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The Effects of Insulin on 3ß-Hydroxysteroid Dehydrogenase Expression in Human Luteinized Granulosa Cells

Chiravudh Sawetawan, MD

Ian Bird, PhD

William E. Rainey, PhD

Bruce R. Carr, MD

Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75235

Objective: We determined the relative effects of insuling and FSH on progesterone accumulation as well as activity, protein content, and mRNA expression of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in human luteinized granulosa cells.

Methods: Luteinized granulosa cells obtained from women undergoing in vitro fertilization were plated and grown to near confluence and treatd with FSH, insulin, or a combination of insulin and FSH. Progesterone production as well as enzyme activity, protein content, and mRNA expression for 3ßHSD were evaluated.

Results: Progesterone production was not affected by insulin alone but increased threefold in the presence of FSH (50 ng/µL) alone. The presence of FSH plus insulin (100 nmol/L) caused a significant increase in progesterone accumulation greater than that of FSH alone. The already high basal levels of 3ßHSD activity were unaffected by insulin alone but increased 1.7-fold in the presence of FSH. The combination of FSH (50 ng/mL) and insulin (100 nmol/L) increased activity 1.3-fold over FSH alone (P < .02) Insulin (greater than 100 nmol/L) alone increased 3ßHSD protein content as measured by Western analysis 1.8-2fold over basal levels, whereas FSH alone increased protein contetn 2.8-fold, and was further augmented by the addition of insulin in a dose-related fashion up to 3.5-fold over basal levels. Insulin increased 3ßHSD mRNA twofold over basal levels; FSH alone increased mRNA expression of 3ßHSD 3.2-fold. In the presence of insulin plus FSH, 3ßHSD mRNA expression increased 7.6-fold over basal levels. For comparison, insulin also stimulated cytochrome P450 aromatase activity, P450 aromatase protein, and mRNA but to a greater degree than that seen for 3ßHSD.

Conclusion: Insulin is a regular of both 3ßHSD and aromatase expression in human granulosa cells. Elevated insulin levels could therefore affect steroid production in human granulosa cells and presumably alter the menstrual cycle and fertility.

Key Words: 3ß-hydroxysteroid dehydrogenase • granulosa cells • insulin • aromatase • FSH • luteinization

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