Endocannabinoids And Appetitive Functions

内源性大麻素及其CB1受体在控制体重中发挥重要作用，CB1受体阻断药物利莫那班最近在欧洲被发布用于治疗肥胖/代谢综合征。我们之前提供的证据表明，肝脏是内源性大麻素在饮食诱导的肥胖小鼠中代谢作用的主要靶点。本研究表明，内源性大麻素通过CB1受体刺激肝脏脂肪生成，高脂肪饮食对这种作用的增加在饮食性肥胖的发展中起着重要作用。CB1受体缺陷（CB1-/-）小鼠对饮食引起的肥胖的抵抗，尽管热量摄入相似，进一步表明这些小鼠也必须增加能量消耗。能量消耗的主要机制是脂肪酸β -氧化，其限速酶是肉碱棕榈酰转移酶1 （CPT1）。在一项正在进行的研究中，我们发现用CB1受体激动剂对小鼠进行体内急性治疗会降低肝脏CPT1活性，而CB1的药物阻断或基因消融会增加肝脏CPT1活性。为了确定位于肝脏中的CB1受体与位于肝外部位（如中枢神经系统或脂肪组织）的相对重要性，我们通过将CB1粘接小鼠与白蛋白Cre小鼠杂交，开发了仅在肝细胞中选择性消融CB1受体的转基因小鼠。结果表明，这些肝细胞特异性敲除小鼠（LCB1-/-小鼠）在高脂肪饮食下确实会变得肥胖，但对脂肪变性、血浆脂质改变（甘油三酯和低密度脂蛋白胆固醇升高，高密度脂蛋白胆固醇降低）、高胰岛素血症和高瘦素血症有抵抗力。这些发现强调了外周限制性CB1拮抗剂在脂肪肝和胰岛素抵抗治疗中的潜在作用，它可能具有较少的中枢介导的副作用（焦虑、抑郁）。

Endogenous cannabinoids and their CB1 receptors play 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 in mice. This study 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 diet-induced obesity. The resistance of CB1 receptor deficient (CB1-/-) mice to diet-induced obesity despite similar caloric intake further suggested that these mice must also have increased energy expenditure. A major mechanism of energy expenditure is fatty acid beta-oxidation, for which the rate limiting enzyme is carnitine palmitoyl transferase 1 (CPT1). Ijn an ongoing study we have found that in vivo acute treatment of mice with a CB1 receptor agonist decreases hepatic CPT1 activity, whereas pharmacologic blockade or genetic ablation of CB1 results in increased hepatic CPT1 activities. To determine the relative importance of CB1 receptors located in the liver vs in extraheptic sites such as the CNS or adipose tissue, we have developed gentically modified mice with selective ablation of CB1 receptors in hepatocytes only, by crossing CB1 floxed mice with albumin Cre mice. Results indicate that these hepatocyte-specific knockouts (LCB1-/- mice) do become obese on a high fat diet, but are resistant to the steatosis, plasma lipid changes (increased triglycerides and LDL cholesterol and decreased HDL cholesterol), hyperinsulinemia and hyperleptinemia caused by the same diet. These findings highlight the potential usefulness of peripherally resticted CB1 antagonists, which may have fewer centrally mediated side effects (anxiety, depression), in the treatment of fatty liver and insulin resistance. Using the same mouse model of liver specific knockout of CB1 receptors (LCB1-/- mice), we have completed a study that demonstrates 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, the rate limiting enzyme in fatty acid beta-oxidation. Alcohol feeding induces 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 submitted for publication (Jeong et al.) In a separate study just published (Liu et al., Neuropharmacology, 2007), 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 had earlier started 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 is a double-blind, placebo controlled study involving forty heavy drinking subjects between the ages of 21 and 45 years who take 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 is evaluated. This study has just been completed and preliminary analysis of the results indicates that a small reduction in the number of drinks in the drug- vs placebo-treated group did not reach statistical significance. The results also indicate that the number of drinks consumed by subjects on placebo was lower than in a study testing the effect of naltrexone using the same laboratory drinking paradigm. This suggests that our study population had lower than optimal drinking drive, which may have reduced the chance to document a significant drug-induced decrease in drinking. In a collaborative study with Dr. Robert Schwabe at Columbia Univ., we found that hepatic levels of 2-AG were significantly elevated in two models of experimental fibrogenesis and reached concentrations that are sufficient to induce death in HSCs. These findings suggest that 2-AG may act as an antifibrogenic mediator in the liver by inducing cell death in activated HSCs but not hepatocytes.