Endocannabinoids And Appetitive Functions

内源性大麻素和食欲功能

• 批准号: 7591941
• 负责人: GEORGE KUNOS
• 金额: $ 114.99万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7591941
• 关键词: 2-arachidonylglycerol; Ablation; Acute; Adipose tissue; Adverse effects; Age; Agonist; Albumins; Alcohol consumption; Alcohols; Anabolism; Animals; Anxiety; Biological; Body Weight; Brain; CNR1 gene; Carnitine; Cell Death; Cell Line; Cells; Central obesity; Cessation of life; Cholesterol; Clinical Trials; Coculture Techniques; Desire for food; Development; Diet; Double-Blind Method; Drunk driving; Endocannabinoids; Endotoxins; Energy Intake; Energy Metabolism; Enzymes; Ethanol; Ethanolamines; Europe; Experimental Models; Fatty Acids; Fatty Liver; Fatty acid glycerol esters; Food; Gene Expression; Genetic; Glycerophosphates; Goals; Heavy Drinking; Hepatic; Hepatic Stellate Cell; Hepatocyte; High Density Lipoprotein Cholesterol; Homeostasis; Hormonal; Hydrolase; Hydrolysis; Hyperinsulinism; Hypertriglyceridemia; Hypotension; Insulin; Insulin Resistance; Knock-out; LDL Cholesterol Lipoproteins; Laboratories; Leptin; Lipids; Liquid substance; Liver; Liver Cirrhosis; Mediating; Mediator of activation protein; Membrane; Mental Depression; Metabolic; Metabolic syndrome; Mus; Naltrexone; Neuropharmacology; Numbers; Obesity; PTPN22 gene; Pathway interactions; Pharmaceutical Preparations; Phase; Phospholipase C; Phospholipase D; Phosphoric Monoester Hydrolases; Physiological; Placebos; Plasma; Play; Population Study; Production; Protein Tyrosine Phosphatase; Publications; Publishing; Rate; Regulation; Relative (related person); Resistance; Rewards; Rodent Model; Role; Safety; Septic Shock; Site; System; Testing; Transferase; Triglycerides; Up-Regulation; Week; Work; alcohol exposure; anandamide; base; day; deacylation; desire; drinking; drug seeking behavior; ethanolamine; feeding; fibrogenesis; hospital laboratories; human PTPN22 protein; in vivo; inositol-1,4,5-trisphosphate 5-phosphatase; interest; lipid biosynthesis; macrophage; mouse model; novel; obesity treatment; oxidation; paracrine; placebo controlled study; psychologic; receptor; research study; response; rimonabant; stellate cell

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.

