Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography.

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Abstract

Quantitative electron microscopic autoradiographic studies in cultured human lymphocytes and isolated rat hepatocytes have demonstrated that labeled insulin initially localizes to the plasma membrane and is subsequently internalized to a limited region of the peripheral cytoplasm. When 0.5 nm 125I-insulin is incubated with isolated rat hepatocytes, binding to the plasma membrane occurs at both 20 degrees C and 37 degrees C. Under steady-state binding conditions approximately equal to 30--40% of the labeled hormone is internalized to a distance of approximately equal to 15% of the radius of the cell. When the localization of the internalized labeled material is analyzed, by 2--5 min of incubation at 37 degrees C there is a fivefold preferential association of autoradiographic grains with lysosomal structures, and by 30--60 min of incubation at 37 degrees C there is a 10-fold preferential association. When the cell-associated radioactivity is extracted and filtered on Sephadex G-50 at each time point of incubation, radioactivity elutes predominantly in the position of 125I-insulin and is predominantly in the position of 125I-insulin and is predominantly trichloracetic acid precipitable, bindable to talc, and rebindable to liver membranes. With increasing time of association at 37 degrees C the initial rate and absolute amount of labeled material dissociable from the cell is reduced. With increasing time of dissociation both the cell-associated radioactivity and the radioactivity released into the incubation medium is progressively degraded. These data demonstrate that in isolated rat hepatocytes labeled insulin initially localizes to the plasma membrane, is progressively internalized, and associates preferentially with lysosomal structures. These events may provide a mechanism that links cell surface binding to the degradation of insulin and to insulin-induced loss of its specific receptor.

Authors

J L Carpentier, P Gorden, P Freychet, A Le Cam, L Orci