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The mitochondrial permeability transition initiates autophagy in rat hepatocytes¹

Department of Cell and Developmental Biology and Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599, USA

³Correspondence: Department of Cell Biology and Anatomy, School of Medicine, University of North Carolina at Chapel Hill, CB#7090, 236 Taylor Hall, Chapel Hill, NC 27599-7090, USA. E-mail: lemaster{at}med.unc.edu

SPECIFIC AIMS

We set out to determine the relationship between mitochondrial depolarization, induction of the mitochondrial permeability transition, and mitochondrial autophagy in rat hepatocytes stimulated by glucagon and nutrient deprivation.

PRINICIPAL FINDINGS

1. Autophagic stimulation induces mitochondrial depolarization
The fluorophores MitoTracker Green (MTG) and tetramethylrhodamine ethylester (TMRM) both accumulate into polarized mitochondria, but only TMRM is released after depolarization. Since TMRM quenches MTG fluorescence by fluorescence resonance energy transfer (FRET), release of TMRM from mitochondria after depolarization leads to unquenching of green MTG fluorescence. Thus, MTG fluorescence uniquely identifies newly depolarized mitochondria. Using this technique, we showed that autophagic stimulation of rat hepatocytes with glucagon and nutrient deprivation caused a fivefold increase in depolarized mitochondria (see Figs. 1 and 2 ).

View larger version (58K): [in this window] [in a new window]   Figure 1. Entry of spontaneously depolarizing mitochondria into autophagosomes after autophagic stimulation. Cultured hepatocytes were labeled with MTG and TMRM in complete growth medium. After autophagic stimulation, images of red and green fluorescence excited with blue light were collected. Note the green punctate structures that do not fluoresce red (arrows, upper panels): these are depolarized mitochondria. Subsequently, LTR was added, and green and red fluorescence was imaged again. After LTR labeling, note that some green fluorescing, depolarized mitochondria colocalized with LTR-labeled lysosomal autophagosomes (lower panels, arrows). Others did not (arrowheads). Bar is 10 µm.

View larger version (84K): [in this window] [in a new window]   Figure 2. Induction of mitochondrial depolarization by nutrient deprivation plus glucagon and its inhibition by cyclosporin A. Cultured hepatocytes were loaded with MTG and TMRM. Shown are overlay confocal images of green and red fluorescence before and 60 min after changing the medium to either fresh growth medium (Serum), nutrient-free buffer plus glucagon (KRH+G), or buffer plus glucagon and cyclosporin A (KRH+G+CsA). Note that the number of green fluorescing, depolarized mitochondria increased when the medium was changed to buffer plus glucagon, but not when changed to fresh growth medium or if cyclosporin A was present.

2. After depolarization, mitochondria enter a lysosomal/autophagosomal compartment
By loading hepatocytes with LysoTracker Red (LTR) after autophagic stimulation, we showed that depolarized mitochondria moved in acidic lysosomal/autophagosomal vacuoles (Fig. 1 ). Simultaneously, the number of these acidic lysosomes/autophagosomes increased by fivefold.

3. Cyclosporin A, a blocker of the mitochondrial permeability transition, inhibited mitochondrial depolarization and autophagosomal proliferation
Opening of high-conductance permeability transition pores in the mitochondrial inner membrane causes the mitochondrial permeability transition, which leads to mitochondrial depolarization and uncoupling of oxidative phosphorylation. The immunosuppressant cyclosporin A is a specific blocker of mitochondrial permeability transition; it inhibited the proliferation of depolarized mitochondria and autophagosomes after autophagic stimulation (Fig. 2 ). Tacrolimus, an immunosuppressant and calcineurin inhibitor that does not block the mitochondrial permeability transition, had no effect on autophagosomal proliferation after autophagic stimulation.

CONCLUSION

These findings support the conclusion that onset of the mitochondrial permeability transition is responsible for mitochondrial depolarization after autophagic stimulation and the subsequent movement of these depolarized mitochondria into autophagic vacuoles (Fig. 3) . Thus, the mitochondrial permeability transition appears to be a key event in the signaling pathway for mitochondrial autophagy in hepatocytes treated with glucagon and nutrient deprivation. The findings also strengthen the link between autophagy and apoptosis, since the mitochondrial permeability transition also promotes apoptosis.

View larger version (58K): [in this window] [in a new window]   Figure 3. Schematic diagram of mitochondrial autophagy. After autophagic stimulation, onset of the mitochondrial permeability transition (MPT) occurs in individual mitochondria with consequent inner membrane permeabilization and depolarization. Depolarized mitochondria are then selected for autophagic sequestration and autophagosomes form. Primary lysosomes fuse with autophagosomes to create autolysosomes, where mitochondrial digestion and degradation occur. By blocking the MPT, cyclosporin A (CsA) prevents mitochondrial depolarization and subsequent autophagic processing.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0206fje; to cite this article, use FASEB J. (August 17, 2001) 10.1096/fj.01-0206fje

² Present address: Laboratory for Physiology, Vrije University, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands.

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