Probing the mechanisms of signal transduction via adiponectin and its receptors

描述（由申请人提供）：肥胖与2型糖尿病的发生相关，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连接的有希望的候选者。低循环脂联素和低脂联素受体（AdipoR 1和AdipoR 2）表达与胰岛素抵抗相关。因此，脂联素受体如何传递信号的详细表征对于更好地理解肥胖如何导致胰岛素抵抗以及针对脂联素生理学的2型糖尿病治疗的未来发展至关重要。脂联素受体在细胞内传递信号的机制的现有理论表明，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，而不是作为脂联素激活神经酰胺酶。此外，提出这些受体通过产生鞘氨醇第二信使激活已知参与葡萄糖传感的PDK 1和下游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，提出了三个具体的目标，使用酵母，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，人类的鞘脂依赖性信号转导和葡萄糖信号转导途径在酵母中高度保守。第二，AdipoR 1已被证明在酵母中异源表达时起作用。第三，AdipoR 1在酵母中表达的生理效应可以很容易地研究，因为在这种生物体中进行实验很容易。在特定目标1中，将通过测量其磷酸化状态和通过测量激酶失活对已知响应AdipoR 1过表达的启动子-报告基因构建体的影响，确认PDK 1和下游AGC激酶的酵母同系物的鞘脂和脂联素受体依赖性激活。在特定 目的2，证实脂联素受体表达对体内鞘脂代谢的影响。最后，在特定目标3中，脂联素受体的神经酰胺酶活性将通过体外生物化学试验进行确认。这一假设，如果被证明是正确的，将重新研究脂联素信号转导机制。

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.