ABSTRACT

Autophagic flux is a critical cellular process that is vastly under-appreciated in terms of its importance to human health. Preclinical studies have demonstrated that reductions in autophagic flux cause cancer and exacerbate chronic diseases, including heart disease and the pathological hallmarks of dementia. Autophagic flux can be increased by targeting nutrition-related biochemical signaling. To date, translation of this knowledge has been hampered because there has been no way to directly measure autophagic flux in humans. In this study we detail a method whereby human macroautophagic/autophagic flux can be directly measured from human blood samples. We show that whole blood samples can be treated with the lysosomal inhibitor chloroquine, and peripheral blood mononuclear cells isolated from these samples could be used to measure autophagic machinery protein LC3B-II. Blocking of autophagic flux in cells while still in whole blood represents an important advance because it preserves genetic, nutritional, and signaling parameters inherent to the individual. We show this method was reproducible and defined LC3B-II as the best protein to measure autophagic flux in these cells. Finally, we show that this method is relevant to assess intra-individual variation induced by an intervention by manipulating nutrition signaling with an ex vivo treatment of whole blood that comprised leucine and insulin. Significantly, this method will enable the identification of factors that alter autophagic flux in humans, and better aid their translation in the clinic. With further research, it could also be used as a novel biomarker for risk of age-related chronic disease.

Abbreviations: AMPK: AMP-activated protein kinase; ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A₁; CQ: chloroquine; DMSO: dimethyl sulfoxide; DPBS: Dulbecco’s phosphate-buffered saline; EDTA: ethylenediaminetetraacetic acid; KO: knockout; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; PBMCs: peripheral blood mononuclear cells; PMNs: polymorphonuclear cells; RPMI: Roswell Park Memorial Institute; SQSTM1: sequestosome 1; TBST: Tris-buffered saline containing 0.1% (v:v) Tween 20; TEM: transmission electron microscopy.

摘要

自噬通量是一个关键的细胞过程，就其对人类健康的重要性而言，它的重要性被大大低估了。临床前研究表明，自噬通量的减少会导致癌症并加剧慢性疾病，包括心脏病和痴呆症的病理特征。通过靶向与营养相关的生化信号可以增加自噬通量。迄今为止，这些知识的翻译一直受到阻碍，因为没有办法直接测量人类的自噬通量。在这项研究中，我们详细介绍了一种可以直接从人类血液样本中测量人类巨自噬/自噬通量的方法。我们证明全血样本可以用溶酶体抑制剂氯喹处理，从这些样本中分离的外周血单个核细胞可用于测量自噬机制蛋白 LC3B-II。在全血中阻断细胞中的自噬通量代表了一项重要的进步，因为它保留了个体固有的遗传、营养和信号参数。我们证明这种方法是可重复的，并将 LC3B-II 定义为测量这些细胞中自噬通量的最佳蛋白质。最后，我们表明，该方法与通过使用 我们证明这种方法是可重复的，并将 LC3B-II 定义为测量这些细胞中自噬通量的最佳蛋白质。最后，我们表明，该方法与通过使用 我们证明这种方法是可重复的，并将 LC3B-II 定义为测量这些细胞中自噬通量的最佳蛋白质。最后，我们表明，该方法与通过使用对包含亮氨酸和胰岛素的全血进行离体处理。重要的是，这种方法将能够识别改变人类自噬通量的因素，并更好地帮助它们在临床中的转化。随着进一步的研究，它也可以用作与年龄相关的慢性病风险的新型生物标志物。

缩写： AMPK：AMP激活的蛋白激酶；ACTB：肌动蛋白β；ATG5：自噬相关5；BAF：巴弗洛霉素 A ₁；CQ：氯喹；DMSO：二甲亚砜；DPBS：Dulbecco's 磷酸盐缓冲液；EDTA：乙二胺四乙酸；KO：淘汰赛；MAP1LC3A/LC3A：微管相关蛋白 1 轻链 3 α；MAP1LC3B/LC3B：微管相关蛋白1轻链3β；MAP1LC3C/LC3C：微管相关蛋白 1 轻链 3 γ；MTOR：雷帕霉素激酶的机制靶点；NBR1：NBR1 自噬货物受体；PBMCs：外周血单个核细胞；PMNs：多形核细胞；RPMI：罗斯威尔公园纪念研究所；SQSTM1：隔离体 1；TBST：含有 0.1% (v:v) Tween 20 的 Tris 缓冲盐水；TEM：透射电子显微镜。