ROLE OF PTG IN ADIPOCYTIC GLYCOGEN METABOLISM

描述(申请人提供)：蛋白磷酸酶-1(PP1)的激活在胰岛素对糖原代谢酶的调节中起着关键作用。糖原靶向蛋白(PTG)与PP1和糖原结合，从而将磷酸酶靶向糖原颗粒。此外，PTG与调节糖原代谢的特定PP1底物结合，单独增强针对这些酶的磷酸酶活性。PTG在多种细胞类型中的过表达导致糖原合成酶去磷酸化和激活显著增加，从而促进糖原蓄积。我们将检验中心假设，即PTG：PP1复合体是3T3-L1脂肪细胞中糖原合成酶活性的主要、胰岛素敏感的酶调节因子。此外，由于超过70%的胰岛素刺激的葡萄糖摄取在3T3-L1脂肪细胞中以糖原的形式沉积，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性和siRNA结构。因此，我们可以以双向的方式显著调节细胞糖原储存。我们将使用一组PTG突变构建物来充分探讨其在调节PP1活性对抗糖原合成酶方面的作用机制。然后，我们将研究PTG功能下降对糖原合成酶去磷酸化和胰岛素激活的影响。最后，PTG的过表达导致脂联素水平的特异性抑制，而不会降低近端的胰岛素信号通路、2-脱氧葡萄糖转运、葡萄糖储存作为脂质或细胞ATP水平。我们将研究双向调节PTG功能和糖原水平对3T3-L1脂肪细胞糖脂摄取、代谢、储存和动员的调节作用。然后，利用基因芯片分析来评估各种脂肪细胞因子在对照、PTG过表达和PTG缺陷细胞中的表达。通过这些实验，我们将充分研究PTG：PP1复合体在胰岛素调节糖原合成酶活性中的作用，并探索体内影响胰岛素敏感性的碳水化合物和脂肪代谢、能量感知和脂肪细胞因子调节分泌之间的复杂相互作用。

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.