The endocannabinoid system (ECS), which includes endogenous cannabinoids and their receptors, plays an important role in the control of body weight, and the CB1 receptor blocking drug rimonabant has been recently released in Europe for the treatment of obesity/metabolic syndrome. We had earlier provided evidence that the liver is a major target of the metabolic effects of endocannabinoids in diet-induced obesity (DIO) in mice. That study (JCI 115:1298, 2005) has established that endocannabinoids stimulate hepatic lipogenesis via CB1 receptors, and an increase in this effect in response to high fat diet plays an essential role in the development of DIO. DIO is associated with fatty liver, insulin and leptin resistance and dyslipidemia (increased triglycerides and LDL cholesterol and decreased HDL cholesterol), and CB1-/- mice are also resistant to these associated phenotypes. To explore the relative role of heptic vs. extrahepatic CB1 receptors in these effects, we developed genetically modified mice with selective ablation of CB1 receptors in hepatocytes only (LCB1-/- mice), by crossing CB1 floxed mice with albumin Cre mice. LCB1-/- mice fed a high-fat diet developed the same degree of obesity as wild-type mice but, similar to CB1-/- mice, had less steatosis, insulin and leptin resistance and dyslipidemia than wild-type mice on the high-fat diet. In wild-type mice, treatment with a CB1 agonist increased de novo hepatic lipogenesis, decreased the activity of carnitine palmitoyl transferase-1 (CPT-1), the rate limiting enzyme in fatty acid beta-oxidation, and decreased total energy expenditure, and these effects were absent in both Cb1-/- and LCB1-/- mice. These just published findings suggest that peripheral CB1 receptors may be targeted for the treatment of fatty liver, impaired glucose homeostasis, and dyslipidemia in order the minimize the neuropsychiatric side effects (anxiety, depression) of non-selective CB1 blockade during the treatment of obesity-associated conditions. ? ? Using the same mouse model of liver specific knockout of CB1 receptors (LCB1-/- mice), we have demonstrated the role of hepatic CB1 receptors in alcohol-induced fatty liver. LCB1-/- as well CB1-/- are resistant to steatosis induced by a low fat, liquid alcohol diet, and are also resistant to the alcohol-induced increased lipogenic gene expression (SREBP1c, FAS) and decreased expression and activity of CPT-1. Alcohol feeding results in increased production of the endocannabinoid 2-arachidonoyl-glycerol (2-AG) in hepatic stellate cells only, and upregulation of CB1 receptors in hepatocytes. This suggests a paracrine mechanism of action by stellate-cell-derived 2-AG acting at hepatocyte CB1 receptors to increase lipogenesis and decrease fat elimination. This mechanism was supported by the results of co-culture experiments, where the presence of ethanol-primed stellate cells increased lipogenic gene expression in control but not in LCB1-/- hepatocytes. This work has been published in Cell Metabolism. ? ? In a separate study (Liu et al., Neuropharmacology, 2008), we continued the characterization of novel biosynthetic pathways of the endocannabinoid anandamide (arachidonoyl ethanolamide) in a mouse macrophage cell line (RAW264.7). In these cells, bacterial endotoxin (LPS) causes a dramatic increase in anandamide levels, which has been implicated in the hypotension of septic shock an advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidyl ethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5 phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD-/- mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer incubations (60 min). ? ? Endocannabinoids and CB1 receptors are essential components of the mesolimbic dopaminergic reward pathway, and CB1 receptor blockade was found to disrupt drug-seeking behavior, including voluntary alcohol drinking in rodent models. Based on such findings in animal studies, including our own, we have completed a phase I/II clinical trial to assess the safety of rimonabant treatment in young, heavy drinking subjects and its efficacy to reduce their desire to drink. This was a double-blind, placebo controlled study involving forty heavy drinking subjects (consuming between 20 and 50 drinks per week) between the ages of 21 and 45 years who took rimonabant (20 mg/day) or placebo for two weeks, followed by an in-hospital laboratory drinking paradigm where their desire to drink as well as their physiological, psychological and hormonal response to exposure to alcohol and drinking was evaluated. Results from both the outpatient call-ins and the alcohol self-administration paradigm failed to show a statistically significant difference between those receiving rimonabant or placebo. This suggests that the daily administration of 20 mg rimonabant for two weeks has no effect on alcohol consumption in heavy social drinkers. A manuscript of these results has been prepared for publication.? ? In a collaborative study with Dr. Michael Meguid's group (SUNY), we reported that in a rat model of Roux-en-Y gastric bypass surgery, anandamide levels in muscle and 2-arachydonoyl glyceride levels in liver were downregualted, suggesting a role for endocannabinoids in the mechanism of sustained weight loss achieved as a result of this procedure. ? ? In another collaborative study (Dr. Palkovits) we analyzed the effects of postmortal delay on endocannabinoid levels in anonymized human brain samples obtained from a brain bank or removed during surgery. We found that the tissue levels of anandamide increased continuously with increasing post-mortal delay reaching a peak of 7-fold by 6 hours (time between death and removal and freezing of samples), whereas 2-arachidonoyl glycerol rapidly dropped within the 1st hour to 25-35% of the 0 time level. These results highlight the pitfall of analyzing endocannabinoid content in brain samples of variable (or undefined) post-mortal delay.? ? In collaboration with Dr. Christoph Buettner (Mt Sinai Sch Med, NY), we found that leptin infused into the mediobasal hypothalamus of rats inhibits white adipose tissue lipogenesis by a STAT3-independent, PI3K-dependent mechanism that results in a sympathetically mediated suppression of adipose tissue anandamide levels. CB1 receptor activation prevents the effect of central leptin on adipose tissue lipogenesis, suggesting that the latter effect is mediated indirectly through reduced endocannabinoid tone in adipose tissue.
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