We established a steroidogenic human ovarian granulosa-like tumor cell line, designated KGN, from a patient with invasive ovarian granulosa cell carcinoma. KGN had a relatively long population doubling time of about 46.4 h and had an abnormal karyotype of 45,XX, 7q-, -22. A steroid analysis of the cultured medium by RIA performed 5 yr after the initiation of culture showed that KGN was able to secrete pregnenolone and progesterone, and both dramatically increased after stimulation with (Bu)(2)cAMP. However, little or no secretion of 17alpha-hydroxylated steroids, dehydroepiandrosterone, androstenedione, or estradiol was observed. The aromatase activity of KGN was relatively high and was further stimulated by (Bu)(2)cAMP or FSH. These findings showed a pattern similar to that of steroidogenesis in human granulosa cells, thus allowing analysis of naturally occurring steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN was also observed, which mimicked the physiological regulation of apoptosis in normal human granulosa cells. Based on these findings, this cell line is considered to be a very useful model for understanding the regulation of steroidogenesis, cell growth, and apoptosis in human granulosa cells.

Obesity is associated with insulin resistance and some reproductive abnormalities. Circulating FFAs are often elevated in obese subjects and are also closely linked to insulin resistance. In this study, we demonstrated that saturated FFAs, such as palmitic acid and stearic acid, markedly suppressed the granulosa cell survival in a time- and dose-dependent manner. Polyunsaturated FFA, arachidonic acid, had no effect on the cell survival, even at supraphysiological concentrations. The suppressive effect of saturated FFAs on cell survival was caused by apoptosis, as evidenced by DNA ladder formation and annexin V-EGFP/propidium iodide staining of the cells. The apoptotic effects of palmitic acid and stearic acid were unrelated to the increase of ceramide generation or nitric oxide production and were also completely blocked by Triacsin C, an inhibitor of acylcoenzyme A synthetase. In addition, acylcoenzyme A, pamitoylcoenzyme A, and stearylcoenzyme A markedly suppressed granulosa cell survival, whereas arachidonoylcoenzyme A had no such effect, and this finding was consistent with the effect of the respective FFA form. Surprisingly, arachidonic acid instead showed a protective effect on palmitic acid- and stearic acid-induced cell apoptosis. A Western blot analysis showed the apoptosis of the granulosa cells induced by palmitic acid to be accompanied by the down-regulation of an apoptosis inhibitor, Bcl-2, and the up-regulation of an apoptosis effector, Bax. These results indicate that saturated FFAs induce apoptosis in human granulosa cells caused by the metabolism of the respective acylcoenzyme A form, and the actual composition of circulating FFAs may thus play a critical role in the apoptotic events of human granulosa cells. These effects of FFAs on granulosa cell survival may be a possible mechanism for reproductive abnormalities, such as amenorrhea, which is frequently observed in obese women.

Abstract.A of serum DHEA-S or the DHEA-S/DHEA ratio between patients with AD and those with CVD. These results suggest that reduced concentrations of serum DHEA-S may not be unique to AD, but instead reflect a common phenomenon in dementing diseases. However, since serum concentration of DHEA in these patients remained unchanged, the significance of DHEA in dementia remains unclear.

To clarify whether adrenal gland secretes inhibin in vivo in physiological or pathological conditions, we measured the levels of inhibin-like immunoreactivity (inhibin-LI) in adrenal veins (A-vein) and compared them with those in inferior vena cava (IVC) using blood samples obtained at catheterization of adrenal vein in the patients with adrenal adenoma manifesting Cushing's syndrome (Cs), aldosterone-producing adenoma, clinically non-functioning adenoma and normal adrenal gland. The tumor sides of A-veins in the patients with adenomas and also both sides of A-veins in subjects with normal adrenal glands showed significantly higher contents of inhibin-LI than their IVC. When the inhibin-LI secretion rate from adrenal gland was estimated by the difference between the levels of A-vein (tumor side) and IVC, Cs adenomas showed the highest secretion rate. Similarly, the tissue inhibin-LI content and the basal secretion rate of inhibit-LI from primary cultured cells were the highest in Cs adenomas. These findings indicated that normal adrenal glands and adrenocortical adenomas produced and secreted inhibin-LI into the general circulation in vivo and Cs adenomas have relatively high capacity for secreting inhibin-LI, and the present study provided the first in vivo evidence for adrenal inhibin-LI production in pathological conditions.

Aromatase activity was studied in cultured human osteosarcoma cell (HOS). HOS was incubated from 12 to 72 hours with 10(-10) M-10(-5) M dexamethasone. Aromatase activity was determined by measuring [3H]H2O released upon the conversion of [1 beta-3H]androstenedione to estrone. HOS showed aromatase activity, and apparent km for [1 beta-3H]androstenedione was 4.46 +/- 0.98 nm (mean +/- SD). The aromatase activity was significantly increased by 10(-9) M-10(-5) M dexamethasone in a dose-dependent manner. Dexamethasone increased Vmax of aromatase activity but did not change its km value. These results suggest that osteoblastic cells have aromatase activity which is regulated by glucocorticoid, and directly convert androgen to estrogen in itself.