CHRONIC EXPOSURE OF HIT CELLS TO HIGH GLUCOSE-CONCENTRATIONS PARADOXICALLY DECREASES INSULIN GENE-TRANSCRIPTION AND ALTERS BINDING OF INSULIN GENE REGULATORY PROTEIN

Chronically culturing HIT-T15 cells in media containing high glucose c oncentrations leads to decreased insulin mRNA levels, insulin content, and insulin secretion. These changes can be prevented by culturing th e cells in media containing lower glucose levels (Robertson, R. P., H. -J. Zhang, K. L. Pyzdrowski, and T. F. Walseth. 1992. J. Clin. Invest. 90:320-325). The mechanism of this seemingly paradoxical phenomenon w as examined by transiently transfecting HIT cells with a chloramphenic ol acetyl transferase (CAT) reporter gene controlled by the 5'-regulat ory domain of the human insulin gene (INSCAT). Early passages of HIT c ells readily expressed INSCAT, whereas late passages of cells chronica lly cultured in 11.1 mM glucose expressed only 28.7 +/- 2.3% (mean +/- SEM) of the CAT activity expressed in early passages. In contrast, la te passages of HIT cells chronically cultured in 0.8 mM glucose retain ed the ability to express the INSCAT reporter gene to 69.6 +/- 10.0% o f the CAT activity observed in early passages. The decrease in INSCAT expression in late passages of cells serially cultured in 11.1 mM gluc ose was associated with the inability to form a specific nuclear prote in-DNA complex with the CT motifs of the human insulin promoter. Forma tion of this specific protein-DNA complex was preserved in late passag es of HIT cells when serially cultured in 0.8 mM glucose. Mutations of the CT motifs caused markedly diminished CAT activity in all passages examined. These data indicate that chronic exposure of the beta cell to high glucose concentrations can paradoxically decrease insulin gene transcription, in part, by altering the ability of a regulatory prote in (GSTF) to interact with the insulin gene promoter. This provides a potential mechanism for glucotoxic effects on the beta cell at the lev el of the insulin gene.