Part I: The Endocannabinoid System, Mechanisms of Action and Functions

Endocannabinoids: biology, mechanism of action and functions

The isolation and identification of endogenous cannabinoid ligands (endocannabinoids) as a family of intercellular signaling molecules that act at the same receptors as the plant-derived cannabinoids has significantly changed our understanding of cellular physiology and metabolism. In the early 1990s, two G-protein coupled receptors termed the cannabinoid CB-1 receptor (CB1) and CB-2 receptor (CB2) were isolated and cloned.1 Their widespread distribution in the central and peripheral nervous system (CB1 receptor) and the immune system (CB2 receptor) led to a search, and subsequent identification, for endogenous ligands of these receptors. In 1992, arachidonoylethanolamide (anandamide) was discovered and shown to be a specific receptor agonist for the CB1 receptor (subsequently it was also found to be a CB2 receptor agonist).2 After 3 years, 2-arachidonoylglycerol (2-AG) was shown to be a second endocannabinoid with efficacy at both CB receptors.3, 4 A number of other putative endocannabinoids have now been proposed. Some 10 years after their isolation, the role of endocannabinoids in regulating food intake and energy balance has been widely recognized, leading to the organization of a symposium, whose proceedings are reported in this issue of International Journal of Obesity. Four speakers, whose presentations are summarized in the reviews following this article, covered the biology, physiology and pharmacology of the endocannabinoids and their receptors; focusing on the role of endocannabinoids in energy homeostasis. In this short overview, a perspective on these topics will be provided.

The endocannabinoid family is expanding and now includes both endogenous agonists and a putative endogenous antagonist, virodhamine.5, 6 Unlike most intercellular signaling systems, the endocannabinoids are made on demand. The biosynthetic pathways for their synthesis and subsequent degradation have now been identified and the distribution of these enzymes suggests, at least in parts of the central nervous system, that they represent a class of key regulators that shape synaptic responses over spatially and temporally restricted areas.7 As pointed out by Matias et al. in their article, many of the biosynthetic enzymes, transporters, intracellular binding proteins (if they exist) and degradative systems have still to be fully described in most of the systems, especially in the periphery, in which endocannabinoids are thought to play key roles. The balance between synthesis, reuptake and degradation represents the limiting factor for the duration of action of endocannabinoids. The conditions for activation of endocannabinoid synthesis are not fully established, particularly concerning the plethora of potential cellular mediators, which may enhance or initiate their synthesis through increasing intracellular calcium. Drugs that increase the apparent extracellular concentrations of the endocannabinoids by inhibiting their uptake into cells have been very valuable tools that have helped establish many physiological or pathophysiological actions of endocannabinoids in vitro and in vivo. However, until the endocannabinoid transporters are finally cloned, doubts will remain as to the specificity of action of these drugs, and more importantly the physiology of endocannabinoid release and reuptake. Along similar lines, it is not clear how most of the components of the endocannabinoid system are altered in pathophysiological states, such as obesity. It is established that endocannabinoid levels are altered in genetically obese animals and that leptin regulates endocannabinoid levels by altering the levels of their biosynthetic precursors.8 However, there are numerous short and long term energy regulating factors whose actions on endocannabinoid synthesis and/or release have yet to be studied.

The relationship between energy regulation and the endocannabinoid system extends to receptor and likely postreceptor mechanisms. CB1 receptor-deficient mice have a lean phenotype and are resistant to diet-induced obesity.9, 10 Furthermore the CB1 receptor antagonists/inverse agonists SR141716A (rimonabant) and AM251 when given to animals and humans reduce food intake and enhance weight loss. Pertwee describes in his article the pharmacology of cannabinoid receptors and discusses how they may be targeted therapeutically now and in the future. The novel observations of an allosteric binding site on the CB1 receptor offers an exciting way to modify receptor interactions and suggests that compounds that may interact with this site have the potential to alter energy homeostasis in the face of dysregulated endocannabinoid production. While the CB1 and not the CB2 receptor appears to play a role in energy metabolism, the possibility of novel CB receptors, an inducible CB receptor (such as a CB2 receptor in the brain) or heteromeric CB receptors (such as a mixed CB1/CB2 receptor) appear likely to be found physiologically or under pathophysiological states, and in the future will expand the role of the endocannabinoid system further. The distribution of CB receptors is being thoroughly investigated. The dogma that CB1 receptors are restricted to the nervous system and CB2 receptors to cells and tissues of the peripheral immune system is now being widely challenged. CB1 receptors on adipocytes likely mediate at least some of the actions of the CB1 receptor antagonists/inverse agonists on energy homeostasis.

