Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes.
In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serumcontaining Waymouth's medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1alpha mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA.
Sara
S
Rodriguez-Enriquez
Yoichiro
Y
Kai
Eduardo
E
Maldonado
Robert T
RT
Currin
John J
JJ
Lemasters
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000588:Amines / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation / Q000187:drug effects; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D045304:Cytochromes c / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000187:drug effects / Q000648:ultrastructure; D008297:Male; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010539:Permeability / Q000187:drug effects; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D000588:Amines; D004272:DNA, Mitochondrial; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; C501429:Ppargc1a protein, rat; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C493330:Red DND-99; D014157:Transcription Factors; C025946:3-methyladenine; D016572:Cyclosporine; D045304:Cytochromes c; C090438:(melle-4)cyclosporin; D003576:Electron Transport Complex IV; D000225:Adenine