Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.

TLDR

Immunolocalization of GLUT4 in rat brown adipose tissue was performed by incubating cryosections with specific antibodies, labeling with Protein A/gold, and examining the distribution by electron microscopy, including markers for early and late endosomes. Insulin stimulation shifts ~40 % of GLUT4 to the cell surface and enriches it in early endosomes, indicating that translocation to the membrane, recycling via coated pits, and exocytosis from tubulo‑vesicular structures mediate insulin‑induced glucose transport.

Abstract

Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.