Internalization and recycling of 125I-photoreactive insulin-receptor complexes in hepatocytes in primary culture

Abstract

When the insulin receptor is tagged with a 125I-photoreactive insulin analogue that can be covalently coupled to it by UV irradiation, the fate of this labeled receptor can be followed both morphologically and biochemically. In the present study we have applied this tool to trace the pathway followed by 125I-photoreactive insulin-receptor complex in hepatocytes in primary culture. As determined by quantitative electron microscopic autoradiography, the internalized labeled material first associates with clear vesicles, second is found in multivesicular bodies, third associates with dense bodies and fourth returns to the cell surface via clear vesicles. This recycling process is inhibited by lysosomotropic agents, i.e. NH 4Cl or chloroquine. These data confirm, in another cell system, our previous observations carried out in freshly isolated rat hepatocytes and demonstrate the feasibility and complementarity of both freshly isolated hepatocytes and hepatocytes in primary culture to study internalization and recycling of the insulin receptor.