There is increasing evidence of endocannabinoids' role in the regulation of appetite. Exogenous cannabinoids (smoked marijuana, A9-THC, dronabinol) have been shown to stimulate eating in humans (Abel, 1971; Foltin et al., 1988; Mattes et al., 1994), and this property may be used to enhance appetite in patients with cancer, AIDS, and Alzheimer's disease, and even in the elderly (Plasse et al., 1991; Volicer et al., 1997; Balog et al., 1998; Berry and Marcus, 2000). In rodents, exogenous and endogenous cannabinoids also promote overeating (see the following text), although some discrepancies exist in this respect (Crawley et al., 1993; Graceffo and Robinson, 1998; Giuliani et al., 2000).
The effects of cannabinoid agonists on feeding behavior are easier to observe when spontaneous eating is low, i.e., in animals fed ad libitum or when they are provided highly palatable food. For example, in free-feeding Lewis rats, given the choice between chow and highly palatable food, A9-THC administered either systemically (0.5 to 1 mg/kg i.p.) or centrally (1 to 25 ^g i.c.v.) induced a short-lasting (30 to 60 min), binge-like pattern of palatable food eating (Koch and Matthews, 2001). However, higher doses of A9-THC (2 or 2.5 mg/kg i.p.) were inactive or even induced hypophagia. In presatiated rats, A9-THC (0.5-2 mg/kg p.o.) induced an early large increase in usual food consumption, which was subsequently offset, so that a 24-h intake was similar to that of controls (Williams et al., 1998). Likewise, acute systemic injections of anandamide (up to 10 mg/kg s.c. or i.p.) or the synthetic CB receptor agonist, WIN55212-2 (0.4-2 mg/kg i.p.), enhanced food intake in presatiated rats (but not in 24-h food-deprived animals) and, curiously, were inactive on central administration (Williams and Kirkham, 1999; Gómez et al., 2002; Kirkham et al., 2002). The stimulation of feeding by A9-THC, anandamide, and 2-AG was mediated by CB1 receptors because it was selectively blocked by rimonabant but not SR144528, a selective CB2 receptor antagonist (Williams and Kirkham, 1999; Kirkham et al., 2002; Williams and Kirkham, 2002). In mice subjected to a 40% diet restriction, a 1-week chronic administration of anandamide at a very low dose (0.001 mg/kg.d i.p.) enhanced food intake during daily 2.5-h feeding periods (Hao et al., 2000), but not at higher doses (0.7 and 4 mg/kg). The biphasic dose-response modulation of feeding, frequently observed in these studies, might be related to motor deficits reported at A9-THC and anandamide doses above 2 to 3 mg/kg (Crawley et al., 1993; Romero et al., 1995). Such kind of effect might explain why acute and subacute administration of high doses of the potent CB receptor agonist HU210 (25 to 100 ^g/kg i.p.) produced dose- and time-dependent decreases of body weight. However, the reduction of food intake induced by the highest dose of HU210 was still present 7 d after the last of 4 daily injections, suggesting that perhaps other factors unrelated to ingestive behavior are involved in this anorectic effect (Giuliani et al., 2000).
On the other hand, the selective blockade of the CBj receptors by acute or subacute administrations of rimonabant reduced feeding in nonobese animals, whether nonfasted or food restricted. In some of these studies, rimonabant selectively reduced the consumption of palatable food (or drink), while having little effects on regular food or water intake (Arnone et al., 1997; Colombo et al., 1998; Simiand et al., 1998; Gallate and McGregor, 1999), suggesting that CBj receptors have a role not only in the consummatory aspect of feeding but also in the hedonic evaluation of food.
However, other studies showed that food deprivation or high palatability are not necessary to observe suppressive effects of rimonabant on food intake (Freedland et al., 2000; Rowland et al., 2001; Gómez et al., 2002; McLaughlin et al., 2003; Verty et al., 2004). Another selective CBj receptor antagonist, AM281 (20 to 40 ^g i.c.v.), also blocked chow intake in fasting rats (Werner and Koch, 2003). This indicates that an endogenous cannabinoid tone might normally stimulate feeding behavior (Berry and Mechoulam, 2002). However, the control of ingestive behavior by endocan-nabinoids might be age dependent, because the usual food intake following an 18-h deprivation period was reduced by rimonabant in young (6 to 10 weeks) but not old (26 to 48 weeks) mice (Wang et al., 2003).
