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.

Type 2 diabetes mellitus (T2DM) progresses from compensated insulin resistance to beta ceil failure resulting in uncompensated hyperglycemia, a process replicated in the Zucker diabetic fatty (ZDF) rat. The Nlrp3 inflammasome has been implicated in obesity-induced insulin resistance and beta cell failure. Endocannabinoids contribute to insuiin resistance through activation of peripheral CB1 receptors (CB1Rs) and also promote beta cell failure. Here we show that beta cell failure in adult ZDF rats is not associated with CB1R signaling in beta ceils, but rather in M1 macrophages infiltrating into pancreatic islets, and that this leads to activation of the Nlrp3-ASC inflammasome in the macrophages. These effects are replicated in vitro by incubating wild-type human or rodent macrophages, but not macrophages from CB1R-deficient [Cnr1−/−) or Nlrp3−/− mice, with the endocannabinoid anandamide. Peripheral CB1R blockade, in vivo depletion of macrophages or macrophage-specific knockdown of CB1R reverses or prevents these changes and restores normoglycemia and glucose-induced insulin secretion. These findings implicate endocannabinoids and inflammasome activation in beta cell failure and identify macrophage-expressed CB1R as a therapeutic target in T2DM.

SUMMARY Obesity-related leptin resistance manifests in loss of leptin’s ability to reduce appetite and increase energy expenditure. Obesity is also associated with increased activity of the endocannabinoid system, and CB1 receptor (CB1R) inverse agonists reduce body weight and the associated metabolic complications, although adverse neuropsychiatric effects halted their therapeutic development. Here we show that in mice with diet-induced obesity (DIO), the peripherally restricted CB1R inverse agonist JD5037 is equieffective with its brain-penetrant parent compound in reducing appetite, body weight, hepatic steatosis, and insulin resistance, even though it does not occupy central CB1R or induce related behaviors. Appetite and weight reduction by JD5037 are mediated by resensitizing DIO mice to endogenous leptin through reversing the hyperleptinemia by decreasing leptin expression and secretion by adipocytes and increasing leptin clearance via the kidney. Thus, inverse agonism at peripheral CB1R not only improves cardiometabolic risk in obesity but has antiobesity effects by reversing leptin resistance.

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

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

Endocannabinoids are lipid mediators of the same cannabinoid (CB) receptors that mediate the effects of marijuana. The endocannabinoid system (ECS) consists of CB receptors, endocannabinoids, and the enzymes involved in their biosynthesis and degradation, and it is present in both brain and peripheral tissues, including the liver. The hepatic ECS is activated in various liver diseases and contributes to the underlying pathologies. In patients with cirrhosis of various etiologies, the activation of vascular and cardiac CB 1 receptors by macrophage-derived and platelet-derived endocannabinoids contributes to the vasodilated state and cardiomyopathy, which can be reversed by CB 1 blockade. In mouse models of liver fibrosis, the activation of CB 1 receptors on hepatic stellate cells is fibrogenic, and CB 1 blockade slows the progression of fibrosis. Fatty liver induced by a high-fat diet or chronic alcohol feeding depends on the activation of peripheral receptors, including hepatic CB 1 receptors, which also contribute to insulin resistance and dyslipidemias. Although the documented therapeutic potential of CB 1 blockade is limited by neuropsychiatric side effects, these may be mitigated by using novel, peripherally restricted CB 1 antagonists. (HE-PATOLOGY 2011;53:346-355)

BACKGROUND & AIMS Obesity-related insulin resistance contributes to cardiovascular disease. Cannabinoid receptor-1 (CB1) blockade improves insulin sensitivity in obese animals and people, suggesting endocannabinoid involvement. We explored the role of hepatic CB1 in insulin resistance and inhibition of insulin signaling pathways. METHODS Wild-type mice and mice with disruption of CB1 (CB1−/− mice) or with hepatocyte-specific deletion or transgenic overexpression of CB1 were maintained on regular chow or a high-fat diet (HFD) to induce obesity and insulin resistance. Hyperinsulinemic-euglycemic clamp analysis was used to analyze the role of the liver and hepatic CB1 in HFD-induced insulin resistance. The cellular mechanisms of insulin resistance were analyzed in mouse and human isolated hepatocytes using small interfering or short hairpin RNAs and lentiviral knockdown of gene expression. RESULTS The HFD induced hepatic insulin resistance in wild-type mice, but not in CB1−/− mice or mice with hepatocyte-specific deletion of CB1. CB1−/− mice that overexpressed CB1 specifically in hepatocytes became hyperinsulinemic as a result of reduced insulin clearance due to down-regulation of the insulin-degrading enzyme. However, they had increased hepatic glucose production due to increased glycogenolysis, indicating hepatic insulin resistance; this was further increased by the HFD. In mice with hepatocytes that express CB1, the HFD or CB1 activation induced the endoplasmic reticulum stress response via activation of the Bip-PERK-eIF2α protein translation pathway. In hepatocytes isolated from human or mouse liver, CB1 activation caused endoplasmic reticulum stress-dependent suppression of insulin-induced phosphorylation of akt-2 via phosphorylation of IRS1 at serine-307 and by inducing the expression of the serine and threonine phosphatase Phlpp1. Expression of CB1 was up-regulated in samples from patients with nonalcoholic fatty liver disease. CONCLUSIONS Endocannabinoids contribute to diet-induced insulin resistance in mice via hepatic CB1-mediated inhibition of insulin signaling and clearance.

Cannabinoids enhance the function of glycine receptors (GlyRs). However, little is known about the mechanisms and behavioral implication of cannabinoid-GlyR interaction. Using mutagenesis and NMR analysis, we have identified a serine at 296 in the GlyR protein critical for the potentiation of IGly by Δ9-tetrahydrocannabinol (THC), a major psychoactive component of marijuana. The polarity of the amino acid residue at 296 and the hydroxyl groups of THC are critical for THC potentiation. Removal of the hydroxyl groups of THC results in a compound that does not affect IGly when applied alone but selectively antagonizes cannabinoid-induced potentiating effect on IGly and analgesic effect in a tail-flick test in mice. The cannabinoid-induced analgesia is absent in mice lacking α3GlyRs but not in those lacking CB1 and CB2 receptors. These findings reveal a new mechanism underlying cannabinoid potentiation of GlyRs, which could contribute to some of the cannabis-induced analgesic and therapeutic effects.