Possible Dissociation Between Insulin Binding and Insulin Action in Isolated Fetal Rat Hepatocytes

Abstract

To directly examine the relationship between insulin receptors and insulin action in fetal tissue, we compared insulin receptor characteristics and insulin-mediated 14C-glucose incorporation into glycogen, as well as glycogen synthase activity, in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of hepatocytes was documented by trypan blue exclusion (> 90%), time-dependent 14C-leucine incorporation into protein, and dose-related incorporation of glucose into glycogen. Percent specific binding of 125I-insulin per unit protein was significantly higher in F than A liver plasma membranes (32.2 ± 0.3 versus 18 ± 2.4; P < 0.01) and Scatchard plots revealed twice the number of receptors in F. Similarly, receptor number per cell surface area was threefold higher in F than in A (150 versus 50 sites/μm2). At a fixed medium glucose concentration of 11.2 mM, insulin stimulated 14C-glucose incorporation into glycogen in a dose-related manner in A with an apparent Km of 1.0 ng/ml and Vmax at 5–10 ng/ml corresponding to 30–40% of total receptor occupancy; no effect was obtained in F with insulin up to 100 ng/ml. Net glucose incorporation into glycogen (nmol/106 cells/h) increased progressively with increasing medium glucose concentrations ranging from 1.4 to 27.8 mM; incorporation by F was significantly greater than by A at each glucose concentration. However, whereas insulin at 100 ng/ml significantly augmented net glucose incorporation at each glucose concentration in A, no effect of insulin was apparent in F. Finally, insulin at 10 and 100 ng/ml significantly increased the active (a form) and total (a + b form) glycogen synthase activity in A; the ratio of active to inactive forms (% I) also increased significantly in A. In contrast, there was no demonstrable augmentation by insulin of glycogen synthase activity in F hepatocytes. Thus, insulin-mediated incorporation of glucose into glycogen and insulin-stimulated glycogen synthase activity was not demonstrable in fetal rat hepatocytes despite the presence of an increased number of insulin receptors.