DELAYED GLUCOCORTICOID-DEPENDENT DEVELOPMENT OF THE INSULIN-RESPONSE AFTER HEAT-SHOCK IN CULTURED FETAL HEPATOCYTES - CORRELATION WITH A TRANSIENT DEFECT IN GLUCOCORTICOID RECEPTOR-BINDING PROPERTY

The effects of a mild heat shock were investigated on glucocorticoid-i nduced maturation of the insulin glycogenic response and on glucocorti coid receptor (GR) in cultured 15-day-old fetal rat hepatocytes, In al l experiments, cell heating at 42.5 degrees C from 30 to 90 min was ap plied at Day 1 of culture before exposure to glucocorticoids, In never heated cells, the insulin stimulation of glycogenesis, measured by [C -14]glucose incorporation into glycogen for 3 h, developed to be maxim al after 32 h in the presence of 100 nM dexamethasone (2.4-fold), In c ells preheated at 42.5 degrees C for times equal to or greater than 60 min before being returned to 37 degrees C in dexamethasone-containing medium, the insulin response was not seen after 32 h (1.3-fold versus 2.4-fold) but was clearly expressed after 48 h, Heat treatment induce d a progressive decrease in intact cell GR binding, reaching 40% after 1 h. When cells were returned to 37 degrees C, GR binding following a lag time of 8 h increased up to complete restoration after 24 h. Such heat shock-induced variations affected the number of GR binding sites with little change in GR binding affinity, while no modifications wer e seen in the 97-kDa GR level from whole cell extracts as revealed by Western immunoblotting using an anti-GR antibody (BuGR2). [S-35]Methio nine metabolic labeling showed reversible heat-stimulated synthesis of 70- and 90-kDa heat shock proteins (Hsps). Variations in Hsp90 level revealed by Western immunoblotting using an anti-Hsp90 antibody (AC88) were inversely correlated with time with GR binding. Therefore, a mil d heat shock applied to cultured fetal hepatocytes led to a delayed de velopment of the glycogenic response to insulin linked to a defect in GR binding property with no alteration in the GR protein level. (C) 19 96 Academic Press, Inc.