Mutations in the hepatocyte nuclear factor (HNF)-1␣ gene have been linked to subtype 3 of maturity-onset diabetes of the young (MODY), a disease characterized by a primary defect in insulin secretion. Here we show that the human GLUT2 gene is closely regulated by HNF-1␣ via sequences downstream of the transcriptional start site by interaction with transcriptional co-activator p300. The promoter region of the human GLUT2 gene was subcloned into luciferase expression plasmids that were transfected together with HNF-1␣ expression plasmid into a pancreatic ␤-cell line, HIT-T15, to evaluate transcriptional activities. HNF-1␣ enhanced human GLUT2 promoter activity sixfold. Sitedirect mutagenesis and footprint analyses showed that the HNF-1␣ binding site (؉200 to ؉218) is critical in human GLUT2 gene expression. Furthermore, mammalian two-hybrid and immunoprecipitation studies revealed the transactivation domain of HNF-1␣ (amino acids 391-540) to interact with both the NH 2 -terminal region (amino acids 180 -662) and the COOH-terminal region (amino acids 1,818 -2,079) of p300. These findings demonstrated that HNF-1␣ binds to the 5-untranslated region of GLUT2 and that p300 acts as a transcriptional co-activator for HNF-1␣. In addition, these results provided new insight into the regulatory function of HNF-1␣ by suggesting a molecular basis for human GLUT2 gene expression.

We analyzed a mechanism of transcriptional regulation of the human insulin gene by cyclic AMP response element modulator (CREM) through four cyclic AMP response elements (CREs). We isolated two novel CREM isoforms (CREM⌬Q1 and CREM⌬Q2), which lack one of the glutamine-rich domains, Q1 and Q2 respectively, and six known isoforms (CREM␣, CREM␣, inducible cyclic AMP early repressor (

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Cytosolic Ca(2+) elevations are known to be involved in triggering apoptosis in many tissues, but the effect of sustained enhancement of Ca(2+) influx on apoptosis in beta cells remains unknown. We have found that the viability of RINm5F cells is decreased dose-dependently by continuous exposure to glibenclamide at concentrations from 10(-7) to 10(-4) M, and that this effect is partially ameliorated by pretreatment with cycloheximide. Electrophoresis of the cells exposed to glibenclamide revealed ladder-like fragmentation characteristic of apoptosis, and which also is suppressed by cycloheximide pretreatment. By using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, we detected increased DNA fragmentation in the nuclei of the cells exposed to glibenclamide, and staining with Hoechst 33342 and propidium iodide showed a dose-dependent increase in the number of cells with the chromatin condensation and fragmentation in their nuclei that is characteristic of apoptosis. The effects of glibenclamide on cell viability and apoptotic cell death were partially inhibited by treatment with Ca(2+) channel blocker, and by reducing the extracellular Ca(2+) concentration during glibenclamide exposure, suggesting that they may be derived from increased Ca(2+) influx. Furthermore, only the percentage of apoptotic cells, and not that of necrotic cells, increased with the increasing intracellular Ca(2+) concentration during glibenclamide exposure. In conclusion, we have demonstrated that the sustained enhancement of Ca(2+) influx caused by glibenclamide exposure can induce apoptotic cell death in a pure beta cell line.

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