Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4.

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Abstract
Polyclonal antibodies to the amino- or carboxyl-terminated peptide sequences of the GLUT4 transporter protein were used in immunoelectron microscopic studies to demonstrate the location and insulin-induced translocation of GLUT4 in intact isolated rat adipocytes. Labeling of untreated adipocytes with the amino-terminal antibody revealed 95% of GLUT4 was intracellular, associated with plasma membrane invaginations or vesicles contiguous with or within 75 nm of the cell membrane. Insulin treatment increased plasma membrane labeling approximately 13-fold, to 52% of the total transporters, and decreased intracellular labeling proportionately. In contrast, labeling of untreated adipocytes with the carboxyl-terminal antibody or with a monoclonal antibody (1F8) that binds to the carboxyl terminus of GLUT4 detected fewer transporters, only approximately 40% of which were intracellular. In insulin-treated cells, plasma membrane labeling increased approximately 20-fold, but the total number of labeled transporters also increased approximately 13-fold. The number of intracellular transporters was not changed. The insulin-induced increase in plasma membrane labeling was reversible. Thus, the vast majority of GLUT4 transporters in untreated adipocytes are intracellular in invaginations or vesicles attached or close to the plasma membrane. Insulin treatment causes translocation of transporters to the plasma membrane, which involves flow of transporters from invaginations to the cell surface and possible fusion of subplasma membrane vesicles with the plasma membrane. Differences in the labeling of intracellular transporters by peptide antibodies suggested the carboxyl-terminal epitope of intracellular transporters was masked. The unmasking of the carboxyl terminus during translocation to the plasma membrane may be part of the mechanism by which insulin stimulates glucose transport in rat adipocytes.