Glucose toxicity and paradoxical decrease in insulin gene transcription in β-TC6 cells

We have shown that chronic exposure of HIT cells to high glucose concentrations leads to decreased insulin intracellular content, insulin mRNA levels and insulin promoter activity. These changes are associated with decreased binding of two β-cell specific transcription factors, STF-1 and 3b1. This study was designed to determine whether these observations are specific to the HIT cell or can be generalized to other β-cell lines. βTC-6 cells were cultured from passage 39 to passage 72 in media containing either 0.8 mM or 11.1 mM glucose. βTC cells chronically cultured in 11.1 mM glucose demonstrated a passage-dependent decrease in insulin intracellular content (107,062 ± 8589 μU/mg protein vs 31,446 ± 8900 μU/mg protein, n=6, p<0.02) and insulin mRNA levels. In all instances, late passages of βTC cells chronically cultured in 0.8 mM glucose had higher insulin content (63,506 ± 14262 μU/mg protein vs. 37,683 ± 7624 μU/mg protein, n=5, p<0.02) and insulin mRNA levels (201 ± 44% of control, n=4) than late passage cells chronically cultured in 11.1 mM glucose. βTC cells were transfected with a chloramphenicol acetyl transferase reporter gene controlled by the 5′-regulatory region of the human insulin gene (INSCAT). Late passage βTC cells chronically cultured in 11.1 mM glucose only expressed 63 ± 5% (n=4) of the INSCAT activity found in early passages. In contrast, late passages of cells chronically cultured in 0.8 mM glucose expressed 108 ± 8% (n=4) of the INSCAT activity found in early passages. Electromobility shift assays demonstrated that binding of a specific nuclear protein that recognizes the 3b1 binding motif of the insulin gene was greatly diminished in late passage cells chronically exposed to 11.1 mM glucose. This binding activity was preserved in late passage cells chronically exposed to 0.8 mM glucose. We conclude that adverse effects of glucose toxicity on insulin gene expression can be explained by defective binding of the transactivacting factor 3b1 and that these findings are not peculiar to HIT cells.