). These changes are pre- –/– Trillou et al., 2004). Additionally, rimonabant lowers vented by rimonabant and are absent in CB1 animals, plasma free fatty acid levels in dietary obese mice, suggesting a key role for liver endocannabinoids in the corrects hyperglycaemia, reduces plasma insulin levels development of obesity. and counters insulin resistance (Ravinet-Trillou et al., 2003). Antagonist treatments may therefore cause direct Osei-Hyiamen et al. (2005) have also suggested that CB1- interference with cannabinoid-mediated processes that mediated regulation of FAS within the brain may normally regulate fat deposition in adipose tissues, fatty provide evidence of a common pathway for central acid oxidation, and glucose homeostasis. It is therefore of endocannabinoid regulation of appetite and peripheral great significance that CB1 receptors are expressed by metabolic regulation. In support of this notion is the fact adipocytes in normal, but not CB1 receptor-deficient that lipid metabolism in feeding-related hypothalamic mice, and that stimulation of these receptors can induce neurons is sensitive to nutrient availability, and that lipogenesis (Cota et al., 2003). In this context, it is inhibition of FAS can suppress eating (Loftus et al., noteworthy that exercise (voluntary wheel-running) has 2000; Kim et al., 2002). Osei-Hyiamen et al. (2005) found been shown to augment the actions of AM251 on weight that hypothalamic FAS is upregulated by CB1 agonist loss in lean and obese agouti Ay mice, with lower doses of treatment, which would be compatible with the hyper- the drug required to reduce weight than when animals phagic actions of these drugs. The story is complicated, were not given access to exercise (Zhou and Shearman, however, by the finding that fasting, which is known 2004). This augmentation was attributed to possible to increase hypothalamic cannabinoid activity and to complementary effects on sympathetic activity, which in stimulate eating, had no effect on FAS expression in turn stimulates lipolysis. Jbilo et al. (2005) have also this region: marked changes were only detected after reported that rimonabant enhances lipolysis through re-feeding. induction of enzymes in adipose tissue that catalyse the b-oxidation and tricarboxylic acid (TCA) cycles. Critical These data may be complemented by some of our recent enzymatic regulators of glucose metabolisms are also data in relation to AMP-activated protein kinase (AMPK). upregulated by rimonabant, supporting a proposed action AMPK is an enzyme proposed to function as a fuel sensor, of the drug to increase energy expenditure (Liu et al., contributing to energy balance regulation at both cellular 2005; Jbilo et al., 2005). and whole-body levels (Hardie et al., 2004; Kahn et al., 2005). We found that both THC and 2-AG stimulate Interestingly, there are parallels between the effects of AMPK activity in the hypothalamus, while inhibiting rimonabant and the adipokine adiponectin, suggesting a AMPK activity in the liver and adipose tissue (Kola et al., possible mechanism whereby body weight is reduced by 2005). Given the proposed role of AMPK, these observa- the antagonist. Adiponectin expression varies inversely tions may provide important evidence of interactions with adiposity in animals and humans and, like rimona- between this enzyme and the orexigenic actions of bant, regulates hyperglycaemia, hyperinsulinaemia and cannabinoids. Thus, cannabinoids could potentially in- fatty acid oxidation, and can reduce the body weight of crease appetite by central AMPK stimulation, or by obese animals through a food intake-independent me- facilitating the restorative actions of AMPK as the chanism (Berg et al., 2001; Fruebis et al., 2001; Wolf, hypothalamus senses fuel deprivation. By contrast, 2003). Importantly, chronic rimonabant treatment in- peripheral inhibition of AMPK by cannabinoids may lead creases adiponectin expression through a CB1-mediated to fat storage. The combined effect of both central and mechanism, with greater effects in obese than lean rats peripheral AMPK activation could therefore be both (Bensaid et al., 2003). increased food intake and lipid deposition. Although such energetic models are likely to considerably more complex, As a major site of lipogenesis, the liver has also received these kinds of data are once again indicative of the rapid attention as a possible site of cannabinoid activity. Thus, development in the study of endocannabinoid mechan- in one of the most intriguing recent developments, isms, and of the strengthening support for their Kunos’ group has discovered that not only do hepatocytes contribution to the combined regulation of appetite and express CB1 receptors, but that agonist stimulation exerts energy utilization.

