Transepithelial glucose transport in cell culture

Abstract

Pig kidney cell line LLC-PK1 cultured on a collagen-coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by circumferential occluding junctions. In the presence of 5.5 mM D-glucose, a potential difference (PD) of 2.8 mV, apical bath negative, short-circuit current Isc of 13.2 .mu.A .cntdot. cm-2 and transepithelial resistance of 211 .OMEGA. .cntdot. cm2 were recorded. Isc and PD were reduced by phlorizin added to the apical bath but were unaffected when phlorizin was placed in the basolateral bath. Ouabain or the replacement of Na by tris-(hydroxymethyl)aminomethane or choline abolished the Isc. The sugar concentrations required to produce the half-maximal Isc were 0.13 mM .beta.-methyl-D-glucoside, 0.28 mM D-glucose, 0.65 mM .alpha.-methyl-D-glucoside, 0.77 mM 6-deoxy-D-glucose, 4.8 mM D-galactose and 29 mM 3-O-methylglucose. When [Na] was reduced, the D-glucose required for half-maximal SCC [short-circuit current] increased. Isotopically 3H- and 14C-labeled D-glucose were used to determine simultaneous bidirectional fluxes; a resultant net apical-to-basolateral flux was present and could be abolished by phlorizin. The transported isotope cochromatographed with labeled D-glucose, indicating negligible metabolism. The cell culture model provides advantages for investigation of mechanisms of transepithelial glucose transport.