EFFECTS OF INSULIN-LIKE GROWTH-FACTOR-I AND GROWTH-FACTOR-II AND INSULIN ON THE IMMORTALIZED HYPOTHALAMIC GTI-7 CELL-LINE

Insulin and insulin-like growth factor I (IGF-I) participate in energy metabolism, regulate cellular growth and differentiation, and are tho ught to act locally in a paracrine manner through specific receptors. Systemic levels of these peptides in humans and primates are directly associated with levels of activity of the reproductive axis. To date, it is unclear whether these peptides participate in reproductive funct ion by acting at the level of the GnRH neuron. In this study we examin ed the effects of IGF-I, IGF-II and insulin on immortalized GnRH-secre ting neurons, the GTI-7 cell line. The GTI-7 cells expressed all three members of the insulin receptor family as determined by analysis of I -125-IGF-I, I-125-IGF-II and I-125-insulin binding sites. Insulin rece ptors bound insulin, IGF-II and IGF-I with a ratio of potency of 1:5:2 0. IGF-I and IGF-II receptors bound both IGF-I and IGF-II. The ratio o f potency of IGF-I/IGF-II was 1:5 for the IGF-I receptor and 100:1 for the IGF-II receptor. The binding characteristics of the growth factor s at 22 degrees C suggested the possibility that these cells may secre te IGF binding proteins. To ensure that changes in GnRH levels in the media were due to secretion and not to changes in cell number, the mit ogenic effect of these peptides on GTI cells was evaluated. Both insul in and IGF-I were strong mitogens (48-hour incubation), restoring cell number to that of serum-replete cultures at a dose of 0.1 ng/ml. A 10 0-fold higher dose of IGF-II was required to produce a similar level o f mitogenicity, implicating an action through the IGF-I and/or insulin receptor. Due to these mitogenic effects, the effect of insulin, IGF- I and IGF-II on GnRH secretion was studied after shortterm exposure. I nsulin and IGF-I did not affect GnRH secretion, but IGF-II had a bipha sic effect on GnRH release after 2 h of incubation (a maximum stimulat ory effect occurred with a 0.1 ng/ml dose). In order to examine the si gnal transduction mechanism, the role of cytoplasmic calcium mobilizat ion in IGF-II-induced GnRH secretion was examined in single cells usin g calcium imaging. The effect of IGF-II on GnRH secretion appeared to operate via a calcium-independent mechanism. The studies document an i nsulin/IGF system in the GTI-7 neuronal cell line and show that insuli n and IGFs can exert direct effects on the immortalized GnRH neurons. The ability of these agents to stimulate mitogenesis and/or secretion suggests that these peptides may play some role in the early developme nt or maturation of GnRH neurons in vivo.