Endogenous cannabinoids acting at CB 1 receptors stimulate appetite, and CB 1 antagonists show promise in the treatment of obesity. CB 1 -/-mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB 1 in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB 1 agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB 1 . High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB 1 density, and basal rates of fatty acid synthesis, and the latter is reduced by CB 1 blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB 1 ligands. We conclude that anandamide acting at hepatic CB 1 contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation. IntroductionMaintenance of energy homeostasis and body weight involves the coordinated regulation of appetitive behavior and peripheral energy metabolism (1), as illustrated by the ability of the appetitereducing hormone leptin to regulate fat metabolism in the liver (2). Endocannabinoids are novel lipid mediators that modulate appetitive behavior through the activation of CB 1 (3-11). Sites in the hypothalamus (4, 10, 12), limbic forebrain (11-13), and peripheral sensory nerve terminals (7) have been implicated in mediating the orexigenic effect of endocannabinoids, which is potentiated by hunger or in hyperphagia associated with obesity (4-6, 9) and antagonized by CB 1 blockade. Indeed, CB 1 antagonists show promise in the treatment of obesity (14). A number of recent observations suggest that reduction of food intake alone cannot fully account for the antiobesity effects of CB 1 antagonists. In a mouse model of diet-induced obesity, chronic treatment with the CB 1 antagonist SR141716 caused a transient reduction in food intake and a more prolonged reduction in body weight (15). Mice lacking CB 1 are resistant to diet-induced obesity even though their total caloric intake is similar to that of wild-type littermates, which become obese on the same diet (16). CB 1 -/-mice display a moderately lean phenotype throughout adulthood but only a temporary hypophagia in the first few weeks of life (17). These observations suggest that endocannabinoids and CB 1 also regulate peripheral energy metabolism. Indeed, adipocytes have been found to express

Endogenous cannabinoids acting at CB 1 receptors stimulate appetite, and CB 1 antagonists show promise in the treatment of obesity. CB 1 -/-mice are resistant to diet-induced obesity even though their caloric intake is similar to that of wild-type mice, suggesting that endocannabinoids also regulate fat metabolism. Here, we investigated the possible role of endocannabinoids in the regulation of hepatic lipogenesis. Activation of CB 1 in mice increases the hepatic gene expression of the lipogenic transcription factor SREBP-1c and its targets acetyl-CoA carboxylase-1 and fatty acid synthase (FAS). Treatment with a CB 1 agonist also increases de novo fatty acid synthesis in the liver or in isolated hepatocytes, which express CB 1 . High-fat diet increases hepatic levels of the endocannabinoid anandamide (arachidonoyl ethanolamide), CB 1 density, and basal rates of fatty acid synthesis, and the latter is reduced by CB 1 blockade. In the hypothalamus, where FAS inhibitors elicit anorexia, SREBP-1c and FAS expression are similarly affected by CB 1 ligands. We conclude that anandamide acting at hepatic CB 1 contributes to diet-induced obesity and that the FAS pathway may be a common molecular target for central appetitive and peripheral metabolic regulation. IntroductionMaintenance of energy homeostasis and body weight involves the coordinated regulation of appetitive behavior and peripheral energy metabolism (1), as illustrated by the ability of the appetitereducing hormone leptin to regulate fat metabolism in the liver (2). Endocannabinoids are novel lipid mediators that modulate appetitive behavior through the activation of CB 1 (3-11). Sites in the hypothalamus (4, 10, 12), limbic forebrain (11-13), and peripheral sensory nerve terminals (7) have been implicated in mediating the orexigenic effect of endocannabinoids, which is potentiated by hunger or in hyperphagia associated with obesity (4-6, 9) and antagonized by CB 1 blockade. Indeed, CB 1 antagonists show promise in the treatment of obesity (14). A number of recent observations suggest that reduction of food intake alone cannot fully account for the antiobesity effects of CB 1 antagonists. In a mouse model of diet-induced obesity, chronic treatment with the CB 1 antagonist SR141716 caused a transient reduction in food intake and a more prolonged reduction in body weight (15). Mice lacking CB 1 are resistant to diet-induced obesity even though their total caloric intake is similar to that of wild-type littermates, which become obese on the same diet (16). CB 1 -/-mice display a moderately lean phenotype throughout adulthood but only a temporary hypophagia in the first few weeks of life (17). These observations suggest that endocannabinoids and CB 1 also regulate peripheral energy metabolism. Indeed, adipocytes have been found to express

