Donati Alessio, Cavallini Gabriella, Bergamini Ettore
Centro di Ricerca Interdipartimentale di Biologia e Patologia dell'Invecchiamento, Università di Pisa, Pisa, Italy.
Methods Enzymol. 2009;452:441-55. doi: 10.1016/S0076-6879(08)03626-4.
The functioning of macroautophagy declines with increasing age in the liver of ad libitum fed animals, whereas it is preserved in rats submitted to antiaging caloric restriction. In this perspective, monitoring autophagy during aging may provide a useful biomarker of aging. Here we describe a procedure for the quantification of the ex vivo functioning of autophagy by the use of single-pass liver perfusion to measure the rate of degradation of prelabeled long-lived proteins. The maximum rate of autophagy can be measured after the pharmacological suppression of the supply of free fatty acids during fasting, which intensifies the activation of autophagy by a physiological mechanism. The effects of treatment on the plasma level of branched chain amino acids may be used as a minimally invasive indicator of the intensification of autophagy. The effects of aging on autophagic proteolysis and on amino acid and hormone control can also be assessed by measuring the rate of the 3-methyladenine-sensitive valine released from isolated liver cells incubated in vitro.
在随意进食的动物肝脏中，巨自噬功能会随着年龄增长而下降，而在进行抗衰老热量限制的大鼠中，其功能得以保留。从这个角度来看，在衰老过程中监测自噬可能会提供一个有用的衰老生物标志物。在此，我们描述了一种通过单次肝脏灌注来测量预标记长寿蛋白降解速率，从而对自噬的离体功能进行定量的方法。在禁食期间对游离脂肪酸供应进行药理学抑制后，可以测量出自噬的最大速率，这会通过生理机制增强自噬的激活。治疗对支链氨基酸血浆水平的影响可以用作自噬增强的微创指标。衰老对自噬性蛋白水解以及氨基酸和激素调控的影响，也可以通过测量体外培养的分离肝细胞中释放的3 - 甲基腺嘌呤敏感缬氨酸的速率来评估。
