ROLE OF PTG IN ADIPOCYTIC GLYCOGEN METABOLISM

描述（由申请人提供）：蛋白磷酸酶-1 （PP1）的激活在胰岛素调节糖原代谢酶中起关键作用。蛋白靶向糖原（PTG）结合PP1和糖原，从而将磷酸酶靶向糖原颗粒。此外，PTG结合调节糖原代谢的特定PP1底物，离散地增强磷酸酶对这些酶的活性。PTG在多种细胞类型中过表达，导致糖原合成酶去磷酸化和活化显著增加，导致糖原积累增强。我们将验证核心假设，即PTG: PP1复合物是3T3-L1脂肪细胞中糖原合成酶活性的主要胰岛素敏感酶调节因子。此外，由于超过70%的胰岛素刺激的葡萄糖摄取以糖原的形式沉积在3T3-L1脂肪细胞中，我们将研究细胞糖原水平在脂肪细胞能量感知和功能中的作用。我们最近发现了抑制细胞PTG功能或表达的显性负rna和siRNA结构。因此，我们可以显著调节细胞糖原储存在一个双向的方式。我们将使用PTG突变体构建小组来充分探索其在调节PP1抗糖原合成酶活性中的作用机制。然后，我们将研究PTG功能降低对糖原合成酶去磷酸化和胰岛素激活的影响。最后，PTG过表达导致脂联素水平的特异性抑制，而不降低近端胰岛素信号通路、2-脱氧葡萄糖转运、葡萄糖作为脂质储存或细胞ATP水平。我们将研究以双向方式调节PTG功能和糖原水平对3T3-L1脂肪细胞中葡萄糖和脂质摄取、代谢、储存和动员的影响。然后，我们将使用微阵列分析来评估对照、PTG过表达和PTG缺陷细胞中多种脂肪细胞因子的表达。通过这些实验，我们将充分研究PTG: PP1复合物在胰岛素调节糖原合成酶活性中的作用，并探索体内糖脂代谢、能量感知和影响胰岛素敏感性的脂肪细胞因子的调节分泌之间复杂的相互作用。

DESCRIPTION (provided by applicant): Activation of protein phosphatase-1 (PP1) plays a critical role in the regulation of glycogen metabolizing enzymes by insulin. Protein Targeting to Glycogen (PTG) binds to PP1 and glycogen, thus targeting the phosphatase to glycogen particles. Additionally, PTG binds specific PP1 substrates that regulate glycogen metabolism, discretely enhancing phosphatase activity against these enzymes. Overexpression of PTG in a variety of cell types causes a marked increase in glycogen synthase dephosphorylation and activation, resulting in enhanced glycogen accumulation. We will test the central hypothesis that the PTG: PP1 complex is the primary, insulin-sensitive enzymatic regulator of glycogen synthase activity in 3T3-L1 adipocytes. Further, since over 70% of insulin-stimulated glucose uptake was deposited as glycogen in 3T3-L1 adipocytes, we will investigate the role of cellular glycogen levels in adipocytic energy sensing and function. We have recently identified dominant negative and siRNA constructs that suppress cellular PTG function or expression. Thus, we can significantly modulate cellular glycogen stores in a bi-directional manner. We will use a panel of PTG mutant constructs to fully explore its mechanism of action in the regulation of PP1 activity against glycogen synthase. We will then examine the effects of decreasing PTG function on glycogen synthase dephosphorylation and activation by insulin. Finally, PTG overexpression results in the specific suppression of adiponectin levels, without reducing proximal insulin signaling pathways, 2-deoxyglucose transport, glucose storage as lipid or cellular ATP levels. We will examine the impact of modulating PTG function and glycogen levels in a bi-directional manner on the regulation of glucose and lipid uptake, metabolism, storage and mobilization in 3T3-L1 adipocytes. Then, the expression of a variety of adipocytic factors will be assessed in control, PTG over expressing and PTG-deficient cells using microarray analysis. Through these experiments, we will fully investigate the role of the PTG: PP1 complex in the regulation of glycogen synthase activity by insulin, and explore the intricate interplay between carbohydrate and lipid metabolism, energy sensing and the regulated secretion of adipocytic factors that influence insulin sensitivity in vivo.