Glucose-6-Phosphate-Mediated Activation of Liver Glycogen Synthase Plays a Key Role in Hepatic Glycogen Synthesis.

Alexander von Wilamowitz-Moellendorff, Roger Hunter, Mar Garcia-Rocha, Li Kang, Iliana Lopez-Soldado, Louise Lantier, Kashyap Patel, Mark Peggie, Carlos Martinez-Pons, Martin Voss, Joaquim Calbo, Patricia Cohen, David Wasserman, Joan Guinovart and Kei Sakamoto

Diabetes 2013.

Abstract

The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through GS kinase-3 and glycogen-associated form of protein phosphatase 1. Here we initially performed mutagenesis analysis and identified a key residue (Arg582) required for activation of GYS2 by G6P. We then employed GYS2 Arg582Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation has been profoundly impaired (60-70%), while sparing regulation through reversible phosphorylation. R582A-mutant-expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P towards glycolysis and lipid synthesis. GYS2(+/R582A) mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.

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The aim of the study was to provide evidence for the direct regulatory role of G6P on glycogen synthesis through the phosphorylation-mediated induction of GYS2 activity in the liver. The authors generated liver-specific glycogen synthase (GYS2) mutants containing Ala substitutions in the glucose-6-phosphate (G6P) phosphorylation regions. Compared to the wild type GYS2, targeted mutagenesis yielded several mutants which were insensitive to the phosphorylation-mediated activation of G6P. Specifically, the R582A mutation resulted in complete absence of glucosyltransferase activity in vitro, when lysates where treated with G6P. The authors generated GYS2 knockin mice GYS2 R582A /R582A which displayed normal growth curves and dramatically reduced hepatic glycogen levels. However, the 93% reduction in hepatic GYS2 expression in GYS2 R582A/R582A mice proved to be a limitation in this study, due to the presence of hypomorphic effect, rather than a true G6P-insensitivity being responsible for the 95% reduction in glycogen synthesis. Consequently, the GYS2 R582A/R582A mice represented a GYS2 knockout rather than a G6P-insensitive mouse model. Further analysis using the GYS2+/R582A mice showed a 60-70% reduction in sensitivity to the effects of phosphorylation-dependent activation by G6P. From the heterozygous GYS2+/R582A mice, the authors determined that the G6P-mediated activation of GYS2 is essential for in vivo hepatic glycogen synthesis. This was further substantiated by in vitro experiments using hepatocytes in which the G6P-insensitive GYS2+/R582A mutant did not yield increased glycogen synthesis under hyperglycemic conditions. This was partially reversed by the dephosphorylative-mediated effects of IPP (protein phosphatase-1)-PTG (Protein-Targeting to Glycogen) treatment resulting in robust increase in GYS2 activity. The authors postulated that glycogen itself may have a role in the stability of GYS2 and consequently significant reduction in glycogen synthesis in the GYS2 R582A/R582A mice is not reflective of a knockout phenotype.

Written by Nicole Poritsanos on Nov. 22, 2013.

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