LEUPEPTIN CAUSES AN ACCUMULATION OF LIPOFUSCIN-LIKE SUBSTANCES IN LIVER-CELLS OF YOUNG-RATS

Leupeptin, a thiol protease inhibitor, has previously been shown to cause a dense accumulation of substances resembling age pigment and called ceroid-lipofuscin, in brain cells of young rats [1]. Thus far, however, attempts to produce age pigments in hepatocytes of normal young rats with protease inhibitor(s) have not been successful. The present study provides the first demonstration that leupeptin induces lipofuscin-like substances in normal young rat hepatocytes. Male Fischer-344 rats (age 4-6 weeks) were continuously infused with leupeptin or saline i.p. for 2 weeks by an osmotic minipump (dosage, 1-50 mg/100 g per day). Liver tissues were then examined by light, fluorescence and electron microscopy. Both hepatocytes and non-parenchymal cells of livers treated with leupeptin, but not saline, showed a dense accumulation of pigments which stained deeply with toluidine blue, were PAS-positive and were brightly autofluorescent. After UV excitation the pigments had an emission spectrum with a broad peak at 480-540 nm extending to 650 nm resembling the spectrum of age pigment from livers of normal aged rats. Electron microscopic examination revealed numerous lipofuscin-like deposits with heterogeneous morphology in the cytoplasm of both hepatocytes and non-parenchymal cells; lipid and myelin-like bodies were also present in hepatocytes. The results indicate that the perturbation of proteolytic activity in liver by leupeptin causes an accumulation of substances which by several criteria resemble lipofuscin. These results thus provide further support for the 'Protease Inhibitor Model of Lipofuscin Formation' [2] as well as a potential experimental model for studying hepatocellular aging processes.