The Uptake of 2-deoxy-D-glucose (2dGlc) by the Endogenous Sugar Transporter(s) of Spodoptera frugiperda Clone 21-AE Cells and the Inhibition of 2dGIc Transport in the Insect Cells by Fructose and Cytoc halasin B

다국어 초록 (Multilingual Abstract)

The baculovirus/Spodoptera frugiperda (Sf) cell system has become popular for the production of large amounts of the human erythrocyte glucose transporter, GLUT1, heterologously. However, it was not possible to show that the expressed transporter in insect cells could actually transport glucose. The possible reason for this was that the activity of the endogenous insect glucose transporter was extremely high and so rendered transport activity resulting from the expression of exogenous transporter very difficult to detect. Sf21-AE cells are commonly employed as the host permissive cell line to support the baculovirus AcNPV replication and protein synthesis. The cells grow well on TC-100 medium that contains 0.1 % D-glucose as the major carbon source, strongly suggesting the presence of endogenous glucose transporters. However, unlike the human glucose transporter, very little is known about properties of the endogenous sugar transporter(s) in insect cells. Thus, the uptake of 2-deoxy-D-glucose (2dGlc) by Sf21-AE cells and the inhibition of 2dGlc transport in the insect cells by fructose and cytochalasin B were investigated in the present work. The binding assay of cytochalasin B was also performed, which could be used as a functional assay for the endogenous glucose transporter(s) in the insect cells. Sf21-AE cells were infected with the recombinant virus AcNPV-GT or no virus, at a multiplicity of infection (MOI) of 5. Infected cells were resuspended in PBS plus and minus 300 mM fructose, and plus and minus 20 $\mu$M cytochalasin B for use in transport assays. Uptake was measured at 28$^{\circ}C$ for 1 min, with final concentration of 1 mM deoxy-D-glucose, 2-[1,2-$^3$H]- or glucose, L-[l,$^3$H]-, used at a specific radioactivity of 4 Ci/mol. The results obtained demonstrated that the sugar uptake in uninfected cells was stereospecific, and was strongly inhibited by fructose but only poorly inhibitable by cytochalasin B. It is therefore suggested that the Sf21-AE glucose transporter has very low affinity for cytochalasin B, a potent inhibitor of human erythrocyte glucose transporter.