Cannabinoids in the pathology and treatment significantly associated with overweight and obesity in of obesity and eating disorders individuals with European or African ancestry, but not in a The involvement of endocannabinoids in appetite small group of Asians. Overall, the median body mass regulation raises two important questions: do these index (BMI) was significantly greater in the FAAH 385A/ systems play a significant role in disorders of eating and A genotype group compared to heterozygote and wild- body weight; and can pharmaceutical interventions type groups, with a higher frequency of the FAAH 385A/A targeting endocannabinoids have useful therapeutic genotype with increasing BMI: the strongest relationship applications? was observed in the white cohort. The authors concluded that this mis-sense polymorphism could indicate an The short answer to the first question is that there is endocannabinoid risk factor in the development of little information currently available. However, three overweight and obesity, specifically related to sensitivity recent studies suggest that further enquiry may be to the rewarding effects of food. fruitful. Monteleone et al. (2005) examined plasma levels of anandamide and 2-AG in women with anorexia nervosa, The possibility of manipulating endocannabinoid systems bulimia nervosa or binge-eating disorder. They found that in disease is already established – and not least in relation plasma levels of anandamide were significantly elevated to appetite and body weight. With continuing advances, in both anorexics and women with binge-eating disorder, the number of therapeutic opportunities will also expand. but not in bulimic patients. No significant alterations to We have already mentioned the use of THC to combat 2-AG levels were detected in any of the groups. the reduction in appetite, metabolic dysfunction and Anandamide and 2-AG levels were not reliably correlated wasting in AIDS and cancer (Plasse et al., 1991; Beal et al., with the severity of psychological symptoms or duration 1995; Haney et al., 2005). However, the clinical applica- of illness; nor was there any significant relationship tion of THC in these conditions predates the current between levels of the two endocannabinoids. Addition- knowledge of endocannabinoids and their behavioural and ally, circulating anandamide levels showed a significant metabolic functions. We anticipate that better under- inverse correlation with plasma leptin concentrations in standing of these functions will produce enhanced healthy controls, anorexics and women with binge-eating treatments in future. Cannabinoids may also have disorder. These findings suggest the possibility of some application in the wasting and appetite loss associated derangement in the production of anandamide in women with ageing and the dementias (Volicer et al., 1997). with these particular disorders, and the authors tenta- tively proposed that this reflected endocannabinoid As the data discussed earlier imply, anorexia nervosa could mediation of the rewarding aspects of aberrant eating be a potential target for the application of cannabinoids, behaviors. The full significance of these findings will despite its complex psychopathology. So far only a single depend on verifying the source of anandamide that was study, in which THC was ineffective, has been reported; measured, and the extent to which plasma levels reflect although more carefully designed studies may be more altered endocannabinoid regulation in the brain. Particu- successful (Gross et al., 1983). There may also be useful larly difficult to interpret is the elevation of anandamide interventions to be made in relation to the failure to in both restricting, underweight anorexics and obese, thrive of infants. Recent work strongly suggests that overconsuming binge-eaters; although the authors sug- endocannabinoid systems are vital to the ability of gested a possible link to leptin deficiency in the anorexic neonates to suckle (Fride et al., 2001). Thus when group and to leptin insensitivity in the binge eaters. rimonabant is administered to newborn mice, milk ingestion and subsequent growth is completely inhibited, These complexities are multiplied by the finding that with fatal results. Similarly, CB1 knockout mice display there may be genotypic differences between restricting deficient suckling at birth (Fride et al., 2005). Surpris- and binge-purging subtypes of anorexia nervosa. Specifi- ingly, the initiation of the suckling response is further cally, Siegfried et al. (2004) examined the frequency of a inhibited by rimonabant, suggesting the existence – and polymorphism of the human CB1 receptor gene, CNR1, possible upregulation – of an additional non-CB1/non- –/– and reported preferential transmission of different alleles CB2 receptor in the CB1 mice (Fride et al., 2003). in the patient subgroups. These authors suggested that Fride has proposed that at birth, brain 2-AG is sufficient the specific alleles do not necessarily increase an to stimulate suckling, but that 2-AG present in maternal individual’s susceptibility to developing anorexia nervosa, milk provides an essential additional stimulus to further but may modify the form of its expression. feeding. By extrapolation, the oral-motor deficiency associated with the nursing and growth failure seen after Finally, Sipe et al. (2005) recently reported obesity-related CB1 receptor blockade in neonatal mice may indicate that variations in a naturally occurring mis-sense polymorph- a deficient endocannabinoid-CB1 receptor system may ism in the gene encoding fatty acid amide hydrolase underlie the failure to thrive in human infants. Since (FAAH), the primary enzyme for inactivation of endo- 2-AG has also been detected in bovine and human milk cannabinoids. A homozygous FAAH 385A/A genotype was (Di Marzo et al., 1998), and dietary supplementation with

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