小鼠中自噬激活有氧运动
Summary
自噬活化是在预防许多疾病有益。一种生理方法的诱导自噬体内是体育锻炼。在这里，我们将展示如何通过有氧运动来激活自噬和在小鼠体内测量细胞自噬水平。
Abstract
Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.
Introduction
自噬是一种进化上保守的降解途径，这是响应于各种胁迫条件如饥饿，缺氧1,2诱导。在自噬，双膜囊，称为自噬体，包括不必要的或损坏的亚细胞成分，并将它们运送到溶酶体降解3。基底自噬是在许多疾病，包括神经变性，肿瘤和2型糖尿病4，5，6被牵连于细胞功能和生物体的发展，和受损基底自噬是必不可少的。
最知名的生理自噬诱导剂是饥饿。然而，它有两个主要的限制。首先，饥饿需要很长的时间，以有效地诱导自噬在动物中， 例如 ，在小鼠中食物限制的48小时在大多数器官。第二，饥饿勉强诱导脑自噬，由于大脑中的相对稳定的营养供给。实际上，它也难以通过小分子诱导剂来检测自噬诱导，因为许多药物不能通过血脑屏障。因此，为了更好地分析自噬活化在疾病的发病机制的功能，我们最近发现，运动是诱导自噬在时间7，8，9短时间内更有效的生理方法。与饥饿相比，自噬是有效地跑步机上跑步一样快，30分钟所致。因此，运动是一种方便和有效的生理方法来研究细胞自噬机制在调解医疗福利和预防疾病。
有用于检测自噬活动的，包括LC3和p62的几种蛋白质的标志物。 LC3（微管相关蛋白1A / 1B-光速c海恩3）是胞质蛋白（LC3-I形式）是，在自噬诱导缀合的PE（磷脂酰乙醇胺）。 PE-脂化LC3（LC3-II的形式）被募集到autophagosomal膜，可用于当用GFP标记的可视化自噬。其从细胞溶质向点状在显微镜下自噬体结构易位是自噬诱导的指示。 P62是自噬底物（如泛素化蛋白质）货物的受体，并且被并入自噬体为好。因为蛋白质是与自噬体沿溶酶体降解，其水平可用于测量自噬通量。在这里，我们展示了如何使用这些标志通过有氧运动，包括强迫运动（跑步机）和自愿运动（跑步轮）引起不同的鼠标组织量化自噬。相同的过程也可以治疗其它诱导物后施加到自噬的体内测量。
Protocol
Representative Results
Discussion
自噬是分解代谢过程提供能量和细胞质组件或受损细胞器溶酶体降解减少细胞毒性。自噬研究重要的是要了解细胞稳态的调节和应激反应的机制。新的模式和方法不断涌现的研究领域15，研究如何吞噬受损有助于众多病理过程16,17。
营养缺乏（饥饿）和药理学诱导常用的方法来诱导自噬在体外和体?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).
Materials
| Treadmill | Columbus Instruments | 150-RM Exer 3/6 | |
| Mouse running wheel | Super Pet | 100079365 | diameter 11.4 cm |
| Odometer | Bell | DASHBOARD 100 | |
| Syringe pump | KD Scientific | KDS100 | |
| Fluorescence microscope | Nikon | Model: inverted microscope ECLIPSE | |
| Cryostat | Leica | CM 1850UV | |
| Homogenizer | IKA | 003737001 / Model: T10 Basic S1 | |
| Chloroquine | CAYMAN CHEMICAL COMPANY | 14194 | |
| Parafolmaldehye | SIGMA-ALDRICH | P6148 | Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer |
| Mounting media | Vector Laboratories | H-1200 | |
| p62 antibody | BD Biosciences | 610833 | |
| LC3 antibody | Novus Biologicals | NB100-2220 | |
| 2X Laemmli Sample Buffer | Bio-Rad Laboratories | 161-0737 | |
| ImageJ | NIH |
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