J. Biol. Chem., Vol. 263, Issue 25, 12171-12174, 09, 1988

Labeling of glucose transporters at the cell surface in 3T3-L1 adipocytes. Evidence for both translocation and a second mechanism in the insulin stimulation of transport

DM Calderhead and GE Lienhard
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.

In order to determine whether translocation of intracellular glucose transporters to the plasma membrane upon insulin stimulation accounts entirely for the increase in glucose transport, a method was developed to measure the relative amount of transporter at the cell surface. Glycoproteins at the cell surface, including the glucose transporter, were labeled by oxidation with galactose oxidase and then reduction by borotritide at 4 degrees C; subsequently the glucose transporter was isolated by immunoprecipitation and sodium dodecyl sulfate gel electrophoresis. Insulin stimulation of 3T3-L1 adipocytes resulted in a 2.6-fold increase in transporter at the cell surface. Under the same conditions, the rate of 2-deoxyglucose uptake was increased 11-fold. These results both provide further evidence that translocation of the glucose transporter occurs in response to insulin and indicate that it does not account for the full stimulation. Most likely, insulin also increases the intrinsic activity of the transporter.
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