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

 描述（由申请人提供）：AMPK激活对抗糖尿病中的肝脏葡萄糖产生、肌肉胰岛素抵抗和高脂血症。Metabolic可能间接靶向AMPK，但其机制仍有争议。没有其他糖尿病药物有效靶向AMPK。鉴于许多外源性物质据报道会影响AMPK，最近发现水杨酸盐在作为抗炎药使用几个世纪后会激活它，我们推断目前实践中的其他药物也可能激活AMPK。黄嘌呤氧化酶（XO）抑制剂用于通过降低尿酸水平来治疗痛风。根据基本的生物化学原理，我们推断阻断嘌呤代谢的最后步骤将增加阻断上游的嘌呤代谢物的浓度，包括AMP和AICAR，这将激活AMPK。初步结果表明这是真的。对XOI处理的HFD小鼠肝脏和培养的肝细胞进行的基于LC-MS/MS的代谢组学研究表明，别嘌呤醇和非布司他均降低细胞内尿酸和黄嘌呤，同时增加次黄嘌呤、IMP、AMP和AICAR。代谢组学特征也揭示了与AMPK激活相关的变化。AMPK活化也通过AMPK及其底物ACC和Raptor内的适当磷酸化来证明。与AMPK激活一致，XOI降低了肥胖小鼠的血糖和胰岛素，降低了肝脏TG，改善了葡萄糖和胰岛素耐受性，而不影响体重或食物摄入。由于“嘌呤补充”恢复了肥胖小鼠的代谢平衡，我们推断肥胖可能是一种“嘌呤耗竭”状态，导致AMPK失调。这也得到了初步结果和文献中许多先前报告的支持，尽管XO和AMPK的生理学和病理学以前没有联系。提出的实验将研究扩展到脂肪组织、肌肉、肠和脉管系统，使用遗传修饰或病毒转导来急性上调/下调肥胖小鼠的选定组织中的XO和AMPK。修饰的小鼠和衍生细胞将用XOI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。将使用标准方法评估这些操作的体内代谢和心血管效应，包括但不限于葡萄糖和胰岛素耐受性试验和血液浓度、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定量和组成。该提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（可能继发于炎症）是否促进嘌呤耗竭以抑制AMPK并对心脏代谢稳态产生不利影响？2)XO抑制是否使组织嘌呤充足以促进AMPK激活并恢复心脏代谢稳态？拟议的临床前实验测试这些假设，为临床转化做准备。

 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.