Induction of glycation suppresses glucokinase gene expression in HIT-T15 cells

Aims/hypothesis. Chronic hyperglycaemia in patients with Type II (non-insul in-dependent) diabetes mellitus often leads to a decline in glucose-respons ive insulin secretion from pancreatic beta cells, a phenomenon called gluco se toxicity. Upon hyperglycaemia, glycation reaction occurs in the beta cel ls and induces oxidative stress. To understand the molecular basis of the b eta-cell glucose toxicity, we investigated the possible effects of glycatio n on the expression and enzymatic activity of glucokinase, which plays a cr ucial part in glucose-responsive insulin secretion. Methods. Glycation and reactive oxygen species were induced in HIT-T15 cell s by treatment with D-ribose and effects on glucokinase gene transcription, glucokinase protein amount, glucose phosphorylation activity, and DNA-bind ing activities of putative glucokinase gene transcription factors were eval uated. Results. When glycation was induced in HIT-T15 cells, the activity of the h uman glucokinase gene beta-cell-type promoter was suppressed substantially (83% reduction at 60 mmol/l D-ribose). Also, similar reductions in mRNA and protein amounts of glucokinase and in the Vmax of its enzymatic activity w ere observed. In agreement with the reduction in the promoter activity, the two major transcription factors of the glucokinase gene, the Pal-binding f actor and PDX-1, reduced their binding to their target sequences in the glu cokinase gene promoter in glycation-induced HIT cells. Because these effect s of D-ribose were counteracted by aminoguanidine or N-acetyl-cysteine, rea ctive oxygen species, generated by the glycation reaction, appears to be in volved in the phenomena. Conclusion/interpretation. The induction of the glycation reaction, which i s known to occur in pancreatic beta cells in chronic hyperglycaemia, suppre sses the glucokinase gene transcription and its enzymatic activity. Thus, h yperglycaemia-dependent inhibition of glucokinase activity could in part ex plain beta-cell glucose toxicity.