Although the effect of insulin-like growth factors (IGFs) in fetal adrenocortical cells has been investigated extensively, the role of the IGF system in the adult human adrenal gland remains unclear. In the present study we investigated the effect of recombinant human IGF-I and IGF-II on cortisol, dehydroepiandrosterone sulfate (DHEA-S) and cAMP synthesis in adult human adrenocortical cells in primary culture. Both IGFs stimulate basal as well as adrenocorticotropin (ACTH)-induced steroid secretion in a time- and dose-dependent fashion. While both IGFs (6.5 nM) induced only a moderate 2-fold increase in basal cortisol output after 48 h, the effect on basal DHEA-S secretion was significantly stronger, with a 2.7- and 3.7-fold stimulation by IGF-I and IGF-II respectively. Similarly, IGF-II enhanced ACTH-induced cortisol and DHEA-S secretion more potently than IGF-I. In dose-response experiments, the maximum stimulation of ACTH-induced DHEA-S secretion was induced by 1.6 nM IGF-I (2-fold increase) or IGF-II (2.9-fold increase), while the maximum response of cortisol secretion was elicited only at 13 nM IGF-I (2-fold increase) or IGF-II (2.5-fold increase). This resulted in a significant shift of the DHEA-S dose-response curves to the left, indicating a relative selective stimulation of androgen biosynthesis by physiologically low concentrations (0.4-3.2 nM) of IGF-II, and less potently by IGF-I. At all doses tested, the steroidogenic effect of IGF-II was significantly stronger than the effect of IGF-I. Although both IGF receptors are present in adult human adrenocortical cells, the steroidogenic effect of IGF-II is mediated through the IGF-I receptor, since [Arg54,55]IGF-II, which only binds to the IGF-I receptor, was equipotent with native IGF-II, whereas [Leu27]IGF-II, which preferentially binds to the type II IGF receptor, did not show any effect. In addition, [des1-3]IGF-I, which exhibits only minimal binding to IGFBPs, was significantly more potent than native IGF-I in stimulating adrenal steroid biosynthesis, and elicited almost the same maximum stimulatory effect as IGF-II and [des1-6]IGF-II. By Western ligand blotting of conditioned medium it was shown that adult human adrenocortical cells secrete various IGF-binding proteins (IGFBPs), which are induced differentially by treatment with ACTH. In conclusion, these results demonstrate that: (1)IGF-II stimulates basal as well as ACTH-induced DHEA-S and cortisol secretion from adult human adrenocortical cells more potently than IGF-I; (2) both IGFs predominantly stimulate androgen biosynthesis; (3) the steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor; (4) the different steroidogenic potency of IGF-I and IGF-II might be explained by interaction of these ligands with locally produced IGFBPs. These data indicate that the IGF system plays an important role in the regulation of the differentiated function of adult human adrenocortical cells.

In previous studies we have shown that IGF-II stimulates basal as well as ACTH-induced cortisol secretion from adult human adrenocortical cells more potently than IGF-I, and that both IGFs predominantly stimulate androgen biosynthesis. The steroidogenic effect of IGF-I and IGF-II is mediated through interaction with the IGF-I receptor, and modified by locally produced IGF-binding proteins (IGFBPs). In the present study, we identified and characterized IGFBP synthesis in normal adult human adrenocortical cells in primary culture, and investigated the effect of ACTH and recombinant human IGF-I and -II on the regulation of IGFBP expression and secretion. Using RT-PCR, we identified the mRNA of all six high-affinity IGFBPs, in both adrenocortical tissue and monolayer cell cultures of adrenocortical cells. Using Western ligand and immunoblotting and two-dimensional Western ligand blotting we confirmed the secretion of IGFBP-1, -2, -3, -4 and -5 by adrenocortical cells in primary culture. The quantification of IGFBPs indicated that IGFBP-3 accounts for almost half the binding activity in conditioned medium of unstimulated cells (47%), followed by IGFBP-4 (20%), IGFBP-5 (15%), IGFBP-2 (12%) and IGFBP-1 (6%). After treatment with ACTH, the abundance of IGFBP-1 was upregulated significantly 2.6-fold, while IGFBP-3 was induced only slightly (1.3-fold). IGFBP-2, -4 and -5 remained unchanged. In contrast, IGF-I and -II (6.5 nM) predominantly induced the abundance of IGFBP-5 (2- and 1.6-fold respectively) and IGFBP-3 (2- and 1.7-fold respectively), while IGFBP-1, -2 and -4 were unaltered. The induction of IGFBP-1 and -5 by ACTH and IGFs, respectively, was paralleled by an increase in the amount of IGFBP-1 and -5 mRNA in these cells. In conclusion, all six high-affinity IGFBPs are expressed in the adult human adrenal gland, and the presence of at least five high-affinity IGFBPs has been demonstrated in conditioned medium of adult human adrenocortical cells. Furthermore, the expression and secretion of IGFBP-1 is upregulated by ACTH, whereas IGFBP-5 is induced by IGF-I and -II. Together with earlier findings, these results suggest that IGFBPs play an important modulatory role in the regulation of the differentiated adrenocortical function.