Summary

小鼠中自噬激活有氧运动

Published: February 03, 2017
doi:

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

所有涉及动物的程序进行根据西北大学机构动物护理和使用委员会(IACUC)批准的指导进行。 1.鼠标型号使用8-12周龄小鼠的运动训练。以检测体内行使诱导自噬,用于成像研究和C57BL / 6小鼠进行生化分析的GFP-LC3转基因小鼠(C57BL / 6背景)。 2.运动诱发的自噬 跑步机设置-强制运动 适应和培养在10°上坡开跑步机小鼠2天。在第…

Representative Results

本协议描述两种不同的方法来诱导自噬通过有氧运动小鼠组织:共90分钟由驯化两天进行一个多车道的跑步机锻炼强迫的;两个星期自愿行使对单只装一个用来跑轮。在每个运动方案,我们可以通过测量各个器官荧光显微镜和Western blot分析自噬通量。 我们用表达GFP标记的LC3当记者通过系统的锻炼1,监测自噬的?…

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体外体?…

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

References

  1. Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y. In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker. Mol Biol Cell. 15, 1101-1111 (2004).
  2. Tracy, K., et al. BNIP3 is an RB/E2F target gene required for hypoxia-induced autophagy. Molecular and cellular biology. 27, 6229-6242 (2007).
  3. Mizushima, N., Komatsu, M. Autophagy: renovation of cells and tissues. Cell. 147, 728-741 (2011).
  4. Nikoletopoulou, V., Papandreou, M. E., Tavernarakis, N. Autophagy in the physiology and pathology of the central nervous system. Cell Death Differ. 22, 398-407 (2015).
  5. Rocchi, A., He, C. Emerging roles of autophagy in metabolism and metabolic disorders. Frontiers in biology. 10, 154-164 (2015).
  6. Mathew, R., Karantza-Wadsworth, V., White, E. Role of autophagy in cancer. Nature reviews. Cancer. 7, 961-967 (2007).
  7. He, C., et al. Exercise-induced BCL2-regulated autophagy is required for muscle glucose homeostasis. Nature. 481, 511-515 (2012).
  8. He, C., Sumpter, R. J., Levine, B. Exercise induces autophagy in peripheral tissues and in the brain. Autophagy. 8, 4 (2012).
  9. Kuramoto, K., et al. Autophagy activation by novel inducers prevents BECN2-mediated drug tolerance to cannabinoids. Autophagy. 12, 1460-1471 (2016).
  10. Dougherty, J. P., Springer, D. A., Gershengorn, M. C. The Treadmill Fatigue Test: A Simple, High-throughput Assay of Fatigue-like Behavior for the Mouse. JoVE. , (2016).
  11. Navone, S. E., et al. Isolation and expansion of human and mouse brain microvascular endothelial cells. Nature protocols. 8, 1680-1693 (2013).
  12. Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nature protocols. 10, 1612-1624 (2015).
  13. Eslami, A., Lujan, J. Western blotting: sample preparation to detection. Journal of visualized experiments : JoVE. , (2010).
  14. Bjørkøy, G., et al. Chapter 12 Monitoring Autophagic Degradation of p62/SQSTM1. Methods Enzymol. 452, 181-197 (2009).
  15. Klionsky, D. J., et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 12, 1-222 (2016).
  16. Levine, B., Kroemer, G. Autophagy in the Pathogenesis of Disease. Cell. 132, 27-42 (2008).
  17. Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature. 451, 1069-1075 (2008).
  18. Fang, Y., et al. Duration of rapamycin treatment has differential effects on metabolism in mice. Cell Metab. 17, 456-462 (2013).
  19. Thomson, A. W., Turnquist, H. R., Raimondi, G. Immunoregulatory functions of mTOR inhibition. Nature reviews. Immunology. 9, 324-337 (2009).
  20. Miller, R. A., et al. Rapamycin-mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction. Aging Cell. 13, 10 (2014).
  21. Grumati, P., et al. Physical exercise stimulates autophagy in normal skeletal muscles but is detrimental for collagen VI-deficient muscles. Autophagy. 7, 1415-1423 (2011).
  22. Lira, V. A., et al. Autophagy is required for exercise training-induced skeletal muscle adaptation and improvement of physical performance. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 27, 4184-4193 (2013).
  23. Lo Verso, F., Carnio, S., Vainshtein, A., Sandri, M. Autophagy is not required to sustain exercise and PRKAA1/AMPK activity but is important to prevent mitochondrial damage during physical activity. Autophagy. 10, 1883-1894 (2014).
  24. Kregel, K. C., et al. Resource book for the design of animal exercise protocols. American Physiological Society. , 152 (2006).
  25. Lightfoot, J. T., Turner, M. J., Debated, K. S., Kleeberg, S. R. Interstrain variation in murine aerobic capacity. Med Sci Sports Exerc. 33, 5 (2001).
  26. Rezende, E. L., Chappell, M. A., Gomes, F. R., Malisch, J. L., Garland, T. Maximal metabolic rates during voluntary exercise, forced exercise, and cold exposure in house mice selectively bred for high wheel-running. The Journal of experimental biology. 208, 2447-2458 (2005).
  27. Kayatekin, B. M., Gonenc, S., Acikgoz, O., Uysal, N., Dayi, A. Effects of sprint exercise on oxidative stress in skeletal muscle and liver. European journal of applied physiology. 87, 141-144 (2002).
  28. Kawanaka, K., Tabata, I., Tanaka, A., Higuchi, M. Effects of high-intensity intermittent swimming on glucose transport in rat epitrochlearis muscle. J Appl Physiol. 84, 4 (1998).
  29. Fernando, P., Bonen, A., Hoffman-Goetz, L. Predicting submaximal oxygen consumption during treadmill running in mice. Can J Physiol Pharmacol. 71, 4 (1993).

Play Video

Cite This Article
Rocchi, A., He, C. Activating Autophagy by Aerobic Exercise in Mice. J. Vis. Exp. (120), e55099, doi:10.3791/55099 (2017).

View Video