内源性大麻素及其CB1受体在控制体重方面发挥着重要作用，CB1受体阻断药物利莫那班最近在欧洲上市，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目标。这项研究已经证实，内源性大麻素通过 CB1 受体刺激肝脏脂肪生成，而这种效应的增加对高脂肪饮食的反应在饮食诱发的肥胖的发展中起着至关重要的作用。尽管热量摄入相似，但CB1受体缺陷（CB1-/-）小鼠对饮食引起的肥胖具有抵抗力，这进一步表明这些小鼠的能量消耗也一定增加。能量消耗的一个主要机制是脂肪酸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，而药物阻断或 CB1 基因消除会导致肝脏 CPT1 活性增加。为了确定位于肝脏中的 CB1 受体与中枢神经系统或脂肪组织等肝外部位的 CB1 受体的相对重要性，我们通过将 CB1 floxed 小鼠与白蛋白 Cre 小鼠杂交，开发了仅选择性消除肝细胞中 CB1 受体的基因改造小鼠。结果表明，这些肝细胞特异性敲除小鼠（LCB1-/- 小鼠）确实因高脂肪饮食而变得肥胖，但能够抵抗脂肪变性、血浆脂质变化（甘油三酯和低密度脂蛋白胆固醇增加，高密度脂蛋白胆固醇减少）、高胰岛素血症和高瘦素血症引起的。通过相同的饮食。这些发现强调了外周限制性 CB1 拮抗剂在治疗脂肪肝和胰岛素抵抗方面的潜在用途，这些拮抗剂可能具有较少的中枢介导副作用（焦虑、抑郁）。 使用相同的肝脏特异性敲除 CB1 受体的小鼠模型（LCB1-/- 小鼠），我们完成了一项研究，证明肝脏 CB1 受体在酒精诱导的脂肪肝中的作用。 LCB1-/- 以及 CB1-/- 能够抵抗低脂液体酒精饮食诱导的脂肪变性，并且还能抵抗酒精诱导的脂肪生成基因表达增加（SREBP1c、FAS）以及 CPT- 表达和活性降低1、脂肪酸β-氧化的限速酶。酒精喂养只会导致肝星状细胞中内源性大麻素 2-花生四烯酰甘油 (2-AG) 的产生增加，并上调肝细胞中的 CB1 受体。这表明星状细胞衍生的 2-AG 作用于肝细胞 CB1 受体以增加脂肪生成并减少脂肪消除的旁分泌作用机制。这一机制得到了共培养实验结果的支持，其中乙醇引发的星状细胞的存在增加了对照中的脂肪生成基因表达，但在LCB1-/-肝细胞中却没有增加。这项工作已提交出版（Jeong 等人） 在刚刚发表的另一项研究中（Liu 等人，Neuropharmacology，2007），我们继续表征小鼠巨噬细胞系（RAW264.7）中内源性大麻素 anandamide（花生四烯酰乙醇酰胺）的新生物合成途径。在这些细胞中，细菌内毒素（LPS）导致大麻素水平急剧升高，这与脓毒性休克和晚期肝硬化的低血压有关。 Anandamide 可以由其膜前体 N-花生四烯酰磷脂酰乙醇胺 (NAPE) 通过磷脂酶 D (NAPE-PLD) 裂解而生成。然而，最近的证据表明存在另外两条平行途径。一种是通过 α,β-水解酶 4 (Abhd4) 对 NAPE 进行连续脱酰作用，然后裂解甘油磷酸盐以产生花生四烯酸乙醇胺，另一种是通过磷脂酶 C 介导的 NAPE 水解，产生磷酸花生四烯乙醇胺，然后通过磷酸酶将其去磷酸化，包括酪氨酸磷酸酶 PTPN22 和肌醇 5 磷酸酶 SHIP1。野生型和 NAPE-PLD-/- 小鼠的脑匀浆将合成 NAPE 转化为 AEA 可以通过 PLC/磷酸酶和 Abdh4 途径进行，前者在较短的时间内（<10 分钟）占主导地位，后者在较长的时间内占主导地位孵化（60 分钟）。 内源性大麻素和 CB1 受体是中脑边缘多巴胺能奖赏通路的重要组成部分，CB1 受体阻断被发现会破坏寻药行为，包括啮齿动物模型中的自愿饮酒。基于动物研究（包括我们自己的研究）的这些发现，我们早些时候启动了一项 I/II 期临床试验，以评估利莫那班治疗对年轻酗酒受试者的安全性及其降低饮酒欲望的功效。这是一项双盲、安慰剂对照研究，涉及 40 名年龄在 21 岁至 45 岁之间的重度饮酒受试者，他们服用利莫那班（20 毫克/天）或安慰剂两周，然后根据他们的意愿进行院内实验室饮酒模式饮酒以及他们对接触酒精和饮酒的生理、心理和荷尔蒙反应进行评估。这项研究刚刚完成，结果的初步分析表明，与安慰剂治疗组相比，药物治疗组饮酒次数的小幅减少并未达到统计学显着性。结果还表明，服用安慰剂的受试者饮用的饮料数量低于使用相同实验室饮酒模式测试纳曲酮效果的研究。这表明我们的研究人群的饮酒欲望低于最佳饮酒欲望，这可能减少了记录药物引起的饮酒量显着减少的机会。 在与哥伦比亚大学 Robert Schwabe 博士的一项合作研究中，我们发现两种实验性纤维化模型中 2-AG 的肝脏水平显着升高，并达到足以诱导 HSC 死亡的浓度。这些发现表明，2-AG 可能通过诱导活化的 HSC（而非肝细胞）细胞死亡来充当肝脏中的抗纤维形成介质。