ECSN Faculty

Daniela Cota, MD
Chargée de Recherche (CR1) and Avenir Group Leader
Institut François Magendie
Bordeaux, France

Vincenzo Di Marzo, PhD
Endocannabinoid Research Group
Institute of Biomolecular Chemistry
Consiglio Nazionale delle Ricerche
Pozzuoli, Italy

Kenneth Mackie, MD
Professor of Psychology
Department of Psychological and Brain Sciences
Indiana University
Bloomington, Indiana

Billy R. Martin, PhD
Harris Professor of Medicine
Chairman, Department of Pharmacology & Toxicology
Medical College of Virginia Commonwealth University
Richmond, Virginia

Introduction: Energy Balance and Metabolic Regulation

The high prevalence of obesity represents a major public health concern. Obesity is associated with serious medical conditions, such as type 2 diabetes and cardiovascular disease, which increase mortality and morbidity.¹ The high prevalence of obesity has made understanding the biological mechanisms involved in feeding behavior and metabolic regulation an important focus of biomedical research.² Interactions between the human thrifty genotype and reduced physical activity and increased food intake have been posited as the root cause of the rising prevalence of obesity and its associated complications.³ Understanding the biological mechanisms underlying energy balance may lead to more effective treatments for obesity, type 2 diabetes, and other diseases associated with excessive energy intake and the dysregulation of metabolism.

Appetite regulation and energy homeostasis are complex physiologic processes involving interactions among multiple neuromodulatory systems in the brain.⁴ The hypothalamus and hindbrain are the two key areas that regulate food intake, energy homeostasis, and body weight, while the limbic system is believed to contain the neuronal circuitry determining perceptions of food palatability and appetite.^{5, 6} Two populations of neurons in the lateral hypothalamus project to key cortical, limbic, and basal forebrain areas, indicating these neurons may have an important role in determining the hedonic or motivational aspects of feeding behavior.^7-9 Over the last decade, a large body of experimental and clinical evidence has demonstrated the involvement of the endocannabinoid system (ECS) in this regulatory network. This module reviews the role of the ECS in feeding behavior and energy balance.

1.  The hypothalamus and the hindbrain regulate

Food intake Energy homeostasis Body weight All of the above

Appetite, Motivation, Satiety, and Energy Expenditure

Feeding Behavior: Role of the ECS

The orexigenic effects of cannabinoids were recognized as early as 300 AD.¹⁰ Recent studies have elucidated the components of the ECS and begun to identify both peripheral and central signals that interact with the ECS.

Data from preclinical studies have demonstrated that stimulation of the ECS increases hunger and appetite. Hao et al investigated the effect of low-dose endocannabinoid (anandamide, 0.001 mg/kg) administration on food intake in mice.¹¹ In this study, mice were diet-restricted for 7 days (food access for 2.5 hours/day) and randomly assigned to receive an intraperitoneal injection of anandamide or control solution 10 minutes before each meal. ¹¹ By day 7, the food intake of diet-restricted mice injected with low-dose anandamide was significantly increased over the intake of diet-restricted mice injected with control solution (Figure 1).¹¹

Additional support for the role of the ECS in food intake regulation comes from studies employing compounds able to block CB1 receptor activity. In one such study, 24-hour fasted mice were allowed access to their regular chow for 1 hour.⁴ The CB1 receptor antagonist SR141716A or a CB2 receptor antagonist was administered via intraperitoneal injection 60 minutes before access to the chow. SR141716A dose-dependently decreased food consumption at doses that did not affect motor activity, whereas the CB² receptor antagonist had no effect on either food intake or motor activity. Moreover, SR141716A decreased food consumption in wild-type mice but had no effect in mice lacking functional CB1 receptors (CB₂ receptor knockout mice). ⁴ Administration of the CB_1/ receptor antagonist LH-21 reduced food intake and body weight gain in obese Zucker rats.¹² Interestingly, CB₁ receptor knockout mice weigh less and have significantly less fat mass than wild-type mice and are resistant to diet-induced obesity.^{13, 14} Additional studies are needed to determine the extent to which these effects on energy balance are due to changes in appetite, satiety, and/or energy expenditure. However, other preclinical data demonstrate that the ECS is involved in the hedonic aspects of feeding behavior.¹⁵ In general, CB₁ receptor blockade is associated with a decreased intake of palatable sweet food.¹⁶

