@article{d1f182da940249c9a550eb4d188772c1,
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.",
keywords = "Autophagy, Cycloheximide, Flux, GABARAP, GATE-16, In vivo, LC3, Leupeptin, Macroautophagy, Mice",
author = "Jeffrey Haspel and Shaik, {Rahamthulla S.} and Emeka Ifedigbo and Kiichi Nakahira and Tamas Dolinay and Englert, {Joshua A.} and Choi, {Augustine M.K.}",
note = "Funding Information: commercial kit (Sigma, CS0740) accord-were measured with a Nanodrop spectro- ing to the manufacturer{\textquoteright}s instructions. photometer (Nanodrop Technologies). We thank Beth Levine for the generous gift EM morphometric analysis. RNA quality was assessed in each sample of beclin 1 heterozygous mice and Tamotsu Approximately 2 mm thick liver fragments using the RNA 6000 Nano chip Kit Yoshimori for his GST-LC3b expression were cut from the tip of the left lobe and (Agilent Technologies, 5065-4776) on construct. We also thank Louise Trakimas then fixed by immersion in 2.5% gluter-the Agilent 2100 Bioanalyzer (Agilent for her technical assistance with electron aldehyde overnight. This was followed by Technologies). The RNA Integrity microscopy and Stefan Ryter and Robyn osmication in 1% osmium tetroxide/1.5% Number (RIN) of the samples analyzed Haspel for their critique of this manu-potassium ferrocyanide and staining with was 9.2 ± 0.6 (mean ± SD). script. This work was funded by NIH T32 1% uranyl acetate. The samples were Reverse transcription was carried HL007633, NIH R03HL097005 and a then dehydrated and embedded in Taab out using the Applied Biosystems High FAMRI Clinical Innovator Award. 812 Resin (Marivac Ltd., Nova Scotia, Capacity cDNA Reverse Transcription Canada). 95 nm sections were cut with Kit (Applied Biosystems, 4368813). One the Leica ultracut microtome, picked up microgram total RNA from each sample Supplemental materials can be found at: on 100 μm formvar coated Cu grids, was reverse transcribed into cDNA using www.landesbioscience.com/journals/ stained with 0.2% lead citrate and imaged random hexamer primers in a final vol-autophagy/article/15100 under the Philips Technai BioTwin Spirit ume of 20 μl as per the manufacturer{\textquoteright}s Electron Microscope. For each tissue protocol. References section, 20 random pictures of peripor-Real-time PCR was performed on the ed pathway of cellular degradation. Science 2000; Klionsky DJ, Emr SD. Autophagy as a regulat- tal hepatocytes were digitally captured ABI PRISM 7300 Sequence Detection 290:1717-21. at 1,900x (which allowed capture of an System (Applied Biosystems) using 2. Ericsson JL. Studies on induced cellular autophagy. entire hepatocyte in the photographic TaqMan Gene Expression Master Mix lysosomes. Exp Cell Res 1969; 55:95-106.I. 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year = "2011",
month = jun,
doi = "10.4161/auto.7.6.15100",
language = "English",
volume = "7",
pages = "629--642",
journal = "Autophagy",
issn = "1554-8627",
number = "6",
}