Endocannabinoids and the Control Of Behavior and Cardiovascular Function

内源性大麻素与行为和心血管功能的控制

• 批准号: 10019956
• 负责人: GEORGE KUNOS
• 金额: $ 96.15万
• 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/10019956
• 关键词: Acylation; Adipose tissue; Affect; Agonist; Alcohol consumption; Alcoholism; Alcohols; Antihypertensive Agents; Anxiety; Area; Behavior Control; Biological; Biological Assay; Blood Pressure; Blood Vessels; Brain; CNR1 gene; CNR2 gene; Caprylates; Cardiac; Cardiovascular Physiology; Cardiovascular system; Carnitine; Cells; Cellular Metabolic Process; Coupled; Denervation; Desire for food; Diet; Endocannabinoids; Enzyme Inhibitor Drugs; Enzymes; Ethanol; Feeling suicidal; Hormones; Hypertension; Institutes; Intake; Kidney; Knock-out; Knockout Mice; Laboratories; Leptin; Leptin resistance; Ligands; Lipids; Liver Cirrhosis; Marijuana; Measures; Mediating; Mental Depression; Metabolic; Metabolic syndrome; Mus; Obese Mice; Octanoic Acids; Operative Surgical Procedures; Palmitoylcarnitine; Pathologic; Pathway interactions; Peptides; Peripheral; Pharmacologic Substance; Physiological; Plasma; Post-Traumatic Stress Disorders; Process; Production; Psychiatry; Publishing; Receptor Activation; Regulation; Reporting; Rodent Model; Role; Scheme; Self Administration; Sensory; Septic Shock; Signal Transduction; Site; Stomach; Syndrome; Therapeutic; Therapeutic Uses; Tissues; age related; alcohol craving; alcohol seeking behavior; anxiety states; anxiety-like behavior; cannabinoid receptor; des-n-octanoyl ghrelin; drinking; drinking behavior; endogenous cannabinoid system; experimental study; fatty acid oxidation; ghrelin; ghrelin receptor; in vivo; inhibitor/antagonist; interest; long chain fatty acid; mouse model; neurobehavioral; neuropsychiatry; obesity treatment; post-traumatic stress; preference; prevent; receptor; reduced food intake; rimonabant; side effect

We have earlier demonstrated that endocannabinoids acting via CB1 receptors promote voluntary alcohol drinking in an age-dependent manner, using a mouse model of two bottle/free choice paradigm (PNAS 100:1393, 2003). The brain-penetrant CB1 inverse agonist rimonabant used in this study was subsequently introduced as a treatment of obesity, but had to be wihdrawn from the pharmaceutical market in 2008, due to neuropsychiatric side effects, including anxiety, depression and suicidal ideation. An alternative approach, championed by my laboratory, was the introduction of peripherally restricted CB1 inverse agonists that retained the metabolic efficacy of rimonabant but were devoid of its neurobehavioral effects in rodent models of the metabolic syndrome. Paradoxically, such compounds were also found to reduce food intake, hypothesized to be a centrally mediated effect. This paradox was resolved by the demonstration that peripheral CB1 blockade in diet-induced obese mice rapidly reversed their leptin resistance by reversing their hyperleptinemia, via inhibiting leptin production in adipose tissue and increasing leptin clearance in the kidney. This made us wonder whether the high alcohol preference of C57Bl6 mice, another central function promoted by CB1 activation, may also be affected indirectly through blockade of CB1 in the periphery. A possible mechanism involves ghrelin, a gastric peptide that promotes appetite via ghrelin receptors in the brain. Ghrelin also promotes alcohol craving and self-administration, as demonstrated recently in our Institute by Dr. Leggio and his group (Biol Psychiatry, 2014; Mol Psychiatry, 2017). The preliminary findings reported last year have been extended and the findings clearly indicate that rimonabant and the non brain-penetrant CB1 inverse agonists, JD5037, were equi-effective in markedly reducing total alcohol intake as well as ethanol preference in wild-type but not in CB1R-/- C57BL6 mice, using a 'two bottle, free choice' paradigm as well as a 'drinking in the dark' paradigm. Furthermore, peripheral CB1 blockade significantly reduced plasma levels of the biologically active acetylated ghrelin, with no change in the level of its precursor, desacyl ghrelin, suggesting that the ghrelin acylation process, involving the enzyme GOAT and its substrates ghrelin and octanoate, maybe CB1R target(s). In additional experiments we found that both alcohol preference and absolute intake are lower in ghrelin knockout and ghrelin receptor1 (GHSR-1)knockout mice, with no additional reduction caused by peripheral CB1 receptor blockade. The effects on plasma ghrelin and the finding in the knockout strains are compatible with acylghrelin involvement in the effects of JD5037 on alcohol drinking. We also found that afferent vagal (sensory) denervation of the stomach as well as subdiaphragmatic surgical denervation of the stomach increased alcohol drinking and drinking preference and abolished the efficacy of peripheral CB1 blockade as well as GHSR-1 blockade to reduce drinking. These findings are compatible with a scheme whereby alcohol drinking increases ghrelin acylation and/or acylghrelin secretion through CB1 receptor activation, and the released acylghrelin signals via ghrelin receptors (GHSR1) on vagal sensory terminals to promote ethanol drinking behavior. We next explored the cellular mechanism involved in the selective decrease in acyl-ghrelin production by peripheral CB1 blockade. Ghrelin-producing MGN3-1 stomach cells generate octanoic acid exclusively from long-chain fatty acid precursors, and also generate endocannabinoids and express CB1 receptors. JD5037 suppressed octanoyl-ghrelin but not desacyl-ghrelin production in MGN3-1 cells by reducing the levels of octanoyl-carnitine generated from palmityl-carnitine due to an increase in fatty acid oxidation (FAO), which was further confirmed by measuring FAO directly using a fluorescent FAO assay kit. Furthermore, treating MGN3-1 cells with selective inhibitors of 3 different enzymes in the FAO pathway selectively reduced acyl-ghrelin production and occluded the similar effect of JD5037. These findings have now been published in Cell Metabolism.