The endocannabinoid system may also play a key role in overeating behavior and obesity. In mice with high fat diet-induced obesity, chronic administration of rimonabant induced a transient reduction of food intake and a sustained reduction of body weight gain and adiposity (Ravinet Trillou et al., 2003). In free-feeding Zucker rats, rimonabant caused dose-dependent reductions of eating and weight gain which were greater in genetically obese (fa/fa) animals than in their lean counterparts (Vickers et al., 2003). Interestingly, in both studies, hypophagia was limited to the first week of treatment, whereas the reduction of body weight gain in obese animals was maintained throughout the 4-week (rats) or 5-week (mice) chronic administration (Ravinet Trillou et al., 2003; Vickers et al., 2003). Rimonabant discontinuation resulted in a rebound hyperphagia, and weights rapidly returned to (and perhaps exceeded) vehicle levels (Vickers et al., 2003). On the other hand, unrestricted obese db/db mice given a 7-d rimonabant (3 mg/kg.d i.p.) treatment also exhibited acute reduction of food intake, resulting in weight loss, but tolerance developed rapidly, so that by day six, weights were similar to vehicle-injected db/db controls (Di Marzo et al., 2001).
Studies using CB1 KO mice gave further evidence of the involvement of CB1 receptors in the regulation of feeding, because these mice ate less than their wild-type littermates in response to an 18-h fasting period, or an i.c.v. infusion of the orexigenic neuropeptide Y (NPY) (Di Marzo et al., 2001; Poncelet et al., 2003). CBj KO mice also displayed less weight gain, essentially because of lower body fat mass, regardless of what they were fed ad libitum: with regular chow (Cota et al., 2003); but see Ledent et al., 1999; Zimmer et al., 1999), or with an obesity-promoting diet (Cota et al., 2003; Ravinet Trillou et al., 2003). Moreover, rimonabant reduced the hyperphagic response to fasting, NPY, and high fat diet in wild-type but not CB1 KO mice (Di Marzo et al., 2001; Poncelet et al., 2003; Ravinet Trillou et al., 2003). Altogether, these results indicate that, by activating CB1 receptors, endocannabinoids tonically stimulate eating and participate in the regulation of body weight.
Apparently, endocannabinoid levels in brain are sensitive to a variety of physiological and biochemical changes. In particular, acute food deprivation (20 to 24 h) induced moderate increases (from 50% to 150%) of anandamide and/or 2-AG levels in rat brain areas involved in feeding control (Kirkham et al., 2002), as well as of 2-AG in mouse brain (Hanus et al., 2003). In contrast, a 12-d diet restriction has been reported to lower 2-AG levels in mouse hippocampus and hypothalamus, an effect that might precipitate in some anorectic-like coping strategies when food is scarce (Hanus et al., 2003). In starving rats, a sevenfold increase of anandamide was also found in the small intestine, which was normalized by refeeding (Gómez et al., 2002). Interestingly, a capsaicin-induced sensory deafferentation prevented the stimulation of food intake by systemic administration of anandamide or WIN55212-2 and the anorectic effect of rimonabant (Gómez et al., 2002). According to Cota et al. (2003), the lower body fat mass in CB1 KO mice might be determined by both hypothalamic alterations and impaired adipocytes functions. Together, these results suggest that, in addition to central receptors, peripheral CB1 receptors participated in the regulation of ingestive behavior.
