Interactive stimulation by luteinizing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17 alpha-hydroxylase/17,20-lyase (CYP17) genes in porcine theca cells

LH and insulin are postulated to jointly stimulate theca-cell androgen bios ynthesis in patients with hyperthecosis or polycystic ovarian syndrome. To explore the mechanisms of putative LH and insulin steroidogenic synergy in primary culture of normal theca cells, we have implemented an in vitro seru m-free monolayer culture system of Percoll-purified, porcine theca cells ha rvested from immature ovaries. Initial dose and time course analyses reveal ed that a maximally effective concentration of LH (100 ng/ml) or insulin (1 00 ng/ml) individually will drive androstenedione production (at 6 to 48 h) by 1.5-to 2.6- and 1.1- to 1.7-fold, respectively, while combined agonists act synergistically over the interval 12 to 48 h yielding a 3- to 4-fold j oint effect. Coadministration of LH and insulin can augment theca-cell conc entrations of CYP17 and StAR messenger RNA (mRNA) resulting in 3.4- to 3.9- and 3.8- to 4.1-fold increases at 24 to 48 h, respectively (P < 0.01). Com bined LH and insulin stimulation also amplified the nuclear content of intr on-specific heterogeneous nuclear (hn)RNAs encoding CYP17 and StAR. Insulin significantly enhanced LH-driven but not basal cAMP accumulation (14-18 vs . 3-5.5 pmol/mu g DNA/12-48 h) (P < 0.01). A stable exogenous analog of cAM P, 8 Br-cAMP, mimicked LH's effect on steroidogenesis and StAR and CYP17 ge ne expression and with insulin stimulated StAR mRNA and hnRNA accumulation synergistically. However, unlike LH, 8 Br-cAMP did not synergize with insul in on theca-cell androstenedione biosynthesis or CYP17 mRNA and hnRNA expre ssion. In summary, the present in vitro data identify molecular interaction s of LH and insulin on StAR and CYP17 gene expression, thus establishing po tent signaling interfaces between these distinct hormonal agonists in regul ating theca-cell steroidogenesis.