Abstract
Although glucose is the major physiologic stimulator of insulin secretion and biosynthesis in vivo, prolonged exposure of pancreatic β-cells to high glucose levels is known to cause β-cell dysfunction. Histologically, such damaged β-cells often reveal extensive degranulation, thus suggesting impaired insulin biosynthesis as a cause of the phenomenon. In this study, we established an in vitro experimental model for the evaluation of glycation effects on the β-cell functions. Glycation was introduced into HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and the effects on insulin gene transcription were examined. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control β-actin gene promoter; approximately 50% and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 days in the presence of 40 mM 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/IPF 1/STF-1. These effect 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 mediator 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.
