Endocrine Abstracts

*supported by The Robert H. Smith Faculty of Agriculture, Food and Environment Research, Fund for International Cooperation.

Objective: Sirtuin1 (SIRT1), an NAD+-dependent enzyme, affects diverse cellular processes. Yet the reproductive functions of SIRT1 remain unclear. The aim of the study was to reveal the regulation of SIRT1 expression and its roles in human granulosa-lutein cells (hGLCs).

Methods: hGLCs were retrieved from women undergoing IVF during oocytes retrieval. SV40-transfected, immortalized hGLCs (t-hGLCs) were used as a model for hGLCs. Cells were treated with SIRT1 activators (resveratrol/SRT2104/metformin), cAMP-elevating agents (hCG/forskolin) or their combination. The roles of SIRT1 were examined using exogenous SIRT1 activators, and silencing endogenous SIRT1 with specific siRNA. To identify the cAMP effector proteins regulating SIRT1, t-hGLCs were cultured with PKA inhibitor or EPAC1 activator. Cells were collected for qPCR and Western blot analyses.

Results: SIRT1 activators and cAMP elevating agents effectively upregulated SIRT1 expression. Combining resveratrol with hCG had additive stimulatory effects on SIRT1, suggesting convergent pathways induced by these compounds, possibly on cAMP levels and other cellular events. Moreover, SIRT1 activation augmented VEGFA, while inhibiting endothelin-2 (EDN2) transcripts. In agreement, SIRT1-silenced cells expressed reduced VEGFA and elevated EDN2. Surprisingly, cAMP effector proteins oppositely affected SIRT1; EPAC1 activator significantly increased, while inhibition of PKA enhanced basal and SRT2104 activated SIRT1 levels.

Conclusions: SIRT1 is dynamically regulated in hGLCs, where a positive feedback loop appears to exist between SIRT1 activity and expression. These findings also ascribe a distinct roles for EPAC1 vs. PKA in the cAMP-dependent SIRT1 activation. Moreover, since SIRT1 activators modulate VEGFA and EDN2 expression, SIRT1 may be implicated in the ovulatory process.