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 mellitus patients often leads to a decline in insulin secretion from pancreatic cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the socalled glucose toxicity to pancreatic cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D -ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control -actin gene promoter; 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D -ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycationinduced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D -ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the cell glucose toxicity. ( J. Clin. Invest.