Probing the mechanisms of signal transduction via adiponectin and its receptors

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理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型糖尿病治疗方法至关重要。目前关于脂联素受体在细胞内传递信号的机制理论表明，它们是一种新型的g蛋白偶联受体。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，这些受体通过产生鞘氨醇第二信使，激活PDK1和已知参与葡萄糖感知的下游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，提出了三个具体目标，以酵母，酿酒酵母，作为一个模型系统。在酵母中工作是有益的，有几个原因。首先，人类鞘脂依赖性信号转导和葡萄糖信号转导通路在酵母中高度保守。其次，AdipoR1已被证明在酵母中异种表达时具有功能。第三，由于易于在酵母中进行实验，因此可以很容易地研究AdipoR1在酵母中表达的生理效应。在Specific Aim 1中，将通过测量PDK1和下游AGC激酶的酵母同源物的磷酸化状态和激酶失活对已知对AdipoR1过表达有反应的启动子报告结构的影响来证实鞘脂和脂联素受体依赖的激活。在特定的

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.