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Quantitation of Na+/K+-ATpase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling

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Abstract

We have quantitated and studied the topology of isoforms of the Na+/K+-ATPase and of the glucose transporter in rat adipocyte plasma membranes.

Adipocytes were incubated with or without insulin for 15 min. Sheets of native plasma membrane, with the cytoplasmic face exposed, were prepared by adsorption to EM grids. Grids were incubated in parallel with monoclonal antibodies against the Na+/ K+-ATPase isoforms α1 and α2, and the glucose transporter isoforms GLUT1 and GLUT4, followed by immunogold labeling, negative staining and quantitation by counting of the gold particles in electron micrographs. In addition, the distribution of glucose transporters and Na+/K+-ATPase isoforms in subcellular membrane fractions prepared by an established fractionation procedure was monitored by Western blotting.

We found that the Na+/K+-ATPases and the glucose transporters were confined to the planar part of the plasma membrane, without association to caveolar invaginations.

The vast majority of the Na+/K+-ATPase molecules in the adipocyte plasma membrane were of the α2 isoform; GLUT4 was the dominating glucose transporter isoform.

The total number of Na+/K+-ATPase molecules labeled in the plasma membrane was 3.5×105 per cell, independent of insulin stimulation. Concomitantly, insulin increased GLUT4 labeling sevenfold to a value of 3.5×105 per cell.

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Authors and Affiliations

  1. Department of Medical Physiology, The Panum Institute, University of Copenhagen, Denmark

    Marianne Voldstedlund & Jørgen Vinten

  2. Department of Medical Anatomy, The Panum Institute, University of Copenhagen, Denmark

    Marianne Voldstedlund & Jørgen Tranum-Jensen

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  1. Marianne Voldstedlund
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  2. Jørgen Tranum-Jensen
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The authors wish to thank Ulla Blankensteiner, Jonna Harpøth and Lisette Hansen for their skillful technical assistance and Birgit Risto for excellent work with the photographic prints. The α1 and α2 antibodies were kindly donated by K.W. Sweadner, Boston, and the F18 and F27 antibodies were granted by Peer N. Jørgensen, Novo-Nordisk, Bagsværd, Denmark. This work was supported by The NOVO Foundation, The Nordisk Insulin Foundation, The Danish Diabetes Foundation, The P. Carl Petersen Foundation and the Foundation of 17-12-81.

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Voldstedlund, M., Tranum-Jensen, J. & Vinten, J. Quantitation of Na+/K+-ATpase and glucose transporter isoforms in rat adipocyte plasma membrane by immunogold labeling. J. Membarin Biol. 136, 63–73 (1993). https://doi.org/10.1007/BF00241490

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