Radiolabelled sugars as probes of hepatocytic autophagy

The sugars [14C]sucrose, [14C]lactose and [3H]raffinose have been loaded into isolated rat hepatocytes by means of electropermeabilisation, and used as probes of autophagic-lysosomal function. All three sugars are sequestered autophagically; in addition sucrose and lactose are taken up by mitochondria, and lactose is hydrolysed intralysosomally. Autophagically sequestered radioactivity accumulates in relatively large vacuoles (not in microsomes). These vacuoles initially have a density which, on average, is lower than that of the lysosomes. With time, the lysosomes become lighter and coincident with the radioactive vacuoles in density gradients, a phenomenon that is partially prevented by the autophagy inhibitor 3-methyladenine (3MA). This may indicate that resting, dense lysosomes become active (light) by engaging in autophagy as well as in other processes (endocytosis?). To assess the extent of convergence of the autophagic and endocytic pathways, the effect of endocytosed invertase on autophagically sequestered sucrose has been investigated. Practically all of the sucrose is eventually degraded the enzyme, indicating a complete intermixing of autophagic and endocytic pathways at the level of the lysosome. The density of lysosomes can be increased by treatment of cells with leupeptin. Most of the vaculoes containing sequestered radioactivity also increase their density under these conditions, even in the presence of vinblastine, an inhibitor of autophagosome-lysosome fusion. This indicates that a substantial fraction of the autophagosomes may be physically associated with lysosomes prior to fusion, a hypothesis supported by electron microscopic observations. Both autophagic sequestration (of raffinose) and autophagic-lysosomal degradation (of lactose, measured by an HPLC method) is completely inhibited by 3MA, whereas endocytic-lysosomal degradation (of asialofetuin) is unaffected. Since endogenous protein degradation is only partially (70%) suppressed by 3MA, it is clear that the remaining degradation must be non-autophagic (non-sequestrational), and most likely non-lysosomal.