Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes

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Abstract
In isolated rat, hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestiration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (> 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.