Overexpression of Protein Targeting to Glycogen (PTG) in Rat Hepatocytes Causes Profound Activation of Glycogen Synthesis Independent of Normal Hormone- and Substrate-mediated Regulatory Mechanisms

Abstract

Protein targeting to glycogen (PTG), also known as PPP1R5, is a widely expressed member of a growing family of proteins that target protein phosphatase-1 (PP-1) to glycogen particles. Because PTG also binds to glycogen synthase and phosphorylase kinase, it has been suggested that it serves as a "scaffold" for efficient activation of glycogen synthesis. However, very little is known about the metabolic effects of PTG. In this study, we have used recombinant adenovirus to overexpress PTG in primary rat hepatocytes, a cell type with high glycogenic capacity. We find that overexpression of PTG potently activates glycogen synthesis in cultured hepatocytes. Surprisingly, the glycogenic effect of PTG is observed even in the complete absence of carbohydrates or insulin in the culture medium. Furthermore, glycogenolytic agents such as forskolin or glucagon are largely ineffective at activating glycogen degradation in PTG overexpressing hepatocytes, even though large increases in cAMP levels are demonstrated. These metabolic effects of PTG overexpression are accompanied by a 3.6-fold increase in glycogen synthase activation state and a 40% decrease in glycogen phosphorylase activity. Our results are consistent with a model in which PTG overexpression "locks" the hepatocyte in a glycogenic mode, presumably via its ability to promote interaction of enzymes of glycogen metabolism with PP-1.

The Glycogenic Action of Protein Targeting to Glycogen in Hepatocytes Involves Multiple Mechanisms Including Phosphorylase Inactivation and Glycogen Synthase Translocation

“…G L (R4) is expressed predominantly in liver and to a lesser extent in heart and human skeletal muscle (6,7), PTG is expressed in several tissues including liver and skeletal muscle (8 -10), and R6 is expressed ubiquitously (11). Both G L and PTG show adaptive changes in expression in rat liver in response to changes in insulin status (5), and the physiological role of these changes has been confirmed from studies (12,13) in hepatocytes, which showed glycogen synthase activation and increased glycogen storage when G L and PTG were overexpressed.…”

“…The effects of glucose and PTG were not additive suggesting that PTG mimics the effect of glucose. DISCUSSION PTG expression activates glycogen synthase and stimulates glycogen storage in various cell types (8,12,13,21). Because phosphorylase a has major roles in the control of synthase phosphatase (1) and the rate of glycogen synthesis in hepatocytes (15, 29), we tested the role of inactivation of phosphorylase in the glycogenic action of PTG in hepatocytes.…”

“…Enzyme Translocation during PTG Expression Is Not Secondary to Glycogen Accumulation-Cells treated with adenovirus for expression of PTG show progressive glycogen accumulation (12,13). To test whether the translocation of glycogen synthase and phosphorylase to the pellet is secondary to glycogen accumulation, hepatocytes treated with PTG-adenovirus were cultured with glucagon (2 M) plus dibutyryl cAMP (50 M) to suppress glycogen deposition (Fig.…”

Genetic models rule out a major role of beta cell glycogen in the control of glucose homeostasis

“…3c), reaching values that were nearly three times higher than those in freshly isolated islets from fed mice. Thus, in contrast to other models of PTG overexpression [31], glycogen accumulation in beta cells of PTG OE mice is subject to regulation and can be modulated both in vivo in response to fasting and in vitro by incubation at a range of glucose concentrations.…”

“…It should be noted that in beta cells of PTG OE mice, in contrast to other mouse models of PTG overexpression [31], glycogen stores can be mobilised when extracellular glucose is low. In line with prior work [4], glycogenolysis in beta cells of PTG OE mice after an overnight fast may stimulate insulin release and explain increased insulin levels when these animals are subjected to fasting.…”