INSULIN SIGNALING DEFECTS IN PCOS

多囊卵巢综合征中的胰岛素信号缺陷

• 批准号: 7417475
• 负责人: Ricardo Azziz
• 金额: $ 31.02万
• 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/7417475
• 关键词: 1-Phosphatidylinositol 3-Kinase; 70-kDa Ribosomal Protein S6 Kinases; A kinase anchoring protein; Abdomen; Adipocytes; Adipose tissue; Adrenal Glands; Affect; Age; Androgens; Basic Science; Binding; Binding Proteins; Biological Markers; Biopsy; Classification; Clinical; Collaborations; Consult; Critical Pathways; Cyclic AMP-Dependent Protein Kinases; Data; Defect; Deoxyglucose; Disease; Economic Burden; Endocrinologist; Etiology; FOXO1A gene; FRAT1 gene; Funding; Future; Glycogen Synthase Kinase 3; Hormonal; Hyperandrogenism; Hyperinsulinism; In Vitro; Insulin; Insulin Receptor; Insulin Resistance; Invasive; Laboratories; Longevity; Longitudinal Studies; MAP Kinase Gene; MAPK14 gene; MAPK8 gene; Mediating; Metabolic; Molecular; Nature; Needles; Ovarian; Pathway interactions; Patients; Phenotype; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phosphorylation; Physiology; Play; Polycystic Ovary Syndrome; Polymerase Chain Reaction; Population; Procedures; Protein Kinase C; Proteins; Proto-Oncogene Proteins c-akt; Race; Receptor Signaling; Recruitment Activity; Regulation; Relative (related person); Research; Research Infrastructure; Research Personnel; Reverse Transcription; Role; Sampling; Scientist; Serine; Signal Transduction; Staging; Steroid biosynthesis; Study Subject; Surgeon; Techniques; Testing; Time; Tissue Sample; Tissues; Transfection; Translations; Tyrosine; Up-Regulation; Western Blotting; Woman; adenoviral-mediated; citrate carrier; concept; experience; glucose transport; glucose uptake; human MAP2K1 protein; in vitro Model; in vivo; inhibitor/antagonist; insulin receptor substrate 1 protein; insulin signaling; intravenous glucose tolerance test; novel; programs; receptor binding; reproductive; research study; response; skills; stress-activated protein kinase 1; subcutaneous; uptake

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（由申请人提供）：多囊卵巢综合征（PCOS）影响约7%的女性，约70%表现为胰岛素抵抗，导致高胰岛素血症刺激雄激素过量。据估计，在美国，仅在生育期，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pcos相关胰岛素抵抗的分子病因学。总的来说，对多囊卵巢综合征中胰岛素信号传导的分子方面知之甚少。胰岛素刺激的葡萄糖摄取在PCOS中缺乏，表明IRS/PI-3激酶/Akt级联发生改变，尽管有丝分裂活性和MAPK通路未受影响。胰岛素受体（IR）酪氨酸自磷酸化也较低，丝氨酸磷酸化较高。此外，我们获得的初步数据表明，PCOS脂肪细胞丝氨酸（抑制性）磷酸化不足，酪氨酸（激活性）糖原合成酶激酶3 （GSK3）磷酸化增加，与GSK3作用增强一致。这些数据表明，GSK3失调可能是多囊卵巢综合征胰岛素抵抗的一种新机制。我们提出以下研究：目的1：确定GSK3的缺陷调控在PCOS的异常IR信号和葡萄糖转运中的作用；我们将进行表型分析，包括进行频繁采样的静脉葡萄糖耐量试验，70名PCOS患者和70名匹配的对照组；并在这些受试者的脂肪细胞中测定GSK3活性与2-脱氧葡萄糖摄取的关系；通过RT-PCR和/或Western blot测定GSK磷酸化的调节因子和底物的含量（包括总和磷酸化的IR底物-1和2 [IRS-1/2]、Akt、PI-3激酶亚基p110a和p110p、220-kDa a -激酶锚定蛋白[AKAP220]、蛋白激酶C [PKC]、p70S6K和2个gsk3结合蛋白FRAT1和FRAT2）；特异性PKA、PKB （Akt）和PKC抑制的影响；在PCOS中，特异性GSK3抑制的影响；在对照组中，通过腺病毒介导的转染，研究GSKSbeta上调的效果。目的2：检测PCOS中是否存在IRS/PI-3激酶/Akt异常信号，而不存在MAPK级联；测定IR结合程度和2-脱氧葡萄糖摄取；并通过RT-PCR和/或Western blot检测IR、总glut4和易位glut4以及关键中间蛋白（例如PI-3激酶/Akt级联的FKHR: c-Raf、MEK-1、ERK1/2、pQORSK和翻译调节因子p70S6， ERK1/2级联的FKHR: SAPK/JNK级联的JNK和P38MAPK级联的p38 MAPK）的总含量和对胰岛素的磷酸化反应。总的来说，这些研究有可能阐明多囊卵巢综合征的病因机制，并指导我们寻找治疗方法和分子标记物。