Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells

Abstract

Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic β cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic β 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 β-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 glycation-induced 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 β cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the β cell glucose toxicity.