INSULIN-RECEPTOR MEDIATES INHIBITORY EFFECT OF INSULIN, BUT NOT OF INSULIN-LIKE GROWTH-FACTOR (IGF)-I, ON IGF BINDING-PROTEIN-1 (IGFBP-1) PRODUCTION IN HUMAN GRANULOSA-CELLS

Insulin-like growth factor binding proteins (IGFBPs) may participate i n regulating ovarian function by modifying effects of insulin-like gro wth factors (IGFs) or by directly affecting ovarian steroidogenesis in both normal and pathological circumstances. The latter include hyperi nsulinemic insulin resistant states, such as polycystic ovary syndrome . We examined regulation of IGFBP-1 production in human granulosa cell s by insulin and IGF-I. The cells were obtained during in, vitro ferti lization, plated in McCoy-5A tissue culture medium supplemented with 1 0% fetal calf serum (10(5) cells/0.5 mL), and incubated at 37 C, 90% h umidity, 5% CO2 for 48 h. After additional 24 h incubation without fet al calf serum, 1, 10, or 100 ng/mL of insulin or IGF-I were added with or without 2 h preincubation with 10 mu g/mL monoclonal anti insulin receptor antibody IR-47-9. After 48 h incubation with insulin or IGF-I , the medium was collected and IGFBP-1 and progesterone concentrations were measured, using kits from Diagnostic Systems Laboratories, Webst er, TX. Progesterone concentration ranged between 50-100 ng/mL/10(5) c ells, without consistent stimulatory effect of either insulin or IGF-I . Control cells produced 7.0 +/- 1.7 ng/mL of IGFBP-1. Incubation with 1 or 10 ng/mL of insulin resulted in culture medium IGFBP-1 concentra tions of 7.1 +/- 1.3 ng/mL and 5.4 +/- 0.7 ng/mL, respectively (P = NS ). Incubation with 100 ng/mL of insulin reduced IGFBP-1 culture medium concentration to 1.6 +/- 0.3 ng/mL, (P < 0.01, compared with controls ). 1, 10, and 100 ng/mL of IGF-I inhibited IGFBP-1 concentrations in t he conditioned culture medium to 1.3 +/- 0.3 ng/mL, 0.4 +/- 0.1 ng/mL and 0.3 +/- 0.1 ng/mL, respectively (P < 0.01, compared with controls) . Preincubation with antiinsulin receptor antibody IR-47-9 alleviated inhibitory effect of insulin, but not of IGF-I on IGFBP-1 production. After preincubation with IR-47-9, IGFBP-1 culture medium concentration s were 5.9 +/- 0.8 ng/mL, 4.9 +/- 1.2 ng/mL, and 4.8 +/- 1.3 ng/mL for 1, 10, and 100 ng/mL of insulin, respectively. The latter number was significantly higher than IGFBP-1 concentration in the medium collecte d from cells incubated with 100 ng/mL of insulin without IR-47-9 (1.6 +/- 0.3 ng/mL, P < 0.01) and not significantly different from the cont rol cells. For cells preincubated with IR-47-9 and then incubated with 1, 10, or 100 ng/mL of IGF-I, the IGFBP-1 conditioned culture medium concentrations were 1.7 +/- 0.1 ng/mL, 0.5 +/- 0.2 ng/mL, and 0.3 +/- 0.1 ng/mL, respectively. None of these were significantly different fr om the IGFBP-1 concentrations in the medium collected from cells incub ated with the respective concentrations of IGF-I without preincubation with IR-47-9. We conclude that 1) both insulin and IGF-I inhibit IGFB P-1 production by cultured human granulosa cells; 2) IGF-I is a more p otent inhibitor of IGFBP-1 production than insulin; 3) in the range of hormone concentrations tested, insulin exerts its inhibitory effect o n IGFBP-1 production via insulin receptor, while IGF-I appears to exer t its effect via another receptor.