Endocannabinoids and the Control Of Behavior and Cardiovascular Function

在我之前的年度报告中，我总结了我们于2019年发表在《细胞代谢》（Cell Metabolism）杂志上的研究结果，即外周限制性CB1受体拮抗剂JD5037与脑渗透性CB1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CB1R-/- C57BL6小鼠中却没有，使用“两瓶，自由选择”范式和“在黑暗中饮酒”范式。此外，外周CB1阻断显著降低了具有生物活性的辛烷酰化胃饥饿素的血浆水平，而其前体去酰基胃饥饿素的水平没有变化，这表明由GOAT酶催化的胃饥饿素酰化过程可能是CB1R的靶标。在另外的实验中，我们发现在胃饥饿素敲除小鼠和胃饥饿素受体1 （GHSR-1）敲除小鼠中，酒精偏好和摄入量较低。我们还发现胃的传入迷走神经（感觉）去神经支配增加了饮酒和饮酒偏好，并取消了外周CB1阻断和GHSR-1阻断减少饮酒的功效。这些发现与一种方案相一致，即饮酒通过CB1受体激活增加胃饥饿素酰化，并通过迷走神经感觉末端的胃饥饿素受体（GHSR1）释放乙酰胃饥饿素信号，促进乙醇饮酒行为。至于通过外周CB1阻断选择性减少酰基胃饥饿素产生的细胞机制，我们发现在产生胃饥饿素的胃细胞系中，JD5037通过降低棕榈酰肉毒碱产生的辛酰肉毒碱水平（由于脂肪酸氧化增加）来抑制辛酰胃饥饿素的产生，但不抑制去酰基胃饥饿素的产生。

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.