Diet-induced obesity is associated with fatty liver, insulin resistance, leptin resistance, and changes in plasma lipid profile. Endocannabinoids have been implicated in the development of these associated phenotypes, because mice deficient for the cannabinoid receptor CB 1 (CB1 -/-) do not display these changes in association with diet-induced obesity. The target tissues that mediate these effects, however, remain unknown. We therefore investigated the relative role of hepatic versus extrahepatic CB 1 receptors in the metabolic consequences of a high-fat diet, using liver-specific CB 1 knockout (LCB1 -/-) mice. LCB1 -/-mice fed a high-fat diet developed a similar degree of obesity as that of wild-type mice, but, similar to CB1 -/-mice, had less steatosis, hyperglycemia, dyslipidemia, and insulin and leptin resistance than did wild-type mice fed a high-fat diet. CB 1 agonistinduced increase in de novo hepatic lipogenesis and decrease in the activity of carnitine palmitoyltransferase-1 and total energy expenditure were absent in both CB1 -/-and LCB1 -/-mice. We conclude that endocannabinoid activation of hepatic CB 1 receptors contributes to the diet-induced steatosis and associated hormonal and metabolic changes, but not to the increase in adiposity, observed with high-fat diet feeding. Theses studies suggest that peripheral CB 1 receptors could be selectively targeted for the treatment of fatty liver, impaired glucose homeostasis, and dyslipidemia in order to minimize the neuropsychiatric side effects of nonselective CB 1 blockade during treatment of obesity-associated conditions.

Background-Endocannabinoids are novel lipid mediators with hypotensive and cardiodepressor activity. Here, we examined the possible role of the endocannabinergic system in cardiovascular regulation in hypertension. Methods and Results-In spontaneously hypertensive rats (SHR), cannabinoid-1 receptor (CB 1 ) antagonists increase blood pressure and left ventricular contractile performance. Conversely, preventing the degradation of the endocannabinoid anandamide by an inhibitor of fatty acid amidohydrolase reduces blood pressure, cardiac contractility, and vascular resistance to levels in normotensive rats, and these effects are prevented by CB 1 antagonists. Similar changes are observed in 2 additional models of hypertension, whereas in normotensive control rats, the same parameters remain unaffected by any of these treatments. CB 1 agonists lower blood pressure much more in SHR than in normotensive Wistar-Kyoto rats, and the expression of CB 1 is increased in heart and aortic endothelium of SHR compared with Wistar-Kyoto rats. Conclusions-We conclude that endocannabinoids tonically suppress cardiac contractility in hypertension and that enhancing the CB 1 -mediated cardiodepressor and vasodilator effects of endogenous anandamide by blocking its hydrolysis can normalize blood pressure. Targeting the endocannabinoid system offers novel therapeutic strategies in the treatment of hypertension.

Alcohol-induced fatty liver, a major cause of morbidity, has been attributed to enhanced hepatic lipogenesis and decreased fat clearance of unknown mechanism. Here we report that the steatosis induced in mice by a low-fat, liquid ethanol diet is attenuated by concurrent blockade of cannabinoid CB1 receptors. Global or hepatocyte-specific CB1 knockout mice are resistant to ethanol-induced steatosis and increases in lipogenic gene expression and have increased carnitine palmitoyltransferase 1 activity, which, unlike in controls, is not reduced by ethanol treatment. Ethanol feeding increases the hepatic expression of CB1 receptors and upregulates the endocannabinoid 2-arachidonoylglycerol (2-AG) and its biosynthetic enzyme diacylglycerol lipase beta selectively in hepatic stellate cells. In control but not CB1 receptor-deficient hepatocytes, coculture with stellate cells from ethanol-fed mice results in upregulation of CB1 receptors and lipogenic gene expression. We conclude that paracrine activation of hepatic CB1 receptors by stellate cell-derived 2-AG mediates ethanol-induced steatosis through increasing lipogenesis and decreasing fatty acid oxidation.

Hepatic ischemia-reperfusion (I/R) injury continues to be a fatal complication that can follow liver surgery or transplantation. We have investigated the involvement of the endocannabinoid system in hepatic I/R injury using an in vivo mouse model. Here we report that I/R triggers several-fold increases in the hepatic levels of the endocannabinoids anandamide and 2-arachidonoylglycerol, which originate from hepatocytes, Kupffer, and endothelial cells. The I/R-induced increased tissue endocannabinoid levels positively correlate with the degree of hepatic damage and serum TNF-alpha, MIP-1alpha, and MIP-2 levels. Furthermore, a brief exposure of hepatocytes to various oxidants (H2O2 and peroxynitrite) or inflammatory stimuli (endotoxin and TNF-alpha) also increases endocannabinoid levels. Activation of CB2 cannabinoid receptors by JWH133 protects against I/R damage by decreasing inflammatory cell infiltration, tissue and serum TNF-alpha, MIP-1alpha and MIP-2 levels, tissue lipid peroxidation, and expression of adhesion molecule ICAM-1 in vivo. JWH133 also attenuates the TNF-alpha-induced ICAM-1 and VCAM-1 expression in human liver sinusoidal endothelial cells (HLSECs) and the adhesion of human neutrophils to HLSECs in vitro. Consistent with the protective role of CB2 receptor activation, CB2-/- mice develop increased I/R-induced tissue damage and proinflammatory phenotype. These findings suggest that oxidative/nitrosative stress and inflammatory stimuli may trigger endocannabinoid production, and indicate that targeting CB2 cannabinoid receptors may represent a novel protective strategy against I/R injury. We also demonstrate that CB2-/- mice have a normal hemodynamic profile.

