Evidence That Downregulation of Hexose Transport Limits Intracellular Glucose in 3T3-L1 Fibroblasts

Measurements of initial glucose entry rate and intracellular glucose concentration incultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of [¹⁴C]methylglucose and D-[¹⁴C]glucose with 1) L-[³H]glucose as an extracellular marker together with the [¹⁴C]glucose or [¹⁴C]methylglucose in the substrate mixture, 2) sampling times as short as 2 s, 3) icecold phloretin-containing medium to stop uptake and rinse away the extracellular label, and 4) nonlinear regression of time courses. Methylglucose equilibrated in two phases—the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 μl/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol · s⁻¹ · ml⁻¹ cell water). Steadystate intracellular glucose concentration was 75–90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.