Role of AMP kinase and DAF-16 in Mediating the Effect o*

AMP 激酶和 DAF-16 在介导 o* 效应中的作用

• 批准号: 6533941
• 负责人: MARIA Carmalita ALEXANDER-BRIDGES
• 金额: $ 38.91万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6533941
• 关键词: CHO cells; Caenorhabditis elegans; DNA binding protein; SDS polyacrylamide gel electrophoresis; adenosine monophosphate; autoradiography; caloric dietary content; enzyme activity; gel mobility shift assay; genetic transcription; glucose; hyperinsulinism; insulin; laboratory rat; longevity; mass spectrometry; nutrition related tag; phosphorylation; phosphotransferases; polymerase chain reaction; protein kinase; protein sequence; thin layer chromatography; transcription factor; transfection

DESCRIPTION (provided by applicant): Genetic evidence in worms (C. Elegans) has shown that insulin-like molecules act via PI3 kinase and AKT/protein kinase B to inhibit the function of the forkhead (FKH) transcription factor DAF-16. Insulin signaling mutants with diminished function undergo dauer arrest and show increased longevity and resistance to oxidative stress due to the unimpeded action of DAF-16. Accordingly, DAF-16 has been shown to activate the superoxide dismutase gene (SOD). In mammals, caloric restriction and low insulin signaling has been shown to slow the rate of aging by mechanisms that include increased DNA repair capacity and reduction of oxidative stress. In the presence of low glucose and circulating insulin levels, DAF-16 homologues appear to be transcriptionally active. Several laboratories have shown that in the absence of insulin, mammalian homologues of DAF-16, FKHR, FKHRL1 and AFX activate the transcription of genes that control apoptosis, and gluconeogenesis, and insulin can inhibit this effect. Our goal is to elucidate the mechanisms by which DAF-16 like factors activate transcription in the absence of insulin. Caloric restriction and low glucose activates AMP kinase. We find that AMP kinase can prevent the effect of insulin on DAF-16 in HepG2 cells. Furthermore, AMP kinase can directly phosphorylate DAF-16. We propose to determine whether regulation of DAF-16 by AMP kinase in worms and regulation of its homologue FKHR in mammalian cells, can explain the ability of caloric restriction to slow the aging process. In HepG2 cells, insulin signaling via the AKT sites in DAF-16 inhibits DAF-16 activity. We find that the AKT sites in DAF-16 carry overlapping AMP kinase sites. In Specific Aim 1 of this proposal, we will determine whether AMP kinase regulates DAF-16 activity directly by altering its phosphorylation or indirectly by regulating other elements of the PI3 kinase-signaling pathway. We will examine the effect of AMP kinase on 1) phosph DAF-16 phosphorylation in vitro and in vivo, 2) 14-3-3 binding to DAF-16 in the presence and absence of insulin and 3) the interaction of DAF-16 with other proteins that increase its binding/transcription activity. In Specific Aim II we will determine whether AMP kinase can counteract the effect of insulin signaling to DAF-16 in C. elegans, and prolong life span in the worm. In Specific Aim III, we will examine the effect of caloric restriction and carbohydrate-induced hyperinsulinemia on the activity of AMP kinase and DAF-16 homologues

描述（由申请人提供）：蠕虫中的遗传证据（C。Elegans） 已经表明胰岛素样分子通过PI 3激酶和AKT/蛋白发挥作用 激酶B抑制叉头（FKH）转录因子的功能 -16。 功能减弱的胰岛素信号传导突变体遭受破坏 并显示出增加的寿命和抗氧化应激， 歼-16的行动毫无阻碍 因此，已经证明， 激活超氧化物歧化酶基因（SOD）。 在哺乳动物中， 低胰岛素信号已经被证明可以减缓衰老的速度， 机制，包括增加DNA修复能力和减少 氧化应激 在低血糖和循环胰岛素的情况下 水平，β-16同系物似乎是转录活性。 几 实验室已经表明，在缺乏胰岛素的情况下， FKHR，FKHRL 1和AFX激活基因的转录， 控制细胞凋亡和凋亡的发生，而胰岛素可以抑制这种作用。 我们的目标是阐明β-16样因子激活的机制， 在没有胰岛素的情况下转录。 热量限制和低葡萄糖激活AMP激酶。 我们发现AMP 激酶抑制胰岛素对HepG 2细胞凋亡的抑制作用。 此外，AMP激酶可以直接磷酸化β-16。 我们建议 确定蠕虫中AMP激酶是否调节BMP 16，以及 其同源FKHR在哺乳动物细胞，可以解释的能力，热量 以减缓衰老过程。 在HepG 2细胞中，胰岛素信号通过 β-16中的AKT位点抑制β-16活性。 我们发现AKT位点 在携带重叠AMP激酶位点的β-16中。 具体目标1 我们将确定AMP激酶是否调节β-16活性 直接通过改变其磷酸化或间接通过调节其他 PI 3激酶信号通路的元件。 我们将研究 AMP激酶对1）体外和体内磷酸化16磷酸化，2）14-3-3 在存在和不存在胰岛素的情况下与β-16结合，和3） β-16与其他蛋白质的相互作用， 结合/转录活性。 在具体目标II中，我们将确定 AMP激酶可抵消胰岛素信号对C. elegans，and extend延长life span跨度in the worm蠕虫. 在具体目标三中，我们将 检查热量限制和碳水化合物引起的 高胰岛素血症对腺苷酸激酶和β-16同源物活性的影响