ROLE OF PTG IN ADIPOCYTIC GLYCOGEN METABOLISM

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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.