Basolateral 3-O-methylglucose transport by cultured kidney (LLC-PK1) epithelial cells

Abstract

In previous work we demonstrated the similarity of basolateral sugar transport of LLC-PK1 renal epithelia to basolateral kidney sugar transport using 2-deoxy-D-glucose as a substrate. In this study we first examine a central limitation to use of 2-deoxyglucose for basolateral sugar transport study in LLC-PK1 epithelia, namely, a shift of the rate-limiting step in uptake from transport to phosphorylation. Use of 3-O-methylglucose avoids this complication because it is not phosphorylated. However, use of 3-O-methylglucose requires much shorter incubation periods to examine linear rates of uptake (steady state is reached by 60 s at 22 degrees C for 0.1 mM 3-O-methylglucose). As was true for 2-deoxyglucose, apical uptake of 3-O-methylglucose was only a fraction of total uptake. Basolateral uptake was characteristically more sensitive to phloretin and cytochalasin B inhibition, relative to phlorizin. Inhibition studies indicate a requirement for a free hydroxyl on C-1 carbon of the pyranose ring, as is characteristic for renal basolateral sugar transport. Kinetic analysis indicates a single transport system with a Km of 10.9 mM and Vmax of 17.2 pmol.micrograms DNA-1.15 s-1. Subconfluent, undifferentiated LLC-PK1 cells show a similar Km (12.7 mM) but a ninefold higher Vmax (166.2 pmol.micrograms DNA-1.15 s-1). Stimulation of 3-O-methylglucose transport rate in confluent cultures by phorbol ester is relatively small (less than 100%) compared with effects on other somatic cells. The uptake rate of 3-O-methylglucose is not affected by glucose starvation, but subsequent refeeding with glucose-containing medium does significantly stimulate uptake.