Effect of Insulin and Dinoprostone on D-Glucose and 2-Deoxy-D-Glucose Uptake by Normal and Diabetic Rat Hepatocytes

Abstract

□ Suspensions of deaggregated hepatocytes were prepared by a collagenase perfusion technique from livers of both normal rats and rats rendered diabetic by streptozocin treatment. Uptake of D-glucose and 2-deoxy-D-glucose was estimated by adding 14C- or 3H-labeled hexose to a stirring suspension of cells in Krebs-Henseleit buffer at 37°, separating the cells by rapid centrifugation, and measuring radioactivity in the packed cell pellet. Uptake, calculated after correction for entrapped extracellular fluid, includes any hexose bound to, transported into, or otherwise immobilized in the cells. High concentrations of glucose (5-20mM) establish an intracellular-extracellular distribution ratio near 1.0 within 1min, indicating a facilitated diffusion transport system. In contrast, a low level of glucose (71 nM) almost immediately {<15sec} establishes ratios of between 3 and 4, which suggests a significant amount of glucose binding to cell membrane. Such binding would not be detected at the high glucose levels because of its small magnitude. Hepatocytes from diabetic rats exhibit a decrease in this apparent binding to ~60% of normal; preincubation with 0.1IU/ml insulin increases this toward normal values, although it does not affect the binding by normal hepatocytes themselves. Preincubation with 0.1μM dinoprostone depresses glucose binding in cells from both normal and diabetic rats. A low concentration (1.2 nM) of 2-deoxy-D-glucose establishes even higher intracellular-extracellular distribution ratios of between 4 and 6, but the apparent binding of this sugar is identical in normal and diabetic rat hepatocytes and is not affected by preincubation with either insulin or dinoprostone alone. However, combined treatment with both agents causes a significant increase in 2-deoxyglucose binding. The results suggest that insulin promotes formation of hexose-binding sites and conversion of these to specific glucose-binding sites while dinoprostone may act by blocking the latter conversion.