Roles of mitophagy and the mitochondrial permeability transition in remodeling of cultured rat hepatocytes

Abstract

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 serum-containing 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-1α 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.

Population-level expression variability of mitochondrial DNA-encoded genes in humans

“…These factors include the stage of the cell cycle, the energetic requirements of the cells, the environmental effects on the redox balance of the cells, the stage of differentiation, and/or cell signaling mechanisms. 54,55 An accurate determination of the number of mtDNA copies per cell is technically difficult to achieve because both the number of mitochondria per cell and the number of mtDNA copies per mitochondrion vary. 56,57 Our analysis could only focus on the variability of mtDNA gene expression at the level of per sample, ignoring the details of expression variability at the level of per mtDNA copy.…”

Roles of mitophagy in cellular physiology and development

“…However autophagy is the only known cellular mechanism that can actually mediate the degradation of entire organelles. Selective mitochondrial autophagy was first described by the Lemasters laboratory (who also coined the term Bmitophagy^; Rodriguez- Enriquez et al 2004Enriquez et al , 2006Enriquez et al , 2009. Later, it was confirmed that, in specific types of mammalian cells, depolarized mitochondria are selectively degraded by mitophagy (Narendra et al 2008;Twig et al 2008).…”

Chronic Expression of RCAN1-1L Protein Induces Mitochondrial Autophagy and Metabolic Shift from Oxidative Phosphorylation to Glycolysis in Neuronal Cells

“…autophagy and mitochondrial degradation (24,25), we tested this possibility and discovered that RCAN1-1L overexpression leads to induction of autophagy and significant mitochondrial loss.…”