The Hypothalamus, Brainstem, and Mesolimbic System

Role of the ECS

The ECS appears to play a direct role in the central regulation of feeding behavior and energy balance.⁵ The CB₁ receptor is among the most abundant G protein-coupled receptors in the brain, where its density is similar to that of γ-aminobutyric acid (GABA)- and glutamate-gated ion channels.^{5, 17} CB₁ receptor protein is expressed in the lateral hypothalamus of the rat¹⁸ and hypothalamic CB₁ receptor mRNA is co-localized with neuropeptides known to modulate food intake, such as melanin-concentrating hormone, cocaine-amphetamine-regulated transcript, and prepro-orexin.¹⁸

2.  In the brain, the CB1 receptors are

Among the most abundant G protein–coupled receptors Expressed in the lateral hypothalamus of the rat Colocalized with neuropeptides known to modulate food intake All of the above

Administration of the endocannabinoid 2-arachidonoyl glycerol (2-AG) into the shell subregion of the nucleus accumbens (a limbic forebrain area implicated in eating motivation) induced short-term hyperphagia.¹⁶ CB₁ receptor blockade reversed this effect, suggesting a CB₁ receptor-mediated effect of 2-AG on feeding.¹⁶ Interestingly, endocannabinoid levels are directly modulated by nutritional status. In fact, in the rat limbic forebrain and/or hypothalamus, levels of the endocannabinoids AEA and 2-AG were elevated with fasting and declined with feeding.^{4, 16} In contrast, endocannabinoid levels in the cerebellum-a region not directly involved in food intake-were unaffected by fasting or feeding (Figure 2).¹⁶

3.  Endocannabinoid levels are directly modulated by nutritional status. In the rat limbic forebrain and/or hypothalamus, levels of the endocannabinoids anandamide and 2-AG

Were elevated with fasting and declined with feeding Declined with fasting and increased with feeding Were elevated with both fasting and feeding Declined with both fasting and feeding

As noted earlier, CB₁ receptor protein is co-localized in lateral hypothalamic neurons with neuropeptides that modulate feeding. These neurons, in turn, project to cortical, limbic, and basal forebrain areas implicated in the regulation of arousal and motivated aspects of feeding.¹⁹ In a recent electrophysiological study, depolarization of perifornical lateral hypothalamic neurons resulted in a suppression of inhibitory postsynaptic currents.¹⁹ This depolarization protocol has been shown to cause release of endocannabinoids in other brain areas. The depolarization-induced decrease in inhibitory tone was blocked with a CB₁ receptor antagonist and mimicked the effect of treatment with a CB₁ receptor agonist. These findings provide support for the hypothesis that endocannabinoids are involved in modulating the excitability of hypothalamic circuits involved in the motivational aspects of feeding.

Implications

Hypothalamic endocannabinoids may be considered to belong to the family of orexigenic mediators that includes neuropeptide Y (NPY), the orexins, and melanin-concentrating hormone.²⁰ Data from experimental studies indicate that CB₁ receptor blockade may produce anorectic effects.⁴ Moreover, electrophysiological evidence suggests that endocannabinoids suppress inhibition of lateral hypothalamic neurons involved in appetitive motivation and that this effect is blocked by CB₁ receptor antagonism.¹⁹ Thus, selectively blocking the CB₁ receptor may hold promise as a treatment for clinical conditions characterized by dysregulated energy balance and metabolism, such as obesity and type 2 diabetes.