The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released ''on demand'' by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC͞phosphatase pathway, which is up-regulated by LPS, whereas NAPE-PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC͞phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC͞phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target.biosynthesis ͉ phosphatase ͉ phospholipase C ͉ phosphoanandamide T he endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) is a lipid transmitter synthesized and released ''on demand'' by neurons in the brain (1). AEA is also generated by macrophages (2), where its bacterial endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock (3, 4) and liver cirrhosis (5, 6). Macrophage-derived AEA has been also implicated in antiinflammatory effects both in the periphery (7) and in the central nervous system (8, 9). AEA is thought to be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE), through cleavage by a phospholipase D (NAPE-PLD) (10, 11), upregulation of which can result in increased tissue levels of AEA (12). We earlier reported that LPS potently stimulates AEA synthesis in RAW264.7 mouse macrophages, in which it increases both the generation of NAPE from [ 14 C]diarachidonoyl phosphatidylcholine and the conversion of NAPE to AEA (4). Because these effects could be prevented by inhibitors of RNA transcription or protein synthesis (4), we hypothesized that LPS induces the expression of proteins involved in the biosynthesis of AEA, and a subtraction cloning strategy using resting and LPS-treated macrophages may help identifying such proteins. Although a specific N-acyltransferase (NAT) involved in the generation of NAPE has not yet been discovered, a NAPEspecific PLD has been identified and its ability to generate AEA from NAPE has been established (11). The results presented here indicate that, unexpectedly, NAPE-PLD is not involved in the stimulated synthesis of AEA in RAW264.7 macrophages. Instead, we identified the lipid phosphoanandamide (pAEA), which is also presen...

Obesity and its metabolic consequences are a major public health concern worldwide. Obesity is associated with overactivity of the endocannabinoid system, which is involved in the regulation of appetite, lipogenesis, and insulin resistance. Cannabinoid-1 receptor (CB 1 R) antagonists reduce body weight and improve cardiometabolic abnormalities in experimental and human obesity, but their therapeutic potential is limited by neuropsychiatric side effects. Here we have demonstrated that a CB 1 R neutral antagonist largely restricted to the periphery does not affect behavioral responses mediated by CB 1 R in the brains of mice with genetic or diet-induced obesity, but it does cause weight-independent improvements in glucose homeostasis, fatty liver, and plasma lipid profile. These effects were due to blockade of CB 1 R in peripheral tissues, including the liver, as verified through the use of CB 1 R-deficient mice with or without transgenic expression of CB 1 R in the liver. These results suggest that targeting peripheral CB 1 R has therapeutic potential for alleviating cardiometabolic risk in obese patients. IntroductionEndocannabinoids are endogenous lipid mediators that interact with the same G protein-coupled receptors - CB 1 R and CB 2 R - that recognize plant-derived cannabinoids, and they regulate a broad range of physiological functions. CB 1 Rs are expressed at very high levels in the brain but are also present at much lower yet functionally relevant concentrations in various peripheral tissues, whereas the expression of CB 2 Rs is largely limited to cells of the immune and hematopoietic systems. Activation of CB 1 R results in increased appetite, insulin resistance, and increased hepatic lipogenesis, which suggests the involvement of the endocannabinoid/ CB 1 R system in obesity and its metabolic consequences (1). Indeed, obesity and its metabolic complications are characterized by an overactive endocannabinoid system (2-5), and chronic treatment with CB 1 R antagonists leads to weight loss and improved cardiometabolic risk profile in obese rodents (6, 7) and humans (8-11). However, concern over neuropsychiatric side effects, including anxiety, depression, and suicidal ideation (12), prevented approval of the first-in-class CB 1 R antagonist rimonabant in the United States and led to its withdrawal from the European market as well as the withdrawal of related compounds from preclinical development (13). Although the exact role of the endocannabinoid system in the control of mood and anxiety-like behaviors is not clear, CB 1 R in the prefrontal cortex, amygdala, and the mesolimbic dopaminergic reward pathway have been linked to the control of these behaviors (14). On the other hand, CB 1 Rs are also present in peripheral tissues including the liver (15-17), skeletal muscle (18,19), endocrine