Formation of autophagosomes during degradation of excess peroxisomes induced by administration of dioctyl phthalate.

The formation of autophagosomes in rat hepatocytes was investigated during degradation of excess peroxisomes. Rat liver peroxisomes were markedly proliferated by administration of dioctyl phthalate (DEHP) for 2 weeks. When the animals were fed on normal diet for a week further, the number and size of the peroxisomes recovered to normal. The recovery process was confirmed by the assay and immunoblot analysis of acyl-CoA oxidase and catalase. During the recovery process, only a few autophagosomes were noted. However, when leupeptin (2 mg/100 g body weight) was injected into these animals, there was a marked accumulation of autophagosomes in the hepatocytes. Using this as an experimental model, the early stage of the autophagosome formation was analyzed by electron microscopy. Twenty minutes after the injection, isolation membranes surrounding the target organelles appeared. They were characterized by double layers with a narrow cisternal space and were sometimes continuous with the rough endoplasmic reticulum. Between the inner membrane of the isolation membranes and the enclosed organelles, electron-dense bridges were noted. Forty minutes after leupeptin injection, the lumen of the isolation membranes were enlarged and the inner membrane attached to the entrapped material. Enzyme cytochemical staining showed that the isolation membranes were negative for acid phosphatase and thiamine pyrophosphatase, but were strongly positive for glucose-6-phosphatase (G6Pase). The enlarged cisternae of the isolation membranes of the early autophagic vacuoles were in part positive for this enzyme, but gradually became negative with time. Similarly, the G6Pase activity was lost when the inner membrane was degraded. The results suggest 1) that the process of degradation of excess peroxisomes is rapid and carried out by the autophagic system in hepatocytes and 2) that the isolation membranes enclosing the target organelles are derived from the endoplasmic reticulum.