Endocannabinoids and the Control Of Behavior and Cardiovascular Function

在我之前的年度报告中，我总结了我们的研究结果，发表在2019年的《细胞代谢》杂志上，外周限制性CB 1受体拮抗剂JD 5037与脑渗透性CB 1拮抗剂利莫那班在显著减少野生型小鼠的总酒精摄入量和乙醇偏好方面同样有效，但在CB 1 R-/-C57 BL 6小鼠中则不然。自由选择“范式以及”在黑暗中饮酒“范式。此外，外周CB 1阻断显著降低了具有生物活性的辛酰化生长素释放肽的血浆水平，而其前体去酰基生长素释放肽的水平没有变化，这表明由GOAT酶催化的生长素释放肽酰化过程可能是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，酒精偏好和摄入量较低。我们还发现，传入迷走神经（感觉）胃去神经增加饮酒和饮酒偏好，并取消外周CB 1阻滞以及GHSR-1阻滞的疗效，以减少饮酒。这些发现与饮酒通过CB 1受体激活增加ghrelin酰化的方案是一致的，并且通过迷走神经感觉末梢上的ghrelin受体（GHSR 1）释放酰基ghrelin信号以促进饮酒行为。至于通过外周CB 1阻断选择性降低酰基-生长素释放肽产生所涉及的细胞机制，我们发现，在生长素释放肽产生的胃细胞系JD 5037中，通过降低由棕榈酰-肉碱产生的辛酰-肉碱水平（由于脂肪酸氧化的增加）抑制辛酰-生长素释放肽，但不抑制去酰基-生长素释放肽的产生。 虽然JD 5037消耗GOAT底物辛酰肉碱是体内观察到的饮酒偏好降低的合理机制，但产生ghrelin的胃细胞上的CB 1受体可能不是其唯一的靶点。具体地说，已知CB 1受体存在于迷走神经传入神经元的外周末梢上，并且它们的阻断可能以抑制饮酒冲动的方式影响迷走神经传入信号。作为探索这种可能性的第一步，我们已经产生了条件性CB 1基因敲除小鼠，在结状神经节的传入神经元的特定子集中选择性遗传缺失Cnr 1。这是通过将CB 1 floxed小鼠与avilin-cre或Trpv 1-cre小鼠杂交来实现的，advilin和Trpv 1是迷走神经传入神经元的不同子集的标记物。这些动物和它们的野生型同窝仔目前正在自愿饮酒的不同范例中进行测试。 在单独的实验中，我们正在测试我们的混合CB 1 R/iNOS抑制剂MRI-1867对饮酒和偏好的影响，并与单一靶点CB 1或iNOS抑制剂的影响进行比较。这些实验的目的是测试iNOS的额外靶向作用和所产生的抗炎作用是否有助于减少饮酒，这是基于在暴饮暴食模型中测试的小鼠胃中M1巨噬细胞浸润的已发表证据。

In my previous annual report I summarized our findings, published in 2019 in Cell Metabolism, that the peripherally restricted CB1 receptor antagonist JD5037 was equally effective with the brain-penetrant CB1 antagonist rimonabant 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 octanoylated ghrelin, with no change in the level of its precursor, desacyl ghrelin, suggesting that the ghrelin acylation process, catalyzed by the enzyme GOAT maybe a CB1R target. In additional experiments we found that alcohol preference and intake are lower in ghrelin knockout and ghrelin receptor1 (GHSR-1)knockout mice. We also found that afferent vagal (sensory) 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 through CB1 receptor activation, and the released acylghrelin signals via ghrelin receptors (GHSR1) on vagal sensory terminals to promote ethanol drinking behavior. As for the cellular mechanism involved in the selective decrease in acyl-ghrelin production by peripheral CB1 blockade, we found that in a ghrelin-producing stomach cell line JD5037 suppressed octanoyl-ghrelin but not desacyl-ghrelin production by reducing the levels of octanoyl-carnitine generated from palmityl-carnitine due to an increase in fatty acid oxidation. While depletion of the GOAT substrate octanoyl carnitine by JD5037 is a plausible mechanism for the reduced drinking preference observed in vivo, CB1 receptors on ghrelin-producing stomach cells may not be its only targets. Specifically, CB1 receptors are known to be present on the peripheral terminals of vagal afferent neurons, and their blockade may affect vagal afferent signaling in a way that would inhibit the drive to drink. As a first step to explore this possibility, we have generated conditional CB1 knockout mice with selective genetic deletion of Cnr1 in specific subsets of afferent neurons of the nodose ganglion. This was achieved by crossing CB1 floxed mice with avilin-cre or Trpv1-cre mice, advilin and Trpv1 being markers of distinct subsets of vagal afferent neurons. These animals and their wild-type littermates are currently being tested in different paradigms of voluntary alcohol drinking. In separate experiments, we are testing the effects on alcohol drinking and preference of our hybrid CB1R/iNOS inhibitor MRI-1867, in comparison with the effects of single target CB1 or iNOS inhibitors. The purpose of these experiments is to test whether the additional targeting of iNOS and the resulting anti-inflammatory effect may contribute to the reduction in alcohol drinking, based on published evidence of M1 macrophage infiltration in the stomach of mice tested in models of binge drinking.