Mechanism of Metformin Action in Normal and PCOS Theca Cells

描述（由申请人提供）：双胍类药物二甲双胍的使用是2型糖尿病年轻女性的标准药物，并且在治疗多囊卵巢综合征（PCOS）女性的高雄激素血症和无排卵方面越来越普遍。体外研究表明，二甲双胍对抑制卵巢卵泡膜雄激素生物合成具有直接作用，这对两组女性都具有临床意义。然而，二甲双胍抑制卵巢雄激素生物合成的机制目前尚不清楚。我们的初步研究表明，二甲双胍治疗对抑制正常周期和PCOS妇女卵泡膜细胞中雄激素生物合成有不同的作用。对经典的二甲双胍激活的LKB 1/AMP激活的蛋白激酶（AMPK）信号级联的检查提供了新的发现，即LKB 1磷酸化以及AMPK活性在PCOS卵泡膜细胞中降低。二甲双胍治疗纠正了PCOS卵泡膜细胞中的这些信号缺陷，同时抑制雄激素生物合成。在这个建议中，我们将测试的假设，在PCOS卵泡膜细胞中的LKB 1/AMPK信号的缺陷有助于增加雄激素的生物合成和破坏细胞生长的PCOS卵巢。在具体目标1中，我们将研究二甲双胍激活LKB 1/AMPK信号通路以抑制PCOS卵泡膜细胞中雄激素生物合成的潜在机制。我们将研究LKB 1，AMPK，和盐诱导激酶，SIK 1和SIK 2，二甲双胍抑制CYP 17和CYP 11 A1基因表达和雄激素生物合成在正常和PCOS卵泡膜细胞在长期培养繁殖的作用。PCOS卵巢的病理特征是多个、小、生长停滞的卵泡。然而，还没有报道比较正常和PCOS卵泡膜细胞的细胞生长或细胞周期控制。我们已经确定了正常和PCOS卵泡膜细胞中细胞周期调控的差异，并观察到二甲双胍治疗PCOS卵泡膜细胞可改变PCOS和细胞周期进展至正常表型。在具体目标2中，我们将鉴定PCOS卵泡膜细胞中的细胞生长和细胞周期缺陷，并探索二甲双胍纠正这些缺陷的能力所涉及的信号传导机制。在正常生理和高胰岛素血症条件下，将评价LKB 1、AMPK和SIK 1/2在二甲双胍依赖性细胞生长和细胞周期进程中的作用。我们相信，对二甲双胍信号介导雄激素生物合成抑制和调节PCOS卵泡膜细胞生长的机制的清楚理解将有助于阐明PCOS的病理生理学，并为PCOS新的治疗靶点的开发提供见解。 公共卫生相关性：多囊卵巢综合征（PCOS）的特征在于卵巢囊肿、不排卵、高雄激素血症和子宫内膜增生，并且影响6-10%的育龄妇女，仅在美国就有大约300 - 500万妇女。PCOS女性的高雄激素血症和无排卵在临床上可以通过胰岛素增敏药物二甲双胍（一种治疗2型糖尿病的药物）治疗，但二甲双胍直接调节卵巢的机制尚未研究。对二甲双胍激活LKB 1/AMPK信号通路以纠正PCOS卵泡膜细胞中雄激素生物合成和细胞生长缺陷的机制进行严格评估，将为PCOS新治疗模式的开发提供重要见解。

DESCRIPTION (provided by applicant): The use of the biguanide drug, metformin, is standard for young women with type 2 diabetes and is becoming prevalent for the treatment of hyperandrogenism and anovulation in women with polycystic ovary syndrome (PCOS). In vitro studies have shown that metformin has direct effects on inhibiting ovarian thecal androgen biosynthesis, which is of clinical relevance to both groups of women. However, the mechanism(s) by which metformin inhibits ovarian androgen biosynthesis is presently unclear. Our preliminary studies suggest that metformin treatment has differential effects on inhibiting androgen biosynthesis in theca cells propagated from normal cycling and PCOS women. An examination of the classical metformin activated LKB1/ AMP-activated protein kinase (AMPK) signaling cascade, provided the novel findings that LKB1 phosphorylation, as well as AMPK activity are decreased in PCOS theca cells. Metformin treatment corrected these signaling defects in PCOS theca cells, while inhibiting androgen biosynthesis. In this proposal we will test the hypothesis that defects in LKB1/AMPK signaling in PCOS theca cells contribute to increased androgen biosynthesis and disrupted cell growth in the PCOS ovary. In Specific Aim 1, we will investigate the underlying mechanism(s) by which metformin activates the LKB1/AMPK signaling pathway(s) to inhibit androgen biosynthesis in PCOS theca cells. We will examine the role(s) of LKB1, AMPK, and the salt inducible kinases, SIK1 and SIK2, in metformin inhibition of CYP17 and CYP11A1 gene expression and androgen biosynthesis in normal and PCOS theca cells propagated in long-term culture. The pathological characteristic of the PCOS ovary is that of multiple, small, growth arrested follicles. However, there have been no reports comparing cell growth or cell cycle control in normal and PCOS theca cells. We have identified differences in cell cycle regulation in normal and PCOS theca cells, and observed that metformin treatment of PCOS theca cells was observed to change PCOS and cell cycle progression to a normal phenotype. In Specific Aim 2, we will identify cell growth and cell cycle defects in PCOS theca cells and explore the signaling mechanism(s) involved in metformin's ability to correct these defects. The roles of LKB1, AMPK, and SIK1/2 in metformin-dependent cell growth and cell cycle progression, under normal physiological and hyperinsulinemic conditions, will be evaluated. We believe a clear understanding of mechanism(s) by which metformin signals to mediate inhibition of androgen biosynthesis and modulate cell growth in PCOS theca cells will help elucidate the pathophysiology of PCOS, and provide insight for the development of new therapeutic targets for PCOS. PUBLIC HEALTH RELEVANCE: Polycystic ovary syndrome (PCOS) is characterized by ovarian cysts, anovulation, hyperandrogenism, and endometrial hyperplasia, and affects 6-10% of reproductive age women, approximately 3-5 million women in the United States alone. Hyperandrogenism and anovulation in women with PCOS can be clinically treated with administration of the insulin sensitizing drug, metformin, a treatment for type 2 diabetes, however the mechanisms by which metformin directly modulates the ovary have not been examined. A rigorous evaluation of the mechanism(s) by which the metformin activates the LKB1/AMPK signaling pathway(s) to correct defects in androgen biosynthesis and cell growth in PCOS theca cells, will provide essential insight for the development of new therapeutic modalities for PCOS.