GLYCATION-DEPENDENT, REACTIVE OXYGEN SPECIES-MEDIATED SUPPRESSION OF THE INSULIN GENE PROMOTER ACTIVITY IN HIT CELLS

Prolonged poor glycemic control in non-insulin-dependent diabetes mell itus patients often leads to a decline in insulin secretion from pancr eatic beta cells, accompanied by a decrease in the insulin content of the cells, As a step toward elucidating the pathophysiological backgro und of the so-called glucose toxicity to pancreatic beta cells, we ind uced glycation in HIT-TIS cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcrip tion. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control p-actin gene promoter; similar to 50 and 80% of the insulin gene promoter acti vity was lost when the cells were kept for 3 d in the presence of 40 a nd 60 mM D-ribose, respectively. In agreement with this, decrease in t he insulin mRNA and insulin content was observed in the glycation-indu ced cells, Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene trans cription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed al most irreversible but could be prevented by addition of 1 mM aminoguan idine or 10 mM N-acetylcysteine, thus suggesting that glycation and re active oxygen species, generated through the glycation reaction, serve as mediators of the phenomena, These observations suggest that protei n glycation in pancreatic beta cells, which occurs in vivo under chron ic hyperglycemia, suppresses insulin gene transcription and thus can e xplain part of the beta cell glucose toxicity.