Permissive Effect of Insulin on the Adenosine 3′,5′-Monophosphate-Dependent Up-Regulation of Low Density Lipoprotein Receptors and the Stimulation of Steroid Release in Bovine Adrenal Cortical Cells

Abstract

Bovine adrenal cortical cell growth on extracellular matrix-coated dishes in the presence of F-12 medium supplemented with high density lipoprotein (30 .mu.g protein/ml), insulin (50 ng/ml), transferrin (1 .mu.g/ml) and fibroblast growth factor (100 ng/ml) expressed high affinity low density lipoprotein (LDL) receptors (Kd = 2.0 .times. 10-8 M) present at a density of 3 .times. 104 receptors/cell. The density of LDL receptors per cell could be increased 3-fold (9 .times. 104 receptors/cell) by incubating the cells for 24 h with cholera toxin (CT; 10 ng/ml), but not with insulin (100 ng/ml). This correlated with increased LDL internalization (10-fold) and degradation (5-fold). 11-Deoxycortisol (11 DOC) release was increased by 2.7-fold. The addition of insulin (100 ng/ml) together with CT (10 ng/ml) markedly potentiated the effects of CT on LDL binding, internalization and degradation as well as on steroid release. Preincubation (24 h) of the cells with insulin (100 ng/ml) together with CT resulted in a 12.7-fold increase in high affinity LDL receptor density (3.6 .times. 108 receptors/cell), while LDL internalization and degradation were increased by 45- and 22-fold, respectively. This correlated with a 9.6-fold increase in the 11 DOC released into the medium. The effects of insulin on the induction of high affinity LDL receptors as well as increased internalization and degradation of [125I]LDL were both time and concentration dependent in cultures exposed to CT. Secretion of 11 DOC paralleled in a time-dependent manner the amount of internalized [125I]LDL. Exposure of the cells to chloroquine (100 .mu.M) resulted in a 95% inhibition of [125I]LDL degradation correlating with an 80% decrease in 11 DOC released in cells preexposed to CT and insuln. Apparently, in bovine adrenal cortex grown in chemically defined medium, insulin modulates steroidogenic cAMP-induced response by increasing both LDL receptor pathway activity and 11 DOC release.