Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.
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 sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 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.
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D004072:Digitonin / Q000494:pharmacology*; D066298:In Vitro Techniques; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000378:metabolism*; D010587:Phagocytosis / Q000187:drug effects*; D051381:Rats; D011919:Rats, Inbred Strains; D013395:Sucrose / Q000378:metabolism*
D000596:Amino Acids; C025946:3-methyladenine; D013395:Sucrose; D000225:Adenine; D004072:Digitonin