title: Characterization of macroautophagic flux in vivo using a leupeptin-based assay.
abstract: Macroautophagy is a highly conserved catabolic process that is crucial for organ homeostasis in mammals. However, methods to directly measure macroautophagic activity (or flux) in vivo are limited. In this study we developed a quantitative macroautophagic flux assay based on measuring LC3b protein turnover in vivo after administering the protease inhibitor leupeptin. Using this assay we then characterized basal macroautophagic flux in different mouse organs. We found that the rate of LC3b accumulation after leupeptin treatment was greatest in the liver and lowest in spleen. Interestingly we found that LC3a, an ATG8/LC3b homologue and the LC3b-interacting protein p62 were degraded with similar kinetics to LC3b. However, the LC3b-related proteins GABARAP and GATE-16 were not rapidly turned over in mouse liver, implying that different LC3b homologues may contribute to macroautophagy via distinct mechanisms. Nutrient starvation augmented macroautophagic flux as measured by our assay, while refeeding the animals after a period of starvation significantly suppressed flux. We also confirmed that beclin 1 heterozygous mice had reduced basal macroautophagic flux compared to wild-type littermates. These results illustrate the usefulness of our leupeptin-based assay for studying the dynamics of macroautophagy in mice.
authors: Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Boston, MA USA.
authors: Jeffrey
authors: J
authors: Haspel
authors: Rahamthulla S
authors: RS
authors: Shaik
authors: Emeka
authors: E
authors: Ifedigbo
authors: Kiichi
authors: K
authors: Nakahira
authors: Tamas
authors: T
authors: Dolinay
authors: Joshua A
authors: JA
authors: Englert
authors: Augustine M K
authors: AM
authors: Choi
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001343:Autophagy; D000071186:Beclin-1; D004305:Dose-Response Relationship, Drug; D006579:Heterozygote; D006706:Homeostasis; D007790:Lactosylceramides / Q000378:metabolism; D007976:Leupeptins / Q000737:chemistry*; D008099:Liver / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000378:metabolism; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D051017:Apoptosis Regulatory Proteins; D000071186:Beclin-1; C115010:Becn1 protein, mouse; D007790:Lactosylceramides; D007976:Leupeptins; C025575:lactotriaosylceramide; C032854:leupeptin