The effects of glucose and the hexosamine biosynthesis pathway on glycogen synthase kinase-3 and other protein kinases that regulate glycogen synthase activity.
BACKGROUND
Glycogen synthase (GS) activity is determined by its phosphorylation state. We have previously demonstrated that high glucose (HG) downregulates both basal and insulin-stimulated GS activity in rat-1 fibroblasts and that the hexosamine biosynthesis pathway (HBP) may be involved in mediating some of the effects of glucose. In this study we investigate the influence of high glucose and glucosamine (GlcN) on the activity of several kinases that phosphorylate and inactivate GS.


METHODS
Glycogen synthase kinase (GSK) 3, cAMP-dependent protein kinase (PKA), protein kinase C (PKC), casein kinase (CK) 1, and phosphorylase kinase (PhK) activities were assayed in cellular extracts from control rat-1 fibroblasts and those that overexpress human cDNA for glutamine:fructose 6-phosphate amidotransferase (GFA), the rate-limiting enzyme in the HBP.


RESULTS
Culturing rat-1 fibroblasts in HG (20 mmol/L) or GlcN (3-5 mmol/L) for 16-20 hours increases GSK-3 activity by 23.9 and 50%, respectively, when compared to activity at low glucose (LG, 1 mmol/L). The effects of HG on GSK-3 activity are greater in cells overexpressing GFA (38.8% increase). Insulin (1.7 nmol/L) treatment leads to a 20-25% decrease in GSK-3 activity that is not affected by HG, GlcN, or GFA overexpression. Culturing control cells in HG increases PKA and CK-1 activities by 56 and 95%, respectively, and HG diminishes insulin action on CK-1 activity. GlcN inhibits insulin action on both PKA and CK-1 activities. HG, GlcN, and GFA overexpression blunted insulin's ability to downregulate PhK activity in LG conditions. PKC activity is not significantly altered in either cell line in the above conditions.


CONCLUSIONS
These results suggest that HG alters both basal and insulin-regulated activity of several kinases that phosphorylate GS, and some of the effects of glucose may be mediated by its metabolism via the HBP.
Department of Medicine, University of Mississippi Medical Center, Jackson 39216, USA.
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LP
Singh
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Crook
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D047388:Casein Kinases; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005944:Glucosamine / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D006006:Glycogen Synthase / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D006595:Hexosamines / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010764:Phosphorylase Kinase / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits
D006595:Hexosamines; D006006:Glycogen Synthase; D011494:Protein Kinases; D010764:Phosphorylase Kinase; D038341:Glycogen Synthase Kinases; D047388:Casein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D005947:Glucose; D005944:Glucosamine