The Gut

Role of the ECS

The intestine and associated organs of the gastrointestinal (GI) tract play a well-defined role in the physiology of energy balance, predominately by communicating with centers in the brain through neural and endocrine pathways.²¹ Signals arising from the gut act in concert with central mechanisms to influence eating behavior (Figure 3).⁵ Among the most important of these are a number of gut-derived hormones which decrease food intake, such as cholecystokinin (CCK). Conversely, ghrelin, another gut-derived peptide, exerts an orexigenic effect.⁵ These hormones function as satiety/hunger signals by triggering nerve impulses in sensory nerves traveling to the hindbrain.⁵ However, they are also transported in the blood and are able to cross the blood-brain barrier, providing signals that are integrated in the hindbrain and the hypothalamus. ⁵

ECS Interactions with Satiety Signals

In the GI tract, CB₁ receptors are present in neurons of the enteric nervous system and in sensory terminals of vagal and spinal neurons, activation of which has been shown to modulate several important aspects of nutrient processing, including gastric secretion, gastric emptying, and intestinal motility. ²² Endocannabinoids are synthesized in the GI tract and their administration, like that of the gastric hormone ghrelin, causes an increase in food intake.⁵ Moreover, endocannabinoid levels in certain areas of the GI tract appear to be modulated by feeding.²³ For example, anandamide levels were shown to increase sevenfold in the small intestine of rats in response to 24 hours of food deprivation, whereas levels normalized when feeding was resumed. In addition, systemic, but not central, blockade of the ECS with SR141716 led to a reduction in food intake both in rats deprived of food for 24 hours and in rats that were partially satiated.²³ This study also showed that chemical destruction of sensory terminals innervating the gut-the same nerves expressing CCK receptors and that mediate CCK-induced satiety-abolished these CB₁ receptor-mediated effects, essentially inhibiting the anorectic action of SR141716A.^{23, 24}

Clearly, there are data that support a role for the ECS in the regulation of food intake at the central level, such as the direct administration of endocannabinoids into the hypothalamus or into brain reward areas, such as the nucleus accumbens. Also, hypothalamic levels of endocannabinoids are modulated by leptin; and endocannabinoids and leptin reciprocally modulate the activity of neuronal hypothalamic populations implicated in the regulation of food intake. Although the data from the study of Gomez et al point to an involvement of the gut,²³ they do not exclude a role for the CNS. It should be taken into consideration, for example, that specific experimental conditions were used, such as pre-satiation.

4.  CB1 receptors in the gastrointestinal tract have been shown to modulate

Gastric secretion Gastric emptying Intestinal motility All of the above

The vagus nerve may be another target through which the ECS modulates food intake.²⁴ The vagus connects the gastrointestinal (GI) tract with medulla and brainstem nuclei that are intimately involved in the control of satiety.²⁴ The gut hormone CCK is secreted during a meal from cells lining the duodenum, and interacts with specific CCK receptors located on the afferent terminals of the vagus nerve.⁵ From there, information is transmitted via vagal axons and ultimately relayed to the hypothalamus, where it is integrated with other signals to decrease food intake.⁵ Receptors for the hormone leptin have also been located on these same nerve terminals. ²⁵

Recent studies have shown that the expression of CB₁ receptor mRNA on vagal afferent neurons projecting into the duodenum is decreased in rats fed ad libitum, while its expression is increased when rats are food deprived.²⁵ Importantly, renewed feeding in previously fasted rats or the administration of CCK leads to decreased levels of CB₁ receptor mRNA in the same vagal afferents. ²⁵ Thus, reduced ECS activity may mediate the induction of satiety by CCK.²⁴