The mechanism of action of endocannabinoids depends on the signal transduction pathways from the receptor. In his article, Ken Mackie explores what we know of the intracellular mechanisms of action of endocannabinoids at CB1 receptors. Interactions of CB1 receptors through Gi/o G proteins coupled to calcium and potassium channels in the cell membrane are likely to be involved in regulation of neurotransmitter release. Through inhibition of adenyl cyclase and a reduction of cAMP, or through MAP kinase pathways, endocannabinoids could also have many long-term cellular actions. In terms of energy metabolism and homeostasis these have yet to be explored. Whether new signal transduction pathways will also emerge as targets of CB receptors remains to be determined, but it seems unlikely that we know the full range of intracellular targets in all the tissues where CB receptors will be found.

In the brain, Mackie describes how CB1 activation shapes the synaptic response in space and time. The hypothalamus and the dorsal vagal complex of the brainstem are well recognized as the central autonomic nuclei essential for the regulation of energy homeostasis. Currently there have been few mechanistic studies examining the action of endocannabinoids that have focused on these central structures. An insightful model by Horvath,11 based on work of Cotta et al.,9 remains to be fully explored, especially in terms of detailed synaptic physiology, at the levels that have been accomplished in the hippocampus and cerebellum. The interactions between the various peptidergic and classical transmitter systems with the CB receptor systems will be important to understand. Similarly, we are well aware that endocannabinoids are capable of activating not only CB receptors, but also the ligand-gated TRPV1 receptors, in the brain and periphery.12 In the hypothalamus, the TRPV1 receptor is densely distributed in regions involved in energy homeostasis,13 suggesting the strong possibility that endocannabinoid interactions play a regulatory role through these receptors as well as the CB receptors.

The spectrum of biological actions of endocannabinoids has increased dramatically in recent years. With a focus on food intake, including suckling, Mechoulam et al. highlight the complexity of the integrative actions of these pleiotropic molecules. They also provide an insightful illustration of the limitations of knockout mice. Investigating food intake from birth, they established that mice given a CB1 receptor antagonist are severely affected by this treatment. In contrast, CB1 receptor-deficient mice compensate within a few days after birth from the loss of the receptor, albeit with some increase in infant mortality rate. Whether CB1 receptor-deficient mice have compensated for other deficiencies remains to be established, but this highlights the need for the cautious interpretation of data in genetically altered animals lacking essential signaling elements.

It has long been established that cannabinoids increase food intake, and, as discussed above, antagonists of the CB1 receptor lead to reduced food intake. The conditions under which examination of food intake occurs can lead to substantially different doses at which endocannabinoids enhance food intake. The efficacy of antagonists to suppress ingestive behavior is also dependent on the state of satiety of the animal (or human). Nevertheless, CB1 receptor antagonists consistently reduce body weight even if food intake is not greatly reduced. The mechanism of action of the CB1 receptor antagonists have yet to be fully elucidated. It seems likely that central and peripheral actions will contribute to their mechanism of action. These interesting compounds target the brain centers involved in hedonistic and appetitive aspects of ingestion, clearly offering an exciting therapeutic potential for the treatment of obesity. Their beneficial actions also rely on peripheral receptors that are currently being more fully characterized.

The endocannabinoid system is an essential regulator of energy balance. Through receptors in the brain and periphery, which have become an important therapeutic target in the treatment of obesity and its complications, endocannabinoids exert powerful effects on the integrative systems of energy homeostasis. A complete understanding of these actions will lead to a new appreciation of the complexity of this critical body system. The articles that follow cross disciplinary boundaries to provide an overview of the panoply of actions of endocannabinoids and their receptors in the context of obesity and its ultimate prevention.

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Acknowledgements

KAS is an Alberta Heritage Foundation for Medical Research Medical Scientist. Thanks to Adam Chambers, Marnie Duncan and Marja van Sickle for their valuable comments.

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Correspondence to K A Sharkey.

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