Probing the mechanisms of signal transduction via adiponectin and its receptors

脂联素及其受体信号转导机制的探讨

• 批准号: 7079917
• 负责人: THOMAS J LYONS
• 金额: $ 13.37万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7079917
• 关键词: Saccharomyces cerevisiae; adiponectin; aminohydrolases; biological signal transduction; enzyme activity; glucose metabolism; hormone receptor; hormone regulation /control mechanism; insulin sensitivity /resistance; lipid metabolism; mass spectrometry; phosphorylation; receptor expression; second messengers; serine threonine protein kinase; sphingolipids; western blottings

DESCRIPTION (provided by applicant): Obesity correlates with the development of type 2 diabetes, however, the molecular mechanisms behind this correlation are inadequately characterized. The adipocyte-derived anti-diabetic hormone, adiponectin, has emerged as a promising candidate for such a molecular link. Low circulating adiponectin and low adiponectin receptor (AdipoR1 and AdipoR2) expression correlate with insulin resistance. Therefore, a detailed characterization of how adiponectin receptors transmit signals is critical for a better understanding of how obesity results insulin resistance and for the future development of therapeutics for type 2 diabetes that target the physiology of adiponectin. Current theories of the mechanism by which adiponectin receptors transmit signals inside the cell suggest that they function as a novel type of G-protein-coupled receptor. This grant proposal challenges that assignment and hypothesizes that these receptors function, instead, as adiponectin-activated ceramidase enzymes. Furthermore, it is proposed that these receptors, via the generation of a sphingosine second messenger, activate PDK1 and downstream AGC kinase pathways known to be involved in glucose sensing (protein kinase A, protein kinase B or AMP-dependent protein kinase). To confirm this hypothesis, three specific aims are proposed using the yeast, Saccharomyces cerevisiae, as a model system. Working in yeast is beneficial for several reasons. First, the pathways of sphingolipid-dependent signal transduction and glucose signaling in humans are highly conserved in yeast. Second, AdipoR1 has been shown to function when expressed heterologously in yeast. And third, the physiological effects of AdipoR1 expression in yeast can be readily investigated due to the ease of experimentation in this organism. In Specific Aim 1, the sphingolipid- and adiponectin receptor-dependent activation of the yeast homologues of PDK1 and downstream AGC kinases will be confirmed by measuring their phosphorylation state and by measuring the effect of kinase inactivation on a promoter-reporter construct that is known to respond to AdipoR1 overexpression. In Specific Aim 2, the effect of adiponectin receptor expression on in vivo sphingolipid metabolism will be confirmed. And lastly, in Specific Aim 3, the ceramidase activity of the adiponectin receptors will be confirmed by in vitro biochemical assay. This hypothesis, if proven correct, will redirect research into the mechansims of adiponectin signaling.

描述（由申请人提供）：肥胖与 2 型糖尿病的发展相关，然而，这种相关性背后的分子机制尚未得到充分表征。脂肪细胞衍生的抗糖尿病激素脂联素已成为这种分子联系的有希望的候选者。低循环脂联素和低脂联素受体（AdipoR1 和 AdipoR2）表达与胰岛素抵抗相关。因此，详细表征脂联素受体如何传递信号对于更好地了解肥胖如何导致胰岛素抵抗以及未来开发针对脂联素生理学的 2 型糖尿病疗法至关重要。目前关于脂联素受体在细胞内传递信号的机制的理论表明，它们作为一种新型的 G 蛋白偶联受体发挥作用。这项拨款提案挑战了这一分配，并假设这些受体的功能是脂联素激活的神经酰胺酶。此外，有人提出，这些受体通过生成鞘氨醇第二信使，激活 PDK1 和已知参与葡萄糖传感的下游 AGC 激酶途径（蛋白激酶 A、蛋白激酶 B 或 AMP 依赖性蛋白激酶）。为了证实这一假设，使用酿酒酵母作为模型系统提出了三个具体目标。出于多种原因，在酵母中工作是有益的。首先，人类中鞘脂依赖性信号转导和葡萄糖信号传导途径在酵母中高度保守。其次，AdipoR1 在酵母中异源表达时已被证明能够发挥作用。第三，由于在酵母中进行实验很容易，因此可以很容易地研究 AdipoR1 表达在酵母中的生理效应。在具体目标 1 中，PDK1 和下游 AGC 激酶的酵母同源物的鞘脂和脂联素受体依赖性激活将通过测量它们的磷酸化状态以及通过测量激酶失活对已知的启动子报告基因构建体的影响来确认。对 AdipoR1 过度表达做出反应。具体来说 目标2，将证实脂联素受体表达对体内鞘脂代谢的影响。最后，在具体目标 3 中，脂联素受体的神经酰胺酶活性将通过体外生化测定来证实。如果这一假设被证明是正确的，将把研究转向脂联素信号传导机制。