INSULIN SIGNALING DEFECTS IN PCOS

多囊卵巢综合征中的胰岛素信号缺陷

• 批准号: 7148632
• 负责人: Ricardo Azziz
• 金额: $ 32.6万
• 依托单位: CEDARS-SINAI MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-05 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7148632
• 关键词: A kinase anchoring protein; JUN kinase; binding proteins; clinical research; female; glucose tolerance test; glucose transport; glucose transporter; hormone regulation /control mechanism; human subject; insulin; insulin receptor; insulin sensitivity /resistance; kinase inhibitor; mitogen activated protein kinase; molecular pathology; patient oriented research; phosphatidylinositol 3 kinase; polycystic ovary syndrome; polymerase chain reaction; protein kinase A; protein kinase C; receptor expression; serine threonine protein kinase; western blottings

DESCRIPTION (provided by applicant): The polycystic ovary syndrome (PCOS) affects ~7% of women and ~70% demonstrate insulin resistance, with the resulting hyperinsulinemia stimulating androgen excess. The economic burden of the disorder is estimated to exceed 4 billion dollars annually, in the U.S. and during the reproductive life span alone. The broad long-term objective of our studies is to establish the molecular etiology(s) of the PCOS-associated insulin resistance. Overall, little is know about the molecular aspects of insulin signaling in PCOS. Insulin-stimulated glucose uptake is deficient in PCOS, suggesting an alteration along the IRS/PI-3 kinase/Akt cascade, although the mitogenic activity and MAPK pathway appears unaffected. Insulin receptor (IR) tyrosine autophosphorylation also appears to be lower, and serine phosphorylation higher. In addition, we have obtained preliminary data indicating that PCOS adipocytes have deficient serine (inhibitory) and increased tyrosine (activating) glycogen synthase kinase-3 (GSK3) phosphorylation, consistent with enhanced GSK3 action. This data suggests that GSK3 dysregulation may represent a novel mechanism for insulin resistance in PCOS. We propose the following studies: Aim 1: To determine the role that defective regulation of GSK3 plays in mediating the abnormal IR signaling and glucose transport of PCOS; we will phenotype, including performing a frequently sampled intravenous glucose tolerance test, 70 PCOS patients and 70 matched controls; and in the adipocytes of these subjects determine the association of GSK3 activity with 2-deoxyglucose uptake; the content of regulators and substrates for GSK phosphorylation, determined by RT-PCR and/or Western blot (including total and phosphorylated IR substrate-1 and 2 [IRS-1/2], Akt, the PI-3 kinase subunits p110a and p110p, the 220-kDa A-kinase anchoring protein [AKAP220], protein kinase C [PKC], p70S6K, and the 2 GSK3-binding proteins known as FRAT1 and FRAT2); the impact of specific PKA, PKB (Akt), and PKC inhibition; in PCOS, the impact of specific GSK3 inhibition; and, in controls, the effect of GSKSbeta upregulation, using adenoviral-mediated transfection. Aim 2: To test whether abnormal signaling of the IRS/PI-3 kinase/Akt, but not the MAPK cascade, is present in PCOS; determining the degree of IR binding and 2-deoxyglucose uptake; and by RT-PCR and/or Western blot, the total content and phosphorylation in response to insulin of the IR, total and translocated GLUT-4, and of critical intermediate proteins (e.g. FKHR of the PI-3 kinase/Akt cascade: c-Raf, MEK-1, ERK1/2, pQORSK, and the translational regulator p70S6, of the ERK1/2 cascade: JNK of the SAPK/JNK cascade; and p38 MAPK of the P38MAPK cascade). Overall, these studies have the potential of elucidating the etioloaic mechanism(s) in PCOS, and guiding our search for therapies and molecular markers.

描述(申请人提供)：多囊卵巢综合征(PCOS)影响约7%的女性，约70%表现出胰岛素抵抗，导致高胰岛素血症刺激雄激素过剩。据估计，仅在美国，仅在生殖生命期间，这种疾病每年造成的经济负担就超过40亿美元。我们研究的长期目标是建立多囊卵巢综合征相关胰岛素抵抗的分子病因学(S)。总体而言，对多囊卵巢综合征中胰岛素信号的分子方面知之甚少。在PCOS中，胰岛素刺激的葡萄糖摄取不足，这表明IRS/PI-3激酶/Akt级联反应发生了变化，尽管丝裂原活性和MAPK通路似乎没有受到影响。胰岛素受体(IR)酪氨酸自动磷酸化水平也较低，丝氨酸磷酸化水平较高。此外，我们还获得了初步的数据，表明PCOS脂肪细胞缺乏丝氨酸(抑制)，增加了酪氨酸(激活)糖原合成酶激酶-3(GSK3)的磷酸化，这与增强的GSK3作用一致。这一数据表明，GSK3失调可能代表了PCOS胰岛素抵抗的一种新机制。我们提出了以下研究：目的1：确定GSK3调节缺陷在PCOS异常IR信号和葡萄糖转运中的作用；我们将对70例PCOS患者和70名匹配的对照组进行表型分析，并在这些受试者的脂肪细胞中确定GSK3活性与2-脱氧葡萄糖摄取的关系；通过RT-PCR和/或Western印迹法检测GSK磷酸化调节因子和底物的含量(包括总的和磷酸化的IR底物-1和2[IRS-1/2]，Akt，PI-3激酶亚基p110a和p110p，220 kDa的A-激酶锚定蛋白[AKAP220]，蛋白激酶C[PKC]，p70S6K，以及2种GSK3结合蛋白FRAT1和FRAT2)；特定的PKA、PKB(Akt)和PKC抑制的影响；在PCOS中，特定的GSK3抑制的影响；以及在对照中，使用病毒介导的GSKSbeta上调的影响。目的：检测PCOS是否存在IRS/PI-3激酶/Akt信号的异常，但不存在MAPK信号通路的异常；测定IR结合和2-脱氧葡萄糖摄取的程度；通过RT-PCR和/或Western印迹，检测ERK1/2信号通路中IR、总的和转位的GLUT-4以及关键中间蛋白(如PI-3激酶/Akt级联信号通路的FKHR：C-Raf、MEK-1、ERK1/2、pQORSK和翻译调节因子p70S6)的总含量和对胰岛素反应的磷酸化程度；和p38MAPK级联的p38MAPK)。总之，这些研究有可能阐明多囊卵巢综合征的发病机制(S)，并指导我们寻找治疗方法和分子标记。