D-Glucose Transport into Suspended Human Fibroblasts. Rapid Measurement of Uptake by Silicone Oil Filtration Centrifugation, and Comparison of Different Cell Detachment Procedures

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Abstract
The uptake of 14C-labeled D-glucose into the cellular space of human diploid fibroblasts (Flow 2000), grown to confluency and detached with trypsin-EDTA, was studied using silicone-oil-layer-filtering centrifugation. This method is rapid enough to enable the determination of initial transport rates, which are not complicated by subsequent metabolism of the hexose taken up into the cells. D-Glucose uptake shows saturation kinetics with a Km of 1.8 mM and maximal transport capacity of 4-8 nmol/(106 cells .times. min) at 20.degree. C. This saturable transport system is responsible for at least 80% of the total glucose taken up into the cells in the concentration range tested (0.1-10 mM D-glucose in incubation medium). The glucose carrier is stereospecific, is independent of Na and K ions, and is inhibited by cytochalasin B. Its temperature dependence reveals an activation energy of 31 kJ/mol (7.5 kcal/mol; Q10 .apprxeq. 1.5). As detachment of the cells from the culture flasks is necessary for applying silicone-layer-filtering centrifugation, various detachment procedures were tested. In the enzymatic procedure cells were treated with either trypsin or pronase. In the chelating method, Ca2+ and Mg2+ were chelated by EDTA and K+ with sodium tetraphenylborate. For mechanical detachment, cells were grown initially on plastic foil. After each of these detachment procedures the transport of D-glucose was the same. This method of rapid measurement of D-glucose uptake in suspended human fibroblasts may serve as an alternative to the uptake measurement with glucose analogs in attached cells when studying the hexose transport system in human diploid fibroblasts.