我们先前已经证明，内源性大麻素通过CB 1受体作用，以年龄依赖性方式促进自愿饮酒，使用两瓶/自由选择范例的小鼠模型（PNAS 100：1393，2003）。本研究中使用的脑渗透剂CB 1反向激动剂利莫那班随后被引入作为肥胖症的治疗，但由于神经精神副作用，包括焦虑，抑郁和自杀意念，不得不在2008年从制药市场撤出。另一种方法，由我的实验室倡导，是引入外周限制性CB 1反向激动剂，保留利莫那班的代谢疗效，但没有其在代谢综合征的啮齿动物模型中的神经行为影响。巧合的是，还发现这些化合物减少食物摄入，假设是中枢介导的作用。这一矛盾通过以下证明得到解决：在饮食诱导的肥胖小鼠中，外周CB 1阻断通过逆转其高瘦素血症（通过抑制脂肪组织中瘦素的产生和增加肾脏中瘦素的清除）来迅速逆转其瘦素抵抗。这让我们想知道C57 B16小鼠的高度酒精偏好，另一个由CB 1激活促进的中枢功能，是否也可能通过外周CB 1的阻断而间接受到影响。一个可能的机制涉及生长激素释放肽，一种胃肽，通过大脑中的生长激素释放肽受体促进食欲。Ghrelin还促进酒精渴望和自我管理，最近在我们的研究所由博士证明Leggio和他的小组（Biol Psychiatry，2014; Mol Psychiatry，2017）。 去年报道的初步研究结果已经得到了扩展，研究结果清楚地表明，利莫那班和非脑渗透性CB 1反向激动剂JD 5037在显著降低野生型小鼠的总酒精摄入量和乙醇偏好方面具有同等效果，但在CB 1 R-/-C57 BL 6小鼠中则不然，使用“两瓶自由选择”范例以及“黑暗中饮酒”范例。此外，外周CB 1阻断显著降低了具有生物活性的乙酰化ghrelin的血浆水平，而其前体去酰基ghrelin的水平没有变化，这表明涉及酶GOAT及其底物ghrelin和辛酸的ghrelin酰化过程可能是CB 1 R靶点。在另外的实验中，我们发现，在ghrelin基因敲除和ghrelin受体1（GHSR-1）基因敲除小鼠中，酒精偏好和绝对摄入量都较低，外周CB 1受体阻断剂没有引起额外的减少。对血浆生长素释放肽的影响和在敲除菌株中的发现与酰基生长素释放肽参与JD 5037对饮酒的影响是一致的。我们还发现，胃的传入迷走神经（感觉）去神经支配以及胃的蛛网膜下手术去神经支配增加了饮酒和饮酒偏好，并取消了外周CB 1阻滞以及GHSR-1阻滞减少饮酒的功效。这些发现是兼容的一个方案，饮酒增加胃饥饿素酰化和/或酰基胃饥饿素分泌通过CB 1受体激活，并通过胃饥饿素受体（GHSR 1）迷走神经感觉末梢上释放的酰基胃饥饿素信号，以促进乙醇饮酒行为。接下来，我们探讨了通过外周CB 1阻断选择性降低酰基-ghrelin产生所涉及的细胞机制。产生Ghrelin的MGN 3 -1胃细胞仅从长链脂肪酸前体产生辛酸，还产生内源性大麻素并表达CB 1受体。JD 5037通过降低由棕榈酰-肉毒碱产生的辛酰-肉毒碱的水平而抑制MGN 3 -1细胞中辛酰-生长素释放肽的产生，但不抑制去酰基-生长素释放肽的产生，这是由于脂肪酸氧化（FAO）的增加，这通过使用荧光FAO测定试剂盒直接测量FAO而进一步证实。此外，用FAO途径中的3种不同酶的选择性抑制剂处理MGN 3 -1细胞选择性地减少酰基-生长素释放肽的产生并阻断JD 5037的类似作用。这些发现现已发表在Cell Metabolism上。