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 in regulating ovarian function by modifying effects of insulin-like growth factors (IGFs) or by directly affecting ovarian steroidogenesis in both normal and pathological circumstances. The latter include hyperinsulinemic insulin resistant states, such as polycystic ovary syndrome. We examined regulation of IGFBP-1 production in human granulosa cells by insulin and IGF-I. The cells were obtained during in vitro fertilization, plated in McCoy-5A tissue culture medium supplemented with 10% fetal calf serum (10(5) cells/0.5 mL), and incubated at 37 C, 90% humidity, 5% CO2 for 48 h. After additional 24 h incubation without fetal calf serum, 1, 10, or 100 ng/mL of insulin or IGF-I were added with or without 2 h preincubation with 10 micrograms/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, Webster, TX. Progesterone concentration ranged between 50-100 ng/mL/10(5) cells, 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 concentrations 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 the 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 concentrations 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 collected 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 control 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 from the IGFBP-1 concentrations in the medium collected from cells incubated with the respective concentrations of IGF-I without preincubation with IR-47-9. We conclude that 1) both insulin and IGF-I inhibit IGFBP-1 production by cultured human granulosa cells; 2) IGF-I is a more potent inhibitor of IGFBP-1 production than insulin; 3) in the range of hormone concentrations tested, insulin exerts its inhibitory effect on IGFBP-1 production via insulin receptor, while IGF-I appears to exert its effect via another receptor.