In Vitro Effect of Insulin and Insulin-Like Growth Factor-I on Cell Multiplication and Adrenocorticotropin Responsiveness of Fetal Adrenal Cells1

The present study examined the effects of both insulin and insulin-like growth factor-I (JGF-I) on cell division and specific functions of cultured adrenocortical cells from 100- to 122-day-old ovine fetuses. When culture was performed in a serum-free medium containing transferrin and ascorbic acid, the number of cells increased only slightly (1.2-fold) over a 4-day period. Addition of insulin or IGF-I in the culture medium enhanced the number of cells counted on Day 5. The effect of both peptides was dose-dependent, but 10 ng/ ml IGF-I was as potent as 10 μg/ml insulin. The acute cyclic adenosine 3′, 5′-monophosphate (cAMP) and steroidogenic responses to adrenocorticotropin (ACTH1–24) decreased in fetal cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1–24-induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells/or 4 days with increasing concentrations of insulin or IGF-I enhanced the acute cAMP and steroidogenic responses (3- to 4-fold) to ACTH1–24 over that of control cells. The ED50 of IGF-I was about 3 ng/ml (≃ 0.4 nM) whereas that of insulin was about 10 ng/ml (1.7 nM). However, a second plateau was apparent at concentrations of insulin above 1 μg/ml. The acute cholera toxin stimulation of cAMP production of cells cultured in the absence of insulin or ACTH. Insulin at micromolar concentrations not only prevented this decrease but enhanced the acute ACTH1–24 -induced cAMP output on Day 5 over that observed on Day 2. Treatment of fetal cells for 4 days with response to forskolin did not change during culture, irrespective of the culture conditions. The positive effect of both insulin and IGF-I on the acute ACTH1–24 stimulation of steroidogenesis was also observed when cells were stimulated by 8Br-cAMP. By contrast, cells cultured in the presence of 10 μg/mI insulin or 10 ng/ml IGF-I resulted in lower amounts of corticosteroids produced when cells were incubated with 22R-hydroxycholesterol or exogenous pregnenolone. The acute cAMP and steroidogenic responses to ACTH1–24 of cells cultured in the continuous presence of 10−11 M ACTH1–24 in insulin-free medium increased during the culture period. Insulin and IGF-I were able to enhance both the cAMP and steroidogenic response of these cells to ACTH1–24. The present data suggest that both insulin and IGF-I are involved in the maintenance and the maturation of ovine fetal adrenocortical cell differentiated functions, whereas, at physiological concentrations only, IGF-I has a growth-promoting effect on these cells.