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

Abstract

Expression of the glycogen-targeting protein PTG promotes glycogen synthase activation and glycogen storage in various cell types. In this study, we tested the contribution of phosphorylase inactivation to the glycogenic action of PTG in hepatocytes by using a selective inhibitor of phosphorylase (CP-91149) that causes dephosphorylation of phosphorylase a and sequential activation of glycogen synthase. Similar to CP-91194, graded expression of PTG caused a concentration-dependent inactivation of phosphorylase and activation of glycogen synthase. The latter was partially counteracted by the expression of muscle phosphorylase and was not additive with the activation by CP-91149, indicating that it is in part secondary to the inactivation of phosphorylase. PTG expression caused greater stimulation of glycogen synthesis and translocation of glycogen synthase than CP-91149, and the translocation of synthase could not be explained by accumulation of glycogen, supporting an additional role for glycogen synthase translocation in the glycogenic action of PTG. The effects of PTG expression on glycogen synthase and glycogen synthesis were additive with the effects of glucokinase expression, confirming the complementary roles of depletion of phosphorylase a (a negative modulator) and elevated glucose 6-phosphate (a positive modulator) in potentiating the activation of glycogen synthase. PTG expression mimicked the inactivation of phosphorylase caused by high glucose and counteracted the activation caused by glucagon. The latter suggests a possible additional role for PTG on phosphorylase kinase inactivation.Regulation of liver glycogen metabolism by hormones and substrates is mediated by changes in the phosphorylation state of glycogen synthase and phosphorylase and by subcellular translocation of these proteins (1-3). Dephosphorylation is catalyzed by PP1C 1 in association with glycogen-targeting proteins (4), of which three isoforms are expressed in liver, designated G L , PTG, and R6 (5). All isoforms have glycogensynthase phosphatase and phosphorylase phosphatase activity when assayed in vitro; however, the synthase phosphatase/ phosphorylase phosphatase ratio is higher for G L -PP1 than for PTG-PP1, suggesting that G L may be the predominant synthase phosphatase (5). 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.A unique property of G L is its allosteric binding site for phosphorylase a, which accounts for the inhibition of glycogensynthase phosphatase activity by low concentrations of phosphorylase a (5). This mechanism ...

Transgenic overexpression of protein targeting to glycogen markedly increases adipocytic glycogen storage in mice

“…One such protein, termed protein targeting to glycogen (PTG), was first identified from 3T3-L1 adipocytes (57) and remains the only PP1 glycogen-targeting subunit reported to be expressed in adipocytes. Importantly, as reported by several groups, overexpression of PTG in a variety of cell types in vitro and in rodent liver in vivo markedly enhanced cellular glycogen levels (17,20,22,24,37,50,57,72).…”

“…Previously, transient overexpression of the PP1 glycogen-targeting subunit PTG had been shown to increase intracellular glycogen levels in a number of cell types and rodent livers (17,20,22,24,37,50,57,72). PTG, originally identified in the 3T3-L1 adipocyte cell line, is the only reported PP1 glycogen-targeting subunit expressed in adipose tissue (57).…”

Inhibition of glycogen synthesis by increased lipid availability is associated with subcellular redistribution of glycogen synthase

“…Similar protocols have been employed to study glycogen synthase translocation in response to other treatments in skeletal muscle (Nielsen et al 2001), hepatocytes (Fernandez-Novell et al 1992, Green et al 2004) and adipocytes (Brady et al 1999). The Triton-insoluble fraction (TIF) typically contains cytoskeletal elements and also glycogen.…”

“…Altered cellular localization of glycogen synthase may represent a third level of regulation, and has been reported upon insulin stimulation (Brady et al 1999), increased glucose availability (Fernandez-Novell et al 1992), glycogen accumulation (Nielsen et al 2001) and alterations in the expression of glycogen-targeting proteins (Green et al 2004). It appears that glycogen synthase can translocate to and from glycogen storage compartments depending on hormonal stimulation (Brady et al 1999) and metabolic requirements, as after exercise (Nielsen et al 2001).…”

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

“…These subtle differences may be masked by the characteristic hyperglycaemia and hyperinsulinaemia of this Alternatively, temporal differences between the in vivo (16 h) and in vitro (1.5 h) assays may explain the divergence in insulin output. PTG is known to modify the phosphorylation and activity of glycogen phosphorylase [39], resulting in reduced glycogenolytic rates [18]. It is thus plausible that the short incubation times of the in vitro insulin secretion assays precludes detection of the effects of glycogenolysis in this setting.…”