Click to Enlarge Figure 4

Click to Enlarge Figure 5

Hypothalamic endocannabinoids appear to be under negative control by leptin, a hormone predominantly produced by the adipose tissue that elicits anorexigenic signaling in the hypothalamus. Di Marzo et al showed that obese rats or mice with disrupted leptin signaling (obese Zucker rats and obese db/db mice) have higher levels of endocannabinoids in the hypothalamus compared with wild-type animals. ²⁰ Moreover, the influence of leptin on endocannabinoids appears to occur specifically in the hypothalamus, as levels of cerebellar endocannabinoids did not differ between db/db mice and controls.²⁰ When leptin was systemically administered to normal animals or to obese animals lacking the hormone, it decreased hypothalamic endocannabinoid levels. These data thus suggest that leptin exerts negative control over hypothalamic endocannabinoids.²⁰ Consistent with this possibility, leptin attenuated the CB₁ receptor-mediated suppression of inhibitory postsynaptic currents in perifornical lateral hypothalamic neurons (Figure 4).¹⁹ In addition, leptin-deficient obese mice (ob/ob) showed an increase in steady-state voltage-gated calcium currents in lateral hypothalamic neurons and a 6-times longer depolarization-induced suppression of inhibition than lean littermates.¹⁹ These findings indicate that integration of endocannabinoid and leptin signaling may regulate the excitability of appetite-related neural circuits. Endocannabinoids also appear to be involved in modulating the orexigenic effect of the gastric hormone ghrelin. ²⁶ In rats, infusion of ghrelin into the paraventricular nucleus of the hypothalamus was associated with a near doubling of food intake (Figure 5). This effect was blocked by pretreatment with the CB₁ receptor antagonist SR141716 at a dose that had no effect on feeding when administered alone. ²⁶

5.  The ECS appears to be modulated by several gut-derived hormones. These hormones include

Adiponectin Ghrelin Leptin B and C only All of the above

Implications

CB₁ receptors are present in neurons associated with the GI tract²² and endocannabinoids are synthesized within the GI tract; their levels being responsive to nutritional status.²³ The induction of satiety by CCK is determined by the decrease in ECS activity. ²⁴ Fasting appears to overcome satiety by elevating endocannabinoid levels in the small intestine, thereby releasing vagal CB₁ receptors from CCK inhibition. ²⁴ Other evidence reviewed in this section indicates that the ECS in the brain is controlled by the anorexigenic hormone leptin and the orexigenic hormone ghrelin, both of which are also found in the GI tract. The gut is an important source of signals that control meal size and participate in metabolic regulation.^{5, 23} The ECS appears to be modulated by these gut-derived signals.

6.  The family of orexigenic hormones includes

Ghrelin Glucagon Leptin None of the above

7.  Hypothalamic endocannabinoids are negatively regulated by

Ghrelin Glucagon Insulin Leptin None of the above

8.  ECS activity in the gastrointestinal tract is inversely correlated with levels of

CCK Glucagon Insulin Leptin Leptin

Peripheral Metabolic Regulation

Liver

Role of the ECS

Activation of the ECS in the liver appears to be an early step in the development of diet-induced obesity. This was shown in experiments in which wild-type and CB₁ receptor knockout mice were fed a high-fat diet.²⁷ Caloric intake did not differ significantly between wild-type mice and CB₁ receptor knockout mice. After 3 weeks, before obesity was detectable, hepatic anandamide levels were significantly elevated in both wild-type and CB₁ receptor knockout mice exposed to the high-fat diet as compared with mice receiving a standard diet. However, the increase was much smaller in the CB₁ receptor knockout mice.²⁷ In addition to the increase in the levels of CB₁ receptor ligands, the expression of hepatic CB₁ receptor mRNA was increased.²⁷ Other experiments by the same investigators showed that basal rates of hepatic fatty-acid synthesis were significantly increased in wild-type animals fed a high-fat diet for 3 weeks but not in CB₁ knockout mice receiving the same diet, and that this effect was blunted in wild-type mice, but not knockout mice, treated with a CB₁ receptor antagonist.²⁷

In additional experiments, injection