Xanthine oxidase inhibition raises intracellular purines to activate AMPK, improve glucose control and decrease fatty liver and atherosclerosis in type 2 diabetes

 说明(申请人提供)：AMPK激活可对抗糖尿病患者的肝脏葡萄糖生成、肌肉胰岛素抵抗和高脂血症。二甲双胍可间接靶向AMPK，但其作用机制尚有争议。没有其他糖尿病药物有效地针对AMPK。据报道，许多外源药物影响AMPK，而水杨酸盐在作为消炎药使用了几个世纪后，最近被发现激活了AMPK，我们推测目前实践中的其他药物也可能激活AMPK。黄嘌呤氧化酶(XO)抑制剂可通过降低尿酸水平来治疗痛风。根据基本的生化原理，我们推断，阻断嘌呤代谢的最后步骤会增加阻断上游的嘌呤代谢产物的浓度，包括AMP和AICAR，这将激活AMPK。初步结果表明，这是真的。基于LC-MS/MS的代谢组学研究表明，别嘌醇和非布司坦均可降低细胞内尿酸和黄嘌呤，同时增加次黄嘌呤、IMP、AMP和AICAR。代谢组学也揭示了与AMPK激活有关的变化。AMPK及其底物ACC和Raptor的适当磷酸化也证明了AMPK的激活。与AMPK激活一致，XOIS降低了肥胖小鼠的血糖和胰岛素，降低了肝脏甘油三酯，并改善了葡萄糖和胰岛素耐量，而不影响体重或食物摄入量。由于“嘌呤补充”恢复了肥胖小鼠的代谢动态平衡，我们推测肥胖可能是一种导致AMPK调节失调的“嘌呤补充”状态。这也得到了初步结果和文献中许多先前报道的支持，尽管XO和AMPK的生理学和病理学以前没有联系。建议的实验通过基因修饰或病毒转导将研究扩展到脂肪组织、肌肉、肠道和血管系统，以显著上调/下调肥胖小鼠选定组织中的XO和AMPK。修改后的小鼠和衍生细胞将用XOIS处理。代谢组学研究将记录嘌呤和AMPK相关代谢物的浓度，XO和AMPK活性将通过生物化学和蛋白质磷酸化和基因表达来证明。这些手法的体内代谢和心血管影响将使用标准方法进行评估，包括但不限于葡萄糖和胰岛素耐量测试和血液浓度、高胰岛素-正常血糖钳夹、组织甘油三酯含量和组织病理学，以及血管斑块的量化和组成。这一建议解决了两个主要问题/假设：1)肥胖动物组织中XO活性的增加(可能发生于炎症)是否促进了嘌呤的消耗，从而抑制了AMPK，并对心脏代谢稳态产生了不利影响？2)XO抑制是否会使组织中的嘌呤充满，以促进AMPK的激活和恢复心脏代谢稳态？拟议的临床前实验测试这些假说，为临床翻译做准备。

 DESCRIPTION (provided by applicant): AMPK activation counters hepatic glucose production, muscle insulin resistance and hyperlipidemia in diabetes. Metformin may indirectly target AMPK, but its mechanism is debated. No other diabetes drug effectively targets AMPK. Given that many xenobiotics reportedly influence AMPK, and salicylate was recently found to activate it after centuries of use as anti-inflammatory drug, we reasoned that other drugs in current practice may also activate AMPK. Xanthine oxidase (XO) inhibitors are used to treat gout by lowering uric acid levels. Following fundamental biochemical principles, we reasoned that blocking the final steps in purine metabolism would increase concentrations of purine metabolites upstream of the blockade, including AMP and AICAR, which would activate AMPK. Preliminary results show this is true. LC-MS/MS based metabolomics studies, conducted with livers from XOI-treated HFD mice and cultured hepatocytes, show that both allopurinol and febuxostat decrease intracellular uric acid and xanthine and concomitantly increase hypoxanthine, IMP, AMP and AICAR. The metabolomics profiles also revealed changes readily linked to AMPK activation. AMPK activation was also demonstrated by appropriate phosphorylations within AMPK and its substrates, ACC and Raptor. Consistent with AMPK activation, the XOIs lowered blood glucose and insulin, reduced hepatic TG, and improved glucose and insulin tolerance in obese mice, without affecting body weight or food intake. And since "purine repletion" restored metabolic homeostasis to the obese mice, we reasoned that obesity might be a state of "purine depletion" leading to dysregulated AMPK. This too is supported by preliminary results and many previous reports in the literature, even though the physiology and pathology of XO and AMPK have not been linked previously. Proposed experiments expand the studies to adipose tissue, muscle, intestine, and the vasculature using genetic modifications or viral transduction to acutely up/down regulate XO and AMPK in selected tissues of obese mice. The modified mice and derived cells will be treated with XOIs. Metabolomics studies will document concentrations of purine and AMPK-relevant metabolites, and XO and AMPK activities will be demonstrated biochemically and through protein phosphorylation and gene expression. The in vivo metabolic and cardiovascular effects of these manipulations will be assessed using standard methods, including but not limited to glucose and insulin tolerance tests and blood concentrations, hyperinsulinemic-euglycemic clamps, tissue triglyceride content and histopathology, and quantification and composition of vascular plaque. This proposal addresses two major questions/hypotheses: 1) Does the increased XO activity in tissues of obese animals (possibly occurring secondary to inflammation) promote purine depletion to suppress AMPK and adversely impact cardiometabolic homeostasis? 2) Does XO inhibition replete tissue purines to promote AMPK activation and restore cardiometabolic homeostasis? Proposed preclinical experiments test these hypotheses in preparation for clinical translation.