Experimental characterization of the autophagic-lysosomal pathway in isolated rat hepatocytes

Abstract

When isolated rat hepatocytes are incubated under amino-acid-free conditions autophagy is maximally activated and overall lysosomal proteolysis is greatly enhanced. To facilitate this, cytoplasmic material is sequestered by autophagic membranes, and the resulting autophagosomes enter the lysosomes by a fusion process to have their contents degraded. The various steps in the autophagic-lysosomal pathway have been investigated by utilizing reversible electropermeabilization to introduce radioactive sugar probes into the hepatocyte cytosol. The inert disaccharide sucrose and the inert trisaccharide raffinose have been used as sequestration probes to study the sequestrational step of autophagy, while the hydrolysable disaccharide lactose has been used to study the fusion and intralysosomal hydrolysis steps. Sucrose and lactose have been used to map the extent of convergence between the autophagic and endocytic (heterophagic) pathways. Sequestration of native cytosolic enzymes has been measured to investigate the question of selectivity in the autophagic process. Finally, the effect of insulin on the sequestrational step has been evaluated.