Glucose 6-Phosphate Produced by Glucokinase, but Not Hexokinase I, Promotes the Activation of Hepatic Glycogen Synthase

Joan Seoane,Anna M Gómez-Foix,Robert M O'Doherty,Cristina Gómez-Ara,Christopher B Newgard,Joan J Guinovart

doi:10.1074/jbc.271.39.23756

Abstract

In a previous study (O'Doherty, R. M., Lehman, D. L., Seoane, J., Gómez-Foix, A. M., Guinovart, J. J., and Newgard, C.B. (1996) J. Biol. Chem. 271, 20524-20530), we demonstrated that adenovirus-mediated overexpression of glucokinase but not hexokinase I has a potent enhancing effect on glycogen synthesis in primary hepatocytes. In an effort to understand the underlying mechanism of this differential effect of the two hexokinase isoforms, we have investigated changes in key intracellular metabolites and the activation state of glycogen synthase in cells treated with recombinant adenoviruses expressing the liver isoform of glucokinase (AdCMV-GKL) or hexokinase I (AdCMV-HKI). Glucose 6-phosphate (Glu-6-P) levels are elevated from approximately 1.5 nmol/mg protein to 8-10 nmol/mg protein in both AdCMV-GKL- and AdCMV-HKI-treated hepatocytes as glucose is raised from 1 to 5 mM, levels four times higher than those in untreated cells. In AdCMV-GKL-treated cells, Glu-6-P continues to accumulate at glucose levels greater than 5 mM, reaching a maximum of 120 nmol/mg protein in cells incubated at 25 mM glucose, a value 10 and 50 times greater than the maximal levels achieved in AdCMV-HKI-treated and untreated cells, respectively. In parallel with the changes observed in Glu-6-P levels, increases in UDP-Glc in AdCMV-HKI- and AdCMV-GKL-treated cells were most pronounced at low (1-5 mM) and high (25 mM) glucose levels, respectively. Despite the significant increases in Glu-6-P and UDP-Glc achieved in AdCMV-HKI-treated cells, only AdCMV-GKL-treated cells exhibited increases in glycogen synthase activity ratio and translocation of the enzyme from a soluble to a particulate form relative to untreated control cells. We conclude that Glu-6-P produced by overexpressed glucokinase is glycogenic because it effectively promotes activation of glycogen synthase. Glu-6-P produced by overexpressed hexokinase, in contrast, appears to be unable to exert the same regulatory effects, probably due to the different subcellular distribution of the two glucose-phosphorylating enzymes.

Highlights

In mammals in the postabsorptive state, glycogen synthesis is potently activated in the liver in response to increased circulating glucose levels
When assayed by the radiometric method, the glucose phosphorylation capacity was found to be increased by 9- and 8-fold at 20 mM glucose and by 10- and 23-fold in 3 mM glucose in extracts from AdCMV-GKL- and AdCMV-hexokinase I (HKI)-treated hepatocytes, respectively, relative to extracts from untreated cells assayed at the same glucose concentrations
Untreated and transduced cells preincubated for 42 h at 1 mM glucose and transferred to media containing variable glucose concentrations in the range of 1–25 mM for 2 h had glucose 6-phosphate (Glu-6-P) levels that increased in a glucose concentration-dependent manner (Fig. 1)

Summary

Introduction

In mammals in the postabsorptive state, glycogen synthesis is potently activated in the liver in response to increased circulating glucose levels. Increases in the glycogen synthase activation state are proportional to the intracellular glucose 6-phosphate (Glu-6-P) level [7, 8]. These observations suggest that glucose phosphorylation is a key step in the activation of glycogen synthesis. The current study was undertaken to elucidate the mechanism of this surprising differential effect of the two glucose-phosphorylating enzymes, with particular emphasis on their impact on Glu-6-P levels and the activation state of glycogen synthase

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