The effects of glucose and the hexosamine biosynthesis pathway on glycogensynthase kinase-3 and other protein kinases that regulate glycogen synthase activity

Background: Glycogen synthase (GS) activity is determined by its phosphoryl ation state. We have previously demonstrated that high glucose (HG) downreg ulates both basal and insulin-stimulated GS activity in rat-1 fibroblasts a nd that the hexosamine biosynthesis pathway (HBP) may be involved in mediat ing some of the effects of glucose. In this study we investigate the influe nce of high glucose and glucosamine (GlcN) on the activity of several kinas es that phosphorylate and inactivate GS. Methods: Glycogen synthase kinase (GSK) 3, cAMP-dependent protein kinase (P KA), protein kinase C (PKC), casein kinase (CK) 1, and phosphorylase kinase (PhK) activities were assayed in cellular extracts from control rat-1 fibr oblasts and those that overexpress human cDNA for glutamine:fructose 6-phos phate 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, wh en compared to activity at low glucose (LG, 1 mmol/L). The effects of HC on GSK-3 activity are greater in cells overexpressing GFA (38.8% increase). I nsulin (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 ac tion on both PKA and CK-1 activities. HG, GlcN, and GFA overexpression blun ted insulin's ability to downregulate PhK activity in LG conditions, PKC ac tivity is not significantly altered in either cell line in the above condit ions. Conclusions: These results suggest that HG alters both basal and insulin-re gulated activity of several kinases that phosphorylate GS, and some of the effects of glucose may be mediated by its metabolism via the HBP.