Mechanisms involved in the repression of human insulin gene promoter activity mediated by chronic hyperglycemia

Type II diabetes is characterized by insulin resistance and failure of pancreatic β-cells to secrete sufficient amounts of insulin to overcome hyperglycemia. Once diabetes is established, chronic hyperglycemia has been postulated to cause adverse alterations in β-cell function, thus exacerbating the disease state. Some of the characteristics of hyperglycemia-induced β-cell damage include both suppression of insulin gene expression and glucose-induced insulin secretion. The later is associated with decreased insulin gene promoter activity. Mechanisms accounting for chronic hyperglycemia-induced suppression of insulin promoter activity still remain unclear. Studies in other cell systems suggest that stress associated with hyperglycemia such as oxidation could be involved in tissue damage. Therefore, experiments were performed to understand the role of c-Jun N-terminal kinase (JNK), a stress-activated kinase, in chronic hyperglycemia-induced insulin promoter repression.; These studies demonstrated that JNK activity is significantly increased, in parallel with increased AP-1 transcription factor activity, in INS-1 cells cultured in 16.7 mM glucose compared to 4.0 mM glucose. Over-expression of JNK repressed insulin promoter activity in INS-1 cells cultured in 4.0 mM glucose. Over-expression of MLK3, an upstream activator of JNK, increased JNK activity, AP-1 transcription factor activity, and repressed insulin promoter activity in cells cultured in 4.0 mM glucose. These data show that activation of JNK either by high levels of glucose or upstream activators leads to functional activation of downstream JNK target proteins and causes reduced insulin promoter activity. These findings suggest that glucose-induced insulin promoter repression might be mediated by the JNK signaling pathway.; Pdx-1, an important regulatory transcription factor of insulin gene expression, partially mediates the repression of glucose-induced insulin promoter activity. Because over-expression of Pdx-1 in insulinoma cells cultured in high levels of glucose is insufficient to prevent insulin promoter repression, we further characterized other potential promoter targets. Truncation analysis and functional studies demonstrated that high levels of glucose mediate promoter repression between −327 and −261 nucleotides. Mobility shift assays showed that three glucose-sensitive complexes bind to the A5/Core, palindrome, and E3 elements, respectively within the insulin promoter. Site-specific mutations of all three elements in the insulin promoter gene partially prevented glucose-induced insulin promoter repression.; Collectively these studies suggest that elevated extracellular glucose mediates insulin promoter repression possibly through the JNK signaling pathway and through novel mechanisms that target the distal promoter region.