Abstract
Glycogen-targeting subunits of protein phosphatase-1 facilitate interaction of the phosphatase with enzymes of glycogen metabolism. We have shown that overexpression of one member of the family, protein targeting to glycogen (PTG), causes large increases in glycogen storage in isolated hepatocytes or intact rat liver. In the current study, we have compared the metabolic and regulatory properties of PTG (expressed in many tissues), with two other members of the gene family, G(L) (expressed primarily in liver) and G(M)/R(Gl) (expressed primarily in striated muscle). Adenovirus-mediated expression of these proteins in hepatocytes led to the following key observations. 1) G(L) has the highest glycogenic potency among the three forms studied. 2) Glycogen synthase activity ratio is much higher in G(L)-overexpressing cells than in PTG or G(M)/R(Gl)-overexpressing cells. Thus, at moderate levels of G(L) overexpression, glycogen synthase activity is increased by insulin treatment, but at higher levels of G(L) expression, insulin is no longer required to achieve maximal synthase activity. In contrast, cells with high levels of PTG overexpression retain dose-dependent regulation of glycogen synthesis and glycogen synthase enzyme activity by insulin. 3) G(L)- and G(M)/R(Gl)-overexpressing cells exhibit a strong glycogenolytic response to forskolin, whereas PTG-overexpressing cells are less responsive. This difference may be explained in part by a lesser forskolin-induced increase in glycogen phosphorylase activity in PTG-overexpressing cells. Based on these results, we suggest that expression of either G(L) or G(M)/R(Gl) in liver of diabetic animals may represent a strategy for lowering of blood glucose levels in diabetes.
Highlights
An emerging concept of recent years is that glycogen metabolism in mammalian cells is regulated by familiar enzymes such as glycogen phosphorylase and glycogen synthase, and by a family of glycogen-targeting subunits of protein phosphatase-1 (PP1)1 [1,2,3,4,5,6,7,8,9]
Endogenous GL was detectable in untreated rat hepatocytes, whereas endogenous protein targeting to glycogen (PTG) was not detected, since amplification of PTG was performed with primers specific to the mouse cDNA, which is the form contained in the AdCMV-PTG virus
In cells incubated at 1 mM glucose and 1 nM insulin, we found that GL overexpression caused a 6.7-fold increase in glycogen synthase activity ratio relative to control hepatocytes, significantly greater than the 3.1- and 1.9-fold increases observed in cells overexpressing PTG or GM/RGl, respectively (Fig. 4A)
Summary
An emerging concept of recent years is that glycogen metabolism in mammalian cells is regulated by familiar enzymes such as glycogen phosphorylase and glycogen synthase, and by a family of glycogen-targeting subunits of protein phosphatase-1 (PP1)1 [1,2,3,4,5,6,7,8,9]. Increased expression of glycogentargeting subunits could conceivably represent an alternative strategy, since these proteins should enhance glucose disposal by activating glycogen synthesis. Consistent with this idea, we recently reported that adenovirus-mediated overexpression of PTG in hepatocytes isolated from fasted rats results in profound activation of glycogen synthesis [6]. The purpose of the current study was to investigate whether glycogen-targeting subunits of PP1 other than PTG might be more suitable candidates for improving hepatic glucose disposal This was accomplished by construction of recombinant adenoviruses containing the cDNAs encoding PTG, GL, and GM/RGl and comparison of the metabolic and regulatory properties of the expressed gene products in isolated rat hepatocytes
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