A possible reciprocal link may exist between endocannabinoid mechanisms and leptin, an appetite-suppressing peptide hormone. Leptin, released from adipocytes, is believed to signal the nutritional status to brain areas controlling appetite. It directly stimulates the action of anorexigenic agents (such as a-melanocyte-stimulating hormone) and suppresses the effects of orexigenic peptides (primarily, NPY), thereby decreasing appetite. Defective leptin signaling — as observed in genetically obese fa/fa Zucker rats (non functional leptin receptor), ob/ob mice (leptin deficient) or db/db mice (defective leptin receptor) — is associated with elevated 2-AG levels in the hypothalamus but not the cerebellum (an area not directly related to feeding), as compared to respective lean controls, and hypothalamic anandamide seems also marginally enhanced in db/db mice (Di Marzo et al., 2001). The administration of leptin, in the range of doses active to reduce food intake and body weight in nonobese animals fed ad libitum (Halaas et al., 1997), lowered the hypothalamic levels of 2-AG and anandamide in both normal rats and ob/ob mice (Di Marzo et al., 2001), suggesting that endocannabinoids may be under the negative control of this hormone. Consistent with 2-AG's role in feeding or nutritional status, in nonobese rats, hypothalamic levels of this endocannabinoid undergo feeding-related changes. They were enhanced in fasting rats, reduced in free-feeding animals engaged in eating a highly palatable diet, and identical to those of controls fed ad libitum, when rats were allowed to eat a palatable diet to satiety. In contrast, hypothalamic levels of anandamide did not change in any experimental condition (Kirkham et al., 2002). The blockade of CBj receptors by rimonabant reduced food intake in nonfasted ob/ob or db/db mice (3 mg/kg i.p.), and fa/fa Zucker rats (10 to 30 mg/kg p.o.), suggesting that the hypothalamic levels of endocannabinoids may contribute to overeating, which leads to obesity in these animals (Di Marzo et al., 2001; Vickers et al., 2003). Because a deficient leptin function has been reported in diet-induced obesity (Lin et al., 2000), it would be interesting to determine the levels of endocannabinoids in eating-related brain structures of animals subjected to such food regimen. Taken altogether, these data suggest that hypothalamic endocannabinoids may play an important role in mediating the appetite-suppressant action of leptin.
Evidence also exists of interactions between the cannabinoid and opioid systems in the regulation of ingestive behavior. The reduction by A9-THC of electrical thresholds for hypothalamic stimulation-induced feeding in satiated rats was attenuated by naloxone (Trojniar and Wise, 1991). Overeating produced by A9-THC or anandamide was reversed by naloxone (Williams and Kirkham, 1999, 2002), and the orexigenic effect of morphine was attenuated by rimonabant (Verty et al., 2003). In addition, subanorectic doses of rimonabant and naloxone acted synergistically to depress food intake (Kirkham and Williams, 2001; Rowland et al., 2001). Leptin is one of the postulated factors (together with CCK, MSH, etc.) that could be involved in the regulation of cannabinoid and opioid interactions, and it has been hypothesized that CBj receptors may control opioid-regulated feeding through an action on hypothalamic leptin levels (Verty et al., 2003).
The endocannabinoid system may be of critical importance in the initiation of suckling. Animal and human milk contains 2-AG and large amounts of 2-palmitoyl glycerol and 2-linoleoyl glycerol, two fatty acid glycerol esters, which do not bind to CB receptors but enhance the activity of 2-AG by a so-called "entourage effect" (Ben-Shabat et al., 1998). Recent studies by Fride and co-workers have shown that a high dose of rimonabant, administered once to one-d-old mice, or twice a day between postnatal days two and eight, completely impaired milk ingestion, thus causing death within few days. The first postnatal day seems the most critical period since almost 100% of the pups died after a single injection on day one, and 50% when the injection was done on day two. Such an effect was almost completely suppressed by the coadministration of A9-THC, and 2-AG injected with its entourage molecules delayed the mortality rate. This indicates that endocannabinoids, through the activation of CB1 receptors, are critical to the initiation of suckling (Fride et al., 2001). Interestingly, CB1 KO mouse pups also failed to ingest milk during their first day of life, but this effect was transient and suckling was initiated by days two to three (Fride et al., 2003). Despite the absence of CB1 receptors, rimonabant had a partial detrimental effect on milk intake and survival in these animals, suggesting the existence of a third CB receptor, which can be blocked by rimonabant. Overall, these data indicate that a compensatory mechanism enables the CB1 KO newborn mice to start drinking (Fride et al., 2003).
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