Defining the molecular roles of peripheral CB1 and CB2 cannabinoid receptors in age-induced changes in energy and metabolic homeostasis.

目前，人们对一类被称为大麻素的化合物在治疗肥胖和代谢相关疾病方面的作用有着极大的兴趣。大麻素存在于大麻中，但我们的身体会自然产生类似大麻素的化学物质，即内源性大麻素，它会锁定在被称为大麻素受体（即CB1和CB2）的细胞表面的蛋白质分子上。在肥胖、糖尿病和我们最近发现的衰老过程中，CB1明显被身体产生的内源性大麻素过度激活，导致胰岛素作用受损，肌肉、脂肪和肝脏等重要代谢组织的能量平衡失调。CB1过度激活与胰岛素抵抗和肥胖增加密切相关。相反，新出现的数据表明CB2可能具有保护性生理作用。根据这一观点，我们发现CB2抑制剂（拮抗剂）增强了内源性大麻素、阿南达明（AEA）在肌肉细胞中的胰岛素脱敏作用，而CB2激活剂（激动剂）改善了胰岛素信号传导的损失。此外，值得注意的是，虽然CB1的表达在衰老肌肉中增强，但CB2的表达却显著降低，这与衰老骨骼肌中普遍存在的胰岛素敏感性降低相一致。有趣的是，CB1拮抗剂（如利莫那班）通过独立于其食欲抑制作用的机制促进肥胖动物的葡萄糖耐量，刺激能量消耗和减轻体重。与这些发现一致，我们发现利莫那班可以改善老年动物的年龄相关组织胰岛素抵抗和脂肪增加。确切地说，CB1拮抗剂是如何引起这些有益效果的尚不清楚，但我们最近的工作表明，CB1阻断诱导AMPK的激活——AMPK分子不仅能感知细胞能量，还能促进线粒体（细胞的能量工厂）中脂肪的分解和燃烧。CB1阻断与AMPK激活之间的分子事件，以及后者对参与脂肪分解/燃烧和线粒体动力学的酶的影响仍然知之甚少。拟议的研究将利用培养的肌肉和脂肪细胞来了解CB1抑制或CB2激活如何影响与胰岛素作用，能量平衡和线粒体功能和完整性相关的分子。我们的分子分析将涉及生化和最先进的成像技术，用于可视化肌肉和脂肪细胞中的线粒体染色。这些以细胞为基础的研究将辅以对年轻和老年CB2基因缺陷小鼠的组织分析，或对服用CB1拮抗剂或CB2激动剂药理活性剂量两周的年轻和老年小鼠进行实验。在此期间，我们将监测食物摄入量、葡萄糖耐量、能量消耗、身体活动和脂肪量，然后采集血液/组织进行实验分析，旨在剖析CB1阻断或CB2激活改善老年动物代谢状态的机制。我们还旨在测试运动对老年动物的影响，因为已知体育活动有助于维持组织对胰岛素等合成代谢激素的敏感性，并在衰老过程中保持组织功能。这些研究将有助于揭示运动是否会减少与年龄相关的组织CB1和CB2表达的变化，如果是这样，这些变化是否与全身能量测量（即体脂、葡萄糖耐量和能量消耗）有利相关。拟议的研究将特别扩展我们对调节外周CB受体活性如何影响能量平衡和胰岛素作用的基本理解。即将出现的发现将推进我们对这些关键问题的认识，并证明在设计选择性靶向外周ECS治疗年龄和肥胖相关代谢紊乱的治疗方法方面是无价的。

There is immense interest at present in targeting the action of a class of compounds, termed cannabinoids, in the treatment of obesity and metabolic-related disorders. Cannabinoids are present in cannabis, but our bodies naturally create cannabinoid-like chemicals, known as endocannabinoids that lock-on to protein molecules found on the surface of cells called cannabinoid receptors (i.e. CB1 and CB2). During obesity, diabetes and, as we have recently discovered, during ageing the CB1 is notably over-activated by endocannabinoids produced by the body resulting in impaired insulin action and dysregulation of energy balance in metabolically important tissues such as muscle, fat and liver. CB1 over-activation has been linked strongly with development of insulin resistance and increased adiposity. In contrast, emerging data indicates that CB2 may confer a protective physiological effect. In line with this idea, we find that CB2 inhibitors (antagonists) augment the insulin-desensitising effects of the endocannabinoid, anandamide (AEA) in muscle cells, whereas CB2 activators (agonists) ameliorate the loss in insulin signalling. Moreover, it is noteworthy that whilst CB1 expression is enhanced in ageing muscle that of CB2 is significantly decreased, consistent with the reduced insulin sensitivity that prevails in ageing skeletal muscle. Intriguingly, CB1 antagonists (e.g. rimonabant) promote glucose tolerance, stimulate energy expenditure and reduce body weight in obese animals by mechanisms independent of their appetite-reducing effect. Consistent with such findings, we find rimonabant ameliorates age-related tissue insulin resistance and fat mass gain in older animals. Precisely how CB1 antagonism elicits these beneficial effects is unclear, but our recent work indicates that CB1 blockade induces activation of AMPK - a molecule that not only senses cellular energy but promotes the breakdown and burning of fat in mitochondria (the cell's energy factory). The molecular events linking CB1 blockade to AMPK activation and the effect of the latter on enzymes involved in fat breakdown/burning and mitochondrial dynamics remain poorly understood. The proposed studies will utilise cultured muscle and fat cells to understand how CB1 inhibition or CB2 activation impacts upon molecules implicated in insulin action, energy balance and mitochondrial function and integrity. Our molecular analyses will involve biochemical and state-of-the-art imaging techniques for visualising mitochondrial staining in muscle and fat cells. These cell-based studies will be complemented with analysis of tissues from young and old mice genetically deficient in CB2 or experiments in young and aged mice administered a pharmacologically active dose of a CB1 antagonist or CB2 agonist for two weeks. During this period we will monitor food intake, glucose tolerance, energy expenditure, physical activity and fat mass before sampling blood/tissue for experimental analyses designed to dissect the mechanisms by which CB1 blockade or CB2 activation improves the metabolic status of aged animals. We also aim to test the effects of exercise in aged animals given that physical activity is known to help sustain tissue sensitivity to anabolic hormones such as insulin and preserve tissue functionality during aging. These studies will help unveil whether exercise curtails age-related changes in tissue CB1 and CB2 expression and, if so, whether these correlate favourably with measures of whole body energetics (i.e. body fat, glucose tolerance and energy expenditure). The proposed research will specifically expand our fundamental understanding of how modulating peripheral CB receptor activity influences energy balance and insulin action. The findings that will emerge will advance our knowledge of these key issues and prove invaluable in designing therapies that selectively target the peripheral ECS for treatment of age and obesity-related metabolic disorders.